Research ArticleStanniocalcin-1 Alleviates Contrast-Induced Acute KidneyInjury by Regulating Mitochondrial Quality Control via theNrf2 Pathway

Fei Zhao Li-Xin Feng Qian Liu Hong-Shen Wang Cheng-Yuan Tang Wei ChengYin-Hao Deng Xi Wu Ping Yan Xiang-Jie Duan Jin-Cheng Peng and Shao-Bin Duan

Department of Nephrology The Second Xiangya Hospital Central South University Hunan Key Laboratory of Kidney Disease andBlood Purification Changsha Hunan 410011 China

Correspondence should be addressed to Shao-Bin Duan duansb528csueducn

Received 27 December 2019 Revised 27 February 2020 Accepted 10 March 2020 Published 31 March 2020

Academic Editor Viacutector M Mendoza-Nuacutentildeez

Copyright copy 2020 Fei Zhao et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Contrast-induced acute kidney injury (CI-AKI) is the third common cause of acute kidney injury (AKI) which is associated withpoor short- and long-term outcomes Currently effective therapy strategy for CI-AKI remains lacking Stanniocalcin-1 (STC1) is aconserved glycoprotein with antiapoptosis and anti-inflammatory functions but the role of STC1 in controlling CI-AKI isunknown Here we demonstrated a protective role of STC1 in contrast-induced injury in cultured renal tubular epithelial cellsand CI-AKI rat models Recombinant human STC1 (rhSTC1) regulated mitochondrial quality control thus suppressingcontrast-induced mitochondrial damage oxidative stress inflammatory response and apoptotic injury Mechanisticallyactivation of the Nrf2 signaling pathway contributes critically to the renoprotective effect of STC1 Together this studydemonstrates a novel role of STC1 in preventing CI-AKI and reveals Nrf2 as a molecular target of STC1 Therefore this studyprovides a promising preventive target for the treatment of CI-AKI

1 Introduction

Acute kidney injury has become a worldwide public healthproblem [1] The third most common cause of AKI is con-trast medium-induced acute kidney injury which happensto 30 of patients who were administered intravascularlywith contrast media [2] CI-AKI is characterized by a sharpdecrease in glomerular filtration rate (GFR) followed by anincrease in serum creatinine concentration or oliguria afterintravascular administration of contrast media [3 4] A largeand growing body of researches show that CI-AKI is relatedto serious short- and long-term adverse outcomes such asshort-term mortality prolonged hospitalization increasedhospital-related costs and chronic kidney disease risk [4 5]

Many pathophysiological processes contribute to thedevelopment of CI-AKI such as renal medullary ischemiadirect tubular cytotoxicity of contrast overproduction ofreactive oxygen species (ROS) mitochondrial damage andmitophagy [1 6ndash9] Most guidelines suggest that expanding

intravascular volume is more reasonable in preventing CI-AKI [10 11] A recent study shows that no prophylaxis isnoninferior and cost-saving in preventing CI-AKI comparedwith a common intravenous hydration prevention strategy[12] To date no optional pharmaceutical has been demon-strated to effectively prevent or treat CI-AKI

STC1 an evolutionarily conserved glycoprotein is ubiq-uitously expressed in some tissues such as the kidney [13]STC1 is considered to function in an autocrineparacrine orbloodborne manner [14ndash16] Then it targets the inner mito-chondrial membrane and more than 90 of cellular STC1immunoreactivity is in mitochondria [17] Evidences suggestthat mammalian STC-1 has various biological effects on theprevention of cell ischemia [18] suppression of inflamma-tory and reduction of ROS generation apoptosis and necro-sis [19] Moreover emerging evidences present that STC1can function as an intracrine factor targeting and workingin the mitochondria [18] Exogenous STC1 can be internal-ized quickly by the mitochondria in inflammatory cells

HindawiOxidative Medicine and Cellular LongevityVolume 2020 Article ID 1898213 17 pageshttpsdoiorg10115520201898213

[19] Therefore STC1 may be a potential therapeutic path-way for example through acting directly on the mitochon-dria of the kidney

In the present study CI-AKI animal and cell injurymodels were used to clarify the protective role of STC1 incontrast-induced damage to renal tubular epithelial cellsAdditionally we explored the molecular mechanisms aboutwhether the STC1-Nrf2 pathway participated in this protec-tive role by regulating the imbalance of mitochondrial qualitycontrol The results of this study present that the STC1-Nrf2pathway contributes to resisting contrast medium-inducedacute kidney injury through downregulating mitochondrialdamage suppressing oxidant stress and inhibiting inflam-mation and apoptosis in the kidney

2 Methods

21 Cell Culture Studies The human proximal tubular celllines (HK-2 cells) were treated with rhSTC1 (50ngml or100ngml) or (and) TBHQ (40ngml) or (and) ML385(10μM) at the same time of iohexol (GE Healthcare Shang-hai China) intervention to illuminate the effects of rhSTC1on mitochondrial damage ROS inflammation and cell apo-ptosis rhSTC1 (Cat No 9400-SO-050) was purchased fromRampD Systems (Minneapolis MN USA) Diluted by usingdistilled water rhSTC1 protein was produced at a final con-centration of 100μgml Tert-butylhydroquinone (TBHQ)and ML385 were purchased from Selleck (State of TexasUSA) STC1 siRNA and negative siRNA control were trans-fected into the HK-2 cells through using the Lipofectamine2000 reagent (Life Technologies USA)

22 Animal Experimental Design Before our studies maleSprague-Dawley rats were maintained for 7 days to acclima-tize according to the animal care rules of the Second XiangyaHospital of Central South University The rats were dividedinto four groups control group (n = 6) CI-AKI group(n = 6) CI-AKI group receiving a tail vein injection ofrhSTC1 (5μl) (n = 6 rhSTC1 05μgrat) and CI-AKI groupreceiving a tail vein injection of rhSTC1 (10μl) (n = 6rhSTC1 1μgrat) The CI-AKI rat model was built accordingto our previous article [6] The rhSTC1 is a human-derivedrecombinant protein but its function has been testified inother previous studies [20 21] The nonionic low-osmolarcontrast medium iohexol (GE Healthcare Shanghai China)was administrated by intravenous injection via the tail veinfurosemide (Shanghai China) was intramuscularly injectedas previously described In brief all rats were dehydratedfor 48 h then received furosemide (10mlkg) 30 minutesbefore iohexol (15mlkg) injection and the rhSTC1 wasadded at the same time of iohexol injection Twenty-fourhours later CI-AKI rat models were built The control groupdid not receive iohexol injection

23 Assessment of Renal Function and Histopathology Waysof measuring blood urea nitrogen (BUN) serum creatinine(Scr) and renal pathology were described in our recentstudies [6] Briefly kidney tissue was fixed and embeddedand tissue sections (4μm thick) were then stained with

hematoxylin-eosin for histopathological analysis Forsemiquantitative analysis of the changes of kidney tissue15 high-magnification (times200) fields of the cortex and outermedulla were selected randomly The specimens werescored as previously described [22] no injury (0) mild lt25 (1) moderate lt 50 (2) severe lt 75 (3) and verysevere gt 75 (4)

24 dUTP Nick-End Labeling Assay The terminal deoxynu-cleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay was performed with a commercial kit (In SituCell Death Detection Kit Roche Basel Switzerland) to detectDNA strand breaks according to the manufacturerrsquos proto-col The number of TUNEL-positive and total cells in differ-ent kidney sections was counted in five different views persection by using a fluorescence microscope (MoticBA410E) TUNEL-positive cells were expressed as a percent-age of total cells

25 Western Blotting Cells or renal cortical tissues (n = 3from each group) were isolated as described previously[6 7] Nuclear and cytoplasm protein were extractedaccording to the operating instructions Protein concentrationwas measured by using the BCA Protein Assay Reagent Kit(Beyotime Institute of Biotechnology Shanghai China) Equalamounts of proteins were separated on SDS-polyacrylamidegel before transferred to nitrocellulose membranes thenprobed with primary antibodies against cleaved caspase 3LC3II (1 1000 Cell Signaling Technology Boston USA)nuclear factor- (erythroid-derived 2-) like 2 (Nrf2) Kelch-like erythroid cell-derived protein with CNC homology-(ECH-) associated protein1 (Keap1) TOMM20 (1 1000Proteintech Chicago USA) [NOD]-like pyrin domain con-taining protein 3 (NLRP3) dynamin-related protein 1(Drp1) heme oxygenase-1 (HO-1) PTEN-induced putativekinase 1 (PINK1) inflammasome and high mobility groupbox 1 (HMGB1) mitofusin-2 (mfn2) RBR E3 ubiquitin pro-tein ligase (Parkin) sequestosome 1 (P62) (1 1000 AbcamCambridge UK) and β-actin (1 1000 Proteintech ChicagoUSA) then horseradish peroxidase-conjugated secondaryantibodies (1 5000 Proteintech Chicago USA) was used assecondary antibody Tanon 5200 Multi image analysis soft-ware (Tanon Shanghai China) was used to analyze theresults

26 Immunohistochemistry Staining For immunohistochem-istry staining kidney tissues were fixed deparaffinizedblocked and reduced nonspecific binding by 01 TritonX-100 The slides were then incubated with primary antibod-ies at 4degC overnight and counteracted with HRP-conjugatedsecondary antibody for 1 hour at room temperature Thecolor was developed with a DAB kit (Beijing ZhongshanJinqiao Biotechnology ZLI-9018)

27 Quantification of Mitochondrial DNA Content Renaltissues were separately collected from the control group theCI-AKI group and the rhSTC1 treatment group mtDNAwas extracted and measured as previously described [6]Plasma DNA from rats of each group was collected by usingthe QIAamp DNA Mini and Blood Mini Kit (Qiagen

2 Oxidative Medicine and Cellular Longevity

Germantown MD) and plasma mtDNA were calculated byquantitative PCR through a LightCycler480 System withSYBR Premix Ex Taq II (Takara Japan) The relative abun-dances of plasma mtDNA were indicated as threshold cycles(Tc) And higher Tc represents lower levels of mtDNA

28 Determination of Reactive Oxygen SpeciesMitochondrialROS in HK-2 cells was evaluated by using MitoSOX Red(Invitrogen Corporation USA) as previously described [7]according to the manufacturerrsquos instruction Briefly cellsgrowing in cell culture plates were rinsed and incubated inMitoSOX (Invitrogen Corporation USA) for 8 minutes at37degC in the dark DAPI (Invitrogen Corporation USA) wasused to label nucleus Dihydroethidium (DHE) (ThermoFisher Scientific D11347) was used to stain ROS in kidneytissues The kidney was cut and rinsed in cold PBS and thenwas placed in Tissue-Tek optimal cutting temperature com-pound followed by snapping frozen in liquid nitrogenTwenty μm thick kidney slices isolated freshly were incu-bated in 10μM DHE in a humidifieddark chamber at 37degCfor half an hour and then counterstained with DAPI (Invitro-gen Corporation USA) The distribution of ROS was mea-sured by a confocal laser scanning microscope LSM780(Carl Zeiss Jena German) In order for quantification fluo-rescent density within 10 random optical sections was deter-mined with ImageJ software

29 Transmission Electron Microscope Analysis (TEM)HK-2cells were treated as mentioned above Then the cells werecollected and undergone dehydration osmosis embeddingsectioning and staining as previously described [7] Typicalimages were captured by a Hitachi H7700 electronmicroscope

210 Immunofluorescence Colocalization Analysis Mito-chondrial morphology and mitophagy were evaluated as pre-viously described [7] The cells incubating in MitoTrackerRed for 8min then were washed fixed rinsed andpermeabilized 1 BSA was added to block the cells for 60minutes at room temperature and the cells then were incu-bated with the primary antibodies anti-LC3II (1 100) anti-Drp1 (1 100) anti-dsDNA antibody (1 100) and anti-Nrf2(1 100) and secondary antibodies conjugated with FITCin vitro DAPI (Invitrogen Corporation USA) was stained inthe dark Significant images were measured by a confocal laserscanning microscope LSM780 (Carl Zeiss Jena German)

211 Statistical Analysis Statistical analysis was conducted byusing the GraphPad Prism and SPSS 190 software All quan-titative variables were presented as themean plusmn standard errorof mean (mean plusmn SE) and were compared by using a pairedor unpaired Studentrsquos t-test or a one-way analysis of variance(ANOVA) followed by the LSD test for post hoc compari-sons A p value of lt005 was considered significant

3 Results

31 Iohexol Induces Cytotoxicity and MitochondrialDysfunction In Vitro In order to maintain mitochondrialnetwork balance mitochondria undergo unceasing fission

and fusion processes which are regulated by profission pro-teins (Drp1) and profusion proteins (mfn2) Dysfunctionalmitochondria exhibit fragmentation increased the expres-sion of Drp1 and ROS and decreased the expression ofmfn2 As we previously reported iohexol could induce adecrease in cell viability and an increase in ROS generation[7] As shown in Figure 1 we further demonstrated thatiohexol induced the increased expression of Drp1 andcleaved caspase 3 and decreased expression of mfn2 Addi-tionally iohexol treatment induced mitochondrial swellingmitochondrial fragmentation and mitophagy (Figure 1(e))These results indicated that iohexol led to mitochondrialdamage mitophagy and cell apoptosis

32 rhSTC1 Treatment Attenuates Inflammation Injury andMitochondrial ROS Generation in HK-2 CellsMitochondrialdysfunction leads to an increase in mitochondrial ROS gen-eration and the release of mitochondrial DNA (mtDNA)Extensive mtDNA lesions exacerbate mitochondrial oxida-tive stress and act as damage-associated molecular patternsto trigger the inflammatory response [9] To examinewhether exogenous rhSTC1 played a role in anti-inflammation and protected mitochondrial function in theHK-2 cells rhSTC1 was added into the HK-2 cells as anintervention at the same time of iohexol treatment As shownin Figure 2 iohexol treatment induced increased expressionof NLRP3 and HMGB1 (Figure 2(a)) overgeneration ofmitochondrial ROS (Figure 2(d)) and mitochondrial DNArelease (Figure 2(e)) Treatment with rhSTC1 could decreasethe expression of NLRP3 and HMGB1 generation of mito-chondrial ROS and mitochondrial DNA release Theseresults demonstrated that treatment with rhSTC1 signifi-cantly can suppress iohexol-induced inflammation and mito-chondrial ROS generation in HK-2 cells

33 rhSTC1 Treatment Reduces Mitochondrial Damage andMitophagy in HK-2 Cells after Iohexol Exposure As reportedin our previous studies [7] iohexol could induce mitochon-drial damage and mitophagy The colocalization of LC3IIwith mitochondria and the expression of P62 are usually usedto indicate mitophagy Mitophagy can be regulated by Drp1PINK1 and Parkin Evidences show that PINK1-Parkin-mediated mitophagy plays an important role in AKI [6 23]Our results showed that iohexol induced increased expres-sion of Drp1 PINK1 Parkin and LC3II and decreasedexpression of mfn2 TOMM20 and P62 which could bereversed by rhSTC1 treatment except the expression ofLC3II and P62 (Figures 3(a) and 3(b)) In addition the fluo-rescent confocal of LC3II-FITC or Drp1-FITC with Mito-Tracker Red was more significantly upregulated comparedwith the control group but rhSTC1 treatment dramaticallyreversed these increases (Figures 3(c) and 3(d)) We alsofound that iohexol led to abnormal mitochondrial morphol-ogy mitochondrial fragmentation and an increase in mito-phagy generation which could also be reversed by rhSTC1treatment (Figure 3(e)) These results suggested that rhSTC1treatment could alleviate mitochondrial damage and thusreduced mitophagy In addition there was no statistical

3Oxidative Medicine and Cellular Longevity

significance of different concentrations of rhSTC1 function-ing on HK-2 cells

34 Silencing STC1 Significantly Increases InflammationResponse and Mitochondrial Damage in HK-2 Cells In orderto further identify the role of STC1 in anti-inflammation andpreventing mitochondrial damage we observed the changesof inflammation and mitophagy-related proteins The abovestudy indicated no difference at different concentrations ofrhSTC1 in alleviating HK-2 cell injury Therefore 50 ngml

rhSTC1 was used to intervene in HK-2 cells after silencingSTC1 Our results showed that iohexol induced increasedexpression of Drp1 NLRP3 HMGB1 PINK1 Parkin andLC3II and decreased expression of mfn2 TOMM20 andP62 compared with the control group which were aggra-vated by silencing STC1 (Figures 4(a) and 4(b)) We alsofound that iohexol-induced mitochondrial fragmentationwas aggravated by silencing STC1 (Figure 4(c)) In sum theseresults indicated the anti-inflammation and protecting mito-chondrial role of STC1 on the opposite side

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Figure 1 Iohexol induces cytotoxicity andmitochondrial dysfunction inHK-2 cells HK-2 cells were treated with iohexol (200mg Iml) at indicatedtime points (a) Drp1 mfn2 and cleaved caspase 3 weremeasured byWestern blot assay (n = 3) Iohexol treatment caused a decreased expression ofmfn2 and increased expression of Drp1 and cleaved caspase 3 (bndashd) Quantification of average Western blot band intensities (e) Representativeimages HK-2 cells were treated with iohexol (200mg Iml) for 4 hours and then collected for transmission electron microscope analysisMitophagy was labeled with red arrow Values were presented as mean plusmn SE lowastp lt 005 compared with the control group

4 Oxidative Medicine and Cellular Longevity

35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells To exam-ine the effect of rhSTC1 in preventing cell apoptosis theexpression level of cleaved caspase 3 and apoptosis cells was

surveyed by Western blot and fluorescence microscopyIohexol induced more significantly increased expression ofcleaved caspase 3 compared with the control group which

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Figure 2 rhSTC1 treatment attenuates inflammation injury and mitochondrial ROS generation in HK-2 cells HK-2 cells were treated withiohexol (200mg Iml) with or without rhSTC1 at different concentrations (50 ngml 100 ngml) at indicated time courses respectively (a)Western blot analysis of the expression of HMGB1 and NLRP3 (n = 3) (b c) Quantification of average Western blot band intensities (dndashf)Representative images of mitochondrial ROS generation (d) and mitochondrial DNA release (e) (n = 3) DAPI staining was performed tolabel nuclear (blue) Mitochondrial ROS was labeled by MitoSOX (red) Mitochondrial DNA was labeled by the indicated antibody (green)Fluorescence images were taken by a LSM780 confocal microscope (magnification times630) Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group

5Oxidative Medicine and Cellular Longevity

was alleviated or aggravated by rhSTC1 treatment orsilencing STC1 respectively (Figures 5(a) and 5(b)) Asshown in Figures 5(c) and 5(d) iohexol markedly led tocell apoptosis which was further alleviated or aggravatedby rhSTC1 treatment or STC1 siRNA Similar resultswere obtained from the analysis of apoptosis rate(Figures 5(e)ndash5(h)) Taken together these results furtherindicated that rhSTC1 suppressed iohexol-induced apo-ptosis in HK-2 cells

36 The Protective Effects of rhSTC1 Treatment on RenalFunction and Pathological Injury We have already con-

structed a highly effectively novel CI-AKI rat model [6] Asshown in Figures 6(e) and 6(f) Scr and BUN significantlyincreased after iohexol injection which were partiallyrestored by rhSTC1 treatment Kidney histopathologicalchanges were also examined Iohexol treatment inducedmore serious detachment and foamy degeneration in tubularcells which were fewer in the rhSTC1 treatment group(Figures 6(b) and 6(d)) Furthermore the number of apopto-tic cells and expression of cleaved caspase 3 in the iohexolgroup were significantly higher compared with the controlgroup Clearly rhSTC1 treatment dramatically amelioratedthese changes (Figures 6(a) and 6(b))

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Figure 3 rhSTC1 treatment reduces mitochondrial damage and mitophagy in HK-2 cells after iohexol exposure (a b) Western blot analysisof the expression of mitophagy and mitophagy-related protein (n = 3) Cells were treated with iohexol (200mg Iml) with or without rhSTC1(50 ngml 100 ngml) at an indicated time course and then the whole cell lysates were collected for Western blot analysis (c d)Representative images HK-2 cells were exposed to iohexol (200mg iodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hoursand then were treated with LC3II-FITC or Drp1-FITC (green) and MitoTracker (red) respectively (n = 3) The distribution of LC3II-FITC Drp1-FITC and MitoTracker was analyzed by a confocal microscope Colocalization of LC3II-FITC or Drp1-FITC andMitoTracker was presented as overlapped red and green peaks (e) Representative images HK-2 cells were exposed to iohexol (200mgiodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hours and then were treated with MitoTracker (red) to label mitochondria(n = 3) or collected for electron micrograph analysis Mitophagy was labeled with red arrow (fndashl) Quantification of average Western blotband intensities (m n) Semiquantitative fluorescence analysis (o) Semiquantitative analysis of number of mitophagy Values werepresented as mean plusmn SE (n = 3) and n = 3 referred to number of replicates lowastp lt 005 compared with the control group p lt 005compared with the iohexol group

6 Oxidative Medicine and Cellular Longevity

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

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Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

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Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

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Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

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Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

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Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

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Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

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Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

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Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

Germantown MD) and plasma mtDNA were calculated byquantitative PCR through a LightCycler480 System withSYBR Premix Ex Taq II (Takara Japan) The relative abun-dances of plasma mtDNA were indicated as threshold cycles(Tc) And higher Tc represents lower levels of mtDNA

28 Determination of Reactive Oxygen SpeciesMitochondrialROS in HK-2 cells was evaluated by using MitoSOX Red(Invitrogen Corporation USA) as previously described [7]according to the manufacturerrsquos instruction Briefly cellsgrowing in cell culture plates were rinsed and incubated inMitoSOX (Invitrogen Corporation USA) for 8 minutes at37degC in the dark DAPI (Invitrogen Corporation USA) wasused to label nucleus Dihydroethidium (DHE) (ThermoFisher Scientific D11347) was used to stain ROS in kidneytissues The kidney was cut and rinsed in cold PBS and thenwas placed in Tissue-Tek optimal cutting temperature com-pound followed by snapping frozen in liquid nitrogenTwenty μm thick kidney slices isolated freshly were incu-bated in 10μM DHE in a humidifieddark chamber at 37degCfor half an hour and then counterstained with DAPI (Invitro-gen Corporation USA) The distribution of ROS was mea-sured by a confocal laser scanning microscope LSM780(Carl Zeiss Jena German) In order for quantification fluo-rescent density within 10 random optical sections was deter-mined with ImageJ software

29 Transmission Electron Microscope Analysis (TEM)HK-2cells were treated as mentioned above Then the cells werecollected and undergone dehydration osmosis embeddingsectioning and staining as previously described [7] Typicalimages were captured by a Hitachi H7700 electronmicroscope

210 Immunofluorescence Colocalization Analysis Mito-chondrial morphology and mitophagy were evaluated as pre-viously described [7] The cells incubating in MitoTrackerRed for 8min then were washed fixed rinsed andpermeabilized 1 BSA was added to block the cells for 60minutes at room temperature and the cells then were incu-bated with the primary antibodies anti-LC3II (1 100) anti-Drp1 (1 100) anti-dsDNA antibody (1 100) and anti-Nrf2(1 100) and secondary antibodies conjugated with FITCin vitro DAPI (Invitrogen Corporation USA) was stained inthe dark Significant images were measured by a confocal laserscanning microscope LSM780 (Carl Zeiss Jena German)

211 Statistical Analysis Statistical analysis was conducted byusing the GraphPad Prism and SPSS 190 software All quan-titative variables were presented as themean plusmn standard errorof mean (mean plusmn SE) and were compared by using a pairedor unpaired Studentrsquos t-test or a one-way analysis of variance(ANOVA) followed by the LSD test for post hoc compari-sons A p value of lt005 was considered significant

3 Results

31 Iohexol Induces Cytotoxicity and MitochondrialDysfunction In Vitro In order to maintain mitochondrialnetwork balance mitochondria undergo unceasing fission

and fusion processes which are regulated by profission pro-teins (Drp1) and profusion proteins (mfn2) Dysfunctionalmitochondria exhibit fragmentation increased the expres-sion of Drp1 and ROS and decreased the expression ofmfn2 As we previously reported iohexol could induce adecrease in cell viability and an increase in ROS generation[7] As shown in Figure 1 we further demonstrated thatiohexol induced the increased expression of Drp1 andcleaved caspase 3 and decreased expression of mfn2 Addi-tionally iohexol treatment induced mitochondrial swellingmitochondrial fragmentation and mitophagy (Figure 1(e))These results indicated that iohexol led to mitochondrialdamage mitophagy and cell apoptosis

32 rhSTC1 Treatment Attenuates Inflammation Injury andMitochondrial ROS Generation in HK-2 CellsMitochondrialdysfunction leads to an increase in mitochondrial ROS gen-eration and the release of mitochondrial DNA (mtDNA)Extensive mtDNA lesions exacerbate mitochondrial oxida-tive stress and act as damage-associated molecular patternsto trigger the inflammatory response [9] To examinewhether exogenous rhSTC1 played a role in anti-inflammation and protected mitochondrial function in theHK-2 cells rhSTC1 was added into the HK-2 cells as anintervention at the same time of iohexol treatment As shownin Figure 2 iohexol treatment induced increased expressionof NLRP3 and HMGB1 (Figure 2(a)) overgeneration ofmitochondrial ROS (Figure 2(d)) and mitochondrial DNArelease (Figure 2(e)) Treatment with rhSTC1 could decreasethe expression of NLRP3 and HMGB1 generation of mito-chondrial ROS and mitochondrial DNA release Theseresults demonstrated that treatment with rhSTC1 signifi-cantly can suppress iohexol-induced inflammation and mito-chondrial ROS generation in HK-2 cells

33 rhSTC1 Treatment Reduces Mitochondrial Damage andMitophagy in HK-2 Cells after Iohexol Exposure As reportedin our previous studies [7] iohexol could induce mitochon-drial damage and mitophagy The colocalization of LC3IIwith mitochondria and the expression of P62 are usually usedto indicate mitophagy Mitophagy can be regulated by Drp1PINK1 and Parkin Evidences show that PINK1-Parkin-mediated mitophagy plays an important role in AKI [6 23]Our results showed that iohexol induced increased expres-sion of Drp1 PINK1 Parkin and LC3II and decreasedexpression of mfn2 TOMM20 and P62 which could bereversed by rhSTC1 treatment except the expression ofLC3II and P62 (Figures 3(a) and 3(b)) In addition the fluo-rescent confocal of LC3II-FITC or Drp1-FITC with Mito-Tracker Red was more significantly upregulated comparedwith the control group but rhSTC1 treatment dramaticallyreversed these increases (Figures 3(c) and 3(d)) We alsofound that iohexol led to abnormal mitochondrial morphol-ogy mitochondrial fragmentation and an increase in mito-phagy generation which could also be reversed by rhSTC1treatment (Figure 3(e)) These results suggested that rhSTC1treatment could alleviate mitochondrial damage and thusreduced mitophagy In addition there was no statistical

3Oxidative Medicine and Cellular Longevity

significance of different concentrations of rhSTC1 function-ing on HK-2 cells

34 Silencing STC1 Significantly Increases InflammationResponse and Mitochondrial Damage in HK-2 Cells In orderto further identify the role of STC1 in anti-inflammation andpreventing mitochondrial damage we observed the changesof inflammation and mitophagy-related proteins The abovestudy indicated no difference at different concentrations ofrhSTC1 in alleviating HK-2 cell injury Therefore 50 ngml

rhSTC1 was used to intervene in HK-2 cells after silencingSTC1 Our results showed that iohexol induced increasedexpression of Drp1 NLRP3 HMGB1 PINK1 Parkin andLC3II and decreased expression of mfn2 TOMM20 andP62 compared with the control group which were aggra-vated by silencing STC1 (Figures 4(a) and 4(b)) We alsofound that iohexol-induced mitochondrial fragmentationwas aggravated by silencing STC1 (Figure 4(c)) In sum theseresults indicated the anti-inflammation and protecting mito-chondrial role of STC1 on the opposite side

Drp1mfn2

Cleaved caspase3

120573-Actin

0 h 2 h 4 h 6 hIohexol

83 kDa86 kDa19 kDa17 kDa43 kDa

(a)

0

1

2

3

4

5

Rela

tive

Drp

1120573

-act

in d

ensit

y

0 h 2 h 4 h 6 h

(b)

00

05

10

15

20

25

Rela

tive c

leav

edca

spas

e 3120573

-act

in d

ensit

y

0 h 2 h 4 h 6 h

(c)

0

1

2

3

4

5

6 hRela

tive m

fn2120573

-act

in d

ensit

y

0 h 2 h 4 h

(d)

1 120583m

1 120583m

Control

Iohexol

(e)

Figure 1 Iohexol induces cytotoxicity andmitochondrial dysfunction inHK-2 cells HK-2 cells were treated with iohexol (200mg Iml) at indicatedtime points (a) Drp1 mfn2 and cleaved caspase 3 weremeasured byWestern blot assay (n = 3) Iohexol treatment caused a decreased expression ofmfn2 and increased expression of Drp1 and cleaved caspase 3 (bndashd) Quantification of average Western blot band intensities (e) Representativeimages HK-2 cells were treated with iohexol (200mg Iml) for 4 hours and then collected for transmission electron microscope analysisMitophagy was labeled with red arrow Values were presented as mean plusmn SE lowastp lt 005 compared with the control group

4 Oxidative Medicine and Cellular Longevity

35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells To exam-ine the effect of rhSTC1 in preventing cell apoptosis theexpression level of cleaved caspase 3 and apoptosis cells was

surveyed by Western blot and fluorescence microscopyIohexol induced more significantly increased expression ofcleaved caspase 3 compared with the control group which

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

NLRP3

HMGB1

120573-Actin

118 kDa

25 kDa

43 kDa

(a)

02

00

04

06

Rela

tive N

LRP3

120573

-act

in d

ensit

y

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(b)

00

02

04

06

Rela

tive H

MG

B1

120573-a

ctin

den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(c)

Control Iohexol 50 ngml 100 ngmlIohexol +rhSTC1

DA

PIM

itoSO

XM

erge

10 120583m

(d)

DAPI MitoTracker DNA-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(e)

000510152025

Rela

tive M

itoSO

Xflu

ores

cenc

e den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(f)

Figure 2 rhSTC1 treatment attenuates inflammation injury and mitochondrial ROS generation in HK-2 cells HK-2 cells were treated withiohexol (200mg Iml) with or without rhSTC1 at different concentrations (50 ngml 100 ngml) at indicated time courses respectively (a)Western blot analysis of the expression of HMGB1 and NLRP3 (n = 3) (b c) Quantification of average Western blot band intensities (dndashf)Representative images of mitochondrial ROS generation (d) and mitochondrial DNA release (e) (n = 3) DAPI staining was performed tolabel nuclear (blue) Mitochondrial ROS was labeled by MitoSOX (red) Mitochondrial DNA was labeled by the indicated antibody (green)Fluorescence images were taken by a LSM780 confocal microscope (magnification times630) Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group

5Oxidative Medicine and Cellular Longevity

was alleviated or aggravated by rhSTC1 treatment orsilencing STC1 respectively (Figures 5(a) and 5(b)) Asshown in Figures 5(c) and 5(d) iohexol markedly led tocell apoptosis which was further alleviated or aggravatedby rhSTC1 treatment or STC1 siRNA Similar resultswere obtained from the analysis of apoptosis rate(Figures 5(e)ndash5(h)) Taken together these results furtherindicated that rhSTC1 suppressed iohexol-induced apo-ptosis in HK-2 cells

36 The Protective Effects of rhSTC1 Treatment on RenalFunction and Pathological Injury We have already con-

structed a highly effectively novel CI-AKI rat model [6] Asshown in Figures 6(e) and 6(f) Scr and BUN significantlyincreased after iohexol injection which were partiallyrestored by rhSTC1 treatment Kidney histopathologicalchanges were also examined Iohexol treatment inducedmore serious detachment and foamy degeneration in tubularcells which were fewer in the rhSTC1 treatment group(Figures 6(b) and 6(d)) Furthermore the number of apopto-tic cells and expression of cleaved caspase 3 in the iohexolgroup were significantly higher compared with the controlgroup Clearly rhSTC1 treatment dramatically amelioratedthese changes (Figures 6(a) and 6(b))

PINK1

Parkin

LC3I

IIp62

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

63 kDa

52 kDa16 kDa14 kDa

62 kDa

43 kDa

(a)

120573-Actin

Drp1

mfn2TOMM20

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

83 kDa86 kDa

20 kDa43 kDa

(b)

DAPI MitoTracker Drp1-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(c)

DAPI MitoTracker LC3II-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(d)

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

Mito-Tracker TEM

10 120583m 1 120583m

1 120583m

1 120583m

1 120583m

(e)

00

01

02

03

04

05

Rela

tive P

INK1

120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(f)

00

01

02

03

04

Rela

tive P

arki

n120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(g)

0

1

2

3

4

Rela

tive L

C3II

120573

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Figure 3 rhSTC1 treatment reduces mitochondrial damage and mitophagy in HK-2 cells after iohexol exposure (a b) Western blot analysisof the expression of mitophagy and mitophagy-related protein (n = 3) Cells were treated with iohexol (200mg Iml) with or without rhSTC1(50 ngml 100 ngml) at an indicated time course and then the whole cell lysates were collected for Western blot analysis (c d)Representative images HK-2 cells were exposed to iohexol (200mg iodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hoursand then were treated with LC3II-FITC or Drp1-FITC (green) and MitoTracker (red) respectively (n = 3) The distribution of LC3II-FITC Drp1-FITC and MitoTracker was analyzed by a confocal microscope Colocalization of LC3II-FITC or Drp1-FITC andMitoTracker was presented as overlapped red and green peaks (e) Representative images HK-2 cells were exposed to iohexol (200mgiodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hours and then were treated with MitoTracker (red) to label mitochondria(n = 3) or collected for electron micrograph analysis Mitophagy was labeled with red arrow (fndashl) Quantification of average Western blotband intensities (m n) Semiquantitative fluorescence analysis (o) Semiquantitative analysis of number of mitophagy Values werepresented as mean plusmn SE (n = 3) and n = 3 referred to number of replicates lowastp lt 005 compared with the control group p lt 005compared with the iohexol group

6 Oxidative Medicine and Cellular Longevity

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

mfn2Drp1

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HMGB1120573-Actin

siRNA control Control STC1Iohexol

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tive

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Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

Cleavedcaspase 3

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Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

Cleaved caspase 319 kDa

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Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

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Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

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Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

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Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

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998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

significance of different concentrations of rhSTC1 function-ing on HK-2 cells

34 Silencing STC1 Significantly Increases InflammationResponse and Mitochondrial Damage in HK-2 Cells In orderto further identify the role of STC1 in anti-inflammation andpreventing mitochondrial damage we observed the changesof inflammation and mitophagy-related proteins The abovestudy indicated no difference at different concentrations ofrhSTC1 in alleviating HK-2 cell injury Therefore 50 ngml

rhSTC1 was used to intervene in HK-2 cells after silencingSTC1 Our results showed that iohexol induced increasedexpression of Drp1 NLRP3 HMGB1 PINK1 Parkin andLC3II and decreased expression of mfn2 TOMM20 andP62 compared with the control group which were aggra-vated by silencing STC1 (Figures 4(a) and 4(b)) We alsofound that iohexol-induced mitochondrial fragmentationwas aggravated by silencing STC1 (Figure 4(c)) In sum theseresults indicated the anti-inflammation and protecting mito-chondrial role of STC1 on the opposite side

Drp1mfn2

Cleaved caspase3

120573-Actin

0 h 2 h 4 h 6 hIohexol

83 kDa86 kDa19 kDa17 kDa43 kDa

(a)

0

1

2

3

4

5

Rela

tive

Drp

1120573

-act

in d

ensit

y

0 h 2 h 4 h 6 h

(b)

00

05

10

15

20

25

Rela

tive c

leav

edca

spas

e 3120573

-act

in d

ensit

y

0 h 2 h 4 h 6 h

(c)

0

1

2

3

4

5

6 hRela

tive m

fn2120573

-act

in d

ensit

y

0 h 2 h 4 h

(d)

1 120583m

1 120583m

Control

Iohexol

(e)

Figure 1 Iohexol induces cytotoxicity andmitochondrial dysfunction inHK-2 cells HK-2 cells were treated with iohexol (200mg Iml) at indicatedtime points (a) Drp1 mfn2 and cleaved caspase 3 weremeasured byWestern blot assay (n = 3) Iohexol treatment caused a decreased expression ofmfn2 and increased expression of Drp1 and cleaved caspase 3 (bndashd) Quantification of average Western blot band intensities (e) Representativeimages HK-2 cells were treated with iohexol (200mg Iml) for 4 hours and then collected for transmission electron microscope analysisMitophagy was labeled with red arrow Values were presented as mean plusmn SE lowastp lt 005 compared with the control group

4 Oxidative Medicine and Cellular Longevity

35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells To exam-ine the effect of rhSTC1 in preventing cell apoptosis theexpression level of cleaved caspase 3 and apoptosis cells was

surveyed by Western blot and fluorescence microscopyIohexol induced more significantly increased expression ofcleaved caspase 3 compared with the control group which

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

NLRP3

HMGB1

120573-Actin

118 kDa

25 kDa

43 kDa

(a)

02

00

04

06

Rela

tive N

LRP3

120573

-act

in d

ensit

y

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(b)

00

02

04

06

Rela

tive H

MG

B1

120573-a

ctin

den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(c)

Control Iohexol 50 ngml 100 ngmlIohexol +rhSTC1

DA

PIM

itoSO

XM

erge

10 120583m

(d)

DAPI MitoTracker DNA-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(e)

000510152025

Rela

tive M

itoSO

Xflu

ores

cenc

e den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(f)

Figure 2 rhSTC1 treatment attenuates inflammation injury and mitochondrial ROS generation in HK-2 cells HK-2 cells were treated withiohexol (200mg Iml) with or without rhSTC1 at different concentrations (50 ngml 100 ngml) at indicated time courses respectively (a)Western blot analysis of the expression of HMGB1 and NLRP3 (n = 3) (b c) Quantification of average Western blot band intensities (dndashf)Representative images of mitochondrial ROS generation (d) and mitochondrial DNA release (e) (n = 3) DAPI staining was performed tolabel nuclear (blue) Mitochondrial ROS was labeled by MitoSOX (red) Mitochondrial DNA was labeled by the indicated antibody (green)Fluorescence images were taken by a LSM780 confocal microscope (magnification times630) Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group

5Oxidative Medicine and Cellular Longevity

was alleviated or aggravated by rhSTC1 treatment orsilencing STC1 respectively (Figures 5(a) and 5(b)) Asshown in Figures 5(c) and 5(d) iohexol markedly led tocell apoptosis which was further alleviated or aggravatedby rhSTC1 treatment or STC1 siRNA Similar resultswere obtained from the analysis of apoptosis rate(Figures 5(e)ndash5(h)) Taken together these results furtherindicated that rhSTC1 suppressed iohexol-induced apo-ptosis in HK-2 cells

36 The Protective Effects of rhSTC1 Treatment on RenalFunction and Pathological Injury We have already con-

structed a highly effectively novel CI-AKI rat model [6] Asshown in Figures 6(e) and 6(f) Scr and BUN significantlyincreased after iohexol injection which were partiallyrestored by rhSTC1 treatment Kidney histopathologicalchanges were also examined Iohexol treatment inducedmore serious detachment and foamy degeneration in tubularcells which were fewer in the rhSTC1 treatment group(Figures 6(b) and 6(d)) Furthermore the number of apopto-tic cells and expression of cleaved caspase 3 in the iohexolgroup were significantly higher compared with the controlgroup Clearly rhSTC1 treatment dramatically amelioratedthese changes (Figures 6(a) and 6(b))

PINK1

Parkin

LC3I

IIp62

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

63 kDa

52 kDa16 kDa14 kDa

62 kDa

43 kDa

(a)

120573-Actin

Drp1

mfn2TOMM20

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

83 kDa86 kDa

20 kDa43 kDa

(b)

DAPI MitoTracker Drp1-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(c)

DAPI MitoTracker LC3II-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

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Figure 3 rhSTC1 treatment reduces mitochondrial damage and mitophagy in HK-2 cells after iohexol exposure (a b) Western blot analysisof the expression of mitophagy and mitophagy-related protein (n = 3) Cells were treated with iohexol (200mg Iml) with or without rhSTC1(50 ngml 100 ngml) at an indicated time course and then the whole cell lysates were collected for Western blot analysis (c d)Representative images HK-2 cells were exposed to iohexol (200mg iodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hoursand then were treated with LC3II-FITC or Drp1-FITC (green) and MitoTracker (red) respectively (n = 3) The distribution of LC3II-FITC Drp1-FITC and MitoTracker was analyzed by a confocal microscope Colocalization of LC3II-FITC or Drp1-FITC andMitoTracker was presented as overlapped red and green peaks (e) Representative images HK-2 cells were exposed to iohexol (200mgiodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hours and then were treated with MitoTracker (red) to label mitochondria(n = 3) or collected for electron micrograph analysis Mitophagy was labeled with red arrow (fndashl) Quantification of average Western blotband intensities (m n) Semiquantitative fluorescence analysis (o) Semiquantitative analysis of number of mitophagy Values werepresented as mean plusmn SE (n = 3) and n = 3 referred to number of replicates lowastp lt 005 compared with the control group p lt 005compared with the iohexol group

6 Oxidative Medicine and Cellular Longevity

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

mfn2Drp1

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HMGB1120573-Actin

siRNA control Control STC1Iohexol

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tive

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Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

Cleavedcaspase 3

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Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

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Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

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Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

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Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

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Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

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HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells To exam-ine the effect of rhSTC1 in preventing cell apoptosis theexpression level of cleaved caspase 3 and apoptosis cells was

surveyed by Western blot and fluorescence microscopyIohexol induced more significantly increased expression ofcleaved caspase 3 compared with the control group which

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

NLRP3

HMGB1

120573-Actin

118 kDa

25 kDa

43 kDa

(a)

02

00

04

06

Rela

tive N

LRP3

120573

-act

in d

ensit

y

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(b)

00

02

04

06

Rela

tive H

MG

B1

120573-a

ctin

den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(c)

Control Iohexol 50 ngml 100 ngmlIohexol +rhSTC1

DA

PIM

itoSO

XM

erge

10 120583m

(d)

DAPI MitoTracker DNA-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(e)

000510152025

Rela

tive M

itoSO

Xflu

ores

cenc

e den

sity

Cont

rol

Iohe

xol

50 n

gm

l

100

ngm

l

Iohexol + rhSTC1

(f)

Figure 2 rhSTC1 treatment attenuates inflammation injury and mitochondrial ROS generation in HK-2 cells HK-2 cells were treated withiohexol (200mg Iml) with or without rhSTC1 at different concentrations (50 ngml 100 ngml) at indicated time courses respectively (a)Western blot analysis of the expression of HMGB1 and NLRP3 (n = 3) (b c) Quantification of average Western blot band intensities (dndashf)Representative images of mitochondrial ROS generation (d) and mitochondrial DNA release (e) (n = 3) DAPI staining was performed tolabel nuclear (blue) Mitochondrial ROS was labeled by MitoSOX (red) Mitochondrial DNA was labeled by the indicated antibody (green)Fluorescence images were taken by a LSM780 confocal microscope (magnification times630) Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group

5Oxidative Medicine and Cellular Longevity

was alleviated or aggravated by rhSTC1 treatment orsilencing STC1 respectively (Figures 5(a) and 5(b)) Asshown in Figures 5(c) and 5(d) iohexol markedly led tocell apoptosis which was further alleviated or aggravatedby rhSTC1 treatment or STC1 siRNA Similar resultswere obtained from the analysis of apoptosis rate(Figures 5(e)ndash5(h)) Taken together these results furtherindicated that rhSTC1 suppressed iohexol-induced apo-ptosis in HK-2 cells

36 The Protective Effects of rhSTC1 Treatment on RenalFunction and Pathological Injury We have already con-

structed a highly effectively novel CI-AKI rat model [6] Asshown in Figures 6(e) and 6(f) Scr and BUN significantlyincreased after iohexol injection which were partiallyrestored by rhSTC1 treatment Kidney histopathologicalchanges were also examined Iohexol treatment inducedmore serious detachment and foamy degeneration in tubularcells which were fewer in the rhSTC1 treatment group(Figures 6(b) and 6(d)) Furthermore the number of apopto-tic cells and expression of cleaved caspase 3 in the iohexolgroup were significantly higher compared with the controlgroup Clearly rhSTC1 treatment dramatically amelioratedthese changes (Figures 6(a) and 6(b))

PINK1

Parkin

LC3I

IIp62

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

63 kDa

52 kDa16 kDa14 kDa

62 kDa

43 kDa

(a)

120573-Actin

Drp1

mfn2TOMM20

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

83 kDa86 kDa

20 kDa43 kDa

(b)

DAPI MitoTracker Drp1-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(c)

DAPI MitoTracker LC3II-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(d)

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

Mito-Tracker TEM

10 120583m 1 120583m

1 120583m

1 120583m

1 120583m

(e)

00

01

02

03

04

05

Rela

tive P

INK1

120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(f)

00

01

02

03

04

Rela

tive P

arki

n120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(g)

0

1

2

3

4

Rela

tive L

C3II

120573

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Figure 3 rhSTC1 treatment reduces mitochondrial damage and mitophagy in HK-2 cells after iohexol exposure (a b) Western blot analysisof the expression of mitophagy and mitophagy-related protein (n = 3) Cells were treated with iohexol (200mg Iml) with or without rhSTC1(50 ngml 100 ngml) at an indicated time course and then the whole cell lysates were collected for Western blot analysis (c d)Representative images HK-2 cells were exposed to iohexol (200mg iodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hoursand then were treated with LC3II-FITC or Drp1-FITC (green) and MitoTracker (red) respectively (n = 3) The distribution of LC3II-FITC Drp1-FITC and MitoTracker was analyzed by a confocal microscope Colocalization of LC3II-FITC or Drp1-FITC andMitoTracker was presented as overlapped red and green peaks (e) Representative images HK-2 cells were exposed to iohexol (200mgiodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hours and then were treated with MitoTracker (red) to label mitochondria(n = 3) or collected for electron micrograph analysis Mitophagy was labeled with red arrow (fndashl) Quantification of average Western blotband intensities (m n) Semiquantitative fluorescence analysis (o) Semiquantitative analysis of number of mitophagy Values werepresented as mean plusmn SE (n = 3) and n = 3 referred to number of replicates lowastp lt 005 compared with the control group p lt 005compared with the iohexol group

6 Oxidative Medicine and Cellular Longevity

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

mfn2Drp1

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HMGB1120573-Actin

siRNA control Control STC1Iohexol

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tive

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Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

Cleavedcaspase 3

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Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

Cleaved caspase 319 kDa

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Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

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Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

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Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

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Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

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998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

was alleviated or aggravated by rhSTC1 treatment orsilencing STC1 respectively (Figures 5(a) and 5(b)) Asshown in Figures 5(c) and 5(d) iohexol markedly led tocell apoptosis which was further alleviated or aggravatedby rhSTC1 treatment or STC1 siRNA Similar resultswere obtained from the analysis of apoptosis rate(Figures 5(e)ndash5(h)) Taken together these results furtherindicated that rhSTC1 suppressed iohexol-induced apo-ptosis in HK-2 cells

36 The Protective Effects of rhSTC1 Treatment on RenalFunction and Pathological Injury We have already con-

structed a highly effectively novel CI-AKI rat model [6] Asshown in Figures 6(e) and 6(f) Scr and BUN significantlyincreased after iohexol injection which were partiallyrestored by rhSTC1 treatment Kidney histopathologicalchanges were also examined Iohexol treatment inducedmore serious detachment and foamy degeneration in tubularcells which were fewer in the rhSTC1 treatment group(Figures 6(b) and 6(d)) Furthermore the number of apopto-tic cells and expression of cleaved caspase 3 in the iohexolgroup were significantly higher compared with the controlgroup Clearly rhSTC1 treatment dramatically amelioratedthese changes (Figures 6(a) and 6(b))

PINK1

Parkin

LC3I

IIp62

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

63 kDa

52 kDa16 kDa14 kDa

62 kDa

43 kDa

(a)

120573-Actin

Drp1

mfn2TOMM20

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

83 kDa86 kDa

20 kDa43 kDa

(b)

DAPI MitoTracker Drp1-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(c)

DAPI MitoTracker LC3II-FITC Merge

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

10 120583m

(d)

Cont

rol

Iohe

xol

Iohe

xol+

rhST

C1(5

0 ng

ml)

Iohe

xol+

rhST

C1(1

00 n

gm

l)

Mito-Tracker TEM

10 120583m 1 120583m

1 120583m

1 120583m

1 120583m

(e)

00

01

02

03

04

05

Rela

tive P

INK1

120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(f)

00

01

02

03

04

Rela

tive P

arki

n120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(g)

0

1

2

3

4

Rela

tive L

C3II

120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(h)

0

1

2

3

Rela

tive P

62

β-ac

tin d

ensit

yControl Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(i)

00

05

10

15

20

25

Rela

tive D

rp1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(j)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

00

05

10

15

Rela

tive m

fn2

120573-a

ctin

den

sity

(k)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

00

05

10

15

20

Rela

tive T

OM

M20

120573

-act

in d

ensit

y

(l)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

0

10

20

30

40

50

Colo

caliz

atio

n co

effici

ent o

fLC

3II-

FITC

and

Mito

Trac

ker (

)

(m)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

0

20

40

60

80

100

Colo

caliz

atio

n co

effici

ent o

f D

rp1-

FITC

and

Mito

Trac

ker (

)

(n)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

0

10

20

30

40

Aut

opha

goso

mal

cont

aini

ngm

itoch

ondr

ia p

er 5

0 ce

ll se

ctio

ns (

)

(o)

Figure 3 rhSTC1 treatment reduces mitochondrial damage and mitophagy in HK-2 cells after iohexol exposure (a b) Western blot analysisof the expression of mitophagy and mitophagy-related protein (n = 3) Cells were treated with iohexol (200mg Iml) with or without rhSTC1(50 ngml 100 ngml) at an indicated time course and then the whole cell lysates were collected for Western blot analysis (c d)Representative images HK-2 cells were exposed to iohexol (200mg iodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hoursand then were treated with LC3II-FITC or Drp1-FITC (green) and MitoTracker (red) respectively (n = 3) The distribution of LC3II-FITC Drp1-FITC and MitoTracker was analyzed by a confocal microscope Colocalization of LC3II-FITC or Drp1-FITC andMitoTracker was presented as overlapped red and green peaks (e) Representative images HK-2 cells were exposed to iohexol (200mgiodineml) with or without rhSTC1 (50 ngml 100 ngml) for 4 hours and then were treated with MitoTracker (red) to label mitochondria(n = 3) or collected for electron micrograph analysis Mitophagy was labeled with red arrow (fndashl) Quantification of average Western blotband intensities (m n) Semiquantitative fluorescence analysis (o) Semiquantitative analysis of number of mitophagy Values werepresented as mean plusmn SE (n = 3) and n = 3 referred to number of replicates lowastp lt 005 compared with the control group p lt 005compared with the iohexol group

6 Oxidative Medicine and Cellular Longevity

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

mfn2Drp1

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Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

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Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

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Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

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Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

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Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

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Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

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Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

37 rhSTC1 Restores the Expression Level of Drp1 and mfn2and Reduces ROS Production In Vivo The expression degreesof Drp1 mfn2 and TOMM20 were assessed by using IHCand Western blot Drp1 expression was upregulated in thetubules of rat kidney while mfn2 and TOMM20 expressionwas downregulated compared with the control grouprhSTC1 treatment presented decreased expression of Drp1and increased expression of mfn2 and TOMM20 comparedwith the CI-AKI group (Figure 7(b)) The consistent results

were obtained by using Western blot assessment(Figure 7(a)) DHE assays exhibited significant increases inoxidative stress in the CI-AKI rat kidney tubular cells andrhSTC1 treatment significantly reduced these effects(Figure 7(b)) mtDNA copy numbers decreased in the CI-AKI kidney tissues and rhSTC1 treatment reversed thesechanges (Figure 7(g)) All the results suggested that rhSTC1played a major role in reducing tubular oxidative stress andmitochondrial damage in rat kidney

mfn2Drp1

TOMM20NLRP3

HMGB1120573-Actin

siRNA control Control STC1Iohexol

86 kDa83 kDa

20 kDa118 kDa25 kDa43 kDa

(a)

120573-Actin

ParkinPINK1LC3 I

II

p62

52 kDa63 kDa16 kDa14 kDa

62 kDa

43 kDa

siRNA control Control STC1Iohexol

(b)

siRNA control Control STC1Iohexol

10 120583m

(c)

00

05

10

15

siRNA Control Control STC1Iohexol

Rela

tive

mfn

2120573

-act

in d

ensit

y

(d)

siRNA Control Control STC1Iohexol

00

02

04

06

08

Rela

tive

Drp

1120573

-act

in d

ensit

y

(e)

siRNA control Control STC1Iohexol

0

1

2

3

4

Rela

tive

TOM

M20

120573-a

ctin

den

sity

(f)

siRNA control Control STC1Iohexol

00

05

10

15

20

Rela

tive

HM

GB1

120573-a

ctin

den

sity

(g)

siRNA control Control STC1Iohexol

00

01

02

03

04

Rela

tive

NLR

P3120573

-act

in d

ensit

y

(h)

siRNA control Control STC1Iohexol

00

05

10

15

20

25

Re

lativ

ePI

NK

1120573

-act

in d

ensit

y

(i)

siRNA control Control STC1Iohexol

00

05

10

15

20

Rela

tive

Park

in120573

-act

in d

ensit

y

(j)

siRNA control Control STC1Iohexol

00

05

10

15

Rela

tive

LC3I

I120573

-act

in d

ensit

y

(k)

siRNA control Control STC1Iohexol

0

1

2

3

4

5

Rela

tive

p62120573

-act

in d

ensit

y

(l)

Figure 4 Silencing STC1 significantly increases inflammation response and mitochondrial damage in HK-2 cells HK-2 cells were firstlytransfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 (50 ngml) Then the whole cell lysates were collected for Western blot analysis or fixed for confocal microscopyanalysis (a) Western blot analysis of the expression of inflammation and mitochondrial damage-associated proteins (n = 3) (b) Westernblot analysis of the expression of mitophagy-associated proteins (n = 3) (c) Representative images HK-2 cells were treated as mentionedabove MitoTracker (red) was used to label mitochondria (n = 3) (dndashl) Quantification of average Western blot band intensities Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

7Oxidative Medicine and Cellular Longevity

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

Cleavedcaspase 3

120573-Actin

19 kDa17 kDa

43 kDa

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(a)

Cleavedcaspase 3

120573-Actin

19 kDa17 kDa

43 kDa

siRNA control Control STC1Iohexol

(b)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

Light field

Hoechst

100 120583m

(c)

siRNA control Control STC1Iohexol

Light field

Hoechst

100 120583m

(d)

00

05

10

15

20

25

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

(e)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

siRNA control Control STC1Iohexol

(f)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

0

20

40

60

80

Apo

ptos

is (

)

(g)

siRNA control Control STC1Iohexol

0

20

40

60

80

100

Apo

ptos

is (

)

(h)

Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

Cleaved caspase 319 kDa

43 kDa

17 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

HE

Merge

TUNEL

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

0

1

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5

The s

core

of t

ubul

ar in

jury

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(d)

0

5

10

15

BUN

(mm

oll)

BaselineEnd

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(e)

0

10

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30

40

Sc

r (120583

mol

l)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

BaselineEnd

(f)

Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

Drp1

TOMM20

120573-Actin

86 kDa

83 kDa

20 kDa

43 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

DHE

Nucleus

Drp1

mfn2

TOMM20

100 120583m

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive m

fn2120573

-act

in d

ensit

y

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

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20

25

Rela

tive T

OM

M20

120573-a

ctin

den

sity

(d)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

01

02

03

04

05

Rela

tive D

rp1120573

-act

in d

ensit

y

(e)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

0

1

2

3

Rela

tive D

HE

dens

ity (f

old)

(f)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

Thre

shol

d cy

cle o

f pla

sma m

tDN

A

(g)

Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

LC3 III

p62

120573-Actin

63 kDa

52 kDa16 kDa14 kDa62 kDa

43 kDa

Control CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

(a)

Control CI-AKI 05 120583g 1 120583gHO-1Keap1

Nrf2HMGB1

120573-Actin

28 kDa70 kDa68 kDa25 kDa

43 kDa

Iohexol + rhSTC1

(b)

00

05

10

15

20

Rela

tive

PIN

K1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(c)

00

05

10

15

Rela

tive

Park

in120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(d)

0

1

2

3

4

Rela

tive

HM

GB1

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(e)

00

05

10

15

Rela

tive

HO

-1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(f)

00

02

04

06

08

10

Rela

tive

LC3I

I120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(g)

00

05

10

15

20

Rela

tive

p62120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(h)

00

05

10

15

20

Rela

tive

Nrf2

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(i)

00

01

02

03

04

05

Rela

tive

Keap

1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(j)

PINK1

Parkin

LC3 II

P62

Nrf2

Keap1

Control CI-AKI 05 120583g

Iohexol + rhSTC1

1 120583g

(k)

Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

38 Mitophagy-Associated Proteins and Keap1 and Nrf2Expression Levels Are Regulated by rhSTC1 in Rat KidneyThe mitophagy-associated proteins PINK1 Parkin P62and LC3II were also evaluated through IHC to identify thepotential effect of rhSTC1 on CI-AKI rat tubular cell mito-phagy Increased PINK1 Parkin and LC3II protein expres-sion levels and decreased expression of P62 were observedin the tubules of CI-AKI rat kidney All of those changes werereversed by rhSTC1 treatment (Figure 8(k)) except theexpression of LC3II and P62 Similar results were obtainedconcerning PINK1 Parkin LC3II and P62 demonstratedby Western blot assays (Figure 8(a)) Notably Nrf2 andHO-1 expression decreased and rhSTC1 administration

largely restored this change (Figures 8(b) and 8(k)) In con-trast the negative regulator of Nrf2 and Keap1 was upregu-lated in the CI-AKI rat kidney and significantly decreasedby rhSTC1 treatment (Figures 8(b) and 8(k)) These resultssuggested that rhSTC1 regulated mitochondrial functionand ROS generation in CI-AKI kidneys probably via theNrf2 pathways And in vivo we also revealed that two differ-ent dosages of rhSTC1 had similar effects in alleviating con-trast medium-induced kidney injury

39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic andMitochondrial Protective Role Partly by Upregulating theExpression of Nrf2 Although we have illuminated that

Cleavedcaspase 3

120573-Actin

19 kDa17 kDa

43 kDa

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

(a)

Cleavedcaspase 3

120573-Actin

19 kDa17 kDa

43 kDa

siRNA control Control STC1Iohexol

(b)

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

Light field

Hoechst

100 120583m

(c)

siRNA control Control STC1Iohexol

Light field

Hoechst

100 120583m

(d)

00

05

10

15

20

25

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

(e)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

siRNA control Control STC1Iohexol

(f)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

0

20

40

60

80

Apo

ptos

is (

)

(g)

siRNA control Control STC1Iohexol

0

20

40

60

80

100

Apo

ptos

is (

)

(h)

Figure 5 Antiapoptotic effects of rhSTC1 in HK-2 cells (a c and g) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hourswith or without rhSTC1 at different concentrations (50 ngml 100 ngml) (b d and h) HK-2 cells were firstly transfected with STC1-siRNAor control siRNA and 24 hours later these cells were treated with iohexol (200mg iodineml) for 4 hours (a b) Western blot analysis ofcleaved caspase 3 expression in the whole cells (n = 3) (c d) Representative images Cells were stained with Hoechst 33342 Cellular andnuclear morphology was recorded by phase-contrast and fluorescence microscopy (e f) Quantification of average Western blot bandintensities (g h) The percentage of apoptosis was estimated by counting the cells with typical apoptotic morphology Values werepresented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the iohexol group

8 Oxidative Medicine and Cellular Longevity

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

Cleaved caspase 319 kDa

43 kDa

17 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

HE

Merge

TUNEL

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

0

1

2

3

4

5

The s

core

of t

ubul

ar in

jury

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(d)

0

5

10

15

BUN

(mm

oll)

BaselineEnd

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(e)

0

10

20

30

40

Sc

r (120583

mol

l)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

BaselineEnd

(f)

Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

Drp1

TOMM20

120573-Actin

86 kDa

83 kDa

20 kDa

43 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

DHE

Nucleus

Drp1

mfn2

TOMM20

100 120583m

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive m

fn2120573

-act

in d

ensit

y

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

20

25

Rela

tive T

OM

M20

120573-a

ctin

den

sity

(d)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

01

02

03

04

05

Rela

tive D

rp1120573

-act

in d

ensit

y

(e)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

0

1

2

3

Rela

tive D

HE

dens

ity (f

old)

(f)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

Thre

shol

d cy

cle o

f pla

sma m

tDN

A

(g)

Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

LC3 III

p62

120573-Actin

63 kDa

52 kDa16 kDa14 kDa62 kDa

43 kDa

Control CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

(a)

Control CI-AKI 05 120583g 1 120583gHO-1Keap1

Nrf2HMGB1

120573-Actin

28 kDa70 kDa68 kDa25 kDa

43 kDa

Iohexol + rhSTC1

(b)

00

05

10

15

20

Rela

tive

PIN

K1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(c)

00

05

10

15

Rela

tive

Park

in120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(d)

0

1

2

3

4

Rela

tive

HM

GB1

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(e)

00

05

10

15

Rela

tive

HO

-1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(f)

00

02

04

06

08

10

Rela

tive

LC3I

I120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(g)

00

05

10

15

20

Rela

tive

p62120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(h)

00

05

10

15

20

Rela

tive

Nrf2

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(i)

00

01

02

03

04

05

Rela

tive

Keap

1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(j)

PINK1

Parkin

LC3 II

P62

Nrf2

Keap1

Control CI-AKI 05 120583g

Iohexol + rhSTC1

1 120583g

(k)

Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

rhSTC1 treatment ameliorated HK-2 cell mitochondrialdamage and apoptosis the specific mechanism was stillunclear It is also observed that the expression of Nrf2 andKeap1 played an important role in antioxidative responseWe assumed that Nrf2 might be a connecting tie betweenthe expression of STC1 and the happening of apoptosis andmitochondrial damage HO-1 a downstream effector mole-cule of Nrf2 also has an antioxidant effect As shown inFigure 9(a) iohexol treatment induced increased expressionof Keap1 and decreased expression of Nrf2 and HO-1 whichcould be reversed by rhSTC1 treatment Confocal micro-scopic images also revealed decreased Nrf2 (green) intensitycompared with the control group These effects were reversedby rhSTC1 treatment (Figure 9(d)) Additionally rhSTC1

treatment also induced increased expression of Nrf2 mRNA(Figure 9(i)) To further illustrate the relationship betweenthe protective effect of rhSTC1 and the expression of Nrf2Tert-butylhydroquinone (TBHQ) the inducer of Nrf2 activ-ity [24] was used to observe the expression of cleaved caspase3 and HO-1 by rhSTC1 treatment or transfection with STC1siRNA The results demonstrated that rhSTC1-mediatedprotection was not abrogated or enhanced by TBHQ treat-ment and TBHQ treatment could reverse the increasedexpression of cleaved caspase 3 and decreased expression ofHO-1 induced by STC1 siRNA treatment (Figures 9(b) and9(c)) A specific Nrf2 inhibitor ML385 was used to furtherlyilluminate the relationship between Nrf2 and STC1 Asshown in Figure 10(c) the expression of nuclear and

120573-Actin

Cleaved caspase 319 kDa

43 kDa

17 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

HE

Merge

TUNEL

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

0

1

2

3

4

5

The s

core

of t

ubul

ar in

jury

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(d)

0

5

10

15

BUN

(mm

oll)

BaselineEnd

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(e)

0

10

20

30

40

Sc

r (120583

mol

l)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

BaselineEnd

(f)

Figure 6 The protective effects of rhSTC1 on renal function and pathological injury in rat kidneys The way to build CI-AKI rat model wasdescribed in our previous article rhSTC1 at different concentrations (05μgrat 1μgrat) and iohexol (200mg iodineml) were administratedthrough the caudal vein at the same time (a) Western blot analysis of the expression of cleaved caspase 3 in the whole cells (n = 3) (b)Representative images of tubular cell injury in rat kidney tissue and immunofluorescent labeling for TUNEL in rat kidney tissue sectionsof the four groups (times200) Rat kidney tissue was stained for TUNEL (green) Nuclei were stained with DAPI (blue) (c) Quantification ofaverage Western blot band intensities (d) Quantitative analysis of histologic scoring (e f) Changes in the levels of Scr and BUN Valueswere presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

9Oxidative Medicine and Cellular Longevity

mfn2

Drp1

TOMM20

120573-Actin

86 kDa

83 kDa

20 kDa

43 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

DHE

Nucleus

Drp1

mfn2

TOMM20

100 120583m

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive m

fn2120573

-act

in d

ensit

y

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

20

25

Rela

tive T

OM

M20

120573-a

ctin

den

sity

(d)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

01

02

03

04

05

Rela

tive D

rp1120573

-act

in d

ensit

y

(e)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

0

1

2

3

Rela

tive D

HE

dens

ity (f

old)

(f)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

Thre

shol

d cy

cle o

f pla

sma m

tDN

A

(g)

Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

LC3 III

p62

120573-Actin

63 kDa

52 kDa16 kDa14 kDa62 kDa

43 kDa

Control CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

(a)

Control CI-AKI 05 120583g 1 120583gHO-1Keap1

Nrf2HMGB1

120573-Actin

28 kDa70 kDa68 kDa25 kDa

43 kDa

Iohexol + rhSTC1

(b)

00

05

10

15

20

Rela

tive

PIN

K1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(c)

00

05

10

15

Rela

tive

Park

in120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(d)

0

1

2

3

4

Rela

tive

HM

GB1

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(e)

00

05

10

15

Rela

tive

HO

-1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(f)

00

02

04

06

08

10

Rela

tive

LC3I

I120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(g)

00

05

10

15

20

Rela

tive

p62120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(h)

00

05

10

15

20

Rela

tive

Nrf2

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(i)

00

01

02

03

04

05

Rela

tive

Keap

1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(j)

PINK1

Parkin

LC3 II

P62

Nrf2

Keap1

Control CI-AKI 05 120583g

Iohexol + rhSTC1

1 120583g

(k)

Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

mfn2

Drp1

TOMM20

120573-Actin

86 kDa

83 kDa

20 kDa

43 kDa

CI-AKI 05 120583gIohexol + rhSTC1

1 120583gControl

(a)

CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

DHE

Nucleus

Drp1

mfn2

TOMM20

100 120583m

50 120583m

Control

(b)

00

05

10

15

20

Rela

tive m

fn2120573

-act

in d

ensit

y

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

(c)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

20

25

Rela

tive T

OM

M20

120573-a

ctin

den

sity

(d)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

01

02

03

04

05

Rela

tive D

rp1120573

-act

in d

ensit

y

(e)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

0

1

2

3

Rela

tive D

HE

dens

ity (f

old)

(f)

Control CI-AKIIohexol + rhSTC105 120583g 1 120583g

00

05

10

15

Thre

shol

d cy

cle o

f pla

sma m

tDN

A

(g)

Figure 7 rhSTC1 treatment restores the changes in Drp1 and mfn2 expression and reduces ROS production in the rat kidneys Rats weretreated as mentioned above (a) Western blot was used to detect the expression of Drp1 mfn2 and TOMM20 (n = 3) (b) Kidney sectionsfrom the mentioned groups were stained with Drp1 mfn2 and TOMM20 antibodies for IHC analysis (magnification times400) Oxidativestress in kidney tubular cells was assessed by using DHE (cndashe) Quantification of average Western blot band intensities (f) Quantificationof tissues stained with DHE (g) Plasma mitochondrial DNA Tc number decreased after iohexol treatment which was reversed byrhSTC1 Values were presented as mean plusmn SE lowastp lt 005 compared with the control group p lt 005 compared with the CI-AKI group

10 Oxidative Medicine and Cellular Longevity

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

LC3 III

p62

120573-Actin

63 kDa

52 kDa16 kDa14 kDa62 kDa

43 kDa

Control CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

(a)

Control CI-AKI 05 120583g 1 120583gHO-1Keap1

Nrf2HMGB1

120573-Actin

28 kDa70 kDa68 kDa25 kDa

43 kDa

Iohexol + rhSTC1

(b)

00

05

10

15

20

Rela

tive

PIN

K1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(c)

00

05

10

15

Rela

tive

Park

in120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(d)

0

1

2

3

4

Rela

tive

HM

GB1

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(e)

00

05

10

15

Rela

tive

HO

-1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(f)

00

02

04

06

08

10

Rela

tive

LC3I

I120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(g)

00

05

10

15

20

Rela

tive

p62120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(h)

00

05

10

15

20

Rela

tive

Nrf2

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(i)

00

01

02

03

04

05

Rela

tive

Keap

1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(j)

PINK1

Parkin

LC3 II

P62

Nrf2

Keap1

Control CI-AKI 05 120583g

Iohexol + rhSTC1

1 120583g

(k)

Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

cytoplasm Nrf2 decreased after iohexol treatment whichcould be restored by rhSTC1 treatment and furtherdecreased after rhSTC1 and ML385 intervention The roleof rhSTC1 in anti-inflammation (decreased expression ofNLRP3 and HMGB1) and antiapoptosis (decreased expres-sion of cleaved caspase 3) was partly abolished by inhibitingthe activity of Nrf2 (Figure 10(b)) Additionally treatmentwith rhSTC1 significantly alleviated mitochondrial swellingfragmentation vacuoles and loss of cristae by an electronmicroscope which was also partly counteracted by the inhi-bition of Nrf2 activity (Figure 10(a)) The changes of expres-sion level of mitochondrial damage associated protein (Drp1mfn2 and TOMM20) further presented that the protectiveeffect of rhSTC1 on mitochondria partly was associated withthe activation of Nrf2 (Figure 10(b)) All of these results indi-cated that rhSTC1 treatment shared some common protec-

tive ways with Nrf2 expression As mentioned the resultsdemonstrated that rhSTC1 could guard HK-2 cells againstcontrast medium-induced cytotoxicity partly through theactivation of Nrf2

4 Discussion

The pathophysiological effects of STC1 in CI-AKI remainunclear In this study we firstly demonstrated the protectiverole of exogenous rhSTC1 in the contrast-induced cell injuryand CI-AKI rat model Our researches indicated that rhSTC1treatment dramatically attenuated CI-AKI by reducing mito-chondrial damage reactive oxidative stress inflammationand cell apoptosis We further provided enough evidencesand demonstrated that the activation of Nrf2 signaling path-way contributed to the protective role of STC1 in

PINK1Parkin

LC3 III

p62

120573-Actin

63 kDa

52 kDa16 kDa14 kDa62 kDa

43 kDa

Control CI-AKI 05 120583gIohexol + rhSTC1

1 120583g

(a)

Control CI-AKI 05 120583g 1 120583gHO-1Keap1

Nrf2HMGB1

120573-Actin

28 kDa70 kDa68 kDa25 kDa

43 kDa

Iohexol + rhSTC1

(b)

00

05

10

15

20

Rela

tive

PIN

K1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(c)

00

05

10

15

Rela

tive

Park

in120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(d)

0

1

2

3

4

Rela

tive

HM

GB1

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(e)

00

05

10

15

Rela

tive

HO

-1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(f)

00

02

04

06

08

10

Rela

tive

LC3I

I120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(g)

00

05

10

15

20

Rela

tive

p62120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(h)

00

05

10

15

20

Rela

tive

Nrf2

120573-a

ctin

den

sity

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(i)

00

01

02

03

04

05

Rela

tive

Keap

1120573

-act

in d

ensit

y

Control CI-AKI 05 120583g 1 120583g

Iohexol + rhSTC1

(j)

PINK1

Parkin

LC3 II

P62

Nrf2

Keap1

Control CI-AKI 05 120583g

Iohexol + rhSTC1

1 120583g

(k)

Figure 8 Expression of mitophagy-associated proteins Keap1 and Nrf2 is regulated by rhSTC1 in rat kidney Rats were treated as mentionedabove (a b) Western blot was used to detect the expression of mitophagy-associated proteins Keap1 and Nrf2 (n = 3) (cndashj) Quantification ofaverage Western blot band intensities (k) Kidney sections from the mentioned groups were stained with PINK1 Parkin LC3II P62 Nrf2and Keap1 antibodies for IHC analysis (magnification times400) Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the CI-AKI group

11Oxidative Medicine and Cellular Longevity

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

HO-1

Keap1

Nrf2

Control Iohexol 50 ngml 100 ngmlIohexol + rhSTC1

120573-Actin

28 kDa

70 kDa

68 kDa

43 kDa

(a)

120573-Actin

HO-1

Cleaved caspase 3

43 kDa28 kDa

19 kDa

17 kDa

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Cleaved caspase 3

HO-1

120573-Actin

siRNA control Control STC1 STC1+TBHQIohexol

43 kDa

28 kDa

19 kDa

17 kDa

(c)

DAPI MergeNrf2-FITC

Control

Iohexol

Iohexol+rhSTC1(50 ngml)

Iohexol+rhSTC1(100 ngml)

10 120583m

(d)

0

1

2

3

Rela

tive H

O-1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(e)

00

02

04

06

08

10

Rela

tive K

eap1

120573-a

ctin

den

sity

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(f)

00

02

04

06

08

Rela

tive N

rf2120573

-act

in d

ensit

y

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

(g)

00

05

10

15

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

Rela

tive N

rf2-F

ITC

fluor

esce

nce i

nten

sity

(h)

Control Iohexol 50 ngml 100 ngml

Iohexol + rhSTC1

00

05

10

15

20

25

Nrf2

mRN

Aex

pres

sion

(rel

ativ

e to

cont

rol)

(i)

00

05

10

15

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

02

04

06

08

Rela

tive H

O-1

120573-a

ctin

den

sity

Iohx

ol+r

hSTC

1+T

BHQ

Iohe

xol+

TBH

Q

Iohexol+rhST

C1

Iohe

xol

Cont

rol

(k)

00

05

10

15

20

Rela

tive c

leav

ed ca

spas

e 3

120573-a

ctin

den

sity

998770

siRNA control Control STC1 STC1+TBHQ

Iohexol

(l)

siRNA control Control STC1 STC1+TBHQ

Iohexol

00

05

10

15

Rela

tive H

O-1

120573-a

ctin

den

sity

998770

(m)

Figure 9 STC1 plays an antiapoptotic role partly by upregulating the expression of Nrf2 (a) HK-2 cells were then exposed to iohexol (200mgiodineml) for 4 hours with or without rhSTC1 at different concentrations (50 ngml 100 ngml) Western blot analysis of Nrf2 Keap1 andHO-1 in the whole cells (n = 3) (b) HK-2 cells were then exposed to iohexol (200mg iodineml) for 4 hours with or without rhSTC1(50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase 3 and HO-1 in the whole cells (n = 3) (c) HK-2 cells were firstly transfected with STC1-siRNA or control siRNA and 24 hours later these cells were treated with iohexol (200mgiodineml) for 4 hours with or without rhSTC1 (50 ngml) or TBHQ (40 ngml) Western blot analysis of the expression of cleaved caspase3 and HO-1 in the whole cells (n = 3) (d) Representative images HK-2 cells were exposed to iohexol (200mg iodineml) for 4 hours andthen treated with Nrf2-FITC (green) to label the distribution of Nrf2 Fluorescence images were taken by a LSM780 confocal microscope(magnification times630) (e f h j l and m) Quantification of average Western blot band intensities (g) Semiquantitative fluorescenceanalysis (i) Expression of STC1 mRNA level Scale bar 10 μM Values were presented as mean plusmn SE lowastp lt 005 compared with the controlgroup p lt 005 compared with the iohexol group p lt 005 compared with the STC1-siRNA treatment group

12 Oxidative Medicine and Cellular Longevity

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

Control Iohexol Iohexol+rhSTC1 Iohexol+rhSTC1+ML385

1 120583m 1 120583m 1 120583m 1 120583m

(a)

mfn2Drp1

TOMM20

NLRP3

HMGB1

HO-1

Cleaved caspase 3

GAPDH

86 kDa

83 kDa

20 kDa

118 kDa

25 kDa

28 kDa

19 kDa17 kDa

36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(b)

Nuclear Nrf2

Lamin B1

Cytoplasm Nrf2

GAPDH

68 kDa

66 kDa

68 kDa36 kDa

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(c)

00

01

02

03

Rela

tive m

fn2

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Con

trol

(d)

00

01

02

03

04

Rela

tive D

rp1

GA

PDH

den

sity

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(e)

00

02

04

06

08

998770

Rela

tive T

OM

M20

G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(f)

000

005

010

015

020

025

Rela

tive N

LRP3

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(g)

00

05

10

15

Rela

tive H

MG

B1

GA

PDH

den

sity 998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(h)

Figure 10 Continued

13Oxidative Medicine and Cellular Longevity

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

ameliorating the contrast-induced cell injury and CI-AKI ratmodel Thus our results indicated exogenous STC1 had thepotential as a novel preventive agent in CI-AKI

The wide administration of iodinated contrast media inmedical practice remains a common cause of AKI and amajor reason of iatrogenic nephropathy [25] The impor-tance of preventing CI-AKI has been demonstrated byemerging studies showing a relation between CI-AKI andmitochondrial quality control Previous studies suggestedthat the imbalance of mitochondrial quality control (exces-sive ROS generation mitochondrial damage and mitophagy)played an important role in apoptosis induced by iodinatedcontrast media [6 7 23 26] However the detailed mecha-nism remains unknown It is also unclear whether inhibitingmitochondrial ROS generation and alleviating mitochondrialdamage can reverse tubular cell damage via regulating mito-chondrial quality control

STC1 is a stress gene responding to various stimulationssuch as hypoxia and cytokines [27 28] Some studies haveshown a protective role of STC1 in antiglomerular basementmembrane glomerulonephritis [19] and LPS-induced lunginjury models [21] Other studies also demonstrated thatoverexpression of STC1 could protect from hypoxia- orhypercalcemia-induced neuron injury [29] However therole of STC1 in CI-AKI is still unknown Our and other stud-ies showed that mitochondrial damage overgeneration ofmitochondrial ROS and inflammation played an importantrole in CI-AKI and uncontrolled oxidative stress could causesevere tissue damage In our present study treatment with

rhSTC1 could suppress mitochondrial damage and mito-chondrial ROS generation in the kidney in vivo and vitrosuggesting an antioxidative effect of rhSTC1 And inflamma-tion factors such as NLRP3 and HMBG1 could also decreasecaused by rhSTC1 treatment More importantly rhSTC1treatment could lead to a decrease in the expression ofcleaved caspase 3 and apoptosis in HK-2 cells and rat kidneySerum creatinine level and kidney histological changes inCI-AKI were attenuated by rhSTC1 treatment Takentogether our results firstly suggested that exogenousrhSTC1 attenuated contrast medium-induced acute kidneyinjury by suppressing the mitochondrial damage attenuat-ing inflammatory response and reducing cell apoptosis inthe kidney Moreover our study further demonstrated thattwo different concentrations of rhSTC1 showed similareffects on CI-AKI Therefore it will be more economical byusing fewer dosages of rhSTC1 to treat CI-AKI in the future

Meanwhile we found that rhSTC1 treatment caused adecrease in mitochondrial damage and further reduced mito-phagy It seems to contrast to our and other previous resultsthat PINK1-Parkin-mediated mitophagy plays a protectiverole in CI-AKI [7 23] In fact our present results are consis-tent with our previous results First priming of mitochondriais a determining event to induce autophagic recognition anddegradation In normal pathophysiology the information ofmitochondrial priming for mitophagy is scarce [30] Oncemitochondria exhibit damage and dysfunction mitophagyis induced to clear damaged or dysfunctional mitochondriafor degradation Our study suggested that rhSTC1 treatment

00

05

10

15

20

25

998770

Rela

tive

HO

-1G

APD

H d

ensit

y

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(i)

00

02

04

06

08

Rela

tive c

ytop

lasm

Nrf2

G

APD

H d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(j)

00

05

10

15

998770

Rela

tive c

leav

ed ca

spas

e 3

GA

PDH

den

sity

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(k)

00

02

04

06

08

10

Rela

tive n

ucle

ar N

rf2 d

ensit

y

998770

Iohe

xol+

rhST

C+M

L385

Iohe

xol+

rhST

C1

Iohe

xol

Cont

rol

(l)

Figure 10 Inhibition of Nrf2 activity partly abolishes the anti-inflammatory antiapoptotic and mitochondrial protective role of rhSTC1HK-2 cells were treated with iohexol (200mg iodineml) for 4 hours with or without rhSTC1 (50 ngml) or ML385 (10 μM) (a)Representative images of transmission electron microscope analysis (b) Western blot analysis of the expression of inflammationapoptosis and mitochondrial damage was associated with proteins (n = 3) (c) Western blot analysis of the expression of nuclear andcytoplasm Nrf2 (n = 3) (dndashl) Quantification of average Western blot band intensities Values were presented as mean plusmn SE lowastp lt 005compared with the control group p lt 005 compared with the iohexol group p lt 005 compared with the rhSTC1 treatment group

14 Oxidative Medicine and Cellular Longevity

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

decreased mitochondrial fragmentation and damage demon-strated by confocal and electron microscopy analysis Sec-ond our results showed that rhSTC1 treatment reduced theexpression of PINK1 Parkin which were important regula-tor molecules in mitophagy [23 31] Third evidences dem-onstrated that translocation of Drp1 to mitochondriapromoted mitophagy generation Specific knockdown ofDrp1 decreased the mitophagy level [32 33] Our resultsindicated that rhSTC1 treatment decreased the expressionof Drp1 and the translocation of Drp1 to mitochondriaFourth other researchers found that ROS as a trigger forthe generation of PINK1-Parkin mediated mitophagy andthe inhibition of ROS generation significantly decreased theexpression of Parkin and its translocation to mitochondria[34] This was also consistent with our results that rhSTC1treatment decreased ROS generation and then probablydecreased mitophagy and mitophagy-related proteins

We verified the protective effects of rhSTC1 in CI-AKIcell injury and rat models In order to further explore the pos-sible mechanism we investigated the effect of the STC1-Nrf2pathway in contrast medium-induced renal tubular epithelialcell injury Nrf2 is ubiquitously found in most tissues andacts as an antioxidant and a pivotal factor in regulating oxi-dative stress and metabolism [35] Nrf2 is relatively inactiveunder unstressed conditions by interacting with an inhibitorprotein Keap1 sequestered in the cytosol [36] Activation ofNrf2 can inhibit the expression of Drp1 and mitochondrialfission thus promoting mitochondrial fusion and survivalin vivo and in vitro studies [37 38] Other studies also showthat increased activity of Nrf2 could decrease mitochondrialdysfunction ROS generation and apoptosis and is associatedwith decreased mitophagy level [39 40] In our study iohexolintervention led to decreased expression of nuclear and cyto-plasm Nrf2 and increased expression of Keap1 which werereversed by rhSTC1 treatment In addition Nrf2 expressionwas decreased in HK-2 cell exposure to iohexol by confocalmicroscopic image analysis compared with the controlgroup However both activation of Nrf2 and rhSTC1 treat-ment had the same effect on alleviating iohexol-inducedcytotoxicity compared with rhSTC1 treatment alone inHK-2 cells On the other side STC1 siRNA aggregated HK-2 cell apoptosis compared to the iohexol intervention groupwhich was partially alleviated by activation of Nrf2 HMGB1is a key inflammatory cytokine involved in kidney diseasesand Nrf2 is considered a master regulator of cellular redoxsignaling inflammatory pathway Inhibition of the inflam-matory mediator HMGB1 along with upregulation ofNrf2HO-1 has been demonstrated to significantly improvehypoxia-induced cell injury [41 42] Another study alsoshows that increased Nrf2 activity can attenuate hyperoxia-induced inflammatory by inhibiting the accumulation ofextracellular HMGB1 [43] Additionally the protective rolesof STC1 in preventing mitochondrial damage apoptosis andinflammation response were partly abolished by specificinhibition of Nrf2 activity demonstrated by Western blotand electron microscope analysis All the data suggested thatthe STC1-Nrf2 pathway played a protective role in contrastmedium-induced renal tubular epithelial cell injury by regu-lating mitochondrial quality control imbalance

In conclusion our researches found a novel beneficialrole of STC1 on renal damage in CI-AKI by usingin vitro and in vivo models Treatment with rhSTC1 sig-nificantly regulated mitochondrial quality control bydecreasing the contrast medium-induced mitochondrialdamage oxidative stress inflammation and cell apoptoticinjury in CI-AKI Its protective role was partly attributedto the upregulation of Nrf2 expression and reduction inROS production Our finding identifies STC1 as a regula-tor of Nrf2 in the kidney and brings new insight intothe role of STC1 in renal physiology suggesting that exog-enous STC1 has the potential as a novel preventive agentin CI-AKI

Abbreviations

AKI Acute kidney injuryCI-AKI Contrast-induced acute kidney injuryrhSTC1 Recombinant human stanniocalcin-1ROS Reactive oxygen speciesNrf2 Nuclear factor- (erythroid-derived 2-) like 2Keap1 Kelch-like erythroid cell-derived protein with

CNC homology- (ECH-) associated protein1GFR Glomerular filtration rateCKD Chronic kidney diseaseHK-2 cells Human proximal tubular epithelial cell lineDrp1 Dynamin-related protein 1mfn2 Mitofusin-2TOMM20 Translocase of outer mitochondrial membrane

20 homolog (yeast)mtDNA Mitochondrial DNAHMGB1 High-mobility group box-1NLRP3 NOD-like receptor pyrin domain containing-3P62 Sequestosome 1siRNA Small interfering RNAHO-1 Heme oxygenase-1TBHQ Tert-butylhydroquinoneDHE DihydroethidiumIHC ImmunohistochemistryPINK1 PTEN-induced putative kinase 1Parkin RBR E3 ubiquitin protein ligaseTUNEL Terminal deoxynucleotidyl transferase-

mediated dUTP nick-end labelingBUN Blood urea nitrogenScr Serum creatinine

Data Availability

The data used to support the results of this study are includedin the article The related materials are available from the cor-responding author upon reasonable request

Conflicts of Interest

There were no potential conflicts of interest that needed to bedisclosed

15Oxidative Medicine and Cellular Longevity

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

Authorsrsquo Contributions

FZ performed the experiments FZ and WC analyzed thedata FZ and SBD wrote the manuscript and conceived theideas LXF XJD JCP and YHD assisted with animal studiesXW PY QL CYT and HSW reviewed the manuscript Allauthors read and approved the final manuscript

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No 81873607 and No 81570618)the Development and Reform Commission of Hunan Prov-ince (2014-658) the Scientific Foundation of Hunan Prov-ince China (S2013F1022) and the Clinical MedicalTechnology Innovation Guide Project of Hunan Province(2017SK50117) We thank Miss Tian-Jiao Chen (MA Man-agement and International Development of University ofEdinburgh UK) for reviewing this manuscript

References

[1] L Yang G Xing LWang et al ldquoAcute kidney injury in Chinaa cross-sectional surveyrdquo The Lancet vol 386 no 10002pp 1465ndash1471 2015

[2] K Nash A Hafeez and S Hou ldquoHospital-acquired renalinsufficiencyrdquo American Journal of Kidney Diseases vol 39no 5 pp 930ndash936 2002

[3] M Fahling E Seeliger A Patzak and P B Persson ldquoUnder-standing and preventing contrast-induced acute kidneyinjuryrdquo Nature Reviews Nephrology vol 13 no 3 pp 169ndash180 2017

[4] F Zhao R Lei S K Yang et al ldquoComparative effect of iso-osmolar versus low-osmolar contrast media on the incidenceof contrast-induced acute kidney injury in diabetic patients asystematic review and meta-analysisrdquo Cancer Imagingvol 19 no 1 p 38 2019

[5] S D Weisbord and P M Palevsky ldquoContrast-induced acutekidney injury short- and long-term implicationsrdquo Seminarsin Nephrology vol 31 no 3 pp 300ndash309 2011

[6] W Cheng F Zhao C Y Tang X W Li M Luo and S BDuan ldquoComparison of iohexol and iodixanol induced nephro-toxicity mitochondrial damage and mitophagy in a newcontrast-induced acute kidney injury rat modelrdquo Archives ofToxicology vol 92 no 7 pp 2245ndash2257 2018

[7] R Lei F Zhao C Y Tang et al ldquoMitophagy plays a protectiverole in iodinated contrast-induced acute renal tubular epithe-lial cells injuryrdquo Cellular Physiology and Biochemistryvol 46 no 3 pp 975ndash985 2018

[8] D B Ward and M A Valentovic ldquoContrast induced acutekidney injury and direct cytotoxicity of iodinated radiocon-trast media on renal proximal tubule cellsrdquo The Journal ofPharmacology and Experimental Therapeutics vol 370 no 2pp 160ndash171 2019

[9] X Zhang X Wu Q Hu et al ldquoMitochondrial DNA in liverinflammation and oxidative stressrdquo Life Sciences vol 236 arti-cle 116464 2019

[10] R Mehran and E Nikolsky ldquoContrast-induced nephropathydefinition epidemiology and patients at riskrdquo Kidney Interna-tional vol 100 pp S11ndashS15 2006

[11] E Seeliger M Sendeski C S Rihal and P B Persson ldquoCon-trast-induced kidney injury mechanisms risk factors andpreventionrdquo European Heart Journal vol 33 no 16pp 2007ndash2015 2012

[12] E C Nijssen R J Rennenberg P J Nelemans et al ldquoProphy-lactic hydration to protect renal function from intravasculariodinated contrast material in patients at high risk ofcontrast-induced nephropathy (AMACING) a prospectiverandomised phase 3 controlled open-label non-inferioritytrialrdquo The Lancet vol 389 no 10076 pp 1312ndash1322 2017

[13] R Varghese C K Wong H Deol G F Wagner and G EDiMattia ldquoComparative analysis of mammalian stanniocalcingenesrdquo Endocrinology vol 139 no 11 pp 4714ndash4725 1998

[14] G J Block S Ohkouchi F Fung et al ldquoMultipotent stromalcells are activated to reduce apoptosis in part by upregulationand secretion of stanniocalcin-1rdquo Stem Cells vol 27 no 3pp 670ndash681 2009

[15] L Huang G Garcia Y Lou et al ldquoAnti-inflammatory andrenal protective actions of stanniocalcin-1 in a model of anti-glomerular basement membrane glomerulonephritisrdquo TheAmerican Journal of Pathology vol 174 no 4 pp 1368ndash1378 2009

[16] CW Luo K Kawamura C Klein and A J Hsueh ldquoParacrineregulation of ovarian granulosa cell differentiation by stannio-calcin (STC) 1 mediation through specific STC1 receptorsrdquoMolecular Endocrinology vol 18 no 8 pp 2085ndash2096 2004

[17] C R McCudden K A James C Hasilo and G F WagnerldquoCharacterization of mammalian stanniocalcin receptorsMitochondrial targeting of ligand and receptor for regulationof cellular metabolismrdquo Journal of Biological Chemistryvol 277 no 47 pp 45249ndash45258 2002

[18] R N Re and J L Cook ldquoThe mitochondrial component ofintracrine actionrdquo American Journal of Physiology Heart andCirculatory Physiology vol 299 no 3 pp H577ndashH583 2010

[19] Y Wang L Huang M Abdelrahim et al ldquoStanniocalcin-1suppresses superoxide generation in macrophages throughinduction of mitochondrial UCP2rdquo Journal of Leukocyte Biol-ogy vol 86 no 4 pp 981ndash988 2009

[20] G W Roddy R H Rosa Jr J Y Oh et al ldquoStanniocalcin-1rescued photoreceptor degeneration in two rat models ofinherited retinal degenerationrdquo Molecular Therapy vol 20no 4 pp 788ndash797 2012

[21] S E Tang C P Wu S Y Wu et al ldquoStanniocalcin-1 amelio-rates lipopolysaccharide-induced pulmonary oxidative stressinflammation and apoptosis in micerdquo Free Radical Biologyand Medicine vol 71 pp 321ndash331 2014

[22] L C Racusen and K Solez ldquoNephrotoxic tubular and intersti-tial lesions morphology and classificationrdquo ToxicologicPathology vol 14 no 1 pp 45ndash57 1986

[23] Q Lin S Li N Jiang et al ldquoPINK1-parkin pathway of mito-phagy protects against contrast-induced acute kidney injuryvia decreasing mitochondrial ROS and NLRP3 inflammasomeactivationrdquo Redox Biology vol 26 article 101254 2019

[24] M Lin L Li Y Zhang et al ldquoBaicalin ameliorates H2O2induced cytotoxicity in HK-2 cells through the inhibition ofER stress and the activation of Nrf2 signalingrdquo InternationalJournal of Molecular Sciences vol 15 no 7 pp 12507ndash12522 2014

[25] R Mehran G D Dangas and S D Weisbord ldquoContrast-asso-ciated acute kidney injuryrdquo The New England Journal of Med-icine vol 380 no 22 pp 2146ndash2155 2019

16 Oxidative Medicine and Cellular Longevity

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments

[26] X Yang X Yan D Yang J Zhou J Song and D YangldquoRapamycin attenuates mitochondrial injury and renal tubularcell apoptosis in experimental contrast-induced acute kidneyinjury in ratsrdquo Bioscience Reports vol 38 no 6 2018

[27] J A Westberg M Serlachius P Lankila M PenkowaJ Hidalgo and L C Andersson ldquoHypoxic preconditioninginduces neuroprotective stanniocalcin-1 in brain via IL-6 sig-nalingrdquo Stroke vol 38 no 3 pp 1025ndash1030 2007

[28] H Y Yeung K P Lai H Y Chan N K Mak G F Wagnerand C K Wong ldquoHypoxia-inducible factor-1-mediated acti-vation of stanniocalcin-1 in human cancer cellsrdquo Endocrinol-ogy vol 146 no 11 pp 4951ndash4960 2005

[29] K Zhang P J Lindsberg T Tatlisumak M Kaste H S Olsenand L C Andersson ldquoStanniocalcin a molecular guard ofneurons during cerebral ischemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 97no 7 pp 3637ndash3642 2000

[30] C Tang H Han M Yan et al ldquoPINK1-PRKNPARK2 path-way of mitophagy is activated to protect against renalischemia-reperfusion injuryrdquo Autophagy vol 14 no 5pp 880ndash897 2018

[31] R Li and J Chen ldquoSalidroside protects dopaminergic neuronsby enhancing PINK1Parkin-mediated mitophagyrdquo OxidativeMedicine and Cellular Longevity vol 2019 Article ID9341018 11 pages 2019

[32] Y S Park S E Choi and H C Koh ldquoPGAM5 regulatesPINK1Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunctionrdquo Toxicology Lettersvol 284 pp 120ndash128 2018

[33] C Zhao Z Chen J Qi et al ldquoDrp1-dependent mitophagyprotects against cisplatin-induced apoptosis of renal tubularepithelial cells by improving mitochondrial functionrdquo Onco-target vol 8 no 13 pp 20988ndash21000 2017

[34] B Xiao J Y Goh L Xiao H Xian K L Lim and Y C LiouldquoReactive oxygen species trigger ParkinPINK1 pathway-dependent mitophagy by inducing mitochondrial recruitmentof Parkinrdquo Journal of Biological Chemistry vol 292 no 40pp 16697ndash16708 2017

[35] A Uruno Y Furusawa Y Yagishita et al ldquoThe Keap1-Nrf2system prevents onset of diabetes mellitusrdquoMolecular and Cel-lular Biology vol 33 no 15 pp 2996ndash3010 2013

[36] B R Imhoff and J M Hansen ldquoTert-butylhydroquinoneinduces mitochondrial oxidative stress causing Nrf2 activa-tionrdquo Cell Biology and Toxicology vol 26 no 6 pp 541ndash551 2010

[37] R Sabouny E Fraunberger M Geoffrion et al ldquoThe Keap1-Nrf2 stress response pathway promotes mitochondrial hyper-fusion through degradation of the mitochondrial fission pro-tein Drp1rdquo Antioxidants amp Redox Signaling vol 27 no 18pp 1447ndash1459 2017

[38] Y Zhu M Li Y Lu J Li Y Ke and J Yang ldquoIlexgenin Ainhibits mitochondrial fission and promote Drp1 degradationby Nrf2-induced PSMB5 in endothelial cellsrdquo Drug Develop-ment Research vol 80 no 4 pp 481ndash489 2019

[39] D M Miller I N Singh J A Wang and E D Hall ldquoNrf2-ARE activator carnosic acid decreases mitochondrial dysfunc-tion oxidative damage and neuronal cytoskeletal degradationfollowing traumatic brain injury in micerdquo Experimental Neu-rology vol 264 pp 103ndash110 2015

[40] Y Ren Y Li J Yan et al ldquoAdiponectin modulates oxidativestress-induced mitophagy and protects C2C12 myoblasts

against apoptosisrdquo Scientific Reports vol 7 no 1 article3209 2017

[41] L Zhang X Qi G Zhang Y Zhang and J Tian ldquoSaxagliptinprotects against hypoxia-induced damage in H9c2 cellsrdquo Che-mico-Biological Interactions vol 315 article 108864 2020

[42] Y Faridvand S Nozari V Vahedian et al ldquoNrf2 activationand down-regulation of HMGB1 and MyD88 expression byamnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cellsrdquo Biomedicine amp Pharma-cotherapy vol 109 pp 360ndash368 2019

[43] V Patel K Dial J Wu et al ldquoDietary antioxidants signifi-cantly attenuate hyperoxia-induced acute inflammatory lunginjury by enhancing macrophage function via reducing theaccumulation of airway HMGB1rdquo International Journal ofMolecular Sciences vol 21 no 3 p 977 2020

17Oxidative Medicine and Cellular Longevity

• Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway
• 1 Introduction
• 2 Methods
• • 21 Cell Culture Studies
  • 22 Animal Experimental Design
  • 23 Assessment of Renal Function and Histopathology
  • 24 dUTP Nick-End Labeling Assay
  • 25 Western Blotting
  • 26 Immunohistochemistry Staining
  • 27 Quantification of Mitochondrial DNA Content
  • 28 Determination of Reactive Oxygen Species
  • 29 Transmission Electron Microscope Analysis (TEM)
  • 210 Immunofluorescence Colocalization Analysis
  • 211 Statistical Analysis
  • • 3 Results
    • • 31 Iohexol Induces Cytotoxicity and Mitochondrial Dysfunction In Vitro
      • 32 rhSTC1 Treatment Attenuates Inflammation Injury and Mitochondrial ROS Generation in HK-2 Cells
      • 33 rhSTC1 Treatment Reduces Mitochondrial Damage and Mitophagy in HK-2 Cells after Iohexol Exposure
      • 34 Silencing STC1 Significantly Increases Inflammation Response and Mitochondrial Damage in HK-2 Cells
      • 35 Antiapoptotic Effects of rhSTC1 in HK-2 Cells
      • 36 The Protective Effects of rhSTC1 Treatment on Renal Function and Pathological Injury
      • 37 rhSTC1 Restores the Expression Level of Drp1 and mfn2 and Reduces ROS Production In Vivo
      • 38 Mitophagy-Associated Proteins and Keap1 and Nrf2 Expression Levels Are Regulated by rhSTC1 in Rat Kidney
      • 39 rhSTC1 Plays an Anti-Inflammatory Antiapoptotic and Mitochondrial Protective Role Partly by Upregulating the Expression of Nrf2
      • • 4 Discussion
        • Abbreviations
        • Data Availability
        • Conflicts of Interest
        • Authorsrsquo Contributions
        • Acknowledgments