As high as 60% of all in-hospital cases of acute kidney injury events are a result of nephrotoxic drug exposure. In injury, studies have demonstrated that susceptible endothelial cells are capable of transdifferentiating to a pathogenic myofibroblast phenotype through a process known as endothelial to mesenchymal transition (EndMT). This transition is difficult to study in vivo due to the highly regulated spatiotemporal morphological changes of individual cells and modified cell-cell communications with resident epithelial cells. In vitro human kidney organoid models are well suited for studying kidney disease, due to their capacity to recapitulate nephron epithelial cell types, however, organoids have notoriously lacked endothelial cells, which renders modeling endothelial injury moot. Our group has developed a novel method for generating highly dense endothelialized human kidney organoids using a transgenic vasculogenic induced pluripotent stem cell line. These organoids contain a robust endothelial network that closely interacts with all nephron cell types previously found in human kidney organoids. Based on preliminary immunofluorescence, electron microscopy and transcriptomic data, I hypothesize that kidney organoid co-culture of engineered endothelial cells enables the induction of kidney-specific endothelial maturation (Specific Aim 1). To examine this, I will use electron microscopy, immunofluorescence, and western blotting to understand whether there is a morphological presence of fenestrations and an endothelial glycocalyx layer. I will also compare transcriptomically, engineered endothelial cells to published datasets of human kidney endothelial cells, examining pathways of endothelial maturity and kidney specificity, while simultaneously determining whether kidney organoid co-culture enables advanced alignment with human clinical samples. In addition, based on preliminary immunofluorescence data, I hypothesize that following nephrotoxic exposure, endothelialized human kidney organoid endothelial cells undergo EndMT, and this process is critically regulated by TGFb (Specific Aim 2). Endothelialized organoids injured with nephrotoxin, doxorubicin, will be examined with immunofluorescence and FACS by tracking GFP+ engineered endothelial cells through injury to understand quantitatively the percentage change of mesenchymal transition. TGFb inhibition will also be used to understand whether this process can be ameliorated. Uninjured, injured, and injured + TGFb inhibition endothelialized human kidney organoids will be compared transcriptomically with snRNAseq to determine computational alignment to existing published datasets of injured endothelial cells having undergone EndMT and to determine upregulated pathways of TGFb as well as master transcription factor regulators. The studies in this proposal represent a significant and innovative step forward for the field of kidney disease because these studies uncover critical knowledge in under...