LAUSR

Abstract Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension and is caused in the majority of cases by an aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia. Different somatic mutations have been identified in APA and in other aldosterone producing structures, which can be distinct within the same adrenal, suggesting multiple mechanisms underlying APA development. Also, APA show important cellular and molecular heterogeneity which may be due to interaction of different signaling pathways involved in adrenal cortex cell differentiation and function. The aim of this study was to investigate the role of Wnt/β-catenin and ACTH signaling as well as elements of paracrine regulation of aldosterone biosynthesis and vascularization in the development of APA and aldosterone producing cell clusters (APCC) and their relationship with intratumoral heterogeneity and mutational status. We performed immunohistochemistry and multiplex immunofluorescence (CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34) multispectral image analysis on 11 adrenals with APA and one with micronodular hyperplasia from patients with PA. CYP11B2 (aldosterone synthase) IHC guided RT-qPCR was performed on RNA extracted from formalin-fixed paraffin-embedded tissues in 7 adrenals. Multiplex immunofluorescence revealed high abundance of tryptase positive mast cells and a dense vascular component in APA, which were independent of the mutational status. Within APA, mast cells were mainly localized in zones expressing CYP11B2, but not in areas expressing CYP17A1, and were rarely colocalized with nerve fibers, suggesting that their activity is not controlled by innervation. In cells expressing aldosterone synthase, β-catenin was activated, i.e. shows nuclear and/or cytoplasmic staining, features suggestive of a zona glomerulosa cell identity; MC2R was found at the cell membrane. Expression of MC2R mRNA was observed at different levels in APA, similar to expression of MRAP and VEGFA; MRAP2 was not detected. Within heterogeneous APA carrying KCNJ5 mutations, both MC2R and VEGFA expression was higher in areas expressing CYP11B2. Remarkably, this pattern was maintained in APCC, where cells show high CYP11B2 expression, together with activated β-catenin, independently of the mutation status. In addition, a high number of mast cells was detected around APCC, with a reorganization of the capillaries around the CYP11B2 positive cells. Our results suggest that aldosterone producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status. Mast cells appear to be closely associated with cells expressing aldosterone synthase, both in APA and APCC, and their role in regulating aldosterone biosynthesis in the context of somatic mutations in PA remains to be established.