线粒体内膜结构调控关键复合物核心蛋白Mic60在肾小球足细胞损伤中的作用及机制

Podocytes are specialized glomerular epithelial cells which cover the outermost layer of the glomerular filtration barrier in the kidney. Podocyte damage is a common feature of chronic kidney disease. At present, the mechanism underlying podocyte injury is incompletely understood. Recent studies from several laboratories have implicated mitochondria exert a critical role for maintaining podocyte homeostasis and mitochondrial dysfunction could lead to podocyte injury. Our previous study demonstrated that mitochondrial dysfunction was an early event in aldosterone-induced podocyte injury. In addition, we found that the outer mitochondrial membrane protein Miro1 (mitochondrial Rho GTPase-1) was critical for mitochondrial movement in podocyte. Loss of Miro1-directed mitochondrial movement resulted in aldosterone-induced mitochondrial dysfunction and apoptosis in podocytes. Recently, increasing number of studies has investigated the important functions of some novel mitochondrial inner membrane proteins. .The inner membrane of mitochondria is organized in two morphologically distinct domains, the inner boundary membrane and the cristae membrane, which are connected by pore-like structures termed cristae junctions. Cristae junctions are essential for maintaining the cristae structure and stability of OXPHOS complexes in mitochondria. Moreover, it was found that the mitochondrial contact site and cristae organizing system (MICOS) was a major factor in the formation of cristae junctions. Mic60 is the core component of MICOS. Previous study demonstrated that Mic60 had a primary role in maintaining crista structure and loss of Mic60 caused loss of Miro and mitochondrial arrest. Our preliminary study found that Mic60 activator inhibited cisplatin-induced mitochondrial dysfunction and apoptosis in cultured rat renal proximal tubular cells (RPTC). Furthermore, the expression of Mic60 decreased at the early time in both aldosterone-treated podocytes and cisplatin-treated RPTC. Mic60 degradation was regulated by OMA1 (overlapping with the m-AAA protease 1), which is a membrane-bound metallopeptidase in mitochondria..Therefore, we hypothesize here that aldosterone induced the activation of OMA1, which mediated the degradation of Mic60. Then, the reduction of Mic60 impaired crista structure via destabilizing the MICOS complex and disturbed mitochondrial movement via affecting Miro1, finally leading to podocytes mitochondrial dysfunction and injury. To verify our main hypothesis, proposed studies are designed to identify the roles of Mic60 and its upstream and downstream signals on podocyte injury both in cultured podocytes and in animal experiments. Our research findings might be of great help in the establishment of a novel therapeutic approach to podocyte injury.

肾小球足细胞损伤是蛋白尿发生的主要机制，线粒体功能障碍是足细胞损伤的早期事件。近年来，线粒体内膜结构调控关键复合物MICOS在稳定线粒体嵴进而控制能量代谢中的作用受到关注。Mic60是MICOS的关键蛋白，既能稳定MICOS结构调控线粒体嵴，也能影响外膜移动调控蛋白影响线粒体移动。我们的预实验结果发现Mic60激动剂抑制顺铂诱导的肾小管上皮细胞线粒体损伤及细胞凋亡。另外，Mic60在损伤早期即发生降解，线粒体内膜蛋白酶OMA1对其发挥降解作用。课题组推测：足细胞损伤因素早期激活OMA1降解Mic60，进而通过影响MICOS使线粒体嵴紊乱以及干扰外膜移动调控蛋白使线粒体移动障碍，线粒体功能障碍最终导致足细胞损伤。本研究将从体外培养足细胞系、动物实验、特异性转基因小鼠及肾脏靶向活体转染基因敲低等方面阐述Mic60为中心的上下游信号在足细胞损伤中的作用，为寻找保护足细胞方法提供新思路。

目的：线粒体内膜结构调控关键复合物MICOS在稳定线粒体嵴进而控制能量代谢中的作用受到关注。Mic60是MICOS的关键蛋白，既能稳定MICOS结构调控线粒体嵴，也能影响外膜移动调控蛋白影响线粒体移动。本研究观察线粒体内膜复合物关键蛋白Mic60在急性肾损伤中肾小管细胞损伤以及慢性肾脏病中足细胞损伤中的作用，探讨Mic60对线粒体稳态的调控作用。.方法：（1）在体外培养的肾小管上皮细胞系中，运用过表达、shRNA干扰技术以及激动剂调控Mic60的表达，观察其对顺铂诱导的肾小管细胞以及线粒体功能损伤的影响，运用免疫共沉淀探索线粒体内膜蛋白酶OMA1与Mic60相互结合。（2）通过单侧输尿管结扎构建肾组织纤维化模型，观察Mic60激动剂对慢性肾脏病纤维化的作用。（3）通过构建体内外阿霉素足细胞损伤模型，观察Mic60对足细胞损伤的影响。.结果：（1）Mic60过表达能够改善顺铂诱导的线粒体功能损伤，抑制细胞凋亡以及caspase-3的激活。运用shRNA技术将Mic60敲低后则加重肾小管上皮细胞凋亡及线粒体损伤。Mic60激动剂MA-5抑制顺铂诱导的肾小管上皮细胞线粒体功能障碍及凋亡，并且MA-5对顺铂诱导肾小管细胞线粒体功能障碍以及细胞损伤的保护作用是由Mic60介导的。免疫共沉淀结果显示线粒体内膜蛋白酶OMA1与Mic60相互结合，OMA1对Mic60发挥降解作用。（2）Mic60激动剂MA-5减轻UUO所致的肾纤维化，对整个线粒体稳态发挥重要作用，包括改善线粒体合成功能、提高线粒体融合蛋白的表达、恢复线粒体抗氧化蛋白表达以及调控MICOS的核心蛋白表达。（3）在阿霉素诱导的小鼠模型中，Mic60激动剂MA-5可抑制ADR所导致的肾损伤；MA-5以及衍生物MA-15、Mic60过表达能够抑制阿霉素诱导的足细胞损伤以及线粒体功能障碍。.结论：Mic60在急性肾损伤肾小管细胞损伤以及慢性肾脏病足细胞损伤中发挥保护作用，其保护作用机制可能与调控线粒体功能稳态密切相关。