ABSTRACT
Background: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 was first reported in Wuhan in 2019 and reached pandemic proportions. SARS-CoV-2-related respiratory failure and acute kidney injury (AKI) are major complications of infection. Kidney Injury Molecule-1 (KIM-1) is a scavenger receptor expressed by kidney epithelial cells and was previously reported to be a receptor for Hepatitis virus A. We hypothesized that KIM-1 is a receptor for SARS-CoV-2 and may play an important role in COVID-19 lung and kidney injury. Methods: Human lung and kidney autopsy samples were immunostained and analyzed. Liposomal nanoparticles displaying the SARS-CoV-2 spike protein on their surface (virosomes) were generated. Virosome uptake by A549 lung epithelial cells, mouse primary lung epithelial cells and human kidney tubuloids (3D structures of kidney epithelial cells) was evaluated in the presences or absence of anti-KIM-1 antibody or TW-37, a small molecule inhibitor of KIM-1-mediated endocytosis that we discovered. Protein-protein interaction characteristics between purified SARS-CoV-2 spike protein and purified KIM-1 were determined using microscale thermophoresis. HEK293 cells expressing human KIM-1 but not angiotensin-converting enzyme 2 (ACE2) were infected with live SARS-CoV-2 or pseudovirions expressing the SARS-CoV-2 spike protein. Results: KIM-1 was expressed in lung and kidney epithelial cells in COVID-19 patient autopsy samples. Human and mouse lung and kidney epithelial cells expressed KIM-1 and endocytosed spike-virosomes. Both anti-KIM-1 antibodies and TW-37 inhibited uptake. Enhanced KIM-1 expression by human kidney tubuloids increased virosome uptake. KIM-1 positive cells expressed less ACE2. Using microscale thermophoresis, the EC50 for interaction between KIM-1 and SARS-CoV-2 spike protein and the receptor binding domain were 56.2±28.8 nM and 9.95±3.10 nM, respectively. KIM-1-expressing HEK293 cells without ACE2 expression had increased susceptibility to infection by live SARS-CoV-2 and pseudovirions expressing spike when compared with control cells. Conclusions: KIM-1 is a receptor for SARS-CoV-2 in the lung and kidney and thus, KIM-1 inhibitors such as TW-37 can be potential therapeutics and/or prophylactic agents for COVID-19.
ABSTRACT
Background: Kidney injury molecule-1 (KIM-1), a type-1 transmembrane glycoprotein, has been well studied as a specific injury marker for proximal tubules (PT). KIM-1 functions as a receptor for apoptotic fragments through a phagocytic process. KIM-1 (also called TIM-1) serves as a receptor for hepatitis A virus and Ebola virus, and possibly for severe respiratory syndrome-coronavirus (SARS-CoV-1). During the pandemic spread of coronavirus disease 2019 (COVID-19), many patients have suffered from acute kidney injury (AKI) as well as lung damage, Viral upkake has been attributed to interactions with ACE2, a receptor for the virus. The goal of this study was to investigate whether there is kidney histological data that KIM-1 may also serve as a receptor for SARS-CoV-2 to infect the PT. Methods: Two patients (one adult and one child) who died of COVID19 and 10 patients with AKI but no COVID19 (control group) were included in the study. All kidney tissue sections were stained for KIM-1 (monoclonal AKG7 antibody) and scored from 0 to 3+. Electron microscopy was conducted using kidney tissue of the COVID19+ patients. Results: Both COVID19+ patients had normal pre-mortem levels of serum creatinine (sCr) (adult 0.63 and child 0.17 mg/dl), whereas the control cases all had elevated sCr (1.9 to 10.7 mg/dl). Control renal biopsies revealed positive KIM-1 staining ranging from 1+ to 3+ along the surface of PT in a patchy pattern involving 20 to 80% of the cortex;no cytoplasmic granular materials were identified. By contrast, the KIM-1 staining in COVID19+ kidneys revealed spotty granular staining in the cytoplasm and diffuse surface 2+ to 3+ staining in most PTs, while glomeruli stained negatively for KIM-1 as internal negative controls. In the two COVID19+ patients, SARS-CoV-2 particles showed spiking-crown appearances with sizes ranging from 70 to 110 nm in the PT cytoplasm by ultrastructural studies. Conclusions: Our initial evidence suggests there is an atypical staining pattern of KIM-1 in the PT of COVID19+ patients, raising a possibility that KIM-1 may serve as a receptor for SARS-CoV-2. KIM-1 may also serve to internalize the virus into the PT. In addition the two COVID+ patients had normal sCr levels but positive KIM-1 staining, indicating that sCr underestimates renal injury caused by SAR-CoV-2 infection.
ABSTRACT
Background: Human kidney organoids have been utilized as a model to study genetic kidney diseases and kidney development. Innate or adaptive immune responses in organoids are currently poorly defined. Kidney transplant rejection and activation of complement pathways are two common renal immune phenomena. SARS-CoV-2 virus, the pathogen of the recent pandemic, leads to complement pathway activation in human kidneys and can infect kidney organoids. Here, we investigated (i) the alloimmunogenicity of kidney organoids in a humanized mice model, and (ii) the responses to exogenous complement C5a and spike protein (S1) of SARS-CoV-2 in kidney organoids. Methods: Kidney organoids were generated from human embryonic stem cells using protocols developed in our laboratory, and transplanted under the kidney capsule in humanized (BLT) mice. Immunophenotype, mixed lymphocyte reaction, and intracellular cytokine staining were analyzed from grafts and mouse splenocytes collected after 30 days of transplantation. In other experiments organoids were treated with S1 protein and human recombinant C5a for 24 hours or 3 days respectively, followed by qPCR and immunofluorescence analysis. Results: Transplanted organoids were extensively infiltrated by lymphocytes. Graft CD8+ T cells demonstrated a switch from naïve to memory T cells. Splenocytes isolated from transplanted BLT mice showed increased IFN-γ and TNF-α. Splenocytes proliferated after exposure to 2D kidney organoids (MLR) for 72 hours ex vivo, and organoids were markedly injured as reflected by DNA damage (γ-H2AX) and cleaved caspase 3. Reflecting innate responses, robust interstitial fibrosis was found in nontransplanted organoids after direct activation of C5aR by exogenous C5a. We confirmed ACE2 expression on proximal tubules and parietal epithelium of glomeruli, consistent with human autopsy results. Non-transplanted organoids treated with S1 protein showed transcriptionally upregulated C5a1 receptors. Conclusions: Our results indicate the alloimmunogenicity of kidney organoids and the deleterious effects of C5a in kidney organoids. Human kidney organoids represent a novel platform to study renal immunology including adaptive and innate immunity and the inflammatory responses to coronavirus disease (COVID-19).
ABSTRACT
Background: Acute kidney injury (AKI) is a common feature of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. Kidney Injury Molecule-1 (KIM-1) has been reported to be a receptor for Hepatitis A virus. KIM-1 is a scavenger receptor in kidney epithelial cells. We hypothesized that KIM-1 is a receptor for SARS-CoV-2 and may play an important role in COVID-19-associated AKI. Methods: Liposomal nanoparticles displaying the SARS-CoV-2 spike protein trimer (S1 and S2) on their surface (virosomes) were generated. We evaluated spike protein and virosome uptake by human KIM-1 expressing kidney epithelial cells and human kidney tubuloids, 3D structures of kidney epithelial cells. KIM-1-mediated uptake was compared to uptake by ACE2, a well-known receptor for SARS-CoV-2. Our recently discovered specific KIM-1 uptake inhibitor, JB-1 was tested for its ability to block virosomes uptake by KIM-1 expressing cells. KIM-1 expression was augmented in the tubuloids by infection with adenovirus vector carrying human KIM-1 cDNA to examine if the virosome uptake was enhanced. Protein-protein interaction characteristics between purified SARS-CoV-2 spike protein and S1 binding domain and purified KIM-1 were determined using microscale thermopheresis. Results: KIM-1 expression on kidney epithelial cells markedly enhanced virosome uptake, despite no change in ACE2 expression. This KIM-1 specific uptake was inhibited by JB-1. Human kidney tubuloids also endocytosed virosomes, and tubuloids with enhanced KIM-1 expression secondary to infection of KIM-1-adenovirus had increased uptake of virosomes. Using microscale thermopheresis the Kd for interaction between KIM-1 and SARS-CoV-2 spike protein and the receptor binding domain were 56.2+/-28.8 nM and 9.95+/-3.10 nM respectively. Conclusions: KIM-1 is a receptor for SARS-CoV-2. KIM-1 specific uptake of the SARS-CoV-2- virosomes suggests that KIM-1 confers efficient SARS-CoV-2 binding in kidney epithelial cells when these cells are expressing KIM-1. The KIM-1 dependent virosome uptake by 3D tubuloids indicates that this can be a valuable human cell model for studying SARS-CoV-2 interactions and testing for inhibitors. KIM-1 inhibitors, such as JB-1, can be potential therapeutics SARS-CoV-2 for COVID-19. Kidney tubular intraluminal and systemic circulating levels of KIM-1 ectodomain may be protective by acting as decoy receptor for the virus.