线粒体融合蛋白2增多致线粒体内质网结构偶联钙调控紊乱参与足细胞损伤机制

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.

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。

蛋白尿是慢性肾脏病进展的独立危险因素，足细胞损伤是蛋白尿发生的关键环节，足细胞损伤机制尚未充分阐明。本课题从位于线粒体内质网结构偶联的肌醇-1，4，5-三磷酸受体（inositol-1, 4, 5-trisphosphate receptor，IP3R）-葡萄糖调节蛋白75（glucose-regulated protein 75，Grp75）-电压依赖性阴离子通道1（voltage-dependent anion channel 1，VDAC1）-线粒体钙单向转运体（Mitochondrial calcium uniporter，MCU）钙轴角度探讨足细胞损伤机制。..首先探讨了IP3R-Grp75-VDAC1-MCU钙轴在小鼠足细胞损伤时的作用。进而，在阿霉素大鼠肾病模型分析了IP3R-Grp75-VDAC1-MCU钙轴表达变化，分析钙轴拮抗剂钌红对足细胞损伤和蛋白尿的作用。之后，探讨肾病综合征患儿钙轴分子表达变化。最后，分别从线粒体动力相关蛋白（包括线粒体融合蛋白2和线粒体分裂相关蛋白）和哺乳动物雷帕霉素靶蛋白复合物1（Mammalian target of rapamycin complex 1，mTORC1）角度探讨钙轴上游调控机制。..研究发现：（1）IP3R-Grp75-VDAC1-MCU钙轴分子高表达介导足细胞损伤，激活钙轴导致线粒体钙超载和足细胞凋亡，拮抗钙轴显著减少足细胞凋亡。（2）IP3R-Grp75-VDAC1-MCU钙轴分子高表达参与阿霉素大鼠肾病模型蛋白尿的发生，钙轴拮抗剂显著改善了足突融合和蛋白尿。（3）部分肾病综合征患儿肾小球IP3R-Grp75-VDAC1-MCU钙轴分子表达增强。（4）在阿霉素大鼠肾病模型中，线粒体动力学蛋白和mTORC1高表达与IP3R-Grp75-VDAC1-MCU钙轴分子高表达伴随。在阿霉素诱导的小鼠足细胞损伤时，mTORC1过度活化与钙轴分子高表达伴随，拮抗mTORC1显著抑制了阿霉素诱导的IP3R-Grp75-VDAC1-MCU钙轴分子表达上调，减少足细胞凋亡，提示mTORC1可能是IP3R-Grp75-VDAC1-MCU钙轴的上游调控机制。..本研究证实IP3R-Grp75-VDAC1-MCU钙轴紊乱是新的足细胞损伤路径，为进一步理解足细胞损伤机制，开发新的足细胞保护药物提供了理论参考。