钙稳态失衡是足细胞损伤的关键环节，参与蛋白尿发生机制。胞内钙池通过线粒体内质网结构偶联进行的钙调控普遍存在于各种细胞，影响细胞存亡，然而其在足细胞钙稳态失衡中的作用及调控机制不清。我们前期发现线粒体融合蛋白2增多参与足细胞损伤，且其过表达致线粒体钙离子增多，提出：线粒体融合蛋白2增多通过影响线粒体内质网结构偶联钙调控致足细胞钙紊乱。拟在小鼠足细胞分别干预线粒体内质网结构偶联处钙通道蛋白，分析对钙稳态及下游路径如内质网应激、钙调磷酸酶活性、线粒体凋亡途径和足细胞损伤的影响；进而过表达线粒体融合蛋白2，分析对线粒体内质网结构偶联钙调控功能的影响；再通过阻断相应钙通道及敲低MITOL和过表达mitostatin拮抗线粒体融合蛋白2证实该路径；最后通过阿霉素大鼠肾病模型证实阻断该损伤路径对蛋白尿的控制作用。研究结果有助于从线粒体内质网结构偶联和线粒体融合蛋白2等全新角度揭示足细胞钙稳态失衡机制。

Disturbance of calcium homeostasis is key to podocyte injury, which play a major role in the development of proteinuria. The mechanism by which intracellular calcium pools modulate calcium homeostasis in podocyte is unclear. We previously found that over-expression of mitofusin 2 was involved in the mechanism of podocyte injury, and over-expression of mitofusin 2 could induce increase of mitochondrial calcium concentration. We propose that over-expression of mitofusin 2 will lead to imbalance of calcium in podocyte by effect on calcium modulation of endoplasmic reticulum mitochondria coupling. Firstly, we will intervene the calcium channel proteins located at the endoplasmic reticulum mitochondria coupling in mouse podocytes, and analyze the calcium homeostasis of endoplasmic reticulum, cytoplasm and mitochondria. The corresponding downstream targets or events including endoplasmic reticulum stress, calcineurin activity, mitochondrial apoptotic pathway and podocyte injury will also be analyzed. Secondly, mitofusin 2 will be over-expressed in podocytes, and its impact on the calcium modulating function of endoplasmic reticulum mitochondria coupling will be analyzed. Thirdly, interventions including corresponding calcium channel protein regulation, knock down of MITOL which can modulate mitofusin 2 by ubiquitination, and over-expression of mitostatin which is the antagonistic protein of mitofusin 2 will be performed to confirm the pathway induced by mitofusin 2. Finally, drugs which can block mitofusin 2 induced calcium disturbance by endoplasmic reticulum mitochondria coupling will be given to adriamycin nephropathy in order to confirm the effect on proteinuria.The results will help to understand the mechanism of calcium disturbance from view of endoplasmic reticulum mitochondria coupling and mitofusin 2.