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Aldosterone and Mineralocorticoids
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Chapter 12
Aldosterone and Mineralocorticoids John W. Funder
DEFINITIONS
Regulation of Aldosterone Secretion
Aldosterone
A number of positive (e.g., adrenocorticotropic hormone [ACTH]) and negative (e.g., nitric oxide [NO]) factors have been shown to affect aldosterone secretion in a variety of experimental situations, but there is consensus that angiotensin II (Ang II) and plasma potassium concentration ([K+]) are the two major determinants of aldosterone secretion. The renin-angiotensin-aldosterone system (RAAS) has evolved to defend organ perfusion and blood pressure (BP), in response to reduced circulating volume monitored by the kidney, and to increased renal sympathetic drive. This level of integration provides a powerful counter-regulatory mechanism in situations of acute volume loss, such as major hemorrhage or massive gastrointestinal fluid and electrolyte loss. Although the primary role for aldosterone in the RAAS is usually considered to be that of volume expansion by epithelial sodium and water retention, it is now clear that aldosterone has additional sites of action. These include the amygdala, to stimulate salt appetite; the circumventricular region within the hypothalamus, to raise BP; and the vascular wall, acting as a vasoconstrictor. Ang II is commonly considered the major determinant of aldosterone secretion, but this is not necessarily the case. Mice in which the gene for angiotensinogen has been knocked out are incapable of producing Ang II in response to physiologic stimuli such as salt restriction. In an elegant series of studies, however, angiotensinogen−/− mice were shown to respond indistinguishably from wild-type in terms of elevating aldosterone in response to a low-salt diet for 2 weeks.2 When mice are placed on a low-[Na+], low[K+] diet, two things are seen. First, even in wild-type mice the aldosterone response is less than to low [Na+] alone. Second, for the first time, the angiotensinogen−/− mice no longer match the wild type in terms of aldosterone response, evidence for the importance of [K+] in the process. In the clinical context there are a number of factors that modify the evolutionary drives of catastrophic volume loss, restricted salt intake, or dietary potassium overload. Western diets are commonly sodium rich; increased sympathetic drive is similarly common, and manifests as essential hypertension. Although the dangers of diuretic-induced hypokalemia have been appropriately recognized, those of a modest and contained degree of hyperkalemia often appear exaggerated. The development of effective angiotensin-converting enzyme (ACE) inhibitors and Ang II receptor (AT1) blockers has proven of immense clinical utility. The recent development of second-generation MR antagonists, and their side-effect–free therapeutic profile, promise to add an additional dimension to the treatment of cardiovascular disorders, including hypertension (see Chapter 70).
Aldosterone is a steroid hormone produced primarily if not exclusively in the adrenal cortex. It is derived from cholesterol by sequential enzymatic reactions, including a final modification of the methyl (CH3) group at carbon 18 (C18) to produce a unique aldehyde (CHO) group, whence the name aldosterone. Aldosterone is the physiologic mineralocorticoid hormone in terrestrial vertebrates; other steroids, most notably deoxycorticosterone (DOC), can also act as mineralocorticoids, but their secretion is not regulated in such a way that they are physiologic regulators of salt and water balance.
Mineralocorticoid Mineralocorticoid is defined in effector terms, as a hormone promoting unidirectional transepithelial sodium transport. Aldosterone was isolated from fractionated bovine adrenal glands half a century ago1 on the basis of this mineralocorticoid activity, and not surprisingly its physiology has been almost exclusively described in epithelial terms. More recently, however, the definition of mineralocorticoid has had to be broadened, to accommodate physiologic actions of aldosterone on blood vessels and in the central nervous system, as detailed later in this chapter. The emerging pathophysiologic roles of mineralocorticoid receptors (MR), also dealt with toward the end of this chapter, similarly call for continuing refinement of their definition.
ALDOSTERONE STRUCTURE AND SYNTHESIS As noted in the preceding definition, aldosterone is characterized by an aldehyde group at C18. Aldehyde groups are chemically very reactive, and in solution the C18 aldehyde cyclizes with the hydroxyl (OH) group at C11 to form an 11,18 hemiacetal. In common with other adrenal steroids, aldosterone is produced by sequential enzymatic steps (side chain cleavage, 3β reduction, 21-hydroxylation) from cholesterol. Unlike other hormonal steroids, aldosterone synthesis in the adrenal gland is confined to its outermost layer, the zona glomerulosa. The final enzymatic step is catalyzed in most species by the enzyme aldosterone synthase (CYP11B2), by a multistep process with DOC as substrate; aldosterone synthase shares 11β hydroxylase activity with the closely related enzyme CYP11B1 (11β hydroxylase), responsible for the defining step in glucocorticoid (cortisol, corticosterone) synthesis. In some species (e.g., bovine) a single CYP11B enzyme appears responsible for both glucocorticoid and aldosterone synthesis, with the mechanism(s) determining zonal specificity yet to be determined.
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Aldosterone Transport and Metabolism Aldosterone can be secreted rapidly in response to elevation in Ang II or plasma [K+], and circulates in the blood loosely bound to albumin (50%-60%) and free (40%-50%). This contrasts with most other adrenal steroids, which are commonly ≥95% bound, in considerable part (and with high affinity) to corticosteroid binding globulin (CBG, transcortin). Metabolism occurs both in the liver (glucuronidation) and in the kidney (reduction) to water soluble products that are largely excreted in urine, where free aldosterone represents only ~1% of the total product. The metabolic clearance rate for aldosterone in humans of the order of 1200 L/day, equivalent to the hepatic blood flow, and consistent with the albuminbound fraction being extracted as equivalent to free in long transit time organs such as the liver.
PHYSIOLOGIC ACTIONS OF ALDOSTERONE Aldosterone was isolated on the basis of its effect on epithelial sodium transport, and this is commonly considered to be its principal physiologic role. Receptors for aldosterone were first identified in classical target tissues such as the kidney,3 and subsequently in a variety of nonepithelial tissues. In some of the latter aldosterone appears to have physiologic actions,4 whereas in others the effects are clearly pathophysiologic.5 Common to both epithelial and nonepithelial actions of aldosterone are MR and the enzyme 11β hydroxysteroid dehydrogenase type 2 (11βHSD2). Mineralocorticoid receptors are members of the steroid/thyroid/retinoid/orphan receptor family of nuclear transactivating factors, closely related to receptors for glucocorticoids (GR), androgens (AR), and progestins (PR).6 MR are unusual in that they have equivalent high affinity for aldosterone and the physiologic glucocorticoid cortisol (and corticosterone, in mice and rats); in fact, cortisol and corticosterone have >30-fold higher affinity for MR than for GR. In addition, MR are found in fish, for example, which do not secrete aldosterone, suggesting the possibility of (patho)physiologic roles for MR occupied by glucocorticoids.7 Circulating plasma levels of glucocorticoids are commonly ~1000-fold higher than those of aldosterone, and plasma-free levels ~100-fold higher. Given their equivalent affinity for MR, a time-honored question is that of the mechanism allowing aldosterone to occupy and activate MR in its physiologic target tissues. Selectivity of the target-tissue response to aldosterone is vested in the enzyme 11βHSD2, which is coexpressed at very high levels (3.5–4 × 106 molecules per renal principal cell, for example) with MR in aldosterone target tissues. 11βHSD2 converts cortisol and corticosterone to their receptor-inactive 11-keto analogs cortisone and 11-dehydrocorticosterone; aldosterone is not similarly metabolized, as its 11-OH group is protected from enzymatic attack by its cyclization to the 11,18 hemiketal.8,9 Although from clinical studies the operation of 11BHSD2 is crucial to allow aldosterone to selectivity activate target tissue MR, conversion of cortisol to cortisone is only one part of the specificity-conferring mechanism. The other action of 11βHSD2 is to stoichometrically convert NAD to NADH. This action also appears crucial in preventing glucocorticoids
from activating MR in aldosterone target tissues under normal circumstances. The NADH/redox state does play a role in inappropriate MR activation under pathophysiologic conditions, as will be discussed later. The postreceptor events following MR activation have been relatively lightly explored. In common with other members of the superfamily, MR can act as transcription factor, binding to response elements in the promoter regions of particular genes, and binding an increasing array of coregulators serving to modulate the rate of gene transcription. A variety of candidate MR-regulated genes have been reported—Na+,K+-ATPase subunits, epithelial sodium channel (ENaC) subunits, CHIF (corticosteroid hormone induced factor), GILZ (glucocorticoid induced leucine zipper) protein—of which the most thoroughly explored has been SGK-1 (serum- and glucocorticoidinduced kinase-1). SGK-1 is constitutively expressed in glomeruli and in response to aldosterone in principal cells in the distal tubule.10 SGK-1, when phosphorylated by insulin (probably inter alia), is ultimately responsible for the phosphorylation of ENaC subunits, thereby blocking ENaC internalization and thus increasing intracellular Na+. Intracellular Na+ is substrate for Na+,K+-ATPase on the basolateral surface of the cell membrane, which pumps Na+ out of the cell into the interstitial space and ultimately the blood. These postreceptor studies have been largely done in kidney, renal cell lines, and to some extent distal colon; comparable studies in nonepithelial aldosterone target tissues are currently in progress. Nonclassical aldosterone target tissues include the vasculature, the amygdala, and the A3V3 region of the hypothalamus. Vascular smooth muscle expresses both MR and 11βHSD2, and both rapid and prolonged effects of aldosterone at physiologic or near physiologic concentrations have been reported.11-13 The amygdala similarly expresses both MR and 11βHSD2, but in terms of selectivity is at a disadvantage compared with peripheral tissues in that aldosterone has a very high reflection coefficient at the blood-brain barrier. The A3V3 region lies outside the blood-brain barrier, and expresses MR but not 11βHSD2. Experimental studies in rat and dog (but not sheep) show that aldosterone clearly can raise BP by acting on MR in the A3V3 region, though the extent to which this reflects a physiologic response in humans is yet to be determined.14 Finally, it is now clear that aldosterone has rapid nongenomic effects. Initially such effects were ascribed to interaction with a membrane receptor for aldosterone, distinct from the classical MR.15 Subsequent studies in both vascular smooth muscle12 and cardiomyocytes16 have shown such rapid nongenomic effects to be mediated via classical MR; whether or not membrane-receptor mediated effects can be shown in these or other tissues remains to be systematically explored. Unlike the estrogen receptor (ER), MR do not have a myristoylation site in their sequence, and are thus unlikely to be plasma membrane located.
CLINICAL SYNDROMES There are various ways of categorizing clinical disorders, and for simplicity this section will begin with a consideration of disorders of aldosterone secretion, followed by disorders of mineralocorticoid receptor activation, with a final section on essential hypertension. Though some of the syndromes described are
Aldosterone and Mineralocorticoids
very rare, they have often been illustrative, with the pathophysiology providing insight into the normal physiology.
Aldosterone Synthase Deficiency Aldosterone deficiency may be part of a generalized hypoadrenal state, may follow deficiency in biosynthetic pathways shared with other adrenal steroids (e.g., 3βHSD, 21 hydroxylase), or may be “pure” aldosterone synthase deficiency. The condition commonly presents in infancy, and is characterized by the signs and symptoms of uncompensated sodium loss— failure to thrive, hyponatremia, hyperkalemia, hyperreninemia, and low or undetectable plasma and urinary aldosterone levels; the latter finding clearly distinguishes the syndrome from pseudohypoaldosteronism. The subject has been comprehensively reviewed.17
Glucocorticoid Remediable Aldosteronism Of more relevance to hypertension is the condition of glucocorticoid remediable aldosteronism (GRA), also known as glucocorticoid suppressible hypertension. GRA reflects the transcription of a chimeric gene, located at 8q24, which contains the 5′ end of CYP11B1 (11β hydroxylase) and the 3′ end of CYP11B2 (aldosterone synthase). Such a gene is not only transcriptionally activated by ACTH, but also is expressed throughout the adrenal cortex. The diagnosis should be suspected in patients with early onset familial hypertension, and can be confirmed or excluded by PCR for the chimeric gene. Treatment is optimally low dose (0.25-0.5 mg/day) dexamethasone. The occurrence of a chimeric CYP11B1/B2 gene reflects the two genes being located in tandem, and sharing 94% nucleotide identity, thus allowing the possibility of an unequal crossing over at meiosis.18 The most common explanation for two highly homologous genes in tandem is a relatively recent gene duplication, consistent with the relatively recent appearance of aldosterone, in terrestrial vertebrates.
Primary Aldosteronism A year after the isolation and characterization of aldosterone in 1953, Jerome Conn reported resolution of hypertension and hypokalemia in a patient after removal of an aldosteroneproducing adenoma.19 For the next 40 years primary aldosteronism was considered to be a rare (<1%) cause of hypertension, despite Conn’s estimate that up to 20% of patients with elevated BP may have primary aldosteronism. Over the past decade, thanks to the wider application of the aldosterone:renin ratio as a screening test, and adrenal venous catheterization to lateralize (or not) the source of the aldosterone, it has become clear that 8% to 15% of unselected hypertensives have autonomous aldosterone production; in a recent general practice population study, 30% of patients with moderate hypertension had elevated aldosterone: renin ratios.20 In terms of diagnosis, patients increasingly are found to have bilateral adrenal hyperplasia rather than a discrete adenoma, and are commonly normokalemic, so that hypokalemia is no longer pathognomonic. Treatment is laparoscopic adrenalectomy, or mineralocorticoid receptor blockade in those with bilateral hyperplasia or who are unsuitable for surgery.
Pseudohypoaldosteronism Although more than 20 years ago defects in MR binding of aldosterone were postulated as the cause of pseudohypoaldosteronism (PHA) from studies on affected patients’ leukocytes,21 in subsequent studies no abnormalities in gene sequence were found.22,23 This apparent conundrum has been resolved by the distinction between PHA type 1 and type 2, where one is caused by an epithelial sodium channel (ENaC) defect, and the other a defect in MR. The phenotype varies, but severe cases can be distinguished from aldosterone synthase deficiency by the often marked elevation in aldosterone as well as renin levels. Treatment is rigorous salt supplementation in infancy, with even severe cases appearing to improve with age, by mechanisms that remain poorly understood.
Pregnancy-Associated Hypertension In contrast to the inactivating MR mutations that may be found in PHA, a recently discovered point mutation24 has been shown to result in a constitutively partially activated MR, in which both progesterone and spironolactone act as agonists, rather than as antagonists as is the case for the wild-type MR. The syndrome was discovered in a young male hypertensive, whose two sisters suffered severe exacerbations of hypertension in pregnancy, presumably reflecting the agonist effect of progesterone on the mutant MR. The syndrome is rare, and abnormal MR do not appear to underlie the relatively common finding of hypertension in pregnancy.
Apparent Mineralocorticoid Excess A more common, though still comparatively rare, condition of inappropriate MR activation is that of apparent mineralocorticoid excess (AME), first described by New and Ulick in 1977.25 In this syndrome—of juvenile hypertension, sodium retention, and hypokalemia, in the presence of suppressed renin and aldosterone levels—epithelial MR are activated by cortisol, reflecting deficient activity of the specificity-conferring enzyme 11βHSD2. The finding of an elevated ratio of urinary free cortisol:cortisone is diagnostic, and patients are treated by MR blockade, on occasion with the suppression of cortisol by dexamethasone administration. Whereas previously it had been assumed that epithelial 11βHSD2 excluded glucocorticoids from MR, this has been shown not to be the case.26 The enzyme acts to debulk intracellular glucocorticoid levels, from ~100-fold those of aldosterone to ~10-fold, consistent with a role for the forgotten cosubstrate (NAD, from which NADH is generated stoichometrically) in activation of glucocorticoid-MR complexes, as briefly discussed subsequently and elsewhere in detail.27,28
Essential Hypertension Although an increasing number of patients with essential hypertension appear to have autonomous aldosterone secretion, the potential role of aldosterone, 11βHSD2, and MR in the majority of hypertensive patients remains unclear. A number of studies have linked allelic variation in 11βHSD2 or CYP11B2 with a higher incidence of elevated BP,29,30 and there have similarly been sporadic reports of a subgroup of essential hypertensives with impaired conversion of cortisol to cortisone.
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The selective MR antagonist eplerenone has been shown to be of equivalent potency to ACE inhibitors, Ca2+ channel blockers, β-blockers, or angiotensin receptor blockers (ARBs) in terms of BP reduction.31,32 In titration-to-effect studies, a wide (4- to 10-fold) dose range of eplerenone was needed in moderate hypertensives to reduce diastolic BP to <90 mm Hg, with comparable falls in BP (~16/12 mm Hg) at each dose level,33 further evidence for a significant role for aldosterone in essential hypertension.
Pathophysiology Whereas the physiologic role of aldosterone in epithelia to retain Na+ and water, and to excrete K+, are well accepted, its other physiologic roles (as a vasoconstrictor, by a direct action on vascular smooth muscle cells (VSMCs), and in the brain to stimulate salt appetite) are less well explored. In other nonepithelial tissues it is unclear what, if any, are physiologic roles for aldosterone. What is clear, however, is that aldosterone may have direct pathophysiologic effects on blood vessels and cardiomyocytes, almost certainly inter alia, in the context of inappropriate salt status.
Mineralocorticoid/Salt Imbalance In physiologic terms, aldosterone and sodium have a reciprocal relationship; when salt status falls aldosterone rises, and vice versa. When, however, aldosterone secretion is no longer responsive to normal negative feedback—in primary aldosteronism, GRA or in animals infused with aldosterone and given only 0.9% NaCl solution to drink—this reciprocal nexus is broken, and aldosterone levels are inappropriate for salt status, and vice versa. Under these circumstances very marked cerebral, renal, and coronary vascular inflammation can be shown in experimental animals,34,35,38 progressing to perivascular and interstitial cardiac fibrosis.36,37 Importantly, if infused animals are on a low-salt diet with water as drinking fluid, these changes are not seen; in the human situation of prolonged Na+ deficiency, very high levels of aldosterone coexist with no cardiac or vascular toxicity. The mechanisms involved in the deleterious synergy between aldosterone and inappropriate Na+ status are currently unclear; their clarification would constitute a major advance in cardiovascular endocrinology.
Vascular Inflammation and Cardiac Fibrosis As noted previously, inappropriate aldosterone (or other mineralocorticoid) for salt status is followed by progressive vascular inflammation in a variety of organs. Animal models used include the stroke-prone spontaneously hypertensive rat (SHR-sp), where eplerenone has been shown to be very protective of both cerebral and renal vasculature, and tissue architecture35; the aldosterone infused rat on 0.9% NaCl solution to drink, in which eplerenone is similarly protective of renal and coronary vessels, and kidney/heart architecture34,38; and the AngII infused/0.9% NaCl drinking rat, in which BP is Ang II driven, and unaffected by eplerenone or adrenalectomy.34,38 On the other hand, adrenalectomy or eplerenone administration completely reverses the vascular and perivascular inflammatory response produced by Ang II/salt, which is
restored in adrenalectomized rats by aldosterone infusion. In all these models, markers of inflammation—ED-1, MCD-1, COX-2, IL-1β, IL-6, osteopontin—increase over the first 2 to 4 weeks of study, and their levels are returned toward or to baseline by MR blockade.
MR Activation by Glucocorticoids In tissues coexpressing MR and 11βHSD2, intracellular glucocorticoid levels are 10 times those of aldosterone, and in unprotected tissues (e.g., cardiomyocytes, most neurons) levels of glucocorticoids are ~100 times higher. Under normal circumstances these glucocorticoid-MR complexes appear inactive, and the glucocorticoid appears to act in tonic inhibitory mode.5,39 In other circumstances, however, glucocorticoids act as MR agonists. The first of these is when 11βHSD2 is deficient or blocked, leading to the syndromes of AME or licorice intoxication. Under these circumstances intracellular cortisol levels rise from ~10 times those of aldosterone toward ~100 times; more importantly, however, levels of intracellular NADH fall precipitously. In other systems NADH has been shown to regulate transcription factor activity by activating corepressors.40,41 Secondly, when 11βHSD2 is blocked by administration of carbenoxolone, an identical pattern of vascular inflammation is seen as with mineralocorticoid/salt administration; importantly, these effects, presumably of glucocorticoids on MR occur when NADH levels fall as 11β hydroxysteroid dehydrogenase is blocked by carbenoxolone.42 Third, experimental angioplasty in pigs is followed by a constriction in coronary luminal diameter, a constriction blocked by eplerenone treatment.43 These animals were not receiving aldosterone or on a high-salt intake; our interpretation of these data is that under conditions of tissue damage and reactive oxygen species (ROS) generation, intracellular redox status changes—just as it does when 11βHSD2 is blocked—allowing normal levels of glucocorticoids to activate vascular MR and thus mimic the aldosterone/salt effects. In this context it should be remembered that in both Randomized Aldactone Evaluation Study (RALES)44 and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS)45 aldosterone levels were normal and salt status unremarkable. In the circumstances of heart failure—progressive or postmyocardial infarction— cardiomyocyte levels of ROS are known to be elevated. Under these circumstances, then, it would appear that spironolactone/ eplerenone are not acting primarily as aldosterone blockers, but truly as MR antagonists blocking the effects of cortisol via MR in the context of tissue damage.
FUTURE DIRECTIONS Any prognostication is necessarily speculative, and thus these will be mentioned only briefly, and in no detail.
Ectopic Aldosterone Synthesis For the past decade there have been claims—commonly but not uniquely based on PCR of steroidogenic enzymes—that aldosterone can be synthesized in neurons, vascular wall and heart. Clinical data are conflicting—aldosterone is elaborated46
Aldosterone and Mineralocorticoids
or extracted47 by the failing heart; in unpublished studies we were unable to find any consistent arteriovenous differences, in normal persons or those with severe heart failure. The Ang II/salt studies by Rocha et al.,34,38 in which adrenalectomy totally reversed the vascular inflammation, argue powerfully against paracrine secretion of cardiac aldosterone of any consequence. Finally, the low levels of enzyme expression found by PCR, commonly between 0.01% and 1% of adrenal levels, means that each step of ectopic steroid synthesis becomes ratelimiting. Unless the enzymes are concentrated in merely a few cells, their contribution to even local aldosterone concentrations is likely to be negligible.
MR-Independent Effects Any molecule that circulates at subnanomolar concentrations can only act effectively by relatively high affinity binding. Many acute nongenomic effects of aldosterone have now been shown to be mediated via classical MR, in for example VSMC12 and cardiomyocytes.16 This does not exclude the possibility of another receptor for aldosterone, distinct from the classic MR, binding aldosterone and other mineralocorticoids with high affinity (and some degree of specificity, given their low circulating concentrations). It is unlikely that this is one of the 48 members of the steroid superfamily of nuclear transactivating factors identified in the human genome; it may, for example, be an analog of the membrane receptor for progesterone.48
Therapeutic Implications If aldosterone/salt imbalance is followed by inflammatory vascular and perivascular responses, and downstream tissue damage, then MR blockade assumes a particular therapeutic importance. If cortisol can activate MR in the context of tissue damage and ROS generation, leading to further ROS generation and exacerbation of tissue damage, MR blockade should prove of utility in breaking this potentially vicious cycle in conditions in addition to those characterized by mineralocorticoid/salt imbalance. MR blockade, therefore, may prove beneficial not only in the context of obvious cardiovascular disease (atherosclerosis/hypertension/myocarditis/heart failure), but also in conditions as diverse as diabetes, cerebrovascular protection and the prevention of premature labor. These possibilities need to be critically examined at the preclinical level, and if found of interest, transferred into the arena of clinical trials.
References 1. Simpson S, Tait J, Wettstein A, et al. Isolierung eines neuen kristallisierten Hormons aus Nebennerien mit besonders hoher Wirksamkeit auf den Mineralsoffwechsel. Experientia 9:333335, 1953. 2. Okubo S, Niimura F, Nishimura H, et al. Angiotensin-independent mechanism for aldosterone synthesis during chronic extracellular fluid volume depletion. J Clin Invest 99:855-860, 1997. 3. Rousseau G, Baxter J, Funder J, et al. Glucocorticoid and mineralocorticoid receptors for aldosterone. J Steroid Biochem 3:219-227, 1972. 4. Funder JW, Pearce PT, Smith R, et al. Vascular type I aldosterone binding sites are physiological mineralocorticoid receptors. Endocrinology 125:2224-2226, 1989.
5. Qin, W, Rudolph A, Bond B, et al. A transgenic model of aldosterone-driven cardiac hypertrophy and heart failure. Circ Res 93:69-76, 2003. 6. Arriza JL, Weinberger C, Cerelli G, et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268-275, 1987. 7. Greenwood A, Butler P, White R, et al. Multiple corticosteroid receptors in a teleost fish: Distinct sequences. Endocrinology 144:4226-4236, 2003. 8. Edwards CR, Stewart PM, Burt D, et al. Localisation of 11 betahydroxysteroid dehydrogenase—tissue specific protector of the mineralocorticoid receptor. Lancet 2:986-989, 1988. 9. Funder JW, Pearce P, Smith R, et al. Mineralocorticoid action: target-tissue specificity is enzyme, not receptor, mediated. Science 242:583-585, 1988. 10. Chen S, Bhargava A, Mastroberardino L, et al. Epithelial sodium channel regulated by aldosterone-induced protein SGK. Proc Natl Acad Sci USA 96:2514-2519, 1999. 11. Kornel L. Colocalization of 11 beta-hydroxysteroid dehydrogenase and mineralocorticoid receptors in cultured vascular smooth muscle cells. Am J Hypertens 7:100-103, (1994). 12. Alzamora R, Michea L, Marusic ET. Role of 11betahydroxysteroid dehydrogenase on nongenomic aldosterone effects in human arteries. Hypertension 35:1099-1104, 2000. 13. Romagni P, Rossie F, Guerrini L, et al. Aldosterone induces contraction of the resistance arteries in man. Atherosclerosis 166:345-349, 2003. 14. Gomez-Sanchez E, Funder J. Central mineralocorticoid receptors in the development of hypertension. Curr Opin Nephrol Hypertens in press, 2004. 15. Christ M, Douwes K, Eisen C, et al. Rapid effects of aldosterone on sodium transport in vascular smooth muscle cells. Hypertension 25:117-125, 1995. 16. Mihailidou A, Mardini M, Funder JW. Rapid, nongenomic effects of aldosterone in the heart mediated by epsilon protein kinase C. Endocrinology 145:773-780, 2004. 17. Dunlop F, Crock P, Montalto J, et al. A compound heterozygote case of type II aldosterone synthase deficiency. J Clin Endocrinol Metab 88:2518-2526, 2003. 18. Lifton R, Dluhy R, Powers M, et al. Hereditary hypertension caused by chimaeric gene duplications and ectopic expression of aldosterone synthase. Nat Genet 2:66-74, 1992. 19. Conn J. Primary aldosteronism: A new clinical syndrome. J Lab Clin Med 45:3-17, 1955. 20. Olivieri O, Ciacciarelli A, Signorelli D, et al. Aldosterone to renin ratio in a primary care setting: the Bussolengo study. J Clin Endocrinol Metab in press, 2004. 21. Armanini D, Kuhnle U, Strasser T, et al. Aldosterone-receptor deficiency in pseudohypoaldosteronism. New Engl J Med 313:1178-1181, 1985. 22. Komesaroff PA, Verity K, Fuller PJ. Pseudohypoaldosteronism: molecular characterization of the mineralocorticoid receptors. J Clin Endocrinol Metab 79:27-31, 1994. 23. Zennaro M-C, Borensztein P, Jeunemaitre X, et al. No alteration in the primary structure of the mineralocorticoid receptors in a family with pseudohypoaldosteronism. J Clin Endocrinol Metab 79:32-38, 1994. 24. Geller D, Farhi A, Pinkerton N, et al. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289:119-123, 2000. 25. New MI, Levine LS, Biglieri EG, et al. Evidence for an unidentified steroid in a child with apparent mineralocorticoid hypertension. J Clin Endocrinol Metab 44:924-933, 1977. 26. Funder JW, Myles K. Exclusion of corticosterone from epithelial mineralocorticoid receptors is insufficient for selectivity of aldosterone action: in vivo binding studies. Endocrinology 137:5264-5268, 1996.
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27. Funder JW. The role of mineralocorticoid receptor antagonists in the treatment of cardiac failure. Expert Opin Investig Drugs 12:1963-1969, 2003. 28. Funder JW. Is aldosterone bad for the heart? Trends Endocrinol Metab 15:139-142, 2004. 29. Lim P, Macdonald T, Holloway C, et al. Variation at the aldosterone synthase (CYP11B2) locus contributes to hypertension in subjects with a raised aldosterone-to-renin ratio. J Clin Endocrinol Metab 87:4398-4402, 2002. 30. Connell JM, Fraser R, MacKenzie SM, et al. The impact of polymorphisms in the gene encoding aldosterone synthase (CYP11B2) on steroid synthesis and blood pressure regulation. Mol Cell Endocrinol 217:243-247, 2004. 31. Krum H, Nolly H, Workman D, et al. Efficacy of eplerenone added to renin-angiotensin blockade in hypertensive patients. Hypertension 40:117-123, 2002. 32. Weinberger M, Roniker B, Krause S, et al. Eplerenone, a selective aldosterone blocker, in mild-to-moderate hypertension. Am J Hypertens 15:709-716, 2002. 33. Levy D, Rocha R, Funder JW. Distinguishing the antihypertensive and electrolyte effects of eplerenone. J Clin Endocrinol Metab 89:2736-2740, 2004. 34. Rocha R, Rudolph A, Frierdich G, et al. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol 283:H1802-H1810, 2002. 35. Chander P, Rocha R, Ranaudo J, et al. Aldosterone plays a pivotal role in the pathogenesis of thrombotic microangiopathy in SHRSP. J Am Soc Nephrol 14:1990-1997, 2003. 36. Brilla CG, Weber KT. Mineralocorticoid excess, dietary sodium, and myocardial fibrosis. J Lab Clin Med 120:893-901, 1992. 37. Young M, Fullerton M, Dilley R, et al. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest 93:2578-2583, 1994. 38. Rocha R, Martin-Berger C, Yang P, et al. Selective aldosterone blockade prevents angiotensin II/salt-induced vascular inflammation in the rat heart. Endocrinology 143:4828-4836, 2002.
39. Sato A, Funder JW. High glucose stimulates aldosteroneinduced hypertrophy via Type I mineralocorticoid receptors in neonatal rat cardiomyocytes. Endocrinology 137:4145-4153, 1996. 40. Zhang Q, Piston D, Goodman R. Regulation of corepressor function by nuclear NADH. Science 295:1895-1897, 2002. 41. Fjeld C, Birdsong W, Goodman R. Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor. Proc Natl Acad Sci USA 100:9202-9207, 2003. 42. Young M, Moussa L, Dilley R, et al. Early inflammatory responses in experimental cardiac hypertrophy and fibrosis: Effects of 11 beta-hydroxysteroid dehydrogenase inactivation. Endocrinology 144:1121-1125, 2003. 43. Ward MR, Kanellakis P, Ramsey D, et al. Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries. Circulation 104(4): 467-472, 2001. 44. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med 341:709-717, 1999. 45. Pitt B, Remme W, Zannad F, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348:1309-1321, 2003. 46. Mizuno Y, Yoshimura M, Yasue H, et al. Aldosterone production is activated in failing ventricle in humans. Circulation 103:72-77, 2001. 47. Tsutamoto T, Wada A, Maeda K, et al. Spironolactone inhibits the transcardiac extraction of aldosterone in patients with congestive heart failure. J Am Coll Cardiol 36:838-844, 2000. 48. Falkenstein E, Meyer C, Eisen C, et al. Full-length DNA sequence of a progesterone membrane-binding protein from porcine vascular smooth muscle cells. Biochem Biophys Res Commun 229:86-89, 1996.
Chapter 12
Aldosterone and Mineralocorticoids John W. Funder
DEFINITIONS
Regulation of Aldosterone Secretion
Aldosterone
A number of positive (e.g., adrenocorticotropic hormone [ACTH]) and negative (e.g., nitric oxide [NO]) factors have been shown to affect aldosterone secretion in a variety of experimental situations, but there is consensus that angiotensin II (Ang II) and plasma potassium concentration ([K+]) are the two major determinants of aldosterone secretion. The renin-angiotensin-aldosterone system (RAAS) has evolved to defend organ perfusion and blood pressure (BP), in response to reduced circulating volume monitored by the kidney, and to increased renal sympathetic drive. This level of integration provides a powerful counter-regulatory mechanism in situations of acute volume loss, such as major hemorrhage or massive gastrointestinal fluid and electrolyte loss. Although the primary role for aldosterone in the RAAS is usually considered to be that of volume expansion by epithelial sodium and water retention, it is now clear that aldosterone has additional sites of action. These include the amygdala, to stimulate salt appetite; the circumventricular region within the hypothalamus, to raise BP; and the vascular wall, acting as a vasoconstrictor. Ang II is commonly considered the major determinant of aldosterone secretion, but this is not necessarily the case. Mice in which the gene for angiotensinogen has been knocked out are incapable of producing Ang II in response to physiologic stimuli such as salt restriction. In an elegant series of studies, however, angiotensinogen−/− mice were shown to respond indistinguishably from wild-type in terms of elevating aldosterone in response to a low-salt diet for 2 weeks.2 When mice are placed on a low-[Na+], low[K+] diet, two things are seen. First, even in wild-type mice the aldosterone response is less than to low [Na+] alone. Second, for the first time, the angiotensinogen−/− mice no longer match the wild type in terms of aldosterone response, evidence for the importance of [K+] in the process. In the clinical context there are a number of factors that modify the evolutionary drives of catastrophic volume loss, restricted salt intake, or dietary potassium overload. Western diets are commonly sodium rich; increased sympathetic drive is similarly common, and manifests as essential hypertension. Although the dangers of diuretic-induced hypokalemia have been appropriately recognized, those of a modest and contained degree of hyperkalemia often appear exaggerated. The development of effective angiotensin-converting enzyme (ACE) inhibitors and Ang II receptor (AT1) blockers has proven of immense clinical utility. The recent development of second-generation MR antagonists, and their side-effect–free therapeutic profile, promise to add an additional dimension to the treatment of cardiovascular disorders, including hypertension (see Chapter 70).
Aldosterone is a steroid hormone produced primarily if not exclusively in the adrenal cortex. It is derived from cholesterol by sequential enzymatic reactions, including a final modification of the methyl (CH3) group at carbon 18 (C18) to produce a unique aldehyde (CHO) group, whence the name aldosterone. Aldosterone is the physiologic mineralocorticoid hormone in terrestrial vertebrates; other steroids, most notably deoxycorticosterone (DOC), can also act as mineralocorticoids, but their secretion is not regulated in such a way that they are physiologic regulators of salt and water balance.
Mineralocorticoid Mineralocorticoid is defined in effector terms, as a hormone promoting unidirectional transepithelial sodium transport. Aldosterone was isolated from fractionated bovine adrenal glands half a century ago1 on the basis of this mineralocorticoid activity, and not surprisingly its physiology has been almost exclusively described in epithelial terms. More recently, however, the definition of mineralocorticoid has had to be broadened, to accommodate physiologic actions of aldosterone on blood vessels and in the central nervous system, as detailed later in this chapter. The emerging pathophysiologic roles of mineralocorticoid receptors (MR), also dealt with toward the end of this chapter, similarly call for continuing refinement of their definition.
ALDOSTERONE STRUCTURE AND SYNTHESIS As noted in the preceding definition, aldosterone is characterized by an aldehyde group at C18. Aldehyde groups are chemically very reactive, and in solution the C18 aldehyde cyclizes with the hydroxyl (OH) group at C11 to form an 11,18 hemiacetal. In common with other adrenal steroids, aldosterone is produced by sequential enzymatic steps (side chain cleavage, 3β reduction, 21-hydroxylation) from cholesterol. Unlike other hormonal steroids, aldosterone synthesis in the adrenal gland is confined to its outermost layer, the zona glomerulosa. The final enzymatic step is catalyzed in most species by the enzyme aldosterone synthase (CYP11B2), by a multistep process with DOC as substrate; aldosterone synthase shares 11β hydroxylase activity with the closely related enzyme CYP11B1 (11β hydroxylase), responsible for the defining step in glucocorticoid (cortisol, corticosterone) synthesis. In some species (e.g., bovine) a single CYP11B enzyme appears responsible for both glucocorticoid and aldosterone synthesis, with the mechanism(s) determining zonal specificity yet to be determined.
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Aldosterone Transport and Metabolism Aldosterone can be secreted rapidly in response to elevation in Ang II or plasma [K+], and circulates in the blood loosely bound to albumin (50%-60%) and free (40%-50%). This contrasts with most other adrenal steroids, which are commonly ≥95% bound, in considerable part (and with high affinity) to corticosteroid binding globulin (CBG, transcortin). Metabolism occurs both in the liver (glucuronidation) and in the kidney (reduction) to water soluble products that are largely excreted in urine, where free aldosterone represents only ~1% of the total product. The metabolic clearance rate for aldosterone in humans of the order of 1200 L/day, equivalent to the hepatic blood flow, and consistent with the albuminbound fraction being extracted as equivalent to free in long transit time organs such as the liver.
PHYSIOLOGIC ACTIONS OF ALDOSTERONE Aldosterone was isolated on the basis of its effect on epithelial sodium transport, and this is commonly considered to be its principal physiologic role. Receptors for aldosterone were first identified in classical target tissues such as the kidney,3 and subsequently in a variety of nonepithelial tissues. In some of the latter aldosterone appears to have physiologic actions,4 whereas in others the effects are clearly pathophysiologic.5 Common to both epithelial and nonepithelial actions of aldosterone are MR and the enzyme 11β hydroxysteroid dehydrogenase type 2 (11βHSD2). Mineralocorticoid receptors are members of the steroid/thyroid/retinoid/orphan receptor family of nuclear transactivating factors, closely related to receptors for glucocorticoids (GR), androgens (AR), and progestins (PR).6 MR are unusual in that they have equivalent high affinity for aldosterone and the physiologic glucocorticoid cortisol (and corticosterone, in mice and rats); in fact, cortisol and corticosterone have >30-fold higher affinity for MR than for GR. In addition, MR are found in fish, for example, which do not secrete aldosterone, suggesting the possibility of (patho)physiologic roles for MR occupied by glucocorticoids.7 Circulating plasma levels of glucocorticoids are commonly ~1000-fold higher than those of aldosterone, and plasma-free levels ~100-fold higher. Given their equivalent affinity for MR, a time-honored question is that of the mechanism allowing aldosterone to occupy and activate MR in its physiologic target tissues. Selectivity of the target-tissue response to aldosterone is vested in the enzyme 11βHSD2, which is coexpressed at very high levels (3.5–4 × 106 molecules per renal principal cell, for example) with MR in aldosterone target tissues. 11βHSD2 converts cortisol and corticosterone to their receptor-inactive 11-keto analogs cortisone and 11-dehydrocorticosterone; aldosterone is not similarly metabolized, as its 11-OH group is protected from enzymatic attack by its cyclization to the 11,18 hemiketal.8,9 Although from clinical studies the operation of 11BHSD2 is crucial to allow aldosterone to selectivity activate target tissue MR, conversion of cortisol to cortisone is only one part of the specificity-conferring mechanism. The other action of 11βHSD2 is to stoichometrically convert NAD to NADH. This action also appears crucial in preventing glucocorticoids
from activating MR in aldosterone target tissues under normal circumstances. The NADH/redox state does play a role in inappropriate MR activation under pathophysiologic conditions, as will be discussed later. The postreceptor events following MR activation have been relatively lightly explored. In common with other members of the superfamily, MR can act as transcription factor, binding to response elements in the promoter regions of particular genes, and binding an increasing array of coregulators serving to modulate the rate of gene transcription. A variety of candidate MR-regulated genes have been reported—Na+,K+-ATPase subunits, epithelial sodium channel (ENaC) subunits, CHIF (corticosteroid hormone induced factor), GILZ (glucocorticoid induced leucine zipper) protein—of which the most thoroughly explored has been SGK-1 (serum- and glucocorticoidinduced kinase-1). SGK-1 is constitutively expressed in glomeruli and in response to aldosterone in principal cells in the distal tubule.10 SGK-1, when phosphorylated by insulin (probably inter alia), is ultimately responsible for the phosphorylation of ENaC subunits, thereby blocking ENaC internalization and thus increasing intracellular Na+. Intracellular Na+ is substrate for Na+,K+-ATPase on the basolateral surface of the cell membrane, which pumps Na+ out of the cell into the interstitial space and ultimately the blood. These postreceptor studies have been largely done in kidney, renal cell lines, and to some extent distal colon; comparable studies in nonepithelial aldosterone target tissues are currently in progress. Nonclassical aldosterone target tissues include the vasculature, the amygdala, and the A3V3 region of the hypothalamus. Vascular smooth muscle expresses both MR and 11βHSD2, and both rapid and prolonged effects of aldosterone at physiologic or near physiologic concentrations have been reported.11-13 The amygdala similarly expresses both MR and 11βHSD2, but in terms of selectivity is at a disadvantage compared with peripheral tissues in that aldosterone has a very high reflection coefficient at the blood-brain barrier. The A3V3 region lies outside the blood-brain barrier, and expresses MR but not 11βHSD2. Experimental studies in rat and dog (but not sheep) show that aldosterone clearly can raise BP by acting on MR in the A3V3 region, though the extent to which this reflects a physiologic response in humans is yet to be determined.14 Finally, it is now clear that aldosterone has rapid nongenomic effects. Initially such effects were ascribed to interaction with a membrane receptor for aldosterone, distinct from the classical MR.15 Subsequent studies in both vascular smooth muscle12 and cardiomyocytes16 have shown such rapid nongenomic effects to be mediated via classical MR; whether or not membrane-receptor mediated effects can be shown in these or other tissues remains to be systematically explored. Unlike the estrogen receptor (ER), MR do not have a myristoylation site in their sequence, and are thus unlikely to be plasma membrane located.
CLINICAL SYNDROMES There are various ways of categorizing clinical disorders, and for simplicity this section will begin with a consideration of disorders of aldosterone secretion, followed by disorders of mineralocorticoid receptor activation, with a final section on essential hypertension. Though some of the syndromes described are
Aldosterone and Mineralocorticoids
very rare, they have often been illustrative, with the pathophysiology providing insight into the normal physiology.
Aldosterone Synthase Deficiency Aldosterone deficiency may be part of a generalized hypoadrenal state, may follow deficiency in biosynthetic pathways shared with other adrenal steroids (e.g., 3βHSD, 21 hydroxylase), or may be “pure” aldosterone synthase deficiency. The condition commonly presents in infancy, and is characterized by the signs and symptoms of uncompensated sodium loss— failure to thrive, hyponatremia, hyperkalemia, hyperreninemia, and low or undetectable plasma and urinary aldosterone levels; the latter finding clearly distinguishes the syndrome from pseudohypoaldosteronism. The subject has been comprehensively reviewed.17
Glucocorticoid Remediable Aldosteronism Of more relevance to hypertension is the condition of glucocorticoid remediable aldosteronism (GRA), also known as glucocorticoid suppressible hypertension. GRA reflects the transcription of a chimeric gene, located at 8q24, which contains the 5′ end of CYP11B1 (11β hydroxylase) and the 3′ end of CYP11B2 (aldosterone synthase). Such a gene is not only transcriptionally activated by ACTH, but also is expressed throughout the adrenal cortex. The diagnosis should be suspected in patients with early onset familial hypertension, and can be confirmed or excluded by PCR for the chimeric gene. Treatment is optimally low dose (0.25-0.5 mg/day) dexamethasone. The occurrence of a chimeric CYP11B1/B2 gene reflects the two genes being located in tandem, and sharing 94% nucleotide identity, thus allowing the possibility of an unequal crossing over at meiosis.18 The most common explanation for two highly homologous genes in tandem is a relatively recent gene duplication, consistent with the relatively recent appearance of aldosterone, in terrestrial vertebrates.
Primary Aldosteronism A year after the isolation and characterization of aldosterone in 1953, Jerome Conn reported resolution of hypertension and hypokalemia in a patient after removal of an aldosteroneproducing adenoma.19 For the next 40 years primary aldosteronism was considered to be a rare (<1%) cause of hypertension, despite Conn’s estimate that up to 20% of patients with elevated BP may have primary aldosteronism. Over the past decade, thanks to the wider application of the aldosterone:renin ratio as a screening test, and adrenal venous catheterization to lateralize (or not) the source of the aldosterone, it has become clear that 8% to 15% of unselected hypertensives have autonomous aldosterone production; in a recent general practice population study, 30% of patients with moderate hypertension had elevated aldosterone: renin ratios.20 In terms of diagnosis, patients increasingly are found to have bilateral adrenal hyperplasia rather than a discrete adenoma, and are commonly normokalemic, so that hypokalemia is no longer pathognomonic. Treatment is laparoscopic adrenalectomy, or mineralocorticoid receptor blockade in those with bilateral hyperplasia or who are unsuitable for surgery.
Pseudohypoaldosteronism Although more than 20 years ago defects in MR binding of aldosterone were postulated as the cause of pseudohypoaldosteronism (PHA) from studies on affected patients’ leukocytes,21 in subsequent studies no abnormalities in gene sequence were found.22,23 This apparent conundrum has been resolved by the distinction between PHA type 1 and type 2, where one is caused by an epithelial sodium channel (ENaC) defect, and the other a defect in MR. The phenotype varies, but severe cases can be distinguished from aldosterone synthase deficiency by the often marked elevation in aldosterone as well as renin levels. Treatment is rigorous salt supplementation in infancy, with even severe cases appearing to improve with age, by mechanisms that remain poorly understood.
Pregnancy-Associated Hypertension In contrast to the inactivating MR mutations that may be found in PHA, a recently discovered point mutation24 has been shown to result in a constitutively partially activated MR, in which both progesterone and spironolactone act as agonists, rather than as antagonists as is the case for the wild-type MR. The syndrome was discovered in a young male hypertensive, whose two sisters suffered severe exacerbations of hypertension in pregnancy, presumably reflecting the agonist effect of progesterone on the mutant MR. The syndrome is rare, and abnormal MR do not appear to underlie the relatively common finding of hypertension in pregnancy.
Apparent Mineralocorticoid Excess A more common, though still comparatively rare, condition of inappropriate MR activation is that of apparent mineralocorticoid excess (AME), first described by New and Ulick in 1977.25 In this syndrome—of juvenile hypertension, sodium retention, and hypokalemia, in the presence of suppressed renin and aldosterone levels—epithelial MR are activated by cortisol, reflecting deficient activity of the specificity-conferring enzyme 11βHSD2. The finding of an elevated ratio of urinary free cortisol:cortisone is diagnostic, and patients are treated by MR blockade, on occasion with the suppression of cortisol by dexamethasone administration. Whereas previously it had been assumed that epithelial 11βHSD2 excluded glucocorticoids from MR, this has been shown not to be the case.26 The enzyme acts to debulk intracellular glucocorticoid levels, from ~100-fold those of aldosterone to ~10-fold, consistent with a role for the forgotten cosubstrate (NAD, from which NADH is generated stoichometrically) in activation of glucocorticoid-MR complexes, as briefly discussed subsequently and elsewhere in detail.27,28
Essential Hypertension Although an increasing number of patients with essential hypertension appear to have autonomous aldosterone secretion, the potential role of aldosterone, 11βHSD2, and MR in the majority of hypertensive patients remains unclear. A number of studies have linked allelic variation in 11βHSD2 or CYP11B2 with a higher incidence of elevated BP,29,30 and there have similarly been sporadic reports of a subgroup of essential hypertensives with impaired conversion of cortisol to cortisone.
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The selective MR antagonist eplerenone has been shown to be of equivalent potency to ACE inhibitors, Ca2+ channel blockers, β-blockers, or angiotensin receptor blockers (ARBs) in terms of BP reduction.31,32 In titration-to-effect studies, a wide (4- to 10-fold) dose range of eplerenone was needed in moderate hypertensives to reduce diastolic BP to <90 mm Hg, with comparable falls in BP (~16/12 mm Hg) at each dose level,33 further evidence for a significant role for aldosterone in essential hypertension.
Pathophysiology Whereas the physiologic role of aldosterone in epithelia to retain Na+ and water, and to excrete K+, are well accepted, its other physiologic roles (as a vasoconstrictor, by a direct action on vascular smooth muscle cells (VSMCs), and in the brain to stimulate salt appetite) are less well explored. In other nonepithelial tissues it is unclear what, if any, are physiologic roles for aldosterone. What is clear, however, is that aldosterone may have direct pathophysiologic effects on blood vessels and cardiomyocytes, almost certainly inter alia, in the context of inappropriate salt status.
Mineralocorticoid/Salt Imbalance In physiologic terms, aldosterone and sodium have a reciprocal relationship; when salt status falls aldosterone rises, and vice versa. When, however, aldosterone secretion is no longer responsive to normal negative feedback—in primary aldosteronism, GRA or in animals infused with aldosterone and given only 0.9% NaCl solution to drink—this reciprocal nexus is broken, and aldosterone levels are inappropriate for salt status, and vice versa. Under these circumstances very marked cerebral, renal, and coronary vascular inflammation can be shown in experimental animals,34,35,38 progressing to perivascular and interstitial cardiac fibrosis.36,37 Importantly, if infused animals are on a low-salt diet with water as drinking fluid, these changes are not seen; in the human situation of prolonged Na+ deficiency, very high levels of aldosterone coexist with no cardiac or vascular toxicity. The mechanisms involved in the deleterious synergy between aldosterone and inappropriate Na+ status are currently unclear; their clarification would constitute a major advance in cardiovascular endocrinology.
Vascular Inflammation and Cardiac Fibrosis As noted previously, inappropriate aldosterone (or other mineralocorticoid) for salt status is followed by progressive vascular inflammation in a variety of organs. Animal models used include the stroke-prone spontaneously hypertensive rat (SHR-sp), where eplerenone has been shown to be very protective of both cerebral and renal vasculature, and tissue architecture35; the aldosterone infused rat on 0.9% NaCl solution to drink, in which eplerenone is similarly protective of renal and coronary vessels, and kidney/heart architecture34,38; and the AngII infused/0.9% NaCl drinking rat, in which BP is Ang II driven, and unaffected by eplerenone or adrenalectomy.34,38 On the other hand, adrenalectomy or eplerenone administration completely reverses the vascular and perivascular inflammatory response produced by Ang II/salt, which is
restored in adrenalectomized rats by aldosterone infusion. In all these models, markers of inflammation—ED-1, MCD-1, COX-2, IL-1β, IL-6, osteopontin—increase over the first 2 to 4 weeks of study, and their levels are returned toward or to baseline by MR blockade.
MR Activation by Glucocorticoids In tissues coexpressing MR and 11βHSD2, intracellular glucocorticoid levels are 10 times those of aldosterone, and in unprotected tissues (e.g., cardiomyocytes, most neurons) levels of glucocorticoids are ~100 times higher. Under normal circumstances these glucocorticoid-MR complexes appear inactive, and the glucocorticoid appears to act in tonic inhibitory mode.5,39 In other circumstances, however, glucocorticoids act as MR agonists. The first of these is when 11βHSD2 is deficient or blocked, leading to the syndromes of AME or licorice intoxication. Under these circumstances intracellular cortisol levels rise from ~10 times those of aldosterone toward ~100 times; more importantly, however, levels of intracellular NADH fall precipitously. In other systems NADH has been shown to regulate transcription factor activity by activating corepressors.40,41 Secondly, when 11βHSD2 is blocked by administration of carbenoxolone, an identical pattern of vascular inflammation is seen as with mineralocorticoid/salt administration; importantly, these effects, presumably of glucocorticoids on MR occur when NADH levels fall as 11β hydroxysteroid dehydrogenase is blocked by carbenoxolone.42 Third, experimental angioplasty in pigs is followed by a constriction in coronary luminal diameter, a constriction blocked by eplerenone treatment.43 These animals were not receiving aldosterone or on a high-salt intake; our interpretation of these data is that under conditions of tissue damage and reactive oxygen species (ROS) generation, intracellular redox status changes—just as it does when 11βHSD2 is blocked—allowing normal levels of glucocorticoids to activate vascular MR and thus mimic the aldosterone/salt effects. In this context it should be remembered that in both Randomized Aldactone Evaluation Study (RALES)44 and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS)45 aldosterone levels were normal and salt status unremarkable. In the circumstances of heart failure—progressive or postmyocardial infarction— cardiomyocyte levels of ROS are known to be elevated. Under these circumstances, then, it would appear that spironolactone/ eplerenone are not acting primarily as aldosterone blockers, but truly as MR antagonists blocking the effects of cortisol via MR in the context of tissue damage.
FUTURE DIRECTIONS Any prognostication is necessarily speculative, and thus these will be mentioned only briefly, and in no detail.
Ectopic Aldosterone Synthesis For the past decade there have been claims—commonly but not uniquely based on PCR of steroidogenic enzymes—that aldosterone can be synthesized in neurons, vascular wall and heart. Clinical data are conflicting—aldosterone is elaborated46
Aldosterone and Mineralocorticoids
or extracted47 by the failing heart; in unpublished studies we were unable to find any consistent arteriovenous differences, in normal persons or those with severe heart failure. The Ang II/salt studies by Rocha et al.,34,38 in which adrenalectomy totally reversed the vascular inflammation, argue powerfully against paracrine secretion of cardiac aldosterone of any consequence. Finally, the low levels of enzyme expression found by PCR, commonly between 0.01% and 1% of adrenal levels, means that each step of ectopic steroid synthesis becomes ratelimiting. Unless the enzymes are concentrated in merely a few cells, their contribution to even local aldosterone concentrations is likely to be negligible.
MR-Independent Effects Any molecule that circulates at subnanomolar concentrations can only act effectively by relatively high affinity binding. Many acute nongenomic effects of aldosterone have now been shown to be mediated via classical MR, in for example VSMC12 and cardiomyocytes.16 This does not exclude the possibility of another receptor for aldosterone, distinct from the classic MR, binding aldosterone and other mineralocorticoids with high affinity (and some degree of specificity, given their low circulating concentrations). It is unlikely that this is one of the 48 members of the steroid superfamily of nuclear transactivating factors identified in the human genome; it may, for example, be an analog of the membrane receptor for progesterone.48
Therapeutic Implications If aldosterone/salt imbalance is followed by inflammatory vascular and perivascular responses, and downstream tissue damage, then MR blockade assumes a particular therapeutic importance. If cortisol can activate MR in the context of tissue damage and ROS generation, leading to further ROS generation and exacerbation of tissue damage, MR blockade should prove of utility in breaking this potentially vicious cycle in conditions in addition to those characterized by mineralocorticoid/salt imbalance. MR blockade, therefore, may prove beneficial not only in the context of obvious cardiovascular disease (atherosclerosis/hypertension/myocarditis/heart failure), but also in conditions as diverse as diabetes, cerebrovascular protection and the prevention of premature labor. These possibilities need to be critically examined at the preclinical level, and if found of interest, transferred into the arena of clinical trials.
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39. Sato A, Funder JW. High glucose stimulates aldosteroneinduced hypertrophy via Type I mineralocorticoid receptors in neonatal rat cardiomyocytes. Endocrinology 137:4145-4153, 1996. 40. Zhang Q, Piston D, Goodman R. Regulation of corepressor function by nuclear NADH. Science 295:1895-1897, 2002. 41. Fjeld C, Birdsong W, Goodman R. Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor. Proc Natl Acad Sci USA 100:9202-9207, 2003. 42. Young M, Moussa L, Dilley R, et al. Early inflammatory responses in experimental cardiac hypertrophy and fibrosis: Effects of 11 beta-hydroxysteroid dehydrogenase inactivation. Endocrinology 144:1121-1125, 2003. 43. Ward MR, Kanellakis P, Ramsey D, et al. Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries. Circulation 104(4): 467-472, 2001. 44. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med 341:709-717, 1999. 45. Pitt B, Remme W, Zannad F, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348:1309-1321, 2003. 46. Mizuno Y, Yoshimura M, Yasue H, et al. Aldosterone production is activated in failing ventricle in humans. Circulation 103:72-77, 2001. 47. Tsutamoto T, Wada A, Maeda K, et al. Spironolactone inhibits the transcardiac extraction of aldosterone in patients with congestive heart failure. J Am Coll Cardiol 36:838-844, 2000. 48. Falkenstein E, Meyer C, Eisen C, et al. Full-length DNA sequence of a progesterone membrane-binding protein from porcine vascular smooth muscle cells. Biochem Biophys Res Commun 229:86-89, 1996.