key: cord-0829506-pgsp8mnr
authors: Wu, Congqing; Ye, Dien; Mullick, Adam E.; Li, Zhenyu; Danser, A.H. Jan; Daugherty, Alan; Lu, Hong S.
title: Effects of Renin-Angiotensin Inhibition on ACE2 and TMPRSS2 Expression: Insights into COVID-19
date: 2020-06-18
journal: bioRxiv
DOI: 10.1101/2020.06.08.137331
sha: 7789866d4927965efc3e6f24f21bb14852e2f553
doc_id: 829506
cord_uid: pgsp8mnr

Angiotensin-converting enzyme 2 (ACE2), a component of the renin-angiotensin system, is a receptor for SARS-CoV-2, the virus that causes COVID-19. To determine whether the renin-angiotensin inhibition regulates ACE2 expression, either enalapril (an angiotensin-converting enzyme inhibitor) or losartan (an AT1 receptor blocker) was infused subcutaneously to male C57BL/6J mice for two weeks. Neither enalapril nor losartan changed abundance of ACE2 mRNA in lung, ileum, kidney, and heart. Viral entry also depends on transmembrane protease serine 2 (TMPRSS2) to prime the S protein. TMPRSS2 mRNA was abundant in lungs and ileum, modest in kidney, but barely detectable in heart. TMPRSS2 mRNA abundance was not altered by either enalapril or losartan in any of the 4 tissues. Next, we determined whether depletion of angiotensinogen (AGT), the unique substrate of the renin-angiotensin system, changes ACE2 and TMPRSS2 mRNA abundance. AGT antisense oligonucleotides (ASO) were injected subcutaneously to male C57BL/6J mice for 3 weeks. Abundance of ACE2 mRNA was unchanged in any of the 4 tissues, but TMPRSS2 mRNA was significantly decreased in lungs. Our data support that the renin-angiotensin inhibition does not regulate ACE2 and hence are not likely to increase risk for COVID-19.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang)I and II, and additionally is a cellular receptor for SARS-CoV-2, the virus that causes COVID-19. Viral entry into host cells occurs through binding of the viral spike (S) protein and ACE2. 1 Preclinical data suggest that renin-angiotensin system (RAS) blockers upregulate ACE2. 2, 3 As a consequence, RAS blockers have been suggested to increase the risk of developing a severe and fatal SARS-CoV-2 infection. However, recent large retrospective studies strongly argue against this hypothesis and rather suggest that RAS blockers might be protective in such patients. 4 Since the findings on RAS blocker-induced ACE2 upregulation are inconsistent, and differed not only per type of RAS inhibitors (ACE inhibitors versus angiotensin receptor blockers [ARB]), 3 between blockers of a certain type (i.e., between various ARBs 5 ), but also per organ, and required high doses, one further option is that this ACE2 upregulation is not the unavoidable consequence of RAS suppression, but rather reflects the non-specific effects of a certain RAS blocker when applied at a high dose. Applying antisense oligonucleotides (ASO) as a tool to suppress the RAS would circumvent the latter. In the present study we therefore compared the effects of an ACE inhibitor (enalapril) and an ARB (losartan), infused subcutaneously to male C57BL/6J mice via mini osmotic pumps, with those of subcutaneously injected angiotensinogen (AGT) antisense oligonucleotides on tissue ACE2.

After 14 days of infusion, both enalapril and losartan increased plasma renin >100-fold, confirming effective RAS inhibition (Figure A) . ACE2 mRNA abundance was determined by quantitative PCR (qPCR) in lung, ileum, kidney, and heart tissues. Neither enalapril nor losartan changed the abundance of ACE2 mRNA in any of the tissues (Figure B) . Ferrario et al. 3 reported that administration of lisinopril (an ACE inhibitor) or losartan for 12 days increases ACE2 mRNA abundance approximately 3 -5 old in male rat heart. It is worth noting that ACE2 mRNA is much less abundant in heart compared with lung, ileum, and kidney (data not shown).

Viral entry also depends on transmembrane protease serine 2 (TMPRSS2) to prime S protein. 1 TMPRSS2 mRNA was highly abundant in lung and ileum, moderately in kidney, while barely detectable in heart (data not shown). Thus, ACE2 and TMPRSS2 are co-expressed most abundantly in lung and ileum, consistent with their roles in ARS-CoV-2 infection. As with ACE2, TMPRSS2 mRNA abundance was not altered by either enalapril or losartan (Figure C) .

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

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