Marinka A.H. Bakker

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.

6-Mercaptopurine: Cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues.

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.

Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate

SummaryIn the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p<0.01) and effective renal plasma flow (p<0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42±13 vs control rats (C): 0.5±0.2 mg/ 24 h, p<0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20±0.04 vs C: 0.39±0.17, p<0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Giomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152–941) vs C: 262 (244–467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712–2014) vs C: 672 (515–1208) ng/mg glomerulus, p<0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14–1.16) vs C: 0.39 (0.30–0.47), p<0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

Circulating Apoptotic Microparticles in Systemic Lupus Erythematosus Patients Drive the Activation of Dendritic Cell Subsets and Prime Neutrophils for NETosis

Circulating chromatin‐containing apoptotic material and/or neutrophil extracellular traps (NETs) have been proposed to be an important driving force for the antichromatin autoimmune response in patients with systemic lupus erythematosus (SLE). The aim of this study was to determine the exact nature of microparticles in the circulation of SLE patients and to assess the effects of the microparticles on the immune system.

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy

SummaryHeparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- and sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2–62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2–0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites × mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Sequence-, time- and dose-dependent synergism of methotrexate and 6-mercaptopurine in malignant human T-lymphoblasts☆

Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.

Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats

Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GBM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these proteolytic enzymes along the glomerular capillary wall may induce glomerular injury. Here, we investigated the effects of PR3 and elastase on the induction of proteinuria and glomerular injury after renal perfusion of these enzymes in Brown–Norway rats. Perfusion of active elastase induced a dose‐dependent proteinuria 24 h after perfusion, while inactivated elastase did not. Perfusion of comparable amounts of active PR3 did not induce proteinuria. Light and electron microscopy showed no morphological abnormalities in any experimental group. However, immunohistology revealed that proteinuria occurring after perfusion of active elastase was associated with a strong reduction in intraglomerular expression of the heparan sulphate side chain and, to a lesser extent, of the protein core of heparan sulphate proteoglycans (HSPG). In vitro, both elastase and PR3 digested HSPG. However, PR3 bound to a lesser extent to HSPG than elastase. We conclude that elastase, but not PR3, induces proteinuria after in vivo renal perfusion. This differential effect probably relates to different binding to the GBM of those enzymes due to differences in their isoelectric points. Degradation of heparan sulfate proteoglycans, leading to the disappearance of their side chains that contribute to the polyanionic structure of the GBM, appears to be involved in the induction of proteinuria after perfusion of elastase.

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.