YTHDF1 Promotes Renal Fibrosis Progression Via Up-regulating YAP

Objective: Kidney fibrosis is the common pathological manifestation of end-stage renal disease developed from all chronic kidney diseases,but there is no effective strategy to retard this progress at present.Kidney fibrosis is pathologically characterized by the transformation of a variety of cells into the myofibroblasts that produce and secrete excess extracellular matrix(ECM)in the renal interstitium and glomeruli,accompanied by the infiltration of immune cells and release of immune factors.Although YAP has been regarded as a crucial modulator in myofibroblast transformation and blocking the function of YAP can suppress the process of kidney fibrosis,its upstream regulators remain unknown.Studies have shown that m6 A modulators affect tumor growth and metastasis by regulating the expression of YAP in non-small cell lung carcinoma,however,whether m6 A is involved in renal fibrosis via regulating YAP remains unclear.Among these m6 A modulators,the YTH domain family(YTHDF),as the ‘reader' of m6 A,has been identified to recognize and bind m6 A modified mRNA,thereby regulating the splicing,translation,and stability of target mRNA.The present study revealed that the expression of the YTHDF1 was up-regulated in human fibrotic kidneys and strongly correlated with YAP via bioinformatics analysis.Therefore,we aimed to further investigate the results of bioinformatics analysis that YTHDF1 participates in the progression of renal interstitial fibrosis by regulating YAP through in vivo and in vitro knockdown experiments.Methods: 1.The present study firstly visualized the expression of m6 A modulators in the human fibrotic kidneys from GEO database via R software and Perl programming language,and further analyzed the correlation of the screened modulator(YTHDF1)with YAP.2.Gene set enrichment analysis(GSEA)was used to predict the relation of YTHDF1 to YAP.3.Immunohistochemical staining and immunofluorescent staining show the expression and colocalization of YTHDF1 and YAP in human healthy and fibrotic kidneys.4.To investigate the role of YTHDF1 in kidney fibrosis progression,we then established three types of mouse kidney fibrosis models,including unilateral ureteral obstruction(UUO),high-dose folic acid(FA),and unilateral ischemic reperfusion injury(U-IRI).5.The alteration of kidney pathological feature and function during different stages in three models was studied by HE,Masson,PAS staining,and immunohistochemical staining.6.Based on three models,we then detected YTHDF1 in control and fibrotic kidneys via immunohistochemical staining,q RT-PCR,and western blotting.Meanwhile,enzyme-linked immunosorbent assay(ELISA)was used to determine the total m6 A level in the UUO model.7.In vitro,YTHDF1 was silenced by small interfering RNA in the kidney fibrosis cell model,and the expression of myofibroblast biomarker,fibronectin,and YAP was also detected.8.In vivo,YTHDF1 was also knocked down in UUO mice via si RNA,and we used immunohistochemical staining,q RT-PCR,and western blotting to detect the expression and localization of YAP in mRNA and protein levels.Meanwhile,we utilized HE,Masson staining,immunohistochemical staining,western blotting,and serum biochemical analysis to observe the pathological and functional change,as well as the expression of ?-SMA,collagen I,and fibronectin in UUO mice that were injected si RNA through the tail vein.9.RIP detected the interaction between YTHDF1 and YAP mRNA.10.In vitro,gene overexpression assay was conducted to detect the effects of YTHDF1 on ?-SMA,fibronectin and YAP,and the expression of fibrosis related proteins(?-SMA and fibronectin)was detected when YAP was knocked down and YTHDF1 was overexpressed.Results: 1.Bioinformatic analysis identified that YTHDF1,a modulator of m6 A methylation,were highly expressed in human fibrotic kidneys.GSEA revealed that YTHDF1 was involved in the up-regulation of YAP.Meanwhile,the correlation analysis revealed that YTHDF1 was positively correlated with YAP.2.Immunohistochemical staining revealed that YTHDF1 and YAP were up-regulated and colocalized at renal tubular cells and glomerulus of human fibrotic kidneys.3.The detection of renal tubular transport function showed that the damage of proximal tubules appeared earlier than the collecting ducts in the kidney fibrosis models induced by high-dose FA and UUO.The expression of aquaporin(AQP)-1 and-2 in tubular cells increased at the acute injury stage,while AQP-1 and AQP-2 were no longer expressed in the severely damaged tubular cells at the later stage(14d and later).4.ELISA showed that the total m6 A level of model groups was higher than that of the control in UUO mice.5.In three types of mouse models,numerous ?-smooth muscle actin positive myofibroblasts and excess extracellular matrix formed in the peritubular interstitial region on day 14.Meanwhile,YTHDF1 was significantly up-regulated in the model group and positively correlated with the expression of myofibroblast markers(?-SMA)and ECM components,such as collagen I and fibronectin.6.YTHDF1 knockdown inhibited the expression of myofibroblast biomarker,fibronectin,and YAP in the kidney fibrosis cell model.7.Consistent with this notion,YTHDF1 knockdown significantly downregulated the expression of YAP in protein level and its downstream connective tissue growth factor(CTGF)in UUO mice.In addition,the expression of myofibroblast biomarker and ECM components was significantly downregulated when YTHDF1 was inhibited.Meanwhile,YTHDF1 knockdown improved renal pathological injury and reduced serum creatinine and urea nitrogen levels in UUO mice.8.RIP revealed YTHDF1 interacted with YAP mRNA.9.In vitro,YTHDF1 overexpression promoted the expression of ?-SMA,fibronectin and YAP,but the expression of fibrosis related proteins(?-SMA and fibronectin)was also inhibited when YAP was knocked down.Conclusion: The present study suggests that YTHDF1 and YAP are highly expressed in human and mouse fibrotic kidneys,and their expression shows a positive correlation.Meanwhile,YTHDF1 knockdown significantly inhibits the expression of YAP and improves the renal pathology and function of UUO-induced fibrotic kidneys.Combined RIP assay and YTHDF1 overexpression in vitro,the present study suggests that YTHDF1 binds to YAP mRNA,thereby upregulating the expression of YAP and promoting the progression of renal fibrosis.Accordingly,the present study provides experimental basis for the target therapy of renal fibrosis.