Miro1调控线粒体移动在肾小球足细胞损伤中的作用及机制

Podocytes are highly specialized glomerular visceral epithelial cells that cover the outside of the glomerular basement membrane. Podocyte injury plays a pivotal role in the onset of proteinuria and progression of glomerular diseases. The mechanisms that underlie podocyte injury are a focus of intense research. Mitochondria are cellular organelles that produce the energy required for cellular processes through synthesis of ATP. Mitochondrial morphology regulated by fission and fusion is important for normal mitochondrial function through maintenance of the metabolite and protein balance between internal compartments. Our previous study has showed that mitochondrial fission played a crucial role in podocyte injury. Recent studies showed that defective mitochondrial movement could induce mitochondrial fission..Mitochondria movement, which serves to distribute mitochondrial functions in a dynamic and non-uniform fashion, has attracted special interest in recent years. Mitochondrial movement is regulated by a series of molecular adaptors that mediate the attachment of mitochondria to molecular motors. Miro1 belongs to the mitochondrial Rho GTPase family and are critical for mitochondrial movement. Miro1 links mitochondria to kinesin motor proteins allowing mitochondria to move along microtubules. Miro1 is the key Ca2+ sensor for Ca2+-dependent mitochondrial stopping in axons and dendrites, which are dependent on its EF-hand domains. Increased cytoplasmic Ca2+ halts mitochondrial movement by binding to Miro1 EF-hand domains, thus leading to altered interactions between Miro1 and the kinesin motor. Furthermore, one of the key proteins responsible for calcium influx in the podocytes is transient receptor potential cation channel, subfamily C, member 6 (TRPC6). Both increased TRPC6 channel activity and expression lead to a pathologically high Ca2+ influx in podocytes, which eventually causes podocyte injury. The role of mitochondrial movement in podocyte injury is poorly investigated. Our preliminary studies found that aldosterone induced calcium influx and reduced the interaction of Miro1 with kinesin in podocytes..Therefore, we hypothesize here that TRPC6-Ca2+-Miro1 signaling may be involved in the process of aldosterone induced podocyte injury by targeting mitochondrial movement. To establish our main hypothesis, proposed studies are designed to investigate the effects of TRPC6-Ca2+-Miro1 signaling and mitochondrial movement in podocyte injury both in cultured podocytes and in animal experiments. These studies are expected to reveal the molecular mechanism of mitochondrial movement in podocyte injury, which may provide us with unprecedented opportunities for therapeutic interventions of glomerular diseases.

足细胞损伤与慢性肾脏病蛋白尿的发生关系密切。申请人前期工作证实线粒体分裂在足细胞损伤中作用重要。线粒体移动障碍可介导线粒体分裂。线粒体移动在足细胞中尚无报道。线粒体外膜的GTPase家族成员Miro1是介导线粒体移动的关键蛋白，当Miro1与马达分子Kinesin发生解离将导致线粒体移动障碍。我们预实验也证实，醛固酮可引起Miro1与Kinesin解离。Miro1主要受细胞内钙离子调控，而瞬时受体电位阳离子通道蛋白6（TRPC6）是介导足细胞钙内流主要途径。我们推测：醛固酮激活TRPC6-钙离子内流使Miro1与Kinesin解离，线粒体移动发生障碍，使线粒体分裂增加，最终导致线粒体功能损伤及足细胞损伤的发生。本研究将从体外培养肾小球足细胞系、动物实验、足细胞特异性Miro1转基因小鼠三方面阐述Miro1介导的线粒体移动及其调控机制在足细胞损伤中的作用，为解析足细胞损伤机制提供新的思路。

目的：观察线粒体移动关键蛋白Miro1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Miro1对线粒体稳态的调控作用。.方法：在体外培养的肾小管上皮细胞系中，运用siRNA干扰技术敲低以及过表达技术增强Miro1的表达，观察其对CoCl2所模拟的体外缺血再灌注以及顺铂损伤的影响。在体内外急性肾损伤模型中加用Miro1激动剂金丝桃苷，观察其对肾小管损伤的作用。在体外培育的肾小球足细胞中，观察醛固酮对Miro1表达的影响，将Miro1过表达后观察其对足细胞损伤以及线粒体稳态的作用，最后，运用Miro1激动剂金丝桃苷干预体内外阿霉素足细胞损伤模型。.结果：1、Miro1的表达在糖尿病肾病IV期中表达较IIa、IIb以及III期中低，Miro1表达与血肌酐、血尿素氮、胱抑素c、尿酸呈负相关，与eGFR呈正相关。2、在体外培养的肾小管上皮细胞系中，运用siRNA干扰技术敲低Miro1的表达，进一步加重CoCl2所模拟的体外缺血再灌注以及顺铂损伤。相反，运用基因过表达技术构建Miro1过表达肾小管上皮细胞系，发现Miro1过表达可抑制IR以及Cisplatin所诱导的细胞凋亡。进一步，Miro1基因敲低加重IR所导致的肾小管细胞线粒体分裂，同时加重线粒体活性氧的产生。3、金丝桃苷可上调Miro1表达，作用于OMA1-OPA1信号阻断线粒体分裂，对缺血再灌注肾损伤发挥保护作用。4、在足细胞中，醛固酮抑制Miro1表达，诱导Miro1与Kinesin结合减少，醛固酮诱导Miro1表达减少。免疫共沉淀结果发现醛固酮刺激30 min，Miro1与Kinesin就开始发生解离。过表达Miro1可阻断阿霉素诱导的足细胞损伤以及线粒体代谢的重编程。醛固酮在刺激后可瞬时引起细胞内钙浓度增高，并且TRPC6的抑制剂能够改善阿霉素所导致的线粒体代谢的重编程。5、Miro1激动剂金丝桃苷通过改善线粒体功能，对足细胞损伤发挥保护作用。.结论：线粒体移动蛋白Miro1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。.