FSGS小鼠模型中YAP与足细胞凋亡的相关性及其激动剂的保护作用

Correlation between YAP and podocyte apoptosis and the protective effect of YAP agonist in FSGS mice model

目的探究YAP与局灶节段性肾小球硬化症(focal segmental glomerulosclerosis,FSGS)中足细胞凋亡的相关性及其激动剂的作用。方法收取复旦大学基础医学院病理学系肾病研究组人FSGS肾穿标本,使用阿霉素刺激构建FSGS小鼠模型和体外足细胞损伤模型,并联合YAP激动剂1-油酰基溶血磷脂酸(1-Oleoyl lysophosphatidic acid,LPA)处理上述体内外模型,使用免疫组化、免疫荧光双染、Western blot及Hoechst 33258染色检测足细胞凋亡和足细胞中YAP表达的相关性;使用LPA处理转染YAP siRNA的足细胞系后,检测足细胞中YAP表达和凋亡的变化。结果在人FSGS、FSGS小鼠模型和体外阿霉素诱导足细胞损伤模型中足细胞凋亡越多,YAP出核越多;LPA处理可以改善FSGS小鼠模型的肾脏形态(t=17.68,P<0.0001)和肾功能(血尿素氮:t=4.576,P=0.0102;尿白蛋白/肌酐:t=2.51,P=0.0456),减少足细胞中p-YAP(S127)的表达,促进YAP入核,减少阿霉素诱导的足细胞凋亡(Cleaved Caspase-3的表达及Hoechst 33258染色结果均为P<0.001);敲减足细胞中YAP后,YAP的总蛋白和磷酸化水平均降低,足细胞凋亡增加,LPA处理减少了p-YAP(S127)的表达,促进YAP入核,减少YAP敲减所致的足细胞凋亡(Cleaved Caspase-3的表达及Hoechst 33258染色结果均为P<0.0001)。结论FSGS模型中活化YAP减少及YAP出核,足细胞凋亡增多;LPA能够通过抑制YAP在Ser127位点的磷酸化,促进足细胞中YAP的入核,减少细胞凋亡,延缓FSGS的疾病进程。

Objective To investigate the correlation between YAP and podocytes apoptosis in focal segmental glomerulosclerosis(FSGS)and the role of YAP activator on the podocytes apoptosis.Methods The renal biopsy specimens of human FSGS were collected from the Nephrology Research Group of the Department of Pathology,School of Basic Medical Sciences,Fudan University.Adriamyc-ininduced FSGS mice model and Adriamycin-induced podocytes injury model in vitro were generated,and the above in vivo or in vitro model were treated in combination with YAP activator 1-Oleoyl lysophosphatidic acid(LPA).Immunohistochemistry(IHC)or double immunofluorescence staining,Western blot and Hoechst 33258 staining were used to examine the correlation between podocytes apoptosis and the expression of YAP in podocytes.Podocytes transfected with YAP siRNA were treated with LPA,and then YAP expression and podocytes apoptosis were examined.Results The more apoptotic podocytes,the more nuclear exclusion of YAP in FSGS were observed either in vivo or in vitro.YAP activator LPA improved glomerular morphology(t=17.68,P<0.0001)and renal function(BUN:t=4.576,P=0.0102;UACR:t=2.51,P=0.0456)of Adriamycin-induced FSGS mice model,reduced the expression of p-YAP(S127),increased YAP nuclear entry and decreased podocyte apoptosis(P<0.001 for both expression of Cleaved Caspase-3 and Hoechst 33258 staining)induced by Adriamycin both in vivo and in vitro.RNA interference of YAP decreased both total and phosphorylated YAP expression,and increased podocytes apoptosis.LPA treatment decreased the expression of p-YAP(S127),promoted YAP nuclear localization and decreased the podocytes apoptosis(P<0.0001 for both expression of Cleaved Caspase-3 and Hoechst 33258 staining)induced by YAP interference.Conclusion There are decrease in YAP activation,nuclear exclusion of YAP,and increased podocytes apoptosis in FSGS model.LPA can inhibit the phosphorylation of YAP at Ser127,promote its nuclear translocation,thus to protect podocytes from apoptosis to ameliorate the progression of FSGS.