线粒体稳态调控：肾脏纤维化干预的新靶标

Renal fibrosis is the final common pathway to end-stage renal disease. Understanding the mechanisms of renal fibrosis is essential in establishing novel therapeutic strategies for the prevention or arrest of progressive kidney diseases. Among the renal residential cells, tubular epithelial are the principal effectors in initiating and mediating renal fibrosis. Our previous study showed that impaired mitochondrial homeostasis was the early event in tubular epithelial cell injury. However, the mechanism involved in the impaired mitochondrial homeostasis remains unclear. Our preliminary data showed that mitochondrial Lon protease expression/activity were significantly down-regulated in the kidney from unilateral ureteral obstruction (UUO) mice and patients with CKD and in the cultured renal tubular epithelial cells stimulated with TGF-beta or angiotension II (Ang II). Transcription factor Oct-1, as transcriptional repressor for Lon protease, inhibited its expression, whereas mitochondrial deacetylase Sirt3 regulated the activity by deacetylation of Lon protease. Therefore, we hypothesized that Lon protease dysfunction induced the impaired mitochondrial homeostasis, which play an important role in the mechanism of renal fibrosis. To test this hypothesis, we determine the effect of Lon protease on maintaining mitochondrial homeostasis and blocking renal fibrosis by using the renal tubular epithelial Lon protease or Oct-1 conditional knockout mice and transgenic mice and cultured renal tubular epithelial cell models. We will investigate the role of transcription factor Oct-1 and deacetylase Sirt3 in the transcriptional and post-transcriptional regulation of Lon protease. The present study will not only enrich the theory of mitochondrial homeostasis and the occurrence of renal fibrosis, also provide the new targets for intervention in renal fibrosis.

肾脏纤维化是CKD进展到ESRD的共同途径，肾小管上皮细胞损伤是启动和推进纤维化的关键环节。我们前期揭示线粒体稳态失衡是肾小管上皮细胞损伤的起始事件，深入探讨该机制有望为干预肾脏纤维化提供新的靶标。前期结果进一步发现，Lon蛋白酶的表达/活性下降与肾脏纤维化进展密切相关；Lon蛋白酶负责线粒体蛋白质量控制，参与维持线粒体的稳态。由此设想，Lon蛋白酶功能障碍引发线粒体稳态失衡是肾脏纤维化进展的核心机制；拟利用肾小管上皮细胞条件性Lon蛋白酶敲除及转基因小鼠和细胞模型，明确Lon蛋白酶在维持线粒体稳态、阻断肾脏纤维化发生中的作用及分子机制。在此基础上，我们还发现转录因子Oct-1和去乙酰化酶Sirt3分别调控Lon蛋白酶的转录表达及乙酰化修饰的活性；故结合体内模型进一步探讨Lon蛋白酶的调控机制，及其在肾脏纤维化中的意义。本研究将丰富肾脏纤维化发生的线粒体稳态学说，为向临床转化提供理论依据。

肾脏纤维化是各种原因导致的慢性肾脏病（CKD）进展到终末期肾病（ESRD）的共同途径，我们前期研究发现线粒体功能障碍是CKD的早期事件，但其在肾脏纤维化中的作用及其机制尚不清除。我们在伴肾脏纤维化的患者肾组织中发现线粒体出现显著的损伤，且与肾功能、肾脏纤维化程度呈显著负相关。通过经典的肾脏纤维化模型-单侧输尿管梗阻（UUO）以及5/6肾切除（5/6Nx）模型均证实，线粒体功能障碍介导了肾脏纤维化的发生。保护线粒体功能能够显著减轻肾组织中NLRP3炎症小体活化及肾脏纤维化，而NLRP3基因敲除则减轻肾脏线粒体损伤，表明线粒体与NLRP3炎症小体对话介导了肾脏纤维化的发生发展。我们提取肾脏纤维化小鼠肾组织线粒体，应用microRNA芯片发现miR-214在线粒体中表达显著升高，过表达miR-214可诱导肾小管细胞线粒体功能障碍及凋亡，肾小管条件性miR-214敲除可显著减轻白蛋白负荷、UUO、单侧IR导致的肾脏纤维化。Lon蛋白酶（LONP1）是线粒体内含量最多、最为重要的蛋白酶，参与降解线粒体内不正常的蛋白，能够调控线粒体稳态，保护线粒体免受氧化应激等的损伤，从而在线粒体功能障碍介导的疾病中发挥着重要的作用。我们的研究发现LONP1的表达及活性随着肾脏纤维化程度的加重而降低；进一步研究发现转录因子Oct-1可转录抑制LONP1表达，同时LONP1存在转录后修饰调节。肾小管细胞条件性Lonp1基因敲除加重UUO和5/6Nx诱导的线粒体损伤、肾脏纤维化以及肾功能的进一步恶化；足细胞条件性Lonp1基因敲除小鼠出生2周开始出现蛋白尿，3周出现肾脏纤维化，4-5周开始死亡。通过蛋白质组学筛选鉴定出HMGCS2可能为LONP1的底物。另外，我们也筛选出三个潜在的LONP1小分子激动剂，目前正在探索其在肾脏纤维化中的作用。综上所述，本项目的完成丰富了线粒体稳态调节与肾脏纤维化发生的学说，促进相关的基础研究向临床应用转化，为肾脏纤维化早期诊断及干预药物的研发提供新的理论依据。