BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes

Abstract

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood.

Method

In this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing.

Result

Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes.

Conclusion

These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro . These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

Significant statement

Many patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

SciScore for 10.1101/2021.11.16.468893: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Cell culture conditions: All cell lines used for this study were obtained under appropriate material transfer agreements and approved by all involved institutional review boards.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: All cells were tested for and shown to be devoid of mycoplasma contamination (Mycoplasma PCR Detection Kit from abm,

Table 2: Resources

Antibodies
Sentences	Resources
, goat anti-rabbit (CST; 7074) or goat anti-mouse (CST; 7076) antibody was added, and the blot was incubated for 1 hour at room temperature.	anti-rabbit suggested: (Assay Biotech Cat# B7076, RRID:AB_10684…

SciScore for 10.1101/2021.11.16.468893: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Cell culture conditions: All cell lines used for this study were obtained under appropriate material transfer agreements and approved by all involved institutional review boards.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: All cells were tested for and shown to be devoid of mycoplasma contamination (Mycoplasma PCR Detection Kit from abm,

Table 2: Resources

Antibodies
Sentences	Resources
, goat anti-rabbit (CST; 7074) or goat anti-mouse (CST; 7076) antibody was added, and the blot was incubated for 1 hour at room temperature.	anti-rabbit suggested: (Assay Biotech Cat# B7076, RRID:AB_10684258) anti-mouse ( CST; 7076 suggested: None
We performed the blocking experiment using an ACE2 polyclonal goat antibody (Cat # AF933; R&D systems) and CD147 (BSG) mouse monoclonal antibody (Human TRA-1-85/CD147 MAb (Clone TRA-1-85)-MAB3159; R&D systems).	CD147 suggested: None
The human iPS cell-induced podocytes were pre-treated with serial dilutions of ACE2 antibody, BSG/CD147 antibody or both for 1 hour.	ACE2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Human colon epithelial (Caco-2) (ATCC, HTB-37) and human embryonic kidney (HEK 293T) (ATCC, CRL-3216	Caco-2 suggested: None
Production of SARS-CoV-2 S-pseudotyped lentiviral particles: The S-pseudotyped lentiviral particles were generated by transfecting HEK 293T cells as illustrated in Figure 1C.	HEK 293T suggested: None
Appropriate volumes of transfection mixture was used to transfect HEK 293Tcells in each flask and incubated in a 37 °C incubator with 5% CO2 for 6 hours.	HEK 293Tcells suggested: None
SARS-CoV-2 USA-WA1/2020 (BEI Resources; NR-52281) was propagated in Vero E6 cells at a MOI of 0.001 in DMEM supplemented with 2% FBS, 1X Penicillin/Streptomycin, 1 mM Sodium pyruvate and 1X Non-Essential Amino Acid (NEAA) at 37 °C in 5% CO2.	Vero E6 suggested: RRID:CVCL_XD71)
Recombinant DNA
Sentences	Resources
(Spike envelope plasmid; Sinobiologicals – Cat #: VG40589-UT) and pLJM1-EGFP (reporter; Addgene plasmid #19319) in a ratio of 1:1:2 respectively.	pLJM1-EGFP suggested: RRID:Addgene_19319)
Lentivirus pseudotyped with the vesicular stomatitis virus spike G (pCMV-VSV-G; Addgene plasmid # 8454) was used as a positive control and the bald lentivirus having no coat was used as a negative control.	pCMV-VSV-G suggested: RRID:Addgene_8454)
podocin (Abcam, ab50339); anti-SARS-CoV2 spike (ProSci, 3525); anti-SARS-CoV-2 N protein (Sinobiological, 40143-R019); VSV-G (Santa Cruz Biotechnology, sc-365019); Human/Mouse/Rat/Hamster ACE-2 (R&D systems, AF933), Human TRA-1-85/CD147 (R&D systems, MAB3195)	VSV-G suggested: RRID:Addgene_138479)
Software and Algorithms
Sentences	Resources
Briefly, human induced pluripotent stem (iPS) cells cultured on Matrigel-coated plates were dissociated with warm enzyme-free dissociation buffer (Gibco, 13150-016) and centrifuged twice at 200xg for 5 min each in advanced DMEM/F12 (Gibco; 12634010).	Gibco suggested: None
BioGRID is an expansive database of experimentally verified protein-protein and genetic interactions as assembled and curated from tens of thousands of studies.	BioGRID suggested: (BioGrid Australia, RRID:SCR_006334)
Briefly, the raw expression data were normalized by robust multiarray averaging 58 and the Human Gene 2.0 ST Affymetrix array mapping obtained from the ENSEMBL mart database was used to map probe IDs to gene IDs.	Human Gene suggested: (Human Gene Connectome, RRID:SCR_002628) ENSEMBL suggested: (Ensembl, RRID:SCR_002344)
The podocyte transcriptomic data was analyzed using the Bioconductor packages, oligo (v3.11), biomaRt, and pd.hugene.2.0.st.59 The expression data for these proteins were then used to annotate a network visualization of these interactions on Cytoscape.	Bioconductor suggested: (Bioconductor, RRID:SCR_006442) Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
For all statistical analyses, the GraphPad Prism 9 software package was used (GraphPad Software).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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