PROJECTSUMMARY/ABSTRACT Potassium [K+]-deficient diets have contributed to the global epidemic of hypertension and chronic kidney disease(CKD).Giventhelowcostandeaseofincreasingdietary[K+],moreresearchisneededtounderstand how[K+]imbalanceleadstothesediseases.Thekidneyshandle90%of[K+],andthedistalconvolutedtubule (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 hyperkalemiabyactivationofthethiazide-sensitivesodium/chlorideco-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, whichwecall?WNKbodies?,remainedamystery.Dr.Boyd-Shiwarski?sinitialworkhasidentifiedthattheseDCT- specificpunctaare(i)dependentupontheexpressionofkidneyspecific(KS)-WNK1(ii)potassium-sensitive;?(iii) membrane-less;?(iv)notassociatedwithconventionalorganelles;?and(v)associatedwithWNK-SPAKproteins. Basedonthesefindings,wehypothesizethatWNKbodiesaremembrane-lessmicrodomainsthatsequesterthe WNK-SPAKpathwaytomodulateWNKsignalingduringpotassiumimbalance.Thishypothesiswillbetestedin twoaimsthatevaluatethephysiologicalsignificanceandbiologicalbasisofWNKbodyformation.Thisproposal?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. Theseskillswillbereinforcedbyateamofmentors,advisors,collaborators,andcoreresourcesavailableatthe 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 thePittsburghCenterforKidneyResearch,andhasmentoredninecareerdevelopmentawardeesandfiveR01 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 progressthroughbiannualmeetings.Dr.Boyd-Shiwarskiwillusethisproposaltoaccomplishhershort-termgoal ofscientificindependenceandherlong-termgoalofbecomingatenuretrackphysician-scientistwithexpertise inpotassiumhomeostasis,hypertension,andCKD.TheresultsfromthisproposalwillformthebasisforanR01 studyingthetranslationalroleofKS-WNK1-dependentWNKbodiesinhumanmodelsofnephropathy.
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