Membranous glomerulonephritis in the mouse

Membranous glomerulonephritis in the mouse. Glomerulonephritis was induced in C57.B110 mice by a single injection of rabbit IgG against homologous, pronase-digested, renal tubular antigens. The heterologous phase was characterized by a transient increase of glomerular permeability with fixation of rabbit IgG to the capillary walls, in a linear or fine-granular pattern, and to the brush borders of the proximal tubuli. The autologous phase was marked by the immune response to the injected protein, during which subepithelial immune deposits, consisting of mouse IgG1, rabbit IgG, and mouse C3 developed. Small amounts were still present at 1 year after the injection of antiserum. The antibody response of the mice correlated with the development and resolution of the deposits. None of the mice developed a nephrotic syndrome. Control mice treated with normal rabbit IgG did not show immune deposits in their kidneys at any stage despite a comparable antibody response to rabbit IgG. Immunoelectronmicroscopy showed that the rabbit antibodies fixed directly to an antigen in the cell membrane of the glomerular visceral epithelium. It seems, therefore, likely that in situ formation of subepithelial immune complexes occurred in the autologous phase by fixation of mouse immunoglobulins to rabbit IgG already present in the glomerular wall.Glomérulonéphrite extra-membraneuse chez la souris. Une glomérulonéphrite a été induite chez des souris C57.B110 par une injection unique d'IgG de lapin contre des antigènes tubulaires rénaux homologues, digérés par de la pronase. La phase hétérologue était caractérisée par une augmentation transitoire de la perméabilité glomérulaire avec fixation d'IgG de lapin aux parois capillaires, d'une façon linéaire ou finement granuleuse, et aux bordures en brosse des tubules proximaux. La phase autologue était marquée par la réponse immune à la protéine injectée, pendant laquelle des dépôts immuns sous-épithéliaux, consistant en de l'IgG1 de souris, de l'IgG de lapin et du C3 de souris, se sont développés. Il en restait encore de faibles quantités 1 an après l'injection de l'antisérum. La réponse anticorps des souris était corrélée avec le développement et la disparition des dépôts. Aucune des souris n'a développé de syndrome néphrotique. Les souris contrôles traitées avec de l'IgG de lapin normal n'ont pas eu de dépôts immuns dans le rein à aucun stade, malgré une réponse anticorps aux IgG de lapin comparable. La microscopie immuno-électronique a montré que les anticorps de lapin se fixaient directement à un antigène situé sur la membrane des cellules de l'épithélium viscéral glomérulaire. Il semble donc probable que la formation in situ de complexes immuns sous-épithéliaux est survenue à la phase autologue par fixation d'immunoglobulines de souris à de l'IgG de lapin déjà présente dans la paroi glomérulaire

A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats

A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG, HS and hyaluronic acid, but not with the core protein of human GBM HSPG, and not with chondroitin sulfate A and C, dermatan sulfate, keratan sulfate and heparin. Furthermore, it did not bind to laminin, collagen type IV or fibronectin. Specificity of JM-403 for HS was also suggested by results of inhibition studies, which found that intact HSPG and HS, but not the core protein, inhibited the binding of JM-403 to HS. In indirect immunofluorescence on cryostat sections of rat kidney, a fine granular to linear staining of the GBM was observed, along with a variable staining of the other renal basement membranes. Pretreatment of the sections with heparitinase completely prevented the binding of mAb JM-403, whereas pretreatment with chondroitinase ABC or hyaluronidase had no effect. The precise binding site of mAb JM-403 was investigated by indirect immunoelec-tron microscopy. It revealed a diffuse staining of the whole width of the GBM. One hour after intravenous injection of JM-403 into rats, the mAb was detected along the glomerular capillary wall in a fine granular pattern, which shifted towards a more mesangial localization after 24 hours. No binding was observed anymore by day 15. Intravenous injection induced a dose-dependent, transient and selective proteinuria that was maximal immediately after the injection. Administration of 2 mg of JM-403 increased the urinary albumin excretion within the first 24 hours after injection from (mean ± SD) 177 ± 19 to 20,755 ± 10,310 µg/24 hr (P < 0.01); the urinary IgG excretion increased from 5.8 ± 2.9 to 236.1 ± 132.2 µg/24 hr (P < 0.03); the selectivity index (clearance IgG/clearance albumin) decreased from 0.33 ± 0.12 to 0.12 ± 0.05 (P < 0.004)

A Role for Mac-1 (CDIIb/CD18) in Immune Complex–stimulated Neutrophil Function In Vivo: Mac-1 Deficiency Abrogates Sustained Fcγ Receptor–dependent Neutrophil Adhesion and Complement-dependent Proteinuria in Acute Glomerulonephritis

Mac-1 (αmβ2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcγ receptors to facilitate immune complex (IC)–stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1–FcγR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti–glomerular basement membrane (GBM) nephritis in wild-type and Mac-1–deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1– deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1–null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5–12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1–FcγR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1–FcγR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3–deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1–null mice

Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases

Distribution of GBM heparan sulfate proteoglycan core protein and side chains in human glomerular diseases. Using monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising twelve different glomerulopathies. Four normal kidney specimens served as controls. A homogenous to linear staining of the GBM was observed in the normal kidney with anti-HSPG-core mAb (JM-72) and anti-HS mAb (JM-403). In human glomerulopathies the major alteration was a segmental or total absence of GBM staining with anti-HS mAb JM-403, which is most pronounced in lupus nephritis, membranous glomerulonephritis (GN), minimal change disease and diabetic nephropathy, whereas the HSPG-core staining by mAb JM-72 was unaltered. In addition we found HSPG-core protein in the mesangial matrix when this was increased in membranoproliferative GN Type I, Schonlein-Henoch GN, IgA nephropathy, lupus nephritis, diabetic nephropathy and in focal glomerulosclerosis. Also staining with the anti-HS mAb JM-403 became positive within the mesangium, although to a lesser extent. Furthermore, amyloid deposits in AL and AA amyloidosis clearly stained with anti-HSPG-core mAb JM-72, and to a lesser degree with anti-HS mAb JM-403. Finally, in membranous GN (stage II and III), the GBM staining with anti-HSPG-core mAb JM-72 became irregular or granular, probably related to the formation of spikes. In conclusion, major alterations were observed in the glomerular distribution of HS and HSPG-core in various human glomerulopathies. The mAbs can be useful to further delineate the significance of HSPG and HS for glomerular diseases

Heparin and heparinoids prevent the binding of immune complexes containing nucleosomal antigens to the GBM and delay nephritis in MRL/lpr mice

Heparin and heparinoids prevent the binding of immune complexes containing nucleosomal antigens to the GBM and delay nephritis in MRL/lpr mice. Monoclonal anti-nucleosome antibodies (mAbs) complexed to nucleosomal antigens can bind to DNA and to heparan sulfate (HS) in ELISA and to the GBM in vivo in a rat renal perfusion system, whereas non-complexed mAbs do not bind [1]. In this study, we analyzed whether heparin (HEP) or N-desulfated/acetylated heparins (DSA-HEP), structurally and functionally strongly related to HS, are able to prevent the binding of these complexed mAbs to DNA and to HS in vitro and to rat GBM in vivo. In ELISA the binding of nucleosome complexed anti-nucleosome antibodies to DNA and HS was inhibited dose-dependently by HEP, DSA-HEP and low molecular weight (LMW) DSA-HEP. Intravenous injection of nucleosome/anti-nucleosome immune complexes without heparin/heparinoids in BALB/c mice led to GBM binding, while simultaneous injection of heparin/heparinoids with complexed antibodies or pretreatment with heparin subcutaneously prior to injection of complexes prevented this binding. Subsequently, we tested the preventive effect of HEP, DSA-HEP and LMW-DSA-HEP on progression of renal disease in MRL/lpr mice. Treatment was started at an age of eight weeks in a dose of 50 µg daily. With all three drugs albuminuria was significantly delayed compared to PBS treated controls (cumulative incidence of proteinuria at 20 weeks in controls 60% vs. 13%, 14% and 6% respectively for HEP, DSA-HEP and LMW-DSA-HEP; P < 0.05). At week 21 the glomerulonephritis was histologically less severe in heparin/heparinoid treated animals (P = 0.02). In immunofluorescence the amount of immunoglobulin and C3 deposits in the glomerular capillary wall tended to be less in heparin/heparinoid treated mice compared to PBS treated controls (P = 0.07). Furthermore, at 20 weeks anti-HS levels in plasma of heparin/heparinoid treated mice were significantly lower (P < 0.05). We conclude that interaction of heparin or heparin analogs with HS reactive immune complexes containing nucleosomal antigens prevents the binding of these immune complexes to the GBM and delays nephritis in MRL/lpr mice