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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.

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

Summary Essentials During contact system activation, factor XII is progressively cleaved by plasma kallikrein.We investigated the role of factor XII truncation in biochemical studies.Factor XII contains naturally occurring truncating cleavage sites for a variety of enzymes.Truncation of factor XII primes it for activation in solution through exposure of R353. Summary Background The contact activation system and innate immune system are interlinked in inflammatory pathology. Plasma kallikrein (PKa) is held responsible for the stepwise processing of factor XII (FXII). A first cleavage activates FXII (into FXIIa); subsequent cleavages truncate it. This truncation eliminates its surface‐binding domains, which negatively regulates surface‐dependent coagulation. Objectives To investigate the influence of FXII truncation on its activation and downstream kallikrein‐kinin system activation. Methods We study activation of recombinant FXII variants by chromogenic assays, by FXIIa ELISA and western blotting. Results We demonstrate that FXII truncation primes it for activation by PKa in solution. We demonstrate this phenomenon in three settings. (i) Truncation at a naturally occurring PKa‐sensitive cleavage site, R334, accelerates FXIIa formation in solution. A site‐directed mutant FXII‐R334A displays ~50% reduced activity when exposed to PKa. (ii) A pathogenic mutation in FXII that causes hereditary angioedema, introduces an additional plasmin‐sensitive cleavage site. Truncation at this site synergistically accelerates FXII activation in solution. (iii) We identify new, naturally occurring cleavage sites in FXII that have so far not been functionally linked to contact system activation. As examples, we show that non‐activating truncation of FXII by neutrophil elastase and cathepsin K primes it for activation by PKa in solution. Conclusions FXII truncation, mediated by either pathogenic mutations or naturally occurring cleavage sites, primes FXII for activation in solution. We propose that the surface‐binding domains of FXII shield its activating cleavage site, R353. This may help to explain how the contact system contributes to inflammatory pathology.

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 (M(358)R; 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.