Cellular communication network 2 (connective tissue growth factor) aggravates acute DNA damage and subsequent DNA damage response-senescence-fibrosis following kidney ischemia reperfusion injury

Chronic allograft dysfunction with progressive fibrosis of unknown cause remains a major issue after kidney transplantation, characterized by ischemia-reperfusion injury (IRI). One hypothesis to account for this is that spontaneous progressive tubulointerstitial fibrosis following IRI is driven by cellular senescence evolving from a prolonged, unresolved DNA damage response (DDR). Since cellular communication network factor 2 ((CCN2), formerly called connective tissue growth factor), an established mediator of kidney fibrosis, is also involved in senescence-associated pathways, we investigated the relation between CCN2 and cellular senescence following kidney transplantation. Tubular CCN2 overexpression was found to be associated with DDR, loss of kidney function and tubulointerstitial fibrosis in both the early and the late phase in human kidney allograft biopsies. Consistently, CCN2 deficient mice developed reduced senescence and tubulointerstitial fibrosis in the late phase; six weeks after experimental IRI. Moreover, tubular DDR markers and plasma urea were less elevated in CCN2 knockout than in wild-type mice. Finally, CCN2 administration or overexpression in epithelial cells induced upregulation of tubular senescence–associated genes including p21, while silencing of CCN2 alleviated DDR induced by anoxia-reoxygenation injury in cultured proximal tubule epithelial cells. Thus, our observations indicate that inhibition of CCN2 can mitigate IRI-induced acute kidney injury, DNA damage, and the subsequent DDR-senescence-fibrosis sequence. Hence, targeting CCN2 might help to protect the kidney from transplantation-associated post-IRI chronic kidney dysfunction. Chronic allograft dysfunction with progressive fibrosis of unknown cause remains a major issue after kidney transplantation, characterized by ischemia-reperfusion injury (IRI). One hypothesis to account for this is that spontaneous progressive tubulointerstitial fibrosis following IRI is driven by cellular senescence evolving from a prolonged, unresolved DNA damage response (DDR). Since cellular communication network factor 2 ((CCN2), formerly called connective tissue growth factor), an established mediator of kidney fibrosis, is also involved in senescence-associated pathways, we investigated the relation between CCN2 and cellular senescence following kidney transplantation. Tubular CCN2 overexpression was found to be associated with DDR, loss of kidney function and tubulointerstitial fibrosis in both the early and the late phase in human kidney allograft biopsies. Consistently, CCN2 deficient mice developed reduced senescence and tubulointerstitial fibrosis in the late phase; six weeks after experimental IRI. Moreover, tubular DDR markers and plasma urea were less elevated in CCN2 knockout than in wild-type mice. Finally, CCN2 administration or overexpression in epithelial cells induced upregulation of tubular senescence–associated genes including p21, while silencing of CCN2 alleviated DDR induced by anoxia-reoxygenation injury in cultured proximal tubule epithelial cells. Thus, our observations indicate that inhibition of CCN2 can mitigate IRI-induced acute kidney injury, DNA damage, and the subsequent DDR-senescence-fibrosis sequence. Hence, targeting CCN2 might help to protect the kidney from transplantation-associated post-IRI chronic kidney dysfunction. Translational StatementOur data suggest that anti–cellular communication network factor 2 (CCN2) therapy may help to prevent chronic allograft dysfunction by limiting ischemia-reperfusion injury (IRI)–induced acute DNA damage, senescent cell accumulation, and subsequent tubulointerstitial fibrosis. Anti-CCN2 therapy could therefore improve graft function and survival outcomes in patients. Several pharmacologic inhibitors of CCN2 have proven safe and tolerable in phase 1 and/or 2 clinical trials for several indications, including senescence- and fibrosis-associated diseases. The next steps toward clinical application of anti-CCN2 therapy in patients who underwent kidney transplantation include experimental IRI studies using pharmacologic inhibitors of CCN2 and addressing the right timing of therapy. Chronic allograft dysfunction (CAD) due to tubulointerstitial fibrosis is the leading cause of kidney allograft loss and may develop without identifiable cause, despite adequate immunosuppression. Ischemia-reperfusion injury (IRI), in which ischemia is followed by reoxygenation injury, is the main cause of acute kidney injury associated with transplantation surgery and considered a major cause of CAD. CAD is defined by progressive tubulointerstitial fibrosis, functional decline, and eventual loss of the kidney graft.1Situmorang G.R. Sheerin N.S. 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Brandt R.M.C. Putavet D.A. et al.Targeted apoptosis of senescent cells restores tissue homeostasis in response to chemotoxicity and aging.Cell. 2017; 169 (e16): 132-147Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar In unilateral IRI, treatment with the “senolytic” agents dasatinib and quercetin reduced tubulointerstitial fibrosis.16Li C. Shen Y. Huang L. et al.Senolytic therapy ameliorates renal fibrosis postacute kidney injury by alleviating renal senescence.FASEB J. 2021; 35: e21229PubMed Google Scholar Cellular communication network factor 2 (CCN2), previously known as connective tissue growth factor, is a matricellular protein involved in IRI and fibrosis. It contributes to tubulointerstitial fibrosis in CAD and has successfully been targeted to limit fibrosis.17Falke L.L. Goldschmeding R. Nguyen T.Q. A perspective on anti-CCN2 therapy for chronic kidney disease.Nephrol Dial Transplant. 2014; 29: i30-i37Crossref PubMed Scopus (21) Google Scholar CCN2 is involved in various processes, including cell proliferation, differentiation, adhesion, and angiogenesis, and promotes inflammation and fibrosis.17Falke L.L. Goldschmeding R. Nguyen T.Q. A perspective on anti-CCN2 therapy for chronic kidney disease.Nephrol Dial Transplant. 2014; 29: i30-i37Crossref PubMed Scopus (21) Google Scholar, 18Ramazani Y. Knops N. Elmonem M.A. et al.Connective tissue growth factor (CTGF) from basics to clinics.Matrix Biol. 2018; 68-69: 44-66Crossref PubMed Scopus (152) Google Scholar, 19Sánchez-López E. Rayego S. Rodrigues-Díez R. et al.CTGF promotes inflammatory cell infiltration of the renal interstitium by activating NF-kappaB.J Am Soc Nephrol. 2009; 20: 1513-1526Crossref PubMed Scopus (97) Google Scholar CCN2 is also implicated in cellular senescence. It is not only a prominent SASP factor but can also function by itself as a cellular survival factor and as an inducer of cellular senescence in vitro.20Yang 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: 143-535Crossref Scopus (892) Google Scholar, 21Wahab N. Cox D. Witherden A. Mason R.M. Connective tissue growth factor (CTGF) promotes activated mesangial cell survival via up-regulation of mitogen-activated protein kinase phosphatase-1 (MKP-1).Biochem J. 2007; 406: 131-138Crossref PubMed Scopus (42) Google Scholar, 22Jun J.I. Lau L.F. CCN2 induces cellular senescence in fibroblasts.J Cell Commun Signal. 2017; 11: 15-23Crossref PubMed Scopus (30) Google Scholar Observing colocalization of CCN2 with DDR and senescence markers in an early and late biopsy of the same human kidney allograft led us to hypothesize that CCN2 could contribute to senescent cell accumulation and tubulointerstitial fibrosis during CAD development after IRI. To elucidate this hypothesis, we investigated the relation between CCN2 and cellular senescence in the early and late phase of human kidney allograft biopsies, in a bilateral IRI model in CCN2 knockout (KO) mice, in CCN2-treated mice and cultured cells, and finally, in an anoxia-reoxygenation (AR) model in CCN2-silenced cultured cells. Tissue sections were derived from routine clinical kidney allograft biopsies at the University Medical Center Utrecht. Follow-up biopsies were taken 7 days and 8 months after transplantation from a 55-year-old man, with a creatinine at biopsy of 1498 μmol/l and 779 μmol/l, respectively. Indication biopsies for delayed graft function (DGF) 6–8 days after transplantation and protocol biopsies 5–29 years after transplantation served as early and late phase specimens. All transplants were derived from nonliving donors. All patient samples were leftover body material from clinical biopsies and were collected according to the institutional ethical guidelines. Samples were anonymized, which allowed us to use this redundant tissue for research purposes, without requirement of informed patient consent.23van Diest P.J. No consent should be needed for using leftover body material for scientific purposes.BMJ. 2002; 325: 648-651Crossref PubMed Google Scholar All animal procedures were performed according to the Animal Research: Reporting of In Vivo Experiments guidelines and with consent of the local Experimental Animal Ethics Committees.24Kilkenny C. Browne W.J. Cuthill I.C. et al.Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.Osteoarthritis Cartilage. 2012; 20: 256-260Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar Generation of tamoxifen-inducible CCN2-full KO mice is described elsewhere.25Fontes M.S. Kessler E.L. van Stuijvenberg L. et al.CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload.J Mol Cell Cardiol. 2015; 88: 82-90Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar In brief, CCN2Flox/Flox mice were crossbred with ROSA26CreERT2 mice (Gt(ROSA)26Sortm(cre/ERT2)Tyj/J, the Jackson Laboratory), both on a C57Bl6/J background. For i.p injection, tamoxifen citrate was dissolved in corn oil (Sigma Aldrich) in a 10 mg/ml concentration. To induce recombination, 12- to 14-week-old male mice received 4 i.p. injections on alternate days over a course of 7 days with 100 μl of tamoxifen-corn oil solution. Littermates injected with vehicle corn oil using the same regimen were used as control mice (referred to as wild-type [WT] mice). Treatments were performed in a blinded fashion. After the last injection, a 14-day washout period was followed by the IRI operation. IRI was executed as previously described.26Kinashi H. Falke L.L. Nguyen T.Q. et al.Connective tissue growth factor regulates fibrosis-associated renal lymphangiogenesis.Kidney Int. 2017; 92: 850-863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Renal pedicles were located through an abdominal midline incision and bilaterally clamped for 25 minutes with neurovascular clamps. Clamping and subsequent reperfusion-associated color changes were visually confirmed. IRI mice without color changes were excluded. Sham-operated mice underwent the same procedure without the pedicle clamping. Mice were anesthetized with 2% isoflurane, and body temperature was maintained at 37 °C. The operator was blinded for the treatment group. After 3 days or 6 weeks, mice were euthanized by lethal dose ketamine-xylazine-acepromazine injection, and plasma and organs were collected and stored at −80 °C. Mouse primary renal proximal tubule epithelial cells (RPTECs; C57-6015; Cell Biologics) were cultured as described previously using 100 ng/ml recombinant CCN2 (Peprotech, 120-19) and a GasPak EZ Anaerobe Container System with indicator (cat. no. 26001; BD Biosciences).27Eleftheriadis T. Pissas G. Golfinopoulos S. et al.Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells.Mol Med Rep. 2021; 23: 472Crossref PubMed Google Scholar Etoposide (1.5 μg/ml; E1383-25MG; Sigma-Aldrich) was used as positive control for senescence. Gene silencing was performed using predesigned small, interfering RNA corresponding to CCN2 (small, interfering RNA ID MSS274358; Thermo-fisher) according to the manufacturer’s protocol. In short, subconfluent cells were transfected in Opti-MEM–reduced serum medium (Invitrogen) for 24 hours with 5 ng/ml small, interfering RNA using 50 nM Lipofectamine RNAi-MAX (Invitrogen), followed by 24 hours of incubation in 20% fetal bovine serum medium and 24 hours of incubation in serum-free medium. Controls were nontransfected cells treated with Lipofectamine vehicle. Cells were subjected to 24 hours of anoxia and 2 hours of reoxygenation. The human kidney-2 (HK-2) human tubular epithelial cell line was cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. Semiconfluent HK-2 cells were infected with lentiviruses bearing a cytomegalovirus promoter-driven human CCN2 cDNA construct (LPP-L5140-Lv105) or control vector (GeneCopoeia) using 5 μg/ml polybrene in Dulbecco’s modified Eagle’s medium/5% fetal bovine serum for 24 hours. After a 24-hour recovery period, stable cultures were selected in 5 μg/ml puromycin containing media. To maintain selection pressure, media were changed every 3 days. Plasma urea (DiaSys) and plasma creatinine (Arbor Assays) were measured using a colorimetric assay that conformed to the manufacturer’s protocol. Tissue was fixed in a buffered 4% formalin solution for 24 hours and embedded in paraffin. Three-micrometer sections were mounted on adhesive slides (Leica Xtra), rehydrated through xylene and alcohol washes, and rinsed in distilled water. For periodic acid–Schiff and Masson’s trichrome staining, standard procedures were used (Dako). Immunohistochemistry for gamma H2AX (γH2AX) and CCN2 was performed as described previously, and immunohistochemistry for p21CIP1 (p21) was set up based on the manufacturer’s protocol.28Lachaud C. Slean M. Marchesi F. et al.Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice.Genes Dev. 2016; 30: 639-644Crossref PubMed Scopus (27) Google Scholar,29Falke L.L. Dendooven A. Leeuwis J.W. et al.Hemizygous deletion of CTGF/CCN2 does not suffice to prevent fibrosis of the severely injured kidney.Matrix Biol. 2012; 31: 421-431Crossref PubMed Scopus (26) Google Scholar First, endogenous peroxidase was blocked using H2O2, followed by antigen retrieval by boiling in pH6 citrate buffer and primary antibody incubation (for human and mouse: anti-γH2AX [pSer139], Novus Biologicals NB100-2280, 1:250; anti-CCN2, Santa Cruz sc-14939, 1:200; for human: anti-p21, Cell Signaling Technology #2947, 1:100; for mouse: anti-p21, Abcam ab188224, 1:4000; anti-Ki-67, Thermofisher RM9106S, 1:100; anti-cleaved caspase 3, BD Pharmingen 559565, 1:500) diluted in 1% bovine serum albumin blocking solution. For p21, CCN2, and Ki-67, secondary horseradish peroxidase–conjugated antibodies were applied and visualized using a Nova Red substrate (Vector Laboratories). For γH2AX and cleaved caspase 3, an alkaline phosphatase–conjugated antibody and liquid permanent red substrate (Dako) were used. Slides were counterstained with Mayer’s hematoxylin. Images were acquired using a Nikon Eclipse E800 microscope or scanned (Hamamatsu NanoZoomer) and digitally photographed in ImageScope to allow manual aligning of serial slides. For assessment of histopathologic damage, a kidney pathologist (TQN) blinded for experimental conditions graded acute tubular injury (ATI) on periodic acid–Schiff slides. ATI was graded on a scale from 0 to 3 as a percentage of the total cortical area of the tissue section (0 = 0%; 1 = <25%; 2 = <50%; 3 = >50%). ATI was defined as tubular dilatation, epithelial necrosis, cast formation, and loss of brush border.30Stokman G. Leemans J.C. Claessen N. et al.Hematopoietic stem cell mobilization therapy accelerates recovery of renal function independent of stem cell contribution.J Am Soc Nephrol. 2005; 16: 1684-1692Crossref PubMed Scopus (79) Google Scholar,31Pieters T.T. Falke L.L. Nguyen T.Q. et al.Histological characteristics of Acute Tubular Injury during Delayed Graft Function predict renal function after renal transplantation.Physiol Rep. 2019; 7: e14000Crossref PubMed Scopus (17) Google Scholar For Masson’s trichrome, positive area percentages were calculated based on 10 random microscopy images with original magnification ×200 using Photoshop CS6 (Adobe) and ImageJ1 (National Institutes of Health). For nuclear stains (γH2AX and p21), whole slides were scanned and the number of positive cells and total cells was calculated in QuPath.32Bankhead P. Loughrey M.B. Fernández J.A. et al.QuPath: open source software for digital pathology image analysis.Sci Rep. 2017; 7: 16878Crossref PubMed Scopus (1733) Google Scholar For all scores, the score is displayed as the mean of the left and right kidney. In human biopsies, cortical tubules containing 1 or more positive cell(s) were annotated and manually counted using QuPath. For assessing the localization of positive staining, tubular structures were identified by kidney pathologists (TQN and RG). Tubular epithelial cells were defined as cross sections through tubular structures with basal membranes. DNA and full RNA were extracted from kidney cortical poles using Trizol (Thermo-Fisher). Purity and quantity were determined using Nanodrop 2000 (Thermo-Fisher). For RNA analysis, a cDNA library was synthesized using 3 μg of RNA per kidney with SuperScript III reverse transcriptase (Thermo-Fisher). Quantitative real-time polymerase chain reactions were run on a ViiA 7 real-time polymerase chain reaction system (Applied Biosystems). The SYBR green primer sequences and Taqman probe quantitative real-time polymerase chain reaction are shown in Supplementary Table S1. To assess CCN2 DNA expression, we used intron/exon spanning SYBR green primers complementary to CCN2 intron2-exon3 and CCN2 exon4-intron4. For mRNA analysis, TATA-box binding protein was used as internal reference. Samples were run in duplicate. Samples free of mRNA and reverse transcriptase were used to control for potential contamination. The ΔΔCT method was used to calculate relative expression levels. RPTECs were lysed, and Western blot and ELISA analyses were performed as described previously.27Eleftheriadis T. Pissas G. Golfinopoulos S. et al.Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells.Mol Med Rep. 2021; 23: 472Crossref PubMed Google Scholar Experiments were repeated 3 times. Membranes were incubated with antibodies for the following proteins: CCN2 (E2W5M; 1:1000; Cell Signaling Technology #10095), γH2AX (1:1000; Novus Biologicals NB110-2280), p21Waf1/Cip1 (1:1000, Cell Signaling Technology #37543), Ki-67 (1:1000, NBP2-22112, Novus Biologicals), GLB1 (1:1000, ab55176, Abcam), and β-actin (1:2500; Cell Signaling Technology #4967) for murine cultures and CCN2 (L-20 goat polyclonal; 1:1000; Santa Cruz-14939), p21 (1:1000; Cell Signaling-2947), plasminogen activator inhibitor-1 (PAI-1; 1:1000; #9163), and β-tubulin (1:1000; Abcam-ab6046) for human cultures. Furthermore, a mouse interleukin-6 ELISA Kit (Elabscience E-EL-M0044) was used. Senescence-associated–β-galactosidase activity was performed following the manufacturer’s protocol (Senescence Cells Histochemical Staining Kit, CS0030-1KT, 069M4101V, Sigma) after 72 hours of CCN2 stimulation. Two-way analysis of variance with post hoc Tukey correction was used to compare the means of continuous variables in the 4 IRI groups. Discrete dependent variables in the IRI experiment and CCN2 administration experiments were tested nonparametrically with Mann-Whitney and Kruskal-Wallis with post hoc Dunn’s test. Correlation of 2 independent variables was assessed using Pearson for continuous variables and Spearman for discrete variables. Values exceeding >1.5 interquartile ranges from the mean of a group were labeled as outliers and excluded. Data that showed abnormal distribution (i.e., right skewness) were log-transformed. Homogeneity of variances was tested with Levene’s test because of unequal sample sizes. All statistical analyses were executed using the statistical program SPSS (IBM SPSS Statistics 25). Error bars represent SEM. P values < 0.05 were considered statistically significant. Phosphorylation of H2AX (γH2AX) marks DDR and induces CCA via the p53/p21 pathway.33Fragkos M. Jurvansuu J. Beard P. H2ax is required for cell cycle arrest via the p53/p21 pathway.Mol Cell Biol. 2009; 29: 2828-2840Crossref PubMed Scopus (120) Google Scholar P21 activation halts the cell cycle, usually transient in case of efficient DNA repair, but permanent in senescence.34Romanov V.S. Pospelov V.A. Pospelova T.V. Cyclin-dependent kinase inhibitor p21(Waf1): contemporary view on its role in senescence and oncogenesis.Biochemistry (Mosc). 2012; 77: 575-584Crossref PubMed Scopus (102) Google Scholar Index case: histologic and immunohistochemical examination of a kidney allograft biopsy 8 months after transplantation revealed colocalization of tubular γH2AX, p21, and CCN2 in fibrotic areas (Figure 1a ). Evaluation of a previous biopsy from that same allograft obtained for DGF 7 days after transplantation revealed colocalization of tubular γH2AX, p21, and CCN2 in areas with ATI (i.e., tubular dilatation, epithelial necrosis, cast formation, and loss of brush borders; Figure 1b). Tubular expression of γH2AX, p21, and CCN2 were assessed in kidney allograft biopsies taken for CAD (5–29 years after transplantation) or for DGF (6–8 days after transplantation) with no apparent cause other than tubulointerstitial fibrosis or IRI, respectively. Patient characteristics are shown in Supplementary Table S2. In CAD biopsies, estimated glomerular filtration rate correlated with γH2AX (r = −0.73; P = 0.02) and p21 (r = −0.68; P = 0.04) expression (Figure 2a and b ). In addition, tendencies were observed between CCN2 and DDR in late biopsies (Supplementary Figure S1A and B), and in DGF biopsies between estimated glomerular filtration rate, DDR, and CCN2 (Supplementary Figure S1C–E) and CCN2 and DDR (Supplementary Figure S1F and G). Finally, CCN2 mRNA colocalized with CCN2 protein, as well as γH2AX and p21 in CAD and DGF biopsies, indicating a tubular source of CCN2 transcription (Supplementary Figure S2). To study the association between CCN2 and kidney function, damage, fibrosis, and senescence, a 6-week IRI model was conducted using ROSA26Cre tamoxifen inducible CCN2 full KO mice. Tamoxifen-induced recombination resulted in a 99% reduction in CCN2 mRNA in both sham and IRI kidneys (P < 0.005; Figure 3a ), as well as reduced CCN2 protein expression (P = 0.03 and P = 0.04, respectively; Figure 3b and Supplementary Figure S3). The plasma urea increase upon IRI was reduced by CCN2 KO (P = 0.01; Figure 3c). Similar trends for plasma creatinine were observed (Supplementary Figure S4A). Furthermore, tubulointerstitial fibrosis in Masson’s trichrome staining was impeded by CCN2 KO (P < 0.005; Figure 3d). Concordantly, IRI-induced upregulation of gene expression levels of tubular injury markers Kim-1 and Ngal, and of regeneration factor Sox9 was reduced in CCN2 KO IRI mice (P = 0.02, P = 0.009, and P < 0.005; Figure 3e–g). Thus, CCN2-deficient mice had reduced tubular damage and fibrosis 6 weeks after IRI. To elucidate if senescence might be implicated in the fibrosis mitigating effect of CCN2 reduction, markers related to DDR, proliferation, p53 signaling, and SASP were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction. Reduced numbers of γH2AX-positive cells in CCN2 KO IRI mice indicated reduced DDR (P < 0.005; Figure 4a ). The great majority of these cells were not cycling as evidenced by absent co-staining with Ki-67 (not shown). In addition, p21 mRNA expression and the numbers of p21-positive cells in IRI kidneys were reduced by CCN2 KO (P = 0.02 and P = 0.04, respectively; Figure 4b). In CCN2 KO IRI kidneys, mRNA levels of SASP factors Interleukin-1β, CC chemokine ligand 2, and Pai-1 were all reduced compared with WT IRI kidneys (P < 0.005, P < 0.005, and P < 0.005; Figure 4c–e). Of note, in WT IRI kidneys, γH2AX- and p21-positive cells were mainly PTECs in fibrotic areas of the outer cortex (Figure 4a and b). Overall, this indicated that CCN2-deficient mice had a reduced senescence phenotype 6 weeks after IRI. To evaluate possible CCN2 involvement in early phases of the transition from acute IRI to the long-term profibrotic senescent state, a 3-day IRI model was performed. Tamoxifen administration resulted in a near total reduction of CCN2 mRNA in sham and IRI kidneys (P < 0.005; Figure 5a ). The IRI-induced increases of plasma urea and creatinine were reduced in CCN2 KO mice (P < 0.005 and P < 0.005; Figure 5b and Supplementary Figure S4B, respectively). However, histologic examination of the cortices revealed similar degrees of acute tubular damage in WT and CCN2 KO IRI mice (Figure 5c). Concordantly, gene upregulation of tubular injury markers Kim-1 and Ngal and regeneration factor SOX9 were equally upregulated in CCN2 KO as in WT kidneys (Figure 5d–f). In addition, IRI-induced proliferation and apoptosis of PTECs were similar in WT and CCN2 KO mice (Supplementary Figure S5). CCN2 KO IRI kidneys had lower numbers of γH2AX-positive cells (P = 0.005; Figure 6a ) and lower p21 mRNA and protein expression (P = 0.02 and P < 0.005, respectively; Figure 6b), indicating reduced DNA damage and DDR. In WT IRI kidneys, γH2AX- and p21-positive cells were mainly PTECs localized in the inner cortex (Figure 6a and b). IRI-induced mRNA levels of SASP factors Interleukin-1β, CC chemokine ligand 2, and Pai-1 were similar in KO and WT mice (Figure 6c–e). Furthermore, in CCN2 KO IRI mice, KO efficiency marked by CCN2 exon 3 DNA levels containing the floxed genomic site correlated with plasma urea, ATI grade, Ngal mRNA, p21 mRNA, number of p21-positive cells, and CC chemokine ligand 2 mRNA, and a trend was observed for Ccn2 mRNA, Kim-1 mRNA, Sox9 mRNA, and Pai-1 mRNA (Supplementary Figure S6). Overall, this indicated that CCN2-deficient kidneys had reduced DNA damage and DDR 3 days after IRI. CCN2-induced DDR was evaluated in RPTECs and HK-2 cells. Stimulation of RPTECs with 100 ng/ml CCN2 increased the expression of γH2AX and p21 protein and had an antiproliferative effect marked by reduc