Glomerular heparan sulfate alterations: Mechanisms and rele-During glomerular ultrafiltration, the barrier restrictvance for proteinuria. Heparan sulfate (HS) is the anionic ing the passage of plasma proteins into the urine is the polysaccharide side chain of HS proteoglycans (HSPGs) presglomerular capillary wall (GCW). The GCW consists of ent in basement membranes, in extracellular matrix, and on the fenestrated endothelium, the glomerular basement cell surfaces. Recently, agrin was identified as a major HSPG membrane (GBM), and the foot processes of the glomerpresent in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion ular visceral epithelial cells or podocytes with in-between of HS by heparitinase or after antibody binding to HS demonthe slit diaphragms. The fenestrae of the endothelial cells strated the importance of HS for the permselective properties allow direct contact of blood with the GBM and hardly of the GBM. With recently developed antibodies directed restrict passage of macromolecules [1]. Some data show against the GBM HSPG (agrin) core protein and the HS side that podocytes are involved in the permeability of the chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as sys-GCW. Cross-linking of the integrin receptors for extratemic lupus erythematosus (SLE), minimal change disease, cellular matrix (ECM) components on the podocyte remembranous glomerulonephritis, and diabetic nephropathy, sulted in a decreased adhesion of podocytes to the GBM whereas the staining of the agrin core protein remained unaland an increased passage of macromolecules in vitro [2]. tered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into Furthermore, in saponin-induced podocyte injury in the the mechanisms responsible for this observation, we studied single nephron model, passage of albumin through the GBM HS(PG) expression in experimental models of protein-GCW was localized to regions of detachment [3]. An uria. Similar HS changes were found in murine lupus nephritis, injection of a monoclonal antibody directed against a adriamycin nephropathy, and active Heymann nephritis. In component of the slit diaphragm resulted in an acute these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigamassive proteinuria [4, 5]. Nevertheless, the GBM is tions, four new and different mechanisms have emerged. First, probably the most important component of this barrier in lupus nephritis, HS was found to be masked by nucleosomes and restricts the passage of macromolecules by both size complexed to antinuclear autoantibodies. This masking was and charge. Tracer studies have shown that the GCW due to the binding of cationic moieties on the N-terminal parts behaves like a sieve, through which small molecules, of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS...

Heparan sulfate, the polysaccharide side chain of heparan sulfate proteoglycan, is important for the permselective properties of the glomerular basement membrane. In this report, we show a role for hydroxyl radicals in heparan sulfate degradation and an enhanced glomerular basement membrane permeability. First, in enzyme-linked immunosorbent assay, exposure of coated heparan sulfate (proteoglycan) to reactive oxygen species resulted in a ؎50% decrease of binding of a monoclonal antibody against heparan sulfate, whereas binding of an antibody against the core protein remained unaltered. Second, on polyacrylamide gel electrophoresis, the molecular weight of heparan sulfate exposed to radicals was reduced which indicates depolymerization. Both in enzyme-linked immunosorbent assay and gel electrophoresis, hydroxyl radicals are instrumental for heparan sulfate degradation as shown by the addition of various radical scavengers. Third, in an experimental model for human nephrotic syndrome (Adriamycin nephropathy in rats), glomerular basement membrane staining of two recently described anti-heparan sulfate antibodies (JM403 and KJ865) was reduced by 24 and 43%. Treatment of Adriamycin-exposed rats with the hydroxyl radical scavenger dimethylthiourea both reduced albuminuria by 37% (p < 0.01) and partly prevented loss of heparan sulfate staining by 53% (JM403) and 39% (KJ865) (p < 0.03). In contrast to the heparan sulfate side chains, the core protein expression and the extent of glycanation did not change in Adriamycin nephropathy. We conclude that glomerular basement membrane heparan sulfate is susceptible to depolymerization by hydroxyl radicals leading to loss of glomerular basement membrane integrity and albuminuria.Heparan sulfate (HS) 1 is the anionic polysaccharide side chain of heparan sulfate proteoglycan (HSPG) present in basement membranes, extracellular matrix, and on the cell surface of many (if not all) cell types (1-3). Several investigators have shown that HS plays an important role in the permselective properties of the glomerular basement membrane (GBM) (4, 5). Enzymatic digestion of HS by heparitinase resulted in an increased passage of native ferritin and albumin through the GBM (6, 7). Furthermore, intravenous injection of a monoclonal antibody (mAb) directed against HS induces acute, selective proteinuria in rats (8). A reduction in GBM HS-associated anionic sites was found with cationic probes in several human and experimental proteinuric glomerulopathies (9, 10). With recently developed antibodies directed against GBM HSPG core protein and the HS side chain (11, 12), we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, whereas the staining of the HSPG core protein remained unaltered (13). The mechanism responsible for this observation remains to be elucidated. In lupus nephritis, masking of HS by autoantibodies complexed to nucleosomes is proposed as a mechanism for the decrease in HS staining and albuminuria (14). In human and experimental diabe...

The dystrophin-glycoprotein complex, which comprises ␣-and ␤-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against ␣-and ␤-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane. (Am J Pathol 2000, 156:1749 -1765) Dystroglycan (DG) is an important member of the dystrophin-glycoprotein complex (DGC) which links the subsarcolemmal cytoskeleton to the basal lamina in skeletal muscle.1 The importance of this link becomes clear from the severe muscular dystrophies resulting from mutations in genes that encode different members of the DGC. 2-5

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane ( Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.

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Ciclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulopathies. This antiproteinuric effect can be the result of a decrease of immunological damage, a decrease in the glomerular filtration rate (GFR), or a change in the permselective properties of the glomerular capillary wall. In this study we investigated the effect of CsA on Adriamycin-induced nephropathy in rats. A single intravenous injection of Adriamycin (5 mg/kg body weight) induced a severe nephrotic syndrome with a massive albuminuria ( ± 400 mg/24 h from 3 weeks onwards) and a hypoalbuminemia ( ± 7 mg/ml after 5 weeks). The IgG/albumin selectivity index was 0.16 ± 0.05, indicating a preferential loss of albumin. A 5-day treatment with CsA reduced the albumin excretion by almost 50% (from 336 ± 91 to 178 ± 58 mg/24 h; p = 0.002) and induced an increase in the serum albumin level (from 7.1 ± 4.1 to 12.8 ± 3.2 mg/ml; p = 0.002) in contrast to the vehicle olive oil (OO). CsA also decreased the GFR by 40% (from 0.74 ± 0.11 to 0.41 ± 0.11 mg/ml/100 g body weight; p = 0.002). Albuminuria corrected for the GFR (fractional excretion of albumin, FE_alb) was still significantly lower in CsA-treated than in OO-treated animals (FE_alb CsA: 1.35 ± 0.88, FE_albOO: 3.17 ± 2.29%; p = 0.0005). This suggests that other factors are also involved in the reduction of albuminuria. To exclude that CsA has an effect on the tubular reabsorption of albumin, we evaluated the blockade of the tubular reabsorption by lysine and found no difference in albuminuria between the CsA- and OO-treated groups. These experiments suggest that the antiproteinuric effect of CsA is not (only) due to a decrease in the GFR, but also to a decrease of the enhanced permeability of the glomerular capillary wall for albumin.

Abstract. In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274 ± 68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (rs = -0.82 and rs = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.