Inactivation of Endocannabinoid‐metabolizing Enzyme FAAH Represses TGF‐β1‐induced Epithelial‐mesenchymal Transition in Renal Proximal Tubular Epithelial Cells

Abstract

Epithelial-mesenchymal transition (EMT) of proximal tubular epithelial cells plays a very important role in the development of renal fibrosis and progressive chronic kidney disease (CKD). No effective therapy for renal fibrosis currently exists. Endocannabinoid system has been described to be involved in renal fibrosis. Anandamide and 2-arachidonoylglycerol, two major endocannabinoids, have been shown to participate in the regulation of renal physiology via cannabinoid receptors (CB1 and CB2). CB1 and CB2 are demonstrated to also participate in CKD. However, the role of fatty acid amide hydrolase (FAAH), a major anandamide-metabolizing enzyme, in CKD still remains poorly understood. We previously reported that FAAH knockout mice showed improved renal function and kidney fibrosis following post ischemia-reperfusion injury. The present study tested the hypothesis that FAAH participates in TGF-β1–induced EMT in renal cells. In HK2 cells, a human proximal tubular epithelial cell line, treatment of FAAH inhibitor PF-04457845 significantly attenuated TGF-β1–induced expression of the mesenchymal marker α-smooth muscle actin (α-SMA, 1.6-fold in TGF-β1 vs. 0.7-fold in TGF-β1+PF to the control group, p<0.05) and tubular epithelial injury protein NGAL (2.1-fold in TGF-β1 vs. 0.95-fold in TGF-β1+PF to the control group, p<0.05). Similarly, siRNA-mediated knockdown of FAAH also showed reduction of TGF-β1–induced expression of ɑ-SMA (0.94-fold vs. 1.8-fold to Ctrl group, p<0.05) and NGAL (1.3-fold vs. 2.8-fold to Ctrl group, p<0.05). The immunocytochemistry staining results demonstrated that TGF-β1-induced loss of epithelial tight junction protein ZO-1 was restored by inhibition of FAAH (46% reduction vs. 17% reduction to the control group, p<0.05). Because CB2 has been shown to mediate renoprotection, HK-2 cells were treated with CB2 antagonist (SR144528) in combination with FAAH inhibitor (PF-04457845). Blockage of CB2 receptor by SR showed no effect on the reduction of α-SMA expression by FAAH inhibitor, suggesting that the protective effect of FAAH inhibition on tubular EMT is independent of CB2 receptor activation. Overall, the present data suggest that suppression of FAAH attenuates TGF-β1-induced EMT in renal proximal tubular epithelial cells. Thus, FAAH could be a new target for developing an effective treatment for CKD.