The Roles And Mechanisms Of FGF2 And DNA Methylation In Post-injury Repair Under Cisplatin Exposure

BackgroundAcute kidney injury(AKI)is a common clinical disease caused by pathogenic factors such as ischemia and nephrotoxicity.It has epidemiological characteristics of high morbidity and mortality,which seriously threatens human health.The severity of AKI is closely related to the prognosis.Mild AKI can make the kidneys recover completely,while severe or repeated AKI can lead to kidney repair disorders,leading to renal interstitial fibrosis and chronic inflammation,which can progress to chronic kidney disease(CKD).Therefore,kidney repair is the key to AKI recovery.Currently,no effective treatment can improve repair impairment after AKI,so conducting preclinical research related to postAKI repair is imperative.Cisplatin is a nephrotoxic chemotherapy drug.Patients receiving multiple cycles of cisplatin therapy repeatedly experienced AKI injury and developed CKD,so repeated low-dose cisplatin(RLDC)mouse model better simulated the progression of AKI to CKD in clinical settings.Multiple cell types in the kidney are involved in the progression of AKI to CKD,among which the proximal tubule is thought to be the driving force and plays a central role.Studies have reported that fibroblast growth factor 2(FGF2)is significantly increased after kidney injury.However,whether it is secreted by renal tubular cells in RLDC and the role it plays in RLDC remains to be explored.Damaged renal tubules can also change DNA methylation patterns to regulate renal injury and fibrosis,which is mediated by DNA methyltransferases(DNMTs).In cisplatin-induced AKI,proximal tubular Dnmt1 knockout can aggravate renal injury.In multiple CKD models,DNMTs inhibitors can hypomethylate the renoprotective protein Klotho in renal tubular cells,thereby protecting against renal fibrosis.However,the role of DNA methylation in RLDC-induced post-injury repair remains unknown.ObjectiveThe objective of this article is to investigate the roles and mechanisms of FGF2 and DNA methylation in post-injury repair under cisplatin exposure to explore the therapeutic targets.Methods1.In vivo,the 12 w male C57BL/6J mice were treated with cisplatin for four consecutive weeks(8mg/kg cisplatin was injected intraperitoneally once/week)to establish the RLDC mouse model.The renal function and pathological changes were observed.In vitro,the proximal renal tubular epithelial cells were treated with cisplatin for four consecutive days(2μM cisplatin for 7 hours/day)to establish the RLDC cell model.The cell morphology and profibrotic phenotype were observed.2.The m RNA and protein levels of FGF2 in the kidneys and proximal tubular cells after RLDC were detected.In vivo,a global Fgf2 knockout mouse model was established to observe its effects on renal fibrosis,fibroblast activation,and inflammatory cell infiltration of RLDC.In vitro,the secretion of FGF2 in the medium was detected,and the recombinant FGF2 antibody was used to observe the effect of blocking FGF2 on the activation and proliferation of fibroblasts.3.The RRBS sequencing was used to detect genome-wide DNA methylation pattern change after RLDC based on the single nucleotide level.The m RNA and protein levels of DNMTs in mouse kidneys and proximal tubular cells are detected.In vivo,the DNMTs inhibitor 5-aza was administered to mice after RLDC treatment to observe its effect on the long-term outcome of cisplatin nephrotoxicity.The mouse model of proximal tubule-specific Dnmt3 a knockout was established to observe its effects on tubular dedifferentiation,proliferation,regeneration,interstitial fibrosis,and inflammation infiltration.In vitro,the pharmacological DNMT inhibitor 5-aza and Dnmt3 a knockdown were used to observe cell injury and profibrotic phenotype shift after RLDC.Results1.In vivo,RLDC induced pathological changes such as renal tubular injury and degeneration,renal tubular atrophy,interstitial fibrosis,and inflammation in mice,resulting in decreased renal function.In vitro,RLDC induced typical profibrotic phenotype changes but few apoptosis in renal tubular cells.2.RLDC induces the production and secretion of FGF2 in BUMPT cells,and its secretion can promote the activation and proliferation of fibroblasts,which the FGF2-neutralizing antibody can reverse.Fgf2 knockout partially attenuated RLDC-induced renal function decline,renal atrophy,fibrosis,and inflammation in mice.3.RLDC induced DNA hypermethylation changes in mouse kidney and DNMTs increase in mouse kidney and tubular cells.In vivo,DNMTs inhibitor 5-aza and proximal tubular Dnmt3 a knockout both promoted the proliferation and repair of renal tubular cells after injury,alleviated RLDC-induced renal fibrosis and inflammation,and improved renal function.In vitro,both the pharmacological DNMT inhibitor 5-aza and Dnmt3 a knockdown significantly attenuated the profibrotic phenotype transition of renal tubular cells.ConclusionsThis study simulated the clinical repeated cisplatin renal injury model in vivo and in vitro.FGF2 is produced by injured renal tubular cells to act as a paracrine factor in maladaptive kidney repair after cisplatin injury.Specific DNMT3 A in proximal tubular epithelial cells regulates tubular cell dedifferentiation,proliferation,and profibrotic phenotype transition after cisplatin injury,promoting renal maladaptive repair and fibrosis.This study provides a theoretical basis to search for therapeutic targets in clinical cisplatin renal injury in the future.