Scott M. MacKenzie

A Lifetime of Aldosterone Excess: Long-Term Consequences of Altered Regulation of Aldosterone Production for Cardiovascular Function

Up to 15% of patients with essential hypertension have inappropriate regulation of aldosterone; although only a minority have distinct adrenal tumors, recent evidence shows that mineralocorticoid receptor activation contributes to the age-related blood pressure rise and illustrates the importance of aldosterone in determining cardiovascular risk. Aldosterone also has a major role in progression and outcome of ischemic heart disease. These data highlight the need to understand better the regulation of aldosterone synthesis and its action. Aldosterone effects are mediated mainly through classical nuclear receptors that alter gene transcription. In classic epithelial target tissues, signaling mechanisms are relatively well defined. However, aldosterone has major effects in nonepithelial tissues that include increased synthesis of proinflammatory molecules and reactive oxygen species; it remains unclear how these effects are controlled and how receptor specificity is maintained. Variation in aldosterone production reflects interaction of genetic and environmental factors. Although the environmental factors are well understood, the genetic control of aldosterone synthesis is still the subject of debate. Aldosterone synthase (encoded by the CYP11B2 gene) controls conversion of deoxycorticosterone to aldosterone. Polymorphic variation in CYP11B2 is associated with increased risk of hypertension, but the molecular mechanism that accounts for this is not known. Altered 11beta-hydroxylase efficiency (conversion of deoxycortisol to cortisol) as a consequence of variation in the neighboring gene (CYP11B1) may be important in contributing to altered control of aldosterone synthesis, so that the risk of hypertension may reflect a digenic effect, a concept that is discussed further. There is evidence that a long-term increase in aldosterone production from early life is determined by an interaction of genetic and environmental factors, leading to the eventual phenotypes of aldosterone-associated hypertension and cardiovascular damage in middle age and beyond. The importance of aldosterone has generated interest in its therapeutic modulation. Disadvantages associated with spironolactone (altered libido, gynecomastia) have led to a search for alternative mineralocorticoid receptor antagonists. Of these, eplerenone has been shown to reduce cardiovascular risk after myocardial infarction. The benefits and disadvantages of this therapeutic approach are discussed.

Extra‐adrenal production of corticosteroids

1. The major corticosteroids aldosterone and cortisol (corticosterone in rodents) are secreted from the adrenal cortex under the regulation of the renin–angiotensin system and the hypothalamic–pituitary–adrenal axis.

Is Altered Adrenal Steroid Biosynthesis a Key Intermediate Phenotype in Hypertension

Approximately 10% of patients with hypertension have a high ratio of aldosterone to renin, but the reason for this and the relationships among low-renin essential hypertension, elevation of the ratio, and true primary aldosteronism are unclear. We have previously reported that a polymorphism of the gene (C-to-T conversion at position -344) encoding aldosterone synthase is associated with hypertension, particularly in patients with a high ratio. However, the most consistent association with this variant is a relative impairment of adrenal 11beta-hydroxylation. In this review, we propose that altered conversion of deoxycortisol to cortisol leads to a subtle, chronic increase in adrenocortrophin drive to the adrenal cortex, with eventual development of hyperplasia. In combination with other genetic or environmental factors (such as dietary sodium intake), we suggest that this might be responsible for the long-term development of a resetting of the aldosterone response to angiotensin II, giving rise to the phenotype of hypertension with a raised ratio. In some subjects, this may progress further to true primary aldosteronism with a dominant adrenal nodule. Thus, there may be a genetically influenced continuum from hypertension with a normal ratio, through hypertension with a raised ratio, and primary aldosteronism.

Depot-specific steroidogenic gene transcription in human adipose tissue

Context  Sex steroids (androgens and oestrogens) and corticosteroids (glucocorticoids and mineralocorticoids) have a major impact on fat distribution. Several genes involved in steroid synthesis and metabolism, such as 11β‐hydroxysteroid dehydrogenase type 1 and aromatase, are known to be expressed within adipose tissue, thus modulating local steroid levels; however, our knowledge of which genes are expressed and at what level is incomplete.

Programming of Hypertension Associations of Plasma Aldosterone in Adult Men and Women With Birthweight, Cortisol, and Blood Pressure

Abstract—Animal models suggest that explanations for the association of low birthweight with adult hypertension may include chronic activation of the hypothalamic-pituitary-adrenal or renin-angiotensin-aldosterone axes. In humans, low birthweight predicts elevated plasma cortisol, but associations with aldosterone have not been reported. We measured aldosterone in serum samples from 205 men and 106 women from 67 to 78 years of age, from Hertfordshire, UK, for whom birthweight was recorded. Participants underwent an overnight low-dose (0.25 mg) dexamethasone suppression test and a low-dose (1 &mgr;g) ACTH (corticotropin) stimulation test and were genotyped for the -344 C/T polymorphism of the CYP11B2 gene encoding aldosterone synthase. Median aldosterone was 6.22 ng/dL (range 0.15 to 38.74) and was higher in men than women (P<0.0001). Higher aldosterone levels after both dexamethasone and ACTH stimulation were associated with higher blood pressure (r=0.20, P=0.001; r=0.33, P<0.0001, respectively) and with lower birthweight (r=−0.16, P=0.008; r=−0.21, P=0.001, respectively). These associations remained significant after adjusting for age, gender, obesity, and genotype. Our findings supplement previous evidence that aldosterone is an important regulator of blood pressure and suggest that factors in early life that retard fetal growth and program activation of the hypothalamic-pituitary-adrenal axis in humans result not only in higher glucocorticoid activity but also in increased mineralocorticoid activity.

MicroRNA-24 Is a Novel Regulator of Aldosterone and Cortisol Production in the Human Adrenal Cortex

Dysregulation of aldosterone or cortisol production can predispose to hypertension, as seen in aldosterone-producing adenoma, a form of primary aldosteronism. We investigated the role of microRNA (miRNA) in their production, with particular emphasis on the CYP11B1 (11&bgr;-hydroxylase) and CYP11B2 (aldosterone synthase) genes, which produce the enzymes responsible for the final stages of cortisol and aldosterone biosynthesis, respectively. Knockdown of Dicer1, a key enzyme in miRNA maturation, significantly altered CYP11B1 and CYP11B2 expression in a human adrenocortical cell line. Screening of nondiseased human adrenal and aldosterone-producing adenoma samples yielded reproducible but distinctive miRNA expression signatures for each tissue type, with levels of certain miRNA, including microRNA-24 (miR-24), differing significantly between the 2. Bioinformatic analysis identified putative binding sites for several miRNA, including miR-24, in the 3′ untranslated region of CYP11B1 and CYP11B2 mRNAs. In vitro manipulation of miR-24 confirmed its ability to modulate CYP11B1 and CYP11B2 expression, as well as cortisol and aldosterone production. This study demonstrates that Dicer-dependent miRNA, including miR-24, can post-transcriptionally regulate expression of the CYP11B1 and CYP11B2 genes. Normal adrenal tissue and aldosterone-producing adenoma differ significantly and reproducibly in their miRNA expression profiles, with miR-24 significantly downregulated in the latter. Adrenal miRNA may, therefore, be a novel and valid target for the therapeutic manipulation of corticosteroid biosynthesis.

Effects of ACTH, dexamethasone, and adrenalectomy on 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in the rat central nervous system

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 μg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either genes expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels.

Polymorphic Variation in the 11β-Hydroxylase Gene Associates With Reduced 11-Hydroxylase Efficiency

The −344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11β-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11β-hydroxylase gene (CYP11B1) and arises through linkage disequilibrium between CYP11B1 and CYP11B2. The pattern of variation across the entire CYP11B locus was determined by sequencing 26 normotensive subjects stratified by and homozygous for the −344 and intron conversion variants. Eighty-three variants associated with −344 and intron conversion were identified. Haplotype analysis revealed 4 common haplotypes, accounting for 68% of chromosomes, confirming strong linkage disequilibrium across the region. Two novel CYP11B1 polymorphisms upstream of the coding region (−1889 G/T and −1859 A/G) were identified as contributing to the common haplotypes. Given the potential for such mutations to affect transcriptional regulation of CYP11B1, these were analyzed further. A total of 512 hypertensive subjects from the British Genetics of Hypertension Study population were genotyped for these polymorphisms. A significant association was identified between the −1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11β-hydroxylase efficiency. A similar pattern was observed for the −1859 polymorphism, but this did not achieve statistical significance. Functional studies in vitro using luciferase reporter gene constructs show that these polymorphisms significantly alter the transcriptional response of CYP11B1 to stimulation by adrenocorticotropic hormone or forskolin. This study strongly suggests that the impaired 11β-hydroxylase efficiency associated previously with the CYP11B2 −344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1.

Corticosteroid Production by Fetal Rat Hippocampal Neurons

11β-hydroxylase and aldosterone synthase catalyse the final stages of corticosterone and aldosterone synthesis respectively. Previously, we established that they are expressed in the rat brain, particularly the cerebellum and the hippocampus. Primary cultures of fetal rat neurons were studied. RT-PCR and immunohistochemistry established that neurons express 11β -hydroxylase and aldosterone synthase mRNAs and protein. After incubating the cells with 10μM DOC for 24 hours, medium was analysed for aldosterone and corticosterone. Median % conversion of DOC to corticosterone was 7.6% compared to 0.4% in controls. Median % conversion of DOC to aldosterone was 6.2% compared to 0.06% in controls. Corticosteroids mediate a number of functions of mammalian brain, including blood pressure homeostasis, salt appetite and neuronal excitability. Local production of these steroids could have significant effects on these processes.

Aldosterone synthase gene variation and adrenocortical response to sodium status, angiotensin II and ACTH in normal male subjects

objective  Aldosterone synthase, a key enzyme in the terminal steps of aldosterone synthesis, is encoded by the CYP11B2 gene. A polymorphism in the 5′ coding region of this gene (−344 C/T) is associated with hypertension, particularly with elevation of the aldosterone to renin ratio. A second polymorphism (a conversion in intron 2 to resemble that of the neighbouring 11β‐hydroxylase (CYP11B1) gene) is found in close linkage dysequilibrium with the variant at −344 C/T. The mechanism by which these variants predispose to cardiovascular disease and the precise intermediate phenotype associated with them remains speculative.