Type IV collagen is the most abundant protein present in the glomerular basement membrane of the kidney. The basic unit of type IV collagen is a triple helical protomer derived from three a-chains. With six known isoforms of type IV collagen (alpha1-alpha6), 56 theoretical combinations of protomers are possible. Emerging data however, suggests a tissue specific preference for certain protomers. Type IV collagen in the glomerular basement membrane (GBM) is predominantly composed of alpha3, alpha4 and alpha5 isoforms of type IV collagen, with alpha3 chain of type IV collagen being most abundant. Mutations in any one of these isoforms in Alport syndrome (a condition associated with progressive kidney disease, occasional hearing loss and eye defects) leads to an absence of all three isoforms from the GBM of these patients, suggesting a molecular association between the three isoforms in the kidney GBM. While in the past five years, biochemical and cell biological experiments have provided further causal support for such protein-protein interactions, molecular drivers that determine specific type IV collagen protomer assembly are still unknown. During the first funding period of this grant application, using human Alport kidneys and kidneys from mice with alpha3(IV) collagen deletion, the relationship between the structure of GBM type IV collagen and its susceptibility of degradation in Alport syndrome, was established. Specific type IV collagen isoforms targets for post-transplant alloantibody response in Alport patients were determined in a multi-center study. Further, genetic and biochemical studies with type IV collagen NC1 domains revealed novel insights into the assembly and organization of type IV collagen in the GBM and elsewhere. Collectively, these studies provide evidence that premature turnover of GBM involving altered composition of type IV collagen may contribute to the early pathogenesis of Alport syndrome. In this renewal application, we now propose to continue our studies to understand the molecular drivers that determine specific assembly of type IV collagen involving the a3 chain in the GBM, and analyze the impact of disease-associated a3 (IV) NC1 domain mutations on the GBM composition, turnover, and assembly. We will generate gene-targeted mice to probe the contribution of GBM instability for the initiation of progressive renal failure associated with Alport Syndrome. We expect this grant application to provide basic understanding of the assembly and function of type IV collagen in the GBM.
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