Thesis (Ph.D.)--University of Washington, 2022Chronic kidney disease (CKD) affects 1 in 7 adults and is the 10th leading cause of death in the United States in 2022. Kidney transplant and dialysis remain the leading treatment strategies for kidney failure despite their expensive and outdated technological innovation. Stem cell derived human kidney organoids aim to provide a vital tool to study complex diseases ranging from infectious to genetic, and a translationally relevant system to discover and probe novel therapeutic pathways to improve our ability to treat kidney disease. CKD is the greatest risk factor for developing severe COVID-19, the 3rd leading cause of death in the US in 2022, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported to cause acute kidney injury in 1 in 4 hospitalized COVID-19 patients. Utilizing genome-edited kidney organoids, SARS-CoV-2 variants, and clinical data, we investigated viral tropism, mechanism, and therapeutic approaches in the context of the kidney. SARS-CoV-2 infected proximal tubules in kidney organoids via angiotensin converting enzyme 2 (ACE2). Infected organoids produced replication competent virus and displayed apoptotic responses in the context of polycystic kidney disease (PKD), a genetic cause of CKD. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in kidney organoids. Replication is reduced by remdesivir treated and infection blocked by treatment with de novo–designed spike binder peptides. This work clarifies the impact SARS-CoV-2 infection has on the kidney and enables the assessment of viral fitness and emerging therapies in the context of infectious disease. Autosomal dominant PKD is a genetic kidney disease affecting 1 in every 400-1000 people worldwide, causing progressive fluid-filled cyst production followed by fibrosis in the kidneys, liver, and other organs, resulting in organ failure. PKD is caused by mutations in the polycystin proteins polycystin-1 (PC1) or polycystin-2 (PC2), but the molecular pathway causing cystogenesis remains elusive. Genome-edited PKD organoids phenocopy cystogenesis and previously identified that the myosin inhibitor blebbistatin resulted in cyst enlargement. I discovered that treatment with the myosin activator, EMD 57033 (EMD), prevented cyst growth and that treatment when cysts were already established was able to slow cyst expansion. Live-imaging of EMD-treated organoids expressing fluorescently-tagged non-muscle myosin II B (NMIIB-GFP) revealed increased apical-basal tubule contractility of PKD organoid tubules compared to controls, indicating that the PKD organoid tubules were poised to contract and may have intrinsic contractile dysfunction. Analysis of the slowly progressing Pkd1RC/RC mouse model reveals a concomitant expansion of phosphorylated myosin light chain 2 (pMLC2) expressing stromal pericytes and cyst growth, suggesting that a therapeutic reducing pMLC2 expression may rescue kidney fibrosis later in disease. Together, this work suggests both a tubular and stromal myosin contribution to PKD pathogenesis that can be therapeutically targeted using myosin activators early in disease and pMLC2 inhibitors in later stage disease progression. In conclusion, our studies of PKD and COVID-19 reveal great utility of kidney organoids for disease modeling and therapeutic development, advancing the translational applications of organoid technology
