Negative feedback is a key mechanism controlling the

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1 JCEM ONLINE Advances in Genetics Endocrine Research Activation of the Hypothalamic-Pituitary-Adrenal Axis in Adults With Mineralocorticoid Receptor Haploinsufficiency Brian R Walker, Ruth Andrew, Brigitte Escoubet,* and Maria-Christina Zennaro* British Heart Foundation Centre for Cardiovascular Science (B.R.W., R.A.), Queen s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom; Assistance Publique-Hôpitaux de Paris (B.E.), Hôpital Bichat-Claude Bernard, Paris, France; Unité Mixte de Recherche 1138 (B.E.), INSERM, Centre de Recherche des Cordeliers, Paris, France; Université Paris Diderot (B.E.), Sorbonne Paris Cité, Paris, France; Unité Mixte de Recherche en Santé 970 (M.-C.Z.), INSERM, Paris Cardiovascular Research Center, Paris, France; University Paris Descartes (M.-C.Z.), Sorbonne Paris Cité, Paris, France; and Assistance Publique-Hôpitaux de Paris (M.-C.Z.), Hôpital Européen Georges Pompidou, Service de Génétique, Paris, France Context: Mineralocorticoid receptors (MRs) contribute to the negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis in rodents. Studies with MR antagonists suggest a similar role in humans. Objective: The objective of the study was to establish whether loss-of-function mutations in NR3C2, encoding MR, cause activation of the HPA axis. Design and Setting: This was a case-control study in members of pedigrees from the PHA1.NET cohort, comprising patients with pseudohypoaldosteronism type 1 (PHA1) who are heterozygous for loss-of-function mutations in NR3C2 and healthy controls who are unaffected family members. Participants: Twelve adult patients with PHA1 (six men, six women) and 20 age-matched healthy controls (seven men, 13 women) participated in the study. Results: Patients with PHA1 had higher morning plasma cortisol ( vs nmol/l, P.02) and increased 24-hour urinary excretion of cortisol metabolites ( vs g/mmol creatinine, P.03), independently of gender. After adjustment for gender, age, PHA1 diagnosis, and percentage body fat, higher plasma cortisol was associated with higher plasma renin, lower serum high-density lipoprotein-cholesterol, and higher waist circumference but not with blood pressure, carotid intima-media thickness, or echocardiographic parameters. Conclusions: Haploinsufficiency of MR in PHA1 causes HPA axis activation, providing genetic evidence that MR contributes to negative feedback in the human HPA axis. With limited sample size, initial indications suggest the resulting hypercortisolemia is related to the severity of MR deficiency and has adverse effects mediated by glucocorticoid receptors on liver lipid metabolism and adipose tissue distribution but does not adversely affect cardiac and vascular remodeling in the absence of normal signaling through the MR. (J Clin Endocrinol Metab 99: E1586 E1591, 2014) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received February 12, Accepted April 1, First Published Online April 8, 2014 Negative feedback is a key mechanism controlling the hypothalamic-pituitary adrenal (HPA) axis. Both glucocorticoid (GR) and mineralocorticoid (MR) receptors are expressed in central feedback sites. In the absence of local expression of the cortisol inactivating enzyme 11 -hydroxysteroid dehydrogenase type 2 in the adult * B.E. and M.-C.Z. contributed equally to this work. Abbreviations: BMI, body mass index; BP, blood pressure; GR, glucocorticoid receptor; HDL, high-density lipoprotein; HPA, hypothalamic-pituitary-adrenal; MR, mineralocorticoid receptor; PHA1, pseudohypoaldosteronism type 1. E1586 jcem.endojournals.org J Clin Endocrinol Metab, August 2014, 99(8):E1586 E1591 doi: /jc

2 doi: /jc jcem.endojournals.org E1587 central nervous system, both GR and MR bind glucocorticoids. A substantial body of evidence in rodents suggests that MR interacts with GR in mediating the negative feedback signal (1, 2). Global MR knockout mice, when rescued from early death by a high-salt diet, exhibit elevated corticosterone (3). Although the basal HPA axis function appears to be normal in mice with forebrain-specific MR knockdown (4) or transgenic MR overexpression (5), in the latter case, there is suppression of stress-induced glucocorticoid responses (6). There is also evidence for a role of MR in the HPA negative feedback in humans (7). Acute administration of the MR agonist fludrocortisone suppresses ACTH and cortisol secretion (8). Acute administration of the MR antagonist spironolactone, or its metabolite canrenoate, elevates cortisol levels (9), especially in the presence of a GR antagonist (10). Moreover, polymorphisms in NR3C2, some of which induce subtle MR dysfunction in vitro, have been associated with altered cortisol levels and altered suppression by dexamethasone in humans (11 14). A variety of loss-of-function mutations in NR3C2 cause the renal form of pseudohypoaldosteronism type 1 (PHA1), characterized by renal unresponsiveness to aldosterone resulting in sodium wasting despite elevated renin and aldosterone concentrations in plasma (15). The condition is autosomal dominant, caused by haploinsufficiency of the MR, and is distinct from generalized PHA1, a more severe autosomal recessive condition caused by mutations in the epithelial sodium channel (15). However, any consequences of MR dysfunction for the HPA axis negative feedback in renal PHA1 have not been addressed. We recently reported the characteristics of a cohort of adults with renal PHA1, compared with unaffected controls recruited from the same families (16). We have used this cohort to test the hypothesis that global MR deficiency in humans causes elevated cortisol secretion. Moreover, because cortisol signaling through the GR is preserved in renal PHA1, we have explored the hypothesis that adults with MR haploinsufficiency exhibit features of glucocorticoid excess, including obesity, hyperglycemia, and dyslipidemia. Materials and Methods As recently reported (16), participants in the Cardiovascular Evaluation of Adult PHA 1 Patients study were recruited from the PHA1.NET pedigrees at a single center between 2008 and Local ethical committee approval and written informed consent were obtained. Eligible participants were adults aged older than 18 years in whom mutations in NR3C2 were either confirmed (cases) or excluded by DNA sequencing (controls). Detailed clinical, cardiovascular imaging, body composition (by multifrequency spectroscopic bioimpedance; Analycor XF; Spengler), and biochemical phenotyping was performed, including collection of plasma obtained in the morning after an overnight fast and a 24-hour urine sample. Suitable blood and/or urine samples were available for only a subset of the full cohort. Cortisol was measured in plasma samples by RIA (MP Biomedicals). Cortisol metabolites were measured in urine by gas chromatography/triple quadrupole mass spectrometry, as previously described (17). Total cortisol metabolites in urine were calculated as the sum of 5 -tetrahydrocortisol 5 -tetrahydrocortisol 5 -tetrahydrocortisone and expressed as micrograms per millimole of creatinine. Statistical analysis Data from cases vs controls and men vs women were compared by Student s t tests. Independent effects of diagnosis and gender were assessed by a two-way ANOVA. Associations with blood pressure (BP), body mass index (BMI), waist circumference, percentage body fat, supine plasma renin and aldosterone, fasting blood glucose, glycated hemoglobin, fasting lipid profile, and results obtained from an ultrasound of the carotid artery and echocardiography were explored initially by simple Pearson correlations and then by multiple regression analyses, with inclusion of a PHA1 diagnosis (scored as 0 for cases or 1 for controls), gender (scored as 0 for women or 1 for men), age, percentage body fat, and either plasma cortisol or total urinary cortisol metabolite excretion. Results From the 39 case-control pairs of adults included in the original study (16), 24-hour urine samples were available from 12 PHA1 cases and 20 controls and suitably timed plasma samples from six PHA1 cases and 12 controls. Characteristics of cases and controls are shown in Table 1. They were well matched for age and BMI, although females were somewhat overrepresented in the control group. These participants came from 17 of the original 41 pedigrees; 10 pedigrees provided one or two cases, and 14 pedigrees provided one to three controls. All cases had mutations predicted to alter amino acid sequence in the N terminus (n 3), DNA-binding (n 7), or ligand-binding (n 3) domains of MR and had substantially elevated renin and aldosterone levels (Table 1). Plasma cortisol was elevated in PHA1 cases compared with controls (Table 1). Total daily urinary excretion of the major metabolites of cortisol was higher in PHA1 cases than controls (Table 1), this difference being most striking for 5 -tetrahydrocortisone, although there were no differences in the ratios of cortisol to cortisone metabolites (not shown). Plasma ACTH, however, did not differ between cases and controls. Given the trend for overrepresentation of women among the controls, plasma cortisol and urinary cortisol metabolite excretion were compared between men and women; the only difference was that men excreted more 5 -tetrahydrocortisone ( vs

3 E1588 Walker et al Cortisol in Pseudohypoaldosteronism 1 J Clin Endocrinol Metab, August 2014, 99(8):E1586 E1591 Table 1. Clinical and Biochemical Characteristics of Cases and Controls PHA1 Cases Controls P Value n (M; F) 12 (6; 6) 20 (7; 13).40 a Age, y BMI, kg/m Waist circumference, mm Body fat, % Carotid intima/media thickness, mm hour average daytime SBP, mm Hg hour average daytime DBP, mm Hg Supine plasma renin, ng/l Supine plasma aldosterone, pmol/l Plasma cortisol, nmol/l Plasma ACTH, pmol/l Fasting plasma glucose, mmol/l Fasting plasma triglycerides, mmol/l Plasma LDL-cholesterol, mmol/l Plasma HDL-cholesterol, mmol/l Urine creatinine, mmol per 24 h Urinary steroids, g/mmol creatinine 5 -Tetrahydrocortisol Tetrahydrocortisol Tetrahydrocortisone Cortisol Cortisone Total cortisol metabolites Abbreviations: DBP, diastolic BP; F, female; M, male; LDL, low-density lipoprotein; SBP, systolic BP. Data are mean SEM. P values are from Student s unpaired t tests except the one marked with a superscript letter. a 2 test g/mmol creatinine, P.02). However, in a two-way ANOVA, categorized by diagnosis (PHA1 case or control) and by gender (male or female), the effects of PHA1 diagnosis and gender were independent, with PHA1 cases having higher plasma cortisol (P.04) and total urinary cortisol metabolites (P.05) than controls, even after adjustment for gender. There were no detectable differences in plasma cortisol or urinary cortisol metabolite excretion between cases with mutations affecting different domains of the MR (not shown). In Pearson correlation analyses, plasma cortisol was inversely associated with age (r 0.65, P.003) and percentage body fat (r 0.40, P.02) (Figure 1) and positively associated with supine plasma renin (r 0.74, P.0001). In multiple regression analyses, with adjustment for effects of PHA1 diagnosis, gender, age, and percentage body fat, higher plasma cortisol was independently associated with higher plasma renin (partial 0.82, P.02), lower plasma high-density lipoprotein (HDL)-cholesterol (partial 0.91, P.006), and higher waist circumference (partial 0.73, P.005) (Figure 1). These associations persisted when only PHA1 cases were included, albeit losing statistical significance with the small sample number (for renin, partial 0.54, P.42; for HDL-cholesterol, partial 0.34, P.28; for waist circumference, partial 0.43, P.06). Plasma cortisol was not associated with BP, blood glucose, total cholesterol, or triglycerides or structural measurements of the carotid artery or heart. Total urinary cortisol metabolite excretion was not associated with any of these cardiometabolic variables in similar regression analyses. Af- A Age (y) C Plasma cortisol (nmol/l) Predictors of waist circumference Beta P Gender Age Diagnosis % Body fat Plasma cortisol B Body fat (%) D Plasma cortisol (nmol/l) Predictors of HDL Cholesterol Beta P Gender Age Diagnosis % Body fat Plasma cortisol Figure 1. Correlation of plasma cortisol with GR-dependent variables. A and B, Pearson correlation of plasma cortisol with age (r 0.65, P.003) (A) and percentage body fat (r 0.40, P.02) (B) in PHA1 cases (filled symbols) and controls (unfilled symbols), with separate regression lines also shown for cases (solid line) and controls (dotted line). C and D, Partial correlation coefficients ( ) and corresponding P values (P) for the independent variables included in multiple regression analyses with waist circumference (C) or HDL cholesterol (D) as dependent variables.

4 doi: /jc jcem.endojournals.org E1589 ter adjustment for confounders in multiple regression analyses, waist circumference was higher (P.03) and HDL-cholesterol lower (P.006) in PHA1 cases than controls (Figure 1). Discussion These data show that deleterious mutations in NR3C2 encoding the MR, even in the presence of a normal second NR3C2 allele, are sufficient to cause activation of the HPA axis, with elevated plasma cortisol and daily cortisol metabolite excretion. This is consistent with previous observations that MR antagonists raise plasma cortisol levels in humans (18) and supports the hypothesis that the MR is important in negative feedback control of the HPA axis in humans, as it is in rodents (2). The hypercortisolemia in PHA1 was associated with biochemical severity, as judged by plasma renin concentrations, and with some evidence of harmful effects on metabolic function, including lower HDL-cholesterol and central adiposity (higher waist circumference) but no evidence of cardiac or vascular structural changes. The power of the primary case-control design used here is limited by the small numbers of PHA1 patients available, even in this national cohort that is the largest yet collected. Any risk of confounding of urinary steroid data by incomplete 24-hour urine sample collections, or by differences in lean body mass between groups, was ameliorated by correcting urinary steroid excretion for creatinine excretion. Previous studies of common genetic variants in NR3C2 (encoding MR) suggested gender-specific effects on salivary cortisol suppression by dexamethasone (14), but here we show that the effect of MR haploinsufficiency is similar in men and women. Specifically, differences in steroid measurements between men and women did not account for the differences between PHA1 cases and controls. Our evaluation of HPA axis function was limited to measurement of morning plasma cortisol and ACTH and 24-hour urinary cortisol metabolite excretion. Additional dynamic testing of the HPA axis would likely be informative, but for practical reasons we were limited in the number of measurements we could make in these unusual patients. We have previously reported that morning plasma ACTH is normal in PHA1 cases (16), although ACTH measurements are known to be relatively insensitive to mild HPA axis activation (10) and are often within the reference range, even in patients with generalized glucocorticoid resistance syndrome. Importantly, elevated plasma cortisol was not accompanied by suppressed plasma ACTH, as is observed when altered peripheral metabolism contributes to elevated plasma cortisol. It has been suggested that variations in MR are more important in negative feedback during the diurnal nadir of ACTH and cortisol secretion, when glucocorticoid levels are low and relatively low affinity GRs are empty of ligand; if so, then elevations in the plasma cortisol in PHA1 patients might be even greater in the evening and ACTH levels might be different then. The observation that 5 -tetrahydrocortisone is the predominant urinary cortisol metabolite that is increased in patients with PHA1 is also consistent with primary activation of the HPA axis; in patients with Cushing s syndrome, there is a relatively high excretion of 5 -reduced cortisol metabolites (19). In recent years, MR expression has been shown to be widespread, including in heart, blood vessels, and cells of the innate immune system. In most sites, 11 -hydroxysteroid dehydrogenase type 2 is not present to inactivate cortisol and allows selective access of the less abundant ligand, aldosterone, to MR. Occupation of MR by cortisol has been confirmed, for example by displacement from the myocardium after administration of a MR antagonist (20). However, the contribution of MR in mediating the actions of cortisol remains uncertain. In many circumstances, MR and GR act in opposition. Our data in PHA1 patients suggest that the lack of association of hypercortisolemia with evidence of target organ damage in the vessel wall and heart is consistent with the interpretation that MR deficiency protects tissues from the adverse effects of glucocorticoids. Deleterious GR-mediated glucocorticoid action may be exaggerated in tissues in which MRs are less likely to be important, including in adipose tissue and liver, potentially explaining the association of plasma cortisol with central adiposity and with HDL-cholesterol in this cohort. Moreover, after adjustment for confounding factors (gender and percentage body fat in particular), PHA1 cases had higher waist circumference and lower HDL-cholesterol than controls. However, this was a secondary analysis in a small sample, and the absence of associations of urinary cortisol metabolites with metabolic variables means that its reproducibility is unproven. It will be valuable in future to investigate other glucocorticoidsensitive outcomes in PHA1 patients, including bone density and cognition. It may also be revealing to study other central processes thought to be affected by MR, including emotional expression. In summary, PHA1 is associated with elevated plasma cortisol and the cortisol production rate. This adds significant genetic evidence to the existing pharmacological evidence that MR is involved in feedback control of the HPA axis in humans and may be significant for metabolic outcomes in patients with PHA1. Subtle changes in MR

5 E1590 Walker et al Cortisol in Pseudohypoaldosteronism 1 J Clin Endocrinol Metab, August 2014, 99(8):E1586 E1591 function or expression may modulate the HPA axis and metabolic risk in the general population. Acknowledgments We are indebted to Estelle Marcault and Naima Beldjoudi (Unitéde Recherche Clinique Paris Nord, Assistance Publique-Hôpitaux de Paris) for the implementation and monitoring of the study; to the nurse team (Department of Physiology, Hôpital Bichat, Assistance Publique-Hôpitaux de Paris) for the care of the patients; to the technical staff (Department of Genetics, Hôpital Européen Georges Pompidou, Assistance Publique- Hôpitaux de Paris) for the genetic analysis of the NR3C2 gene; and to Jill Harrison and Sanjay Kothiya (British Heart Foundation Centre for Cardiovascular Science and Wellcome Trust Clinical Research Facility Mass Spectrometry Core Laboratory) for excellent technical support for the cortisol and cortisol metabolite assays. The study could not have been conducted without the contribution of the members of the PHA1 families and their referring clinicians in the PHA1.NET network, who include the following: Mohamed Amani, Hôpital Docteur Duchenne, Boulogne sur Mer; Alina Arion, Centre Hospitalier Universitaire Caen, Caen; Marion Beaumesnil, Centre Hospitalier Universitaire Angers, Angers; Khahil Benchekroun-Krimi, Centre Hospitalier de Châteauroux, Châteauroux; Laurence Bérard, Hôpital Armand Trousseau, Paris; Catherine Boegner, Centre Hospitalier Universitaire Lapeyronie, Montpellier; Elisabeth Bonnemaison, Centre Hospitalier Régional Universitaire de Tours-Hôpital Gatien de Clocheville, Tours; Jean-Bernard Bonte, Centre Hospitalier Universitaire Caen, Caen; Patricia Bretones, Hôpital Mère Enfant, Bron; A. L. Cuvelier, Centre Hospitalier de Calais, Calais; Fabienne Dalla Vale, Centre Hospitalier Universitaire de Montpellier, Montpellier; Michèle Dechaux, Hôpital Necker- Enfants Malades, Paris; Georges Deschenes, Hôpital Robert Debré, Paris; Catherine Fernando, Hôpital Armand Trousseau, Paris; Chantal Gagliardone, Institut Hospitalier Franco-Britannnique, Levallois-Perret; Emmanuelle Ginglinger, Service de Génétique, Mulhouse; Alice Goldenberg, Centre Hospitalier Universitaire de Rouen, Rouen; Jean-Michel Halimi, Centre Hospitalier Régional Universitaire Tours, Tours; Muriel Houang, Hôpital Trousseau, Paris; Assia Khammar, Hôpital d Enfants de la Timone, Marseille; Michel Legagneur, Service de Pédiatrie et Néonatologie, Forbach; Juliane Leger, Hôpital Robert Debré, Paris; Aurelia Liutkus-Bertholet-Thomas, Hôpital Femme-Mère-Enfant, Bron; Chantal Loirat, Hôpital Robert Debré, Paris; Chantal Metz, Centre Hospitalier Universitaire de Brest-Hôpital Augustin Morvan, Brest; Jean-Jacques Montseny, Hôpital de Pontoise-Centre René Dubos, Cergy Pontoise; Dr Mozelle-Nivoix, Centre Hospitalier de Troyes, Troyes; Patrick Niaudet, Hôpital Necker-Enfants Malades, Paris; François Nobili, Centre Hospitalier Universitaire Saint Jacques, Besancon; Sylvie Odent, Hôpital Sud, Rennes; Christine Pietrement, Hopital Américain-Centre Hospitalier Universitaire Reims, Reims; Rachel Reynaud, Hôpital d Enfants de la Timone, Marseille; Sabine Sigaudy, Assistance Publique-Hôpitaux de Marseille, Marseille; Sylvie Soskin, Hôpitaux Universitaires de Strasbourg, Strasbourg; Pierre-François Souchon, Centre Hospitalier Régional Universitaire de Reims, Reims; Anne Spiteri, Centre Hospitalier Universitaire de Grenoble, Grenoble; C. Stuckens, Hôpital Jeanne de Flandres-Clinique de Pédiatrie, Lille; Alain Verloes, Hôpital Robert Debré, Paris; and Hubert Ythier, Centre Hospitalier de Roubaix-Hôpital Victor Provo, Roubaix. This study had a clinical trial registration ( clinicaltrials.gov) with the unique identifier of NCT Address all correspondence and requests for reprints to: Professor Brian R. Walker, British Heart Foundation Centre for Cardiovascular Science, Queen s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. b.walker@ed.ac.uk. This work was supported by the British Heart Foundation, the French Ministry of Health, and the Département à la Recherche Clinique et au Développement, Assistance Publique Hôpitaux de Paris (Cardiovascular Evaluation of Adult PHA 1 Patients, Programme Hospitalier de Recherche Clinique Grant AOM07017-P07139). 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