Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Instillations of low pH lactate buffer, a high glucose concentration and glucose degradation products contribute differently and often cumulatively to peritoneal injury in vivo.

We previously demonstrated that polymorphisms in the carnosinase-1 gene (CNDP1) determine the risk of nephropathy in type 2 diabetic patients. Carnosine, the substrate of the enzyme encoded by this gene, is considered renoprotective and could possibly be used to treat diabetic nephropathy (DN). In this study, we examined the effect of carnosine treatment in vivo in BTBR (Black and Tan, BRachyuric) ob/ob mice, a type 2 diabetes model which develops a phenotype that closely resembles advanced human DN. Treatment of BTBR ob/ob mice with 4 mM carnosine for 18 weeks reduced plasma glucose and HbA1c, concomitant with elevated insulin and C-peptide levels. Also, albuminuria and kidney weights were reduced in carnosine-treated mice, which showed less glomerular hypertrophy due to a decrease in the surface area of Bowman’s capsule and space. Carnosine treatment restored the glomerular ultrastructure without affecting podocyte number, resulted in a modified molecular composition of the expanded mesangial matrix and led to the formation of carnosine-acrolein adducts. Our results demonstrate that treatment with carnosine improves glucose metabolism, albuminuria and pathology in BTBR ob/ob mice. Hence, carnosine could be a novel therapeutic strategy to treat patients with DN and/or be used to prevent DN in patients with diabetes.

However, such residues were scarce in cell surface glypican-1. Brefeldin A-arrested glypican-1, which was non-S-nitrosylated and carried side chains rich in Nunsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of Snitrosylated glypican-1 in caveolin-1-rich compartments. Inhibition of deaminative cleavage did not prevent heparanase from generating heparan sulfate oligosaccharides that colocalized strongly with caveolin-1. Growthquiescent cells displayed extensive NO-dependent deaminative cleavage of heparan sulfate-generating anhydromannose-terminating fragments that were partly associated with acidic vesicles. Proliferating cells generated such fragments during polyamine uptake. We conclude that recycling glypican-1 that is associated with caveolin-1-containing endosomes undergoes sequential N-desulfation/N-deacetylation, heparanase cleavage, S-nitrosylation, NO release, and deaminative cleavage of its side chains in conjunction with polyamine uptake.Mammalian glypican-1 (Gpc-1) 1 is a member of a glycosylphosphatidylinositol (GPI)-linked cell-surface proteoglycan (PG) family with six known members to date. These PG, like other cell surface PG, are selective regulators of ligand-receptor encounters and thereby control growth and development (1-4). Gpc proteins are post-translationally modified by the addition of the glycosaminoglycan heparan sulfate (HS) at sites located close to the C-terminal GPI-membrane anchor (see Scheme 1). The central part of the protein consists of a cysteine-rich domain containing information that ensures a high level of HS substitution (5). Many of the functions of Gpc are dependent on the HS side chains, which are capable of binding and/or activating and/or transporting a variety of growth factors, cytokines, enzymes, viral proteins, and polyamines (6 -11).GPI-anchored proteins are usually associated with sphingolipid-and cholesterol-rich plasma membrane domains. Such enriched domains may exist either as small phase-separated "rafts" or, when associated with caveolin-1 (Cav-1), form flaskshaped plasmalemmal invaginations called caveolae, which are involved in signal transduction and special forms of non-clathrindependent endocytosis mediated by Cav-1-containing endosomes, also called caveosomes (12)(13)(14)(15)(16)(17).Biochemical studies using radioactively labeled precursors have demonstrated recycling of newly made Gpc-1 in normal fibroblasts as well as in transformed cells (18,19). During recycling, the HS side chains are degraded both by heparanase and by NO-dependent deaminative cleavage at N-unsubstituted glucosamine residues (GlcNH 3 ϩ ) (20). New HS chains can then be synthesized on the stubs remaining on the core protein (see Scheme 1). Biosynthesis of HS takes place in the Golgi and involves many interacting enzymes (21,22). The stubs should first be extended with a GlcNAc-hexuronic acid (HexUA) repeat backbone. The GlcNH 3 ϩ residues are either a result of inadequate sulfation dur...

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)