三种不同药物作用下足细胞分子的变化

目的 从足细胞分子的角度探讨抗蛋白尿药物作用的细胞分子机制。方法 建立阿霉素肾病大鼠模型。注射阿霉素后次日分别给予利生普利、泼尼松以及全反式维甲酸(ATRA)干预蛋白尿。注射阿霉素后第3、7、14、28 天每组处死6 只大鼠，留取肾脏标本。应用间接免疫荧光染色、实时PCR、Western印迹分别检测各个时间点nephrin、podocin、CD2相关蛋白(AP)、α辅肌动蛋白(actinin)-4 的分布、mRNA 和蛋白表达量的变化。应用免疫沉淀检测nephrin 与podocin、nephrin与CD2AP 分子间作用以及nephrin 磷酸化水平。结果 与对照组相比，第14天时肾病组尿蛋白显著增加(P < 0.01)。与肾病组相比，利生普利、泼尼松和ATRA干预后均显著降低了蛋白尿( P < 0.05)， 减轻了足突融合。通过分析不同时间点足细胞分子的表达，显示3种干预药物均引起了nephrin、podocin、CD2AP 表达的变化，维持了正常的nephrin 磷酸化水平，而且利生普利和泼尼松首先抑制了podocin 分子，而ATRA首先抑制了CD2AP 分子的异常变化。与此同时，nephrin、podocin、CD2AP 和α-actinin-4 分子的分布在干预后也趋于正常。此外，无论在肾病组还是干预组大鼠，nephrin 与podocin、nephrin 与CD2AP 分子间一直保持着共沉淀关系。 结论 利生普利、泼尼松和ATRA 都通过稳定重要的足细胞分子nephrin、podocin、CD2AP 来发挥它们的抗蛋白尿作用。

Changes of podocyte molecules in the intervention of three different drugs

Objective To explore the molecular mechanisms underlying therapeutic responses of the anti-proteinuria drugs from the view of podocyte molecule. Methods Adriamycin (ADR) nephropathy was induced by a single tail intravenous injection of adriamycin. Lisinopril， prednisone and all-trans retinoic acid (ATRA) were administered once a day to the adriamycin-induced nephrotic rats at the first day after adriamycin injection respectively. Renal tissue samples were collected at day 3， 7， 14， and 28 after adriamycin injection respectively. The distribution， mRNA expression and protein expression of nephrin， podocin， CD2AP and α-actinin-4 were examined by indirect immunofluorescence， real-time PCR and Western blotting， respectively. The interactions among nephrin and podocin， nephrin and CD2AP， as well as the nephrin phosphorylation were detected by immunoprecipitation， respectively. Results Compared to the control rats， 24 h urinary protein of the ADR rats increased significantly at day 14 (P < 0.01). Compared with the ADR rats， lisinopril， prednisone and ATRA significantly reduced the excretion of urinary protein and alleviated the fusion of foot process at day 14 and 28 (P < 0.05). By analyzing the expression changes of podocyte molecules at different time points， three intervention drugs all resulted in the expression change of nephrin， podocin， CD2AP and increased the tyrosine phosphorylation of nephrin. Lisinopril and prednisone firstly diminished the abnormal change of podocin， whereas ATRA firstly diminished the abnormal change of CD2AP. Simultaneously， the staining of nephrin， podocin， CD2AP and α- actinin-4 also revealed the normal pattern after the intervention of lisinopril， prednisone and ATRA. The interactions between nephrin and podocin， nephrin and CD2AP were always maintained either in ADR rats or in three drug-intervention rats. Conclusion The anti-proteinuria effect of lisinopril， prednisone and ATRA is achieved by stabilizing the expression and distribution of important podocyte molecules， nephrin， podocin and CD2AP.