转录因子Foxo3在镉致肾小管上皮细胞线粒体自噬中的作用

Mitophagy is involved in mechanism of kidney injury, but the role of mitophagy in cadmium-induced renal damage has not been clarified yet. The transcription factor Foxo3 is involved in the regulation of mitophagy, and previous studies have found that cadmium causes abnormal protein expression of Foxo3 and mitophagy in proximal tubular cells. Accordingly, we propose here the scientific hypothesis that Foxo3 may participate in regulating the mitophagy in proximal tubular cells exposed to cadmium, and present the following research proposal to validate it. In present study, primary rat proximal tubular (rPT) cells and NRK-52E cells will be applied for the in vitro toxicology model. Partial cells will be treated with either gene overexpression or gene silence technology to make some genes overexpressed or silenced, respectively. Then, all of the model cells will be exposed to cadmium. After the treatments, the levels of protein expression, phosphorylation, nuclear translocation of Foxo3, and mitophagy level will be assessed to clear the role of Foxo3 in cadmium-induced mitophagy of proximal tubular cells. Next, the expression of proteins in mitochondrial dynamic, PINK1/Parkin signaling pathway, ubiquitination and formation of autophagosome will be assessed, to clear the role of miRNAs located downstream of Foxo3 via mitochondrial homeostasis-, damaged mitochondria recognition and trafficking-mediated cadmium-induced mitophagy in proximal tubular cells. This study will elucidate the regulatory role of Foxo3 in cadmium-induced mitophagy of rPT cells, which can provide the theoretical evidence for prevention of nephrotoxicity induced by cadmium from the perspective of mitochondrial protection.

线粒体自噬与肾脏损伤机制密切相关，但线粒体自噬在镉致肾脏损伤中的作用尚不明确。转录因子Foxo3参与调控线粒体自噬，前期研究发现镉导致肾小管上皮细胞Foxo3蛋白表达异常和线粒体自噬。因此，本项目提出Foxo3参与调控镉致肾小管上皮细胞线粒体自噬这一科学假说，开展如下研究：对大鼠肾小管上皮细胞特定基因/miRNA的表达正负调控后，进行镉染毒。检测Foxo3蛋白表达、磷酸化、核转位及线粒体自噬变化，明确Foxo3在镉致肾小管上皮细胞线粒体自噬中的作用；检测线粒体动力蛋白表达、PNK1/Parkin信号通路蛋白表达、泛素化水平和自噬小体关键蛋白表达，明确Foxo3下游miRNA通过介导线粒体融合-分裂稳态、受损线粒体识别和转运调控镉致肾小管上皮细胞线粒体自噬的机制。预期结果将阐明Foxo3在镉致肾小管上皮细胞线粒体自噬中的调控作用，为从线粒体保护的角度防治镉的肾脏毒性提供理论依据。

线粒体自噬是肾脏损伤过程中一种重要反应机制，研究表明其参与氧化应激导致的肾脏损伤过程。但线粒体自噬在镉致肾脏损伤中的作用尚不明确。前期研究发现镉暴露会导致肾小管上皮细胞FOXO3蛋白表达和线粒体自噬异常，本项目提出FOXO3参与调控镉致肾小管上皮细胞线粒体自噬这一科学假说，开展如下研究：原代大鼠肾小管上皮细胞经基因过表达/沉默技术对特定基因的表达进行调控，或经特异性抑制剂/诱导剂处理后进行CdCl2染毒，检测FOXO3蛋白表达和磷酸化修饰，Nrf2蛋白表达和核转位，线粒体融合蛋白OPA1和MFN2及线粒体分裂蛋白FIS1和DNM1L表达，miRNA Let-7f-2-3p表达，及线粒体自噬。研究结果如下：① 发现镉暴露导致FOXO3蛋白表达量降低，磷酸化FOXO3与总FOXO3比值显著升高，线粒体结构损伤，及线粒体网络异常。②镉暴露导致Nrf2核转位增加，FOXO3基因敲减进一步促进Nrf2核转位水平，线粒体损伤加剧，同时线粒体自噬增强。③镉暴露导致的Nrf2核转位改变线粒体融合蛋白OPA1和MFN2及线粒体分裂蛋白FIS1和DNM1L的表达，导致线粒体稳态失衡。④FOXO3靶分子miRNA Let-7f-2-3p在镉暴露的肾小管上皮细胞内表达降低，负调控线粒体自噬相关蛋白PARKIN的表达，参与调控线粒体自噬。本项目发现FOXO3靶基因Nrf2和miRNA Let-7f-2-3p分别参与调控线粒体稳态，受损线粒体识别和线粒体自噬。证实FOXO3通过Nrf2 - 线粒体稳态和Let-7f-2-3p - Parkin分别调控线粒体稳态和线粒体自噬参与镉暴露导致的大鼠肾小管上皮细胞病理损伤过程。本研究结果为从线粒体保护的角度防治镉的肾脏毒性提供理论依据。