OKHSC COBRE: PROTECTING THE RETINA FROM OXIDATIVE STRESS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 糖尿病肾病是糖尿病的主要并发症，也是终末期肾病的主要原因。糖尿病肾病最早的形态学改变是肾小球和肾小管的血流动力学和结构改变。在细胞水平上， 系膜细胞是这些早期变化的标志。多个实验证据表明，血管紧张素II受体1A型（AT 1A）和p27 Kip 1在糖尿病系膜细胞肥大的相互作用的突出作用。p27 Kip 1是一种细胞周期抑制剂，通常必须降解才能使 从G1期进入S期。在高血糖症期间，p27 Kip 1通过来自ATlA的信号传导事件不适当地稳定。AT 1A的一个效应子RCBTB 1是诱导细胞肥大所必需的，但RCBTB 1的功能及其如何在高血糖后导致肥大尚不清楚。我们已经发现RCBTB 1是泛素系统的一个组成部分，并且与AT 1A结合的RCBTB 1的相同结构域也与泛素缀合酶UBE 2 E3相互作用。我们的假设是UBE 2 E3和RCBTB 1以泛素依赖的方式调节AT 1A，从而促进p27 Kip 1依赖的系膜细胞肥大。我们的模型系统是大鼠系膜细胞，一种特征性良好的肾小球细胞模型，其重现了在糖尿病大鼠中观察到的肥大。 肾病我们的具体目标是：（1）对AT 1A、RCBTB 1和UBE 2 E3之间形成的复合物进行生物化学作图，和（2）确定破坏RCBTB 1和UBE 2 E3的表达/功能是否改变响应于高葡萄糖的p27 Kip稳定和细胞肥大。这些研究将在组织培养细胞中进行，并通过STZ-啮齿动物糖尿病肾病模型中的研究进行补充。将对这些动物的肾脏进行组织学、免疫组织学和生化分析，以确定肥大和p27 Kip 1蓄积。为了补充这些目标，我们将产生UBE 2 E3无效小鼠 或RCBTB 1。这些动物将为我们提供强有力的工具，通过遗传和生理学方法在经验证的糖尿病动物模型中研究UBE 2 E3和RCBTB 1。总的来说，从这些研究中获得的信息可能有助于合理设计糖尿病肾病的新治疗方案。随着糖尿病和肾衰竭发病率的上升，迫切需要这种治疗方法

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Diabetic nephropathy is a major complication of diabetes and the leading cause of end-stage renal disease. The earliest morphological changes accompanying diabetic nephropathy are hemodynamic and structural changes in the renal glomerular and tubular compartments. At the cellular level, hypertrophy of the mesangial cells is a hallmark of these early changes. Multiple lines of experimental evidence have revealed a prominent role for the interplay of the angiotensin II receptor type 1A (AT1 A) and p27Kip1 in diabetic mesangial cell hypertrophy. p27Kip1 is a cell cycle inhibitor that normally must be degraded to enable progression from G1 to S phase. During hyperglycemia, p27Kip1 is inappropriately stabilized through signaling events from the AT1 A. An effector of the AT1A called RCBTB1 is required for the induction of cellular hypertrophy, but the function of RCBTB1 and how it contributes to hypertrophy following hyperglycemia are unknown. We have discovered that RCBTB1 is a component of the ubiquitin system and that the same domain of RCBTB1 that binds to the AT1A also interacts with UBE2E3, a ubiquitin conjugating enzyme. Our hypothesis is that UBE2E3 and RCBTB1 modulate the AT1A in a ubiquitin-dependent fashion to contribute to p27Kip1-dependent, mesangial cell hypertrophy. Our model system is rat mesangial cells, a well-characterized glomerular cell model that recapitulates the hypertrophy observed in diabetic nephropathy. Our specific aims are: (1) to biochemically map the complexes formed between the AT1 A, RCBTB1, and UBE2E3, and (2) to determine if disrupting the expression/function of RCBTB1 and UBE2E3 alters p27Kip stabilization and cellular hypertrophy in response to high glucose. These studies will be done in tissue culture cells and complemented by studies in an STZ-rodent model of diabetic nephropathy. Kidneys from these animals will be analyzed histologically, immunohistologically, and biochemically for hypertrophy and p27Kip1 accumulation. To complement these aims, we will generate mice null for UBE2E3 or RCBTB1. These animals will provide us with powerful tools to study UBE2E3 and RCBTB1 by genetic and physiological methods in validated animal models of diabetic disease. Collectively, the information learned from these studies may contribute to the rational design of new treatment options for diabetic nephropathy. Such therapies are desperately needed as the incidence of diabetes and renal failure escalate