Previous work showed that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This present report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme, N-deacetylase/N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 (Ndst1-/-) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4 and for a-actinin-4 when compared to controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1-/- podocytes attached, spread and migrated less efficiently compared to Ndst1+/+ podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1-/- cells had decreased clustering of syndecan 4, and decreased recruitment of protein kinase Ca, a-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1-/- compared to Ndst1+/+ cells. A significant decrease in the abundance of activated integrin a5ß1 on the cell surface of Ndst1-/-cells compared to Ndst1+/+ cells was seen. These results serve to highlight the critical role of HS N-sulfation in facilitating normal podocyte-matrix interactions.

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