NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury

NADPH oxidase 4 (NOX4) is highly expressed in kidney proximal tubular cells. NOX4 constitutively produces hydrogen peroxide, which may regulate important pro-survival pathways. Renal ischemia reperfusion injury (IRI) is a classical model mimicking human ischemic acute tubular necrosis. We hypothesized that NOX4 plays a protective role in kidney IRI. In wild type (WT) animals subjected to IRI, NOX4 protein expression increased after 24 hours. NOX4 KO (knock-out) and WT littermates mice were subjected to IRI. NOX4 KO mice displayed decreased renal function and more severe tubular apoptosis, decreased Bcl-2 expression and higher histologic damage scores compared to WT. Activation of NRF2 was decreased in NOX4 KO mice in response to IRI. This was related to decreased KEAP1 oxidation leading to decreased NRF2 stabilization. This resulted in decreased glutathione levels. In vitro silencing of NOX4 in cells showed an enhanced propensity to apoptosis, with reduced expression of NRF2, glutathione content and Bcl-2 expression, similar to cells derived from NOX4 KO mice. Overexpression of a constitutively active form of NRF2 (caNRF2) in NOX4 depleted cells rescued most of this phenotype in cultured cells, implying that NRF2 regulation by ROS issued from NOX4 may play an important role in its anti-apoptotic property.

induced in IRI and NRF2 knockout animals display higher tubular injury scores that can be reversed by gluthatione or N-acetylcysteine. In human chronic kidney disease, NRF2 activation was to be promising for diabetic nephropathy treatment, but revealed associated with elevated cardiovascular events 21 .
AKI is defined by a rapid loss of kidney function, resulting in increased levels of nitrogenous waste products and dysregulation of volume and electrolyte homeostasis 22 . Although usually in part reversible, this syndrome is associated with an elevated morbidity and mortality in patients, and most frequently associated to ischemia reperfusion injury. In addition, AKI may also promote progression of chronic kidney disease 23 . No therapy currently exists to prevent or cure AKI and the role of oxidative stress in its pathogenesis is debated. Indeed, enhanced oxidative stress was described after IRI in animal models 24 . However, baseline ROS production may also be protective in IRI and increased baseline superoxide production has been described to improve resistance to IRI in mice 25,26 . The role of NOX4 in kidney IRI is not known. In the heart, NOX4 deletion does not protect from IRI and may even have a deleterious effect on microvascularisation when combined with NOX2 deletion 27,28 .
In this manuscript, we demonstrate that NOX4 KO mice are more prone to kidney tubular injury during ischemia reperfusion. This is due to enhanced propensity to tubular cell apoptosis probably via regulation of NRF2 and modulation of cellular glutathione content, Bcl-2 expression and mitochondrial function.

NOX4 expression increases 24 hours after IRI.
WT mice subjected to 22 minutes of warm ischemia displayed tubular necrosis as opposed to sham-operated littermates 24 hours after reperfusion as assessed by histology (Fig. 1A). Histological kidney injury correlated with increased serum creatinine levels (Fig. 1B). Immunochemistry showed an increase in NOX4 expression, particularly in injured proximal tubular cells (Fig. 1C), which was confirmed by Western blot analysis (Fig. 1D-F). The specificity of NOX4 antibody was demonstrated in NOX4 KO mice (Supp. Fig. 1).

NOX4 KO mice display increased tubular apoptosis and creatinine levels under IRI.
We previously demonstrated that NOX4 KO animals do not display enhanced kidney tubular cell apoptosis at baseline, but were more susceptible to tubular injury during unilateral uretral obstruction (UUO) 13 . In order to test a protective role of NOX4 against apoptosis in IRI, tubular apoptosis was assessed by TUNEL assay 24 hours after 22 minutes bilateral IRI in WT and NOX4 KO mice ( Fig. 2A). Tubular cell apoptosis in the cortex and outer medulla was more pronounced in NOX4 KO animals compared to their WT littermates (Fig. 2B). In addition, tubular injury score quantified at the corticomedullary junction was higher in NOX4 KO animals (Fig. 2C), in line with increased serum creatinine levels (Fig. 2D). Tubular cells depleted of NOX4 display enhanced apoptosis via the classical pathway 13 . In parallel, NOX4 KO mouse embryonic fibroblasts (MEF) cells derived from our animals displayed an increased late apoptosis in vitro compared to WT MEF as assessed by Annexin V/PI assay (Fig. 2E). In addition, enhanced PARP cleavage was observed in NOX4 KO MEF cells, as previously observed in kidney tubular cells depleted with NOX4 13 , both at baseline and in condition of hypoxia (Supp. Fig. 2). Finally, since no reliable proximal derived tubular cell line exists, we used kidney tubule suspension issued from both the cortex of both WT and NOX4 KO mice, therefore containing a majority of proximal tubules. During preparation, suspensions are exposed to some degree of stress and hypoxia and we clearly observed a decrease of the full length isoform of PARP and an increase of its cleaved isoform in the tubules suspension from the NOX4 KO mice compared to WT, implying enhanced apoptosis in the absence of NOX4 (Fig. 2F). The cortical suspensions used here are mainly representative of proximal tubular cells found in mice kidney as confirmed by high NaPi-IIa protein expression. The lack of NOX4 in our suspension issued from KO mice was also confirmed by western-blot (Supp. Fig. 3). Thus we observed enhanced apoptosis of tubular cells deficient in NOX4 in vivo, in vitro in a tubular cell line depleted with NOX4, in NOX4 deficient MEFs, as well as in tubule suspension of proximal tubular cells issued from mouse kidneys. We thereby confirm that NOX4 depletion increases apoptosis in different cell lines, including primary suspensions of tubular cells. In order to further study the molecular mechanisms of these observations, we further used both tubular cell lines depleted in NOX4 and MEF cells from NOX4 KO mice.

NOX4 depletion decreases the expression of Bcl-2.
Bcl-2 is a major and well characterized anti-apoptotic factor. We hypothesize that NOX4 deletion may induce apoptosis through decreased Bcl-2 expression. In vitro Bcl-2 expression was decreased in NOX4 KO MEF cells (Fig. 3A) and in mCCD cl1 cells transfected with a siRNA targeting NOX4 (Fig. 3B), respectively in comparison to WT MEF and scrambled transfected mCCD cl1 cells, likely participating to the enhanced apoptosis observed in these two cell lines. Bax, another pro-apoptotic protein, was not regulated. Downstream proteins such as caspase 3 appeared down-regulated, in line with Bcl-2 regulation and enhanced apoptosis (Supp. Fig. 4). Finally, in mice subjected to IRI, Bcl-2 protein expression was largely decreased in NOX4 KO mice compared to WT, which presumably is at least partially responsible for to the increased apoptosis and tubular injury observed in these mice (Fig. 3C). NRF2 and NRF2 target genes are down-regulated in NOX4 KO mice subjected to IRI. We previously observed that NOX4 KO mice display decreased baseline NRF2 expression 13 . Bcl-2 expression is both regulated by NRF2 as well as by KEAP1 [29][30][31] . We hypothesized that NRF2 down-regulation was also observed after IRI and may participate to enhanced tubular apoptosis in NOX4 KO mice. The NRF2 transcription factor and its target genes were down-regulated in NOX4 KO animals compared to their WT littermates, as assessed by Western blot and real time PCR analysis after IRI ( Fig. 4A-E). We previously demonstrated that NRF2 protein and target gene expression were down-regulated in tubular cells treated with interfering siRNA targeting NOX4 13 . In order to use a second cellular model to study the pathway of this regulation, we verified that NOX4 KO MEF cells also showed decreased NRF2 expression as well as target gene expression (GSTα 2 (Gluthation-s-transferase), GCLC (Glutamate-cysteine ligase catalytic subunit)) compared to WT cells (Supp. Fig. 5).
NOX4 regulates NRF2 by modifying KEAP1 oxidation. We further tried to decipher how NOX4 regulates the NRF2 pathway in vivo and in vitro. KEAP1 is the main regulator of NRF2 in the cytosol. KEAP1 oxidation can be assessed under non-reducing conditions 32 . KEAP1 oxidation as well as expression was assessed by Western blot under non-reducing and reducing conditions in WT and NOX4 KO mice as well as in cell lines ( Fig. 5A,C,E,G). KEAP1 oxidation was decreased in NOX4 KO animals compared to their WT littermates as assessed by the appearance of high molecular weight oxidized KEAP1 band under non-reducing conditions (Fig. 5A,B), whereas total KEAP1 expression was unchanged (Fig. 5C,D). KEAP1 oxidation was also decreased in NOX4 KO MEF cells compared to WT MEFs (Fig. 5E,F) as well as in mCCD cl1 cells transfected with siRNA targeting NOX4 in comparison to scramble RNA transfected cells (Fig. 5G,H). We further performed immunoprecipitation followed by western blotting to determine whether KEAP1 and NOX4 physically interact. There was no evidence for a physical interaction between NOX4 and KEAP1 (Supp. Fig. 6), although the Western blot data obtained for KEAP1 were difficult to interprete, since KEAP1 co-migrates in SDS-PAGE with the immunoglobulin heavy chain (Supp. Fig. 6). These data suggest that NOX4 may regulate NRF2 activity by altering KEAP1 oxidation status. Regulation of the NRF2-Keap pathway may lead to modulation of pro-inflammatory genes 33 . Coherently, using kidney tissue from both WT and KO NOX4, we observed that IRI induced expression of pro-inflammatory genes, that was even more pronounced in NOX4 KO mice compared to WT (Supp. Fig. 7).  NOX4 regulates intracellular glutathione synthesis. NRF2 regulates the antioxidant glutathione pathway. Glutathione is an important tripeptidic cofactor involved in the cytoprotection against oxidative and xenobiotics stresses. Protein glutathionylation decreases in NOX4 KO animals and MEF cells in comparison respectively to their WT littermates (Fig. 6A,B) and WT derived MEF cells (Fig. 6C,D). Total glutathione amount as measured by a luciferase kit decreases in mCCD cl1 cells treated with an interfering RNA targeting NOX4 in comparison to scramble treated cells (Fig. 6E). This observation is consistent with mRNA expression of enzymes involved in glutathione biosynthesis previously demonstrated to be down-regulated in the absence of NOX4 in mCCD cl1 cells 13 as well as in MEF cells (Supp. Fig. 5).

Mitochondrial integrity is altered by NOX4 depletion. Several studies reported mitochondrial or
ER NOX4 localization without a clear insight into its role [34][35][36] . Mitochondrial glutathione depletion may lead to enhanced ROS mitochondrial production. Bcl-2 and NRF2 expression also affects mitochondrial membrane function 37,38 . We therefore assessed mitochondrial potential as a read out for mitochondrial health. MEF mitochondrial membrane potential was assessed by JC-1 assay, based on a dye exhibiting a potential dependent accumulation in mitochondria indicated by a fluorescence emission shift from red to green. NOX4 KO MEF cells displayed a decrease of mitochondrial membrane potential compared to the WT MEF cells (Fig. 7A,B), indicating instability of mitochondrial membranes in the absence of NOX4. In mCCD cl1 cells transfected with a siRNA targeting NOX4, NOX4 depletion induces a basal decrease of mitochondrial membrane potential compared to scramble cells (Fig. 7C,D). We further assessed different subunits of the NADH dehydrogenase (or mitochondrial complex I). We observed no downregulation of subunits tested in Complex I (NDUFS1, NDUFA9, NDUFV1), complex III (MTCO1) and IV (UQCRQ) in MEF cells (Supp. Fig. 8), nor in mCCD cells (data not shown). NADPH dehydrogenase quinone 1 (NQO1) and thioredoxin 2 (TXN), two major mitochondrial ROS scavengers, were also down-regulated in NOX4 KO MEF cells (Fig. 7E,F) as well as in mCCD cl1 cells 33 , probably also contributing to this mitochondrial dysfunction 39 . Overexpression of active NRF2 protein partly reverses the increased propensity to apoptosis in cells deficient in NOX4. We observed that NRF2 was down-regulated in the absence of NOX4. To determine whether NRF2 down-regulation plays a role in the enhanced propensity to apoptosis in NOX4 depleted cells, we transfected NOX4 KO MEF cells with an constitutively active form of the NRF2 protein 19 (Fig. 8A,B).  caNRF2 overexpression in MEF increased the expression levels of antioxidant NRF2 target gene GSTα 2 whereas trends were observed for NQO1 and GCLC C (Fig. 8E). caNRF2 also rescued Bcl-2 downregulation (Fig. 8C,D). Furthermore, caNRF2 overexpression increased glutathione abundance in NOX4-deleted MEF beyond basal values of WT cells (Fig. 8F). Finally caNRF2 overexpression decreased apoptosis by Annexin V assay in NOX4 KO cells, supporting an important role for NRF2 down-regulation in the pro-apoptotic NOX4 KO phenotype (Fig. 8G).

Discussion
In this study, we demonstrate that NOX4 deletion enhances tubular apoptosis in response to IRI in the kidney. We further show that part of this phenotype is in part related to regulation of Bcl-2 via changes in NRF2 regulation and oxidation of KEAP1. NRF2 regulation further induced alterations of glutathione cycling and mitochondrial function.
We first demonstrate that NOX4 protein increases IRI. NOX4 expression has been described to be elevated in different diseases types, but often using poorly specific antibodies 12 . Using an antibody that we characterized in NOX4 KO mice, we demonstrate that NOX4 protein expression increases 24 hours after ischemia in injured proximal tubular cells. The increased NOX4 may play a protective role in IRI. Indeed NOX4 KO mice subjected to IRI display enhanced tubular apoptosis as well as increased creatinine levels 24 hours after injury compared to controls, in conjunction with enhanced tubular lesions. This finding is consistent with our previous observation showing that NOX4 KO mice display enhanced tubular cell apoptosis in response to urinary tract obstruction 13 . In addition, a recent study shows that NOX4 promotes cell survival in stress condition via modulation of elF2α mediated signaling 40 , thereby protecting tubular cells from ER stress mediated tunicamicin apoptosis. These studies confirm that NOX4 complete deletion is detrimental in vivo under conditions of acute or chronic tubular injury. Since NOX4 produces low levels of ROS, mainly in the form of hydrogen peroxide, our observation is also in line with recent work demonstrating that exposure to ROS before IRI is protective 25,26,28 and that ROS scavenging under conditions of ischemia may aggravate kidney injury 41 . Indeed, low levels of baseline ROS production may be critical for the regulation of major antioxidant pathways, such as NRF2, that will then be able to buffer acute ROS production by ischemia, likely issued in great part by mitochondria 42 .
Bcl-2 is a major anti-apoptotic protein. We observed a NOX4-dependent regulation of Bcl-2 protein in two cellular models and in mice under IRI. This regulation likely contributes to enhanced tubular apoptosis observed in vivo and in vitro and to the altered mitochondrial function 38 . Recently, activation of Bcl-2 transcription by NRF2 via ARE binding sites has been described 29,30 . In addition, KEAP1 may alter Bcl-2 stability 31 . Our results tend to show that NOX4 depletion regulates Bcl-2 transcription via altered NRF2 stability since transfection with an active from of NRF2 rescued this regulation in vitro.
As observed following unilateral urinary tract obstruction 13 , NOX4-depleted animals display decreased NRF2 protein expression as well as target gene expression. Here, we demonstrate that NOX4 modulates KEAP1 oxidation in both animals and cell lines, leading to increased NRF2 activity, and enhanced antioxidant defense. This results in decreased glutathione levels in NOX4-depleted cells in vitro and in animals that is rescued by NRF2 overexpression in vitro. Decrease in glutathione, a very potent anti-oxidant, renders kidney tubular cells vulnerable to injury under stress conditions and may disrupt several ubiquitous functions, such as mitochondrial function and cellular redox homeostasis. Previous work demonstrated that NOX4 overexpression may be deleterious for mitochondrial function and enhance apoptosis in podocytes and mesangial cells 43 . In human umbilical vein endothelial cells, NOX4 depletion was associated with stabilization of mitochondrial membrane potential and decreased H 2 O 2 production 36 . In our experiments, embryonic fibroblasts issued from NOX4 KO mice and tubular cells depleted in NOX4 display decreased mitochondrial membrane potential, in line with decreased NRF2 expression 37 , depletion of NQO1, an important regulator of mitochondrial integrity 39 , Bcl-2 and glutathione in cells derived from NOX4 KO mice and in NOX4-depleted cells by siRNA. The enhanced propensity to tubular apoptosis of NOX4 KO mice under IRI appears related in part to regulations of major antiapoptotic and antioxidant pathways via NOX4 dependent regulation of the NRF2 pathway. Our results are consistent with published observations, including ours, that NOX4 KO mice are more susceptible to chronic tubular injury such as urinary obstruction 12,13 and to tunycamycin mediated cellular injury 40 . However, they contrast with recently published observations in diabetic nephropathy where NOX4 deletion appears to be protective 14 . NOX4 expression appears to increase largely during diabetic nephropathy in podocytes and mesangial cells 44 . Therefore, it is possible that NOX4 overexpression in podocytes, where it is poorly expressed under basal conditions, is detrimental and contributes to apoptosis in conditions of hyperglycemia 45 . In mesangial cells, NOX4 accumulation may hypothetically also decrease apoptosis and lead to their accumulation, as described for lung fibroblasts 11 , although this is not demonstrated. On the other hand, NOX4 appears to be important for tubular cells, where basal expression levels are high and may participate in the global REDOX balance and pro-survival pathway regulation of these cells which are frequently subjected to stress stimuli. Since tubular cells are essential to kidney function, this pro-apoptotic effect is detrimental in conditions of acute or chronic injuries, which are relatively frequent events.
In conclusion, we demonstrate that NOX4 deletion enhances kidney tubular cell susceptibility to apoptosis by IRI, a classical model of human AKI. We further demonstrate that in the absence of NOX4, cytoprotective and antioxidant pathways are down-regulated in the kidney. NOX4 appears to be an important regulator of NRF2 in kidney tubular cells via oxidation of KEAP1 and this may explain some of the consequences of NOX4 depletion. Finally, this raises important questions regarding therapies that aim to completely abrogate ROS production in the kidney, which may alter tubular defenses in acute tubular stresses.

Methods
Ischemia reperfusion. WT and KO NOX4 mice on a C57BL6 background were generated as previously described 46 . Littermates were used for experiments. All animal experiments were approved by the Institutional Scientific RepoRts | 6:38598 | DOI: 10.1038/srep38598 Ethical Committee of Animal Care in Geneva and Cantonal authorities. The methods were carried out in accordance with the approved guidelines of the Swiss federal office for animal studies. Briefly, 22-30 gr mice mice were anesthetized by intra-peritoneal injection of ketamine and xylazine (100 mg/kg and 5 mg/kg, respectively) (GRAEUB, BayerHealthcare) and shaved. They were then laterally placed on a thermostatic working station. A 1 centimeter incision at 0.5 centimeter to the limit of the last rib and of the vertebral column was performed. The skin was separated from the muscular layer using a sterile cotton bud. A second incision of the muscular layer was performed. The kidney and renal artery were then exposed and after careful dissection, 22 minutes renal occlusion was performed using an atraumatic vascular clamp. Similar procedure was performed on the contralateral side. The animal ischemia-reperfusion and recovery was done and on a heating pad and in a thermostatic cage respectively (37 °C for both). 24 h after renal artery occlusion release, the animal was sacrificed and the two kidneys harvested for histological, biochemistry and molecular biology analysis.
Mouse kidney histology and immunohistochemistry. Kidneys from IRI and sham operated animals were fixed in 4% paraformaldehyde (Alfa Aesar), paraffin embedded and 5 μ m section cut with a microtome. Sections were stained with Haematoxylin Eosin and PAS. Immuno-histostaining were performed using citrate buffer (10 mM, pH 6) microwave based antigen target retrieval technique and the EnvisionFlex kit from Dako. TUNEL assay. Terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein Nick End Labelling (TUNEL) was performed to detected apoptotic (necrotic) cells using DeadEnd TM fluorometric TUNEL system (Promega) according to the manufacturer's instructions. The slides were visualized and analyzed using fluorescent microscope coupled to a camera (Axiocam) and MIRAX midi slide scanning system (Zeiss) with a MIRAX viewer respectively. Quantitative analysis of TUNEL assay was carried out on slides using metamorph software. Briefly, all slides were scanned with MIRAX midi (Zeiss) coupled to Axiocam MRm (Zeiss) or photographed on a fluorescent microscope coupled to Axiocam (Zeiss). 5 random images were taken in different regions of renal cortex and Cortico medullary junction and analyzed with Metamorph image analysis software. The positive staining were matched and reported to the total tissue area of each animal under ischemia reperfusion or sham conditions. TUNEL positive cells re reported to the total tissue area respectively for apoptosis. Data were expressed as ratio of positive area by the total area examined and the mean for all animals in each group. Serum creatinine measurement. Creatinine was measured using the Jaffe colorimetric method.

Tubular injury score. Haematoxylin
Cell culture and transfection. mCCD cl1 cells were seeded and cultured as described previously 47 . WT and NOX4 KO MEF cells were seeded and cultured in complete medium (DMEM, 10%FCS and 1% penicillin streptomycin). mCCD cl1 and MEF Cells were seeded at low confluence (30-40%) in 6 well plates in complete medium 24 h before transfection. mCCD cl1 cells were transfected with 120 pmol of Stealth RNAi against NOX4 and 5 μ L of lipofectamin 2000 (Invitrogen) for 72 h. The siRNA duplexes (Invitrogen) were provided as purified and annealed duplexes. The following sequences were used: siNOX4 5′ -UUUAGGGACAGCCAAAUGAGCAGGC-3′ and scramble 5′ -GCCACUCGUUUGUCGCCCUUGUAAA-3′ . MEF cells were transfected with FuGene HD (Roche) Transfection Reagent using a 3:2 ratio (100 μ L DMEM, 3 μ L FuGene Transfection Reagent and 2 μ g DNA) and a plasmid DNA expressing active NRF2 (a gift from Matthias Schaffer, Zurich) for 72 h. For the co-immunoiprecipitation experiments, cells overexpressing human NOX4 in an inducible fashion were used 4 . These cells express both KEAP1 and human NOX4 (data not shown).
In vitro mitochondrial membrane potential assessment (Δψm) by JC-1 assay. mCCD cl1 and MEF cells were seeded at low confluence in 6 well-plate 24 h before JC-1 assay (Life Technologies) to assess mitochondrial membrane potential. To determine the electric potential of the inner mitochondrial membrane, cells were suspended in warm medium (DMEM phenol red free) at approximately 1 × 10 6 cells/mL and incubated 5 minutes at 37 °C (5% CO 2 ) then 30 minutes in the presence of 2 μ M final of JC-1 (5′ ,6,6′ -tetrachloro-1,1′ ,3,3′ -tetraethylbenzimidazolylcarbocyanine iodide). 50 μ M CCCP (Carbonyl cyanide -3-chlorophenylhydrazone), a mitochondrial membrane potential disrupter was used as a positive control. After the incubation, cells were pelleted, washed and suspended in 500 μ L of medium before flow cytometer analysis (488 nm excitation, emission filter appropriate for Alexa Fluor 488 dye and R-phycoerythrin).
Apoptosis assessment by Annexin V/PI assay. Cells were detached, washed with cold PBS and resuspended at 1 × 10 6 cells/mL in 1x binding medium (10 mM Hepes [pH7.4], 140 mM NaCl and 2.5 mM CaCl 2 ). Then 100 μ L of the solution (10 5 cells) were incubated with 5 μ L of Annexin APC and 1 μ L of PI (1 mg/mL) 15 minutes at RT (25 °C) in the dark. 400 μ L of 1x binding medium were added in each tube before flow cytometer analysis.
In vitro glutathione measurement by GSH-GLO assay. Cells were plated as previously described for transfection assay. Cells were then incubated 30 minutes with a GSH-GLO reagent containing a Luciferin NT-substrate and Glutathione S transferase according to the manufacturer procedure (GSH-GLO Promega). In the presence of glutathione, the GST catalyzes the reaction generating the luminogenic substrate, luciferin proportionally to the amount of glutathione. Addition of Luciferin detection reagent containing a stabilized Scientific RepoRts | 6:38598 | DOI: 10.1038/srep38598 luciferase (Ultra-Glo Luciferase) allows the luminescent reaction. The luminescent signal, proportional to the glutathione availability, is read in a 96 well plate luminometer.
Western blot analysis. Cells or kidney tissue samples were homogenized in 100 μ l or 1 ml respectively in cold lysis buffer (20 mmol/L Tris-HCl; 2 mmol/L EDTA; 30 mmol/L NaF; 30 mmol/L NaPPi; 0.5 mol/L Na 3 VO 4 ; 20% SDS; 10% Triton-X-100 and Roche-Complete mini protease inhibitor mixture) on ice. Protein concentration assay was performed using both BCA protein Assay (Thermo Scientific, PIERCE) and Coomassie Blue gel staining (Thermo Scientific GelCode blue stain Reagent). For protein oxidation analysis in non-reduced condition, lysis buffer is completed with 40 mM of NEM (N-ethylmaleinide) in absence of β -mercaptoethanol. Equal amount of protein were separated by 4-12% Bis-Tris pre Cast protein Gel (Invitogen) or 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Immobilon-P, Milipore). Rabbit polyclonal anti-PARP (Cell Signaling), Rabbit anti-NRF2 (Santa Cruz), Rabbit anti-Keap1 (Proteintech), Rabbit anti Bcl-2 (Cell signaling and Santa Cruz), Mouse anti-glutathionylated proteins (Virogen) and mouse anti-β -actin (Sigma) antibodies were used. HRP labeled polyclonal anti-mouse or rabbit Ig (BD Biosciences Pharmingen) were used and the antigen-antibody complexes were detected by the chemiluminescent HRP substrate method (Immobilon, Milipore). Bands were quantified using a Java-based image processing software (ImageJ). A rabbit monoclonal antibody against NOX4 was used as described 48 Freshly Isolated Renal Cortical Tubule Suspensions. Renal cortex was isolated using a modification of the method described by Guder 49 previously described by Fenton 50 ). Cortex from both kidneys was dissected, sliced into ∼ 1-mm pieces, and then placed in an enzyme solution containing 0.5 mg collagenase type IIand 0.5 mg/mL protease inhibitor pronase (Roche Diagnostics) at 37 °C in buffer B (125 mM NaCl, 0.4 mM KH2PO4 1.6 mM K2HPO4, 1 mM MgSO4, 10 mM Na-acetate, 1 mM α -ketogluterate, 1.3 mM Ca-gluconate, glucose 30 mM, 5 mM glycine, 48 μ g/mL trypsin inhibitor, and 25 μ g/mL DNase, pH 7.4). Two milliliters of enzyme solution was used for each kidney cortex. Samples were mixed continuously at 850 × g at 37 °C. After 10 min, half of the enzyme solution was removed and replaced with buffer B, and samples were incubated for a further 10 min. This procedure was repeated for another 10 min. Samples were then spun at 200 × g for 2 min, buffer B was removed and samples were resuspended in HamF12/DMEM cell medium. The cortical tubular suspensions were kept at 37 °C under 5% CO 2 and 19% O2 during 4 hours.
Co-Immunoprecipitation. Co-Immunoprecipitation was performed as previously described 51 . In brief, cells were harvested under non denaturing conditions with 500 uL of RIPA buffer and then centrifuge for 15 minutes at 13,000 RPM, 4 °C. A pre-clearing step was conducted on the resulting supernatant with 100 μ l of GammaBind Plus Sepharose beads for 60 minutes, 4 °C. The primary antibody was incubated on a tube roller for 3 hours at 4 °C. GammaBind Plus Sepharose beads were added for another 30 minutes at 4 °C with gentle rocking. After a 2 min centrifugation at 5000 RPM, 4 °C the protein pellet was re-suspended with 50 μ l of SDS-PAGE sample buffer for later use.

Statistics. Groups statistics were analyzed by t test and two way ANOVA/Bonferroni's Multiple Comparison
Test respectively for two or multiple groups.