In vivo and ex vivo molecular analyses of aldosterone producing cells of the adrenal gland, in patients with primary aldosteronism

Abstract

Aldosterone producing micronodules (APM) are the predicted precursor to aldosterone producing adenomas (APA) and can cause primary aldosteronism (PA) in their own right. PA is the commonest cause of curable hypertension, and has higher morbidity and mortality than matched patients with essential hypertension. This thesis aimed to identify specific markers of APMs, and their potential for translational applications, by investigating orphan transmembrane transporter SLC35F1 and exploring APM aldosterone regulation, by establishing the role and presence of Connexin-43 (Cx43) gap junctions (GJ). A comparison between radiolabelled ligand, para-chloro-2-[18F]fluoroethyl-etomidate (CETO), with previously validated 11C-metomidate (MTO), as PET ligands for the lateralisation of PA, was also made. Immunochemistry studies of both proteins, and transfection or pharmacological inhibition of SLC35F1 and Cx43, respectively, were undertaken in adrenal sections taken from 32 patients, and in cultured cell lines, in order to study function. Bioinformatic analysis was used to predict the structure and function of SLC35F1. Thirty one patients underwent CETO, MTO and AVS, as a planned extension of the MATCH study (1). SLC35F1 was co-expressed with CYP11B2, and colocalised with the plasma membrane, and microtubules, of APM cells, but stimulation experiments demonstrated no direct relationship with aldosterone production. Its transport substrate has not been established, but bioinformatic analysis does not support a role in nucleotide sugar transport. Expression and turnover of Cx43 GJs were demonstrated in human zona glomerulosa, which was less abundant in adjacent APMs. CETO and MTO predicted high probability of unilateral PA in 17 and 19 patients respectively, with (mildly) discrepant scores in only four of 31 patients. SLC35F1 is an attractive target for APM identification, for in vitro enrichment of primary cell culture, and in vivo clinical applications, due to its accessible subcellular localisation. A role in tumourigenesis is proposed. The active turnover and regulatory role of GJs in APMs have important mechanistic implications for our understanding of steroidogenesis regulation. CETO could replace MTO, and improve access to optimal care.