Title: Glomerular heparan sulfate alterations: Mechanisms and relevance for proteinuria
Abstract: Glomerular heparan sulfate alterations: Mechanisms and relevance for proteinuria. 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. Glomerular heparan sulfate alterations: Mechanisms and relevance for proteinuria. 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. During glomerular ultrafiltration, the barrier restricting the passage of plasma proteins into the urine is the glomerular capillary wall (GCW). The GCW consists of the fenestrated endothelium, the glomerular basement membrane (GBM), and the foot processes of the glomerular visceral epithelial cells or podocytes with in-between the slit diaphragms. The fenestrae of the endothelial cells allow direct contact of blood with the GBM and hardly restrict passage of macromolecules1Dworkin L.D. Brenner B.M. Biophysical basis of glomerular filtration.The Kidney: Physiology and Pathophysiology. edited by Seldin DW, Giebisch G. Raven Press, New York1992: 979Google Scholar. Some data show that podocytes are involved in the permeability of the GCW. Cross-linking of the integrin receptors for extracellular matrix (ECM) components on the podocyte resulted in a decreased adhesion of podocytes to the GBM and an increased passage of macromolecules in vitro2Adler S. Sharma R. Savin V.J. Abbi R. Eng B. Alteration of glomerular permeability to macromolecules induced by cross-linking of beta-1 integrin receptors.Am J Pathol. 1996; 149: 987-996PubMed Google Scholar. Furthermore, in saponin-induced podocyte injury in the single nephron model, passage of albumin through the GCW was localized to regions of detachment3Laurens W. Battaglia C. Foglieni C. De Vos R. Malanchini B. Van Damme B.J.C. Vanrenterghem Y.F. Remuzzi G. Remuzzi A. Direct podocyte damage in the single nephron leads to albuminuria in vivo.Kidney Int. 1995; 47: 1078-1086Abstract Full Text PDF PubMed Google Scholar. 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HS contains many negative charges, which are important determinants for the charge-dependent permeability of the GBM. HS is an unbranched chain of repeating disaccharide units, which contain an uronic acid (glucuronic or iduronic acid) and the aminosugar glucosamine. After formation of the precursor molecule heparosan, extensive modifications take place starting with N-deacetylation/N-sulfation of the glucosamine residue, followed by C-5 epimerization of glucuronic acid into iduronic acid, and O-sulfation at various positions36Lindahl U. Lidholt K. Spillmann D. Kjellen L. More to “heparin” than anticoagulation.Thromb Res. 1994; 75Abstract Full Text PDF Scopus (297) Google Scholar. For most reactions, several isoenzymes exist that have subtle differences in substrate-specificity; furthermore, these reactions run incompletely, leading to a great variation in the degree of sulfation along the stretch of the side chains. Because of the sequential order of the modification reactions and the substrate specificity of the enzymes involved, the initial distribution of N-sulfate groups will greatly determine the location of iduronic acid residues and O-sulfate groups. This results in a block-like structure in which alternating N-acetylated, mixed N-acetylated/N-sulfated and N-sulfated (heparin-like) domains are found Figure 237Turnbull J.E. Gallagher J.T. Molecular organization of heparan sulphate from human skin fibroblasts.Biochem J. 1990; 265Crossref Google Scholar, 38Lindblom A. Bengtsson Olivecrona G. Fransson L.A. Domain structure of endothelial heparan sulphate.Biochem J. 1991; 279Crossref Google Scholar, 39Schmidt A. Lemming G. Yoshida K. Buddecke E. Molecular organization and antiproliferative domains of arterial tissue heparan sulfate.Eur J Cell Biol. 1992; 59Google Scholar, 40Lyon M. Deakin J.A. Gallagher J.T. 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Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.J Biol Chem. 1997; 272Crossref Scopus (85) Google Scholar. This suggested that in certain forms of glomerular pathology, changes occur in the HS side chains of agrin that may be related to the development of proteinuria. This review focuses on four different mechanisms that are identified up to now for these changes in the HS polysaccharide side chain, masking by immune complexes, depolymerization by radicals, degradation by proteolytic enzymes or metabolically-induced biochemical changes in the HS structure. Systemic lupus erythematosus is an autoimmune disease characterized by the formation of autoantibodies, mainly directed against nuclear antigens. Currently, it is clear that this autoimmune response is T-cell dependent and antigen driven. 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Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: Characterization and immunohistological application in human tissues.J Histochem Cytochem. 1994; 42Crossref Google Scholar, we found a strong decrease or even total absence of staining for the HS side chain in patients with lupus nephritis, whereas the staining for the HSPG core protein was unaltered58van den Born J. Van Den Heuvel L.P.W.J. Bakker M.A.H. Veerkamp J.H. Assmann K.J.M. Weening J.J. Berden J.H.M. Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases.Kidney Int. 1993; 43Abstract Full Text PDF Google Scholar. This observation extended previous reports in which a decrease of anionic sites within the GBM was reported in lupus nephritis85Melnick G.F. Ladoulis C.T. Cavallo T. D