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

描述（由申请人提供）：USRDS将糖尿病性肾小球硬化（DG）列为美国ESRD的主要原因。据估计，美国有2000万糖尿病患者（DM），其中35-40%患有DG，这表明这种并发症发生在遗传上有风险的个体中。野生型（WT） p66ShcA基因被认为是长寿的遗传决定因素，它控制线粒体代谢，以及细胞对氧化应激、衰老和凋亡的反应。本研究的主要目的是确定突变秋田小鼠基因组中WTp66ShcA的缺失是否赋予抗氧化表型，从而保护驻留的肾小球细胞免受活性氧（ROS）依赖信号的影响，活性氧依赖信号启动并促进DG和目标基因组DNA的进展，激活凋亡和细胞衰老的遗传程序。我们提出了一个模型，在该模型中，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，导致强效应激反应调节因子FOXO3a易位到细胞核中，在那里它协调应激反应程序。特异性目标1的重点将是这种新型应激反应程序的分子成分的身份。实验方法将采用体外条件永生化人足细胞培养系统来测试沉默WTp66ShcA是否可以从高血糖诱导的ROS死亡信号中拯救这种终末分化、高度特化的细胞群。据报道，缓激肽1和2受体位点（B1/B2-/-）的突变会增加糖尿病秋田小鼠DG和衰老表型的风险和严重程度。在Specific Aims 2和3中，将从秋田小鼠的基因组中删除WTp66ShcA，以测试这种功能缺失突变是否通过抑制ROS危险信号的产生和传递，诱导DM中持续的肾保护，ROS危险信号对GFB造成不可逆损伤，触发肾小球重塑和靶向基因组DNA，诱导细胞进入凋亡和衰老程序。提出的研究是基于基因的策略发展的基础，目标是阻止或预防DG。多项证据表明，遗传易感性和氧化应激（ROS）是糖尿病肾病发展的关键因素。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的细胞损伤。该应用的中心假设是p66ShcA位点的纯合突变，在具有糖尿病肾病遗传风险的小鼠中，会减弱或阻止早期糖尿病肾病的生物标志物和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.