Atypical hypoadrenocorticism with intact zona glomerulosa of the adrenal cortex after long-term observation: a case report of a dog.

An 8-year-old intact male pointer presented with lethargy and hypoalbuminemia. On abdominal ultrasonography, both adrenal glands were reduced in thickness. Based on the ACTH stimulation test results and the absence of electrolyte abnormalities, the dog was diagnosed with atypical hypoadrenocorticism. After treatment with low-dose prednisolone, his general condition improved, and blood tests normalized. The dog died 818 days later, and a complete autopsy was performed. Histologically, the architecture of the zonae fasciculata and reticularis was disrupted in both adrenal glands; however, the zona glomerulosa remained relatively normal. In summary, in this study, we detailed the pathological presentation of atypical hypoadrenocorticism without electrolyte abnormalities.

Cellular Senescence in Adrenocortical Biology and Its Disorders.

Cellular senescence is considered a physiological process along with aging and has recently been reported to be involved in the pathogenesis of many age-related disorders. Cellular senescence was first found in human fibroblasts and gradually explored in many other organs, including endocrine organs. The adrenal cortex is essential for the maintenance of blood volume, carbohydrate metabolism, reaction to stress and the development of sexual characteristics. Recently, the adrenal cortex was reported to harbor some obvious age-dependent features. For instance, the circulating levels of aldosterone and adrenal androgen gradually descend, whereas those of cortisol increase with aging. The detailed mechanisms have remained unknown, but cellular senescence was considered to play an essential role in age-related changes of the adrenal cortex. Recent studies have demonstrated that the senescent phenotype of zona glomerulosa (ZG) acts in association with reduced aldosterone production in both physiological and pathological aldosterone-producing cells, whereas senescent cortical-producing cells seemed not to have a suppressed cortisol-producing ability. In addition, accumulated lipofuscin formation, telomere shortening and cellular atrophy in zona reticularis cells during aging may account for the age-dependent decline in adrenal androgen levels. In adrenocortical disorders, including both aldosterone-producing adenoma (APA) and cortisol-producing adenoma (CPA), different cellular subtypes of tumor cells presented divergent senescent phenotypes, whereby compact cells in both APA and CPA harbored more senescent phenotypes than clear cells. Autonomous cortisol production from CPA reinforced a local cellular senescence that was more severe than that in APA. Adrenocortical carcinoma (ACC) was also reported to harbor oncogene-induced senescence, which compensatorily follows carcinogenesis and tumor progress. Adrenocortical steroids can induce not only a local senescence but also a periphery senescence in many other tissues. Therefore, herein, we systemically review the recent advances related to cellular senescence in adrenocortical biology and its associated disorders.

Impaired Morphogenesis and Function of Rat Adrenal Zona Glomerulosa by Developmental Low-Dose Exposure to DDT Is Associated with Altered Oct4 Expression.

Dichlorodiphenyltrichloroethane (DDT) is a persistent organic pollutant and one of the most widespread endocrine disrupting chemicals. The impact of low-dose exposure to DDT on the morphogenesis of the adrenal gland is still poorly understood. The development and function of zona glomerulosa in rats has been found to be associated with changes in the expression of the transcription factor Oct4 (Octamer 4), which is the most important player in cell pluripotency. The aim of the study was to investigate the morphogenesis and function of rat adrenal zona glomerulosa in rats exposed to low doses of DDT during prenatal and postnatal development and to determine the possible role of Oct4 in DDT-mediated structural and functional changes. The DDT-exposed rats demonstrated slower development and lower functional activity of the zona glomerulosa during the pubertal period associated with higher expression of Oct4. Further, accelerated growth and restoration of hormone production was associated with, firstly, a decrease in Oct4 expressing cells and secondly, the loss of the inverse relationship between basal aldosterone levels and the number of Oct4 expressing cells. Thus, the transcriptional factor Oct4 exhibited an altered pattern of expression in the DDT-exposed rats during postnatal development. The results of the study uncover a novel putative mechanism by which low doses of DDT disrupt the development of adrenal zona glomerulosa.

Adrenal cortex development and related disorders leading to adrenal insufficiency.

The adult human adrenal cortex produces steroid hormones that are crucial for life, supporting immune response, glucose homeostasis, salt balance and sexual maturation. It consists of three histologically distinct and functionally specialized zones. The fetal adrenal forms from mesodermal material and produces predominantly adrenal C19 steroids from its fetal zone, which involutes after birth. Transition to the adult cortex occurs immediately after birth for the formation of the zona glomerulosa and fasciculata for aldosterone and cortisol production and continues through infancy until the zona reticularis for adrenal androgen production is formed with adrenarche. The development of this indispensable organ is complex and not fully understood. This article gives an overview of recent knowledge gained of adrenal biology from two perspectives: one, from basic science studying adrenal development, zonation and homeostasis; and two, from adrenal disorders identified in persons manifesting with various isolated or syndromic forms of primary adrenal insufficiency.

The Age-Dependent Changes of the Human Adrenal Cortical Zones Are Not Congruent.

BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.

Ultrastructural Mechanisms of Impaired Aldosterone Synthesis in Rats Exposed to DDT during Prenatal and Postnatal Development.

The study is aimed at elucidation of ultrastructural mechanisms underlying impaired aldosterone synthesis by glomerulosa cells in Wistar rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal development. Analysis of rat zona glomerulosa histology and function during the pubertal and postpubertal periods showed that exposure to endocrine disrupter DDT disturbs its development and reduced the production of aldosterone. Electron microscopy showed that changes in the aldosterone synthesis are related to impaired reorganization of the mitochondrial apparatus, one of the leading factors in the regulation of steroidogenesis, in glomerulosa cells in DDT-exposed rats during puberty.

Cellular Pathophysiology of Mutant Voltage-Dependent Ca2+ Channel CACNA1H in Primary Aldosteronism.

The physiological stimulation of aldosterone production in adrenocortical glomerulosa cells by angiotensin II and high plasma K+ depends on the depolarization of the cell membrane potential and the subsequent Ca2+ influx via voltage-activated Ca2+ channels. Germline mutations of the low-voltage activated T-type Ca2+ channel CACNA1H (Cav3.2) have been found in patients with primary aldosteronism. Here, we investigated the electrophysiology and Ca2+ signaling of adrenal NCI-H295R cells overexpressing CACNA1H wildtype and mutant M1549V in order to understand how mutant CACNA1H alters adrenal cell function. Whole-cell patch-clamp measurements revealed a strong activation of mutant CACNA1H at the resting membrane potential of adrenal cells. Both the expression of wildtype and mutant CACNA1H led to a depolarized membrane potential. In addition, cells expressing mutant CACNA1H developed pronounced action potential-like membrane voltage oscillations. Ca2+ measurements showed an increased basal Ca2+ activity, an altered K+ sensitivity, and abnormal oscillating Ca2+ changes in cells with mutant CACNA1H. In addition, removal of extracellular Na+ reduced CACNA1H current, voltage oscillations, and Ca2+ levels in mutant cells, suggesting a role of the partial Na+ conductance of CACNA1H in cellular pathology. In conclusion, the pathogenesis of stimulus-independent aldosterone production in patients with CACNA1H mutations involves several factors: i) a loss of normal control of the membrane potential, ii) an increased Ca2+ influx at basal conditions, and iii) alterations in sensitivity to extracellular K+ and Na+. Finally, our findings underline the importance of CACNA1H in the control of aldosterone production and support the concept of the glomerulosa cell as an electrical oscillator.

Mass Spectrometry Imaging Establishes 2 Distinct Metabolic Phenotypes of Aldosterone-Producing Cell Clusters in Primary Aldosteronism.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

Adrenal cortex hypoxia modulates aldosterone production in heart failure.

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated ß3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced ß3-adrenergic receptor expression. Hypoxia-mediated ß3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Splice-variant-specific effects of primary aldosteronism point mutations on human CaV3.2 calcium channels.

CaV3.2 calcium channels play important roles in both neural excitability and aldosterone secretion. Recent clinical studies found four germline mutations (S196 L, M1549I, V1951E and P2083 L) in CaV3.2 channels. All four mutations caused primary aldosteronism (PA), while only the M1549I mutation resulted in obvious neural malfunctions besides PA. In human, there are two major CaV3.2 channel gene (CACNA1H) splice variants, either with or without exon 26. In this study, we tested the expression of the two CACNA1H splice variants in zona glomerulosa (ZG) cells of human adrenal cortex and the possibility that CaV3.2 (-26) and CaV3.2 (+26) channels have different functional responses to the four PA mutations. We found that human ZG cells only express long form CaV3.2(+26) channels. The M1549I mutation slowed the inactivation of CaV3.2(+26) more than 5 fold, and CaV3.2(-26) more than 2 fold. The S196 L, V1951E and P2083 L mutations accelerated channel recovery from inactivation for CaV3.2(+26), but not CaV3.2(-26) channels. All four mutations resulted in gain of function of CaV3.2(+26) channels, leading to overproduction of aldosterone. In conclusion, the four PA mutations caused more profound changes on CaV3.2 (+26) currents than on CaV3.2 (-26) currents, and except the M1549I mutation, the S196 L, V1951E and P2083 L have little effect on the electrophysiological properties of CaV3.2(-26) currents, which may partially explain the limitation of the phenotype associated with the V1951E, S196 L and P2083 L germline mutations to PA.

ANO4 (Anoctamin 4) Is a Novel Marker of Zona Glomerulosa That Regulates Stimulated Aldosterone Secretion.

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10-24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.

Decrease in Lipid Droplets in Adrenal Cortex of Male Wistar Rats after Chronic Exposure to Energy Drinks.

Background and objectives: Energy drinks are popular non-alcoholic beverages. They are consumed in large amounts, mainly by active, young people. Although they are easily accessible and marketed as safe, numerous cases of adverse effects have been published, including cardiac arrest, arrythmias, acute hepatitis, and renal failure. The aim of the current study is the assessment of energy drink influence on the histological structure of adrenal cortex in rats. Material and Methods: 15 male young Wistar rats were equally divided into three groups: control (C), experimental (E) and reversibility control (RC). C group received water and standard rodent food ad libitum while both E and RC groups had additionally unlimited access to energy drinks. C and E groups were decapitated after 8 weeks and RC was given another 8 weeks without energy drinks. Adrenal glands were embedded in paraffin blocks and 5 µm slides were prepared and stained according to standard H&E and Masson's trichrome protocols. Additionally, immunohistochemical stainings against Ki-67, p53, CTGF and caspase-3 were prepared. Results: Decreased vacuolization and numerous pyknotic nuclei were noted in E and RC groups. Overexpression of caspase-3 was noted both subcapsular in zona glomerulosa and along sinusoids in zona fasciculata. Increased collagen deposition in zona glomerulosa and zona fasciculata of E and RC was observed. Insular and irregular overexpression of CTGF was noted. The overall picture of CTGF expression matched the Masson's trichrome. No significant difference was observed in Ki-67 expression. Conclusions: The results of the current study suggest that the stimulation is so intense that it causes significant damage to adrenal cortical cells, resulting in their apoptosis. It seems, however, that the observed effects are at least partially reversible.

Cellular and Genetic Causes of Idiopathic Hyperaldosteronism.

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.

The Biology of Normal Zona Glomerulosa and Aldosterone-Producing Adenoma: Pathological Implications.

The identification of several germline and somatic ion channel mutations in aldosterone-producing adenomas (APAs) and detection of cell clusters that can be responsible for excess aldosterone production, as well as the isolation of autoantibodies activating the angiotensin II type 1 receptor, have rapidly advanced the understanding of the biology of primary aldosteronism (PA), particularly that of APA. Hence, the main purpose of this review is to discuss how discoveries of the last decade could affect histopathology analysis and clinical practice. The structural remodeling through development and aging of the human adrenal cortex, particularly of the zona glomerulosa, and the complex regulation of aldosterone, with emphasis on the concepts of zonation and channelopathies, will be addressed. Finally, the diagnostic workup for PA and its subtyping to optimize treatment are reviewed.

Stress-induced changes in the aged-rat adrenal cortex. Histological and histomorphometric study.

BACKGROUND: Stress exposure exerts direct effects on the morphology and functionality of the adrenal cortex. In addition, ageing effects growth, differentiation, apoptosis and cellularity of the cortex. The missing data is the combined effect of stress and ageing on the adrenal cortex. The aim of this study was to demonstrate the structural changes in the adrenal cortex following the exposure to stress in the adult and aged albino rats. MATERIALS AND METHODS: Forty rats were divided into groups I and II (adult and senile). Each group was further subdivided into subgroups a and b (control and stressed). Light and electron microscopic studies were done. Area per cent of collagen fibres (Masson's trichrome-stained sections), number of proliferating cells (optical density immunoreactivity in the Ki67 stained sections) and thickness of the three adrenal zones were also measured. RESULTS: Lamellar separation of the capsule with subcapsular spindle cell hyperplasia and areas of ghost cells were observed in zona glomerulosa (ZG) and zona fasciculata (ZF) in group I-b. Separation and indentation of the capsule with its lamellar separation were observed in group II-a with the existence of multiple scattered degenerative foci in ZF and zona reticularis (ZR). Similar and aggressive was the architectural pattern of ZF in group II-b with the presence of areas of homogenous degeneration. The nuclei of ZG had marginated chromatin in group I-b and were pyknotic with deformed irregular outlines in group II-b. Multiple lysosomes and vacuolar degeneration mitochondria were also seen in group I-b. The nuclei of ZF were irregular with condensed marginated heterochromatin in group I-b, irregular with scattered chromatin in group II-a and indented with areas of chromatin destruction in group II-b. Mitochondria with disrupted cristae and cristolysis were also detected in group I-b. Numerous lipofuscin granules and dilated smooth endoplasmic reticulum were revealed in group II-b. The mean collagen fibre area per cent and the mean number of the proliferating cells in group II-b were significantly higher by 39% and 23%. The thickness of ZG decreased significantly by 20% in group I-b. Contrary, the thickness of both ZF and ZR increased significantly by 10% in group I-b. CONCLUSIONS: Histological alterations occurred in the adrenal cortex in response to stress, especially when coupled with the advance of age. This was accompanied by increase in the area per cent of collagen fibres and increase in the mean number of the proliferating cells in the adrenal cortex.

Changes in Canonical ß-Catenin/Wnt Signaling Activation in the Adrenal Cortex of Rats Exposed to Endocrine Disruptor Dichlorodiphenyltrichloroethane (DDT) during Prenatal and Postnatal Ontogeny.

Prenatal and postnatal exposure to low doses of the endocrine disruptor dichlorodiphenyltrichloroethane (DDT) leads to delayed activation of the canonical ß-catenin/Wnt signaling in zona glomerulosa and zona reticularis of the adrenal cortex in rats, which changed the rate of their postnatal development. Suppression of the Wnt pathway in zona fasciculata promotes its regeneration after DDT-induced blood circulation disorders and cell death.

The angiotensin type 2 receptor in the human adrenocortical zona glomerulosa and in aldosterone-producing adenoma: low expression and no functional role.

The angiotensin II (Ang II) type 2 receptor (AT2R) and the angiotensin-(1-7) (Ang-(1-7)) receptor (MasR) play a cardiovascular protective role by counter-regulating Ang II type 1 receptor (AT1R)-mediated effects, but whether this involves blunting of adrenocortical hormone secretion is unknown. We investigated the presence of AT1R, AT2R, and MasR in aldosterone-producing adenoma (APA), a condition featuring hyperaldosteronism, and in APA-adjacent tissue. The effect of Compound 21 (C21), an AT2R agonist, on CYP11B1 (cortisol synthase) and CYP11B2 (aldosterone synthase) gene expression in NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, was also assessed using the AT1R antagonist irbesartan to ascertain the specificity of C21 effect. We found that the AT1R, AT2R, and MasR were expressed in APA and APA-adjacent tissue, albeit heterogeneously. The gene expression of AT1R and AT2R was lower, and that of the MasR higher in APAs than in APA-adjacent tissue. In steroid-producing NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, C21 was ineffective at nanomolar concentrations, but increased CYP11B1 and CYP11B2 gene expression at micromolar concentrations through AT1R, as this effect was blunted by irbesartan. The scant expression of the AT2R, along with the lack of any effect of C21 at low concentrations on CYP11B2, do not support the contention that the protective arm of renin-angiotensin system (RAS) blunts aldosterone synthase in the normal adrenal cortex and primary aldosteronism.

A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel 1 , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl- currents that were abolished in Clcn2-/- mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain'2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl- conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.

The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion.

Primary and secondary hypertension are major risk factors for cardiovascular disease, the leading cause of death worldwide. Elevated secretion of aldosterone resulting from primary aldosteronism (PA) is a key driver of secondary hypertension. Here, we report an unexpected role for the ubiquitin ligase Siah1 in adrenal gland development and PA. Siah1a-/- mice exhibit altered adrenal gland morphology, as reflected by a diminished X-zone, enlarged medulla, and dysregulated zonation of the glomerulosa as well as increased aldosterone levels and aldosterone target gene expression and reduced plasma potassium levels. Genes involved in catecholamine biosynthesis and cAMP signaling are upregulated in the adrenal glands of Siah1a-/- mice, while genes related to retinoic acid signaling and cholesterol biosynthesis are downregulated. Loss of Siah1 leads to increased expression of the Siah1 substrate PIAS1, an E3 SUMO protein ligase implicated in the suppression of LXR, a key regulator of cholesterol levels in the adrenal gland. In addition, SIAH1 sequence variants were identified in patients with PA; such variants impaired SIAH1 ubiquitin ligase activity, resulting in elevated PIAS1 expression. These data identify a role for the Siah1-PIAS1 axis in adrenal gland organization and function and point to possible therapeutic targets for hyperaldosteronism.