miRNA-214通过诱导线粒体功能障碍促进肾小管间质纤维化

Renal tubulointerstitial fibrosis is the final common pathway to end-stage renal disease. Understanding the mechanisms of tubulointerstitial 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 initiating and mediating tubulointerstitial fibrosis. Our previous study showed that mitochondrial dysfunction was the early event in tubular epithelial cell injury. However, the mechanism involved in the mitochondrial dysfunction remains unclear. Our preliminary data showed that albumin upregulated miRNA-214 expression and translocated to the mitochondia. Overexpression of miRNA-214 induced mitochondrial dysfunction and cell injury. Informatics analysis predicted 3’-UTR of NADH dehydrogenase 1 (ND1) and ND5 contains conserved critical nucleotides that may serve as a legitimate target of miRNA-214. Therefore, we hypothesized that after renal tubular epithelial cell injury, miRNA-214 expression increases and transloactes to the mitochondria, decreases ND1 and ND5 expression, and induces mitochondrial dysfunction and cell injury. To test this hypothesis, we identify that miRNA-214 directly targets the 3’-UTR of ND1 and ND5. Using miRNA-214 knockout mice and RNAi strategy, we determine the effect of miRNA-214 on the mitochondrial dysfunction and renal tubular epithelial cell injury , which may guide us in therapeutic strategies for renal tubulointerstitial fibrosis.

肾小管间质纤维化是多种慢性肾脏病进展到终末期肾衰竭的共同病理基础，肾小管上皮细胞损伤是启动和推进肾间质纤维化的主要因素。我们前期研究发现线粒体功能障碍是肾小管上皮细胞损伤的起始事件，并介导细胞损伤。但线粒体功能障碍的发生机制尚不清楚。预实验发现白蛋白促进肾小管上皮细胞中miRNA-214表达，且富集于线粒体中。过表达miR-214诱导线粒体功能障碍和细胞损伤，生物信息学分析提示线粒体NADH脱氢酶ND1和ND5是其潜在靶基因。由此设想肾小管上皮细胞在受到损伤刺激后，诱导miRNA-214表达并从胞浆转运、富集到线粒体，抑制线粒体ND1和ND5表达，诱导线粒体功能障碍和细胞损伤。本研究将鉴定线粒体内miRNA-214 靶基因，并应用miRNA-214基因敲除小鼠以及过表达和沉默技术阐明其在诱导线粒体功能障碍和肾小管损伤中的作用及其机制，为肾小管间质纤维化发生机制和早期干预的研究提供新的视点。

肾小管间质纤维化是多种慢性肾脏疾病进展到终末期肾衰竭的共同病理基础，其病理生理过程主要包括：细胞激活和损伤阶段、纤维形成的信号传递阶段、纤维形成阶段和肾脏损害阶段。其中，肾小管上皮细胞损伤及损伤后诱发的一系列生物化学反应是启动和推进肾小管间质纤维化的主要因素。在本研究中，我们应用白蛋白诱导的肾小管损伤及肾纤维化模型，系统研究了miR-214在白蛋白引起的肾小管损伤及纤维化中的作用及机制。在白蛋白处理的肾小管上皮细胞、小鼠肾脏，以及蛋白尿患者的肾脏组织，我们均发现了miR-214显著上调。进一步研究显示，在病理情况下，miR-214可以转入肾小管上皮细胞的线粒体，作用于线粒体靶基因ND4L及ND6，导致线粒体功能障碍，进而引起小管上皮损伤，诱导肾脏纤维化的发生和发展。在体内，我们在肾小管条件性敲除miR-214，然后给与白蛋白负灌注诱导小管损伤，结果发现miR-214KO 小鼠可以改善白蛋白诱导的肾小管损伤及线粒体功能障碍，验证了肾小管miR-214的病理作用。接下来我们使用anti-miRs药物体内抑制miR-214的表达，观察是否可以阻断白蛋白诱导的线粒体功能障碍和肾小管损伤，达到治疗的目的。腹腔注射anti-miR-214寡核苷酸，qPCR结果显示肾脏miR-214表达降低了约90%。给与白蛋白关注后，发现anti-miR-214治疗小鼠显著减少了肾小管刷状缘的消失，肾小管变性以及细胞凋亡。同时减轻了肾脏炎症因子IL-1β、TNF-α和MCP-1的表达。上述研究结果显示，miR-214可能是介导肾小管损伤的重要分子机制，靶向调控肾小管miR-214的表达可能为临床治疗肾脏疾病提供新的手段。