Alport syndrome is a common disorder that results in progressive kidney disease, and is occasionally associated with hearing loss and eye defects. The most common form of Alport syndrome is inherited as an X-linked trait (XAS), and is due to mutations in the alpha5 chain, one of six genetically distinct isoforms of type IV collagen. Rare autosomal recessive forms of the disease also exist, and are due to mutations in the alpha3 or alpha4 chains of type IV collagen. Type IV collagen is a major component of the glomerular basement membrane (GBM) and is predominantly composed of alpha3, alpha4 and alpha5 isoforms of type IV collagen. Mutations in any one of these isoforms in Alport syndrome leads to an absence of all three isoforms from the GBM of these patients, making their membranes more susceptible to breakdown by proteases. These results suggest a molecular association between the three isoforms in the kidney GBM. In the first part of this proposal we plan to study the relationship between the structure of GBM type IV collagen and the turnover of this membrane in Alport syndrome, using human Alport kidneys and kidneys from mice with Alport-like disease. These studies will determine the structural organization of type IV collagen in the human GBM and how such organized networks are altered by mutations in Alport syndrome. In the second part of this proposal, we will identify specific populations of NC1 hexamer in the human GBM and how mutations in Alport syndrome effect their assembly. We will use recombinant NC1 domains of alpha3, alpha4 and alpha5 chains produced in NIH 3T3 cells for in vitro hexamer assembly experiments. The role of mutated alpha3 chain in the in vivo assembly of GBM type IV collagen will be evaluated in transgenic mice. Lastly, the role of alpha5 chain (X-linked Alport gene) will be studied in mice, made deficient for this gene by gene targeting technology. These mice will be valuable in understanding the molecular events leading to defects in the Alport GBM due to mutations in the alpha5 chain. Successful completion of this project will determine the role of type IV collagen in the GBM turnover and identify molecular parameters for the assembly of type IV collagen NC1 hexamer which turns defective in Alport syndrome. A consideration of the differential sensitivity of GBM in XAS to degradation might perhaps at some point lead to therapeutic trials with new generation protease inhibitors now emerging for clinical use.
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