The function of kidney specific (KS)-WNK1 condensates during potassium stress

Potassium [K+]-­deficient diets have 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 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 includeimplementation  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 meetings. Dr. Boyd-­Shiwarski will use this proposal to accomplish her short-­term goal  of scientific independence and her long-­term goal of becoming a tenure track physician-­scientist with expertise  in potassium homeostasis, hypertension, and CKD. The results from this proposal will form the basis for an R01  studying the translational role of KS-­WNK1-­dependent WNK bodies in human models of nephropathy.