No Change in Glomerular Heparan Sulfate Structure in Early Human and Experimental Diabetic Nephropathy

Abstract

Heparan sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro)albuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies, glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in rat renal tissue after 5 months diabetes duration. Glomerular staining, however, revealed no differences between control and diabetic specimens. A significant ( p < 0.05) ϳ60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli. Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is not caused by loss of glomerular HS expression or sulfation and suggest other mechanisms to be responsible for increased glomerular albumin permeability.One of the first signs of diabetic nephropathy (DNP) 2 is a discrete increase in urinary excretion of albumin called microalbuminuria (1, 2). In this early phase of DNP mainly the charge-dependent permeability of the glomerular capillary wall seems to be affected, while during progression to overt proteinuria also the size-dependent permeability becomes disturbed (3). These findings suggest an early loss of glomerular basement membrane (GBM) charge with a subsequent increase in pore size. This charge-dependent permeability of the GBM is probably related to the presence of anionic constituents of the GBM, mainly heparan sulfates (HS), the glycosaminoglycan side chains of heparan sulfate proteoglycans (HSPGs) (4). Some years ago, we identified agrin as a major HSPG present in the GBM (5, 6), using antibodies toward either the core protein or the HS polysaccharide side chains (7-9). The relevance of HS for the charge-dependent permeability is illustrated by several observations: (i) enzymatic removal of HS from the GBM leads to albuminuria (10); (ii) an acute selective albuminuria was induced in rats by a monoclonal antibody to HS (4); (iii) a reduction of HS expression in the GBM was documented in several human and experimental proteinuric glomerular diseases, which inversely correlated with proteinuria (11,12).In overt DNP, the expression of HS in the GBM was decreased and correlated with proteinuria (13,14). However, these studies were performed in advanced stages of DNP. In a rat diabetes model resembling early human DNP, we documented a significant loss in HS-related anionic sites in the GBM; howeve...

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

“…51,52 Although these studies support the theory that reductions in GBM-HS contribute directly to loss of barrier function, labeling with mAb JM-403 was recently reported to be normal in diabetic humans and rats with microalbuminuria, and it was concluded that loss of GBM-HS may be a secondary event that occurs in advanced disease. 53 Here, immunostaining with both antibodies revealed that adult conditional mutants lack GBM-HS. This confirms that mouse agrin exists as a HSPG in vivo but questions the importance of GBM-HS for kidney function.…”

Size and charge selectivity of the glomerular filter in early experimental diabetes in rats

“…Furthermore, although the enzyme heparanase was found to be increased in STZ-induced diabetic rats, this was actually not found to imply a significant reduction in the GBM HS content (37). Furthermore, no change in HS sulfation or charge density has been detected in early STZ-induced diabetes (35). Hence, the contribution of a decreased charge selectivity in the urinary hyperexcretion of albumin in early DNP is controversial.…”

Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy

“…However, several recent studies suggest that heparan sulphate in the GBM may not play a primary decisive role in the chargeselective permeability properties and development of proteinuria. For example, van den Born recently reported that there was no change in glomerular heparan sulphate content in early human and experimental diabetic nephropathy, although in both cases albuminuria was present [15]. Additional studies have shown that rats did not develop acute albuminuria after in vivo degradation of heparan sulphate by heparinase III [35].…”

“…Production of the core protein of agrin, the major heparan sulphate proteoglycan in the GBM, was not aberrant [11,13]. However, these heparan sulphate changes occur at the macroalbuminuric stage of diabetic nephropathy, since no glomerular heparan sulphate alterations were found at the microalbuminuric stage [15]. Recently, we showed that glomerular production of heparanase, an endo-β-1,4-D-glucuronidase involved in the hydrolytic cleavage of the β-1,4-glycosidic bond between glucuronic acid and glucosamine residues within N-sulphated and/or Nacetylated/N-sulphated domains of heparan sulphate [16,17], was increased in patients with overt diabetic nephropathy [11,18].…”