大鼠系膜细胞醛固酮的合成及其对细胞外基质生成的影响

目的　证明肾脏系膜细胞能够自身合成醛固酮 ,并探讨醛固酮对系膜细胞细胞外基质生成的影响。方法　用RT PCR法检测大鼠系膜细胞醛固酮合成酶CYP11B2mRNA表达 ,PCR产物存化后直接测序 ;放免法测定系膜细胞培养上清液中醛固酮的浓度。用RT PCR半定量法比较 (3 磷酸甘油醛脱氢酶theenzymeglyceraldehyde 3 phosphatedehydrogenase ,GAPDH为内参照 )经 10 -10 ～ 10 -6mol/LAngⅡ或 7mmol/L、9mmol/LKCl干预 4 8h ,系膜细胞CYP11B2mRNA的表达量。大鼠系膜细胞醛固酮特异性受体 (mineralocorticoidreceptor,MR)和保护其配体特异性的 11β -羟类固醇脱氢酶 2 (11β -HSD2 )的mRNA和蛋白质表达分别用RT PCR法和细胞免疫化学法检测。以ELISA和Western印迹方法检测经 10 -7mol/L醛固酮干预 2 4h ,细胞培养上清液中层黏蛋白 (FN)和Ⅳ型胶原的浓度。结果　大鼠系膜细胞能表达CYP11B2mRNA ,且细胞培养上清液中检测到醛固酮 ,浓度为 1 0 6 5pg/ 10 6个细胞。 10 -8、10 -7mol/LAngⅡ (10 -8mol/L :2 15± 0 10 ,10 -7mol/L :1 16± 0 0 4vs非干预组 0 77± 0 0 3,P <0 0 0 1;)和 9mmol/L的KCl (1 2 7± 0 11vs非干预组 0 89± 0 12 ,P <0 0 5 )均能显著提高CYP11B2mRNA的表达。大鼠系膜细胞有MR和 11β -H

Production of aldosterone by rat mesangial cell and the accumulation of extracellular matrix induced by aldosterone

OBJECTIVE: To investigate whether aldosterone (Aldo) may be synthesized by glomerular mesangial cells and whether Aldo promotes the synthesis of fibronectin (FN) and type IV collagen in mesangial cells. METHODS: Rat mesangial cells (RMC) were cultured and then divided into 4 groups: control group, AngII group (AngII of the concentrations of 10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) mol/L was added), losartan group (AngII 1 type inhibitor losartan and AngII were added), and KCL group (KCL of the concentrations of 7 and 9 mmol/L was added). 48 hours after the RNAs of the cells in different groups were collected to detect the Aldo synthesized by the RMCs themselves. Another RMCs were cultured and divided into 4 groups: control group, Aldo group (Aldo of the concentration of 10(-7) mol/L was added), spironolactone + Aldo group (spironolactone 10(-9) mol/L and Aldo 10(-7) mol/L were added), and spironolactone group (spironolactone 10(-9) mol/L was added). RT-PCR was used to detect the expression of the Aldo synthase CYP11B2, mineralocorticoid receptor (MR), and 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2 (an enzyme required for MR ligand specificity) mRNAs and proteins. The level of Aldo in the incubation medium was detected by radioimmunoassay. After being exposed to 10(-7) mol/L Aldo for 24 h, the level of FN and type IV collagen in the incubation medium were examined by ELISA and Western blotting respectively. RESULTS: CYP11B2, MR, and 11 beta-HSD2 mRNA expressions were detectable in RMCs by RT-PCR and confirmed by sequencing. The concentration of Aldo in the incubation medium was 1.605 pg/10(6) cells. The mRNA expression of CYP11B2 was significantly up-regulated by both AngII and potassium. Immunocytochemistry showed that MR and 11 beta-HSD2 were distributed in both the cytoplasm and the nucleolus. After treatment of 10(-7) mol/L Aldo for 24 hours, the concentration of FN was 74 +/- 16 ng/ml, significantly higher than the baseline value (12.4 +/- 1.9 ng/ml, P < 0.05) and those of spironolactone group and spironolactone + Aldo groups (17.8 +/- 3.6 ng/ml and 25.9 +/- 5.3 ng/ml respectively); and the level of type IV collagen was 136% +/- 14%, significantly higher than the baseline level (100%, P < 0.05), and those of spironolactone group and spironolactone + Aldo groups (112% +/- 33% and 102% +/- 10% respectively). CONCLUSION: RMCs synthesize Aldo. Aldosterone also acts on RMCs, leading to extracellular matrix accumulation AngII and potassium are involved in the regulation of local production of aldosterone.