Effect and mechanism of intermedin in NRK- 52E cellular fibrosis induced by angiotensin II

Abstract

Objective To investigate the effect of intermedin (IMD) on angiotensin Ⅱ- induced rat tubular cell (NRK- 52E) fibrosis and its possible mechanisms. Methods NRK- 52E cells were cultured and randomly allotted to the following 4 groups: control group, Ang Ⅱ (10-6 mol/L) group, IMD+ Ang Ⅱ group, and empty plasmid+ Ang Ⅱ group. Cells in IMD+ Ang Ⅱ group and empty plasmid+ Ang Ⅱ group were transfected with pIRES2- IMD or pIRES2- empty vector, respectively, by transfection complex comprising optimal proportion of plasmid and Fugene HD reagents. The transfer efficiency was detected by flow cytometry. The mRNA expression of collagen I (Col I) was detected by real- time RT- PCR. Protein levels of Col I and E- cadherin were examined by Western blot analysis. The contents of eNOS and cGMP in the supernatant were determined by ELISA. Results Compared with the control group, the expression of Col I was significantly increased while E- cadherin markedly decreased in the Ang Ⅱ group (Col I mRNA level t=4.835, P=0.008; Col I protein level P<0.001; E- cadherin protein level t=4.284, P=0.013); eNOS and cGMP levels in the supernatant of Ang Ⅱ- stimulated cells were significantly higher than those in the control cells (eNOS t=20.718, P<0.001; cGMP t=8.324, P=0.001). IMD gene transfer reversed the above changes (Col I mRNA level t=3.302, P=0.039; Col I protein level P<0.001; E- cadherin protein level t=6.045, P=0.004; eNOS t=10.682, P<0.001; cGMP t=18.974, P<0.001 ), while the empty plasmid had no effects on them (Col I mRNA level t=0.951, P=0.395; Col I protein level t=1.208, P=0.294; E- cadherin protein level t=1.613, P=0.182; eNOS t=2.039, P=0.111; cGMP t=1.469, P=0.216). Compared with the Ang Ⅱ group, the eNOS and cGMP levels of the IMD plasmid+ Ang Ⅱ group were further increased (eNOS t=10.682, P<0.001; cGMP t=18.974, P<0.001); and in the empty plasmid+ Ang Ⅱ group, the eNOS and cGMP levels were not significantly different from those in the Ang Ⅱ group (eNOS t=2.039, P=0.111; cGMP t=1.469, P=0.216). Conclusion IMD inhibited the Ang Ⅱ- induced tubular cell fibrosis, which might be achieved, at least partly, by blocking the effects of Ang Ⅱ through the eNOS- cGMP pathway. Key words: Angiotensin Ⅱ; Fibrosis; Intermedin; eNOS; cGMP