Role of NG2 in diabetic nephropathy

描述(申请人提供)：糖尿病肾病是1型和2型糖尿病的常见并发症。慢性肾功能衰竭的预防是肾病学家和肾脏内科研究人员面临的最重要的医学挑战之一。糖尿病肾病的特征是肾小球基底膜结构的改变和系膜基质的进行性扩张。肾小球系膜细胞被认为是包裹大多数大小血管的修饰周细胞样细胞(血管周围细胞)。NG2是一种细胞表面硫酸软骨素蛋白多糖，是周细胞的特异性标志物，在人和小鼠肾小球系膜细胞中特异表达。与其他类型的肾小球细胞相比，我们的初步研究表明，NG2在糖尿病肾病小鼠模型中的表达增加。这种NG2表达的增加与链脲佐菌素(STZ)诱导的高血糖小鼠肾小球系膜细胞的增殖和细胞外基质的产生直接相关。利用新型的NG2特异性小鼠转基因试剂，我们的目的之一是研究NG2阳性的系膜细胞在糖尿病肾病的发生和发展中的作用。我们的第二个目标是通过删除NG2阳性系膜细胞中受转化生长因子β调节的信号分子来获得对这种疾病进展的机械性见解。为了实现目标一，我们将通过多次小剂量注射STZ在转基因小鼠中诱导糖尿病肾病，在转基因小鼠中，系膜细胞被黄色荧光蛋白或病毒胸苷激酶转基因标记(在特定时间点用更昔洛韦去除增殖的系膜细胞)，并评估NG2在糖尿病肾病早期的功能贡献。此外，我们将通过流式细胞仪分离糖尿病小鼠和对照组小鼠的NG2阳性系膜细胞，并在培养系统中研究它们的增殖、迁移和ECM沉积特性。转化生长因子β是糖尿病肾病中高血糖诱导系膜细胞活性的关键调节因子，从而进一步从机制上了解这一信号轴。第二个目标是在NG2阳性系膜细胞中特异性缺失转化生长因子受体II及其下游的细胞内信号调节因子Smad4。我们计划通过将NG2-CRE小鼠与转化生长因子-1R11和Smad4系小鼠交配来实现这一点。STZ诱导的小鼠糖尿病将决定转化生长因子-β信号通路在这些新的小鼠模型中的作用。总的来说，这些研究将阐明与早期糖尿病肾病相关的新机制，并开辟新的治疗途径。

DESCRIPTION (provided by applicant): Diabetic nephropathy is a common complication of diabetes mellitus types 1 and 2. Prevention of chronic renal failure is one of the most important challenges in medicine for nephrologists and nephrology researchers, Diabetic nephropathy is characterized by changes in the structure of glomerular basement membranes, and progressive expansion of the mesangial matrix. Glomerular mesangial cells are considered to be modified pericyte-like cells (perivascular cells) that envelope most small and large blood vessels. NG2, a cell surface chondroitin sulfate proteoglycan is a specific marker for pericytes and specifically expresses in mesangial cells of the human and mouse kidneys when compared to other cell types of the glomeruli, Our preliminary studies suggest that NG2 expression increases in the mouse models of diabetic nephropathy. Such increase in NG2 expression directly correlates with an increased proliferation of mesangial cells and production of extracellular matrix in the glomeruli of mice with hyperglycemia induced by streptozotocin (STZ). Utilizing novel NG2 specific mouse transgenic reagents, our aim one in this grant application proposes to study the contribution of NG2 positive mesangial cells in the initiation and progression of diabetic nephropathy. Our second aim is to gain mechanistic insights into the progression of this disease by deleting TGF-¿ regulated signaling molecules in NG2 positive mesangial cells. To achieve aim one, we will induce diabetic nephropathy by multiple low dose injection of STZ in transgenic mice in which mesangial cells are transgenically tagged with yellow fluorescent protein or viral thymidine kinase (to ablate proliferating mesangial cells at specific time points using ganciclovir) and evaluate the functional contribution of NG2 to early phases of diabetic nephropathy. Additionally, NG2 positive mesangial cells from both diabetic and control mice will be isolated by FACS sorting and their proliferation, migration, and ECM deposition properties will be investigated in the culture system, TGF-¿ is a key modulator of hyperglycemia induced mesangial cells acivity in diabetic nephropathy and thus to further gain mechanistic insight into this signaling axis, in our second aim we will specifically delete TGF-¿ receptor II and its downstream intracellular signaling modulator Smad4 in NG2 positive mesangial cells. We plan to achieve this by mating NG2-Cre mice with TGF-¿ Rll floxed and Smad4 floxed mice. STZ induced diabetes in these mice will determine the contribution of TGF-¿ signaling pathway in these novel mouse models. Collectively these studies will elucidate novel mechanisms associated with early diabetic nephropathy and open new avenues to explore therapies.