The Role Of Na~+-H~+ Exchange Isoform 1 In The Progression Of Aldosterone-induced-Glomerulosclerosis And Its Involved Mechanisms

PARTâ… The 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. PARTâ…¡Effect 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. PARTâ…¢Aldosterone 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.