Coordinated cellular heterogeneity in the thick ascending limb and distal tubule - In this proposal, we will define cell states in the kidney, and specifically along the thick ascending limb, distal convoluted tubule, and connecting tubule in health, and during stress. In preliminary work, we have used an technique called INTACT to enrich specific cell types, allowing us to study their transcriptome in unparalleled detail. We have combined this approach with immunohistochemistry, allowing us to find new cell types in these tubule segments. We recently used this approach in a publication defining the distal convoluted tubule Here, we will use new mouse lines that allow us to enrich connecting tubules to test how potassium stress and diuretic drugs alter cell state, leading to both homeostatic adaptation and to dysfunction. We will then examine thick ascending limb cells, where our preliminary data suggest the presence of two previously unrecognized cell types. One of these generates a transepithelial voltage and mediates salt reabsorption. The other is electrically neutral, but mediates the reabsorption of calcium and magnesium. We will then test whether water stress and calcium stress activate these cell types selectively. To achieve this, we have planned the following Specific Aims: Aim 1: Define the role of cell types and states in DCT and CNT function Characterize cell types along DCT and CNT Determine how dietary K+ intake alters cell state to modulate Ca2+ excretion Determine how diuretic drugs alter cell state to modulate Mg2+ and Ca2+ excretion Aim 2: Define the role of cell types and states in TAL function Characterize TALa and TALb cells and develop a new physiological model Determine how water stress and arginine vasopressin affect TALa cell states and function Determine how calcium stress affects TALb cell states and function
