Apelin对单侧输尿管结扎小鼠肾脏纤维化的影响研究

Effects of Apelin on Renal Fibrosis in Unilateral Ureteral Obstruction Mice

目的探讨生物活性肽Apelin对单侧输尿管结扎(UUO)小鼠肾脏纤维化的影响及相关机制,分析在肾脏纤维化时血清和肾脏局部Apelin/APJ系统的代偿性变化。方法 2014年1月选取7周龄雄性C57Bl/6j小鼠共60只,构建UUO的小鼠模型,并设立假手术组(Sham)作为对照。术后每天给小鼠腹腔注射Apelin-13(0.1μmol/kg)或等量0.9%氯化钠溶液(vehicle)。根据小鼠手术方式和术后用药情况分为4组(15只/组):Sham+vehicle组、Sham+Apelin组、UUO+vehicle组、UUO+Apelin组。术后2周将小鼠处死,取手术侧肾脏进行检测。采用Masson染色观察肾脏纤维化程度;采用实时荧光定量PCR(RT-PCR)法检测肾脏细胞外基质胶原〔纤维连接蛋白(Fibronectin)、Ⅰ型胶原(Collagen I)、Ⅲ型胶原(CollagenⅢ)〕表达水平以及肾脏Apelin、APJ表达水平;采用Western blotting法检测肾小管上皮细胞标志物上皮细胞钙黏蛋白(E-cadherin)、间充质细胞标志物α平滑肌肌动蛋白(α-SMA)表达水平以及转化生长因子β(TGF-β)/Smads信号通路的主要信号分子(TGF-β1、TGF-βR1、p-Smad2)表达水平;采用ELISA法检测小鼠血清Apelin表达水平。结果 4组小鼠肾脏纤维化程度比较,差异有统计学意义(P<0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏纤维化程度均高于Sham+vehicle组和Sham+Apelin组(P<0.05);UUO+Apelin组小鼠肾脏纤维化程度低于UUO+vehicle组(P<0.05)。4组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平比较,差异均有统计学意义(P<0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平高于Sham+vehicle组和Sham+Apelin组(P<0.05);UUO+Apelin组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平低于UUO+vehicle组(P<0.05)。4组小鼠肾脏E-cadherin、α-SMA表达水平比较,差异均有统计学意义(P<0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏E-cadherin表达水平低于Sham+vehicle组和Sham+Apelin组,α-SMA表达水平高于Sham+vehicle组和Sham+Apelin组(P<0.05);UUO+Apelin组小鼠肾脏E-cadherin表达水平高于UUO+vehicle组,α-SMA表达水平低于UUO+vehicle组(P<0.05)。4组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平比较,差异均有统计学意义(P<0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平均高于Sham+vehicle组和Sham+Apelin组(P<0.05);UUO+Apelin组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平均低于UUO+vehicle组(P<0.05)。4组小鼠肾脏Apelin表达水平比较,差异无统计学意义(P>0.05);4组小鼠肾脏APJ、血清Apelin表达水平比较,差异有统计学意义(P<0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏APJ表达水平高于Sham+vehicle组和Sham+Apelin组,血清Apelin表达水平低于Sham+vehicle组和Sham+Apelin组(P<0.05);UUO+Apelin组小鼠肾脏APJ表达水平低于UUO+vehicle组,血清Apelin表达水平高于UUO+vehicle组(P<0.05)。结论 Apelin通过干扰经典的TGF-β/Smads信号通路抑制UUO小鼠肾脏的肾小管上皮-间充质转分化,进而减轻肾脏纤维化。在肾脏纤维化时,小鼠血清Apelin表达水平降低,肾脏APJ表达水平升高,可能对慢性肾脏病的进展起到一定的代偿作用。

Objective To investigate the effect of biological active peptide Apelin and its mechanism and analyze the compensatory change of partial Apelin / APJ system of serum and kidney in the process of renal fibrosis in unilateral ureteral obstruction（ UUO） mice. Methods In January 2014,a total of 60 seven- week- old male C57 Bl /6j mice were selected. We established mice models of UUO and made Sham operated mice as controls. After surgery,intraperitoneal injection of Apelin- 13（ 0. 1 μmol/kg） or the same amount of 0. 9% sodium chloride solution（ vehicle） was administrated on mice everyday. The mice were divided into four experimental groups（ 15 mice / group） according to operation mode and postoperative drug administration：Sham ＋ vehicle group,Sham ＋ Apelin group,UUO ＋ vehicle group and UUO ＋ Apelin group. Two weeks after surgery,mice were killed and the operative kidneys were taken for various tests. Masson＇s trichrome staining was performed to evaluate the extent of renal fibrosis. RT-PCR analysis was performed to evaluate the expression of kidney extracellular matrix collagens,including Fibronectin,Collagen Ⅰ and Collagen Ⅲ,as well as the expression of Apelin and APJ in kidneys. Western blotting method was performed to determine the expression of E- cadherin and α- SMA, as well as the expression of TGF-β / Smads signaling molecules TGF-β1, TGF-βR1, and p- Smad2. ELISA method was performed to determine the expression of Apelin in serum. Results The 4 groups were significantly different in the degree of renal fibrosis（ P〈0. 05）. UUO ＋ vehicle group and UUO ＋ Apelin group were higher than Sham ＋ vehicle group and Sham ＋ Apelin group in the degree of renal fibrosis（ P〈0. 05）; UUO ＋ Apelin group was lower than UUO ＋ vehicle group in the degree of renal fibrosis（ P〈0. 05）. The 4 groups were significantly different in the expression of Fibronectin,CollagenⅠand Collagen Ⅲ（ P〈0. 05）. UUO ＋ vehicle group and UUO ＋ Apelin group were higher than Sham ＋ vehicle group and Sham ＋ Apelin group in the expression of Fibronectin,CollagenⅠ and Collagen Ⅲ in kidney（ P〈0. 05）; UUO ＋ Apelin group was lower than UUO ＋ vehicle group in the expression of Fibronectin, Collagen Ⅰ and Collagen Ⅲ（ P〈0. 05）. The 4 groups were significantly different in the expression of E- cadherin and α- SMA in kidney（ P〈0. 05）. UUO ＋ vehicle group and UUO ＋ Apelin group were lower in the expression of E- cadherin and higher in the expression of α- SMA than Sham ＋ vehicle group and Sham ＋ Apelin group（ P〈0. 05）; UUO＋ Apelin group was higher in the expression of E- cadherin and lower in the expression of α- SMA than UUO ＋ vehicle group（ P〈0. 05）. The 4 groups were significantly different in the expression of TGF-β1,TGF-βR1 and p- Smad2（ P〈0. 05）.UUO ＋ vehicle group and UUO ＋ Apelin group were higher than Sham ＋ vehicle group and Sham ＋ Apelin group in the expression of TGF-β1 、TGF-βR1、p- Smad2（ P〈0. 05）; UUO ＋ Apelin group was lower than UUO ＋ vehicle group in the expression of TGF-β1,TGF-βR1,p- Smad2（ P〈0. 05）. The 4 groups were significantly different in the expression of Apelin in kidney（ P〈0. 05）; the 4 groups were significantly different in the expression of APJ in kidney and Apelin in serum（ P〈0. 05）. UUO ＋ vehicle group and UUO ＋ Apelin group were higher in the expression of APJ in kidney and lower in the expression of Apelin in serum than Sham ＋ vehicle group and Sham ＋ Apelin group（ P〈0. 05）; UUO ＋ Apelin group was lower in the expression of APJ in kidney and higher in the expression of Apelin in serum than UUO ＋ vehicle group（ P〈0. 05）.Conclusion Apelin is able to inhibit epithelial- mesenchymal transdifferentitation in UUO mice by interfering TGF-β / Smads signal pathway,thus alleviating renal fibrosis. During the process of renal fibrosis,the expression of serum Apelin decreases and the expression of kidney APJ increases in mice,which may have certain compensatory effect on the development of chronic kidney diseases.