Fatty acid amide hydrolase (FAAH) inhibition mitigates TGF‐β1‐induced fibrogenesis via the anandamide‐COX‐2‐dependent pathway

Abstract

Chronic proximal tubular injury with subsequent fibrosis is the key factor in the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). An increasing body of evidence has shown that endocannabinoids such as anandamide (AEA) and 2‐arachidonoylglycerol (2AG) and their cannabinoid receptors (CBs) play important roles in renal diseases. It remains unclear whether substrates or metabolites of AEA‐metabolizing enzyme FAAH participate in fibrogenesis of kidneys. Though AEA binds the CB1 receptor with high binding affinity, its in vivo pharmacological effects mediated by cannabinoid receptors are of short duration. Likely, active metabolites produced from the rapid catabolism of AEA exert physiological actions. We have previously showed that inhibition of FAAH protects against TGF‐β1‐induced damages via actions independent of CBs in renal tubular cells. Therefore, our aims are to study the effects of downstream metabolites derived from FAAH as well as AEA‐ cyclooxygenase‐2 (COX‐2) pathways on TGF‐β1‐induced renal fibrogenesis. To produce the AKI to CKD transition model, male wild type (WT) C57BL6 and Faah−/− mice were subjected to post ischemia‐reperfusion injury (PIR) by unilateral kidney ischemia‐reperfusion and contralateral nephrectomy 10 days after ischemia‐reperfusion. Our previous studies showed that Faah−/− mice protected against PIR‐induced renal fibrogenesis. Here, LC‐MS/MS assay demonstrated that PIR‐injured WT mice had a significant downregulation of renal AEA level, which was blocked in Faah−/−‐PIR mice (68% reduction in WT‐PIR versus 12% in Faah−/−‐PIR mice compared with WT‐sham group, p<0.001), whereas 2AG showed no difference between WT‐PIR and Faah−/−‐PIR mice (1.2‐fold versus 1.1‐fold increase, p>0.9). In addition, Faah−/− mice did not show difference in the levels of arachidonic acid (AA) (1.13‐fold to WT‐sham, p=0.25), the downstream metabolites of FAAH. Our previous in vitro studies showed that TGF‐β1‐induced profibrotic effects in HK‐2 cells (human kidney proximal tubular epithelial cells) were abolished after the treatment of FAAH inhibitor (PF‐04457845, PF, 1μM) in a CB2‐independent manner. In the current study, blockage of AEA‐COX‐2 derived prostamides pathway by a substrate‐selective COX‐2 inhibitor (LM‐4131, 300nM) reversed the decreased level of a profibrotic marker α‐smooth muscle actin (α‐SMA) caused by PF (1.9‐fold in TGF‐β1, 0.83‐fold in PF+TGF‐β1 and 2.88‐fold in PF+LM‐4131+TGF‐β1 compared to the control group, p<0.0001). Further, supplement of AA did not reverse the protection of FAAH inhibition against TGF‐β1‐induced expression of α‐SMA (2.53‐fold in TGF‐β1, 0.62‐fold in PF+TGF‐β1 versus 0.48‐fold in AA+PF+TGF‐β1 compared to the control group, p>0.99). The AEA‐COX‐2 metabolite, prostamide E2 mimicked the protective effect of FAAH inhibition against TGF‐β1‐induced α‐SMA expression (2.17‐fold in TGF‐β1 vs. 0.9‐fold in prostamide E2 +TGF‐β1 compared to the control group, p<0.0001). Overall, the present study suggests that inactivation of FAAH augments the actions of downstream metabolites of AEA‐COX‐2, which mediates the protection against TGF‐β1‐induced renal fibrogenesis.