Modeling of acute kidney injury in organoids.

Of patients admitted to VA intensive care units, nearly one quarter have or develop acute kidney injury (AKI), which is associated with an approximately five-fold increase in mortality. AKI survivors can develop end-stage renal disease, subsequently requiring dialysis or transplantation to survive. Patients in the Veteran population are diagnosed with AKI at a rate that is higher than that of the civilian population. Current AKI modeling is largely in animal models and dissociated human tubule cells. Animal models have species-specific challenges and human proximal tubule cells grown in 2D on hard plastic surfaces readily lose their epithelial characteristics. Both of these models have advanced our understanding of AKI, which is difficult to study in human patients due to lack of biopsy tissue samples. Despite years of study in these traditional models of AKI, the clinical situation for Veterans has not changed, as we still rely on supportive care for AKI treatment. Human kidney organoid in vitro modeling is a robust and highly innovative approach with numerous advantages. First, the amount of drug that is needed to perform tests in the human kidney organoid model is substantially lower than in mouse models. Secondly, species matching of model and drug is an important issue in many areas of medicine including gene and cell therapy. Finally, testing of therapeutics in human kidney organoid models of kidney injury first would reduce animal usage and ensure that animal use is well-justified. Validation of advanced human kidney organoid reporter models is necessary to further demonstrate to the research community the potential of human kidney organoids. Our lab and others have shown that human kidney organoids treated with cisplatin have consistent upregulation of kidney injury molecule 1 (KIM-1, also known as hepatitis A virus cellular receptor 1 or HAVCR1). In this pilot award, we will edit the genome of a line of human induced pluripotent stem cells (iPSC) for high-throughput screening of human kidney organoids with live readout of kidney injury. In Aim 1, iPSC will be gene edited with the same Cas9 guide RNA and homologous recombination cassette as in our previous study to target reporter genes into the KIM-1 locus. Additionally, we will edit a marker of proximal tubule cell type specification to control for organoid to organoid and batch variation. These iPSC-derived kidney organoids will express mTagBFP2 from proximal tubule cells and upregulate GFP and luciferase in response to injury. All lines generated will be confirmed for iPSC pluripotency, whole genome sequencing, and functional testing. In Aim 2A, we will fully validate cellular differentiation in 96-well plates. In Aim 2B, organoids grown in 96-well plates will be tested for dose-dependent injury response. The equipment for confocal high-throughput fluorescent and luminescent screening is available through the Vanderbilt High-throughput Screening Facility. At the conclusion of this pilot award, we will possess a fully characterized and validated reporter cell line for live, quantitative assessment of human AKI in vitro. This line will enable high-throughput screening of libraries to uncover unknown sources of nephrotoxicity as well as discover new AKI therapeutics. Project Number: 1I21BX006554-01A1 | Fiscal Year: 2025 | NIH Institute/Center: Veterans Affairs (VA) | Principal Investigator: Lauren Woodard | Institution: VETERANS HEALTH ADMINISTRATION, NASHVILLE, TN | Activity Code: I21 | Study Section: Special Emphasis Panel[ZRD1 NEPH-N (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11047118