Impaired ACE2 glycosylation and protease activity lowers COVID‐19 susceptibility in Gitelman's and Bartter's syndromes

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Impaired ACE2 glycosylation and protease activity lowers COVID-19 susceptibility in Gitelman's and Bartter's syndromes
Dear Editor, The SARS-CoV-2 pandemic (COVID-19) has focused attention on the renin angiotensin system (RAS), specifically angiotensin-converting enzyme 2 (ACE2) as it serves as the entry point of the SARS-CoV-2 virus [1]. The SARS-CoV-2 virus attaches to its target cell via its surface spike (S) protein binding to ACE2 [2], which is then followed by subsequent fusion of the viral envelop with the host cell membrane through the action of specific proteases, such as cathepsin (Cat)-L [3]. ACE2, part of the RAS counter-regulatory system, opposes the activity of the regulatory RAS ACE/angiotensin II/AT1R axis by inducing endothelial-dependent vasodilation, and antiproliferative and anti-inflammatory effects [4]. However, ACE2's role in the cellular entry of the virus raised clinical concerns regarding increased SARS-CoV-2 infection in patients treated with angiotensin receptor blockers or ACE inhibitors that increase ACE2 levels, although it is now clear that ACE2 upregulation has a protective impact on COVID-19 morbidity and mortality [5,6].
During the first Italian wave of the COVID-19 pandemic in early 2020, we assessed via telephone survey the impact of COVID-19 on our cohort of 128 GS/BS patients living in the main northern Italy COVID-19 hotspots. We found that none * Giovanni Bertoldi and Verdiana Ravarotto contributed equally to this work.
[Correction included on 01 August 2022 after original online publication: The copyright and legal statement has been updated.] of them had COVID-19 symptoms compared to the adjusted northern Italian general population's COVID-19 prevalence (p < 0.008), which again suggests that increased risk of COVID-19 due to increased ACE2 is unlikely [8]. A second survey on the same cohort 1 year later found that only eight patients tested positive for COVID-19, of which four were asymptomatic and four had very mild symptoms. Based on this and considering GS/BS patients' increased ACE2 levels, we sought to investigate the possible factors that render GS/BS patients at a minimum resistant to COVID-19. Given that blocking ACE2/viral S protein interaction is effective against SARS-COV-2 infection and that increased pH, a feature of GS/BS, has been shown to interfere with ACE2 glycosylation (Refs. 1 and 2 in the Supporting Information), we recruited 20 GS/BS patients from the previous survey (13 females, 7 males, 32-68 years), with either GS (n = 19) or BS (n = 1) and 15 healthy controls (seven females, eight males, 29-52 years) and assessed the levels of mononuclear ACE2 and its glycosylation alongside plasma Cat-L activity (Supporting Information Methods). GS/BS patients had higher nonglycosylated ACE2 levels (0.82 ± 0.19 d.u. vs. 0.67 ± 0.13 p = 0.01) and lower Cat-L activity (3.91 ± 1.13 r.f.u. vs. 5.31 ± 0.8 p < 0.001) (Fig. 1A,B) compared to healthy subjects. In addition, GS/BS's Cat-L activity inversely correlated (p < 0.001, r = 0.78) (Fig. 1C) with blood bicarbonate (HCO 3 − ), while a negative correlation between ACE2 glycosylated isoform and HCO 3 − approaches statistical significance (p = 0.08).
The genetic defects of GS/BS inducing metabolic alkalosis alter chloride transport. Chloride anion (Cl − ) is a key factor in cellular homeostasis as changes in intracellular Cl − concentration drive gene and protein expression, post-translational modification, and intracellular/extracellular pH [9]. Endo-lysosomal pH plays a critical role for the and Cat-L activityalongside the trend toward a negative correlation between blood HCO 3 − and the glycosylated isoform of ACE2-also suggests that GS/BS patients' metabolic alkalosis underlies these effects.

Fig. 1 (A) Protein expression of glycosylated (120 kDa) and nonglycosylated (90 kDa) isoforms of ACE2 in Gitelman's and Bartter's syndromes (GS/BS) and healthy control subjects (CTRL). Densitometric analysis of western blot products shows higher expression of nonglycosylated form in GS
GS/BS's higher level of nonglycosylated ACE2 alongside the reduced Cat-L activity, which has also been shown to be pH dependent, suggests that the endosomal processing system in GS/BS patients is impaired.
Both glycosylated ACE2 and Cat-L activity are critical for SARS-CoV-2 binding and infection [8]. The increased nonglycosylated ACE2 and decreased Cat-L activity found in GS/BS patients provide a mechanistic explanation for the near absence of COVID-19, and the very small number of GS/BS SARS-CoV-2 positives found either asymptomatic or with minimal symptoms. In addition, our find-ings 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 that avoid the potentially deleterious heart rhythm effects of hydroxychloroquine and chloroquine.