肾小球内皮细胞表达的swiprosin-1在早期糖尿病肾病发病中的作用及机制研究

The pathogenesis of diabetic nephropathy remain unclear. It was demonstrated that the glomerular endothelial cells dysfunction played a vital role in diabetic nephropathy and caused mesangial cells lesions and glomerular sclerosis. Our previous studies found that swiprosin-1, a novel calcium-binding protein, was highly expressed in renal glomerular endothelial cell of mice. Swiprosin-1 expression increased in renal cortical tissue of rat after streptozotocin-induced diabetes mellitus 4 weeks. Over-expression of swiprosin-1 in human renal glomerular endothelial cell significantly increased cell permeability. These results suggested that swiprosin-1 might play an important role in pathogenesis of diabetic nephropathy. However the mechanisms of swiprosin-1 involved in the early stage of diabetic nephropathy are still unclear. To further demonstrate that swiprosin-1 involves in the early stage of diabetic nephropathy, the streptozotocin-induced diabetic nephropathy is planned to investigate in swiprosin-1 knockout mice and wild type mice. The cell permeability is intended to observe after RNA interfering swiprosin-1 in human renal glomerular endothelial cells. To explore the signal transduction pathway of swiprosin-1 in diabetic nephropathy, the swiprosin-1 expression is observed in diabetic nephropathy mice and human renal glomerular endothelial cells after inhibition or RNA interference of protein kinase C-beta. To explore the mechanisms of swiprosin-1 in regulation of human renal glomerular endothelial cells permeability, the expression and function of cytoskeleton protein, F-actin, is intended to examine when over-expression of swiprosin-1 in human renal glomerular endothelial cell. Our studies will contribute to clarigy the mechanisms of early diabetic nephropathy, to find the potential target for therapeutic intervention diabetic nephropathy, and provide a theoretical basis for design a novel drug for prevent of diabetic nephropathy.

糖尿病肾病（DN）的确切发病机制至今尚未完全阐明，近年研究发现肾脏内皮细胞功能障碍在DN的发生发展中起重要作用。我们前期研究发现一种新型蛋白swiprosin-1（SWP）在肾小球内皮细胞高表达；糖尿病大鼠模型4w后肾皮质组织增加SWP表达；人肾小球内皮细胞（HRGEC）在高糖诱导后增加SWP表达；过表达SWP基因后HRGEC的通透性增加，表明SWP可能是影响早期DN发病的重要蛋白。 本项目拟进一步观察SWP基因敲除小鼠DN的发病情况及高糖诱导干扰SWP基因表达的HRGEC通透性，明确SWP在DN早期中的重要作用；抑制或干扰蛋白激酶C对DN小鼠早期肾脏和HRGEC表达SWP的影响及过表达SWP对细胞骨架蛋白的影响，从而明确SWP影响早期DN的信号通路及调节细胞通透性机制。本项目将有助完善阐明DN早期发病机制，发现调控DN新分子靶点，为设计延缓DN新型药物提供一定的理论基础。

糖尿病肾病是糖尿病患者主要的致死性微血管并发症之一。糖尿病肾病最显著的临床特征是尿蛋白排泄增加。蛋白尿的发生与肾小球滤过屏障的结构和功能改变有着密切的关系。肾小球内皮细胞是构成肾小球滤过屏障主要一种细胞。目前研究发现，肾小球内皮细胞损伤是导致糖尿病肾病持续进展的重要因素。.Swiprosin-1是2004年首次报道发现的小分子量蛋白，目前关于该蛋白的研究主要集中见于B淋巴细胞、T淋巴细胞等免疫细胞调节方面，该蛋白在其他方面作用知之甚少。我们研究发现swiprosin-1主要在肾小球内皮细胞高表达；糖尿病小鼠模型4w，8w和16w后肾皮质组织增加swiprosin-1表达；人肾小球内皮细胞（HRGEC）在高糖诱导后增加swiprosin-1表达；同时伴随着肾小球内皮细胞凋亡数目增加；给药PKCβ抑制剂LY333531可明显抑制肾小球内皮细胞凋亡和swiprosin-1表达上调；肾小球内皮细胞过表达swiprosin-1增加caspase-9, caspase-3和Bax表达，促进凋亡；相反敲减swiprosin-1减弱高糖或PKCβ激动剂诱导的肾小球内皮细胞凋亡；过表达swiprosin-1促进swiprosin-1和caspase-9相互作用，增加凋亡小体形成；和swiprosin-1+/+糖尿病小鼠相比，swiprosin-1-/-糖尿病小鼠的肾重/体重和尿白蛋白增加，肾小球肥大， 线粒体依赖的凋亡相关蛋白增加，肾小球内皮细胞凋亡增加。.总之，本研究证实了肾小球内皮细胞表达的swiprosin-1是影响糖尿病肾病进展的一个重要蛋白；swiprosin-1介导高糖诱导的肾小球内皮细胞凋亡；高糖刺激增加PKCβ1的表达，上调swiprosin-1表达，促进细胞色素C释放，激活caspase9和caspase3，引发肾小球内皮细胞凋亡；swiprosin-1缺失可减少糖尿病小鼠早期肾小球内皮细胞的凋亡，延缓糖尿病肾病的发展。本研究有助于完善早期糖尿病肾病发病的机制，发现调控糖尿病肾病新的分子靶点，为设计新型抗糖尿病肾病药物提供一定的理论依据。