LABORATORY STUDY

Effect of Nephrotoxins on Tubulointerstitial Injury and NF-kB Activation inAdriamycin Nephropathy

Gopala K. Rangan, Yiping Wang, Yuet-Ching Tay, Jason D. Coombes, and David C. H. HarrisCenter for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Westmead Hospital,

Westmead, Sydney, Australia

In a previous study we found that an episode of acute

subclinical nephrotoxicity with gentamicin (G) (but not that

induced by another proximal tubular cell nephrotoxin: ferric

nitrilotriacetate, FeNTA), paradoxically reduced the progression

of renal function and injury in uninephrectomized rats with

nephrotic glomerular disease due to Adriamycin nephropathy

(AN). Here, we hypothesized that subclinical exposure to G

reduces early renal cortical tubulointerstitial inflammation and

NF-kB activation in AN. To test this hypothesis, male Wistar

rats with established AN received either G (10, 40, or 80 mg/kg

by daily s.c.i. for 3 days), FeNTA (1.25, 5, or 10 mg/kg by a

single i.p.i.), or vehicle (n=8 per group), 13 to 15 days after

disease induction. Although G and FeNTA caused acute tubular

necrosis in a dose-dependant manner (day 17), only the highest

doses (10 mg/kg and 80 mg/kg) produced an acute elevation in

the serum creatinine. On day 33, chronic tubulointerstitial

inflammation (tubular atrophy, interstitial ED-1+/CD8+ cell

accumulation) and NF-kB activation were exacerbated only in

the groups that caused functional nephrotoxicity. These data

suggest that: 1) the protective effect of subclinical G

nephrotoxicity in chronic AN does not involve early changes

in interstitial inflammation or NF-kB activation; and 2) a single

episode of G exposure must be accompanied by clinically

apparent nephrotoxicity in order to accelerate progression in a

nonuremic model of chronic glomerular disease.

Keywords nephrotoxin, chronic tubulointerstitial inflammation,

glomerular disease

INTRODUCTION

Nephrotoxins such as aminoglycosides, radiocontrast,

amphotericin, and anaesthetic compounds cause acute re-

nal failure, in part, by direct injury to proximal tubular

epithelial cells (PTECs).[1] It widely accepted that expo-

sure to nephrotoxins during the course of chronic glo-

merular disease (CGD) could hasten progression to

end-stage kidney failure by exacerbating tubulointerstitial

injury.[2,3] Paradoxically, in a previous longterm study,

we found that an episode of subclinical nephrotoxicity

(defined as pathological renal injury without a rise in the

serum creatinine) induced by gentamicin [but not ferric

nitrilotriacete (FeNTA); another proximal tubular cell

nephrotoxin] reduced the decline in renal function,

glomerulosclerosis, and tubulointerstitial injury in a rat

model of CGD (Adriamycin nephropathy, AN).[4] The

mechanisms involved in this novel type of acquired resis-

tance were not known.

Nuclear factor (NF)-kB is a highly conserved family

of transcription factors that has a critical role in mediating

inflammation, apoptosis, and growth in chronic disease.[5]

In early AN, the renal cortical activation of NF-kB is in-

creased and correlated with the severity of tubulointerstitial

injury.[6] Moreover, suppression of NF-kB activation in

established AN, with the antioxidant, pyrrolidine dithio-

carbamate (PDTC), attenuated tubulointerstitial inflam-

mation.[6] These data suggest that NF-kB has an important

role in mediating disease progression in this model.

In other experimental systems (such as the repeated

exposure of monocytes to lipopolysaccharide) attenua-

tion of NF-kB has been postulated to be one of the

mechanisms involved in mediating acquired resistance to

further tissue injury.[7] These observations led us to

hypothesize that an episode of subclinical gentamicin

nephrotoxicity reduces renal cortical NF-kB activation in

AN. To investigate this hypothesis, the dose-dependant

Address correspondence to Dr. Gopala K. Rangan, Depart-

ment of Renal Medicine and Transplantation, Westmead

Hospital, Westmead, Sydney 2145, Australia; Fax: (61-2)

9633-9351; E-mail: g.rangan@wmi.usyd.edu.au

609

Renal Failure, 27:609–614, 2005

Copyright D 2005 Taylor & Francis Inc.

ISSN: 0886-022X print / 1525-6049 online

DOI: 10.1080/08860220500200437

Order reprints of this article at www.copyright.rightslink.com

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effects of a single episode of gentamicin- and FeNTA-

induced nephrotoxicity on renal cortical tubulointerstitial

injury and NF-kB activation in early AN were examined

in this study.

MATERIALS AND METHODS

Male Wistar rats (6–8 weeks old) (n=56) were

supplied by the Animal Care Facility, Westmead Hospital

and allowed free access to food and water. The AN was

induced on day 0, by a single intravenous injection of

doxorubicin hydrochloride (7.5 mg/kg).[6] On day 10,

baseline 24-hour urinary protein and creatinine were

measured. On day 13, rats were stratified into seven groups

(n=8 per group) according to body weight, baseline

proteinuria, and endogenous creatinine clearance (CrCl).

The groups received: 1) vehicle (saline, day 14 to day 16,

by s.c. or i.p.i.); 2) gentamicin (G) (10, 40, or 80 mg/kg,

day 14 to day 16, by s.c.i.) or FeNTA (1.25, 5, or 10 mg/kg,

by a single i.p.i. on day 16). Sterile FeNTA was prepared

as previously described[4] and gentamicin was obtained

from David Bull Laboratories (Melbourne, Australia). The

doses of nephrotoxins were determined from preliminary

experiments and a previous study,[4] and designed to

induce mild, moderate, or severe acute tubular necrosis in

AN. Higher doses of the nephrotoxins (>80 mg/kg

gentamicin or >10 mg/kg FeNTA) caused diffuse tubular

necrosis and early mortality due to severe acute renal

failure in AN, whereas doses less than 1.25 mg/kg FeNTA

or 10 mg/kg gentamicin caused no tubular necrosis.

Animals were pair fed, and body weight and food

intake were measured daily. Proteinuria and CrCl were

determined on days 17, 21, and 33. Groups of animals

were sacrificed either on day 17 (n=3 per group) or day

33 (n=5 per group) for analysis of acute and chronic

effects on tubulointerstitial injury, respectively. On the

day of sacrifice, animals were anesthetized with ketami-

ne:xylazine, a mid-line laparotomy was performed and

both kidneys were removed, as previously described.

Table 1

Serum creatinine (mmol/L) in the experimental groups

Time AN+vehicle

AN+gentamicin (mg/kg) AN+FeNTA (mg/kg)

10 40 80 1.25 5 10

Day 10 49±1 50±2 47±2 50±1 46±1 44±1 48±2

Day 17 58±3 56±3 81±22 253±33* 50±3 54±2 161±38*

Day 21 57±2 57±3 65±5 236±50* 54±2 56±2 66±4

Day 33 64±4 64±4 59±4 85±9 45±1 48±2 58±2

NB: Gentamicin and FeNTA were administered on days 14 to 16.*p<0.05 vs. AN+vehicle.

Figure 1. The acute effect of gentamicin (G, 10–80 mg/kg) and ferric nitrilotriacetate (Fe, 1.25–10 mg/kg) on cortical

tubulointerstitial injury in Adriamycin nephropathy (AN) on day 17. Areas of focal acute tubular necrosis are present, in a dose-

dependant manner, in the groups treated with nephrotoxins (Magnification, �400, PAS stain).

G. K. Rangan et al.610

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The Animal Ethics Committee, University of Sydney at

Westmead Hospital, approved all experimental protocols.

Formalin-fixed paraffin sections (3 mm), were stained

with periodic-acid Schiff (PAS). Cortical tubulointerstitial

injury was assessed by both a semiquantitative scoring

system and quantitative morphometric analysis. For

semiquantitative analysis, 20 random cortical fields were

viewed at �200 magnification and graded according to the

percentage area occupied by tubular injury (tubular

atrophy, dilatation) and interstitial inflammation (0=nor-

mal; 1=less than 25%; 2=26%–50%; 3=51%–75%;

4=76%–100%). For quantitative morphometry, 10 ran-

dom cortical fields were viewed on a computer screen

using a video camera (�400). The cross-sectional cell

height of cortical tubules and interstitial volume, was

measured by line and area morphometric measurements

respectively, using image analysis software (Optimas

version 5.2, Optimas Corporation, Seattle, WA).[6] The

number of interstitial monocyte (ED-1) and cytotoxic

T-lymphocytes (OX-8) were assessed by immunohisto-

chemistry on acetone-fixed frozen sections. Nonspecific

staining was blocked with normal rabbit serum (1:5)

followed by either mouse anti-rat ED-1 (1:400, Serotec,

Oxford, England) or mouse anti-rat OX8 (1:100, Serotec,

Oxford, England) for 1 h at room temperature. Anti-mouse

rabbit antibody (1:50 in 1% rat serum, Dako Australia,

Sydney, Australia) and mouse peroxidase-antiperoxidase

complex (PAP, Z0259, 1:100; Dako Australia, Sydney,

Australia) were applied sequentially for 25 minutes each at

room temperature. The mean numbers of 3,3-diamino-

benzidine tetrahydrochloride positive ED-1 or OX-8 pos-

itive cells were determined from 10 nonoverlapping

cortical fields (�400, measuring 0.075 mm2 each).

Extraction of nuclear protein and electrophoretic

mobility shift assay for NF-kB and activator protein (AP)-

1 were performed as previously described.[6,8] Competi-

tion and supershift assay were performed to confirm the

specificity of the retarded bands.

All data were expressed as mean±SEM. Compar-

isons between experimental groups were performed using

Figure 2. The subacute effect of gentamicin (G) and ferric nitrilotriacetate (Fe) on tubulointerstitial inflammation in Adriamycin

nephropathy (AN) on day 33. Chronic tubulointerstitial inflammation in AN is exacerbated by high-dose G and Fe treatment (PAS

stain).

Figure 3. Semiquantitative cortical tubulointerstitial injury

score in vehicle (C)-, gentamicin- and ferric nitrilotriacetate

(FeNTA)-treated rats with Adriamycin nephropathy on day 33.

*P<0.05 vs. C.

611Nephrotoxins and Adriamycin Nephropathy

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the independent t-test and Mann-Whitney U test for

parametric and nonparametric data respectively. A P

value less than 0.05 indicated a significant difference

between groups.

RESULTS

No animals died during the study. Groups adminis-

tered the highest doses of FeNTA (10 mg/kg) and G (80

mg/kg), gained less body weight than vehicle (Day 33:

AN+vehicle: 295±18; AN+G: 244±13; AN+FeNTA:

272±15 g; P=0.05). Body weight in the other groups was

similar to vehicle-treated animals. Serum creatinine was

elevated only in groups that received the highest doses of

FeNTA and G (Table 1). The recovery of the serum

creatinine occurred earlier in the FeNTA group (day 22),

whereas it was only partial by day 33 in the G group

(Table 1). The serum creatinine (or CrCl, data not shown),

did not change significantly in the other groups receiving

nephrotoxins (compared to vehicle) (Table 1), even

though acute tubular necrosis did occur at these lower

doses in AN (see the following information). There were

no differences in urinary protein excretion in the experi-

mental groups at any timepoint (urinary protein:creatinine

ratio on day 33: AN+vehicle: 72.2±6.5; AN+G: 76.4±

8.1; AN+FeNTA: 77.8±5.1 mg/mmol; P=not significant).

Histological examination on day 17 confirmed that

animals treated with either nephrotoxin developed dose-

dependant degrees of acute tubular necrosis (Figure 1).

On day 33 the histological changes of AN consisted of

cortical tubular atrophy, interstitial cell accumulation, and

Table 2

Quantitative morphometric analysis of the tubulointerstitial inflammation in the experimental groups on day 33

Group n

Dose

mg/kg

Interstitial

volume %

Mean tubule

cell height (mm)

Interstitial

ED-1 cells/hpf

Interstitial

CD8 cells/hpf

AN+vehicle 5 — 12.2±1.6 14.9±3.0 6.64±0.68 8.16±1.30

AN+gentamicin 5 80 33.0±6.5* 8.6±0.6* 35.78±3.56* 11.90±0.70*

AN+FeNTA 5 10 26.9±5.8* 9.4±0.3* 18.92±3.32* 11.58±2.45*

*p<0.05 vs. AN+vehicle.

Figure 4. The DNA-binding activity of nuclear factor (NF)-kB (A) and activator protein (AP)-1 (B) in renal cortical nuclear

extracts from vehicle (C)-, gentamicin-, and ferric nitrilotriacetate (FeNTA)-treated rats with Adriamycin nephropathy on day 33 of

the study. Top and lower panels show representative autoradiographs and mean densitometry (n=5 per group) respectively. *P<0.05

vs. C.

G. K. Rangan et al.612

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volume expansion (Figure 2). By semiquantitative anal-

ysis, cortical tubulointerstitial injury was exacerbated

only in the groups that received the highest doses of either

FeNTA or G (Figures 2 and 3). By quantitative mor-

phometric analysis, interstitial volume and cortical

tubular atrophy (as assessed by a reduction in cross-

sectional tubule cell height) were increased in these

groups, compared to vehicle (Table 2). Similarly,

interstitial ED-1 and CD8 cell accumulation in AN was

worsened by high-dose FeNTA and G (Table 2).

The DNA-binding activity of both AP-1 and NF-kB

were increased in renal cortical nuclear extracts from rats

with AN. On day 33, NF-kB DNA-binding activity (but

not AP-1) was exacerbated only in the groups that

received the highest doses of either FeNTA or G,

compared to vehicle (Figure 4).

DISCUSSION

The results of this study demonstrate that the acute

administration of nephrotoxic doses of two site-specific

proximal tubular cell nephrotoxins (gentamicin and

FeNTA) to rats with established AN exacerbated under-

lying tubulointerstitial inflammation and renal cortical

NF-kB activation. In contrast, lower doses, that caused

mild acute tubular necrosis and subclinical nephrotoxicity,

were not sufficient to alter early tubulointerstitial inflam-

mation or renal cortical NF-kB activation.

Previously, in a uninephrectomized model of Adria-

mycin nephropathy, we reported that an episode of

subclinical nephrotoxicity induced by gentamicin (but

not FeNTA) at week 6, completely prevented the decline

in creatinine clearance and partially reduced glomerulo-

sclerosis and tubulointerstitial fibrosis at week 14.[4] The

present study was performed to investigate the potential

role of NF-kB in mediating the protective effect of

gentamicin in AN. However, we were unable to demon-

strate that an early downregulation in NF-kB activation (as

described in some models of acquired resistance)[7] and

tubulointerstitial inflammation were mechanisms to ex-

plain the protective effect of subclinical gentamicin

nephrotoxicity on the longterm progression of chronic

AN. Other possibilities that could be involved include the

upregulation of antioxidant proteins (such as heme-

oxygenase-1)[9] and/or specific effects of gentamicin on

the lysosomal processing of filtered plasma proteins.[4]

In previous studies, inhibition of renal cortical NF-

kB activation with PDTC reduced cortical tubulointer-

stitial injury in AN.[6] In the present study, exacerbation

of interstitial inflammation and tubular atrophy with

nephrotoxins was associated with augmentation of renal

cortical NF-kB (but not AP-1). These correlative data

provide further evidence that NF-kB activation is

involved in the pathogenesis of chronic tubulointerstitial

injury in progressive renal diseases.

Avoiding exposure to nephrotoxins is one of the main

therapeutic cornerstones in the clinical management of

preventing disease progression in chronic kidney disease.

Our experimental data suggests that an episode of

subclinical nephrotoxicity, at least following a short

exposure to gentamicin, does not have any adverse long-

term effects in a nonuremic nephrotic model of glomerular

disease. However, the clinical scenario in humans is more

complex than that that can be replicated in the laboratory.

In the clinical setting, patients have impaired renal

function and are exposed to multiple nephrotoxic insults,

sometimes on repeated occasions. This may be an

explanation for the divergence of our experimental data

with retrospective clinical studies. For example, Aubia and

colleagues showed that subclinical nephrotoxin exposure

(including aminoglycosides and possibly anesthesia)

accelerated the decline in renal function by more than 2-

fold in humans with impaired renal function due to

diabetic nephropathy.[10] Similarly, aminoglycoside expo-

sure accelerated the decline in loss of residual renal

function in peritoneal dialysis patients with end-stage

renal failure.[11,12]

In summary, the cytoprotective effect of subclinical

gentamicin nephrotoxicity in AN is not due to an early

downregulation in renal cortical NF-kB activation, and

involves other mechanisms that require further investiga-

tion. In addition, the acute exposure to clinically

nephrotoxic doses of gentamicin and FeNTA worsened

renal cortical tubulointerstitial injury in AN through

mechanisms that may involve the upregulation of NF-kB.

ACKNOWLEDGMENTS

This study was supported by the Australian Kidney

Foundation (GKR) and project grant 970721 from the

National Health Medical Research Council of Austra-

lia (DCH).

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