Poly(ADP-ribose) polymerase in diabetic nephropathy

描述（由申请人提供）：氧化应激是糖尿病肾病（PDN）发病机制中的重要因素。氧化损伤的后果之一是激活核酶，聚（ADP-核糖）聚合酶（PARP）。它由自由基和氧化剂诱导的DMA单链断裂触发，并导致1）能量衰竭; 2）甘油醛3-磷酸脱氢酶下调，导致糖尿病并发症的三种途径的激活，即非酶糖化、蛋白激酶C和己糖胺途径; 3）转录调节和基因表达的变化。PARP激活在糖尿病并发症发病机制中作用的证据，内皮功能障碍、外周和自主神经病变以及视网膜病变正在出现;然而，PARP在糖尿病肾病中的作用仍然未被探索。该建议的总体假设是PARP激活是糖尿病肾病的重要机制。具体目的是1）确定两种结构上不相关的PARP抑制剂是否对抗链脲佐菌素-糖尿病大鼠中糖尿病肾病的发展; 2）比较PARP-1缺陷（PARP-/-）小鼠和PARP含量正常的野生型小鼠中糖尿病肾病的严重程度;和3）鉴定山梨醇途径的两种酶，醛糖还原酶和山梨醇脱氢酶的贡献，和单个活性氧对高糖诱导的人肾小球系膜细胞PARP活化的影响。在动物实验中，我们将测定肾皮质肾小球和肾小管隔室中的PARP活性，以及糖尿病肾病的以下标志物：尿白蛋白排泄量、肾小球体积、肾脏内皮素-1含量以及内皮素-1和内皮素受体mRNA表达、肾脏血管内皮生长因子表达、肾小球转化生长因子-β和胶原蛋白α 1（IY）免疫反应性，血浆肌酐水平、肾小球系膜基质扩张和肾小球硬化。在细胞培养研究中，我们将评估细胞内氧化应激（DCF荧光试验/流式细胞术）、PARP激活（CELISA）和山梨醇途径中间体（酶荧光分光光度法测定）。这些研究将产生关于PARP在糖尿病肾病发病机制中作用的新信息，并将为开发PARP抑制剂预防和治疗糖尿病这一毁灭性并发症提供理论依据。

DESCRIPTION (provided by applicant): Oxidative stress is an important factor in the pathogenesis of diabetic nephropathy (PDN). One of the consequences of oxidative injury is activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). It is triggered by free radical and oxidant-induced DMA single-strand breakage and leads to 1) energy failure; 2)down-regulation of glyceraldehyde 3-phosphate dehydrogenase with resulting activation of three pathways contributing to diabetic complications i.e. non-enzymatic glycation, protein kinase C and hexosamine pathway; and 3) changes in transcriptional regulation and gene expression. Evidence for the role for PARP activation in the pathogenesis of diabetic complications e.g., endothelial dysfunction, peripheral and autonomic neuropathy, and retinopathy, is emerging; however, the role for PARP in diabetic nephropathy remains unexplored. The overall hypothesis of this proposal is that PARP activation is an important mechanism in diabetic renal disease. The specific Aims are 1) to determine whether two structurally unrelated PARP inhibitors counteract development of diabetic nephropathy in streptozotocin-diabetic rats; 2) to compare severity of diabetic nephropathy in PARP-1 deficient (PARP-/-) mice and the wild-type mice with normal PARP content; and 3) to identify contribution of two enzymes of the sorbitol pathway, aldose reductase and sorbitol dehydrogenase, and individual reactive oxygen species to high glucose-induced PARP activation in human mesangial cells. In animal experiments, we will determine PARP activity in glomerular and tubular compartments of the renal cortex, as well as the following markers of diabetic nephropathy: urinary albumin excretion, glomerular volume, renal endothelin-1 content and endothelin-1 and endothelin receptor mRNA expression, renal vascular endothelial growth factor expression, glomerular transforming growth factor-beta and collagen alpha1(IY) immunoreactivities, plasma creatinine levels, glomerular mesangial matrix expansion and glomerulosclerosis. In cell culture studies, we will evaluate intracellular oxidative stress (DCF fluorescence test/flow cytometry), PARP activation (CELISA) and sorbitol pathway intermediates (enzymatic spectrofluorometric assays).The studies will generate novel information regarding the role for PARP in the pathogenesis of diabetic nephropathy and will provide rationale for development of PARP inhibitors for prevention and treatment of this devastating complication of diabetes mellitus.