Impaired ACE2 glycosylation and protease activity lowers COVID-19 susceptibility in Gitelman’s and Bartter’s syndromes

Introduction: Gitelman's and Bartter's syndromes (GS/BS) are two rare genetic tubulopathies which present with metabolic alkalosis and increased ACE2 levels. ACE2 serves as the entry point of the SARS‐CoV‐2 virus. The virus attaches to its target cell via its surface spike (S) protein binding to ACE2 and spreads into the cells through the action of specific proteases, such as cathepsin (Cat‐L), whose activity relies on an acidic enviroment. During the first two years of the COVID 19 pandemic it has been assessed via telephone surveys the impact of COVID‐19 on a cohort of 128 GS/BS patients living in the main northern Italy: none of them suffered major syntoms suggesting a natural protection from the disease. 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 and protease activity, we recruited 20 GS/BS patients (13 females, 7 males, 32–68 years), with either GS (n = 19) or BS (n = 1) and 15 healthy controls (7 females, 8 males, 29–52 years) and assessed the levels of mononuclear ACE2 and its glycosylation alongside plasma Cat-L activity. Material and methods: ACE2 profile of protein expression was assessed using Western Blot analysis. Cat-L activity was measured using a commercially available fluorescence-based assay. Metabolic alkalosis, in terms of bicarbonate blood levels, was assessed through hemogasanalysis. Results 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) compared to healthy subjects. In addition, GS/BS's Cat‐L activity inversely correlated (p < 0.001, r = 0.78) with blood bicarbonate (HCO3 −), while a negative correlation between ACE2 glycosylated isoform and HCO3 − approaches statistical significance (p = 0.08). Discussion/Conslusions: Endo‐lysosomal pH plays a critical role for the endocytic uptake of SARS‐CoV‐2. Increased intracellular organelle pH interferes with both ACE2 glycosylation and the binding via S protein as observed in experiments with chloroquine (CQ). The inverse correlation observed in GS/BS between blood HCO3 – and Cat‐L activity, alongside the trend toward a negative correlation between blood HCO3 − and the glycosylated isoform of ACE2 suggest that GS/BS patients’ metabolic alkalosis underlies these effects; this explains the protection against COVID 19 that has been observed in telephone surveys. These data have been replicated in patients with Fabry disease (FD) confirming that an alteration in the endosomal system can determine protection against the virus. The altered endosomal processing provides a robust mechanistic rationale for the effects of the combination of nirmatrelvir-ritonavir (Paxlovid), which exerts its effect via inhibition of proteins involved in lysosomal processes key for SARS-CoV-2 cell entry and replication; furthermore it provides an “in vivo” human model where the effects of endosomal pH, ACE2 glycosylation status and Cat-L activity alter SARS-CoV-2 infection rate and severity and point to these as new potential targets to fight COVID-19.