key: cord-0720605-tg8ymfgv
authors: Ichimura, T.; Mori, Y.; Aschauer, P.; Padmanabha Das, K. M.; Padera, R. F.; Weins, A.; Nasr, M. L.; Bonventre, J. V.
title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney
date: 2020-09-18
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.09.16.20190694
sha: f6a4726e1b9790e928b0c8ddb14cb15ff7f64f90
doc_id: 720605
cord_uid: tg8ymfgv

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited both by anti-KIM-1 antibodies and by TW-37, our newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 positive cells express less angiotensin-converting enzyme 2 (ACE2), the well-known receptor for SARS-CoV-2. Using microscale thermophoresis, the EC50 for KIM-1-SARS-CoV-2 spike protein, and receptor binding domain (RBD) interactions, were 19 and 10 nM respectively. Thus KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

, and T-cell immunoglobulin and mucin 11 domain 1 (TIM-1), has been reported to be a receptor for Ebola virus (Kondratowicz et al., 2011), and 12 Dengue virus (Meertens et al., 2012) . KIM-1 also facilitates cellular uptake of West Nile virus (Jemielity et 13 al., 2013). KIM-1 specifically mediates internalization and transduction of Marburg virus glycoprotein (GP) 14 and full-length Ebola virus GP pseudovirions into human mucosal epithelia from the trachea (Kondratowicz 15 et al., 2011). KIM-1-mediated infection was efficiently inhibited by anti-human KIM-1 IgV domain specific 16 monoclonal antibody ARD5 (Bailly et al., 2002) . In the kidney, KIM-1 acts as a receptor for 17 phosphatidylserine exposed on the surface of apoptotic cells and for oxidized lipids (Ichimura et al., 2008) . 18 Once ligands bind to KIM-1, they are internalized by phagocytosis or endocytosis. Recently we have 19 identified TW-37 as an inhibitor of KIM-1 mediated oxidized lipid and fatty acid (bound to albumin) uptake 20 (Mori et al., 2020) . 21 The SARS-CoV-2 envelope has a spike (S) glycoprotein, consisting of S1 and S2 subunits (Du et al., 22 2009). After infection the trimeric S protein is cleaved into the two subunits and S1, which contains the 23 receptor binding domain, binds to angiotensin-converting enzyme 2 (ACE2) and is internalized by lung 24 epithelium. In addition to ACE2, carcinoembryonic antigen-related cell adhesion molecule (CEACAM) is 25 known to be a receptor for SARS-CoV (Krueger et al., 2001) . KIM-1 has a N-terminus conserved IgV 26 domain with has high homology with CEACAM's IgV domain where the S1 subunit binds (Lewicki and 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint virosomes in a KIM-1 dependent manner, whereas there was no uptake of unconjugated empty liposomes 1 (Fig. 3A) as assessed by confocal microscopy. Both hKIM-1-LLC-PK1 and pcDNA3-LLC-PK1 cells stained 2 positively for ACE2; however, the ACE2 was not membrane associated. pcDNA3-LLC-PK1 control cells 3 appeared to have more intense and defuse ACE2 staining, even though the cells did not demonstrate 4 significant uptake of the virosomes when compared to the hKIM-1-LLC-PK1 cells (Fig. 3B) . ACE2 protein 5 expression was confirmed by western blot analysis of the cell lysates from both cell lines whereas KIM-1 6 was expressed only in the hKIM-1-LLC-PK1 cells (Fig. 3C) . When ACE2 and KIM-1 were co-stained and 7 analyzed in high magnification confocal microscope images ACE2 is found mainly in cytoplasm with small 8 amounts on the cell surface, while KIM-1 expression is primarily on the cell surface (arrows) (Fig. 3D) . 9 TW-37 (10 µM) pretreatment for 30 min markedly reduced uptake of SARS-CoV-2 virosomes into hKIM-1-10 LLC-PK1 cells (Fig. 3E) . 11 12 Three dimensional human renal epithelial tubuloids take up virosomes. 13 We have developed a method to make epithelial KIM-1 expressing "tubuloids" from kidney tissue derived 14 from human subjects (Mori et al., 2020) . Human primary epithelial cell cultures were established from the 15 non-tumor kidney tissue removed from patients with renal cell carcinoma. Those primary cells were 16 cultured in suspension with growth factors and Matrigel in non-adherent dishes (see Methods). KIM-1 17 expressing cells of human tubuloids took up DiI-labeled virosomes (Fig 3F) . With uptake of spike 18 conjugated virosomes, the tubuloids maintained their tubule-like three dimensional structures as seen in 19 phase contrast images (Fig. 3G) . Since well-developed tubuloids with polar epithelium reduce expression 20 of KIM-1, likely due to progressive differentiation of the tubular epithelium, we enhanced human KIM-1 21 expression by infecting tubuloids with an adenovirus expressing KIM-1 (Adenovirus-KIM-1) or the control 22 vector expressing β-galactosidase (β-GAL) (Adenovirus-β-GAL). Both viral vectors contained a GFP 23 expression cDNA for tracing of viral infection and transgene expression in the infected cells. After 48 hours 24 of infection, we added DiI-labelled virosomes to the tubuloids (Fig. 3H) . Adenovirus-KIM-1 infected GFP- 25 positive tubuloid cells showed a high level of uptake of virosomes (Fig 3H, left) and robust expression of 26 KIM-1 protein (Fig. 3H, right) . The KIM-1 infected tubuloids showed polarized expression of KIM-1 similar 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint to proximal tubules in vivo and also expressed ACE2 (Fig. 3I) . In comparison to the Adenovirus-KIM-1 1 infected cells, control Adenovirus-β-GAL infected tubuloids showed no KIM-1 staining and no uptake of 2 virosomes or oxidized LDL (data not shown). Uptake of virosomes by the tubuloids overexpressing KIM-1 3 was dose dependent (Fig. 3J) . Quantification of ACE2 expression by flow cytometry on tubuloids infected 4 by Adenovirus-KIM-1 or control Adenovirus-β-GAL, after digestion into single cells, revealed a mean 29.8% 5 decrease on single cell ACE2 expression secondary to Adenovirus-KIM-1 infection (Fig. 3K) . To test 6 potential involvement of KIM-1 mediated viral uptake in the pro-inflammatory phenotype often seen with 7 SARS-CoV-2 infection we treated tubuloids with the S1 subunit of the Spike protein and found KIM-1 8 dependent upregulation of intracellular interleukin-1β (IL-1β) (Fig. 3L) . 9 10 KIM-1 binds SARS-COV2 Spike protein and receptor binding domain (RBD) in vitro. 11 KIM-1 binding to SARS-Cov2 spike protein ectodomain and GST-RBD was tested using Micro Scale 12 Thermophoresis. The EC50 for interaction between KIM-1 ectodomain and the Spike ectodomain was 19 13 nM with a CI (95%) of 9 to 39 nM ( Fig. 4A and 4C) . Interaction of the KIM-1 ectodomain with showed an EC50 of 10 nM with a confidence interval (CI) (95%) from 6 to 16 nM ( Fig. 4B and 4C) . 15 16 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We have found that KIM-1 serves as a receptor for SARS-CoV-2 virosomes in alveolar and kidney 2 proximal tubule epithelial cells and uptake is inhibited by anti-KIM-1 antibodies or TW-37, an inhibitor for 3 KIM-1-mediated endocytosis (Mori et al., 2020) . Inhibition of this KIM-1 mediated uptake may be 4 therapeutically useful in SARS-CoV-2 infection. The fact that KIM-1 binds to the spike protein of SARS-5

CoV-2 and virosomes stimulated IL-1β, an inflammatory mediator, in a KIM-1 dependent fashion suggests 6 that KIM-1 may be involved in the pro-inflammatory state that can be so profound and has been associated 7 with a "cytokine storm" (Moore and June, 2020). KIM-1 protein is detectable in partially autolyzed autopsy 8 samples due to the protein's stability (Yin et al., 2018) . KIM-1 expressing proximal tubules have reduced 9 expression of ACE2, the well-known receptor for SARS-CoV-2 (Hoffmann et al., 2020), suggesting that 10 KIM-1 may alter ACE2-mediated viral entry into cells. 11 While the role of SARS-CoV-2 viral infection of kidney cells remains controversial, the implications of 12 our findings extend beyond the kidney tubule cells since the ectodomain of KIM-1 enters the blood and 13 can interact with the virus there. KIM-1 is upregulated by many insults to the kidney which result in AKI. 14 We have shown that acute upregulation of KIM-1 with injury is protective likely due to an anti-inflammatory 15 and anti-obstructive response associated with phagocytosis of apoptotic debris in the tubule (Yang et al., 16 2015). Our findings that KIM-1 is a receptor for SARS-CoV-2 indicate that KIM-1 could interact with 17 exfoliated and virus infected cells in the lung and kidney. Furthermore, the KIM-1 ectodomain may bind to 18 the virus in the pulmonary alveoli and kidney tubular lumen and could also act as a decoy receptor. 19 KIM-1 facilitated endocytosis of SARS-CoV-2 virosomes is both KIM-1 and spike protein dependent. 20 Jemielity et al. demonstrated that the phosphatidylserine binding characteristics of human KIM-1/TIM-1 21 mediate its activity as a SARS-CoV-1 pseudovirus receptor (Jemielity et al., 2013) . It is important to 22 recognize, however, that the virosomes we used do not contain phosphatidylserine, so the interaction of 23 KIM-1 with this phospholipid cannot explain the KIM-1 mediated facilitation of uptake. The high affinity 24 interaction between the receptor binding domain of the S1 subunit with KIM-1 confirms that the KIM-1 25 mediated binding interaction is with the spike protein of SARS-CoV-2. 26 Our recently discovered KIM-1 small molecule inhibitor, TW-37, blocked KIM-1-mediated endocytosis 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint of SARS-CoV-2 virosomes in renal epithelial cells. TW-37 was originally discovered as a bcl-2 inhibitor in 1 cancer drug screening, and has been found safe to use in animals (Ahn et al., 2019; Lei et al., 2017) . Our 2 finding that TW-37 blocked entry of the SARS-CoV-2 virosomes suggests prevention of viral internalization 3 as an approach to anti-SARS-CoV-2 viral treatment. This would be reminiscent of the use of Enfuvirtide, 4 Maraviroc and Ibalizumab for HIV where administration of agents that will bind to the virus in the circulation 5 will compete for tissue binding (Henrich and Kuritzkes, 2013). 6

Using our newly established three-dimensional renal epithelial tubuloid system we demonstrated 7 uptake of the virosomes and enhancement of IL-1β production after tubuloid exposure to the SARS-CoV- 20 We found clear evidence for viral infection in one patient but the infection was quite focal and could easily 21 have been missed if the specimen was a biopsy during to sampling bias. It is possible there is early 22 infection of the kidney but then the virus is cleared by the time the kidney specimen is obtained later in the 23 course of illness in the patient who develops AKI or succumbs to the COVID-19 disease. This viral 24 clearance has been reported to occur in the lung also (Schaefer et al., 2020) . It is also possible that KIM-25 1-bound virus may shunt the virus to degradation pathways to destroy them, as occurs with HIV in 26 macrophages (Gobeil et al., 2012). This is compatible with KIM-1 uptake of injurious material by 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . phagocytosis followed by degradation in a special autophagic pathway (Brooks et al., 2015). While KIM-1 1/TIM-1 mediated SARS-CoV-1 entry to the human cells has been reported to be facilitated by KIM-1, KIM-2 1 does not enhance infectivity (Jemielity et al., 2013) . Our finding that the uptake of the spike protein 3 promotes pro-inflammatory signaling, suggests, however, that even if the binding of the virus by KIM-1 4 does not promote infectivity it may be detrimental contributing the cytokine storm and worsening of the 5 lung and kidney disease. 6

In conclusion, KIM-1 is a receptor for SARS-CoV-2 with a high affinity interaction with the receptor 7 binding domain of the virus S1 subunit of the spike protein. KIM-1 dependent uptake by lung and kidney 8 cells can be inhibited by anti-KIM-1 antibodies and TW-37. This may have important implications for viral 9 entry, triggering of the cytokine storm, and/or inactivation of the virus through viral degradation or decoy 10 function in the kidney and respiratory mucosa. Targeted treatment directed at the KIM-1-SARS-CoV-2 11 interaction may be both therapeutic and prophylactic for this devastating disease that occurs secondary to 12 infection. 13 14 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Correspondence and requests for materials should be addressed to J.V.B.. 20 21 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this this version posted September 18, 2020. Laboratories, Burlingame, CA) and were incubated with primary antibodies for 1 hour at room temperature. 11 After washing with PBS, sections were incubated with secondary antibodies for 30 minutes. Vectashield 12 (Vector Laboratories, Burlingame, CA) containing DAPI (12.5 μg/ml) was applied and the slides were 13 cover-slipped. All images were obtained by confocal microscopy (C1 Eclipse from Nikon). 

Laboratories. All mouse work was performed in accordance with the animal use protocol approved by 25 the Institutional Animal Care and User Committee of the Harvard Medical School. Mice aged 8-12 weeks 26 and weighing 20-22 g were subjected to IRI according to procedures as described previously (Brooks et 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint al., 2015; Park et al., 2003) . Briefly, both kidneys were exposed by flank incisions, and the renal pedicles 1 were clamped for 25 min at 37°C. Following surgery, 1 ml of warm saline (37°C) was injected 2 intraperitoneally for volume supplement. Sham operations were performed by exposing both kidneys 3 without clamping of renal pedicles. Kidney tissue was collected at 24 hours or 48 hours post IRI. Women's Hospital in Boston, US, by modifying a previously established protocol (Ichimura et al., 2008) . 8 The protocol was approved by the Institutional Review Board of the Ethics Committee of Partners 9

Healthcare. Mouse primary lung epithelial cells were obtained and cultured by using the same protocol 10 (Ichimura et al., 2008) . Briefly, human renal cortex was diced, or mouse lung was taken out after sacrificed 11 and was diced. They were digested with collagenase (0.5 mg/ml) in DMEM/F12 50:50 media for 40 minutes. 12

The enzyme reaction was terminated with fetal bovine serum (FBS). Glomeruli and other remaining tissue 13 clumps were separated from tubules by decanting the samples after gravity sedimentation (2 minutes). 14 The remaining sample was washed 2 times in media and tubules were resuspended in tubular cell culture 15 medium (DMEM/F12 with BSA, transferrin, insulin, selenium, hydrocortisone, and epidermal growth factor 16 (EGF)). The epithelial cells were cultured for 7 to 14 days before being used for experiments. His tags at the C-terminal. The gene was cloned into pGS21a vector at BamH1/NotI restriction sites. The 24 resulting '6XHis-GST-TEV-RBD-Avi-6XHis' construct was transformed into BL21-Codon Plus (DE3) 25 competent cells and the cultures were grown at 37 °C in LB medium containing Kanamycin to an OD600 of 26 0.6. Cells were cooled down to 16 °C and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside. 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint Following overnight induction, cells were lysed in lysis buffer (20 mM Sodium phosphate buffer pH 7.4, 1 250 mM NaCl, 0.1% Titron X-100, 2.5 mM β mercaptoethanol) and purified using Ni affinity chromatography. 2 The GST fusion protein was further purified by binding to Resource S ion-exchange column (GE 3 Healthcare, USA) in the presence of (20 mM phosphate buffer pH 6.0, 50 mM NaCl, 1 mM DTT, 1mM 4 EDTA), and eluting with a salt gradient, before loading onto a Superdex 200 column (GE Healthcare, USA) 5 in a buffer containing 20 mM phosphate buffer pH 7.4, 137 mM NaCl, 1 mM DTT, 1 mM EDTA. 6 7 Virosomes assembly and purification. Purified SARS-CoV-2 spike ectodomain with deletion of the furin 8 cleavage site, two proline mutations, a foldon trimerization domain and a C terminal His tag was used in 9 the virosome assembly. Chloroform lipid stocks were mixed to accomplish the following ratio 15% 1,2- His-tagged Spike ectodomain to achieve a ratio of 40:1 (spike-trimer: liposome) The mixture was incubated 18 for 30 min on 4 °C to allow the spike His-tag to bind to the Ni-NTA lipids. Free spike protein was separated 19 from spike loaded liposomes using a Superose 6 10/300 size exclusion chromatography column. loaded in coated capillary (coated in house) and measured using a Monolith Pico (Nano Temper). The 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint samples were measured using the pico-red channel on medium MST power and excitation power 20%. 1 For GST-RBD interaction with KIM-1, we followed the same procedure as above with the difference that 2 GST-RBD was labeled was RED-tris-NTA and KIM-1 was used for the serial dilution. The excitation power 3 used for GST-RBD was 60%. The highest concentration of the dilution series was 3.3 μM. Processing of 4 the raw data was done in Microsoft Excel, data points between 5 s and 10 s were averaged. The results 5 were normalized between 0 and 1 and exported to GraphPad where nonlinear fitting and calculation of the 6 EC50 was done. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . Biosciences). Data were analyzed using FLOWJO (FLOWJO). 1 2 Immunofluorescence staining of cells. After fixation with 4% PFA-PBS, the cells were permeabilized 3 with 0.1% Triton X-100-PBS and blocked with 3% BSA-PBS for 30 minutes. Primary antibodies were 4 applied and the slides were incubated for 1 hour at room temperature or overnight at 4 degree. After 5 washing with PBS, the slides were exposed to secondary antibodies and incubated for 30 minutes at room 6 temperature. All images were obtained by confocal microscopy (C1 Eclipse from Nikon). Western blotting. Cells and kidneys were lysed and protein was purified as previously described (Ichimura 25 et al., 1998) . Bands were visualized by chemiluminescence (Western Lightning, PerkinElmer, Waltham, 26

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The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint 1 Human renal tubuloids. The manuscript on the protocol to make human renal tubuloids is in preparation. 2 Briefly, human primary renal epithelial cells were cultured on ultra-low attachment plates with 5% FBS-3 RPMI. After 2-3 days incubation, Matrigel was added and media was changed to 5% FBS-Advanced RPMI 4 containing EGF, bFGF and HGF. Media was changed once or twice a week. The tubuloids are ready for 5 use after 2 weeks. Adenoviruses for expression of human KIM-1 or control β-galactosidase (β-GAL) were 6 produced and stored as described previously (Zhang et al., 2007) . For detection of IL-1β induced by 7 treatment with Spike S1 subunit protein, tubuloids infected by adenovirus for GFP and KIM-1 or for control 8 GFP and β-GAL were treated with biotinylated 2019-nCoV S1 protein, His, Avitag (AcroBiosystems, DE) 9 for 24 hours and immunostained. was used for multiple comparisons. p<0.05 was considered to represent a statistically significant difference. 19 Prism 8 (GraphPad Software, LLC) was used for all the statistical analysis. 20

Further information and requests for resources and reagents should be directed to and will be fulfilled by 24 the Lead Contact, Joseph V. Bonventre (joseph_bonventre@hms.harvard.edu). 25

This study did not generate new unique reagents. 27 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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All other data are available from the Lead Contact on reasonable request. 2 3 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Spike S1 subunit naive GFP (Adeno-infection marker) IL-1β KIM-1 DAPI

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The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.16.20190694 doi: medRxiv preprint A Figure 4 B C All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells. J Clin 45Invest 118, 1657-1668. 46 Ichimura, T., Bonventre, J.V., Bailly, V., Wei, H., Hession, C.A., Cate, R.L., and Sanicola, M. (1998). Kidney injury 47