N-sulfation of heparan sulfate is critical for syndecan-4-mediated podocyte cell-matrix interactions.

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This 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 α-actinin-4 compared with 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 than 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-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1(-/-) compared with Ndst1(+/+) cells. A significant decrease in the abundance of activated integrin α5ß1 on the cell surface of Ndst1(-/-) cells compared with Ndst1(+/+) cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.

Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo.

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

The glomerular basement membrane as a model system to study the bioactivity of heparan sulfate glycosaminoglycans.

The glomerular basement membrane and its associated cells are critical elements in the renal ultrafiltration process. Traditionally the anionic charge associated with several carbohydrate moieties in the glomerular basement membrane are thought to form a charge selective barrier that restricts the transmembrane flux of anionic proteins across the glomerular basement membrane into the urinary space. The charge selective function, along with the size selective component of the basement membrane, serves to limit the efflux of plasma proteins from the capillary lumen. Heparan sulfate glycosaminoglycans are anionically charged carbohydrate structures attached to proteoglycan core proteins and have a role in establishing the charge selective function of the glomerular basement membrane. Although there are a large number of studies in the literature that support this concept, the results of several recent studies using molecular genetic approaches to minimize the anionic charge of the glomerular basement membrane would suggest that the role of heparan sulfate glycosaminoglycans in the glomerular capillary wall are still not yet entirely resolved, suggesting that this research area still requires new and novel exploration.

Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria.

Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.