p66/Insulin Like Growth Factor-1 Reno-Protection in Diabetes

描述(由申请人提供)：USRDS将糖尿病肾小球硬化(DG)列为美国终末期肾病的主要原因。据观察，在大约2000万美国糖尿病患者中，有35%-40%的人会患上DG，这表明这种并发症是在一部分遗传高危人群中发生的。野生型(WT)p66ShcA基因已成为长寿的遗传决定因素，它控制着线粒体的代谢，以及细胞对氧化应激、衰老和凋亡的反应。这项建议的主要目标将是确定WTp66ShcA从突变的秋田小鼠基因组中删除是否提供了一种抗氧化表型，从而保护常驻肾小球细胞免受依赖于ROS的信号的影响，这些信号启动和促进DG和靶基因组DNA的进展，激活凋亡和细胞衰老的遗传程序。我们已经提出了一个模型，在这个模型中，通过与p66ShcA-/-小鼠的同源重组，沉默WTp66ShcA或从秋田基因组中删除WTp66ShcA，导致强大的应激反应调节因子FOXO3a移位到细胞核，在那里它协调应激反应程序。具体目标1的重点将是识别这一新的应激反应程序的分子成分。该实验方法将使用条件永生化的人足细胞的体外细胞培养系统来测试沉默WTp66ShcA是否可以从高血糖诱导的ROS死亡信号中拯救这个终末分化的高度特化的细胞群体。据报道，缓激肽1和2受体基因(B1/B2-/-)突变会增加糖尿病秋田鼠DG和衰老表型的风险和严重程度。根据特定的目标2和3，WTp66ShcA将从秋田小鼠的基因组中删除，以测试这种功能突变是否通过抑制ROS危险信号的产生和传递而诱导糖尿病的持续肾脏保护，ROS危险信号对GFB造成不可逆转的损伤，触发肾小球重塑和靶基因组DNA，诱导细胞进入细胞凋亡和衰老程序。拟议的研究是发展以基因为基础的策略的基础，目的是阻止或预防DG。公共卫生相关性多条证据表明，遗传易感性和氧化应激(ROS)是糖尿病肾病发生的关键因素。P66ShcA蛋白在高血糖ROS信号的产生中起关键作用，ROS信号驱动糖尿病肾病的病理生物学，并造成不可逆转的细胞损伤。这项应用的中心假设是，在具有糖尿病肾病遗传风险的小鼠中，p66ShcA基因座上的纯合子突变将减弱或阻止早期糖尿病肾病的生物标记物以及细胞凋亡和细胞衰老的ROS表型。

DESCRIPTION (provided by applicant): The USRDS lists diabetic glomerulosclerosis (DG) as the leading cause of ESRD in the United States. The observation that 35-40% of the estimated 20 million Americans with diabetes (DM) develop DG, is indicative that this complication develops in a subset of genetically at risk individuals. The wild type (WT) p66ShcA gene has emerged as a genetic determinant of longevity, that controls mitochondrial metabolism, and cellular responses to oxidative stress, aging and apoptosis. The major objective of this proposal will be to determine if deletion of WTp66ShcA from the genome of mutant Akita mice, confers an oxidant resistant phenotype that protects resident glomerular cells from reactive oxygen species (ROS) dependent signals that initiate and promote progression of DG and target genomic DNA, activating genetic programs for apoptosis and cell senescence. We have proposed a model in which silencing WTp66ShcA or deleting WTp66ShcA from the Akita genome by homologous recombination with p66ShcA-/- mouse, results in the translocation of the potent stress response regulator FOXO3a to the nucleus, where it orchestrates the stress response program. The focus of Specific Aim 1 will be the identity of the molecular components of this novel stress response program. The experimental approach will employ in vitro cell culture system of conditionally immortalized human podocytes to test if silencing WTp66ShcA can rescue this terminally differentiated, highly specialized cell population from hyperglycemia-induced ROS death signal. Mutations at the bradykinin 1 and 2 receptor loci (B1/B2-/-) have been reported to increase the risk and severity of DG and aging phenotypes in diabetic Akita mice. Under Specific Aims 2 & 3, WTp66ShcA will be deleted from the genome of Akita mice, to test if this loss of function mutation induces sustained renoprotection in DM, by inhibiting the generation and transmission of ROS danger signals that inflict irreversible injury to the GFB, trigger glomerular remodeling and target genomic DNA, inducing cell entry to apoptosis and senescence programs. The proposed studies are fundamental to the development of gene based strategies with the goal of to arresting or preventing DG. PUBLIC HEALTH RELEVANCE Multiple lines of evidence indicate genetic susceptibility and oxidative stress (ROS) are critical factors in development of diabetic nephropathy. The p66ShcA protein plays a pivotal role in the generation of hyperglycemic ROS signals that drive the pathobiology of diabetic nephropathy and inflict irreversible cell injury. The central hypothesis of this application is homozygous mutation at the p66ShcA locus, in mice genetically at risk for diabetic nephropathy, will attenuate or prevent biomarkers of incipient diabetic nephropathy and ROS phenotypes of apoptosis and cell senescence.