Part2: Kidney Injury Molecule-1 Is A Potential Receptor For SARS-CoV-2

For more info. contact tina.xiang@wecistanche.com

Clinical manifestations of coronavirus 

1. The incubation period is long, and the main symptoms are fever, fatigue, sore throat, and cough. The fever can be high fever, moderate fever, or low fever;

2. Infants and the elderly are not typical and are often accompanied by wheezing and dyspnea. In severe cases, multi-system damage may occur;

3. The dry cough in the early stage is mainly paroxysmal and severe, similar to whooping cough, which affects sleep and activities; in the later stage, the cough is sputum, the sputum is vicious, occasionally contains a small amount of blood, and some are accompanied by wheezing;

4. Some patients have mild symptoms and may have no fever. Most patients have a good prognosis, and a small number of patients are critically ill or even die;

5. Acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, and coagulation dysfunction.

How to prevent the new coronavirus, improving one's own immunity is the most effective means for people to fight the virus. Cistanche, which has been used as a nourishing treasure since ancient times, is known as "desert ginseng" and is the king of boosting immunity. Cistanche - The King of Boosting Immunity

Modern medical research has proved that in addition to the functions of nourishing and strengthening the body, Cistanchehas significant curative effects in preventing tumors, anti-aging, anti-arrhythmia, inhibiting apoptosis, and improving immunity. favor.

The total glucosides of Cistanche are the active components of ginseng to regulate immune function, which can improve immune function not only for normal people but also for people with low immune function.

Clinical observation has confirmed that Cistanche has a certain curative effect on malignant tumors, and has the obvious effect of increasing leukocytes. Taking ginseng during radiotherapy and chemotherapy can reduce the toxic and side effects of radiotherapy and chemotherapy, accelerate the repair of damaged tissue, and prevent leukopenia;

Discussion

To fight against the COVID-19 pandemic, a deep understanding of how SARS-CoV-2 invades human cells is warranted. Studies have indicated direct infection of SARS-CoV-2 in the kidney in addition to the lung (Braun et al., 2020; Farkash et al.,2020). However, ACE2 remains the only well-recognized receptor that may mediate this invasion. Furthermore, the renal tropism of SARS-CoV-2 and associated kidney injury seem unexplainable by the relatively decreased level of ACE2 upon viral invasion(Kuba et al.,2005). Here, our study suggests that KIM1, a drastically upregulated biomarker for kidney injury (Yang et al., 2015), mediates SARS-CoV-2 kidney invasion as a receptor.

We also found that SARS-CoV-2-RBD binds to KIM1 with a higher affinity than that of SARS-CoV-RBD and MERS-COV-RBD, which probably underlies the stronger contagion of SARS-CoV-2 (Rabaan et al,2020); therefore, the renal infection and the roles of KIM1 in these severe respiratory diseases worth revisit-ing. Notably, our results suggest distinct binding sites of KIM1 and ACE2 on viral RBD, thus it is worth investigating whether and how KIM1 and ACE2 mediate SARS-CoV-2 invasion in these organs. In addition, since KIM1 is endocytosed via clathrin-dependent pathways (Zhao et al.,2016), it would also be interesting to further explore the KIM1-dependent process after viral attachment to the cell membrane.

ACE2 is the most well-studied receptor for SARS-CoV-2, yet it is not an ideal therapeutic target for COVID-19, since it is widely expressed in multiple organs and plays crucial roles in regulating blood pressure and preventing heart/kidney injury (Imai et al.,2005; Li et al.,2020d).In contrast, KIM1 has a stronger association with kidney function and is highly expressed only after renal injury(Kondratowicz et al,2011; Yuan et al, 2015; Costafreda and Kaplan,2018), which makes it a more specific and maybe also safer therapeutic target for COVID-19 patients with kidney diseases.

In summary, our data suggest a crucial role of KIM1 in SARS-CoV-2 renal tropism as a potential receptor for SARS-CoV-2. Here, we propose a model of a 'vicious cycle' mediated by KIM1 and ACE2 (Figure 5G), which may explain the renal tropism of SARS-CoV-2 in COVID-19 patients. During the initial stage of SARS-CoV-2 invasion, the higher physiological level (Supplementary Figure S1) and binding affinity (Supplementary Table S1) make ACE2 the primary target, which is not kidney-specific. However, after the onset of virus-induced AKl, the resulting drastically upregulated KIM1 rapidly promotes a secondary viral infection mediated by KIM1 and ACE2, which is more kidney-specific, and consequently exacerbates kidney damage in a vicious cycle (Figure 5G). Approaches that can break the interaction between SARS-CoV-2 and KIM1, including anti-KIM1 antibodies, small-molecule inhibitors, and KIM1-derived antagonist peptides, may shed light on COVID-19 treatment.

Materials and methods 

Materials

Recombinant SARS-CoV-2-RBD (T80302) was obtained from Genscript. Antagonist peptide 1 (AP1, SCSLFTCQNGIV, purity >95%) and antagonist peptide 2 (AP2, SCSLFTCQNGGGWF, purity >95%) were chemically synthesized by Genscript. Anti-mouse-lgG antibody (p/n 18-8816-33) and anti-rabbit-lgG anti-body (p/n 18-8817-33) were obtained from Rockland. IgG with SureBeads" Protein G magnetic beads (J2112LB-02) was purchased from Bio-Rad. DAPI (D9542) was from Sigma. Alex Flour 594  labeled phalloidin  (C2205S)  was from  Beyotimes. Antibodies against KIM1 (NBP1-76701, Novus Biologicals), Flag (F1804, Sigma), HA (H6908, Sigma), and ACE2 (2115-1-AP, Proteintech) were used.

Acquisition and analysis of expression profiles of KIM1 and ACE2

To obtain the comprehensive transcriptome and protein profiles of KIM1 and ACE2 for human tissues, we collected and analyzed the transcriptome data and immunohistochemistry-based protein profiles from Human Protein Atlas (HPA, https://www.proteinatlas.org), which showed the expression and localization of human proteins across tissues and organs, based on deep sequencing of RNA (RNA-seq) from 37 normal tissue and immunohistochemistry on tissue microarrays containing 44 tissue types (Uhlen et al., 2015). HPA RNA-seq tissue of the protein-coding gene was recorded as mean protein-coding transcripts per million (pTPM), corresponding to the mean values of samples from each tissue. Histology-based protein expression levels were analyzed manually into four levels (not detected, low, medium, and high). In Supplementary Figure S1, the top 10 tissular transcriptional levels and histology-based protein expression levels of KIM1 and ACE2 are listed, respectively, and the overlapped expression profile of KIM1 and ACE2 is summarized.

Molecular docking and dynamics simulations

Dockings were conducted via Z-Dock (http://zdock. umassmed.edu/). Crystal structures of SARS-CoV-RBD (PBD ID 2AJF), SARS-CoV-2-RBD (PDB ID 6MOJ), MERS-COV-RBD (PDB ID 4L3N), ACE2(PDB ID 1R42), and KIM1 Ig V domain (PDB ID 5DZO) were used to seek potential binding models. The best-scored protein complexes were selected for the following molecular dynamics simulations, which were conducted by the Desmond server and analyzed by   Pymol2.3   and Maestro11.8.012.The 50 ns dynamics simulations diagram was applied to study the dynamic parameters of the protein complexes. MM-GBSA binding free energy was calculated by HawkDock (Misini lgnjatovic et al.,2016).

Root mean square deviation (RMSD) and root mean square fluctuation (RMSF)

RMSD was utilized to estimate the average change in displacement of a selection of atoms for a particular frame as described (Li et al.,2011).RMSF was conducted to study the displacement changes in the protein chain (Li et al.,2011).

AKI mouse models and qPCR

I/R injury was performed on C57BL/6 mice as we previously described (Chen et al,2015, 2017). For cisplatin-induced AKI, 30 mg/kg bodyweight cisplatin was injected intraperitoneally into 8-week-old male mice, and mice were sacrificed 3 days later. Blood and kidney samples were collected for further analysis, with n=4 for each experimental animal group. Total RNA was isolated from kidneys by RNAiso Plus (TaKaRa) and reverse-transcribed into cDNA using the M-MLV first-strand synthesis system (Invitrogen). The abundance of specific gene transcripts was assessed by qPCR. Primers used in the study are provided (Supplementary Table S4).

Constructs

Mammalian expression plasmids for human KIM1, KIM1 Ig V (KIM1 residues aa 20-127), KIM1 △lg V(truncated KIM1 with-out residues aa 20-127), KIM1-CFP, KIM1 lg V-CFP, ACE2, SARS-CoV-2-RBD(SARS-CoV-2-S residues aa 319-541), SARS-CoV-2-S, SARS-CoV-2-RBD-YFP, and TIM4-YFPwere constructed. PCR amplification products of the corresponding cDNA fragments were cloned into a pRK promoter-based vector containing either HA or FLAG tag. SARS-CoV-2-related plasmids were kind gifts from Dr. P.H.Wang at Shandong University.

Cell culture and transfection

Human kidney tubular cell line HK-2 (obtained from China Center for Type Culture Collection) was cultured in DMEM/F12 media (Hyclone) containing 17.5 mM glucose and 10% fetal bovine serum. To evaluate the impact of SARS-CoV-2 on cells, HK-2 cells were transfected with SARS-CoV-2-S and SARS-CoV-2-RBD plasmids and then collected for further detection.

Co-IP

Indicated HK-2/HEK293T cells (1×10) were lysed in 1 ml pre-lysis buffer(25 mM Tris-HCl,pH7.4,150mM NaCl,1% NP-40, 1mM EDTA, 5% glycerol), which is formulated for pulldown and IP assays and as a wash buffer for beads. For IP, cell lysate was immunoprecipitated with the indicated antibody or respective gG with SureBeadsTM Protein G magnetic beads overnight at 4C. After washing with a pre-lysis buffer containing 500 mM NaCl, the beads were boiled in a loading buffer and subjected to immunoblotting (Wan et al.,2017).

FRET assay

Intracellular interaction between KIM1 and SARS-CoV-2-RBD was detected by a standard FRET-based assay(Karpova and McNally,2006)using KIM1-CFP and SARS-CoV-2-RBD-YFP. Briefly, mammalian expression plasmids expressing KIM1-CFP or KIM1 Alg V-CFP were cotransfected with SARS-CoV-2-RBD-YFP into-293T cells. For fluorescence spectrophotometer-based detection, cells were collected and lysed 24h after transfection, and the lysate was detected by an F-2700 Fluorescence Spectrophotometer (Hitachi) via wavelength scan(500-600nm) and time scan (435/527 nm, excitation/emission).For confocal-based detection, cells were imaged with a Leica TCS SP8 confocal microscope under high-power objective lens(40×)(CFP channel:435/485 nm, excitation/emission; YFP channel:485/527nm, excitation/emission; FRET channel:435/527 nm, excitation/emission) (Li et al,2020b). Cotransfection of unconjugated CFP and YFP was included as a negative control as described(Karpova and McNally, 2006). Interaction between KIM1 and its ligand TIM4 was detected by FRET assay as a positive control (Rong et al, 2011).

CRISPR-Cas9-mediated knockout of KIM1

The CRISPR-Cas9-based protocols for genome engineering were used as described (Zhang et al.,2017). Guide RNA target sequences for KIM1 are provided (Supplementary Table S4).

FITC labeling and confocal microscopy

FITC label was performed as we previously described(Li et al, 2020b; Zhang et al.,2020). Briefly, SARS-CoV-2-RBD was incubated with FITC (molar ratio 1:5)overnight, and then 5 mM NHCl was added to stop the reaction and quench the unreacted FITC. The solution was dialyzed twice and lyophilized for further use.

HEK293T cells (5×109)or HK-2 cells (1×107) were incubated with free FITC or FITC-SARS-CoV-2-RBD （100μg/ml for 2. For peptide-based internalization assays， AP1 or AP2（50μM）was co-added with FITC-SARS-CoV-2-RBD （100 μg/m）. After fix-ing with 4%(w/v) formaldehyde, cell membranes were stained with Alex Flour 594 labeled phalloidin （2μg/ml）, and the nuclei were stained by DAPI（1μg/m）， and then imaged with a Leica TCS SP8 confocal microscope. For each group, at least 100 cells from five fields under a high-power objective lens (64×) were included in the assessment. Representative images were presented. Quantification of images was conducted by ImageJ1.8.0.

Cell viability assays

Cells were plated at 3000-4000 cells per well in 96-well plates. At 80% confluence, cells incubated with SARS-CoV-2-RBD （100 μg/ml） were treated with or without AP1 or AP2（10，50，100 μM）. After that，10μl MTT （5 mg/ml was added to each

well for 4h, the medium was removed, and DMSO was added. Absorbance measured at 490nm was normalized to the respective control group.

Statistical analysis

Data were expressed as mean±SD. Significant differences were assessed by a two-tailed Student's test. A two-sided P-value <0.05 was considered statistically significant. Analyses were performed with Excel 2017 and GraphPad Prism 8.0.