Role of NG2 in diabetic nephropathy

描述（由申请人提供）：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脏病学家和肾脏病研究人员在医学上最重要的挑战之一，糖尿病肾病的特征在于肾小球基底膜结构的变化和系膜基质的进行性扩张。肾小球系膜细胞被认为是修饰的周细胞样细胞（血管周细胞），包裹大多数大小血管。NG 2是一种细胞表面硫酸软骨素蛋白聚糖，是周细胞的特异性标志物，与肾小球的其他细胞类型相比，NG 2在人和小鼠肾脏的系膜细胞中特异性表达。我们的初步研究表明，NG 2在糖尿病肾病小鼠模型中表达增加。NG 2表达的这种增加与链脲佐菌素（STZ）诱导的高血糖小鼠肾小球中系膜细胞增殖和细胞外基质产生的增加直接相关。利用新型NG 2特异性小鼠转基因试剂，我们的目标之一是在这个授权申请中提出研究NG 2阳性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NG 2阳性系膜细胞中TGF-β调节的信号分子来获得对这种疾病进展的机制见解。为了实现目标一，我们将通过在转基因小鼠中多次低剂量注射STZ来诱导糖尿病肾病，其中肾小球系膜细胞用黄色荧光蛋白或病毒胸苷激酶转基因标记（以在特定时间点使用更昔洛韦消融增殖的肾小球系膜细胞），并评估NG 2对糖尿病肾病早期阶段的功能贡献。此外，将通过FACS分选分离来自糖尿病小鼠和对照小鼠的NG 2阳性系膜细胞，并将在培养系统中研究它们的增殖、迁移和ECM沉积特性。TGF-β是糖尿病肾病中高血糖诱导的系膜细胞活性的关键调节剂，因此为了进一步获得对该信号传导轴的机制认识，在我们的第二个目的中，我们将特异性地删除TGF-β。受体II及其下游细胞内信号调节因子Smad 4在NG 2阳性系膜细胞中的表达我们计划通过将NG 2-Cre小鼠与TGF-β R11 floxed和Smad 4 floxed小鼠交配来实现这一目标。STZ在这些小鼠中诱导的糖尿病将决定TGF-²信号通路在这些新型小鼠模型中的贡献。总之，这些研究将阐明与早期糖尿病肾病相关的新机制，并为探索治疗方法开辟新途径。

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.