Targeted Deletion of P53 in the Proximal Tubule Prevents Ischemic Renal Injury

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Targeted Deletion of p53 in the Proximal Tubule Prevents Ischemic Renal Injury

† ‡ Yuan Ying,* Jinu Kim,* Sherry N. Westphal,* Kelly E. Long,* and Babu J. Padanilam*

Departments of *Cellular and Integrative Physiology and ‡Internal Medicine, Section of Nephrology, University of Nebraska Medical Center, Omaha, Nebraska; and †Department of Anatomy, Jeju National University School of Medicine, Jeju, Republic of Korea

ABSTRACT The contribution of p53 to kidney dysfunction, inflammation, and tubular cell death, hallmark features of ischemic renal injury (IRI), remains undefined. Here, we studied the role of proximal tubule cell (PTC)-specific p53 activation on the short- and long-term consequences of renal ischemia/reperfusion injury in mice. After IRI, mice with PTC-specific deletion of p53 (p53 knockout [KO]) had diminished whole-kidney expression levels of p53 and its target genes, improved renal function, which was shown by decreased plasma levels of creatinine and BUN, and attenuated renal histologic damage, oxidative stress, and infiltration of neutrophils and macrophages compared with wild-type mice. Notably, necrotic cell death was attenuated in p53 KO ischemic kidneys as well as oxidant-injured p53-deficient primary PTCs and pifithrin-a–treated PTC lines. Reduced oxidative stress and diminished expression of PARP1 and Bax in p53 KO ischemic kidneys may account for the decreased necrosis. Apoptosis and expression of proapoptotic p53 targets, including Bid and Siva, were also significantly reduced, and cell cycle arrest at the G2/M phase was attenuated in p53 KO ischemic kidneys. Furthermore, IRI-induced activation of TGF-b and the long-term development of inflammation and interstitial fibrosis were significantly reduced in p53 KO mice. In conclusion, specific deletion of p53 in the PTC protects kidneys from functional and histologic deterioration after IRI by decreasing necrosis, apoptosis, and inflammation and modulates the long-term sequelae of IRI by preventing interstitial fibrogenesis.

J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013121270

AKI is a clinical syndrome characterized by a rapid The transcription factor p53, which was first decline in GFRover a period of hours to days, leading to identified as a tumor suppressor, performs many retention of metabolic waste products and disrupted essential cell functions, such as halting the cell cycle, fluid, electrolyte, and acid–base balance.1 Ischemic re- promoting senescence and apoptosis, and regulat- nal injury (IRI), which results from compromised per- ing cell metabolism.15 In response to various cell fusion of renal tissues, is the leading cause of AKI.2,3 stresses and DNA damage, p53 controls the tran- Persistent perfusion deficitinthemedulla,limitedan- scription of target genes that are usually key factors aerobic glycolytic capacity, and targeted inhibition of in cellular stress pathways.16 Although the transcrip- glycolysis in the proximal tubule cell (PTC) make the tional activation of p53 was considered the major proximal straight tubule the most vulnerable tubular mechanism by which p53 responds to cell stress, sev- segment to ischemic injury.4–7 Pathologically, IRI is eral studies suggest that transcription-independent characterized by apoptotic/necrotic cell death and inflammation in the outer medulla, which are propor- tional to the severity of renal ischemia.8,9 Pro- and Received December 5, 2013. Accepted March 11, 2014. antiapoptotic signaling pathways in the PTC are pre- Published online ahead of print. Publication date available at cisely regulated by essential factors, such as p53 and www.jasn.org. its proapoptotic targets, BCL2 family proteins and Correspondence: Dr. Babu J. Padanilam, Department of Cellular caspases.9–14 Therefore, it is crucial to understand and Integrative Physiology, 985850 Nebraska Medical Center, the roles of these molecules in regulating tubular cell Omaha, NE 68198-5850. Email: [email protected] apoptosis in the pathogenesis of IRI. Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: ccc–ccc, 2014 ISSN : 1046-6673/2512-ccc 1 BASIC RESEARCH www.jasn.org activity of p53 can potentiate apoptosis by directly interacting IRI (Figure 2B). No significant difference in renal function oc- with members of BCL2 family proteins.12,17 curred among sham-operated WTand KO mice (Figure 2, A and The involvement of p53 has been reported in nephrotoxic B). Moreover, there were no significant differences in BUN or injury and IRI.18–20 Although previous studies showed that serum creatinine values between WT and heterozygote mice af- p53 levels are significantly increased in the medulla after ter IRI (data not shown). These data suggest that specificde- IRI,9 the contribution of p53 to tubular cell death and kidney letion of the p53 gene in the PT leads to renal functional pro- dysfunction is still unclear.8,9,20–22 Thus, considering the im- tection after IRI in mice. portance of p53 in promoting apoptotic/necrotic cell death, we hypothesized that knockout (KO) of p53 in the proximal PT-Specific KO of p53 Decreased Renal Histologic tubule significantly reduces tubular cell death and kidney dys- Damage after IRI function after IRI. We tested this hypothesis using KO mice Ischemic kidneys from WTmice showed widespread necrosis, with the p53 gene specifically deleted in the proximal tubule. brush border blebbing, and sloughed cells in the proximal straight tubule, whereas these features were much less apparent in ischemic kidneys from p53 KO mice. The histologic changes RESULTS after IRI were quantified by counting and scoring the percentage of tubules that displayed tubular necrosis, cast formation, Deletion of p53 in the Proximal Tubule Reduced p53 and tubular dilation (Figure 2, C and D). The cumulative Expression after IRI scores of histologic damage in the outer medulla at 1, 5, and Weanalyzed the expressionofp53 and one ofits target genes, p21, 16 days as well as necrosis (Figure 2E) at 1 day were signifi- after injury in whole-kidney tissues. Indeed, 24 hours after IRI, cantly lower in p53 KO kidneys compared with WT kidneys the expression levels of p53 (Figure 1, A and B) and p21 (Figure 1, post-IRI, showing that gene ablation of p53 reduced tubular A and C) were significantly increased in wild-type (WT) male damage and cellular necrosis. littermates compared with sham-operated mice kidneys. How- ever, the expression level of p53 was only slightly increased and Renal Inflammation Was Reduced after IRI in p53 KO p21 expression was not altered in p53 KO mice compared with Mice sham-operated mice after IRI. The slightly increased levels of p53 The infiltration of leukocytes in the outer medulla of WT and in IRI-induced p53 KO may be because of p53 expression in cells p53 KO mice at 1, 5, and 16 days post-IRI was assessed by other than the proximal tubules (PTs). Be- cause of the difficulty in immunodetection of p53 in renal tissue, we carried out Western blot analysis for p53 in PTs isolated from p53 KO mice. Our data further confirmed suc- cessful deletion of p53 in the PT (Supple- mental Figure 1). These data suggest that p53 is induced and involved in transcrip- tional regulation after IRI.

PT-Specific KO of p53 Reduced Deterioration of Renal Function after IRI Totest whether the absence of p53 in the PTC changes the course of IRI, kidney function after IRI in p53 KO mice and WT littermates was assessed. BUN levels were increased in WTmice 6 hours after IRI and peaked during 24–48 hours compared with WT sham- operated mice. The increase in BUN level was significantly less in p53 KO mice 6–48 hours after IRI compared with WT mice (Figure 2A). Serum creatinine levels were – also increased in WT mice 6 48 hours after Figure 1. PT-specific KO of p53 reduced p53 and p21 expression after IRI. (A) Repre- IRI compared with WTsham-operated mice. sentative images of Western blot analysis showing expression levels of p53 and p21 at 24 Similar to the BUN levels, the increase of hours (1 d) after IRI. (B and C) The expression levels of p53 and p21 in sham and IRI-induced serum creatinine was less in p53 KO com- kidneys were quantified (n=4 in each group). *P,0.05 compared with WT IRI. GAPDH pared with WT at 6, 24, and 48 hours after served as a loading control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Figure 2. PT-specific KO of p53 reduced renal function deterioration and histologic damage after IRI. (A) Plasma creatinine and (B) BUN levels from WT and p53 KO mice (n=6) at 6 hours (h) and 1, 2, and 3 days after IRI. *P,0.05 compared with WT IRI. (C) WT or p53 KO mice underwent sham surgery or IRI. Renal histologic changes in the outer medulla after IRI were assessed by Periodic acid–Schiff (PAS) staining at 1, 5, and 16 days after IRI. Original magnification, 3400. (D) Histologic damage in the outer medulla assessed in PAS-stained kidney sections was scored by counting the percentage of tubules that displayed tubular necrosis, cast formation, and tubular dilation as follows: 0, normal; 1, ,10%; 2, 10%–25%; 3, 26%–50%; 4, 51%–75%; 5, .75%. Ten fields (3200 magnification) per kidney were used for counting. *P,0.05 compared with WT IRI (n=6). (E) The number of necrotic tubules was counted in PAS-stained kidney sections at 1 day after IRI. *P,0.05 compared with WT IRI 1 day (n=6).

J Am Soc Nephrol 25: ccc–ccc, 2014 p53 Deficiency Reduces Ischemic Renal Injury 3 BASIC RESEARCH www.jasn.org immunostaining for neutrophils and macrophages. As shown significantly increased in WT kidneys after IRI, but its expression in representative photographs (Supplemental Figure 2), WT was downregulated in p53 KO mice (Figure 3D). This novel mice exhibited increased infiltration of neutrophils and mac- finding suggests that increased PARP1 function may be one of rophages in the outer medulla, which was attenuated in p53 the mechanisms by which p53 activation regulates necrosis. KO mice. The numbers of positively stained cells were counted in a blinded manner, and quantitative data indicate that the PT-Specific KO of p53 Decreased Apoptosis after IRI accumulation of neutrophils and macrophages was reduced in Todetermine whether thereisa changeof apoptotic levelsin the the outer medulla of p53 KO mice compared with WTmice at outer medulla of WT mice compared with p53 KO mice, all time points after IRI (Figure 3, A and B). terminal deoxynucleotidyl transferase-mediated digoxigenin- deoxyuridine nick-end labeling (TUNEL) assay was performed Attenuated Oxidative Stress after IRI in p53 KO Mice together with Western blot analysis for proapoptotic proteins. Oxidative stress was assessed by lipid hydroperoxide levels in The outer medulla of WTmice exhibited increased numbers of the kidney. Quantification of the whole-kidney lipid peroxide TUNEL-positive cells compared with that of p53 KO mice (Fig. levels at 5 hours and 1 and 2 days post-IRI shows that its levels 4A, Supplemental Figure 3). Cleaved Caspase-3 expression were significantly decreased in p53 KO mice compared with was increased in WT kidney 24 hours after IRI compared WT mice at all time points (Figure 3C). with p53 KO mice (Figure 4, B and C), further confirming increased apoptosis. Similarly, Western blot analysis showed PT-Specific Deletion of p53 Decreased PARP1 enhanced activation of Bax in the kidney of WT mice com- Expression after IRI pared with that of p53 KO mice 24 hours after IRI, whereas PARP1can induce necroticcell deathafterIRI.PARP1expression total Bax was not changed. The expression of proapoptotic was examined by Western blot analysis. PARP1 expression was proteins Bid and Siva was also increased in WT mice after IRI compared with p53 KO mice (Figure 4, B and D–F). These data suggest that specific deletion of p53 in the PT decreases apoptotic cell death after IRI by decreasing the levels of proapoptotic proteins.

Loss of p53 Reduced Renal Fibrosis after IRI To investigate whether deletion of p53 reduces renal fibrosis, collagen deposition in the kidneys of WT and p53 KO mice was measured using Sirius red staining and a-SMA immunofluorescence staining. Six- teen days after IRI, WT mice showed a dra- matic increase of Sirius red-positive area in the kidneys compared with p53 KO mice, indicating that deletion of p53 reduces renal fibrosis in the later stage of IRI (Figure 5, A and B). No change in the Sirius red staining was seen at 5 days postinjury. a-SMA expression, however, was decreased in p53 KO mice compared with WT mice at 5 days after IRI, which was shown by immunostaining and Western blot analysis (Figure 5, C–F). p-Smad3, a downstream signaling molecule of TGF-b, was also increased in WT mice but significantly reduced in p53 KO mice at fi fi Figure 3. PT-speci c KO of p53 decreased leukocyte in ltration, oxidative stress, and 16 days post-IRI (Figure 5, E and G). PARP-1 expression. The number of (A) neutrophils and (B) macrophages accumulated in the outer medulla of WT and p53 KO kidneys post-IRI was measured at 1, 5, and 16 days after IRI in high-magnification (3200) fields. *P,0.05 compared with WT IRI (n=4). Loss of p53 Reduced Cell Cycle Arrest (C) Lipid hydroperoxide levels in the WT and p53 KO mice after IRI. *P,0.05 com- after IRI pared with WT IRI (n=6). (D) Western blots showing increased expression of PARP1 24 h Cell cycle arrest at G2/M phase is associated after IRI in WT kidneys. *P,0.05 compared with WT IRI (n=3). GAPDH, glyceraldehyde- with late kidney fibrosis in IRI. Histone H3 3-phosphate dehydrogenase. is phosphorylated during mitosis at Ser-10

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p-H3–positive cells were arrested at G2 phase (Supplemental Figure 4, B and C). The number of cells at M phase was negligi- ble at 5 and 16 days in both WTand p53 KO mice tissues.

Loss of p53 Reduced Necrotic Cell Death of PTC To further study the role of p53 in necrotic cell death, we used an in vitro H2O2- induced necrosis model. Necrosis was as- sayed using trypan blue staining and lactate dehydrogenase (LDH) release assay. After 1 hour of treatment of 1 or 2 mM H2O2,p53 KO primary PTCs had fewer trypan blue- positive cells compared with WT PTCs (Figure 6A). Pharmacological inhibition of p53 using piﬁthrin-a also reduced LDH release in LLC-PK1 cells after 5 mM H2O2 treatment (Figure 6B). These data indicate that deletion or inhibition of p53 reduces H2O2-induced necrosis.

DISCUSSION

Several studies have previously investigated the role of p53 in IRI. Kelly et al.9 first showed that p53 expression is increased in the renal medulla after IRI and that pharmacological inhibition of p53 using Figure 4. Apoptosis and pro-apoptotic molecule expression were reduced after IRI in pifithrin-a can reduce renal injury in rats. p53 KO mice. (A) TUNEL assay to detect apoptotic cells was done using the In Situ Cell Molitoris et al.20 showed that systemic ad- Death Detection Kit in kidneys derived from WT or p53 KO at 1, 5, and 16 days after IRI. ministration of p53-targeted small inter- The number of TUNEL-positive cells was measured in 10 randomly chosen high- magnification (3200) fields per kidney. *P,0.05 compared with WT IRI (n=5). Repre- fering RNA in mice attenuates ischemic sentative Western blot images showing increased expression of cleaved Caspase-3 (B, C), and cisplatin-induced AKI. In a recent re- Bax (B, D), Bid (B, E), and Siva (B, F) in WT mice after 24 h of IRI. *P,0.05 compared with port, Yang et al.26 administered pifithrin-a WT IRI (n=3–5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. in a single dose 3 and 14 days after unilateral IRI in mice and showed that it relieved epithelial cell cycle arrest and inhibited fi- 23 in the G2/M phase. Phospho-H3 (p-H3) staining was per- brogenesis. However, two recent reports showed that p53 in- formed to detect G2/M phase arrest. In WT mice, the number hibition is detrimental to renal function in IRI. Dagher et al.21 of p-H3–positive cells was significantly higher compared with administered pifithrin-a daily, starting at the time of bilateral the number of p-H3–positive cells in p53 KO mice at 1, 5, and 16 IRI in rats and continuing for 7 days, which ultimately in- days after IRI (Fig. 5H, Supplemental Figure 4A). To confirm creased renal fibrosis. To add to the paradox, Sutton et al.22 that the cells are truly arrested at the G2 phase and not in mitotic showed that global p53 deficiency or pharmacological inhibi- phase, we assessed the number of mitotic cells at the above time tion of p53 exacerbates the injury by increasing and prolong- points. Mitotic entry is accompanied by the phosphorylation of ing leukocyte infiltration into the renal parenchyma. These several molecules, including mitotic protein monoclonal 2 differing results suggest that the role of p53 in the pathophys- (MPM-2), that may regulate the mitotic processes.24,25 Immu- iology of IRI is much more complicated and still incompletely nostaining using antiphospho-Ser/Thr-Pro and MPM-2 anti- understood. body (05–368; EMD Millipore, Bedford, MA) showed that the In this study, we used KO mice with p53 gene specifically number of cells at M phase was increased in WTmice 1 day after deleted in the PTC to study its effect on the kidney damage after IRI compared with in KO mice, but it only accounted for a small IRI. This strategy not only specifically targets the PT, the major fraction of the cells arrested at G2/M phase, indicating that most injury site of IRI,5 but also excludes the normal function of p53

J Am Soc Nephrol 25: ccc–ccc, 2014 p53 Deﬁciency Reduces Ischemic Renal Injury 5 BASIC RESEARCH www.jasn.org

Figure 5. Loss of p53 in PT reduced renal fibrosis and cell cycle arrest after IRI. (A) Collagen deposition detected by Sirius red staining in WT and p53 KO kidneys at 5 and 16 days after IRI. (B) The Sirius red-positive area was measured in four randomly chosen high-power (3200) fields per kidney using the National Institutes of Health ImageJ software. *P,0.05 compared with WT IRI (n=3). (C and D) Immunofluorescence staining of a-SMA in the outer medulla. The a-SMA–positive area was measured in four randomly chosen high-power (3200) fields per kidney. *P,0.05 compared with WT IRI (n=3). (E and F) Representative Western blot images and quantified data for expression of a-SMA in WT and p53 KO kidneys at 1, 5, and 16 days after IRI. *P,0.05 compared with WT IRI (n=3). (E and G) Representative Western blot films and quantified data for expression of p-Smad3 in WT and p53 KO kidneys at 1, 5, and 16 days after IRI. *P,0.05 compared with WT IRI (n=3). GAPDH served as a loading control. (H) Kidney sections were immunostained using p-H3 antibody. The number of p-H3–positive cells in WT and p53 KO kidneys at 1, 5, and 16 days after IRI was counted in 10 randomly selected high-power (3200) fields per kidney. *P,0.05 compared with WT IRI (n=4). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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in the outer medulla of p53 KO mice kidneys compared with WTafter IRI (Figure 2). The role of CypD in necrotic cell death after IRI is established by us and other groups.31,32 Two key factors involved in CypD activation are redox stress and calcium mishandling. Our data indicate that p53 deletion can decrease oxidative stress, which could be a potential mechanism by which p53 inhibits necrosis in renal PTCs. Can p53 translocate to mitochondrial matrix and interact with CypD to open the MPTP after IRI? Our attempts to locate p53 to the mitochondria in various in vitro and in vivo models failed, suggesting Figure 6. Loss of p53 reduced PTC necrosis. (A) Percentage of trypan blue-positive cells that it may not be a prominent mechanism after H2O2 treatment in primary PTCs derived from p53 KO compared with WT mice kidneys. *P,0.05 (n=4). (B) Percentage of LDH activity after 5 mM H O treatment in by which p53 induces necrosis in renal PTC 2 2 fi LLC-PK1 cells with or without different concentrations of the p53 inhibitor pifithrin-a. (data not shown). However, our novel nd- *P,0.05 (n=4). Con, control with PBS or DMSO (dimethyl sulfoxide) treatment. ing that p53 inhibition can downregulate PARP1expressioninrenalPTCsuggests that it could be an alternate mechanism by in other cells within the kidney, like inflammatory cells re- which p53 regulates necrosis after IRI. Activation of PARP1 is cruited after IRI, in which absence of p53 may prolong in- required for DNA repair, but excessive activation leads to ne- flammatory responses and increase renal injury.22 Our data crotic cell death by depletion of intracellular ATP.33,34 We pre- show that specific deletion of p53 gene in the PT significantly viously reported that pharmacological and genetic inhibition of improves kidney function, preserves renal histology, and re- PARP1 can prevent kidney dysfunction, oxidative stress, inflam- duces necrosis and apoptosis after IRI along with attenuated mation, and tubular necrosis but not apoptosis after ischemia/ inflammatory response and late-term fibrogenesis. These reperfusion7,35 and cisplatin nephrotoxicity.36 Furthermore, al- findings support the concepts that p53 is a critical mediator though bax/bak is generally regarded as an apoptosis inducer by of IRI and that its inhibition or its downstream signaling path- regulating mitochondrial outer membrane permeabilization, re- way prevents IRI and is a suitable therapeutic target. cent evidence suggests that they may form the outer membrane A role for p53 in apoptosis is well established. p53 can channels of MPTP and contribute to necrotic cell death.37,38 Bax upregulate the expression of several proapoptotic genes, in- expression is attenuated after IRI as a function of p53 and thus, cluding Bax, Fas/Apo-1, PERP, Siva, PUMA, and Noxa, under could play a role in attenuating necrosis. stress situations.16 Recently, Dong and colleagues,27 using two It is well established that neutrophils, monocytes/macro- Bax-deficient mouse models, found that only conditional Bax phages, and T cells play major roles in the pathophysiology of deletion specifically from PTs could ameliorate IRI. Systemic renal ischemia-reperfusion injury in animal models and deletion of Bax enhanced neutrophil infiltration without signif- human AKI.39–42 Blocking the activation or trafficking of icant effect on kidney injury.27 Our data indicate that expression proinflammatory leukocytes into the kidney is shown to pre- of Bax, Bid, and Siva are induced in ischemic kidneys but atten- vent renal function deterioration and histologic damage. Re- uated in p53 KO mice. The significance of Bid and Siva expres- cently, Sutton et al.22 used bone marrow transplantation to sion in IRI was previously reported. In a previous report, Dong produce chimeric mice lacking p53 in leukocytes and showed and colleagues28 showed that Bid deficiency attenuated tubular that p53 deletion prevents leukocyte apoptosis and increases disruption, tubular cell apoptosis, and caspase-3 activation dur- their potential for cytokine secretion, thus worsening the ing 48 hours of reperfusion. We previously reported that the pathophysiology of IRI. This study also showed that systemic expression of the proapoptotic p53 target Siva and its cognate deletion of p53 can worsen the injury.22 Our studies using receptor CD27 is highly upregulated and coexpressed in the mice lacking p53 in PT, however, showed that it can pro- ischemic rat and mouse renal tissues,29 indicating that Siva foundly decrease the infiltration of leukocytes and thus, pro- plays a critical role in apoptosis after IRI. It is likely that synergic tect the kidneys from IRI. The apparent paradox in the data functions of these proapoptotic molecules may lead to the exe- from the two studies could be because of the variations in the cution of apoptosis after IRI. experimental protocol and the differing effects of inhibition of Recently, Vaseva et al.30 showed that p53 may also be implicated p53 in specific cell types within the kidney. in necrotic cell death through its interaction with cyclophilin D The role of cell cycle arrest and tubular apoptosis on the (CypD) in the inner membrane of mitochondria and subsequent fibrogenic response of kidney tissue has recently been estab- opening of mitochondrial permeability transition pore (MPTP). lished in different injury models of CKD.26 Defects in pro- Indeed, our data showed significantly reduced tubular cell necrosis gression of the cell cycle after injury, such as arrest at G1/G0 or

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G2/M phase, cause tubular cells to switch to a profibrotic phe- induced by bilateral renal pedicle clamping using microaneurysm notype with increased expression and release of TGF-b, clamps (Roboz Surgical Instrument, Gaithersburg, MD), and the core thereby promoting fibrosis.26 p53 is a major player in cell cycle body temperature of mice was kept at 37°C. After 30 minutes of occlu- regulation. The effect of pharmacological inhibition of p53 sion, the clamps were removed, and kidney reperfusion was verified using pifithrin-a in fibrosis development after IRI was pre- visually. Sham-operated control animals underwent the same surgical viously examined by two groups21,26 (the opposing results are procedure, except for the occlusion of the renal arteries. During the detailed above). Although the exact cause of these differing surgery, all animals were placed on a heating pad to maintain body results is not clear, the different species and the timing of p53 temperature at 37°C. Blood samples were collected at the time of eu- inhibition in the two experiments may have contributed. Our thanasia or from the orbital sinus under isoflurane anesthesia at 0, 6, 24, data indicate that G2/M cell cycle arrest occurs after IRI in WT and 48 hours post-IRI for measurement of serum creatinine and BUN. mice but not p53 KO mice, which was shown by reduced p-H3 At the end of each time point (1, 5, or 16 days), renal tissue was collected, staining, and may contribute to the attenuation of fibrosis. PT- fixed in Bouins solution or snap frozen using liquid nitrogen, and stored specific deletion of p53 downregulated p21 expression, sug- at 280°C for future experiments. gesting that it may titrate the level of p21, which allows the cells to progress through G2 into M phase. However, our data show Measurement of Serum Creatinine and BUN that p53 deletion can prevent inflammation and both necrotic Serum creatinine and BUN were measured to evaluate renal function and apoptotic cell deaths. Although it is unclear if the severity using a Quantichrom assay kit (BioAssay Systems, Hayward, CA) of the injury after an ischemic episode directly affects the pro- according to the manufacturer’s protocol. gression of CKD, it is likely that the attenuated inflammation and injury in the p53 KO mice may contribute to the decreased Morphologic Studies fibrosis. Additional studies are needed to answer this question. WTand KO mice that underwent IRI were euthanized at 1 or 16 days. In summary, our data show that p53 has profound effects on The kidneys thenwereprocessed at the UniversityofNebraskaMedical tubular cell necrosis and apoptosis after IRI. Absence of p53 in Center histology core facility. Briefly, kidneys were fixed in formalin, the PT significantly preserves renal function and markedly embedded in paraffin, and cut into 5-mm sections. The tissue sections reduces kidney damage, renal oxidative stress, inflammation, were then stained with Periodic acid–Schiff. and long-term fibrosis. Targeting proximal tubular cell death by modulating p53 or its downstream signaling pathways may Immunofluorescence for Neutrophils provide a more efficient therapeutic strategy for IRI. Formalin-fixed mouse kidney sections were processed for immunostaining as described previously.44 The slides were sequentially incubated with rabbit anti-mouse neutrophil antibody (Accu- CONCISE METHODS rate, Westbury, NY) at a 1:100 dilution overnight at 4°C followed by FITC- conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) Generation of PTC-Specific p53 KO Mice at a 1:200 dilution for 1 hour at room temperature. Neutrophil infiltration Homozygous p53-floxed mice (C57BL/6J background) were obtained was quantified by counting the number of stained cells per field. from The Jackson Laboratories (Bar Harbor, ME). The breeding strategy for transgenic mice that expressed Cre recombinase under the control of Immunohistochemistry for Macrophages and p-H3 kidney-specific Pepck promoter (Pepck-Cre) was reported elsewhere.43 Staining KO mice with the p53 gene specifically disrupted in renal proximal tubular Formalin-fixed mouse kidney sections were processed for immunostaining fl fl 2 epithelial cells (genotype p53 / ,Cre+/ ) were developed by mating by sequential incubation with rabbit anti-F4/80 antibody (18705–1-AP; p53-floxed mice with Pepck-Cre transgenic mice. A routine PCR pro- Proteintech, Chicago, IL) and anti–p-H3 antibody (sc-8656-R; Santa tocol was used for genotyping from tail DNA samples with the following Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution overnight at primer pairs: Cre, 59-CGGTGCTAACCAGCGTTTTC-39 and 59- 4°C followed by horseradish peroxidase-conjugated goat anti-rabbit TGGGCGGCATGGTGCAAGTT-39 and p53, 59-GGTTAAACC- IgG (Vector Laboratories) at a 1:200 dilution for 1 hour at room tem- CAGCTTGACCA-39 and 59-GGAGGCAGAGACAGTTGGGAG-39. perature. The color development was induced by diaminobenzamide fl fl 2 2 Male littermates of the genotype p53 / ,Cre / were used as controls reagent (Vector Laboratories) according to the manufacturer’sin- (WT). All animals were born at the expected Mendelian frequency and structions.Macrophageinfiltration and p-H3–positive cells were did not display any gross physical or behavioral abnormalities. Animal quantified by counting the number of stained cells per field. experiments were approved by the Institutional Animal Care and Use Lipid hydroperoxide assays were performed in the kidney extracts Committee of the University of Nebraska Medical Center. using kits (BioVision, Mountain View, CA) according to the manufacturer’sinstructions. Induction of IRI in Mice IRI was induced in male mice as described previously.31,44 All animals Apoptosis Detection by TUNEL Staining were given free access to food and water. The mice were anesthetized TUNEL staining of kidney tissue sections was carried out using the In by intraperitoneal administration of a cocktail containing ketamine Situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IN) (200 mg) and xylazine (16 mg) per 1 kg body wt. Ischemic injury was according to the manufacturer’s protocol.

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Collagen Deposition by Sirius Red This study was supported by a University of Nebraska Medical The rehydrated paraffin sections were stained with Sirius red solution Center predoctoral fellowship (to Y.Y.) and National Institute of (0.1% Direct Red 80 and 1.3% picric acid; Sigma-Aldrich, St. Louis, Diabetes and Digestive and Kidney Diseases Grant DK-083291 (to MO) and washed two times in acidified water (0.5% acetic acid; B.J.P.). Sigma-Aldrich). Then, the sections were dehydrated and cleared before being observed under the microscope.45 DISCLOSURES a-SMA Immunofluorescence Staining None. Formalin-fixed mouse kidney sections were processed for immunostaining as described previously.44 The slides were sequentially incubated with mouse anti–a-SMA antibody (A5228; Sigma-Aldrich) at a 1:500 dilution REFERENCES overnight at 4°C followed by FITC-conjugated horse anti-mouse IgG (Vector Laboratories) at a 1:200 dilution for 1 hour at room temperature. 1. 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10 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 WT KO

p53

GAPDH

p53

Figure S1. Western blot showing p53 levels in proximal tubules of WT and KO mice before IRI.

Sham IRI 1 d IRI 5 d IRI 16 d A

Figure S2. (A) Typical neutrophil and macrophage accumulation in the outer medulla of WT and p53 KO kidneys at 1, 5 and 16 days post IRI. Neutrophils (PMN) were identified by immunofluorescence using a neutrophil-specific antibody. (B) Macrophages were identified by DAB staining using anti-F4/80 antibody. Images are representative of four independent experiments.

A Sham IRI 1 d IRI 5 d IRI 16 d

Figure S3. (A) Apoptosis detection by TUNEL staining in WT and p53 KO kidneys at 1, 5 and 16 days post IRI. Images are representative of four independent experiments.

A Sham IRI 1 d IRI 5 d IRI 16 d

Figure S4. (A) Cell cycle arrest at G2/M phase was analyzed by p-H3 staining. Representative images of p- H3 staining in WT and p53 KO kidneys at 1, 5 and 16 days after IRI. Images are representative of five independent experiments. (B, C) Cells at M phase were marked by anti-phospho-Ser/Thr-Pro MPM-2 antibodies. * P < .05 compared with WT-IRI 1 d; n=4.