The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders

Genome-wide association study for renal traits in the Framingham Heart and Atherosclerosis Risk in Communities Studies

Background: The Framingham Heart Study (FHS) recently obtained initial results from the first genome-wide association scan for renal traits. The study of 70,987 single nucleotide polymorphisms (SNPs) in 1,010 FHS participants provides a list of SNPs showing the strongest associations with renal traits which need to be verified in independent study samples. Methods: Sixteen SNPs were selected for replication based on the most promising associations with chronic kidney disease (CKD), estimated glomerular filtration rate (eGFR), and serum cystatin C in FHS. These SNPs were genotyped in 15,747 participants of the Atherosclerosis in Communities (ARIC) Study and evaluated for association using multivariable adjusted regression analyses. Primary outcomes in ARIC were CKD and eGFR. Secondary prospective analyses were conducted for association with kidney disease progression using multivariable adjusted Cox proportional hazards regression. The definition of the outcomes, all covariates, and the use of an additive genetic model was consistent with the original analyses in FHS. Results: The intronic SNP rs6495446 in the gene MTHFS was significantly associated with CKD among white ARIC participants at visit 4: the odds ratio per each C allele was 1.24 (95% CI 1.09–1.41, p = 0.001). Borderline significant associations of rs6495446 were observed with CKD at study visit 1 (p = 0.024), eGFR at study visits 1 (p = 0.073) and 4 (lower mean eGFR per C allele by 0.6 ml/min/1.73 $\text{m}^2$, p = 0.043) and kidney disease progression (hazard ratio 1.13 per each C allele, 95% CI 1.00–1.26, p = 0.041). Another SNP, rs3779748 in EYA1, was significantly associated with CKD at ARIC visit 1 (odds ratio per each T allele 1.22, p = 0.01), but only with eGFR and cystatin C in FHS. Conclusion: This genome-wide association study provides unbiased information implicating MTHFS as a candidate gene for kidney disease. Our findings highlight the importance of replication to identify common SNPs associated with renal traits

K007 Functional impact of mineralocorticoid receptor overexpression in embryonic stem cell-derived cardiomyocytes

Mineralocoticoid receptor (MR or NR3C2) is expressed in a wide range of tissues including in the heart, where its role is not fully understood. Recent clinical trials had underscored the importance of aldosterone, the main mineralocorticoid hormone, in the cardiovascular system based on the beneficial effect of mineralocorticoid antagonists to improve the prognosis of heart failure patients. We generated transgenic mice models overexpressing the human MR (hMR) under the control of its own P1 proximal (P1.hMR) or P2 distal (P2.hMR) promoters. Both transgenes were expressed in the heart among other mineralocorticoid target tissues. P1.hMR mice present with a dilated cardiomyopathy associated with tachycardia and arrhythmia. To further investigate the role of MR and aldosterone in the heart using an in vitro cellular model, we established several embryonic stem (ES) cell lines from wild type (WT), P1.hMR and P2.hMR blastocysts. Cardiac differentiation was initiated by culturing ES cells in hanging drops to derive embryoid bodies (EB). Patches of spontaneously beating cardiomyocytes appeared from day 7 and were detected in 70 % of EB at day 16. Recombinant and endogenous MR mRNA were expressed concomitantly with cardiac specific markers such as Nkx2.5, alphaMHC and Troponin T both during differentiation and in excised beating patches. hMR overexpression was confirmed by immunocytochemistry. In WT ES cells, aldosterone sharply enhanced cardiomyocyte differentiation efficiency since up to 100 % of EB exhibited beating areas. The most striking phenotype was the 2 fold-increase of basal cardiomyocyte beating rate induced by hMR overexpression, using several independent cell lines (WT: 1.09±0.2Hz, P1.hMR: 1.8±0.3Hz, P2.hMR: 1.7±0.5Hz, p<0.005). The beta-adrenergic-stimulated beating rate remained significantly higher in overexpressing cardiomyocytes. We are currently studying the expression of key players of cardiac physiology and ion channels such as Kir2.1, Cav1.2 and Cav3.2 in order to decipher the molecular events underlying hormonal and receptor-induced actions on cardiomyocyte differentiation and function. These versatile and original models of overexpression allowed to discriminate between MR and hormonal effects and represented useful cell-based systems to study the role of MR in other cellular contexts, such as neuronal differentiation

Hibernoma development in transgenic mice identifies brown adipose tissue as a novel target of aldosterone action.

Aldosterone is a major regulator of salt balance and blood pressure, exerting its effects via the mineralocorticoid receptor (MR). To analyze the regulatory mechanisms controlling tissue-specific expression of the human MR (hMR) in vivo, we have developed transgenic mouse models expressing the SV40 large T antigen (TAg) under the control of each of the two promoters of the hMR gene (P1 or P2). Unexpectedly, all five P1-TAg founder animals died prematurely from voluminous malignant liposarcomas originating from brown adipose tissue, as evidenced by the expression of the mitochondrial uncoupling protein ucp1, indicating that the proximal P1 promoter was transcriptionally active in brown adipocytes. No such hibernoma occurred in P2-TAg transgenic mice. Appropriate tissue-specific usage of P1 promoter sequences was confirmed by demonstrating the presence of endogenous MR in both neoplastic and normal brown adipose tissue. Several cell lines were derived from hibernomas; among them, the T37i cells can undergo terminal differentiation into brown adipocytes, which remain capable of expressing ucp1 upon adrenergic or retinoic acid stimulation. These cells possess endogenous functional MR, thus providing a new model to explore molecular mechanisms of mineralocorticoid action. Our data broaden the known functions of aldosterone and suggest a potential role for MR in adipocyte differentiation and regulation of thermogenesis