IL-22 is secreted by proximal tubule cells and regulates DNA damage response and cell death in acute kidney injury

Acute kidney injury (AKI) affects over 13 million people worldwide annually and is associated with a 4-fold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces cell death and worsens AKI. The factors that trigger DDR to switch from pro-repair to pro-cell death remain to be resolved. Here we investigated the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identified PTCs as a novel source of urinary IL-22. Functionally, IL-22 binding IL-22RA1 on PTCs amplified the DDR. Treating primary PTCs with IL-22 alone induced rapid activation of the DDR. The combination of IL-22 and either cisplatin- or AA-induced cell death in primary PTCs, while the same dose of cisplatin or AA alone did not. Global deletion of IL-22 protected against cisplatin- or AA-induced AKI, reduced expression of DDR components, and inhibited PTC cell death. To confirm PTC IL-22 signaling contributed to AKI, we knocked out IL-22RA1 specifically in kidney tubule cells. IL-22RA1ΔTub mice displayed reduced DDR activation, cell death, and kidney injury compared to controls. Thus, targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with repair of damaged DNA. Acute kidney injury (AKI) affects over 13 million people worldwide annually and is associated with a 4-fold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces cell death and worsens AKI. The factors that trigger DDR to switch from pro-repair to pro-cell death remain to be resolved. Here we investigated the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identified PTCs as a novel source of urinary IL-22. Functionally, IL-22 binding IL-22RA1 on PTCs amplified the DDR. Treating primary PTCs with IL-22 alone induced rapid activation of the DDR. The combination of IL-22 and either cisplatin- or AA-induced cell death in primary PTCs, while the same dose of cisplatin or AA alone did not. Global deletion of IL-22 protected against cisplatin- or AA-induced AKI, reduced expression of DDR components, and inhibited PTC cell death. To confirm PTC IL-22 signaling contributed to AKI, we knocked out IL-22RA1 specifically in kidney tubule cells. IL-22RA1ΔTub mice displayed reduced DDR activation, cell death, and kidney injury compared to controls. Thus, targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with repair of damaged DNA. Translational StatementAcute kidney injury, which affects 10% to 20% of hospitalized patients, is associated with a 4-fold increase in mortality and predisposes patients to chronic kidney disease. In the present study, we identify interleukin-22 as a cofactor that worsens acute kidney injury. Interleukin-22 activates the DNA damage response, which, in combination with nephrotoxic drugs, amplifies the injury response in kidney epithelial cells and increases cell death. Deletion of interleukin-22 from mice or its receptor from mouse kidneys ameliorates cisplatin- or aristolochic acid–induced nephropathy. These findings may help clarify the molecular mechanisms of DNA damage–induced kidney injury and identify interventions that can help treat acute kidney injury. Acute kidney injury, which affects 10% to 20% of hospitalized patients, is associated with a 4-fold increase in mortality and predisposes patients to chronic kidney disease. In the present study, we identify interleukin-22 as a cofactor that worsens acute kidney injury. Interleukin-22 activates the DNA damage response, which, in combination with nephrotoxic drugs, amplifies the injury response in kidney epithelial cells and increases cell death. Deletion of interleukin-22 from mice or its receptor from mouse kidneys ameliorates cisplatin- or aristolochic acid–induced nephropathy. These findings may help clarify the molecular mechanisms of DNA damage–induced kidney injury and identify interventions that can help treat acute kidney injury. Acute kidney injury (AKI) occurs in 21.6% of hospitalized adults worldwide.1Susantitaphong P. Cruz D.N. Cerda J. et al.World incidence of AKI: a meta-analysis.Clin J Am Soc Nephrol. 2013; 8: 1482-1493Crossref PubMed Scopus (957) Google Scholar DNA damage is one of the major manifestations of AKI.2Yan M. Tang C. Ma Z. et al.DNA damage response in nephrotoxic and ischemic kidney injury.Toxicol Appl Pharmacol. 2016; 313: 104-108Crossref PubMed Scopus (54) Google Scholar,3Tiong H.Y. Huang P. Xiong S. et al.Drug-induced nephrotoxicity: clinical impact and preclinical in vitro models.Mol Pharm. 2014; 11: 1933-1948Crossref PubMed Scopus (138) Google Scholar Cells respond to DNA damage by activating a sensitive and complex DNA damage response (DDR) pathway, which detects lesions in damaged chromatin, delays cell cycle progression, and repairs the lesions, or, in the case of severe injury, promotes cell death. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3–related protein (ATR) are major DDR sensor kinases that sense DNA damage and transmit DDR signals to downstream effector proteins, such as p53. ATM can stabilize p53 through inhibition of the degrader mouse double minute 2 homolog (MDM2), while both ATM and ATR can activate p53 through phosphorylation. Activation of p53 ultimately regulates cell fate by inducing cell cycle regulators, such as p21, to promote cell cycle arrest or pro-apoptotic genes, such as p53 upregulated modulator of apoptosis (PUMA) or BCL2-associated X (Bax), to induce apoptosis.4Tanida S. Mizoshita T. Ozeki K. et al.Mechanisms of cisplatin-induced apoptosis and of cisplatin sensitivity: potential of BIN1 to act as a potent predictor of cisplatin sensitivity in gastric cancer treatment.Int J Surg Oncol. 2012; 2012862879PubMed Google Scholar Our laboratory and others have shown that inhibition of p53 is protective against multiple forms of AKI whereas inhibition of ATM or ATR dramatically worsens AKI.5Kishi S. Brooks C.R. Taguchi K. et al.Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses.J Clin Investig. 2019; 129: 4797-4816Crossref PubMed Scopus (65) Google Scholar, 6Uehara M. Kusaba T. Ida T. et al.Pharmacological inhibition of ataxia-telangiectasia mutated exacerbates acute kidney injury by activating p53 signaling in mice.Sci Rep. 2020; 10: 4441Crossref PubMed Scopus (12) Google Scholar, 7Tang C. Ma Z. Zhu J. et al.P53 in kidney injury and repair: mechanism and therapeutic potentials.Pharmacol Ther. 2019; 195: 5-12Crossref PubMed Scopus (69) Google Scholar Thus, balanced DDR signaling is necessary for recovery from AKI and prevention of excessive cell death in AKI. Interleukin-22 (IL-22) is a member of the IL-10–related family of cytokines.8Dumoutier L. Van Roost E. Ameye G. et al.IL-TIF/IL-22: genomic organization and mapping of the human and mouse genes.Genes Immun. 2000; 1: 488-494Crossref PubMed Scopus (185) Google Scholar,9Ouyang W. Rutz S. Crellin N.K. et al.Regulation and functions of the IL-10 family of cytokines in inflammation and disease.Annu Rev Immunol. 2011; 29: 71-109Crossref PubMed Scopus (1337) Google Scholar IL-22 is predominantly expressed by T cell subsets, such as CD4+, T helper 17 cells, T helper 22 cells, T helper, or group 3 innate lymphoid cells.10Arshad T. Mansur F. Palek R. et al.A double edged sword role of interleukin-22 in wound healing and tissue regeneration.Front Immunol. 2020; 11: 2148Crossref PubMed Scopus (46) Google Scholar The IL-22 receptor complex consists of a ligand-binding chain, IL-22 receptor subunit alpha 1 (IL-22RA1), and a signal-transducing chain, interleukin-10 receptor subunit 2 (IL-10R2). In contrast to ubiquitous expression of IL-10R2,11Donnelly R.P. Sheikh F. Kotenko S.V. et al.The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain.J Leukoc Biol. 2004; 76: 314-321Crossref PubMed Scopus (250) Google Scholar expression of IL-22RA1 is restricted to epithelial cells, thus allowing immune-to-epithelial signaling. The binding of IL-22 to the IL-22 receptor leads to activation of the transcriptional factor signal transducer and activator 3 (STAT3) via phosphorylation.12Gurney A.L. IL-22, a Th1 cytokine that targets the pancreas and select other peripheral tissues.Int Immunopharmacol. 2004; 4: 669-677Crossref PubMed Scopus (105) Google Scholar,13Nagem R.A. Ferreira Júnior J.R. Dumoutier L. et al.Interleukin-22 and its crystal structure.Vitam Horm. 2006; 74: 77-103Crossref PubMed Scopus (13) Google Scholar The primary biological function of IL-22 is regulation of epithelial antibacterial responses through strengthening epithelial barrier integrity.14Fumagalli S. Torri A. Papagna A. et al.IL-22 is rapidly induced by pathogen recognition receptors stimulation in bone-marrow-derived dendritic cells in the absence of IL-23.Sci Rep. 2016; 633900Crossref PubMed Scopus (16) Google Scholar,15Keir M. Yi Y. Lu T. et al.The role of IL-22 in intestinal health and disease.J Exp Med. 2020; 217e20192195Crossref PubMed Google Scholar In this way, IL-22 is thought to provide protection against acute injuries, such as ischemia, acetaminophen, or alcohol-induced liver injuries by accelerating wound healing.10Arshad T. Mansur F. Palek R. et al.A double edged sword role of interleukin-22 in wound healing and tissue regeneration.Front Immunol. 2020; 11: 2148Crossref PubMed Scopus (46) Google Scholar,16Kleinschmidt D. Giannou A.D. McGee H.M. et al.A protective function of IL-22BP in ischemia reperfusion and acetaminophen-induced liver injury.J Immunol. 2017; 199: 4078-4090Crossref PubMed Scopus (31) Google Scholar,17Lucke J. Sabihi M. Zhang T. et al.The good and the bad about separation anxiety: roles of IL-22 and IL-22BP in liver pathologies.Semin Immunopathol. 2021; 43: 591-607Crossref PubMed Scopus (13) Google Scholar On the contrary, IL-22 can induce cell death in intestinal stem cells or erythroid precursors18Gronke K. Hernandez P.P. Zimmermann J. et al.Interleukin-22 protects intestinal stem cells against genotoxic stress.Nature. 2019; 566: 249-253Crossref PubMed Scopus (215) Google Scholar,19Raundhal M. Ghosh S. Myers S.A. et al.Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.Nat Immunol. 2021; 22: 520-529Crossref PubMed Scopus (8) Google Scholar and plays a pathological role in irritable bowel disease, Crohn disease, dermatitis, and lupus nephritis.20Jin M. Yoon J. From bench to clinic: the potential of therapeutic targeting of the IL-22 signaling pathway in atopic dermatitis.Immune Netw. 2018; 18: e42Crossref PubMed Scopus (22) Google Scholar, 21Powell N. Pantazi E. Pavlidis P. et al.Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells.Gut. 2020; 69: 578-590Crossref PubMed Scopus (66) Google Scholar, 22Hu L. Hu J. Chen L. et al.Interleukin-22 from type 3 innate lymphoid cells aggravates lupus nephritis by promoting macrophage infiltration in lupus-prone mice.Front Immunol. 2021; 12584414Google Scholar In the clinic, IL-22 inhibition has been shown to be safe and effective at reducing severe atopic dermatitis.23Guttman-Yassky E. Brunner P.M. Neumann A.U. et al.Efficacy and safety of fezakinumab (an IL-22 monoclonal antibody) in adults with moderate-to-severe atopic dermatitis inadequately controlled by conventional treatments: a randomized, double-blind, phase 2a trial.J Am Acad Dermatol. 2018; 78: 872-881.e876Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar Thus, IL-22 signaling can be protective or pathological, depending on the context. The role of IL-22 in the kidney remains controversial. Earlier studies using high concentrations of IL-22 or IL-22-Fc found IL-22 to be protective against diabetic nephropathy, acetaminophen, and ischemia-induced kidney injury,24Xu M.J. Feng D. Wang H. et al.IL-22 ameliorates renal ischemia-reperfusion injury by targeting proximal tubule epithelium.J Am Soc Nephrol. 2014; 25: 967-977Crossref PubMed Scopus (73) Google Scholar,25Ma Q. Luan J. Bai Y. et al.Interleukin-22 in renal protection and its pathological role in kidney diseases.Front Immunol. 2022; 13851818Google Scholar whereas more recent studies have shown that endogenous levels of IL-22 or untagged IL-22 worsens lupus nephritis, IgA nephropathy, or hypertensive kidney injury.22Hu L. Hu J. Chen L. et al.Interleukin-22 from type 3 innate lymphoid cells aggravates lupus nephritis by promoting macrophage infiltration in lupus-prone mice.Front Immunol. 2021; 12584414Google Scholar,26Wang W. Lu Y. Hu X. et al.Interleukin-22 exacerbates angiotensin II-induced hypertensive renal injury.Int Immunopharmacol. 2022; 109108840Crossref Scopus (4) Google Scholar Previous animal studies, however, have largely relied on systemic overexpression, injection, or global knockout (KO) models that modulate IL-22 signaling on the systemic level. Thus, the kidney-specific role of IL-22 signaling has yet to be resolved. Also, despite the fact that kidney IL-22RA1 is restricted to the apical surface of proximal tubule cells (PTCs), urinary IL-22 concentration is not monitored in most studies. In the present study, we aim to determine the kidney-specific role of IL-22 in nephrotoxin-induced AKI. Mouse colonies were derived from commercial sources and maintained on a 12-hour light/dark cycle at room temperature with free access to food and water under protocols approved by the institutional animal care and use committee at Vanderbilt University Medical Center. IL-22RA1flox/flox (IL-22RA1fl/fl; B6.Cg-Il22ra1tm1.1Koll/J) and Six2-TGCtg (Six2Cre; STOCK Tg(Six2-EGFP/Cre)1Amc/J) mice were purchased from the Jackson Laboratory and crossed to create kidney tubule–specific IL-22RA1 KO mice (IL-22RA1ΔTub). IL-22RA1fl/fl was used in the present study as a control. C57BL/6-Il22tm1.1(iCre)Stck/J (IL-22Cre) were purchased from the Jackson Laboratory. Exon 1 of the Il22 gene was replaced with a codon-optimized Cre recombinase (iCre), which abolishes expression of IL-22 globally in homozygotes. Floxed-stop tdTomato mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) were crossed with IL-22Cre to generate an IL-22 reporter line, IL-22Cre:tdTomato. Wild-type (WT) and IL-22RA1fl/fl control mice were from littermates. No developmental phenotypes were observed in IL-22 global KO (IL-22KO) or IL-22RA1ΔTub mice. Experimental mice aged 8 to 12 weeks were divided into control and cisplatin or aristolochic acid (AA) groups at random. Male mice were chosen for the study because we and others have observed a reduced response to both cisplatin- and AA-induced kidney injury in female mice.27Shi M. Ma L. Zhou L. et al.Renal protective effects of 17β-estradiol on mice with acute aristolochic acid nephropathy.Molecules. 2016; 21: 1391Crossref PubMed Scopus (20) Google Scholar,28Hwang D.B. Cha M.H. Won D.H. et al.Transcriptomic analysis of rat kidney reveals a potential mechanism of sex differences in susceptibility to cisplatin-induced nephrotoxicity.Free Radic Biol Med. 2021; 174: 100-109Crossref PubMed Scopus (5) Google Scholar For cisplatin-induced AKI, mice were administered cisplatin (Sigma, catalogue number 4394) dissolved in sterile saline (K•D Medical; catalogue number RGC-3290) at 30 mg/kg in a single i.p. injection. The same amount of sterile saline was injected as a control. Blood was taken and body weight measured every 24 hours. For cisplatin studies, WT mice were found to reach human end points—20% weight loss, inability to reach food/water, hunched posture, and/or abnormal mentation—by day 4 to 5. For this reason, most mice were sacrificed on day 3 after administration of cisplatin and kidneys, blood, and urine were collected. AA nephropathy, a model of AKI and subsequent chronic kidney disease transition, was induced by an i.p. injection of 3 doses of AA (5 mg/kg body weight; Sigma; catalogue number A5512) dissolved in sterile saline. Control mice were injected the same amount of saline as a control. Blood was collected and body weight measured on day 3, day 7, and every 7 days after that until day 42. Mice were sacrificed and kidneys were isolated on day 7 as acute phase and on day 42 as chronic phase. RNAscope fluorescent in situ hybridization was performed according to the manufacturer's protocol (ACDBio, RNAscope Multiplex Fluorescent Assay v2). Briefly, 4- to 6-μm paraffin-embedded kidney sections from patient biopsies or mouse kidneys were deparaffinized and incubated with H2O2 to inhibit endogenous peroxidase activity. RNA was retrieved followed by incubation with proteinase to enable the access to target RNA. Then, sections were hybridized with Il-22 probes for 2 hours at room temperature, followed by incubation with appropriate fluorescent probes to visualize target probe RNA. Images were obtained with a Zeiss LSM 710 confocal microscope, and RNA dots were counted by ImageJ (National Institutes of Health). To identify the source of secreted cytokines, cytokine secretion was inhibited using brefeldin A (BFA) injection and kidneys were stained for cytokines using immunofluorescence staining as previously described.29Taguchi K. Sugahara S. Elias B.C. Brooks C.R. Identification of the source of secreted proteins in the kidney by brefeldin A injection.J Vis Exp. 2021; : 177Google Scholar Briefly, BFA was dissolved with dimethylsulfoxide at a concentration of 10 mg/ml as a stock solution and further diluted with sterile phosphate-buffered saline (PBS) at the final concentration of 1.25 mg/ml immediately before use; 200 μl of a 1.25 mg/ml BFA solution was injected into mice through the tail vein 6 hours before sacrifice. Kidneys were harvested and processed for immunofluorescence staining as described above. De-identified human biopsies were obtained from 3 patients with cisplatin-induced nephropathy as part of standard clinical practice (Supplementary Table S1). De-identified human samples were obtained from 3 patients with chronic kidney disease undergoing a partial or radical nephrectomy procedure for urological indication. Interstitial fibrosis was evaluated by a trained senior pathologist, and tissue with >10% interstitial fibrosis was categorized as fibrotic kidney (Supplementary Table S2). Kidney biopsies were obtained as part of standard clinical practice. IL-22 staining was performed using RNAscope as described above. All animal experiments were approved by the Institutional Animal Care & Use Committee of Vanderbilt University Medical Center. Human samples were obtained under protocols approved by the Hôpital Necker Enfants Malades, Université de Paris Institutional Review Board and Boston University Medical Center and Brigham and Women's Hospital. All necessary patient/participant written informed consent was obtained. All data were presented as mean ± SD. One-way analysis of variance and subsequent Tukey post hoc test was used to determine statistical differences among 3 groups. When 2 groups were compared, the unpaired 2-tailed t test was conducted. Survival curves were derived using the Kaplan-Meier method and compared using the log-rank test. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software). For all comparisons, P < 0.05 was considered to indicate statistical significance. Expanded methods are provided in the Supplementary Methods. To investigate the role of IL-22 in DNA damage–induced kidney injury, we stained patient biopsies from patients with cisplatin-induced nephropathy for IL-22 RNA using RNAscope (patient characteristics in Supplementary Tables S1 and S2). We were surprised to find that kidney tubule cells express IL-22 RNA (Figure 1a). To confirm these results, we treated mice with cisplatin or AA and stained for mouse IL-22 RNA and the PTC marker lotus lectin. Similar to human samples, both cisplatin- and AA-induced expression of IL-22 RNA in mouse kidneys (Figure 2b). To confirm IL-22 gene expression in injured kidneys, we bred IL-22Cre mice with floxed-stop tdTomato reporter mice to generate mice that express tdTomato under the control of the IL-22 promotor. Injuring IL-22Cre;tdTomato mice with cisplatin or AA injection induced tdTomato expression in kidney tubule cells (Supplementary Figure S1A).Figure 2Proximal tubule cell (PTC) interleukin-22 receptor subunit alpha 1 (IL-22RA1) expression is increased with injury. (a) Representative image of IL-22RA1 and lotus lectin (LTL) in the S1–S2 and S3 segments of the normal kidney cortex. Bar = 20 μm. (b) Large scan image of the IL-22RA1- and LTL-labeled normal kidney and quantification of IL-22RA1+/LTL+ (%) in the S1–S2 and S3 segments of the normal kidney cortex. n = 4. Bar = 500 μm. (c) Schematic diagram of cisplatin- and aristolochic acid (AA)–induced acute kidney injury (AKI) mouse models. (d) Representative images of IL-22RA1 expression in S1–S2 PTC segments after AA- or cisplatin (Cis)-induced injury, and quantification of IL-22RA1+ area/high-power field (HPF) (%). n = 3. Bar = 50 μm. (e) Schematic diagram of the changes in IL-22RA1 expression after injury. Created with BioRender.com. Data are presented as mean ± SD. An unpaired 2-tailed t test was performed to identify statistical difference (b) and 1-way analysis of variance and subsequent Tukey post hoc test (e). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. DAPI, 4ʹ,6-diamidino-2-phenylindole; HM, high magnification; IL-22KO, interleukin-22 global knockout;s WT, wild-type. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As IL-22 is a secreted protein, it is difficult to detect IL-22 protein within the source cells. To overcome this challenge, we injected mice with the secretion inhibitor BFA 6 hours before sacrifice and stained for IL-22 protein as shown previously.29Taguchi K. Sugahara S. Elias B.C. Brooks C.R. Identification of the source of secreted proteins in the kidney by brefeldin A injection.J Vis Exp. 2021; : 177Google Scholar After cisplatin + BFA or AA + BFA treatment, IL-22 was detected in lotus lectin–positive PTCs (Figure 1c). To confirm PTC production of IL-22, we isolated PTCs from mice and treated with cisplatin or AA with or without BFA and stained for IL-22. Primary PTCs upregulated IL-22 in response to both cisplatin and AA, and staining was enhanced with BFA (Figure 1d). The next question became: Where is PTC IL-22 secreted? To answer this question, we performed enzyme-linked immunosorbent assay (ELISA) analysis on serum, urine, and kidney lysates. Cisplatin or AA upregulated IL-22 predominantly in urine (Figure 1e). To confirm that urinary IL-22 was from PTCs, we induced kidney injury in immune-deficient Rag1 KO mice, which lack mature T cells and B cells, using cisplatin or AA. Rag1 KO mouse kidneys stain positive for IL-22 in PTCs after AA + BFA injection (Figure 1f). Rag1 KO mice expressed a similar level of urinary IL-22 as WT mice (Figure 1g). Serum IL-22 was not increased in Rag1 KO mice (data not shown). These data suggest that PTCs are a novel source of IL-22 and contribute to urinary IL-22 levels. Given high urinary expression of IL-22, we next sought to identify which kidney cell type presented the IL-22 receptor to urine in uninjured mouse kidneys and kidneys after AKI. Immunofluorescent staining for IL-22RA1 revealed that it was expressed on the brush boarder of PTCs, with staining most apparent in the S3 segment of the proximal tubule in uninjured kidneys (Figure 2a and b). Both cisplatin- and AA-induced injury upregulated IL-22RA1 in the S1 and S2 segments (Figure 2c–e). IL-22RA1 was also upregulated at the RNA level after injury (Supplementary Figure S1B). Costaining for IL-22RA1 and the kidney injury marker kidney injury molecule-1 (KIM-1) revealed that expression was lower in KIM-1+ PTCs (Supplementary Figure S1C). These data indicate that IL-22RA1 is constitutively expressed on the apical surface of S3 segment PTCs and is upregulated in S1 + S2 segments with injury. To evaluate the role of IL-22 in AKI, we treated WT and IL-22KO mice with cisplatin and AA (Figure 3a) and followed mice over time. No IL-22 RNA was detected in IL-22KO mice with or without injury (Supplementary Figure S1D). Kaplan-Meier analysis showed that cisplatin administration led to significantly less mortality in IL-22KO mice than in WT mice (Figure 3b). Kidney dysfunction, assessed by plasma creatinine, and weight loss were attenuated in IL-22KO in cisplatin- and AA-induced AKI (Figure 3c and d; Supplementary Figure S1E and F). KIM-1, a marker of tubular injury, was upregulated in WT and IL-22KO mice after cisplatin- or AA-induced injury, but the increase was significantly less in IL-22KO (Figure 3e and f). Similarly, the RNA levels of KIM-1, neutrophil gelatinase–associated lipocalin (NGAL), and IL-6 were significantly lower in IL-22KO mice after injury than in WT mice (Supplementary Figure S1G and H). Cleaved caspase 3 (C-casp3), a marker of apoptosis, was reduced in IL-22KO mice compared with WT mice after cisplatin- or AA-induced injury (Figure 3g and h; Supplementary Figure S1I). The PTC differentiation marker Na/K-adenosine triphosphatase transporter (Na/K-ATPase) was better preserved in injured IL-22KO mice than in WT mice (Figure 3i and j). Periodic acid–Schiff staining demonstrated that the kidney structure was better preserved in IL-22KO mice (Supplementary Figure S1J). Taken together, these data suggest that deletion of IL-22 prevents AKI induced by cisplatin or AA. In addition to acute injury, AA is known to induce chronic kidney disease and progressive fibrosis. We also examined the effect of IL-22 KO on the chronic phase of AA-induced injury. Chronic injury, as measured by serum creatinine, and weight loss up to 42 days after injury were significantly reduced in IL-22KO mice compared with WT mice (Supplementary Figure S2A and B). Kidney fibrosis, as measured by picrosirius red staining, was also significantly reduced in IL-22KO mice compared with WT mice (Supplementary Figure S2C). Consistent with a reduction in kidney injury, less immune cell infiltration was observed in IL-22KO mice after AA injury (Supplementary Figure S2D). These data suggest that the protective effect of IL-22 deletion in the acute phase is associated with reduced chronic injury. To investigate the mechanism by which IL-22 worsens kidney injury, we analyzed the phosphorylation and activation of DDR sensor proteins H2A histone family member X (H2AX) and ATM in injured kidneys. The number of cells positive for H2AX phosphorylated on serine 139 (γH2AX), a specific marker of DNA damage, was increased in both WT and IL-22KO mice in cisplatin- and AA-induced AKI. However, the number of γH2AX+ cells was significantly lower in AA-injured IL-22KO mice (Figure 4a). γH2AX+ cells were also reduced at day 42 after AA injection (Supplementary Figure S3A and B). Phosphorylated-ATM+ cells were lower in IL-22KO mice than in WT mice after cisplatin or AA exposure (Figure 4b). STAT3, one of the primary IL-22/IL-22RA1 signaling molecules, was upregulated in both injured WT and IL-22KO mice, but to a lesser extent in the IL-22KO group (Figure 4c). Phosphorylated p53 and total p53 were increased in the kidneys from WT mice following cisplatin-induced AKI, which were reduced in IL-22KO mice in parallel with MDM2 levels (Figure 4c and d; Supplementary Figure S3C and D). These data suggest that IL-22 upregulates DDR activity in vivo. Although our data suggest that IL-22 worsens AKI by acting on intrinsic kidney epithelial cells, it remains possible that it functions in an extrarenal manner. To confirm that IL-22 promotes kidney injury by directly acting on PTCs in vivo, we deleted IL-22RA1 specifically in kidney epithelial cells by crossing IL-22RA1flox/flox with Six2Cre (IL-22RA1ΔTub). IL-22RA1ΔTub mice developed normally and had no observed kidney phenotype before injury. We confirmed that IL-22RA1 deletion abolished IL-22 signaling in PTCs by treating IL-22RA1ΔTub primary PTCs with saline or IL-22 and/or cisplatin or AA. IL-22 incubation did not promote STAT3 phosphorylation, a primary target of IL-22RA1 (Supplementary Figure S4A). IL-22RA1ΔTub mice were injected with cisplatin to induce AKI (Figure 5a). Immunostaining revealed that IL-22RA1 expression was significantly reduced in the kidneys from IL-22RA1ΔTub mice (Figure 5b and c). After cisplatin injection, kidney function, as measured by plasma BUN, was better preserved in IL-22RA1ΔTub mice compared to IL-22RA1fl/fl (Figure 5d). IL-22RA1ΔTub mice demonstrated greater survival and decreased inflammation compared with IL-22RA1fl/fl mice after cisplatin injection (Supplementary Figure S4B and C). Periodic acid–Schiff staining demonstrated that the kidney structure was better preserved in IL-22KO mice (Supplementary Figure S4D). Immunostaining revealed that C-casp3 levels were increased in IL-22RA1fl/fl mice but not in IL-22RA1ΔTub mice (Fi