Thrombin induces Epithelial-Mesenchymal Transition through the activation of PAR-1 in Tubular Epithelial Cells

Abstract

BACKGROUND: Fibrin deposition is commonly observed in tubulointerstitial injury of chronic kidney diseases. Recent studies have suggested that local activation of thrombin contributes to renal fibrosis. However, the effect of thrombin on tubular cells is not fully understood. Since epithelial-mesenchymal transition (EMT) is an important mechanism of renal tubulointerstitial fibrosis, we investigated whether thrombin plays a role in tubular EMT and dissected the underlying mechanism. METHODS: Rat kidney tubular epithelial cells (NRK52E) were treated with thrombin (1-4U/ml) for 72 h and were examined for the expression of EMT and profibrotic markers. To determine the potential role of protease activated receptor (PAR) in tubular EMT, selective PAR antagonists, SCH7979 for PAR-1 and tcY-NH2 for PAR-4 were co-incubated with thrombin. RESULTS: After 72-h treatment, cells underwent a transition from an epithelial to a mesenchymal phenotype as evidenced by reduced expression of E-cadherin, and increased expression of α-smooth muscle actin in a dose-dependent manner. Expression of snail, the known inducer of EMT, was also upregulated by thrombin. Blockade of PAR-1, but not PAR-4 partially restored the altered expression of thrombin-induced EMT markers. Besides, thrombin also stimulated the expression of profibrotic growth factors (TGFβ1 and CTGF) and extracellular matrix (ECM) proteins (fibronectin and collagen IV). The induction of TGFβ1 and collagen IV were suppressed by PAR-1 antagonist; while the induction of fibronectin was inhibited by both PAR-1 and PAR-4 antagonists. CONCLUSIONS: Our data demonstrated that thrombin promoted EMT and increased the production of profibrotic factors and ECM proteins in tubular epithelial cells. These effects were partially mediated by PAR-1 and, to a lesser extent, PAR-4. These results suggest that modulation of PAR signaling may provide a potential therapeutic strategy for the treatment of renal fibrosis. Fund support: Research Grants Council of Hong Kong (GRF grant number 7796/11M) and Small Project Funding (project code 201309176032) from the University of Hong Kong.