LAMININ BETA2 AND GLOMERULAR FILTRATION

描述(由申请人提供)： 项目摘要/摘要 肾脏疾病是一个正在变得越来越普遍的巨大的世界性健康问题。原发性肾小球疾病，包括获得性和遗传性，在这些病例中占相当大的比例，部分原因是糖尿病肾病的发生率。我们感兴趣的是了解肾小球滤过屏障的组成以及它是如何受损并渗漏到血浆蛋白中的。在过去的十年里，我们的重点一直是研究肾小球基底膜(GBM)的组成和功能，GBM是一种特殊的细胞外基质，是滤过屏障的组成部分。基底膜含有层粘连蛋白、IV型胶原、尼德原和硫酸乙酰肝素蛋白多糖凝集素。最近对缺乏层粘连蛋白22链的小鼠的分析表明，基底膜本身是白蛋白的主要屏障，因为即使足细胞具有明显正常的足突和裂隙横隔膜结构，受影响的小鼠也表现出蛋白尿。层粘连蛋白22链是正常基底膜中唯一存在的层粘连蛋白2链。此外，体内的GBM通透性研究表明，突变的GBM对铁蛋白的通透性比对照更强。最近，人类LAMB2基因突变被证明会导致肾脏疾病。零突变导致Pierson综合征(伴有眼睛和神经系统异常的先天性肾病综合征)，而一些错义突变导致孤立的先天性肾病综合征。这项建议的重点是了解人类LAMB2的这些突变导致肾小球滤过缺陷的机制。为此，我们将1)将人LAMB2错义突变导入小鼠，以便能够详细跟踪突变蛋白的行为、疾病进展和肾小球超微结构；2)使用生化方法研究突变形式在体外组装和聚合的能力；3)测试在缺少22的情况下基底膜中总层粘连蛋白数量减少会导致滤过屏障缺陷的假设；4)检测层粘连蛋白异三聚体和集聚蛋白在基底膜平面内扩散的能力；5)测定层粘连蛋白22和集聚蛋白在基底膜中的半衰期。综上所述，这些研究结果将为层粘连蛋白和基底膜生物学提供重要的新见解，并有助于更好地理解肾小球滤过的机制和肾小球疾病的病因。 项目说明/相关性 肾脏疾病是一个正在变得越来越普遍的巨大的世界性健康问题。原发性肾小球疾病在这些病例中占相当大的比例，部分原因是糖尿病肾病和肥胖症的发病率增加。这项建议的目的是为了更好地了解肾小球滤过屏障的组成以及它是如何受损并泄漏到血浆蛋白质中的，重点是肾小球基底膜的重要组成部分层粘连蛋白22。

DESCRIPTION (provided by applicant): Project Summary/Abstract Kidney disease is huge worldwide health problem that is becoming increasingly prevalent. Primary glomerular disease, both acquired and genetic, represents a significant proportion of these cases, due in part to the incidence of diabetic nephropathy. We are interested in understanding the makeup of the glomerular filtration barrier and how it becomes damaged and leaky to plasma proteins. Our focus over the last decade has been to investigate the composition and function of the glomerular basement membrane (GBM), a specialized extracellular matrix that is an integral component of the filtration barrier. The GBM contains laminin, collagen IV, nidogen, and the heparan sulfate proteoglycan agrin. Recent analysis of mice lacking the laminin 22 chain, the only laminin 2 chain present in normal GBM, suggests that the GBM itself serves as the primary barrier to albumin, as the affected mice exhibit proteinuria even when podocytes have apparently normal foot process and slit diaphragm architecture. In addition, in vivo GBM permeability studies show that the mutant GBM is more permeable to ferritin than is control. Recently, mutations in human LAMB2 have been shown to cause kidney disease. Null mutations cause Pierson syndrome (congenital nephrotic syndrome with ocular and nervous system abnormalities), whereas some missense mutations cause isolated congenital nephrotic syndrome. The focus of this proposal is to understand the mechanism whereby these mutations in human LAMB2 cause glomerular filtration defects. To accomplish this, we will 1) engineer human LAMB2 missense mutations into mice so that behavior of the mutant proteins, disease progression, and glomerular ultrastructure can be followed in detail; 2) use biochemical methods to investigate the ability of the mutant forms to assemble and polymerize in vitro; 3) test the hypothesis that a reduction in the amount of total laminin in the GBM in the absence of 22 contributes to filtration barrier defects; 4) assay the ability of laminin heterotrimers and agrin to diffuse in the plane of the GBM; 5) determine the half-lives of laminin 22 and agrin in the GBM. Together, the results of these studies will provide important new insights into laminin and basement membrane biology and lead to a better understanding of the mechanism of glomerular filtration and the etiology of glomerular disease. Project Narrative/Relevance Kidney disease is huge worldwide health problem that is becoming increasingly prevalent. Primary glomerular disease represents a significant proportion of these cases, due in part to the increasing incidence of diabetic nephropathy and obesity. The goal of this proposal is to provide a better understanding the makeup of the glomerular filtration barrier and how it becomes damaged and leaky to plasma proteins, with a focus on laminin 22, an important component of the glomerular basement membrane.