ΥΠΟΔΟΧΕΑΣ ΑΛΑΤΟΚΟΡΤΙΚΟΕΙΔΩΝ ΥΠΟΔΟΧΕΑΣ ΑΛΑΤΟΚΟΡΤΙΚΟΕΙΔΩΝ ΧΑΡΑΚΤΗΡΙΣΤΙΚΑ & ΠΛΕΙΟΤΡΟΠΕΣ ΔΡΑΣΕΙΣ 23 Ιανουαρίου 2014 Κωνσταντίνος Π. Μακαρίτσης Παθολογική Κλινική Πανεπιστημίου Θεσσαλίας 2

ΕΙΣΑΓΩΓΗ Η αλδοστερόνη απομονώθηκε για πρώτη φορά το 1953 και παράγεται στη σπειροειδή ζώνη του φλοιού των επινεφριδίων. Μείζονα ερεθίσματα για έκκριση αλδοστερόνης: Αγγειοτενσίνη ΙΙ Επίπεδα του Κ+ του πλάσματος Η αλδοστερόνη αυξάνει την επαναρρόφηση Να+ και ύδατος από το τελικό άπω εσπειραμένο και τη φλοιώδη μοίρα του αθροιστικού σωληναρίου του νεφρού.

NCCT ENaC Aldosterone NHEs NKCC2 Figure 1. Sites of Diuretic Action in the Nephron. The percentage of sodium reabsorbed in a given region is indicated in parentheses. “K+-sparing agents” collectively refers to the epithelial sodiumchannel inhibitors (e.g., amiloride and triamterene) and mineralocorticoidreceptor antagonists (e.g., spironolactone and eplerenone). Sodium is reabsorbed in the distal tubule and collecting ducts through an aldosterone- sensitive sodium channel and by activation of an ATP-dependent sodium– potassium pump. Through both mechanisms, potassium is secreted into the lumen to preserve electroneutrality. Sodium-channel inhibitors preserve potassium by interfering with the sodium–potassium pump, whereas mineralocorticoid-receptor antagonists spare potassium through their inhibitory effect on aldosterone. NaHCO3 denotes sodium bicarbonate. N Engl J Med 4

Sodium Channels and Transporters Approximately 2-3% of filtered sodium is reabsorbed in the cortical collecting tubule via the epithelial Na channel (ENaC). ENaC is composed of 3 subunits, α, β, γ. All 3 subunits are required for a fully functional channel.

Aldosterone-Regulated Transport - Cortical Collecting Tubule 6

Aldosterone-Regulated Transport - Cortical Collecting Tubule X X Spironolactone Eplerenone Amiloride Triamterene N Engl J Med 1999;340:1177-87. 7

Sodium Channels and Transporters Regulation of Na reabsorption depends on the number of channels inserted in the cell membrane. Vasopressin (via increased cAMP) and aldosterone (via serum and glucocorticoid-regulated kinase [SGK]) increase the density of channels at the cell surface.

Regulation of ENaC membrane expression α β γ Insulin MR 9

Mineralocorticoid Receptor-MR Στοιχεία Παθοφυσιολογίας

Στοιχεία Παθοφυσιολογίας Η συγκέντρωση της αλδοστερόνης στο πλάσμα είναι πολύ μικρή (<1nmol/L) και κυκλοφορεί συνδεδεμένη με την αλβουμίνη σε ποσοστό περίπου 50%. Αντιθέτως, τα φυσικά γλυκοκορτικοειδή – κορτιζόλη και κορτικοστερόνη – κυκλοφορούν συνδεδεμένα με την τρανσκορτίνη (CBG) και την αλβουμίνη σε ποσοστό περίπου 95% Τα επίπεδα της κορτιζόλης στο πλάσμα είναι από 100-1000 φορές υψηλότερα από τα επίπεδα της αλδοστερόνης.

Στοιχεία Παθοφυσιολογίας Ο Mineralocorticoid Receptor-MR είναι μέλος της οικογένειας των πυρηνικών υποδοχέων των στεροειδών/θυρεοειδικών/ρετινοϊκών/λιπιδικών/ ”ορφανών” υποδοχέων, που απαρτίζεται από 49 μέλη στον άνθρωπο. Ο MR μεταβάλλει την έκφραση συγκεκριμένων γονιδίων, αλλά έχει δειχθεί ότι συμμετέχει και στις καλούμενες ταχείες μη γονιδιωματικές δράσεις (rapid non-genomic effects). Hypertension. 2011;57:1019-1025.

Aldosterone signaling PI3K Rapid non-Genomic Effects Hypothetical model describing membrane-initiated rapid aldosterone signaling. Classic mineralocorticoid receptor (MR) is translocated to and associated with the membrane in a signaling complex including striatin, caveolin (Cav) 1, src, and the epidermal growth factor receptor (EGF-R). Similar associations have been described for estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR). On aldosterone stimulation, MR is released from the membrane-signaling complex and EGF-R is transactivated by aldosterone, initiating c-src–mediated mitogen-activated protein kinase (MAPK) activation. In addition, G protein–coupled receptor (GPR) 30 may function as aldosterone receptor mediating rapid MR-independent signaling. Genomic Effects Hypertension. 2011;57:1019-1025. 13

Στοιχεία Παθοφυσιολογίας Ο MR εκφράζεται στα επιθηλιακά κύτταρα νεφρού κατιόντος κόλου σιελογόνων και ιδρωτοποιών αδένων Ωστόσο, ο MR έχει εντοπιστεί και σε μη-επιθηλιακά κύτταρα ιπποκάμπου καρδιάς(καρδιακά μυϊκά κύτταρα, ενδοθηλιακά,ινοβλάστες, μακροφάγα) αγγείων (ενδοθηλιακά και λεία μυϊκά κύτταρα)

Mineralocorticoid Receptor-MR Molecular and Cellular Endocrinology 350 (2012) 289–298.

Mineralocorticoid Receptor-MR Η απάντηση στο ερώτημα αυτό προέκυψε με την ανακάλυψη του ρόλου του ενζύμου 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2), το οποίο εκφράζεται σε υψηλές συγκεντρώσεις μαζί με τον MR στα επιθηλιακά κύτταρα, αλλά και στο τοίχωμα των αγγείων και στον πυρήνα της μονήρους δεσμίδας (NTS). Το ένζυμο αυτό (11βHSD2) καταλύει τη μετατροπή της κορτιζόλης σε κορτιζόνη, ενώ δεν επηρεάζει την αλδοστερόνη. Η κορτιζόνη δεν ενεργοποιεί τον MR, οπότε διευκολύνεται η επίδραση της αλδοστερόνης στον MR. Molecular and Cellular Endocrinology 350 (2012) 289–298.

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GRA 11β-HSD2* SAME Syndrome Glycyrrhizic acid Carvenoxolone CHIMERIC GENE GRA Congenital Adrenal Hyperplasia 11β-HSD2* * 11β-Hydroxy Steroid Dehydrogenase2 SAME Syndrome Glycyrrhizic acid Carvenoxolone 18

Mineralocorticoid Receptor-MR Hypertens Res 2004; 27: 781–789.

Mineralocorticoid Receptor-MR It has been subsequently shown that under normal conditions most epithelial MRs are occupied (~ 90%), but not activated by normal levels of endogenous glucocorticoids. MR–glucocorticoid complexes are presumably held inactive under normal conditions by the obligate co-generation of high levels of NADH, shown to be an inhibitor of transcription by co-repressor activation in other transcriptional systems. Biochimica et Biophysica Acta 1802 (2010) 1188–1192.

Mineralocorticoid Receptor-MR Under conditions of tissue damage, reactive oxygen species generation and intracellular redox change, cortisol becomes a mineralocorticoid receptor agonist, in the vessel wall and heart, mimicking the deleterious effects of elevated aldosterone inappropriate for salt status. Biochimica et Biophysica Acta 1802 (2010) 1188–1192.

MR and Evolution Aldosterone is postulated to have played a key role in the phylogenic transition from aquatic fishes to terrestrial tetrapods, given its major epithelial effects on sodium retention and potassium excretion. Thus, the aldosterone/MR pathway enabled animals to retain sodium in the body to sustain life on land, where there was little salt. In our modern industrialized societies, however, an abundance of salt and a pandemic of obesity synergistically cause inappropriate activation of the aldosterone/MR system, that causes salt-sensitive hypertension and cardiorenal disease. Hypertension 2010;55:813-818.

Phylogenetic perspectives on the aldosterone/MR system Phylogenetic perspectives on the aldosterone/ MR system. a Ald/MR, a regulator of renal Na reabsorption, played an important role in the transition from aquatic fishes to terrestrial tetrapods. b In modern society, obesity and salt cause Ald/MR overactivation, leading to disease CVD Clin Exp Nephrol (2010) 14:303–314.

Effects of Aldosterone in Relation to Sodium Status High levels of aldosterone in response to dietary salt restriction, promotes renal sodium conservation, but has no cardiovascular consequences. When aldosterone is produced in inappropriate amounts for the level of sodium status, it results in excessive renal sodium retention, potassium wasting, hypertension, and cardiovascular damage. N Engl J Med. 2004;351:8-10.

Physiologic and Pathophysiologic Effects of Aldosterone on the Kidney and Heart in Relation to Dietary Salt levels High Low N Engl J Med. 2004;351:8-10.

Is aldosterone a cardiovascular risk factor? In primary aldosteronism and chronic high salt intake, aldosterone levels are inappropriate high for sodium status and aldosterone is clearly a cardiovascular risk factor. In essential hypertension and heart failure it might be cortisol which activates mineralocorticoid receptors. Thus, mineralocorticoid receptor activation, not aldosterone, is the risk factor. Biochimica et Biophysica Acta 1802 (2010) 1188–1192.

Increased MR Activation Deleterious actions of Increased MR Activation Increased MR Activation Cardiology in Review 2005;13:118–124.

MR, Aldosterone and Blood Pressure

MR, Aldosterone and blood pressure Aldosterone secretion is raised in response to sodium deficiency. Secretion of endogenous ouabain is raised in response to sodium loading. The role of aldosterone is to retain sodium in the face of chronic deficiency. The role of endogenous ouabain is to excrete sodium, via a pressure natriuresis effect. Endogenous ouabain increases blood pressure.

MR, Aldosterone and blood pressure Aldosterone secretion is raised in response to sodium deficiency. Secretion of endogenous ouabain is raised in response to sodium loading. The role of aldosterone is to retain sodium in the face of chronic deficiency. The role of endogenous ouabain is to excrete sodium, via a pressure natriuresis effect. Endogenous ouabain increases blood pressure.

MR, Aldosterone and blood pressure It is possible that the blood pressure elevating effects of aldosterone reflect not only direct effects on the vessel wall but also sodium retention with the resultant elevation of endogenous ouabain secretion. The combined elevation of aldosterone and endogenous ouabain levels in response to salt/mineralocorticoid imbalance may thus be an explanation of the hypertension produced.

Pathways of salt-sensitive hypertension Ouabain NCX1 ___ Nature Medicine, Nov 2004

Figure 3. Molecular Pathways Implicated in the Generation of Increased Arterial and Arteriolar Smooth-Muscle Tone by an Excess of Sodium and a Deficit of Potassium in Primary Hypertension. Solid arrows indicate an increase or stimulation, and broken arrows indicate a decrease or inhibition. The inhibition of the sodium pump and the resulting stimulation of the sodium–calcium exchanger type 1 (NCX1) increase the intracellular concentration of calcium that in turn triggers actin–myosin interaction and stimulation of vascular contraction. Na+i denotes intracellular sodium concentration, K+i intracellular potassium concentration, Ca2+i intracellular calcium concentration, Vm membrane potential, and RyR ryanodine-receptor calcium channel. PST 2238 (rostafuroxin) antagonizes the effect of digitalis-like factor on the sodium pump. SEA-0400 is a specific inhibitor of the bidirectional NCX1 preferentially blocking the calcium influx pathway. ___ Ouabain N Engl J Med. 2007;356:1966-78. Aldosterone

MR in vascular constriction and relaxation

MR in vascular constriction and relaxation In all studies, the effects of Aldo are MR-dependent, implicating vascular MR in direct regulation of vascular tone. MR activation in vascular SMC and EC increases ROS and decreases bioavailable NO and thus would be expected to promote VSMC contraction by decreasing GC activity. Molecular and Cellular Endocrinology 350 (2012) 256–265. British Journal of Pharmacology (2011) 163 1163–1169.

MR in vascular constriction and relaxation Interestingly, when Aldo is infused into vessels intraluminally to target the endothelium a vasodilator response was found, that required the presence of the endothelium, MR, and NO generation via NOS. Co-incubation with NOS inhibitors resulted in a loss of vasodilation and/or enhanced contraction, again implicating endothelial MR in vasodilation and SMC MR in vasoconstriction. Molecular and Cellular Endocrinology 350 (2012) 256–265. British Journal of Pharmacology (2011) 163 1163–1169.

MR in vascular constriction and relaxation The effects of MR activation on vascular reactivity in “healthy” humans also remains somewhat controversial due to conflicting results from clinical studies with many demonstrating a constrictive response and some showing vascular relaxation. The discrepancies may be due to differences in the vascular health of the study participants in addition to differences in dose and duration of Aldo infusion. Molecular and Cellular Endocrinology 350 (2012) 256–265. British Journal of Pharmacology (2011) 163 1163–1169.

MR in vascular constriction and relaxation When patients with underlying cardiovascular diseases are studied, including patients with atherosclerosis, heart failure, and hypertension, the data are quite consistent with MR-activation promoting increased systemic vascular resistance and reduced forearm blood flow. Molecular and Cellular Endocrinology 350 (2012) 256–265. British Journal of Pharmacology (2011) 163 1163–1169.

MR in vascular constriction and relaxation

MR in vascular constriction and relaxation Taken together, these data support that in healthy vessels, acute MR activation may evoke endothelium - dependent, NO - mediated vasodilation while, in the presence of endothelial dysfunction, vascular injury, or high vascular oxidative stress (as in patients with cardiovascular risk factors), MR activation promotes vasoconstriction. Molecular and Cellular Endocrinology 350 (2012) 256–265. British Journal of Pharmacology (2011) 163 1163–1169.

MR and Vascular Oxidative Stress

MR, Aldosterone and Vascular Oxidative Stress The interaction of ROS with NO also decreases the bioavailability of NO resulting in impaired EC-dependent vasorelaxation and the peroxinitrite formed can directly alter many vascular cell functions. Aldosterone also produces oxidative stress and endothelial dysfunction by decreasing the expression of G6PD, which reduces NADP+ to NADPH. Molecular and Cellular Endocrinology 350 (2012) 256–265. Clinical Science (2007) 113, 267–278.

MR, Aldosterone and Vascular Oxidative Stress peroxinitrite Molecular and Cellular Endocrinology 350 (2012) 256–265. Clinical Science (2007) 113, 267–278.

MR, Aldosterone and Vascular Oxidative Stress

MR and Vascular Inflammation

MR, Aldosterone and Vascular Inflammation Direct activation of MR has been shown to promote inflammatory gene expression. MR activation promotes expression of: adhesion molecules ICAM1 and VCAM1 interleukin-16 cytotoxic T-lymphocyte-ass. Protein 4 Infusion of Aldo increased circulating IL-6 Treatment with spironolactone reduced MCP-1 and PAI-1 levels Molecular and Cellular Endocrinology 350 (2012) 256–265.

MR, Aldosterone and Vascular Inflammation Vascular MR activation participates in the inflammatory response by up-regulating adhesion molecules, chemokines, cytokines, and growth factors that promote the recruitment and activation of inflammatory cells. Molecular and Cellular Endocrinology 350 (2012) 256–265.

MR and Vascular Remodeling

MR, Aldosterone and Vascular Remodeling Multiple animal models support that Aldo exacerbates vascular remodeling in association with endothelial damage in vivo and these effects are reversed by MR antagonists. Human studies have shown that patients with primary aldosteronism have significantly increased vascular medial thickness and narrowed vessel lumens compared to patients with similar degrees of essential hypertension and other forms of secondary hypertension. Molecular and Cellular Endocrinology 350 (2012) 256–265.

MR, Aldosterone and Vascular Remodeling Molecular and Cellular Endocrinology 350 (2012) 256–265. 50

Mineralocorticoid receptors in vascular dysfunction and disease Molecular and Cellular Endocrinology 350 (2012) 256–265.

MR and Myocardial Remodeling

MR, Aldosterone and Myocardial Remodeling Cardiac tissue remodeling is characterised by: Accumulation of collagen fibers types I & III Cardiomyocyte hypertrophy Fibroblast proliferation Remodeling of the structural electrical coupling components of the myocardium Molecular and Cellular Endocrinology 350 (2012) 248–255.

MR, Aldosterone and Myocardial Remodeling It is widely accepted that collagen synthesis is stimulated by a number of signaling molecules, including: Cytokines (IL-13, IL-21, TGF-b1) Chemokines (MCP-1 and MIP-1b) VEGF Osteopontin PAI-1 Endothelin-1 Molecular and Cellular Endocrinology 350 (2012) 248–255.

MR, Aldosterone and Myocardial Remodeling Numerous studies have shown that, in the presence of a high salt diet, aldosterone increases interstitial and perivascular cardiac fibrosis. Conversely, aldosterone-infused rats on a low salt diet did not. The cardiac response to aldosterone is a direct, MR-dependent response, that is independent of the effect on blood pressure and the circulating and tissue RAS. Molecular and Cellular Endocrinology 350 (2012) 248–255.

Aldosterone effect on the expression of profibrotic factors Clinical Science (2007) 113, 267–278.

Macrophage MR are critical for the activation of tissue macrophages and the onset of fibrosis whereas vascular MR (endothelial cell and vascular smooth muscle cell, VSMC) and macrophage MR contribute to the increased systolic blood pressure response. Molecular and Cellular Endocrinology 350 (2012) 248–255.

MR and Heart Failure

MR, Aldosterone and Heart Failure Consistent with experimental studies, several clinical trials (RALES, EPHESUS, EMPHASIS-HF), have demonstrated a reduced mortality and morbidity when MR antagonists are included in the treatment of moderate–severe heart failure. The guidelines of American (ACC/AHA) and of the European (ESC) Societies of Cardiology recommend ACE inhibitors and beta-blockers as class I indication, then angiotensin receptor antagonists (ARBs) and MRAs. Molecular and Cellular Endocrinology 350 (2012) 266–272.

Characteristics of studies with MR antagonists Curr Heart Fail Rep (2011) 8:7–13. 61

Eur J Clin Invest 2012 DOI: 10.1111/j. 1365-2362.2012.02676.x

MR, Aldosterone and Heart Failure Available evidence favors addition of MRA as the next step in heart failure on ACE inhibitor therapy rather than an ARB. In two recent meta-analyses, mortality was reduced by 25% (P=0.00001) with the addition of MRA vs. to no significance with added ARB. Thus, the data in aggregate (and cost) seem to favor the addition of MRAs over ARBs. However, despite the clear benefit of MRAs in several classes of HF they remain underused. Molecular and Cellular Endocrinology 350 (2012) 266–272.

MR and Kidney Disease

MR, Aldosterone and Chronic Kidney Disease In 1996 a landmark study by Greene et al. reported that the protective effects of ACEI and ARB in renal ablation model are reversed by exogenous aldosterone infusion, clearly demonstrating that aldosterone plays a major role in causing kidney injury independent of angiotensin II. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Chronic Kidney Disease Previous studies have shown the presence of 11bHSD2 in the glomeruli and cultured podocytes, implying that aldosterone can directly modulate the glomerular cell function through MR. Aldosterone/salt-treated animals exhibit heavy proteinuria because of severe glomerular injury resulting in glomerulosclerosis. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Chronic Kidney Disease Almost all renal parenchyma are affected Vasculature Glomeruli Tubulointerstitium Renal vascular changes significantly contribute Transmural fibrinoid necrosis Intimal thickening Adventitial fibrosis Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Chronic Kidney Disease Aldosterone causes glomerular injury, especially in podocytes that serve as the key filtration barrier in the glomeruli. Decreased glomerular expression of nephrin podocin Increased glomerular expression of desmin, a marker for podocyte damage In addition, these changes were almost completely prevented by the coadministration of eplerenone. Molecular and Cellular Endocrinology 350 (2012) 273–280. Hypertension 2007;49:355–364.

Involvement of podocyte damage in the renal dysfunction of aldosterone/salt-treated rats 24h Urinary Protein Hypertension 2007;49:355–364.

Mechanisms of aldosterone/MR-induced kidney injury Taken together, aldosterone can exert its effects on non-aldosterone-sensitive distal nephron (ASDN) cells, including podocytes, mesangial cells and renal fibroblasts, and the injurious actions of aldosterone on kidney function can be at least in part attributable to these cellular effects (Fig. 2). Molecular and Cellular Endocrinology 350 (2012) 273–280. 70

MR blockade and Chronic Kidney Disease Although hyperkalemia limits its use in renal insufficiency, accumulating data indicate that MR blockade can confer renoprotection. Clinical studies involving relatively small numbers of subjects reported that MR blockade effectively reduces proteinuria in subjects with hypertension, diabetes, and chronic kidney diseases. Other studies have shown that the combination of an ACEI with spironolactone decreases albuminuria more than the combination of an ACEI with an ARB. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR and the Metabolic Syndrome

MR, Aldosterone and Metabolic Syndrome Plasma aldosterone in women correlated directly with visceral adipose tissue, and higher plasma aldosterone values have also been reported in patients with metabolic syndrome, which is independent of plasma renin activity. Accumulating studies have elucidated the close relationship between aldosterone and obesity. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Metabolic Syndrome The adipose tissue is an endocrine organ that secretes a variety of adipokines. Adipocytes are capable of stimulating adrenal aldosterone synthesis through the secretion of potent aldosterone-releasing factors (ARFs), which are not yet identified. Nonetheless, the adipose tissue does not express 11βHSD2, and MR in adipocytes are predominantly occupied by glucocorticoids which have an essential function in adipocytes. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Metabolic Syndrome Aldosterone-releasing factors (ARFs) Cross-talk between adipocyte and adrenocortical cell. Aldosterone (black) and cortisol (white) secreted by the adrenocortical cell, bind and activate adipocyte MR, which controls transcription of several target genes, such as PPARc, C/EBPa, IL-6, MCP-1, PAI-1, TNF-a, involved in white adipocyte differentiation and inflammation. Moreover, MR activation up-regulates the expression of 11bHSD1, which amplifies local availability of glucocorticoids. On the other hand, unknown factors derived from adipocytes directly stimulate aldosterone secretion by the adrenal gland, in addition to the canonical control by RAAS activation. Molecular and Cellular Endocrinology 350 (2012) 281–288. 75

MR, Aldosterone and Metabolic Syndrome There is a worse control of BP in obese than lean hypertensives, which can also be related to excessive aldosterone. Aldosterone overproduction is an important cause of resistant hypertension and MR blockade has been shown to effectively reduce BP in such patients. Molecular and Cellular Endocrinology 350 (2012) 273–280.

MR, Aldosterone and Metabolic Syndrome Adipocyte type 1 Hypertension 2010;55:813-818.

Summary - MR and CardioRenal Disease For 50 years aldosterone has been thought to act primarily on the renal epithelia to regulate fluid and electrolyte homeostasis. The discovery in the 1980s that aldosterone had a range of extrarenal MR receptors and actions, especially in the heart and blood vessels, has certainly renewed interest in the field of MR antagonists. Further studies will provide a clear understanding of the mechanisms of the MRAs beneficial effects in CV and Renal disease. Molecular and Cellular Endocrinology 350 (2012) 266–272.

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A hypothetical model of MR pathway activation in type 2 metabolic syndrome (no hyperaldosteronism) Metabolic syndrome type 2 A hypothetical model of aldosterone/MR pathway activation in the development of chronic kidney disease (CKD) and cardiovascular disease (CVD) in salt-sensitive hypertension and metabolic syndrome. Molecular and Cellular Endocrinology 350 (2012) 273–280. Hypertension 2010;55:813-818. 81