【摘要】 目的探讨醛固酮能否在肾小球系膜细胞(GMC)激活钠氢交换子1(NHE1),以及能否通过NHE1诱导GMC细胞外基质增生。方法体外培养大鼠GMC,并按如下分组:对照组(Con,普通培养液);醛固酮组(Aldo,10-7mol／L);醛固酮+安体舒通组(Aldo+Spir 10-9 mol／L);醛固酮+NHE1抑制剂DMA组(Aldo+DMA 25μmol／L)。24 h后收集培养上清液和各组细胞,抽提总RNA。用酸负荷后钠依赖的pHi(细胞内pH值)的变化来检测NHE1活性,同时采用实时PCR以及流式细胞术检测NHE1基因和蛋白的表达。采用实时PCR和ELISA方法检测纤连蛋白(FN)基因和蛋白的表达。结果与对照组比较,Aldo组肾小球系膜细胞NHE1的活性(△pHi／100S)显著增高[Con(4．48±0．25)％,Aldo(5．29±0．11)％,P<0．05]。加入安体舒通和DMA后,上述NHE1活性均有所降低[Aldo+Spir(4．92±0．35)％,Aldo+DMA(4．07±0．23)％,与Aldo组比较,P<0．05]。实时PCR结果显示Aldo组NHE1 mRNA水平有所增高,是对照组的1．16倍(P<0．05),而Aldo+Spir组NHE1 mRNA水平明显下降,是Aldo组的81．9％(P<0．05)。流式细胞术结果显示Aldo组NHE1蛋白水平显著增高,是对照组的2．9倍(P<0．01),而Aldo+Spir组NHE1蛋白水平明显下降,仅为Aldo组的52．3％(P<0．05),安体舒通本身对NHE1的基因和蛋白表达没有显著影响。实时PCR结果显示Aldo组FN mRNA水平有所增高,是对照组的1．03倍(P<0．05); Aldo+Spir组和Aldo+DMA组FN mRNA水平有所下降,分别是Aldo组的91．21％(P<0．01)和92．30％(P<0．01)。ELISA结果显示培养上清液中分泌的FN(ng／ml)也表现为相同的趋势[Con(17．84±3．77),Aldo(51．66±1．40),P<0．01;Aldo+Spir(29．60±1．99),Aldo+DMA(25．75±4．66),与Aldo组比较,P<0．01]。结论醛固酮能够刺激肾小球系膜细胞分泌细胞外基质成分FN,其作用可能是由于醛固酮激活系膜细胞NHE1活性、上调其基因和蛋白表达所致。更多还原

【Abstract】 Objective To investigate whether aldosterone(Aldo)can activate Na+-H+ exchange isoform 1 and increase its expression in rat mesangial cells (MC),and this effect on the proliferation of exracellular matrix (ECM) in rat mesangial cells. Methods Rat mesamgial cells were cultured and then divided as followings: control group (Con), Aldo group (Aldo of the concentration of 10-7mol/L was added),Aldo+spironolactone group(spironolactone 10-9mol/L and Aldo 10-7mol/L were added), Aldo+dimethylamiloride (DMA) group (DMA 25μmol/L and Aldo 10-7 mol/L were added) and spironolactone group ( spironolactone 10-9 mol/L was added). Twenty-four hours later, the mesangial cells, the supematants and the RNAs of the cells in different groups were collected. The NHE activity was calculated from the initial rate of Na+ dependent pHi recovery after acid load.NHE 1 mRNA expression and protein abundance were measured by real-time PCR and flow cytometry analysis. The mRNA expression and protein level of FN were examined by real-time PCR and EOSA respectively. Results After 24 h exposure of MCs to aldosterone(10-7 mmol/L), NHE1 activity was increased compared to control[Con(4.48±0.25)%, Aldo (5.29±0.11)%, P < 0.05], the antagonists of mineralocorticoid receptor (MR) spironolactone and the NHE-1 inhibitor DMA could inhibit this effect[Aldo+Spir(4.92±0.35)%, Aldo+DMA (4.07±0.23)% , vs Aldo, P<0.05]. NHE1 mRNA expression was increased by 1.16 fold of Con(P< 0.05), and spironolactone could inhibit this effect to 81.9% of Aldo(P< 0.05).NHE1 protein abundance was increased by 2.9 fold of Con(P< 0.01), and spironolactone could inhibit this effect to only 52.3% of Aldo (P < 0.05). Spironolactone itself had no effect on NHE1 mRNA expression and protein abundance. FN mRNA expression was increased by 1.03 fold of Con (P<0.05), spironolactone and DMA could inhibit this effect to 91.21% and 92.30% of Aldo respectively (P<0.01). ELISA showed the similar result in the concentration of FN(ng/ml) (Con 17.84±3.77, Aldo 51.66±1.40,P < 0.01; Aldo+Spir 29.60±1.99,Aldo+DMA 25.75±4.66, vs Aldo, P<0.01). Conclusion Aldosterone can activate NHE1 and induce NHE-1-dependent proliferation of ECM in RMCs.更多还原