Kidney Microphysiological Analysis Platforms (MAP) to Explore SARS-CoV-2 Receptors and Inhibitors. A supplement to Parent Grant: Kidney Microphysiological Analysis Platforms (MAP) to Optimize Function

摘要 2019冠状病毒病（COVID-19）已达到大流行程度。肺和 肾脏疾病是SARS-CoV-2感染高度流行的严重后果。肾 损伤分子-1（KIM-1）被Bonventre博士和Ichimura博士鉴定为最上调的 蛋白质在受损的肾脏近端小管。KIM-1，也称为TIM-1，是肝炎的受体 埃博拉病毒、登革热病毒，可能还有SARS-CoV 1病毒。我们假设KIM-1是一种受体， 在肾小管上皮细胞和气道上皮细胞中均存在SARS-CoV-2， JB 1，我们新发现的KIM-1的小分子抑制剂，和/或纳米盘- 掺入的KIM-1胞外域可以是COVID-19的预防剂和治疗剂。 我们还假设，我们可以利用KIM-1和ACE 2的高亲和力结合， 病毒创造新的诊断设备的病毒。在具体目标1中，我们将描述 KIM-1在促进SARS冠状病毒2经肾脏进入肾脏和肺上皮细胞中作用 微生理分析平台（MAP）的芯片，并开发一个超灵敏的芯片， 潜在SARS-CoV-2结合抑制剂的高通量评价。KIM-1和ACE 2 将在肾脏中比较SARS-CoV-2仿生病毒（病毒体）介导的内吞作用 和肺上皮细胞。我们将评估KIM-1介导的刺突蛋白或 仿生病毒细胞粘附和摄取对产生旁分泌因子的影响， 使用肾-肺MAP检测内皮细胞。为了了解结合和/或摄取动力学 的SARS-CoV-2和表征潜在的抑制剂，我们将开发一种超灵敏的 用于快速和灵敏抑制的基于纳米等离子体激元三联体的快速侧向流诊断芯片 使用肾-肺MAP进行测定。这种方法也可以用于床旁诊断 检测病毒在具体目标2中，我们将评估JB 1、可溶性KIM-1 胞外域和纳米盘掺入的KIM-1或ACE 2抑制KIM-1或ACE 2的结合和内化。 肺-肾整合MAP法在肾和肺细胞上模拟SARS-CoV-2病毒 在芯片上将对潜在抑制剂进行检测，以评价它们是否与S蛋白竞争和/或 仿生病毒结合并减少IL-6的产生。KIM-1之间的结合亲和力和动力学 变体或ACE 2作为游离胞外域或掺入纳米盘中， 将使用微尺度热泳（MST）和生物层干涉测量法进行测量。

Abstract Coronavirus disease 2019 (COVID-19) has reached pandemic proportions. Pulmonary and kidney disease are highly prevalent serious consequences of infection with SARS-CoV-2. Kidney Injury Molecule-1 (KIM-1) was identified by Drs Bonventre and Ichimura as the most upregulated protein in the injured kidney proximal tubule. KIM-1, also called TIM-1, is a receptor for hepatitis A, Ebola, Dengue and possibly SARS-CoV1 viruses. We hypothesize that KIM-1 is a receptor for SARS-CoV-2 both in renal tubule epithelial cells and in airway epithelial cells and that JB1, our newly discovered small molecule inhibitor of KIM-1, and/or nanodisc- incorporated KIM-1 ectodomain can be prophylactic and therapeutic agents for COVID-19. We also hypothesize that we can use the high-affinity binding of KIM-1 and ACE2 to the virus to create novel diagnostic devices for the virus. In Specific Aim 1 we will characterize the role of KIM-1 in promoting SARS-CoV-2 entry into kidney and lung epithelia using kidney microphysiological analysis platforms (MAPs) on chip and develop an ultrasensitive chip for the high-throughput evaluation of potential SARS-CoV-2 binding inhibitors. KIM-1 and ACE2 mediated endocytosis of SARS-CoV-2 biomimetic viruses (virosomes) will be compared in kidney and lung epithelial cells. We will evaluate the effects of KIM-1-mediated spike proteins or biomimetic virus cellular adhesion and uptake on production of paracrine factors which activate endothelial cells using a kidney-lung MAP. In order to understand binding and/or uptake kinetics of SARS-CoV-2 and characterize potential inhibitors we will develop an ultrasensitive nanoplasmonic triplets-based rapid lateral flow diagnostic chip for a rapid and sensitive inhibition assay using the kidney-lung MAP. This approach can also be used for point of care diagnostic testing for the virus. In Specific Aim 2 we will evaluate the efficacy of JB1, soluble KIM-1 ectodomain and nanodisc-incorporated KIM-1 or ACE2 to inhibit binding and internalization of SARS-CoV-2 biomimetic viruses by kidney and lung cells using an integrated lung-kidney MAP on chip. Potential inhibitors will be tested to evaluate whether they compete with S-protein and/or biomimetic virus binding and reduce IL-6 production. Binding affinity and kinetics between KIM-1 variants or ACE2 either as free ectodomains or incorporated into nanodiscs and the Spike protein will be measured using MicroScale Thermophoresis (MST) and Biolayer Interferometry.