heparitin-sulfate and Glomerulonephritis

The primary filtration of blood occurs in the glomerulus in the kidney. Destruction of any of the layers of the glomerular filtration barrier might result in proteinuric disease. The glomerular endothelial cells and especially its covering layer, the glycocalyx, play a pivotal role in development of albuminuria. One of the main sulfated glycosaminoglycans in the glomerular endothelial glycocalyx is heparan sulfate. The endoglycosidase heparanase degrades heparan sulfate, thereby affecting glomerular barrier function, immune reactivity and inflammation. Increased expression of glomerular heparanase correlates with loss of glomerular heparan sulfate in many glomerular diseases. Most importantly, heparanase knockout in mice prevented the development of albuminuria after induction of experimental diabetic nephropathy and experimental glomerulonephritis. Therefore, heparanase could serve as a pharmacological target for glomerular diseases. Several factors that regulate heparanase expression and activity have been identified and compounds aiming to inhibit heparanase activity are currently explored.

Topics: Albuminuria; Animals; Diabetic Nephropathies; Endothelial Cells; Glomerulonephritis; Glucuronidase; Heparitin Sulfate; Humans; Kidney Diseases; Kidney Glomerulus

Topics: Animals; Collagen; Doxorubicin; Epithelium; Glomerulonephritis; Heparitin Sulfate; Humans; Kidney Glomerulus; Nephrosis; Puromycin Aminonucleoside

The GBM is an important structure for the development of immunologic renal disease. It may serve as the primary antigen or as the site of deposition of immune complexes. The GBM is recognized as the major protein filtration barrier of the glomerulus. The composition of the GBM includes collagen, predominantly but not exclusively type IV, which is present as procollagen. Two glycoproteins, laminin and fibronectin, are either intrinsic components of the GBM or intimately associated with the membrane. Heparan sulfate, a glycosaminoglycan, is contained within the substance of the GBM and seems to comprise the primary anion of the structure. Antibodies to these individual GBM components are being recognized with increasing frequency in patients with glomerulonephritis. The recognition of these antibodies may serve to expand our definition of both immunologic and degenerative diseases of basement membrane.

Topics: Anti-Glomerular Basement Membrane Disease; Antibodies; Antigen-Antibody Complex; Antigens; Basement Membrane; Collagen; Fibronectins; Glomerulonephritis; Glycoproteins; Heparitin Sulfate; Humans; Immune Complex Diseases; Kidney Glomerulus; Laminin

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Topics: Animals; CD11b Antigen; Chemokine CXCL1; Fibrin; Gene Expression Regulation; Glomerulonephritis; Heparitin Sulfate; Intercellular Adhesion Molecule-1; Interleukin-6; Kidney Glomerulus; L-Selectin; Mice; Single-Chain Antibodies

Heparanase, a heparan sulfate (HS)--specific endoglucuronidase, mediates the onset of proteinuria and renal damage during experimental diabetic nephropathy. Glomerular heparanase expression is increased in most proteinuric diseases. Herein, we evaluated the role of heparanase in two models of experimental glomerulonephritis, being anti-glomerular basement membrane and lipopolysaccharide-induced glomerulonephritis, in wild-type and heparanase-deficient mice. Induction of experimental glomerulonephritis led to an increased heparanase expression in wild-type mice, which was associated with a decreased glomerular expression of a highly sulfated HS domain, and albuminuria. Albuminuria was reduced in the heparanase-deficient mice in both models of experimental glomerulonephritis, which was accompanied by a better renal function and less renal damage. Notably, glomerular HS expression was preserved in the heparanase-deficient mice. Glomerular leukocyte and macrophage influx was reduced in the heparanase-deficient mice, which was accompanied by a reduced expression of both types 1 and 2 helper T-cell cytokines. In vitro, tumor necrosis factor-α and lipopolysaccharide directly induced heparanase expression and increased transendothelial albumin passage. Our study shows that heparanase contributes to proteinuria and renal damage in experimental glomerulonephritis by decreasing glomerular HS expression, enhancing renal leukocyte and macrophage influx, and affecting the local cytokine milieu.

Topics: Acute Disease; Animals; Diabetic Nephropathies; Glomerular Basement Membrane; Glomerulonephritis; Glucuronidase; Heparitin Sulfate; Mice, Inbred C57BL; Proteinuria; Tumor Necrosis Factor-alpha

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

Topics: Animals; Binding Sites; Cell Line; Disease Models, Animal; Gene Expression Regulation; Glomerulonephritis; GTP-Binding Proteins; Heparitin Sulfate; Kidney Function Tests; Mice; NIH 3T3 Cells; Protein Glutamine gamma Glutamyltransferase 2; Rats; Syndecan-4; Transforming Growth Factor beta1; Transglutaminases

Hyperlipidemia accelerates the progression of glomerular disease, and lipoproteins bind glomerular mesangial cells (MC) and induce proliferation and cytokine expression. In the vessel wall, the binding of lipoproteins to endothelial cells is markedly enhanced by lipoprotein lipase (LpL), synthesized by the underlying smooth muscle cells. While it is known that LpL is localized to the glomerulus, it is not known if and how it modulates the lipoprotein-mesangial interaction.. Very low-density lipoprotein (VLDL) was isolated from rats and was used to treat cultured primary rat MCs. Binding studies were done with and without LpL and with/without pretreatment with heparanase, which degrades cell surface heparan sulfate proteoglycan (HSPG), known to modulate the LpL-lipoprotein interaction in blood vessels. VLDL/LpL was also used to assess MC proliferation and gene expression of the cytokine platelet-derived growth factor (PDGF).. LpL enhanced VLDL binding to MCs by as much as 200-fold, and most of this effect was blocked by pretreatment with heparanase. LpL amplified VLDL-driven MC proliferation and increased VLDL-induced PDGF expression. Heparanase pretreatment of cells eliminated both of these amplifications. LpL alone increased MC proliferation and PDGF gene expression.. As in the vessel wall, LpL enhances VLDL binding to MCs. MCs respond to LpL binding by proliferating and expressing cytokines such as PDGF. LpL may be a crucial paracrine mediator of the glomerular response to circulating lipoproteins, amplifying a response that includes cytokine elaboration, influx of circulating monocytes, and eventual sclerosis.

Topics: Animals; Cell Division; Cells, Cultured; Female; Gene Expression; Glomerular Mesangium; Glomerulonephritis; Glucuronidase; Heparitin Sulfate; Hyperlipidemias; Lipoprotein Lipase; Lipoproteins, VLDL; Paracrine Communication; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Rats, Zucker; RNA, Messenger

Crescentic glomerulonephritis leads to a rapid loss of renal function. Although glomerular crescents are rich in extracellular matrix (ECM), the composition and genesis of the ECM are incompletely understood. Heparan sulfate (HS) is a major ECM molecule and has polymeric structure of great variability. Recent findings that alterations in HS epitopes are associated with renal pathology prompted us to hypothesize that specific HS epitopes might be expressed in the evolution of crescents. We reviewed clinical records of 724 patients who underwent renal biopsy and found 21 patients with rapidly progressive glomerulonephritis. Immunohistochemistry was performed using monoclonal antibodies (mAbs) against well-defined HS epitopes. One mAb was directed against unsaturated uronic acid residues generated during the selective removal of HS by heparitinase (a), and a further two different mAbs against N-sulfate-enriched and O-sulfate-poor portions of HS (b). Results showed that mAb (a) reacted to ECM of normal, sclerosed and crescentic glomeruli and that mAbs (b) reacted strongly to ECM of fibrocellular crescents but not to fibrous crescents, the periglomerular areas and noncrescentic intraglomerular areas. We concluded there are regional differences in HS epitope expression, although HS are ubiquitous components of glomerular ECM. N-sulfate-enriched and O-sulfate-poor portions of HS might play a role in crescent formation.

Topics: Animals; Antibodies, Monoclonal; Epitopes; Glomerulonephritis; Heparitin Sulfate; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar

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 (r(s) = -0.82 and r(s) = -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.

Topics: Albuminuria; Animals; Autoantibodies; Basement Membrane; Capillaries; Complement System Proteins; Female; Free Radicals; Glomerulonephritis; Heparitin Sulfate; Immunoglobulin G; Kidney Glomerulus; Nitrogen; Proteoglycans; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Staining and Labeling

Experimental systemic lupus erythematosus (SLE)-like disease was induced in BALB/c mice by immunization with heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. Following booster injections with heparan sulfate (HS), high levels of anti-HS, anti-dsDNA, and anti-cardiolipin antibodies were detected in the sera of the immunized mice. An enzyme-linked immunospot (ELISPOT) assay indicted that IgG anti-HS and anti-dsDNA antibody-secreting cells were present in the kidneys and most likely contributed to antibody localization. Antibodies eluted from the kidneys of immunized mice were found to react strongly with HS and dsDNA when tested in vitro. The HS-immunized mice developed moderate to severe levels of proteinuria. Histologic examination of kidneys from HS-immunized mice revealed deposition of immunoglobulin in the kidneys. Our results describe the induction of SLE-like disease in normal mice following immunization with HS. This experimental model may be useful for understanding the immunologic basis for autoimmunity to HS.

Topics: Animals; Antibodies, Antinuclear; B-Lymphocytes; Glomerulonephritis; Heparitin Sulfate; Immunization; Immunoglobulin G; Immunoglobulin Isotypes; Kidney; Lupus Erythematosus, Systemic; Male; Mice; Mice, Inbred BALB C; Spleen

Most glomerular pathologies are associated with alterations of the matrix compartment. Using reagents directed against the alpha 1/alpha 2 and alpha 3 chains of type IV collagen [alpha 1/alpha 2(IV), alpha 3(IV)], laminin, heparan sulphate proteoglycan (HPG), fibronectin, collagen I, and collagen III, we investigated the modifications of the glomerular matrix components in several human glomerular lesions compared with normal kidney. In type I membranous glomerulonephritis (MGN) (nine cases), we did not observe alterations in the matrix component distribution. In MGN types II and III (five cases), the spikes and chainettes were made of the alpha 3(IV) chain, laminin, and HPG, while the alpha 1/alpha 2(IV) chains were localized along the subendothelial side of the glomerular basement membrane (GBM). In focal and segmental glomerulosclerosis (six cases), fibronectin, alpha 1/alpha 2(IV) chains, laminin, and small amounts of interstitial collagens were detected within the collapsed capillary loops; the newly formed matrix material between the podocytes and the GBM contained the alpha 1/alpha 2(IV) chains, laminin, and HPG but not the alpha 3(IV) chain. In crescentic glomerulonephritis (six cases), fibronectin was the most abundant and, in purely cellular crescents, the unique component. A basement membrane-like network containing laminin, HPG, alpha 1/alpha 2(IV) chains, and interstitial collagens developed in a second step between the crescent cells. Interstitial collagens were present in the crescent framework, even when the integrity of Bowman's capsule was preserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Collagen; Extracellular Matrix Proteins; Fibronectins; Glomerulonephritis; Glomerulonephritis, Membranoproliferative; Glomerulonephritis, Membranous; Glomerulosclerosis, Focal Segmental; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Kidney Glomerulus; Laminin; Proteoglycans

We investigated alterations in the main components of glomerular extracellular matrices, including heparansulfate proteoglycan (HS-PG), laminin, type IV collagen and fibronectin in the renal tissues of 61 patients with various types of glomerulonephritis. Indirect immunofluorescence stainings with polyclonal antibodies of these extracellular matrix components were performed. In minimal change nephrotic syndrome, no remarkable changes were observed. In membranous glomerulonephritis, non-collagenous components, such as HS-PG and laminin altered in distribution, forming spikes in stage II and circles between or around the immune deposits in stage III. These changes were observed more clearly in HS-PG and laminin than in type IV collagen. These results suggested that non-collagenous components played an important role in repairing the GBM. In membranoproliferative glomerulonephritis and IgA nephropathy, type IV collagen and fibronectin expanded in the proliferated mesangial area. These findings showed that there was a close relationship between these extracellular matrix and the progression of glomerulosclerosis. In dense deposit disease, double contour of staining of HS-PG, laminin and type IV collagen was observed along the capillary wall. These findings suggested that there were no main extracellular matrix components within the dense materials.

Topics: Collagen; Extracellular Matrix; Fibronectins; Fluorescent Antibody Technique; Glomerulonephritis; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Laminin; Proteoglycans

Binding of 125I-heparan sulphate to the cell surface of Streptococcus pyogenes is mediated by proteins, that could be released from the streptococcal cell wall by using alkaline buffer. SDS-electrophoresis revealed two bands with molecular weights of 63 and 58 kDa. Binding of the 125I-labelled heparan sulphate probe to streptococci seems to be due to charge interactions, as the same probe was displaced by unlabelled heparan sulphate, other negatively charged molecules such as heparin, dextran sulphate, dermatan sulphate or by high ionic strength. The interaction was also strongly influenced by pH. The binding constant at pH 7.2 was estimated to be 9.8 x 10(6) mol/l, suggesting a moderate affinity. The presence of collagen of different types enhanced binding of 125I-labelled heparan sulphate to streptococci, whereas fibronectin and vitronectin had an inhibitory effect. The cooperation between heparan sulphate and collagen could be important for the adhesion of streptococci to connective tissue.

Topics: Bacterial Outer Membrane Proteins; Binding, Competitive; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Glomerulonephritis; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Humans; Protein Binding; Radioligand Assay; Streptococcus pyogenes

Only a fraction (12%) of 268 "autoreactive" T cell clones derived from lupus-prone mice can selectively induce the production of pathogenic anti-DNA autoantibodies in vitro and accelerate the development of lupus nephritis when transferred in vivo. The CDR3 loops of T cell receptor beta chains expressed by these pathogenic T helper (Th) clones contain a recurrent motif of anionic residues suggesting that they are selected by autoantigens with cationic residues. Herein, we found that approximately 50% of these pathogenic Th clones were specific for nucleosomal antigens, but none of them responded to cationic idiopeptides shared by variable regions of pathogenic anti-DNA autoantibodies. Nucleosomes did not stimulate the T cells as a nonspecific mitogen or superantigen. Only the pathogenic Th cells of lupus responded to nucleosomal antigens that were processed and presented via the major histocompatibility class II pathway. Although the presentation of purified mononucleosomes to the Th clones could be blocked by inhibitors of endosomal proteases, neither of the two components of the nucleosomes--free DNA or histones by themselves--could stimulate the Th clones. Thus critical peptide epitopes for the Th cells were probably protected during uptake and processing of the nucleosome particle as a whole. The nucleosome-specific Th clones preferentially augmented the production of IgG autoantibodies to histone-DNA complex in vitro. In vivo, nucleosome-specific, CD4+ T cells were not detectable in normal mice, but they were found in the spleens of lupus-prone mice as early as 1 mo of age, long before other autoimmune manifestations. Immunization of young, preautoimmune lupus mice with nucleosomes augmented the production of autoantibodies and markedly accelerated the development of severe glomerulonephritis. Previously, crude preparations containing nucleosomes were shown by others to have polyclonal mitogenic activity for B cells from normal as well as lupus mice. Identification here of pure mononucleosome as a lupus-specific immunogen for the Th cells that selectively help the pathogenic anti-DNA autoantibody producing B cells of lupus could lead to the design of specific therapy against this pathogenic autoimmune response.

Topics: Amino Acid Sequence; Animals; Autoantibodies; Glomerulonephritis; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunoglobulin G; Lupus Erythematosus, Systemic; Major Histocompatibility Complex; Mice; Mice, Inbred Strains; Molecular Sequence Data; Nucleosomes; Proteoglycans; Spleen; T-Lymphocytes, Helper-Inducer

Kidneys of Balb/c mice intravenously injected with rabbit antibodies to heparan sulfate proteoglycan (HSPG) derived from PYS-2 cells were studied for 14 weeks. Antibodies were found to bind to the glomerular basement membrane (GBM) as early as 15 min after the injection. Binding of antibodies was observed over the whole thickness of the GBM. The lamina rara interna and foot process base appeared to be more intensely stained. This pattern did not change throughout the experiments. Mild inflammatory changes (infiltration of polymorphonuclear cells and swelling of the glomerular endothelium) were seen at the initial stage (approximately 1 day), and mesangial expansion followed (1-4 weeks). In mice pre- and booster-immunized with normal rabbit IgG, a moderate autologous response was observed by immunofluorescence microscopy, but no significant inflammatory changes were noted. At the late stage (6 weeks approximately), irregular GBM thickening was observed. The thickening was due mainly to expansion of the lamina rara externa. These findings suggest that the anti-HSPG antibodies caused mild glomerulonephritis at the initial stage, and later caused thickening of the GBM possibly by disturbing the assembly, production and degradation of GBM components.

Topics: Animals; Antibodies; Chondroitin Sulfate Proteoglycans; Female; Glomerulonephritis; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Mice; Proteoglycans; Tumor Cells, Cultured

Authors studied the dispersion of immunoreactive HSPG with monoclonal antibody specific to protein nucleus of HSPG in renal diseases associated with nephrosis syndrome. Their results showed that the dispersion of HSPG in GBM does not fully agree with dispersion of anion linkage places known in literature. In membrane and diffuse proliferative lupus glomerulonephritis immunoreactive HSPG cannot be demonstrated in the place of immune deposits, concurrent with dispersion of anion places, while it appears in GBM newly developed round the deposits. Inconsistent with this, in glomerulonephritis having minimal changes, the anion loss of GBM is not associated with absence of nuclear protein, the immunoreactive HSPG remains intact. These observations reflected in literature indicate that--similarly to proteinuria--presumably, deficiency of anion linkage places of GBM is caused by different pathomechanisms.

Topics: Basement Membrane; Glomerulonephritis; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Proteoglycans

Monoclonal antibodies (mAbs) (4F2 and 7E12) were prepared against heparan sulfate proteoglycan (HSPG) isolated from bovine glomeruli. Enzyme linked immunosorbent assays (ELISA) and immunoblotting demonstrated that the mABs reacted with HSPG. Indirect immunofluorescence (IF) showed that the mAbs stained renal basement membranes (BMs) and BMs in other organs of normal bovine and human tissues in patterns typical of HSPG. Immunoinhibition studies, and immunoblotting of heparan lyase digested HSPG, indicated that the mAbs recognize HSPG core protein. In kidney biopsies from patients with acute poststreptococcal GN, intact linear glomerular BM (GBM) staining for HSPG was noted despite markedly widened capillary loops. In membranous and in diffuse proliferative lupus GN, loss of HSPG staining was demonstrated at sites of immunodeposition of IgG or C3, while increased staining for HSPG was noted in areas of newly formed GBM. Extensive loss of HSPG was seen in areas of glomerular sclerosis and necrosis. In biopsies from patients with minimal change glomerulonephritis (GN) and mesangioproliferative lupus GN, a normal linear GBM distribution of HSPG was noted. The findings are discussed in the context of current knowledge regarding the pathogenesis of glomerular injury. MAbs to BM HSPG should prove useful for future immunochemical studies, and for the study of diseases of the basement membrane.

Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Biopsy; Cattle; Chondroitin Sulfate Proteoglycans; Glomerulonephritis; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Kidney; Proteoglycans

We investigated nephritogenic potential of antibodies to heparan sulfate-proteoglycan of glomerular basement membrane. Glomeruli were isolated, basement membranes were prepared, proteoglycans extracted, and purified core protein was obtained. We immunized rabbits with the core protein, IgG fraction prepared from the antisera and specificity of the antibody determined. A single immunoprecipitin line in agar diffusion plate and a single band (approximately 18,000 mol wt) on the immunoblot autoradiograms were visualized. The antibody showed precise reactivity with the glomerular basement membranes. The clearance studies indicated that approximately 75% of the radioiodinated antibody disappeared from circulation within 1 h and 1-2% bound to the kidney. For nephritogenicity experiments, the antibody was intravenously administered into rats and we examined their kidneys at 1 h to 24 d later. A linear immunofluorescence of glomerular basement membranes was observed with rabbit IgG at all times while that of C3 until the 10th day. Early morphologic changes included glomerular infiltration of polymorphonuclear leukocytes with focal exfoliation of endothelium. The leukocytic infiltration subsided by the third day and was followed by progressive thickening of basement membranes, focal mesangial cell proliferation, increase in mesangial matrix, and accumulation of monocytes. Focal knob-like thickening of glomerular basement membrane was observed from the 15th day onward. Regularly-spaced electrondense deposits were seen in the lamina rara interna and externa of glomerular basement membranes and persisted throughout the investigatory period. No significant proteinuria was observed at any stage of the experiment. These findings suggest that the antibodies to the basement membrane heparan sulfate-proteoglycan are nephrotoxic but possess weak nephritogenic potential.

Topics: Animals; Antibody Specificity; Antigens; Autoantibodies; Autoantigens; Basement Membrane; Binding Sites, Antibody; Chondroitin Sulfate Proteoglycans; Glomerulonephritis; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Metabolic Clearance Rate; Proteinuria; Proteoglycans; Rabbits; Rats

Topics: Chondroitin; Chondroitin Sulfates; Glomerulonephritis; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney Tubules; Nephritis, Interstitial

In 30 of 33 human systemic lupus erythematosus (SLE) sera and in 10 sera from MRL/l mice with spontaneous SLE, antibodies against heparan sulfate were detected. The anti-heparan sulfate titers showed a significant correlation with the anti-DNA antibody titers. By inhibition studies it was demonstrated that heparan sulfate could inhibit the binding of anti-DNA antibodies to DNA, whereas DNA could block the binding to heparan sulfate. That this reaction is due to crossreactivity of anti-DNA antibodies was further substantiated by the finding that two monoclonal anti-DNA antibodies also bound to heparan sulfate. Antibodies eluted from human and mouse kidneys with diffuse SLE glomerulonephritis showed a similar binding to DNA and heparan sulfate when these eluted antibodies were tested in vitro. Heparan sulfate is the major glycosaminoglycan constituent of the glomerular basement membrane. Our findings suggest that heparan sulfate might serve as a target antigen in vivo for cross-reactive anti-DNA antibodies.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Basement Membrane; Cross Reactions; DNA; Enzyme-Linked Immunosorbent Assay; Glomerulonephritis; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney; Kidney Glomerulus; Lupus Erythematosus, Systemic; Mice; Osmolar Concentration

Topics: Child; Chondroitin Sulfates; Diabetes Mellitus; Glomerulonephritis; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidoses; Nephritis, Hereditary; Nephrotic Syndrome; Proteinuria

Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.

Topics: Anti-Glomerular Basement Membrane Disease; Autoimmune Diseases; Basement Membrane; Collagen; Extracellular Matrix; Fibronectins; Glomerulonephritis; Heparitin Sulfate; Humans; Laminin; Proteoglycans; Streptococcal Infections

Antibodies, found in human sera from patients with poststreptococcal glomerulonephritis, against proteoglycans (PG) derived from bovine and human glomeruli were investigated. PG were isolated by 4 M guanidine-HCl extraction of whole glomeruli, followed by DEAE-Sepharose CL-6B ion exchange chromatography. The anionic fractions were further purified by chromatography on Sepharose CL-4B. Biochemical analysis of the two resulting peaks revealed the presence of high molecular weight anionic material containing protein, uronic acid, glucosamine, and galactosamine. Enzymatic and chemical susceptibilities indicated the presence of heparan sulfate PG and a galactosamine-containing PG. Immunologic studies revealed the presence of anti-PG antibodies to both PG peaks of the Sepharose CL-4B column in glomerulonephritis sera. Inhibition studies using an ELISA demonstrated that heparan sulfate was a major antigenic determinant. Cross-reactivity with both mammalian and streptococcal hyaluronate was noted. Inhibition studies also indicated the presence of a second antigenic site containing N-acetylgalactosamine, possibly representing chondroitin or dermatan sulfate PG.

Topics: Acute Disease; Animals; Antigen-Antibody Reactions; Autoantibodies; Cattle; Chemical Phenomena; Chemistry, Physical; Chondroitin Sulfate Proteoglycans; Enzyme-Linked Immunosorbent Assay; Glomerulonephritis; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hexosamines; Humans; Kidney Glomerulus; Proteins; Proteoglycans; Rabbits; Streptococcal Infections; Uronic Acids

The ultrastructural and the biophysical and biochemical qualities of glomerular permeability to protein molecules are reviewed. With regard to differently located immune deposition in human glomerulonephritis, description and discussion are addressed in a fixed order of layers: 1. endothelial-subendothelial, i.e. the endothelial cells with fenestrate and the lamina rara interna of the basement membrane (bm), 2. membranous, i.e. the lamina densa of the bm, 3. subepithelial-epithelial, i.e. the lamina rara externa of the bm and the podocytes with food processes and slit diaphragms. It is emphasized that the 3 layers act as gradually (coarse to fine) filter barriers. On the basis of well known structural peculiarities, in the last 10 years experimental studies revealed that the meshwork of type IV collagen and the negatively charged heparan sulfate-proteoglycans - "the glomerular polyanion" - are integrated in sieving of protein molecules. These components are differently located in the stratified cellular and extracellular layers of the glomerular filter and their combined action is the basis of a size, charge and configuration depended filtration of macromolecules. In this way the passage of the mostly negative charge blood proteins, especially albumin, is prevented under normal conditions.

Topics: Animals; Basement Membrane; Blood Proteins; Capillaries; Capillary Permeability; Endothelium; Ferritins; Glomerular Filtration Rate; Glomerular Mesangium; Glomerulonephritis; Heparitin Sulfate; Humans; Intercellular Junctions; Kidney Glomerulus; Membrane Potentials; Microscopy, Electron, Scanning; Molecular Weight; Proteoglycans