Development of a fluidic chip model of PKD to elucidate cystogenic signals using kidney organoids

点击翻译按钮获取中文摘要

Project Summary Kidney organoids derived from human pluripotent stem cells (hPSCs) can be a novel tool to study kidney diseases. Polycystic kidney disease (PKD) is the most common genetic kidney disease which results in end-stage kidney disease, afflicting over 12 million people worldwide. Animal and static kidney organoid models have been developed to understand the pathomechanisms of cystogenesis for therapeutic development. However, renal phenotypes in ARPKD mice are often absent, and when present, it is a mild lesion that typically involves proximal tubules rather than distal nephrons. In contrast, human kidney organoid models could provide new insights into PKD pathogenesis, yet current ARPKD models are developed with a chemical inducer, forskolin, in static culture, exhibiting proximal tubular cysts. Recently, our collaborative team has developed a new model of organoid-on-the-chip by which fluidic shear stress can be applied to whole kidney organoids. Interestingly, our preliminary results using the fluidic chips showed clinically relevant phenotypes with distal nephron dilatation in PKHD1-/- organoids cultured under flow. These novel tools marrying kidney organoid, kidney-on-a-chip, and CRISPR genome editing might offer new opportunities to efficiently explore the signal pathways that are involved in cyst formation and signal molecules that can be new therapeutic targets for ARPKD patients. To develop a physiologic model of ARPKD, in Specific Aim 1, we will generate kidney organoids from PKHD1-mutant hPSCs. Then we will optimize culture conditions on millifluidic chips to apply shear stress to kidney organoids in vitro. Flow-induced mechanical stress including ciliary stress and cellular stretching will be evaluated by ELISA, biosensors, and transcriptomics. We will validate the fluidic ARPKD model by comparing cystic phenotypes to human ARPKD kidney samples. For mechanistic studies, in Specific Aim 2, we will generate additional tools: 1. primary cilia knock-out lines by CRISPR genome editing in PKHD1-/- hPSCs, and 2. primary cilia reporter lines using human SSTR3-GFP. Cilia knock-out lines will enable mechanistic studies involving ciliary stress-mediated pathogenic signals in ARPKD kidney and other organoids. The reporter lines will enable real-time imaging of primary cilia movement in varied experimental settings including organoid-on-chip and intravital imaging in transplanted animals. Our proposed work will generate novel in vitro tools for PKD and ciliopathy research communities to investigate pathomechanisms of cystogenesis induced by ciliary stress in kidney and other organoids. Transcriptomic data from perfused kidney organoids will also provide mechanistic insights into cystogenesis in ARPKD. All tools and data developed by this study will be disseminated through NIH PKD and RBK consortiums to facilitate therapeutic development for PKD patients.