Mineralocorticoid receptor antagonists in cardiovascular translational biology

This review examines the role of mineralocorticoid receptor antagonists (MRAs) in cardiovascular biology and the molecular mechanisms involved in mineralocorticoid receptor antagonism. The data discussed suggest that MRAs can play an important role in decreasing the impact of inflammation and fibrosis on cardiorenal outcomes. Evidence derived from major randomized clinical trials demonstrates that steroidal MRAs reduce mortality in patients with heart failure and reduced ejection fraction. Initial positive findings observed in patients with chronic kidney disease and type 2 diabetes (T2D) indicate the possible mechanisms of action of nonsteroidal MRAs, and the clinical benefits for patients with cardiorenal disease and T2D. This article supports the application of basic science concepts to expand our understanding of the molecular mechanisms of action involved in pathophysiology. This approach encourages the development of treatment options before diseases clinically manifest. Video Abstract: http://links.lww.com/CAEN/A42


Introduction
Fibrosis is a major component of ventricular remodeling observed in patients with heart failure [1], and targeting fibrosis is an important therapeutic approach in heart failure management.However, there are limited treatment options for addressing fibrosis and inflammation associated with cardiovascular diseases (CVDs).Over the past two decades, research in this area has targeted specific disease phenotypes, such as hypertension, heart-kidney-liver failure and aging-related vascular stiffness.Endothelial dysfunction [2] and inflammation [3] are key underlying pathologies in CVDs, where inappropriate mineralocorticoid receptor activation plays a significant role.Together with oxidative stress and impaired cardiac calcium signaling, these consequences of mineralocorticoid receptor activation have been shown in preclinical studies to be attenuated with mineralocorticoid receptor antagonists (MRAs).
Although the mechanisms of action of MRAs are not fully understood, first-generation MRAs such as spironolactone [4,5] and eplerenone [6] demonstrated positive clinical benefits in patients with heart failure in the RALES [5], EPHESUS [7] and EMPHASIS-HF [8] trials, including reductions in hospitalizations for heart failure [5,7,8] and in all-cause mortality.Results from the more recent FIDELIO-DKD [9] and FIGARO-DKD [10] trials investigating the nonsteroidal MRA finerenone, have shown consistent cardiovascular improvements in patients with type 2 diabetes (T2D) and chronic kidney disease (CKD).
The aim of this review is to discuss the role of MRAs in CVDs, considering their interaction with the mineralocorticoid receptor when it becomes overactivated.As such, this article will explore how the binding abilities of nonsteroidal MRAs, such as finerenone [9,[11][12][13][14] and esaxerenone [15,16], impact selectivity and ability to bind to the mineralocorticoid receptor compared with those of steroidal MRAs.Evidence supporting a lower risk of hyperkalemia with nonsteroidal MRAs compared with steroidal MRAs will also be discussed [9,11,12].
Eplerenone and spironolactone are currently approved by the US Food and Drug Administration (FDA) as fourth-line treatments for heart failure and will be examined alongside finerenone, the only nonsteroidal MRA currently approved by the FDA [17] and the European Medicines Agency (EMA) [18].Other novel nonsteroidal MRAs [19][20][21] in earlier stages of clinical development will also be evaluated, noting key differences between the two classes of MRAs.

Molecular basis of mineralocorticoid receptor being a driver of cardiac remodeling
Our understanding of the role of the mineralocorticoid receptor and its main ligand, aldosterone, in CVD has evolved over the past 30 years.As research has advanced, so has our knowledge of the role of aldosterone.It is one of the physiologic ligands for mineralocorticoid receptor and raises blood pressure primarily by affecting the kidney vasculature and central nervous system.Aldosterone is the main mineralocorticoid hormone.It serves a fundamental role in the control of extracellular volume homeostasis by stimulating renal sodium reabsorption and potassium excretion [22].
Aldosterone acts on the mineralocorticoid receptor in the epithelial cells of the kidney [22].However, the mineralocorticoid receptor is also expressed in numerous cell types and tissues, including the heart and vascular tissues [23], and is a driver of cardiac remodeling [22].Vascular tissues express 11-beta-hydroxysteroid dehydrogenase 2, whereas the heart does not [24,25].Glucocorticoids and aldosterone can activate the mineralocorticoid receptor in the heart (Fig. 1) [23,26].Mineralocorticoid receptor activation impacts cardiovascular regulation as well as metabolic function through mechanisms such as oxidative stress, inflammation, interstitial fibrosis and endothelial dysfunction [26].
Fibrosis is a critical aspect of tissue repair that arises after tissue injury [27] and can be viewed as a pathologic aspect of most chronic inflammatory diseases [28].Fibrosis is defined by the buildup of excess extracellular matrix components, such as collagen and fibronectin [28].In nonpathological tissue repair, accumulation of connective tissue through fibrosis maintains tissue architecture [27,28].Progressive fibrosis, however, indicates disease and leads to scarring, function impairment and organ damage [27], and can affect all tissues [28] including the myocardium, arteries, kidney and liver (Fig. 1).There is increasing evidence that overactivation of the mineralocorticoid receptor encourages inflammation and fibrosis, impacting the progression of CKD and CVD [29] (Fig. 1), which can affect patient morbidity and mortality associated with these diseases [30].
The culmination of mineralocorticoid receptor activation in various cell types [vascular smooth muscle cells (VSMCs), endothelial cells, macrophages, cardiomyocytes] contributes to the development of cardiac diastolic dysfunction and heart failure [23].In endothelial cells, mineralocorticoid receptor contributes to elevated levels of reactive oxygen species and increased oxidative stress, which is associated with vascular inflammation [26] as well as coronary microvascular dysfunction [23].In VSMCs, mineralocorticoid receptor activation results in increases in smooth muscle cell contraction and oxidative stress [23].Mineralocorticoid receptor-mediated actions in macrophages increase macrophage activation, chemotaxis and vascular infiltration [23].Vascular dysfunction in the form of vascular fibrosis can result in reduced elasticity and stiffening (Fig. 1) [26].Vascular stiffness due to increased accumulation of interstitial collagen is clinically related to fibrosis in many vascular diseases, such as hypertension [23].In hypertensive heart disease, increased accumulation of interstitial collagen leads to increased left ventricular stiffness, chamber remodeling and ventricular, atrial diastolic and systolic dysfunction (Figs. 1 and 2) [33,34].Other organs may also experience significant functional impairment owing to reduced blood vessel compliance, seen particularly in the elderly as aging progresses [35,36].Changes in collagen synthesis and degradation result in the buildup of collagen in blood vessel walls, deterioration in vascular compliance and endothelial dysfunction, which in turn increase the cardiovascular risk in individuals with hypertension (Figs. 1 and 2) [35,36].Mineralocorticoid receptor activation in cardiomyocytes [23] contributes to overactive fibrosis, which may also lead to unfavorable cardiac remodeling in patients following myocardial infarction, myocardial dysfunction and the development of heart failure [37].

The molecular structure of mineralocorticoid receptor has impacted the evolution of mineralocorticoid receptor antagonists
Aldosterone was first identified as a mineralocorticoid in 1954 [38], and the role of excessive aldosterone secretion in heart failure and liver cirrhosis with volume overload was soon realized [39].This led to the discovery by Kagawa et al. [40] in 1957 of the first MRA molecule, spironolactone.An oral, bioavailable form of spironolactone known as SC-9420 [41] was approved by the FDA in 1960 for primary hyperaldosteronism and edema of heart failure or cirrhosis.Spironolactone remains approved for the management and treatment of hypertension and heart failure.
The initial development of spironolactones reportedly stemmed from an effort to combine structures of progesterone (an endogenous aldosterone antagonist) and digitoxin [6,42].As a result, spironolactones were soon recognized as progesterone agonists with anti-androgenic properties [43].However, the clinical implications of the nonspecific action of spironolactones were first reported in the form of painful gynecomastia [44].Endocrine dysfunction associated with spironolactone included impotence in men and menstrual irregularities and sore breasts in women [45].These observations were followed by extensive research aimed toward the development of spironolactones devoid of such side effects.This process led to the development of mexrenone [46], spirorenone [47] and mespirenone [48].
Differences exist between nonsteroidal and steroidal MRAs, not only in their structure but also in their mode of mineralocorticoid receptor antagonism.Preclinical data have shown that the nonsteroidal MRA finerenone is able to exert key antifibrotic effects through its selectivity and nuclear recruitment.
It has been shown to demonstrate greater selectivity for the mineralocorticoid receptor than eplerenone and spironolactone while being equally as potent [6].The risk of hyperkalemia is a key clinical consideration in MRA treatment.The molecular structure of nonsteroidal MRAs in terms of their tissue distribution could provide evidence for a potential link to reduced risk of hyperkalemia [6,13,14].

Preclinical studies evaluating nonsteroidal mineralocorticoid receptor antagonists in cardiovascular fibrosis reduction
Evidence indicates that mineralocorticoid receptor is implicated in cardiovascular pathophysiology and this has led to studies of the effects of mineralocorticoid receptor blockade.Susic et al. [49] found that long-term mineralocorticoid receptor blockade using eplerenone in male, 22-week-old, spontaneously hypertensive rats improved left ventricular diastolic function and coronary hemodynamics.This improvement was associated with an enhancement in cardiac function, independent of changes in blood pressure [49].In recent decades, advances in research, such as cloning of the human mineralocorticoid receptor and sophisticated robotics (ultra-high throughput screening), have accelerated the development of nonsteroidal MRAs [6,50].As a result, dihydropyridine calcium channel blockers were determined to have mineralocorticoid receptor antagonistic effects [51], and this observation led to the development of dihydronaphthyridine finerenone (previously known as BAY 94-8862).
Preclinical studies investigating antifibrotic activity revealed that finerenone exhibited higher potency/efficacy and inverse agonism when compared with eplerenone in mineralocorticoid receptor transcriptional co-factor binding assays [52].Differential mineralocorticoid receptor co-factor modulation has been reported and suggests that finerenone may have properties that contribute to the lessening of cardiac fibrosis effects observed in mice models [52].Finerenone was also shown to improve cardiovascular dysfunction in ovariectomy-induced left ventricular diastolic dysfunction with preserved ejection fraction [53].
Other nonsteroidal MRAs being investigated in preclinical animal models include the nonsteroidal MRA KBP-5074 (KBP Biosciences; Princeton, New Jersey, USA), which was shown to restrict albuminuria in aldosterone-induced injury in rat models [54].The main aim of the study mentioned was to compare KBP-5074 with the classical steroidal MRA eplerenone.However, further investigations comparing other nonsteroidal MRAs, such as esaxerenone and finerenone, would offer further insight into the efficacy and safety of this compound [14,54].Furthermore, these favorable mechanisms of action and characteristics support clinical applications of newer nonsteroidal MRAs in cardiovascular biology.

The impact of mineralocorticoid receptor antagonist utilization in cardiovascular diseases
Once it was discovered that myocardial fibrosis could be prevented through mineralocorticoid receptor blockade [55,56], the medical community began using spironolactone therapy to improve long-term prognosis in patients with heart failure with reduced ejection fraction (HFrEF), rather than just as a potassium-sparing diuretic.Spironolactone was also proposed to be the 'renal aspirin', considering its beneficial effects in terms of preventing proteinuria [4].Forty years later, the RALES trial demonstrated the efficacy of spironolactone in improving 'hard' clinical outcomes and long-term prognosis in patients with HFrEF, including a significant 3% absolute reduction in the risk of sudden cardiac death (P = 0.02) [5].
Eplerenone, initially known as the epoxy derivative of mexrenone, is a well-known second-generation MRA [6].
Despite its short half-life and low affinity to the mineralocorticoid receptor, eplerenone was shown to be associated with a significant reduction in cardiovascular deaths in the EPHESUS trial, which involved patients with acute coronary syndrome with left ventricular dysfunction [7].In the EMPHASIS trial, eplerenone reduced the risk of death from cardiovascular causes or hospitalization for heart failure among patients with HFrEF and mild symptoms compared with placebo [8].
In clinical studies involving MRAs, reductions have been observed in both N-terminal pro-B-type natriuretic peptide and procollagen type 1 C-terminal propeptide, without changes in procollagen type 3 N-terminal peptide [57].These findings suggest that MRAs may have a favorable impact on collagen turnover (reducing synthesis and increasing degradation) with improved cardiac remodeling [57] (Fig. 1).The greatest impact observed in the HOMAGE trial, which investigated the effects of spironolactone on fibrosis and cardiac function in people with increased risk of developing heart failure was the change in vascular wall fibrosis through a reduction in collagen type 1 alpha 1 chain (COL1A1) [57].In addition, it has been demonstrated that MRAs reduce endothelin 1 production and oxidative stress [58].Proteomic analyses assessing the effect of spironolactone on plasma protein  [32].BNP, brain natriuretic peptide; LV, left ventricle; MMP2, matrix metalloproteinase 2; RAAS, renin-angiotensin-aldosterone system; VEGF, vascular endothelial growth factor.
biomarkers indicated that spironolactone reduced levels of collagen metabolism biomarkers (COL1A1, matrix metalloproteinase 2 and brain natriuretic peptide) [32] (Fig. 2).Following 1 month of spironolactone treatment, levels of two other plasma protein biomarkers associated with increased risk of CVD -vascular endothelial growth factor D (VEGFD) and neurogenic locus notch homolog protein 3 (NOTCH3) [32] -also decreased.VEGFD is a powerful lymphangiogenic and angiogenic growth factor [59], and important for endothelial cell growth, while NOTCH3 has been found to affect cell proliferation and apoptosis [32].Though initial data are promising, the potential impact of fibrosis suppression in CVDs such as heart failure, and in other solid organs, will require more extensive research.
Esaxerenone is approved for the treatment of hypertension in Japan [19].Various other unapproved nonsteroidal MRAs are currently under investigation, including KBP-5074, which is being studied as a treatment for lowering blood pressure in patients with CKD [20,21], and apararenone, which is being investigated in patients with kidney disease and diabetes [62].Clinical differences between nonsteroidal MRAs, such as association with hypotension and hyperkalemia, may appear to exist, but direct comparisons between compounds are required for these to be truly determined.
The nonsteroidal MRA finerenone is approved by the FDA [17] to reduce the risk of cardiovascular death, non-fatal myocardial infarction and hospitalization for heart failure in adult patients with CKD and T2D.This nonsteroidal MRA is also approved and indicated for the treatment of CKD associated with T2D in adults by the EMA [18].Finerenone differs from the steroidal MRAs spironolactone (Fig. 2) and eplerenone in its mechanism of action of mineralocorticoid receptor antagonism, pharmacokinetics and effect on inflammation and fibrosis in models of cardiac fibrosis and CKD (Fig. 1 and 2) [30].Finerenone has a short half-life and no active metabolites, whereas spironolactone is a prodrug with multiple active metabolites with long half-lives; eplerenone has no active metabolites but has a half-life of 4-6 h [30].
The FIDELIO-DKD and FIGARO-DKD trials investigated the efficacy and safety of finerenone in reducing kidney failure and cardiovascular outcomes in patients with CKD and T2D [9,10].In FIDELIO-DKD, finerenone treatment resulted in lower risk of CKD progression and cardiovascular events compared with placebo [9].Given that the majority of patients in FIDELIO-DKD were receiving an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB), this was the first study to show the validity of using the combination of two renin-angiotensin-aldosterone system (RAAS) inhibitors in reducing hard kidney and cardiovascular outcomes [9,63].By contrast, in the ONTARGET, VA-NEPHRON-D, and ALTITUDE trials, which involved RAAS inhibition in combination with non-MRA-based treatments, increased risk of serious adverse events was observed with no significant clinical benefit [63].
The FIGARO-DKD trial built upon the findings from FIDELIO-DKD, and together the two trials covered patient populations with multiple comorbidities.These patients had varying degrees of CKD in T2D [9,10], meaning that they were at high risk for both renal and cardiovascular events.The prognosis benefit of finerenone was observed early in FIDELIO-DKD [9,61], and was maintained for the duration of the trial; kidney outcome benefits were evident at 12 months, and cardiovascular outcome benefits were observed as early as at 1 month [9,30].FIGARO-DKD demonstrated consistent cardiovascular benefits of finerenone independent of baseline urinary albumin : creatinine ratio and estimated glomerular filtration rate (eGFR) [10].A subanalysis of the FIDELIO-DKD trial indicated that, in a similar patient cohort (CKD and T2D), finerenone reduced the risk of new-onset atrial fibrillation and flutter (AFF) [61].The risk of kidney or cardiovascular events was reduced, irrespective of history of AFF at baseline [61].

Mineralocorticoid receptor antagonist therapy safety precautions: hyperkalemia
Hyperkalemia has been a major concern with older steroidal MRAs [64].This is particularly the case in view of the increased use of ACE inhibitors and ARBs in patients with kidney disease and heart failure.The increased risk of hyperkalemia associated with steroidal MRAs also increases with age, which is a concern given the aging population [64].Meta-analyses have consistently shown a greater risk (approximately two to three times higher) of clinically relevant hyperkalemia in study cohorts receiving steroidal MRA compared with those receiving placebo [65,66].In a small cohort study of patients aged >75 years, 36% (23/64) of patients developed hyperkalemia (serum potassium ≥5.5 mmol/l) and 10% had severe hyperkalemia (serum potassium >6.0 mmol/l) during the 11-month follow-up period [67].
The large variation in hyperkalemia occurrence seen in studies will have to be addressed by trials with a larger number of participants.The development of nonsteroidal agents may significantly reduce the risk of hyperkalemia [68].A significant proportion of patients with heart failure who have poor creatinine have been deprived of the benefits of mineralocorticoid receptor blockade, owing to genuine concerns of life-threatening hyperkalemia.Both the RALES [5] and EPHESUS [8] trials excluded patients with serum creatinine >2.5 mg/dl or with initial potassium levels >5.0 mmol/l.Despite these exclusion criteria, the incidence of severe hyperkalemia remained significantly higher in patients receiving steroidal MRA compared with those receiving placebo (1% in the spironolactone arm vs. 2% placebo in RALES) [5].
The limitations of steroidal MRAs prompted extensive basic and clinical research to support the development of nonsteroidal MRAs.In preclinical research, nonsteroidal MRA finerenone has been shown to exhibit weaker potassium-sparing effects than earlier MRAs.Though potassium measurements could not be taken in these animal model studies, results indicated that finerenone was associated with a low risk of hyperkalemia related to renal injury in animal models [69].Barrera-Chimal et al. [69] noted that finerenone was also able to prevent renal dysfunction and tubular injury induced by ischemia-reperfusion in mice.
Finerenone reduced albuminuria in T2D patients with CKD and has been linked to a lower risk of hyperkalemia compared with currently available steroidal MRAs [70] from smaller phase 2 trials with limited numbers.In the FIDELIO-DKD trial, finerenone was shown to be associated with a low incidence of hyperkalemia-related treatment discontinuation (2.3% with finerenone vs. 0.8% with placebo in patients with CVD, and 2.2% with finerenone vs. 1.0% with placebo in patients without CVD) [9].The FIGARO-DKD trial [10] revealed a lower incidence of hyperkalemia associated with finerenone treatment compared with that in FIDELIO-DKD (10.8% vs. 18.3%) [9].Given that the risk of hyperkalemia doubles per 15 ml/min decrease in eGFR in patients with CKD, this difference could be explained by the baseline mean eGFR being higher in FIGARO-DKD compared with FIDELIO-DKD (68 vs. 44 ml/min/1.73m 2 ) [9,10].It is important to note that the encouraging results achieved with finerenone in FIDELIO-DKD were observed despite the fact the lack of restrictions on dietary potassium or potassium supplements, even in patients with hyperkalemia [9].Overall, newly developed nonsteroidal MRAs indicate promising results and provide novel opportunities for this class of agents.

Conclusion
Overactivation of mineralocorticoid receptor may stimulate inflammation and fibrosis, resulting in CVD progression.The evidence discussed suggests that MRAs can play an important role in reducing the molecular impact of inflammation and fibrosis, and thereby reduce cardiovascular and renal morbidity.Preclinical findings and randomized clinical trials reinforce this hypothesis by demonstrating that MRAs can reduce the risk of progression of cardiorenal diseases and their associated morbidities.Additionally, recent trials involving patients with T2D and CKD suggest that there may be further, hitherto undiscovered cardiovascular applications for this class of agents.These initial positive cardiovascular effects could potentially be linked to MRA-induced molecular changes and fibrosis reduction.While these initial results may support a new role for nonsteroidal MRAs such as finerenone, additional research is required to validate this hypothesis.