[Effect of aldosterone and its antagonist spironolactone on epithelial-mesenchymal transition of normal rat kidney epithelial cells in high glucose].
OBJECTIVE
To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton.


METHODS
NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4,500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4,500 mg/L) DMEM plus 10(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and alpha smooth muscle actin(alpha-SMA) mRNA expression.


RESULTS
RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and alpha-SMA mRNA expression was significantly increased(P<0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of alpha-SMA mRNA was significantly increased in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P<0.05;r=0.67, P<0.05). E-cadherin mRNA in the SP group was significantly higher,while alpha-SMA mRNA expression was lower than that in the HG group(P<0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and alpha-SMA expression was significantly increased in the HG group(P<0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and alpha-SMA protein expression was significantly higher than that in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P<0.05;r=0.81, P<0.05). In the SP group, E-cadherin protein expression was higher, and alpha-SMA protein level was lower than that in the HG group(P<0.05).


CONCLUSIONS
Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.
Liu
Kanghan
K
Department of Nephrology, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhou
Qiaoling
Q
Ao
Xiang
X
Pouranan
Veeraragoo
V
Hong
Xuemin
X
Xiao
Zhou
Z
Yuan
Mingxia
M
D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003928:Diabetic Nephropathies / Q000517:prevention & control; D004847:Epithelial Cells / Q000166:cytology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D007684:Kidney Tubules / Q000166:cytology*; D008648:Mesoderm / Q000166:cytology; D051381:Rats; D013148:Spironolactone / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D013148:Spironolactone; D000450:Aldosterone; D005947:Glucose