355 A Novel Role for Cd2Ap in Normal Podocyte Differentiation

Abstract

Background: CD2AP is a multifunctional adaptor molecule, binding other key podocyte proteins within the slit-diaphragm complex. Methods: The cellular phenotype of conditionally immortalized wild-type (WT) and CD2AP mutant podocytes was compared by light microscopy. Immunoflourescence and western blot was then used to examine the expression of characteristic podocyte markers nephrin, podocin, CD2AP, PAX2 WT1 as well as WTIP and mesenchymal markers fibronectin and α-SMA in CD2AP mutant podocytes. To investigate differences in localisation of WTIP further, nuclear and cytoplasmic fractions were isolated from each cell line and nuclear/cytoplasmic expression ratios compared. Results: In contrast to WT podocytes, CD2AP mutant demonstrated spindle shaped fibroblast like morphology similar to WT1 mutatant podocytes and were unable to form recognizable cell-cell contacts. Although nephrin, CD2AP podocin and WT1 were expressed at comparable levels in both cell lines, mesenchymal markers fibronectin and α-SMA were significantly overexpressed in CD2AP mutants compared with WT. Western blot and immunoflourescence data showed that PAX-2 was also upregulated in CD2AP mutant cells in keeping with a WT1-related defect. Immunofluorescence staining confirmed that WTIP co-localized at the cell membrane in WT podocytes, whereas in mutant cells WTIP expression was primarily nuclear. This observation was confirmed by a significant increase. (p=0.03) in the nuclear/cytoplasmic ratio of WTIP expression in CD2AP mutants. Conclusion: a role for CD2AP in maintainance of the normal differentiated podocyte phenotype. This may be partly mediated through WT1. Our findings have clear implications for a novel role for CD2AP in development and progression of glomerular disease.