Reduced CXCL1 production by endogenous IL-37 expressing dendritic cells does not affect T cell activation.

The dendritic cell (DC)-derived cytokine profile contributes to naive T cell differentiation, thereby directing the immune response. IL-37 is a cytokine with anti-inflammatory characteristics that has been demonstrated to induce tolerogenic properties in DC. In this study we aimed to evaluate the influence of IL-37 on DC-T cell interaction, with a special focus on the role of the chemokine CXCL1. DC were cultured from bone marrow of human IL-37 transgenic (hIL-37Tg) or WT mice. The phenotype of unstimulated and LPS-stimulated DC was analyzed (co-stimulatory molecules and MHCII by flow cytometry, cytokine profile by RT-PCR and ELISA), and T cell stimulatory capacity was assessed in mixed lymphocyte reaction. The role of CXCL1 in T cell activation was analyzed in T cell stimulation assays with anti-CD3 or allogeneic DC. The expression of the co-stimulatory molecules CD40, CD80 and CD86, and of MHCII in LPS-stimulated DC was not affected by endogenous expression of IL-37, whereas LPS-stimulated hIL-37Tg DC produced less CXCL1 compared to LPS-stimulated WT DC. T cell stimulatory capacity of LPS-matured hIL-37Tg DC was comparable to that of WT DC. Recombinant mouse CXCL1 did not increase T cell proliferation either alone or in combination with anti-CD3 or allogeneic DC, nor did CXCL1 affect the T cell production of interferon-γ and IL-17. Endogenous IL-37 expression does not affect mouse DC phenotype or subsequent T cell stimulatory capacity, despite a reduced CXCL1 production. In addition, we did not observe an effect of CXCL1 in T cell proliferation or differentiation.

Role of syndecan-1 in the interaction between dendritic cells and T cells.

Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that can bind cytokines and chemokines via its heparan sulfate side chains, and has immunomodulatory properties in experimental models. Sdc-1 expression has been reported on dendritic cells (DC) and T cells. The potential role of Sdc-1 in DC-T cell interaction has not been investigated yet. We postulate that Sdc-1 is involved in DC-T cell interaction and may influence graft survival in an allogeneic transplant model. Sdc-1 expression on bone marrow-derived DC and T cells was analyzed by flow cytometry. Unstimulated and LPS stimulated Sdc-1 deficient DC were evaluated in vitro for phenotype and stimulatory capacity in mixed lymphocyte reaction. Sdc-1 deficient T cells were evaluated for proliferative capacity and differentiation in a mixed lymphocyte reaction and a proliferation assay. Allograft survival was evaluated in a fully MHC mismatched heterotopic heart transplant model, with either Sdc-1 deficient donors or recipients. Sdc-1 was expressed on the cell surface of unstimulated and LPS matured DC. Sdc-1 deficiency had no effect on expression of co-stimulatory molecules, cytokine production or T cell stimulatory capacity as compared to WT DC. Sdc-1 expression was not detectable on WT T cells, although intracellular Sdc-1 expression could be demonstrated after ConA activation. Sdc-1 deficient T cells showed reduced proliferation upon DC or ConA stimulation and reduced IL-17 production upon ConA stimulation, compared to WT T cells. Sdc-1 deficiency of either allograft or recipient did not prolong allograft survival. In conclusion, Sdc-1 is expressed on the cell surface of DC, where its absence does not affect DC phenotype or T cell stimulatory capacity. Sdc-1 is intracellularly expressed in ConA activated T cells. Sdc-1 deficiency in T cells results in a reduced proliferative response in vitro, as induced by DC and ConA. Sdc-1 deficiency in donor or recipient does not affect allograft survival.

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.

Dynamic Expression of Genes Involved in Proteoglycan/Glycosaminoglycan Metabolism during Skin Development.

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

Urinary MicroRNA as Biomarker in Renal Transplantation.

Urine represents a noninvasive source in which proteins and nucleic acids can be assessed. Such analytes may function as biomarkers to monitor kidney graft pathology at every desired frequency, thereby providing a time window to prevent graft damage by therapeutic intervention. Recently, several proteins have been measured in urine as markers of graft injury. However, the specificity is limited, and measuring urinary proteins generally lacks the potential to predict early kidney graft damage. Currently, urinary mRNA and microRNA are being investigated to evaluate the prognostic value of changes in gene expression during the initial stages of graft damage. At such time point, a change in treatment regimen and dosage is expected to have maximum potency to minimize future decline in graft function. Both mRNA and microRNAs have shown promising results in both detection and prediction of graft injury. An advantage of microRNAs compared to mRNA molecules is their stability, a characteristic that is beneficial when working with urine samples. In this review, we provide the current state of urinary biomarkers in renal transplantation, with a focus on urinary microRNA. In addition, we discuss the methods used to study urinary microRNA expression.

Allostimulatory Effects of Dendritic Cells with Characteristic Features of a Regulatory Phenotype.

INTRODUCTION: Tolerogenic dendritic cells (DCs) have the potential to prolong graft survival after transplantation. Tolerogenic DCs are in general characterized by a low expression of co-stimulatory molecule and a high IL-10:IL-12 production ratio. Based on promising results with earlier used alternatively activated DCs, we aimed to generate in culture potentially tolerogenic DC by simultaneously blocking GSK3 by lithium chloride (LiCl) and stimulating TLR2 by PAM3CysSerLys4. MATERIALS AND METHODS: Bone marrow-derived LiClPAM3 DCs were generated by the addition of LiCl 24 hours before harvesting, and one hour later PAM3CysSerLys4. The phenotype of the DCs was assessed by determining the expression of co-stimulatory molecules in flow cytometry and cytokine production in ELISA, whereas their functional properties were tested in a mixed lymphocyte reaction. A fully MHC mismatched heterotopic heart transplant preceded by infusion of donor-derived LiClPAM3 DC was performed to assess the tolerogenic potential of LiClPAM3 DCs in vivo. RESULTS: LiClPAM3 DCs displayed a tolerogenic phenotype accompanied with a low expression of co-stimulatory molecules and a high IL-10:IL-12 production ratio. However, in mixed lymphocyte reaction, LiClPAM3 DCs appeared superior in T cell stimulation, and induced Th1 and Th17 differentiation. Moreover, mice pretreated with LiClPAM3 DC displayed a reduced graft survival. Analysis of LiClPAM3 DC culture supernatant revealed high levels of CXCL-1, which was also found in supernatants of co-cultures of LiClPAM3 DC and T cells. Nevertheless, we could not show a role for CXCL-1 in T cell proliferation or activation in vitro. DISCUSSION: LiClPAM3 DCs display in vitro a tolerogenic phenotype with a high IL-10:IL-12 ratio, but appeared to be highly immunogenic, since allograft rejection was accelerated. As yet unidentified LiClPAM3 DC-derived factors, may explain the immunogenic character of LiClPAM3 DCs in vivo.

Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus.

In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.

Chromatin remodelling initiation in spermatids: differences among human males.

During the last phase of spermatogenesis, called spermiogenesis, the nucleosome-based chromatin structure is replaced by a protamine-based DNA packaging. Not much is known about the chromatin remodelling involved in humans and animals. Here, we have investigated initiation of chromatin remodelling over seven probands of which five were diagnosed with non-obstructive azoospermia (NOA) and two with obstructive azoospermia (OA) (failed vaso-vasostomy patients with proven fertility prior to vasectomy, Johnsen scores ≥9). Chromatin remodelling was studied evaluating the presence of nucleosomes, histone H3, pre-protamine 2 and protamine 1. This approach was feasible since the local initiation of nucleosome eviction in the sub acrosomal domain, which was visible in alkaline nuclear spread preparations. The patterns of nucleosome and H3 loss were largely congruent. Nucleus wide incorporation of protamine 1 could already be observed at the late round spermatid stage. Both for nucleosome loss and for protamine 1 incorporation, there was distinct variation within and between probands. This did not relate to the efficiency of sperm production per meiocyte. Pre-protamine 2 was always confined to the subacrosomal domain, confirming the role of this area in chromatin remodelling.

Improvement in chromatin maturity of human spermatozoa selected through density gradient centrifugation.

A two-step gradient density centrifugation system has been set up to isolate two contrasting sperm populations of normozoospermic and oligoasthenoteratozoospermic (OAT) men. High- and low-density fractions were characterized by total and free thiol fluorescence as determined by monobromobimane-flow cytometry and by protamine/DNA ratios after protamine extraction and polyacrylamide acid-urea gel electrophoresis. Further chromatin characterization was performed through immunofluorescence (IF) with specific antibodies to nucleosomes, histone subtypes (H3.1/H3.2 and TH2B), histone modifications (KM-2 and H4K8ac) and precursor protamine 2. The native sperm samples from normozoospermic and OAT patients showed a biphasic distribution of total thiol levels, which changed in the sperm fractions obtained using the density isolation protocol presented here. Moreover, significant differences were detected in the protamine content in the different fractions of OAT and fertile donor samples. In addition, in the high-density fractions from OAT and normozoospermics, higher IF levels for H4K8ac and TH2B were seen. These results would be consistent with the intended beneficial effect on chromatin maturity of the density selection techniques currently being used in assisted fertilization procedures. However, most nucleosome and related proteins/modifications differ between OAT and normozoospermic men, even after gradient centrifugation, providing evidence for incomplete nuclear maturity in OAT patients.

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.

Apoptosis in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototype inflammatory autoimmune disease resulting from autoimmune responses against nuclear autoantigens. During apoptosis many lupus autoantigens congregate inside the cells and are susceptible to modifications. Modified nuclear constituents are considered foreign and dangerous. Therefore, apoptotic cells have to has to be efficiently removed to avoid the accumulation of apoptotic debris and the subsequently development of autoimmune responses. Hence, apoptosis and clearance of apoptotic cells/material are considered key processes in the aetiology of SLE. Clearance deficiencies may account for the development of autoimmunity by inducing a loss of tolerance in lymphoid tissues. Furthermore, phagocytosis of apoptotic cells may lead to a pro-inflammatory response in the presence of autoantibodies. This may sustain inflammatory conditions and the pathology found in overt lupus.

Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K.

Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes. Here, we focused on the apoptotic processing of one of the essential components of the mRNA splicing machinery, the U1-70K snRNP protein. We found that at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3, the basal phosphorylation of the Ser140 residue located within the RNA recognition motif, increases very significantly. A caspase-dependent, PP1-mediated dephosphorylation of other serine residues takes place in a subset of U1-70K proteins. The U1-70K protein phosphorylated at Ser140 is clustered in heterogeneous ectopic RNP-derived structures, which are finally extruded in apoptotic bodies. The elaborate processing of the spliceosomal U1-70K protein we identified might play an important role in the regulated breakdown of the mRNA splicing machinery during early apoptosis. In addition, these specific changes in the phosphorylation/dephosphorylation balance and the subcellular localization of the U1-70K protein might explain why the region encompassing the Ser140 residue becomes a central autoantigen during the autoimmune disease systemic lupus erythematosus.

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.