Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis.

Background: The glomerular endothelial glycocalyx is degraded during inflammation. The glycocalyx plays a pivotal role in endothelial function and is involved in many processes including binding of chemokines and cytokines, leukocyte trafficking, and preventing proteinuria. HS-based therapeutics are a promising novel class of anti-inflammatory drugs to restore a compromised endothelial glycocalyx under inflammatory conditions. Recently, we demonstrated that treatment with HS extracted from unstimulated glomerular endothelial glycocalyx (unstimulated HSglx) reduced albuminuria during anti-GBM induced glomerulonephritis. Since endothelial HS domains are distinct in unstimulated versus inflammatory conditions, we hypothesized that 1) unstimulated HSglx, 2) LPS-stimulated HSglx, 3) the HS-mimetic fucoidan and 4) the glycosaminoglycan preparation sulodexide, which is a mixture of low molecular weight heparin and dermatan sulfate, might have different beneficial effects in experimental glomerulonephritis. Methods: The effect of unstimulated HSglx, LPS HSglx, Laminaria japonica fucoidan, or sulodexide on experimental glomerulonephritis was tested in LPS-induced glomerulonephritis in mice. Analyses included urinary albumin creatinine measurement, cytokine expression in plasma and renal cortex, and renal influx of immune cells determined by flow cytometry and immunofluorescence staining. Furthermore, the observed in vivo effects were evaluated in cultured glomerular endothelial cells and peripheral blood mononuclear cells by measuring cytokine and ICAM-1 expression levels. The ability of the compounds to inhibit heparanase activity was assessed in a heparanase activity assay. Results: Treatment of mice with LPS HSglx or sulodexide near-significantly attenuated LPS-induced proteinuria. All treatments reduced plasma MCP-1 levels, whereas only fucoidan reduced IL-6 and IL-10 plasma levels. Moreover, all treatments reversed cortical ICAM-1 mRNA expression and both fucoidan and sulodexide reversed cortical IL-6 and nephrin mRNA expression. Sulodexide decreased renal influx of CD45+ immune cells whereas renal influx of macrophages and granulocytes remained unaltered for all treatments. Although all compounds inhibited HPSE activity, fucoidan and sulodexide were the most potent inhibitors. Notably, fucoidan and sulodexide decreased LPS-induced mRNA expression of ICAM-1 and IL-6 by cultured glomerular endothelial cells. Conclusion: Our data show a potentially protective effect of glycosaminoglycans and fucoidan in experimental glomerulonephritis. Future research should be aimed at the further identification of defined HS structures that have therapeutic potential in the treatment of glomerular diseases.

Heparanase-2 protein and peptides have a protective effect on experimental glomerulonephritis and diabetic nephropathy.

Introduction: The endothelial glycocalyx degrading enzyme heparanase-1 (HPSE1) is a major contributor to kidney diseases, such as glomerulonephritis and diabetic nephropathy. Therefore, inhibition of HPSE1 could be an interesting therapeutic strategy to treat glomerular diseases. A possible HPSE1 inhibitor is heparanase-2 (HPSE2) because HPSE2 is a structural homolog of HPSE1 without enzymatic activity. The importance of HPSE2 has been recently demonstrated in HPSE2-deficient mice, since these mice developed albuminuria and died within a few months after birth. We postulate that inhibition of HPSE1 activity by HPSE2 is a promising therapeutic strategy to target albuminuria and resulting renal failure. Methods: First, we evaluated the regulation of HPSE2 expression in anti-GBM and LPS-induced glomerulonephritis, streptozotocin-induced diabetic nephropathy, and adriamycin nephropathy by qPCR and ELISA. Second, we measured the HPSE1 inhibiting capacity of HPSE2 protein and 30 different HPSE2 peptides and assessed their therapeutic potential in both experimental glomerulonephritis and diabetic nephropathy using kidney function and cortical mRNA expression of HPSE1 and cytokines as outcome parameters. Results: HPSE2 expression was downregulated under inflammatory and diabetic conditions, whereas this effect on HPSE2 expression was absent with HPSE1 inhibition and in HPSE1-deficient mice. Both HPSE2 protein and a mixture of the three most potent HPSE1 inhibitory HPSE2 peptides could prevent LPS and streptozotocin induced kidney injury. Discussion: Taken together, our data suggest a protective effect of HPSE2 in (experimental) glomerular diseases and support the therapeutic potential of HPSE2 as HPSE1 inhibitor in glomerular diseases.

Peroxisome proliferator-activated receptor ɣ agonist mediated inhibition of heparanase expression reduces proteinuria.

BACKGROUND: Proteinuria is associated with many glomerular diseases and a risk factor for the progression to renal failure. We previously showed that heparanase (HPSE) is essential for the development of proteinuria, whereas peroxisome proliferator-activated receptor É£ (PPARÉ£) agonists can ameliorate proteinuria. Since a recent study showed that PPARÉ£ regulates HPSE expression in liver cancer cells, we hypothesized that PPARÉ£ agonists exert their reno-protective effect by inhibiting glomerular HPSE expression. METHODS: Regulation of HPSE by PPARÉ£ was assessed in the adriamycin nephropathy rat model, and cultured glomerular endothelial cells and podocytes. Analyses included immunofluorescence staining, real-time PCR, heparanase activity assay and transendothelial albumin passage assay. Direct binding of PPARÉ£ to the HPSE promoter was evaluated by the luciferase reporter assay and chromatin immunoprecipitation assay. Furthermore, HPSE activity was assessed in 38 type 2 diabetes mellitus (T2DM) patients before and after 16/24 weeks treatment with the PPARÉ£ agonist pioglitazone. FINDINGS: Adriamycin-exposed rats developed proteinuria, an increased cortical HPSE and decreased heparan sulfate (HS) expression, which was ameliorated by treatment with pioglitazone. In line, the PPARÉ£ antagonist GW9662 increased cortical HPSE and decreased HS expression, accompanied with proteinuria in healthy rats, as previously shown. In vitro, GW9662 induced HPSE expression in both endothelial cells and podocytes, and increased transendothelial albumin passage in a HPSE-dependent manner. Pioglitazone normalized HPSE expression in adriamycin-injured human endothelial cells and mouse podocytes, and adriamycin-induced transendothelial albumin passage was reduced as well. Importantly, we demonstrated a regulatory effect of PPARÉ£ on HPSE promoter activity and direct PPARy binding to the HPSE promoter region. Plasma HPSE activity of T2DM patients treated with pioglitazone for 16/24 weeks was related to their hemoglobin A1c and showed a moderate, near significant correlation with plasma creatinine levels. INTERPRETATION: PPARÉ£-mediated regulation of HPSE expression appears an additional mechanism explaining the anti-proteinuric and renoprotective effects of thiazolidinediones in clinical practice. FUNDING: This study was financially supported by the Dutch Kidney Foundation, by grants 15OI36, 13OKS023 and 15OP13. Consortium grant LSHM16058-SGF (GLYCOTREAT; a collaboration project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships).

Selective Binding of Heparin/Heparan Sulfate Oligosaccharides to Factor H and Factor H-Related Proteins: Therapeutic Potential for C3 Glomerulopathies.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.

Hydroxychloroquine Inhibits the Trained Innate Immune Response to Interferons.

Hydroxychloroquine is being investigated for a potential prophylactic effect in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but its mechanism of action is poorly understood. Circulating leukocytes from the blood of coronavirus disease 2019 (COVID-19) patients show increased responses to Toll-like receptor ligands, suggestive of trained immunity. By analyzing interferon responses of peripheral blood mononuclear cells from healthy donors conditioned with heat-killed Candida, trained innate immunity can be modeled in vitro. In this model, hydroxychloroquine inhibits the responsiveness of these innate immune cells to virus-like stimuli and interferons. This is associated with a suppression of histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation of inflammation-related genes, changes in the cellular lipidome, and decreased expression of interferon-stimulated genes. Our findings indicate that hydroxychloroquine inhibits trained immunity in vitro, which may not be beneficial for the antiviral innate immune response to SARS-CoV-2 infection in patients.

Increased Plasma Heparanase Activity in COVID-19 Patients.

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues.

Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step.

Ankyrin-3 is a novel binding partner of the voltage-gated potassium channel Kv1.1 implicated in renal magnesium handling.

The voltage-gated potassium channel, Kv1.1, was recently identified as a causative gene in isolated dominant hypomagnesemia. The channel is situated in the distal convoluted tubule, where it participates in maintaining a favorable electrical gradient for driving magnesium ion into the cell through the transient receptor potential melastatin 6 channel. Pull-down experiments coupled to mass spectrometry using the carboxy-terminal domain of Kv1.1 as bait were used in mouse kidney lysates. Ankyrin-3 (ANK3) was identified as a binding partner of Kv1.1 and was enriched in isolated distal convoluted tubules as compared to whole kidney. Electrophysiology studies performed in HEK293 cells expressing Kv1.1 showed that ANK3 significantly inhibited Kv1.1-mediated currents (267 compared to 125 pA/pF) for control and ANK3, respectively. Finally, to evaluate a potential role of ANK3 in magnesium handling, the intrarenal abundance of ANK3 was measured in mice fed a low-, normal-, or high-magnesium diet for 10 days. Mice maintained on high dietary magnesium significantly doubled their fractional urinary excretion of magnesium, which coincided with a 1.8-fold increase in the renal expression of ANK3 compared to mice on a normal- or low-magnesium diet. Thus, our observations demonstrate a novel role for ANK3 in modulating the biophysical properties of Kv1.1. Such regulation appears to be particularly important in conditions of high dietary magnesium.

The identification of genetic pathways involved in vascular adaptations after physical deconditioning versus exercise training in humans.

Physical inactivity and exercise training result in opposite adaptations of vascular structure. However, the molecular mechanisms behind these adaptations are not completely understood. We used a unique study design to examine both vascular characteristics of the superficial femoral artery (using ultrasound) and gene expression levels (from a muscle biopsy) in human models for physical deconditioning and exercise training. Initially, we compared able-bodied control subjects (n = 6) with spinal cord-injured individuals (n = 8) to assess the effects of long-term deconditioning. Subsequently, able-bodied control subjects underwent short-term lower limb deconditioning using 3 weeks of unilateral limb suspension. Spinal cord-injured individuals were examined before and after 6 weeks of functional electrical stimulation exercise training. Baseline femoral artery diameter and hyperaemic flow were lower after short- and long-term deconditioning and higher after exercise training, whilst intima-media thickness/lumen ratio was increased with short- and long-term deconditioning and decreased with exercise training. Regarding gene expression levels of vasculature-related genes, we found that groups of genes including the vascular endothelial growth factor pathway, transforming growth factor ß1 and extracellular matrix proteins were strongly associated with vascular adaptations in humans. This approach resulted in the identification of important genes that may be involved in vascular adaptations after physical deconditioning and exercise.

γ-Adducin stimulates the thiazide-sensitive NaCl cotransporter.

The thiazide-sensitive NaCl cotransporter (NCC) plays a key role in renal salt reabsorption and the determination of systemic BP, but the molecular mechanisms governing the regulation of NCC are not completely understood. Here, through pull-down experiments coupled to mass spectrometry, we found that γ-adducin interacts with the NCC transporter. γ-Adducin colocalized with NCC to the distal convoluted tubule. (22)Na(+) uptake experiments in the Xenopus laevis oocyte showed that γ-adducin stimulated NCC activity in a dose-dependent manner, an effect that occurred upstream from With No Lysine (WNK) 4 kinase. The binding site of γ-adducin mapped to the N terminus of NCC and encompassed three previously reported phosphorylation sites. Supporting this site of interaction, competition with the N-terminal domain of NCC abolished the stimulatory effect of γ-adducin on the transporter. γ-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, γ-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that γ-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that γ-adducin may influence BP homeostasis by modulating renal NaCl transport.

Methionine sulfoxide reductase B1 (MsrB1) recovers TRPM6 channel activity during oxidative stress.

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.