Inhibition of the H3K4 methyltransferase MLL1/WDR5 complex attenuates renal senescence in ischemia reperfusion mice by reduction of p16
Authors: Hironori Shimoda, Shigehiro Doi, Ayumu Nakashima, Kensuke Sasaki, Toshiki Doi, Takao Masaki
Journal: Kidney International (2019)

Renal function declines with aging and is pathologically characterized by chronic inflammation and fibrosis. Renal senescence is induced not only by aging but also by various stimuli, including ischemia reperfusion injury. Recently, the accumulation of p16INK4a-positive cells in the kidney has been considered a molecular feature of renal senescence, with the p16INK4a gene reportedly regulated by mixed-lineage leukemia 1 (MLL1)/WD-40 repeat protein 5 (WDR5)-mediated histone 3 lysine 4 trimethylation (H3K4me3). Here, we determined whether inhibition of MLL1/WDR5 activity attenuates renal senescence, inflammation, and fibrosis in mice with ischemia reperfusion injury and in cultured rat renal fibroblasts. MM-102 or OICR-9429, both MLL1/WDR5 protein–protein interaction inhibitors, and small interfering RNA (siRNA) for MLL1 or WDR5 suppressed the expression of p16INK4a in mice with ischemia reperfusion injury, accompanied by downregulation of H3K4me3 expression. MM-102 attenuated renal fibrosis and inflammation in the kidney of mice with ischemia reperfusion injury. Moreover, in vitro study showed that transforming growth factor-β1 induced the expression of MLL1, WDR5, H3K4me3, and p16INK4a. Finally, chromatin immunoprecipitation identified the p16INK4a promoter at an H3K4me3 site in renal fibroblasts. Thus, our findings show that H3K4me3 inhibition ameliorates ischemia reperfusion-induced renal senescence along with fibrosis and inflammation. Renal function declines with aging and is pathologically characterized by chronic inflammation and fibrosis. Renal senescence is induced not only by aging but also by various stimuli, including ischemia reperfusion injury. Recently, the accumulation of p16INK4a-positive cells in the kidney has been considered a molecular feature of renal senescence, with the p16INK4a gene reportedly regulated by mixed-lineage leukemia 1 (MLL1)/WD-40 repeat protein 5 (WDR5)-mediated histone 3 lysine 4 trimethylation (H3K4me3). Here, we determined whether inhibition of MLL1/WDR5 activity attenuates renal senescence, inflammation, and fibrosis in mice with ischemia reperfusion injury and in cultured rat renal fibroblasts. MM-102 or OICR-9429, both MLL1/WDR5 protein–protein interaction inhibitors, and small interfering RNA (siRNA) for MLL1 or WDR5 suppressed the expression of p16INK4a in mice with ischemia reperfusion injury, accompanied by downregulation of H3K4me3 expression. MM-102 attenuated renal fibrosis and inflammation in the kidney of mice with ischemia reperfusion injury. Moreover, in vitro study showed that transforming growth factor-β1 induced the expression of MLL1, WDR5, H3K4me3, and p16INK4a. Finally, chromatin immunoprecipitation identified the p16INK4a promoter at an H3K4me3 site in renal fibroblasts. Thus, our findings show that H3K4me3 inhibition ameliorates ischemia reperfusion-induced renal senescence along with fibrosis and inflammation. Translational StatementRenal senescence is induced not only by aging but also by various stimuli, leading to renal fibrosis and inflammation. Recently, the accumulation of p16INK4a-positive cells in the kidney has been considered a molecular feature of renal senescence. In this study, we show that transforming growth factor-β1 induces p16INK4a expression through histone 3 lysine 4 trimethylation (H3K4me3), which ultimately contributes to renal fibrosis and inflammation. Accordingly, the inhibition of H3K4me3 methyltransferase is a potential therapeutic target for chronic kidney disease progression. Renal senescence is induced not only by aging but also by various stimuli, leading to renal fibrosis and inflammation. Recently, the accumulation of p16INK4a-positive cells in the kidney has been considered a molecular feature of renal senescence. In this study, we show that transforming growth factor-β1 induces p16INK4a expression through histone 3 lysine 4 trimethylation (H3K4me3), which ultimately contributes to renal fibrosis and inflammation. Accordingly, the inhibition of H3K4me3 methyltransferase is a potential therapeutic target for chronic kidney disease progression. Chronic kidney disease (CKD) is a health problem worldwide and contributes to an increased risk of not only end-stage kidney failure but also all-cause death. Clinically, in CKD patients, renal function decreases with advancing age,1Rule A.D. Amer H. Cornell L.D. et al.The association between age and nephrosclerosis on renal biopsy among healthy adults.Ann Intern Med. 2010; 152: 561-567Crossref PubMed Scopus (329) Google Scholar and their frequency of acute kidney injury (AKI) and renal cancer is increased compared with that in the general population.2Grams M.E. Sang Y. Ballew S.H. et al.A meta-analysis of the association of estimated GFR, albuminuria, age, race, and sex with acute kidney injury.Am J Kidney Dis. 2015; 66: 591-601Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3Lowrance W.T. Ordoñez J. Udaltsova N. et al.CKD and the risk of incident cancer.J Am Soc Nephrol. 2014; 25: 2327-2334Crossref PubMed Scopus (154) Google Scholar Pathologically, interstitial fibrosis and inflammation are commonly observed in the kidney of patients with CKD, regardless of the primary disease.4Meng X.M. Nikolic-Paterson D.J. Lan H.Y. Inflammatory processes in renal fibrosis.Nat Rev Nephrol. 2014; 10: 493-503Crossref PubMed Scopus (423) Google Scholar These clinical and pathological features of CKD are similar to those in the aging kidney,2Grams M.E. Sang Y. Ballew S.H. et al.A meta-analysis of the association of estimated GFR, albuminuria, age, race, and sex with acute kidney injury.Am J Kidney Dis. 2015; 66: 591-601Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 5Hommos M.S. Glassock R.J. Rule A.D. Structural and functional changes in human kidneys with healthy aging.J Am Soc Nephrol. 2017; 28: 2838-2844Crossref PubMed Scopus (177) Google Scholar suggesting that the same molecular mechanisms underlie the aging process and CKD progression. At the cellular level, senescence is the phenomenon in which cells cease to divide,6van Deursen J.M. 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Four faces of cellular senescence.J Cell Biol. 2011; 192: 547-556Crossref PubMed Scopus (1393) Google Scholar However, cell cycle–arrested cells exhibit a senescence-associated secretory phenotype, which contributes to fibrosis, chronic inflammation, increased susceptibility to AKI, and a higher risk of kidney cancer.10Coppé J.P. Desprez P.Y. Krtolica A. et al.The senescence-associated secretory phenotype: the dark side of tumor suppression.Annu Rev Pathol. 2010; 5: 99-118Crossref PubMed Scopus (2638) Google Scholar, 11Campisi J. Aging, cellular senescence, and cancer.Annu Rev Physiol. 2013; 75: 685-705Crossref PubMed Scopus (1651) Google Scholar These reports suggest the value of identifying a therapeutic target for renal senescence to prevent CKD progression. Recent studies have demonstrated that the AKI-to-CKD transition is involved in CKD progression.12Venkatachalam M.A. Griffin K.A. Lan R. et al.Acute kidney injury: a springboard for progression in chronic kidney disease.Am J Physiol Renal Physiol. 2010; 298: F1078-F1094Crossref PubMed Scopus (391) Google Scholar, 13Chawla L.S. Eggers P.W. Star R.A. et al.Acute kidney injury and chronic kidney disease as interconnected syndromes.N Engl J Med. 2014; 371: 58-66Crossref PubMed Scopus (1140) Google Scholar The expression of cell cycle–arrested cells has also been shown to increase in mouse kidney after ischemia reperfusion injury (IRI), a mouse model of AKI.14Yang L. Besschetnova T.Y. Brooks C.R. et al.Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.Nat Med. 2010; 16: 535-543Crossref PubMed Scopus (906) Google Scholar Among cell cycle regulators, p16INK4a, also known as cyclin-dependent kinase inhibitor 2A, is reported to inhibit cyclin-dependent kinase 4 and play a central role in decelerating cell progression from the G1 to S phase.15Sherr C.J. Roberts J.M. Inhibitors of mammalian G1 cyclin-dependent kinases.Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3209) Google Scholar, 16Rayess H. Wang M.B. Srivatsan E.S. Cellular senescence and tumor suppressor gene p16.Int J Cancer. 2012; 130: 1715-1725Crossref PubMed Scopus (434) Google Scholar, 17Gire V. Dulic V. Senescence from G2 arrest, revisited.Cell Cycle. 2015; 14: 297-304Crossref PubMed Scopus (144) Google Scholar We previously found that p16INK4a expression is upregulated with the progression of renal fibrosis in patients with IgA nephropathy.18Yamada K. Doi S. Nakashima A. et al.Expression of age-related factors during the development of renal damage in patients with IgA nephropathy.Clin Exp Nephrol. 2015; 19: 830-837Crossref PubMed Scopus (15) Google Scholar Recent studies have also revealed that renal fibrosis and tubular atrophy are improved in p16INK4a knockout mice after IRI.19Braun H. Schmidt B.M. Raiss M. et al.Cellular senescence limits regenerative capacity and allograft survival.J Am Soc Nephrol. 2012; 23: 1467-1473Crossref PubMed Scopus (122) Google Scholar, 20Lee D.H. Wolstein J.M. Pudasaini B. et al.INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury.Am J Physiol Renal Physiol. 2012; 302: F183-F191Crossref PubMed Scopus (47) Google Scholar Therefore, pharmacological inhibition of AKI-induced p16INK4a expression may prevent CKD development. Epigenetics describes the regulatory system of gene expression that occurs without changes of the primary nucleotide sequence.21Dupont C. Armant D.R. Brenner C.A. Epigenetics: definition, mechanisms and clinical perspective.Semin Reprod Med. 2009; 27: 351-357Crossref PubMed Scopus (393) Google Scholar Among a number of epigenetic modifications, the methylation of histone tails is regulated by specific enzymes,22Lanouette S. Mongeon V. 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Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries.Science. 2011; 293: 1150-1155Crossref Scopus (599) Google Scholar, 30Santos-Rosa H. Schneider R. Bannister A.J. et al.Active genes are tri-methylated at K4 of histone H3.Nature. 2002; 419: 407-411Crossref PubMed Scopus (1597) Google Scholar Indeed, according to previous studies, deficiency of H3K4 methyltransferase extends the life span in Caenorhabditis elegans,31Greer E.L. Maures T.J. Hauswirth A.G. et al.Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans.Nature. 2010; 466: 383-387Crossref PubMed Scopus (366) Google Scholar whereas inactivation of H3K4 demethylase shortens the life span in flies.32Li L. Greer C. Eisenman R.N. et al.Essential functions of the histone demethylase lid.PLoS Genet. 2010; 6e1001221Crossref PubMed Scopus (105) Google Scholar Altogether, these findings led us to the hypothesis that inhibiting MLL1/WDR5 activity suppresses renal senescence by reducing p16INK4a expression. To test this, we determined whether administering MLL1/WDR5 inhibitors or small, interfering RNA (siRNA) for MLL1 or WDR5 suppresses renal senescence by reducing H3K4me3 in IRI mice and renal fibroblast cells. Accordingly, in this study, we show that the inhibition of MLL1/WDR5 complex suppresses H3K4me3 levels, along with p16INK4a expression, in mice at 7 days after IRI. We also show that pharmacological inhibition of the MLL1/WDR5 complex suppresses renal fibrosis and inflammation in IRI mice. In a cell line of renal fibroblasts, we found that transforming growth factor (TGF)-β1 induces the expression of MLL1, WDR5, and H3K4me3, as well as p16INK4a expression, and that inhibition of the MLL1/WDR5 complex suppresses TGF-β1–induced expression of these proteins. Finally, we demonstrate that promoter expression of the p16INK4a gene is regulated by H3K4me3. Overall, our data suggest that TGF-β1–induced H3K4me3 is a candidate target for preventing renal senescence after IRI. The H3K4me3 methyltransferase MLL1 is activated on forming a complex with WDR5.24Dou Y. Milne T.A. Ruthenburg A.J. et al.Regulation of MLL1 H3K4 methyltransferase activity by its core components.Nat Struct Mol Biol. 2006; 13: 713-719Crossref PubMed Scopus (567) Google Scholar, 25van Nuland R. Smits A.H. Pallaki P. et al.Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes.Mol Cell Biol. 2013; 33: 2067-2077Crossref PubMed Scopus (150) Google Scholar Furthermore, both MM-102 and OICR-9429 are known to inhibit activity of the MLL1/WDR5 complex.33Karatas H. Townsend E.C. Cao F. et al.High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction.J Am Chem Soc. 2013; 135: 669-682Crossref PubMed Scopus (132) Google Scholar, 34Li D.D. Chen W.L. Xu X.L. et al.Structure-based design and synthesis of small molecular inhibitors disturbing the interaction of MLL1-WDR5.Eur J Med Chem. 2016; 118: 1-8Crossref PubMed Scopus (29) Google Scholar, 35Grebien F. Vedadi M. Getlik M. et al.Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia.Nat Chem Biol. 2015; 11: 571-578Crossref PubMed Scopus (172) Google Scholar To identify their effects on MLL1, WDR5, and H3K4me3, we examined their expression in IRI mice. Male C57BL/6 mice were subjected to IRI, with the left renal pedicle clamped for 30 minutes. Immediately after surgery, mice were i.p. administered MM-102 (15 mg/kg per day), OICR-9429 (1.5 mg/kg per day), or vehicle (distilled water for MM-102, dimethylsulfoxide/distilled water at 1:24 for OICR-9429). The same treatments were repeated every 24 hours for 7 days after IRI (Figure 1a). Western blotting revealed increased expression of MLL1, WDR5, and H3K4me3 in IRI mice. Moreover, MM-102 and OICR-9429 inhibited the expression compared with that in the vehicle group (Figure 1b and c). Immunohistochemical staining also revealed an H3K4me3-positive area in nuclei. The number of H3K4me3-positive cells increased in IRI mice, but decreased in mice with MM-102 treatment (Figure 1d). Cell cycle arrest is a major feature of cellular senescence.9Rodier F. Campisi J. Four faces of cellular senescence.J Cell Biol. 2011; 192: 547-556Crossref PubMed Scopus (1393) Google Scholar, 36Childs B.G. Gluscevic M. Baker D.J. et al.Senescent cells: an emerging target for diseases of ageing.Nat Rev Drug Discov. 2017; 16: 718-735Crossref PubMed Scopus (557) Google Scholar, 37Collado M. Blasco M.A. Serrano M. Cellular senescence in cancer and aging.Cell. 2007; 130: 223-233Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar Therefore, we examined the effect of H3K4 methyltransferase inhibitor on p16INK4a expression in IRI mice. Protein levels of p16INK4a increased in IRI mice following vehicle treatment compared with those in sham-operated mice, whereas they decreased in IRI mice with MM-102 or OICR-9429 injection (Figure 2a). Similarly, gene expression of p16INK4a also increased in IRI mice and was suppressed by MM-102 or OICR-9429 administration (Figure 2b). Meanwhile, expansion of the p16INK4a-positive area was observed mainly in the interstitial region of the kidney in IRI mice following vehicle treatment, but this area shrank with MM-102 treatment. To identify the localization of p16INK4a, S100A4—also called fibroblast-specific protein-1—was used as a mesenchymal marker that is located in the kidneys' interstitial area. Double staining for p16INK4a and S100A4 revealed that S100A4 was coexpressed with p16INK4a (Figure 2c). Renal fibrosis is a common pathological feature of CKD, regardless of the primary disease, which is also exhibited in aging kidney. During renal fibrosis, TGF-β1–induced upregulation of α-smooth muscle actin (α-SMA)-positive myofibroblasts is responsible for the production of extracellular matrix proteins such as collagen types I and III. Thus, we investigated the effect of MLL1/WDR5 inhibitors on TGF-β1, α-SMA, and collagen in IRI mice. Western blotting revealed increased α-SMA protein expression in IRI mice with vehicle treatment, which was suppressed in IRI mice with MM-102 or OICR-9429 treatment (Figure 3a). In addition, areas immunopositive for α-SMA were rarely observed in sham-operated mice. Although α-SMA expression increased in IRI mice with vehicle treatment, MM-102 ameliorated this (Figure 3b). The expression levels of actin alpha 2 (Acta2), TGF-β1, collagen type 1 alpha 1 (Col1a1), and collagen type 3 alpha 1 (Col3a1) genes were increased in IRI mice, but decreased by MM-102 treatment (Figure 3c). Senescent cells exhibit senescence-associated secretory phenotype, which is characterized by the secretion of proinflammatory molecules such as cytokines, chemokines, and growth factors through nuclear factor-κB (NF-κB) activation.36Childs B.G. Gluscevic M. Baker D.J. et al.Senescent cells: an emerging target for diseases of ageing.Nat Rev Drug Discov. 2017; 16: 718-735Crossref PubMed Scopus (557) Google Scholar To identify the effect of MLL1/WDR5 complex inhibitors on inflammation, we investigated the expression of phosphorylated NF-κB (p-NF-κB) (p65), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and CD11b. Western blotting showed that p-NF-κB (p65) was increased in IRI mice, while MM-102 or OICR-9429 suppressed its expression (Figure 4a). Similarly, IL-1β, TNF-α, and IL-6 mRNA expression was increased in IRI mice, but this upregulation decreased in IRI mice with MM-102 treatment (Figure 4b). In addition, Western blotting showed increased CD11b expression in IRI mice, which was suppressed with MM-102 or OICR-9429 treatment (Figure 4c). Immunohistochemistry revealed a CD11b-positive area in the interstitial region in IRI kidneys (Figure 4d). To determine more accurately the role of MLL1 and WDR5 in the regulation of p16INK4a, 50 μg of siRNA of MLL1 or WDR5 was injected i.v. into mice after IRI surgery (Figure 5a). Western blotting showed that knockdown of MLL1 significantly decreased protein levels of WDR5, H3K4me3, and p16INK4a at day 7 after IRI (Figure 5b and d). Similarly, knockdown of WDR5 by siRNA injection decreased protein levels of MLL1, H3K4me3, and p16INK4a (Figure 5c and e). To confirm the effect of MM-102 on MLL1, WDR5, H3K4me3, and p16INK4a expression in renal fibroblasts, we examined their expression in TGF-β1–stimulated normal rat kidney–49F (NRK-49F) cells. Western blotting showed markedly increased protein levels of MLL1, WDR5, H3K4me3, and p16INK4a in TGF-β1–stimulated NRK-49F cells (Figure 6a). However, preincubation of MM-102 at a dose of 50 μmol/l resulted in significant reductions of MLL1, WDR5, H3K4me3, and p16INK4a expression (Figure 6b–e). To confirm the role of MLL1 and WDR5 in H3K4me3 and p16INK4a expression in TGF-β1–stimulated renal fibroblasts, NRK-49F cells were transfected with siRNA of MLL1 or WDR5 before TGF-β1 stimulation. Western blotting analyses revealed that MLL1 or WDR5 siRNA transfection silenced the expression in NRK-49F cells (Figure 7a). As shown in Figure 7b, knockdown of MLL1 significantly inhibited the H3K4me3 and p16INK4a expression compared with the negative control siRNA treatment (Figure 7b). Similarly, knockdown of WDR5 also blocked H3K4me3 and p16INK4a expression compared with the negative control siRNA treatment (Figure 7c). A previous study found that MLL1-mediated H3K4me3 regulates p16INK4a in lung fibroblast cells26Kotake Y. Zeng Y. Xiong Y. DDB1-CUL4 and MLL1 mediate oncogene-induced p16INK4a activation.Cancer Res. 2009; 69: 1809-1814Crossref PubMed Scopus (57) Google Scholar and pancreatic β cells.27Dhawan S. Tschen S.I. Bhushan A. Bmi-1 regulates the Ink4a/Arf locus to control pancreatic β-cell proliferation.Genes Dev. 2009; 23: 906-911Crossref PubMed Scopus (190) Google Scholar To determine whether H3K4me3 directly regulates p16INK4a gene transcription, we performed ChIP assays using anti-H3K4me3 antibody and rat p16INK4a primers. Expression of the p16INK4a gene immunoprecipitated with H3K4me3 increased in TGF-β1–stimulated NRK-49F cells, but this upregulation was suppressed on MM-102 preincubation (Figure 8). We also performed ChIP assay using rat albumin primers as a negative control (Supplementary Figure S1). We have shown that the H3K4me3 methyltransferases, MLL1 and WDR5, are upregulated in IRI mice, resulting in increased expression of H3K4me3. Moreover, administration of the MLL/WDR5 complex inhibitors, MM-102 or OICR-9429, resulted in decreased H3K4me3 levels and reduced p16INK4a expression. These inhibitors also suppressed renal fibrosis and inflammation in IRI mice. In vitro experiments using a rat cell line of renal fibroblasts identified TGF-β1 as a cytokine responsible for the upregulation of MLL1, WDR5, H3K4me3, and p16INK4a. Finally, we showed that the p16INK4a gene promoter is immunoprecipitated with H3K4me3. These findings suggest that inhibition of H3K4me3 can act as a therapeutic target for preventing AKI-induced renal senescence. Expression of p16INK4a is positively regulated by MLL1/WDR5 complex–mediated H3K4me3, and negatively regulated by the polycomb-group protein, BMI1 (also called polycomb-group RING finger protein 4 [PCGF4]).38Cao L. Bombard J. Cintron K. et al.BMI1 as a novel target for drug discovery in cancer.J Cell Biochem. 2011; 112: 2729-2741Crossref PubMed Scopus (124) Google Scholar, 39Bhattacharya R. Mustafi S.B. 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Pudasaini B. et al.INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury.Am J Physiol Renal Physiol. 2012; 302: F183-F191Crossref PubMed Scopus (47) Google Scholar These findings indicate that, although p16INK4a expression itself has the ability to protect against the renal fibrogenesis, the accumulation of p16INK4a-positive cells promotes renal fibrosis at the chronic phase after kidney injury. In fact, the abolition of p16INK4a not only improves mouse models of chronic renal disease46Baker D.J. Wijshake T. Tchkonia T. et al.Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.Nature. 2011; 479: 232-236Crossref PubMed Scopus (2164) Google Scholar but also prolongs life span.47Baker D.J. Childs B.G. Durik M. et al.Naturally occurring p16Ink4a-positive cells shorten healthy lifespan.Nature. 2016; 530: 184-189Crossref PubMed Scopus (1516) Google Scholar Thus, the deletion of accumulated p16INK4a-positive cells can be considered a therapeutic target for AKI-induced chronic renal fibrosis. Many studies have demonstrated that chronic inflammation plays a pivotal role in the progression of CKD.4Meng X.M. Nikolic-Paterson D.J. Lan H.Y. Inflammatory processes in renal fibrosis.Nat Rev Nephrol. 2014; 10: 493-503Crossref PubMed Scopus (423) Google Scholar, 48Lan H.Y. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.Int J Biol Sci. 2011; 7: 1056-1067Crossref PubMed Scopus (485) Google Scholar, 49Nikolic-Paterson D.J. Wang S. Lan H.Y. Macrophages promote renal fibrosis through direct and indirect mechanisms.Kidney Int Suppl (2011). 2014; 4: 34-38Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar Cell cycle arrest serves as protection from acute injury as well as tumorigenesis, but also results in a secretory phenotype that causes inflammation, known as senescence-associated secretory phenotype.10Coppé J.P. Desprez P.Y. Krtolica A. et al.The senescence-associated secretory phenotype: the dark side of tumor suppression.Annu Rev Pathol. 2010; 5: 99-118Crossref PubMed Scopus (2638) Google Scholar Therefore, the accumulation of cell cycle–arrested cells in tissue leads to chronic inflammation, contributing to increased susceptibility to AKI and cancer.10Coppé J.P. Desprez P.Y. Krtolica A. et al.The senescence-associated secretory phenotype: the dark side of tumor suppression.Annu Rev Pathol. 2010; 5: 99-118Crossref PubMed Scopus (2638) Google Scholar, 11Campisi J. Aging, cellular senescence, and cancer.Annu Rev Physiol. 2013; 75: 685-705Crossref PubMed Scopus (1651) Google Scholar Chronic inflammation also contributes to renal fibrosis through TGF-β1 production, as well as inflammation-induced tissue damage.6van Deursen J.M. The role of senescent cells in ageing.Nature. 2014; 509: 439-446Crossref PubMed Scopus (1429) Google Scholar, 48Lan H.Y. Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.Int J Biol Sci. 2011; 7: 1056-1067Crossref PubMed Scopus (485) Google Scholar Thus, the accumulation of cell cycle–arrested cells causes inflammation, which then participates in further accumulation of cell cycle–arrested cells.50Muñoz-Espín D. Serrano M. Cellular senescence: from physiology to pathology.Nat Rev Mol Cell Biol. 2014; 15: 482-496Crossref PubMed Scopus (1449) Google Scholar, 51Ferenbach D.A. Bonventre J.V. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD.Nat Rev Nephrol. 2015;