Glomerular endothelial glycocalyx-derived heparan sulfate inhibits glomerular leukocyte influx and attenuates experimental glomerulonephritis.

Proliferative forms of glomerulonephritis are characterized by the influx of leukocytes, albuminuria, and loss of kidney function. The glomerular endothelial glycocalyx is a thick carbohydrate layer that covers the endothelium and is comprised of heparan sulfate (HS), which plays a pivotal role in glomerular inflammation by facilitating endothelial-leukocyte trafficking. We hypothesize that the exogenous glomerular glycocalyx may reduce the glomerular influx of inflammatory cells during glomerulonephritis. Indeed, administration of mouse glomerular endothelial cell (mGEnC)-derived glycocalyx constituents, or the low-molecular-weight heparin enoxaparin, reduced proteinuria in mice with experimental glomerulonephritis. Glomerular influx of granulocytes and macrophages, as well as glomerular fibrin deposition, was reduced by the administration of mGEnC-derived glycocalyx constituents, thereby explaining the improved clinical outcome. HSglx also inhibited granulocyte adhesion to human glomerular endothelial cells in vitro. Notably, a specific HSglx fraction inhibited both CD11b and L-selectin binding to activated mGEnCs. Mass spectrometry analysis of this specific fraction revealed six HS oligosaccharides, ranging from tetra- to hexasaccharides with 2-7 sulfates. In summary, we demonstrate that exogenous HSglx reduces albuminuria during glomerulonephritis, which is possibly mediated via multiple mechanisms. Our results justify the further development of structurally defined HS-based therapeutics for patients with (acute) inflammatory glomerular diseases, which may be applicable to non-renal inflammatory diseases as well.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Interleukin-6 is essential for glomerular immunoglobulin A deposition and the development of renal pathology in Cd37-deficient mice.

Immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is characterized by IgA depositions in the kidney. Deficiency of CD37, a leukocyte-specific tetraspanin, leads to spontaneous development of renal pathology resembling IgAN. However, the underlying molecular mechanism has not been resolved. Here we found that CD37 expression on B cells of patients with IgAN was significantly decreased compared to B cells of healthy donors. Circulating interleukin (IL)-6 levels, but not tumor necrosis factor-α or IL-10, were elevated in Cd37-/- mice compared to wild-type mice after lipopolysaccharide treatment. Cd37-/- mice displayed increased glomerular neutrophil influx, immune complex deposition, and worse renal function. To evaluate the role of IL-6 in the pathogenesis of accelerated renal pathology in Cd37-/-mice, we generated Cd37xIl6 double-knockout mice. These double-knockout and Il6-/- mice displayed no glomerular IgA deposition and were protected from exacerbated renal failure following lipopolysaccharide treatment. Moreover, kidneys of Cd37-/- mice showed more mesangial proliferation, endothelial cell activation, podocyte activation, and segmental podocyte foot process effacement compared to the double-knockout mice, emphasizing that IL-6 mediates renal pathology in Cd37-/- mice. Thus, our study indicates that CD37 may protect against IgA nephropathy by inhibition of the IL-6 pathway.

Endothelial Nitric Oxide Synthase Prevents Heparanase Induction and the Development of Proteinuria.

Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria.

Cathepsin L is crucial for the development of early experimental diabetic nephropathy.

Proteinuria is one of the first clinical signs of diabetic nephropathy and an independent predictor for the progression to renal failure. Cathepsin L, a lysosomal cysteine protease, can be involved in the development of proteinuria by degradation of proteins that are important for normal podocyte architecture, such as the CD2-associated protein, synaptopodin, and dynamin. Cathepsin L also activates heparanase, a heparan sulfate endoglycosidase previously shown to be crucial for the development of diabetic nephropathy. Here, we evaluated the exact mode of action of cathepsin L in the development of proteinuria in streptozotocin-induced diabetes. Cathepsin L-deficient mice, in contrast to their wild-type littermates, failed to develop albuminuria, mesangial matrix expansion, tubulointerstitial fibrosis, and renal macrophage influx and showed a normal renal function. In wild-type mice the early development of albuminuria correlated with the activation of heparanase and loss of heparan sulfate expression, whereas loss of synaptopodin expression and podocyte damage occurred at a later stage. Thus, cathepsin L is causally involved in the pathogenesis of experimental diabetic nephropathy. Most likely, cathepsin L-dependent heparanase activation is crucial for the development of albuminuria and renal damage.

Endothelin-1 Induces Proteinuria by Heparanase-Mediated Disruption of the Glomerular Glycocalyx.

Diabetic nephropathy (DN) is the leading cause of CKD in the Western world. Endothelin receptor antagonists have emerged as a novel treatment for DN, but the mechanisms underlying the protective effect remain unknown. We previously showed that both heparanase and endothelin-1 are essential for the development of DN. Here, we further investigated the role of these proteins in DN, and demonstrated that endothelin-1 activates podocytes to release heparanase. Furthermore, conditioned podocyte culture medium increased glomerular transendothelial albumin passage in a heparanase-dependent manner. In mice, podocyte-specific knockout of the endothelin receptor prevented the diabetes-induced increase in glomerular heparanase expression, consequent reduction in heparan sulfate expression and endothelial glycocalyx thickness, and development of proteinuria observed in wild-type counterparts. Our data suggest that in diabetes, endothelin-1 signaling, as occurs in endothelial activation, induces heparanase expression in the podocyte, damage to the glycocalyx, proteinuria, and renal failure. Thus, prevention of these effects may constitute the mechanism of action of endothelin receptor blockers in DN.

1,25-Vitamin D3 Deficiency Induces Albuminuria.

Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.

Heparanase Is Essential for the Development of Acute Experimental Glomerulonephritis.

Heparanase, a heparan sulfate (HS)--specific endoglucuronidase, mediates the onset of proteinuria and renal damage during experimental diabetic nephropathy. Glomerular heparanase expression is increased in most proteinuric diseases. Herein, we evaluated the role of heparanase in two models of experimental glomerulonephritis, being anti-glomerular basement membrane and lipopolysaccharide-induced glomerulonephritis, in wild-type and heparanase-deficient mice. Induction of experimental glomerulonephritis led to an increased heparanase expression in wild-type mice, which was associated with a decreased glomerular expression of a highly sulfated HS domain, and albuminuria. Albuminuria was reduced in the heparanase-deficient mice in both models of experimental glomerulonephritis, which was accompanied by a better renal function and less renal damage. Notably, glomerular HS expression was preserved in the heparanase-deficient mice. Glomerular leukocyte and macrophage influx was reduced in the heparanase-deficient mice, which was accompanied by a reduced expression of both types 1 and 2 helper T-cell cytokines. In vitro, tumor necrosis factor-α and lipopolysaccharide directly induced heparanase expression and increased transendothelial albumin passage. Our study shows that heparanase contributes to proteinuria and renal damage in experimental glomerulonephritis by decreasing glomerular HS expression, enhancing renal leukocyte and macrophage influx, and affecting the local cytokine milieu.

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.

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-ß), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.

Vitamin D attenuates proteinuria by inhibition of heparanase expression in the podocyte.

The glomerular filtration barrier consists of podocytes, the glomerular basement membrane, and endothelial cells covered with a glycocalyx. Heparan sulphate (HS) in the glomerular filtration barrier is reduced in patients with proteinuria, which is associated with increased expression of the HS-degrading enzyme heparanase. Previously, we showed that heparanase is essential for the development of proteinuria in experimental diabetic nephropathy. Vitamin D supplementation reduces podocyte loss and proteinuria in vitro and in vivo. Therefore, we hypothesize that vitamin D reduces proteinuria by reducing glomerular heparanase. Adriamycin-exposed rats developed proteinuria and showed increased heparanase expression, which was reduced by 1,25-dihydroxyvitamin D3 (1,25-D3) treatment. In vitro, adriamycin increased heparanase mRNA in the podocyte, which could be corrected by 1,25-D3 treatment. In addition, 1,25-D3 treatment reduced transendothelial albumin passage after adriamycin stimulation. In line with these results, we showed direct binding of the vitamin D receptor to the heparanase promoter, and 1,25-D3 dose-dependently reduced heparanase promoter activity. Finally, 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice developed proteinuria and showed increased heparanase, which was normalized by 1,25-D3 treatment. Our data suggest that the protective effect of vitamin D on the development of proteinuria is mediated by inhibiting heparanase expression in the podocyte.

The role of heparan sulfate as determining pathogenic factor in complement factor H-associated diseases.

Complement factor H (FH) systemically inhibits excessive complement activation in the microenvironment of host cells, but for instance not on microbes. This self-recognition is mediated by two binding sites that recognize distinctly sulfated heparan sulfate (HS) domains. The interaction with HS not only concentrates FH on host cells, but directly affects its activity, evoking novel models of conformational activation. Genetic aberrations in the HS-binding domains systemically disturb the protective function of FH, yet the resulting loss of complement control affects mainly ocular and renal tissues. Recent results suggest that the specific expression of HS domains in these tissues restricts the interaction of HS to a single binding site within FH. This lack of redundancy could predispose eyes and kidneys to complement-mediated damage, making HS a central determinant for FH-associated diseases.

Modulation of heparan sulfate in the glomerular endothelial glycocalyx decreases leukocyte influx during experimental glomerulonephritis.

The glomerular endothelial glycocalyx is postulated to be an important modulator of permeability and inflammation. The glycocalyx consists of complex polysaccharides, the main functional constituent of which, heparan sulfate (HS), is synthesized and modified by multiple enzymes. The N-deacetylase-N-sulfotransferase (Ndst) enzymes initiate and dictate the modification process. Here we evaluated the effects of modulation of HS in the endothelial glycocalyx on albuminuria and glomerular leukocyte influx using mice deficient in endothelial and leukocyte Ndst1 (TEKCre+/Ndst1flox/flox). In these mice, glomerular expression of a specific HS domain was significantly decreased, whereas the expression of other HS domains was normal. In the endothelial glycocalyx, this specific HS structure was not associated with albuminuria or with changes in renal function. However, glomerular leukocyte influx was significantly reduced during antiglomerular basement membrane nephritis, which was associated with less glomerular injury and better renal function. In vitro decreased adhesion of wild-type and Ndst1-deficient granulocytes to Ndst1-silenced glomerular endothelial cells was found, accompanied by a decreased binding of chemokines and L-selectin. Thus, modulation of HS in the glomerular endothelial glycocalyx significantly reduced the inflammatory response in antiglomerular basement membrane nephritis.

Chemokine cooperativity is caused by competitive glycosaminoglycan binding.

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.

Association of kidney function with changes in the endothelial surface layer.

BACKGROUND AND OBJECTIVES: ESRD is accompanied by endothelial dysfunction. Because the endothelial glycocalyx (endothelial surface layer) governs interactions between flowing blood and the vessel wall, perturbation could influence disease progression. This study used a novel noninvasive sidestream-darkfield imaging method, which measures the accessibility of red blood cells to the endothelial surface layer in the microcirculation (perfused boundary region), to investigate whether renal function is associated with endothelial surface layer dimensions. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Perfused boundary region was measured in control participants (n=10), patients with ESRD (n=23), participants with normal kidney function after successful living donor kidney transplantation (n=12), and patients who developed interstitial fibrosis/tubular atrophy after kidney transplantation (n=10). In addition, the endothelial activation marker angiopoietin-2 and shed endothelial surface layer components syndecan-1 and soluble thrombomodulin were measured using ELISA. RESULTS: Compared with healthy controls (1.82 ± 0.16 µm), ESRD patients had a larger perfused boundary region (+0.23; 95% confidence interval, 0.46 to <0.01; P<0.05), which signifies loss of endothelial surface layer dimensions. This large perfused boundary region was accompanied by higher circulating levels of syndecan-1 (+57.71; 95% confidence interval, 17.38 to 98.04; P<0.01) and soluble thrombomodulin (+12.88; 95% confidence interval, 0.29 to 25.46; P<0.001). After successful transplantation, the perfused boundary region was indistinguishable from healthy controls (without elevated levels of soluble thrombomodulin or syndecan-1). In contrast, however, patients who developed interstitial fibrosis and tubular atrophy showed a large perfused boundary region (+0.36; 95% confidence interval, 0.09 to 0.63; P<0.01) and higher levels of endothelial activation markers. In addition, a significant correlation between perfused boundary region, angiopoietin-2, and eGFR was observed (perfused boundary region versus GFR: Spearman's ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearman's ρ=-0.33; P<0.05). CONCLUSION: Reduced renal function is strongly associated with low endothelial surface layer dimensions. After successful kidney transplantation, the endothelial surface layer is indistinguishable from control.

The role of heparanase and the endothelial glycocalyx in the development of proteinuria.

Proteinuria is a hallmark of many glomerular diseases and an independent risk factor for the progression of renal failure. Proteinuria results from damage to the glomerular filtration barrier (GFB), which plays a critical role in size- and charge-selective filtration. The GFB consists of three layers, which is the fenestrated endothelium that is covered by the glycocalyx, the podocytes and the intervening glomerular basement membrane. Defects in one of the three layers in the GFB can lead to the development of proteinuria. Heparan sulphate (HS) is a negatively charged polysaccharide that is abundantly expressed in all layers of the GFB. HS expression in the GFB is reduced in the majority of patients with proteinuria, which is associated with an increased glomerular expression of the HS-degrading enzyme heparanase. The primary role of HS in the development of proteinuria has been challenged after the establishment of several genetically engineered mouse models with an altered HS expression that did not display development of overt proteinuria. However, in a recent study, we showed that heparanase is essential for the development of proteinuria in diabetic nephropathy, which suggests that loss of HS contributes to the development of proteinuria. Recent studies also further highlight the importance of the glomerular endothelial glycocalyx in charge-selective filtration and the development of proteinuria. This review aims to summarize our current knowledge on the role of in particular HS and heparanase in the development of proteinuria.

Glomerular endothelial surface layer acts as a barrier against albumin filtration.

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of native ferritin [corrected] in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

Urinary heparanase activity in patients with Type 1 and Type 2 diabetes.

BACKGROUND: A reduced heparan sulphate (HS) expression in the glomerular basement membrane of patients with overt diabetic nephropathy is associated with an increased glomerular heparanase expression. We investigated the possible association of urinary heparanase activity with the development of proteinuria in patients with Type 1 diabetes (T1D), Type 2 diabetes (T2D), or membranous glomerulopathy (MGP) as non-diabetic disease controls. METHODS: Heparanase activity, albumin, HS and creatinine were measured in the urine of patients with T1D (n=58) or T2D (n=31), in patients with MGP (n=52) and in healthy controls (n=10). Heparanase messenger RNA (mRNA) expression in leukocytes was determined in a subgroup of patients with T1D (n=19). RESULTS: Urinary heparanase activity was increased in patients with T1D and T2D, which was more prominent in patients with macroalbuminuria, whereas no activity could be detected in healthy controls. Albuminuria levels were associated with increased urinary heparanase activity in diabetic patients (r=0.20; P<0.05) but not in patients with MGP (r=0.11; P=0.43). A lower urinary heparanase activity was observed in diabetic patients treated with inhibitors of the renin-angiotensin-aldosterone system (RAAS), when compared to diabetic patients treated with other anti-hypertensives. Additionally, urinary heparanase activity was associated with age in T1D and MGP. In MGP, heparanase activity and ß2-microglobulin excretion correlated. In patients with T1D, no differences in heparanase mRNA expression in leukocytes could be observed. CONCLUSIONS: Urinary heparanase activity is increased in diabetic patients with proteinuria. However, whether increased heparanase activity is a cause or consequence of proteinuria requires additional research.

The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HS(NS4F5)), which we have rigorously identified as (GlcNS6S-IdoA2S)(3). HS(NS4F5) exhibits a restricted expression in healthy adult tissues. Blocking HS(NS4F5) on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HS(NS4F5) is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HS(NS4F5), which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HS(NS4F5) was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HS(NS4F5) and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

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.