CAJ | 학술논문

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10-8 mol/L AngⅡ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 -mNPHS1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18 h,显著降低AngⅡ单独刺激的足细胞凋亡率(P＜0.05).(2)10-8 mol/L AngⅡ刺激12 h后,nephrin mRNA和蛋白较对照组显著降低,24 h nephrin mRNA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nephrin表达逐渐降低.(3)10-8 mol/L AngⅡ刺激15 min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12 h后,细胞凋亡率显著高于AngⅡ和LY294002单独刺激(均P＜0.05).(5)pcDNA3.1-mNPHS1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PI3K-Akt信号通路调节足细胞存活状态.
목적 연구nephrin재혈관긴장소Ⅱ(AngⅡ)유도족세포조망중적작용,이급가능적분자궤제.방법 체외배양영생화소서족세포( MPC),이불동농도AngⅡ처리MPC화10-8 mol/L AngⅡ자격불동시간,용류식세포의검측세포조망솔；실시정량PCR、면역형광화Western인적법검측nephrin적표체화분포；Western인적법검측Akt린산화수평.지질체법전염pcDNA3.1-mNPHS1질립,G418사선은정전염세포계.용Akt억제제LY294002혹여AngⅡ공부육자격MPC화pcDNA3.1 -mNPHS1전염세포,검측Akt린산화수평화세포조망솔.결과 (1)AngⅡ이제량화시간의뢰방식유도MPC조망.AngⅡ수체길항약록사탄여AngⅡ공부육18 h,현저강저AngⅡ단독자격적족세포조망솔(P＜0.05).(2)10-8 mol/L AngⅡ자격12 h후,nephrin mRNA화단백교대조조현저강저,24 h nephrin mRNA약위정상대조적50％(P＜0.05).정상족세포nephrin주요분포우핵막주위적포질화세포막,수자격시간연장,포막화포질nephrin표체축점강저.(3)10-8 mol/L AngⅡ자격15 min후,Akt린산화현저강저,약위정상대조세포적50％(P＜ 0.01).(4)AngⅡ여LY294002공부육12 h후,세포조망솔현저고우AngⅡ화LY294002단독자격(균P＜0.05).(5)pcDNA3.1-mNPHS1은정전염현저상조족세포Akt린산화수평(P＜0.05),억제AngⅡ유도적세포조망(P＜0.05).결론 AngⅡ통과AngⅡ수체유도소서족세포조망,억제족세포nephrin표체.nephrin통과PI3K-Akt신호통로조절족세포존활상태.
Objective To evaluate the effects of Ang Ⅱ on apoptosis of podocytes and explore the signaling pathway of nephrin in preventing Ang Ⅱ-induced podocyte 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 pedocytes 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 PcDNA3.1-mNPHS1 were exposed to Ang Ⅱ (10-8 mol/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 Westem blotting. Results (1) AngⅡ promoted podocyte apoptosis in a dose-and time-dependent manner. Pretreatment with losartan significantly prevented Ang Ⅱ -induced apoptosis. (2) Nephfin mRNA and protein were obviously decreased in podocytes exposed to 10-8 mol/L Ang Ⅱ for at least 12 h than those in vehicle-treated cells (P＜0.05).(3) Ang Ⅱ exposure for more than 15 min 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.