Cell Cycle Dependent Mechanisms of Renal Hypertrophy

肾肥大的细胞周期依赖性机制

• 批准号: 6623538
• 负责人: DANIEL J RILEY
• 金额: $ 20.17万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623538
• 关键词: AP1 protein; cell cycle; cell growth regulation; cyclin dependent kinase; diabetic nephropathy; gel mobility shift assay; gene expression; genetic regulation; genetically modified animals; glomerulosclerosis; glucose; immunoprecipitation; insulin dependent diabetes mellitus; kidney cell; kidney hypertrophy; laboratory mouse; mesangium; phosphorylation; protooncogene; retinoblastoma protein; site directed mutagenesis; western blottings

DESCRIPTION (Provided by applicant): Early in diabetic nephropathy glomerular mesangium and renal tubules hypertrophy. Mesangial hypertrophy is an important, perhaps reversible, step preceding glomeruloscierosis and overt nephropathy. Using unique transgenic mice overexpressing retinoblastoma protein (Rb) and mesangial cells expressing Rb from a tetracycline-confrollable promoter system, we have discovered that renal and glomerular mesangial cell hypertrophy occur with both diabetes mellitus and with over expression of hypophosphorylated Rb. Preliminary data has shown that when Rh is over expressed, neither kidneys nor mesangial cells hypertrophy further in diabetic conditions. Our hypothesis to explain these observations is that excess glucose results in specific, cyclin-dependent phosphorylation of Rb protein during early Gi phase, and that Rb is involved distally in a pathway of glomerular mesangial cell hypertrophy. To test this hypothesis, three Specific Aims are proposed: (1) Determine whether renal and glomerular hypertrophy caused by diabetes mellitus in vivo and mesangial cell hypertrophy caused by high glucose concentrations ex vivo are dependent on activation of specific C1 phase cyclin-cdk complexes and specific phosphorylations of Rb protein. (2) Determine how high glucose regulates cdk4-cyclin Dl activity and Rb phosphorylation in mesangial cells by examining patterns of gene regulation including transcription of the cyclin D1 gene itself and of events controlled by relevant transcription factors including AP-1. (3) Test the requirement for Cl cyclin dependent kinase activation and specific cdk4-dependent phosphorylations of Rb protein for diabetic renal hypertrophy in vivo and mesangial cell hypertrophy in culture. Transgenic mice with strategic phosphorylation sites in Rb inactivated by site-directed mutagenesis will be generated and the effects of type 1 diabetes mellitus will be examined in them. Primary mesangial cells will also be cultured from the mice and more detailed studies on hypertrophy and GI cell cycle regulation by cdks and Rb conducted in vitro.

描述(申请人提供)：早期糖尿病肾病肾小球 系膜和肾小管肥大。系膜肥大是一种重要的、 可能是可逆的，先于肾小球硬化和临床肾病。 使用过表达视网膜母细胞瘤蛋白(Rb)和 系膜细胞表达来自四环素-可控制启动子系统的Rb， 我们发现肾和肾小球系膜细胞肥大。 既有糖尿病，又有低磷酸化Rb的过度表达。 初步数据显示，当Rh过度表达时，无论是肾脏还是 糖尿病时系膜细胞进一步肥大。我们的假设是 解释这些观察结果的原因是过量的葡萄糖会导致特定的， 依赖细胞周期蛋白的Rb蛋白在胃肠道早期的磷酸化 Rb在远端参与了肾小球系膜细胞肥大的途径。 为了验证这一假设，提出了三个具体目标：(1)确定 糖尿病是否导致体内肾脏和肾小球肥大 体外高糖致系膜细胞肥大 依赖于特定的C1期细胞周期蛋白-cdk复合体的激活和 Rb蛋白的特异性磷酸化。(2)确定血糖有多高 人肾小球系膜细胞CDK4-Cyclin D1活性和Rb磷酸化的调节 检测基因调控的模式，包括细胞周期蛋白D1的转录 基因本身和由相关转录因子控制的事件 包括AP-1。(3)检测对氯化细胞周期蛋白依赖性激酶的要求 Rb蛋白的激活及其依赖于CDK4的特异性磷酸化 体内糖尿病肾肥大和体外培养的系膜细胞肥大。 Rb策略磷酸化位点的转基因小鼠被灭活 将产生定点突变和1型糖尿病的影响 将在他们身上检查糖尿病。原代系膜细胞也将 小鼠的培养及对肥大和胃肠道细胞更详细的研究 CDKs和Rb在体外进行的周期调节。