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

Tyrone L R Humphries,Kunyu Shen,David A Vesey,David Nikolic-Paterson,David W Johnson,Abishek Iyer,Glenda C Gobe,David P Fairlie

doi:10.3390/ijms22147532

Abstract

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.

Highlights

We show that protease-activated receptor 2 (PAR2) activation in human kidney tubular epithelial cells (HTECs) induces Tissue factor (TF) synthesis, which is enhanced by the elevated availability of glucose and reduced by inhibiting glycolysis and/or glycosylation or by a PAR2 antagonist
We found that PAR2 activation, in HTECs, with a potent synthetic PAR2 agonist peptide, 2F, induced enhanced synthesis and secretion of TF by 4-fold and 9-fold, respectively, over a 24 h experimental period (Figure 1A,B)
We have shown that PAR2 activation induces synthesis and secretion of tissue factor by

Summary

Introduction

Diabetic kidney disease (DKD) develops in approximately 40% of diabetics and is the leading cause of chronic kidney disease (CKD) worldwide [1,2]. Patients with diabetes and CKD often have blood coagulation disorders [5,6,7,8] associated with enhanced synthesis and premature activation of the coagulation system components. This may present as thrombotic microangiopathy or disseminated intravascular coagulation [9,10,11]. TF is an integral transmembrane glycoprotein with a molecular weight of ~45 kDa (by immunoblotting) [12]

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Discussion

Conclusion