Heparan Sulfate–Rich Anionic Sites in the Human Glomerular Basement Membrane

Abstract

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. Thefindings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and tes-ticular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific or heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent ma'or components of these sites since the latter are not affected by digestion with neuraminidase and other gly cosaminoglycan-specific enzymes. Identical findings were o, tained on basement membranes in other locations (Bowman's capsule, tubule epithelium and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury. Physiologic studies have established that the glomerular cap-illaries function as a size-and charge-selective barrier in the production of the glomerular filtrate and in the retention of plasma proteins in the circulation (1-3). By inference it has been assumed (1-3) that charge selectivity is due to the presence in the glomerulus of fixed negative charges, but their precise nature and location in the capillary wall have not been established. Since macromolecular compounds carrying sialic acid residues have been-up to recently-the only known polyanions present in the glomerulus, they have been considered the most likely candidates responsible for the charge barrier (1, 2, 4-7). Sialyl residues have been found in all three layers of the capillary wall. They have been detected in isolated glomerular basement membranes (GBM) (8-10) and in association with the cell membranes of both the endothelium (11-12) and epithelium (4-7, 11), where they are part of the sialic acid-rich, cell-surface coats. A colloidal iron-stainable, cell-coat material is particularly concentrated on the epithelium. Accordingly, it has been repeatedly suggested (1, 2, 4-7) that this "epithelial polyanion"...

Disruption of Glomerular Basement Membrane Charge through Podocyte-Specific Mutation of Agrin Does Not Alter Glomerular Permselectivity

“…Agrin mutants showed a ϳ50% reduction in the number of subepithelial anionic sites, and on a qualitative basis, those that remained appeared less intensely stained by PEI. This charge defect is, in both respects, as profound as that typically attributed to be a causative factor in human and experimental kidney disease; reported alterations range from a 63% reduction in human congenital nephrotic syndrome, 54 to 57% in membranous nephritis, to 52 to 20% in focal segmental glomerulosclerosis, to 21% in minimal change disease, 55,56 and to 28 to 18% in rat models of diabetic nephropathy and puromycin nephrosis. 57,58 The key finding of this work is that agrin mutant mice have normal renal function despite a severe disruption of GBM charge.…”

Reduced Heparan Sulfate Accumulation in Enterocytes Contributes to Protein-Losing Enteropathy in a Congenital Disorder of Glycosylation

“…30 The involvement of HS in PLE was also seen in a study of three infants with normal small bowel biopsies, who had massive PLE from birth and loss of HS from the basolateral enterocyte membrane. 31 HS loss from the glomerular basement membrane leads to albumin leakage across the vascular endothelium in kidney [45][46][47][48] and thus enterocyte HS deficiency may be analogous to congenital nephrotic syndrome. 47 The findings here suggest that CDG-1c causes a similar loss of HS from the enterocyte basolateral membrane.…”

“…31 HS loss from the glomerular basement membrane leads to albumin leakage across the vascular endothelium in kidney [45][46][47][48] and thus enterocyte HS deficiency may be analogous to congenital nephrotic syndrome. 47 The findings here suggest that CDG-1c causes a similar loss of HS from the enterocyte basolateral membrane. Loss of syndecan-1 is also seen in reparative cells from patients with inflammatory bowel disease, and this is thought to compromise their ability to bind basic fibroblast growth factor.…”

Effect of Concanavalin A on Nephrotic Peripheral Blood Mononuclear Cells Mediated Increased 35Sulfate Uptake in Rat Glomerular Basement Membrane

“…Its removal is associated with an increase in GBM anionic sites (6). Heparan sulfate has been shown to be decreased in the GBM of patients with congenital nephrotic syndrome and in rats with aminonucleoside nephrosis (18,19). Furthermore, a decrease in the number of anionic sites (thought to be composed of glycosaminoglycans) in the lamina rarae interna of the GBM of patients with IMLNS has been reported (20).…”