| Objectives To investigate the preventive effects of spironolactone on renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction(UUO) in rats.Methods (1)32 healthy male Sprague-Dawley rats were randomly divided into sham-operated group, UUO group, low dose spironolactone-treated group(20mg.kg-1.d-1) and high dose spironolactone-treated group(50mg.kg-1.d-1). The left ureter was exposed via an abdominal midline incision under anesthesia. UUO was created by placing 2 sutures around the left mid ureter. Then, the abdomen was closed in 2 layers. In sham-operated animals the ureter was mobilized and slightly manipulated but not ligated. Rats in low and high dose spironolactone-treated group were treated with a daily dose of spironolactone (20mg/kg or 50mg/kg) by gastric gavage at 24 hours before surgery until being sacrificed. Correspondingly, rats in UUO group or sham-operated group were daily administered with the same amount of water as controls. The experiment was terminated at the 14th day after operation. (2)Biochemical parameters and blood pressure measurement: On the day 14, after measurement of systolic blood pressure, animals were decapitated. Blood was taken from abdominal aorta of all rats. Serum potassium, sodium, creatinine and blood pressure were measured on the 14th day after operation. (3) The left kidney was rapidly removed and processed for light microscopic evaluation of renal histology. Fixed sections of the left kidney were cut and stained with hematoxylin-eosin stain and Masson Trichrome staining. Coded slides were examined using light microscopy to determine the degree of renal fibrosis. (4) Immunohistochemistry was performed to examine the tubulointerstitial expression of transforming growth factor-β1 (TGF-β1),α-smooth muscle actin (α-SMA) and fibronectin (FN).Results (1) Biochemical parameters and blood pressure measurement: There were no significant changes in the level of serum potassium, sodium concentrations among the four groups. After 14 days of UUO, the creatinine and blood pressure values were significantly higher compared with sham-operated group(P<0.05), but there was not significantly different between low and high dose spironolactone-treated group(P>0.05).(2) Histological studies: Fourteen days after UUO, Masson Trichrome staining displayed severe tubular dilation and interstitial expansion with collagen accumulation and deposition compared with the sham-operated group (assessed by the percentage area of green-stained region by Masson Trichrome staining (P<0.01)). By contrast, rats in low and high dose spironolactone-treated group exhibited obvious attenuation of these morphological lesions (P<0.05). There were no significant differences between low and high dose spironolactone-treated group (P>0.05). (3) Immunohistochemical studies: The expression of tubulointerstitial TGF-β1,α-SMA and FN of UUO group was markedly up-regulated in contrast to the sham-operated group (P<0.01), whereas the expression of tubulointerstitial TGF-β1,α-SMA and FN in low and high dose spironolactone-treated group was significantly less than that of UUO group(P<0.05). The expression of tubulointerstitial TGF-β1,α-SMA and FN was not statistically different between two spironolactone-treated group (P>0.05). (4)Correlation analysis: Correlation analysis was performed between the degree of renal interstitial fibrosis and the expression of TGF-β1,α-SMA and FN. There was a significant positive correlation between the collagen deposition measured by Masson Trichrome staining and the expression of TGF-β1 ,α-SMA and FN(P<0.01). The interstitial expression of TGF-β1,α-SMA correlated significantly with the expression of FN (P<0.01). The interstitial expression of TGF-β1, correlation withα-SMA also reached statistical significance (P<0.01).Conclusions Spironolactone ameliorates renal fibrosis presumably via the inhibition of cytokine release and tubular-epithelial-myofibroblast transdifferentiation independent of its blood pressure-lowing effect. |
PDF Full Text Request