依普利酮抑制慢性肾脏病妊娠大鼠梗阻对侧肾脏血管新生并减轻肾间质纤维化

Eplerenone alleviates renal angiogenesis and interstitial fibrosis in contralateral kidney of obstructed pregnant rats with chronic kidney disease

目的:探讨依普利酮抑制慢性肾脏病妊娠大鼠梗阻对侧肾脏血管新生的机制及作用途径。方法:未经产雌性Wistar大鼠60只随机分为假手术组、假手术+妊娠组、模型组和依普利酮组,每组15只。模型组和依普利酮组大鼠采用单侧输尿管梗阻(UUO)手术造成肾脏损伤,9周后除假手术组外所有处于动情周期的雌鼠与雄鼠合笼,建立肾损伤妊娠大鼠模型。依普利酮组自UUO术后开始给药,给予依普利酮(100 mg·kg^(-1)·d^(-1))治疗。妊娠第19天处死母鼠,留取血清标本,摘取梗阻对侧肾脏。采用HE、Masson和天狼星红染色观察大鼠肾脏组织病理形态学改变。ELISA法测定血清及肾组织中醛固酮含量,免疫组化、免疫荧光、RT-qPCR和Western blot法检测肾组织中炎症因子、血管新生标志物及低氧诱导因子1α(HIF-1α)/Slit2/Robo1信号通路相关蛋白的表达。结果:肾脏病理切片显示模型组大鼠肾小管扩张明显,有大量胶原纤维沉积及炎症细胞浸润。ELISA结果显示模型组大鼠血清及肾组织中醛固酮含量显著升高(P<0.01)。免疫组化和RT-qPCR结果显示模型组大鼠肾脏盐皮质激素受体(MR)和核因子κB(NF-κB)表达显著升高(P<0.01)。免疫组化和Western blot结果显示模型组大鼠肾脏HIF-1α、分泌型糖蛋白Slit2和跨膜受体Robo1表达显著增多(P<0.01)。免疫荧光和RT-qPCR结果显示模型组大鼠血管新生标志物CD34和CD105表达显著增多(P<0.01)。依普利酮可以减轻肾脏病理损伤,抑制醛固酮分泌,下调MR、NF-κB、HIF-1α、Slit2、Robo1、CD34和CD105表达(P<0.05)。结论:依普利酮可阻断醛固酮与MR的结合,调控HIF-1α/Slit2/Robo1信号通路,抑制慢性肾脏病妊娠大鼠梗阻对侧肾脏血管新生,从而延缓肾间质纤维化进展。

AIM:To investigate the mechanism of eplerenone inhibiting angiogenesis in the contralateral kidney of obstructed pregnant rats with chronic kidney disease.METHODS:Sixty nulliparous female Wistar rats were randomly divided into 4 groups:sham group,sham+pregnancy group,model group and eplerenone group,with 15 rats in each group. The rats in model group and eplerenone group were injured by unilateral ureteral obstruction(UUO)operation. After 9 weeks,the female and male rats in the estrous cycle in sham+pregnancy group,model group and eplerenone group were caged together to establish a kidney injury pregnant rat model. The rats in eplerenone group were given eplerenone(100 mg·kg^(-1)·d^(-1))after UUO operation. On the 19th day of gestation,the female rats were sacrificed,the serum samples were collected,and the contralateral kidney of the obstruction was removed. Histopathological changes in rat kidneys were observed by HE,Masson and Sirius red staining. Serum and renal tissue aldosterone levels were determined by ELISA. Immunohistochemistry,immunofluorescence,RT-qPCR and Western blot were used to detect the expression of inflammatory factors,angiogenesis markers and hypoxia-inducible factor-1α(HIF-1α)/Slit2/Robo1 signaling pathway-related proteins.RESULTS:Renal pathology showed that the renal tubules in model group were significantly dilated,with a large number of collagen fiber deposition and inflammatory cell infiltration. The serum and renal tissue aldosterone levels in model group were significantly increased(P<0. 01). The expression levels of mineralocorticoid receptor(MR)and nuclear factor-κB(NF-κB)in the kidney of rats in model group were significantly increased(P<0. 01). The expression levels of HIF-1α,secreted glycoprotein Slit2,and transmembrane receptor Robo1 were significantly increased in the kidney of rats in model group(P<0. 01). Angiogenesis markers such as CD34 and CD105 were significantly increased in model group(P<0. 01). Eplerenone alleviated renal pathological damage,inhibited the secretion of aldosterone,and down-regulated the expression levels of MR,NF-κB,HIF-1α,Slit2,Robo1,CD34 and CD105(P<0. 05).CONCLUSION:Eplerenone attenuates renal angiogenesis and interstitial fibrosis in the contralateral kidney of obstructed pregnant rats with chronic kidney disease via HIF-1α/Slit2/Robo1 signaling pathway.