Catecholamines and adrenocortical steroids are important regulators of blood pressure. Bidirectional relationships between adrenal steroids and catecholamines have been established but whether this is relevant to patients with pheochromocytoma is unclear.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

Identification of patients with endocrine forms of hypertension (EHT) (primary hyperaldosteronism [PA], pheochromocytoma/paraganglioma [PPGL], and Cushing syndrome [CS]) provides the basis to implement individualized therapeutic strategies. Targeted metabolomics (TM) have revealed promising results in profiling cardiovascular diseases and endocrine conditions associated with hypertension.

Reduced or absent compliance to anti-hypertensive treatment is a major obstacle to the achievement of blood pressure target in patients with arterial hypertension. Current available methods for therapeutic adherence assessment display low accuracy, limited applicability in clinical practice and/or high costs. We designed a prospective study to evaluate the accuracy of serial measurement of ARR to assess the therapeutic compliance to RAAS inhibitors. We prospectively enrolled 80 subjects: 40 patients with arterial hypertension and 40 normotensive controls. The ARR was evaluated at baseline and 2 and 8 week after initiation of a RAAS inhibitor in patients with hypertension, and at baseline and 2 weeks for the control group. Adherence to the prescribed therapy was confirmed by therapeutic drug monitoring. We observed a significant increase of renin levels and reduction of aldosterone levels after RAAS inhibitors initiation, with consequent reduction of ARR. Delta ARR (ΔARR), defined as relative change in ARR before and after treatment initiation, provided high accuracy for determination of therapeutic compliance, with an AUC of 0.900 at 2 weeks and 0.886 at 8 weeks. A cut-off of -48% of ΔARR provided 90% sensitivity and 75% specificity, at 2 and 8 weeks. In conclusion, the measurement of ΔARR is a powerful test, cheap and widely available to accurately identify the non-adherence to RAAS inhibitors treatment. Herein we propose the implementation of ΔARR in clinical practice through a multi-step flow-chart for the management of patients with uncontrolled blood pressure, with identification of those suspected of non-adherence, reserving therapeutic drug monitoring for non-adherence confirmation.

Mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) display a phenotype of hyperaldosteronism, due to the upregulation of type VI 3β-hydroxyl-steroid dehydrogenase (Hsd3b6), the murine counterpart to the human type I 3β-hydroxyl-steroid dehydrogenase (HSD3B1) gene. In the present study, we evaluated the role of CRY1 and CRY2 genes, and their potential interplay with HSD3B isoforms in adrenal pathophysiology in man. Forty-six sporadic aldosterone-producing adenomas (APAs) and 20 paired adrenal samples were included, with the human adrenocortical cells HAC15 used as the in vitro model. In our cohort of sporadic APAs, CRY1 expression was 1.7-fold [0.75⁻2.26] higher (p = 0.016), while CRY2 showed a 20% lower expression [0.80, 0.52⁻1.08] (p = 0.04) in APAs when compared with the corresponding adjacent adrenal cortex. Type II 3β-hydroxyl-steroid dehydrogenase (HSD3B2) was 317-fold [200⁻573] more expressed than HSD3B1, and is the main HSD3B isoform in APAs. Both dehydrogenases were more expressed in APAs when compared with the adjacent cortex (5.7-fold and 3.5-fold, respectively, p < 0.001 and p = 0.001) and HSD3B1 was significantly more expressed in APAs composed mainly of zona glomerulosa-like cells. Treatment with angiotensin II (AngII) resulted in a significant upregulation of CRY1 (1.7 ± 0.25-fold, p < 0.001) at 6 h, and downregulation of CRY2 at 12 h (0.6 ± 0.1-fold, p < 0.001), through activation of the AngII type 1 receptor. Independent silencing of CRY1 and CRY2 genes in HAC15 cells resulted in a mild upregulation of HSD3B2 without affecting HSD3B1 expression. In conclusion, our results support the hypothesis that CRY1 and CRY2, being AngII-regulated genes, and showing a differential expression in APAs when compared with the adjacent adrenal cortex, might be involved in adrenal cell function, and in the regulation of aldosterone production.

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence among patients with resistant hypertension. Despite the recent discovery of somatic variants in aldosterone-producing adenoma (APA)-associated PA, causes for PA due to bilateral aldosterone production (bilateral hyperaldosteronism; BHA) remain unknown. Herein, we identified rare gene variants in ATP2B4, in a cohort of patients with BHA. ATP2B4 belongs to the same family of Ca-ATPases as ATP2B3, which is involved in the pathogenesis of APA. Endogenous ATP2B4 expression was characterized in adrenal tissue, and the gene variants were functionally analyzed for effects on aldosterone synthase (CYP11B2) expression, steroid production in basal and agonist-stimulated conditions, and for changes in biophysical properties of channel properties. Knockdown of ATP2B4 in HAC15 exhibited reduced angiotensin II stimulation in one of four shRNA clones. Stable HAC15 cell lines with doxycycline (dox) - inducible wild-type and variant forms of ATP2B4 - were generated, and dox-induced upregulation of ATP2B4 mRNA and protein was confirmed. However, ATP2B4 variants did not alter basal or agonist-stimulated CYP11B2 expression. Whole-cell recordings in HAC15 cells indicated robust endogenous ATP2B4 conductance in native cells but reduced conductance with overexpressed WT and variant ATP2B4. The previously defined PA-causing ATP2B3 variant served as a positive control and exhibited elevated CYP11B2 mRNA. In conclusion, while this study did not confirm a pathogenic role for ATP2B4 variants in BHA, we describe the sequencing analysis for familial and sporadic BHA and outline a template for the thorough in vitro characterization of gene variants.

In primary aldosteronism (PA) the differentiation of unilateral aldosterone-producing adenomas (APA) from bilateral adrenal hyperplasia (BAH) is usually performed by adrenal venous sampling (AVS) and/or computed tomography (CT). CT alone often lacks the sensitivity to identify micro-APAs. Our objectives were to establish if steroid profiling could be useful for the identification of patients with micro-APAs and for the development of an online tool to differentiate micro-APAs, macro-APAs and BAH. The study included patients with PA (n = 197) from Munich (n = 124) and Torino (n = 73) and comprised 33 patients with micro-APAs, 95 with macro-APAs, and 69 with BAH. Subtype differentiation was by AVS, and micro- and macro-APAs were selected according to pathology reports. Steroid concentrations in peripheral venous plasma were measured by liquid chromatography-tandem mass spectrometry. An online tool using a random forest model was built for the classification of micro-APA, macro-APA and BAH. Micro-APA were classified with low specificity (33%) but macro-APA and BAH were correctly classified with high specificity (93%). Improved classification of micro-APAs was achieved using a diagnostic algorithm integrating steroid profiling, CT scanning and AVS procedures limited to patients with discordant steroid and CT results. This would have increased the correct classification of micro-APAs to 68% and improved the overall classification to 92%. Such an approach could be useful to select patients with CT-undetectable micro-APAs in whom AVS should be considered mandatory.

Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃)]--vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [³H]thymidine cell incorporation after treatment with 1α,25(OH)₂D₃ at increasing doses. The effect of 1α,25(OH)₂D₃ on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle transition phase, was evaluated in cells treated with 1α,25(OH)₂D₃ before and after VDR gene silencing. 1α,25(OH)₂D₃ treatment inhibited cell proliferation by 20% at a dose of 1 nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10 nM 1α,25(OH)₂D₃ cell exposure. CDK4 activation was reduced by 10 nM 1α,25(OH)₂D₃ but was not affected by 1α,25(OH)₂D₃ after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues.