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) approximately 60% 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.
Highlights
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 study we evaluated whether the development ofalbuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular heparan sulfates (HS) expression or structure
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
Summary
Ated of enalapril, nifedipine retard, or placebo on the progression of diabetic nephropathy in normotensive insulin-de-. HS was isolated according to standard procedures, which comprises papain digestion, DEAE-Sephacel ion-exchange chromatography, and chondroitinase ABC treatment followed by Mono Q fast protein liquid chromatography Both HS samples were subjected to two different labeling procedures. The second part of the glomerular HS samples was partly deacetylated for 1 h by hydrazinolysis, followed by N-acetylation using [3H]acetic anhydride [26, 27] These radiolabeled HS preparations were used to perform protein binding experiments (see below) and to estimate the length of the chains by Sephacryl S-300 high resolution (Pharmacia, Uppsala, Sweden) gel chromatography, 1 ϫ 70-cm column, in 0.5 M NaCl. In vivo [35S]sulfate-radiolabeled glomerular HS (in a separate group of four control and four diabetic rats) was isolated according to standard procedures. Significance ( p Ͻ 0.05) was tested by the unpaired Mann Whitney U test
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