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C J Raats

Publications and source records attributed to C J Raats.

9 recordsLinked to original sources
PubMed2000

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies.

The dystrophin-glycoprotein complex, which comprises alpha- and beta-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 alpha- and beta-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.

Agrin↗
PubMed2000

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.

Animals↗
PubMed1999

Reduction in glomerular heparan sulfate correlates with complement deposition and albuminuria in active Heymann nephritis.

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.

Albuminuria↗
PubMed1998

Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.

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 (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. 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.

Agrin↗
PubMed1997

Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.

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.

Animals↗
PubMed1997

Antiproteinuric effect of ciclosporin A in adriamycin nephropathy in rats.

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(alb) OO: 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.

Albuminuria↗
PubMed1995

Mycobacterial 65-kilodalton heat shock protein induces tumor necrosis factor alpha and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages.

The 65-kDa heat shock protein (Hsp65) is supposed to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. These effects are thought to result mainly from an Hsp65-specific T-lymphocyte-mediated immune response that recognizes conserved epitopes. The aim of the present study was to assess whether mycobacterial Hsp65 has a direct effect on resident murine peritoneal macrophages, independent of Hsp65-sensitized T lymphocytes. Exposure of peritoneal macrophages from naive C57BL/6 mice to the mycobacterial Hsp65 in vitro induced an enhanced release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6. These cells also produced large amounts of reactive nitrogen intermediates (RNI) and inhibited the intracellular proliferation of Toxoplasma gondii. Small amounts of gamma interferon acted synergistically with Hsp65. Thus, exposure of murine macrophages to Hsp65 results in activation of these cells. The acquisition of these characteristics by peritoneal macrophages occurred in the absence of sensitized T lymphocytes. Addition of anti-TNF-alpha antiserum resulted in an attenuation of the Hsp65-induced release of RNI and toxoplasmastatic activity, indicating that endogenous TNF-alpha is involved in the Hsp65-induced macrophage activation. The conclusion of this study is that in vitro exposure of peritoneal macrophages to the mycobacterial Hsp65 induces the release of proinflammatory cytokines and RNI and results in inhibition of the intracellular proliferation of T. gondii. These effects on murine macrophages occur independently of Hsp65-specific T lymphocytes. The proinflammatory effect of Hsp65 demonstrated in this study suggests that this heat shock protein may play a role in the initiation of inflammation that adds to a non-species-specific resistance in the early stages of infections.

Animals↗
PubMed1994

Mycobacterial heat-shock protein 65 induces proinflammatory cytokines but does not activate human mononuclear phagocytes.

The 65 kDa heat-shock protein (Hsp65), a well-conserved and immunodominant antigen which elicits a cellular and humoral immune response, may play a role in host defence against invading microorganisms and autoimmune disorders. The aim of the present study was to assess the effects of Hsp65 on the functional activities of human mononuclear phagocytes in the absence of lymphocytes. Incubation with Hsp65 resulted in an enhanced release of TNF-alpha and IL-1 beta by human monocytes and monocyte-derived macrophages (MDM). The amount of cytokines released by these cells in response to Hsp65 was similar to that released in response to IFN-gamma together with LPS. Incubation with ovalbumin did not stimulate the release of these cytokines. In vitro stimulation of monocytes with Hsp65 enhanced the membrane expression of complement receptor III but did not influence either the expression of Fc gamma-receptor I and HLA class-II antigens or the release of reactive oxygen intermediates. Therefore, Hsp65-stimulated monocytes cannot be considered to be activated according to classical criteria. The release of the proinflammatory cytokines TNF-alpha and IL-1 beta by human mononuclear phagocytes in response to Hsp65 indicates that this protein can contribute to both host defence and tissue damage in inflammatory lesions characterized by an abundant expression of Hsp65.

Antigens, Surface↗