中文摘要：

目的通过建立血管紧张素Ⅱ（AngⅡ）输注大鼠模型及体外足细胞培养，观察AngⅡ刺激对足细胞nephrin磷酸化水平的影响。方法30只SPF级Wistar大鼠皮下埋置渗透性微泵，随机分为AngⅡ组（AngⅡ400ng·kg-1·min-1，n=12）、替米沙坦（Tel）组（AnglI＋Tel3mg·kg-1·d-1，n=12）和正常对照组（生理盐水代替AngⅡ，n=6），于实验0、7、14、21、28d测量大鼠尾动脉收缩压，收集24h尿液，检测尿白蛋白。分别在14、28d处死动物，收集血液标本，检测血肌酐；收集肾脏标本，透射电镜观察肾小球足细胞形态，放免法检测血浆及肾组织AngⅡ水平；Western印迹检测nephrin表达及其磷酸化水平。体外培养小鼠永生化足细胞，AngⅡ（10μmol／L）刺激不同时间，并行洛沙坦（10μmol／L）干预，Western印迹法检测足细胞nephrin表达及其磷酸化水平。异硫氰酸荧光素（FITC）．鬼笔毒环肽（phalloidin）染色标记足细胞F-actin，用外周F—actin环评分系统（CFS）半定量分析足细胞骨架运动。结果（1）与正常对照组同时间点相比，AngⅡ组大鼠自7d起尾动脉收缩压开始升高（P〈O．05），随着作用时间的延长，血压持续升高。AngⅡ输注7d大鼠开始出现蛋白尿，并持续增加。各组大鼠血肌酐、尿肌酐及内生肌酐清除率无明显变化。AngⅡ输注大鼠血浆及肾组织AngⅡ浓度明显增高（均P〈0．05）。（2）与正常对照组相比，AngⅡ输注组大鼠肾小球足细胞nephrin表达明显减少，其磷酸化水平亦显著下降（P〈0．05）。（3）与正常对照组相比，AngⅡ刺激早期（3～6h），足细胞nephrin磷酸化表达即明显下调（P〈0．05），刺激12—24h维持低水平表达。（4）AngⅡ刺激后，F-actin排列紊乱，逐渐向细胞外周分布形成F-actin环，CFS评分显著高于正常对照组（P〈0．05）。结论正常状态下足细胞nephrin维持一定水平磷酸化状态，An

英文摘要：

Objective To evaluate the effect of angiotensin Ⅱ （Ang Ⅱ ） on the change of nephrin phosphorylation both in Ang 11-infused rat model and cultured podocytes. Methods Thirty Wistar rats were subcutaneously embedded with osmotic minipumps and randomly divided into 3 groups according to receiving either Ang Ⅱ at a dose of 400 ng· kg-1· min-1 or Ang Ⅱ ＋ telmisartan at a dose of 3 mg· kg-1· d-1, or normal saline as a control group. Blood pressure and 24-hour urinary albumin were measured at 0 d, 7 d, 14 d, 21 d and 28 d of the experiment. Renal histomorphology was evaluated through electron microscopy. The concentrations of Ang Ⅱ both in blood plasma and kidney were detected by radioimmunoassay. In vitro, cultured murine podocytes were exposed to Ang Ⅱ （10-6 mol/L） pretreated with or without losartan （10-5 mol/L） for different time periods. Nephrin and its phosphorylation expression were analyzed by Western blotting. The distribution of F-actin was presented by FITC-phallodin labeling. The change in phenomenon of F-actin was evaluated by cortical F-actin score index （CFS）. Results （1）Ang Ⅱ- infused rats exhibited increased Ang Ⅱ concentration, significant hypertension and marked albuminuria. （2）In Ang Ⅱ-infusion group, nephrin expression was decreased （P〈0.05）. Ang Ⅱ- receiving rats displayed diminished phosphorylation of nephrin. （3）In vitro, the phosphorylation of nephrin was significantly reduced after Ang Ⅱ stimulation for 3-6 hours （P〈O.05）. （4）Ang Ⅱ stimulatation resulted in irregularly arrangement of F-actin followed by the redistribution of F-actin to podocyte periphery and formation of F-actin ring, in which the CFS obviously increased compared to control （P〈0,05）. Conclusions Phosphorylation of nephrin is important for the survival status of podocytes. Ang Ⅱ-induced nephrin dephospholylation may be an important molecular mechanism for Ang Ⅱ-induced podocyte cytoskeleton rearrangement and foot process effacement.