【作者】 张敏敏；

【导师】 顾勇； 陈靖； 郝传明；

【作者基本信息】 复旦大学 ， 肾脏病学， 2008， 博士

【摘要】 第一部分钠氢交换子1在醛固酮所致大鼠肾小球硬化中的作用体内研究目的:近年来研究发现,醛固酮与脏器纤维化等病变密切相关,并且可作为一个独立的致病因素直接参与纤维化过程。钠氢交换子1（NHE1）是一种糖蛋白,除了调节酸碱平衡、稳定细胞内pH的作用外,目前有研究发现NHE1还能对细胞周期进行调节,NHE1也被证实参与了肾脏纤维化的发生,但是在肾脏纤维化过程中醛固酮的致纤维化作用是否与NHE1有关尚不清楚。为了证实NHE1在醛固酮所致大鼠。肾小球硬化中的作用,我们在本部分的研究中拟通过体内实验证明大鼠肾小球也是醛固酮作用的靶目标,并在体内环境直接探讨醛固酮是否参与慢性肾小球损伤的发展以及NHE1在其中的作用和相关机制。（一）MR和11β—HSD2在大鼠肾小球中的表达方法:用正常SD大鼠的肾脏分别制备石蜡和冰冻切片,并用筛网法提取肾小球蛋白,同时分别提取肾脏皮质和髓质蛋白。选择醛固酮作用的特异性受体MR和保证醛固酮和其受体特异性结合的酶11β—HSD2作为局部醛固酮系统存在的关键指标,分别用免疫组化、免疫荧光和Western blot检测上述指标在肾脏的分布区域和表达量。结果:1.免疫组化结果显示阳性11β—HSD2表达在肾皮质的远曲小管和集合管,染色阳性细胞在上皮细胞的胞核和胞浆中都有大量分布,但是在肾小球和近曲小管也可见到少量表达,高倍镜下可见在肾小球中的阳性部位主要位于肾小球系膜区和近曲小管的胞浆;采用免疫荧光的方法,发现11β—HSD2主要表达在肾脏的远曲小管和集合管上皮细胞,在肾小球也有少量表达;2.免疫组化结果显示阳性MR表达在肾皮质的远曲小管和集合管,染色阳性细胞在上皮细胞的胞核和胞浆中都有大量表达,但是在肾皮质的肾小球和近曲小管也可见到少量表达;采用免疫荧光的方法,用绿色荧光FITC标记,发现MR主要表达在。肾脏的远曲小管和集合管上皮细胞,在肾小球的系膜区也有少量表达;3.采用免疫荧光的方法,用绿色荧光FITC标记,发现11β—HSD2和MR主要表达在肾脏的远曲小管和集合管上皮细胞,在肾小球的系膜区也有少量表达;4.Western blot检测发现,MR在髓质中表达量最高,在皮质中由于有大量肾小管的存在,也有所表达,我们发现新鲜肾脏获得的肾小球蛋白中能检测到MR的少量表达。结论:在本部分的研究中,我们在体内证实大鼠肾小球内存在MR和11β—HSD2的阳性表达,大鼠肾小球也是醛固酮体内作用的靶目标。（二）钠氢交换子1在醛固酮所致大鼠肾小球硬化中的作用体内研究方法:用体重180—200g雄性SD大鼠建立5/6肾大部切除模型,实验分组如下:（1）假手术组（SHAM组）;（2）5/6肾切组（SNX组）;（3）SNX+醛固酮组（ALDO组）;（4）ALDO+NHE1抑制剂DMA组（DMA组）,成模后12周处死大鼠,进行常规血、尿生化检测及尾动脉收缩压测定,PAS和Masson染色观察大鼠肾小球硬化和肾小管间质纤维化程度;免疫组化和Western blot检测各组大鼠肾组织的FN、TGF—β1和PCNA的表达情况。结果:1.成模后12周,5/6肾大部切除大鼠均出现明显的高血压,蛋白尿和肾功能严重损害。和SNX组相比,ALDO组的血压（mmHg）升高更显著（ALDO∶224±9.4 vs.SNX∶194±6.36,p＜0.01）,蛋白尿（mg/24h）（ALDO∶237.93±10.06 vs.SNX∶82.76±4.65,p＜0.01）和肾功能损害（血清肌酐值:μmol/L）（ALDO∶123.57±11.12 vs.SNX∶85.5±4.96）更严重,DMA组大鼠比ALDO组大鼠蛋白尿有明显减轻（DMA∶163.73±10.15 vs.ALDO∶237.93±10.06,p＜0.01）,但是对高血压和肾功能损害并没有明显的改善作用;2.PAS染色显示,除SHAM组外的四个实验组大鼠肾小球均呈局灶节段性硬化甚至全小球硬化,其中以ALDO组最为显著;与ALDO相比,DMA组的肾小球增殖和硬化程度明显减轻（DMA∶3.1±0.176 vs.ALDO∶3.35±0.15,p＜0.01）。Masson染色显示与SHAM组相比,手术实验组均有明显的的肾小管间质纤维化,伴小箭扩张和蛋白管型。ALDO组和SNX组相比,大鼠的间质纤维化程度明显加重（ALDO∶2.225±0.117 vs.SNX∶1.9±0.085,p＜0.05）,DMA对ALDO所致间质纤维化和蛋白管型没有明显改善作用;3.免疫组化指数评分结果表明,FN在肾大部切除大鼠肾小球和小管间质内均有大量表达,表达水平均显著高于SHAM组。ALDO组和DMA组FN表达明显高于SNX组（ALDO∶0.733±0.04,DMA∶0.725±0.062 vs.SNX∶0.617±0.047,p＜0.01）,但是这两组之间没有明显差异;4.免疫组化和Western blot结果均提示各组大鼠肾组织TGF-β1表达水平显著高于SHAM组,免疫组化指数的评分结果表明,ALDO组TGF-β1表达水平高于SNX组,但是无明显统计学差异（ALDO∶0.73±0.024 vs.SNX∶0.69±0.03,p＞0.05）,和ALDO组相比,DMA组的TGF-β1表达有所下降（DMA∶0.64±0.047 vs.ALDO∶0.73±0.024,p＜0.01）。Western blot结果提示ALDO组TGFβ1蛋白水平比SNX组进一步升高（ALDO∶431.33±19.04 vs.SNX∶207.83±9.61,p＜0.01）。和ALDO组相比,DMA组的TGF-β1表达有所下降（DMA∶0.64±0.047 vs.ALDO∶0.73±0.024,p＜0.05）;5.免疫组化和Western blot结果均提示,肾大部切除后PCNA表达明显高于SHAM组,其中ALDO组表达最高,DMA组的PCNA表达和ALDO组相比有所下降。结论:我们的体内研究结果提示,NHE1介导了醛固酮所致大鼠肾小球硬化的作用,而且不依赖于血流动力学作用。其抑制剂DMA能显著改善5/6肾大切动物模型的肾小球硬化,其机理可能是通过抑制细胞外基质的积聚以及肾小球系膜细胞的增殖。第二部分醛固酮通过钠氢交换子1诱导肾小球系膜细胞外基质增生的作用探讨目的:我们在前面第一部分的体内研究中已经发现,NHE1介导了醛固酮所致大鼠肾小球硬化的作用,而且不依赖于血流动力学作用。其抑制剂DMA能显著改善5/6肾大切动物模型的肾小球硬化,且其机理可能是通过抑制系膜细胞外基质的积聚以及系膜细胞的增殖。由于体内环境比较复杂,为了排除众多体内因素的干扰,我们在本部分的研究中,选择与肾小球硬化细胞外基质分泌密切有关的重要固有细胞——系膜细胞,旨在探讨在肾小球系膜细胞（mesangial cell,MC）上醛固酮是否能够激活NHE1,以及是否能通过NHE1诱导肾小球系膜细胞外基质增生。从而进一步在体外实验中证实NHE1介导了醛固酮所致大鼠肾小球硬化的作用。方法:体外培养大鼠系膜细胞（MC）,并进行如下分组:对照组（普通培养液）,醛固酮组(10^-7mol/L)（Aldo）,醛固酮+安体舒通(10^-9mol/L)组（Aldo+Spir）,醛固酮+NHE1抑制剂Dimethylamiloride（DMA,25μmol/L）组（Aldo+DMA）。24小时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Real-time PCR检测NHE1基因表达,采用流式细胞术和Western blot检测NHE1蛋白表达;采用ELISA方法检测细胞外基质成分纤连蛋白FN的蛋白表达。结果:1.与对照组相比,醛固酮组肾小球系膜细胞NHE1的活性（△pHi/100S）与对照组相比显著增高（Cont 4.48±0.25%vs.Aldo 5.29±0.11%,p＜0.05）,加入安体舒通和DMA后活性均有所降低（Aldo+Spir 4.92±0.35%,Aldo+DMA 4.07±0.23%,vs.Aldo 5.29±0.11%,p＜0.05）;2.Real-time 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的mRNA水平明显下降,是Aldo组的81.9%（p＜0.05）,安体舒通本身对NHE1的基因表达没有影响;3.Western blot结果显示Aldo组NHE1蛋白水平显著增高（Cont 401.74±17.96;vs.Aldo 535.84+8.67,p＜0.01）,而Aldo+Spir组NHE1蛋白水平明显下降（Aldo535.84±8.67 vs.Aldo+Spir 457.64±11.27,p＜0.05）,安体舒通本身对NHE1的蛋白表达没有影响。流式细胞术检测结果与Western blot相似;4.ELISA方法检测纤连蛋白（Fibronectin,FN）的水平,结果显示Aldo组FN水平有所增高（Cont 17.84±3.77 vs.Aldo 51.66±1.40,p＜0.01）;和Aldo组相比,Aldo+Spir组和Aldo+DMA组FN水平（ng/ml）有所下降（Aldo+Spir 29.60±1.99,Aldo+DMA 25.75±4.66,vs.Aldo 51.66±1.40,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,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。第三部分ERK1/2通路介导钠氢交换子1在醛固酮诱导大鼠肾小球系膜细胞外基质增生中的作用目的:在第二部分的研究中,我们的结果提示醛固酮能够刺激肾小球系膜细胞分泌细胞外基质,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。而ERK1/2通路在肾小球系膜细胞的细胞外基质积聚的过程中又发挥了极为重要的作用。因此本部分研究拟通过构建NHE1的短发夹状RNA抑制NHE1表达,从而探讨ERK1/2通路是否介导了醛固酮通过NHE1致大鼠系膜细胞外基质增生的作用。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天、3天、4天采用荧光定量PCR（Real-time PCR）和Western blot法从mRNA和蛋白水平观察对NHE1的抑制作用。转染4天成功抑制NHE1的表达后,实验分组如下:（1）对照组:只加入培养液;（2）醛固酮组:培养液中加入醛固酮10^-7mol/L;（3）醛固酮+shRNA—NHE1组:shRNA-NHE1质粒DNA转染96小时后加入醛固酮10^-7mol/L;（4）醛固酮+安体舒通组:用安体舒通10^-9mol/L先孵育3小时后加用醛固酮10^-7mol/L;（5）醛固酮+PD98059（ERK1/2拮抗剂）组:同时加入醛固酮10^-7mol/L和25μM PD98059。干预24h后,分别提取各组细胞蛋白以及上清液,用于NHE1,ERK1/2和phosphor—ERK1/2的Western blot检测以及ELISA方法检测培养上清液中细胞外基质成分纤连蛋白的表达。结果:1.Real-time PCR结果显示转染后24小时shRNA-NHE1组NHE1的mRNA表达即开始下降36.9%,转染3天、4天后NHE1的基因表达可进一步降低,分别下降69.2%和77.9%;2.Western blot显示转染后24小时NHE1的蛋白表达无明显变化（Control426.56±12.17,Negative 434.9±23.91 vs.1day 368.47±13.7,p＞0.05）;3天后蛋白水平显著下降（negative 434.9±23.91 vs.3day 260.59±12.04,p＜0.01）,4天后抑制作用更明显（negative 434.9±23.91 vs.4day 107.585±7.66,p＜0.01）;3.ELISA方法显示在无该质粒转染的系膜细胞中,醛固酮刺激后可导致上清液中FN水平明显升高,而当细胞转染shRNA-NHE1质粒,醛固酮的上述作用被明显抑制（Control 17.74±1.38ng/ml,Aldo+shRNA-Negative 51.78±1.15ng/ml Vs.Aldo+shRNA-NHE 128.07±1.73ng/ml,p＜0.01）;安体舒通或PD98059干预24h后也可拮抗醛固酮刺激所刺激的FN增生的作用（Aldo+Spir 29.60±1.99ng/ml,Aldo+PD98059 29.82±1.39 ng/ml,vs.Aldo+shRNA-Negative 51.78±1.15ng/ml,p＜0.01）;4.Western blot结果发现醛固酮刺激24h后系膜细胞NHE1的表达增强（Control139.31±9.95 vs.Aldo 351.4±42.16,P＜0.01）可以被PD98059所抑制（Aldo351.4±42.16,vs.Aldo+PD98059 225.68±21.13,p＜0.05）;相反,醛固酮作用24h后,大鼠系膜细胞磷酸化ERK1/2的水平明显增高（Control 280.52±33.46vs.Aldo 513.22±24.77,p＜0.01）,且醛固酮对ERK1/2磷酸化的激活作用在转染shRNA—NHE1成功抑制NHE1表达后依然存在（Aldo 513.22±24.77 vs.Aldo±shRNA-NHE1 462±24.34,p＞0.05）。结论:通过构建靶向NHE1的shRNA真核表达载体导入细胞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细胞外基质增生,且该作用可能是通过ERK1/2通路的激活所导致,因此我们认为ERK1/2通路介导了醛固酮通过NHE1致大鼠系膜细胞外基质增生的作用。更多还原

【Abstract】 PARTⅠThe Role of NHE1 in the Progression of Aldosterone-induced-Glomerulosclerosis in vivoObjectiveAldosterone has been suggested to be involved in the progression of fibrosis of multiple organs.Besides the basic role of the pHi and volume regulation of the cell,many studies suggested that NHEI also had an important role in cell proliferation,division or migration.But whether NHE1 is involved in the aldosterone induced glomerulosclerosis is not clear.This study,therefore,is to further the research on the NHEl-dependent glomerulosclerosis induced by aldosterone in vivo.1.The Expression of MR and 11β-HSD2 in rat glomeruliMethodsThe nomal SD rats are killed for the preparation of the kidney specimen.The cortical and medullary part of the kidneys are separated and the protein of the isolated glomeruli was extracted for the detection of MR and 11β-HSD2 by immunochemistry immunofluorescence and Western blot respectively.Results1.By immunochemistry and immunofluorescence,when rat kidney sections were incubated with MR and 11β-HSD2 antibody,intense immunoreactivity was observed in distal convoluted tubules and collecting ducts,where the reactivity seemed to be localized in the cytoplasm and the nucli.In addition,the antibody also detected moderate immunoreactivity in the glomeruli and proximal tubules.2.To demonstrate the presence of MR protein in the glomerulus,we performed immunoblotting,the MR antibody clearly detected a single band at 102 KD with samples of freshly isolated rat glomeruli as well as whole kidney cortex.ConclusionThe result of this study in vivo showed that MR and 11-HSD2 was expressed not only in distal convoluted tubules and collecting ducts,but also in the glomeruli of the rat kidney. Therefore,we concluded that rat glomeruli are also the target of aldosterone.2.The Role of NHE1 in the Progression of Aldosterone-induced-Glomerulosclerosis in vivoMethodsSD male rats were operated by 5/6 renal ablation and randomly divided as followings: SHAM rats;5/6 nephrectomy rats（SNX group）;SNX rats infused with exogenous aldosterone（ALDO group） or plus DMA（DMA group） by osmotic mini-pump respectively.They were allowed free access to saline and sacrificed at the 12th weeks. Proteinuria,renal function and artery blood pressure of the rats were measured at that time.Glomerulosclerosis,tubulointerstitial fibrosis and tubular proteinaceous cast were evaluated by PAS and Masson stainesss.The expression of FN,TGFβ1 and PCNA were assayed by renal immunohistochemical staining and western blot.Results1.Renal ablation rats had marked proteinuria,hypertension and glomerulosclerosis. Rats treated by aldosterone became more serious than those of SNX group in blood pressure（ALDO:224±9.4 mmHg vs.SNX:194±6.36 mmHg,p＜0.01）,proteinuria （ALDO:237.93±10.06 mg/24h vs.SNX:82.76±4.65 mg/24h,p＜0.01 ） and renal function（SCr:ALDO:123.57±11.12μmol/L vs.SNX:85.5±4.96μmol/L,p＜0.01）. Compared to the ALDO group,the DMA group improved much in proteinuria（DMA: 163.73±10.15 mmHg vs.ALDO:237.93±10.06 mmHg,p＜0.01） but had no effect on the hypertension and the renal function;2.The glomerulosclerosis scores,tubulointerstitial fibrosis and tubular proteinaceous cast were higher in all renal ablation rats groups than the SHAM group,among them the ALDO group showed the most significant.DMA group showed improved glomerulosclerosis compared to the ALDO group（DMA:3.1±0.176 vs. ALDO:3.35±0.15,p＜0.01）.But there was no effect of DMA on the aldosterone-induced tubulointerstitial fibrosis and tubular proteinaceous cast;2.Immunochemistry data demonstrated that the FN level raised much higher in all renal ablation rats than that of SHAM.The expression of FN level was higher in ALDO and the DMA group than the SNX group（ALDO:0.733±0.04,DMA:0.725±0.062 vs. SNX:0.617±0.047,p＜0.01）.But there was no significance between these two groups;3.Immunochemistry and Western blot data demonstrated that the TGFβ1 level raised much higher in all renal ablation rats than that of SHAM.The expression of TGFβ1 level was higher in ALDO group than the SNX group（ALDO:431.33±19.04 vs. SNX:207.83±9.61,p＜0.01）.The expression of TGFβ1 level was lower in DMA group than the ALDO group by western blot（DMA:0.64±0.047 vs.ALDO: 0.73±0.024,p＜0.05）;4.Immunochemistry and Western blot data demonstrated that the PCNA level raised much higher in all renal ablation rats than that of SHAM,among them the ALDO group showed the highest level.The expression of PCNA in the DMA group decreased significantly than that of ALDO group measured either by immunohistoassay or western blot.ConclusionOur data in vivo suggested that NHE1,contributed to the progression of aldosterone induced glomerulosclerosis in the rat through the mechanism of more than systolic blood pressure.DMA,the antagonist of NHE1 ameliorates remnant nephropathy in the rat through the mechanism of inhibiting the ECM accumulation and the proliferation of mesangial cells. PARTⅡEffect of NHE-1-Dependent Accumulation of ECM Induced by Aldosterone on Rat Mesangial CellsObjectiveOur data in vivo suggested that NHE1,contributed to the progression of aldosterone induced glomerulosclerosis in the rat through the mechanism of more than systolic blood pressure.DMA,the antagonist of NHEI ameliorates remnant nephropathy in the rat through the mechanism of inhibiting the ECM accumulation and the proliferation of mesangial cells.The second part of this study,therefore,is to investigate whether aldosterone（Aldo） can activate Na⁺-H⁺ exchange isoform 1（NHE1） and increase its expression in rat mesangial cells（MC）,and its effect on the accumulation of exracellular matrix（ECM） in rat mesangial cells.MethodsRat mesamgial cells were cultured and then divided as followings:control group,Aldo group(Aldo of the concentration of 10^-7 mol/L was added),Aldo+ spironolactone group(spironolactone 10^-9mol/L and Aldo 10^-7 mol/ L were added), Aldo+Dimethylamiloride（DMA） group(DMA25μmol/L and Aldo 10^-7 mol/ L were added) and spironolactone group(spironolactone 10^-9 mol/L was added).24 hours later, the mesangial cells,the supernatants and the RNA of the cells in different groups were collected.The NHE1 activity was calculated from the initial rate of Na⁺ dependent pHi recovery after acid load.NHE1 mRNA expression was measured by real-time PCR and its protein abundance was detected by flow cytometry analysis and Western blot.The mRNA expression and protein level of FN were examined by real-time PCR and ELISA respectively.Results1.After 24h exposure of MCs to aldosterone(10^-7 M),NHE1 activity was increased compared to Cont（Aldo 5.29±0.11%,vs.Cont 4.48±0.25%,p＜0.05）,the antagonists of mineralocorticoid receptor（MR） spironolactone and the NHE1 inhibitor DMA can inhibit this effect（Aldo+Spir 4.92±0.35%,Aldo+DMA 4.07±0.23%,vs.Aldo 5.29±0.11%,p＜0.05）;2.NHE1 mRNA expression was increased by 1.16 fold of Control group（p＜0.05）,and spironolactone can inhibit this effect to 81.9%of Aldo（p＜0.05）.Spironolactone itself had no effect on NHE1 mRNA expression;3.NHE1 protein abundance was increased after the treatmet ofaldosternoe for 24 hours （control 401.74±17.96;vs.Aldo 535.84±8.67,p＜0.01）,and spironolactone can inhibit this effect（Aldo 535.84±8.67 vs.Aldo+Spir 457.64±11.27,p＜0.05）.The result of FACS showed the similar result.Spironolactone itself had no effect on NHE1 protein abundance;4.ELISA shows the similar result in the concentration ofFN（ng/ml）（Cont 17.84±3.77, Aldo 51.66±1.40,p＜0.01;Aldo+Spir 29.60±1.99,AIdo+DMA 25.75±4.66,vs Aldo 51.66±1.40,p＜0.01 ）.ConclusionOur results therefore demonstrated that aldosterone can activate NHE1 and increase its expression in MCs.NHE-1-dependent accumulation of ECM in MCs was also suggested in this study. PARTⅢAldosterone Promoted Extracellular Matrix Synthesis in Rat Mesangial Cells via ERK1/2 Stimulated NHE1ObjectiveThe results of the second part of our study demonstrated that aldosterone can activate NHE1 and induce NHE1-dependent proliferation of ECM in MCs.It is well-known that ERK1/2 signaling pathway has also been shown to increase the accumulation of ECM.In this study,We investigated whether vector-based short--hairpin RNA（shRNA） could efficiently inhibit the expression of NHEI in rat mesangial cells,and the signaling pathway of ERK1/2 in mediating the effect of NHEI in rat mesangial cells.MethodsThe eukaryotic vector of shRNA with insert targeting on the sequence of NHE1 were successfully constructed and transfected into rat mesangial cells.Cells were cultured till day 4 after transfection compared with control group and non-specific transfected group. The mRNA of NHE1 was reverse transcribed and quantified by real-time PCR and protein expression was assessed by Western blot.On day 4 after transfection,the expression of NHE1 was successfully inhibited in MCs.Then the cells were divided as followings:control group,Aldo group（Aldo of the concentration of 10-7 moll L was added）,Aldo+shRNA-NHE1 group（aldosterone with the same concentration was added after transfection of shRNA-NHE1 for 4 days）,Aldo+ spironolactone group (spironolactone 10^-9mol/L and Aldo 10^-7 mol/ L were added) and Aldo+PD98059 group(PD98059 25μmol/L and Aldo 10^-7 mol/L were added).24 hours later,the proteins and the supernatants in different groups were collected.We measured the protein expression of NHE1 and ERK1/2,phosphor-ERKl/2 by western blot and the level of FN by ELISA.Results1.After the transfection of shRNA-NHE1,mRNA expression of NHE1 decreased on day 1（by 36.9%）,and it decreased progressively on day 3 and day 4（by 69.2%and 77.9%respectively）; 2.The suppression of NHE1 protein abundance didn’t appear until day 3（Negative 434.9±23.91 vs.3day 260.59±12.04,p＜0.01）.Its protein abundance was gradually decreased on day 4（Negative 434.9±23.91 vs.4day 107.585±7.66,p＜0.01）;3.ELISA shows the fibronectin expression was increased compared with the control group,and shRNA-NHE1 can inhibit this effect remarkbly（Cont 17.74±1.38ng/ml, Aldo+shRNA-Negative 51.78±1.15ng/ml vs.Aldo+ shRNA-NHEI 28.07±1.73ng/ml, p＜0.01）.Importantly,spironolactone and PD98059 can also inhibit the aldosteroneinduced fibronectin accumulation（Aldo+Spir 29.60±1.99ng/ml,Aldo+PD98059 29.82±1.39 ng/ml,vs.Aldo+shRNA-Negative 51.78±1.15ng/ml,p＜0.01）;4.ERK inhibition by PD98059 significantly blunted the induction of NHEI expression by aldosterone treatment（Aldo 351.4±42.16,vs.Aldo+PD98059 225.68±21.13, p＜0.05）.By contrast,knocking-down of NHE1 did not alter aldosterone stimulated-phospho-ERK1/2 expression（Aldo 513.22±24.77 vs.Aldo±shRNA-NHE1 462±24.34,p＞0.05）.ConclusionOur results showed that vector-based shRNA is a potential tool to inhibit the expression of NHE1 and shRNA-NHE1 can inhibit the alsosterone-mediatd accumulation of ECM in rat mesangial cells.Also ERK1/2 pathway was involved in the up-regulation of NHE1 induced by aldosterone in MCs.Therefore we deduced that aldosterone promoted ECM synthesis in rat mesangial cells via ERK 1/2 stimulated NHE1.更多还原