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

doi:10.3877/cma.j.issn.2095-3216.2016.01.006

Abstract

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

Yun Fan, Xi Qiao, Lihua Wang. Effect and mechanism of intermedin in NRK- 52E cellular fibrosis induced by angiotensin Ⅱ[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2016, 05(01): 23-28.

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Figures/Tables 6

References 18

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基因	引物序列
Col I	5′- GACATGTTCAGCTTTGTGGACCTC- 3′
Col I	5′- AGGGACCCTTAGGCCATTGTGTA- 3′
β- actin	5′- CCCATCTATGAGGGTTACGG- 3′
β- actin	5′- TTTAATGTCACGCACGATTC- 3′

基因	引物序列
Col I	5′- GACATGTTCAGCTTTGTGGACCTC- 3′
Col I	5′- AGGGACCCTTAGGCCATTGTGTA- 3′
β- actin	5′- CCCATCTATGAGGGTTACGG- 3′
β- actin	5′- TTTAATGTCACGCACGATTC- 3′

分组	eNOS(ng/L)	cGMP(nmol/L)
正常对照组	732.70±11.64	16.17±0.66
Ang Ⅱ组	1247.31±28.76^a	33.03±3.44^a
IMD质粒＋Ang Ⅱ组	1532.12±36.13^ab	71.98±0.89^ab
空质粒＋Ang Ⅱ组	1319.65±54.29^a	37.04±3.24^a

分组	eNOS(ng/L)	cGMP(nmol/L)
正常对照组	732.70±11.64	16.17±0.66
Ang Ⅱ组	1247.31±28.76^a	33.03±3.44^a
IMD质粒＋Ang Ⅱ组	1532.12±36.13^ab	71.98±0.89^ab
空质粒＋Ang Ⅱ组	1319.65±54.29^a	37.04±3.24^a

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