The Role And Mechanism Of Murine Double Microsome 2 (MDM2) In Aldosterone-induced Mesangial Cell Proliferation

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. 11?-hydroxysteroid dehydrogenases (11?-HSD) type 2, by converting cortisol to inactive cortisone, serves to "protect" the MR. So both MR and 11?-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 11?-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 11?-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,11?-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.