miR-455通过AMPK调控能量代谢防治糖尿病肾病的分子机制

Diabetic nephropathy (DN) is one of the most common causes of chronic kidney disease, leading to premature death and end-stage renal disease (ESRD). None of the new agents tested during the past decade have proved effective in late-stage clinical trials. Inhibitors of the renin-angiotensin-aldosterone system (RAAS) have lowered the risk of albuminuria but have not reduced the risk of ESRD. Increasing data suggest that abnormal lipid accumulation in the kidneys contributes to the kidney damage caused by diabetes and other kinds of chronic kidney diseases,which hint that the improvement of energy metabolism in kidneys may be a promise target for the treatment of nephropathy. MicroRNAs (miRs) are a family of small, noncoding RNAs that regulate gene expression in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis. Recently miRs correlated to TGF-β emerged as major area of biomedical research with relevance to DN. However miRs characterized by regulation of energy metabolism have not been reported to prevent the progression of DN. In our prior study we found that miR-455, which has selectively high expression in the kidney, was significantly upregulated during brown adipogenesis. Overexpression of miR-455 in mice help to resist the obesity induced by high fat diet via AMPK activation. Thus, in the present study, we will further explored the protective roles of miR-455 in the progression of DN. First, the mesangial cells with overexpression or knock-down of miR-455 incubated with high glucose will be employed to analyze the function of miR-455. The proliferation of cells and the excretion of FN、Col IV will be analyzed by MMT and ELISA respectively. The mRNA expressions of FN, Col IV, Col I, Col III, OPN, αSMA, TGF-β, AMPK, Sirt1,PGC1α, Nrf1 and Tfam will be analyzed by RT-qPCR. The protein expressions of TGF-β, Smad3, t-AMPK, p-AMPK and ACC will be analyzed by western blot. We will further verify the importance of AMPK pathway in the protective effects of miR-455 by AMPK-knock down using siAMPK in mesangial cells transfected by lenti-miR-455. Then we will further find out the target genes of miR-455. After verifying the protective effects and the mechanisms of miR-455 in vitro, the miR-455-mimic and LNA-miR-455 will be respectively injected into DN mice models to understand the roles of miR-455 on the progression of DN. In this study, we will elucidate the protective effects of miR-455 on the progression of DN via activation of AMPK-PGC1α signaling pathway in DN.

我们在前期研究中发现，miR-455选择性高表达于野生型小鼠的肾皮质，ap2-miR-455转基因小鼠能抵抗高脂饮食诱导的肥胖和代谢异常，其机制主要与miR-455激活能量感受器AMPK有关，而后者能改善高糖刺激下系膜细胞功能及高脂饮食诱导的apoE基因敲除小鼠的肾损害。我们推测miR-455可能通过抑制靶基因而激活AMPK发挥对糖尿病肾病（DN）的肾脏保护作用。本课题拟首先观察过表达和抑制miR-455对高糖刺激下系膜细胞功能及AMPK信号通路的影响；进一步采用AMPK基因沉默探讨miR-455保护系膜细胞的机制，并采用RNA-ChIP技术验证miR-455与候选靶基因的直接作用关系。本课题将阐明miR-455通过抑制靶基因进而激活AMPK改善代谢，可能是防治DN潜在的新靶点和治疗途径。

糖尿病肾病是导致终末期肾病（ESRD）的主要原因之一，如何有效延缓或防止糖尿病肾病进展仍然是目前的研究热点和难点。研究显示，约30%的糖尿病肾病患者尽管并无白蛋白尿，但肾功能仍呈进行性下降；尽管应用肾素-血管紧张素-醛固酮系统（RAAS）阻断剂能有效降低尿白蛋白水平，但并未完全阻止糖尿病肾病进展至ESRD。因此，深入研究糖尿病肾病的发病机制，寻找调控糖尿病肾病进展的关键靶点，探索更为有效而安全的防治手段十分重要。近年的研究揭示了能量代谢异常在糖尿病肾病的发病过程中具有十分重要的作用，并贯穿整个肾病进展过程的始终。细胞能量代谢感受器——腺苷酸活化蛋白激酶（AMPK）激活能通过改善能量代谢发挥对糖尿病肾病的肾脏保护作用。microRNA是一种内源性的仅有18~24个核苷酸的短链非编码RNA，通过降解靶基因的mRNA或抑制其翻译为蛋白质而达到对该基因功能的调控作用。由于microRNA的作用属于转录后的调控机制，合成方便，且在脏器安全性方面优于化合物，具有很好的临床应用前景，目前已成为肿瘤和丙肝等难治性疾病的新的防治手段，并已进入临床研究。. 课题组与丹麦哥本哈根大学的研究人员合作，发现microRNA-455在小鼠肾脏呈高表达，提示miR-455可能在肾脏生理和病理过程中发挥了重要作用。在前期研究基础上，课题组在体外实验中模拟了糖尿病肾病发生的重要病理基础，高糖刺激系膜细胞增殖及分泌ECMs增加等，并在系膜细胞中过表达miR-455，结果发现miR-455可保护高糖刺激下的系膜细胞功能，而敲减miR-455可加剧高糖引起的细胞损伤；进一步抑制AMPK信号通路可阻断了miR-455对系膜细胞的保护作用，提示miR-455可能通过激活AMPK信号通路而改善系膜细胞功能。研究结果揭示了代谢调控型microRNA——miR-455可通过激活AMPK信号途径改善能量代谢进而防治DN进展，从贯穿DN发生和进展整个过程的代谢紊乱着手，从改善肾脏局部代谢的角度，阐明了miR-455在DN防治中的价值，今后有望成为糖尿病防治的可能的新型手段。