Previous work showed that cell surface heparan sulfate (HS) glycosaminoglycans (GAG) modulate podocyte (podo) organization on the glomerular basement membrane (Kid. Int. 74: 289–299). This study examined the role of HSGAG sulfation in this process by podo‐specific deletion of NDST1, the enzyme responsible for N‐sulfation of HSGAG. A novel mutant mouse (2.5P‐Cre/NDST1fl/fl) and immortalized podo line (NDST1‐null) were developed using a Cre‐lox approach. The kidneys in mutant mice appeared normal compared to controls, the only measurable difference in routine LM studies was a slight glomerular hypertrophy in the mutant mice. Electron micrographs from mutant animals showed mild podo foot process effacement and abnormal podo attachment to Bowman's capsule, suggesting that podo‐matrix interactions were affected. NDST1‐null podo had a significantly decreased ability to adhere, spread, and migrate upon fibronectin compared to controls. Mutant podo also failed to assemble focal adhesions as efficiently as control cells. These results indicate that HSGAG sulfation is a critical mediator of cell surface proteoglycan (PG)‐matrix interaction, highlighting the importance of HSPGs in orchestrating podo cell behavior.This work is supported by: NIDDK RO1‐DK077860Grant Funding Source: NDDK RO1‐DK077860
Heparan sulfate (HS) proteoglycans (PG) are considered important in GBM ultrafiltration and are synthesized primarily by podocytes. The EXT1 gene encodes for the glycosyltransferase that is the limiting step in HS‐GAG polymerization on PG core proteins. PEXTKO (podocyte EXT1 knockout) mice were developed by breeding EXT1fl/fl with mice transgenic for CRE‐recombinase expression driven by the podocin promoter. Podocytes from PEXTKO mice lack HS‐GAG, yet renal development and maturation occurs. H&E stained sections from kidneys of PEXTKO animals were similar to control animals. Electron microscopy (EM) studies showed podocyte foot process effacement occurred PEXTKO mice at all ages. GBM ultrastructure was compromised in 1 month old animals, with GBM splitting in some areas. In adult animals there was evidence of enhanced GBM synthesis yet overall GBM thickness appeared normal. Immunostaining kidney sections from PEXTKO mice with core protein specific antibodies against perlecan showed no difference in the pattern of staining between control animals and PEXTKO animals, however agrin core protein staining was increased. Albuminuria was not seen in 3 month old PEXTKO animals but mild microalbuminuria was seen in 8 month old animals. The data indicate that the presence of HS chains on GBM proteoglycan core proteins may not exclusively function as a passive sieve, but rather modulate podocyte behavior.
Podocytes adhere to the glomerular basement membrane via cell surface receptors which include heparan sulfate (HS) proteoglycans (PG). Podocytes unable to assemble HS glycosaminoglycan chains have defective cell attachment and spreading, and cytoskeletal disruption in vitro and in vivo. The current report explores the importance of heparan sulfation in regulating podocyte cell‐matrix interactions, using an immortalized podocyte cell line having the genomic deletion of N‐deacetylase/ N‐sulfotransferase 1 (NDST1), the enzyme responsible for N‐sulfation of HS. Compared to wild type podocytes, mutant podocytes attached, spread and migrated less efficiently in assays than control cells. In the process of cell‐matrix interaction, the mutant cells showed decreased clustering of syndecan 4, the cell surface HSPG involved in focal adhesion formation, and decreased recruitment of PKCα, α‐actinin 4, vinculin, and pFAK to focal adhesions. Cell surface integrin expression and integrin activation/signaling was significantly diminished in the mutant cells. These results serve to highlight the critical role of HS N‐sulfation in regulating the ability of podocytes to interact with the extracellular matrix.Grant Funding Source: NIDDK RO1‐DK07786
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