PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

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

Frontiers in Physiology ◽

10.3389/fphys.2021.615428 ◽

2021 ◽

Vol 12 ◽

Author(s):

Abishek Iyer ◽

Tyrone L. R. Humphries ◽

Evan P. Owens ◽

Kong-Nan Zhao ◽

Paul P. Masci ◽

...

Keyword(s):

Epithelial Cells ◽

Tissue Factor ◽

Blood Clotting ◽

Kidney Diseases ◽

Human Kidney ◽

Tubular Epithelial Cells ◽

Interacting Protein ◽

Thrombosis Risk ◽

Increased Risk ◽

Protease Activated Receptor 2

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.

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Underlying Histopathology Determines Response to Oxidative Stress in Cultured Human Primary Proximal Tubular Epithelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21020560 ◽

2020 ◽

Vol 21 (2) ◽

pp. 560

Author(s):

Muhammad Ali Khan ◽

Xiangju Wang ◽

Kurt T.K. Giuliani ◽

Purba Nag ◽

Anca Grivei ◽

...

Keyword(s):

Oxidative Stress ◽

Kidney Disease ◽

Epithelial Cells ◽

Functional Response ◽

Mitochondrial Function ◽

Human Kidney ◽

Tubular Epithelial Cells ◽

Cortical Tissue ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular

Proximal tubular epithelial cells (PTEC) are key players in the progression of kidney diseases. PTEC studies to date have primarily used mouse models and transformed human PTEC lines. However, the translatability of these models to human kidney disease has been questioned. In this study, we investigated the phenotypic and functional response of human primary PTEC to oxidative stress, an established driver of kidney disease. Furthermore, we examined the functional contribution of the underlying histopathology of the cortical tissue used to generate our PTEC. We demonstrated that human primary PTEC from both histologically ‘normal’ and ‘diseased’ cortical tissue responded to H2O2-induced oxidative stress with significantly elevated mitochondrial superoxide levels, DNA damage, and significantly decreased proliferation. The functional response of ‘normal’ PTEC to oxidative stress mirrored the reported pathogenesis of human kidney disease, with significantly attenuated mitochondrial function and increased cell death. In contrast, ‘diseased’ PTEC were functionally resistant to oxidative stress, with maintenance of mitochondrial function and cell viability. This selective survival of ‘diseased’ PTEC under oxidizing conditions is reminiscent of the in vivo persistence of maladaptive PTEC following kidney injury. We are now exploring the impact that these differential PTEC responses have in the therapeutic targeting of oxidative stress pathways.

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Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646535 ◽

1989 ◽

Vol 61 (01) ◽

pp. 101-105 ◽

Cited By ~ 27

Author(s):

Bonnie J Warn-Cramer ◽

Fanny E Almus ◽

Samuel I Rapaport

Keyword(s):

Molecular Weight ◽

Endothelial Cells ◽

Tissue Factor ◽

Phorbol Ester ◽

Umbilical Vein ◽

Primary Cultures ◽

Factor Xa ◽

Extrinsic Pathway ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

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