Poly(ADP-ribose) polymerase in diabetic nephropathy

描述(申请人提供)：氧化应激是糖尿病肾病(PDN)发病机制中的一个重要因素。氧化损伤的后果之一是核酶聚腺苷二磷酸核糖聚合酶(PARP)的激活。它由自由基和氧化剂诱导的DNA单链断裂触发，导致1)能量衰竭；2)甘油醛3-磷酸脱氢酶下调，导致导致糖尿病并发症的三条途径的激活，即非酶糖基化、蛋白激酶C和氨基己糖途径；以及3)转录调节和基因表达的变化。PARP激活在糖尿病并发症如内皮功能障碍、周围神经和自主神经病变以及视网膜病变的发病机制中的作用的证据正在涌现；然而，PARP在糖尿病肾病中的作用仍未被探索。这一建议的总体假设是，PARP激活是糖尿病肾病的一个重要机制。其具体目的是1)确定两种结构无关的PARP抑制剂是否能对抗链脲佐菌素糖尿病大鼠糖尿病肾病的发展；2)比较PARP-1缺陷(PARP-/-)小鼠和PARP含量正常的野生型小鼠糖尿病肾病的严重程度；以及3)确定山梨醇途径的两种酶，即醛糖还原酶和山梨醇脱氢酶，以及个别活性氧在高糖诱导的人肾小球系膜细胞PARP激活中的作用。在动物实验中，我们将检测肾皮质肾小球和肾小管区的PARP活性，以及下列糖尿病肾病的标志物：尿白蛋白排泄、肾小球体积、肾脏内皮素-1含量和内皮素-1及其受体mRNA的表达、肾血管内皮生长因子的表达、肾小球转化生长因子-β和胶原α1(IY)的免疫反应、血肌酐水平、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荧光测试/流式细胞术)、PARP激活(CELISA)和山梨醇途径中间产物(酶荧光光谱分析)。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基础。

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.