PROJECT SUMMARY/ABSTRACT Potassium [K+]-deficient diets have contributed to the global epidemic of hypertension and chronic kidney disease (CKD). Given the low cost and ease of increasing dietary [K+], more research is needed to understand how [K+] imbalance leads to these diseases. The kidneys handle 90% of [K+], and the distal convoluted tubule (DCT) acts as a [K+] sensor via the WNK-SPAK (With-No-Lysine/Ste20/SPS-1-related proline-alanine-rich protein kinase) pathway. Gain-of-function mutations to this pathway lead to severe hypertension and hyperkalemia by activation of the thiazide-sensitive sodium/chloride co-transporter (NCC). Curiously, dietary [K+] depletion or loading causes the WNK-SPAK kinases to assemble into large DCT-specific cytoplasmic puncta, that are not seen in mice on normokalemic diets. For years, the structure and function of these condensates, which we call “WNK bodies”, remained a mystery. Dr. Boyd-Shiwarski’s initial work has identified that these DCT- specific puncta are (i) dependent upon the expression of kidney specific (KS)-WNK1 (ii) potassium-sensitive;; (iii) membrane-less;; (iv) not associated with conventional organelles;; and (v) associated with WNK-SPAK proteins. Based on these findings, we hypothesize that WNK bodies are membrane-less microdomains that sequester the WNK-SPAK pathway to modulate WNK signaling during potassium imbalance. This hypothesis will be tested in two aims that evaluate the physiological significance and biological basis of WNK body formation. This proposal’s physiology-based aim will provide Dr. Boyd-Shiwarski with the opportunity to work with animal models and (i) implement ex vivo microscopy techniques, (ii) quantify changes in urine, serum, and blood pressure, and (iii) develop transgenic mouse models. Whereas, the biology-based portion of this proposal will include implementation of (i) molecular cloning, (ii) protein biochemistry, and (iii) mass spectrometry and RNA Seq. These skills will be reinforced by a team of mentors, advisors, collaborators, and core resources available at the University of Pittsburgh. The primary mentor, Dr. Arohan Subramanya, is an established NIH R01-funded physician-scientist with 13 years of experience in WNK signaling biology and prior experience mentoring over 20 trainees. The co-mentor, Dr. Tom Kleyman, is an internationally recognized physician scientist who directs the Pittsburgh Center for Kidney Research, and has mentored nine career development awardees and five R01 recipients within the last 10 years. In addition, an advisory committee of accomplished investigators with expertise in hypertension, WNK-SPAK signaling, and renal tubular transport will monitor Dr. Boyd-Shiwarski’s progress through biannual me...