钠氢交换子1短发夹RNA抑制醛固酮引起的系膜细胞纤连蛋白增生

shRNA-mediated knockdown of NHE1 expression inhibits the fibronectin proliferation induced by aidosterone in rat mesangiai cells

目的构建钠氢交换子1（NHE1）的短发夹RNA（shRNA）,用来抑制NHE1表达,进一步明确NHE1是否直接介导醛固酮引起的细胞外基质增生。方法构建靶向NHE1的短发夹状双链RNA的真核表达质粒（shRNA-NHE1）,并将该质粒转染体外培养的大鼠肾小球系膜细胞。分别于转染后1、3、4 d用荧光定量PCR（real-time PCR）和Western印迹观察NHE1 mRNA和蛋白的表达。选择shRNA-NHE1质粒转染后第4天,给予醛固酮（10^-7mol/L）干预24 h,用ELISA法检测培养上清液中细胞外基质成分纤连蛋白（FN）的表达。结果PCR结果显示转染后24 h,shRNA-NHE1组NHE1 mRNA表达下降36.9%;转染第3、4天NHE1 mRNA表达降低69.2%和77.9%。Western印迹显示转染后24 h NHE1的蛋白表达无明显变化;3 d后蛋白水平显著下降,4 d后抑制作用更明显。ELISA结果显示,在未转染的系膜细胞中,醛固酮刺激后上清液中FN水平比对照组明显升高[（51.78±1.15）比（17.74±1.38）μg/L,P〈0.05],而当细胞转染shRNA-NHE1质粒后,醛固酮的上述作用被明显抑制[（28.07±1.73）μg/L,P〈0.05]。结论靶向NHE1的shRNA真核表达载体导入可以特异性抑制大鼠肾小球系膜细胞NHE1的表达,同时显著抑制醛固酮引起的FN增生,提示NHE1在醛固酮诱导的肾小球硬化中起重要作用。

Objective To investigate whether vector-based short-hairpin RNA （shRNA） could efficiently inhibit the expression of NHE1 in rat mesangial cells （MC） and the effect on aldosterone-mediatd proliferation of fibronectin （FN） in rat mesangial cells. Methods The eukaryotic vector of shRNA with insert targeting on the sequence of Na/H exchanger-1 （NHE1） was successfully constructed and transfected into rat mesangial cells. MCs were cultured till day 4 after transfection and the results were compared with those of the control group and non-specific transfected group. The mRNA of NHE1 were reverse transcribed and quantified by real-time PCR. Protein expression was assessed by Western blot. On day 4 after transfection with shRNA-NHE1, aldosterone （10^-7mol/L） was added to the medium, 24 hours later, the supernatant level of FN was examined by ELISA. Results After transfection of shRNA-NHE1, the mRNA expression of NHE1 decreased on day 1, and it decreased progressively on day 3 and day 4, while the suppression of NHE1 protein abundance did not appear until day 3. Its protein abundance was further decreased on day 4. ELISA showed the FN expression induced by aldosterone was increased compared with the control group [（51.78±1.15） vs. （17.74±1.38） μg/L, P 〈 0.05], and shRNA- NHE1 could inhibit this effect remarkably [FN （28.07 ±1.73） μg/L, P 〈 0.05]. Conclusion Vector-based shRNA is a potential tool to inhibit expression of NHE1 and shRNA-NHE1 can inhibit aldosterone-mediatd proliferation of FN in rat mesangial cells, which indicates that NHE1 may play an important role in aldosterone-mediated glomerular sclerosis.