Podocytes and oxidative stress in diabetic kidney

描述(由申请人提供)：美国终末期肾病的最大原因是糖尿病肾病(DN)。我们最近在一种名为OVE26的I型糖尿病转基因小鼠模型上表征了糖尿病肾病的进展。该模型是对以前的糖尿病肾病模型的重大改进，因为9月龄OVE26小鼠表现出许多晚期糖尿病肾病的特征。他们显示蛋白尿增加了100倍，广泛的间质纤维化和适度的GFR减少。我们将使用这个模型来评估肾小球足细胞中的活性氧物种(ROS)在糖尿病肾病发生中的作用。OVE26小鼠还将允许我们通过激光捕获和Affymetrix基因阵列分析来评估OVE26小鼠从2个月龄的早期糖尿病肾病到14个月龄的重度糖尿病肾病过程中肾小球基因表达的变化。此外，OVE26动物的严重糖尿病肾病将使我们能够对口服抗氧化剂治疗阻止进展的糖尿病肾病的能力进行第一次测试。 ROS已被证明是糖尿病肾病的关键组成部分。抗氧化剂或普遍表达的抗氧化剂转基因基因的系统治疗可以保护糖尿病动物免受早期糖尿病肾病的影响。然而，通过系统治疗，不可能确定肾小球中的哪些细胞受到ROS的损伤或受到抗氧化治疗的保护。足细胞是一个重要的候选细胞。它们构成了选择性肾小球滤过膜的关键成分，许多足细胞在糖尿病肾病的进展过程中丢失。此外，它们已被证明能产生RO。我们开发了转基因小鼠，使用足细胞选择性neparin启动子过表达有效抗氧化蛋白Metaliothionein的转基因。我们通过免疫组织化学和Western blotting证实了转化的Metalithiionein基因在足细胞中表达增加。在足细胞中过表达金属硫蛋白的小鼠被培育成OVE26 I型糖尿病小鼠。我们的初步结果表明，足细胞过表达金属硫蛋白显著减少了糖尿病引起的大量蛋白尿，并显著减少了肾小球肥大。这些小鼠将被用来评估ROS对足细胞的损伤在导致糖尿病终末期肾脏疾病的基因表达变化和结构和功能恶化中的作用。

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.