ABSTRACT
Objective: ACE2, part of the counterregulatory arm of the ennin-angiotensin system, serves both as protective toward oxidative stress and cardiovascular ennin ling and as key entry for SARS-CoV-2. ACE2 has two isoforms, non-glycosylated and glycosylated, being this latter accountable for the binding with SARS-CoV-2. After the binding, viruses use proteases as cathepsin-L (Cat-L) to entry the cells. Both ACE2 glycosylation and Cat-L activity are pH-dependent. Gitelman and Bartter syndromes (GS/BS), rare genetic tubulopathies, are characterized by electrolytic alterations, activation of the ennin-angiotensin system, yet normo-hypotension, increased levels of ACE2 and metabolic alkalosis with likely increased intracellular pH. We reported that during the first wave of COVID-19 in early 2020 none of our cohort of 128 GS/BS patients from the major hotspots in Northern Italy had been infected or suffered any major COVID-19 symptoms and in a second survey on the same cohort in 2021 we reported only 8 positives, 4 asymptomatic and 4 with very light symptoms This study aims to investigate potential mechanisms as ACE2 glycosylation and Cat-L activity related to patients' metabolic alkalosis and viral entry/infection. Design and method: Mononuclear cells ACE2 glycosylation (Western blot) and blood Cat-L activity (ELISA) from 20 GS/BS patients have been compared to those from 15 heathy subjects. Results: Non-glycosylated ACE2 was higher in GS/BS (0.82 ± 0.19 d.u. vs 0.67 ± 0.13 p = 0.01);glycosylated ACE2 was not different (0.85 ± 0.28 in GS/BS vs 0.73 ± 0.23 p = 0.19). Cat-L activity was lower in GS/BS (3.90 ± 1.13 r.f.u. vs 5.31 ± 0.8 p < 0.001) and inversely correlated with blood bicarbonate (HCO3-), while a negative correlation between glycosylated ACE2 and HCO3- approaches statistical significance (p = 0.08). Conclusions: GS/BS's metabolic alkalosis, likely by increasing intracellular pH, influences the glycosylation of ACE2 and the activity of Cat-L, providing a mechanistic explanation for the near complete absence of COVID-19 or its symptoms reported in our cohort. These findings provide a rationale for pursuing the identification and/or synthesis of new drugs that specifically target ACE2 glycosylation and/or proteases involved in SARS-CoV-2 infection.
ABSTRACT
BACKGROUND AND AIMS: ACE2, part of the counter-regulatory arm of the renin-angiotensin system, serves both as protective toward oxidative stress and cardiovascular remodeling and as a key entry for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 has two isoforms, non-glycosylated and glycosylated, the latter being accountable for the binding with SARS-CoV-2. After the binding, viruses use proteases as cathepsin-L (Cat-L) to entry the cells. Both ACE2 glycosylation and Cat-L activity are pH-dependent. Gitelman and Bartter syndromes (GS/BS), rare genetic tubulopathies, are characterized by electrolytic alterations, activation of the renin-angiotensin system, yet normo-hypotension, increased levels of ACE2 and metabolic alkalosis with likely increased intracellular pH. We reported that during the first wave of COVID-19 in early 2020 none of our cohort of 128 GS/BS patients from the major hotspots in Northern Italy had been infected or suffered any major COVID-19 symptoms and in a second survey on the same cohort in 2021, we reported only eight positives, four asymptomatic and four with very light symptoms This study aims to investigate potential mechanisms as ACE2 glycosylation and Cat-L activity related to patients' metabolic alkalosis and viral entry/infection. METHOD: Mononuclear cells ACE2 glycosylation (Western blot) and blood Cat-L activity (ELISA) from 20 GS/BS patients have been compared with those from 15 healthy subjects. RESULTS: Non-glycosylated ACE2 was higher in GS/BS (0.82 ± 0.19 d.u. versus 0.67 ± 0.13, P = 0.01);glycosylated ACE2 was not different (0.85 ± 0.28 in GS/BS versus 0.73 ± 0.23, P = 0.19). Cat-L activity was lower in GS/BS (3.90 ± 1.13 r.f.u. versus 5.31 ± 0.8, P <0 0.001) and inversely correlated with blood bicarbonate (HCO3 -), while a negative correlation between glycosylated ACE2 and HCO3 - approaches statistical significance (P = 0.08). CONCLUSION: GS/BS's metabolic alkalosis, likely by increasing intracellular pH, influences the glycosylation of ACE2 and the activity of Cat-L, providing a mechanistic explanation for the near-complete absence of COVID-19 or its symptoms reported in our cohort. These findings provide a rationale for pursuing the identification and/or synthesis of new drugs that specifically target ACE2 glycosylation and/or proteases involved in SARS-CoV-2 infection.
ABSTRACT
BACKGROUND AND AIMS: COVID-19 is spreading globally with Angiotensin Converting Enzyme (ACE)-2 serving as the entry point of SARS-CoV-2 virus. This raised concerns how ACE2 and Renin-Angiotensin (Ang)-System (RAS) are to be dealt with given their involvement in COVID-19's morbidity and mortality. Specifically, increased ACE2 expression in response to treatment with ACE inhibitors (ACEi) and Ang II receptor blockers (ARBs) might theoretically increase COVID-19 risk by increasing SARS-CoV-2 binding sites. However, ACE2 is part of the protective counter-regulatory ACE2-Ang1-7-MasR axis, which opposes the classical ACE-AngIIAT1R regulatory axis. We used Gitelman's and Bartter's syndromes (GS/BS) patients, rare genetic tubulopathies, who have endogenously increased levels of ACE2, to provide more insight on these issues. METHOD: 128 genetically confirmed GS/BS patients, living in Lombardia, Emilia Romagna and Veneto, the Northern Italy hot spots for COVID-19, were surveyed via telephone survey regarding COVID-19. RESULTS: The survey found no COVID-19 infection and absence of COVID-19 symptoms in any patient. Comparison analysis with the prevalence of COVID-19 in those Regions [8.96% (95% CI 8.96-8.99% vs 0.00% (95% IC 0.00-3.62%)] showed statistical significance (p<0.01). CONCLUSION: The results of the study contribute to suggest that increased ACE2 does not increase risk of COVID-19 and that ACEi and ARBs by blocking excessive AT1Rmediated Ang II activation, might favour the increase of ACE2-derived Ang 1-7. The GS/BS patients' increased ACE2 and Ang 1-7 levels and their characteristic chronic metabolic alkalosismight suggest for SARS-COV-2 amechanism similar to that of chloroquine/ hydroxychloroquine effect altering ACE2 glycosylation which resulted, in previous studies, in SARS-COV binding inhibition and block/inhibition of viral entry. Studies from our laboratory are ongoing to explore in GS/BS ACE2 glycosylation.
ABSTRACT
Objective: ACE2 in addition to be the counterregulatory enzyme in RAS system is the entry point of SARS-CoV-2. Several studies have reported its protective role in SARS-CoV-2-induced Acute Distress Syndrome (ARDS) and a relationship between Rho kinase (ROCK), ARDS and increased ACE2 levels induced by ROCK inhibitors. Cells' ACE2 expression is in two different immunoreactive forms, glycosylated and unglycosylated, but only the glycosylated is associated with viral binding. ACE2-glycosylation is driven by the acidic pH of intracellular endosomes/lysosomes/Golgi vesicles and this represents the rational to the prophylactic/therapeutic use of antimalarial drugs as hydroxychloroquine/chloroquine weak bases that increase the pH of vesicles, which leads to the production of unglycosylated ACE2 and therefore reduction/inhibition of viral binding. Gitelman's and Bartter's syndrome patients (GS/BS) (rare genetic tubulopathies) have activated RAS, high Ang II yet normotension/hypotension, metabolic alkalosis increased and related ACE2 and its product Ang 1-7 levels, reduced ROCK activity therefore may provide information on the protective role of increased ACE2 levels and ROCK inhibition. This study has assessesed the impact of COVID-19 on GS/BS Design and method: We performed a telephone survey on more than 100 GS/ BS from the hotspots of COVID-19 pandemic in Northern Italy (Veneto, Lombardia Emilia Romagna). COVID-19 prevalence in the general population was obtained from Civil Protection's official data and the estimated prevalence from published data. 95% CI values processed with Clopper-Pearson method using Rsoftware Results: None of GS/BS reported infection/symptoms of COVID-19, which was statistically not signi ficant vs the prevalence of COVID-19 in the general population in the Italian hotspots due to the low prevalence of GS/ BS (rare diseases) (95% CI 0.0-3.0% vs 0.65%, 95% CI 0.6-0.7%). It becomes signi ficant vs the estimated prevalence in the general population in the hotspot regions (8.7%, 95% CI 8.7-8.8%, (C2), p=0.004) Conclusions: GS/BS might serve as a human model of increased ACE2 for exvivo mechanistic investigation on viral binding;provide additional evidence on the absence of high ACE2 levels-increased risk of COVID-19;demonstrate that suspension of treatments with ACE inhibitors/ARBs, which increase ACE2 expression to reduce the risk of infection is not rationale.