鼠双微体2（MDM2）在醛固酮诱导的系膜细胞增殖中的作用及机制研究

【作者】 王海云；

【导师】 李学旺； 李雪梅；

【作者基本信息】 北京协和医学院 ， 内科肾脏病学， 2009， 博士

【摘要】 背景醛固酮(Aldosterone)是一种类固醇激素,在机体生理及病生理活动中起重要作用。除影响水盐代谢及循环容量的经典作用,近年的研究表明醛固酮还有许多重要的病生理作用,包括激活氧化应激、促进细胞趋化因子的产生及促使组织纤维化等。使用ACEI及血管紧张素受体拮抗剂后产生的“醛固酮逃逸”现象,使得醛固酮在RAS特别是局部RAS激活等一系列致病机制中的作用和地位进一步提高。醛固酮在人体内的作用方式包括基因组调节与非基因组调节两种。基因组调节是指醛固酮与盐皮质激素受体(MR)结合形成复合物入核,诱导靶基因转录翻译,行使其生物效应。非基因方式是指醛固酮与胞浆中的盐皮质激素受体或细胞膜上的受体结合后通过触发一系列第二信使的级联反应行使其生物效应。由于人体内糖皮质激素、醛固酮与MR的亲和力相当,而血浆中糖皮质激素的浓度可达醛固酮的100-1000倍,所以能将糖皮质激素转化以保证醛固酮与MR作用的高度特异性的11β-羟类固醇脱氢酶(11β-HSD2)的表达是醛固酮基因组调节的必要条件。醛固酮与肾脏病变关系密切,盐皮质激素受体拮抗剂具有抗蛋白尿、抗肾脏硬化的肾脏保护作用。肾小球系膜细胞增殖是目前较为公认的醛固酮肾脏损害主要表现之一。细胞增殖周期的调控受促进周期进程的因子(如细胞周期蛋白)及抑制性因子(如p21、p53)共同影响,当促进周期进程的因子增加或抑制性因子减少,系膜细胞即可增殖。有研究表明,醛固酮可通过与盐皮质激素受体结合,激活一系列磷酸激酶使得S期细胞周期蛋白表达增加并激活相应Cdk酶活性,从而使细胞增殖。但醛固酮对于细胞周期抑制性因子如p53是否也存在调节作用,目前并无相关报告。从肿瘤学的研究中得知鼠双微体2(MDM2)蛋白是一个可与p53形成复合物的核蛋白,该蛋白可以通过介导p53经过泛素—蛋白酶体迅速降解而阻止p53介导的调亡并逆转p53诱导的细胞周期锁定。有研究指出,在醛固酮介导的血管平滑肌细胞增殖中存在MDM2的表达增加。MDM2是否同样参与了醛固酮诱导的系膜细胞增殖是本研究将探讨的主要问题。方法以RT-PCR方法检测人传代系膜细胞(HMCs)及人系膜细胞株(HMCLs)有无MR、11β-HSD2、MDM2 mRNA的表达。用Western Blot及流式细胞术检测不同浓度、不同作用时间醛固酮对HMCs及HMCLs MDM2表达及S期细胞百分比的影响。将HMCLs分组以醛固酮、针对MDM2设计的小干扰RNA(siMDM2)、醛固酮+siMDM2刺激,检测MDM2蛋白表达及S期细胞百分比的改变以明确MDM2在醛固酮诱导系膜细胞增殖中的作用。以螺内酯(盐皮质激素受体拮抗剂)抑制试验检测盐皮质激素受体是否参与了醛固酮诱导MDM2表达增加。以放线菌酮(蛋白合成抑制剂)抑制试验检测醛固酮诱导MDM2表达是否为非基因组机制。为了解体内醛固酮水平与系膜细胞MDM2表达关系,分别选取高醛固酮血症患者肾活检组织及正常肾组织,行MDM2及WT1(一种足细胞标记性抗原)免疫组织化学染色,分析两组间平均单位面积MDM2阳性细胞个数及平均单位面积WT1阳性细胞个数的差异。结果HMCs及HMCLs有MR、11β-HSD2、MDM2的表达,且MDM2蛋白主要表达于细胞核,少量表达于胞浆。醛固酮刺激可使传代培养的人肾小球系膜细胞MDM2蛋白表达增加,这种增加随剂量的增加而增加,在醛固酮浓度为10-8mo1/L时达峰,为阴性对照的2.725倍,P值小于0.05,具有显著性差异。醛固酮刺激可使HMCLs的MDM2表达随时间及剂量增加,在醛固酮浓度为10-8mol/L、作用时间为24小时达峰,分别为阴性对照的2.27及1.91倍,具有显著性差异。醛固酮作用5min及1小时不能使MDM2蛋白的表达显著增加。醛固酮刺激可使HMCLs的S期细胞百分比增加,在醛固酮浓度为10-8M时达峰,为阴性对照的2.77倍,具有显著性差异。针对MDM2不同DNA片段设计的siMDM2可使MDM2蛋白表达明显下降、醛固酮+siMDM2刺激,不能使MDM2蛋白的表达明显增加,相应S期细胞百分比亦不升高。螺内酯可抑制醛固酮刺激导致的MDM2 mRNA及蛋白表达增加,与醛固酮刺激组(阳性对照)比,螺内酯的抑制率为44.44%。两者间具显著性差异,P值小于0.05。蛋白合成抑制剂放线菌酮可抑制醛固酮刺激导致的MDM2表达增加,与醛固酮刺激组(阳性对照)比,两者间具显著性差异。在高醛固酮血症及正常肾脏肾小球切片中,足细胞、系膜细胞MDM2染色均呈阳性。高醛固酮血症患者的肾小球中平均单位面积MDM2阳性细胞数目较正常对照者明显增多,两组间差异有统计学意义(P=0.004)。而两者WT1平均单位面积阳性细胞数目大致相仿(P=0.699)。结论HMCs及HMCLs有MR、11β-HSD2表达。醛固酮刺激可导致HMCs及HMCLs MDM2表达增加,这种作用在一定范围内呈剂量及时间依赖性。同时醛固酮可使HMCL细胞S期细胞百分比剂量依赖性地提高。针对MDM2不同DNA片段设计的siMDM2可抑制醛固酮刺激导致的S期细胞百分比增加,进一步提示MDM2可能参与了醛固酮诱导的HMCLs细胞周期与增殖的调节。盐皮质激素受体拮抗剂螺内酯可拮抗醛固酮刺激导致的MDM2表达增加,说明这种作用需经过盐皮质激素受体介导。蛋白合成抑制剂放线菌酮可抑制醛固酮刺激导致的MDM2表达增加,说明醛固酮对MDM2的作用方式不属于醛固酮的非基因组作用方式。高醛固酮血症患者的肾小球中平均单位面积MDM2阳性细胞数目较正常对照者明显增多,以系膜细胞为主。更多还原

【Abstract】 BackgroundAldosterone has been recognized as a steroid hormone which binds to the mineralocorticoid receptor (MR) to increase systemic blood pressure by regulating systemic electrolytes and volume balance in kidney. It has also been demonstrated that aldosterone plays an important role in the pathogenesis of tissue fibrosis, oxidative stress, and inflammation in recent years. The importance of aldosterone in the process of RAS activation, especially to local RAS was improved by aldosterone escape after the use of ACEI and/or ARB.Although the aldosterone-MR complex primarily acts as a transcription factor (genomic regulation), recent evidence suggests that it may also mediate rapid nongenomic (or nonnuclear) activation of second messenger pathways. Cortisol can bind and activates the MR, which, given that its concentration is at least an order of magnitude greater than that of aldosterone, would see it fully occupying the MR. 11β-hydroxysteroid dehydrogenases (11β-HSD) type 2, by converting cortisol to inactive cortisone, serves to "protect" the MR. So both MR and 11β-HSD2 are important to the pathogenesis of aldosterone.Aldosterone involved in the pathogenesis of kidney diseases. Some studies indicated MR antagonist therapy could prevent renal function by reducing proteinuria and retarding renal fibrosis. Glomerular mesangial cell proliferation was one of the most confirmed evidence that aldosterone contributes to the progression of renal injury. Some evidence has shown relationship between aldosterone and mesangial cell proliferation. The cell cycle regulation depends on the balance between positive regulation proteins including cyclins and cyclin dependent kinases and cyclin dependent kinases inhibitors which act as negative regulation proteins (p21, p53 et al). The positive regulation factors increase and/or the negative regulation factors reduced, the cells may proliferate. Yoshio al et. reported that aldosterone could stimulate proliferation of mesangial cells by activating a series of cyclins, but there was no study on whether the negative regulation factors, such as p53, involved in aldosterone inducing proliferation of mesangial cells. MDM2 was known as a p53 binding protein to regulate the biological activity of p53 by preventing p53-mediated apoptosis or reversing p53-induced G1 block of the cell cycle and thus promoting the entry of cells into S phases through formation of these complexes. Some evidence showed MDM2 has involved in aldosternone inducing proliferation of cells. In this study, whether and how MDM2 involved in aldosternone inducing proliferation of mesangial cells was assessed.MethodsMesangial cells isolated and cultured from human beings (HMCs) and HMCLs were used in these experiments. Expression of both MR and 11β-HSD type 2 were determined by RT-PCR. Expression of MDM2 was determined by RT-PCR and immunoflourescence. Both RT-PCR and western blot were used to estimate the relationship among time, dose and MDM2 expression. Proliferation of HMCLs was evaluated by flow cytometry. Small interference RNA of MDM2 was used to confirm the relationship among aldosterone, MDM2 expression and proliferation of HMCLs.Spironolactone, a MR blocker was used to estimate the role of whether MR was involved in aldosterone inducing MDM2 elevation. Cycloheximide, a protein synthesis inhibitor was inducted to estimate whether the rapid nongenomic mechanisms regulate aldosterone inducing MDM2 expression directly. In vivo study, immunohistochemical analysis was performed. Renal biopsy specimens from hyperaldosteronemia patients and some other specimens from normal as control were selected. Mean numbers of MDM2 and WT1 positive cells were counted to estimate the role of aldosterone induced MDM2 expression.ResultsBoth MR and 11β-HSD type 2 mRNAs were detected in HMCs and HMCLs. MDM2 protein expression was detectable in both the nucleus and the cytoplasm by immunoflourescence. Aldosterone significantly increased MDM2 expression in a dose and time-dependent manner compared with control ones. Flow cytometry showed that the percentage of S phase increased in HMCLs stimulated with different concentrations of aldosterone for 24 hours (P<0.05). A reduction of MDM2 protein was dose-dependent confirmed by transfection of MDM2 siRNAs. Under the transfection of MDM2 siRNA, aldosterone did not promote the cell proliferation of HMCLs compared with control ones. Aldosterone with spironolactone did not promote expression of MDM2 mRNA and protein. Aldosterone with CHX did not increase expression of MDM2 protein. Mean number of MDM2 positive cells was expressed higher in glomeruli of hyperaldosteronemia renal biopsy specimens than that of normal or minor lesion patiens’.ConclusionMR,11β-HSD type 2 and MDM2 were expressed by HMCs and HMCLs. Aldosterone increased MDM2 expression in a dose and time-dependent manner. The percentage of S phase increased in HMCLs after stimulated with aldosterone. Under the transfection of MDM2 siRNA, aldosterone do not promote the cell proliferation of HMCLs compared with control one. These results confirm that MDM2 participates in aldosterone inducing HMCLs proliferation. MDM2 is significantly induced by aldosterone via MR and inhibited by spironolactone. Aldosterone with CHX did not increase expression of MDM2 protein indicated that the increase expression of MDM2 protein induced by aldosterone is not directly regulated by the rapid nongenomic mechanisms. Immunohistochemical analysis demonstrated MDM2 were expressed higher in glomeruli of hyperaldosteronemia renal biopsy specimens than that of normal ones.更多还原