The transcription factor Fosl1 preserves Klotho expression and protects from acute kidney injury

Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. In vivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI. Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. In vivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI. Translational StatementFosl1 is 1 of 7 canonical components of the activator protein-1 (AP-1) transcription factor. While it is known that AP-1 is activated early in the course of acute kidney injury, the function of individual components is unknown. We have now uncovered evidence that supports that Fosl1 is increased in an adaptive manner in proximal tubular cells during acute kidney injury where it plays a kidney protective role, promoting the expression of the kidney protective protein Klotho and decreasing proinflammatory factors. These findings identify a kidney protective Fosl1-Klotho axis that may be exploited therapeutically in acute kidney injury and raises the spectrum of nephrotoxicity for cancer therapy targeting Fosl1. Fosl1 is 1 of 7 canonical components of the activator protein-1 (AP-1) transcription factor. While it is known that AP-1 is activated early in the course of acute kidney injury, the function of individual components is unknown. We have now uncovered evidence that supports that Fosl1 is increased in an adaptive manner in proximal tubular cells during acute kidney injury where it plays a kidney protective role, promoting the expression of the kidney protective protein Klotho and decreasing proinflammatory factors. These findings identify a kidney protective Fosl1-Klotho axis that may be exploited therapeutically in acute kidney injury and raises the spectrum of nephrotoxicity for cancer therapy targeting Fosl1. Acute kidney injury (AKI) is characterized by a sudden decline of kidney function.1Bellomo R. Kellum J.A. Ronco C. Acute kidney injury.Lancet. 2012; 380: 756-766Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar AKI increases the risk of chronic kidney disease, which is among the fastest-growing global causes of death.2Chawla L.S. Eggers P.W. 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Acute kidney injury.Lancet. 2019; 394: 1949-1964Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar An improved understanding of the molecular mechanisms of AKI will allow developing novel therapeutic approaches. AKI is characterized by tubular cell injury leading to a proinflammatory stress response and cell dedifferentiation followed by tubular cell proliferation and recovery of kidney structure and function.5Ronco C. Bellomo R. Kellum J.A. Acute kidney injury.Lancet. 2019; 394: 1949-1964Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar Inflammatory mediators such as tumor necrosis factor superfamily, member 12 (TWEAK) amplify kidney injury through recruitment of inflammatory cells, induction of regulated necrosis, and downregulation of kidney protective factors such as Klotho.6Moreno J.A. Izquierdo M.C. 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Grantham J.J. et al.Sequential protooncogene expression in regenerating kidney following acute renal injury.J Biol Chem. 1989; 264: 8389-8393Abstract Full Text PDF PubMed Google Scholar However, the precise role of the AP-1 transcription factor and of individual components in AKI remained poorly understood. We used kidney transcriptomics to identify novel players in the pathogenesis of AKI that may be targeted therapeutically. Specifically, we focused on highly expressed and active transcription factors and identified AP-1 as a highly active transcription factor and Fosl1 as having functions in tissue injury and repair and being highly expressed in AKI and in murine proximal tubular cells exposed to a mediator of AKI. We now report that Fosl1 is part of an adaptive kidney protective response during AKI in proximal tubular cells that preserves Klotho and limits the inflammatory response and the severity of tubular cell injury and AKI. Figure 1 summarizes the experimental approach. Kidney and kidney cell transcriptomics identified key transcription factors upregulated in nephrotoxic AKI and differentially expressed genes encoding key transcription factors relevant for proximal tubular cells. The AP-1 transcription factor was upregulated in AKI (Supplementary Tables S1 and S2) and Fosl1 was the only Fos family gene upregulated >1.5-fold by the cytokine TWEAK in cultured proximal tubule cells and during both cisplatin and folic acid (FA)–induced AKI (Supplementary Table S3). TWEAK is a key mediator of AKI.6Moreno J.A. Izquierdo M.C. Sanchez-Niño M.D. et al.The inflammatory cytokines TWEAK and TNFα reduce renal klotho expression through NFκB.J Am Soc Nephrol. 2011; 22: 1315-1325Crossref PubMed Scopus (296) Google Scholar, 7Sanz A.B. Izquierdo M.C. Sanchez-Niño M.D. et al.TWEAK and the progression of renal disease: clinical translation.Nephrol Dial Transplant. 2014; 29: i54-i62Crossref PubMed Scopus (80) Google Scholar, 8Martin-Sanchez D. Guerrero-Mauvecin J. Fontecha-Barriuso M. et al.Bone marrow-derived RIPK3 mediates kidney inflammation in acute kidney injury.J Am Soc Nephrol. 2022; 33: 357-373Crossref PubMed Scopus (12) Google Scholar, 9Martin-Sanchez D. Fontecha-Barriuso M. Carrasco S. et al.TWEAK and RIPK1 mediate a second wave of cell death during AKI.Proc Natl Acad Sci U S A. 2018; 115: 4182-4187Crossref PubMed Scopus (95) Google Scholar Fosl1 was prioritized for detailed expression and functional studies. Extended Supplementary Methods provide further detail. C57BL/6 mice carrying the floxed Fosl1 gene (Fosl1f/f mice, Erwin F. Wagner MGI:3055991 aka Fosl1tm2Wag)33Cowley Jr., B.D. Chadwick L.J. Grantham J.J. et al.Sequential protooncogene expression in regenerating kidney following acute renal injury.J Biol Chem. 1989; 264: 8389-8393Abstract Full Text PDF PubMed Google Scholar,34Eferl R. Hoebertz A. 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Calvanese V. et al.Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells.PLoS One. 2010; 5e10192Crossref PubMed Scopus (90) Google Scholar Statistical analysis was performed using GraphPad Prism Software 8. Results are expressed as mean ± SD. Significance (P < 0.05) was assessed by Student's t-test for 2 groups of data and analysis of variance for 3 or more groups with Bonferroni post hoc correction. Upstream regulator analysis of kidney transcriptomics from murine cisplatin-induced AKI identified AP-1 as being among the top upregulated transcription factors (Supplementary Table S1). Furthermore, transcription factor ChIP-X enrichment analysis 3 identified 7 of 20 top-ranked transcription factors as AP-1 heterodimer components (Supplementary Table S2). Of these, Fosl1 and Fosl2 mRNA were upregulated in both FA- and cisplatin-induced AKI, and Fosl1 was additionally upregulated in proximal tubular cells exposed to TWEAK, a cytokine that contributes to nephrotoxic and IRI-AKI9Martin-Sanchez D. Fontecha-Barriuso M. Carrasco S. et al.TWEAK and RIPK1 mediate a second wave of cell death during AKI.Proc Natl Acad Sci U S A. 2018; 115: 4182-4187Crossref PubMed Scopus (95) Google Scholar,49Ruiz-Andres O. Suarez-Alvarez B. Sánchez-Ramos C. et al.The inflammatory cytokine TWEAK decreases PGC-1α expression and mitochondrial function in acute kidney injury.Kidney Int. 2016; 89: 399-410Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar (Supplementary Table S3). No Jun gene was upregulated simultaneously in cisplatin-AKI, FA-AKI, and TWEAK-MCT cells at false discovery rate <0.05 level. Thus, we focused on unraveling the regulation of the expression and activation as well as function of Fosl1 in proximal tubular cells and in AKI. Reverse transcription quantitative polymerase chain reaction confirmed transcriptomics findings of increased kidney Fosl1 mRNA expression in cisplatin-induced AKI (Figure 2a). Fosl1 mRNA was already increased at 6 hours, preceding the increase in plasma creatinine and urea (Figure 2b and c), and peaked at 72 hours. Kidney Fosl1 protein levels (Figure 2d) and nuclear Fosl1 and phosphorylated Fosl1 (Figure 2e) were also increased in cisplatin-induced AKI consistent with Fosl1 activation.50Young M.R. Nair R. Bucheimer N. et al.Transactivation of Fra-1 and consequent activation of AP-1 occur extracellular signal-regulated kinase dependently.Mol Cell Biol. 2002; 22: 587-598Crossref PubMed Scopus (104) Google Scholar Immunohistochemistry localized the increased expression of Fosl1 to tubular cell nuclei (Figure 2f) and nuclear Fosl1 also increased progressively from 6 hours (Supplementary Figure S1A). Specifically, Fosl1 was observed in tubular cell nuclei staining for the proliferation marker PCNA (Supplementary Figure S1B). Kidney c-Jun protein also increased during cisplatin-induced AKI and increased mRNA preceding increased protein levels (Supplementary Figure S2A and B). Reverse transcription quantitative polymerase chain reaction confirmed increased kidney Fosl1 mRNA expression in FA-AKI (Supplementary Figure S3A) where Fosl1 also localized to tubular cell nuclei (Supplementary Figure S3B) and was associated to increased c-Jun (Supplementary Figure S2C and D). Supporting the clinical translation of the findings, immunohistochemistry showed multiple FOSL1-positive tubular cells in human AKI but not in normal kidney tissue (Supplementary Figure S4A). In the urinary sediment of patients with AKI, FOSL1 mRNA levels were up to 40-fold higher than in controls, and FOSL1 protein colocalized with proximal tubular cell markers while FOSL1-positive proximal tubular cells were not observed in control urine sediments (Supplementary Figure S4B and C). To test the functional relevance of Fosl1 in kidney disease, we generated mice with proximal tubule–specific Fosl1 deletion, in which Fosl1 mRNA could not be amplified in healthy kidneys (Supplementary Figure S5). Fosl1 immunohistochemistry confirmed selective deletion of Fosl1 in proximal tubules from Fosl1f/f PEPCKCre/− (Fosl1Δtub) mice, even during AKI, compared to multiple proximal tubular cells expressing Fosl1 in WT (Fosl1f/f) mice with AKI (Supplementary Figure S6). The overall baseline kidney histology of Fosl1Δtub mice was unremarkable and plasma creatinine and urea were normal. However, Fosl1Δtub mice developed a more severe cisplatin-induced AKI characterized by higher plasma creatinine and urea levels and more severe histological injury than in WT littermates with AKI at 72 hours (Figure 3a and b, Supplementary Figure S7A). Furthermore, Fosl1Δtub mice with AKI also displayed kidney expression of chemokine (monocyte chemoattractant protein-1; regulated on activation, T-cell expressed, and secreted [RANTES]) mRNA (Figure 3c and d), tubular cell tumor necrosis factor-α (TNF-α) immunostaining (Supplementary Figure S7A) and infiltration by interstitial macrophages (Figure 3e). These results suggest that Fosl1 upregulation in nephrotoxic AKI is an adaptive response and that precluding this adaptive response activates an amplification loop of inflammation and kidney injury. Next, we examined whether proximal tubular Fosl1 deficiency modulated AP-1 transcriptional activity in vivo. Confirming bioinformatics predictions, AKI was associated with increased whole kidney AP-1 transcriptional activity that was already observed at 24 hours and peaked at 72 hours. In Fosl1Δtub mice with AKI, peak AP-1 transcriptional activity at 72 hours was higher than in WT littermates with AKI (Figure 4a). While this increased AP-1 activity was not associated to increased c-Jun mRNA levels (Figure 4b), increased nuclear p-Ser73-c-Jun staining was observed at 72 hours (Figure 4c). Thus, proximal tubular cell Fosl1 deficiency did not result in an overall decrease in kidney AP-1 activity, rather it was associated with an increased AP-1 and c-Jun activity. The impact of proximal tubular cell Fosl1 deficiency was studied in murine nephrotoxic AKI induced by FA (72-hour time point). As for cisplatin-induced AKI, Fosl1Δtub mice had more severe FA-AKI as assessed by plasma creatinine or urea (Supplementary Figure S3C and D). To provide insight into the drivers of the more severe AKI in Fosl1Δtub mice, we generated kidney transcriptomics profiles of Fosl1Δtub and WT mice with cisplatin AKI. RNA-sequencing revealed 146 differentially expressed genes (P < 0.05), 73 upregulated and 73 downregulated, in Fosl1Δtub mice with AKI when compared to WT littermates with AKI (Supplementary Table S4, Supplementary Figure S8A). Principal component analysis clearly separated Fosl1Δtub AKI and WT AKI mice (Supplementary Figure S8B). The top-10 downregulated genes, all fold-change <0.50, included Kl encoding Klotho (fold-change 0.48) and Cyp24a1 (fold-change 0.28) encoding the 25-hydroxy-vitamin-D-24-hydroxylase, an enzyme downstream of FGF23/Klotho signaling in proximal tubular cells whose expression is known to decrease under conditions of Klotho deficiency (Supplementary Figure S8C). None of the other top-10 downregulated genes has a known function in AKI (according to a PubMed search on August 31, 2022). Upregulated genes included c-Fos and FosB, potentially contributing to higher AP-1 activity. AKI is characterized by decreased expression of the anti-inflammatory, nephroprotective, and antiaging factor Klotho, and preventing Klotho downregulation prevents AKI.11Liao H.K. Hatanaka F. Araoka T. et al.In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation.Cell. 2017; 171: 1495-1507.e15Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 12Hu M.C. Shi M. Gillings N. et al.Recombinant α-Klotho may be prophylactic and therapeutic for acute t