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.

Novel mouse strains to study circulating permeability factor(s) in primary focal segmental glomerulosclerosis.

Recurrence of proteinuria after kidney transplantation in primary focal segmental glomerulosclerosis (FSGS) is unpredictable. Several putative circulating permeability factors (CPFs) have been suggested, but none have been validated. A clinically relevant experimental model is required that demonstrates the presence of CPF(s) in patient material, to study CPF(s) and possibly predict recurrence in patients. We aimed to develop a FSGS-prone Thy-1.1 transgenic mouse model with accelerated proteinuria after injection of samples from patients with FSGS. The Thy-1.1 transgene was backcrossed into 5 mouse strains. The age of onset and severity of spontaneous proteinuria varied between the different genetic backgrounds. 129X1/SvThy-1.1 and 129S2/SvPasThy-1.1 mice displayed proteinuria at 4 weeks, whereas Balb/cThy-1.1 and C57BL/6JThy-1.1 mice developed proteinuria from 6 weeks, and were used further. We determined the maximum protein dose that could be injected without causing protein overload in each background. Balb/cThy-1.1 and C57BL/6JThy-1.1 males and females were injected with presumably CPF-containing plasmapheresis effluent from 6 FSGS patients, which induced albuminuria particularly in Balb/cThy-1.1 males. Unfortunately, no response could be detected when using sera instead of plasmapheresis effluent, serum being more clinically relevant in the context of predicting FSGS recurrence. Considering the differences between responses elicited by serum and plasmapheresis effluent, simultaneously collected serum, plasma, and plasmapheresis effluent were tested. Whereas we could detect responses using a validated in vitro model, none of these presumably CPF-containing samples induced proteinuria in Balb/cThy-1.1 males. Thus, we have extensively tested the Thy-1.1 mouse model on different genetic backgrounds with proteinuria after injection of FSGS patient material as clinically relevant readout. The Balb/cThy-1.1 male mouse strain demonstrated the most promising results, but to detect CPF activity in FSGS serum e.g. prior to kidney transplantation, this strain clearly lacks sensitivity and is therefore not yet clinically applicable. It could, however, still be used as research tool to study CPFs in patient samples that did induce proteinuria.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling.

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Primary Focal Segmental Glomerulosclerosis Plasmas Increase Lipid Droplet Formation and Perilipin-2 Expression in Human Podocytes.

Many patients with primary focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation. Several circulating permeability factors (CPFs) responsible for recurrence have been suggested, but were never validated. We aimed to find proteins involved in the mechanism of action of CPF(s) and/or potential biomarkers for the presence of CPF(s). Cultured human podocytes were exposed to plasma from patients with FSGS with presumed CPF(s) or healthy and disease controls. Podocyte proteomes were analyzed by LC-MS. Results were validated using flow cytometry, RT-PCR, and immunofluorescence. Podocyte granularity was examined using flow cytometry, electron microscopy imaging, and BODIPY staining. Perilipin-2 protein expression was increased in podocytes exposed to presumed CPF-containing plasmas, and correlated with the capacity of plasma to induce podocyte granularity, identified as lipid droplet accumulation. Elevated podocyte perilipin-2 was confirmed at protein and mRNA level and was also detected in glomeruli of FSGS patients whose active disease plasmas induced podocyte perilipin-2 and lipid droplets. Our study demonstrates that presumably, CPF-containing plasmas from FSGS patients induce podocyte lipid droplet accumulation and perilipin-2 expression, identifying perilipin-2 as a potential biomarker. Future research should address the mechanism underlying CPF-induced alterations in podocyte lipid metabolism, which ultimately may result in novel leads for treatment.

Thrombotic Events in COVID-19 Are Associated With a Lower Use of Prophylactic Anticoagulation Before Hospitalization and Followed by Decreases in Platelet Reactivity.

Background: Coronavirus disease of 2019 (COVID-19) is associated with a prothrombotic state and a high incidence of thrombotic event(s) (TE). Objectives: To study platelet reactivity in hospitalized COVID-19 patients and determine a possible association with the clinical outcomes thrombosis and all-cause mortality. Methods: Seventy nine hospitalized COVID-19 patients were enrolled in this retrospective cohort study and provided blood samples in which platelet reactivity in response to stimulation with ADP and TRAP-6 was determined using flow cytometry. Clinical outcomes included thrombotic events, and all-cause mortality. Results: The incidence of TE in this study was 28% and all-cause mortality 16%. Patients that developed a TE were younger than patients that did not develop a TE [median age of 55 vs. 70 years; adjusted odds ratio (AOR) = 0.96 per 1 year of age, 95% confidence interval (CI) 0.92-1.00; p = 0.041]. Furthermore, patients using preexisting thromboprophylaxis were less likely to develop a thrombotic complication than patients that were not (18 vs. 54%; AOR = 0.19, 95% CI 0.04-0.84; p = 0.029). Conversely, having asthma strongly increased the risk on TE development (AOR = 6.2, 95% CI 1.15-33.7; p = 0.034). No significant differences in baseline P-selectin expression or platelet reactivity were observed between the COVID-19 positive patients (n = 79) and COVID-19 negative hospitalized control patients (n = 21), nor between COVID-19 positive survivors or non-survivors. However, patients showed decreased platelet reactivity in response to TRAP-6 following TE development. Conclusion: We observed an association between the use of preexisting thromboprophylaxis and a decreased risk of TE during COVID-19. This suggests that these therapies are beneficial for coping with COVID-19 associated hypercoagulability. This highlights the importance of patient therapy adherence. We observed lowered platelet reactivity after the development of TE, which might be attributed to platelet desensitization during thromboinflammation.

Working memory and visual discrimination distraction tasks improve cold pressor pain tolerance in children.

OBJECTIVE: Distraction is a well-established pain management technique for children experiencing acute pain, although the mechanisms underlying the effectiveness of distraction are not well understood. It has been postulated that engagement of executive functions, such as working memory, may be a critical factor in attenuating pain via distraction. To test this hypothesis, we compared a 1-back task requiring engagement of working memory with a simple visual discrimination task demanding focused attention, but lower cognitive load (0-back). METHOD: Seventy-nine children (6-12 years old) underwent a baseline cold pressor trial followed by cold pressor trials in which they completed the visual discrimination and 1-back tasks in counterbalanced order. Executive functioning ability was assessed via the Wechsler Intelligence Scale for Children (5th Edition) working memory subscales and by parent report on the Behavior Rating Inventory of Executive Function, Second Edition (BRIEF®2). RESULTS: Children's pain tolerance improved in both the visual discrimination and 1-back conditions though a differential response to the 2 tasks was not observed. Age moderated the relation between executive functioning and response to distraction; older children with better executive functioning skills demonstrated greater improvements in both distraction interventions. CONCLUSIONS: Findings demonstrate the benefits of both visual discrimination and working memory distraction tasks for elementary-aged children experiencing acute pain. Further research is required in order to elucidate the role of executive functioning skills and cognitive load in enhancing distraction analgesia in children, with particular focus on determining optimal load and task difficulty in light of emerging executive functioning abilities in this age group. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

A mutation in the kringle domain of human factor XII that causes autoinflammation, disturbs zymogen quiescence, and accelerates activation.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

Design and characterization of α1-antitrypsin variants for treatment of contact system-driven thromboinflammation.

The contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although α1-antitrypsin (α1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; α1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of α1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two α1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. α1AT-SMTR/S no longer inhibits thrombin, but residually inhibits APC. In contrast, α1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P1' position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both α1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, α1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of α1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation.

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib, but unaffected by bevacizumab.

INTRODUCTION: At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings. MATERIALS AND METHODS: In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet-EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC-MS/MS and ELISA. RESULTS: In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 µM sunitinib: 71.3%, p < 0.001; 25 µM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high concentrations, but not ex vivo. CONCLUSION: Sunitinib significantly inhibits platelet aggregation in patients already after 24 h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.

Peer emotion socialization and somatic complaints in adolescents.

Somatic symptoms tend to increase during early adolescence and although youth's social environments and emotional functioning play a role in somatic symptoms, few studies have examined mechanisms through which social interaction could influence youth's somatic wellbeing. Participants were 132 youth (61.6% girls, Mage = 12.61 years, 84.7% Caucasian) and their mothers. Reciprocated best-friend dyads participated in a video-taped problem discussion task to assess peer emotion socialization responses. Two supportive friend responses (i.e., emotion-focused, problem-focused) and two unsupportive responses (i.e., punitive, neglect) were examined. Mothers reported on their child's somatic complaints. Friends who provided emotion-focused, problem-focused, punitive, and neglect responses to their close friend's emotional disclosures had significantly fewer somatic symptoms. However, youth who received punitive responses to their emotional disclosures from their close friends had more somatic complaints. These findings provide initial evidence of a link between emotion socialization responses within close friendships and somatic complaints in early adolescence.