C末端Src激酶在血管紧张素Ⅱ诱导足细胞骨架重构中的作用

Role of C-terminal Src kinase in angiotensin Ⅱ -induced cytoskeletal rearrangement in glomerular podocytes

目的观察血管紧张素Ⅱ（AngⅡ）刺激对大鼠肾脏及条件永生性小鼠足细胞C末端Src激酶（C—terminal Srckinase，Csk）表达的影响，探讨Csk在Angll诱导足细胞骨架重构中的作用。方法24只无特定病原（SPF）级雄性Wista大鼠皮下埋置渗透性微泵，随机分为正常对照组和AnglI输注组（AngⅡ以400ng·kg^-1·min^-1持续输注），于造模后2周和4周留取肾组织用于后续实验。采用透射电镜观察各组大鼠肾脏足细胞超微结构改变；Western印迹检测各组大鼠Csk蛋白表达水平；免疫荧光法检测肾脏Csk表达及分布改变。体外培养条件永生性小鼠足细胞（MPC），Western印迹法检测不同浓度AngⅡ（10^-9、10^-8、10^-7、10^-6、10^-5mol／L）刺激MPC细胞6h及10^-6mol／LAngⅡ刺激不同时间（0、1．5、3、6、12、24h）后足细胞Csk蛋白表达水平。CsksiRNA转染足细胞下调Csk表达，异硫氰酸荧光素（FITC）-鬼笔环肽（phalloidin）标记F-actin评价AngⅡ、细胞松弛素D（CytochalasinD）诱导的足细胞骨架重构改变。结果（1）透射电镜观察发现，AngⅡ输注组大鼠足细胞出现足突融合；（2）免疫荧光及Western印迹显示，AngⅡ输注组肾小球Csk表达增加（P〈0．05）；（3）AngⅡ可诱导培养的小鼠足细胞Csk蛋白表达增加（P〈0．05），且呈时间和剂量依赖性；（4）CsksiRNA转染足细胞下调Csk表达，可减轻AngⅡ诱导的足细胞F—actin重构，可减轻细胞松弛素D诱导的足细胞骨架破坏。结论AngⅡ可诱导Csk表达上调，Csk可能参与了AngⅡ诱导的足细胞骨架重构及足突融合。

Objective To investigate the effects of angiotensin Ⅱ （Ang Ⅱ） on the expression of C-terminal Src kinase （Csk） in Ang Ⅱ-infused rat model and cultured podocytes, and to explore the role of Csk in Ang II-induced cytoskeletal rearrangement of podoeytes. Methods Twenty-four Wista rats were randomly subjected to normal saline infusion, or Ang II infusion at 400 ng· kg^-1·min^-1 （via subcutaneous osmotic minipumps） for 2 or 4 weeks. Renal histomorphology was evaluated through electron microscopy. The expression of glomerular Csk was analyzed by immunofluorescenee and Western blotting. In vitro, conditionally immortalized mouse podocytes were cultured and treated with Ang II doses ranging from 10-9 mol/L to 10-5mol/L and for different hours. The expression of podoeytes Csk was assessed by Western blotting. After transfection to podocytes with Csk siRNA, FITC- conjugated phalloidin was used to stain F- actin, to investigate the role of Csk in Ang Ⅱ-induced or eytochalasin D- induced cytoskeletal rearrangement. Results （1） Examination of Ang Ⅱ infusion rats glomerular and podoeyte uhrastructure by electron microscopy revealed foot process effacement and fusion; （2） In Ang Ⅱ infusion rats, the expression of glomerular Csk was increased （P 〈 0.05）; （3） In vitro, Ang Ⅱ- stimuli up- regulated the expression of Csk （P 〈 0.05）, and the effects of Ang II were on dose-dependent and time-dependent manner; （4） Ang Ⅱ-induced disruption of F-actin was alleviated by Csk siRNA transfection in cultured podocytes; furthermore, cytochalasin D depolymerized the F-actin cytoskeleton, while Csk siRNA stabilized the actin filaments. Conclusion The enhanced expression of Csk may be involved in Ang Ⅱ-induced podocytes cytoskeletal rearrangement and foot process fusion.