BACKGROUND:heparanase Inhibition of the renin-angiotensin-aldosterone system (RAAS) provides renoprotection in adriamycin nephropathy (AN), along with a decrease in overexpression of glomerular or heparanase. Angiotensin II (AngII) and reactive oxygen species (ROS) are known to regulate heparanase expression in vivo. However, it is Treatment unknown whether this is also the case for aldosterone. Therefore, we further assessed the role of aldosterone, AngII and ROS Therefore, in the regulation of glomerular heparanase expression. METHODS: Six weeks after the induction of AN, rats were treated with vehicle resulted (n = 8), lisinopril (75 mg/L, n = 10), spironolactone (3.3 mg/day, n = 12) or the combination of lisinopril = and spironolactone (n = 14) for 12 weeks. Age-matched healthy rats served as controls (n = 6). After 18 weeks,expression renal heparanase and heparan sulfate (HS) expression were examined by immunofluorescence staining. In addition, the effect of aldosterone, AngII and induction ROS on heparanase expression in cultured podocytes was determined. RESULTS: Treatment with lisinopril, spironolactone or their combination significantly blunted the their increased glomerular heparanase expression and restored the decreased HS expression in the GBM. Addition of aldosterone to cultured podocytes resulted of in a significantly increased heparanase mRNA and protein expression, which could be inhibited by spironolactone. Heparanase mRNA and protein expression protein in podocytes were also significantly increased after stimulation with AngII or ROS. CONCLUSIONS: Our in vivo and in vitro results protein show that not only AngII and ROS, but also aldosterone is involved in the regulation of glomerular heparanase expression.

Rationale:of Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by severe, progressive fibrosis. Roles for inflammation and oxidative stress fluid have recently been demonstrated, but despite advances in understanding the pathogenesis, there are still no effective therapies for IPF. This Increases study investigates how extracellular superoxide dismutase (EC-SOD), a syndecan-binding antioxidant enzyme, inhibits inflammation and lung fibrosis. Objective: We hypothesize that inhibits EC-SOD protects the lung from oxidant damage by preventing syndecan-1 shedding. Methods: Wild-type or EC-SOD null mice were exposed to and an intratracheal instillation of asbestos or bleomycin. Western blot was used to detect syndecans in the bronchoalveolar lavage fluid and were lung. Human lung samples (normal and IPF) were also analyzed. Immunohistochemistry for syn-1 and EC-SOD was performed on human and in mouse lungs. In vitro, alveolar epithelial cells were exposed to oxidative stress and EC-SOD. Cell supernatants were analyzed for shed EC-SOD syndecan-1 by western blot. Syn-1 ectodomain was assessed in wound healing and neutrophil chemotaxis. Results: Increases in human syndecan-1 are in detected in lung homogenates and lavage fluid of IPF lungs. Syndecan-1 is also significantly elevated in the lavage fluid of by EC-SOD null mice after asbestos and bleomycin exposure. On IHC, syndecan-1 staining increases within fibrotic areas of human and mouse oxidant-induced lungs. In vitro, EC-SOD inhibits oxidant-induced loss of syndecan-1 from A549 cells. Syndecan-1 ectodomains induce neutrophil chemotaxis, inhibit alveolar epithelial oxidant-induced wound healing, and promote fibrogenesis. Conclusion: Oxidative shedding of syndecan-1 is an underlying cause of neutrophil chemotaxis and aberrant wound performed healing that may contribute to pulmonary fibrosis.

The growth extracellular compartments of most biological tissues are significantly less well protected against oxidative damage than intracellular sites and there is consequences considerable evidence for such compartments being subject to a greater oxidative stress and an altered redox balance. However, with some recognized notable exceptions (e.g., plasma and lung lining fluid) oxidative damage within these compartments has been relatively neglected and is poorly redox understood. In particular information on the nature and consequences of damage to extracellular matrix is lacking despite the growing realization such that changes in matrix structure can play a key role in the regulation of cellular adhesion, proliferation, migration, and cell is signaling. Furthermore, the extracellular matrix is widely recognized as being a key site of cytokine and growth factor binding, and matrix modification of matrix structure might be expected to alter such behavior. In this paper we review the potential sources of these oxidative matrix damage, the changes that occur in matrix structure, and how this may affect cellular behavior. The role of site such damage in the development and progression of inflammatory diseases is discussed.

BACKGROUND:- .381, Oxidative stress plays a role in the mechanism of chronic kidney disease (CKD), and antioxidant regimes are regarded as promising weeks, treatment modalities. We compared the effects of cilazapril, simvastatin, and hyperbaric oxygen (HBO) treatment on proteinuria and on oxidative stress SOD in adriamycine (ADR)-induced proteinuria. METHODS: Seventy male Sprague-Dawley rats were housed, and 60 were injected with ADR to induce nephrosis.nephrosis. After the stabilization of proteinuria, rats were treated for 6 weeks with simvastatin (n = 10, 4 mg/kg/day), cilazapril (n simvastatin = 10, 10 mg/kg/day), HBO (n = 10, 2.8 athmosphere absolute, 90 min/daily), HBO + cilazapril (n = 10), HBO (GPx) + simvastatin (n = 10), and vehicle (n = 10). After euthanization at 12 weeks, protein carbonyl (PCO), superoxide dismutase p (SOD), and glutathion peroxidase (GPx) levels were analyzed from tissues. The histological alterations in the kidneys were determined by semiquantitative 10, scoring. RESULTS: Protein carbonyl (PCO) levels were higher (p < .001), and the GPx and SOD levels were lower (p .001 < .001 for all) in the nephrotic rats. Proteinuria was correlated to PCO (r = .483), GPx (r =- .686),in or SOD (r =- .620)(p < .001 for all). Superoxide dismutase (SOD)(beta =- .381, p = .02) and Both GPx (beta =- .509, p < .001) were independently related to proteinuria levels. Both cilazapril and simvastatin significantly improved GPx,Both SOD, PCO, and proteinuria. When HBO was combined with either drug, the above markers further improved (p < .001). Both kidneys regimens caused distinct histological features, while the combination of HBO made much significant histological improvement. CONCLUSION: Both cilazapril and simvastatin induce regimens improve oxidative stress and proteinuria, while the effects significantly increase with the combination of HBO treatment. HBO seems to GPx be a candidate antioxidant strategy in glomerular diseases.

Heparan to sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression in of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different sulfate heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by different the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified membrane and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about sulfated fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate of domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in were the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective chains filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases whereas contributes to proteinuria.Kidney International advance online publication, 28 November 2007; doi:10.1038/sj.ki.5002706.

Following can removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast.This Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise an long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important by role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of no a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This upregulation radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane the type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also before increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor antibody. and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release role of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line absence MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is absence an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.British expression Journal of Cancer advance online publication, 6 November 2007; doi:10.1038/sj.bjc.6604072 www.bjcancer.com.

Asbestosis scavenging is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis.in Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme chamber, that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such has as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in In the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with investigated heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced to neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis as in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice the at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate heparin that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative asbestos fragmentation.

Recent the advances in the field of glycobiology have exposed a multitude of biological processes that are controlled or influenced by proteoglycans,fibrosis in both physiological and pathological conditions ranging from early embryonic development, inflammation, and fibrosis to tumor invasion and metastasis. The roles first part of this article reviews the biosynthesis of proteoglycans and their multifunctional roles in health and disease; the second influenced part of this review focuses on their putative roles in peritoneal homeostasis and peritoneal inflammation and fibrosis in the context focuses of chronic peritoneal dialysis and peritonitis.

Heparanase of 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 in has been established. Recent data suggest a role for heparanase in several proteinuric diseases and an increased glomerular heparanase expression (iii) is associated with loss of HS in the glomerular basement membrane (GBM). Furthermore, an increase in heparanase activity was detected of in urine from proteinuric patients. Mice with transgenic heparanase overexpression developed mild proteinuria. Glomerular heparanase activity is proposed to lead in to loss of HS in the GBM and proteinuria. Because the primary role of GBM HS for charge-selective permeability has role been questioned recently, heparanase may induce or enhance proteinuria by (i) changes in the glomerular cell-GBM interactions, due to loss research. of HS;(ii) release of HS-bound factors and HS fragments in glomeruli; or (iii) intracellular signaling by binding of heparanase patients. to glomerular cells. Which of these mechanisms is prevailing requires further research. The precise mechanisms leading to increased heparanase expression of in the different glomerular cell types remain elusive, but may involve hyperglycemia, angiotensin II, aldosterone, and reactive oxygen species. This HS; review focuses on the role of heparanase in HS degradation in proteinuric diseases and the possibility/feasibility of heparanase inhibitors, such oxygen as heparin(oids), as treatment options.Kidney International advance online publication, 23 May 2007; doi:10.1038/sj.ki.5002337.

Despite (GBM) recent discoveries of molecules in podocytes, the mechanisms behind most conditions of proteinuria are still poorly understood. In order to the understand more about this delicate barrier, we studied the functional and morphological effects of mild (15 min) renal ischemia reperfusion did injury, IRI. Renal function was studied in rats in vivo, followed by a more detailed analysis of the glomerular barrier a in cooled (8 degrees C) isolated perfused kidneys (cIPK). Renal blood flow was quickly restored, whereas GFR remained halved 30 renal min after IRI. Tubular cell activity was intact as judged from the unaffected Cr-EDTA U/P concentration ratio. In vivo, the In fractional clearance (theta) for albumin increased 16 times. In rats subjected to cIPK starting 30 min after in vivo IRI,the thetaalbumin was 15 times and thetaFicoll_36A 1.8 times higher than in control cIPKs. According to the heterogeneous charged fiber model,GFR IRI reduced the fiber charge density to 38% of control (P< .01, N=7). Morphometric analysis with electron microscopy did not reveal the any changes of the podocytes or the glomerular basement membrane (GBM) after IRI, suggesting more subtle changes of the GBM fiber and/or the endothelial glycocalyx. We conclude that mild renal IRI induce formation of reactive oxygen species, massive proteinuria, and loss morphology. of charged fibers with no apparent change in morphology. These novel findings stress the importance of other components of the morphology. barrier such as proteoglycans produced by the glomerular cells and provide a tentative explanation for the mechanisms behind proteinuria in in e.g. glomerulonephritis. Key words: Endothelium, Glycocalyx, Basement membrane, Kidney, oxidative stress.

The with dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal agrin lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement Immunoelectron membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and agrin, cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and passive beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found was in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was the localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining membrane. for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused for podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein and complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the and glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton the to the glomerular basement membrane.

In = a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane,attached was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria.was Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of 2 complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274+/-68 mg/18 h at week due 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs normal. that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive mAb decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide at side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P =staining .016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (r(s)=- .82 and r(s)of =- .75, respectively, both mAb P < .0001). This decrease in heparan sulfate staining was due to a progressive reduction week of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the week disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis seen is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.from

We and determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat antibodies glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan cells sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa that core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate that proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der ovary Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J.of H., and van den Heuvel, L. P. W. J.(1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our van antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or not the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum the clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct tubular encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly basement stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and agrin the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution we of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement epitopes membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we whereas hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a J., truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative components splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Ciclosporin animals A (CsA) can reduce proteinuria in various forms of human and experimental glomerulopathies. This antiproteinuric effect can be the result almost of a decrease of immunological damage, a decrease in the glomerular filtration rate (GFR), or a change in the permselective the properties of the glomerular capillary wall. In this study we investigated the effect of CsA on Adriamycin-induced nephropathy in rats.intravenous A single intravenous injection of Adriamycin (5 mg/kg body weight) induced a severe nephrotic syndrome with a massive albuminuria (+/-in 400 mg/24 h from 3 weeks onwards) and a hypoalbuminemia (+/- 7 mg/ml after 5 weeks). The IgG/albumin selectivity index h; was .16 +/- .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 = .002) and induced an increase in 7 the serum albumin level (from 7.1 +/- 4.1 to 12.8 +/- 3.2 mg/ml; p = .002) in contrast to the still vehicle olive oil (OO). CsA also decreased the GFR by 40%(from .74 +/- .11 to .41 +/- .11 mg/ml/100 .74 g body weight; p = .002). Albuminuria corrected for the GFR (fractional excretion of albumin, FE(alb)) was still significantly lower tubular in CsA-treated than in OO-treated animals (FE(alb) CsA: 1.35 +/- .88, FE(alb) OO: 3.17 +/- 2.29%; p = .0005). This tubular suggests that other factors are also involved in the reduction of albuminuria. To exclude that CsA has an effect on albumin the tubular reabsorption of albumin, we evaluated the blockade of the tubular reabsorption by lysine and found no difference in single 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 tubular for albumin.

Heparan histones sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and glomerular on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased This permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the developed importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin)prevented core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human observation, proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of HS the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases.lupus To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of cationic proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an different inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms in have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking in was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in changes HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be With prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of Second, purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein renal near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular proteinuric cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a glomerular reduction in the degree of HS sulfation.

BACKGROUND:decrease To investigate whether albuminuria in streptozotocin-induced diabetes mellitus in the rat is a symptom of a more generalized vessel wall liver, permeability, and whether changes in vessel wall heparan sulfate (HS) are associated with alterations in vessel wall permeability. METHODS: The and transcapillary escape rate of albumin (TERalb) was calculated from the disappearance rate from the circulation of i.v. injected radiolabeled albumin,clearance together with the regional clearance of albumin (RCalb) in several tissues using a double isotope technique. These measurements were performed .004), in seven rats one year after diabetes induction and in seven sex- and age-matched control rats. To evaluate the association antibody between vessel wall HS and the transcapillary passage of albumin, we determined the content of basement membrane HS in tissue and homogenates of heart, liver, kidneys, and lungs with a sensitive inhibition-ELISA using a monoclonal antibody (JM-403), which specifically recognizes basement control membrane HS. RESULTS: Diabetic rats developed albuminuria (31.7 +/- 10.8 mg/24 h) in contrast to control animals (2.2 +/- 1.5 significantly mg/24 h; P = .0006). TERalb was increased from 13.3 +/- 1.7 in control rats to 15.6 +/- 2.6%/h in +/- diabetic rats, P = .02. RCalb was significantly increased in heart, liver, skeletal muscle and aorta, unchanged in kidneys and changes skin, and significantly decreased in lung tissue. We found a decrease in HS content in heart tissue of diabetic rats,in and a correlation between HS content and RCalb (r =- .72, P = .004), in contrast with an increase in developed lung HS content that correlated with a decrease in RCalb (r =- .64, P = .014). No changes in HS regional content were found in kidney and liver tissue. CONCLUSIONS: These data indicate that in one-year diabetic rats albuminuria coincides with heart an increased TERalb and RCalb in most, but not all tissues, and that alterations in basement membrane HS content correlate decrease with changes in the RCalb, which suggests a functional relationship.

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

Heparan site sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin sites and in age- adn sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was the performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes,was showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of basement anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular rara basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly anionic smaller than those in the lamina rara externa of the glomerular basement membrane (p< .001 and p< .01 for diabetic and control anionic rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2-62. ) mg/24 h after 8 months in contrast to the control sulphate animals ( .8 ( .2- .9) mg/24 h, p< .002). At the same time, the number of heparan sulphate anionic sites and the total after anionic site surface (number of anionic sites x mean anionic site surface) in the lamina rara externa of the glomerular part basement membrane was reduced by 19%(p< .021) and by 26%(p< .02), respectively. Number and total anionic site surface in the of remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that lamina in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina staining rara externa of the glomerular basement membrane.

Immunohistochemical from application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate stubs showed the presence of heparan sulfate increasing proteoglycan in all basement membranes of the rat kidney. However, a monoclonal antibody (JM-403) against native heparan sulfate (van den by Born, J., van den Heuvel, L. P. W. J., Bakker, M. A. H., Veerkamp, J. H., Assmann, K. J. M.,and and Berden, J. H. M.(1992) Kidney Int. 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence the of heparan sulfate chains lacking the specific JM-403 epitope. Heparan sulfate preparations from various sources differed markedly with regard to epitope JM-403 binding, as demonstrated by liquid phase inhibition in enzyme-linked immunosorbent assay, the interaction decreasing with increasing sulfate contents of reduction the polysaccharide. Mapping of the JM-403 epitope indicated that it was dominated by one or more N-unsubstituted glucosamine unit(s), since epitope. treatments that destroyed or altered the structure of such units in heparan sulfate preparations (cleavage at N-unsubstituted glucosamine units with glucosamine HNO2 at pH 3.9 and N-acetylation with acetic anhydride, respectively), abolished antibody binding. Conversely, immunoreactivity could be induced in a with (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl-1,4) polysaccharide by the generation of N-unsubstituted glucosamine N-unsubstituted glucosamine in a JM-403-binding heparan sulfate (preparation HS-II from human aorta)units was demonstrated by an approximately 3-fold reduction in molecular size following HNO2 (pH 3.9) treatment. Further characterization of the epitope are recognized by JM-403, based on enzyme-linked immunosorbent assay inhibition tests with chemically/enzymatically modified polysaccharides, indicated that one or more N-sulfated N-unsubstituted glucosamine units are invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity. It is concluded that M., the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the by heparan sulfate chain composed of mixed N-sulfated and N-acetylated disaccharide units.

Using AL monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate membranous proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising positive twelve different glomerulopathies. Four normal kidney specimens served as controls. A homogenous to linear staining of the GBM was observed homogenous 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 JM-403. segmental or total absence of GBM staining with anti-HS mAb JM-403, which is most pronounced in lupus nephritis, membranous glomerulonephritis whereas (GN), minimal change disease and diabetic nephropathy, whereas the HSPG-core staining by mAb JM-72 was unaltered. In addition we found mAb HSPG-core protein in the mesangial matrix when this was increased in membranoproliferative GN Type I, Schönlein-Henoch GN, IgA nephropathy, lupus the nephritis, diabetic nephropathy and in focal glomerulosclerosis. Also staining with the anti-HS mAb JM-403 became positive within the mesangium, although a to a lesser extent. Furthermore, amyloid deposits in AL and AA amyloidosis clearly stained with anti-HSPG-core mAb JM-72, and to diabetic a lesser degree with anti-HS mAb JM-403. Finally, in membranous GN (stage II and III), the GBM staining with anti-HSPG-core probably mAb JM-72 became irregular or granular, probably related to the formation of spikes. In conclusion, major alterations were observed in probably the glomerular distribution of HS and HSPG-core in various human glomerulopathies. The mAbs can be useful to further delineate the In significance of HSPG and HS for glomerular diseases.

OBJECTIVES:expression To investigate the expression of glomerular heparin sulfate (HS) in paediatric patients with minimal change nephritic syndrome (MCNS). METHODS: The species kidyney tissues were collected by biopsy from 13 paediatric patients with MCNS, while 5 normal renal biopsy samples were used Children as control. HS in glomeruli was analysed by indirect immunofluorescence staining using four different monoclonal antibodies, Hepss1, 3G10, JM403 and normal 10E4, which all recognize distinct HS species and each interacts with a specific HS domain. The concentrations of urine heparan urinary sulfate also were measured by enzyme-linked immunosorbent assay (Elisa). RESULTS: Expression of HS fine domains was aberrant in paediatric patients specific compared with control subjects. Children with MCNS in replase showed a decreased glomerular expression of 10E4, JM403 and Hepss1 (P Hepss1 < .05). The level of urinary HS was significantly increased in peadiatric patients with MCNS when compared with that in staining control subjects (P < .01). CONCLUSION: These results suggest that loss of heparan sulphate in renal tissue may play a replase role in the pathogenesis of MCNS proteinuria.

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

Heparan to sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression in of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different sulfate heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by different the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified membrane and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about sulfated fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate of domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in were the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective chains filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases whereas contributes to proteinuria.Kidney International advance online publication, 28 November 2007; doi:10.1038/sj.ki.5002706.

Heparan this sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of "open" the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example,neuraminic in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state.the We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall confirmed in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid-but not HS-from the GBM, and this potential led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in removal response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage that of proteins through the glomerular capillary wall.

BACKGROUND:MCNS, Minimal change nephrotic syndrome (MCNS) is the most frequent form of nephrotic syndrome in childhood. In the glomerular basement membrane with (GBM) of adult patients with MCNS, a reduced expression of a specific heparan sulphate (HS) domain has been reported. In addition, children with MCNS, urinary activity of the HS-degrading enzyme heparanase was increased. It is, therefore, possible that a decreased GBM of HS expression is associated with the pathogenesis of proteinuria in patients with MCNS. METHODS: In this study, HS in glomeruli adult of five adult and six paediatric patients with MCNS were analysed by immunofluorescence staining using four different antibodies, each defining four a specific sulphated HS domain. The pediatric patients were subdivided into three groups depending on the presence or absence of Expression podocyte foot process effacement, the level of proteinuria and prednisone administration at the time of the biopsy. In addition, kidneys pathogenesis of rats with adriamycin nephropathy (ADRN), a model for MCNS, were included in the study. RESULTS: Expression of sulphated HS adriamycin domains was not aberrant in adult or paediatric patients compared with control subjects. Children with and without proteinuria had the level same HS content. In contrast, rats with ADRN showed a decreased glomerular expression of sulphated HS domains. CONCLUSIONS: These results content. suggest that in patients with MCNS proteinuria is not associated with major changes in glomerular expression of sulphated HS domains.In

Heparan in sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane various (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases,after such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with associated the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria.in Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control).experiment, Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed.other Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies Rats and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in agrin, HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in concentration the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in associated glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal resulted of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas associated removal of neuraminic acid does.

Heparan HS sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability rat properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro)albuminuria in early human and in experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies,diabetic glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in as rat renal tissue after 5 months diabetes duration. Glomerular staining however revealed no differences between control and diabetic specimens. A duration. significant (p< .05)~60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli.HS Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or HS charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and radiolabeling diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is sulfation not caused by loss of glomerular HS expression or sulfation, and suggest other mechanisms to be responsible for increased glomerular that albumin permeability.

In laminin the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily of glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively In charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to Reactive play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG.loss By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay,assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding nephropathy, of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro evaluated and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was decreased found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that loss deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the agrin-DG detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.Kidney agrin-DG International (2006) 69, 1526-1534. doi:10.1038/sj.ki.5000138; published online 15 March 2006.

The cell molecular mechanisms of heparan sulfate proteoglycan downregulation in the glomerular basement membrane (GBM) of the kidneys with diabetic nephropathy remain culture controversial. In the present study, we showed that the expression of heparanase-1 (HPR1), a heparan sulfate-degrading endoglycosidase, was upregulated in sulfate the renal epithelial cells in the kidney with diabetic nephropathy. Urinary HPR1 levels were elevated in patients with diabetic nephropathy.(HPR1), In vitro cell culture studies revealed that HPR1 promoter-driven luciferase reporter gene expression, HPR1 mRNA, and protein were upregulated in grown renal epithelial cells under high glucose conditions. Induction of HPR1 expression by high glucose led to decreased cell surface heparan luciferase sulfate expression. HPR1 inhibitors were able to restore cell surface heparan sulfate expression. Functional analysis revealed that renal epithelial cells Functional grown under high glucose conditions resulted in an increase of basement membrane permeability to albumin. Our studies suggest that loss diabetic of heparan sulfate in the GBM with diabetic nephropathy is attributable to accelerated heparan sulfate degradation by increased HPR1 expression.were