Molecular changes in slit diaphragm related proteinuria

描述（由申请人提供）： 蛋白尿是肾小球疾病的主要表现，减少蛋白尿已明确显示可延缓肾衰竭的进展。我们的研究工作是针对研究蛋白尿的发病机制，长期目标是开发基于疾病机制的特异性抗蛋白尿疗法。狭缝隔膜在维持肾小球渗透性特征中起主要作用。我们最近发现，将抗neph 1和抗nephrin抗体以单独的亚致肾炎剂量组合注射到大鼠中导致蛋白尿/白蛋白尿。在这个提议中，我们计划使用这个新的模型来研究足细胞基因和蛋白质在选择性横膈膜损伤和蛋白尿过程中的表达变化。 具体目标1：为了表征由于使用亲和纯化的抗neph 1和抗nephrin抗体的组合在大鼠中诱导的完全范围的补体和白细胞非依赖性异源相蛋白尿，并研究其对裂膈蛋白的表达和磷酸化的影响。 具体目标2：采用抑制消减杂交、真实的时间PCR、原位杂交、Western印迹和培养细胞转染研究相结合的方法，鉴定和表征在该模型中足细胞中差异表达的基因。 这项研究将使我们更接近了解蛋白尿的发病机制，也将有助于我们现有的知识与体内功能的裂膜蛋白的体外特征。

DESCRIPTION (provided by applicant): Proteinuria is a major manifestation of glomerular disease, and reducing proteinuria has been conclusively shown to retard the progression of kidney failure. Our research effort is directed towards investigating the pathogenesis of proteinuria, with the long term goal of developing specific anti-proteinuric therapies based disease mechanisms. The slit diaphragm plays a major role in maintaining glomerular permeability characteristics. We have recently shown that injecting a combination of anti-neph1 and anti-nephrin antibodies in individual sub-nephritogenic doses into rats results in proteinuria / albuminuria. In this proposal, we plan to use this new model to study changes in the expression of podocyte genes and proteins during selective slit diaphragm injury and proteinuria. Specific aim1: To characterize the full range of complement- and leukocyte-independent heterologous phase proteinuria induced in rats as a result of slit diaphragm injury using a combination of affinity purified anti-neph1 and anti-nephrin antibodies, and study its effect on the expression and phosphorylation of slit diaphragm proteins. Specific aim 2: To identify and characterize genes that are differentially expressed in the podocyte in this model using a combination of supression subtractive hybridization, real time PCR, in situ hybridization, Western blot and cultured cell transfection studies. This study will bring us one step closer to understanding the pathogenesis of proteinuria, and will also help to correlate our existing knowledge of the in vitro characteristics of slit diaphragm proteins with in vivo function.