Functional and Phenotypic Characterization of a New FSGS Gene

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DESCRIPTION (provided by applicant): Focal segmental glomerulosclerosis (FSGS), a common cause of kidney failure, is the result of pathogenic changes that alter the functional integrity of the glomerular filtration barrier (GFB). The study of familial FSGS cases points to a central role of the podocyte in its pathogenesis. Our long term goals are to understand the molecular pathogenesis of FSGS by identifying pathways that are critical for the maintenance of the functional integrity of the GFB and identify novel therapeutic targets for FSGS. The overall objective of this application is to study the mechanisms by which mutations in an F-actin binding cell cycle gene, ANLN, causes FSGS. Our approach is feasible because we recently identified a mutation in F-actin binding domain of ANLN, R431C, as a cause of familial FSGS. We showed that anillin is upregulated in kidney biopsies of subjects with collapsing FSGS compared with normal kidney. Anillin co-localizes with key podocyte proteins that are important in maintaining the integrity of the actin cytoskeleton. We also showed that knockdown of anillin in zebrafish embryos disrupts the GFB. Our overarching hypothesis is that mutations in the F-actin binding domain of anillin affect F-actin cytoskeleton polymerization and lead to aberrant podocyte proliferation and migration; disruption of podocyte homeostasis then disrupts normal GFB function and leads to the pathogenesis of FSGS. We will explore this hypothesis through the following specific aims: 1) To determine the mechanisms by which ANLN R431C causes disruption of podocyte homeostasis. We will characterize the effect of R431C variant on a) podocyte motility and proliferation in vitro and b) the subcellular localization of anillin and its interactions with known and newly identified podocyte proteins. 2) To determine the functional effect of the R431C mutation and other ANLN variants on the GFB of zebrafish embryos. We will use an in vivo complementation assay to determine allele pathogenicity of R431C ANLN mutations and other new variants using glomerular filtration as a physiologically relevant readout. 3) To analyze mutations of the ANLN gene in a cohort of patients with FSGS. We will sequence the exons of ANLN in our cohort of FSGS patients and compare the disease phenotype in subjects with and without mutations. The pathogenicity of all new variants will be measure in zebrafish. Innovation: This proposal represents the first study designed to define the mechanisms by which anillin variants cause FSGS. Significance: Unraveling the mechanisms by which mutations in ANLN cause FSGS may identify pathways that are important for maintaining the functional integrity of the podocyte cytoskeleton. Furthermore, by probing the role of anillin in cell proliferation and motility, we will provide insight into the mechanisms of podocyte renewal in health and disease. Our genetic and mechanistic approaches will advance our understanding of the molecular pathogenesis of podocyte phenotype changes in FSGS and lead to identification of novel therapeutic targets and less toxic pharmacologic approaches.