vorapaxar and Reperfusion-Injury

Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis.. We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition.. During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation.. Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Topics: Acute Kidney Injury; Animals; Endothelial Cells; Fibrosis; Humans; Hypoxia; Inflammation; Kidney; Mice; Receptor, PAR-1; Renal Insufficiency, Chronic; Reperfusion Injury; Thrombin; Vascular Endothelial Growth Factor A; Vascular System Injuries

Coagulation disturbances in several liver diseases lead to thrombin generation, which triggers intracellular injury via activation of protease-activated receptor-1 (PAR-1). Little is known about the thrombin/PAR-1 pathway in hepatic ischemia-reperfusion injury (IRI). The present study aimed to clarify whether a newly selective PAR-1 antagonist, vorapaxar, can attenuate liver damage caused by hepatic IRI, with a focus on apoptosis and the survival-signaling pathway.. A 60-min hepatic partial-warm IRI model was used to evaluate PAR-1 expression in vivo. Subsequently, IRI mice were treated with or without vorapaxar (with vehicle). In addition, hepatic sinusoidal endothelial cells (SECs) pretreated with or without vorapaxar (with vehicle) were incubated during hypoxia-reoxygenation in vitro.. In naïve livers, PAR-1 was confirmed by immunohistochemistry and immunofluorescence analysis to be located on hepatic SECs, and IRI strongly enhanced PAR-1 expression. In IRI mice models, vorapaxar treatment significantly decreased serum transaminase levels, improved liver histological damage, reduced the number of apoptotic cells as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining (median: 135 versus 25, P = 0.004), and induced extracellular signal-regulated kinase 1/2 (ERK 1/2) cell survival signaling (phospho-ERK/total ERK 1/2: 0.96 versus 5.34, P = 0.004). Pretreatment of SECs with vorapaxar significantly attenuated apoptosis and induced phosphorylation of ERK 1/2 in vitro (phospho-ERK/total ERK 1/2: 0.66 versus 3.04, P = 0.009). These changes were abolished by the addition of PD98059, the ERK 1/2 pathway inhibitor, before treatment with vorapaxar.. The results of the present study revealed that hepatic IRI induces significant enhancement of PAR-1 expression on SECs, which may be associated with suppression of survival signaling pathways such as ERK 1/2, resulting in severe apoptosis-induced hepatic damage. Thus, the selective PAR-1 antagonist attenuates hepatic IRI through an antiapoptotic effect by the activation of survival-signaling pathways.

Topics: Animals; Apoptosis; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Humans; Lactones; Liver; Male; MAP Kinase Signaling System; Mice; Pyridines; Receptor, PAR-1; Reperfusion Injury; Thrombin

Protease-activated receptor (PAR)-1 has emerged as a key profibrotic player in various organs including kidney. PAR-1 activation leads to deposition of extracellular matrix (ECM) proteins in the tubulointerstitium and induction of epithelial-mesenchymal transition (EMT) during renal fibrosis. We tested the anti-fibrotic potential of vorapaxar, a clinically approved PAR-1 antagonist for cardiovascular protection, in an experimental kidney fibrosis model of unilateral ureteral obstruction (UUO) and an AKI-to-chronic kidney disease (CKD) transition model of unilateral ischemia-reperfusion injury (UIRI), and dissected the underlying renoprotective mechanisms using rat tubular epithelial cells. PAR-1 is activated mostly in the renal tubules in both the UUO and UIRI models of renal fibrosis. Vorapaxar significantly reduced kidney injury and ameliorated morphologic changes in both models. Amelioration of kidney fibrosis was evident from down-regulation of fibronectin (Fn), collagen and α-smooth muscle actin (αSMA) in the injured kidney. Mechanistically, inhibition of PAR-1 inhibited MAPK ERK1/2 and transforming growth factor-β (TGF-β)-mediated Smad signaling, and suppressed oxidative stress, overexpression of pro-inflammatory cytokines and macrophage infiltration into the kidney. These beneficial effects were recapitulated in cultured tubular epithelial cells in which vorapaxar ameliorated thrombin- and hypoxia-induced TGF-β expression and ECM accumulation. In addition, vorapaxar mitigated capillary loss and the expression of adhesion molecules on the vascular endothelium during AKI-to-CKD transition. The PAR-1 antagonist vorapaxar protects against kidney fibrosis during UUO and UIRI. Its efficacy in human CKD in addition to CV protection warrants further investigation.

Topics: Animals; Biomarkers; Cell Hypoxia; Endothelial Cells; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Inflammation; Kidney; Kidney Tubules; Lactones; Macrophages; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxidative Stress; Pyridines; Rats; Reactive Oxygen Species; Receptor, PAR-1; Reperfusion Injury; Smad3 Protein; Thrombin; Transforming Growth Factor beta; Up-Regulation; Ureteral Obstruction