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.

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A monoclonal antibody was used in early studies to identify a novel chondroitin sulfate proteoglycan, secreted by L-2 cells, the core protein of which was approximately 100 kDa. To characterize this proteoglycan core protein at the molecular level, an L-2 cell cDNA library was probed by expression screening and solution hybridization. Northern blot analysis assigned transcript size to approximately 3.1 kilobases and, after contig assembly, the coding region of the mRNA corresponded to 2.18 kilobases. Immunoassays were performed to confirm the identity of this sequence, using a polyclonal antibody raised against an expressed fusion protein encoded by sequence representing the carboxyl half of the molecule. The antibody recognized the core protein in Western blots after prior digestion of the intact proteoglycan with chondroitinase ABC. Immunostaining tissue sections with the same antibody localized the proteoglycan to basement membranes, and expression of the entire sequence in Chinese hamster ovary K-1 cells showed that the protein encoded by the sequence secreted as a chondroitin sulfate proteoglycan. The core protein not only has motifs permitting glycosylation as a proteoglycan, but also possesses the endoplasmic reticulum retrieval signal, KDEL, which suggests that, in addition to its role as a basement membrane component, it may also participate in the secretory pathway of cells.

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

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.