Angelique L. Rops

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway.

Induction of glomerular heparanase expression in rats with Adriamycin nephropathy is regulated by reactive oxygen species and the renin-angiotensin system

Heparan sulfate (HS) in the glomerular basement membrane (GBM) is important for regulation of the charge-dependent permeability. Heparanase has been implicated in HS degradation in several proteinuric diseases. This study analyzed the role of heparanase in HS degradation in Adriamycin nephropathy (AN), a model of chronic proteinuria-induced renal damage. Expression of heparanase, HS, and the core protein of agrin (to which HS is attached) was determined on kidney sections from rats with AN in different experiments. First, expression was examined in a model of unilateral AN in a time-course study at 6-wk intervals until week 30. Second, rats were treated with the hydroxyl radical scavenger dimethylthiourea (DMTU) during bilateral AN induction. Finally, 6 wk after AN induction, rats were treated with angiotensin II receptor type 1 antagonist (AT1A) or vehicle for 2 wk. Heparanase expression was increased in glomeruli of rats with AN, which correlated with HS reduction at all time points and in all experiments. Treatment with DMTU prevented the increased heparanase expression, the loss of GBM HS, and reduced albuminuria. Finally, treatment of established proteinuria with AT1A significantly reduced heparanase expression and restored glomerular HS. In conclusion, an association between heparanase expression and reduction of glomerular HS in AN was observed. The effects of DMTU suggest a role for reactive oxygen species in upregulation of heparanase. Antiproteinuric treatment by AT1A decreased heparanase expression and restored HS expression. These results suggest involvement of radicals and angiotensin II in the modulation of GBM permeability through HS and heparanase expression.

Protein-Bound Uremic Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

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: Spearmans ρ=0.31; P<0.05; perfused boundary region versus angiopoietin-2: Spearmans 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.

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.

In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

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

Localization and Functional Characterization of Glycosaminoglycan Domains in the Normal Human Kidney as Revealed by Phage Display-Derived Single Chain Antibodies

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.

Role of the Heparan Sulfate Proteoglycan Syndecan-1 (CD138) in Delayed-Type Hypersensitivity

The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) modulates the activity of chemokines, cytokines, integrins, and other adhesion molecules which play important roles in the regulation of inflammation. We have previously shown that syndecan-1-deficient murine leukocytes display increased interactions with endothelial cells and increased diapedesis in vivo and in vitro. In this study, we demonstrate that syndecan-1 has an important function as a negative modulator in the murine contact allergy model of oxazolone-mediated delayed-type hypersensitivity (DTH). Following elicitation of the DTH response, syndecan-1-deficient mice showed an increase in leukocyte recruitment, resulting in an increased and prolonged edema formation. Expression of the cytokines TNF-α and IL-6 of the chemokines CCL5/RANTES and CCL-3/MIP-1α and of the adhesion molecule ICAM-1 were significantly increased in syndecan-1-deficient compared with wild-type mice. In wild-type mice, syndecan-1 mRNA and protein expression was reduced during the DTH response. The differentially increased adhesion of syndecan-1-deficient leukocytes to ICAM-1 was efficiently inhibited in vitro by CD18-blocking Abs, which emerges as one mechanistic explanation for the anti-inflammatory effects of syndecan-1. Collectively, our results show an important role of syndecan-1 in the contact DTH reaction, identifying syndecan-1 as a novel target in anti-inflammatory therapy.

Regulation of glomerular heparanase expression by aldosterone, angiotensin II and reactive oxygen species

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

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 β2-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.