The permissive role of protease-activated receptor-1 in kidney fibrosis

Abstract

Chronic kidney disease (CKD) affects 10 to 15% of the world’s population with limited treatment options. Health-related quality of life and longevity decrease as kidney function declines. A better understanding of its pathogenesis is crucial in the development of new therapy. In CKD, there is robust generation of thrombin with an aberrant activation of the coagulation system, indicating a role of protease-activated receptor-1 (PAR-1), the thrombin receptor, in kidney fibrosis, the final common pathway to kidney failure. This work aims to define the involvement of PAR-1 in CKD and to establish a scientific basis for antagonizing PAR-1 as a treatment strategy. In vitro, PAR-1 activation led to extracellular matrix (ECM) proteins accumulation and epithelial-mesenchymal transition (EMT) in rat tubular epithelial cells under a hypercoagulable condition. Mechanistically, PAR-1 mediated thrombin-induced oxidative stress, inflammatory and fibrotic responses via ERK MAPK and TGF-β/Smad signaling. In vivo, pharmacological inhibition of PAR-1 by vorapaxar, a clinically approved PAR-1 antagonist in cardiovascular disease, against kidney fibrosis was investigated in two murine models, namely, unilateral ureteral obstruction (UUO) and unilateral ischemia reperfusion injury (UIRI)-induced CKD. Vorapaxar significantly ameliorated kidney injury and tubulointerstitial fibrosis with reduction of fibronectin, collagen and α-smooth muscle actin in the injured kidney. ERK MAPK and TGF-β/Smad pathway-mediated oxidative events, overexpressed pro-inflammatory cytokines and macrophage infiltration were suppressed with vorapaxar during UUO and UIRI. These beneficial effects of vorapaxar were recapitulated in thrombin- and hypoxia-induced cultured tubular epithelial cells. To further explore the role of PAR-1 in acute kidney injury (AKI)-to-CKD transition, a longitudinal study of UIRI-induced CKD using PAR-1 deficient mice was conducted. PAR-1 deficiency mitigated capillary loss and leukocyte adhesion during the early stage of AKI, and thereafter alleviated progression to tubulointerstitial fibrosis with abated macrophage recruitment in the later phase. At day 3 post UIRI, PAR-1 deficiency reduced vascular injury and preserved endothelial integrity and capillary permeability. When the observation was extended for up to 28 days, there was continuous tubular injury with loss of kidney function. PAR-1 deficiency diminished tubulointerstitial fibrosis and preserved kidney function during the transition phase to CKD at day 7 and through to the late phase at day 28 post UIRI. Chronic inflammation was also prevented in PAR-1 deficient mice with reduced recruitment of infiltrating macrophages as shown by flow cytometry analysis. Finally, the detrimental role of PAR-1 in vascular injury was demonstrated in thrombin-induced cultured human dermal microvascular endothelial cells. PAR-1 induced a pro-inflammatory phenotype, and mediated an angiogenic overactivity by promoting leukocyte-endothelial cell adhesion in cells incubated under a hypercoagulable state. Silencing of PAR-1 arrested vascular inflammation with suppressed expression of the angiogenesis marker vascular endothelial growth factor and leukocyte adhesion molecules in microvascular endothelial cells. Collectively, these findings suggest that PAR-1 activation mediates thrombin-induced profibrotic responses and vascular dysfunction upon tissue injury, and subsequently accelerates the transmigration of chronic inflammatory cells and the progression of tubulointerstitial fibrosis to culminate in AKI-to-CKD transition. Targeting PAR-1 by using vorapaxar may provide a promising treatment approach for both AKI and progressive kidney fibrosis.