PAR2 Activation on Human Kidney Tubular Epithelial Cells Induces Tissue Factor Synthesis, That Enhances Blood Clotting.

Abishek Iyer,David Nikolic-Paterson,Tyrone L R Humphries,David P Fairlie,Evan P Owens,Glenda C Gobe,Kong-Nan Zhao,David W Johnson,Paul P Masci,David A Vesey

doi:10.3389/fphys.2021.615428

Abstract

Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.

Highlights

Patients with chronic kidney disease (CKD) often have blood coagulation disorders, associated with activation of the coagulation system and the local presence of activated clotting factors (Kincaid-Smith, 1972; Wendt et al, 1995; Wang et al, 1996, 1997)
We investigated whether Protease-activated receptor-2 (PAR2) activation, reported to be involved in coagulation, inflammation, fibrosis and infection, could potentially promote the Tissue factor (TF)-mediated clotting cascade in the vicinity of the kidneys (Vesey et al, 2007a, 2013; Adams et al, 2011; Chung et al, 2013; Oe et al, 2016)
Using primary cultures of human kidney tubular epithelial cells (HTEC), we have identified a potential link between PAR2 expressed on the surface of these cells with the induction of TF expression and TF stimulation of blood clotting

Summary

Introduction

Patients with chronic kidney disease (CKD) often have blood coagulation disorders, associated with activation of the coagulation system and the local presence of activated clotting factors (Kincaid-Smith, 1972; Wendt et al, 1995; Wang et al, 1996, 1997). Tissue factor (TF) is the primary physiological initiator of the injury-induced blood coagulation cascade (Grover and Mackman, 2018). In terms of CKD, local dysregulated TF expression in the kidneys can increase the risk of thrombosis and lead to chronic fibrin deposition (Grover and Mackman, 2018). TF is localized to both the glomerulus and kidney tubules during various inflammatory CKDs, such as glomerular and tubulointerstitial nephritis (Lwaleed and Bass, 2006; Moussa et al, 2007) In these conditions, elevated levels of TF can be found in the urine and are associated with phospholipid vesicles that appear to originate from cells within the kidney itself, rather than the glomerular blood filtrate (Lwaleed et al, 1999). Some studies have suggested measuring urinary TF as a clinical biomarker for glomerulonephritis (Lwaleed et al, 1997, 1998)

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