BASIC RESEARCH www.jasn.org

Tubular Regulates Multiple to Mediate AKI

† † † † † Dongshan Zhang,* Yu Liu,* Qingqing Wei, Yuqing Huo, Kebin Liu, Fuyou Liu,* and † Zheng Dong*

*Departments of Emergency Medicine and Nephrology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China; and †Department of Cellular Biology and Anatomy, Vascular Biology Center and Department of Biochemistry and Molecular Biology, Georgia Regents University and Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia

ABSTRACT A pathogenic role of p53 in AKI was suggested a decade ago but remains controversial. Indeed, recent work indicates that inhibition of p53 protects against ischemic AKI in rats but exacerbates AKI in mice. One intriguing possibility is that p53 has cell type-specific roles in AKI. To determine the role of tubular p53, we generated two conditional knockout mouse models, in which p53 is specifically ablated from proximal tubules or other tubular segments, including distal tubules, loops of Henle, and medullary collecting ducts. Proximal tubule p53 knockout (PT-p53-KO) mice were resistant to ischemic and cisplatin nephrotoxic AKI, which was indicated by the analysis of renal function, histology, , and inflammation. However, other tubular p53 knockout (OT-p53-KO) mice were sensitive to AKI. Mechanis- tically, AKI associated with the upregulation of several known p53 target genes, including Bax, p53- upregulated modulator of apoptosis-a, , and Siva, and this association was attenuated in PT-p53-KO mice. In global expression analysis, ischemic AKI induced 371 genes in wild-type kidney cortical tissues, but the induction of 31 of these genes was abrogated in PT-p53-KO tissues. These 31 genes included regu- lators of cell death, , , oxidative stress, and mitochondria. These results suggest that p53 in proximal tubular cells promotes AKI, whereas p53 in other tubular cells does not.

J Am Soc Nephrol 25: 2278–2289, 2014. doi: 10.1681/ASN.2013080902

AKI, often resulting from ischemic, nephrotoxic, clinical trials (http://clinicaltrials.gov/ct2/results? and septic insults, is a devastating clinical condition term=I5NP&Search=Search). In AKI, p53 was that is associated with unacceptably high rates of shown to regulate several genes involved in cell mortality and morbidity.1,2 Moreover, AKI is fre- death and survival, including Bcl-2–associated x quently associated with and may contribute to the (Bax), p53 upregulated modulator of apoptosis development of CKD.3–6 The pathogenesis of AKI is (PUMA)-a, CD27 binding (Siva), and multifactorial, involving multiple cell types, cellu- p21.12,21–23 However, a global analysis of p53-regulated lar processes, and molecular mediators and regula- genes in AKI is not available. tors.7–9 Among them, p53 seems to play a patho- Despite these findings, recent studies suggest logic role.10,11 In 2003, Dagher and colleagues12 thatthepathologicroleofp53inAKIisfarmore showed the first evidence for the involvement of p53 in a rat model of ischemic AKI. We and others Received August 27, 2013. Accepted January 30, 2014. established a critical role of p53 and associated DNA damage response in cisplatin-induced AKI Published online ahead of print. Publication date available at using both cell culture and animal models includ- www.jasn.org. ing global p53 knockout mice.13–16 p53 was also Correspondence: Dr. Zheng Dong, Department of Nephrology, implicated in kidney injury induced by folic acid, Second Xiangya Hospital, Central South University, Changsha, Hunan, China, or Department of Cellular Biology and Anatomy, 17–19 aristolochic acid, and glycerol injection. As a Georgia Regents University and Charlie Norwood Veterans Af- result, inhibition of p53 may offer an effective ther- fairs Medical Center, 1459 Laney Walker Boulevard, Augusta, GA apy for AKI,12,16,20 and small interfering RNA 30912. Email: [email protected] (siRNA) targeting p53 has been tested for AKI in Copyright © 2014 by the American Society of Nephrology

2278 ISSN : 1046-6673/2510-2278 JAmSocNephrol25: 2278–2289, 2014 www.jasn.org BASIC RESEARCH complicated and that its inhibition may not always be beneficial. bearing the floxed p53 alleles (p53f/f XY) were crossed with In this regard, the pharmacological p53 inhibitor pifithrin-a, female phosphoenolpyruvate carboxykinase-cAMP-response when given after initial injury in AKI, promoted renal fibrosis element (PEPCK-Cre) transgenic mice (p53+/+XcreXcre)that in rats.24 Even more surprisingly, pifithrin-a and global p53 de- express Cre recombinase under the control of a modified letion exacerbated ischemic AKI in mice.25 Although this study PEPCK promoter, which directs Cre expression predomi- indicates that the action of p53 is animal species-dependent, nantly in kidney proximal tubular cells.26 After the first round mechanistically, it is puzzling how p53 may be injurious to of breeding, heterozygous female progenies (p53f/+XcreX) AKI in rats but protective in mice. One explanation is that were selected to further breeding with p53f/fXY males to gen- AKI in rats depends largely on renal tubular injury, whereas erate PT-p53-KO (p53f/fXcreY) and proximal tubule p53 wild- AKI in mice depends more on inflammation and inflammatory type (PT-p53-WT) littermate mice for testing. damage. This possibility is based on the assumption that p53 in To verify the genotypes, three sets of PCR were conducted for different cell/tissue types may have distinct or opposite roles each mouse. The genotype of PT-p53-KO mice was indicated by in the pathogenesis of AKI: whereas leukocyte p53 is anti- (1)amplification of the 390-bp fragment of the floxed allele, (2) inflammatory and thus, renoprotective, tubular p53 is a critical lack of amplification of the 270-bp fragment of the wild-type trigger and/or mediator of AKI. The anti-inflammatory function allele, and (3)amplification of the 370-bp fragment of the Cre of leukocyte p53 was recently suggested by the experiments gene (Figure 2B, lanes 2, 6, and 7). The absence of the Cre gene using chimeric mouse models.25 However, the pathogenic ensured the genotype of wild-type (PT-p53-WT) mice (Figure 2B, role of tubular p53 has yet to be established by using kidney lanes 1, 3, and 5). At the protein level, because p53 in control tubule-specific p53 knockout models. kidney tissues was very low (Figure 1), we compared p53 expres- In the present study, we established two conditional knockout sion in PT-p53-KO and wild-type tissues after AKI. After ischemic mouse models, in which p53 was specifically ablated from AKI, wild-type mice showed p53 expression in both cortical and proximal tubules or other tubular segments. Knockout of p53 outer medullary tissues (Figure 2C, lanes 1 and 2) that was sup- from proximal tubules but not other tubules protected against pressed in PT-p53-KO tissues (Figure 2C, lanes 3 and 4). Similarly, ischemic and cisplatin nephrotoxic AKI. AKI-associated up- p53 expression during cisplatin-induced AKI was significantly regulation of several known p53 target genes was shown to be lower in PT-p53-KO kidney tissues than wild-type tissues (Figure attenuated in proximal tubule p53 knockout (PT-p53-KO) 2D,lanes7and8versus5and6).Inimmunohistochemistry kidney tissues.Additionalglobalgene expressionanalysisshowed (Figure 2, E and F), p53 was induced during AKI in the nuclei the induction of 371 genes by ischemic AKI inwild-type kidneys, of wide-spread tubular cells (Figure 2, E, inset and F, inset) and the of which the induction of 31 genes was abrogated in PT-p53-KO of a subset of tubules in wild-type kidney tissues, and tissues. These 31 genes included regulators of cell death, again, p53 expression was largely attenuated in PT-p53-KO tis- metabolism, signal transduction, oxidative stress,and mitochon- sues, verifying tubular p53 ablation in this conditional model. drial carriers. Together, the results suggest that p53 in proximal Without AKI treatment, these mice showed similarly low tubules contributes critically to AKI by regulating multiple genes levels of BUN and serum creatinine, indicating normal renal involved in kidney tissue injury, remodeling, and repair. function.Renalischemia-reperfusioninducedsevererenalfailure inwild-typemice, raising BUNandserumcreatinine levelsto295 and 3.02 mg/dl, respectively (Figure 3, A and B). In contrast, after RESULTS the same treatment, PT-p53-KO littermate mice had 175 mg/dl BUN and 1.8 mg/dl serum creatinine (significantly lower than We first verified p53 expression in kidney tissues during AKI. the levels of the wild type). Similarly, at day 3 of cisplatin treat- Bilateral renal ischemia-reperfusion induced AKI in C57/Bl6 ment, wild-type mice developed severe renal failure, with 287 mice as indicated by marked increases in BUN and serum mg/dl BUN and 2.34 mg/dl serum creatinine, whereas PT-p53- creatinine (Figure 1, A and B); p53 expression was very low in KO mice had 122 mg/dl BUN and 0.92 mg/dl serum creatinine sham control (day 0) but induced by ischemic AKI in renal (Figure 3, C and D). Histologic analysis confirmed that both cortex and outer medulla (Figure 1C), and p53 induction cisplatin and ischemia-reperfusion induced severe kidney tissue seemed significantly higher in outer medulla than renal cor- damage in PT-p53-WT mice, which was significantly amelio- tex. Temporally, p53 induction peaked at day 1 of reperfusion rated in PT-p53-KO mice (Figure 3, C and G). In wild-type and then decreased by day 2. In cisplatin nephrotoxic AKI, p53 mice, the tubular damage scores were 2.9 and 3.9 after ischemic was induced in kidneys gradually from day 1 to day 3 and and cisplatin AKI, respectively, whereas the scores were mark- accompanied by increases in BUN and serum creatinine (Fig- edly decreased to 1.2 and 1.0 after ischemic and cisplatin AKI, ure 1, D–F). These data, confirming previous studies,12–16 in- respectively, for PT-p53-KO tissues (Figure 3, D and H). dicate the induction of p53 in AKI. Apoptosis contributes significantly to the pathogenesis of Todetermine the pathologic role of tubular p53, we initially AKI,27 and p53 is known as a trigger of apoptosis under cell established a conditional knockout mouse model, inwhich p53 stress.28 We, therefore, analyzed apoptosis in kidney cortical was deleted specifically from kidney proximal tubules. The tissues by terminal deoxynucleotidyl -mediated di- breeding protocol is shown in Figure 2A. Briefly, male mice goxigenin-deoxyuridine nick-end labeling (TUNEL) assay.

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Figure 1. p53 is induced in ischemic and cisplatin nephrotoxic AKI in mice. Male C57BL/6 mice were (A–C) subjected to 28 minutes of bilateral renal ischemia followed by 0–2 days of reperfusion (n=20) or (D–F) injected with 30 mg/kg cisplatin (n=28) for 0–3daysof examination. In A, B, D, and E, blood samples were collected at indicated time points to measure BUN and serum creatinine. Data were expressed as means6SDs; the bars with different superscripts (a–c) in each panel were significantly different (P,0.05). In C and F, kidneys were harvested to extract cortical and outer medulla tissues at indicated time points for immunoblot analysis of p53 and cyclophilin B (loading control). I/R, ischemia/reperfusion.

Representative TUNEL staining is presented in Figure 4, A and B. p53-floxed mice with kidney-specific (Ksp-Cre) mice. No apoptosis was detected in the kidney tissues of sham- Ksp promoter drives Cre expression (and therefore, floxed gene operated or saline-injected mice. However, after renal ischemia- deletion) in distal tubules, loops of Henle, and medullarycollecting reperfusion and cisplatin treatment, significant apoptosis was ducts with significantly fewer effects on proximal tubules (http:// induced in kidney cortical tissues in wild-type mice. Impor- jaxmice.jax.org/strain/012237.html).30 As shown in Figure 5A, tantly, much less apoptosis was induced in PT-p53-KO mice. p53 expression during ischemic AKI seemed lower in OT-p53- This observation was further verified by counting apoptotic KO kidney tissues than wild-type tissues (Figure 5A, lanes 3 and 4 cells in cortical and outer medulla regions from independent versus 1 and 2). However, ischemic AKI shown by BUN, serum experiments (Figure 4, C and D). We further examined the creatinine, and tubular damage was not significantly ameliorated infiltration of inflammatory cells in ischemic AKI. As shown in OT-p53-KO mice (Figure 5, B–E). It was noticed that 28 in Supplemental Figure 1, renal ischemia-reperfusion in- minutes of renal ischemia-reperfusion induced moderate AKI duced the infiltration of leukocytes, including neutrophils in OT-p53-WT mice (Figure 5) but severe AKI in PT-p53-WT and macrophages, into wild-type kidney tissues, which was mice (Figure 3). For comparison, we subjected the PT-p53 mice to suppressed in PT-p53-KO tissues. The decrease of inflamma- 23 minutes of ischemia-reperfusion and showed that PT-p53-KO tion in PT-p53 KO mice correlated with lower apoptosis, sup- mice were protected in this model of moderate AKI (Figure 5, B porting the inference that tubular apoptosis may be a driving and C, Supplemental Figure 2). Together, these results suggest that factor for inflammation.29 p53 in proximal tubules (and not p53 in other tubular segments) To determine if p53 in renal tubular segments other than plays a critical pathogenic role in ischemic AKI. proximal tubules contributes significantly to AKI, we established p53 regulates cell death and survival through both the other tubular p53 knockout (OT-p53-KO) model by crossing transcription-dependent and -independent mechanisms.29

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Figure 2. Creation and characterization of the PT-p53-KO mouse model. (A) Breeding protocol for generating PT-p53-KO mice. After confirming genotype, male littermate mice at 8–10 weeks of age were used for experiments. (B) Representative gel images of PCR- based genotyping. Genomic DNA was extracted from tail biopsy and amplified to detect wild-type and floxed alleles of p53 and PEPCK-Cre allele as indicated. (C and D) Kidney cortex and outer medulla were collected from PT-p53-KO and PT-p53-WT littermate mice after I/R injury or cisplatin injection for immunoblot analysis of p53 and cyclophilin B. (E and F) Immunohistochemical staining of p53 in kidney cortical tissues of wild-type and PT-p53-KO mice after I/R or cisplatin injury. The selected areas are shown as insets at a higher magnification. Original magnification, 3200. DAPI, 49,6-diamidino-2-phenylindole; I/R, ischemia/reperfusion.

Interestingly, several apoptotic genes subjected to p53 regula- to determine their dependence on proximal tubular p53. As tion have been reported to be upregulated in AKI. For example, shown in Figure 6, both p53 and its -15 phosphorylated PUMA-a is induced by cisplatin in kidney tissues,21,31,32 form were induced by cisplatin in kidney cortical tissues in PT- whereas Bax and Siva are induced in ischemic AKI.12,23 In ad- p53-WT mice. Concomitantly, Bax, PUMA-a, and p21 were dition, p21, a p53 target gene involved in arrest and induced. In PT-p53-KO mice, the induction of p53 as well as cytoprotection, is induced markedly in various AKI mod- Bax, PUMA-a, and p21 was suppressed (Figure 6A). In ische- els.22,33,34 We, therefore, analyzed the expression of these genes mic AKI, Bax, PUMA-a, Siva, and p21 were markedly induced

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Figure 3. Ischemic and cisplatin-induced AKI is attenuated in PT-p53-KO mice. (A–D) Wild-type and PT-p53-KO littermate mice were subjected to 28 minutes of bilateral renal ischemia followed by 2 days of reperfusion or sham operation as control. (E–H) Wild-type and PT-p53-KO littermate mice were injected with 30 mg/kg cisplatin or saline as control for 3 days. (A, B, E, and F) Blood samples were collected for measurements of BUN and serum creatinine levels. Data were expressed as means6SDs (n=8); the bars with different

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Figure 4. AKI-associated renal apoptosis is suppressed in PT-p53-KO mice. Wild-type and PT-p53-KO littermate mice were subjected to 28 minutes of bilateral renal ischemia followed by 2 days of reperfusion or injected with 30 mg/kg cisplatin for 3 days. Control mice were sham-operated or injected with saline. Kidney cortical tissues were examined by TUNEL assay to reveal apoptosis. (A and B) Repre- sentative images of TUNEL staining. Original magnification, 3200. (C and D) Quantification of TUNEL-positive cells in the tissues from each condition. Data were expressed as means6SDs (n=8); #P,0.05, significantly different from the PT-p53-WT group. I/R, ischemia/ reperfusion. along with p53 in wild-type kidney tissues, and the inductive Compared with wild type, the expression of 204 and 257 genes response was largely diminished in PT-p53-KO tissues (Figure at 24 and 48 hours of reperfusion, respectively, was suppressed 6B). The dependence of Bax, PUMA-a, Siva, and p21 induc- in PT-p53-KO kidneys (Supplemental Tables 4 and 5). In these tion on proximal tubule p53 was further substantiated by den- genes, 83 were suppressed at both reperfusion time points sitometric analysis of the immunoblots (Figure 6, C and D). (Supplemental Table 6). Notably, 31 of 83 suppressed genes Together, these data suggest that p53 in proximal tubules is were among 371 genes that were induced by both 24 and 48 responsible for the induction of key cell death regulatory genes hours of reperfusion in wild-type kidneys (Figure 7A). These during AKI. 31 genes included the regulators of cell death, signal trans- To gain a comprehensive understanding of p53-regulated duction, metabolism, oxidative stress, and mitochondrial car- in AKI, we conducted a global gene expression riers (Table 1). Seven of thirty-one genes have been reported to analysis. To this end, RNA samples were isolated from kidney be induced by ischemia-reperfusion in kidneys, brain, heart, cortex, amplified, and hybridized to a cDNA microarray or lung. Interestingly, the analysis with the JASPAR CORE containing 26,515 genes. In wild-type mice, renal ischemia database (http://jaspar.genereg.net/) showed that 7 of 31 genes followed by 24 and 48 hours of reperfusion led to the induction contain p53 binding sites in their promoters (Table 2). By of 769 and 800 genes, respectively, of which 341 genes were immunoblot analysis, we further confirmed the induction of induced at both time points (Supplemental Tables 1–3). thioredoxin-interacting protein (Txnip), mixed lineage

superscripts (a–c) were significantly different from each other (P,0.05). In C and G, kidney cortical tissues were stained with hematoxylin- eosin to show histology. Original magnification, 3200. In D and H, tubular damage in I/R and cisplatin-treated cortical tissues was semiquantified as pathologic scores. Data were expressed as means6SDs (n=8). #P,0.05, significantly different from PT-p53-WT group. I/ R, ischemia/reperfusion.

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protective during cisplatin AKI in mice.13–16 Based on these findings, recent clinical trials have tested the therapeutic effect of p53 siRNA in human patients (http://clinicaltrials. gov/ct2/results?term= I5NP&Search=Search). However, recent studies have suggested a much more complex role of p53 in AKI. Specifically, it was shown that, although sys- temic inhibition of p53 protects against is- chemic AKI in rats,12 it enhances AKI in mice.25 One intriguing possibility is that AKI in rats depends largely on p53-mediated renal tubular injury, whereas AKI in mice depends more on inflammation and inflam- matory damage that is suppressible by p53 in leukocytes. The anti-inflammatory function of leukocyte p53 was recently suggested by using chimeric mouse models.25 Our study has now shown the injurious role of proxi- mal tubular p53 in AKI. Together, these studies indicate that p53 in different cell/ tissue types plays distinguished roles in the pathogenesis of AKI. Moreover, by global gene expression analysis, we have identi- fied several classes of genes that are upreg- ulated through p53 in ischemic AKI, gaining new insights into the mechanism of p53 regulation in the disease condition. This study has established two renal tu- Figure 5. Ischemic AKI is diminished in PT-p53-KO mice, but not in OT-p53-KO mice. bular p53 knockout mouse models. One OT-p53-KO and wild-type littermate mice were subjected to 28 minutes of bilateral model was generated by crossing PEPCK- renal ischemia, whereas PT-p53-KO and wild-type littermate mice were subjected to 23 Cre mice, and the other was generated by minutes of bilateral renal ischemia. Samples were collected after 2 days of reperfusion. crossing Ksp-Cre with p53-floxed mice. (A) p53 expression in kidney tissues of OT-p53-KO and wild-type littermate mice. (B fl and C) BUN and serum creatinine, respectively. Data were expressed as means6SDs; PEPCK-Cre results in the deletion of oxed – bars with different superscripts (a–c) in each panel were significantly different genes in the majority (70% 80%) of prox- (P,0.05). (D) Representative histology of cortical tissues from OT-p53-KO and wild- imal tubules without deletion in other tu- type littermate mice after hematoxylin-eosin staining. Original magnification, 3200. bular segments.26 In contrast, Ksp-Cre (E) Tubular damage score in renal cortical tissues from OT-p53-KO and wild-type lit- leads to floxed gene deletion mainly in col- termate mice. I/R, ischemia/reperfusion. lecting ducts, loops of Henle, and distal tu- bules but very weak or limited deletion in proximal tubules.30 Using these two mod- domain–like protein (MLKL), dual specificity phosphatase 5 els, our study determined the effects of specificp53deletion (DUSP5), and suppressors of cytokine signaling 2 (SOCS2) from proximal tubules or other tubules on AKI. Considering during renal ischemia-reperfusion in wild-type kidneys, the pleiotropic role of p53 in cell cycle, differentiation, and which was suppressed in PT-p53-KO tissues (Figure 7B). apoptosis, one may expect significant effects of tubular p53 ablation on kidney development, histology, and function. However, these effects were not observed in either PT-p53- DISCUSSION KO or OT-p53-KO mice. This result is not surprising, because global p53 knockout does not result in notable abnormalities A pathologic role of p53 in AKI has been supported by several in kidneys, although some newborn pups were reported to lines of evidence. p53 is upregulated and activated in avarietyof have an aberrant renal phenotype.35 It is possible that p53 AKI models accompanied by the induction of downstream knockout delays kidney development, but at adult age, the target genes.12–23 p53 inhibitors and siRNA afford protection kidneys have completed the development process and gained against ischemic and cisplatin nephrotoxic AKI in rats.12,16,20 normal histology and function. Regardless, PT-p53-KO mice In addition, genetic and pharmacologic inhibition of p53 is were shown to be resistant to AKI. Of note, p53 ablation from

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most likely caused by the variations in the animal models. Notably, p53-related gene expression was shown in both studies, sup- porting the significance of transcriptional regulation by p53 in AKI. Previous studies showed the induction of several p53-regulated genes in AKI.12,21–23 Consistently, we confirmed the induction of Bax, PUMA-a, Siva, and p21 during is- chemic and cisplatin AKI. Importantly, the inductive response was largely diminished in PT-p53-KO mice, indicating that proxi- mal tubular p53 is the key to the expression of these genes. Interestingly, although Bax, PUMA-a, and Siva are known to be proa- poptotic, p21 arrests the cell cycle and has a critical cytoprotective role in kidneys.27,37 The paradoxical gene induction by p53 Figure 6. Induction of p53 target genes in AKI is suppressed in PT-p53-KO mice. Wild- in AKI remains puzzling. It is plausible type and PT-p53-KO littermate mice were subjected to 28 minutes of bilateral renal that, in response to stress, the cells initially ischemia followed by 2 days of reperfusion or injected with 30 mg/kg cisplatin for 3 activate defensive or cytoprotective mech- days. Control mice were subjected to sham operation or injected with saline. The lysate anisms, including p21 induction through of kidney cortex and outer medulla was collected for immunoblot analysis of p53, p53; however, as the insult continues and/ p-p53 (ser15), p21, PUMA-a, Bax, Siva, and Cyclophilin B by using specific antibodies. or intensifies, prodeath gene expression is (A and B) Representative immunoblots. (C and D) Densitometry of immunoblot signals. For densitometric analysis, the protein signal of the wild-type control group (n=5) was triggered. Of note, both p53-dependent arbitrarily set as one, and the signals of other conditions were normalized (n=8). I/R, and -independent mechanisms contribute 33 ischemia/reperfusion. to p21 induction in AKI. To further elucidate the p53-mediated gene expression in AKI, we conducted a proximal tubules in this model was obvious but incomplete global gene expression analysis using a cDNA microarray (Figure 2, C and D), an observation that is consistent with the containing 26,515 genes. In wild-type mice, renal ischemia- 70%–80% recombination efficiency of the PEPCK-Cre reperfusion for 24–48 hours led to the consistent induction of model.26 The notable renoprotection shown in this model 341 genes. The number of genes identified in our microarray further supports a role of proximal tubular p53 in AKI. In analysis is, therefore, much larger than the number in a pre- contrast, OT-p53-KO mice were as sensitive to AKI as wild- vious report,38 in which only 91 genes were shown to be type animals, suggesting that p53 in other tubular segments upregulated in ischemic AKI. The larger number of genes does not seem to have a critical role in AKI. However, these identified in our analysis was likely because of the larger cov- results do not rule out the involvement of other tubular seg- erage (26,515 versus 9000 genes) of the cDNA microarray ments in the pathogenesis of AKI. Other tubules, such as distal used in our study. In addition, the previous study examined tubules and thick ascending limbs, may contribute to AKI the effect of unilateral renal ischemia-reperfusion in Swiss– through p53-independent mechanisms. Webster mice, whereas bilateral ischemia-reperfusion in p53, although known as a tumor suppressor, responds C57/Bl6 mice was analyzed in the current study. Despite rapidly to cellular stress in both and normal cells. these differences, 14 genes were shown to be induced in Depending on the severity of the stress, p53 may result in cell both studies. cycle arrest and/or cell death under various pathophysiologic In PT-p53-KO mice, ischemic AKI was also associated with conditions. Mechanistically, p53 is a key regulator of gene significant changes in gene expression. Notably, compared expression in the nucleus. Nonetheless, p53 may also partic- withwild-type tissues, 83 genes were suppressed in PT-p53-KO ipate in cell regulation through transcription-independent kidneys during ischemic AKI, suggesting that these genes are mechanisms in the cytoplasm.29,36 In a rat model of ischemic subjected to p53 regulation in proximal tubules in AKI. AKI, p53 induction was detected mainly in the cytoplasm of Interestingly, in these 83 genes, 31 genes were among 371 renal tubular cells, with limited signals in the nucleus.12 In our genes that were induced by renal ischemia-reperfusion in wild- study using mouse models, p53 was induced in the nuclei of type kidneys (Figure 7A). The analysis suggests that these 31 widespread tubular cells, although some cytoplasmic staining genes are induced during ischemic AKI in a p53-dependent was revealed in a subset of tubular cells. The subcellular local- manner. These 31 genes include important regulators of cell ization difference in p53 observed in these two studies was death, oxidative stress, metabolism, mitochondrial carriers,

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and others (Table 1). Seven of thirty-one genes have been reported to be induced by ischemia-reperfusion in kidneys or other organs. Moreover, 7 of 31 genes con- tain p53 binding sites in their promoters (Table 2), suggesting a direct transcrip- tional regulation of these genes by p53 in AKI. Immunoblot analysis further con- firmed the induction of several interesting genes (i.e., MLKL, DUSP5, Socs2, and Txnip) during renal ischemia-reperfusion in wild-type mice, which was suppressed in PT-p53-KO mice (Figure 7B). MLKL is a newly identified key regulator of necrosis downstream of the interacting 3.39,40 p53-Dependent in- duction of MLKL suggests that, in addition to apoptosis, p53 may also be involved in tubular cell necrosis in AKI. In support of this possibility, PT-p53-KO mice showed less necrotic damage in renal tubules after ischemic or cisplatin AKI than wild-type mice (Figure 3). SOCS2 is a suppressor of cytokine signaling that blocks the Janus ki- nase/signal transducer and activation of the transcription (JAK/STAT) pathway, which has been implicated in ischemic injury in kidneys.41 DUSP5 is an inducible nuclear phosphatase that functions as both an inactivator of and a nuclear anchor for ex- tracellular signal-regulated kinase in mam- malian cells. p53-mediated induction of DUSP5 may, therefore, block extracellular signal-regulated kinase signaling and its role in ischemic AKI.42 Txnip is a modula- tor of cellular redox state that contributes to cell apoptosis.43 In mice, knockout of Txnip impairs mitochondrial function but protects myocardium from ischemia- reperfusion injury.44 Thus, the pathologic role of p53 in AKI may not be limited to the regulation of tubular cell death/survival; rather, it may be extended to the regulation of metabolism, oxidative stress, and mito- chondria. It is important to recognize that the differential gene expression shown in PT-p53-KO and wild-type kidney tissues Figure 7. The induction of 31 genes during ischemic AKI is suppressed in PT-p53-KO after AKI may also be secondary to the mice. Wild-type (n=4) and PT-p53-KO (n=4) littermate mice were subjected to 28 lower injury associated with the PT-p53- minutes of bilateral renal ischemia followed by 24 or 48 hours of reperfusion or sham KO model. Nonetheless, identification of operation as control. (A) Total RNA samples isolated from renal cortical tissues were these genes lays a foundation for additional analyzed by a cDNA microarray containing 26,515 genes. The amount of the mRNA of investigation to distinguish the genes that each gene from the I/R group was divided by the amount of sham control to calculate are directly regulated by p53 from those the fold change. (B) The lysate of renal cortical tissues was examined by immunoblot genes that are secondary to the effect of analysis of Txnip, MLKL, DUSP5, SOCS2, and cyclophilin B. I/R, ischemia/reperfusion. p53 ablation-related protection.

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Table 1. Thirty-one genes induced by p53 during renal ischemia-reperfusion Cell Death/Survival Signal Transduction Metabolic Processes Mitochondrial Carrier Oxidative Stress Others Baxa,b Dusp5b Acat3 Slc25a25 Cyp4a12b Plk3 PUMAa,b Socs2a,b Acaa1b Slc25a34 Txnipb SYTL5 p21a,b Map3k6 Adm Tomm5 Serpina3na Txnipb Olfr98a Cbr3 Sprr2g MLKL Fbp2 Snord37 Ftl1 Glrxb Gpta Klk1b1 Klk1b27 Klk1b3 Plin2 Ttpa aGenes with p53 binding sites in their promoters. bGenes that have been reported to be induced in ischemia-reperfusion of kidney, brain, heart, or liver.

Table 2. p53 binding sites in the promoter of seven induced anti-SOCS2, anti-MLKL, antineutrophil, and antimacrophage from genes Abcam, Inc.; anti-PUMA, anti-p53, and antiphospho-p53 (ser15) Genes p53 Binding Sites from Cell Signaling Technology; and anti-p21, anti-Bax, and anti- PUMA Site 1 AGGGGGTGCCCGGGCATGT DUSP5 from Santa Cruz. All secondary antibodies were from Thermo PUMA Site 2 ACAGACATGCCCGGGCAGCC Fisher Scientific. p21 Site 1 AGGAACATGTCTTGACATGT p21 Site 2 CTGAACATGTCAAGACATGT Animals fl fl Bax Site 1 GCAGGCCCGGGCTTG Ksp-Cre and p53 ox/ ox mouse lines were obtained from The Jackson Bax Site 2 ACAAGCCCGGGCCTG Laboratory. PEPCK-Cre mice were provided by Volker Haase (Van- fl fl Socs2 Site 1 GCATCCCCAGGCATCTCCTT derbilt University School of Medicine, Nashville, TN). p53 ox/ ox Olfr98 Site 1 CTGGGCATGTCTGGGAAGGT mice were crossed with PEPCK-Cre mice to produce PT-p53-KO Olfr98 Site 2 CCAGACATGCCCAGAGGTGT fl fl (p53 ox/ oxXcreY) and PT-p53-WT littermate mice, which is depicted Gpt Site 1 AGGTGCATGCTCGGGCTTGC fl fl in the breeding protocol in Figure 1A. Similarly, p53 ox/ ox mice were Gpt Site 2 AGGTGCATGCTCGGGCTTGC Serpina3n Site 1 AAGCATATGCCCTGACATGT crossed with Ksp-Cre mice to produce OT-p53-KO mice. The mice Serpina3n Site 2 GGAAACATGTCAGGGCATAT were housed in the animal facility of the Charlie Norwood Veterans Affairs Medical Center under a 12-hour /dark pattern with free access to food and water. All animal experiments were conducted in accordance with a protocol approved by the Institutional Animal In conclusion, this study has established two conditional Care and Use Committee of the Charlie Norwood Veterans Affairs knockout mouse models, in which p53 is specifically deleted Medical Center. from either proximal tubules or other tubular segments. By using these models, the study has shown a critical pathogenic role of proximal tubular p53 (but not other tubular p53) in AKI Models Ischemic and cisplatin nephrotoxic AKI was induced in male mice of AKI. Global gene expression analysis has further identified 31 8–10 weeks of age as described recently.45–47 For cisplatin injury, mice genes induced in ischemic AKI directly or indirectly through were intraperitoneally injected with a single dose of cisplatin at 30 p53, which may regulate cell death, metabolism, oxidative mg/kg. For ischemic AKI, renal pedicles were exposed by flank in- stress, and mitochondrial activity. Additional investigation in cisions for the indicated duration of bilateral clamping followed by these directions will generate significant new insights into p53 release for reperfusion. During ischemic AKI, the body tempera- regulation of kidney injury, tissue remodeling, and repair. tures of the mice were controlled at approximately 36.5°C. Sham control mice underwent the same operation without renal pedicle clamping. CONCISE METHODS Microarray Reagents and Antibodies Total RNA was isolated from kidney cortical tissues for reverse Chemicals, including the 3-hydroxy-49-nitro-2-naphthanilide chlor- transcription using the Ambion WT Expression Kit (Life Technol- oacetate (specific esterase) for leukocyte staining, were purchased ogies). The synthesized cDNAs were fragmented and biotin-labeled from Sigma-Aldrich. Antibodies included anti-cyclophilin B, anti-Txnip, using the GeneChip WT Terminal Labeling Kit (Affymetrix). The

J Am Soc Nephrol 25: 2278–2289, 2014 Tubular p53 in AKI 2287 BASIC RESEARCH www.jasn.org labeled cDNAs were then hybridized onto the Affymetrix Mouse Gene DISCLOSURES 2.0ST Array according to the manufacturer’s protocol. After 16 hours None. of hybridization, the arrays were washed and stained using Affymetrix GeneChip Fluidics Station 450 Systems. The stained arrays were scanned with an Affymetrix GeneChip Scanner 3000. REFERENCES

Renal Function, Histology, and TUNEL Assay 1. Chertow GM, Burdick E, Honour M, Bonventre JV, Bates DW: Acute BUN and serum creatinine were measured using commercial kits kidney injury, mortality, length of stay, and costs in hospitalized pa- tients. J Am Soc Nephrol 16: 3365–3370, 2005 45–47 from Stanbio Laboratory to indicate renal function. Renal his- 2. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, tology was subjected to a blind examination after hematoxylin and Schetz M, Tan I, Bouman C, Macedo E, Gibney N, Tolwani A, Ronco C; eosin staining. Tissue damage was scored according to the percent- Beginning and Ending Supportive Therapy for the Kidney (BEST Kid- age of damaged tubules: 0, no damage; 1, less than 25% damage; 2, ney) Investigators: Acute renal failure in critically ill patients: A multi- – 25%–50% damage; 3, 50%–75% damage; 4, more than 75% dam- national, multicenter study. JAMA 294: 813 818, 2005 3. Hsu CY: Yes, AKI truly leads to CKD. J Am Soc Nephrol 23: 967–969, 2012 age. The criteria of tubular damage included the loss of brush bor- 4. Venkatachalam MA, Griffin KA, Lan R, Geng H, Saikumar P, Bidani AK: der, tubular dilation, cast formation, and cell lysis. TUNEL assay Acute kidney injury: A springboard for progression in chronic kidney was conducted using the In Situ Cell Death Detection Kit from disease. Am J Physiol Renal Physiol 298: F1078–F1094, 2010 Roche Applied Science. For quantification, 10–20 fields were randomly 5. Chawla LS, Kimmel PL: Acute kidney injury and chronic kidney disease: selected from each tissue section to count the TUNEL-positive cells per An integrated clinical syndrome. Kidney Int 82: 516–524, 2012 6. Okusa MD, Chertow GM, Portilla D; Acute Kidney Injury Advisory millimeter2. Group of the American Society of Nephrology: The nexus of acute kidney injury, chronic kidney disease, and World Kidney Day 2009. Clin Immunohistochemistry and Immunoblot Analyses J Am Soc Nephrol 4: 520–522, 2009 For immunohistochemistry, kidney tissues were fixed with 4% 7. Bonventre JV, Yang L: Cellular pathophysiology of ischemic acute kidney injury. JClinInvest121: 4210–4221, 2011 paraformaldehyde and paraffin-embedded to collect tissue sections, 8. Pabla N, Dong Z: Cisplatin nephrotoxicity: Mechanisms and re- fi which were then deparaf nized and incubated with 0.1 M sodium noprotective strategies. Kidney Int 73: 994–1007, 2008 citrate (pH 6.0) at 65°C for antigen retrieval. After the incubation 9. Sharfuddin AA, Molitoris BA: Pathophysiology of ischemic acute kidney with blocking buffers, tissue sections were exposed sequentially to injury. Nat Rev Nephrol 7: 189–200, 2011 the primary antibody, the biotinylated secondary antibody, and the 10. Dagher PC: Apoptosis in ischemic renal injury: Roles of GTP depletion – Tyramide Signal Amplification Biotin System (PerkinElmer). The and p53. Kidney Int 66: 506 509, 2004 11. Jiang M, Dong Z: Regulation and pathological role of p53 in cisplatin signals were developed with the VECTASTAIN ABC Standard Kit nephrotoxicity. J Pharmacol Exp Ther 327: 300–307, 2008 and DAB Peroxidase Substrate Kit (Vector Laboratories) following 12. Kelly KJ, Plotkin Z, Vulgamott SL, Dagher PC: P53 mediates the apo- the protocols of the manufacturer. Cell nuclei were counterstained ptotic response to GTP depletion after renal ischemia-reperfusion: with Hoechst 33342. For immunoblot analysis, tissue lysate from kidney Protective role of a p53 inhibitor. J Am Soc Nephrol 14: 128–138, 2003 cortex and outer medulla was extracted for SDS–polyacrylamide elec- 13. Cummings BS, Schnellmann RG: Cisplatin-induced renal cell apopto- sis: Caspase 3-dependent and -independent pathways. J Pharmacol trophoresis, blotting, and antibody exposure by standard procedures. Exp Ther 302: 8–17, 2002 14. Jiang M, Yi X, Hsu S, Wang CY, Dong Z: Role of p53 in cisplatin-induced Statistical Analyses tubular cell apoptosis: Dependence on p53 transcriptional activity. Am Qualitative data, including immunoblots and tissue histology images, J Physiol Renal Physiol 287: F1140–F1147, 2004 are representatives of at least three experiments. Quantitative data are 15. Pabla N, Huang S, Mi QS, Daniel R, Dong Z: ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apo- expressed as means6SDs. Statistical analysis was conducted using ptosis. J Biol Chem 283: 6572–6583, 2008 the GraphPad Prism software. Multiple groups were compared with 16. Wei Q, Dong G, Yang T, Megyesi J, Price PM, Dong Z: Activation and ANOVA followed by Tukey’s post-tests. Statistical differences be- involvement of p53 in cisplatin-induced nephrotoxicity. Am J Physiol tween two groups were determined by two-tailed unpaired or paired Renal Physiol 293: F1282–F1291, 2007 t tests. P,0.05 was considered significantly different. 17. Zhou D, Li Y, Lin L, Zhou L, Igarashi P, Liu Y: Tubule-specific ablation of endogenous b- aggravates acute kidney injury in mice. Kidney Int 82: 537–547, 2012 18. Zhou L, Fu P, Huang XR, Liu F, Lai KN, Lan HY: Activation of p53 pro- ACKNOWLEDGMENTS motes renal injury in acute aristolochic acid nephropathy. JAmSoc Nephrol 21: 31–41, 2010 19. Homsi E, Mota da Silva S Jr., Machado de Brito S, Bouçada Inácio We thank Dr. Volker Haase at Vanderbilt University School of Peixoto E, Butori Lopes de Faria J, Janino P: p53-Mediated oxidative Medicine for the PEPCK-Cre mouse line. stress and tubular injury in rats with glycerol-induced acute kidney in- The study was supported, in part, by National Natural Science jury. Am J Nephrol 33: 49–59, 2011 Foundation of China Grants 81370791 and 81100507, National Basic 20. Molitoris BA, Dagher PC, Sandoval RM, Campos SB, Ashush H, Fridman E, Brafman A, Faerman A, Atkinson SJ, Thompson JD, Kalinski H, Research Program of China 973 Program 2012CB517600, and the Skaliter R, Erlich S, Feinstein E: siRNA targeted to p53 attenuates is- National Institutes of Health and Department of Veterans Adminis- chemic and cisplatin-induced acute kidney injury. J Am Soc Nephrol tration of the United States. 20: 1754–1764, 2009

2288 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2278–2289, 2014 www.jasn.org BASIC RESEARCH

21. Jiang M, Wei Q, Wang J, Du Q, Yu J, Zhang L, Dong Z: Regulation of 35. Yu F, Megyesi J, Safirstein RL, Price PM: Identification of the functional PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis. Onco- domain of p21(WAF1/CIP1) that protects cells from cisplatin cytotox- gene 25: 4056–4066, 2006 icity. Am J Physiol Renal Physiol 289: F514–F520, 2005 22. Megyesi J, Safirstein RL, Price PM: Induction of p21WAF1/CIP1/SDI1 in 36. Saifudeen Z, Dipp S, El-Dahr SS: A role for p53 in terminal epithelial cell kidney tubule cells affects the course of cisplatin-induced acute renal differentiation. JClinInvest109: 1021–1030, 2002 failure. JClinInvest101: 777–782, 1998 37. Vousden KH, Lane DP: p53 in health and disease. Nat Rev Mol Cell Biol 23. Singaravelu K, Padanilam BJ: p53 target Siva regulates apoptosis in 8: 275–283, 2007 ischemic kidneys. Am J Physiol Renal Physiol 300: F1130–F1141, 38. Price PM, Megyesi J, Safirstein RL: Cell cycle regulation: Repair and 2011 regeneration in acute renal failure. Semin Nephrol 23: 449–459, 2003 24. Dagher PC, Mai EM, Hato T, Lee SY, Anderson MD, Karozos SC, Mang 39. Supavekin S, Zhang W, Kucherlapati R, Kaskel FJ, Moore LC, Devarajan HE, Knipe NL, Plotkin Z, Sutton TA: The p53 inhibitor pifithrin-a can P: Differential gene expression following early renal ischemia/re- stimulate fibrosis in a rat model of ischemic acute kidney injury. Am J perfusion. Kidney Int 63: 1714–1724, 2003 Physiol Renal Physiol 302: F284–F291, 2012 40. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei 25. Sutton TA, Hato T, Mai E, Yoshimoto M, Kuehl S, Anderson M, Mang H, X, Wang X: Mixed lineage kinase domain-like protein mediates ne- Plotkin Z, Chan RJ, Dagher PC: p53 is renoprotective after ischemic crosis signaling downstream of RIP3 kinase. Cell 148: 213–227, 2012 kidney injury by reducing inflammation. J Am Soc Nephrol 24: 113– 41. Wang Z, Jiang H, Chen S, Du F, Wang X: The mitochondrial phos- 124, 2013 phatase PGAM5 functions at the convergence point of multiple ne- 26. Rankin EB, Tomaszewski JE, Haase VH: Renal cyst development in mice crotic death pathways. Cell 148: 228–243, 2012 with conditional inactivation of the von Hippel-Lindau tumor suppres- 42. Yang N, Luo M, Li R, Huang Y, Zhang R, Wu Q, Wang F, Li Y, Yu X: sor. Cancer Res 66: 2576–2583, 2006 Blockage of JAK/STAT signalling attenuates renal ischaemia-reperfusion 27. Havasi A, Borkan SC: Apoptosis and acute kidney injury. Kidney Int 80: injury in rat. Nephrol Dial Transplant 23: 91–100, 2008 29–40, 2011 43. Zhuang S, Kinsey GR, Yan Y, Han J, Schnellmann RG: Extracellular 28. Chipuk JE, Green DR: Dissecting p53-dependent apoptosis. Cell signal-regulated kinase activation mediates mitochondrial dysfunction Death Differ 13: 994–1002, 2006 and necrosis induced by hydrogen peroxide in renal proximal tubular 29. Daemen MA, van ’t Veer C, Denecker G, Heemskerk VH, Wolfs TG, cells. J Pharmacol Exp Ther 325: 732–740, 2008 Clauss M, Vandenabeele P, Buurman WA: Inhibition of apoptosis in- 44. Lu J, Holmgren A: Thioredoxin system in cell death progression. An- duced by ischemia-reperfusion prevents inflammation. JClinInvest tioxid Redox Signal 17: 1738–1747, 2012 104: 541–549, 1999 45. Brooks C, Wei Q, Cho SG, Dong Z: Regulation of mitochondrial dy- 30. Shao X, Johnson JE, Richardson JA, Hiesberger T, Igarashi P: A minimal namics in acute kidney injury in cell culture and rodent models. JClin Ksp-cadherin promoter linked to a green fluorescent protein reporter Invest 119: 1275–1285, 2009 gene exhibits tissue-specific expression in the developing kidney and 46. Jiang M, Wei Q, Dong G, Komatsu M, Su Y, Dong Z: in genitourinary tract. J Am Soc Nephrol 13: 1824–1836, 2002 proximal tubules protects against acute kidney injury. Kidney Int 82: 31. Vousden KH, Prives C: Blinded by the light: The growing complexity of 1271–1283, 2012 p53. Cell 137: 413–431, 2009 47.PablaN,DongG,JiangM,HuangS,KumarMV,MessingRO,DongZ: 32. Han X, Yue J, Chesney RW: Functional TauT protects against acute Inhibition of PKCd reduces cisplatin-induced nephrotoxicity without kidney injury. JAmSocNephrol20: 1323–1332, 2009 blocking chemotherapeutic efficacy in mouse models of cancer. J Clin 33. Tsuruya K, Yotsueda H, Ikeda H, Taniguchi M, Masutani K, Hayashida H, Invest 121: 2709–2722, 2011 Hirakata H, Iida M: Involvement of p53-transactivated Puma in cisplatin- induced renal tubular cell death. Life Sci 83: 550–556, 2008 34. Megyesi J, Udvarhelyi N, Safirstein RL, Price PM: The p53-independent activation of transcription of p21 WAF1/CIP1/SDI1 after acute renal This article contains supplemental material online at http://jasn.asnjournals. failure. Am J Physiol 271: F1211–F1216, 1996 org/lookup/suppl/doi:10.1681/ASN.2013080902/-/DCSupplemental.

J Am Soc Nephrol 25: 2278–2289, 2014 Tubular p53 in AKI 2289 Leukocytes/section Neutrophils/section Macrophages/section

Supplementary Figure 1. Infiltration of neutrophils, macrophages and leukocytes in ischemic AKI in PT-p53-KO and wild-type mice. Wild-type and OT-p53-KO littermate mice were subjected to 28 minutes of bilateral renal ischemia followed by 2 days of reperfusion or sham operation as control. (A) Immunohistochemical staining of neutrophils, macrophages, and esterase-positive leukocytes (original magnification X 200). (B) Quantitation of positive staining cells per cross-sectional area. Data were expressed as mean±SD, n=8; bars with different superscripts were significantly different from each other (p<0.05). Supplementary Figure 2. Renal histology of PT-p53-KO and wild-type mice. Wild-type and PT-p53-KO littermate mice were subjected to 23 minutes of bilateral renal ischemia followed by 2 days of reperfusion (I/R) or sham operation as control. (A) Kidney cortical tissues were stained with hematoxylin-eosin to show histology (original magnification X200). (B) Tubular damage in I/R cortical tissues was semi-quantified as pathological scores. Data were expressed as mean±SD, n=8; # p<0.05, significantly different from PT-p53-WT group. Supplementary Table 1. 769 Genes induced by 28 minutes of renal ischemia

and 24 hours of reperfusion in C57/Bl6 mice

Acetylation Phospho- Membrane Disulfide Cytoplasm Glycoprotein protein 3110003A1 A4galt 1190002H23 bond 7Rik Abcb1b Rik 1190002H23 Adam12 4930572J05Rik Acot2 Acsl4 Rik Adam19 Acot1 A4galt Acsf2 Ada 3110082I17R Adam8 Abcb1b Acsl4 Adam12 Acta2 ik Adamts1 Adam12 Acta2 Adam19 4930506M07 Adamts4 Actg1 Adam19 Actg1 Adam8 Rik Adm Actn1 Adam8 Actn1 Adora2b Abcb1b Adora2b Akap12 Adamts1 Ada Aldh18a1 Acot5 Angptl4 Akr1b8 Adamts4 Aldh1a1 Anxa1 Acta2 B4galnt1 Aldh1a1 Adamtsl4 Anxa1 Arv1 Actg1 Bmp6 Aldh1a2 Adora2b Anxa2 Axl Actn1 C5ar1 Aldh1a7 Angptl4 Anxa3 B4galnt1 Adam12 Cadm1 Anxa1 Anxa1 Anxa5 Bak1 Aff2 Ccbe1 Aprt Axl Aprt Bax Akap12 Ccl2 Arhgdia B4galnt1 Arhgdia C5ar1 Aldh1a2 Cd14 Bmp6 Arpc1b Cadm1 Arpc1b Ampd3 Cd44 C5ar1 Asns Capn2 Atf5 Anxa1 Cd68 Cadm1 Atg7 Cd14 Atg7 Anxa2 Cd9 Ccbe1 Bag2 Cd151 Bax Anxa5 Chi3l3 Ccl2 Bax Cd44 Bcar1 Aprt Chrnb1 Cd14 Cbr1 Cd68 Bin1 Arhgap11a Clic1 Cd151 Cdkn1a Cd9 Blvrb Arpc1b Clu Cd44 Chaf1b Cdc42se2 Camk2n2 Asns Col12a1 Cd68 Clic1 Cdh3 Capn2 Axl Col4a1 Cd9 Cnn2 Cdh6 Carhsp1 Bag2 Col4a2 Cdh3 Coro1c Cftr Cbr1 Bcar1 Cpb2 Cdh6 Cstb Ch25h Ccdc85b Bcl3 Crispld2 Cftr Ctps Chrnb1 Cd2ap Bin1 Crlf1 Ch25h Cttn Ckap4 Cdc42se2 C5ar1 Csf1 Chrnb1 Ddx21 Cldn1 Cdkn1a Cadm1 Ctgf Clcf1 Ddx39 Cldn4 Cep170 Carhsp1 Ctsc Col12a1 Dock5 Clic1 Clic1 Cbr1 Ctsd Col1a1 Cmtm3 Cpeb1 Ccdc21 Cxcl1 Col4a1 Eaf1 Cpeb1 Csda Ccdc41 Cyr61 Col4a2 Eef1a1 Creb3l1 Cstb Cd14 Edn1 Cpb2 Efhd2 Csf1 Cttn Cd2ap Entpd1 Cpe Ehhadh Ctxn1 Des Cd44 Epha2 Creb3l1 Eif4a2 Ctxn3 Dock5 Cd9 F2r Crispld2

Supplementary Table 1 1 Eif4ebp1 Cdh3 Cyp27b1 F2rl1 Dusp8 Crlf1 Emd Cdkn1a Cyp3a13 F3 Eef1a1 Csf1 Ept1 Cep170 Cyp4a14 Fbn1 Eid3 Ctsc Esd Cftr Cyp4a32 Fgb Eif6 Ctsd Flna Chaf1b Cyp4v3 Fgg Enc1 Emp1 Ftsj3 Chrnb1 Efhd2 Gdf15 Esd Emp2 G6pdx Ckap4 Emd Glrx Fam129a Emp3 Gbe1 Cldn1 Emp1 Gp49a Fam82a1 Entpd1 Glrx Clic1 Emp2 Gsr Fblim1 Epha2 Gm6177 Clu Emp3 Havcr1 Fbxo6 F2r Gm6682, Cnn2 Entpd1 Hfe Flna F2rl1 Tuba1c Coro1c Epha2 Icam1 Flnc F3 Gnb2l1 Cpeb1 Ept1 Ifngr1 Frk Fam171b Gnl3 Creb5 F2r Igsf6 Fbn1 Glrx Gpatch4 Crlf1 F2rl1 Il1r1 Fgb Gsr Csda F3 Il1rl2 Gm15453 Fgf18 Gtf2f1 Cstb Fam171b Itga5 Gsr Fgg Hmga1 Ctps Fam57a Itga6 Gsta4 Fndc4 Hmga1-rs1 Cttn Fam82a1 Itgam Hdac6 Fut2 Hmgcs2 Cyp4a14 Flnc Itgb2 Hnrpdl Fxyd5 Hmox1 Cyp4v3 Flot1 Klk1b1 Hpgds G630090E17Ri Hnrpdl D8Ertd82e Fndc4 Klk1b11 Ifi204 k Hspb1 Dctd Fut2 Klk1b24 Gdf15 Igf2bp2 Impdh2 Ddx21 Fxyd5 Klk1b27 Golm1 Ipo5 Ipo5 Ddx39 G630090E1 Klk1b3 Gp49a Ipo7 Ipo7 Dock5 7Rik Klk1b8 Gpnmb Mical2 Kras Dok4 Gdpd1 Klk1b9 Gpr176 Mphosph6 Krt18 Dusp14 Glipr2 Lamc2 Gpr64 Msn Krt8 E2f4 Gnb2l1 Layn H6pd Lgals1 Eaf1 Golm1 Lcn2 Mvp Havcr1 Lgals3 Eef1a1 Gp49a Lgals3 Mybbp1a Hfe Lmna Efhd2 Gpnmb Lif Ncl Hgsnat Lrrc59 Eif4ebp1 Gpr176 Loxl2 Nek6 Icam1 Mcm2 Eif6 Gpr64 Lrp8 Nqo1 Ier3 Mcm4 Emd Havcr1 Masp1 Nt5c1a Ifngr1 Mcm6 Enc1 Hfe Mcam Pa2g4 Il1r1 Mgst1 Entpd1 Hgsnat Mmp14 Pdlim1 Il1rl2 Mki67ip Epha2 Icam1 Mmp19 Pdlim7 Il34 Msn F2rl1 Ier3 Muc13 Pea15a Itga5 Mt1 Fam129a Ifngr1 Ngf Pgd Itga6 Mt2 Fam171b Igsf6 Nid1 Pgm1 Itgam Mthfd1l Fbn1 Il1r1 Npm1 Pgm5 Itgb2 Mvp Fbp2 Il1rl2 Oit1 Plec Kcnmb1 Mybbp1a Fbxo6 Itga5 Osmr Plekho1 Klk1b1

Supplementary Table 1 2 Flna Itga6 Pappa Ppa1 Klk1b11 Myh9 Flnc Itgam Pla2g2e Ppl Klk1b24 Myof Flot1 Itgb2 Plaur Ppp1r14b Klk1b27 Ncl Fntb Kcnmb1 Pros1 Ptp4a1 Klk1b3 Npm1 Fosl2 Kdelr3 Prrg4 Qtrt1 Klk1b8 Nqo1 Frk Kras Ptgfr Rasd1 Klk1b9 Pa2g4 Ftsj3 Large Ptgs2 Rbpms Krt18 Pdlim1 Fxyd5 Layn Ptp4a1 Rfxank Krt8 Pgd G6pdx Lrp8 Pvrl1 Rin1 Lamc2 Pgm1 Gbe1 Lrrc59 Rbp4 Rock2 Large Plec Gm15453 Mall Sema3c Rpl3 Layn Ppa1 Gm6177 Maoa Sema7a Rps12 Lcn2 Ppp1r14b Gm6682, Mboat1 Slc7a11 Rps27a Lgi2 Psat1 Tuba1c Mcam Slc7a5 Rpsa Lif Rcn1 Gnb2l1 Mgst1 Slpi Loxl2 Rdh11 Gnl3 Mmp14 Srxn1 S100a6 Lrp8 Rpl12, Golm1 Mmp19 St3gal1 Serpinb1a Masp1 Gm11425 Gp49a Mospd2 Tacstd2 Sh3bgrl3 Mcam Rpl29 Gpatch4 Ms4a6c Tgfb1 Slc7a5 Mmp19 Rpl3 Gpnmb Msn Tgfb3 Smox Muc13 Rps11 Gpr64 Muc13 Tgfbi Spatc1 Myc Rps12 Grwd1 Mxra7 Thbd Sprr1a Ngf Rps27a Gtf2f1 Myof Timd2 Nipal1 Sprr2f Rpsa H6pd Nipal1 Timp1 Nkain1 Sprr2g Rras2 Hdac6 Nkain1 Tinagl1 Oit1 Srxn1 Rtn4 Hgsnat Osmr Tnc Osmr Stat3 Runx1 Hmga1-rs1, Pcdh18 Tnfrsf10b Pappa Sult1e1 S100a10 Hmga1 Perp Tnfrsf12a Pcdh18 S100a6 Hmgcs2 Pex11a Tnfrsf23 Tagln Pi15 Serpinb1a Hmox1 Plaur Trem1 Taldo1 Plaur Tagln2 Hnrpdl Pld2 Txnrd1 Tars Popdc3 Tfdp1 Hspb1 Plekho1 Vmn1r40 Tax1bp3 Ppap2c Tmem43 Ifngr1 Plin2 Vtcn1 Tes Pros1 Tomm5 Igf2bp2 Plk3 Xdh Tpm3 Ptgfr Tpm3 Il1r1 Plp2 Tpm4 Ptgs2 Cell Tpm4 Impdh2 Popdc3 Tsc22d1 Ptprn Tubb5 Ipo5 Ppap2c adhesion Ttf2 Pvrl1 Ugdh Ipo7 Ppapdc1b Ttpa St3gal1 Ada Vat1 Itga6 Ppl Txnip Timd2 Adam12 Vim Itgb2 Prrg4 Txnrd1 Vtcn1 Bcar1 Jun Ptgfr Xdh Zyx Cadm1 Extracellular Kctd1 Ptgs2 Zc3hav1 Cd44 matrix Klf10 Ptp4a1 Zyx Cd9 Ptprn Adamts1 Cytoskelet Cdh3 Methylat

Supplementary Table 1 3 on Krt18 Pvr Cdh6 ion Adamts4 Krt20 Pvrl1 Col12a1 Angptl4 1190002H2 Acta2 Krt8 Qsox1 Ctgf Anxa2 3Rik Actg1 Lamc2 Rab31 Cyr61 Col12a1 Acta2 Eef1a1 Layn Rap2c Fblim1 Col1a1 Actg1 Gm15453 Lgals3 Rasd1 Icam1 Col4a1 Actn1 Hmga1 Lig1 Rdh11 Itga5 Col4a2 Akap12 Hmga1-rs1 Lmna Rell1 Itga6 Ctgf Arpc1b Hnrpdl Lrp8 Rhbdf2 Itgam Fbn1 Ccdc85b Kras Lrrc59 Rhoc Itgb2 Lamc2 Cd2ap Ncl Lrrfip2 Rhoj Lamc2 Lgals1 Cdc42se2 Rap2c Ltv1 Rin1 Mcam Mmp19 Cep170 Rasd1 Map3k6 Rock2 Nid1 Mmp7 Cttn Rhoc Map4k4 Rpsa Pcdh18 Nid1 Enc1 Rhoj Mapk13 Rras Perp Spp1 Fam82a1 Rps27a Mapk4 Rras2 Pgm5 Tgfbi Fblim1 Rras Mapk6 Rtn4 Pvrl1 Timp1 Flna Rras2 Mcam S100a6 Spp1 Tnc Flnc Runx1 Mcm2 Sema7a Tgfbi Itgb2 Mcm4 Serp2 Tnc Iron-binding Mical2 - Mcm6 Shisa4 Tnfrsf12a Msn binding Mki67ip Slc11a1 Zyx Ch25h Pdlim1 Cyp27b1 Mlkl Slc12a4 Cyp27b1 Pdlim7 Cyp3a13 Mospd2 Slc16a1 GTP-bindi Cyp3a13 Pgm5 Cyp4a12b Mphosph6 Slc20a1 ng Cyp4a12b Plec Cyp4a14 Mrps18b Slc25a20 Arl4c Cyp4a14 Ppl Cyp4a32 Msn Slc25a22 Eef1a1 Cyp4a32 Ptp4a1 Cyp4f16 Mvp Slc25a24 Gnl3 Cyp4f16 Rin1 Cyp4v3 Mxra7 Slc25a25 Kras Cyp4v3 Spatc1 Hmox1 Mybbp1a Slc25a34 Rab31 Ftl1 Tagln Ptgs2 Myc Slc35e4 Rap2c Hmox1 Tpm3 Cell binding Myh9 Slc36a4 Rasd1 Ptgs2 Tpm4 Col4a1 Myof Slc39a6 Rhoc Slc11a1 Col4a2 Zyx Ncl Slc40a1 Rhoj Slc40a1 Nid1 Nek6 Slc7a11 Rras Steap1 Oxidoreduc Spp1 Ngf Slc7a5 Rras2 Steap2 Angiogenesis tase Noc2l Slco2a1 Tuba1a Ccbe1 Cbr1 Nop56 Spns2 outer Col4a1 Cyp4a32 Nop58 Spred3 Secreted membrane Col4a2 Ehhadh Npm1 St3gal1 4930572J Acsl4 Epha2 G6pdx Nuak2 Steap1 05Rik Bax Mmp19 Gsr Pa2g4 Steap2 Adamts1 Maoa Tnfrsf12a H6pd Pdk4 Syt12 Adamts4 Mgst1 -binding Hmox1 Pdlim1 Sytl2 Adamtsl4 Tmem173 Actn1

Supplementary Table 1 4 Hsdl2 Pdlim7 Sytl5 Adm Arpc1b Tomm5 Impdh2 Pea15a Tacstd2 AI413582 Cnn2 Loxl1 Pgm1 Tgm1 Angptl4 Coro1c Acetylated Loxl2 Pgm5 Thbd Anxa2 Emd amino end Maoa Pld2 Timd2 Bmp6 Enc1 Acta2 Pgd Plec Tlr13 C1ql3 Flna Anxa1 Ptgs2 Plekho1 Tlr8 Ccbe1 Flnc Anxa2 Qsox1 Plk3 Tmbim1 Ccl2 Hdac6 Mt1 Rdh11 Ppan Tmed5 Chi3l3 Myh9 Mt2 Smox Ppl Tmem128 Clcf1 Plec Srxn1 Ppm1j Tmem173 Clu Tpm3 Keratinization Steap1 Ppp1r14b Tmem199 Col12a1 Tuba1c Ppl Steap2 Psmd14 Tmem43 Col1a1 Gm6682 Sprr1a Txnrd1 Ptgs2 Tmem45a Col4a1 Tubb2a Sprr2f Vat1 Ptpn23 Tnfrsf10b Col4a2 Tubb5 Sprr2g Xdh Ptprn Tnfrsf12a Cpb2 Tubb6 Tgm1 Pvrl1 Tnfrsf23 Cpe Blood Rab31 Tomm5 Crispld2 coagulation Glucose Rad18 Trem1 Crlf1 Anxa5 metabolism Rap2c Tspan17 Csf1 F2r G6pdx Abcb1b Rbpms Tspan4 Ctgf F3 H6pd Acot1 Rdh11 Ucp2 Cxcl1 Fgb Pdk4 Acot2 Rell1 Vmn1r100 Cyr61 Fgg Pgm1 Acot5 Rfxank Vmn1r112 Edn1 Pros1 Pgm5 Ada Rhbdf2 Vmn1r130 Fbn1 Thbd Adam12 Rhoc Vmn1r143 Fgb Calcium Adam19 Rhoj Vmn1r135 Fgf18 Duplication binding Adam8 Rin1 Vmn1r148 Fgg Abcb1b Anxa1 Adamts1 Riok1 Vmn1r132 Gdf15 Anxa1 Anxa2 Adamts4 Rock2 Vmn1r107 Gm6484 Anxa2 Cdh3 Ampd3 Rpl12, Vmn1r104 Il34 Cdh3 Nid1 Ang4 Gm11425 Vmn1r152 Lamc2 Col4a1 S100a10 Arg2 Rpl29 Vmn1r40 Lcn2 Col4a2 S100a6 Capn2 Rpl3 Vmn1r93 Lgals1 Impdh2 Tnc Cftr Rpp25 Vopp1 Lgi2 Itgb2 Cpb2 Rps11 Vtcn1 Lif Ncl Cleavage Cpe Rps27a Zdhhc15 Loxl1 Plaur on pair of Ctsc Rpsa Loxl2 Rps27a basic residues Ctsd Rras2 Transmem Lrp8 Adam12 Dctd Rtn4 brane Masp1 Nadp Adam19 Ddx21 Runx1 protein Mmp19 Akr1b8 Adamts1 Ddx39 Serpina7 Abcb1b Mmp7 Aldh18a1 Adamts4 Dusp14 Sgtb Adam8 Muc13 Blvrb Adm Dusp5 Sh3bp2 Axl Ngf Cbr1 Bmp6 Dusp8

Supplementary Table 1 5 Eif4a2 Sh3tc2 B4galnt1 Nid1 Cyp27b1 Cdh3 Entpd1 Slc11a1 C5ar1 Oit1 G6pdx Cdh6 Ercc1 Slc12a4 Cd68 Pappa Gsr Cpe Esd Slc16a1 Cdh3 Pi15 H6pd Edn1 Fbp2 Slc20a1 Chrnb1 Pla2g2e Hsdl2 Gdf15 Gdpd1 Slc39a6 Cyp3a13 Plaur Nqo1 Itga5 H6pd Slc40a1 Cyp4a32 Pros1 Pgd Itga6 Hdac6 Slc7a11 F3 Qsox1 Rdh11 Loxl1 Klk1b1 Slc7a5 Ifngr1 Rbp4 Txnrd1 Mmp14 Klk1b11 Socs3 Ifrd1 Sema3c Ngf Klk1b24 Sphk1 Il1r1 Serpina3n Proteas Pros1 Klk1b27 Spp1 Itga5 Serpina7 e Prrg4 Klk1b3 Spred3 Itgam Serpine1 Tgfb1 Klk1b8 Src Muc13 Slpi Adam12 Tgfb3 Klk1b9 Srgap1 Ptgfr Smpdl3b Adam19 Masp1 Stat3 Ptprn Spp1 Adam8 Others Mmp14 Steap1 St3gal1 Adamts1 Fam38a Mmp19 Sytl2 Tgfb1 Adamts4 Fam83c Mmp7 Tacstd2 Tgfb3 Capn2 Fga Nt5c1a Taf1d Tgfbi Cpb2 Gadd45a Nt5dc3 Tagln Timp1 Cpe Gas5 Pappa Tagln2 Tinagl1 Ctsc Gm10021 Pla2g2e Taldo1 Tnc Ctsd Gm10325 Pld2 Tars Xdh Klk1b1 Gm10375 Ppa1 Tax1bp3 Klk1b11 Gm10377 Ppap2c Tcerg1 Klk1b24 BC061237 Ppapdc1b Tes Klk1b27 Gm3676 Ppm1j Tfdp1 Klk1b3 Gm10408 Psmd14 Tgm1 Klk1b8 Gm10409 Ptp4a1 Thbd Klk1b9 Gm10639 Ptpn23 Tmbim1 Masp1 Gm10670

Ptprn Tmem173 Mmp14 Gm10768 Ptrh1 Tmem45a Mmp19 Gm13363 Rpp25 Tnc Mmp7 Gm15706 Smpdl3b Tnfaip1 Pappa Gm3002 Ttf2 Top2a Psmd14 Gm3219 Tpm3 Slp Gm3500 Tpm4 Gm3696 Zymogen Trmt61a Others Gm10413 Adam12 Tsc22d1 1110007C09RGm3776 Adam19 Ttf2 ik Gm5622 Adamts1 Tuba1a 1110038B12RGm5945 Adamts4 Tubb2a ik Gm6121 Cpb2 Tubb5 1300002K09RGm8439

Supplementary Table 1 6 Cpe Tubb6 ik Gsta1 Ctsc Txnip 1300014I06RiGsta2 Ctsd Txnrd1 k Heatr1 Klk1b1 Ugdh 1500012F01RHemt1 Klk1b11 Utp14a ik Hkdc1 Klk1b24 Vim 1700091H14RIfitm7 Klk1b27 Wdr43 ik Ifngr2 Klk1b3 Xdh 1810007I06RiLgals4 Klk1b8 Zc3hav1 k LOC100040196 Klk1b9 Zswim4 2010003K11RLOC100041256 Mmp14 Zwint ik LOC100039753 Mmp19 Zyx 2200002D01RLOC100039574 Mmp7 ik LOC100040744

Pappa 2210403K04RLOC100039810 Pros1 ik Ssty2 2410004N09RMGC107098

ik LOC434960 2410006H16RLOC380994 ik LOC100039675 2610203C20RLrrc32 ik Mad2l2 2610524H06RMir139 ik Mir703 4833422C13RMir7-1 ik Nnat 5430425J12RiNup62cl k Oasl1 5730408K05ROlfr1128 ik Olfr1219 6330406I15RiOlfr98 k Pold4 A130040M12 Pusl1 Rik Raph1 A330021E22RRbmy1a1 ik Rcan1 A930015D03 Ripply3 Rik Rnu3b1 Acat3 Serpinb6b Ahnak Serpinb9e Aim1l Slc16a6 Ammecr1 Smyd5 Astx Snhg1 Atf3 Snhg3 BC021614 Snhg6

Supplementary Table 1 7 Btg3, Gm7334Snhg7 C130026I21RiSnord118 k Snord13 Cad Snord16a Car13 Snord37 Clca1 Snord49a Crct1 Snord88a Csprs Snord88c Cyp4f17 Snord95 Socs2 Taf4b Sox9 Tbc1d8 Spcs3 Tbrg1 Speer4d D19Bwg1357Speer4e e Srsx Dcdc2a Srsy Eif2b3 Stxbp3b Fam124a

Supplementary Table 1 8 Supplementary Table 2. 800 Genes induced by 28 minutes of renal ischemia and 48 hours of reperfusion in C57/Bl6 mice

Apoptosis Methylation Kinase Cell cycle ATP-bindin Cell 4632434I11Rik Acta2 1190002H23 division Akap12 Adamtsl4 Gm15453 Rik g 2610039C1 Aurka Bak1 Hist1h2ab, 2610039C10 Abca8a 0Rik Aurkb Bax Hist1h2an Rik Abca8b 6720463M2 Axl Birc5 Hist1h2af 4632434I11 Abcb1b 4Rik Bub1 Bub1 Hist1h2ak Rik Abcc2 Anln Bub1b Bub1b Hist1h2bb 6720463M24 Abcc3 Aurkb Cdk1 Ckap2 Hist1h2bk Rik Abcc4 Birc5 Cdk6 Clu Hist2h2bb Anln Acsf2 Bub1 Cdkn1a Csrnp1 Hmga1-rs1 Aurka Acsl4 Bub1b Cdkn3 Ddit4 Kras Aurkb Acta2 Casc5 Chek1 Epha2 Ralb Birc5 Asns Ccdc99 Chek2 Krt20 Rap2c Brca1 Atad2 Ccna2 D8Ertd82 Naip1 Rasd1 Bub1 Aurka Ccnb1 e Nek6 Rhod Bub1b Aurkb Ccnb2 Epha2 Pea15a Rhoj Casc5 Axl Ccne2 Ipmk Sgk1 Rnd2 Ccdc99 Blm Ccnf Irak3 Siah1a Rras Ccna2 Bub1 Cdc20 Map3k6 Tmem173 Tubb5 Ccnb1 Bub1b Cdc25b Mapk13 Tnfrsf10b Ccnb2 Cdk1 Cdc25c Mapk4 Tnfrsf12a Kinetochor Ccne2 Cdk6 Cdca5 Mapk6 Trp53inp1 e Ccnf Cenpe Cdca8 Mastl Zmat3 Ndc80 Cdc20 Cftr Cdk1 Melk Nek2 Cdc25b Chek1 Cdk6 Nek2 F630043A0 Cdc25c Chek2 Cenpe Nek6 Mad2l2 4Rik Cdca5 Ctps Cep55 Pbk Ndc80 Spc24 Cdca8 D8Ertd82e Ercc6l Pdgfrb Nek2 Spc25 Cdk1 Ehd2 F630043A0 Pdk4 Nek6 Zwilch Cdk6 Entpd1 4Rik Pi4k2b Oip5 Bub1 Cdkn1a Epha2 Fam83d Plaur 2610039C10Ri Bub1b Cdt1 Ercc6l Hells k Casc5 Cenpe Hells Incenp Plk3 6720463M24Ri Cenpa Cep55 Ipmk Kif11 Plk4 k Cenpe Chaf1b Irak3 Kntc1 Rock2 F630043A04Ri Cenph Chek1 Kif11 Lig1 Sgk1 k Cenpn Chek2 Kif15 Mad2l1 Sphk1 Spc24 Cenpt Ckap2 Kif18a Mad2l2 Tk1 Spc25 Ccdc99 Dbf4 Kif18b Mcm5 Ttk Zwilch Ercc6l Dlgap5 Kif20a Ncapd2

Supplementary Table 2 1 Anln Incenp Extracell Kif22 Ncapg2 Aurkb Kntc1 ular E2f8 Kif23 Ncaph Birc5 Mlf1ip matrix Ercc6l Lig1 Ndc80 Bub1 Plk1 Adamts1 Esco2 Map3k6 Nek2 Bub1b Pmf1 Adamts12 F630043A04 Mapk13 Nek6 Casc5 Sgol1 Adamts2 Rik Mapk4 Nuf2 Cdk1 Sgol2 Angptl4 Fam83d Mapk6 Nusap1 Cdc20 Nuf2 Bgn Fancd2 Mastl Oip5 Cdc25b Mad2l1 Col1a1 G0s2 Mcm2 Plk1 Cdc25c Col1a2 H2afx Mcm3 Pmf1 Cdca5 Phospho- Col3a1 Hells Mcm4 Prc1 Cdca8 protein Col4a1 Incenp Mcm5 Racgap1 Cenpe Agpat9 Col4a2 Kif11 Mcm6 Sgol1 Cep55 1190002H2 Col5a1 Kntc1 Melk Sgol2 Ccdc99 3Rik Col6a2 Lig1 Mthfd1l Smc2 Ccna2 2610039C1 Col6a3 Lin9 Naip1 Spc24 Ccnb2 0Rik Col8a1 Mad2l1 Nek2 Spc25 Ccnf 2810417H1 Col12a1 Mad2l2 Nek6 Ube2c Ercc6l 3Rik Col14a1 Mapk13 Oplah Zwilch Fam83d 4930506M0 Col15a1 Mapk4 Pbk Hells 7Rik Ctgf Mapk6 Pdgfrb Angiogene Incenp 4930534B0 Emilin1 Mcm2 Pdk4 sis Kif11 4Rik Fbn1 Mcm3 Pi4k2b Ccbe1 Kntc1 5430435G2 Fn1 Mcm6 Plk1 Col4a1 Ncapd2 2Rik Lama5 Ncapd2 Plk3 Col4a2 Ncaph 5730590G1 Lamc1 Ncapg2 Plk4 Col8a1 Ncapg2 9Rik Lamc2 Ncaph Rock2 Epha2 Nusap1 6720463M2 Lgals1 Ndc80 Rrm1 Figf Plk1 4Rik Mmp17 Nek2 Sgk1 Fn1 Pmf1 Abcb1b Mmp19 Nek6 Smc2 Mfge8 Ccnb1 Abcc2 Mmp2 Nuf2 Sphk1 Mmp19 Sgol1 Abcc3 Pxdn Nusap1 Srxn1 Mmp2 Nuf2 Acot5 Sparc Oip5 Tk1 Tnfrsf12a Mad2l1 Acta2 Spp1 Plk1 Top2a Smc2 Actn1 Anxa2 Pmf1 Ttk Cytoplasm Ube2c Adam12 Nid1 Prc1 Ube2c 1190002H2 Adamts12 Timp1 Racgap1 3Rik DNA Adra1d Tgfbi Rassf2 2310079F2 replication Aff2 Sgol1 3Rik Blm AI464131 Centrom Sgol2 Acetylation 4632434I11 Cdt1 Akap12 ere Siah1a Rik 3110003A17 Chaf1b Aldh1a2 Nek2 Smc2 4930547N1 Rik Dbf4 Anln Oip5 Spc24 6Rik Acot2 Dtl Anxa1 2610039C Spc25 Acot1 Acsf2

Supplementary Table 2 2 Gins2 Anxa2 10Rik Tfdp1 Acsl4 Acta2 Lig1 Apbb1ip Aurkb Txnip Acta2 Actn1 Mcm2 Arhgap11a Birc5 Ube2c Actn1 Akap12 Mcm3 Arpc1b Cdca8 Uhrf1 Aldh1a1 Aldh1a1 Mcm4 Asf1b Cenpa Zwilch Anln Aldh1a2 Mcm5 Asns Cenpe Anxa1 Aldh1a7 Mcm6 Atad2 Cenpi Glycoprotei Anxa2 Anln Pcna Aurka Cenpn n Anxa3 Anxa1 Pola1 Aurkb Cenpt Arpc1b Apbb1ip Pola2 Axl Ercc6l 4930572J05 Asns Arpc1b Pold4 Bag2 Incenp Rik Atg7 Atg7 Pole Bcar1 Sgol1 Abca8a Aurkb Aurka Prim2 Birc5 Sgol2 Abca8b Bag2 Aurkb Rpa2 Blm Abcb1b Bax Bax Rrm1 Brca1 Zymogen Abcc2 Blm Bcar1 Rrm2 Bub1 Adam12 Abcc3 Bub1b Birc5 Bub1b Adamts1 Adam12 Ccnb1 Blvrb Collagen C330027C0 Adamts12 Adam8 Cdk1 Bub1b C1ql3 9Rik Adamts2 Adamts1 Cdk6 C330027C0 Ccbe1 Card14 Bmp1 Adamts12 Cdkn1a 9Rik Col1a1 Casc5 Cpb2 Adamts2 Cenph Capn2 Col1a2 Ccdc21 Cpe Adamtsl4 Cenpt Card14 Col3a1 Ccdc41 Ctsk Adra1d Chaf1b Ccdc85b Col4a1 Ccdc99 Ctss AI464131 Clic1 Ccdc99 Col4a2 Ccna2 Klk1b1 Angptl4 Cnn2 Ccna2 Col5a1 Ccnb1 Klk1b11 Anxa1 Coro1c Ccnb1 Col6a2 Ccnb2 Klk1b16 Axl Cstb Cdc25b Col6a3 Ccne2 Klk1b24 B4galnt1 Ctps Cdca8 Col8a1 Cd14 Klk1b27 B4galt1 Efhd2 Cdkn1a Col12a1 Cdc20 Klk1b3 Bgn Eif4ebp1 Cenpe Col14a1 Cdc25b Klk1b8 Bmp1 Fabp1 Cep55 Col15a1 Cdc25c Klk1b9 Bmp6 Fkbp5 Chek1 Colec12 Cdca5 Mmp14 Cacna2d1 Flna Cited4 Emilin1 Cdca8 Mmp17 Ccbe1 G6pdx Ckap2 Msr1 Cdk1 Mmp19 Cd14 Gbe1 Clic1 Cdk6 Mmp2 Cd68 Gins2 Cpeb1 Cell Cdkn1a Pappa Cftr Glrx Cry2 binding Cdt1 Pros1 Ch25h Gm6177 Cstb Col4a1 Cenpa Ren1 Chrnb1 Gnl3 Cygb Col4a2 Cenpe Tpsab1 Clcf1 Gpt Ddit4 Col6a2 Cenpi Clstn2 Gsr Des Lama5 Cenpn Secreted Clu H2afx Dlgap5 Lamc1 Cenpt 4930572J Col12a1 Hist1h1a Dtl Nid1 Cep55 05Rik Col14a1 Hist1h1b Dtx4

Supplementary Table 2 3 Spp1 Cftr Adamts1 Col15a1 Hist1h2ab, Dusp8 Chaf1b Adamts12 Col1a1 Hist1h2an Eid3 Signaling Chek1 Adamts2 Col1a2 Hist1h2af Eif6 4930572J0 Chek2 Adamtsl4 Col3a1 Hist1h2ak Eps8l3 Rik Chrnb1 Adm Col4a1 Hist1h2bb F630043A0 Adam12 Ckap2 Angptl4 Col4a2 Hist1h2bk 4Rik Adam8 Ckap2l Anxa2 Col6a2 Hist2h2bb Fabp1 Adamts1 Ckap4 B4galt1 Col6a3 Hmga1-rs1 Fam129a Adamts12 Cldn1 Bgn Colec12 Hmgcs2 Fam83d Adamts2 Clic1 Bmp6 Cpb2 Hmox1 Fblim1 Adamtsl4 Clu C1ql3 Cpe Hspb1 Fkbp5 Adm Cnn2 Ccbe1 Creb3l1 Ipo5 Flna Angptl4 Col3a1 Ccl6 Crispld2 Kif11 Flnc Axl Col6a2 Clcf1 Crlf1 Kif15 Gab2 B4galt1 Colec12 Clu Csf1 Kras Glrx Coro1c Col12a1 Ctsk Krt18 Gm15453 Bgn Cpeb1 Col14a1 Ctss Krt8 Gpt Bmp1 Creb5 Col15a1 Dpep2 Lgals1 Gsr Bmp6 Crlf1 Col1a1 Emilin1 Lgals3 Gsta4 C1ql3 Cry2 Col1a2 Emp2 Lin9 Gtse1 Cacna2d1 Cstb Col3a1 Emp3 Lmna Gucy1a3 Ccbe1 Ctps Col4a1 Entpd1 Mad2l1 Hmmr Ccl17 Cyp4a14 Col4a2 Epha2 Mcm2 Homer3 Ccl6 Cyp4v3 Col5a1 F2r Mcm3 Hpgds Cd14 D8Ertd82e Col6a2 Fam171b Mcm4 Id3 Cd68 Dbf4 Col8a1 Fam198b Mcm5 Ifi204 Chrnb1 Depdc1a Cpb2 Fam20a Mcm6 Igf2bp2 Clcf1 Dlgap5 Cpe Fbln5 Mgst1 Incenp Clstn2 Dnajc9 Crispld2 Fbn1 Mt1 Ipo5 Clu Dok4 Crlf1 Fgb Mt2 Kif15 Col12a1 Dtl Csf1 Fgf18 Mthfd1l Kif18a Col14a1 Dtx4 Ctgf Fgg Mvp Kntc1 Col15a1 E2f8 Cxcl13 Figf Mybl1 Lrig2 Col1a1 Ect2 Cyr61 Fkbp10 Myc Lrp1 Col1a2 Efhd2 Edn1 Fn1 Myl9 Mad2l1 Col3a1 Ehd2 Emilin1 Fstl1 Myof Melk Col4a1 Eif4ebp1 Fam20a Fut2 Ncaph Mical1 Col4a2 Eif6 Fbln5 Gabra3 Nqo1 Mical2 Col5a1 Entpd1 Fbn1 Gdf15 Pbk Mlf1ip Col6a2 Epha2 Fgb Golm1 Pcna Mvp Col6a3 Ercc6l Fgf18 Gpnmb Pcyt1a Ncapd2 Col8a1 Esco2 Fgg Gpr176 Pdlim1 Ncaph Cpb2 F630043A0 Figf Gpr64 Pgm1 Nek6 Cpe 4Rik Fn1 Gpr97 Plec Nqo1 Crispld2

Supplementary Table 2 4 Crlf1 Fam129a Fstl1 Havcr1 Pmf1 Numbl Csf1 Fam171b Gdf15 Hfe Ppp1r14b Nusap1 Ctgf Fam35a Il34 Hmmr Psat1 Pcyt1a Ctsk Fam83d Islr Ier3 Rcn1 Pdlim1 Ctss Fancd2 Lama5 Ifngr1 Rpa2 Pdlim2 Cxcl13 Fbn1 Lamc1 Igsf5 Rpl3 Pdlim7 Cyr61 Fbp2 Lamc2 Il1r1 Rps11 Pea15a Dpep2 Fkbp10 Lbp Il1rl2 Rps12 Per1 Edn1 Fkbp5 Lcn2 Il34 Rpsa Pgm1 Emilin1 Flna Lgals1 Islr Rrm1 Pgm5 Epha2 Flnc Lgi2 Itga5 S100a10 Pi4k2b F2r Fn1 Lox Itga6 S100a6 Pla2g16 Fam171b Fosl2 Loxl1 Itgam Serpinb1a Plec Fam20a Foxm1 Loxl2 Itgb2 Slc25a20 Plekho1 Fbln5 Foxp4 Loxl3 Kcnmb1 Slc25a24 Plk4 Fbn1 G6pdx Lrp8 Kif18a Smc2 Pmm1 Fgb Gab2 Mfge8 Klk1b1 Spc25 Ppl Fgf18 Gabra3 Mmp17 Klk1b11 Sumo3 Ppp1r14b Fgg Gas2l3 Mmp19 Klk1b16 Tagln Prc1 Figf Gata6 Mmp2 Klk1b24 Tagln2 Psrc1 Fkbp10 Gbe1 Muc13 Klk1b27 Taldo1 Racgap1 Fn1 Gins2 Ngf Klk1b3 Tfdp1 Rasd1 Fstl1 Gldc Nid1 Klk1b8 Tmem43 Rbp1 Gabra3 Gm15453 Oit1 Klk1b9 Tomm5 Rerg Gdf15 Gm6177 Pappa Krt18 Top2a Rock2 Gpnmb Gnl3 Pi15 Krt8 Tpm2 Rpl3 Gpr64 Golm1 Pla2g2e Lama5 Tpm4 Rps12 Gpr97 Gpnmb Plaur Lamc1 Tuba1c Rpsa Havcr1 Gpr64 Pros1 Lamc2 Tubb5 Rrm1 Hfe Gtse1 Pxdn Large Txnrd1 Rrm2 Ifngr1 H2afx Ren1 Layn Ube2c S100a6 Igsf5 Hells Scg5 Lbp Uchl1 Serpinb1a Igsf6 Hist1h1b Sema3c Lcn2 Uhrf1 Sgk1 Il1r1 Hist1h2ab, Serpina3n Lgi2 Vat1 Sgol1 Il1rl2 Hist1h2an Serpina7 Lox Vim Sh3bgrl3 Il34 Hist1h2af Serpine1 Loxl2 Wdr62 Siah1a Islr Hist1h2ak Slit3 Loxl3 Zyx Sla Itga5 Hist1h2bb Slpi Lrig2 Smc2 Itga6 Hist1h2bk Smpdl3b Lrp1 Smox Itgam Hist2h2bb Sparc Lrp8 Cleavage Spatc1 Itgb2 Hmga1-rs1 Spp1 Mcam on pair of Sprr2f Klk1b1 Hmgcs2 Tgfb1 Mfge8 Sprr2g Klk1b11 Hmmr Tgfb3 Mmp17 basic Srxn1 Klk1b16 Hmox1 Tgfbi Mmp19 residues Stil

Supplementary Table 2 5 Klk1b24 Homer3 Timp1 Mmp2 Adam12 Ston1 Klk1b27 Hspb1 Tinagl1 Msr1 Adamts1 Sumo3 Klk1b3 Id1 Xdh Muc13 Adamts12 Tacc3 Klk1b8 Ifngr1 Myc Adm Tagln Klk1b9 Igf2bp2 Disulfide Ngf Bmp1 Taldo1 Lama5 Il1r1 bond Nid1 Bmp6 Tk1 Lamc1 Incenp Adam12 Nkain1 Cpe Tpm2 Lamc2 Ipmk Adam8 Nrcam Edn1 Tpm4 Layn Ipo5 Adamts1 Nup210 Figf Trp53inp1 Lbp Irak3 Adamts12 Oit1 Gdf15 Tsc22d1 Lcn2 Itga6 Adamts2 Osmr Itga5 Ttll7 Lgi2 Itgb2 Adm P2ry6 Itga6 Ttpa Lox Itpr3 Angptl4 Pappa Loxl1 Txnip Loxl1 Jun Axl Pdgfrb Mmp14 Txnrd1 Loxl2 Kcnip2 B4galnt1 Pear1 Mmp17 Uchl1 Loxl3 Kif11 B4galt1 Pi15 Ngf Xdh Lrg1 Kif15 Bgn Pigw Pros1 Zc3hav1 Lrig2 Kif18a Bmp1 Plaur Prrg4 Zyx Lrp1 Kif18b Bmp6 Popdc3 Ren1 Lrp8 Kif20a Cacna2d1 Ppap2c Scg5 Oxidoreduc Mcam Kif22 Ccbe1 Pros1 Tgfb1 tase Mfge8 Klf10 Ccl6 Ptgfr Tgfb3 Mmp14 Klf4 Cd14 Ptgfrn Akr1c13 Mmp17 Kntc1 Cd68 Ptprn Aldh1a1 Mmp19 Krt18 Chrnb1 Pvrl1 Aldh1a2 Mmp2 Krt20 Clic1 Pxdn Ckap2 Aldh1a7 Muc13 Krt8 Clu Rcn1 F630043A0 Bcmo1 Ngf Lama5 Col12a1 Ren1 4Rik Blvrb Nid1 Lamc2 Col14a1 Rhbdf1 Gtse1 Cbr3 Nrcam Layn Col15a1 Scarb2 Incenp Ch25h Nup210 Lgals3 Col3a1 Scn1b Kif11 Cyp1b1 Oit1 Lig1 Col4a1 Sema3c Kif15 Cyp24a1 Osmr Lin9 Col4a2 Sema4b Kif18a Cyp27b1 Pappa Lmna Col6a2 Sema7a Kif18b Cyp3a13 Pdgfrb Lrig2 Colec12 Serpina3n Kif20a Cyp4a12b Pear1 Lrp1 Cox6b2 Serpina7 Kif22 Cyp4a14 Pi15 Lrp8 Cpb2 Serpine1 Kif23 Cyp4a32 Pla2g2e Lrrfip2 Crispld2 Slc11a1 Nusap1 Cyp4f16 Plaur Mad2l1 Crlf1 Slc12a4 Prc1 Cyp4v3 Pros1 Map3k6 Csf1 Slc38a3 Tuba1a G6pdx Prrg4 Mapk13 Ctgf Slc43a3 Tuba1c Gldc Ptgfrn Mapk4 Ctsk Slco2a1 Tubb2a Gsr Ptprn Mapk6 Ctss Slit3 Tubb5 Hmox1 Pvrl1 Mastl Cxcl13 Smpdl3b Tubb6 Hsdl2

Supplementary Table 2 6 Pxdn Mcam Cyr61 Sparc Lox Rcn1 Mcm2 Dpep2 Spp1 GTP-bindin Loxl1 Ren1 Mcm3 Edn1 Sulf2 g Loxl2 Scg5 Mcm4 Emilin1 Tacstd2 5430435G2 Loxl3 Scn1b Mcm6 Entpd1 Tgfb1 2Rik Maoa Sema3c Melk Epha2 Tgfb3 Arl4c Mosc1 Sema4b Mical1 F2r Timp1 Gnl3 Nqo1 Sema7a Mlf1ip Fbln5 Tinagl1 Gucy1a3 Pxdn Serpina3n Mlkl Fbn1 Tlr13 Kras Rrm1 Serpina7 Mlph Fgb Tlr7 Rab31 Rrm2 Serpine1 Mmp2 Fgg Tlr8 Ralb Smox Shisa4 Msr1 Figf Tnfrsf23 Rap2c Srxn1 Slit3 Mvp Fn1 Tpsab1 Rasd1 Steap1 Slpi Mxra7 Fstl1 Tshr Rasl12 Txnrd1 Smpdl3b Myc Gabra3 Tspan17 Rerg Vat1 Sparc Myl9 Gdf15 Uchl1 Rhod Xdh Spp1 Myof Glrx Vmn1r40 Rhoj Sulf2 Ncapd2 Gsr Vnn1 Rhou Tacstd2 Ncapg2 Havcr1 Vtcn1 Rnd2 Tgfb1 Ncaph Hfe Xdh Rras Others Tgfb3 Ndc80 Ifngr1 Tuba1a 1300014I06 Tgfbi Nek2 Igsf5 Hydroxylati Tuba1c Rik Timp1 Nek6 Igsf6 on Tubb2a 1700056N1 Tinagl1 Ngf Il1r1 Tubb5 0Rik Tlr13 Npas2 Il1rl2 Tubb6 2010003K1 Tlr7 Nrcam Islr Col12a1 Oxidoreduc 1Rik Tlr8 Nuf2 Itga5 Col14a1 tase 2200002D0 Tmed2 Numbl Itga6 Col15a1 Aurka 1Rik Tnfrsf10b Nup210 Itgam Col1a1 Aurkb 2210403K0 Tnfrsf12a Nusap1 Itgb2 Col1a2 Axl 4Rik Tnfrsf23 Oip5 Klk1b1 Col3a1 Cdk1 3300005D0 Tpsab1 Pbk Klk1b11 Col4a1 Cdk6 1Rik Tshr Pcyt1a Klk1b16 Col4a2 Mapk6 4833419F2 Ulbp1 Pde3a Klk1b24 Col5a1 Pdgfrb 3Rik Vnn1 Pdgfrb Klk1b27 Col6a2 Plk1 4833422C1 Vopp1 Pdk4 Klk1b3 Col8a1 Plk3 3Rik Vtcn1 Pdlim1 Klk1b8 Pros1 Plk4 4833438C0 Pdlim2 Klk1b9 Rock2 2Rik

Cytoskele Pdlim7 Lama5 Cell Tk1 4930502E1 ton Pea15a Lamc1 adhesion Ttk 8Rik 1190002H2 Pear1 Lamc2 Adam12 4930563D2 3Rik Per1 Layn Bcar1 Nucleus 3Rik Acta2 Pgm1 Lcn2 Clstn2 1110007C0 4930579G2 Pgm5 Lgals3 Col12a1 9Rik 4Rik

Supplementary Table 2 7 Pi4k2b Loxl2 Col14a1 1190002H2 9130230L23 Actn1 Pigw Loxl3 Col15a1 3Rik Rik Akap12 Plec Lrig2 Col6a2 2610039C1 9930014A1 Anln Plekhg2 Lrp1 Col8a1 0Rik 8Rik Apbb1ip Plekho1 Lrp8 Ctgf 4632434I11 A130040M1 Arpc1b Plk1 Mcam Cyr61 Rik 2Rik Aurka Plk3 Mfge8 Emilin1 4930547N1 A330040F1 Aurkb Plk4 Mmp14 Fblim1 6Rik 5Rik Birc5 Pmf1 Mmp17 Fbln5 Aff2 A630081J0 Ccdc85b Pola1 Mmp19 Fn1 Alox5ap 9Rik Ccdc99 Pola2 Mmp2 Itga5 Anln Acaa1b Ccnb1 Ppl Msr1 Itga6 Anxa1 Acat3 Cdc25b Ppm1j Muc13 Itgam Asf1b Ahnak Cdca8 Ppp1r14b Ngf Itgb2 Atad2 Aim1 Cenpe Prc1 Nid1 Lama5 Aurkb Aim1l Cep55 Prim2 Nrcam Lamc1 Birc5 Ammecr1 Chek1 Prr11 Oit1 Lamc2 Blm Ap1s2 Psrc1 Osmr Mcam Brca1 Arg2 Ckap2 Ptgfrn P2ry6 Mfge8 Bub1 BC021614 Dlgap5 Ptpn23 Pappa Nid1 Bub1b Btg2 F630043A0 Ptprn Pcyt1a Nrcam Casc5 C2cd2 4Rik Pvrl1 Pdgfrb Pcdh7 Ccdc85b Car13 Fam83d Pxdn Pear1 Pcdhb17 Ccdc99 Ccdc120 Fblim1 Rab31 Pla2g2e Pgm5 Ccna2 Cenpf Flna Racgap1 Plaur Pvrl1 Ccne2 Clca1 Flnc Rad18 Pros1 Spp1 Ccnf Cldn4 Incenp Rap2c Prrg4 Tgfbi Cdc25c Cml5 Itgb2 Rassf2 Ptgfr Tnfrsf12a Cdca5 Cnnm1 Kif15 Reep4 Ptgfrn Zyx Cdca8 Ctxn1 Kif18a Rerg Pvrl1 Cdk1 Ctxn3 Kntc1 Rexo1 Pxdn Nucleotide- Cdkn1a Cxx1c Mical1 Rgs1 Ren1 binding Cdt1 D17H6S56E Mical2 Rhbdf1 Rrm1 5430435G22 Cebpd -5 Nusap1 Rhbdf2 Scg5 Rik Cenpa Ddah2 Pdlim1 Rhoj Scn1b Abca8a Cenph Dusp5 Pdlim2 Rock2 Sema3c Abca8b Cenpi E030003E1 Pdlim7 Rpa2 Sema4b Abcb1b Cenpn 8Rik Pgm5 Rpl3 Sema7a Abcc2 Cenpt E230016M1 Plec Rpp25 Slc7a11 Abcc3 Chaf1b 1Rik Plk4 Rps11 Slit3 Abcc4 Chek1 Fam111a Ppl Rpsa Slpi Acsf2 Chek2 Fam163a Prc1 Rrm2 Sparc Acsl4 Cited4 Fam38a Racgap1 Scg5 Srxn1 Acta2 Clic1 Fam57a Sgol1 Sema4b Tacstd2 Asns Creb3l1 Fga Spatc1

Supplementary Table 2 8 Tagln Serpina7 Tgfb1 Atad2 Creb5 Gsta1 Tpm2 Sgk1 Tgfb3 Aurka Csrnp1 Gsta2 Tpm4 Sgol1 Tgfbi Aurkb D2Ertd750e Ifngr2 Ttll7 Sgol2 Timp1 Axl Dbf4 Iqgap3

Sgtb Tinagl1 Blm Depdc1a Kdelr3 Sh3tc2 Tnfrsf10b Bub1 Dlgap5 Klk1b7-ps Sipa1l2 Tnfrsf12a Bub1b Dtl Lgals4 Sla Tnfrsf23 Cdk1 Dusp8 Lix1l Slc11a1 Tpsab1 Cdk6 E2f3 LOC100036 Slc12a4 Tshr Cenpe E2f8 513 Slc16a1 Txnrd1 Cftr Eid3 Lonrf3 Slc20a1 Vmn1r40 Chek1 Eif6 Lrrc32 Slc38a3 Vtcn1 Chek2 Esco2 Man1c1 Slc7a11 Xdh Ctps Fancd2 Plin2 Sparc D8Ertd82e Fkbp5 Sec24d Spc24 Ehd2 Foxm1 Svopl Sphk1 Entpd1 Foxp4 Tatdn2 Spp1 Epha2 Gata6 Tbc1d8 Spred3 Ercc6l Gins2 Tpx2 Steap1 Gnl3 Gnl3 Stil Gucy1a2 H2afx Sytl2 Gucy1a3 Hells Tacc3 Hells Heyl Tacstd2 Ipmk Hist1h1a Taf1d Irak3 Hist1h1b Tagln Kif11 Hist1h2af Tagln2 Kif15 Hist1h2ak Taldo1 Kif18a Hist1h2an, Tcerg1 Kif18b Hist1h2ab Tcf19 Kif20a Hist1h2bb Tfdp1 Kif22 Hist1h2bk Tk1 Kif23 Hist2h2bb Tlr7 Lig1 Hmga1-rs1 Tmcc3 Mastl Id1 Tmem173 Mcm2 Id3 Tmem45a Melk Ifi204 Top2a Mthfd1l Incenp Tpm2 Naip1 Ipmk Tpm4 Nek2 Ipo5 Trmt61a Nek6 Isg20 Tsc22d1 Oplah Jun Tshr Pbk Kif18a Ttk Rab31 Klf10 Tuba1a Rasd1 Klf4

Supplementary Table 2 9 Tuba1c Rasl12 Klf6 Tubb2a Rerg Mki67 Tubb5 Rnd2 Ndc80 Tubb6 Rock2 Nek2 Txnip Rras Oip5 Txnrd1 Ttk Racgap1 Uhrf1 Rad18 Vim Rasd1 Wdr62 Rexo1 Xdh S100a6 Zc3hav1 Sh3bgrl3 Zfp36 Sox9 Zswim4 Spc24 Zyx Spc25 Taf1d Tsc22d1 Vopp1

Supplementary Table 2 10 Supplementary 3. 341 genes induced by both 24 and 48 hours of reperfusion after 28 minutes of renal ischemia in wild type mice.

Apoptosis Signal Disulfide Acetylation Secreted Glycoprotein Adamtsl4 4930572 bond 3110003- 4930572- 4930572J05Rik Bak1 J05Rik Adam12 A17Rik J05Rik Abcb1b Bax Adam12 Adam8 Acot2 Adamts1 Adam12 Clu Adam8 Adamts1 Acsf2 Adamtsl4 Adam8 Epha2 Adamts1 Adm Acsl4 Adm Adamts1 Krt20 Adamtsl Angptl4 Acta2 Angptl4 Adamtsl4 Nek6 4 Axl Actn1 Anxa2 Angptl4 Pea15a Adm B4galnt1 Aldh1a1 Bmp6 Anxa1 Tmem173 Angptl4 Bmp6 Anxa1 C1ql3 Axl Tnfrsf10b Axl Ccbe1 Anxa2 Ccbe1 B4galnt1 Tnfrsf12a Bmp6 Cd14 Anxa3 Clcf1 Bmp6 C1ql3 Cd68 Arpc1b Clu Ccbe1 Extracellula Ccbe1 Clic1 Atg7 Col12a1 Cd14 r matrix Cd14 Clu Bag2 Col1a1 Cd68 Adamts1 Cd68 Col12a1 Bax Col4a1 Clcf1 Angptl4 Clcf1 Col4a1 Cdkn1a Col4a2 Clu Anxa2 Clu Col4a2 Clic1 Ctgf Col12a1 Col12a1 Col12a1 Ctgf Coro1c Cyr61 Col1a1 Col1a1 Col1a1 Cyr61 Ctps Edn1 Col4a1 Col4a1 Col4a1 Edn1 Efhd2 Fbn1 Col4a2 Col4a2 Col4a2 Entpd1 Eif4ebp1 Fgb Emp2 Ctgf Ctgf Epha2 Flna Fgf18 Emp3 Fbn1 Cyr61 F2r G6pdx Fgg Entpd1 Lamc2 Edn1 Fbn1 Gbe1 Gdf15 Epha2 Lgals1 Epha2 Fgb Glrx Il34 F2r Mmp19 F2r Fgg Gm6177 Lamc2 Fam171b Nid1 Fam171 Gdf15 Gnl3 Lcn2 Fbn1 Spp1 b Glrx Gsr Lgals1 Fgb Tgfbi Fbn1 Gsr Hmga1-rs1 Lgi2 Fgf18 Timp1 Fgb Havcr1 Hmgcs2 Loxl1 Fgg Fgf18 Hfe Hmox1 Loxl2 Fut2 Zymogen Fgg Ifngr1 Hspb1 Lrp8 Gdf15 Adam12 Gdf15 Igsf6 Ipo5 Mmp19 Golm1 Adamts1 Gpnmb Il1r1 Kras Muc13 Gpnmb Klk1b1 Gpr64 Il1rl2 Krt18 Ngf Gpr176 Klk1b11 Havcr1 Itga5 Krt8 Nid1 Gpr64 Klk1b24 Hfe Itga6 Lgals1 Pappa Havcr1 Klk1b27 Ifngr1 Itgam Lgals3 Pi15 Hfe Klk1b3 Igsf6 Itgb2 Lmna Pla2g2e Ier3 Klk1b8 Il1r1 Klk1b1 Mcm2 Plaur Ifngr1

Supplementary Table 1

Klk1b9 Il1rl2 Klk1b11 Mcm4 Pros1 Il1r1 Mmp14 Il34 Klk1b24 Mcm6 Sema3c Il1rl2 Mmp19 Itga5 Klk1b27 Mt1 Serpina3n Il34 Pappa Itga6 Klk1b3 Mt2 Serpina7 Itga5 Pros1 Itgam Klk1b8 Mthfd1l Serpine1 Itga6 Tpsab1 Itgb2 Klk1b9 Mvp Slpi Itgam Klk1b1 Lamc2 Myc Smpdl3b Itgb2 Cleavage Klk1b11 Layn Myof Spp1 Kcnmb1 on pair of Klk1b24 Lcn2 Pdlim1 Tgfb1 Klk1b1 basic Klk1b27 Lgals3 Pgm1 Tgfb3 Klk1b11 residues Klk1b3 Loxl2 Plec Tgfbi Klk1b24 Itga5 Klk1b8 Lrp8 Ppp1r14b Timp1 Klk1b27 Itga6 Klk1b9 Mcam Psat1 Tinagl1 Klk1b3 Loxl1 Lamc2 Mmp14 Rcn1 Xdh Klk1b8 Mmp14 Layn Mmp19 Rpl3 Klk1b9 Ngf Lcn2 Muc13 Rps11 Phospho- Krt18 Pros1 Lgi2 Ngf Rps12 protein Krt8 Prrg4 Loxl1 Nid1 Rpsa 1190002H23 Lamc2 Tgfb1 Loxl2 Pappa S100a10 Rik Large Tgfb3 Lrg1 Pla2g2e S100a6 4930506M07 Layn Lrp8 Plaur Serpinb1a Rik Lcn2 Cell Mcam Pros1 Slc25a20 Abcb1b Lgi2 adhesion Mmp14 Prrg4 Slc25a24 Acot5 Loxl2 Adam12 Mmp19 Ptgfr Tagln Acta2 Lrp8 Bcar1 Muc13 Pvrl1 Tagln2 Actn1 Mcam Col12a1 Ngf Sema3c Taldo1 Adam12 Mmp19 Ctgf Nid1 Sema7a Tfdp1 Aff2 Muc13 Cyr61 Pappa Slc7a11 Tmem43 Akap12 Myc Fblim1 Pi15 Slpi Tomm5 Aldh1a2 Ngf Itga5 Pla2g2e Tgfb1 Top2a Anxa1 Nid1 Itga6 Plaur Tgfb3 Tpm4 Anxa2 Nkain1 Itgam Pros1 Tgfbi Tuba1c Arhgap11a Pappa Itgb2 Prrg4 Timp1 Tubb5 Arpc1b Pi15 Lamc2 Ptprn Tinagl1 Txnrd1 Axl Plaur Mcam Pvrl1 Tnfrsf10b Vat1 Bag2 Popdc3 Nid1 Rcn1 Tnfrsf12a Vim Bcar1 Ppap2c Pgm5 Sema3c Tnfrsf23 Zyx Ccdc21 Pros1 Pvrl1 Sema7a Tpsab1 Ccdc41 Ptgfr Spp1 Serpina3 Txnrd1 Membrane Cd14 Ptprn Tgfbi n Vtcn1 Cdkn1a Pvrl1 Tnfrsf12a Serpina7 Xdh Abcb1b Cldn1 Rcn1 Zyx Serpine1 Acsl4 Clic1 Sema3c Shisa4 Oxidoredu Adam12 Clu Sema7a Acetylated Slpi ctase Adam8 Coro1c Serpina3n

Supplementary Table 2

amino end Smpdl3b Aldh1a1 Anxa1 Ctps Serpina7 Acta2 Spp1 Aldh1a2 Axl Cyp4a14 Serpine1 Anxa1 Tgfb1 Aldh1a7 B4galnt1 Cyp4v3 Slc11a1 Anxa2 Tgfb3 Blvrb Bak1 Efhd2 Slc12a4 Mt1 Tgfbi Cbr3 Bax Eif4ebp1 Slco2a1 Mt2 Timp1 Cyp4a12b Capn2 Eif6 Smpdl3b Tinagl1 Cyp4a14 Cd14 Entpd1 Spp1 Cell Tlr13 Cyp4a32 Cd68 Epha2 Tgfb1 binding Tlr8 Cyp4f16 Cldn1 Fam129a Tgfb3 Col4a1 Tnfrsf10 Cyp4v3 Cldn4 Fam171b Timp1 Col4a2 b G6pdx Clic1 Fbn1 Tinagl1 Nid1 Tnfrsf12 Gsr Ctxn1 Fbp2 Tlr13 Spp1 a Hmox1 Ctxn3 Flna Tlr8 Tnfrsf23 Hsdl2 Cyp4a14 Flnc Tnfrsf23 Glucose Tpsab1 Loxl1 Cyp4a32 Fosl2 Tpsab1 metabolism Vopp1 Loxl2 Cyp4v3 G6pdx Tspan17 G6pdx Vtcn1 Maoa Efhd2 Gbe1 Vtcn1 Pdk4 Smox Emp2 Gm15453 Xdh Pgm1 Angioge Steap1 Emp3 Gm6177 Pgm5 nesis Txnrd1 Entpd1 Gnl3 Ccbe1 Vat1 Epha2 Golm1 1190002H23Ri Col4a1 Xdh F2r Gpnmb k Adam8 Col4a2 Fam171b Gpr64 Acta2 Eif6 Epha2 FAD Fam57a Hmga1-rs1 Actn1 Itga5 Mmp19 Gsr Flnc Hmgcs2 Akap12 Itga6 Tnfrsf12 Maoa Fut2 Hmox1 Arpc1b Itgam Mical2 Golm1 Hspb1 Ccdc85b Itgb2 Growth Smox Gpnmb Ifngr1 Fblim1 factor Steap1 Gpr176 Igf2bp2 Flna GTP-bindin Bmp6 Txnrd1 Gpr64 Il1r1 Flnc g Fgf18 Xdh Havcr1 Ipo5 Itgb2 Arl4c Gdf15 Hfe Itga6 Mical2 Gnl3 Il34 Mitochond Ier3 Itgb2 Pdlim1 Kras Klk1b3 rion outer Ifngr1 Jun Pdlim7 Rab31 Ngf membrane Igsf6 Klf4 Pgm5 Rap2c Tgfb1 Il1r1 Krt18 Plec Rasd1 Tgfb3 Acsl4 Il1rl2 Krt20 Ppl Rhoj Bax Itga5 Krt8 Spatc1 Rras Blood Maoa Itga6 Lamc2 Tagln Tuba1a coagul Tmem173 Itgam Layn Tpm4 Tuba1c ation Tomm5 Itgb2 Lgals3 Zyx Tubb2a F2r Kcnmb1 Lig1 Tubb5 Fgb Transmem Kdelr3 Lmna Others Tubb6 Fgg brane Kras Lrp8 1110007C09Rik

Supplementary Table 3

Methylation Pros1 protein Large Lrrfip2 1300014I06Rik Acta2 Proteas Abcb1b Layn Map3k6 2010003K11Rik Gm15453 e Adam8 Lrp8 Mapk13 2200002D01Ri Hmga1-rs1 Adam12 Axl Maoa Mapk4 k Kras Adam8 B4galnt1 Mcam Mapk6 2210403K04Ri Rasd1 Adamts1 Cd68 Mmp14 Mcam k Rhoj Capn2 Cyp4a32 Mmp19 Mcm2 4833422C13Ri Rras Klk1b1 Ifngr1 Ms4a6c Mcm4 k Klk1b11 Il1r1 Muc13 Mcm6 A130040M12Ri Klk1b24 Itga5 Mxra7 Mlkl k Klk1b27 Itgam Myof Mvp Acat3 Klk1b3 Muc13 Nkain1 Mxra7 Acot1 Klk1b8 Ptgfr Pex11a Myc Aim1l Klk1b9 Ptprn Plaur Myof Ammecr1 Mmp14 Plekho1 Nek6 Arg2 Mmp19 Plin2 Ngf BC021614 Pappa Plk3 Pdk4 Car13 Slpi Plp2 Pdlim1 Cebpd Tpsab1 Popdc3 Pdlim7 Clca1 Ppap2c Pea15a Dusp5 Ppapdc1b Pgm1 Dusp8 Ppl Pgm5 E2f3 Prrg4 Plec Fam38a Ptgfr Plekho1 Fga Ptprn Plk3 Ftl1 Pvr Ppl Gm10325 Pvrl1 Ppm1j Gm10639 Rab31 Ppp1r14b Gm10768 Rap2c Ptpn23 Gm15706 Rasd1 Ptprn Gm3219 Rhbdf2 Pvrl1 Gm3776 Rhoj Rab31 Gm7334, Btg3 Rock2 Rad18 Gm8439 Rpsa Rap2c Gsta1 Rras Rhbdf2 Gsta2 S100a6 Rhoj Gsta4 Sema7a Rock2 Hpgds Shisa4 Rpl3 Ifi204 Slc11a1 Rpp25 Ifitm7 Slc12a4 Rps11 Ifngr2 Slc16a1 Rpsa Klf6 Slc20a1 Serpina7 Lgals4 Slc25a20 Sh3tc2 Lrrc32 Slc25a22 Slc11a1 Mad2l2

Supplementary Table 4

Slc25a24 Slc12a4 Nnat Slc25a25 Slc16a1 Ptrh1 Slc25a34 Slc20a1 Serpinb6b Slc35e4 Slc7a11 Sh3bgrl3 Slc7a11 Sphk1 Slc16a6 Slco2a1 Spp1 Snhg3 Spns2 Spred3 Snord13 Spred3 Steap1 Snord16a Steap1 Sytl2 Snord37 Sytl2 Taf1d Snord49a Sytl5 Tagln Socs2 Tlr13 Tagln2 Sox9 Tlr8 Taldo1 Spcs3 Tmem173 Tfdp1 Tbc1d8 Tmem43 Tmem173 Tchhl1 Tmem45a Tmem45a Ttpa Tnfrsf10b Top2a Vgll3 Tnfrsf12a Tpm4 Zbtb38 Tnfrsf23 Trmt61a Zfp648 Tomm5 Tsc22d1 Tspan17 Tuba1a Ucp2 Tuba1c Vopp1 Tubb2a Vtcn1 Tubb5 Zdhhc15 Tubb6 Txnip Txnrd1 Vim Xdh Zc3hav1 Zswim4 Zyx

Supplementary Table 5

Supplementary 4. 204 genes that were expressed at lower levels in PT-p53-KO kidneys than wild type at 24 hours of reperfusion

Glycoprotein Disulfide Signal Secreted Transport Transmembrane Aqp1 Adamts4 Adamts4 Adamts4 bond Aqp2 protein Agt Adm Adm Adamts4 Aqp4 Aqp1 Angpt2 Agt Agt Adm Aqp6 H2-T23 Aqp1 Angpt2 Angpt2 Angpt2 Cmah Htr1b Aqp2 Cck Cck Cckar Glrx Itga5 Aqp4 Ccl28 Ccl28 Ccl28 Kcnj13 Lepr Aqp6 Chrdl1 Chrdl1 Crispld2 Mcoln3 Muc13 Avpr2 Crispld2 Crispld2 Cyr61 Osta Ptger1 B3gat2 Cyr61 Cyr61 Defb29 Rbp4 Slc6a12 Cckar Defb29 Defb29 Egfl6 Scg5 Ccl28 Egfl6 Egfl6 Gdf15 Slc10a2 Membrane Chrdl1 Gdf15 Gdf15 Glrx Slc14a1 1110032A Crispld2 Gm106 Gm106 Gm106 Slc25a25 04Rik Egfl6 Gm5887 Igfbp5 H2-T23 Slc25a34 Adssl1 Esr1 Gpr64 Lepr Htr1b Slc2a5 Aqp1 Gdf15 H2-T23 Lgi2 Igfbp5 Slc35f1 Aqp2 Gm106 Igfbp5 Muc13 Itga5 Slc6a12 Aqp4 Gm5887 Itga5 Rbp4 Klk1b1 Slc7a11 Aqp6 Gpr64 Klk1b1 Scg5 Klk1b27 Ttpa Avpr2 H2-T23 Klk1b27 Sectm1b Klk1b3 B3gat2 Htr1b Klk1b3 Serpina3n Lepr Oxidoredu Bax Ier3 Lepr Serpinf2 Ly6d ctase Cckar Itga5 Lgi2 Spon1 Muc13 Cldn19 Klk1b1 Ly6d Tgfb3 Npy1r Aldh1a3 Cox8b Klk1b27 Lypd2 Tmem25 Psca Akr1c18 Ctxn3 Klk1b3 Muc13 Vwa2 Ptger1 BC089597 Cyp7b1 Lepr Psca Rbp4 Cbr3 Glipr2 Lgi2 Rbp4 Scg5 Cmah Gm5887 Ly6d Scg5 Zymogen Sectm1b Cox8b Gpr64 Lypd2 Sectm1b Adamts4 Sema5a Cyp4a12b H2-T23 Muc13 Sema5a Klk1b1 Serpinf2 Cyp7b1 Htr1b Npy1r Serpina3 Klk1b27 Slc7a11 Smox Ier3 Osta n Klk1b3 Spon1 Itga5 Psca Serpinf2 Tpsab1 Sycn Kcnj13 Ptger1 Spon1 Tgfb3 Lepr Sectm1b Sycn Thbd Ly6d Sema5a Tgfb3 Tmem25 Lypd2 Serpina3n Thbd Tnn Mcoln3 Serpinf2 Tmem25 Tpsab1 Muc13 Slc10a2 Tnn Supplementary Table 4 1

Slc14a1 Vmn1r40 Tpsab1 Npy1r Slc2a5 Vwa2 Upk3b Osta Slc6a12 Vwa2 Plin2 Spon1 Others Plk3 Syp Psca LOC3821 Tgfb3 Ptger1 33 Thbd Scel LOC1000 Tmem25 Sectm1b 39574 Tnn Sema5a Ssty2 Tpsab1 Slc10a2 LOC1000 Upk3b Slc14a1 40744 Vmn1r40 Slc25a25 Igk-V1 Vwa2 Slc25a34 Ifi44 Slc2a5 Keg1 Slc35f1 Others Mir107 Slc6a12 Alx1 Mir7-1 Slc7a11 Dclre1b Map3k6 Sycn Eid3 MLKL Syp Nanp Nnat Thbd Ranbp3l Nccrp1 Tmem25 Rasl11b Olfr1128 Upk3b 1810007I06Rik Olfr1219 Vmn1r135 2610017I09Rik Olfr98 Vmn1r104 4930506M07Rik Pgm5 Vmn1r143 4932415G12Rik Ftl1 Vmn1r152 5430425J12Rik Gm10664 Vmn1r40 9030619P08Rik Gm10670

9130409J20Rik Vmn1r126

Fcgbp Gm11992 A130040M12Rik Gm15706 A930015D03Rik Gm6177 C130026I21Rik Gm3696 D630023F18Rik Vmn1r117 Rbmy1a1 Gm6300 Rasgef1b Prox1 Srgap1 Rcan1 Trbv13-2 Serpinb9e Ugt2b37 Srsy Xpnpep2 LOC1000 Acat3 39675 Acaa1b Tcl1b2 Acot1 Sim2 Asb9 Sgtb Bcat1 Snhg3 Supplementary Table 4 2

Cidea Snord37 Ccdc85b Snord88c Csrnp1 Sprr2g Dusp5 Slc14a2 Esrrb Slc7a12 AI314278 Sult1e1 Fbp2 Socs2 Gsta2 Stxbp3b Hemt1 Txnip Hkdc1 Tcea3 LOC100040196 Tchhl1 Vgll3 Zfp648

Supplementary Table 4 3

Supplementary 5. 257 genes that were expressed at lower levels in PT-p53-KO kidneys than wild type at 48 hours of reperfusion

Oxidoreductase Signal Secreted Disulfide Mitochon Membrane bond drion Akr1c18 Adm Adm Aadac Bax Akr1d1 Angpt2 Angpt2 Aadac Adm Cbr2 BC089597 Cck Cck Abca8a Angpt2 Cox8b Blvrb Ccl17 Ccl9 Abca8b Ccl9 Cyp27b1 Cbr2 Ccl9 Chrdl1 Agpat9 Clec2d Gatm Cbr3 Chrdl1 Crispld2 Aqp4 Clec2h Gldc Cox8b Crispld2 Defb29 Aqp6 Crispld2 Gm4450 Cyp27b1 Defb29 Egfl6 Atp10b Defb29 Hmgcs2 Cyp2d26 Egfl6 Fam20a Bax Egfl6 Hsd3b2 Cyp3a13 Fam171b Fgf18 Cldn19 Gabra3 Hsd3b4 Cyp4a12b Fam20a Gpx6 Clec2d Glrx Keg1 Cyp4a32 Fgf18 Igf1 Clec2h Htr1b Mosc1 Cyp7b1 Gabra3 Igfbp5 Clic3 Igf1 Mrpl12 Dio1 Gpnmb Il34 Cox8b Igfbp5 Pdk4 Gldc Gpr97 Lbp Cpeb1 Il1rl2 Slc25a22 Gm4450 Gpx6 Lepr Cyp27b1 Itga5 Slc25a25 Gpx6 Igf1 Lgi2 Cyp2d26 Klk1b1 Slc25a34 Hsd3b2 Igfbp5 Pigr Cyp3a13 Klk1b16 Tomm5 Hsd3b4 Il1rl2 Plau Cyp4a32 Klk1b24 Mosc1 Il34 Proz Cyp7b1 Klk1b27 Itga5 Serpina1d Dio1 Klk1b3 Transport Glycoprotein Klk1b1 Serpina1f Fam171b Klk1b8 Abca8a Aadac Klk1b16 Serpina3n Gab2 Lepr Abca8b Abca8a Klk1b24 Serpine1 Gabra3 Lrig2 Aqp4 Abca8b Klk1b27 Slpi Gatm Npy1r Aqp6 Angpt2 Klk1b3 Sostdc1 Gm4450, Pigr Clic3 Aqp4 Klk1b8 Spon1 Hsd3b4 Plau Gabra3 Aqp6 Lbp Vwa2 Gpnmb Proz Glrx Chrdl1 Lepr Gpr97 Psca Hvcn1 Clec2d Lgi2 Transme Hsd3b2 Ptger1 Kcnip2 Clec2h Lrig2 mbrane Htr1b Ptger3 Kcnj13 Crispld2 Pigr protein Hvcn1 Sema7a Lbp Egfl6 Plau Cyp3a13 Il1rl2 Slc34a3 Mcoln3 Fam171b Proz Cyp4a32 Itga5 Slc7a11 Slc10a2 Fam20a Psca Htr1b Kcnip2 Slpi Slc14a1 Fgf18 Sema7a Itga5 Kcnj13 Sostdc1 Slc16a1 Gabra3 Serpina1d Lepr Lepr Spon1 Slc25a22 Gpnmb Serpina1f Ptger1 Lrig2 Sycn Slc25a25 Gpr97 Serpina3n Ptger3 Ltc4s Tnn Slc25a34 Htr1b Serpine1 Slc6a12 Mcoln3

Supplementary Table-5 1

Il1rl2 Slpi Tshr Tshr Slc34a3 Ms4a6c Il34 Sostdc1 Vwa2 Slc38a3 Nkain1 Itga5 Spon1 Slc6a12 Npy1r Klk1b1 Sycn Slc7a11 Nrxn2 Klk1b16 Tlr8 Slc7a13 Pde3a Klk1b24 Tmed2 Svopl Pigr Klk1b27 Tnn Tmed2 Plin2 Klk1b3 Tshr Tomm5 Plk3 Klk1b8 Upk3b Ttpa Popdc3 Lbp Vwa2 Psca Lepr Ptger1 Lgi2 Others Ptger3 Lrig2 E030003E18Rik Rasd1 Nkain1 E230016M11Rik Scel Npy1r Eid3 Sema7a Pigr Fam35a Slc10a2 Plau Fam40b Slc14a1 Popdc3 Fbp2 Slc16a1 Proz Ftl1 Slc25a22 Psca G0s2 Slc25a25 Ptger1 Gata6 Slc25a34 Ptger3 Gm10664 Slc34a3 Sema7a Gm10768 Slc38a3 Serpina1d Gm15706 Slc6a12 Serpina1f Gm3219 Slc7a11 Serpina3n Gm6300 Slc7a13 Serpine1 Gm906 Svopl Slc10a2 Gpt Sycn Slc14a1 Gsta2 Sytl5 Slc34a3 Hspb1 Tlr8 Slc38a3 Hspb7 Tmed2 Slc6a12 Igk-V1 Tomm5 Sostdc1 Ipmk Tshr Spon1 Irak3 Upk3b Tlr8 Isg20 Tnn Krt20 Tshr Lgals4 Upk3b Lonrf3 Vwa2 Lrrc32 Map3k6 Others Mid1 1190002H23Rik Mir145 1190005F20Rik Mir30d 1700027D21Rik Mlkl

Supplementary Table-5 2

1700056N10Rik Myl6 1700109H08Rik Nccrp1 1700113A16Rik Olfr1392 2210403K04Rik Olfr920 2310079F23Rik Olfr98 2310081J21Rik Pdlim7 2610017I09Rik Pfkfb3 4833419F23Rik Ppara 4930506M07Rik Ppm1j 4930563D23Rik Prox1 9130230L23Rik Ranbp3l 9130409J20Rik Rassf2 A130040M12Rik Rgs1 A4gnt Sh2d4a A630081J09Rik Sh3bgrl3 Acaa1b Sim2 Acat3 Sla Acot5 Slc14a2 Aim1l Slc7a12 Alx1 Snord16a B430319H21Rik Snord37 Bag2 Socs2 BC021614 Sox9 BC024386 Spatc1 Bcat1 Sprr2f Btg2 Sprr2g C330021F23Rik Sult1c2 Ccdc41 Tbc1d8 Ccdc85b Trbv13-2 Cebpd Tubb2a Cited4 Txnip Cry2 Ugt2b37 Csrnp1 Xpnpep2 Cxx1c Zbtb38 D630023F18Rik Zfp161 Dusp5 Zfp493 Zfp69 Zscan4c

Supplementary Table-5 3 Supplementary Table 6. 83 genes that were expressed at lower levels in PT-p53-KO kidneys than wild type at both 24 and 48 hours of reperfusion

Disulfide Glyco- Signaling Transport Secreted bond protein Adm Aqp4 Adm BC089597 Adm Angpt2 Angpt2 Aqp6 Angpt2 Cbr3 Angpt2 Aqp4 Cck Glrx Cck Cox8b Crispld2 Aqp6 Chrdl1 Kcnj13 Chrdl1 Cyp27b1 Defb29 Chrdl1 Crispld2 Mcoln3 Crispld2 Cyp4a12b Egfl6 Crispld2 Defb29 Slc10a2 Defb29 Cyp7b1 Glrx Egfl6 Egfl6 Slc14a1 Egfl6 Htr1b Htr1b Igfbp5 Slc25a25 Igfbp5 Others Igfbp5 Itga5 Itga5 Slc25a34 Serpina3 1110032A04Rik Itga5 Klk1b1 Klk1b1 Slc6a12 n 9130409J20Rik Klk1b1 Klk1b27 Klk1b27 Slc7a11 Spon1 A130040M12Rik Klk1b27 Klk1b3 Klk1b3 Ttpa Vwa2 Acaa1b Klk1b3 Npy1r Psca Acat3 Npy1r Psca Serpina3n Developmental Bax Psca Ptger1 Spon1 protein Bcat1 Ptger1 Serpina3n Sycn 4930506M07Ri Cldn19 Slc7a11 Slc10a2 Tnn k Csrnp1 Spon1 Slc14a1 Upk3b Alx1 D630023F18Rik Sycn Slc6a12 Vwa2 Angpt2 Dusp5 Tnn Spon1 Chrdl1 Eid3 Vwa2 Tnn Egfl6 Fbp2 Upk3b Prox1 Ftl1 Vwa2 Sim2 Gm10664

Gm6300 Igk-V1 Keg1 Map3k6 MLKL Nccrp1 Olfr98 Plin2 Plk3 Ranbp3l Scel Slc14a2 Slc7a12 Snord37 Socs2 Sprr2g Trbv13-2

Supplementary Table 6 1 Txnip Ugt2b37 Xpnpep2

Supplementary Table 6 2