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症（DG）列为美国ESRD的主要原因。在估计的2000万患有糖尿病（DM）的美国人中，有35-40%发生DG的观察结果表明，这种并发症在遗传风险个体的子集中发生。野生型（WT）p66 ShcA基因已成为长寿的遗传决定因素，其控制线粒体代谢以及对氧化应激、衰老和凋亡的细胞反应。本提案的主要目的是确定从突变型秋田小鼠基因组中删除WTp 66 ShcA是否赋予抗氧化剂表型，该表型保护常驻肾小球细胞免受启动和促进DG和靶基因组DNA进展的活性氧（ROS）依赖性信号的影响，从而激活细胞凋亡和细胞衰老的遗传程序。我们已经提出了一种模型，其中通过与p66 ShcA-/-小鼠同源重组使WTp 66 ShcA沉默或从秋田基因组中删除WTp 66 ShcA，导致有效的应激反应调节因子FOXO 3a易位到细胞核，在那里它协调应激反应程序。具体目标1的重点将是这种新的应激反应程序的分子组成的身份。实验方法将采用条件永生化人足细胞的体外细胞培养系统来测试沉默WTp 66 ShcA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的高度特化的细胞群。据报道，缓激肽1和2受体基因座（B1/B2-/-）突变可增加糖尿病秋田小鼠DG和衰老表型的风险和严重程度。在特定目标2和3下，将从秋田小鼠的基因组中删除WTp 66 ShcA，以测试这种功能丧失突变是否通过抑制对GFB造成不可逆损伤的活性氧危险信号的产生和传递来诱导DM的持续肾保护作用，触发肾小球重塑并靶向基因组DNA，诱导细胞进入细胞凋亡和衰老程序。提出的研究是基础的发展基因为基础的战略，以逮捕或预防DG的目标。公共卫生相关性多方面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。p66 ShcA蛋白在高血糖ROS信号的产生中起关键作用，高血糖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。本申请的中心假设是，在遗传上具有糖尿病肾病风险的小鼠中，p66 ShcA基因座处的纯合突变将减弱或预防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.