Because a renewed wave of SARS-CoV-2 (COVID-19) infection is underway, but effective prevention is not yet available to all, strategies to limit viral infectivity and disease severity are required urgently. Young, normotensive, female subjects with normal renal and cardiac function are relatively protected from COVID-19 infection and its complications.1–3 Therefore, strategies to improve host defenses against viral infection in those susceptible could have a major impact. However, the underlying mechanisms are poorly understood.1–4 Experimental studies report that angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) can upregulate tissue ACE type 2 (ACE2).2,4 One domain of ACE2 acts as the receptor for SARS-CoV-2 and is therefore essential for viral infectivity and cell entry.5–8 However, most authorities recommend maintaining ARB or ACEI therapy where indicated,9 and epidemiological studies of the association of COVID-19 infection with the use of ACEIs or ARBs have not generally reported an increase risk.8–11 This has been ascribed to the second domain of ACE2 whose enzymic action generates angiotensin 1-7 (Ang 1-7) that acts on the Mas receptor to reduce inflammation and preserve organ function.2,12–15 However, data on cellular ACE2 expression in human subjects are sparse. ACE2 is cleaved from cell membranes by disintegrin and metalloproteinase 17 (ADAMS-17) or by furin that can be upregulated by angiotensin II (Ang II), inflammation,16 and by the binding of the viral spike protein to ACE2.17 Therefore, measurements of the soluble, circulating form of cleaved ACE2 are not necessarily indicative of cellular ACE2 expression.8 Moreover, injection of SARS-CoV-2 spike protein into mice decreases ACE2 expression and worsens lung injury18,19 suggesting that the potential beneficial enzymic activity of ACE2 may become diminished after the binding of the virus. The reports that human recombinant full-length ACE2 can block infection of vascular or kidney organoids20 demonstrate the potential protection provided by circulating ACE2 as a decoy for SARS-CoV-2. However, any increases in circulating ACE2 with ACEIs or ARBs have been generally modest and inconsistent,8,11,21,22 and circulating levels of ACE2 are considered below those required to bind significantly to, and inactivate, SARS-CoV-2.8 By contrast, reports that the overexpression of the human ACE2 gene in mice infected with SARS-CoV-2 enhances disease severity demonstrate the potential importance of cellular ACE2 in viral cell entry.23 These data together suggest that ACE2 plays a complex role both as a receptor for SARS-CoV-2 for cellular entry and, on occasion, as a protective mechanism against viral spread and organ injury.24 By contrast, classical ACE generates Ang II that generally exerts adverse effects on cardiac, pulmonary, renal, and vascular functions25 but is not implicated directly in SARS-CoV-2 cell entry.8 Thus, an ideal therapeutic approach to prevent COVID-19 infection or mortality based on the modulation of ACE2 is difficult to predict because a reduction of ACE2 at critical sites for viral entry into the body in the nose, lung, and gastrointestinal tract might limit infectivity, yet a reduction of ACE2 expression in infected organs in the lung, blood vessels, and kidneys might reduce Ang 1-7 generation and thereby enhances disease severity if some enzymatic functions of ACE2 are maintained in infected cells.
Presently lacking are measurements of the levels of Ang II or aldosterone in patients infected with COVID-19. However, in contrast to an earlier report,26 a current study of patients infected with SARS-CoV-2 in China reported a high incidence of 45% of hypokalemia accompanied by increased urinary potassium and systemic alkalosis, suggesting hyperaldosteronism.27
The mineralocorticosteroid receptor (MR) is activated by mineralocorticosteroids, most notably aldosterone, but also by glucocorticosteroids, such as cortisol. However, the MR is normally protected from cortisol activation by 11-beta-hydroxysteroid dehydrogenase type 2 that is co-expressed with the MR and metabolizes cortisol to cortisone that is not an MR agonist. This confers specificity of the MR for mineralocorticosteroids, unless inhibited by drugs or genetically mutated. Classical MRs are expressed intracellular and dictate transcription of genes that mediate their actions. Other cell types express “nongenomic” MRs. Although controversial, spironolactone generally blocks both genomic and nongenomic signaling.
An alternative approach may be to manipulate the high-affinity binding of SARS-CoV-2 to ACE2. This binding requires proteolytic processing of the spike S protein of SARS-CoV-2 by the transmembrane protease receptor serine type 2 (TMPRSS2) that is co-expressed with ACE2 at several strategic sites.28 Additional proteolytic processing of the S protein of the coronavirus by furin29–31 and plasmin32,33 enhances the binding affinity of SARS-CoV-2 to ACE2 further. Protease nexin 1 (PN1) inhibits the activity of both furin and plasmin. Thus, TMPRSS2, furin, plasmin, and PN1 provide potential targets to modulate host defense against the virus. Our hypothesis is that spironolactone is the preferred renin–angiotensin–aldosterone system inhibitor to favorably modulate the proteins that determine viral processing and cell entry (Fig. 1). Indeed, spironolactone has been proposed as a drug to provide protection against COVID-19.34,35 If proven, this could have an immediate impact because spironolactone is widely available, well tolerated, and inexpensive. Eplerenone does not inhibit the androgen receptor that, in this setting, might be a disadvantage relative to spironolactone.36 Novel, more potent nonsteroidal MR antagoists (MRAs) such as finerenone and KBP-5074 are more specific for the MR than spironolactone but also lack antiandrogenic actions.37,38
Angiotensin-converting Enzyme 2
ACE2 is widely expressed in the vascular endothelium, lung, cardiac myocytes, renal proximal tubules, circulating mononuclear cells,39 and nasopharyngeal epithelium.40–43 ACE2 protects experimental animals from cardiac,44 inflammatory,45 and lung diseases13 and reduces inflammation.46
SARS-CoV-2 mortality is associated with many conditions1,3 for which ACEIs or ARBs are frequently prescribed. Some studies have suggested that ACEIs or ARBs may be harmful for SARS-CoV-2, whereas others report no association47,48 and some report up to a 67% reduction in mortality in patients prescribed with ACEIs.49 Unfortunately, associations of COVID-19 infectivity or outcome with the use of MRAs have not been reported. Most studies suggest that ACEIs increase tissue ACE2 levels and activities,50–55 but the few human studies with ACEIs report that Ang 1-7 is not changed acutely but can increase over time making it uncertain if ACE2 activity is increased significantly.56,57 Plasma ACE2 was unchanged or reduced very modestly by ACEI or ARB treatment in a large study of 3500 patients with heart failure yet was increased by spironolactone.4 Moreover, plasma ACE2 in human subjects can predict mortality or bad outcomes in patients with cardiovascular disease,58–61 making it uncertain if any increase in circulating ACE2 with RASSi therapy would be a therapeutic goal.
ACE2 expression or signaling is reduced by aldosterone, but not by Ang II, in cardiomyocytes,62,63 human neutrophils,64 and in the rat kidney,65 although spironolactone failed to change cardiac ACE2 in Dahl salt-sensitive rats66 perhaps because of their dysregulated RAAS.67 By contrast, ACE expression or signaling is increased by aldosterone in cardiomyocytes.63,68 The one clinical study of cellular ACE2 during renin-angiotensin-aldosterone system inhibitor therapy reported that spironolactone given to patients with heart failure, increased ACE2 expression or activity by 3-fold to 6-fold in circulating monocyte-derived macrophages while halving the expression and activity of ACE.69 In a parallel experimental study in rats, aldosterone reduced cardiac ACE2 and increased ACE activity and expression by increasing reactive oxygen species.69 On balance, these data suggest that MRAs may increase tissue expression of ACE2 yet reduce ACE.7 This might combat inflammation favorably but could potentially enhance SARS-CoV-2 infectivity.
Transmembrane Protease Receptor Serine 2
TMPRSS isoforms cooperate with furin and plasmin to activate epithelial sodium channel (ENaC), thereby increasing its activity at several epithelial sites.70–72 TMPRSS2 is expressed widely73 in the lung,43 the gastrointestinal and urogenital tracts, and the nasal epithelium.41,73 Its gene has an androgen promoter,74 and its expression is enhanced in men.75 TMPRSS2 is the essential first step for the proteolytic activation of SARS-CoV, MERS-CoV, SARS-CoV-2, H1N1, and H7N7.76–78 Indeed, it may promote SARS-CoV-2 cell entry at 2 sites: the cleavage of ACE2 and cleavage of the SARS-S protein.77 Whereas TMPRSS2 and ADAMS-17 both cleave ACE2, only TMPRSS2 augments SARS-CoV-2 cell entry.77 The knockdown TMPRSS2 protects mice from H1N179 or H7N780 influenza. TMPRSS2 is considered a prime target to combat coronavirus infection.43,81,82
ACE2 and TMPRSS2 are co-expressed in cells at several strategic sites for SARS-CoV-2 infection41,43,77,78,83–85 including human and mouse conjunctiva, with increased expression in men and diabetics,84 bronchial transient secretory cells,83 pulmonary type II pneumocytic cells,43,78 human nasal cells,41 and human ileal absorptive enterocytes.85 Half of pulmonary transient secretory cells co-expressed ACE2, TMPRSS2, furin, and Rho-GTPase.83 The Rho-GTPase family are signaling molecules for aldosterone,86 suggesting potential modulation by spironolactone. SARS-CoV-2 cellular entry and infectivity are reduced by blockade of TMPRSS2 by camostat mesylate6,87 or nafamostat88 that are also anticoagulants89. Therefore, a triple benefit of reduced TMPRSS2 could be postulated as follows: to block viral entry; to combat SARS-CoV-2 coagulopathy and to limit ENaC activation and pulmonary edema. However, the regulation of TMPRSS2 by the RAAS has yet to be studied.
Furin is a proprotein convertase that cooperates with aldosterone to enhance the activity of the ENaC within the Golgi apparatus and endoplasmic reticulum of the renal tubular collecting duct cells and the tubular lumen (Fig. 2).90,91
The ENaC is expressed in the kidney, pulmonary epithelium, colon, salivary glands, eye, and vascular endothelium92 where its activation is tightly regulated by proteases, including furin and plasmin.93 Interestingly, aldosterone also targets the lung to regulate alveolar fluid and Na+ uptake by the ENaC94 where furin also cooperates with aldosterone to activate the ENaC.95 Pulmonary ENaC activation by aldosterone and furin may be detrimental because spironolactone improves lung diffusion in patients with CHF.96 Aldosterone increases furin activity,97,98 whereas protease inhibitors inhibit the renal99 and tracheobronchial ENaC.100
Furin is a circulating protein but also is expressed strategically at sites of bodily entry of SARS-Cov-2 in human nasal epithelial cells and lung.101 It cleaves the S1/S2 site of the SARS-CoV-2 spike protein29–31 to enhance viral binding to ACE2 and viral entry into the human lung.31 Furin also cleaves the viral coat protein of other members of the coronavirus family.73,102 Furin and ADAMS-17 are upregulated during inflammation and cleave membrane-bound ACE2.103 Although the actions of ACEIs and ARBs on furin have not been reported, they are likely to increase furin because its expression is reduced by Ang II.104 Likewise, there are no reports of the effects of MRAs on furin, but MRAs are likely to decrease furin because there is an extensive positive interaction between aldosterone and furin in renal tubules and the lung (Fig. 2), and furin activity is increased by aldosterone.97,98 This could be an important point of discrimination between ACEIs/ARBs and MRAs.
Plasmin is generated in the blood stream from plasminogen whose activity is regulated by tissue plasminogen activator (tPA) and in tissues and body fluids by urokinase (uPA). Plasmin, plasminogen, and tPA/uPA generally are elevated in the body fluids of patients with the major COVID-19 risk factors.32 Plasmin mediates fibrinolysis whose D-dimer product levels are strong predictors of mortality in patients with COVID-19.105 Plasmin may be a driver of complex dynamic interactions of hypercoagulation with extensive microthrombi and organ infarction combined with an anticoagulant profile of reduced platelets, tissue hemorrhage, and increased D-dimer levels characteristic of disseminated intravascular coagulation. This syndrome has been termed “hemovascular dysfunction.”106 Plasminogen is expressed in the airways and the alveolar type I and II epithelial cells that are also sites of expression of ACE2 and TMPRSS2.43 Plasmin can cleave the spike protein of SARS-CoV-2 at the furin site, thereby enhancing viral cell entry33 and may be a key factor for COVID-19 susceptibility.32 Thus, plasmin, like furin, can enhance SARS-CoV-2 infectivity and perhaps contribute to impaired host defense against coronavirus in many risk circumstances. Plasmin and furin cooperate with aldosterone to enhance ENaC activity in the renal collecting ducts.107 Aldosterone, but not Ang II, can increase plasmin activity.97,108 tPA is increased modestly by an ARB but not by spironolactone.109 Any differential effects of MRAs versus ARBs/ACEIs on PN1 would predict that MRAs may decrease, but ARBs and ACEIs may increase, the activity of plasmin (Fig. 1). It will be important to test these hypotheses experimentally.
Protease Nexin 1 (PN1; Serpin E2)
This is expressed in human nasal epithelial cells.101 It inhibits the activity of many proteases including α-thrombin,89 plasmin, plasminogen,32,110 and furin.97,111 Although Ang II increases neural and myocardial PN1112–114 and thereby should decrease furin and plasmin activity, aldosterone reduces renal PN1 expression and thereby should increase furin and plasmin activity.97,115 Indeed, Ang II-induced cardiac remodeling and fibrosis are dependent on PN1.114 Thus, PN1 expression is likely to be reduced by ACEIs and ARBs but increased by MRAs, predicting that MRAs, but not ACEIs or ARBs, will reduce plasmin and furin activity and thereby reduce the proteolytic processing and binding of SARS-CoV-2 to ACE2 and reduce the virus infectivity. Because disseminated coagulation is increasingly recognized as an underlying cause of SARS-CoV-2 morbidity and mortality, inhibition of plasminogen/plasmin could be additional benefits of an increased PN1. However, effects of MRAs on PN1 have yet to be studied directly.
Strategies to identify the underlying mechanism of defense against COVID-19 in women, and to promote their expression in men, could have a major impact. Mortality is increased by about 20%–50% in men compared with women both in SARS-CoV-2, as well as SARS-CoV and MERS-CoV infections.116,117 However, this sex dependence is age dependent. Thus, the mortality from SARS-CoV-2 is increased in men versus women by >3-fold at age <70 and >6-fold at age 40–49 years.118,119 This suggests a pathogenic role for testosterone whose levels decline with age. Indeed, androgen receptor activation is required for the transcription of TMPRSS274 as no other human promoter has yet been described120 (NIH 2020 Gene ID:7113). Indeed, TMPRSS2 is upregulated by androgens at both the transcriptional and the posttranscriptional levels.121 ACE2 activity can be enhanced somewhat by estrogens, but effects are less consistent.122 Several lines of evidence implicate androgens in SARS-CoV-2 infectivity or complications. African Americans have a disproportionate mortality with COVID-19 and a genetic variation in the androgen receptor that may enhance its activity.123 The development of benign prostatic hypertrophy depends on androgens. Among COVID-19 infected men, prostatic hypertrophy and androgen levels both predict cardiac involvement with COVID-19.117,124 The relative length of the index and ring fingers is proportional to testosterone levels and correlates in men with death from COVID-19.125 Androgenic alopecia among hospitalized men is more common in those with COVID-19.126 The administration of antiandrogen drugs to male mice reduces the lethality of SARS-CoV-2.117 Spironolactone has an off-target action to inhibit the androgen receptor82 and reduces the expression of ACE2 and TMPRSS2 in cardiac cells through an antiandrogen action.127 Collectively, these results suggest that spironolactone could reduce or prevent COVID-19 infection or complications by its off-target actions as an androgen receptor antagonist aided, perhaps, by its proestrogenic/progestogenic signaling properties that are predicted to enhance ACE2 expression.
The antiandrogen receptor actions of spironolactone to downregulate TMPRSS282 are relatively specific for the parent drug rather than its metabolites.128 The use of spironolactone would confer potential benefit to men alone.129 However, the higher plasma levels of its active metabolite canrenone in women130 that account for much of its action as an MR antagonist131 might confer a potential benefit for women mediated through the MR directly.
Additional Actions of Spironolactone
Whereas a modest immune mediated inflammatory response likely helps to resolve infection early in the course of COVID-19 infection, some develop “hyperinflammation” or a “cytokine storm” with vasculopathy, thrombotic microangiopathy, and intravascular coagulopathy132 that may underlie multiorgan failure.133 The immunothrombosis entails direct endothelial cell infection and dysfunction, activation of plasminogen activator inhibitor-1 (PAI-1), and platelet activation.134 Although aldosterone135 and Ang II136 both can increase PAI-1, spironolactone reduces PAI-1135,137 but ACEIs or ARBs have variable effects.138–140
MRAs have quite prominent anti-inflammatory properties. Eplerenone reduces inflammation in a mouse model of viral myocarditis.141 MRAs modulate “trained immunity,”128 reduce inflammation,127 and prevent vascular dysfunction in models of inflammation or hypertension.142 Thus, spironolactone could have favorable effects on the oxidative stress, hyperinflammation, and widespread coagulopathy that are reported increasingly in severe COVID-19 infections but direct clinical trials are lacking. However, it is not clear if any of these effects of MRAs are superior to those of an ACEI or ARB.
The present evidence allows the prediction that reductions in TMPRSS2, furin, and plasmin and increases in PN1 should convey a benefit against COVID-19. Furin and plasmin expression should be favorably reduced by an MRA but not by an ACEI or ARB. These effects should be amplified by a reduction of PN1 by MRAs but not by ACEI/ARBs. The antiandrogen receptor actions of spironolactone should reduce the expression of TMPRSS2. Thus, MRAs may favorably affect the processing of the SARS-CoV-2 spike protein and thereby reduce viral cell entry through ACE2. In addition, the established effects of spironolactone, augmented by reduced furin and plasmin activity, should limit pulmonary edema, improve pulmonary gas exchange, and limit diffuse coagulation and fibrinolysis. Prospective clinical studies will be required to test these hypotheses and to determine the optimal dose of spironolactone and the target population. The recent availability of patiromer143 and sodium zirconium cyclosilicate (Lokelma)144 to counter hyperkalemia opens the possibility to explore higher doses of MRAs. The antiandrogen actions of spironolactone might be enhanced by twice daily administration because spironolactone itself has a short plasma half-life and it mediates much of the antiandrogen effects, whereas its major metabolite, canrenoate, has a very long plasma half-life and mediates much of the MRA action.128 Because spironolactone often can be combined beneficially with an ACEI or ARB for hypertension,145 CKD,146 or heart failure,147 physicians might consider adding spironolactone for patients at high risk for SARS-CoV-19 infection, providing there are no contraindications, such as hyperkalemia.
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