FGF2 Promotes Renal Interstitial Fibrosis Through The STAT3-transactivated YAP1 Transcription

Background:Tubulointerstitial fibrosis(TIF)is a major factor influencing the progression of chronic Kiney disease(CKD)to end stage renal disease(ESRD).At present,the molecular mechanism causing TIF is not fully understood,and there is a lack of effective treatment in clinical practice.Therefore,further elucidation of the pathogenesis of TIF is of great significance for the prevention and treatment of the progression of CKD.The excessive deposition of extracellular matrix(ECM)is a characteristic of TIF,and the epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells is an important factor in the increase of ECM production.FGF2 is involved in the EMT process of tumor and organ fibrosis,and its expression is increased in a variety of renal diseases.However,its role and mechanism in the pathogenesis of TIF are still unclear.It has been found that FGF2 can activate signal transduction and transcriptional activation factor 3(STAT3)in a variety of disease states,but it has not been reported in TIF.STAT3 is a transcription factor,and biological software was used to predict that STAT3 might have binding sites with the promoter region of Yes-related protein 1(YAP1)to regulate the transcription of YAP1.Therefore,this study intends to take the renal puncture section of TIF and unilateral ureteral obstruction(UUO)rats and renal tubular epithelial cells as the research objects.Therefore,this study used clinical renal biopsy section of TIF,UUO rats and renal tubular epithelial cells to investigate whether FGF2 can regulate the transcription of YAP1 by activating STAT3 to promote the occurrence of EMT in renal tubular epithelial cells and lead to renal interstitial fibrosis.To observe whether knockdown of FGF2 in vivo can delay the progression of TIF in order to further elucidate the pathogenesis of renal tubulointerstitial fibrosis and explore new effective targets for the prevention and treatment of CKD.Objective:1.To observe the expression of FGF2 in renal tissues of TIF to clarify the role of FGF2 in renal tubulointerstitial fibrosis.2.To further explore whether FGF2 can promotes renal interstitial fibrosis through the STAT3-transactivated YAP1 transcription,thereby promoting the EMT transformation of renal tubular epithelial cells and eventually leading to renal interstitial fibrosis.3.To observe whether FGF2 knockdown can inhibit the progression of renal tubulointerstitial fibrosis,so as to explore a new target for the prevention and treatment of CKD.Methods:1.To observe the expression and function of FGF2 in renal tissue of TIFRenal biopsy from TIF patients were collected and unilateral ureteral obstruction(UUO)was conducted to establish rats TIF model.The histological morphology and collagen deposition were observed by HE staining and Masson staining;FGF2 was detected by Immunohistochemistry and Western blot assays.NRK-52 E cells were stimulated by FGF2 cytokine,the expression of α-SMA,Col Ⅲ proteins were detected by Immunofluorescence and Western blot assays to observe the EMT of NRK-52 E cells stimulated by FGF2.Adeno-associated virus carrying FGF2 knockdown plasmid was injected in situ into the kidney of mice,and then UUO was performed,Immunohistochemistry,Immunofluorescence and Western blot were used to observe the changes in the expression of FGF2,Col I,α-SMA proteins in renal tissues after knockdown of FGF2 in vivo,and observe the effect of FGF2 knockdown on the process of TIF.2.To observe whether FGF2 promotes TIF progression by activating the STAT3 signaling pathwayThe expression of p-STAT3 in renal tissue sections of TIF patients by Immunohistochemistry,STAT3 and p-STAT3 proteins in UUO renal tissues were observed by Immunohistochemistry and Western blot.The expression of STAT3 and p-STAT3 protein was detected by Western blot and cell Immunofluorescence in NRK-52 E cells which were stimulated by FGF2 cytokines.After si-STAT3 and STATTIC were used,the effect of knockdown or suppression of STAT3 activation on EMT of NRK-52 E cells stimulated by FGF2 was observed by Western blot.The receptor pathway on STAT3 protein activation stimulated by FGF2 was observed using type I and type I FGFR-receptor inhibitors detected by Western blot.Immunohistochemical,Immunofluorescence and Western blot were used to observe the protein expression changes of STAT3 and p-STAT3 in mice after knockdown of FGF2 in vivo.3.To observe whether FGF2 can finally regulate the process of TIF by promoting the transcription of YAP1 after activating STAT3 signaling pathwayImmunohistochemistry and Western blot were used to observe the expression of YAP1 in renal tissue sections of TIF patients and UUO renal tissues.Western blot and Cell Immunofluorescence assays were used to detect the expression of YAP1 protein in NRK-52 E cells stimulated with FGF2;Western blot was used to observe the effect of si-STAT3 and STATTIC on the expression of YAP1 protein in NRK-52 E cells stimulated by FGF2;The expressions of Total-YAP1 p-YAP1 and Active-YAP1 in the cytoplasm and nucleus of NRK-52 E cells stimulated by FGF2 were detected by Western blot.The m RNA levels of STAT3 and YAP1 after UUO were detected by q PCR and the correlation between them was observed.The binding site and strength between STAT3 and YAP1 promoter in primary renal tubular epithelial cells of rats were detected by Double Luciferase reporter assay and Chromatin Immunoprecipitation assay(Ch IP);Western blot and q PCR were conducted to detect the expression of YAP1 protein and m RNA after overexpression of STAT3 in NRK-52 E cells;In addition,the expression of YAP1,CTGF,α-SMA in NRK-52 E cells stimulated by FGF2 were detected by Western blot after si-YAP1 was transfected into cells.Immunohistochemical,Immunofluorescence and Western blot were used to observe the changes of YAP1 protein expression after knockdown of FGF2 in mice.Results:1.The expression of FGF2 was significantly increased in TIF renal tissues and promoted the progression of TIFBoth HE and Masson staining could observe that there were inflammatory cell infiltration and collagen deposition around the renal tubules in renal sections of TIF patients and rats after UUO operation;The protein of FGF2 was significantly increased in TIF kidneys of patients and rats detected by Immunohistochemistry and Western blot methods(P<0.05).The protein of α-SMA and Col Ⅲ-were significantly higher(P<0.05)in FGF2 cytokine stimulated group by Immunofluorescence and Western blot methods,these results suggested that FGF2 can promote EMT changes of NRK-52 E cells.FGF2 knockdown could significantly reduce the expression of Col I and α-SMA protein(P<0.05)in renal tissues detected by Immunohistochemistry,Immunofluorescence and Western blot,suggesting that FGF2 knockdown could slow down the process of TIF.2.FGF2 promotes TIF progression by activating the STAT3 signaling pathwayThe expression of STAT3 and p-STAT3 in sections of TIF patients and in UUO renal tissue was significantly increased(P<0.05)detected by Immunohistochemical and Western blot.NRK-52 E cells stimulated by FGF2 showed no significant change in STAT3 protein but significantly increased p-STAT3 protein expression(P<0.05)detected by Western blot and Immunofluorescence.Western Bolt showed that si-STAT3 and STATTIC could significantly reduce the EMT transformation in NRK-52 E cells induced by FGF2(P<0.05).We found FGF2 activated STAT3 by FGFR2 receptors on the cell membrane when using type Ⅰ and type Ⅱ FGFR receptor inhibitor.FGF2 knockdown could significantly reduce the expression of STAT3 and p-STAT3 protein(P<0.05)in renal tissues detected by Immunohistochemistry,Immunofluorescence and Western blot.3.FGF2 activates STAT3 signaling pathway,promotes the transcription of YAP1 and ultimately regulates the process of TIFThe expression of YAP1 in both sections of TIF patients and UUO kidney tissue was significantly higher than that of normal group detected by Immunohistochemistry and Western blot analysis(P<0.05).YAP1 protein was significantly increased in NRK-52 E cells stimulated by FGF2 detected by Western blot and Cell Immunofluorescence assay(P<0.05).The protein of Total-YAP1 and Active-YAP1 in cytoplasm and nucleus of NRK-52 E cells were significantly increased using Western blot assay(P<0.05).Western Blot showed that the expression of YAP1 protein in NRK-52 E cells stimulated by FGF2 decreased significantly after treatment with si-STAT3 or STATTIC(P<0.05).The m RNA levels of STAT3 and YAP1 detected by q PCR after UUO were positively correlated(P<0.05).The dual Luciferase reporter gene assay(Luciferase)and chromatin immunoprecipitation(Ch IP)assay were used to detect that STAT3 could bind to the RE1 and RE3 sequences of YAP1 promotor to regulate the transcription of YAP1 in rat primary renal tubular epithelial cells.After STAT3 overexpression in NRK-52 E cells,the expression of YAP1 protein and m RNA was significantly increased(P<0.05).NRK-52 E cells were transfected with si-YAP1,and Western blot showed that knockdown of YAP1 could significantly reduce the increase of CTGF and fibrosis index α-SMA Col Ⅲ induced by FGF2-stimulated cells(P<0.05).After knockdown of FGF2 in mice,the expression of YAP1 protein was also significantly reduced(P<0.05);Conclusions:1.The expression of FGF2 was significantly increased in renal tissues of TIF,which promoted the occurrence of EMT in renal tubular epithelial cells and led to renal interstitial fibrosis.2.FGF2 may regulate the transcription of YAP1 by activating STAT3,thereby promoting EMT transformation in renal tubular epithelial cells,and finally leading to the occurrence of TIF.3.Knocking down FGF2 can inhibit the progression of renal tubulointerstitial fibrosis,which can be used as a potential target for the treatment of CKD...