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. 2019 Oct 11;9:14677. doi: 10.1038/s41598-019-50988-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Adrenal cortex disorganization persists with aging in Rarα^−/− mice. (A) Morphological characterization of adrenals from 52 weeks old male and female Rarα^+/+ and Rarα^−/− mice. HES staining, Dab-2, aldosterone synthase and 11β-hydroxylase immunofluorescence and Ki67 immunohistochemistry were performed. (B) Number of Dab-2 positive cells in the cortex was determined in 3 to 9 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer) and is expressed as a percentage of total number of cells in the entire cortex area. (C) Relative proliferative index of adrenals from male and female Rarα^+/+ and Rarα^−/− mice. Ki67 positive cells were separately counted in the adrenal cortex in 5–6 animals per genotype. (D) Number of nuclei in the adrenal cortex was determined in 3 to 9 animals of each genotype and sex using an automated molecular imaging platform (Vectra, Perkin Elmer). (E–H) Expression of steroidogenic genes in male and female Rarα^+/+ and Rarα^−/− mice. mRNA expression of Star (E), Cyp11a1 (F), Cyp11b1 (G) and Cyp11b2 (H) was assessed by RT-qPCR. RT-qPCR were performed on mRNA extracted from 6–8 adrenals from 52 weeks old male and female Rarα^+/+ and Rarα^−/− mice. (I,J) Measure of plasma aldosterone (I) and corticosterone (J) concentration by mass spectrometry in male mice. (K,L) Plasma renin concentration (PRC) and aldosterone to renin ratio. Measure of plasma aldosterone, plasma corticosterone and plasma renin were done on 5–6 animals per group. Values are presented as means ± SEM. *p < 0.05; **p < 0.01.