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SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

Mark Donowitz, Chung-Ming Tse, Karol Dokladny, Manmeet Rawat, Ivy Horwitz, Chunyan Ye, Alison Kell, Ruxian Lin, Sun Lee, Chenxu Guo, Shang Jui Tsai, Andrea Cox, Stephen Gould, Julie In, Steven Bradfute, Nicholas C. Zachos, Olga Kovbasnjuk
doi: https://doi.org/10.1101/2021.04.27.441695
Mark Donowitz
1Departments of Medicine/GI
2Departments of Physiology
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  • For correspondence: mdonowit@jhmi.edu OKovbasnjuk@salud.unm.edu
Chung-Ming Tse
1Departments of Medicine/GI
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Karol Dokladny
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Manmeet Rawat
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Ivy Horwitz
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Chunyan Ye
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Alison Kell
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Ruxian Lin
1Departments of Medicine/GI
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Sun Lee
1Departments of Medicine/GI
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Chenxu Guo
3Departments of Medicine/ID
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Shang Jui Tsai
3Departments of Medicine/ID
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Andrea Cox
3Departments of Medicine/ID
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Stephen Gould
4Departments of Biological Chemistry
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Julie In
1Departments of Medicine/GI
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Steven Bradfute
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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Nicholas C. Zachos
1Departments of Medicine/GI
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Olga Kovbasnjuk
1Departments of Medicine/GI
5Departments of the Johns Hopkins University School of Medicine and Department of Medicine, University of New Mexico Health Sciences Center
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  • For correspondence: mdonowit@jhmi.edu OKovbasnjuk@salud.unm.edu
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ABSTRACT

Diarrhea occurs in 2-50% of cases of COVID-19 (∼8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for ∼24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ∼8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of ∼10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

Competing Interest Statement

The authors have declared no competing interest.

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Posted April 28, 2021.
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SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels
Mark Donowitz, Chung-Ming Tse, Karol Dokladny, Manmeet Rawat, Ivy Horwitz, Chunyan Ye, Alison Kell, Ruxian Lin, Sun Lee, Chenxu Guo, Shang Jui Tsai, Andrea Cox, Stephen Gould, Julie In, Steven Bradfute, Nicholas C. Zachos, Olga Kovbasnjuk
bioRxiv 2021.04.27.441695; doi: https://doi.org/10.1101/2021.04.27.441695
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SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels
Mark Donowitz, Chung-Ming Tse, Karol Dokladny, Manmeet Rawat, Ivy Horwitz, Chunyan Ye, Alison Kell, Ruxian Lin, Sun Lee, Chenxu Guo, Shang Jui Tsai, Andrea Cox, Stephen Gould, Julie In, Steven Bradfute, Nicholas C. Zachos, Olga Kovbasnjuk
bioRxiv 2021.04.27.441695; doi: https://doi.org/10.1101/2021.04.27.441695

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