Differential binding of chemokines CXCL1, CXCL2 and CCL2 to mouse glomerular endothelial cells reveals specificity for distinct heparan sulfate domains.

INTRODUCTION: Proliferative glomerulonephritis manifests in a range of renal diseases and is characterized by the influx of inflammatory cells into the glomerulus. Heparan sulfate (HS) is an important (co-)receptor for binding of chemokines, cytokines and leukocytes to the endothelial glycocalyx, a thick glycan layer that covers the inside of blood vessels. During glomerulonephritis, HS in the glomerular endothelial glycocalyx plays a central role in chemokine presentation and oligomerization, and in binding of selectins and integrins expressed by leukocytes. We hypothesize that distinct endothelial HS domains determine the binding of different chemokines. In this study we evaluated the interaction of three pro-inflammatory chemokines (CXCL1, CXCL2 and CCL2) with mouse glomerular endothelial cells (mGEnC-1) in ELISA in competition with different HS preparations and anti-HS single chain variable fragment (scFv) antibodies specific for distinct HS domains. RESULTS: HS appeared to be the primary ligand mediating chemokine binding to the glomerular endothelial glycocalyx in vitro. We found differential affinities of CXCL1, CXCL2 and CCL2 for HS in isolated mGEnC-1 glycocalyx, heparan sulfate from bovine kidney or low molecular weight heparin in competition ELISAs using mGEnC-1 as a substrate, indicating that chemokine binding is affected by the domain structure of the different HS preparations. Blocking of specific HS domains with anti-HS scFv antibodies revealed a domain-specific interaction of the tested chemokines to HS on mGEnC-1. Furthermore, chemokines did not compete for the same binding sites on mGEnC-1. CONCLUSION: CXCL1, CXCL2 and CCL2 binding to the glomerular endothelial glycocalyx appears differentially mediated by specific HS domains. Our findings may therefore contribute to the development of HS-based treatments for renal and possibly other inflammatory diseases specifically targeting chemokine-endothelial cell interactions.

Coexistence of systemic lupus erythematosus, tuberous sclerosis and aggressive natural killer-cell leukaemia: coincidence or correlated?

Among patients with systemic lupus erythematosus (SLE) there is an increased risk of haematological malignancies, especially non-Hodgkin lymphoma. However, the association of SLE with aggressive CD3 negative natural killer (NK)-cell leukaemia has not been reported so far. We present a case of a 39-year-old woman with SLE, aggressive NK-cell leukaemia and tuberous sclerosis complex. The prior probability of developing the combination of these three rare diseases by coincidence is extremely low (<10(-13)). Possible underlying immunological, genetic and toxic/environmental pathways are discussed.

Induction therapy with short-term high-dose intravenous cyclophosphamide followed by mycophenolate mofetil in proliferative lupus nephritis.

BACKGROUND: For decades, high-dose intravenous cyclophosphamide (ivCY) given for 24-30 months was regarded as the standard therapy for proliferative lupus nephritis, despite serious side effects. Our aim was to evaluate the effect of induction therapy with short-term high-dose ivCY followed by mycophenolate mofetil (MMF) on disease parameters, mortality and health-related quality of life (HRQoL) in patients with proliferative lupus nephritis. METHODS: Between January 2003 and November 2006, 71 patients with biopsy-proven proliferative lupus nephritis were included in the second Dutch Lupus Nephritis Study. All patients were treated with ivCY (750 mg÷m2, six monthly pulses) plus oral prednisone, followed by MMF (2000 mg÷day) plus oral prednisone for 18 months, and then azathioprine (2 mg÷kg÷day) plus oral prednisone. Study endpoints included the occurrence of renal relapse, end-stage renal disease (ESRD) and mortality. RESULTS: After a median follow-up of 3.8 years (range 0.1-4.5), four (5.6%) of the 71 patients had a renal relapse, one (1.4%) failed treatment, one (1.4%) reached ESRD, and two (2.8%) died. Systemic lupus erythematosus (SLE) Disease Activity Index, serum creatinine, proteinuria and antibodies against anti-dsDNA decreased significantly during treatment and serum levels of complement factor 3 and 4 increased significantly. Furthermore, six of eight domains of the Short Form-36 as well as the number of symptoms and total distress level according to the SLE Symptom Checklist improved significantly over time. CONCLUSIONS: This open-label study shows that induction therapy with short-term (six monthly pulses) high-dose ivCY followed by MMF is effective in preventing renal relapses, ESRD and mortality and improving HRQoL in patients with proliferative lupus nephritis.

Idiopathic giant oesophageal ulcer and leucopoenia after renal transplantation.

A 45-year-old male recipient of a renal allograft was admitted because of a giant oesophageal ulcer coinciding with leucopoenia. An extensive workup revealed no explanation for the ulcer and leucopoenia. Our final diagnosis by exclusion was an idiopathic giant oesophageal ulcer and late-onset neutropenia as consequences of rituximab induction therapy given during the transplant procedure. The patient fully recovered after treatment with prednisone. However, after four months, the ulcer and leucopoenia recurred and again successfully responded to treatment with prednisone.

Dutch guidelines for diagnosis and therapy of proliferative lupus nephritis.

Proliferative lupus nephritis is a strong predictor of morbidity and mortality in patients with systemic lupus erythematosus. Despite improvements in the management of lupus nephritis, a significant number of the patients do not respond to immunosuppressive therapy and progress to end-stage renal failure. In order to optimise the diagnostic strategy and treatment of patients with proliferative lupus nephritis, guidelines are needed. In this review, the Dutch Working Party on Systemic Lupus Erythematosus provides recommendations regarding four important areas in patients with proliferative lupus nephritis: I) indications for a first renal biopsy, II ) definitions of treatment response, III ) selection of treatment options, and IV) indications for a repeat biopsy.

Receptor for advanced glycation end products (RAGE) polymorphisms are associated with systemic lupus erythematosus and disease severity in lupus nephritis.

OBJECTIVE: Interaction of advanced glycation end products (AGEs) with their receptors (RAGE) plays an important role in inflammation in auto-immune diseases. Several functional polymorphisms of RAGE have been described. In this study we analysed the role of RAGE polymorphisms in disease susceptibility for systemic lupus erythematosus (SLE). In addition, we investigated whether these polymorphisms in SLE are associated with serum levels of soluble RAGE (sRAGE), renal involvement (lupus nephritis (LN)) and its outcome. METHODS: For this cross-sectional study DNA samples of 97 SLE patients, 114 LN patients and 429 healthy controls (HC) were genotyped for four RAGE polymorphisms: -429 T/C, -374 T/A, 2184 A/G and Gly82Ser. Differences in genotype frequencies and allele frequencies were tested between patients and HCs. In SLE patients, sRAGE was measured by enzyme-linked immunosorbent assay (ELISA). In addition, association of genotypes with sRAGE and disease severity in LN was analysed. RESULTS: The C allele of -429 T/C, the T allele of -374 T/A and the G allele of 2184 A/G were significantly more prevalent in SLE and LN compared with HC. In LN, the C allele of RAGE -429 T/C, the A allele of -374 T/A and the G allele of RAGE 2184 A/G polymorphism were significantly associated with more proteinuria and worse renal function during the first two years of treatment. No association of genotype with sRAGE was found. CONCLUSION: RAGE polymorphisms are associated with susceptibility to SLE and LN. In addition, some of these polymorphisms are likely to be associated with disease severity and initial response to treatment in LN.

Synchronized turbo apoptosis induced by cold-shock.

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30-90 min at 37°C more than 80-90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.

Both early and late apoptotic blebs are taken up by DC and induce IL-6 production.

During apo blebs, containing nuclear components, are formed at the cells' surfaces. When these blebs separate from the dying cell an apo cell body remains. The contents of apo blebs are modified and can be released, especially in patients with systemic lupus erythematosus (SLE) since impaired clearance of apo material has been observed in this autoimmune condition. Accordingly, autoantibodies present in subjects with SLE bind to apo blebs. Based on AnxA5 binding, and permeability for PI, we show that apo blebs can be categorized as early (AnxA5(+)/PI(- )) or late (AnxA5(+)/PI(+)) apo ones. Both forms of blebs contain apo-induced chromatin modifications and are efficiently phagocytosed by dendritic cell (DC). Uptake by DC of late, but also early apo blebs, stimulate DC to produce IL-6. This bleb-induced effect on DC may be an important step in the initiation of the autoimmune responses in SLE.

The role of reactive oxygen species in apoptosis of the diabetic kidney.

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney.

Pregnancy, chimerism and lupus nephritis: a multi-centre study.

Chimerism occurs twice as often in the kidneys of women with lupus nephritis as in normal kidneys and may be involved in the pathogenesis of systemic lupus erythematosus. Pregnancy is considered the most important source of chimerism, but the exact relationship between pregnancy, the persistence of chimeric cells and the development of systemic lupus erythematosus has not been investigated. Renal biopsies and clinical data from patients in the First Dutch Lupus Nephritis Study were used. Chimeric cells were identified by in-situ hybridization of the Y chromosome. A questionnaire was used to obtain detailed reproductive data including pregnancy history and miscarriages. Chimerism was found in 12 of 26 (46%) renal biopsies. Of the 12 chimeric women, 5 reported a pregnancy; of 14 women who were not chimeric, 8 reported a pregnancy. Chimeric women who had been pregnant reported significantly more pregnancies than non-chimeric women who had been pregnant (P=0.04). The median age of the youngest child was higher in chimeric women (19 years) than in non-chimeric women (6 years). Despite the attention given to pregnancy histories with respect to chimerism, this study shows that in patients with systemic lupus erythematosus, a clear-cut relationship is not apparent. A considerable number of chimeric women did not report a pregnancy: in these women, other sources of chimerism must be considered. Our data support the theory that only certain subsets of chimeric cells persist into the maternal circulation after pregnancy.

Heparan sulfate domains on cultured activated glomerular endothelial cells mediate leukocyte trafficking.

Heparan sulfate (HS) proteoglycans by playing key roles in the leukocyte-endothelial interactions are thought to mediate inflammatory cell influx in proliferative glomerulonephritis. Here, we evaluated the specific features within glomerular endothelial HS that promote leukocyte adhesion. Mouse and human glomerular endothelial cells activated by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta increased expression of inflammatory N- and 6-O-sulfated HS domains. In addition, altered expression of HS-modifying enzymes occurred, a feature also found in mouse kidneys with anti-glomerular basement membrane disease or lupus nephritis. Inhibition of the nuclear factor (NF)-kappaB pathway repressed cytokine-induced alterations in HS and gene expression of modifying enzymes. Firm adhesion of leukocytes to activated mouse glomerular endothelial cells decreased after removal of endothelial HS or addition of sulfated heparinoids. Specific antibodies that block N- and 6-O-sulfated HS domains on activated mouse endothelial cells reduced the number of rolling and firmly adhering leukocytes under dynamic flow conditions, while they increased the average leukocyte-rolling velocity. Our study shows that N- and 6-O-sulfated domains in HS on activated glomerular endothelium are crucial for leukocyte trafficking and are possible therapeutic targets.

Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy.

AIMS/HYPOTHESIS: Recent studies suggest that loss of heparan sulphate in the glomerular basement membrane (GBM) of the kidney with diabetic nephropathy is due to the increased production of heparanase, a heparan sulphate-degrading endoglycosidase. Our present study addresses whether heparan sulphate with different modifications is differentially reduced in the GBM and whether heparanase selectively cleaves heparan sulphate with different domain specificities. METHODS: The heparan sulphate content of renal biopsies (14 diabetic nephropathy, five normal) were analysed by immunofluorescence staining with four anti-heparan sulphate antibodies: JM403, a monoclonal antibody (mAb) recognising N-unsubstituted glucosamine residues; two phage display-derived single chain antibodies HS4C3 and EW3D10, defining sulphated heparan sulphate domains; and anti-K5 antibody, an mAb recognising unmodified heparan sulphate domains. RESULTS: We found that modified heparan sulphate domains (JM403, HS4C3 and EW3D10), but not unmodified domains (anti-K5) and agrin core protein were reduced in the GBM of kidneys from patients with diabetic nephropathy, compared with controls. Glomerular heparanase levels were increased in diabetic nephropathy kidneys and inversely correlated with the amounts of modified heparan sulphate domains. Increased heparanase production and loss of JM403 staining in the GBM correlated with the severity of proteinuria. Loss of modified heparan sulphate in the GBM as a result of degradation by heparanase was confirmed by heparan sulphate staining of heparanase-treated normal kidney biopsy specimens. CONCLUSIONS/INTERPRETATION: Our data suggest that loss of modified heparan sulphate in the GBM is mediated by an increased heparanase presence and may play a role in the pathogenesis of diabetes-induced proteinuria.

Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria.

Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria.

Incomplete nuclear transformation of human spermatozoa in oligo-astheno-teratospermia: characterization by indirect immunofluorescence of chromatin and thiol status.

BACKGROUND Sperm heterogeneity in the human, as observed in oligo-astheno-teratozoospermia (OAT), is associated with hypospermatogenesis. METHODS The chromatin of sperm from OAT and normospermic males was characterized with antibodies specific for nucleosomes, the histone H3.1/H3.2 isoform, histone TH2B, apoptosis-associated H4 acetylation (KM-2) and protamines. Subsequently, sperm samples were stained with the thiol-specific fluorochrome monobromobimane (mBBr) before and after reduction with dithiotreitol (DTT) as most thiol groups reside in the cysteine-rich protamines. We also used fluorescence-activated cell sorter (FACS) for sperm histograms and sorting for high or low free and total thiol levels. These fractions were further analysed for DNA damage with the TdT-UTP nick end-labelling (TUNEL) assay. RESULTS OAT sperm nuclei stained higher for nucleosomes and KM2-epitopes, and lower for TH2B. For free, and total, thiol groups, OAT sperm were characterized by biphasic distributions, reflecting incomplete thiol oxidation as well as overoxidation, and possibly reduced protamine contents. The TUNEL assay on sperm subfractions, for both control and OAT sperm, revealed that a lower level of free thiol groups is associated with a higher TUNEL incidence, and this relationship was also found for total thiol levels. Hence, both overoxidation and a low total number of thiol groups predestine for sperm apoptosis. CONCLUSIONS Chromatin characteristics reflecting an incomplete nucleosome to protamine remodelling were found in subfertile males. Sperm apoptosis is related to both incomplete remodelling and protamine overoxidation.

Syndecan-1 deficiency aggravates anti-glomerular basement membrane nephritis.

During the heterologous phase of experimental anti-glomerular basement membrane (anti-GBM) nephritis, leukocyte influx peaks within hours, whereas albuminuria occurs within 1 day. In the subsequent autologous phase, endogenous anti-GBM IgG develops and albuminuria persists. Heparan sulfate (HS) proteoglycans like syndecan-1 play multiple roles during inflammation and we evaluate its role in experimental anti-GBM disease using syndecan-1 knockout (sdc-1-/-) mice. During the heterologous phase, glomerular leukocyte/macrophage influx was significantly higher in the sdc-1-/- mice and this was associated with higher glomerular endothelial expression of specific HS domains. In the autologous phase, glomerular influx of CD4+/CD8+ T cells was higher in the sdc-1-/- mice and these mice had persistently higher albuminuria and serum creatinine levels than wild-type mice. This resulted in a more sever glomerular injury and increased expression of extracellular matrix proteins. The sdc-1-/- mice developed higher plasma levels and glomerular deposits of total mouse Ig and IgG1 anti-rabbit IgG, whereas the levels of mouse IgG2a anti-rabbit IgG were lower. Furthermore, decreased Th1 and higher Th2 renal cytokine/chemokine expression were found in the sdc-1-/- mice. Our studies show that syndecan-1 deficiency exacerbates anti-GBM nephritis shifting the Th1/Th2 balance towards a Th2 response.

Heparanase in glomerular diseases.

Heparanase is an endo-beta(1-4)-D-glucuronidase that degrades heparan sulfate (HS) polysaccharide side chains. The role of heparanase in metastasis, angiogenesis, and inflammation has been established. Recent data suggest a role for heparanase in several proteinuric diseases and an increased glomerular heparanase expression is associated with loss of HS in the glomerular basement membrane (GBM). Furthermore, an increase in heparanase activity was detected in urine from proteinuric patients. Mice with transgenic heparanase overexpression developed mild proteinuria. Glomerular heparanase activity is proposed to lead to loss of HS in the GBM and proteinuria. Because the primary role of GBM HS for charge-selective permeability has been questioned recently, heparanase may induce or enhance proteinuria by (i) changes in the glomerular cell-GBM interactions, due to loss of HS; (ii) release of HS-bound factors and HS fragments in glomeruli; or (iii) intracellular signaling by binding of heparanase to glomerular cells. Which of these mechanisms is prevailing requires further research. The precise mechanisms leading to increased heparanase expression in the different glomerular cell types remain elusive, but may involve hyperglycemia, angiotensin II, aldosterone, and reactive oxygen species. This review focuses on the role of heparanase in HS degradation in proteinuric diseases and the possibility/feasibility of heparanase inhibitors, such as heparin(oids), as treatment options.

Glomerular binding of anti-dsDNA autoantibodies: the dispute resolved?

The binding of anti-double-stranded DNA (anti-dsDNA) autoantibodies to the glomerular basement membrane (GBM) in lupus nephritis can be explained by two mechanisms: (1) direct crossreactive binding to intrinsic glomerular antigens; (2) nucleosome-mediated binding to heparan sulfate in the GBM. Kalaaji et al. demonstrated using novel techniques that glomerular in vivo-bound antoantibodies bind to nucleosomes/dsDNA derived from apoptotic cells and not to intrinsic glomerular structures.

Increased expression of heparanase in overt diabetic nephropathy.

In overt diabetic nephropathy (DNP), an increase in the permeability of the glomerular basement membrane (GBM) has been associated with a loss of negatively charged heparan sulfates (HS) in the GBM. Heparanase (HPSE), an endo-beta(1-4)-D-glucuronidase, can cleave HS and could be a potential candidate for the degradation of glomerular HS, leading to the development of proteinuria. We analyzed whether changes in HS expression are associated with HPSE expression in overt DNP. Immunofluorescence staining was performed to analyze HS, HPSE, and agrin core protein expression in kidney biopsies from patients with overt DNP and from rats and mice with streptozotocin (STZ)-induced diabetes. We also investigated the effect of transgenic HPSE overexpression in mice on glomerular HS and agrin expression. We demonstrate that the loss of GBM HS (-50%) and tubular HS (-60%) is associated with a four-fold increased HPSE expression in overt DNP. In addition, glomerular HPSE expression is upregulated in rats (messenger RNA (mRNA) 2.5-fold, protein three-fold) and mice (mRNA seven-fold, protein 1.5-fold) with STZ-induced diabetes. Furthermore, transgenic HPSE overexpression results in disappearance of HS, whereas expression of the core protein agrin remains unaltered. Our observations suggest that HPSE is involved in the pathogenesis of proteinuria in overt DNP by degradation of HS.