Heparanase Accelerates Obesity-Associated Breast Cancer Progression.

Obese women have higher risk of bearing breast tumors that are highly aggressive and resistant to therapies. Tumor-promoting effects of obesity occur locally via adipose inflammation and related alterations to the extracellular matrix (ECM) as well as systemically via circulating metabolic mediators (e.g., free fatty acids, FFA) associated with excess adiposity and implicated in toll-like receptor-mediated activation of macrophages-key cellular players in obesity-related cancer progression. Although the contribution of macrophages to proneoplastic effects of obesity is well documented, the role of ECM components and their enzymatic degradation is less appreciated. We show that heparanase, the sole mammalian endoglucuronidase that cleaves heparan sulfate in ECM, is preferentially expressed in clinical/experimental obesity-associated breast tumors. Heparanase deficiency abolished obesity-accelerated tumor progression in vivo. Heparanase orchestrated a complex molecular program that occurred concurrently in adipose and tumor tissue and sustained the cancer-promoting action of obesity. Heparanase was required for adipose tissue macrophages to produce inflammatory mediators responsible for local induction of aromatase, a rate-limiting enzyme in estrogen biosynthesis. Estrogen upregulated heparanase in hormone-responsive breast tumors. In subsequent stages, elevated levels of heparanase induced acquisition of procancerous phenotype by tumor-associated macrophages, resulting in activation of tumor-promoting signaling and acceleration of breast tumor growth under obese conditions. As techniques to screen for heparanase expression in tumors become available, these findings provide rational and a mechanistic basis for designing antiheparanase approaches to uncouple obesity and breast cancer in a rapidly growing population of obese patients. SIGNIFICANCE: This study reveals the role of heparanase in promoting obesity-associated breast cancer and provides a mechanistically informed approach to uncouple obesity and breast cancer in a rapidly growing population of obese patients.

CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.

Autoantibodies against Modified Histone Peptides in SLE Patients Are Associated with Disease Activity and Lupus Nephritis.

Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis.

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro.

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.

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

OBJECTIVE: Circulating chromatin-containing apoptotic material and/or neutrophil extracellular traps (NETs) have been proposed to be an important driving force for the antichromatin autoimmune response in patients with systemic lupus erythematosus (SLE). The aim of this study was to determine the exact nature of microparticles in the circulation of SLE patients and to assess the effects of the microparticles on the immune system. METHODS: We analyzed microparticles isolated from the plasma of patients with SLE, rheumatoid arthritis (RA), and systemic sclerosis (SSc), as well as from healthy subjects. The effects of the microparticles on blood-derived dendritic cells (DCs) and neutrophils were assessed by flow cytometry, enzyme-linked immunosorbent assay, and immunofluorescence microscopy. RESULTS: In SLE patients, we identified microparticles that were highly positive for annexin V and apoptosis-modified chromatin that were not present in healthy subjects or in RA or SSc patients. These microparticles were mostly CD31+/CD45- (endothelial), partly CD45+/CD66b+ (granulocyte), and negative for B and T cell markers. Microparticles isolated from the plasma of SLE patients increased the expression of the costimulatory surface molecules CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines interleukin-6, tumor necrosis factor, and interferon-α by blood-derived plasmacytoid DCs (PDCs) and myeloid DCs (MDCs). SLE microparticles also primed blood-derived neutrophils for NETosis. Microparticles from healthy subjects and from RA or SSc patients exhibited no significant effects on MDCs, PDCs, and NETosis. CONCLUSION: Circulating microparticles in SLE patients include a population of apoptotic cell-derived microparticles that has proinflammatory effects on PDCs and MDCs and enhances NETosis. These results underline the important role of apoptotic microparticles in driving the autoimmune response in SLE patients.

Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway.

The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.

The tetraspanin CD37 protects against glomerular IgA deposition and renal pathology.

The tetraspanin protein CD37 is a leukocyte-specific transmembrane protein that is highly expressed on B cells. CD37-deficient (CD37(-/-)) mice exhibit a 15-fold increased level of immunoglobulin A (IgA) in serum and elevated numbers of IgA+ plasma cells in lymphoid organs. Here, we report that CD37(-/-) mice spontaneously develop renal pathology with characteristics of human IgA nephropathy. In young naïve CD37(-/-) mice, mild IgA deposition in glomeruli was observed. However, CD37(-/-) mice developed high titers of IgA immune complexes in serum during aging, which was associated with increased glomerular IgA deposition. Severe mesangial proliferation, fibrosis, and hyalinosis were apparent in aged CD37(-/-) mice, whereas albuminuria was mild. To further evaluate the role of CD37 in glomerular disease, we induced anti-glomerular basement membrane (GBM) nephritis in mice. CD37(-/-) mice developed higher IgA serum levels and glomerular deposits of anti-GBM IgA compared with wild-type mice. Importantly, glomerular macrophage and neutrophil influx was significantly higher in CD37(-/-) mice during both the heterologous and autologous phase of anti-GBM nephritis. Taken together, tetraspanin CD37 controls the formation of IgA-containing immune complexes and glomerular IgA deposition, which induces influx of inflammatory myeloid cells. Therefore, CD37 may protect against the development of IgA nephropathy.

Expression of sialidase and dystroglycan in human glomerular diseases.

BACKGROUND: alpha-Dystroglycan (alpha-DG) is a negatively charged glycoprotein that covers the surface of podocytes. A decreased glomerular expression of alpha-DG has been described in minimal change nephropathy (MCN), but not in focal segmental glomerulosclerosis (FSGS). This was suggested as a tool to distinguish these diseases. Sialic acid is a negatively charged carbohydrate extensively present on both alpha-DG and podocalyxin, which is also expressed on podocytes. Intrarenal perfusion with bacterial sialidase leads to foot process effacement and proteinuria. This is the first study on the expression of endogenous glomerular sialidase; furthermore, the expression of dystroglycan was re-evaluated. METHODS: The expression of alpha-DG and sialidase was investigated by immunofluorescence in kidney biopsies of patients with MCN (n = 5), FSGS (n = 15), proliferative lupus nephritis (LN, n = 9), membranous glomerulopathy (MG, n = 10) and normal human kidneys (NHK, n = 4). The urinary sialic acid concentration was measured using a newly developed LC-tandem mass spectrometry method. RESULTS: A 3-fold increased glomerular expression of sialidase was found in MG, accompanied with an increased urinary sialic acid concentration in two MG patients. However, we did not observe major changes in the expression of alpha-DG in patients with the above-mentioned glomerular diseases compared to NHK, also not between MCN and FSGS. CONCLUSIONS: Endogenous glomerular sialidase expression is increased in MG, which might represent a novel mechanism for the loss of negative charge in the glomerular capillary filter. The expression of dystroglycan cannot be used as a diagnostic tool to differentiate between glomerular diseases.

Ligation of alpha-dystroglycan on podocytes induces intracellular signaling: a new mechanism for podocyte effacement?

BACKGROUND: Alpha-dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of beta-dystroglycan. At the basolateral side alpha-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of alpha-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture. METHODOLOGY/PRINCIPAL FINDINGS: Conditionally immortalized podocytes were exposed in vitro to antibodies to alpha-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of beta-dystroglycan and podocyte architecture were studied. Antibodies to alpha-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in beta-dystroglycan. Ligation of alpha-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte. CONCLUSIONS/SIGNIFICANCE: We conclude that ligation of alpha-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.

Expression of glomerular heparan sulphate domains in murine and human lupus nephritis.

BACKGROUND: Recently, we identified specific N- and 6-O-sulphated heparan sulphate (HS) domains on activated glomerular endothelial cells. In this study, we evaluated in lupus nephritis the expression of different HS domains on glomerular endothelium and in the glomerular basement membrane (GBM). METHODS: The expression of specific glomerular HS domains and the presence of immunoglobulins (Ig) were determined by immunofluorescence staining of kidney sections of patients with nephritis due to systemic lupus erythematosus (SLE) and MRL/lpr lupus mice. The expression/presence of glomerular HS domains and Ig was also evaluated after eluting Ig from renal sections of lupus mice using two elution methods, and in renal sections of lupus mice treated with heparinoids. RESULTS: Both MRL/lpr mice and patients with lupus nephritis showed a decreased expression of HS in the GBM. The expression of N- and 6-O-sulphated HS domains on glomerular endothelium was decreased in MRL/lpr mice, but increased in SLE patients. MRL/lpr mice had more extensive glomerular Ig deposits than SLE patients. After elution of Ig, the glomerular endothelial expression of N- and 6-O-sulphated HS domains in MRL/lpr mice was recovered and even increased above normal levels, while the expression of HS in the GBM was restored to normal levels. Treatment with heparinoids prevented Ig deposition and preserved the expression of glomerular HS domains at normal levels in lupus mice. CONCLUSION: The expression of specific HS domains on glomerular endothelium and in the GBM is changed during lupus nephritis due to masking by Ig deposits and induction of inflammatory N- and 6-O-sulphated HS domains.

Adult and paediatric patients with minimal change nephrotic syndrome show no major alterations in glomerular expression of sulphated heparan sulphate domains.

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

No change in glomerular heparan sulfate structure in early human and experimental diabetic nephropathy.

Heparan sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro) albuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies, glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in rat renal tissue after 5 months diabetes duration. Glomerular staining, however, revealed no differences between control and diabetic specimens. A significant (p < 0.05) approximately 60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli. Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is not caused by loss of glomerular HS expression or sulfation and suggest other mechanisms to be responsible for increased glomerular albumin permeability.

Localization of alpha-dystroglycan on the podocyte: from top to toe.

alpha-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that alpha-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against alpha-DG. Therefore, we studied the exact localization of glomerular alpha-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of alpha-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that alpha-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that alpha-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.