The Role Of Aldosterone In The Progression Of Glomerulosclerosis And Its Involved Mechanisms

BackgroundThe efficacy of ACEI in slowing the progression of renal disease has become widely accepted. More recently, aldosterone has also been implicated as a deleterious influence on the RAAS. This study, therefore, is tested the hypothesis that adrenalectomy ameliorates remnant nephropathy in the rat which depends on low aldosterone level. MethodsFive groups of male Wistar rats weighing 200 grams were studied: SHAM rats, 5/6 nephrectomy rats (SNX group), nephrectomy and adrenalectomized rats (ADX group), bi-ectomized rats infused with exogenous dexamethasone (DXM group) or plus aldosterone (ALDO group) by osmotic mini-pump at 12μg/kg/d and 40μg/kg/d, respectively. They were allowed free access to saline and sacrificed at 8 weeks. Results5/6 rats had marked proteinuria, hypertension, glomerulosclerosis and heigh expression of TGF-pi in renal cortex as well as a > 4-fold elevation in plasma aldosterone compared to SHAM rats. Above pathology were improved much in bi-ectomised rats with significant lower aldosterone level (urinary albumin (mg/24h): 19.7±2.0 vs. SNX: 31.7+1.7 PO.001; SBP (mmHg): 173.8 + 4.3 vs. SNX: 210.4 + 4.1 P<0.001; glomerulosclerosis scores: 38.2±7.9 vs. SNX: 92.3 + 6.7 P<0.001; TGF-β1: 3.8 + 0.6 vs. SNX: 10.3 + 1.2 P<0.01 ). However, if being constantly infused exogenous aldosterone, bi-ectomized rats would manifeste greater proteinuria (urinary albumin mg/24h: 24.9+1.4 P<0.001), hypertension (SBP mmHg: 201.5+4.5 P<0.001), glomerulosclerosis (scores: 88.1+7.2 P<0.001) and increased level of TGF-β1 (5.8 ±0.6 P<0.01) compared to bi-ectomised rats. Indeed, these features were similar in exogenous aldosterone rats and 5/6 nephrectomy rats. But replacing with dexamethasone didn't deteriorate injury. Furthermore, the expression ofmineralocorticoid receptor (MR) mRNA was remarkable enhanced in SNX group anddecreased in ADX group (SNX (copies/million GAPDH): 39866.7+10579.0 vs.SHAM: 2366.7+446.3 PO.05) (ADX: 22100.0+4435.7 vs. SNX PO.05). However,the mRNA expression of 11 p-hydroxysteroid dehydrogenase II (11P-HSD2) in eachgroup was opposite to that of MRmRNA. In SNX group it was 9150.0 + 969.9 muchlower than that of SHAM group (48100.0 + 9315.2 PO.05) and the expression ofllp-HSD2mRNA was increased in ADX group (30066.7 + 5150.2 vs. SNX PO.05).Otherwise, Ccr and kidney/body weight were not different in four experimentalgroups.ConclusionOur data suggested that aldosterone, not glucocorticoid, contributes to the progressionof ablative nephropathy in the rat through mechanisms more than systolic bloodpressure.BackgroundAldosterone has been suggested to cause vasoconstriction and collagen accumulation.A body of evidences demonstrated that aldosterone is synthesized locally in bloodvessels and heart, which is regulated by angiotensin II (Angll) and potassium.However, there is no available documentation showing the existence of localaldosterone production and function in the kidney. This study, therefore, is conductedto investigate whether aldosterone might also be synthesized by rat glomerularmesangial cells (RMCs).MethodsUsing Western blot, the existence aldosterone synthase (CYP11B2) in rat glomeruliwas examined. Messenger RNA expressions of CYP11B2 in cultured RMCs treatedwith different concentration of Angll and potassium were detected by real-time PCR.The level of aldosterone in the incubation medium was examined byradioimmunoassay.ResultsThe protein comes from rat adrenal was used for positive control and the expressionof aldosterone synthase in rat glomeruli was found. CYPllB2mRNA expression wasdetectable in RMCs by RT-PCR, the product of which was confirmed by sequencing.The mRNA expression of CYPIlB2mRNA was significantly up-regulated by bothAngll (4.6E+07± 1.1E+07 at lO^M Angll, 4.8E+07 + 6.7E+06 at 10'7M vs. 2.2E+07±5.8E+06 at baseline, PO.001, respectively) and potassium (3.90E+07 + 6.30E+06at 7mM, vs. 2.2E+07+2.5E+06 at 5.5mM, PO.05). The concentration of aldosteronein the incubation medium was 1.605pg/106cells.ConclusionOur study indicated that local synthesis of aldosterone exists in rat glomeruli andcultured rat mesangial cells. Angll and potassium are involved in the regulation forthe local production of aldosterone in mesangial cells at the mRNA level.BacgroundIn vivo studies recently have showed that aldosterone seems to play a role in the development of chronic renal failure and proteinuria. As the same, a body of evidences demonstrated that aldosterone could act on non-epithelial cells, leading to extracellular matrix accumulation. Because mesangial cells are responsible for much of the extracellular matrix (ECM) production observed in renal disease and we have recently shown that local synthesis of aldosterone exists in cultured rat mesangial cells which is regulated by Angll and potassium at the mRNA level. This study, therefore, is conducted to investigate whether mineralocorticoid receptors (MR) localize in rat mesangial cells (RMC) and whether aldosterone promote the synthesis of fibronectin(FN) and type IV collagen in RMC. MethodsMessenger RNA expressions of MR, 11P-HSD2 were detected by RT-PCR. The proteins were examined by immunofluoresence. Cells were exposured to 10"7M aldosterone, and the level of FN and type IV collagen in the incubation medium were examined by ELISA and Western blot, respectively. ResultsMR, llp-HSD2mRNA expressions were detectable in RMCs by RT-PCR, the products of which were confirmed by sequencing. Immunofluoresence analysis revealed that MR and 110-HSD2 were diffusely distributed in the cells. The concentration of FN and type IV collagen in the incubation medium were increased after incubation with 10'7M aldosterone (FN(ng/ml):74.55± 16.75 vs control 12.37 + 1.91, PO.05; type IV collagenC %control): 100.00 vs control 136.94+13.76, PO.05) but not in the presence of 10'9M spironolactone. ConclusionWe detected the co-expression of MR and lip-HSD2mRNA in RMCs. Furthermore, by immunofluoresency we examined a diffuse localization of MR and 11|3-HSD2 throughout the cells, which suggested that rat mesangial cell is the target cell of aldosterone. The increased expressions of fibronectin and type IV collagen induced byaldosterone can be ameliorated by its antagonist spironolactone, which means thataldosterone could lead to ECM accumulation.BackgroundAlthough the role of aldosterone in mediating progressive renal disease has been frequently documented these years, the mechanism of which is still unclear. Because mesangial cells are responsible for much of the ECM production observed in renal disease and we have recently shown that local synthesis of aldosterone exists in cultured rat mesangial cells (RMCs) and exogenous aldosterone acts on RMCs leading to ECM accumulation. This study, therefore, is conducted to investigate whether aldosterone promote the synthesis of fibronectin in RMCs through a Smad2-dependent TGF-pi pathway, the key participant in TGF-pl signaling. MethodsThe level of FN in the RMCs incubation medium, the production of TGF-pl and Smad2 in RMCs were detected by ELIS A and Western blot, respectively. The effect of aldosterone on TGF-pi pathway via Smad was examined by a TGF-pi reporter system using luciferase as a reporter. The Smad2 antisense plasmid, constructed by the DNA sequence in the Smad2 MH2 functional region and the vector pEGF-Cl, was transferred into the RMCs with Lipofectin. Because the plasmid encodes the green fluorescent protein, the transfection can be monitered by fluorescence microscopy. ResultsAfter the antisense plasmid transferring into the RMCs, the level of Smad2 protein was 69.1%±8.6% significantly lower than the baseline level (100%, PO.01). The vector pEGF-Cl didn't influence the expression of Smad2. The production of TGF-pi was significantly increased after cells exposeing to 10'7M aldosterone (TGF-pi: 134.2%±13.9% vs. control 100%, PO.05), but decreased in the presence of 10'9M spironolactone. Incubation of cultured RMCs with aldosterone markedly increased the TGF-pi reporter luciferase activity (aldosterone: 1.7 + 0.1 vs. control 0.8 + 0.1, PO.05). However, if the cells were transferred the Smad2 antisense plasmid, aldosterone couldn't promote the production of FN. The concentration of FN in the incubation medium was much lower than that without transfection (ng/ml: 36.1±19.82 vs. 72.3±16.6, PO.05) . Conclusion...