**3. Mechanisms of aldosterone-induced tissue damage**

A growing body of evidence suggests that exposure to inappropriate aldosterone levels for salt status or activation of the mineralocorticoid receptor can produce massive myocardial, vascular, and renal tissue injury with mechanisms that are independent of blood pressure (Marney & Brown, 2007). Landmark experiments demonstrated that chronic aldosterone infusion causes myocardial fibrosis in rats that are maintained on a high-salt diet. Later on, it was demonstrated that aldosterone-induced myocardial fibrosis is preceded by

stages. Also, recent evidence suggests that the benefits of aldosterone antagonists in the context of cardiac failure are not restricted to patients with impaired systolic function but can be extrapolated also to patients with diastolic dysfunction. Finally, some studies support the view that mineralocorticoid receptor blockade may exert an antialbuminuric effect in patients with proteinuria, an effect that occurs independent of blood pressure reduction.

The use of classic mineralocorticoid receptor antagonists, however, has been limited by the high incidence of breast engorgement and gynecomastia and the risk of severe hyperkalemia. To overcome these tolerance problems, new aldosterone blockers have been developed (Garthwaite & McMahon 2004) using two different strategies that include search for non-steroidal antagonists and inhibition of aldosterone synthesis. Inhibition of aldosterone synthesis could have an additional benefit due to blockade of the mineralocorticoid receptor-independent pathways that might account for some of the untoward effects of aldosterone. The new aldosterone blockers are currently having extensive preclinical evaluation, and one of them has passed phase-II trials showing promising results in patients with essential hypertension and primary aldosteronism.

More than 98% of total body potassium is located inside the cells and homeostatic control of extracellular potassium by the intracellular pool is critical in the regulation of plasma potassium concentration. Plasma potassium levels are maintained stable between 3.5 and 5.0 mEq/l despite remarkable variability in potassium intake with meals. This balance is due to mechanisms that operate principally at the renal level and regulate potassium excretion. In normal conditions, daily intake of potassium is entirely eliminated by the body, 90% by the kidney and 10% by the intestine. Therefore, changes in body potassium content are physiologically regulated by the kidney that compensates with increased reabsorption in conditions of hypokalemia, and increased secretion in conditions of hyperkalemia. Potassium transport occurs along the entire nephron, but the major role in potassium secretion is played at the distal site by the connecting tubule and cortical collecting duct. In these sites, principal cells are responsible for regulation of sodium reabsorption and via the amiloride-sensitive epithelial sodium channel (ENaC) (Figure 2) with the associated potassium and hydrogen ion excretion. Sodium entry via ENaC generates an excess of negative charges in the tubular lumen that causes intracellular potassium to leave the cell through the renal outer medullary K channel (ROMK) and the flow-sensitive maxi-K potassium channel (BK K+) (Figure 2). In addition to distal sodium reabsorption through the ENaC, potassium secretion is therefore dependent on distal tubular flow. Aldosterone has direct influence on potassium secretion, activating sodium transport through activation of the ENaC and increasing the

A growing body of evidence suggests that exposure to inappropriate aldosterone levels for salt status or activation of the mineralocorticoid receptor can produce massive myocardial, vascular, and renal tissue injury with mechanisms that are independent of blood pressure (Marney & Brown, 2007). Landmark experiments demonstrated that chronic aldosterone infusion causes myocardial fibrosis in rats that are maintained on a high-salt diet. Later on, it was demonstrated that aldosterone-induced myocardial fibrosis is preceded by

**2. Regulation of potassium excretion at the distal tubular site** 

driving force for potassium secretion into the tubular lumen (Figure 3).

**3. Mechanisms of aldosterone-induced tissue damage** 

inflammatory changes of perivascular tissue, and that both inflammation and fibrosis can be prevented by administration of mineralocorticoid receptor antagonists or adrenalectomy. Similar evidence was obtained in the kidney of uninephrectomized and stroke-prone spontaneously hypertensive rats in which aldosterone produced intrarenal vascular damage, glomerular injury, and tubulointerstitial fibrosis. Elevated aldosterone also caused aortic fibrosis and hypertrophy in different rat models of hypertension, and administration of eplerenone to hypertensive rats corrected vascular remodeling and fibrosis, suggesting a mineralocorticoid receptor-mediated mechanism.

Fig. 2. Sodium, potassium, and water transport in principal cells of connecting tubule and cortical collecting duct. ENaC: amiloride-sensitive epithelial sodium channel; ROMK: renal outer medullary K channel; BK K+: flow-sensitive maxi K channel; AQP-2: apical aquaporin; AQP-3 and 4: basolateral aquaporin.

All these studies consistently indicate that aldosterone causes tissue damage only in the context of inappropriate salt status. It was suggested that untoward effects of high-salt intake are largely dependent on activation of mineralocorticoid receptors and that this activation might reflect increased oxidative stress. Mineralocorticoid receptors are found in epithelial and nonepithelial tissues with high affinity for aldosterone and glucocorticoid hormones, such as cortisol and corticosterone. Under physiological conditions, the majority of mineralocorticoid receptors in nonepithelial tissues are occupied by greater concentrations of cortisol, whereas in epithelial tissues, binding of cortisol to receptors is prevented by 11β-hydroxysteroid dehydrogenase (11β-HSD2), the enzyme that converts cortisol to the receptor-inactive cortisone. In addition to the conversion of cortisol to cortisone, activity of 11β-HSD2 generates NADH from NAD and produces changes in the intracellular redox potential that might, in turn, inactivate the glucocorticoid–receptor complex. 11β-HSD2 is not present in nonepithelial tissues including the heart, but in such tissues, changes of the intracellular redox potential can result from generation of reactive oxygen species (ROS) and thereby affect the activity of the mineralocorticoid receptor.

Potassium-Sparing Diuretics in Hypertension 73

medullary potassium channel (ROMK), and the sodium/potassium ATPase pump. The net effect is sodium reabsorption in the bloodstream and potassium excretion in urine. Steroidal or non-steroidal mineralocorticoid receptor antagonists block the mineralocorticoid receptor activation by aldosterone. Inappropriate activation of mineralocorticoid receptors by cortisol is inhibited by its conversion to the receptor-inactive cortisone by the 11β-hydroxysteroid

In vitro experiments have demonstrated that changes in the redox potential of cardiomyocytes by exposure to oxidized glutathione turn cortisol from being a receptor antagonist to an agonist. More recently, it has been demonstrated that aldosterone itself induces changes in the intracellular redox potential in diverse cell types through an activation of the NOX1 catalytic subunit of NAD(P)H oxidase. This aldosterone-dependent change in the redox potential is amplified by exposure to high concentrations of salt leading

In fact, ROS are responsible for apoptosis of cardiomyocytes and for mesangial cell proliferation and matrix expansion in glomeruli of rats. In vascular tissues a slight increase in sodium concentration in the presence of aldosterone affects the biomechanical properties of the endothelial cells leading to cell swelling and cell stiffening (Figure 4). Both these effects are blunted by amiloride (a selective ENaC inhibitor) or spironolactone demonstrating the involvement of a mineralocorticoid receptor-dependent activation of ENaC pathway also in the vascular tissue (Figure 4). Furthermore, the analysis of gene expression profiling of vascular tissues demonstrated that, in the context of an enhanced oxidative stress, aldosterone can stimulate the expression of several pro-atherogenic genes

Thus, in addition to the well-known effects of salt loading on epithelial swelling, vascular stiffening and blood pressure increase, some effects of salt loading might depend on mineralocorticoid receptor activation and reflect, in different tissues, impairment of 11β-HSD2 activity and/or increased oxidative stress, both mechanisms possibly leading to changes in the intracellular redox state. The distinction of these effects of salt from those generated at the tissue level by elevated aldosterone is complex and even genetic manipulations could not help in the understanding of their respective roles. In fact, both cardiac overexpression of the mineralocorticoid receptor and cardiac-specific induction of aldosterone production do not cause cardiac fibrosis, whereas fibrosis results from

Although the use of mineralocorticoid receptor antagonists can inhibit or reduce aldosterone effects, several rapid actions of aldosterone on vascular tissue, such as regulation of vascular tone are, at least in part, independent of mineralocorticoid receptor blockade. Therefore, a mineralocorticoid receptor-independent pathway and the existence of a new aldosterone receptor have been hypothesized (Figure 4). Recently, it has been shown that in vascular smooth muscle aldosterone action is linked to a mineralocorticoid receptor-independent pathway that is mediated by the G protein coupled receptor (GPR30) (Figure 4). This pathway involves phosphatidylinositol 3-kinase (PI3K) and the extracellular signalregulated kinase (ERK). These findings rise new questions on the role of aldosterone-related

and mineralocorticoid receptor-independent pathways in causing tissue injury.

to increased production of ROS and thereby to cellular and tissue injury.

and this expression can be inhibited by mineralocorticoid receptor antagonists.

knockdown of the cardiac receptor by the use of antisense mRNA.

dehydrogenase (11βHSD2).

Fig. 3. Aldosterone (A) and cortisol (C) synthesis in the adrenal cortex and mechanisms of aldosterone action in the principal cells of the collecting duct. Aldosterone and cortisol originate from the metabolic conversion of cholesterol in the adrenal cortex. Finals steps of this conversion involve the cytochrome P450 enzymes CYP11B2 and CYP11B1 for the synthesis of aldosterone and cortisol, respectively. Aldosterone synthase inhibitors selectively block CYP11B2 and reduce aldosterone levels. In tubular cells, aldosterone activates the mineralocorticoid receptor and thereby induces structural and regulatory proteins that increase the activity of the epithelial sodium channel (ENaC), the renal outer

Fig. 3. Aldosterone (A) and cortisol (C) synthesis in the adrenal cortex and mechanisms of aldosterone action in the principal cells of the collecting duct. Aldosterone and cortisol originate from the metabolic conversion of cholesterol in the adrenal cortex. Finals steps of this conversion involve the cytochrome P450 enzymes CYP11B2 and CYP11B1 for the synthesis of aldosterone and cortisol, respectively. Aldosterone synthase inhibitors selectively block CYP11B2 and reduce aldosterone levels. In tubular cells, aldosterone activates the mineralocorticoid receptor and thereby induces structural and regulatory proteins that increase the activity of the epithelial sodium channel (ENaC), the renal outer

medullary potassium channel (ROMK), and the sodium/potassium ATPase pump. The net effect is sodium reabsorption in the bloodstream and potassium excretion in urine. Steroidal or non-steroidal mineralocorticoid receptor antagonists block the mineralocorticoid receptor activation by aldosterone. Inappropriate activation of mineralocorticoid receptors by cortisol is inhibited by its conversion to the receptor-inactive cortisone by the 11β-hydroxysteroid dehydrogenase (11βHSD2).

In vitro experiments have demonstrated that changes in the redox potential of cardiomyocytes by exposure to oxidized glutathione turn cortisol from being a receptor antagonist to an agonist. More recently, it has been demonstrated that aldosterone itself induces changes in the intracellular redox potential in diverse cell types through an activation of the NOX1 catalytic subunit of NAD(P)H oxidase. This aldosterone-dependent change in the redox potential is amplified by exposure to high concentrations of salt leading to increased production of ROS and thereby to cellular and tissue injury.

In fact, ROS are responsible for apoptosis of cardiomyocytes and for mesangial cell proliferation and matrix expansion in glomeruli of rats. In vascular tissues a slight increase in sodium concentration in the presence of aldosterone affects the biomechanical properties of the endothelial cells leading to cell swelling and cell stiffening (Figure 4). Both these effects are blunted by amiloride (a selective ENaC inhibitor) or spironolactone demonstrating the involvement of a mineralocorticoid receptor-dependent activation of ENaC pathway also in the vascular tissue (Figure 4). Furthermore, the analysis of gene expression profiling of vascular tissues demonstrated that, in the context of an enhanced oxidative stress, aldosterone can stimulate the expression of several pro-atherogenic genes and this expression can be inhibited by mineralocorticoid receptor antagonists.

Thus, in addition to the well-known effects of salt loading on epithelial swelling, vascular stiffening and blood pressure increase, some effects of salt loading might depend on mineralocorticoid receptor activation and reflect, in different tissues, impairment of 11β-HSD2 activity and/or increased oxidative stress, both mechanisms possibly leading to changes in the intracellular redox state. The distinction of these effects of salt from those generated at the tissue level by elevated aldosterone is complex and even genetic manipulations could not help in the understanding of their respective roles. In fact, both cardiac overexpression of the mineralocorticoid receptor and cardiac-specific induction of aldosterone production do not cause cardiac fibrosis, whereas fibrosis results from knockdown of the cardiac receptor by the use of antisense mRNA.

Although the use of mineralocorticoid receptor antagonists can inhibit or reduce aldosterone effects, several rapid actions of aldosterone on vascular tissue, such as regulation of vascular tone are, at least in part, independent of mineralocorticoid receptor blockade. Therefore, a mineralocorticoid receptor-independent pathway and the existence of a new aldosterone receptor have been hypothesized (Figure 4). Recently, it has been shown that in vascular smooth muscle aldosterone action is linked to a mineralocorticoid receptor-independent pathway that is mediated by the G protein coupled receptor (GPR30) (Figure 4). This pathway involves phosphatidylinositol 3-kinase (PI3K) and the extracellular signalregulated kinase (ERK). These findings rise new questions on the role of aldosterone-related and mineralocorticoid receptor-independent pathways in causing tissue injury.

Potassium-Sparing Diuretics in Hypertension 75

the first oral dose. Spironolactone is moderately more potent than eplerenone in blocking mineralocorticoid receptors. Spironolactone remains active when renal function is impaired because it reaches its site of action independent of glomerular filtration. This accounts for the risk of hyperkalemia observed in patients with chronic kidney disease and in patients

The recommended oral dosing range of spironolactone is from 12.5 to 250 mg once or twice a day in primary hypertension and other disease conditions in which the use of this agent is

Spironolactone is a medication that has been used for more than 50 years to treat hypertension, edema, primary aldosteronism, and, more recent evidence indicates benefits

Almost a decade ago, a landmark trial investigated the effects of spironolactone in patients with functional class III-IV systolic heart failure, showing a significant decrease in the mortality rate as compared to patients who received placebo on top of conventional treatment. The Randomized Aldactone Evaluation Study (RALES) (Pitt et al., 1999) was conducted in patients with New York Heart Association (NYHA) class III-IV heart failure who were treated with spironolactone. More recently, it has been suggested that the benefits of spironolactone in the context of cardiac failure are not restricted to patients with impaired systolic function and some studies have tested the effects of spironolactone in patients with heart failure and preserved systolic function (HFPSF). Edwards et al. reported improved diastolic function parameters with use of spironolactone in 112 patients with stage 2-3 chronic renal failure and HFPSF who were included in the Chronic Renal Impairment in Birmingham (CRIB II) study. In this study, the effects of spironolactone on left ventricular function and circulating markers of collagen turnover were compared with those of placebo. After 40 weeks, spironolactone improved significantly markers of left ventricular relaxation and attenuated significantly the increase in aminoterminal propeptide of type-III procollagen that was observed with placebo. This and other studies on HFPSF suggest a possible benefit of spironolactone also on this subtype of cardiac insufficiency. Notably, all these studies have employed doses of spironolactone (from 25 to 50 mg/day) that did not lower blood pressure suggesting that the cardioprotective effects of spironolactone occurs independent of the blood pressure-related hemodynamic load to the heart. Taken together, the findings obtained in the studies that have tested the effects of spironolactone in heart

failure provide indirect evidence of untoward effect of aldosterone on the heart.

Many studies have reported a beneficial effect of blockers of the renin-angiotensinaldosterone system in slowing down progression of renal disease, but the relative contributions of angiotensin II versus aldosterone have been dissociated only recently in animal studies. Clinical studies have supported the view that mineralocorticoid receptor blockade may exert an antialbuminuric effect in patients with proteinuria. In patients with diabetic nephropathy, it was shown that the antiproteinuric effect of angiotensin-converting enzyme (ACE) inhibitors reverts to baseline in patients who manifest aldosterone escape

with congestive heart failure and impaired renal function.

also in patients with congestive heart failure and proteinuria.

**4.2 Dosing** 

indicated.

**4.3 Clinical use** 

Fig. 4. Effect of aldosterone (A) on blood vessels. Aldosterone binds to mineralocorticoid receptors (MR) increasing generation of reactive oxygen species (ROS) and induces endothelial cell swelling and stiffening via a mineralocorticoid receptor-dependent pathway involving the NADPH oxidase and epithelial sodium channel, respectively. Both effects are blocked by mineralocorticoid receptor antagonists. Increased oxidative stress turns cortisol (C) to be an agonist rather than an antagonist of mineralocorticoid receptor. Aldosterone might exert its deleterious cardiovascular effects also through a mineralocorticoid receptor-independent pathway involving the G protein coupled receptor (GPR30) that can induce vascular smooth muscle cell apoptosis and inappropriate vasoconstriction. The pathophysiological role of these "non-genomic" effects of aldosterone needs to be further explored.
