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

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1 ORIGINAL ARTICLE Stress-induced Aldosterone Hyper-Secretion in a Substantial Subset of Patients With Essential Hypertension Athina Markou, Amalia Sertedaki,* Gregory Kaltsas,* Ioannis I. Androulakis, Chrisanthi Marakaki, Theodora Pappa, Aggeliki Gouli, Labrini Papanastasiou, Stelios Fountoulakis, Achilles Zacharoulis, Apostolos Karavidas, Despoina Ragkou, Evangelia Charmandari, George P. Chrousos, and George P. Piaditis Department of Endocrinology and Diabetes Center (A.M., I.I.A., C.M. T.P., A.G., L.P., S.F., D.R., G.P.P.), G. Gennimatas General Hospital, Athens 11527, Greece; Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics (A.S., E.C., G.P.C.), University of Athens Medical School, Aghia Sophia Children s Hospital, Athens 11527, Greece; Department of Pathophysiology (G.K.), University of Athens Medical School, Laikon Hospital, Athens 11527, Greece; and Department of Cardiology (A.Z., A.K.), G. Gennimatas General Hospital, Athens 11527, Greece 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 (). Patients and Methods: One hundred thirteen hypertensives without PA and 61 normotensive controls underwent an ultralow-dose (0.03- g) ACTH stimulation and a treadmill test. Patients with ALD hyperresponse 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 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 ( 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 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 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. (J Clin Endocrinol Metab 100: , 2015) ISSN Print X ISSN Online Printed in USA Copyright 2015 by the Endocrine Society Received January 29, Accepted May 11, First Published Online May 14, 2015 For related article see page 2853 * A.S. and G.K. contributed equally to the study. Abbreviations: AAR, ALD-to-ACTH ratio; ALD, aldosterone; ARR, aldosterone-to-renin ratio; AUC, area under the curve; BMI, body mass index; BP, blood pressure; CT, computed tomography; DBP, diastolic blood pressure;, essential hypertension; F, cortisol; FAR, F-to-ACTH ratio; FDST, fludrocortisone-dexamethasone suppression test; FH, familial hyperaldosteronism;, hyper-response; MBPR, the mean of values of baseline, peak exercise and recovery; MRA, mineralocorticoid receptor antagonist; PA, primary aldosteronism; REN, renin; SBP, systolic blood pressure. doi: /jc J Clin Endocrinol Metab, August 2015, 100(8): press.endocrine.org/journal/jcem 2857

2 2858 Markou et al Aldosterone Hyper-response to ACTH Stimulation J Clin Endocrinol Metab, August 2015, 100(8): Primary aldosteronism (PA) represents the most frequent cause of secondary hypertension with an estimated prevalence of 5 15% (1, 2). Following the introduction of the combined fludrocortisone-dexamethasone suppression test (FDST), which takes into consideration the stimulatory effect of adrenocorticotropic hormone (ACTH) on aldosterone (ALD) secretion, we have demonstrated that the prevalence of PA is probably higher: up to 30% (3, 4). This is particularly important given that patients with hypertension with PA have a higher risk of developing target-organ damage compared with age- and sex-matched patients with essential hypertension () (5). Following the rationale of administering mineralocorticoid receptor antagonists (MRAs) in patients with apparent we have identified a cohort of patients with hypertension without PA and low normal serum potassium concentrations who have an impressive response in blood pressure (BP) control to these agents. We therefore hypothesized that in these subjects a form of ALD excess not directly related to the REN-angiotensin axis might be implicated. Given that ACTH is a potent stimulus of ALD secretion in humans (6) we speculated that repetitive acute and/or continuous (chronic) physical and/or psychological stress might induce ACTH-mediated ALD hyper-secretion and/or prime the adrenal cortex to secrete ALD in response to ACTH, angiotensin II, or other zona glomerulosa stimuli. The CYP11B1/CYP11B2 chimeric gene has been identified in patients with familial hyperaldosteronism (FH) type I and encodes ALD synthase activity under ACTH instead of angiotensin II control; such patients can be either normo- or hypertensive (7 9). Recently, somatic mutations of the KCNJ5 gene encoding the inward rectifier K channel GIRK4 have been identified in ALD-producing adenomas, whereas similar germline mutations have also been described in patients with FH type III. The clinical spectrum ranges from mild to moderate hypertension associated with normal adrenal morphology to severe hypertension and bilateral adrenal hyperplasia or ALD-producing adenoma (10 15). In this study we evaluated the role of stress-induced ACTH-mediated ALD secretion in patients with employing two tests. The ultralow ACTH stimulation test, which simulates low-grade chronic physical and/or psychological stress, and the treadmill exercise stress test, which simulates acute physical stress. In addition, we assessed the antihypertensive effects of MRAs in two subgroups of hypertensives and investigated whether specific genetic variations of ALD synthesis/secretion-regulating genes could be implicated. Patients and Methods We concurrently studied 61 normotensive controls (4) and 113 patients with hypertension who had normal ALD suppression post-fdst and normal adrenal computed tomography (CT). All participants were identified and selected from an outpatient endocrine clinic among patients referred for thyroid goiter or bone mineral density evaluation. Normotensive individuals had been followed up for a mean period of 5 years and as all remained normotensive were used as controls (4). Normal BP was defined as systolic BP (SBP) of less than 135 mm Hg and diastolic BP (DBP) less than 85 mm Hg, measured at the outpatient unit in at least three consecutive visits (mean of three measurements) (16). Eleven of the patients with hypertension were newly diagnosed and 102 under treatment with antihypertensive agents. Exclusion criteria included the presence of cardiovascular, renal, and/or hepatic disease; past or present malignancy; rheumatological disease; as well as the presence of a known adrenal lesion(s). On the basis of the 97.5-percentile values from the control population, the post-fdst cutoff levels for ALD and ALD-to- REN ratio (ARR) were defined as 82 pmol/l and 26 pmol/miu, respectively. The diagnosis of PA was based on the combination of a post-fdst ARR at least 26 pmol/miu and post-fdst ALD at least 82 pmol/l (4). All participants were euthyroid and postmenopausal women did not receive any hormone replacement therapy. The study protocol was approved by the institution s ethics committee and informed consent was obtained from all participants. At baseline, all participants underwent recording of their medical history and a complete physical examination and documentation of anthropometric characteristics [weight (kg), height (cm), waist circumference (cm), and body mass index (BMI) (kg/m 2 )] by the same physician (A.M.). All participants underwent the following tests as outpatients : 1) Day 1: blood sampling for routine testing and 24-hour urinary Na and K concentrations; 2) Day 2: ultralow ACTH test. Plasma ACTH, cortisol (F), ALD, and REN were measured at baseline followed by 0.03 g of ACTH iv; F and ALD were measured and the -F, -ALD, and -ARR was calculated at 15 and 30 minutes g of ACTH were used as this dose has been shown to be the lowest dose to stimulate ALD secretion (17). During the test, participants were in a sitting position and BP was measured at baseline and after completion of the test. 3) Day 3: treadmill test (exercise test as per Bruce protocol) (18). ACTH, F, F-to-ACTH ratio (FAR), REN, ALD, ARR, and ALD-to-ACTH ratio (AAR) were measured at baseline, at peak exercise, and upon recovery (15 min after peak exercise). Peak exercise was optimal when participants reached 80% of maximal cardiac pulse according to their age. 4) Days 4 7: FDST. Fludrocortisone acetate (0.1 mg) was administered orally every 6 hours, and sodium chloride (4 g) was administered three times daily; on Day 7, 2 mg of dexamethasone were administered orally at midnight; 5) Day 8: The concentrations of F, ALD, REN, and ACTH were measured. All blood samples were drawn with the participants remaining seated in a nonstressful environment for at least 30 minutes (0830 h). Supplementation with potassium gluconate (4.68 g 3 d) was given to all during the FDST to maintain normal serum K concentrations ( meq/l). BP was monitored every morning in the outpatient unit in the sitting position. Patients receiving antihypertensive medication had their treatment changed to a

3 doi: /jc press.endocrine.org/journal/jcem 2859 calcium channel blocker (nifedipine or diltiazem) 2 4 weeks before testing. An adrenal CT scan with 2-mm sections using the Philips Brilliance 16 Spiral scanner was performed to all participants. Adrenal adenomas were defined as well-circumscribed adrenal lesions greater than 10 mm with a noncontrast CT attenuation coefficient of less than 10 Hounsfield Units. Hypertensive participants without PA but with ALD hypersecretion following the ultralow ACTH test had their initial antihypertensive treatment changed to an MRA (spironolactone or eplerenone) and were re-evaluated 3 months later. In addition, a subgroup of 22 patients with (all had similar to nonhypertensive controls FDST and ultralow ACTH test results), who were on treatment with calcium channel blockers only, had their antihypertensive treatment changed to MRAs alone (spironolactone or eplerenone) and were also re-evaluated 3 months later. These patients served as controls (MRAs-controls) to evaluate the MRAs effect on BP levels in patients with. Genetic studies Twenty six of the patients gave informed consent and underwent genetic analysis. DNA isolation from peripheral lymphocytes was carried out as previously described (13). Detection of CYP11B1/CYP11B2 chimeric gene The presence/absence of the CYP11B1/CYP11B2 chimeric gene was tested by PCR amplification as previously described (19). Sequencing KCNJ5 The coding regions and the intron-exon junctions of the KCNJ5 gene were sequenced as previously described (11). To exclude the possibility that any mutations found were common polymorphisms, 148 control DNA samples were sequenced. Three in silico programs were used to predict the effect of the amino acid change on the protein function: PolyPhen-2 (Polymorphism Phenotyping v2), SIFT (Sorting Intolerant From Tolerant) and Mutation Taster. Hormonal measurements Hormonal measurements for ALD, REN, F, and ACTH were performed as previously described (3, 4). Statistical analysis Statistical analysis used the SPSS software package (SPSS Inc, version 17.0). One-way ANOVA was performed for the statistical comparison of hormone values in hypertensives and controls. Student t test and the nonparametric Mann-Whitney U test were used to compare continuous variables with and without normal distribution, respectively. The mean SD was used to express the results, and P.05 was considered statistically significant. The 97.5 percentile was used to define the upper-normal cutoffs for ALD hypersecretion. For the ultralow ACTH test, the area under the curve (AUC) was used for the comparison of ALD, ARR, and F among the participants. For the treadmill test the ratios of ARR, ALD/ACTH (AAR), and F/ACTH (FAR) were calculated. In addition, overall responses of ACTH, F, ALD, and REN were expressed as the mean of values of baseline, peak exercise, and recovery (MBPR). Results On the basis of the 97.5-percentile values from controls, the post-ultralow-acth test cutoff levels for ALD and ARR were defined as 47 ng/dl (1300 pmol/l) and 2.7 ng/dl/ U/mL (77 pmol/miu), respectively, at either 15 or 30 minutes post-acth stimulation. Given that hyperresponders were defined patients whose response was above the 97.5 percentile cutoff for both these measurements, 30/113 (27%) participants had a hyper-response to the ultralow ACTH test ( group), whereas the remaining 83/113 (73%) did not, and were considered to have. The characteristics of the three groups (,, and controls) are shown in Table 1. Participants of the and groups had significantly higher baseline SBP (P 2 and P 3 ), and DBP (P 2 and P 3 ) compared with controls. Twenty-four-hour urine K and 24-hour urine K /Na ratio were significantly higher (P 2 ) in the group compared with controls. All groups obtained sufficient suppression of ACTH, F, REN, and ALD concentrations and the ARR post-fdst. Table 2 and Figure 1 show the F, -F, ALD, -ALD, ARR, and -ARR concentrations in the three groups, upon ultralow ACTH testing. There was a clear increase from baseline in F, -F, ALD, -ALD, ARR, and -ARR concentrations at 15 and 30 minutes (P 1 and P 2 )inall three groups. On the basis of the AUC, the group showed significantly higher ALD, -ALD, ARR, and -ARR compared with the and control groups (P 3 and P 4, respectively); however, there was no difference in F concentrations between groups (P 3, P 4, P 5 ). The ACTH, F, REN, and ALD concentrations, and FAR, ARR, and AAR during the treadmill test are shown in Table 3 and Figure 2. There was a significance increase of ACTH, REN, and ALD concentrations (P 1 and P 2 ), and ARR, AAR (P 1 and P 2 ) from baseline in all three groups. Mean ACTH, F, FAR, and REN concentrations did not differ between groups (P 3, P 4, P 5 ). The group showed significantly higher mean ALD, ARR, and AAR compared with the and control groups (P 3, P 4 ), but there was no difference between the group and the control groups (P 5 ). After changing the antihypertensive treatment of the group to a MRA, the mean SBP decreased from 154 to 124 mm Hg (a 19% reduction) (P.0001) and the mean DBP from 92 to 77 mm Hg (a 16% reduction) (P.0001). Eighteen patients were treated with spironolactone only (median dose, 50 mg; dose range, mg daily), two with spironolactone and calcium channel blocker, seven with eplerenone only (median dose, 100 mg; dose range, mg daily) and three with epler-

4 2860 Markou et al Aldosterone Hyper-response to ACTH Stimulation J Clin Endocrinol Metab, August 2015, 100(8): Table 1. Clinical Characteristics and Endocrinological Data in All Three Groups Studied (n 30), Mean (SD) (n 83), Mean (SD) CON (n 61), Mean (SD) P 1 P 2 CON P 3 CON Age, y 53 (8) 54 (7) 52 (7) NS NS NS BMI, kg/m 2 29 (4) 30 (5) 28 (6) NS NS NS Waist, cm 95 (11) 99 (12) 94 (14).0001 NS.0001 SBP, mm Hg 155 (13) 153 (11) 117 (12) NS DBP, mm Hg 92 (5) 90 (9) 77 (8) NS Na, meq/l 141 (3) 141 (3) 142 (2) NS NS NS K, meq/l 4.0 (0.4) 4.1 (0.3) 4.1 (0.3) NS NS NS 24-h urine Na, meq/24 h 130 (54) 137 (61) 140 (50) NS NS NS 24-h urine K, meq/24 h 81 (35) 61 (25) 54 (22) NS.02 NS 24-h urine K /Na ratio 0.7 (0.5) 0.4 (0.1) 0.4 (0.1) NS.02 NS ACTH, pg/ml 25 (16) 23 (15) 21 (16) NS NS NS F, g/dl 18.3 (7.3) 17.5 (7.6) 17.3 (6.3) NS NS NS ALD, ng/dl 16.0 (11.3) 11.7 (6.7) 9.2 (6.4) NS REN, U/mL 15 (7) 21 (21) 19 (16) NS NS NS ARR, ng/dl/ U/mL 1.7 (2.0) 1.1 (0.7) 0.7 (0.5) NS ACTH POST-FDST, pg/ml 7 (3) 7 (4) 6 (2) NS NS NS F POST-FDST, g/dl 1.0 (0.5) 1.4 (1.1) 1.0 (0.4) NS NS NS ALD POST-FDST, ng/dl 2.8 (1.8) 2.4 (1.3) 1.7 (0.6) NS REN POST-FDST, U/mL 7 (6) 8 (8) 5 (2) NS ARR POST-FDST, ng/dl/ U/mL 0.5 (0.2) 0.4 (0.2) 0.4 (0.2) NS NS NS Abbreviations: CON, controls;, essential hypertensives;, hyper-responders; NS, not significant; POST, at end of test. Results are expressed as mean (SD). enone plus one of the following: calcium channel blocker, -blocker, angiotensin II receptor blocker. In contrast, after changing the antihypertensive treatment of the subjects who were considered to be the MRA controls to a MRA alone, (16 on spironolactone and six on eplerenone; dose range, mg), the mean SBP and the mean DBP rebounded from 134 to 143 mm Hg (a 7% increment) (P.002) and 84 to 88 mm Hg (a 5% increment) (P.05), respectively. Genetic studies The CYP11B1/CYP11B2 chimeric gene was not detected in any of the 26 patients examined. Sequencing of the KCNJ5 gene revealed that two of the 26 patients studied harbored two different germline heterozygous mutations. The first mutation was agto A substitution at nucleotide 775 (n.g775a), which alters codon 259 from Valine to Methionine (p.v259m) and the second mutation was attoasubstitution at Table 2. Hormonal Responses in the Ultralow ACTH Test and Their Corresponding AUC Hormonal Responses in Ultralow ACTH Test, Mean (SD) AUC, Mean (SD) Time, min P P AUC P 3 P 4 CON F, g/dl 18.8 (9.0) 30.9 (9.5) 33.9 (10.2) (270) NS NS NS 17.4 (7.2) 28.5 (9.4) 29.9 (9.8) (259) CON 17.3 (6.7) 27.4 (7.5) 29.9 (8.9) (205) ALD, ng/dl 25.0 (14.9) 81.7 (26.7) 69.1 (25.4) (584) NS 11.4 (6.7) 28.6 (10.3) 23.6 (11.1) (263) CON 8.4 (5.7) 22.8 (11.4) 20.0 (10.0) (266) ARR, ng/dl/ U/mL 2.0 (1.6) 7.3 (5.8) 6.0 (4.1) (126) NS 0.8 (0.6) 2.2 (1.3) 1.9 (1.4) (36) CON 0.6 (0.4) 1.6 (1.0) 1.3 (0.6) (22) Abbreviations: CON, controls;, essential hypertensives;, hyper-responders; NS, not significant. Results are expressed as the mean with SDs given in parentheses. P 5 CON

5 doi: /jc press.endocrine.org/journal/jcem 2861 mutation Y348N was damaging by Poly-Phen 2, SIFT, and Mutation Taster. The KCNJ5 Y348N positive hyper-responder had baseline BP, 160/90mmHg;serumK,4.1mEq/L; and urinary K, 78 meq/d. The KCNJ5 V259M -positive hyper-responder had baseline BP, 150/85 mm Hg;serumK,4.2mEq/L;andurinary K, 96 meq/d. Figure 1. Ultralow ACTH-test: -aldosterone, -Aldosterone/Renin ratio; and -cortisol response (left) and the corresponding AUC (right). The results are expressed as mean (SE). nucleotide 1042 (n.t1042a) altering codon 348 from Tyrosine to Asparagine (p.y348n). These two mutations were not detected in 296 chromosomes, thereby excluding the possibility that they are common polymorphisms. In silico analysis revealed that mutation V259M was damaging by PolyPhen-2 and SIFT, whereas Discussion In this study we identified a cohort of hypertensive individuals without PA who exhibited increased ALD secretion following stimulation with 0.03 g ACTH. In addition, these subjects showed a similar ALD hyperresponse to a treadmill exercise test and had higher 24-hour urinary K excretion than controls, in accordance with the observed ALD hypersecretion. Targeted therapy with MRAs effectively normalized their BP. A plausible explanation for these findings is that in such cases, glomerulosa cells are primed by stressinduced ACTH secretion, which makes them more sensitive to any increase in either ACTH or REN/angiotensin II levels, as happens with chronic physical or psychological stressful events. This concept is supported by the findings of the subsequent genetic studies, as the presence of the chimeric gene was not documented in any of the hyper-responders, whereas only two of the 26 hyper-responders (8%) had novel germline mutations of the KNCJ5 gene. In these two cases, KCNJ5 mutations could have sensitized the glomerulosa cells to either ACTH or angiotensin stimulation, also leading to an ALD hyper-response. ACTH and angiotensin II stimulate ALD secretion by activating different postreceptor pathways (20 22). It is likely that under conditions of stress, the actions of ACTH and/or angiotensin II are potentiated by the already increased intracellular Ca concentrations due to the presence of sensitizing KCNJ5 mu-

6 2862 Markou et al Aldosterone Hyper-response to ACTH Stimulation J Clin Endocrinol Metab, August 2015, 100(8): Table 3. Hormonal Responses in the Treadmill Test Hormonal Response in Treadmill Test, Mean (SD) MBPR, Mean (SD) Time, min Basal, Mean (SD) Peak, Mean (SD) Recovery, Mean (SD) P 1 B P P 2 B R MBPR, Mean (SD) P 3 P 4 CON ACTH, pg/ml 21 (16) 32 (28) 26 (21).01 NS 26 (20) NS NS NS 19 (10) 26 (18) 22 (15).01 NS 22 (13) CON 20 (13) 24 (15) 20 (11).0001 NS 21 (11) F, g/dl 17.3 (5.4) 17.4 (6.8) 16.6 (6.7) NS NS 17 (6) NS NS NS 17.4 (7.4) 16.9 (8.2) 16.9 (7.7) NS NS 17 (7) CON 17.4 (6.2) 16.6 (7.3) 15.8 (6.6) NS NS 19 (6) FAR, g/dl/pg/ml 1.2 (0.6) 0.7 (0.4) 0.8 (0.5) NS NS 0.9 (0.5) NS NS NS 1.1 (0.8) 0.9 (1.0) 1.0 (0.7).001 NS 1.0 (0.8) CON 1.1 (0.6) 0.9 (0.5) 0.9 (0.6) (0.5) REN, U/mL 14 (8) 25 (19) 24 (18) (13) NS NS NS 17 (19) 25 (26) 24 (24) (22) CON 14 (10) 25 (23) 22 (18) (20) ALD, ng/dl 16.1 (11.2) 33.5 (22.3) 56.8 (42.5) (23) NS 10.1 (6.7) 21.9 (15.2) 25.6 (18.3) (15) CON 7.2 (6.0) 12.7 (7.5) 16.9 (12.0) (12) ARR, ng/dl/ U/mL 1.8 (2.0) 2.1 (2.5) 4.0 (5.0) NS (3.2) NS 0.9 (0.6) 1.3 (1.3) 1.8 (1.5) (0.7) CON 0.6 (0.5) 0.9 (0.8) 1.0 (0.7) NS (0.7) AAR, ng/dl/pg/ml 1.2 (1.4) 1.5 (1.5) 3.4 (3.6) NS (2.1) NS 0.6 (0.4) 0.9 (0.8) 1.3 (1.0) (0.7) CON 0.5 (0.5) 0.8 (0.8) 1.1 (1.1) (0.7) P 5 CON Abbreviations: CON, controls;, essential hypertensives;, hyper-responders; MBPR, mean values at baseline (B), at peak exercise (P), and upon recovery (R); NS, not significant. Results are expressed as the mean with SDs given in parentheses. tations, leading to ALD hyper secretion. It seems that these new mutations in our two patients in the group induce a milder phenotype than the KCNJ5 mutations seen in patients with FH type III and in APAs, probably representing a forme fruste of the former (10 13). Similarly the germline KCNJ5 mutation G151E has been shown to exhibit a milder phenotype than the somatic G151R mutation, although electrophysiologic studies on channels expressed in 293T cells revealed that the G151E mutants were producing larger Na conductance than G151R and increased Na dependent cell lethality (13). We used the combination of the post ultralow ACTH, ALD concentration, and ARR to define the group. This approach was employed to avoid including false-positive cases such as hypertensive individuals with elevated ALD concentrations due to high REN levels secondary to low salt intake, or hypertensive individuals with normal ALD but elevated ARR due to low REN concentrations. ACTH may exert an important facilitatory and priming role in stimulating mineralocorticoid synthesis and release, as previously described in conditions of stress (17, 23 26). To reproduce a mild physical activity or a response to an acute stressor, all participants underwent a treadmill exercise test and an ultralow ACTH test, respectively. In the treadmill test, the group showed significantly higher ALD concentrations and higher ARR compared with the and the control groups. As expected, during the treadmill test, both ACTH and REN concentrations increased significantly in all participants; however, these elevations did not differ between the groups. Although this suggests that the glomerulosa cells of the group oversecrete ALD, it is still unclear whether ACTH or angiotensin II are responsible. However, the findings from the ultralow ACTH test suggest that ACTH may be a sensitizing factor in a subset of patients with hypertension, as the group showed a significantly higher ALD levels and ARR than the or control groups, despite the absence of any difference in basal ACTH and mean REN concentrations during the

7 doi: /jc press.endocrine.org/journal/jcem 2863 Figure 2. Treadmill test: Aldosterone, Aldosterone/Renin ratio, and cortisol (left), and the corresponding mean values of baseline, peak, and recovery (right). The results are expressed as mean (SE). ultralow ACTH test between groups. This potential ACTH-induced adrenocortical hypersensitivity to stimuli expressed as AAR and FAR, seems not to involve the zona fasciculata cells, given that F response was not different between groups in either test. Furthermore, the absence of a F increase during the treadmill test is possibly related to the inability of this acute stressor to generate F release. Indeed, exercise levels below 70% VO 2 max activate catecholamine and ALD secretion without affecting F (24 26). Targeted treatment with MRAs reduced both SBP and DBP within the normal range (BP 135/85 mm Hg) in most the group, with only five patients requiring the coadministration of a second antihypertensive agent. This finding suggests that ALD excess is probably directly implicated with the development of hypertension in this subgroup of patients particularly as those regarded as having previously adequately controlled with calcium channel blockers, did not respond to MRAs alone. Although Levy et al (27) have documented that up to 80% of patients with apparent may obtain adequate BP control with MRAs, in the present study, both PA and ALD hyper-response to ACTH were excluded in the MRA controls by performing the FDST and ultralow ACTH tests, which was not the case in the previous studies (27). Furthermore, in the present study, the maximum dose of MRAs used was up to 100 mg, whereas other studies have used higher doses of up to 200 mg (27). Previous studies performed in our unit have revealed that the prevalence of PA in unselected patients with hypertension ranges from 29 32% (3, 28), which added to the 27% prevalence of ALD hyper-response to stress, found in the present study, account for approximately 60% of patients with apparent expected to respond to MRAs. This figure is close to that of patients responding to eplerenone in the Levy et al study. In summary, we have identified a cohort of hypertensive individuals with apparent without PA that exhibit ACTH- and/or angiotensin II dependent ALD hyper-secretion, in whom targeted treatment with a MRA improved BP control. We hypothesize that physical and/or psychological stress and/or mutations of the KCNJ5 and potentially other involved genes may sensitize the glomerulosa adrenocortical cells to their excitatory stimuli. It is, therefore, possible that this state of ALD

8 2864 Markou et al Aldosterone Hyper-response to ACTH Stimulation J Clin Endocrinol Metab, August 2015, 100(8): hyper-response to stress-related ACTH secretion in hypertensive individuals constitutes a new clinical entity, with potentially adverse clinical consequences in the long term. Acknowledgments A positive control DNA sample for the presence of the CYP11B1/ CYP11B2 chimeric gene was kindly provided by Dr Shengxin Xu via Professor R. D. Gordon. Address all correspondence and requests for reprints to: Dr Athina Markou, Department of Endocrinology and Diabetes Center, Athens General Hospital, G. Gennimatas, 154 Mesogion Avenue, Athens 11527, Greece. amarkouuk@yahoo. co.uk. This work was supported in part by the University of Athens Medical School Athens, Greece. Disclosure Summary: The authors have nothing to disclose. References 1. Funder JW, Carey RM, Fardella C, et al. 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