LAMININ BETA2 AND GLOMERULAR FILTRATION

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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.