Overexpressed SIRT6 attenuates cisplatin-induced acute kidney injury by inhibiting ERK1/2 signaling

Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase associated with numerous aspects of health and physiology. Overexpression of SIRT6 has emerged as a protector in cardiac tissues against pathologic cardiac hypertrophy. However, the mechanism of this protective effect is not fully understood. Here, both in vivo and in vitro results demonstrated that SIRT6 overexpression can attenuate cisplatin-induced kidney injury in terms of renal dysfunction, inflammation and apoptosis. In addition, SIRT6 knockout aggravated kidney injury caused by cisplatin. We also found that SIRT6 bound to the promoters of ERK1 and ERK2 and deacetylated histone 3 at Lys9 (H3K9) thereby inhibiting ERK1/2 expression. Furthermore, inhibition of ERK1/2 activity eliminated aggravation of kidney injury caused by SIRT6 knock out. Thus, our findings uncover the protective effect of SIRT6 on the kidney and define a new mechanism by which SIRT6 regulates inflammation and apoptosis. This may provide a new therapeutic target for kidney injury under stress. Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase associated with numerous aspects of health and physiology. Overexpression of SIRT6 has emerged as a protector in cardiac tissues against pathologic cardiac hypertrophy. However, the mechanism of this protective effect is not fully understood. Here, both in vivo and in vitro results demonstrated that SIRT6 overexpression can attenuate cisplatin-induced kidney injury in terms of renal dysfunction, inflammation and apoptosis. In addition, SIRT6 knockout aggravated kidney injury caused by cisplatin. We also found that SIRT6 bound to the promoters of ERK1 and ERK2 and deacetylated histone 3 at Lys9 (H3K9) thereby inhibiting ERK1/2 expression. Furthermore, inhibition of ERK1/2 activity eliminated aggravation of kidney injury caused by SIRT6 knock out. Thus, our findings uncover the protective effect of SIRT6 on the kidney and define a new mechanism by which SIRT6 regulates inflammation and apoptosis. This may provide a new therapeutic target for kidney injury under stress. Sirtuin 6 (SIRT6) is a NAD+ (nicotinamide adenine nucleotide positive)-dependent enzyme associated with many biological processes, mainly through 2 distinct enzymatic reactions: adenosine diphosphate-ribosylation and deacetylation of both histone and long-chain fatty acyl groups.1Finkel T. Deng C.X. Mostoslavsky R. Recent progress in the biology and physiology of sirtuins.Nature. 2009; 460: 587-591Crossref PubMed Scopus (1187) Google Scholar, 2Mao Z. Hine C. Tian X. et al.SIRT6 promotes DNA repair under stress by activating PARP1.Science. 2011; 332: 1443-1446Crossref PubMed Scopus (589) Google Scholar, 3Michishita E. McCord R.A. 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Zhong L. et al.The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun.Nat Med. 2012; 18: 1643-1650Crossref PubMed Scopus (356) Google Scholar, 7Zhong L. D’Urso A. Toiber D. et al.The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.Cell. 2010; 140: 280-293Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar, 8Tao R. Xiong X. DePinho R.A. et al.FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J Biol Chem. 2013; 288: 29252-29259Crossref PubMed Scopus (117) Google Scholar Based on these activities, SIRT6 is involved in DNA damage repair and modulating various inflammation and apoptosis-related signaling.9Kugel S. Mostoslavsky R. Chromatin and beyond: the multitasking roles for SIRT6.Trends Biochem Sci. 2014; 39: 72-81Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar Cisplatin (CP) is a common and effective chemotherapeutic drug for the treatment of many kinds of solid tumors.10Wang D. Lippard S.J. Cellular processing of platinum anticancer drugs.Nat Rev Drug Discov. 2005; 4: 307-320Crossref PubMed Scopus (3009) Google Scholar, 11Aran I. Safirstein R.L. Cisplatin nephrotoxicity.Semin Nephrol. 2003; 23: 460-464Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar, 12Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar It is mainly excreted by the kidney and accumulates in renal proximal tubular cells.13Yao X. Panichpisal K. Kurtzman N. Nugent K. Cisplatin nephrotoxicity: a review.Am J Med Sci. 2007; 334: 115-124Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar This results in a positive feedback loop among oxidative stress, inflammation, and apoptosis, which is a major cause of CP-induced renal injury.14Pabla N. Murphy R.F. Liu K. Dong Z. The copper transporter Ctr1 contributes to cisplatin uptake by renal tubular cells during cisplatin nephrotoxicity.Am J Physiol Renal Physiol. 2009; 296: F505-F511Crossref PubMed Scopus (189) Google Scholar, 15Zhang B. Ramesh G. Uematsu S. et al.TLR4 signaling mediates inflammation and tissue injury in nephrotoxicity.J Am Soc Nephrol. 2008; 19: 923-932Crossref PubMed Scopus (235) Google Scholar Inhibition of mitogen-activated protein kinase and tumor necrosis factor-α reduces CP-induced kidney injury.16Jo S.K. Cho W.Y. Sung S.A. et al.MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis.Kidney Int. 2005; 67: 458-466Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Potocnjak I. Skoda M. Pernjak-Pugel E. et al.Oral administration of oleuropein attenuates cisplatin-induced acute renal injury in mice through inhibition of ERK signaling.Mol Nutr Food Res. 2016; 60: 530-541Crossref PubMed Scopus (50) Google Scholar, 18Ramesh G. Reeves W.B. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity.J Clin Invest. 2002; 110: 835-842Crossref PubMed Scopus (711) Google Scholar Exogenous resveratrol and β-lapachone attenuate the CP-induced kidney injury by mediating sirtuins.19Oh G.S. Kim H.J. Choi J.H. et al.Pharmacological activation of NQO1 increases NAD(+) levels and attenuates cisplatin-mediated acute kidney injury in mice.Kidney Int. 2014; 85: 547-560Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20Do Amaral C.L. Francescato H.D. Coimbra T.M. et al.Resveratrol attenuates cisplatin-induced nephrotoxicity in rats.Arch Toxicol. 2008; 82: 363-370Crossref PubMed Scopus (100) Google Scholar More effective interventions need to be explored. SIRT6 overexpression is capable of blocking the development of pathologic cardiac hypertrophy and heart failure from hypertrophic stimuli, although no obvious effect is seen without these stressors.6Sundaresan N.R. Vasudevan P. Zhong L. et al.The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun.Nat Med. 2012; 18: 1643-1650Crossref PubMed Scopus (356) Google Scholar Furthermore, SIRT6 protects the heart from ischemia/reperfusion injury through forkhead box O3α activation.21Wang X.X. et al.SIRT6 protects cardiomyocytes against ischemia/reperfusion injury by augmenting FoxO3alpha-dependent antioxidant defense mechanisms.Basic Res Cardiol. 2016; 111: 13Crossref PubMed Scopus (91) Google Scholar SIRT1 and SIRT3 could protect the kidney from different types of injuries.22Wakino S. Hasegawa K. Itoh H. Sirtuin and metabolic kidney disease.Kidney Int. 2015; 88: 691-698Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar However, the function of SIRT6 on the kidney remains unclear. Our previous study showed that calorie restriction enhances SIRT6 expression, alleviating renal insufficiency caused by aging.23Zhang N. Li Z. Mu W. et al.Calorie restriction-induced SIRT6 activation delays aging by suppressing NF-kappaB signaling.Cell Cycle. 2016; 15: 1009-1018Crossref PubMed Scopus (72) Google Scholar To further clarify the function of SIRT6 in the kidney and discover new therapeutic interventions for CP-induced acute kidney injury, SIRT6-mutated mice and primary kidney epithelial cells were used to investigate the effect of SIRT6 on the kidney under CP stress. Our results confirmed that SIRT6 overexpression attenuates CP-induced acute kidney injury in mice; conversely, SIRT6 deficiency exacerbates CP-induced renal injury. Furthermore, we discovered that SIRT6 could inhibit the expression of both extracellular signal–regulated kinase (ERK) 1 and 2, which may define a new mechanism for the SIRT6 protecting effect. Our study provides a new therapeutic target for kidney injury under stress. We obtained primary kidney epithelial cells from SIRT6 mutant mice and wild-type (WT) mice to investigate the effect of the protein level of SIRT6 on CP-induced apoptosis. Megalin immunostaining demonstrated that >95% of cells were of proximal tubule origin (Supplementary Figure S1). According to Western blot assay, we found that there were significant changes in SIRT6 expression in the mutant cells (Figure 1a). The cells were treated with CP for 24 hours; we then compared the stress response in different groups, including WT cells treated with dimethylsulfoxide (DMSO) or CP (WT+CP), SIRT6 overexpressed cells treated with DMSO (transgenic [TG]) or CP (TG+CP), and SIRT6-deficient cells treated with DMSO (knockout [KO]) or CP (KO+CP). CP treatment caused a significant decrease in cell viability in the WT and SIRT6 KO cells, but this effect was weaker in the SIRT6 overexpression line (Figure 1b and Supplementary Figure S2A). Under the microscope, we could find massive apoptosis in WT+CP mice, and the percentage of apoptotic cells was markedly reduced in TG+CP mice compared with WT+CP mice, whereas KO+CP mice did not show more apoptosis than WT+CP mice (Figure 1c and Supplementary Figure S2B). Flow cytometry was used to measure cell-cycle status and revealed fewer S-phase cells after treatment with CP. This effect was reduced in TG+CP cells compared with WT+CP cells (Figure 1d and e). As for the SIRT6 KO, we did find a reduced amount of S phase in KO compared with WT; however, KO+CP did not show a significant difference from WT+CP (Supplementary Figure S1C and E). Furthermore, we found an increase in the number of propidium iodide–positive cells after CP treatment in WT. This effect was reduced in TG+CP cells compared with WT+CP cells, and no significant difference was found between KO+CP and WT+CP cells (Figure 1f and g and Supplementary Figure S1D and F). Based on the data from primary kidney cells, to determine whether SIRT6 expression would change with the process of CP treatment, quantitative polymerase chain reaction (PCR) and Western blotting were used to show SIRT6 expression in mouse kidneys at different days since injection of CP. The results show that there was a mild increase in SIRT6 expression starting 2 days after injection, and the greatest expression appeared 3 days after injection. The mRNA level was consistent with the protein level (Supplementary Figure S3). To model CP-induced renal injury in vivo, CP was i.p. injected in mice compared with the DMSO control. After 3 days, some of the WT mice and SIRT6 knockout mice were dead; surviving mice were used in the following histological and molecular tests (Supplementary Table S1). Western blot assays confirmed that the expression of SIRT6 was ∼4-fold higher in SIRT6 transgenic mice, and we could hardly detect any expression in SIRT6-deficient mice (Figure 2a and b). We found no difference among groups without CP treatment in relative kidney weight, blood urea nitrogen, and serum creatinine. In all genotypes, CP was associated with an increase in these variables, which indicated kidney hypertrophy and renal dysfunction. These effects were most pronounced in the KO+CP mice and least pronounced in TG+CP mice (Figure 2c–e). Reduction of the mitochondria number in normal renal cells is a result of CP-induced kidney injury and a hallmark of renal dysfunction.24Negishi K. Noiri E. Sugaya T. et al.A role of liver fatty acid-binding protein in cisplatin-induced acute renal failure.Kidney Int. 2007; 72: 348-358Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The mitochondria number, represented by mitochondrial DNA copy number, decreased after CP treatment, but overexpression of SIRT6 attenuates this decrease, whereas knockout of SIRT6 intensifies the decrease (Figure 2f). A decrease in the number of mitochondria was independently confirmed by electron microscopy (Supplementary Figure S4). These results suggest that SIRT6 plays a protective role against CP-induced renal dysfunction. To directly observe the injury levels of different groups, kidney specimens were stained with hematoxylin and eosin. Quantitative statistical analysis was performed as described in the Methods section. After treatment with CP, we could easily find tubular histopathologic changes in WT+CP mice kidneys, whereas the prevalence of tubular necrosis appeared lower in TG+CP mice kidneys. However, KO+CP mice kidneys did not significantly show more tubular necrosis than the WT+CP control (Figure 3). These results suggest that SIRT6 overexpression protects renal tubules from CP-induced damage. Inflammation is a major feature of CP-induced cytotoxicity.12Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar, 25Miller R.P. Tadagavadi R.K. Ramesh G. Reeves W.B. Mechanisms of Cisplatin nephrotoxicity.Toxins (Basel). 2010; 2: 2490-2518Crossref PubMed Scopus (1071) Google Scholar To examine the level of inflammation in the kidney, tumor necrosis factor-α, interleukin-6, and interleukin1-β were tested in different groups. Among all cytokines tested, only tumor necrosis factor-α was increased before CP treatment in KO mice compared with WT mice. Following CP treatment, we found less inflammation in the TG+CP mice and more inflammation in the KO+CP mice compared with the WT+CP control (Figure 4a–c). As an activator of NF-κB signaling, nuclear factor-α upregulation acts as major signal for CP-induced inflammation.17Potocnjak I. Skoda M. Pernjak-Pugel E. et al.Oral administration of oleuropein attenuates cisplatin-induced acute renal injury in mice through inhibition of ERK signaling.Mol Nutr Food Res. 2016; 60: 530-541Crossref PubMed Scopus (50) Google Scholar, 18Ramesh G. Reeves W.B. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity.J Clin Invest. 2002; 110: 835-842Crossref PubMed Scopus (711) Google Scholar, 19Oh G.S. Kim H.J. Choi J.H. et al.Pharmacological activation of NQO1 increases NAD(+) levels and attenuates cisplatin-mediated acute kidney injury in mice.Kidney Int. 2014; 85: 547-560Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar We found less transcriptional activity of a NF-κB–luciferase reporter gene and lower mRNA levels of NF-κB–regulated genes in TG+CP compared with WT+CP (Figure 4d and e), consistent with the previous report that SIRT6 decreases NF-κB signaling activity via H3K9 deacetylation at the chromatin of the target gene.5Kawahara T.L. Michishita E. Adler A.S. et al.SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.Cell. 2009; 136: 62-74Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar In addition, we also found a decreased level of tumor necrosis factor-α and decreased phosphorylation of p65 in TG+CP mice compared with WT+CP mice (Figure 4f and g). These results reveal that SIRT6 attenuates CP-induced inflammation in the kidney partly by inhibiting NF-κB activity. Apoptosis is a major cause of CP-induced renal injury.12Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar To examine the effect of SIRT6 on CP-induced apoptosis, we performed terminal deoxynucleotidyltransferase–mediated dUTP nick end-labeling (TUNEL) staining on SIRT6 overexpression or knockout mouse lines. We found fewer TUNEL-positive cells in TG+CP mice kidneys and more TUNEL-positive cells in KO+CP mice kidneys compared with the WT+CP control (Figure 5a, c, and e). Transmission electron microscopy independently confirmed these results (Figure 5b and d). The expression of cleaved caspase-9/8/3 increased after treatment with CP, and this increase was diminished in TG+CP but was intensified in KO+CP mice (Figure 5f and Supplementary Figure S5). It has been reported that p53 plays a critical role in the expression of apoptosis-related genes under CP-related stress.26Jiang M. Yi X. Hsu S. et al.Role of p53 in cisplatin-induced tubular cell apoptosis: dependence on p53 transcriptional activity.Am J Physiol Renal Physiol. 2004; 287: F1140-F1147Crossref PubMed Scopus (143) Google Scholar, 27Jiang M. Wei Q. Wang J. et al.Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (177) Google Scholar Luciferase reporter experiments showed less transcriptional activity of p53 when SIRT6 was overexpressed with the treatment of CP, and the mRNA levels of p21 and Bax, which are downstream targets of p53, supported this result. Transcriptional activity of p53 was increased in KO+CP mice relative to the WT+CP control (Figure 5g and h). The amounts of phosphorylated p53, p53, p21, and Bax were all decreased, and Bcl-2 was slightly but not significantly increased in TG+CP mice compared with WT+CP mice, and the results in KO+CP mice showed the opposite trend (Figure 5i). These results suggest that SIRT6 protects the kidney from CP-induced apoptosis by inhibiting p53 signaling. To explore the specific modulation of SIRT6 on p53 and p65, we attempted to find molecules that mediate this process. ERK1/2 are known to play a critical role in CP-induced renal inflammation and apoptosis. Exogenous inhibition of ERK1/2 effectively attenuates CP-induced injury.16Jo S.K. Cho W.Y. Sung S.A. et al.MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis.Kidney Int. 2005; 67: 458-466Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 28Kim Y.K. Kim H.J. Kwon C.H. et al.Role of ERK activation in cisplatin-induced apoptosis in OK renal epithelial cells.J Appl Toxicol. 2005; 25: 374-382Crossref PubMed Scopus (152) Google Scholar Activation of ERK1/2 is an upstream regulatory step during inflammation and apoptosis.17Potocnjak I. Skoda M. Pernjak-Pugel E. et al.Oral administration of oleuropein attenuates cisplatin-induced acute renal injury in mice through inhibition of ERK signaling.Mol Nutr Food Res. 2016; 60: 530-541Crossref PubMed Scopus (50) Google Scholar In our results, we found that ERK1/2 could bind with p53 and p65 by immunoprecipitation (Figure 6a). To investigate whether SIRT6 functions through ERK1/2 signaling, we assessed the expression of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) by Western blotting. As shown in Figure 6b, CP treatment does not appear to noticeably affect the expression of total ERK1/2, but the levels of p-ERK1/2 increased by ∼4-fold following treatment. In addition, the levels of ERK1/2 and p-ERK1/2 were significantly lower in TG+CP mice compared with the WT+CP control (∼4-fold and 2-fold, respectively), whereas KO+CP mice showed a concordant increase in ERK1/2 and p-ERK1/2 levels (∼2- and 1.5-fold, respectively). However, we did not find obvious changes in p38, phosphorylated p38, JNK (c-Jun N-terminal kinase), and phosphorylated JNK with different protein levels of SIRT6 by Western blot assay, which suggested that SIRT6 inhibit ERK1/2 specifically in mitogen-activated protein kinase family (Figure 6b and c and Supplementary Figure S6). To investigate whether ERK1/2 were affected by transcription, we tested the mRNA level of ERK1/2 by quantitative PCR, and the result was consistent with the protein level (Figure 6d). These results suggest that SIRT6 represses the expression of ERK1/2. Previous studies showed that SIRT6 binds to mitogen-activated protein kinase promoter in K562 cells.29Cea M. Cagnetta A. Adamia S. et al.Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells.Blood. 2016; 127: 1138-1150Crossref PubMed Scopus (78) Google Scholar To explore whether a similar mechanism occurs in mouse renal cells, we conducted chromatin immunoprecipitation analysis of renal tissues. We found increased binding of SIRT6 at the promoters of ERK1/2 in TG mice compared with the WT control, whereas hardly any binding was detected in KO renal tissue. Based on its specific activity, we next asked whether SIRT6 modulated the expression of ERK1/2 by deacetylating the H3K9 at the promoter of the ERK1/2 gene. Chromatin immunoprecipitation analysis showed that the increased binding of SIRT6 at promotors was associated with decreased acetylation of H3K9. In contrast, KO renal samples showed a significant increase in H3K9 acetylation at the ERK1/2 promoters (Figure 6e–h). These results suggest that SIRT6 suppresses the expression of ERK1/2 by deacetylating H3K9 at promotors of ERK1/2. To further explore whether SIRT6 protects the kidney through ERK1/2 signaling, U0126, an inhibitor of ERK1/2 activity, was used to treat SIRT6 KO mice before CP injection. In our results, U0126 specifically inhibited ERK1/2 phosphorylation, without visible changes in p-p38 and p-JNK (Figure 7a). U0126 has been found to attenuate CP-induced renal injury by decreasing inflammation and apoptosis.16Jo S.K. Cho W.Y. Sung S.A. et al.MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis.Kidney Int. 2005; 67: 458-466Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar To explore whether inhibition of ERK1/2 could eliminate differences between WT+CP and KO+CP mice, we examined some renal injury phenotypes that were aggravated in KO+CP mice compared with WT+CP mice. TUNEL staining showed that treatment with U0126 could alleviate CP-induced kidney cell apoptosis, and the increase in CP-induced apoptosis caused by SIRT6 depletion disappeared after treatment with U0126 (Figure 7b). The levels of BUN and serum creatinine in KO+CP mice were significantly higher than those in WT+CP mice, and no visible differences between KO+CP and WT+CP mice were found after treatment with U0126 (Figure 7c and d). As the downstream targets of ERK1/2 in our proposed mechanism, p53 and p65 were more phosphorylated (activated) in KO+CP mice than in WT+CP mice, and these differences are abolished after treatment with U0126. Furthermore, some common protein factors that are targets of NF-κB and p53 signaling showed no difference between wild-type and SIRT6 KO mice under simultaneous treatment with CP and U0126 (Figure 7e). All in all, these results suggest that the aggravation of renal injury caused by SIRT6 depletion is ERK1/2 dependent. SIRT6 is involved in a variety of critical biological processes, including genomic stability, inflammation, and glucose metabolism, and SIRT6 overexpression has been shown to improve outcomes of diabetes, heart disease, cancer, and aging.9Kugel S. Mostoslavsky R. Chromatin and beyond: the multitasking roles for SIRT6.Trends Biochem Sci. 2014; 39: 72-81Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar However, the function of SIRT6 in kidney disease remains unknown. In this study, we used CP to induce acute kidney injury, and SIRT6-deficient and transgenic mice were used to probe the function of SIRT6 on renal injury caused by CP. In the absence of CP, SIRT6 overexpression did not have an obvious effect on mice physically at 2 months (Supplementary Table S2). SIRT6 deficiency induced loss of subcutaneous fat, low insulin level, hypoglycemia, premature aging, and some slight inflammation at 4 weeks.6Sundaresan N.R. Vasudevan P. 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Notably, CP-treated SIRT6 KO mice did not significantly show abnormal levels of relative kidney weight (Figure 2c) and histopathologic changes (Figure 3b) compared with the WT control, but we could still see a more serious trend in KO mice. SIRT1 has been reported to play an important role in attenuating kidney injury caused by CP and other stressors.31Mortuza R. Feng B. Chakrabarti S. SIRT1 reduction causes renal and retinal injury in diabetes through endothelin 1 and transforming growth factor beta1.J Cell Mol Med. 2015; 19: 1857-1867Crossref PubMed Scopus (47) Google Scholar, 32Jung Y.J. Lee J.E. Lee A.S. et al.SIRT1 overexpression decreases cisplatin-induced acetylation of NF-kappaB p65 subunit and cytotoxicity in renal proximal tubule cells.Biochem Biophys Res Commun. 2012; 419: 206-210Crossref PubMed Scopus (75) Google Scholar, 33Hasegawa K. Wakino S. 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These results suggest that SIRT6 acts as an inhibitor for CP-induced inflammation and apoptosis by modulating the activation of NF-κB and p53. SIRT6 is known to inhibit NF-κB signaling by H3K9 deacetylation at the promoters of target genes.5Kawahara T.L. Michishita E. Adler A.S. et al.SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.Cell. 2009; 136: 62-74Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar ERK1/2 are proapoptotic signaling molecules that are activated in response to DNA damage.39Lu Z. Xu S. ERK1/2 MAP kinases in cell survival and apoptosis.IUBMB Life. 2006; 58: 621-631Crossref PubMed Scopus (483) Google Scholar ERK1/2 activation is crucial to the phosphorylation of p65 and activation of NF-κB.40Kwon H.J. Choi G.E. 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