Podocytes and oxidative stress in diabetic kidney

描述（由申请人提供）：在美国，终末期肾病的最大单一病因是糖尿病肾病（DN）。我们最近在一种称为OVE 26的I型糖尿病转基因小鼠模型中表征了DN的进展。该模型相对于先前的DN模型是显著的改进，因为9月龄OVE 26小鼠表现出晚期DN的许多特征。它们显示白蛋白尿增加100倍，广泛的间质纤维化和GFR适度降低。我们将使用该模型来评估肾小球足细胞中活性氧（ROS）在DN发展中的作用。OVE 26小鼠还将允许我们通过激光捕获和AffyIase基因阵列分析来评估在OVE 26小鼠中从两个月龄的早期DN到十四个月龄的严重DN的进展期间肾小球基因表达的变化。此外，OVE 26动物中的严重DN将使我们能够对口服抗氧化剂治疗阻断晚期DN的能力进行首次测试。 ROS已被证明是DN的关键组分。抗氧化剂或普遍表达的抗氧化剂转基因的全身治疗保护糖尿病动物免于早期DN。然而，通过全身治疗，不可能鉴定肾小球中哪些细胞被ROS损伤或被抗氧化剂治疗保护。足细胞是一种重要的候选细胞。它们构成选择性肾小球滤过膜的关键组分，并且许多足细胞在DN进展期间丢失。此外，它们还能产生ROS。我们开发了转基因小鼠，使用足细胞选择性nephrin启动子过表达一个有效的抗氧化蛋白金属硫蛋白的转基因。我们通过免疫组化和蛋白质印迹证明了金属硫蛋白转基因的足细胞表达升高。将在足细胞中过表达金属硫蛋白的小鼠培育成OVE 26 I型糖尿病小鼠。我们的初步结果表明足细胞金属硫蛋白的过度表达显著减少糖尿病诱导的大量白蛋白尿，并显着减少肾小球肥大。这些小鼠将被用于评估ROS对足细胞的损伤在改变基因表达和恶化结构和功能中的作用，从而导致糖尿病终末期肾病。

DESCRIPTION (provided by applicant): The single largest cause of end stage renal disease in the United States is diabetic nephropathy (DN). We have recently characterized the progression of DN in a transgenic mouse model of Type I diabetes called OVE26. This model is a significant improvement over previous DN models in that 9 month old OVE26 mice exhibit many characteristics of advanced DN. They show a 100 fold increase in albuminuria, extensive interstitial fibrosis and a modest reduction in GFR. We will use this model to assess the role of reactive oxygen species (ROS) in the glomerular podocyte for development of DN. OVE26 mice will also allow us to assess changes in glomerular gene expression by laser capture and Affymetrix gene array analysis during the progression from early DN at two months of age to severe DN at fourteen months of age in OVE26 mice. In addition the severe DN in OVE26 animals will allow us to make the first test of the ability of oral antioxidant therapy to block advance DN. ROS have been shown to be a critical component of DN. Systemic treatment with antioxidants or ubiquitously expressed antioxidant transgenes protect diabetic animals from early stage DN. However by systemic treatment it is not possible to identify which cells in the glomerulus are injured by ROS or protected by antioxidant treatment. Podocytes are an important candidate cell. They make up a critical component of the selective glomerular filtration membrane and many podocytes are lost during the progression of DN. In addition they have been shown to produce ROS. We developed transgenic mice that use the podocyte selective nephrin promoter to overexpress a transgene for the potent antioxidant protein metaliothionein. We demonstrated elevated podocyte expression of the metaliothionein transgene by immunohistochemistry and western blotting. Mice that overexpress metaliothionein in podocytes were bred to OVE26 Type I diabetic mice. Our initial results demonstrate that podocyte overexpression of metaliothionein dramatically reduces diabetes-induced macroalbuminuria and significantly reduces glomerular hypertrophy. These mice will be used to assess the role of ROS damage to the podocyte in the changing gene expression and deteriorating structure and function that lead to diabetic end stage renal disease.