LAMININ BETA2 AND GLOMERULAR FILTRATION

描述（由申请人提供）： 项目概要/摘要 肾脏疾病是一个巨大的全球性健康问题，正变得越来越普遍。原发性肾小球疾病，包括获得性和遗传性，在这些病例中占很大比例，部分原因是糖尿病肾病的发病率。我们有兴趣了解肾小球滤过屏障的组成，以及它是如何受损和泄漏给血浆蛋白的。在过去的十年中，我们的重点一直是研究肾小球基底膜（GBM）的组成和功能，GBM是一种专门的细胞外基质，是滤过屏障的组成部分。GBM含有层粘连蛋白、胶原IV、巢蛋白和硫酸乙酰肝素蛋白聚糖聚集蛋白。最近对缺乏层粘连蛋白22链（正常GBM中存在的唯一层粘连蛋白2链）的小鼠的分析表明，GBM本身充当白蛋白的主要屏障，因为即使足细胞具有明显正常的足突和狭缝隔膜结构，受影响的小鼠也表现出蛋白尿。此外，体内GBM渗透性研究表明，突变GBM比对照组更易渗透铁蛋白。最近，人类LAMB 2的突变已被证明会导致肾脏疾病。突变导致皮尔逊综合征（先天性肾病综合征，伴有眼部和神经系统异常），而一些错义突变导致孤立的先天性肾病综合征。该提案的重点是了解人类LAMB 2中这些突变导致肾小球滤过缺陷的机制。为了实现这一点，我们将1）将人LAMB 2错义突变工程化到小鼠中，以便可以详细地跟踪突变蛋白的行为、疾病进展和肾小球超微结构; 2）使用生物化学方法来研究突变形式在体外组装和组装的能力; 3）检验在不存在22的情况下GBM中总层粘连蛋白的量的减少有助于过滤屏障缺陷的假设; 4）测定层粘连蛋白异源三聚体和聚集蛋白在GBM平面中扩散的能力; 5）测定层粘连蛋白22和聚集蛋白在GBM中的半衰期。总之，这些研究的结果将提供重要的新见解层粘连蛋白和基底膜生物学，并导致更好地了解肾小球滤过的机制和肾小球疾病的病因。 项目叙述/相关性 肾脏疾病是一个巨大的全球性健康问题，正变得越来越普遍。原发性肾小球疾病在这些病例中占很大比例，部分原因是糖尿病肾病和肥胖症的发病率增加。该提案的目标是提供更好地了解肾小球滤过屏障的组成以及它如何受损和泄漏给血浆蛋白，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.