We have recently demonstrated that mutations in ACTN4, encoding a-actinin-4, cause an autosomal dominant form of familial focal and segmental glomerulosclerosis, a-actinin-4 is a member of a group of homodimeric actin crosslinking proteins. Our data suggests that these mutations act in a gain-of-function manner to disrupt normal glomerular epithelial cell function. The precise mechanism by which these mutations cause disease is unclear. We do not know if the mutations cause clinical disease through the altered interaction of actinin with actin we have observed, through an alteration in the interaction of actinin with some other protein or proteins, or by some other effect. In this proposal, we aim to gain additional understanding into the pathobiology of this form of FSGS. There are three basic aims of this proposal. We first will examine the interaction of mutant actinins with actin in both in vitro and in cell culture. We will produce mutant a-actinin-4 and measure its interaction with actin filaments as well as with a-actinin itself. We will observe the effect of transfecting mutant ACTN4 constructs on the actinin localization and the actin cytoskeleton. Next, we will examine the effect of these mutations on the interactions of a-actinin-4 with two other proteins which are thought to be important in podocyte structure and function, b1-integrin and a-catenin. Thirdly, we will develop a mouse model of this form of focal segmental glomerulosclerosis using methods of homologous recombination in embryonic stem cells. We will replace the endogenouse mouse actn4 gene with a gene harboring a missense point mutation identified as disease-causing in one large human pedigree. This model will allow us to investigate the pathobiology of this disease in vivo. The phenotype in patients with this familial form of FSGS is similar to the secondary glomerulosclerosis seen with many underlying conditions, including diabetes and hypertension. We therefore believe that these studies will provide significant insight into both the role of actinin and the actin cytoskeleton in the podocyte as well as in mediating chronic progressive renal dysfunction.
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