Since the first report of coronavirus disease 2019 (COVID-19) in China on 31 December 2019 and isolation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus on January 7, 2020, the number of infections is still growing with an accelerated rate, as well as the number of deaths worldwide [1]. Considering all countries and regions, the number of infections reached > 117 million people and more than 2,600,000 deaths on March 16, 2021 [2]. Even though the illness was considered a viral infection targeting the respiratory system [3], some cases developed into septic shock [4, 5], in which acute kidney injury (AKI) plays a central role [6]. Age, cancer, cardiovascular diseases and metabolic diseases–such as diabetes–are currently considered the main risk factors for the explosive outbreak of the pandemic [7]. Even though some studies have demonstrated an increased risk for infected patients with renal lesions [8, 9], pre-renal mechanisms for AKI-and progression to chronic lesions–must be considered besides the direct interaction of the virus with ACE2. Likely, reduction of blood and O₂ supply aggravate kidney lesions during COVID-19 infection.

With respect to the kidney, it is important to remember that angiotensin converting enzyme 2 (ACE2), which is the receptor for the entrance of SARS-CoV-2 into cells [10, 11], is highly expressed in the membranes of proximal tubule cells [12], the tubular segment especially affected in AKI [13]. ACE2 is a 92 kDa Zn-metalloprotease [14, 15] that catalyzes the conversion of Ang I in Ang-(1-9) and the generation of Ang-(1-7) from Ang II [16, 17]. Due to its great abundance in heart and kidneys, it was early considered as a key regulator of the local renin-angiotensin-system (RAS) resident in these organs [15]. As a part of the ACE2/Ang-(1-7)/Ang-(1-7) receptor (Mas) axis, ACE2 antagonizes the ACE/Ang II/angiotensin type 1 receptor (AT₁R) axis [18].

Levels of ACE2 seem to be important during COVID-19 infections, and this has been reviewed recently in terms of an apparent paradox demonstrating friend-and-foe roles in the evolution of the viral infection [19]. The friend side is exemplified by the decrease in the proinflammatory Ang II when it is hydrolyzed to Ang-(1-7). However it was also demonstrated that binding of SARS-CoV-2 to ACE2 activates the ACE/Ang II/AT₁R axis triggering the cytokines storm [20]. From a historical and epidemiological perspective, it is important to mention that ACE2 has been described as a receptor for the SARS coronavirus at the beginning of the century [21], indicating that both viruses share the same mechanism in the first step of cell invasion.

ACE is another Zn-metalloprotease that catalyzes the proteolysis of Ang I to Ang II and, therefore, the enzyme that feeds downstream the ACE/Ang II/AT₁R axis within the RAS. The metalloprotease catalytic domains of ACE2 are identical [14, 15] and comparison of their genomes suggests that the two genes resulted from duplication [15]. Two isoforms have been described, membrane-bound and soluble, and the main function is attributed to the membrane-bound form, including in kidney [22, 23]. The ACE/ACE2 imbalance– at least in lung and heart–has been proposed to be critical for the outcome of COVID-19 infection [24]. With respect to the kidney, the balance between the two enzymes is considered a key parameter for the regulation of the local RAAS [25] and also for the renal impairment during SARS-CoV-2 infection [26].

The levels of ACE2 decrease with ageing in different tissues [27, 28], but there are no reports regarding the influence of the nutritional status on these levels, especially in obesity and undernutrition, which, as recently emphasized, are considered comorbidities for high risk of unfavorable outcomes [29, 30] and, therefore, must be taken into account to investigate the impact of COVID-19 [29]. The experiments with overweight and undernourished rats were conducted and presented together with data from normonourished juvenile and adult rats, thus allowing comparisons aiming to shed some light on the potential mechanisms underlying the progression, the severity and the outcomes of renal lesions due to COVID-19.

ACE2 levels decrease with age in CTR rats, in contrast to the outcome with ACE (Figure 1A and B). Juvenile rats are the CTR animals from the undernutrition study and, according to Quinn [48], 90 days of age correspond to 21 human years and 162 days (adult) correspond to 38 human years. In contrast, ACE levels remained unmodified (Figure 1A and C).

The next figures (Figures 2 and 3) show that modifications in the nutritional status profoundly modified ACE2 abundance in the renal cortex corticis, which is also modulated in a different manner by antagonizing RAAS by Ang-(3-4) in cases of overweight or undernutrition. The abundance of ACE2 decreased 30% in the HL (overweight) rats (Figure 2A and B), whose body mass was ~ 20% higher than that of the CTR group [42]. Administration of Ang-(3-4) to the overweight rats increased by 75 and 160%, when the ACE2 levels are compared with those in the CTR and HL rats, respectively, with no influence in the normonourished group. The levels of ACE (Figure 2A and C) remained unmodified either by diet or Ang-(3-4) treatment.

The influence of chronic undernutrition on renal ACE2 levels presents some similarity, but also a huge difference, compared with overweight rats (Figure 3). The similarity is in the downregulation of the enzyme level by RBD alone (~ 40%; Figure 3A and B), i.e. a value that did not differ from the overweight-induced downregulation seen in Figure 2A and B (~ 35%). In contrast with the effect observed in the case of overweight rats, the administration of Ang-(3-4): (i) did not restore (i.e. upregulate) ACE2 in undernourished rats, and (ii) provoked a similar decrease in CTR rats.

The main results described in this study are that Ang-(3-4) per se mimics the effect of undernutrition by downregulating the ACE2 abundance, whereas it upregulates the ACE2 abundance in overweight but not in normonourished rats. They also demonstrate the influence of ageing and nutritional status (overweight and undernutrition), as well as the opposite effects of Ang-(3-4) in the renal ACE2 levels in overweight and undernourished rats. The decrease in ACE2 in this short period of life (Figure 1A and B) may represent an increased the risk of kidney proximal tubule damage during SARS-CoV-2 infection in normonourished rats. ACE levels remained unmodified over that relatively long period of life (Figure 1A and C) and, therefore, this could contribute to the ACE/ACE2 imbalance that would worsen kidney injuries, as is the case with lung and heart [24], especially in the presence of comorbidities or modifications in the renal local RAAS.

The observations presented in Figure 2 support the view that, in overweight animals, antagonizing the Ang II/AT₁R axis by Ang-(3-4) might avoid adverse renal injury in SARS-CoV-2 infections. Recently, Lanza et al. [26] proposed that the RAAS imbalance is central in the pathophysiology of COVID-19. The effect of Ang-(3-4) implies that, beyond upregulation of the ACE/Ang II/AT₁R axis, intrarenal RAAS can be counteracted– in the case of obesity/overweight–by the end product of a pathway that involves progressively shorter Ang II-derived peptides, as we demonstrated a decade ago [49]. The hypothesis of ACE/ACE2 imbalance within the renal RAAS [26] in underlying adverse effects of ageing and nutritional status, however, should be considered speculative at the present stage of this study and requires further investigation, notably looking for possible lesions.

The mechanism underlying the effect of Ang-(3-4) in overweight rats might reflect its action as an “allosteric enhancer” that induces a second binding site in AT₂R with a very high affinity for Ang II [50], possibly involved in the modulation of ACE2 formation but not of ACE. Another possibility would be a beneficial formation of heterodimers involving these modified AT₂R and Mas receptors, which would be able to act on ACE2, as in the case of blood pressure [51]. That Ang-(3-4)-mediated upregulation of renal ACE2 occurred in overweight rats, but not in the CTR group, is indicative that a “pro-hypertensive tissular microenvironment” (high Ang II) [41] develops in animals fed with a diet rich in lipids, causing downregulation of ACE2 (Figure 2A and B) and kidney injury during a SARS-CoV-2 attack.

Again, it may be that activation of the Ang II/AT₁R axis now in a “pro-hypertensive tissular microenvironment” induced by the multideficient diet contributes to the notable decrease in ACE2, and, since there was no modification in ACE (Figure 3A and C), to the important ACE/ACE2 imbalance in proximal tubules that might be crucial in the kidney, as seems to be the case in the lung [24]. On this account, we also have strong evidence for the existence of a “pro-hypertensive tissular microenvironment” in chronically undernourished rats: the increased number of Ang II-positive cells in the tubulointerstitium [52] leads to an increase of the local RAAS.

The observation that Ang-(3-4) administration per se mimics the influence of undernutrition in downregulating ACE2 abundance is consistent with the supposition that AT₂R acts in parallel with AT₁R in promoting tissular damage in kidney tissue, as recently reviewed [24]. There is a possibility of an adjuvant and independent AT₂R-associated pathway that, being altered by an amino acid imbalance [53] due to undernutrition, downregulates ACE2 synthesis and levels. A second possibility is the formation of heterodimers between AT₁R and abnormal AT₂R, which could be modulated by Ang II and Ang-(3-4), as in the case of spontaneously hypertensive rats [41]. As noted above, these rats are of a juvenile age. Therefore, undernutrition may favor severe kidney lesions in young people affected by COVID-19, an issue that has not been explored so far.

In conclusion, our evidence is that overweight and undernutrition favor downregulation of the ACE2 level in proximal tubule cells, being a threat during the clinical evolution of the SARS-CoV-2 infection. However, it must be emphasized that, besides the of ACE/ACE2 imbalance, external mechanisms can contribute to the progression of renal lesions: undernutrition and overnutrition provoke local and systemic inflammation that may exacerbate the progression of the renal COVID-19 lesions [20, 30]. The results demonstrate that counteracting RAAS by Ang-(3-4) administration influences ACE2 synthesis in an opposite way in overweight and undernourished rats, indicating different underlying mechanisms, which could modify the type and severity of renal outcomes during SARS-CoV-2 infection.