Zinc finger protein 24-dependent transcription factor SOX9 up-regulation protects tubular epithelial cells during acute kidney injury

Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation. Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation. Translational StatementThe transcription factor SOX9 is upregulated in injured tubular epithelial cells and plays a protective role during acute kidney injury (AKI). Identifying the upstream factors could lead to the development of therapeutic strategies that increase the expression of protective genes such as SOX9, reduce injury, and augment the repair processes. To this end, the present study describes the identification of zinc factor protein ZFP24 as the transcriptional regulator that controls the upregulation of a protective SOX9-mediated transcriptional program in tubular epithelial cells during AKI. The transcription factor SOX9 is upregulated in injured tubular epithelial cells and plays a protective role during acute kidney injury (AKI). Identifying the upstream factors could lead to the development of therapeutic strategies that increase the expression of protective genes such as SOX9, reduce injury, and augment the repair processes. To this end, the present study describes the identification of zinc factor protein ZFP24 as the transcriptional regulator that controls the upregulation of a protective SOX9-mediated transcriptional program in tubular epithelial cells during AKI. Sox (sex-determining region-Y related high-mobility group [HMG]-box) proteins are a conserved family of transcription factors and cell fate regulators with widespread roles in embryonic development and adult tissue homeostasis.1Kamachi Y. Kondoh H. Sox proteins: regulators of cell fate specification and differentiation.Development. 2013; 140: 4129-4144Crossref PubMed Scopus (399) Google Scholar The SOX family, which encompasses about 20 members with highly divergent functions, is defined by the presence of a conserved HMG domain that mediates DNA binding.2Bowles J. Schepers G. Koopman P. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators.Dev Biol. 2000; 227: 239-255Crossref PubMed Scopus (760) Google Scholar SOX9 is a key member of the SOX family and was identified initially as the gene underlying the haploinsufficiency disorder campomelic dysplasia (CD), a human syndrome characterized by defective chondrogenesis and sex reversal.3Lee Y.H. Saint-Jeannet J.P. Sox9 function in craniofacial development and disease.Genesis. 2011; 49: 200-208Crossref PubMed Scopus (78) Google Scholar, 4Pritchett J. Athwal V. Roberts N. et al.Understanding the role of SOX9 in acquired diseases: lessons from development.Trends Mol Med. 2011; 17: 166-174Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 5Jo A. Denduluri S. Zhang B. et al.The versatile functions of Sox9 in development, stem cells, and human diseases.Genes Dis. 2014; 1: 149-161Crossref PubMed Scopus (210) Google Scholar Notwithstanding its critical role in skeletal development and male sex determination, studies with Sox9-deficient mice have revealed the importance of this transcription factor in cell fate determination in multiple organs, such as the pancreas, intestine, kidney, heart, and brain.5Jo A. Denduluri S. Zhang B. et al.The versatile functions of Sox9 in development, stem cells, and human diseases.Genes Dis. 2014; 1: 149-161Crossref PubMed Scopus (210) Google Scholar Sox9 is widely expressed during embryonic development, including in the kidneys. However, in most adult tissues its expression level is low, except in certain cell types such as astrocytes6Sun W. Cornwell A. Li J. et al.SOX9 is an astrocyte-specific nuclear marker in the adult brain outside the neurogenic regions.J Neurosci. 2017; 37: 4493-4507Crossref PubMed Scopus (165) Google Scholar in the brain and adult stem cells in tissues with high turnovers, such as the intestine7Blache P. van de Wetering M. Duluc I. et al.SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (380) Google Scholar and hair follicles.8Kadaja M. Keyes B.E. Lin M. et al.SOX9: a stem cell transcriptional regulator of secreted niche signaling factors.Genes Dev. 2014; 28: 328-341Crossref PubMed Scopus (145) Google Scholar In hair follicles, which undergo cyclical periods of growth, Sox9 is highly expressed and is functionally critical for stem cells that reside at the base of the resting follicle.8Kadaja M. Keyes B.E. Lin M. et al.SOX9: a stem cell transcriptional regulator of secreted niche signaling factors.Genes Dev. 2014; 28: 328-341Crossref PubMed Scopus (145) Google Scholar Sox9 also drives postnatal injury repair,9Zhao J. Patel J. Kaur S. et al.Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.Nat Commun. 2021; 12: 2564Crossref PubMed Scopus (17) Google Scholar as well as fibrosis,10Lacraz G.P.A. Junker J.P. Gladka M.M. et al.Tomo-Seq identifies SOX9 as a key regulator of cardiac fibrosis during ischemic injury.Circulation. 2017; 136: 1396-1409Crossref PubMed Scopus (59) Google Scholar in multiple organs. Thus, in adult tissues, Sox9 seems to be part of a tightly regulated transcriptional program that ensures proper tissue architecture during normal cell turnover and post-injury repair. Within the adult kidneys, the Sox9 expression level is low in the renal tubular epithelial cells (RTECs). However, under conditions of cellular stress and injury, Sox9 is transcriptionally upregulated.11Kumar S. Liu J. Pang P. et al.Sox9 activation highlights a cellular pathway of renal repair in the acutely injured mammalian kidney.Cell Rep. 2015; 12: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar Functionally, Sox9 plays a cytoprotective role during the early phase of ischemia–reperfusion injury (IRI),12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar cisplatin nephrotoxicity,12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar and rhabdomyolysis-associated acute kidney injury (AKI)13Kim J.Y. Bai Y. Jayne L.A. et al.Involvement of the CDKL5-SOX9 signaling axis in rhabdomyolysis-associated acute kidney injury.Am J Physiol Renal Physiol. 2020; 319: F920-F929Crossref PubMed Google Scholar and facilitates repair during the recovery phase.11Kumar S. Liu J. Pang P. et al.Sox9 activation highlights a cellular pathway of renal repair in the acutely injured mammalian kidney.Cell Rep. 2015; 12: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,14Kang H.M. Huang S. Reidy K. et al.Sox9-positive progenitor cells play a key role in renal tubule epithelial regeneration in mice.Cell Rep. 2016; 14: 861-871Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar We have recently identified post-translational regulatory mechanisms that suppress Sox9 protein function during AKI.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar However, the mechanisms that control Sox9 mRNA upregulation in RTECs during AKI remain unknown. In the current study, we have utilized an unbiased RNA interference (RNAi)-mediated functional screening approach to identify transcription factors that regulate stress-induced Sox9 upregulation in RTECs. Our studies have identified zinc finger protein Zfp24,15Elbaz B. Aaker J.D. Isaac S. et al.Phosphorylation state of ZFP24 controls oligodendrocyte differentiation.Cell Rep. 2018; 23: 2254-2263Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar also known as Zfp191 and Znf24, as a crucial upstream regulator of Sox9 in RTECs. Zfp24 is a Cys2-His2 (C2H2) zinc finger protein that contains 4 putative DNA-binding zinc finger domains.15Elbaz B. Aaker J.D. Isaac S. et al.Phosphorylation state of ZFP24 controls oligodendrocyte differentiation.Cell Rep. 2018; 23: 2254-2263Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,16Harper J. Yan L. Loureiro R.M. et al.Repression of vascular endothelial growth factor expression by the zinc finger transcription factor ZNF24.Cancer Res. 2007; 67: 8736-8741Crossref PubMed Scopus (38) Google Scholar Zfp24 plays crucial roles in oligodendrocyte maturation and central nervous system myelination.15Elbaz B. Aaker J.D. Isaac S. et al.Phosphorylation state of ZFP24 controls oligodendrocyte differentiation.Cell Rep. 2018; 23: 2254-2263Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,17Howng S.Y. Avila R.L. Emery B. et al.ZFP191 is required by oligodendrocytes for CNS myelination.Genes Dev. 2010; 24: 301-311Crossref PubMed Scopus (60) Google Scholar but its role in Sox9 regulation and the pathogenesis of AKI has not been explored previously. Our results show that Zfp24 drives Sox9 upregulation in RTECs during AKI. Boston University mouse proximal tubule cells (BUMPT, generated by Drs. Wilfred Lieberthal and John Schwartz18Sinha D. Wang Z. Price V.R. et al.Chemical anoxia of tubular cells induces activation of c-Src and its translocation to the zonula adherens.Am J Physiol Renal Physiol. 2003; 284: F488-497Crossref PubMed Scopus (61) Google Scholar and obtained from Dr. Zheng Dong, Augusta University, Augusta, GA) were grown at 37 °C in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. We used a promoter–reporter lentiviral firefly luciferase vector (Applied Biological Materials, catalog numbers LV653 and LV655) to generate 2 stable BUMPT cell lines expressing empty vector (promoter-less) and murine Sox9 promoter (–2500 base pairs from the transcription start site) driven firefly luciferase. BUMPT stable cells were then reverse-transfected with control small interfering (si)RNA or siRNAs targeting transcriptional regulators, using methods described in our previous work.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar,19Sprowl J.A. Ong S.S. Gibson A.A. et al.A phosphotyrosine switch regulates organic cation transporters.Nat Commun. 2016; 710880Crossref PubMed Scopus (84) Google Scholar Briefly, the Dharmacon mouse siRNA library targeting transcriptional regulators (1887 genes) containing 4 pooled siRNAs for each gene was utilized in the primary screen. Stable BUMPT cells were plated in 96-well plates and reverse-transfected with 25 nM siRNA using Lipofectamine RNAiMAX reagent (Life Technologies). At 48 hours post-transfection, cells were treated with 15 μM cisplatin in fresh media. Subsequently, at 8 hours post-treatment, firefly luciferase assay (Thermo Fischer, catalog number 16176) was performed. The siRNAs that reduced luciferase activity in stable cells that expressed the Sox9 promoter–driven luciferase were selected for secondary screening. The primary screen was carried out in triplicate samples, and data analysis was performed according to established and previously reported methods.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar,19Sprowl J.A. Ong S.S. Gibson A.A. et al.A phosphotyrosine switch regulates organic cation transporters.Nat Commun. 2016; 710880Crossref PubMed Scopus (84) Google Scholar The details of the secondary screening are provided in the Supplementary Methods. All animals were housed and handled, and animal studies conducted, in accordance with approved institutional animal care and use committee procedures. Mice were housed in a temperature-controlled environment with a 12-hour light cycle and given a standard diet and water ad libitum. Conditional gene knockout in RTECs was achieved through breeding of Zfp24-floxed mice (Jackson Laboratory, stock no. 029023) and Sox9-floxed mice (Jackson Laboratory, stock no. 013106) with Ggt1-Cre mice (Jackson Laboratory, stock no. 012841). For all mouse colonies, the pups were ear-tagged and genotyped at 3 weeks of age, as described in our previous studies. Methods used for generating mice in which CRISPR (for clustered regularly interspaced short palindromic repeats)-Cas9 mediated knock-in mutation was introduced in the SOX9 promoter (Prommut mice) are described in detail in the Supplementary Methods. The Zfp24 floxed and Prommut strains were obtained and maintained on a C57BL/6J background. The Ggt1-Cre mice have a mix of BALB/cJ and C57BL/6 background. Given that AKI is associated with distinct disease conditions, multiple mouse models have been developed.20Hukriede N.A. Soranno D.E. Sander V. et al.Experimental models of acute kidney injury for translational research.Nat Rev Nephrol. 2022; 18: 277-293Crossref PubMed Scopus (11) Google Scholar Ischemia, cisplatin, and rhabdomyolysis-associated AKI was triggered in male mice aged 8–12 weeks using methods described in our recent studies.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar,13Kim J.Y. Bai Y. Jayne L.A. et al.Involvement of the CDKL5-SOX9 signaling axis in rhabdomyolysis-associated acute kidney injury.Am J Physiol Renal Physiol. 2020; 319: F920-F929Crossref PubMed Google Scholar,21Kim J.Y. Jayne L.A. Bai Y. et al.Ribociclib mitigates cisplatin-associated kidney injury through retinoblastoma-1 dependent mechanisms.Biochem Pharmacol. 2020; 177113939Crossref Scopus (11) Google Scholar,22Pabla N. Gibson A.A. Buege M. et al.Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.Proc Natl Acad Sci U S A. 2015; 112: 5231-5236Crossref PubMed Scopus (70) Google Scholar Littermate controls were used in all studies. Experiments were carried out in a blinded fashion; the researchers evaluating, quantifying, and analyzing experimental outcomes were blinded to the genotype of the mice. Briefly, to cause IRI, mice were anesthetized by isoflurane and placed on a surgical platform, with body temperature monitored during the procedure. The skin was disinfected, kidneys were exposed, and bilateral renal pedicles were clamped for 30 minutes, followed by clamp removal and suturing to close the muscle and skin around the incision. To compensate for the fluid loss, 0.5 ml of warm sterile saline was administered via intraperitoneal injection. The sham (mock) groups underwent a similar procedure, but without bilateral clamping. Nephrotoxicity was triggered by a single cisplatin (30 mg/kg) intraperitoneal injection, as described previously.21Kim J.Y. Jayne L.A. Bai Y. et al.Ribociclib mitigates cisplatin-associated kidney injury through retinoblastoma-1 dependent mechanisms.Biochem Pharmacol. 2020; 177113939Crossref Scopus (11) Google Scholar The sham (vehicle) groups were injected with equal volumes of normal saline. To induce rhabdomyolysis, mice were injected with either 7.5 ml/kg 50% glycerol intramuscularly to the 2 hind legs or saline as a control. Following injury, blood was collected at 24 hours (IRI and rhabdomyolysis) or 72 hours (cisplatin) via cardiac puncture after carbon dioxide asphyxiation. Renal tissues were collected and processed for immunoblot, quantitative polymerase chain reaction, and histologic analysis, as described previously23Kim J.Y. Bai Y. Jayne L.A. et al.SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in renal tubular epithelial cells.J Biol Chem. 2020; 295: 16328-16341Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,24Bai Y. Kim J.Y. Bisunke B. et al.Kidney toxicity of the BRAF-kinase inhibitor vemurafenib is driven by off-target ferrochelatase inhibition.Kidney Int. 2021; 100: 1214-1226Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar and in the Supplementary Methods. Details regarding the generation of CRISPR/Cas9-mediated knockout cells are provided in the Supplementary Methods. Site-directed mutagenesis was carried out using methods described in our previous studies23Kim J.Y. Bai Y. Jayne L.A. et al.SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in renal tubular epithelial cells.J Biol Chem. 2020; 295: 16328-16341Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,25Zavorka Thomas M.E. Jeon J.Y. Talebi Z. et al.Gilteritinib-induced upregulation of S100A9 is mediated through BCL6 in acute myeloid leukemia.Blood Adv. 2021; 5: 5041-5046Crossref PubMed Scopus (3) Google Scholar,26van Oosterwijk J.G. Buelow D.R. Drenberg C.D. et al.Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia.J Clin Invest. 2018; 128: 369-380Crossref PubMed Scopus (25) Google Scholar and in the Supplementary Methods. Methods used to analyze protein and gene expression in cell culture and renal tissues have been described previously27Pabla N. Bhatt K. Dong Z. Checkpoint kinase 1 (Chk1)-short is a splice variant and endogenous inhibitor of Chk1 that regulates cell cycle and DNA damage checkpoints.Proc Natl Acad Sci U S A. 2012; 109: 197-202Crossref PubMed Scopus (41) Google Scholar, 28Pabla N. Huang S. Mi Q.S. et al.ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis.J Biol Chem. 2008; 283: 6572-6583Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 29Pabla N. Dong G. Jiang M. et al.Inhibition of PKCdelta reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer.J Clin Invest. 2011; 121: 2709-2722Crossref PubMed Scopus (117) Google Scholar and in the Supplementary Methods. Data in all the graphs are presented as mean with SD. Statistical analyses were carried out using GraphPad Prism. A P value < 0.05 was considered statistically significant. To calculate statistical significance between 2 groups, a 2-tailed unpaired Student's t test was performed. For comparisons among 3 or more groups, 1-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's multiple-comparisons test was used. No outliers were excluded, and all experiments were repeated at least 3 times. To identify the transcriptional factors that mediate Sox9 upregulation in RTECs under stress conditions associated with kidney injury, we used a modified version of a previously developed RNAi-based screening method.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar To this end, we used BUMPT cells18Sinha D. Wang Z. Price V.R. et al.Chemical anoxia of tubular cells induces activation of c-Src and its translocation to the zonula adherens.Am J Physiol Renal Physiol. 2003; 284: F488-497Crossref PubMed Scopus (61) Google Scholar because the high transient transfection efficiency (∼95%) of this murine RTEC cell line makes it a suitable model for high-throughput (siRNA) screening assays.12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar We generated stable BUMPT cells in which a luciferase-based reporter plasmid encoding the Sox9 promoter was introduced. Treatment of these cells with cisplatin results in a robust induction of Sox9 promoter activity (Supplementary Figure S1; Figure 1a). We then transiently transfected these cells with control siRNAs (nonspecific) or transcription factor–specific siRNAs (set of 4 pooled per target, 1837 targets), followed by cisplatin treatment, and assessment of Sox9 promoter activity using luciferase reporter assay (Figure 1b). The primary screen was carried out in triplicate, and subsequent data analysis identified 3 hit candidates, namely interferon regulatory factor 1 (Irf1), SMAD family member 4 (Smad4), and Zinc finger protein 24 (Zfp24; Figure 1c and d). Our secondary screening in a human RTEC cell line—human kidney (HK)–2 cells, transfected with a plasmid encoding human SOX9 promoter luciferase plasmid—showed that among these candidates, knockdown of ZFP24 had the highest effect in suppressing stress-induced SOX9 promoter activity (Figure 1e). For final validation, we generated HK-2 cells in which the ZFP24 gene was knocked out using a CRISPR/Cas9 lentiviral construct. We then performed add-back experiments in which empty or wild-type human ZFP24 expression vectors were introduced in the knockout cells (Figure 1f). Subsequent, functional assays showed that ZFP24 knockout suppresses stress-induced SOX9 promoter activity, which is reversed by reintroduction of ZFP24 (Figure 1g). Given that the role of Zfp24 in kidneys remains unknown, we initially examined its expression under normal and disease conditions. Moreover, Sox9 expression is undetectable in the RTECs of adult murine kidneys; however, under conditions of stress, such as ischemia–reperfusion injury (IRI), cisplatin nephrotoxicity, and rhabdomyolysis-associated AKI, a profound increase occurs in the mRNA11Kumar S. Liu J. Pang P. et al.Sox9 activation highlights a cellular pathway of renal repair in the acutely injured mammalian kidney.Cell Rep. 2015; 12: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,30Kirita Y. Wu H. Uchimura K. et al.Cell profiling of mouse acute kidney injury reveals conserved cellular responses to injury.Proc Natl Acad Sci U S A. 2020; 117: 15874-15883Crossref PubMed Scopus (158) Google Scholar and protein12Kim J.Y. Bai Y. Jayne L.A. et al.A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.Nat Commun. 2020; 11: 1924Crossref PubMed Scopus (17) Google Scholar,13Kim J.Y. Bai Y. Jayne L.A. et al.Involvement of the CDKL5-SOX9 signaling axis in rhabdomyolysis-associated acute kidney injury.Am J Physiol Renal Physiol. 2020; 319: F920-F929Crossref PubMed Google Scholar,23Kim J.Y. Bai Y. Jayne L.A. et al.SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in renal tubular epithelial cells.J Biol Chem. 2020; 295: 16328-16341Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar levels of Sox9. So, we first sought to examine the expression of Zfp24 under normal and AKI conditions. To induce AKI, C57BL/6J mice underwent bilateral ischemic surgery or were injected with a single 30-mg/kg intraperitoneal dose of cisplatin, or rhabdomyolysis was induced by glycerol injection (7.5 ml/kg 50% glycerol) in the hind-leg muscles. The extent of renal damage was determined by measurement of blood urea nitrogen and serum creatinine (Figure 2a and b ). With use of ischemia–reperfusion and rhabdomyolysis, renal impairment occurs 24 hours postsurgery, whereas in the cisplatin-associated kidney injury models, renal impairment is observed 72 hours post-injection. Gene and protein analysis of kidney tissues at these time-points showed that Zfp24 expression does not appreciably change during AKI (Supplementary Figure S2; Figure 2c and d). Zfp24 contains a SCAN (named after SRE-ZBP, CTfin51, AW-1, and Number 18 cDNA) domain and 4 DNA-binding C2H2 zinc finger domains that are interspersed by linkers that contain phosphorylation sites.15Elbaz B. Aaker J.D. Isaac S. et al.Phosphorylation state of ZFP24 controls oligodendrocyte differentiation.Cell Rep. 2018; 23: 2254-2263Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Linker phosphorylation is a mechanism that can result in the deactivation of the C2H2 proteins because phosphorylation introduces a negative charge to the DNA-binding domain of the protein and hence reduces its affinity to DNA. To examine the phosphorylation status of Zfp24, we performed immunoprecipitation studies in normal and injured kidneys. Our results (Figure 2e and f) show that Zfp24 is present in the phosphorylated state in normal renal tubular epithelial cells. However, Zfp24 is dephosphorylated and activated early during the development of AKI. Time-course experiments in the IRI model showed that Zfp24 dephosphorylation occurs within 1 hour postreperfusion and precedes Sox9 upregulation (Supplementary Figure S3). We next sought to examine the consequence of RTEC-specific Zfp24 gene deletion on normal renal function and the severity of AKI. To this end, we generated Zfp24 conditional knockout mice (Zfp24PT–/–) by crossing the Zfp24-floxed15Elbaz B. Aaker J.D. Isaac S. et al.Phosphorylation state of ZFP24 controls oligodendrocyte differentiation.Cell Rep. 2018; 23: 2254-2263Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar mice with the Ggt1-Cre31Iwano M. Plieth D. Danoff T.M. et al.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1739) Google Scholar mice. The Ggt1-driven Cre is expressed in RTECs 7–10 days after birth, post–completion of renal development.31Iwano M. Plieth D. Danoff T.M. et al.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1739) Google Scholar Initial studies showed that normal renal function (Supplementary Figure S4) is not affected by Zfp24 deficiency. Remarkably, Zfp24 gene ablation in RTECs (Figure 3a) markedly increased kidney damage during IRI-, cisplatin-, and rhabdomyolysis-associated AKI (Figure 3b–f). Zfp24 deficiency also resulted in increased cell death activation as measured by Caspase-3 activation (Supplementary Figure S5). To determine the effect of RTEC-specific Zfp24 gene ablation on overall survival post-injury, we used the rhabdomyolysis model for long-term studies. As shown in Supplementary Figure S6, rhabdomyolysis-associated AKI was not severely fatal in the control mice, and