High Prevalence of Primary Aldosteronism in Patients with Type 2 Diabetes Mellitus and Hypertension.

Primary aldosteronism (PA) is the most common cause of endocrine hypertension. The prevalence of hypertension is higher in patients with diabetes mellitus-2 (DM-2). Following the limited existing data, we prospectively investigated the prevalence of aldosterone excess either as autonomous secretion (PA) or as a hyper-response to stress in hypertensive patients with DM-2 (HDM-2). A total of 137 HDM-2 patients and 61 non-diabetics with essential hypertension who served as controls (EH-C) underwent a combined, overnight diagnostic test, the Dexamethasone-captopril-valsartan test (DCVT) used for the diagnosis of PA and an ultralow dose (0.3 µg) ACTH stimulation test to identify an exaggerated aldosterone response to ACTH stimulation. Twenty-three normotensive individuals served as controls (NC) to define the normal response of aldosterone (ALD) and aldosterone-to-renin ratio (ARR) to the ultralow dose ACTH test. Using post-DCVTALD and ARR from the EH-C, and post-ACTH peak ALD and ARR from the NC, 47 (34.3%) HDM-2 patients were found to have PA, whereas 6 (10.4%) HDM-2 patients without PA (DCVT-negative) exhibited an exaggerated aldosterone response to stress-a prevalence much higher than ever reported. Treatment with mineralocorticoid receptor antagonists (MRAs) induced a significant and permanent reduction of BP in all HDM-2 patients. Early diagnosis and targeted treatment of PA is crucial to prevent any aggravating effect on chronic diabetic complications.

Genetic screening of hypertensive patients with aldosterone hypersecretion under conditions of stress.

PURPOSE: Although ACTH is considered a secondary regulator of aldosterone production, patients with apparent essential hypertension have been treated with mineralocorticoid receptor antagonists (MRAs). In this study, we aimed to identify potentially damaging variants that might be implicated in the phenotype of a well-characterized cohort of 21 hypertensive patients without PA but with stress-induced aldosterone hypersecretion. The patients' blood pressure was normalized though MRA administration. METHODS: Genetic screening was performed through whole-exome sequencing (WES), and variants in PA-associated or in ion-channels of aldosterone-regulating genes were prioritized. Variants with population frequency < 0.01, predicted to alter protein structure and classified as likely pathogenic by in silico tools, were retained. RESULTS: Qualifying variants were identified in nine of the 21 patients screened. Seven patients were carriers of six potentially damaging variants in six genes associated with PA (KCNK9, KCNK5, ATP13A3, SLC26A2, CACNA1H, and CACNA1D). A novel variant in the KCNK9 gene (p.V221M) is reported. Our analysis revealed two variants in two novel susceptibility genes for aldosterone hypersecretion, namely, KCNK16 (p.P255H) and CACNA2D3 (p.V557I). CONCLUSION: WES revealed potentially damaging germline variants in genes participating in aldosterone synthesis/regulating pathways in 9/21 patients of our cohort. The variants identified might play a role in aldosterone hypersecretion under conditions of stress. The potential pathogenicity of these variants should be examined in future functional studies.

Transcriptomics, Epigenetics, and Metabolomics of Primary Aldosteronism.

INTRODUCTION: Primary aldosteronism (PA) is the most common cause of endocrine hypertension, mainly caused by aldosterone-producing adenomas or hyperplasia; understanding its pathophysiological background is important in order to provide ameliorative treatment strategies. Over the past several years, significant progress has been documented in this field, in particular in the clarification of the genetic and molecular mechanisms responsible for the pathogenesis of aldosterone-producing adenomas (APAs). METHODS: Systematic searches of the PubMed and Cochrane databases were performed for all human studies applying transcriptomic, epigenetic or metabolomic analyses to PA subjects. Studies involving serial analysis of gene expression and microarray, epigenetic studies with methylome analyses and micro-RNA expression profiles, and metabolomic studies focused on improving understanding of the regulation of autonomous aldosterone production in PA were all included. RESULTS: In this review we summarize the main findings in this area and analyze the interplay between primary aldosteronism and several signaling pathways with differential regulation of the RNA and protein expression of several factors involved in, among others, steroidogenesis, calcium signaling, and nuclear, membrane and G-coupled protein receptors. Distinct transcriptomic and metabolomic patterns are also presented herein, depending on the mutational status of APAs. In particular, two partially opposite transcriptional and steroidogenic profiles appear to distinguish APAs carrying a KCNJ5 mutation from all other APAs, which carry different mutations. CONCLUSIONS: These findings can substantially contribute to the development of personalized treatment in patients with PA.

Unravelling the Genetic Basis of Primary Aldosteronism.

Primary aldosteronism (PA), a condition characterized by autonomous aldosterone hypersecretion, constitutes the most common cause of secondary hypertension. Over the last decade, major breakthroughs have been made in the field of genetics underpinning PA. The advent and wide application of Next Generation Sequencing (NGS) technology led to the identification of several somatic and germline mutations associated with sporadic and familial forms of PA. Somatic mutations in ion-channel genes that participate in aldosterone biosynthesis, including KCNJ5, CACNA1D, ATP1A1, and ATP2B3, have been implicated in the development of aldosterone-producing adenomas (APAs). On the other hand, germline variants in CLCN2, KCNJ5, CACNA1H, and CACNA1D genes have been implicated in the pathogenesis of the familial forms of PA, FH-II, FH-III, and F-IV, as well as PA associated with seizures and neurological abnormalities. However, recent studies have shown that the prevalence of PA is higher than previously thought, indicating the need for an improvement of our diagnostic tools. Further research is required to recognize mild forms of PA and to investigate the underlying molecular mechanisms.

Five Reasons for the Failure to Diagnose Aldosterone Excess in Hypertension.

Primary hyperaldosteronism (PA) is a well-known cause of hypertension although its exact prevalence amongst patients with apparent essential hypertension has been a matter of debate. A number of recent studies have suggested that mild forms of PA may be relatively common taking into consideration factors that were previously either overestimated or ignored when developing diagnostic tests of PA and when applying these tests into normotensive individuals. The performance characteristics and diagnostic accuracy of such tests are substantially increased when the adrenocorticotrophin effect, inappropriate potassium levels and their application in carefully selected normotensive individuals are considered. In the present review, we critically analyze these issues and provide evidence that several, particularly mild, forms of PA can be effectively identified exhibiting potentially important clinical implications.

Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion.

BACKGROUND: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K+ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA). OBJECTIVE: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion. DESIGN AND PATIENTS: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out. RESULTS: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected. CONCLUSIONS: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

A new highly sensitive and specific overnight combined screening and diagnostic test for primary aldosteronism.

CONTEXT: Primary aldosteronism (PA) is the most common cause of endocrine hypertension that is diagnosed following a two-step process: an initial screening test, based on the serum aldosterone-to-renin ratio (ARR), followed by a relatively laborious and time-consuming confirmatory test to document autonomous aldosterone (ALD) secretion. OBJECTIVE: The aim of this study is to develop a simple overnight test for the early and definite diagnosis of PA. PATIENTS AND METHODS: Totally, 148 hypertensive patients underwent a fludrocortisone-dexamethasone suppression test (FDST) and the new overnight diagnostic test (DCVT) using pharmaceutical RAAS (renin-angiotensin-aldosterone system) blockade with dexamethasone, captopril and valsartan. RESULTS: Of the 148 patients, 45 were diagnosed as having PA and they all normalized their elevated blood pressure (BP) after administration of spironolactone or eplerenone. The remaining 103 patients were considered as having essential hypertension and served as controls. Using ROC analysis, the estimated sensitivity and specificity were 91 and 100%, respectively, for the post-FDST ARR, whereas 98% and 89% and 100% and 82% for the post-DCVT ARR and post-DCVT ALD, respectively, with selected cutoffs of 0.32ng/dL/µU/mL and 3ng/dL respectively. However, considering these cutoffs simultaneously, the estimated sensitivity and specificity were 98 and 100% respectively. Applying these cutoffs, the diagnosis of PA was confirmed in 44 (98%) of the 45 patients who were considered to have the disease. CONCLUSIONS: In this study, a highly sensitive and specific, low-cost, rapid, safe, and easy-to-perform diagnostic test (DCVT) for PA is described, which could be utilized on an outpatient basis potentially substituting conventional laborious testing.

Stress-induced Aldosterone Hyper-Secretion in a Substantial Subset of Patients With Essential Hypertension.

CONTEXT: Aldosterone (ALD) secretion is regulated mainly by angiotensin II, K(+), and adrenocorticotropic hormone (ACTH). Mineralocorticoid receptor antagonists (MRAs) have effectively been used for the treatment of patients with hypertension who do not have primary aldosteronism (PA). OBJECTIVE: We tested whether chronic stress-related ACTH-mediated ALD hypersecretion and/or zona glomerulosa hypersensitivity could be implicated in the pathogenesis of essential hypertension (ESHT). PATIENTS AND METHODS: One hundred thirteen hypertensives without PA and 61 normotensive controls underwent an ultralow-dose (0.03-µg) ACTH stimulation and a treadmill test. Patients with ALD hyper-response according to the cutoffs obtained from controls received treatment with MRAs and underwent genomic DNA testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. A control group of 22 patients with simple ESHT received treatment with MRAs. RESULTS: Based on the cutoffs of ALD and aldosterone-to-renin ratio (ARR) post-ACTH stimulation obtained from controls, 30 patients (27%) exhibited an ALD but not cortisol (F) hyper-response (HYPER group). This group had no difference in basal ACTH/renin (REN) concentrations compared with controls and the 83 patients with hypertension (73%) without an ALD hyper-response to ACTH stimulation. Patients in the HYPER group demonstrated significantly higher ALD concentrations, ARR, and ALD/ACTH ratio (AAR) in the treadmill test. Treatment with MRAs alone produced normalization of blood pressure in these patients whereas patients with hypertension with neither PA nor ALD hyper-response to ACTH stimulation who served as a control group failed to lower blood pressure. Also, two novel germline heterozygous KCNJ5 mutations were detected in the HYPER group. CONCLUSIONS: A number of patients with hypertension without PA show ACTH-dependent ALD hyper-secretion and benefit from treatment with MRAs. This could be related to chronic stress via ACTH hyper secretion and/or gene-mutations increasing the zona glomerulosa responsiveness to excitatory stimuli.

Patients with apparently nonfunctioning adrenal incidentalomas may be at increased cardiovascular risk due to excessive cortisol secretion.

CONTEXT: Although adrenal incidentalomas (AIs) are associated with a high prevalence of cardiovascular risk (CVR) factors, it is not clear whether patients with nonfunctioning AI (NFAI) have increased CVR. OBJECTIVE: Our objective was to investigate CVR in patients with NFAI. DESIGN AND SETTING: This case-control study was performed in a tertiary general hospital. SUBJECTS: SUBJECTS included 60 normotensive euglycemic patients with AI and 32 healthy controls (C) with normal adrenal imaging. MAIN OUTCOME MEASURES: All participants underwent adrenal imaging, biochemical and hormonal evaluation, and the following investigations: 1) measurement of carotid intima-media thickness (IMT) and flow-mediated dilatation, 2) 2-hour 75-gram oral glucose tolerance test and calculation of insulin resistance indices (homeostasis model assessment, quantitative insulin sensitivity check, and Matsuda indices), 3) iv ACTH stimulation test, 4) low-dose dexamethasone suppression test, and 5) NaCl (0.9%) post-dexamethasone saline infusion test. RESULTS: Based on cutoffs obtained from controls, autonomous cortisol secretion was documented in 26 patients (cortisol-secreting AI [CSAI] group), whereas 34 exhibited adequate cortisol and aldosterone suppression (NFAI group). IMT measurements were higher and flow-mediated vasodilatation was lower in the CSAI group compared with both NFAI and C and in the NFAI group compared with C. The homeostasis model assessment index was higher and quantitative insulin sensitivity check index and Matsuda indices were lower in the CSAI and NFAI groups compared with C as well as in CSAI compared with the NFAI group. The area under the curve for cortisol after ACTH stimulation was higher in the CSAI group compared with the NFAI group and C and in the NFAI group compared with C. In the CSAI group, IMT correlated with cortisol, urinary free cortisol, and cortisol after a low-dose dexamethasone suppression test, whereas in the NFAI group, IMT correlated with area under the curve for cortisol after ACTH stimulation and urinary free cortisol. CONCLUSIONS: Patients with CSAI without hypertension, diabetes, and/or dyslipidemia exhibit adverse metabolic and CVR factors. In addition, NFAIs are apparently associated with increased insulin resistance and endothelial dysfunction that correlate with subtle but not autonomous cortisol excess.

Evidence of primary aldosteronism in a predominantly female cohort of normotensive individuals: a very high odds ratio for progression into arterial hypertension.

CONTEXT: Primary aldosteronism (PA) is an established cause of hypertension, whereas high-normal serum aldosterone levels have been linked to an increased risk for hypertension. OBJECTIVE: We aimed to define the post-fludrocortisone-dexamethasone suppression test (FDST) normal cutoff values of aldosterone and the aldosterone to renin ratio and evaluate the presence of PA in normotensive individuals. DESIGN: This study was designed as a case-control study. SETTING: The study was performed in a tertiary general hospital. PATIENTS: One hundred normotensive participants (80 females), mean age 53 years, were studied. MAIN OUTCOME MEASURES: All participants underwent baseline biochemical and hormonal evaluation, FDST, and adrenal computerized tomography. Blood pressure was assessed at baseline and after 5 years. RESULTS: Sixty-nine participants with normal adrenal computerized tomography who remained normotensive after 5 years were used as a control population to calculate the cutoff values of adequate aldosterone suppression. PA was defined as a combination of post-FDST aldosterone to renin ratio of 0.93 ng/dL · µU/mL or greater (100% sensitivity and 96% specificity) and post-FDST aldosterone of 2.96 ng/dL or greater (100% sensitivity and 61% specificity on receiver-operating characteristic analysis). Thirteen of 100 participants had PA at baseline and 11 (85%) developed hypertension, whereas only 20 of 87 without PA (23%) developed hypertension at 5 years [odds ratio (OR) 18.42, 95% confidence intervals (CI) 3.76-90.10, P < .0001]. Logistic regression analysis showed a positive relation of PA [odds ratio (OR) 16.30, confidence interval (CI) 1.78-150.30, P = .01] and a negative relation of serum potassium (OR 0.39, CI 0.19-0.79, P = .01) with the development of hypertension. CONCLUSIONS: Normotensive PA represents a clinical entity referring to normotensive individuals with PA who are at increased risk for hypertension.

The functional status of incidentally discovered bilateral adrenal lesions.

OBJECTIVE: To investigate autonomous cortisol and aldosterone secretion and insulin resistance (IR) indices, in patients with incidentally discovered bilateral adrenal lesions (BA). PATIENTS: Thirty-six patients with BA, 113 patients with unilateral adrenal incidentalomas (UA) and 89 healthy subjects (C) with normal adrenal imaging. MEASUREMENTS: All participants underwent adrenal imaging, baseline biochemical and hormonal measurements and the following investigations on consecutive days: (i) A 2-h oral glucose tolerance test (OGTT) (75 g) with glucose and insulin measurements every 30 min. (ii) An adrenocorticotrophin (ACTH) stimulation test with intravenous (i.v.) bolus administration of 250 µg of ACTH (1-24) and measurement of serum cortisol and aldosterone before and after 30 and 60 min. (iii) A low-dose dexamethasone suppression test (LDDST) (0·5 mg of dexamethasone every 6-h for 2 days) with cortisol measurement 6 h after the last dexamethasone dose and (iv) A NaCl (0·9%) postdexamethasone saline infusion test (PD-SIT) (2 l of NaCl 0·9% iv in 4 h) following the LDDST, with aldosterone measurement at the end of the test. RESULTS: Cortisol and aldosterone cut-offs based on the mean + 2 SD values obtained from the C group following the LDDST and PD-SITs were calculated (34·11 nm and 74·83 pm, respectively). Based on the above cut-offs, autonomous cortisol and aldosterone secretion was found in 42·5 and 15·9% of patients with UA, and in 41·7 and 19·4% of patients with BA, respectively. In addition, 17·7% of patients with UA and 19·4% of patients with BA had concomitant autonomous cortisol and aldosterone secretion. Cortisol and aldosterone levels following the LDDST and PD-SIT were significantly higher in the BA compared to the UA group, respectively. Furthermore, patients with BA had more pronounced glucose levels and insulin resistance (IR) indices compared to patients with UA. CONCLUSIONS: Patients with BA have more pronounced autonomous cortisol and aldosterone secretion and glucose metabolism alterations than patients with UA. Further studies are needed to evaluate the potential long-term consequences of these findings.