OKHSC COBRE: PROTECTING THE RETINA FROM OXIDATIVE STRESS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。伴随糖尿病肾病的最早的形态改变是肾小球和肾小管腔的血流动力学和结构改变。在细胞水平上，肥大的血管 系膜细胞是这些早期变化的标志。多条实验证据表明血管紧张素II受体1A型(AT1A)和p27Kip1在糖尿病系膜细胞肥大中的相互作用具有显著作用。P27Kip1是一种细胞周期抑制物，通常必须被降解才能使 从G1期进入S期。在高血糖时，p27Kip1通过AT1A的信号事件被不适当地稳定，AT1a的一个称为RCBTB1的效应器是诱导细胞肥大所必需的，但RCBTB1的功能及其在高血糖后如何促进肥大的作用尚不清楚。我们发现RCBTB1是泛素系统的一个组成部分，与AT1a结合的RCBTB1的同一结构域也与泛素结合酶UBE2E3相互作用。我们的假设是，UBE2E3和RCBTB1以泛素依赖的方式调节AT1a，从而促进p27Kip1依赖的系膜细胞肥大。我们的模型系统是大鼠系膜细胞，这是一种具有良好特征的肾小球细胞模型，它概括了糖尿病患者观察到的肥大 肾病。我们的具体目标是：(1)对AT1A、RCBTB1和UBE2E3之间形成的复合体进行生化定位，以及(2)确定干扰RCBTB1和UBE2E3的表达/功能是否改变了p27Kip的稳定性和细胞肥大对高糖的反应。这些研究将在组织培养细胞中进行，并辅之以糖尿病肾病的STZ啮齿动物模型的研究。将对这些动物的肾脏进行组织学、免疫组织学和生化分析，以确定是否存在肥大和p27Kip1积聚。为了补充这些目标，我们将为UBE2E3生成MICE空 或RCBTB1。这些动物将为我们在经过验证的糖尿病动物模型中通过遗传学和生理学方法研究UBE2E3和RCBTB1提供强大的工具。总而言之，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。随着糖尿病和肾功能衰竭发病率的上升，迫切需要这样的疗法

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