Supporting information_Role of transient receptor potential vanilloid 4 in therapeutic anti-fibrotic effects of pirfenidone

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal lung disorder characterized by aberrant extracellular matrix deposition in the interstitium. Pirfenidone is an anti-fibrotic agent used to treat patients with IPF. Pirfenidone shows a pleiotropic mode of action, but its underlying anti-fibrotic mechanism is unclear. Transient receptor potential vanilloid 4 (TRPV4), which is a mechanosensitive calcium channel, was recently shown to be related to pulmonary fibrosis. To clarify the anti-fibrotic mechanisms of pirfenidone, we investigated whether TRPV4 blockade had a pharmacological effect in a murine model of pulmonary fibrosis and whether pirfenidone contributes to suppression of TRPV4. Our synthetic TRPV4 antagonist, as well as pirfenidone treatment, attenuated lung injury in the bleomycin mouse model. TRPV4-mediated calcium influx into cells was inhibited by pirfenidone. Additionally, TRPV4-stimulated interleukin-8 release from cells was reduced and a delay in cell migration was abolished by pirfenidone. Furthermore, pirfenidone decreased TRPV4 endogenous ligands in bleomycin-administrated mouse lungs and their production by microsomes of human lungs. We found TRPV4 expression in the bronchiolar and alveolar epithelium and activated fibroblasts of the lungs in patients with IPF. Finally, we showed that changes in forced vital capacity of patients with IPF treated with pirfenidone were significantly correlated with metabolite levels of TRPV4 endogenous ligands in bronchoalveolar lavage fluid. These results suggest that the anti-fibrotic action of pirfenidone is partly mediated by TRPV4 and that TRPV4 endogenous ligands in bronchoalveolar lavage fluid may be biomarkers for distinguishing responders to pirfenidone.