Arecoline-induced pro-fibrotic proteins in LLC-PK1 cells are dependent on c-Jun N-terminal kinase

Areca nut (AN) chewing is associated with chronic kidney disease (CKD). However, the molecular mechanisms of AN-induced CKD are not known. Thus, we studied the effects of arecoline, a major alkaloid of AN, on proximal tubule (LLC-PK1) cells in terms of cytotoxicity, fibrosis, transforming growth factor-beta (TGF-beta) and c-Jun N-terminal kinase (JNK). We found that arecoline dose (0.1-0.5 mM) and time (24-72 h)-dependently induced cytotoxicity without causing cell death. Arecoline (0.25 mM) also time-dependently (24-72 h) increased fibronectin and plasminogen activator inhibitor-1 (PAI1) protein expressions. Arecoline (0.25 mM) time-dependently (24-72 h) increased TGF-beta gene transcriptional activity and supernatant levels of active TGF-beta 1. Moreover, arecoline (0.25 mM) activated JNK while SP600125 (a JNK inhibitor) attenuated arecoline-induced TGF-beta gene transcriptional activity. SP600125, but not SB431542 (a TGF-beta receptor type I kinase inhibitor), attenuated arecoline-induced fibronectin and PAI1 protein expressions. Finally, tubulointerstitial fibrosis occurred and renal cortical expressions of fibronectin and PAI1 proteins increased in arecoline-fed mice at 24 weeks. We concluded that arecoline induced tubulointerstitial fibrosis in mice while arecoline-induced TGF-beta and pro-fibrotic proteins (fibronectin, PAI1) are dependent on JNK in LLC-PK1 cells. (C) 2016 Elsevier Ireland Ltd. All rights reserved.