中文摘要：

目的研究nephrin在血管紧张素Ⅱ（AngⅡ）诱导足细胞凋亡中的作用，以及可能的分子机制。方法体外培养永生化小鼠足细胞（MPC），以不同浓度AngⅡ处理MPC和10μmol／LAngⅡ刺激不同时间，用流式细胞仪检测细胞凋亡率；实时定量PCR、免疫荧光和Western印迹法检测nephrin的表达和分布；Western印迹法检测Akt磷酸化水平。脂质体法转染pcDNA3．1-mNPHS1质粒，G418筛选稳定转染细胞系。用Akt抑制剂LY294002或与AngⅡ共孵育刺激MPC和pcDNA3．1-mNPHSl转染细胞，检测Akt磷酸化水平和细胞凋亡率。结果（1）AngⅡ以剂量和时间依赖方式诱导MPC凋亡。AngⅡ受体拮抗药氯沙坦与AngⅡ共孵育18h，显著降低AngⅡ单独刺激的足细胞凋亡率（P〈0．05）。（2）10μmol／LAngⅡ刺激12h后，nephrinmRNA和蛋白较对照组显著降低，24hnephrinmRNA约为正常对照的50％（P〈0．05）。正常足细胞nephrin主要分布于核膜周围的胞质和细胞膜，随刺激时间延长，胞膜和胞质neDhrin表达逐渐降低。（5）10μmol／LAngⅡ刺激15min后，Akt磷酸化显著降低，约为正常对照细胞的50％（P〈0．01）。（4）AngⅡ与LY294002共孵育12h后，细胞凋亡率显著高于AngⅡ和LY294002单独刺激（均P〈0．05）。（5）pcDNA3．1-mNPHSl稳定转染显著上调足细胞Akt磷酸化水平（P〈0．05），抑制AngⅡ诱导的细胞凋亡（P〈0．05）。结论AngⅡ通过AngⅡ受体诱导小鼠足细胞凋亡，抑制足细胞nephrin表达。nephrin通过P13K—Akt信号通路调节足细胞存活状态。

英文摘要：

Objective To evaluate the effects of Ang Ⅱ on apoptosis of podocytes and explore the signaling pathway of nephrin in preventing Ang Ⅱ-induced podoeyte apoptosis. Methods Differentiated mouse podocytes were exposed to Ang Ⅱ at different concentrations for 18 h or at 10-8 mol/L for variable incubation times. Undifferentiated mouse podocytes were transfected using lipofectamine 2000 with the pcDNA3.1-mNPHS1 plasmid and stably transfected cell lines were generated with G418 selection. In separated experiments, untransfected mouse podocytes （MPC） and stably transfected podocytes with pcDNA3.1-neo and peDNA3.1-mNPHS1 were exposed to Ang Ⅱ （10-8 tool/L） or LY294002 （a selective Akt inhibitor, 50 μmol/L） for indicated times. Apoptosis was evaluated by flow cytometry. The expression of nephrin was assessed by quantitative real-time PCR,immunofluorescence and Western blotting. The phosphorylation level of Akt was determined by Western blotting. Results （1） Ang Ⅱ promoted podocyte apoptosis in a dose-and time-dependent manner. Pretreatment with losartan significantly prevented Ang Ⅱ -induced apoptosis. （2） Nephrin mRNA and protein were obviously decreased in podocytes exposed to 10-s mol/L Ang H for at least 12 h than those in vehicle-treated cells （P〈0.05）. （3） Ang Ⅱ exposure for more than 15 rain inhibited the phosphorylation of AKT in MPC, which was dramatically reversed by pcDNA3.1-mNPHS1 transfection, but not by pcDNA3.1-neo transfection. （4） Podocyte apoptosis was promoted by LY294002. Conversely, Ang Ⅱ-induced podocyte apoptosis was significantly alleviated by pcDNA3.1-mNPHS1 transfection. Conclusion Ang Ⅱ induces mouse podocyte apoptosis which is suppressed by overexpression of nephrin through PI3K-Akt signaling pathway.