SARS-CoV-2, COVID-19, and the Renin-Angiotensin System: Pressing Needs and Best Research Practices

Abstract

The coronavirus disease 2019 (COVID-19) pandemic is associated with significant morbidity and mortality throughout the world, predominantly due to lung and cardiovascular injury. The virus responsible for COVID-19 — severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) — gains entry into host cells via angiotensin-converting enzyme 2 (ACE2). ACE2 is a primary enzyme within the key counter-regulatory pathway of the renin-angiotensin system (RAS), which acts to oppose the actions of angiotensin (Ang) II by generating Ang-(1–7) to reduce inflammation and fibrosis and mitigate end-organ damage. As COVID-19 spans multiple organ systems linked to the cardiovascular system, it is imperative to understand clearly how SARS-CoV-2 may affect the multifaceted RAS. In addition, recognition of the role of ACE2 and the RAS in COVID-19 has renewed interest in its role in the pathophysiology of cardiovascular disease in general. We provide researchers with a framework of best practices in basic and clinical research to interrogate the RAS using appropriate methodology, especially those who are relatively new to the field. This is crucial, as there are many limitations inherent in investigating the RAS in experimental models and in humans. We discuss sound methodological approaches to quantifying enzyme content and activity (ACE, ACE2), peptides [Ang II, Ang-(1–7)], and receptors (types 1 and 2 Ang II receptors, Mas receptor). Our goal is to ensure appropriate research methodology for investigations of the RAS in patients with SARS-CoV-2 and COVID-19 in order to ensure optimal rigor and reproducibility and appropriate interpretation of results from these investigations.

Introduction

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing the coronavirus disease 2019 (COVID-19) pandemic. Initially described in Wuhan, China in December of 2019, COVID-19 has severely affected human health, the global economy, and day-to-day living worldwide. The primary host protein to which SARS-CoV-2 binds to infect human host cells is angiotensin-converting enzyme 2 (ACE2).¹ ACE2 is a homolog of angiotensin-converting enzyme (ACE), a key component of the renin-angiotensin system (RAS), and functions to counterbalance the increased blood pressure, pro-inflammatory, and pro-fibrotic effects of the ACE pathway by converting angiotensin (Ang) II to Ang-(1–7) (Figure 1).^2–4 Ang II mediates the above effects via the type 1 Ang II receptor (AT₁R), while Ang-(1–7) exerts vasodilatory, anti-inflammatory, and anti-fibrotic effects via its Mas receptor.^5–8 Not only is ACE2 a cellular entry point for the virus, but it is hypothesized that SARS-CoV-2 may reduce membrane-bound ACE2, based on prior investigations of SARS-CoV.⁹ In most patients, the initial infection is limited to the oropharynx or nasopharynx, but in some patients, SARS-CoV-2 can additionally infect pulmonary epithelial cells leading to lung injury.¹⁰ In a subset of patients, viral infection and replication has been postulated, but not yet demonstrated, to occur within ACE2-expressing vascular endothelial, gastrointestinal (GI) epithelial, and kidney proximal tubular cells, as well as cardiac myocytes, which may contribute to endothelial dysfunction and vascular disease, as well as contribute to clinical manifestations such as diarrhea, acute kidney injury (AKI), and myocarditis (Figure 2). What makes SARS-CoV-2 infections unique (compared to other common respiratory pathogens like influenza and parainfluenza viruses) is the use of ACE2, a specific protein, rather than protein-linked sialic acid, as its receptor. Furthermore, the RAS may be indirectly involved in the pathophysiology of these other respiratory infections, but in SARS-CoV-2 infection, the RAS may play an important and direct role in development and progression of COVID-19.

Figure 1. Balance of angiotensin (Ang) II and Ang-(1–7) in the renin-angiotensin system (RAS).

A, In health, the RAS is in balance: blood pressure is normal, and no disease develops. B, With perturbations from various disease states (eg, hypertension or SARS-CoV-2 infection), there is a shift towards the ACE/Ang II pathway and away from the ACE2/Ang-(1–7) pathway. ACE: angiotensin-converting enzyme, ACE2: angiotensin-converting enzyme 2, AT₁R: type 1 Ang II receptor. Figure adapted from South et al.²

Figure 2. Spectrum of possible cardiovascular manifestations of COVID-19.

MIS-C: multisystem inflammatory syndrome in children, EF: ejection fraction, PT: prothrombin time, aPTT: activated partial thromboplastin time, D-dimers: dimers of fragment D.

Given concerns that chronic use of ACE inhibitors or Ang II receptor blockers (ARBs) may upregulate ACE2 expression,^{11, 12} and thereby theoretically increase the risk of SARS-CoV-2 infection, clinical decisions informed by decades of ACE inhibitor/ARB randomized clinical trials (RCTs) in patients with hypertension, kidney disease, and heart disease suddenly were called into question regarding the continuation or initiation of RAS blockade during the COVID-19 pandemic.^{13, 14} Initially, expert opinions and case series, and later, observational studies, recommended to continue the use of ARBs and ACE inhibitors unless compelling indications to discontinue existed. These statements were made with incomplete insights in an attempt to address this dearth of critical information.^{15, 16} While this issue has not been resolved, prospective studies and randomized clinical trials and ongoing. Recently, Wysocki et al demonstrated that mice treated with the ACE inhibitor, captopril, or the ARB, telmisartan resulted in no alteration of lung ACE2 expression.¹⁷

Given the theoretical importance of this topic, we need to ensure that researchers who may be unfamiliar with the RAS have a guide to avoid the common missteps that occur in interrogating this elaborate system. The RAS is an intricate, multi-enzymatic cascade of peptides that results in multiple G-protein-coupled receptor (GPCR) signaling events, leading to physiological responses. Moreover, the RAS is ubiquitously expressed throughout the body. While research in the RAS has been conducted over the last half a century in cardiovascular and kidney disease, relatively little is known about how this system is perturbed in coronavirus infection. We highlight the myriad of possible ways in which the RAS could play a role in COVID-19 pathophysiology.

In this review, we discuss current knowledge and theory regarding the role of the RAS and ACE2 in SARS-CoV-2 infection and COVID-19; present up-to-date understanding of COVID-19 pathophysiology and related multisystem involvement; and provide multidisciplinary best research practices for future research. Strong clinical evidence coupled with novel basic science evidence is required to confirm how SARS-CoV-2 interacts with and modifies the RAS and how this interaction may influence COVID-19 pathogenesis. Furthermore, standardized and validated techniques for evaluation of RAS components in both clinical and basic science studies are paramount to producing research that is rigorous and reproducible.

Virology of SARS-CoV-2

SARS-CoV-2 is a zoonotic coronavirus that is believed to have begun circulating in humans in November 2019.¹⁸ Coronaviruses are enveloped RNA viruses that infect mammals, including humans, and birds, and cause acute and persistent infections.¹⁹ A halo of spike (S) proteins gives the viral particles the appearance of a corona, Latin for crown. The coronaviruses are non-segmented positive-strand RNA viruses whose replication is characterized by the transcription of multiple nested subgenomic RNAs, hence their classification in the Nidovirales (nidus is Latin for nest).²⁰

Figure 3 illustrates the replicative cycle of coronaviruses.²¹ SARS-CoV-2 binds to ACE2 to initiate infection.²² A series of events follows, culminating in the delivery of the encapsidated genome, the nucleocapsid, into the cytoplasm. The genome is unusually long for a RNA virus, about 30,000 nucleotides. The replicase gene codes for a long polyprotein that is cleaved into 16 proteins that include prime targets for antiviral drugs.²³

Figure 3. The SARS-CoV-2 replicative cycle.

(from left to right). Cell entry of SARS-CoV-2 occurs through recognition of its ACE2 receptor, and after cleavage/activation of the spike (S) protein by cellular proteases, including TMPRSS2. After fusion of the viral envelope with the cellular membrane, the viral positive strand genomic RNA, (+) gRNA, is uncoated and translated into a polyprotein, which is cleaved by viral proteases. The processed non-structural proteins assemble to form the replicase-transcriptase complex (RTC, green blobs). Proteins in this complex elicit the remodeling of membranes to form organelles (double-membrane vesicles) that are dedicated to viral RNA synthesis. The proteins of the RTC then execute viral RNA replication. This process includes production of multiple negative stand (−) subgenomic RNAs (sgRNAs), from which the positive strand (+) sgRNAs are produced. These are also messenger RNAs for the four structural protein S, envelope (E), membrane (M), and nucleocapsid (N). The N protein encapsidates the (+) gRNA to form the nucleocapsid. The S, E, and M proteins, which are co-translationally inserted in the membrane of the endoplasmic reticulum (ER), assemble with the nucleocapsid in the ER-Golgi intermediate compartment (ERGIC). After budding into the ERGIC, viral particles are transported to the plasma membrane in smooth-walled vesicles and released by exocytosis. Figure adapted from Howley and Knipe.²³⁸

The other viral components are the membrane (M) protein, which connects the nucleocapsid with the envelope, and the envelope (E) protein, which secures localization of the M protein at the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Following assembly and budding in the ERGIC, exocytosis releases progeny virions.

For SARS-CoV-2, a fraction of the S protein travels to the cell’s plasma membrane, where it can mediate fusion between the infected cell and adjacent uninfected cells that express ACE2. The resultant formation of large, multinucleated syncytiae enables the rapid spread of infection by a route that does not rely on an extracellular phase of completely assembled virions.²⁴

The interaction between the S protein and its receptor is the principal determinant of coronavirus host species range and tissue tropism.^{1, 25} However, initiation of infection critically depends also on the activation of the S protein by cellular proteases. This process requires at least one protease, located either at the cell surface like transmembrane serine protease 2 (TMPRSS2), or in the endosome, like cathepsins. Furin may also activate the SARS-CoV-2 S protein, which may account for enhanced virulence.²⁶

Clinical Impact of COVID-19 on Systems Relevant to Cardiovascular Medicine

Clinical Impact of SARS-CoV-2 on the Lungs

SARS-CoV-2-induced COVID-19 is largely a lower respiratory tract disease; symptoms and signs include dry cough, pneumonia, dyspnea, hypoxia, and ultimately acute respiratory distress syndrome (ARDS).²⁷ Lung computed tomography has demonstrated pleural effusions, ground-glass opacities (filling of the alveolar spaces with macrophages, fibrin, and proliferating pneumocytes), and progressively diffuse pulmonary hemorrhage.²⁸ Pulmonary vessel hypertrophy, diffuse alveolar damage, and formation of hyaline membranes mainly composed of fibrin have been reported,^{29, 30} as have pulmonary emboli.³¹ Moreover, viral load correlates with the ARDS index and the partial pressure of arterial oxygen to percentage of inspired oxygen ratio, and inversely with the lung injury Murray score.²⁸

The pulmonary RAS may mediate COVID-19-related lung injury, as the alveolar epithelium and capillary endothelium express ACE2.^{1, 32} ACE and ACE2 activity from bronchoalveolar lavage fluid correlate with inflammatory markers in patients with ARDS.³³ SARS-CoV-2 infection of epithelial cells and subsequent viral proliferation likely exacerbates alveolar wall destruction and diffuse alveolar damage. SARS-CoV-2 may also target alveolar stem cells.³⁴ Such loss could compromise regenerative processes in the lungs and has stimulated discussion of applying assorted stem cell therapies as therapeutic strategies in COVID-19. Interestingly, viral-induced ACE2 suppression is thought to propagate this acute lung injury by leading to increased lung Ang II content, based on SARS experimental models.⁹ Mast cell recruitment and degranulation releases chymase, which activates matrix metalloproteinases and further increases Ang II.^35–37 Ang II induces alveolar cell apoptosis and lung inflammation and fibrosis, while the ACE2/Ang-(1–7) pathway, and possibly the type 2 Ang II receptor (AT₂R), inhibits this process.^38–42 Experimental models have shown that acute and chronic lung injury reduce membrane-bound ACE2 and initiate de novo expression of the ACE/Ang II pathway to further drive lung injury.^43–45 Thus, direct SARS-CoV-2-induced ACE2/Ang-(1–7) pathway suppression and subsequent ACE/Ang II pathway upregulation may drive COVID-19-related acute lung injury and fibrosis, though this must be confirmed in COVID-19 experimental models and clinical studies.

Clinical Impact of COVID-19 and the Immune System in Cardiovascular Disease

SARS-CoV-2 is thought to magnify interactions between inflammatory responses and cardiovascular disease. Recent data suggests that inflammatory changes within the myocardium are independent of the severity of initial disease presentation and independent of time since disease onset.⁴⁶ Patients with COVID-19 who manifest the systemic inflammatory response syndrome (SIRS) have increased risk of cardiac injury.⁴⁷ However, even less ill, non-hospitalized patients who have tested positive for COVID-19 may demonstrate signs of reduced left ventricular ejection fraction (LVEF), native T1 and T2 by cardiac MRI (i.e., signs of myocardial inflammation) and elevated serum troponin.⁴⁶ Conversely, patients with cardiovascular disease who develop COVID-19 have increased mortality.⁴⁸ Inflammation associated with COVID-19 appears to contribute to myocarditis and arrhythmias, as well as vascular thrombosis.^{49, 50} Given the important role of the immune system in cardiovascular disease, understanding how the host immune response to SARS-CoV-2 may enhance cardiovascular risk is paramount.⁵¹

COVID-19 has profound effects on both the innate and adaptive immune systems that coalesce to provoke a systemic cytokine release and a resultant cytokine storm in select patients with severe disease. Within the innate immune system, monocyte and macrophage hyperactivation may contribute to increased circulating levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1, and interferon (IFN)-γ, that are seen in patients with COVID-19 and ARDS, particularly among those admitted to the Intensive Care Unit.^{52, 53} ACE2 is expressed on macrophages in patients with hypertension,⁵⁴ and ACE2 overexpression suppresses Ang II-mediated macrophage generation of pro-inflammatory cytokines.⁵⁵ While it has been shown that SARS-CoV infection reduces membrane-bound ACE2,⁹ whether macrophages or monocytes contribute to the increased cytokine levels and cardiac risk in COVID-19-associated ARDS is unclear. For example, reductions in ACE2 levels in adipose tissue can secondarily provoke inflammation and cardiac dysfunction.⁵⁶ Among patients who are critically ill with COVID-19, circulating numbers of neutrophils, the other major innate immune system constituent, are increased compared to those with non-critical illness.⁵⁷ Autopsy reports suggest that COVID-19-induced formation of neutrophil extracellular traps, web-like structures of DNA and proteins secreted by neutrophils to capture pathogens, may contribute to cytokine storm, vascular thrombosis, and ARDS.⁵⁸

Within the adaptive immune system, circulating numbers of T cells are similarly reduced in patients with more severe COVID-19.^{57, 59} As demonstrated in case reports and small retrospective cohort studies, the remaining T cells show more markers of activation,⁶⁰ higher percentages of pro-inflammatory Th17 cells⁶¹ and lower proportions of immunosuppressive T regulatory cells.⁶² The reduction in circulating T cell numbers could be due to induction of apoptosis signaling pathways in leukocytes.⁶³ Thus, the innate and adaptive immune responses in patients with overlapping COVID-19 and cardiovascular disease are complex and may differentially influence therapeutic considerations in these patients.

Clinical Impact of SARS-CoV-2 on Coagulation

COVID-19 has been associated with a coagulopathy that appears unlikely to be a direct viral effect but rather a consequence of the subsequent inflammatory response.⁶⁴ Numerous cases of limb ischemia, pulmonary emboli, and strokes in otherwise young, healthy individuals have been reported.^{65, 66} High rates of venous thrombosis⁶⁷ arterial thrombosis,⁶⁶ and microvascular thrombosis⁶⁸ have been reported in patients with COVID-19. Notably, as is increasingly recognized, venous thromboembolic disease in COVID-19 often occurs despite appropriate prophylactic measures. The endothelium, which can express ACE2,^{32, 69} is thought to be dysregulated due to inflammatory cytokines shifting to a pro-adhesive and pro-thrombotic state. Microthrombi have also been reported in patients with COVID-19,⁷⁰ and levels of dimers of fragment D (D-dimers) are markedly increased, with longer prothrombin times and activated partial thromboplastin times in patients with worse outcomes.^{71, 72} Additional clinical signs include a pro-coagulant profile, including increased clot strength, and hyperfibrinogenemia.⁷³

Previous work has shown that ACE inhibitors enhance release of tissue plasminogen activator and reduce the production of its inhibitor, plasminogen activator 1 (PAI-1).⁷⁴ A small study of patients with moderate-to-severe COVID-19, in fact, showed improvement in oxygen saturation and reduced alveolar effusion after treatment with plasminogen.³⁰ Moreover, ACE2 is, in general, an antithrombotic enzyme,⁷⁵ such that loss of ACE2 via internalization with the virus or enhanced shedding⁷⁶ could exacerbate the coagulopathy. Loss of ACE2 promotes higher levels of Ang II, and Ang II potently upregulates PAI-1 expression, among other pro-coagulant actions. Thus, the RAS may play a role in the development of COVID-19-associated coagulopathy via inflammation-induced endothelial injury.

Clinical Impact of SARS-CoV-2 on the Vasculature

COVID-19 has been associated with endothelial dysfunction, which was originally described in terms of altered vascular responses to acetylcholine,⁷⁷ but is now characterized by a localized inflammatory response with increased oxidative stress, infiltration of inflammatory cells, and a pro-thrombotic state.^{78, 79} A recent report studying post-mortem samples obtained from multiple organs of patients with COVID-19 demonstrated inflammatory cell infiltration of the vessel wall, apoptosis, and endothelial cell death.⁸⁰ Components of the RAS, including ACE2, are expressed in the endothelium and play major role in regulating vascular tone and function.^{81, 82} It i theoretically is possible that SARS-CoV-2 could infect endothelial cells, though definitive evidence has not been demonstrated. Thus, direct viral downregulation of endothelial ACE2 or secondarily increased Ang II with decreased Ang-(1–7) could further promote a pro-inflammatory response,⁸³ directly via the AT₁R, and indirectly through phosphorylation of disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), causing subsequent ACE2 cleavage.⁸⁴ Taken together, various components of the RAS could mediate multiple aspects of vascular dysfunction that occur in COVID-19, supporting the concept that the RAS provides viable therapeutic targets for further exploration.

Clinical Impact of SARS-CoV-2 on Blood Pressure

A direct effect of SARS-CoV-2 on blood pressure is incompletely described. While hypotension associated with multi-organ dysfunction can occur in severely ill patients with COVID-19,⁸⁵ it appears to be uncommon at presentation.⁷¹ Initial reports suggest that blood pressure at presentation might be higher in patients who go on to develop more severe COVID-19, independent of the presence of baseline hypertension.^{52, 86} However, this question has not been investigated directly; indeed, most studies have not reported blood pressure.^{47, 87} Multiple factors that influence blood pressure in acute illness must be accounted for, including fever, inflammatory response, fluid status, and kidney function.

The ACE/Ang II pathway is upregulated in acute illness to maintain adequate organ perfusion. However, if this occurs without an adequate compensatory increase in the ACE2/Ang-(1–7) pathway, deleterious hypertension can occur.^{88, 89} Indeed, patients with hypertension, heart disease, diabetes mellitus, and chronic kidney disease often demonstrate RAS dysregulation.^{88, 90} In COVID-19, it is thought that SARS-CoV-2—ACE2 binding and subsequent internalization and downregulation may induce pulmonary RAS dysregulation. Loss of pulmonary endothelial ACE2 could lead to increased circulating Ang II [and decreased Ang-(1–7)] and subsequent unopposed increased vasoconstriction, kidney sodium and water reabsorption (possibly mediated in part by increased aldosterone production and release), and ultimately increased blood pressure.⁸⁸ Concurrent AKI, and possibly early endothelial dysfunction, could magnify this effect through ACE/Ang II upregulation and other mechanisms. Initial reports demonstrating hypokalemia, possibly due to increased Ang II and aldosterone, support this physiologic theory.^{86, 91} However, further investigation into these mechanisms in animal and clinical studies is needed, such as serial measurements of RAS components and blood pressure throughout the disease course, including secondary pathways such as chymase-mediated Ang II generation.^{35, 90}

Clinical Impact of SARS-CoV-2 on the Heart

Approximately 20–30% of hospitalized patients with COVID-19 develop acute cardiac injury that appears to be unrelated to viral load and could be related to either direct myocardial injury or secondary to a hyperinflammatory response to the virus in the days to week after acute infection (Figure 3).⁹² The etiology of COVID-19-related cardiac involvement remains to be determined. COVID-19-related dysfunction manifests as myocardial injury, with elevated serum troponin levels, elevated B-type natriuretic peptide, signs of cardiac ischemia, and decreased left ventricular systolic ejection fraction. The hyperinflammatory response (e.g., stage 3) in adults presents as a myocarditis-like condition.

In its earliest stages, the hyperinflammatory response may be mediated by interferon, and later by activated macrophages and subsequent release of inflammatory cytokines, including IL-1, IL-6, and TNF-α.⁹³ Purported therapies under investigation for the management of cardiac COVID-19 sequelae have included intravenous immunoglobulin, steroids, the anti-IL-6 antibody, tocilizumab,¹⁷ anti-IL-1 anakinra, and the antiviral-drug, remdesivir.^{94, 95} A minority of children appears to be at risk for severe cardiac injury that may be associated with SARS-CoV-2. According to the Centers for Disease Control and Prevention criteria, multisystem inflammatory syndrome in children (MIS-C) is characterized by fever, laboratory evidence of inflammation (≥1 of the following: elevated neutrophil count, C-reactive protein, erythrocyte sedimentation rate, procalcitonin, fibrinogen, D-dimer, ferritin, lactic acid dehydrogenase, or IL-6, reduced lymphocyte count, and hypoalbuminemia), and evidence of multi-organ dysfunction requiring hospitalization (e.g. cardiac, respiratory, hematologic, GI, kidney, dermatologic, or neurologic), as well as evidence of current/recent SARS-CoV-2 infection or recent exposure to a suspected or confirmed SARS-CoV-2 case.^{94, 96–98} Specifically, MIS-C is associated with depressed left ventricular ejection fraction and coronary artery dilatation, ectasia, or aneurysms.^{94, 99}

Transcriptomic analysis of the heart in patients with hypertrophic obstructive cardiomyopathy revealed that ACE2 was the most robustly expressed transcript, a finding corroborated by quantitative real-time polymerase chain reaction and western blot analysis.¹⁰⁰ As with many cardiac diseases, it is generally accepted that ACE2 upregulation is a cytoprotective response that mitigates cardiac compromise. This has several important implications and may explain in part why cardiac disease is a risk factor for COVID-19, as well as why SARS-CoV-2-induced ACE2 suppression could be uniquely damaging to the heart, as this would vitiate this important cytoprotective mechanism. However, it remains unclear if this observation is pertinent to SARS-CoV-2.

Clinical Impact of SARS-CoV-2 on the Central Nervous System

Premorbid cerebrovascular disease is associated with an approximately 2.5-times higher risk of severe COVID-19.¹⁰¹ Conversely, having critical illness due to COVID-19 appears to increase one’s risk of thrombotic events.¹⁰² Of 221 patients with COVID-19 from Wuhan, China, acute stroke occurred in 5.9% of patients (ischemic stroke in 11, hemorrhagic stroke and sinus venous thrombosis in one each), with the majority having severe COVID-19.¹⁰² However, emergency department stroke code activations dramatically decreased at the onset of the outbreak, raising the possibility that social distancing may limit the spread of non-COVID-19 infections as precipitants of acute stroke pathogenesis, or because patients are delaying care due to fear of exposure to COVID-19 in the hospital.^103–105

Numerous mechanisms may contribute to this higher prevalence of cerebrovascular disease during COVID-19.¹⁰⁶ Besides a predilection for thrombosis,¹⁰⁷ cytokine storm syndrome and hypoxia due to ARDS likely increase the risk of ischemic and hemorrhagic stroke.^{106, 108} As noted above, the virus may infect endothelial cells directly,⁸⁰ alter vascular tone of the autonomic nervous system by binding to ACE2,¹⁰⁹ and increase blood pressure.^{106, 109} SARS-CoV-2—ACE2 binding on the capillary endothelial lining may weaken the blood-brain barrier as a means to promote microvascular thrombo-inflammation, as well as allow viral entry into the central nervous system, contributing not only to stroke but also encephalitis.^{106, 108} SARS-CoV-2 could directly infect the central nervous system, as many cells in the brain express ACE2, though this has yet to be adequately demonstrated.⁹¹

Clinical Impact of SARS-CoV-2 on the Kidney

The kidney consequences of COVID-19 are emerging, and AKI is now reported frequently in hospitalized patients.¹¹⁰ In a retrospective analysis of 5449 patients admitted to the hospital in a large New York health care system, 37% had AKI. Of patients admitted to the Intensive Care Unit who required mechanical ventilation, 97% required kidney replacement therapy.¹¹¹ However, initial reports describing AKI lacked sufficient information to gain much pathophysiologic insight.¹¹² Two reports that included kidney histology from autopsy specimens have provided evidence for severe acute tubular necrosis.^{113, 114} The mechanisms are unclear and may predominantly involve tubulointerstitial injury from various causes.¹¹⁰ SARS-CoV-2 may have the potential to infect kidney parenchymal cells, as suggested by the presence of virus-like particles in podocytes and tubular cells¹¹⁴ and the presence of viral RNA, but this has not been definitively demonstrated or confirmed.¹¹⁵ The pathophysiology is likely complex, involving viral-mediated injury, cytokine storm, complement activation, hypercoagulation, and microangiopathy, as well as other common contributors to AKI such as hypotension and nephrotoxic medication exposure.¹¹²

The ACE2/Ang-(1–7) pathway is highly expressed in the kidneys and co-localizes with ACE/Ang II.¹¹⁶ ACE2 is predominantly expressed on the apical brush border of proximal tubular cells.¹¹⁷ Lower ACE2 expression has been reported in the glomerulus (namely podocytes), distal tubule, interlobular arteriole endothelium, and vascular smooth muscle.^117–119 In chronic kidney disease and experimental AKI, ACE expression/activity and Ang II concentration are higher relative to lower ACE2 expression/activity and Ang-(1–7) concentration.^120–126 Thus, the RAS could contribute to COVID-19-associated AKI in several ways.¹¹² ACE/Ang II upregulation at the expense of ACE2/Ang-(1–7) downregulation could enhance AKI, independent of direct viral kidney infection, which would propagate inflammation and fibrosis. While SARS-CoV-2 could infect a variety of cell lineages in the kidney that express ACE2, this would be dependent upon viremia and would require priming by TMPRSS2. TMPRSS2, however, is highly expressed in the distal rather than proximal tubule.^{112, 127} More information clearly is needed to understand AKI pathophysiology in patients with COVID-19.

Clinical Impact of SARS-CoV-2 on the Gastrointestinal Tract

The GI tract is a common target for infection by a variety of coronaviruses, especially in pigs, and the small bowel epithelium abundantly expresses ACE2.^{128, 129} Anorexia, nausea, vomiting, diarrhea, and abdominal pain are common symptoms, occurring in 30–60% of patients infected with SARS-CoV-2.^{130, 131} Liver blood tests are often elevated, especially in patients with severe COVID-19, which may indicate SARS-CoV-2-related liver injury, though this has not been proven and is likely multifactorial in etiology.^{132, 133} Pancreatic blood tests also may be mildly elevated. However, the relationship of abnormal liver and pancreatic tests to the actual infection is unclear.

The gut is the largest immune organ with a tremendous capacity for an inflammatory response that can have a systemic impact. Breakdown of the gut barrier is a common feature of multi-organ failure, accompanying conditions such as ARDS and septic shock, and any infection that involves the gut could amplify inflammation elsewhere. Rapid changes in the microbiome have been described in animal models of infection with delta coronaviruses and recently SARS-CoV-2.^{128, 134} This dysbiosis and the early disruption of the intestinal barrier together with a potent gut immune response to infection could contribute to systemic cytokine release.¹³⁵ The apparent intracellular staining of viral nucleocapsid protein in GI epithelial cells and recovery of coronavirus RNA from stool suggests the possibility of a fecal-oral transfer, though this has not been confirmed.¹³⁶ Similarly, we need to understand if viral ACE2 targeting alters RAS activity in the GI tract, and if that contributes to the GI inflammatory response.¹³⁷ A focus on mitigating or preventing the impact of the virus on the gut early in the disease may mitigate later systemic consequences of the infection.

Clinical Impact of SARS-CoV-2 on Pregnancy

There is no definitive evidence, to date, that pregnancy increases the risk of more severe COVID-19 or that vertical transmission exists.^138–140 However, the RAS could potentially mediate SARS-CoV-2-associated pregnancy and fetal complications. RAS upregulation is crucial to normal pregnancy physiology to maintain maternal hemodynamics and placental blood flow. Both the ACE/Ang II and ACE2/Ang-(1–7) pathways are expressed in the uterus and throughout the placenta.^{141, 142}

Blood pressure tends to be lower early on in the first trimester, in part due to a blunted response to Ang II, increased Ang-(1–7) concentration, and increased production of vasodilatory prostaglandins, but usually increases as pregnancy progresses.^143–145 Correspondingly, syncytiotrophoblasts highly express ACE2 in early gestation, but this declines as pregnancy progresses.¹⁴⁶

In pathologic states such as preeclampsia, there is a shift towards increased ACE/Ang II pathway expression and decreased ACE2/Ang-(1–7) pathway expression in the placenta and maternal circulation.¹⁴⁷ Clinical studies have shown decreased maternal plasma Ang-(1–7) concentration and Ang II hyper-responsiveness in women with preeclampsia.^{145, 148} Thus, SARS-CoV-2 binding to and the consequent downregulation of uterine/placental ACE2, with increased Ang II relative to decreased Ang-(1–7), could contribute to pregnancy-specific pathology, including preeclampsia, fetal growth restriction, and fetal programming of blood pressure and cardio-metabolic disease.⁸⁹ Indeed, a recent systematic review of pregnancy outcomes in women with SARS, Middle East respiratory syndrome, or COVID-19 reported a higher pooled proportion of preeclampsia compared to the general population, though the sample size was small (79 total, with 41 women who had COVID-19).¹⁴⁹ This preliminary analysis demonstrates the need for further investigation into RAS-mediated mechanisms in the clinical context of preeclampsia and other pregnancy and offspring complications.

Factors Associated with Worse Outcomes in COVID-19: Potential Links to the RAS

Several factors have been associated with worse outcomes in COVID-19. While a majority of these associations are likely attributable to social determinants of health, lack of access to healthcare, racial bias, and the compounding effect of prior health disparities,¹⁵⁰ it is possible that biological differences linked to the RAS could play some role. Emerging factors that may be associated with worse COVID-19 outcomes include male sex, racial and/or ethnic minority, obesity, older age, prior history of lung disease, smoking status, and lower socioeconomic status.^151–153 The gene for ACE2 is contained on the X chromosome,¹⁵⁴ thus differential ACE2 expression may occur with respect to sex. However, data reporting sex differences in ACE2 expression are conflicting. A study performed in rats showed that ACE2 protein expression in the lungs decreases with age, especially in older males compared to older females.¹⁵⁵ However, an integrated bioinformatics analysis of single-cell RNA sequencing data in humans indicated that men may have higher ACE2 expression in pulmonary alveolar type II cells compared to women.¹⁵⁶ Smoking and chronic obstructive pulmonary disease, which are more common in men, have been associated with higher ACE2 expression, though there are significant methodological limitations to these studies.^157–159 Patients with obesity may have higher ACE2 expression in adipose tissue, as obesity is associated with increased ACE/Ang II relative to ACE2/Ang-(1–7) expression.^{160, 161} However, it is unclear whether differential RAS expression according to these factors has any impact on SARS-CoV-2 infection or COVID-19 severity. It will be important to continue to conduct careful research to evaluate how each of the factors are perturbed in the context of SARS-CoV-2 and whether the RAS is involved.

Best Practices for Basic/Clinical Research of the RAS

Measuring Angiotensin-Converting Enzyme 2 Abundance and Activity

Whereas ACE is a dicarboxypeptidase that cleaves two amino acids from the decapeptide, Ang I, to form the octapeptide, Ang II, ACE2 is a monocarboxypeptidase that cleaves one amino acid, phenylalanine, from Ang II to form the heptapeptide, Ang-(1–7) (Figure 1).^{3, 162–165} ACE2 also cleaves leucine from Ang I to form Ang-(1–9) (which may have modest biological activity), though with much lower affinity than for Ang II, as well as several other substrates.^{3, 162–165} ACE2 shares 42% sequence identity with the N- and C-terminal domains of somatic ACE and is the only homologue of ACE that is enzymatically active, but ACE2 is unaffected by all of the ACE inhibitors.^{3, 166}

ACE2 exists in two forms: full length and soluble. Full-length ACE2 has a transmembrane domain, which anchors its extracellular domain to the plasma membrane.¹⁶⁷ In polarized epithelia such as in the kidney, lung, and intestine, ACE2 localizes to the apical surface.^{117, 168} Soluble ACE2, released by sheddases such as ADAM17, lacks the cell membrane-anchoring transmembrane domain and circulates in exceptionally small concentrations in the blood.¹⁶⁹ The extracellular domain includes the binding moiety for the S protein of SARS-CoV^{170, 171} and SARS-CoV-2.¹

The relative abundance of full-length ACE2 can be estimated using immunohistochemistry, western blotting, and tissue enzymatic assays (Table 1). Recent studies have provided information on ACE2 localization based on single-cell RNA sequencing analysis.^{172, 173} However, the levels of messenger RNA do not always correlate with protein levels since ACE2 is frequently regulated post-transcriptionally.^{172, 173} ACE2 activity can also be measured using high-performance liquid chromatography (HPLC),^{164, 174} mass spectrometry (MS),^175–177 LC-MS/MS^{165, 178} and an in situ MS-based enzyme activity technique (Table 1).¹⁶² These MS and HPLC-based techniques all have in common that they use a natural substrate for ACE2, the peptide Ang II. The products of enzymatic ACE2 cleavage, i.e. Ang-(1–7) and/or the phenylalanine cleaved from Ang II can be quantitatively or semi-quantitatively assessed. A technically less challenging assay, the phenylalanine assay, also uses Ang II as a substrate and measures the formation of phenylalanine to estimate ACE2 activity in biological samples.^179–181 This assay is practical but is not specific for ACE2.

Table 1.

Principles and methods used to evaluate ACE2

Measured parameter	Principle	Methods used	Information
ACE2 activity	Natural ACE2 substrates: Ang II des-9Arg-bradykinin Apelin	High-performance liquid chromatography (HPLC) Mass spectrometry (MS) LC-MS/MS in-situ MS-based enzyme activity Phenylalanine assay	Quantitative or semiquantitative determination of ACE2 enzyme activity against specific natural substrate
	Artificial ACE2 substrates: Mca-YVADAPK(Dnp) Mca-APK(Dnp) ABZ-S-P-Y(NO2)	Detection of fluorescence formation (fluorescence detecting microplate readers)	Quantitative determination of ACE2 enzyme activity in a high-throughput format
ACE2 protein	Antibody-based assay	Immunohistochemistry Immunofluorescence	ACE2 protein localization in organs and tissues
		Immunogold Electron Microscopy	ACE2 protein subcellular distribution
		ELISA	Quantitative determination of ACE2 protein in a high-throughput format
		Western blot	Semi-quantitative determination of ACE2 protein and its immunoreactive fragments

ACE2 activity assays based on fluorescent substrates are most commonly used. The assays utilize relatively inexpensive intramolecularly quenched fluorescent peptides as the substrate.^182–187 They require only regular fluorescence microplate readers and are relatively simple, fast, and convenient to quantitate plasma and tissue ACE2 activity in a high-throughput format. The substrate peptide in these assays usually contains a fluorescent 7-methoxycoumarin group (Mca), which is quenched by energy transfer to a 2,4-dinitrophenyl moiety (Dnp, quencher group). The cleavage of an amide bond between the fluorescent and quencher groups, results in an increase in fluorescence which is used to measure the activity of ACE2 and of other peptidases.¹⁸⁸ The two most commonly used peptides for ACE2 activity measurements are Mca-YVADAPK(Dnp)^{163, 187, 188} and Mca-APK(Dnp).^{165, 183, 189–192} The Mca-APK(Dnp) substrate is more specific for ACE2 than the Mca-YVADAPK(Dnp) substrate. For evaluation of urine, serum, and kidney ACE2 activity using the intra-molecularly quenched synthetic ACE2-specific substrate, Mca-APK-(Dnp), measurements are performed in black microtiter plates with a 100 μl total volume. MLN-4760 or other inhibitors, such as DX-600, can be used to calculate ACE2-dependent activity. DX-600 is effective in inhibiting human but not rodent ACE2.^{165, 193} Fluorescence is measured using a microplate fluorescence reader at 320 nm excitation and 420 nm emission wavelength, and ACE2 activity is then calculated from plotting the relative fluorescence units against the linear equations generated for a human or mouse recombinant ACE2 standard.¹⁹² It should be emphasized that serum is preferred over plasma for ACE and ACE2 analysis given the potential for assay interference. Many of these substrates are not highly specific for ACE or ACE2, and many of these techniques cannot distinguish between circulating, plasma membrane-bound, and intracellular enzyme activity or content.¹⁹⁴

To maintain Ang II at a steady-state level, the formation driven by ACE and the degradation driven by ACE2 should be synchronized under physiological conditions.¹⁹⁵ When Ang II formation increases as a result of increased Ang I formation and increased ACE, one would anticipate that ACE2 would eventually increase as well to foster Ang II degradation, otherwise Ang II would increase without a checkpoint. Conversely, if Ang II formation decreases, one would expect a decrease in ACE followed by a decrease in ACE2.¹⁹⁵ Overall, the concept of counter-regulation implies that ACE2 changes in the same direction as ACE under physiologic conditions. As such, changes in ACE2 should compensate for changes in ACE in a manner whereby large changes in Ang II are attenuated.¹⁹⁵ This, however, has never been fully tested, and other enzymes and substrates are involved, such as aminopeptidase A (Ang II to Ang III [Ang-(2–8)]), neprilysin [Ang I to Ang-(1–7)], and prolyl oligopeptidase [Ang II to Ang-(1–7)]. What is known is that, in pathophysiologic conditions, ACE and ACE2 often change in the opposite direction, similar to Ang II and Ang-(1–7). Discordance in the activity levels of these two enzymes has been well documented in models of diabetic kidney disease.¹⁶⁵ The use of the ACE/ACE2 ratio [and Ang II/Ang-(1–7)] is therefore a convenient method to capture relative changes between these two pathways. It is important to emphasize, however, that the ratio may result in misleading conclusions because each enzyme can be regulated independently from the other, so appropriate interpretation is important.

Measurement of RAS Peptides

Dating back to the groundbreaking work of Nussberger and Campbell to develop methods for measuring Ang II,^{196, 197} appropriate and validated measurements of Ang II and Ang-(1–7) are critical in assessing the RAS, including in interpreting ACE and ACE2 data (Table 2).^{89, 194, 198} As with ACE and ACE2, Ang II and Ang-(1–7) quantification is difficult and has variable between-laboratory reproducibility. While collection of human samples in hospitalized patients with COVID-19 can be technically challenging, adhering to precise collection, processing, storage, and assay methods by experienced investigators and laboratories whenever possible is necessary to ensure adequate rigor, reproducibility, accuracy, and reliability. Especially relevant to SARS-CoV-2 research, standardized methods and centralized specimen analysis will enable comparisons of RAS measurements across studies and patient populations and enhance the ability to make appropriate inferences.

Table 2.

Comparison of assay limitations to detect Ang peptides

		Limitations
Assay	Low Concen.		Interfering Substances	Ongoing Metabolism	Sequence Homogeneity	Detection Specificity		Posttranslational Modification		Cost and Expertise
RIA/ELISA*		X	X	X	X	X	X
LC-MS/MS HPLC-MS/MS			X	X					X

Appropriate sample handling and extraction are crucial for all methods of angiotensin peptide detection. Animals should be in a calm environment and medications that acutely alter the RAS be avoided. Blood samples for plasma separation should be collected directly into a peptidase inhibitor cocktail validated for the specific assay being used, while urine typically requires acidification (HCl), peptidase inhibitors, or collection on dry ice. An example of a peptidase inhibitor cocktail tailored to HPLC-RIA includes 1,10-phenanthroline monohydrate, pepstatin, and Na p-hydroxymercuribenzoate.¹⁹⁹ Samples should be frozen or extracted immediately. Appropriate sample extraction is required. Particularly for RIA/ELISA, it is critical to perform peptide purification and fractionation (i.e. HPLC-RIA).

^*Commercially available RIA/ELISA’s utilize antibodies directed against the COOH-terminus and cannot distinguish the NH₂-terminus of metabolites.

Limitations to Ang peptide quantification include low concentrations (~fmol/ml), interfering substances, ongoing metabolism (generation and degradation), and sequence homogeneity affecting detection specificity and cross-reactivity (e.g. Ang III with anti-Ang II antibodies).^{89, 194} Samples should be collected in an EDTA tube with a peptidase inhibitor cocktail validated for the intended assay or possibly ice-cold methanol (Table 2).^{161, 194, 199} Urine samples should be collected with acidification or inhibitors.^{161, 194} Samples should be stored at −80°C or immediately extracted. Tissue samples should be rinsed of blood and rapidly frozen in liquid nitrogen or an ethanol/dry ice bath, or immediately extracted.¹⁵⁸

RIA and ELISA, if validated with HPLC/MS, can have the requisite sensitivity and specificity, but require optimal conditions and precise methods, including peptide extraction/purification.^194,158 The antibodies target each peptide’s unique COOH-terminus, but fail to distinguish the NH2-terminus unless coupled to HPLC. LC-MS/MS and HPLC-MS/MS-based methods are the gold standard but require sufficient expertise and are costly.¹⁵⁸ Of note, these assays cannot readily distinguish peptide source (i.e. extracellular vs. intracellular from tissue samples, specific tissue for blood samples). Finally, while normative RAS peptide values do not exist, verified content in various sample sources across species has been established.¹⁹⁴

Angiotensin II Type 1 and Type 2 Receptors/Mas Receptor

The main effector peptide of the RAS, Ang II, mediates the classical actions of the RAS via activation of the GPCR AT₁R.²⁰⁰ AT₁Rs are broadly expressed throughout the body. While humans and primates have a single AT₁R, mice and rats have two AT₁Rs, termed AT_1AR and AT_1BR. These are distinct genes encoded on separate chromosomes and have 91% sequence identity.²⁰¹ The AT_1AR has been identified as the closest homologue to the single human AT₁R based upon physiologic studies using selective knockout of either the AT_1AR or the AT_1BR.^{202, 203} The type 2 Ang II receptor (AT₂R) is a GPCR receptor isoform of the RAS, whose activation results in actions opposite of AT₁Rs.²⁰⁴ Thus, AT₂R activation by Ang II leads to reduced blood pressure, natriuresis, and diminished end organ damage such as fibrosis.^{204, 205}

In the RAS, Ang II generation in humans is largely mediated by ACE and chymase, which are compartmentalized in the blood and tissue.^{35, 206, 207} New evidence has demonstrated chymase within human cardiomyocytes²⁰⁸ in addition to the AT₁R and AT₂R on nuclear^209–212 and mitochondrial membranes.^{213, 214} Intracellular Ang II microinjection directly into single kidney proximal tubular cells increases [Ca²⁺] levels in the presence of extracellular losartan, suggesting activation of intracellular AT₁R and AT₂R.²¹⁵

Finally, Ang-(1–7) activation of the Mas receptor initiates a dynamic response that involves an intricate pathway of internalization by either clathrin-coated pits or caveolae through a dynamin-dependent mechanism.^{216, 217} Endocytic vesicles fuse with early endosomes and stimulate Akt and ERK1/2 phosphorylation, among other signaling events.¹⁷⁸ These and similar studies support the function of an intracrine RAS/chymase process that varies from cell to cell and organ to organ, in addition to the known paracrine and autocrine roles of the RAS. This has supplanted the physiologic relevance of previously utilized receptor-binding assays that homogenize tissue and disrupt this important functional compartmentalization, thus leading to significant bias in those studies, which do not account for appropriate methodology. Measurement of ATR is challenging, as commercially available antibodies show a high degree of non-specificity.²¹⁸ Thus, results from western blot analysis and immunohistochemistry are generally unreliable and proper negative and positive controls should be employed.

Animal Models Relevant to SARS-CoV-2

Gene targeting is a recognized method for establishing the function of a known gene. While more established in mice, genetic manipulation in rats has emerged with new molecular strategies, such as CRISPR/Cas9, but no studies manipulating the ace2 gene in rat models have been published to date. Much has been learned about the components of the RAS by using genetically engineered mice.²¹⁹ As one example, mice lacking ACE2 were first reported in 2002 and demonstrated a marked cardiac defect.¹²⁰ Subsequent ACE2 knockout (KO) mouse lines were not able to reproduce this severe contractility defect, but did clearly demonstrate enhanced susceptibility to Ang II-induced hypertension¹⁷⁶ and cardiac hypertrophy following transverse aortic constriction.²²⁰ These ACE2 KO lines have been further studied in different disease models and to test potential therapeutics, and have been useful in understanding SARS pathology.²²¹ In particular, Kuba et al showed that SARS infection or SARS-CoV S protein dramatically reduced membrane-bound ACE2 in wild-type mice.⁹ Moreover, while mice treated with SARS-CoV S protein (which binds both human and mouse ACE2) and subsequently subjected to acid instillation in their lungs exhibited worse lung injury and enhanced edema, ACE2 KO mice did not exhibit worsening of lung pathology after S protein exposure.⁹

More recently, transgenic mice expressing human ACE2 (hACE2) have been generated ^222,223 and are available for studies related to viral infection. This is important because the SARS-CoV-2 S protein, much like SARS-CoV, binds human ACE2 with much higher affinity than rodent ACE2.^{224, 225} These mice express hACE2 in their upper airway, but not alveolar epithelia, as well as in epithelia from liver, kidney, and the GI tract, and in small amounts in the brain.²²² When infected with SARS-CoV or SARS-CoV-2, they exhibit macrophage and lymphocyte infiltration in the lungs and pro-inflammatory cytokine and chemokine upregulation in lung and brain, as well as interstitial pneumonia, similar to human disease.^222,226 Another novel approach to modeling COVID-19 in mice was recently reported by Hassan et al where wild-type mice were primed for infection by adenoviral delivery of hACE2 to the lungs.²²⁷ Lung infection and inflammation are reversed by neutralizing antibodies (a monoclonal chimeric mouse/human antibody constructed with the Fv-human Fc (IgG1) antibody generated after immunization of C57BL/6J mice with SARS-CoV-2 S protein).²²⁷ As SARS-CoV-2 entry into cells requires priming by TMPRSS2,¹ mice with a deletion of this gene may be of use as well. The range of available models allows investigation of the role of the RAS in COVID-19, and innovation following this pandemic should add to this arsenal.

Best Research Practices for the RAS: Clinical Research

Strong clinical evidence is required to confirm the role of the RAS in COVID-19 and the role of ACE inhibitor and ARB therapy during the pandemic. Key questions include: (1) Does SARS-CoV-2 induce RAS dysregulation; (2) Does RAS dysregulation (e.g. decreased ACE2 and increased Ang II) predict worse outcomes; and (3) Does the RAS mediate the effect of ACE inhibitors/ARBs on outcomes? The answers to these questions may inform risk stratification and treatment strategies. The importance of these questions and need for data in near-real time without the luxury of traditional RCTs has provided a tremendous impetus for collaborations on better-designed observational studies. This section will briefly discuss observational study and RCT methodological considerations.

The explicitly defined study population, exposures, and outcomes of interest inform how one conceptualizes co-morbidities (e.g. hypertension, heart disease) and ACE inhibitor/ARB use as exposures, confounders, effect modifiers, or mediators. Observational studies investigating these topics must adequately address bias to ensure exposure groups are balanced (exchangeability), including confounding (e.g. age), selection, collider, information, and time-dependent biases, including time-varying factors (e.g. kidney function, hypotension).^228–231 Methodological considerations include causal diagrams (in particular, directed acyclic graphs), propensity scores, sensitivity analyses, effect modification, and causal mediation.^232–237 For example, causal mediation analysis allows us to estimate the direct effect of an exposure (e.g. ACE inhibitor/ARB use) on outcomes and the indirect effect mediated through the RAS (e.g. Ang II, ACE2). Robust observational studies are providing meaningful data sooner and can supplement ongoing RCTs centered on the question of ACE inhibitor/ARB use during COVID-19. Within an RCT, RAS measurements can serve as surrogate outcomes to inform whether the RAS is responsible for the observed treatment effects. Critical appraisal of RCT results is crucial, including determining if the results are generalizable beyond the study population and if major sources of bias have been adequately addressed.

Conclusion

The interaction between the SARS-CoV-2 S protein and ACE2 has generated renewed and great interest in the RAS. Many in the research community are asking questions regarding if and how the RAS modulates SARS-CoV-2 infectivity and COVID-19 morbidity and mortality. We need to ensure that researchers and those who read these studies, especially those who may be unfamiliar with the RAS, have a guide to avoid the common missteps that occur in interrogating this elaborate system.. We highlight the myriad possible ways in which the RAS could play a role in COVID-19 pathophysiology.

The clinical manifestations of COVID-19 include detrimental effects to the heart, central nervous system, lungs, vasculature, kidneys, and GI tract. These system-wide effects are further augmented by derangements of hemolysis, thrombosis, and immune system hyperactivation. Components of the RAS, including ACE2, are expressed throughout each of these organ systems by a variety of cells. Unravelling how the RAS participates in SARS-CoV-2 infectivity and organ damage is a crucial goal. Importantly, as many patients are on inhibitors of the RAS for cardiovascular or kidney disease indications, it is of urgent need to better understand how their risks may be altered.

Fortunately, there are many tools at the disposal of the researcher to examine each of the components of the RAS. However, it is important to note the caveats that exist for each of their uses and to fully understand the strengths and weaknesses of each methodological approach, including enzyme and peptide analysis and animal models and clinical studies. Furthermore, many other components of this system, such as aldosterone, should be measured and considered. Thus, the framework we provide in this review will help guide researchers when developing both clinical and pre-clinical research proposals.

In conclusion, the COVID-19 pandemic has resulted in a dramatic shift in RAS research that traditionally has focused on cardiovascular disease to one that now encompasses another important respiratory viral infection and with further emphasis on the ACE2/Ang-(1–7) pathway. It is important to reflect on lessons learned from the last several decades in research on the RAS and juxtapose this with the pressing need to understand how COVID-19 pathogenesis occurs. The resultant focus on the RAS in SARS-CoV-2 biology will also lead to additional insights, tools, and discoveries into how this important regulatory system potentiates kidney and cardiovascular disease.