线粒体动力学在铅镉联合致肾小管上皮细胞自噬中的调控作用

The proximal tubular cells are the target sites of renal damage induced by lead and/or cadmium. It has been demonstrated that mitochondria are the key target damaged organelles following lead and/or cadmium exposure. Moreover, mitophagy plays an important role in the process of clearing damaged mitochondria and renewing mitochondria. It is important to note that mitophagy is closely related to mechanism of kidney injury, but the role of mitophagy in lead and/or cadmium-induced renal damage has not been clarified yet. It is known that imbalance of mitochondrial dynamics is a prerequisite for mitophagy, we have previously found that dysfunction of mitochondrial dynamics has been involved in rat proximal tubular (rPT) cells exposed to lead and/or cadmium. Accordingly, we propose here the scientific hypothesis that mitochondrial dynamics may participate in regulating the mitophagy in proximal tubular cells exposed to lead and/or cadmium, and present the following research proposal to validate it. In this study, primary rPT cells will be applied for the in vitro toxicology model. Some portion of primary cells will be treated with either gene overexpression or gene silence technology to make some genes overexpressed or silenced, respectively. Also, some portion of cells will be incubated with specific chemical inhibitors. Then, all of the model cells will be exposed to lead and/or cadmium. After the treatments, expression levels of mitochondrial dynamics proteins will be assessed by immunoblot assays. Moreover, we will investigate the effect of mitochondrial dynamics proteins on mitochondrial morphology, mitochondrial function and the occurrence of mitophagy in lead/cadmium-exposed rPT cells. Next, we will analyze the protein levels of key signaling molecules, mitochondrial translocation of some proteins from cytoplasm, ubiquitination of some proteins, and protein-protein interactions involving in the PINK1/Parkin signaling pathway, also clarify the link between p62 adapter protein and the balance of mitochondrial dynamics. Finally, this study will form the basis of multiple signaling events that demonstrate the role of PINK1/Parkin pathway in the occurrence of mitophagy induced by lead and/or cadmium. This study will elucidate the regulatory role of mitochondrial dynamics in lead and/or cadmium-induced mitophagy of rPT cells, which can provide the theoretical evidence for prevention of nephrotoxicity induced by lead and/or cadmium from the perspective of mitochondrial protection.

肾小管是铅、镉致肾毒性损伤的靶部位。现已证明线粒体是铅镉肾毒性作用的重要靶标，线粒体自噬在受损线粒体清除和线粒体更新过程中发挥关键作用。线粒体动力学(线粒体分裂与融合的动态平衡过程)是维持线粒体功能的重要保障，线粒体动力学改变是线粒体自噬发生的必要条件。课题组前期研究发现铅镉染毒过程中肾小管上皮细胞线粒体的损伤和线粒体自噬现象，但线粒体动力学在此过程中的作用机制尚不明确。因此，本项目提出线粒体动力学参与调控铅镉致肾小管上皮细胞线粒体自噬的发生这一科学假说，开展如下研究：原代大鼠肾小管上皮细胞经基因过表达与沉默技术对特定基因的表达进行正负调控，或经特异性抑制剂处理后，进行铅、镉单独与联合染毒。检测线粒体动力蛋白表达水平，分析动力蛋白对线粒体形态、功能及线粒体自噬发生的影响；检测PINK1/Parkin信号通路中关键信号分子的蛋白表达、线粒体转位、泛素化水平及蛋白互作，分析p62蛋白与线粒体动力学的内在联系，明确该通路在铅镉致肾小管上皮细胞线粒体自噬发生中的作用。预期结果将阐明线粒体动力学在铅镉联合致肾小管上皮细胞自噬中的调控作用，为从线粒体保护的角度防治铅镉的肾毒性提供理论依据。

线粒体自噬是一种重要的线粒体质量控制机制，通过选择性清除受损的线粒体维持细胞内环境的稳定。线粒体自噬与肾损伤机制密切相关，但线粒体自噬在铅镉致肾损伤中的作用尚不明确。线粒体动力学改变是线粒体自噬发生的必要条件，本项目围绕线粒体动力学蛋白参与调控铅镉致肾小管上皮细胞线粒体自噬发生这一科学假说，开展了如下研究：原代大鼠肾小管上皮细胞经基因过表达与沉默技术对特定基因的表达进行正负调控，或经特异性抑制剂处理后，进行铅、镉染毒。检测线粒体动力蛋白表达水平，分析动力蛋白对线粒体形态、功能及线粒体自噬发生的影响；检测PINK1/Parkin信号通路中关键信号分子的蛋白表达、线粒体转位、泛素化水平及蛋白互作，明确该通路在铅镉致肾小管上皮细胞线粒体自噬发生中的作用；检测了p62蛋白与线粒体动力学蛋白Drp1及受损线粒体的互作，分析该蛋白在线粒体自噬发生中的作用。取得如下研究结果：①发现铅镉染毒导致肾小管上皮细胞线粒体动力学稳态失衡，明确了分裂蛋白Drp1、融合蛋白Mfn2是铅镉诱导线粒体断裂和线粒体自噬活化的关键动力学蛋白；②发现铅镉通过活化PINK1/Parkin信号通路促进线粒体自噬，明确了Parkin的线粒体转位促进Mfn2蛋白泛素化是受损线粒体被自噬体识别的关键环节。③发现铅镉诱导p62与Drp1发生蛋白互作，且p62促进携带Drp1的受损线粒体转运至自噬体，进行自噬降解以维持线粒体动力学平衡，明确了p62是受损线粒体转运至自噬体的重要衔接蛋白。本项目明确了铅镉通过诱导线粒体动力学稳态失衡而激活线粒体自噬的分子机制，阐明了PINK1/Parkin信号通路在铅镉活化线粒体自噬中的关键作用，确证了p62在自噬体识别受损线粒体中的衔接作用。本项目从线粒体动力学稳态的角度详细阐明了铅镉活化肾小管上皮细胞线粒体自噬的分子机制，为从线粒体保护的角度防治铅镉的肾毒性提供了重要理论依据。