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Medical Research Council Blood Pressure Group, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow G12 8TA, United Kingdom
Correspondence: Address all correspondence and requests for reprints to: Professor John M. C. Connell, Division of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, 126 University Place, Glasgow G12 8TA, United Kingdom. E-mail: jmcc1m{at}clinmed.gla.ac.uk
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Abstract |
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 Top Abstract I. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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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 11β-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.
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I. Introduction |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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However, aldosterone has since reemerged as a key hormone in cardiovascular homeostasis that, when present in excess, determines cardiovascular risk. In part, this appreciation followed the discovery of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme, which, at least in part (see Section V), prevents glucocorticoid activation of the mineralocorticoid receptor (MR) (4, 5). The ability of 11β-HSD2, where present, to convert cortisol to cortisone, thus contributing to the selectivity of the MR for aldosterone transactivation, clarified the importance of aldosterone in determining blood pressure and salt and water homeostasis through renal actions. It also revealed the role of aldosterone in the physiological and pathophysiological function of a range of other tissues including vascular endothelium, the central nervous system (CNS), and placenta (6). Studies of the efficacy of MR antagonism in the treatment of heart failure emphasized that aldosterone’s effects may extend far beyond blood pressure regulation (7). Finally, a series of observations in the last decade have confirmed that relative aldosterone excess, as identified by a raised aldosterone to renin ratio (ARR), is common in patients with hypertension (8). Thus, the role of aldosterone in cardiovascular physiological regulation and in the risk of developing cardiovascular dysfunction has advanced from a specialist to a mainstream concern.
In this review, we will explore in detail the regulation of aldosterone synthesis, its role in cardiovascular syndromes, and its contribution to more common forms of vascular dysfunction. We will focus on how variation in the genes that encode key enzymes involved in aldosterone and cortisol synthesis may account for the altered responsiveness of aldosterone to its main regulators; we suggest that this identifies a lifelong, genetically determined trait, to enhance aldosterone secretion in response to regulatory factors such as AngII and potassium, so that a minor rise in production of aldosterone, over many years, leads to high blood pressure with particularly adverse vascular and cardiac consequences. Finally, the reemergence of aldosterone as an important cardiovascular hormone has substantial implications for therapeutic manipulation, and we will consider existing and emerging treatment options.
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II. Aldosterone Biosynthesis |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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A. Translocation of cholesterol
Aldosterone synthesis from cholesterol within the adrenal cortex is the result of a series of enzymatic reactions occurring solely in the ZG (10). First, cholesterol must be taken up and translocated to the inner mitochondrial membrane. This is the rate-limiting stage of aldosterone production and is mediated by the steroidogenic acute regulatory protein (StAR). StAR is present in all steroidogenic tissues (11) and is synthesized as a 37-kDa cytosolic precursor that forms a mature 30-kDa enzyme after hormonal stimulation. The most compelling evidence that StAR plays a key role in steroidogenesis comes from studies of patients with congenital lipoid adrenal hyperplasia, a lethal genetic disorder caused by nonsense mutations in the StAR gene and characterized by the severe impairment of steroid hormone biosynthesis, elevated levels of ACTH, and enlarged adrenal glands that contain high levels of cholesterol and cholesterol esters (12). Although StAR gene expression is increased by most agents known to stimulate steroid biosynthesis, the mechanism by which StAR regulates cholesterol transfer to the inner mitochondrion is unclear, although it may form a channel through the mitochondrial membrane.
B. Side-chain cleavage enzyme
After the translocation of cholesterol to the inner mitochondrial membrane, aldosterone synthesis occurs through a series of reactions catalyzed by dehydrogenases and mixed function oxidases (Fig. 1
). The oxidases belong to the cytochrome P450 (CYP) superfamily of heme-containing enzymes and use the adrenodoxin/adrenodoxin reductase-coupled coenzyme system to transfer electrons to the P450 enzyme as reducing equivalents for the "hydroxylation" reaction (13). The first such reaction is the conversion of cholesterol to pregnenolone, catalyzed by the side-chain cleavage enzyme, located on the inner mitochondrial membrane and encoded by the CYP11A1 gene on human chromosome 15. The enzyme catalyzes three reactions (20
-hydroxylation, 22-hydroxylation, and cleavage of the bond between C-20 and C-22) to produce pregnenolone (14).
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4 by 3β-HSD, which is located on the membrane of the smooth endoplasmic reticulum. Two 3β-HSD isoenzymes have been identified in man (15, 16), and it is the type II isoform, encoded by the HSD3B2 gene on chromosome 1p13.1, that is found in the adrenal gland (17, 18).
D. 21-Hydroxylase
Progesterone is converted by 21-hydroxylase (CYP21A) located on the cytoplasmic surface of the smooth endoplasmic reticulum to 11-deoxycorticosterone (DOC) (19). The CYP21A gene maps to human chromosome 6p21.3 and is adjacent to a pseudogene, CYP21P (20).
E. Aldosterone synthase
The conversion of DOC to aldosterone involves three consecutive reactions catalyzed by a single enzyme, aldosterone synthase. It performs 11β-hydroxylation of DOC to form corticosterone, an 18-hydroxylation step providing 18-hydroxycorticosterone and finally 18-methyloxidation to produce aldosterone. Aldosterone synthase is located on the inner mitochondrial membrane and is encoded by the CYP11B2 gene. DOC is the preferred substrate of aldosterone synthase and remains bound to the active site throughout these reactions, whereas corticosterone and 18-hydroxycorticosterone are only released as by-products (21). Aldosterone synthase is approximately 93% homologous to 11β-hydroxylase, the product of the CYP11B1 gene, which catalyzes the conversion of 11-deoxycortisol to the main glucocorticoid, cortisol (in rodents, it converts DOC to the principal glucocorticoid, corticosterone). The CYP11B1 and CYP11B2 genes are located in tandem on human chromosome 8q21–22 (22, 23, 24) but are not coexpressed in the adrenal cortex; CYP11B2 expression is confined to the ZG, whereas CYP11B1 is expressed in the ZF and ZR. This zonal pattern of expression is accounted for by major differences in the regulatory regions of the two genes. CYP11B2 expression is mainly controlled by AngII and potassium, whereas CYP11B1 is regulated by ACTH.
Two CYP11B genes are present in man, mice, rats, and hamsters. However, in contrast, cows, pigs, sheep, and frogs possess a single multifunctional enzyme encoded by a single gene. Furthermore, rats possess a CYP11B3 gene and a CYP11B4 gene, the latter a pseudogene. CYP11B3 encodes an enzyme possessing 18- and 11β-hydroxylase activities, expressed in the adrenal ZF/ZR for a short period after birth and regulated by ACTH (25, 26).
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III. Regulation of Aldosterone Biosynthesis |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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A. The renin-angiotensin system
Renin is synthesized and released by the juxtaglomerular cells in the afferent arteriole of the kidney as a response to decreased intravascular volume, detected by baroreceptors (mediated by β-adrenoreceptor activation), and by reduced sodium concentration at the macula densa. Renin catalyzes the hydrolysis of angiotensinogen, secreted by the liver, to biologically inert angiotensin I (AngI) which is then converted to the active component of the system, the octapeptide AngII, by the angiotensin-converting enzyme (ACE) (29). ACE is widely distributed in vascular endothelium and other tissues; complete RASs have been described in other tissues including the brain, vasculature, and adrenal cortex (30, 31). There is some evidence that aldosterone may increase ACE tissue expression, thus identifying a positive feedback mechanism that would stimulate local generation of AngII (32), although the concentrations of aldosterone at which this was noted were unphysiological and the contribution of this to increased local AngII generation is somewhat uncertain.
Recently, an additional complementary but contrasting arm of the RAS has been identified (33, 34). The monocarboxypeptidase ACE2, a homolog of ACE, cleaves AngII to form Ang (1, 2, 3, 4, 5, 6, 7). It also hydrolyzes AngI to form Ang (1, 2, 3, 4, 5, 6, 7, 8, 9) which is subsequently converted to Ang (1, 2, 3, 4, 5, 6, 7) by ACE and neutral endopeptidase. Ang (1, 2, 3, 4, 5, 6, 7) is believed to act via a distinct G protein-coupled Mas receptor, although additional effects on the AT1 and AT2 receptors have not been excluded (35, 36). In contrast to AngII, Ang (1, 2, 3, 4, 5, 6, 7) exhibits vasodilatory and antiproliferative properties and is considered to be the protective peptide of the RAS cascade. ACE2 is distributed throughout the cardiovascular system as well as in noncardiovascular tissue. Although structurally similar to ACE, its activity remains unaffected by ACE inhibitors due to differences in the ligand-binding domain (37).
AngII raises blood pressure by direct vasoconstriction, increasing both sympathetic nerve activity and myocardial contractility, and also by enhancing renal salt and water retention through the stimulation of adrenal aldosterone production. The adrenal response to AngII occurs within minutes, a time course that implies no new protein synthesis is required. This acute, AngII-mediated release of aldosterone may result from rapid conversion of intermediate compounds in the steroidogenic pathway or de novo synthesis from cholesterol, possibly as a consequence of StAR protein activation to increase cholesterol availability to the inner mitochondrial membrane. Chronic stimulation by AngII results in ZG hypertrophy and hyperplasia, increased CYP11B2 expression, and consequently increased aldosterone secretion. The resulting increase in sodium and water retention and blood pressure inhibits further renin release.
In the adrenal gland, AngII acts on specific G protein-coupled receptors (AT1 receptors) that cause phospholipase C to stimulate intracellular production of 1,4,5 inositol triphosphate and 1,2-diacylglycerol which, in turn, activate protein kinase C. 1,4,5 Inositol triphosphate also increases intracellular free calcium, causing the phosphorylation of several Ca2+-calmodulin-dependent protein kinases and activation of transcription factors such as ATF-1, ATF-2, and cAMP-responsive element binding protein (38). These bind cAMP-responsive elements and other cis-acting elements (e.g., Ad-5 and NBRE-1) unique to the 5' regulatory region of the CYP11B2 gene. The Ad-5 cis-element binds steroidogenic factor 1 (SF-1), members of the neuronal growth factor IB family, and chicken ovalbumin upstream promoter-transcription factor. In addition, NURR1, a member of the neuronal growth factor IB family of orphan nuclear receptors, appears to play a key role in CYP11B2 transcription. It is found in high levels in the ZG and is increased by AngII treatment. Its expression is also up-regulated in aldosterone-secreting tumors (39, 40).
B. Potassium
Aldosterone production is acutely sensitive to very small changes in [K+]e. Increased [K+]e stimulates aldosterone secretion, thereby helping to maintain K+ homeostasis. The effects of [K+]e and AngII are synergistic, with the level of aldosterone production in response to AngII being determined by [K+]e (41). Increased [K+]e causes depolarization of the ZG cell membrane, opening voltage-dependent L- and T-type Ca2+ channels and facilitating a rapid rise in [Ca2+]i. This causes activation of calmodulin and Ca2+-calmodulin-dependent protein kinases that phosphorylate the transcription factors described above, creating active forms that stimulate CYP11B2 gene transcription (38). AngII and potassium therefore regulate CYP11B2 transcription through common Ca2+-dependent signaling pathways and also through common transcription factors (42). The effects of [K+]e to modulate the response to other agonists (such as ACTH and AngII) have already been mentioned.
C. ACTH
ACTH is the principal effector peptide of the hypothalamic/pituitary adrenal axis and is primarily involved in the regulation of glucocorticoid production. However, ACTH also contributes to the regulation of aldosterone biosynthesis. ACTH is a 39-amino acid peptide and is synthesized as part of the large precursor molecule, proopiomelanocorticotrophin (POMC). ACTH release from the anterior pituitary follows a diurnal rhythm with the levels being highest in the morning and lowest at night (43). Underlying this is a continuous pulsatility that is reflected in plasma cortisol concentrations.
Acutely, ACTH stimulates adrenal blood flow and modestly increases aldosterone production by interacting with specific G protein-coupled receptors in the adrenal ZG that in turn activate adenylate cyclase, thus increasing [cAMP]i and activating protein kinase A (44, 45). ACTH-responsive genes contain cAMP-responsive elements within their 5' regulatory regions; the cAMP-mediated increase in steroidogenic gene transcription takes several hours and is inhibited by cycloheximide, suggesting that protein synthesis, probably StAR, is an absolute requirement. cAMP-independent signaling pathways also appear to be induced by ACTH, including protein kinase C, calcium influx via calcium channels, and the lipooxygenase pathway (46, 47, 48, 49)
Chronic administration of high doses of ACTH results in adrenal hyperplasia and hypertrophy of the adrenal ZF (27, 50, 51, 52), but CYP11B2 gene expression and plasma aldosterone are suppressed in both humans and animal models (53, 54, 55). The mechanism of this chronic inhibition is unclear. One possibility is that cAMP may down-regulate the expression of AngII receptors in ZG cells (56, 57). Alternatively, ACTH may transform proliferating ZG cells into ZF cells or divert precursors from the mineralocorticoid to the glucocorticoid pathway (51, 58, 59).
Although ACTH has contrasting acute and chronic effects on aldosterone secretion, there is substantial evidence to suggest that it is a key physiological regulator of aldosterone production. Firstly, aldosterone secretion displays a diurnal variation with higher levels in the morning and lower levels later in the day; this pattern is regulated by ACTH (60). Recent evidence shows that the POMC knockout mouse has abnormal adrenocortical morphology and reduced, but detectable levels of aldosterone, further suggesting that ACTH is required for normal aldosterone secretion (61). Finally, humans with mutations in genes encoding the adrenal ACTH receptor or its essential accessory protein demonstrate altered regulation of aldosterone (62). Thus, although ACTH in pharmacological doses does inhibit aldosterone production, its likely physiological role is to modulate the response of aldosterone to other major regulatory factors such as AngII and potassium.
In summary, the control of aldosterone is established to preserve two major physiological requirements, the circulating volume, via the RAAS, and potassium homeostasis. There is interaction between potassium and AngII so that potassium status amplifies the effect of AngII in altering aldosterone production, thus illustrating the primary importance of defending the organism against hyperkalemia. Other influences such as ACTH modify the acute and chronic secretion of aldosterone, and we will describe later how alterations in the usual relationship between aldosterone and its major regulatory systems may lead to cardiovascular dysfunction.
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IV. Extraadrenal Synthesis of Aldosterone |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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A. CNS
mRNA from all of the genes required for aldosterone biosynthesis have been detected in the rat brain (64, 65, 66), with relatively high levels of CYP11B2 transcripts in the hippocampus and cerebellum where MR levels are also highest (67, 68). Aldosterone synthase itself has been detected in these brain regions using immunohistochemistry and, although levels are low compared with the adrenal gland, expression is strongest in the Purkinje cells of the cerebellum and the hippocampal dentate gyrus and CA1–2 neurons. We also found expression of 11β-hydroxylase to localize to the same regions, suggesting that these two enzymes would have to compete for their substrate, DOC (67). CYP11B2 transcription in the hippocampus and cerebellum is subject to regulation by dietary sodium, with low-salt diet causing an increased CYP11B2 transcription (69). Subsequent studies in human brain tissue detected transcripts for StAR, side-chain cleavage enzyme, 3β-HSD2, and 21-hydroxylase in hippocampus and cerebellum, but no evidence of CYP11B2 or CYP11B1 transcription although their mRNA have been detected in other regions such as corpus callosum, thalamus, and spinal cord (70) (S. M. MacKenzie, unpublished observations). The lack of aldosterone synthase expression effectively excludes the possibility of aldosterone synthesis, but the de novo synthesis of DOC is possible (Fig. 1
), and this steroid is known to possess mineralocorticoid properties, like aldosterone, albeit with lower potency (71).
B. The cardiovascular system
Takeda et al. (72) performed much of the early work on vascular aldosterone, detecting both aldosterone secretion and CYP11B2 transcription in the mesenteric arteries of WKY rats. Later, they reported the same findings in human vascular endothelial cells in vitro (73), although another group could not reproduce this (74).
Silvestre et al. (75) reported CYP11B2 transcription in the rat heart, finding that cardiac expression could be increased by the administration of AngII. Another group claimed that rat cardiac CYP11B2 transcription was increased after adrenalectomy (76), whereas yet another showed cardiac expression to be strain-specific, with CYP11B1 and CYP11B2 transcripts detected in WKY but not Sprague Dawley rats (77). In the years since these studies, however, there has been growing skepticism that the rat heart is capable of significant aldosterone production. Gòmez-Sánchez et al. failed to detect significant cardiac aldosterone levels after adrenalectomy, whereas our own studies could not consistently demonstrate CYP11B2 transcription in stroke-prone spontaneously hypertensive rats/Wistar Kyoto rats either with or without induced heart failure, transgenic Ren-2 rats or ventricular myocytes cultured from the Sprague Dawley strain (78, 79).
In the human heart, a thorough study by Kayes-Wandover and White (80) detected transcription of genes for side-chain cleavage enzyme, 3β-HSD, 21-hydroxylase, and MR throughout the nondiseased human adult and fetal heart, but CYP11B2 transcription was only seen in the fetal heart, although another study of fetal tissues was negative (81). Young et al. (82) were also unable to find evidence for CYP11B2 transcription in the normal human heart and could not consistently detect CYP11B2 transcripts in samples of failing human heart removed during cardiac transplantation. Various groups proposed that cardiac expression of CYP11B2 may only be induced in the human heart in pathological circumstances, but evidence has been contradictory. Several studies have attempted to use cardiac catheterization to measure cardiac aldosterone levels. These have yielded contradictory results, with some reports suggesting that patients can secrete aldosterone into the coronary circulation (83, 84) and others concluding that aldosterone is being sequestered by the heart (85). Such contradictory evidence has become typical of this topic. Nevertheless, as with the hippocampus and cerebellum, the heart does seem to express the machinery required to synthesize DOC, and evidence of its synthesis in the human heart should be sought (86).
To summarize, the evidence from studies concerning the local synthesis of aldosterone in the heart or peripheral vasculature is contradictory. There is stronger evidence that aldosterone can be produced locally in discrete regions of the rodent CNS and that this may be a regulated process. However, steroidogenic enzyme expression within the human brain is very different from that of the rat; there is strong potential for the production of the mineralocorticoid DOC in areas where MR are highly expressed but none for aldosterone production. The role that local steroidogenesis might play in the regulation of normal physiological function remains the subject of speculation, although recent evidence that overexpression of aldosterone synthase in the rat CNS raises blood pressure suggests that a local mineralocorticoid-producing system is involved in the control of cardiovascular homeostasis (87).
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V. Cellular Actions of Aldosterone |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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Aldosterone’s major action on epithelial cells is to regulate the reabsorption of Na+, thereby also influencing the transport of water, K+, and H+ across the membrane (Fig. 2). The epithelial cell monolayers form barriers between the internal environment (the blood) and the external environment (the lumen and, eventually, urine). An electrochemical gradient permits the passage of sodium from the lumen into the epithelial cell through the amiloride-sensitive epithelial sodium channel (ENaC). From there, active transport by the Na+/K+-ATPase carries the Na+ across the basolateral membrane, from the epithelial cell into the bloodstream, while simultaneously excreting K+; water follows the movement of the Na+.
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Aldosterone induces the expression of ENaC’s -, β-, and 
-subunits, although its major effect appears to be achieved either by increasing the number of channels in the plasma membrane or by increasing the probability that the channels are open and therefore permit the passage of Na+. This regulation of ENaC is achieved via the expression of a wide range of aldosterone-induced proteins, some of which appear to act by negatively regulating the tonic inhibition of ENaC activity.
The best characterized of these proteins is the serine-threonine kinase SGK1 (92). SGK1 is activated by phosphorylation, mediated by the phosphatidylinositol 3-kinase pathway, which is believed to be a central point of integration of the signaling pathways of aldosterone, insulin, and vasopressin (93, 94, 95, 96). Active SGK1 activates ENaC by phosphorylating a range of substrates. These include: 1) the ENaC channel itself, thereby increasing its open probability (97); 2) the neuronal precursor cells expressed developmentally down-regulated protein 4-2 (Nedd4-2), thus preventing its binding to the channel and subsequent inhibitory effects on channel internalization and degradation (98, 99); and 3) ALL1-fused gene from chromosome 9, which inhibits its transcriptional repression of the ENaC subunit (100).
Recently, a second protein, which acts in parallel to SGK1 to mediate the action of aldosterone, has also been characterized. Glucocorticoid-induced leucine zipper appears to inhibit the ERK signaling cascade, which under normal circumstances tonically limits sodium reabsorption by facilitating the interaction of ENaC with Nedd4 proteins. A more complete review of this mechanism is given elsewhere (101).
Aldosterone is not a specific ligand for the MR, which has an equal affinity for cortisol; given its 1000-fold greater levels in plasma, the vast majority of these receptors would be expected to be transactivated by glucocorticoid, particularly at cortisol’s diurnal peak concentrations. That this does not result in constant MR activation in aldosterone-selective tissues such as the target epithelia is due, at least in part, to the presence within these tissues of the 11β-HSD2 enzyme complex, which catalyzes the conversion of cortisol into cortisone in human subjects (4, 5). Cortisone has little affinity for MR, making the receptor available to activation by aldosterone in tissues where 11β-HSD2 is also expressed.
The mechanism of action described above, whereby the receptor-steroid complex acts as a transcription factor, entails an inevitable latent period between the binding of MR and any eventual physiological effect of the functional protein, owing to the time required for transcription of the gene, translation of the mRNA, and posttranslational modification of the protein. Certain effects attributed to aldosterone occur too rapidly (<15 min) for the "classical" or "genomic" mechanism described above to be plausible. These include cardiovascular effects such as changes to baroreceptor function that alter heart rate response and cellular actions that include rapid alterations to calcium flux. These are often unaffected by the presence of cycloheximide, an inhibitor of transcription, and have led to them being termed "nongenomic" or "rapid" effects. No specific membrane-bound aldosterone receptor has yet been identified, but several of these rapid responses can be blocked by the MR antagonist Spironolactone, suggesting that at least some of these nongenomic effects may be mediated via the classic MR.
In addition to aldosterone’s actions on epithelial cells, it also acts on certain nonepithelial tissues, including the cardiovascular system and the CNS. The widespread potential for aldosterone to affect these sites is illustrated by the extensive distribution of MR, which is present in vascular (endothelial and smooth muscle), cardiac (vascular and myocyte), and nervous (vascular and neurological) tissues. However, whereas vascular MR are accompanied by 11β-HSD2 (see below), receptors in other tissues often are not, implying that the receptors will be constitutively bound by glucocorticoids that block aldosterone’s access. Furthermore, the access of circulating aldosterone to the brain is more restricted than that of circulating glucocorticoid (102). This creates a paradox—aldosterone appears to activate MR despite much higher available concentrations of glucocorticoid which ought, in theory, to dominate receptor binding. In those tissues where there are MR but no 11β-HSD2, it may be that low-level aldosterone activation against a background of predominant glucocorticoid occupation is sufficient to elicit a physiological effect within that tissue. Nevertheless, as described later in this article, it is clear that excess aldosterone promotes cardiac hypertrophy and fibrosis, as well as abnormal vascular endothelial function. Subsequent studies have shown that these effects result directly from aldosterone’s actions, that they are mediated via the MR, that they occur independently of blood pressure, and that they involve interactions with sodium. For example, infusion of rats with corticosterone (the major rodent glucocorticoid) and aldosterone together significantly reduces the increase in blood pressure and cardiac fibrosis observed with aldosterone alone, suggesting that glucocorticoids may act as MR antagonists in cardiomyocytes (103). If the levels of active glucocorticoid are significantly reduced, as in the transgenic mouse overexpressing 11β-HSD2 in cardiomyocytes, then cardiac hypertrophy, fibrosis, and heart failure result, even on a normal-sodium diet. Treatment with the MR antagonist eplerenone reverses these effects (104). Thus, what may be important in these tissues is not so much the absolute level of aldosterone but the balance between glucocorticoid- and mineralocorticoid-occupied MR.
Effects of aldosterone in the CNS are also complex and likely to be determined by the balance of MR occupancy by glucocorticoid and mineralocorticoid. Thus, administration of aldosterone intracerebroventricularly to the rat CNS, at doses too low to induce an effect when administered sc, increases systemic blood pressure (105). When corticosterone or the MR antagonist RU-28318 is coinfused intracerebroventricularly with the aldosterone, its hypertensive effect is attenuated (106). Although there are high levels of MR in the rat CNS, particularly in the hippocampus and cerebellum, significant 11β-HSD2 expression is apparently limited to the nucleus of the solitary tract (NTS) and to the subcommissural organ (107, 108, 109), a region involved with the central regulation of aldosterone secretion and sodium homeostasis. Recent studies have identified aldosterone-sensitive neurons within the NTS that appear to play an important role in driving sodium appetite (109). These neurons border a blood-brain barrier-deficient area within the NTS, thus permitting access to circulating aldosterone (110).
The actions of aldosterone at classical epithelial targets involves, as described above, regulation of key genes such as SGK1. However, it is now apparent that the effects of the hormone in other nonclassic target tissues, such as the vasculature and adipose tissue, includes the regulation of genes whose products modulate levels of a wide range of molecules that are involved in cardiovascular homeostasis and, potentially, dysfunction. These include proinflammatory molecules such as monocyte chemoattractant protein 1, adhesion molecules, including vascular cell adhesion molecule I and intercellular adhesion molecule I, and cytokines such as IL-6 (111, 112, 113). Additionally, aldosterone increases production of reactive oxygen species, possibly through increased expression of key intracellular regulators such as nuclear factor-
B (114, 115, 116). Many of the deleterious actions of aldosterone are attributed to these proinflammatory effects (see Section VII).
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VI. Aldosterone and Cardiovascular Regulation |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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Although much attention in previous decades centered on the consequences of excessive aldosterone production, it is now clear that, within the normal population, differing capacity to synthesize aldosterone between individuals has an impact on the rise of blood pressure with age and on the risk of developing hypertension. For example, in a recent study from the Framingham cohort, it was noted that subjects with aldosterone values within the top quartile of the population distribution showed a steeper blood pressure rise over a 5-yr period than subjects with aldosterone measurements in the bottom quartile; subjects with aldosterone measurements between these extremes showed intermediate rises in blood pressure (120). Correspondingly, subjects with aldosterone measurements in the top quartile showed the highest risk of developing overt hypertension. These data are supported by our recent studies showing that aldosterone levels in an elderly population have a positive correlation with systolic blood pressure; subjects in the top tertile of aldosterone levels had, on average, a blood pressure reading 10 mm Hg higher than those in the bottom tertile, independent of antihypertensive treatment (121). The factors that might lead to altered aldosterone secretory rates within the general population are discussed in detail in Section VIII, but stated briefly, the level of aldosterone will be determined by interactions among environmental factors (such as sodium and potassium intake), genetic factors, and other influences such as early life programming. Little is known about this last influence, although we have recently presented data that show a strong inverse correlation between birth weight and aldosterone in an older adult population (121). The mechanism of this apparent programming effect is, at present, unclear.
Regardless of the mechanisms that determine altered aldosterone regulation, it is evident from the above studies that higher levels of aldosterone contribute to the risk of developing hypertension in the general population. However, in recent years the major focus of research into aldosterone’s role in hypertension has involved the syndrome of primary aldosteronism (PA): excessive aldosterone production independent of its normal regulators. PA was previously regarded as a relatively rare syndrome (from 1–4% of hypertensives); the majority of patients were thought to have solitary aldosterone-producing adenomas (APAs). Over the last 15 yr, these estimates have been radically revised, with the introduction of more extensive screening of hypertensive patients for altered aldosterone secretion, using the now widely accessible ARR as an index of abnormal regulation of aldosterone production. A high ARR is described in 8–15% of hypertensive patients (8, 122, 123). The variation in the absolute proportion of patients with a raised ARR largely reflects local assay criteria and performance. Elegant studies from the Mayo Clinic have shown the absolute dependence of the ratio on renin measurements, so that the value of the ARR is principally determined by the sensitivity of the renin assay (124). Nevertheless, despite the reservations, it is clear that raised ARR values are found in a substantial proportion of patients with hypertension. The most recent and careful study from Italy reports that the frequency of a raised ARR is 11.2%, and this is likely to be an accurate estimate of incidence, at least in a European population (123). Higher frequencies of abnormal aldosterone levels are reported in specially selected populations; for example, several studies have reported that the frequency of inappropriately elevated aldosterone (interpreted as synonymous with PA) is around 22% in patients with resistant hypertension (125).
The majority of studies have regarded the presence of a raised ARR and the subsequent failure of aldosterone to suppress upon a variety of sodium- or volume-loading maneuvers as being diagnostic of PA. However, this does not imply that these patients all harbor an APA. In a review of experiences at the Mayo Clinic, Young and colleagues (126) reported that the proportion of patients diagnosed with PA who harbored an APA diminished over the time period during which ARR became more widely used to detect relative aldosterone excess. This experience is mirrored by the Primary Aldosteronism Prevalence in Italy study; of the patients identified as having a raised ARR, fewer than 50% (or 
4% of the total population of hypertensives) were found to have a solitary APA (123).
Thus, the majority of patients with a raised ARR have excess aldosterone production from both adrenal glands (bilateral adrenal hyperplasia). Whether this represents true PA (i.e., autonomous secretion) is largely a semantic concern. Earlier studies in the 1970s noted that patients with apparent bilateral adrenal hyperplasia (sometimes called idiopathic hyperaldosteronism) most closely resembled patients with low renin essential hypertension (127). In particular, although renin and AngII levels in these patients were suppressed, they were less markedly abnormal than in patients with definite APAs. Furthermore, aldosterone retained its ability to respond to AngII—indeed, the relationship between AngII and aldosterone was steeper than in patients with essential hypertension or in normal subjects, although these patients undoubtedly show markedly reduced suppressibility of aldosterone during sodium or volume loading. If these tests are used to diagnose PA, then patients with bilateral adrenal hyperplasia will be classified as having the disorder. However, if it is accepted that they retain responses to AngII (and potassium) that are steeper than normal, it may be argued that they have inappropriate regulation of aldosterone rather than autonomous secretion of the hormone. Again, this argument is largely one of terminology; what is important is that excess aldosterone production in this circumstance is a contributor to hypertension and cardiovascular damage and is therefore deleterious. In this regard, there are no data that examine whether relative aldosterone excess is more common in older hypertensive patients. Laragh and colleagues (128), in the 1970s, pointed out that low renin hypertension was more common in elderly subjects, a finding corroborated by other studies (129). Furthermore, studies on body sodium content in older hypertensive patients showed this was increased, consistent with a long-term tendency to retain sodium, probably as a consequence of relative aldosterone excess (130), leading to suggestions that low renin hypertension and idiopathic hyperaldosteronism were variants of essential hypertension. There are no studies that examine whether individual hypertensive subjects change volume (and renin) status with time, but it is reasonable to speculate that genetically determined increases in aldosterone secretion will, over many years, lead to hypertension with volume expansion (reflected by low renin) and a persistently increased aldosterone response to AngII and potassium.
Studies that examine the frequency of types of relative aldosterone excess in different age groups and that investigate longitudinal changes in blood pressure, renin, and aldosterone are required to address these issues. However, we describe data below that suggest that aldosterone is subject to genetic regulation so that a long-term but minor increase in production is likely to occur; this, coupled to evidence that aldosterone levels in the normal range predict blood pressure rise with age, supports the proposal that the late development of a volume-expanded form of hypertension driven by relative aldosterone excess may have its origins in early life.
There is now very convincing evidence that excess aldosterone is a cause of cardiovascular damage. Initial concepts that aldosterone is important in cardiovascular function principally in relation to sodium and volume homeostasis have been superseded by an appreciation that the hormone also contributes to the regulation of cardiac structure and function, vascular reactivity, and regulation of CNS sympathetic outflow. In relation to blood pressure outcomes, Milliez et al. (131) reported that patients with PA had substantially higher rates of atrial fibrillation, stroke, and myocardial infarction (MI) compared with patients with essential hypertension who were matched according to their level of blood pressure elevation. Furthermore, there is a clear positive correlation between the level of aldosterone and the degree of left ventricular hypertrophy in patients with PA that reflects the impact of aldosterone on cardiac structure (132). More detailed discussion of the effects of aldosterone on a range of target tissues is presented in Section VII.
The importance of aldosterone in other pathophysiological circumstances is also clear. In patients with heart failure, secondary hyperaldosteronism driven by activation of the RAAS contributes to the spiral of deterioration in cardiorenal function that characterizes this syndrome. Aldosterone excess has been implicated in poor outcomes after heart failure as well as in the development of adverse events after MI (133). The degree of neurohumoral activation is associated with increased mortality; both the SAVE trial (post-MI) and CONSENSUS trial (heart failure) demonstrated that high aldosterone levels predict poor outcome (134, 135, 136). In addition, this association with increased mortality is present in patients across all New York Heart Association classifications of heart failure. The significance of this is further illustrated by the marked impact of MR blockade noted in the RALES study where patients allocated to low-dose spironolactone showed a substantial (30%) improvement in cardiovascular morbidity and mortality compared with those given placebo (7). In addition, recent data from patients with acute MI show that aldosterone levels predict outcome over the first year after the event. For example, patients with aldosterone values in the highest quartile of the distribution have a significantly increased risk of death or further cardiovascular event compared with those in the lowest quartile; this risk persists after correction for all other factors (133). The importance of aldosterone here is further strengthened by the beneficial effect of use of the MR antagonist, eplerenone. In the EPHESUS study, its administration led to a significant decrease in the risk of death or reinfarction in comparison to those subjects given placebo (137). Although benefit from use of an antagonist at the MR does not necessarily imply that aldosterone activation of the receptor is responsible for all aspects of cardiovascular dysfunction, the majority of the data cited above are consistent with the notion that increased availability of aldosterone to the MR is an important cause of morbidity and mortality in patients who develop acute coronary syndromes or who progress to heart failure. Nevertheless, there are no studies that examine whether hypertensive subjects with relative aldosterone excess (or PA) who then progress to develop heart failure or acute coronary syndromes have a particularly adverse outcome. It is worth recalling that data from the RESOLVD trial demonstrated that even the combination of an ACE inhibitor and angiotensin receptor blocker does not suppress aldosterone secretion long term—a phenomenon described as "aldosterone breakthrough" (note that this term is used here to denote the persistent production of aldosterone despite inhibition of generation of AngII or of activation of the AT1 receptor; this is likely to reflect the important action of potassium to regulate aldosterone production) (138). Are those patients with a high ARR who subsequently develop heart failure more likely to develop the phenomenon of aldosterone breakthrough? If we accept that the majority of patients with a raised ARR retain excess sensitivity to AngII, then it would seem likely that this is a long-term phenomenon that would, in the context of heart failure, lead to inappropriate aldosterone production that is difficult to suppress fully. Clarification would require longitudinal studies of patients with a raised ARR. However, regardless of mechanism, a high proportion of patients treated with ACE inhibitors and AngII antagonists have elevated aldosterone levels identifying the option of MR antagonism as an additional therapeutic approach in cardiovascular syndromes (see also Section XI).
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VII. Adverse Tissue Consequences of Aldosterone Excess |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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In 1990, Weber and colleagues (139), using a rat model of renovascular hypertension, observed fibrosis not only of the hypertrophied left ventricle but also of the nonhypertrophied right ventricle. This implied that fibrosis might be a result of humoral rather than hemodynamic factors; aldosterone was identified as a possible cause. Cardiac fibrosis also occurs in rat models of PA (uninephrectomized salt-fed rats infused with aldosterone) (140). Moreover, despite severe persistent hypertension, cardiac hypertrophy and fibrosis are blocked by doses of spironolactone insufficient to ameliorate the hypertension (141). Other investigators have confirmed and developed these findings to show that aldosterone causes cardiac fibrosis independently of its effect on blood pressure or on the development of ventricular hypertrophy (142). Crucially, these profibrotic actions only develop in animals fed a high-salt diet (143). Dietary salt restriction inhibits the effect, emphasizing the vital requirement of both salt and mineralocorticoid in this pathophysiological effect, although how this interaction results in damage is unknown. Young and Funder (142) suggest that cardiovascular damage is accounted for not by aldosterone per se but by inappropriate activation of the MR by normal levels of glucocorticoids because 11β-HSD2 fails to reduce local cortisol levels sufficiently to allow aldosterone access to the MR. Furthermore, a by-product of 11β-HSD2 activity is the generation of nicotinamide adenine dinucleotide hydroxide (NADH). They propose that high levels of NADH (produced by high 11β-HSD2 activity) inhibit the activity of glucocorticoid-occupied MR. When intracellular NADH falls, in response to enzyme blockade or generation of reactive oxygen species (as a result of tissue damage), the MR is activated by glucocorticoids, mimicking the cardiovascular effects of inappropriate aldosterone (144). However, although this is an attractive hypothesis, it has not yet been confirmed by direct experimental evidence, but in a very recent study, it was noted that salt loading itself increased reactive oxygen species availability; the cardiovascular and renal damage seen in the animal model subject to salt loading was reduced by MR antagonism, although aldosterone levels were partially inhibited. This may imply that MR activation by either aldosterone or glucocorticoid is greater in the presence of altered reactive oxygen species state, but it does not demonstrate conclusively that the MR activation is exclusively due to one or the other ligand (145, 146). Moreover, it is clear that the major histological abnormalities that arise in response to aldosterone excess (particularly in animal models where salt loading is combined with activation of aldosterone secretion) include intense perivascular fibrosis and an acute inflammatory response. The perivascular nature of these changes may suggest that the changes are a consequence of aldosterone acting on MR in cardiac vascular tissue, rather than on cardiac myocytes; vascular MR is protected from occupancy by cortisol (or corticosterone) by 11β-HSD2 (143, 147, 148). Aldosterone also increases collagen I and III synthesis in cardiac fibroblasts (149), but animal models clearly demonstrate that these deleterious effects of aldosterone are also dependent on a high sodium intake. Although the effects occur over several weeks, increases in collagen III deposition and inflammatory markers have been reported within 2–3 d in certain animal models (150).
Several mechanisms have been proposed to account for aldosterone-related tissue damage, and all may contribute to varying extents. Firstly, evidence from animal and human subjects suggests that aldosterone is a proinflammatory hormone that causes cardiac perivascular inflammation (86). After several weeks of treating rats with aldosterone and salt, macrophages, lymphocytes, and proliferating endothelial and vascular smooth muscle cells and fibroblasts are found in the perivascular space of intramural coronary arteries as well as areas of cardiomyocyte necrosis in both ventricles. This is accompanied by fibrosis, as demonstrated by a significant increase in ventricular collagen volume fraction. Moreover, there are concurrent increases in mRNA for proinflammatory mediators and cytokines such as intercellular adhesion molecule-1, monocyte chemoattractant protein 1, and TNF (152). Importantly, development of all these changes can be prevented by coadministration of selective aldosterone receptor antagonists such as eplerenone (153).
Similar perivascular changes to those reported in cardiac tissue are reported in the kidneys of animals exposed to aldosterone excess and are likely to be due to similar mechanisms (154). These changes are accompanied by increased glomerular protein loss. Although there are no data on renal histology in human subjects, it is known that treatment of hypertensive, diabetic patients with eplerenone causes a significant reduction in proteinuria (155).
In the CNS of animals exposed to excess aldosterone, perivascular inflammatory changes are also noted, whereas MR antagonist treatment reduces the rate of stroke in the stroke-prone spontaneously hypertensive rat model without significantly lowering blood pressure, again reinforcing the notion that the hormone has widespread adverse tissue effects (156). In subjects with long-term aldosterone excess as a consequence of glucocorticoid-remediable aldosteronism (GRA), there is a reported increase in cerebral aneurysm formation, which is associated with cerebral hemorrhage (157).
In addition to the histological changes described above, aldosterone excess is associated with functional changes in the vasculature; there is abnormal endothelial nitric oxide availability, probably due to increased production of reactive oxygen species after activation of membrane-bound nicotinamide adenine dinucleotide phosphate oxidase. Treatment with spironolactone restores the vasorelaxation response of blood vessels to acetylcholine (158). The decrease in large vessel compliance noted in aldosterone excess may be a reflection of this change in responsiveness. The adverse effects of aldosterone on vessels are probably additive to those of AngII; a positive feedback loop is described whereby aldosterone increases tissue ACE levels leading to increased generation of AngII and further exacerbating vascular damage (158, 159).
Finally, aldosterone effects on adipose tissue have been described. There is an association between the metabolic syndrome and aldosterone excess (160, 161), and MR is present in adipose cells. Aldosterone increases secretion of adipocytokines such as IL-6 in vitro (162); adipocytokines are held to account for the increased risk of insulin resistance and diabetes and to affect vascular endothelial function. This could represent an additional mechanism by which aldosterone increases cardiovascular risk.
In summary, it is now clear that aldosterone levels within the normal population distribution contribute to blood pressure and its rise with age. Hypertensive subjects with relative aldosterone excess have a particularly poor cardiovascular outcome. The majority of subjects with aldosterone excess have idiopathic hyperaldosteronism due to bilateral adrenal hyperplasia where the abnormality is likely to be disordered regulation of aldosterone production in relation to a range of trophins. This may reflect altered genetic regulation of aldosterone secretion, a concept that is discussed in Sections VIII and IX. It is relevant that a recent report on the Framingham population suggests that the ARR is a heritable phenotype (163). In the context of acute coronary syndrome or heart failure, aldosterone is an important predictor of outcome, and blockade of the MR has substantial therapeutic benefit. For these reasons, a better understanding of the regulation of aldosterone secretion throughout life may identify new markers of cardiovascular risk and better ways of stratifying patients in relation to risk and potential therapeutic manipulation. In Section VIII, we will consider how variation in aldosterone secretion within the population might be controlled.
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VIII. Genetic and Environmental Factors in the Regulation of Aldosterone Secretion |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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Drug therapy affects aldosterone as well as the ARR. Antihypertensive treatments have well-recognized effects on the RAS. In the Framingham offspring cohort, aldosterone levels were higher in subjects using diuretics (166); the ARR was positively associated with beta-blockers and hormone replacement therapy and negatively associated with diuretics and ACE inhibitors (163). Aldosterone levels also vary with age (167, 168, 169, 170), sex (171), ethnicity (171, 172), hypertension (173), and body mass index (174). However, it is clear that there remains significant basal variation in aldosterone levels in the population, probably reflecting an interaction of genetic influences with the environmental factors mentioned above.
A. Genetic determination of aldosterone levels
In defining whether a phenotype is subject to genetic regulation, it is useful, in the first instance, to identify whether it demonstrates familial clustering or heritability. In the Framingham population—a large normotensive cohort of 3326 subjects—heritability of multivariate adjusted logARR was 0.4 (P < 10–4). Inglis et al. (175) reported in a study of normotensive monozygotic and dizygotic twins that there was no evidence of a significant genetic influence on plasma aldosterone, although urinary excretion of aldosterone was clearly heritable. In contrast, modest heritability of plasma aldosterone has been reported in the Framingham offspring population (H2 = 0.1, P < 0.04) (166) and in a study of 43 hypertensive sibling pairs (H2 = 0.19) (176). However, in all of these studies, it was acknowledged that, in measuring a single plasma aldosterone sample, the heritability of aldosterone could be obscured by factors such as dietary salt, potassium, and posture. Better measurements of mineralocorticoid status, uninfluenced by short-term environmental changes, such as 24-h urinary excretion of its principal metabolite, tetrahydroaldosterone (THaldo), may be required to estimate heritability reliably. The initial finding of a high degree of heritability of urinary aldosterone excretion rate by Inglis et al. has been replicated for THaldo in a large collection of nuclear families (H2 = 0.52, P < 10–6), confirming a strong genetic influence on this corticosteroid phenotype (177).
Thus, there is now considerable evidence of a modest genetic influence on aldosterone status as assessed by its urinary excretion and plasma levels. The most obvious candidate gene is CYP11B2, encoding aldosterone synthase. Variation at this locus affects blood pressure and aldosterone synthesis in rodent models of hypertension such as the Dahl salt-sensitive rat and rare human monogenic syndromes, and these are described in the remainder of this section.
B. The Dahl salt-sensitive rat
The Dahl rat offers a classic example of how an environmental factor—in this case, salt—and genetic factors interact to cause a hypertensive phenotype. Furthermore, it highlights the importance of the CYP11B1 and CYP11B2 two-gene locus in altering corticosteroid biosynthesis in the pathogenesis of high blood pressure. The Dahl rat strains were originally selected according to the blood pressure they achieved on high-salt diets. In one strain, blood pressure was salt-sensitive and rats became severely hypertensive upon salt loading. In the other, salt-resistant (SR) strain, blood pressure did not increase so dramatically upon sodium loading. In rodents, the principal glucocorticoid is corticosterone, synthesized from DOC by 11β-hydroxylase. This enzyme is also able to catalyze corticosteroid 18-hydroxylation of deoxycorticosterone to form 18-hydroxydeoxycorticosterone (18-OH-DOC), a weak mineralocorticoid. Salt-sensitive animals synthesized proportionally greater levels of 18-hydroxydeoxycorticosterone relative to corticosterone when compared with SR animals (178). Genetic analysis of the rats’ CYP11B1 gene revealed five mutations in exons 2, 6, 7, and 8 of the SR strain (R127C, V351A, V381L, I384L, and V443M), which associated with altered 11β-hydroxylase activity and blood pressure (179).
The contribution of the CYP11B2 locus to the hypertensive phenotype in this model is less clear. Two nonsynonymous mutations (Glu136Asp and Glu251Arg) have been identified in CYP11B2 of the Dahl SR rat (180), but these have been shown to cause an increase in aldosterone synthase activity in the SR animal.
C. Human monogenic disorders
GRA is an autosomal dominant condition characterized by elevated aldosterone levels, suppressed PRA, hypertension, hypokalemia, and high levels of 18-hydroxy-cortisol and 18-oxo-cortisol (181, 182). GRA is caused by the presence of a chimeric gene resulting from unequal crossover of the 11β-hydroxylase (CYP11B1) and the aldosterone synthase (CYP11B2) genes during meiosis (183). Gene expression and enzyme activity of aldosterone synthase is controlled by ACTH rather than AngII and does not respond to Na+ status.
D. 11β-Hydroxylase deficiency
11β-Hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia. It is an autosomal recessive condition characterized by hypertension, hypokalemia, and low renin activity. The condition results from inactivating mutations that have been reported across the entire CYP11B1 gene, leading to reduced or absent cortisol biosynthesis. In an effort to maintain circulating cortisol levels, there is increased secretion of ACTH, which causes adrenal hyperplasia. The deficiency in 11β-hydroxylase results in the accumulation of precursors in the steroid biosynthetic pathway such as DOC, which exhibits mineralocorticoid properties. Under normal circumstances, the circulating levels of DOC are too low to activate the MR but the high levels associated with this condition result in mineralocorticoid excess and subsequent hypertension. The accumulating precursor steroids are also redirected toward androgen biosynthesis, causing female virilization and hyperandrogenism (184).
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IX. The CYP11B2/CYP11B1 Locus in Common Human Cardiovascular Variation |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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Aside from these differences between the CYP11B1 and CYP11B2 genes, much work has gone into characterizing polymorphic variations within their coding and regulatory regions of the individual genes. The most studied single nucleotide polymorphism (SNP) is at the –344 position of the CYP11B2 gene, much of the early interest being due to the fact that this polymorphism lies within an Ad4 cis-element that is capable of binding SF-1 (185). It was proposed that the two variant forms at this position (either a T or a C, occurring with roughly equal frequency) might result in differential transcription of CYP11B2, but, although subsequent in vitro studies showed the C form of the gene to bind SF-1 more strongly than the T form, they could not demonstrate any differences in gene transcription rates (186).
A. Association with blood pressure
We and others have noted that the –344T and intron 2 conversion alleles are more frequent in patients with essential hypertension (187, 188, 189, 190). Additionally, we have reported that the frequency of the –344T allele is increased in hypertensive patients with an elevated ARR but not in subjects with a normal ratio (191). However, other groups have reported contrary findings, with some reporting no effect and others that the C-allele is associated with increased blood pressure (192, 193). Despite the variation in these findings, a recent meta-analysis designed to address this controversy suggested that the –344T allele is indeed associated with a higher risk of hypertension, albeit modest, but has little influence on aldosterone excretion (194). However, it must be pointed out that all studies reported to date (including those within the meta-analysis) are relatively small; it is salutary that much larger case/control series have been required to identify effects of genes that exert relatively minor effects (generally conferring odds ratios of around 1.2) on other common complex traits such as obesity (195). For this reason, a large, appropriately powered, case control analysis that uses suitably defined tag SNPs that identify common haplotypes with independent confirmation in a separate population is required to definitively implicate this locus in essential hypertension.
B. Association with aldosterone synthesis
If there is an association between the locus and hypertension, it is necessary to speculate on the potential mechanism, and the link between genotype and aldosterone synthesis needs to be considered. Despite the absence of any evidence of altered regulation of expression noted above, several studies have shown associations between aldosterone production and allelic variants at the CYP11B2 locus, including this –344C/T SNP. It is now known to be in tight LD with two other variants, the intron 2 conversion and K173R (185, 196, 197). We initially reported that the –344T allele was associated with increased THaldo excretion rate (187). Subsequent investigations have reported that plasma levels of aldosterone are raised in subjects with the –344T allele (189). The coding region polymorphism (K173R) was first described in a Chilean population in 1996; the R variant was found to be more common in hypertensive subjects with suppressed renin, although it has no effect on enzymatic activity in vitro (197). More recent studies assessing levels of mRNA transcripts in adrenal tissue suggest that the –344T/K173 haplotype is associated with higher gene expression than the opposite –344C/R173 haplotype (198).
Thus, although not all data are concordant, it does appear that the –344T allele as well as the intron 2 conversion and K173 variants of CYP11B2 associate with increased aldosterone synthesis. However, our recent studies of association between urinary THaldo excretion and a number of variants across both the CYP11B2 and CYP11B1 genes revealed a more complex picture (177). In this large family-based study derived from hypertensive probands, we demonstrated a relatively weak (although statistically significant) association between THaldo excretion and the –344 and intron 2 variants (R2 1.1 and 1.4%, respectively). However, the strongest association with variation in THaldo excretion occurred with SNPs within the CYP11B1 gene encoding 11β-hydroxylase (199). This study demonstrated that, although highly heritable, variation in CYP11B2 and CYP11B1 could only account for up to 10% of variability in THaldo excretion. In addition, a recent study found no association between plasma aldosterone levels and variation in the CYP11B2 and MR genes (166). Clearly, other genetic factors—possibly encoding other elements of the RAS—must contribute to variability in aldosterone status, although this has not yet been studied in detail. However, it is also important to consider how these data on aldosterone regulation relate to other alterations in corticosteroid secretion in cardiovascular disease.
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X. Association of Other Corticosteroid Phenotypes and Hypertension |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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The use of heritability studies to identify a possible genetic component to a phenotype has been discussed above in relation to aldosterone. Twin and other family studies also show that the S:F ratios in plasma and urine are heritable traits. Moreover, CYP11B2 and CYP11B1, which are logical candidate genes for essential hypertension, are also implicated in the phenotype of apparent altered 11β-hydroxylation efficiency. We have reported that the –344T allele of CYP11B2, implicated in hypertension with a raised ARR, is also associated with a higher S:F ratio in normal subjects (191, 205). More recently, Ganapathipillai et al. (204) reported a similar finding in relation to variation in CYP11B1, a finding that we corroborated in a large population of hypertensive patients and in a separate family-based population study (199).
Thus, there are convincing data that the phenotype of a raised ratio of 11-deoxysteroid (either deoxycortisol or deoxycorticosterone) to product (cortisol or corticosterone, respectively) is accounted for by variation at CYP11B1. The recent studies of Barr et al. (206) provide a possible explanation for this. Briefly, two SNPs in the 5' untranslated region of CYP11B1 (–1889G/T and –1859A/G) are associated with variation in expression of a reporter gene construct in vitro. The –1889T and –1859G alleles, which drive reduced expression in vitro, are associated with higher S:F ratios in vivo, consistent with lesser 11β-hydroxylase efficiency. Moreover, these SNPs are in close LD with the –344T allele of CYP11B2. The above data allow the definition of a haplotype associated with altered S:F ratio, increased aldosterone production, and increased risk of hypertension and raise the question of whether the parallel blood pressure-associated changes in aldosterone and cortisol synthesis are coincidental—and therefore independent—or whether they are causally linked. Although either explanation is plausible, we suggest that current evidence favors the hypothesis that genetically determined, variable 11β-hydroxylase efficiency is the primary intermediate phenotype and that changes in aldosterone levels and blood pressure are consequences of this, mediated by long-term—in fact, lifelong—changes in ACTH drive to the adrenal cortex.
Is it plausible that a very mildly raised ACTH drive can, over a long period of time (lifelong), raise aldosterone levels by the small amount seen in the blood pressure/aldosterone association studies and, in addition, alter the relationship between aldosterone and renin to account for the differences in ARR? Aldosterone synthesis depends, like other corticosteroids, on mobilization of cholesterol for initial side-chain cleavage, and it is clear that the principal agent, StAR, is activated during mineralocorticoid synthesis in the rat (207). ACTH is a powerful activator of StAR. Nevertheless, it is widely believed that ACTH is not an important trophin for aldosterone secretion (see Section III). This belief is due mainly to studies in which ACTH administration was shown to stimulate aldosterone levels initially, before they returned to basal levels and were eventually suppressed. However, these studies tended to involve the administration of high levels of ACTH over short time periods (53, 208, 209, 210). The response of aldosterone to all its trophins is attenuated by high sodium status; a likely explanation of the decreasing effectiveness of ACTH is that high doses promote sodium retention (211). This conclusion is supported by the fact that ACTH infusion into sodium-restricted subjects resulted in prolonged stimulation of aldosterone production (209). Interestingly, Pratt et al. (212) found a sustained increase in THaldo excretion rate over the 4 d of ACTH infusion, and Kem et al. (213) and Daidoh et al. (214) found plasma aldosterone levels to be more sensitive to ACTH than either cortisol or dehydroepiandrosterone.
Additional evidence derives from comparisons of aldosterone and cortisol levels in human subjects receiving no exogenous ACTH. In an early study, Biglieri et al. (215) found aldosterone to be within the normal range, but not increased, in Cushing’s syndrome. Several studies show a strong similarity in the circadian and pulsatile pattern of cortisol and aldosterone concentrations, suggesting that ACTH is an important component of the minute-to-minute control of aldosterone secretion (171, 216, 217), but others have found no such correlation (218). It is pertinent that the hypophysectomized rat progressively loses its ability to increase aldosterone production in response to sodium depletion. However, this cannot be restored by ACTH administration, and other pituitary factors might be involved; Griffing et al. suggest -MSH (219). Finally, in a large population survey, Freel et al. (220) found a close correlation between urinary cortisol metabolites, ACTH-dependent adrenocortical androgen metabolites, and THaldo excretion rates. Intriguingly, this correlation could be demonstrated only in subjects homozygous for the –344T CYP11B2 polymorphism, which is consistent with the hypothesis that, in subjects with relative reduction in 11β-hydroxylase efficiency, a minor but chronic increase in ACTH drive to the adrenal results in an increased dependence of aldosterone secretion on POMC-derived peptides. Thus, it seems legitimate to conclude that ACTH plays a significant role in aldosterone control and that the genetic variations in CYP11B1 may initiate the increased levels of ACTH and, indirectly, aldosterone. Chronically elevated ACTH drive might alter ARR in two ways: 1) as an alternative trophin to AngII, ACTH might subsume part of renin’s function so that less is required to maintain normal steroid levels; 2) alternatively, as a growth factor for the adrenal cortex, ACTH might promote ZG hypertrophy and thereby increase sensitivity to AngII and potassium. Indeed, it would be reasonable to speculate that, in this circumstance, adrenal hyperplasia might develop over a period of many years. This could account for the development of nontumorous bilateral adrenal hyperplasia with aldosterone excess, the etiology of which is currently unknown. This theory offers a testable hypothesis for the genesis of hypertension with relative aldosterone excess.
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XI. Aldosterone as a Therapeutic Target |
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The use of MR antagonists has been limited, until recently, by their side effect profile. Spironolactone is a weak antagonist of the androgen receptor and in male subjects, particularly at high dose, can cause gynecomastia and loss of libido. The recently developed aldosterone receptor antagonist, eplerenone, does not bind to the androgen or other steroid hormone receptors. In animal studies, it lowered blood pressure in uninephrectomized, salt-loaded rats treated with aldosterone for 2 wk. Proinflammatory molecule expression was also substantially reduced (113). It reversed myocardial injury in rats treated with AngII and N-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthesis) (224), independently of any blood pressure-lowering effect and reduced histological damage and protein loss in animal models of hypertension (225, 226) and diabetes (227). In clinical trials, it was well tolerated: the incidence of adverse events was similar to placebo, and there were no reports of gynecomastia or menstrual irregularity (228). It is as effective an antihypertensive agent as the calcium channel blocker (amlodipine) (229), ACE inhibitor (enalapril) (230), and AngII receptor blocker (losartan) (231, 232) and provides additive benefits when combined with other inhibitors of the RAAS. For example, in the recent 4E trial (233), eplerenone reduced end organ damage and blood pressure. When combined with enalapril, substantial additive benefits were seen. Furthermore, combined enalapril and eplerenone reduced microalbumin excretion by 52.6% more than enalapril (37.4%, P = 0.038) or eplerenone (24.9%, P = 0.001) alone.
Other options for reducing aldosterone levels include inhibition of renin; the new orally active renin inhibitor, aliskerin, lowers aldosterone levels and reduces blood pressure (234). However, it is likely that patients given this drug will show aldosterone breakthrough in the same way as patients given an ACE inhibitor or an AngII receptor blocker (235). Specific inhibition of aldosterone synthase could be achieved by developing agents that target aldosterone synthase, but the similarity of this enzyme to 11β-hydroxylase makes identification of agents that are entirely specific for aldosterone synthesis difficult. Nevertheless, data on the use of a specific inhibitor of aldosterone synthase, fadrazole, have shown that the drug can block the production of aldosterone, but its in vivo efficacy in man has not been tested (236). A more promising approach might be to utilize drugs that have a more tissue-selective antagonist effect at the MR (selective MR modulators), but at present such agents are not available for clinical use.
B. MR blockade in other cardiovascular syndromes
There is extensive information supporting the use of MR antagonists in other cardiovascular syndromes. Aldosterone breakthrough is a common phenomenon with reports of aldosterone levels returning to baseline as soon as 3 d after initiation of ACE inhibitor therapy (237). In the RESOLVD Pilot study, heart failure patients given both enalapril and candesartan had at 17 wk aldosterone levels that were significantly lower than patients given either agent alone, but mean aldosterone levels had returned to baseline by 43 wk, even with maximum doses of both agents (138). This is not surprising given the importance of plasma potassium as well as AngII in the regulation of aldosterone production. The relationship between aldosterone levels during escape from RAS inhibition and eventual clinical outcomes has been assessed in large-scale clinical studies. A subpopulation of 534 post-MI patients participating in the SAVE trial were randomized to captopril or placebo; mean aldosterone levels were found to be lower among patients who remained free of cardiovascular events over 2 yr compared with those who died, developed heart failure, or had a further MI (136). In the CONSENSUS trial, among patients who received enalapril, those with a large decrease in aldosterone levels at 6 wk had approximately half the 6-month mortality rate of those with smaller reductions in aldosterone (135). It has also been reported that aldosterone breakthrough is associated with reduced exercise capacity in patients with congestive heart failure (238) and decreased vascular compliance (225), despite ACE inhibitor therapy. Taken together, these data suggest that insufficient suppression of plasma aldosterone with ACE inhibitor treatment correlates with poor outcome and that more specific blockade of aldosterone’s effects could be beneficial.
The first evidence of MR blockade’s additional benefit in heart failure came from the Randomized Aldactone Evaluation Study (RALES) published in 1999 (7). In this population of patients with chronic moderate-to-severe heart failure, spironolactone, given in relatively low dose in addition to ACE inhibitor therapy, significantly lowered mortality (relative risk 0.70; 95% CI, 0.60–0.82; P < 0.001) through a reduced risk of death from progressive heart failure and sudden cardiac death. A substudy of sodium retention demonstrated that the dose of spironolactone used in the study (25–50 mg) had no major diuretic effect. The authors concluded, therefore, that the cardioprotective effect of MR blockade contributed to the reduction in mortality rate. However, this is not without risk. Subsequent to the RALES publication, general prescription of spironolactone increased, and this was noted to result in a rise in frequency in hyperkalemia often with adverse consequences (239). This caution is required in extending MR antagonist use.
To provide a mechanistic explanation for these benefits, a further substudy of the RALES population looking at serological markers of collagen turnover [procollagen type I carboxy-terminal peptide (PICP), procollagen type I amino-terminal peptide (PINP), and procollagen type II amino peptide (PIIINP)] was performed (240). The procollagen markers decreased in the spironolactone group over 6 months but remained unchanged in the placebo cohort, indicating that the benefits of MR blockade paralleled the reduction in cardiac fibrosis.
More recently, the eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival (EPHESUS) study evaluated treatment with eplerenone (137) in 6632 patients with acute MI complicated by heart failure. A major difference from RALES was that 75% of patients received beta-blockers compared with 10% in RALES. At a mean follow-up of 16 months, treatment with eplerenone was associated with a 15% reduction in overall mortality and a 17% reduction in cardiovascular mortality. Hence, the myocardial protective effect of MR blockade is maintained even in the presence of optimal therapy and in patients close to the acute phase of MI.
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XII. Conclusion |
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online February 21, 2008
Abbreviations: ACE, Angiotensin-converting enzyme; Ang, angiotensin; APA, aldosterone-producing adenoma; ARR, aldosterone to renin ratio; CNS, central nervous system; DOC, 11-deoxycorticosterone; DOC:B, DOC to corticosterone ratio; ENaC, epithelial sodium channel; GRA, glucocorticoid-remediable aldosteronism; HRE, hormone response element; HSD, hydroxysteroid dehydrogenase; 11β-HSD2, 11β-HSD type 2; [K+]e, extracellular potassium concentration; LD, linkage disequilibrium; MI, myocardial infarction; MR, mineralocorticoid receptor; NADH, nicotinamide adenine dinucleotide hydroxide; Nedd4-2, neuronal precursor cells expressed developmentally down-regulated protein 4-2; NTS, nucleus of the solitary tract; PA, primary aldosteronism; POMC, proopiomelanocorticotrophin; RAAS, renin-angiotensin-aldosterone system; RAS, renin-angiotensin system; S:F, 11-deoxycortisol to cortisol ratio; SF-1, steroidogenic factor 1; SNP, single nucleotide polymorphism; SR, salt-resistant; StAR, steroidogenic acute regulatory protein; THaldo, tetrahydroaldosterone; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis.
Received for publication August 29, 2007. Accepted for publication February 11, 2008.
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TopAbstractI. IntroductionII. Aldosterone BiosynthesisIII. Regulation of Aldosterone...IV. Extraadrenal Synthesis of...V. Cellular Actions of...VI. Aldosterone and...VII. Adverse Tissue Consequences...VIII. Genetic and Environmental...IX. The CYP11B2/CYP11B1 Locus...X. Association of Other...XI. Aldosterone as a...XII. ConclusionReferences |
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