AIM; To investigate the role of nephrin, a slit diaphragm -associated protein, in angiotensin II ( Ang II ) - induced cytoskeleton rearrangement in podocytes . METHODS: Immortalized mouse podocytes were exposed to Ang II (10 -8 mol/L) with or without Ang Ⅱ receptor antagonist lorsatan and Akt inhibitor LY 294002. FITC - conjugated phalloidin was used to stain F - actin, and semi - quantitative system with cortical F - actin score ( CFS) was introduced to analyze the degree of actin cytoskeleton arrangement . The expression of nephrin was assessed by quantitative real - time RTrn- PCR, RT - PCR and Western blotting. Undifferentiated podocytes were transfected with pcDNA 3.1- mNPHSl plasmid containing the full length of nephrin . The stably transfected cell line was generated by G418 selection. Phosphorylation level of Akt was assessed by Western blotting , and F - actin distribution was further evaluated in transfected cells exposed to AngⅡ or not. RESULTS; Cytoskeletal rearrangements including cortical F -actin ring formation and stress fiber attenuation were observed in Ang Ⅱ- and LY294002 - stimulated podocytes. Pretreatment with losartan significantly prevented Ang Ⅱ - induced actin cytoskeleton reorganization. The mRNA and protein levels of nephrin and phosphorylation of Akt were obviously decreased in the podocytes exposed to Ang Ⅱ , which were dramatically reversed by pcDNA3. 1 -mNPHSl transfection. Transfection of pcDNA3.1 - mNPHSl induced the formation of short filopodia and partially prevented Ang IIrn-induced F -actin remodeling. CONCLUSION; PBK/Akt signaling is a common downstream pathway of nephrin and Ang II. Nephrin is able to stabilize Ang II - induced cytoskeletal rearrangement via PI3K/Akt signaling pathway.%目的:研究足细胞裂孔膜分子nephrin调节血管紧张素Ⅱ(AngⅡ)诱导的足细胞骨架分布改变的分子机制.方法:用AngⅡ及AngⅡ受体拮抗剂氯沙坦或Akt抑制剂LY294002刺激足细胞,FITC-phalloidin染色标记F-actin,分析足细胞骨架运动.Real-time RT-PCR、RT-PCR和Western blotting检测nephrin mRNA和蛋白表达.转染nephrin全长表达质粒(pcDNA3.1-mNPHS1),建立稳定转染足细胞系,Western blotting检测转染细胞的Akt磷酸化水平,FITC-phalloidin染色分析高表达nephrin对F-actin分布影响.结果:AngⅡ和LY294002刺激后,足细胞的F-actin重组,应力纤维减少,形成F-actin外周环.氯沙坦显著抑制F-actin重排.AngⅡ刺激后nephrin mRNA和蛋白表达显著降低,Akt磷酸化水平降低.pcDNA3.1-mNPHS1转染显著上调足细胞Akt磷酸化水平,促进足细胞短线状足突形成,部分抑制AngⅡ诱导的骨架重排.结论:PI3K/Akt是nephrin和AngⅡ的共同下游通路.Nephrin能通过PI3K/Akt途径部分稳定AngⅡ诱导的足细胞细胞骨架改变.
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