Activating Nrf-2 Signaling by Sulforaphane Attenuates Unilateral Ureteral Obstruction-Enhanced Apoptosis and Pyroptosis in Kidney

林威佑、戴槐青、鄭劍廷 1 、余宏政

台灣大學醫學院附設醫院 泌尿部1 國立臺灣師範大學生命科學系

Introduction

Clinically, ureteral obstruction is a common problem.Incidence: 3.9% Cause:

○ Ureteral stone○ Benign prostate hyperplasia○ Urothelial tumor○ Other causes such as ureteral stricture

Hegarty et al., 2001

Increased reactive oxygen species (ROS) may promote two types of programmed cell death in the obstructed kidneyapoptosis and pyroptosis

Sulforaphane (SA) via activating Nrf-2 antioxidant signaling may protect UUO-induced renal damage

Method Unilateral ureteral obstruction (UUO)

A well animal model of mimic renal injury leading to interstitial fibrosis

Nagatoya et al., 2002

Left kidney of Female Wistar rats NormalUUOUUO and sulforaphane

Reactive oxygen species (ROS) Fibrosis, ED-1 (macrophage/monocyte)

infiltration, oxidative stress, autophagy, apoptosis and pyroptosis

Immunohistochemisty and Western blot

Result

H.E.Stain

Normal UUO 7day SA UUO 7day

ED1 Stain Macrophage

Normal UUO 7day SA UUO 7day

4HNE Stain Lipid Oxide

Normal UUO 7day SA UUO7day

Beclin Stain Autophagy

SA UUO7day

Normal

UUO 7day

偵測腎臟組織細胞自噬初期蛋白 Beclin-1表現，觀察時間點為 Normal、阻塞七天、阻塞七天 +DL-蘿蔔硫素 (n=3)。＊表示與 Normal組有顯著差異 (p<0.05)。

Normal UUO 7day UUO & SA 7day

Beclin 1

control UUO UUO+SA

0.0

0.2

0.4

0.6

0.8

1.0

1.2 *

β-actin

N Engl J Med 2012

ATG5-ATG12 Stain Autophagy

SA UUO7day

Normal

UUO 7day

Atg5-Atg12

偵測腎臟組織細胞自噬過程促自噬泡形成之 Atg5-Atg12蛋白表現，觀察時間點為 Normal、阻塞七天、阻塞七天 + DL-蘿蔔硫素 (n=3)。＊表示與 Normal組有顯著差異 (p<0.05)。

Normal UUO 7day UUO & SA 7day

β-actin

control UUO UUO+SA

0.0

0.2

0.4

0.6

0.8

1.0

1.2 *

N Engl J Med 2012

LC3II Stain Autophagy

Normal

UUO 7day

SA UUO7day

LC3II

Normal UUO 7day UUO & SA 7day

偵測腎臟組織細胞自噬之自噬泡形成代表性蛋白 LC3 typeⅡ表現，觀察時間點為 Normal、阻塞七天、阻塞七天 + DL-蘿蔔硫素 (n=3)。＊表示與 Normal組有顯著差異 (p<0.05)。

β-actin

control UUO UUO+SA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

N Engl J Med 2012

Caspase1 Stain pyropotosis

SA UUO7day

Normal

UUO 7day

IL1-βStain pyropotosis

SA UUO7day

Normal

UUO 7day

Nrf2 Stain

SA UUO7day

Normal

UUO7day

偵測腎臟組織細胞自噬過程 Nrf2蛋白表現，觀察時間點為 Normal、阻塞七天、阻塞七天 + DL-蘿蔔硫素(n=3)。＊表示與 control組有顯著差異 (p<0.05)。

Normal UUO 7day UUO & SA 7day

β-actin

Nrf2

control UUO UUO+SA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

UOO

Nuclear Nrf-2 translocation

Cytosolic Bax translocation to mitochondria

MOMP

Enhance mitochondrial Cytochrome c release into the cytosol

Cytosolic Nrf-2

sulforaphane

Cytochrome C Stain

Control UUO 7day SA UUO7day

Masson Stain Hydroxylproline

Normal UUO 7day SA UUO7day

Normal UUO7days UUO7&SA days

Hyd

roxylp

rolin

e c

on

ce

nt (m

g/m

l)

0

20

40

60

80

100Normal n=3UUO7daysUUO7days&SA

*

*

Conclusion

Conclusion: Sulforaphane via activating Nrf-2 signaling to preserve mitochondrial function and suppressed UUO-induced renal apoptosis and pyroptosis

Thank you for your attention!!