Establishing and Mimicking Patterning Mechanisms in the Distal Nephron Tubule and Kidney Organoid

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During embryonic and fetal stages, the kidneys develop millions of nephrons that generate highly specialized cells. These cells ensure that blood flowing into the kidney is filtered and required substances are reabsorbed while unwanted metabolites and solutes are led to the bladder for excretion. Birth defects are common in the kidney, ~ 1/100 of all births have a so-called Congenital Anomaly of the Kidney and Urinary Tract (CAKUT). At the most severe end of CAKUT, newborns are missing kidney functionality, and their life expectancy is less than one year. Most abnormalities have no current effective interventions and genetic changes lack context. There is thus a critical need to understand where developmental defects arise and to generate new therapies restoring or replacing kidney function. In our work we have used single cell omics and molecular characterizations of human and mouse kidneys to provide a blueprint for how nephrons form and maps for to replicate this in human stem cell-derived kidney organoids. In doing so we provide a genetic and developmental context to genes identified in CAKUT patients. In this proposal we will follow these leads and address three outstanding questions in developmental nephrology. In Aim 1, we investigate the embryonic origins of distal nephron tubule segments. We will perform the first single cell omic analysis linking developing and adult kidneys. This provides a roadmap for how cells differentiate. We will use new genetic mouse lineage-tracing tools to test how cells in the early distal nephron relate to functional cells in mature kidneys. These experiments will map where genes are required as the nephron develops. In Aim 2, we will investigate how proteins that turn genes on and off control the development of the distal nephron. We will use a technique called Cut&Run to analyze how genes often mutated in CAKUT, control DNA and gene expression. We will also activate signaling pathways and alter the expression of genes linked to CAKUT. This will allow us to directly study how distal nephron cells form provide causality between gene expression and regulation. We will use our new system to generate hundreds of nephrons from human stem cells in - synchronized nephroids. In this system, nephrons develop at the same time and pace, unlike in the body where nephrons from many developmental stages form near each other. Our system provides a unique advantage to study, manipulate, and isolate cells from nephrons at the same developmental stage. The data we collect will show how genes are activated. In Aim 3 we address a fundamental question in developmental nephrology - how is the nephron initially patterned? To do this we will use synthetic cellular organizers that secrete signaling proteins to pattern our synchronized nephroids. We will study how signal ligands control nephron formation and patterning. This also has a practical application as we can gain control over nephroid patterning. Our system will inform our efforts to build massive parallel arrays of nephroids for replacement therapies and disease modeling. A strength of the proposal is the unique expertise intersecting human and mouse kidney genetics, a novel system of stem cell derived kidney organoids, and synthetic biology.