microRNA-217/Wnt5a介导多巴胺D2受体对肾脏纤维化的保护机制研究

Besides its actions in the maintenance of normal renal function and blood pressure the renal dopaminergic system has remarkable effects in the regulation of inflammatory reaction and tissue injury. Lack or downregulation of the dopamine D2 receptor (D2R) activates TGFβ1 pathway and induces renal fibrosis, but the mechanism is undetermined. D2R single nucleotide polymorphisms (SNPs) are common in human and associated with decreased receptor expression/function. People carrying D2R SNPs may be vulnerable to chronic renal failure. We have shown in human renal proximal tubule cells from subjects carrying D2R SNPs that miR-217 mediates the protective effect of D2R on renal fibrosis, probably through regulating Wnt5a/Ror2 pathway. This study aims to verify Wnt5a as the target of miR-217 by structural and functional tests; to study the effects and mechanism of miR-217 on renal fibrosis, and the therapy in models of diabetic nephropathy and unilateral obstructive nephropathy using miRNA sponge, ultrasonic microbubble-mediated kidney transfection. We will clarify the role of miR-217/Wnt5a in renal fibrosis and provide new targets for early intervention.

肾内多巴胺系统对维持正常肾功能与血压起重要作用，近期发现多巴胺D2受体(D2R)缺失可激活TGFβ1，导致肾纤维化，但作用通路未明。D2R编码基因的单核苷酸多态性(SNPs)在人群中高发，可导致D2R表达与功能下调，携带该SNPs位点者可能成为慢性肾衰竭的易感人群。课题组已在携带D2R SNPs位点的人近端肾小管细胞中发现miR-217可介导D2R对肾纤维化的抑制作用，且可能通过下调Wnt5a/Ror2通路而抑制TGFβ1表达。本研究进一步从基因结构与功能角度验证Wnt5a为miR-217靶基因；并通过miRNA sponge、超声微泡介导肾脏定向转染等技术，研究下调miR-217的致肾纤维化作用；上调miR-217逆转D2R敲除小鼠肾纤维化的作用，及对糖尿病肾病、单侧梗阻性肾病模型的治疗意义。本研究可明确miR-217/Wnt5a对肾纤维化的作用及机制，有助于开发慢性肾脏病防治的新靶点。

肾脏纤维化是各种肾脏疾病慢性、进行性发展的共同通路，直接导致慢性肾衰竭，尚缺乏有效的预测和治疗办法。本课题在体外实验中，发现人肾小管上皮细胞中D2R抑制TGFβ1信号传导通路，从而抑制该细胞向纤维化表型转化；并且证明其作用通过miR-217下调Wnt5a/Ror2信号通路的活化而介导，同时D2R还可通过Wnt/β-catenin途径干预肾小管上皮细胞凋亡与增殖；但是在动物模型体内实验中，敲除D2R模型未出现明显的肾纤维化表现，而过表达miR-217对糖尿病肾病模型、单侧梗阻性肾病(UUO)模型的肾脏病变进展均未发现明显效果，其原因可能与体内相关通路被抑制后，其它补偿机制的激活有关。因此，我们对实验方案进行了调整，在I/R诱导急性肾损伤后发生肾纤维化（AC模型）小鼠中重新采用RNA-seq进行miRNA筛选，发现miR-874-3p表达下降，而将其过表达可明显减轻AC模型和UUO模型纤维化；并证实ADAM19是miR-874-3p的结合靶蛋白，在正常小鼠中过表达ADAM19可导致明显的肾脏炎症与纤维化；下游机制上，miR-874-3p/ADAM19信号通路可以通过增强肾小管上皮细胞损伤和巨噬细胞浸润调控肾脏纤维化；并在不同病情严重程度分期的IgA肾病患者中证实miR-874、Adam19表达水平具有显著的鉴别价值。由此，我们发现miR-874-3p/ADAM19可能具有较好的临床应用前景，对于预测肾病进展可能具有一定的价值。在机制研究的同时，我们也进行了IgA肾病、狼疮性肾炎、ANCA相关性小血管炎肾损害等肾脏炎性进展性疾病的患者资料与标本库建设，进行了相关特征分析与总结，为临床标志物的筛选提供了病例基础。