线粒体分裂调控在肾小球足细胞损伤中的作用机制研究

Podocytes are specialized visceral epithelial cells that reside on the glomerular basement membrane outside of the glomerular capillaries. It therefore forms the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria and chronic kidney disease (CKD). Previous study has showed that mitochondrial dysfunction played a crucial role in the pathogenesis of proteinuria and podocyte injury. Mitochondrial morphology is an important determinant of mitochondrial function. Mitochondria constantly undergo fusion and fission that are necessary for the maintenance of organelle fidelity. Several studies have indicated that excessive mitochondrial fission is associated with cellular apoptosis. Reports that the precise role of abnormal mitochondrial fission in the initiation of podocyte apoptosis are quite limited. Mitochondrial fission requires the activation of a dynamin related protein-1 (Drp1). Drp1 is a large GTPase of the dynamin superfamily that has an essential role in mitochondrial fission in mammalian cells. Recently, it was reported that p53 could upregulate Drp1 expression in a transcription-dependent manner. Meanwhile, p53 was regulated by SIRT1. Therefore, we hypothesize SIRT1/p53/Drp1 signaling may be involved in the process of podocyte injury by targeting mitochondrial fission. To establish our main hypothesis, proposed studies are designed to investigate the effects of SIRT1/p53/Drp1 signaling and mitochondrial fission in podocyte injury in primary nephrotic syndrome patients, animal experiments and podocytes in vitro. These studies are expected to open a new avenue in the understanding of podocyte injury, which may guide us in therapeutic strategies for CKD.

足细胞损伤与慢性肾脏病（CKD)发生及进展关系密切。项目申请人前期研究发现线粒体功能损伤在足细胞损伤中发挥重要作用，而线粒体分裂作为线粒体功能损伤的始发因素，已证实在一些肾小管损伤模型中起关键作用，然而其在足细胞损伤中的作用及其调控机制目前尚未明确。业已发现，沉默信息调节因子1（SIRT1）可通过阻断线粒体功能损伤而发挥足细胞保护作用，而抑癌基因p53作为SIRT1重要的靶基因，与细胞凋亡密切相关。此外，有报道证实在心肌细胞中p53通过作用于Drp1调控线粒体分裂。由此，我们推测：SIRT1/p53/Drp1信号可能通过调控线粒体分裂参与线粒体功能损伤及足细胞损伤过程。本研究拟在原发性肾病综合征患者、动物实验及体外培养肾小球足细胞系三个层面，观察SIRT1/P53/Drp1通路、线粒体分裂在线粒体损伤及足细胞损伤中的作用，为临床慢性肾脏病足细胞损伤的防治提供新的治疗靶点。

目的：观察线粒体分裂在醛固酮诱导的肾小球足细胞损伤中的作用，探讨调控线粒体分裂主要分子发动相关蛋白1（Drp1）及其上游信号抑癌基因p53、沉默信息调节因子SIRT1在醛固酮诱导足细胞损伤中的作用及其机制。.方法：体外培养小鼠肾小球足细胞，采用荧光探针mitoSOX染料检测线粒体活性氧生成情况；Mitotracker red染料以及电镜检测足细胞内线粒体形态的改变；JC-1染色流式细胞仪检测线粒体膜电位；应用实时定量PCR和Western blot方法检测足细胞Nephrin、podocin、Drp1的表达；流式细胞术检测细胞凋亡情况。体内实验通过皮下埋置含有醛固酮的渗透性微泵建立醛固酮肾损伤模型，线粒体分裂抑制剂Mdivi-1干预小鼠，检测线粒体分裂、线粒体功能及足细胞损伤相关指标。最后，在肾病综合征患者肾组织标本中检测p53以及Drp1的表达。.结果：1、在体外培养肾小球足细胞中，醛固酮呈剂量依赖诱导Drp1表达，Drp1表达抑制阻断醛固酮诱导足细胞线粒体分裂发生，抑制醛固酮诱导线粒体活性氧增加以及膜电位下降，同时，Drp1 siRNA抑制醛固酮诱导的足细胞nephrin、podocin表达减少。2、醛固酮呈剂量依赖诱导足细胞内p53表达。p53表达抑制减少醛固酮诱导Drp1的表达，并且可阻断醛固酮诱导线粒体分裂、线粒体功能障碍及足细胞损伤。3、醛固酮呈剂量依赖抑制足细胞内SIRT1表达，SIRT1过表达可抑制醛固酮诱导p53及Drp1表达，并阻断醛固酮诱导的线粒体分裂、线粒体功能障碍及足细胞损伤。4、SIRT1激动剂白藜芦醇亦可抑制醛固酮诱导p53及Drp1表达，阻断醛固酮诱导的线粒体分裂、线粒体功能障碍及足细胞损伤。5、动物实验中，醛固酮损伤组小鼠足细胞nephrin、podocin表达减少，Drp1表达增加、细胞色素C向胞浆释放增多。应用Mdivi-1处理后Drp1表达较降低，足细胞损伤减轻。6、入组154例肾病综合征患者，在人肾穿刺活检标本组织检测Drp1、P53表达，发现P53以及Drp1在膜性肾病中高表达。.结论：醛固酮引起足细胞内SIRT1表达减少，使其对p53的抑制作用减弱，p53表达增加进一步调控Drp1的表达，使Drp1表达增加，线粒体分裂增加，进而诱导线粒体功能障碍，最终导致足细胞损伤。