A thickening in basement membranes (BM) is a common secondary complication of diabetes and is associated with abnormalities in kidney function when the glomerular BM (GBM) is affected. While the mechanisms underlying GBM thickening are not understood, it is evident that the normal biosynthesis and maintenance of GBM is an important aspect of kidney function. GBM thickening can be explained in terms of problems in the balance of matrix component synthesis, polymerization, and turnover. These processes all involve cell/ECM interactions to some degree and imply that GBM formation is tightly controlled. To understand the mechanisms underlying GBM formation and maintenance under normal and pathological conditions, we propose to utilize the zebrafish (ZF) as a model system. The ZF lends itself to such studies because its renal system develops rapidly and the organism is amendable to cellular, molecular, and genetic approaches. The ZF renal system develops in two stages including: 1) formation of a transient pronephros by 50 hrs of development that is composed of a single glomerulus and 2) formation of a mesonephros by the 3d week that contains multiple glomeruli as seen in mammals. Unlike the pronephros, the ZF mesonephric kidney has not been well studied although it is required for studies related to GBM formation, maturation, and turnover.
The specific aims of this two-year R21 (Exploratory/Developmental Grant) will focus on establishing the ZF as a model for analysis of GBM formation and turnover in the mesonephric kidney under normal conditions and conditions of elevated glucose levels as observed in diabetes.
The Specific Aims will include: 1) to determine if isoform switching of BM components occurs during maturation of the ZF mesonephric glomerulus, 2) development of procedures to target expression of GBM components and associated matrix metalloproteinases (MMP5) in the mesonephric kidney of transgenic ZF, 3) to determine if elevated glucose levels result in glycation of proteins in the GBM and if a thickening of the GBM occurs as observed in rodent kidney models, and 4) initiation of experiments using the above mentioned GBM compositional data and transgenic procedures to begin to test the hypothesis that 'GBM thickening in the ZF mesonphric kidney involves a disruption in the normal balance between the polymerization and turnover of matrix components due to non-enzymatic glycation of matrix components that makes them less susceptible to cleavage by MMPs.""""""""
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