DGKε/SNARE信号通路在糖尿病肾病足细胞胰岛素抵抗中的作用及机制

Despite evidence from multiple clinical and experimental studies, the pathogenesis of diabetic nephropathy is not yet fully understood.Recently reports indicate that podocyte insulin resistance is critical for the production of large amount of proteinuria which contribute to the devolepment of diabetic nephropathy. Diacylglycerol kinase ε (DGKε) is considered as a new target for the research work on kidney diseases according to the latest study. Our preliminary study suggested that DGKε may play an important role in podocyte injury and insulin resistance in diabetic nephropathy，but the mechanism is not yet understood. In this study, we will use various methods of morphology, cellular and molecular biology, GTPase Pull-Down，patch clamp，flow cytometry, Co-IP and confocal to investigate the role of DGKε on podocyte insulin resisitance in diabetic nephropathy by means of both in vivo and in vitro transfection techniques, together with clinical kidney sections. Moreover, we are going to demonstrate that the mechanism of this effect is partly through activating GTPase, Rho protein, then regulating the activity of soluble N-ethylmaleimide -sensitive factor-attachment protein receptor (SNARE) complex regulating protein, Sec/Munc18 like proteins (SM protein), which may in turn increase the stability of SNARE complex to improve the memberane transloction of glucose transporters 4 (GLUT4) from GLUT4 storage vesicles (GSV). In the end, we are aiming at establishing such a signal transduction pathway as diabetic nephropathy →DGKε↓→GTPase Rho protein→SM protein↓→SNAREcomplex↓→GSV vesicle trafficking↓→GLUT4 membrane transloction↓→podocyte insulin resisitance→podocyte injury. Our study may enrich and improve the pathophysiologic mechanisms of diabetic nephropathy, add new results for the signal transduction pathway of podocyte insulin sensitivity. Moreover, our work may provide a new viewpoint for both the basic and clinical research on the pathogenesis and the potential theraputic methods of diabetic nephropathy in the future.

糖尿病肾病病理生理机制未明，足细胞胰岛素抵抗参与了该病的发生发展，而最新研究表明DGKε已成为研究肾脏疾病的重要靶标。我们的前期工作发现DGKε可能在糖尿病肾病足细胞胰岛素抵抗中扮演重要角色，但机制不明。本课题将应用细胞、动物模型及临床标本，借助体内外转染技术，通过形态学、细胞和分子生物学、免疫共沉淀、膜片钳、流式细胞术、激光共聚焦、Pull-Down等方法明确在糖尿病肾病过程中，DGKε能够改善足细胞胰岛素抵抗，进一步证明该作用是通过调控SNARE复合体介导的GLUT4囊泡运输实现的，从而建立糖尿病肾病→DGKε↓→Rho蛋白→SM蛋白↓→SNARE复合体↓→GSV 囊泡运输↓→GLUT4膜转位↓→胰岛素抵抗→足细胞损伤的信号转导通路。本课题将为充实糖尿病肾病的病理生理机制、阐明糖尿病肾病足细胞损伤的信号转导途径增加新的研究基础，并为临床寻找糖尿病肾病有效防治措施提供新的思路。

糖尿病肾病是糖尿病最常见的血管并发症，也是终末期肾功能衰竭死亡的重要原因之一，严重威胁人类健康。迄今为止该病发生发展的确切病理生理机制尚不清楚，亦无理想的防治措施。糖尿病肾病的典型标志之一是大量蛋白尿，这也是疾病持续进展和肾功能衰竭的重要原因。作为肾小球滤过屏障重要组成部分的足细胞损伤是蛋白尿形成的最关键因素之一，也是终末期肾病肾小球损伤最重要的早期事件。自2005年足细胞被确认为胰岛素敏感细胞以来，与足细胞胰岛素抵抗相关的疾病发生发展机制受到广泛关注。最新研究表明二酰甘油激ε (DGKε, DGKE) 功能缺失性基因突变是参与非典型性溶血性尿毒综合征和肾小球微血管病变的重要因素，说明DGKε已成为研究肾脏疾病重要靶标。我们的前期工作发现DGKε可能在糖尿病肾病足细胞胰岛素抵抗中扮演重要角色，但机制不明。本课题应用细胞、动物模型及临床标本，借助体外转染技术和构建足细胞特异性基因敲入模式动物，通过建立1型和2型糖尿病肾病和嘌呤霉素氨基核苷肾病等不同的足细胞损伤模型，利用形态学、细胞和分子生物学、流式细胞术、免疫荧光双标等方法，证明在不同类型的糖尿病肾病中，DGKε的表达变化趋势不同，说明其在不同的疾病状态下发挥着不同的作用，尤其在二型糖尿病肾病模型中，DGKε参与了糖尿病肾病足细胞损伤和胰岛素抵抗的发生发展过程；而体内外足细胞特异性过表达DGKε能够显著减轻嘌呤霉素核苷肾病导致的足细胞损伤，进一步实验证明该作用是通过调控podocin, Nephrin和TRPC6等足细胞结构蛋白的表达实现的。本课题将为充实糖尿病肾病和其他足细胞损伤性肾脏疾病的病理生理机制、阐明足细胞损伤相关肾病的信号转导途径增加新的研究基础，并为临床寻找糖尿病肾病及其他足细胞损伤为特征的肾小球疾病的有效防治措施提供新的思路。