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)

Segmental and global subclasses of class IV lupus nephritis have similar renal outcomes.

Item does not contain fulltextWhether renal outcomes differ between the segmental and global subclasses of diffuse proliferative (class IV) lupus nephritis is unknown. In this meta-analysis, we searched the literature in MEDLINE, EMBASE, five registries of clinical trials, and selected cohort studies and randomized, controlled trials that used the 2003 International Society of Nephrology and Renal Pathology Society classification of lupus nephritis in adult patients. Our endpoint was the composite of doubling of serum creatinine concentration or ESRD. In the eight studies included in the final analysis, the incidence of this endpoint varied between 0% and 67%. A funnel plot and Egger's test did not suggest significant heterogeneity. The meta-analysis did not support a significant difference in renal outcome between the segmental (IV-S) and global (IV-G) subclasses (relative risk for class IV-G versus IV-S, 1.08; 95% confidence interval, 0.68-1.70). Meta-regression did not suggest that ethnicity or duration of follow-up influenced the association between histologic class and renal risk. In conclusion, the rate of doubling of serum creatinine concentration or of ESRD did not differ between patients with class IV-S and those with IV-G lupus nephritis.1 januari 201

A synthetic heparanase inhibitor reduces proteinuria in passive heymann nephritis

Contains fulltext : 57531.pdf (publisher's version ) (Open Access)The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by acting to selectively degrade the negatively charged side chains of heparan sulfate proteoglycans (HSPG) within the glomerular basement membrane (GBM). A loss of the negatively charged HSPG may result in alteration of the permselective properties of the GBM, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors. This study examined the effect of PI-88, a sulfated oligosaccharide heparanase inhibitor, on renal function, glomerular ultrastructure, and proteinuria. Continuous PI-88 infusion at 25 mg/kg per d did not adversely affect animal behavior, growth, or GFR. Cortical tubular vacuolation, however, was observed by light microscopy, and GBM thickness was significantly reduced in these animals (P < 0.0002). Tissue distribution studies using [(35)S]-labeled PI-88 revealed high levels of radioactivity in the kidney after a single subcutaneous injection of 25 mg/kg, suggesting protracted accumulation; moreover, active PI-88 was detected in urine. In passive Heymann nephritis, PI-88 delivered as a continuous infusion at 25 mg/kg per d significantly reduced autologous-phase proteinuria, at day 14 (P < 0.009), in the absence of altered sheep antibody deposition, C5b-9 deposition, and circulating rat anti-sheep antibody titers. Glomerular vascular endothelial growth factor and fibroblast growth factor expression was unaffected by PI-88 administration. However, PI-88 administration significantly prevented glomerular HSPG loss as demonstrated by quantitative immunofluorescence studies (P < 0.0001) in the absence of altered agrin distribution. These data therefore confirm the importance of heparanase in the development of proteinuria

Endothelial nitric oxide synthase prevents heparanase induction and the development of proteinuria

Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria

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

Endothelin-1 Induces Proteinuria by Heparanase-Mediated Disruption of the Glomerular Glycocalyx

NephrologyVascular nephrolog

New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis

Item does not contain fulltextBackgroundFocal segmental glomerulosclerosis (FSGS) is a leading cause of steroid-resistant nephrotic syndrome. Hereditary FSGS is frequently caused by mutations in important structural podocyte proteins, including the slit diaphragm-associated transient receptor potential channel C6 (TRPC6).MethodsIn five patients with biopsy-proven autosomal-dominant FSGS from five different Dutch families, all 13 exons of TRPC6 were sequenced. Upon identification of a novel TRPC6 sequence variant, the resultant amino acid change was introduced in the wild-type TRPC6 protein and functionally tested using patch-clamp analyses and cell-surface biotinylation experiments.ResultsNone of the previously described TRPC6 mutations were found in our cohort. In one family, we identified a novel c.524G>A sequence variant resulting in a p.Arg175Gln (R175Q) substitution in the TRPC6 protein. This sequence variant was absent in 449 control subjects and from public SNP databases. The mutation was located in the third ankyrin repeat domain (ANK3) in the cytoplasmic N-tail of TRPC6, important for protein-protein interaction and regulation of ion channel activity. Patch-clamp analyses of the mutant channel indeed showed an increased TRPC6 channel-mediated current. However, cell-surface expression of the mutant channel was not increased.ConclusionsWe identified a novel TRPC6 p.Arg175Gln gain-of-function mutation that shows increased TRPC6-mediated current, which is not due to altered cell-surface expression. This is the first mutation identified in ANK3 of the TRPC6 N-tail and is most likely responsible for the late-onset autosomal dominant FSGS in this family