WNK-SPAK kinases are integral components of a potassium-dependent signaling system, coined the “potassium switch,” that adjusts the activity of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) and shapes the structure of the entire distal nephron to maintain sodium and potassium balance over a wide range of dietary potassium intakes. Low potassium consumption, common in modern diets, presses the pathway to conserve potassium at the expense of increasing sodium absorption and increasing blood pressure. A decrease in plasma K+, leads to a decrease in intracellular Cl- which stimulates the WNK/SPAK pathway, leading to activation of NCC and hypertrophy/hyperplasia of the DCT. The molecular details of the signaling pathway are still mostly unknown or are controversial, especially the specific role of a kidney-specific isoform of WNK1 and Cab39 (MO25), a kinase adaptor protein. How Na+ transport signals to increase DCT mass remains another great mystery. Here, we tackle these pressing unknowns, challenging prevailing views, and pushing the boundaries of the field with an innovative, stepwise multidisciplinary approach, combining molecular genetics, genomics, cellular biology, imaging, state-of-the-art physiological phenotyping in novel mouse models, and, mechanistically in innovative in vitro systems Aim 1: Newly published and preliminary data challenges the view that KS-WNK1 is an inhibitor of WNK4 and suggests that it acts to enhance WNK4-SPAK signaling in the DCT. We test this idea by specifically targeting KS-WNK1 gene knockout in the DCT and performing a molecule- to-physiology phenotyping analysis; characterize the KS-WNK1 transcript and protein, which our preliminary data indicate are different than previously believed; and define mechanistically how KS-WNK1 modulates NCC function. Aim 2: Recent data, including our preliminary data, indicate that kinase adaptor proteins, Cab39/Cab39l, enhance WNK-SPAK signaling in the DCT. Here, we have engineered a new mouse model, overcoming a roadblock in the field, to test this idea in a mammalian system for the first time, and have developed a new system to understand mechanistically how the adaptor proteins interact with WNK4 and SPAK, and modulate their function. Aim 3: Our RNAseq analysis on our DCT-specific constitutively active SPAK mouse, which is sufficient to activate DCT hypertrophy and hyperplasia, identified a network of DCT-specific transcription factors. We hypothesize that one of these Spalt like transcription factor-3 (Sall3) is the master regulator of the transcription network that defines the epigenetic fingerprint of the DCT and maintains the DCT lineage as it expands. To test this novel idea, we will define how Sall3 is induced in response to activation of SPAK and/or NCC; establish the impact of specifically targeting Sall3 knockout in the DCT on the remodeling response using a innovative new imaging approach; and determine if Sall3 repress...