毒素蓄积诱导慢性肾脏病胰岛素抵抗的分子机制研究

The accumulation of toxins in patients with chronic renal failure induced insulin resistance, oxidative stress may play an important role in these, but the specific mechanism is unknown. This study contains three parts, the first part is the in vivo experiments: establish the 5/6 nephrectomized model of rats, and the parameters of renal function, urinary protein, renal and pancreatic pathology, glucose metabolism, insulin signaling pathway and oxidative stress would be collected to clarify the mechanism of insulin resistance in chronic renal failure, and also the effects and molecular mechanism of valsartan and probucol. The second part is the in vitro experiments: the model of insulin resistance induced by urea in skeletal muscle cells would be established, the parameters of glucose metabolism, insulin signaling pathway and oxidative stress would be collected to clear the mechanism of urea induced insulin resistance of skeletal muscle, and also the role of oxidative stress in the process. The third part: use of proteomics, bioinformatics, RNAi, immunoprecipitation and immunoblotting techniques to find and validate the new biomarkers for the chronic renal failure patients with insulin resistance. The results of this research will provide a theoretical basis and experimental data for the future clinical intervention of insulin resistance in patients with chronic renal failure.

慢性肾衰患者毒素蓄积会诱发胰岛素抵抗，氧化应激可能在其中起重要作用，但具体机制不明。本研究分为三部分，第一部分体内研究：建立5/6肾切除大鼠慢性肾衰模型，通过对各组大鼠肾功能及尿蛋白检查、肾脏及胰腺病理检查、糖代谢相关检查、胰岛素信号通路相关检查和氧化应激水平检查明确慢性肾衰胰岛素抵抗的发生机制以及缬沙坦和普罗布考缓解慢性肾衰胰岛素抵抗的作用效应和分子机制。第二部分体外研究：建立尿素诱导骨骼肌细胞胰岛素抵抗模型，观察各组细胞糖代谢、胰岛素信号通路及氧化应激水平的改变，明确尿素诱导骨骼肌胰岛素抵抗的机制以及在这一过程中氧化应激的作用。第三部分：利用蛋白组学、生物信息学、RNAi、免疫共沉淀和免疫印迹技术筛选并验证与慢性肾衰胰岛素抵抗新的分子标志物。研究结果将为今后临床干预慢性肾衰患者胰岛素抵抗提供理论依据和实验数据支持。

尿素诱导骨骼肌胰岛素抵抗的分子机制及其干预研究.目的：研究胰岛素抵抗（insulin resistance, IR）与慢性肾衰的关系，探索慢性肾衰患者发生IR的分子机制。.方法：通过CKD代表毒素尿素刺激骨骼肌细胞（C2C12细胞系）和大鼠5/6肾切除，建立离体、在体慢性肾衰模型，观察CKD骨骼肌糖摄取改变；同时设立AngⅡ组和缬沙坦组，观察干预药物对IR的作用，并对各组C2C12细胞、大鼠肾组织和骨骼肌进行western blot，检测细胞和组织中胰岛素信号通路上关键蛋白IRS-1、Akt的表达及其磷酸化情况。.结果：尿素刺激和5/6肾切除均能诱发骨骼肌细胞糖摄取障碍（P < 0.01），AngⅡ组骨骼肌IR加重（P < 0.05），而缬沙坦组IR减轻（P < 0.01）。无论在体或离体实验，模型组IRS-1和Akt酪氨酸磷酸化均较假手术组/对照组明显降低（P < 0.01），丝氨酸磷酸化则显著升高（P < 0.01）。在模型组基础上叠加AngⅡ加重异常磷酸化（P < 0.05），反之，叠加缬沙坦在一定程度上减轻异常磷酸化（P < 0.05）。.结论：慢性肾衰毒素蓄积可使机体发生IR，骨骼肌IS下降，AngⅡ加重上述现象而缬沙坦能使其部分缓解。以尿素为代表的毒素作用于IRS-PI3k-Akt通路上关键蛋白，使其磷酸化情况改变可能是慢性肾衰患者IR的发生机制之一。