MiR-130b在嘌呤霉素氨基核苷诱导的足细胞线粒体功能障碍中的作用及白藜芦醇的保护机制研究

It has been clearly established that podocyte lesion is the important reason for occurrence and development of chronic kidney disease (CKD) in both humans and experimental animal models, therapeutic strategies aimed specifically at improving the health and function of the podocyte represent a promising avenue to address CKD. Mitochondrial dysfunction is the main initiator of podocyte lesion by various local physical and chemical factors. In our preliminary experiment, the up-regulated circulating miR-130b has been observed in the lupus nephritis(LN) patients with early CKD stage when compared with healthy controls. The further analysis found that the expression of serum miR-130b was positively correlated with 24-hour proteinuria. These studies demonstrate that the up-regulation of miR- 130b level may be associated with podocyte lesion in CKD patients. The present study is performed in vitro and in vivo: (1)We first determine the effect of miR-130b on mitochondrial function and apoptosis in cultured mouse podocytes injuried by puromycin aminonucleoside(PAN), and then to get further details of target genes and regulation mechanism of miR-130b by multiple molecular biology technology. (2) In pre-test, We found miR-130b could be up-regulated by PAN while reduced by resveratrol(Res), The further study show that NFYc is one of the promoter of miR-130b and can be regulated by PAN and Res in the pre-test of luciferase system. we will outline Whether regulation of NFYc by Res play a crucial role in protective effect of podocyte injury in mitochondrial function induced by PAN; (2) The experiment model mouse with nephrosis will be established by way of intravenous injection of PAN. We will assess the presence of an association between expression of miR-130b and the rate of progression of renal disease or podocyte lesion in PAN mice. The effect of miR-130b on podocyte lesions, mitochondrial dysfunction and some biochemical indexes such as proteinuria and serum creatinine，will be investigated by suppressing the expression of miR-130b. These data will allow us to gain further insight into the mechanism of podocyte injury in CKD patients, which is prerequisite to designing novel therapeutic strategies to preserve the structure and functional properties of the podocyte，and offer the new theory basis for the Res’s treatment in CKD.

足细胞损伤是促进慢性肾脏病（CKD）发生发展的重要原因，线粒体功能障碍是足细胞损伤的使动因素。我们前期的研究显示早期狼疮性肾炎（LN）患者存在血清miR-130b高表达，并且与蛋白尿水平呈正相关，提示miR-130b可能参与足细胞损伤。本研究拟通过嘌呤霉素氨基核苷（PAN）诱导小鼠足细胞损伤，探讨miR-130b对于线粒体功能障碍的影响以及机制；同时，预实验中我们发现白藜芦醇（Res）可以下调足细胞miR-130b的表达，其机制可能通过影响转录因子NFYc与启动子区DNA的结合。本研究中，我们将基于miR-130b的表达探讨Res在改善线粒体功能障碍中的新机制；同时建立PAN诱导的小鼠肾病模型，观察miR-130b表达与疾病进展及Res治疗效果的关系，并探讨干扰miR-130b是否有利于延缓疾病进展。本研究结果可为CKD干预措施提供新的治疗靶点，并为Res的足细胞保护作用提供新的理论依据。

研究表明无论原发病如何，蛋白尿均可加速CKD的进展。因此蛋白尿是CKD防治工作中重要的靶点，减少蛋白尿的发生发展将有助于延缓CKD的进展，改善患者预后。足细胞的损害是肾小球性蛋白尿发生的主要机制，与多种肾小球疾病的发生发展关系密切，本研究主要着眼于慢性肾脏病患者足细胞损伤机制及靶向基因治疗进行检测。在本研究中，我们首先通过加权重共表达网络（WGCNA）分析显示分析多种CKD患者的肾小球部分的表达谱信息和临床资料，并构建出足细胞损伤相关的基因共表达模块，通过计算模块内间距计算出足细胞损伤的核心基因MAGI2，并进一步研究发现肾病综合征（NS）和糖尿病肾病（DN）患者肾小球MAGI2表达量与肾小球滤过率（eGFR）呈正相关（r = 0.575 P = 1.99e-4 和 r = 0.668 P = 0.049），在狼疮性肾炎患者（LN）则与蛋白尿呈反比（r = -0.501，P = 0.007），抑制足细胞内MAGI2的表达则可以逆转阿霉素和嘌呤霉素（PAN）诱导的足细胞骨架排列紊乱和凋亡，同时，生物信息学预测及双荧光素报告基因系统显示MAGI2是miR-130b-3p的靶基因；体外研究显示PAN可以诱导miRNA-130b-3p表达水平上升，并呈现时间依赖型和浓度依赖型，miR-130b-3p mimic加重PAN诱导的足细胞损伤，抑制足细胞内miR-130b-3p的表达可以有效减缓PAN诱导的足细胞损伤；miR-130b-3p mimic加重PAN诱导的足细胞骨架重排，抑制足细胞内miR-130b-3p的表达可以有效减缓PAN诱导的足细胞骨架排列紊乱；抑制足细胞内源性miR-130b-3p表达减少嘌呤霉素氨基核苷（PAN）引起的足细胞内活性氧（Reactive oxygen species，ROS）积累和线粒体膜电位（∆Ψm）下降；抑制足细胞内源性miR-130b-3p表达减少嘌呤霉素氨基核苷（PAN）引起的足细胞凋亡；双荧光素素报告酶基因系统显示PGC-1α、PPAR-γ是miR-130b-3p的直接靶基因。体内实验显示，使用miR-130b抑制可显著降低PAN引起的小鼠蛋白尿，减轻小鼠的肾脏足细胞损伤，并提高Nephrin、synaptoptin的表达。miR-130b-3p具有是CKD足细胞损伤的潜在治疗靶点，并且是白藜芦醇保护足细胞的靶点之一。