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

钾应激期间肾脏特异性 (KS)-WNK1 缩合物的功能

基本信息

批准号：
10397071
负责人：
Cary Ragan Boyd-Shiwarski
金额：
$ 16.25万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10397071
关键词：
Advisory Committees Affect Alanine Animal Model Animals Binding Proteins Biological Biology Blood Blood Pressure Chronic Kidney Failure Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Diet Dietary Potassium Disease Distal convoluted renal tubule structure Duct (organ) structure Epidemic Equilibrium Evolution Foundations Funding Goals Homeostasis Human Hypertension Hypokalemia In Vitro Intake International Kidney Kidney Diseases Knockout Mice Lead Link Lysine Maps Mass Spectrum Analysis Measures Membrane Mentors Metabolic Microscopy Molecular Molecular Cloning Monitor Morbidity - disease rate Mus Mutagenesis Mutation Organelles Pathway interactions Phosphorylation Phosphotransferases Physicians Physiological Physiology Plasma Population Potassium Process Proline Protein Biochemistry Protein Isoforms Protein Kinase Proteins RNA Recommendation Regulation Renal function Research Research Personnel Resources RiboTag Ribosomes Role SLC12A3 gene Scientist Serum Signal Pathway Signal Transduction Slice Sodium Sodium Chloride Stress Structure Techniques Telemetry Testing Time Tissues Transgenic Mice United States National Institutes of Health Universities Urine Work base blood pressure elevation blood pressure regulation career development cost experience gain of function mutation human model hyperkalemia in vivo insight meetings mouse model novel pressure proteotoxicity renal tubular transport response sensor skills symporter tenure track thiazide transcriptome sequencing

项目摘要

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 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.

项目摘要/摘要钾[K+] - 缺乏饮食有助于全球高血压和慢性肾脏流行疾病（CKD）。鉴于成本低廉和饮食习惯的易用性[K+]，需要更多的研究才能理解 [K+]失衡如何导致这些疾病。肾脏处理[K+]的90％和远端曲折的小管（DCT）通过WNK-spak（with-lysine/ste20/sps-1与丙氨酸 - 丙氨酸 - 丙氨酸 - 富含丙氨酸）充当[K+]传感器蛋白激酶）途径。该途径的功能突变导致严重的高血压和通过激活噻嗪类敏感的钠/氯化物共转运蛋白（NCC）来激活高钾血症。奇怪的是，饮食[K+] 耗竭或加载会导致WNK-Spak激酶组装成大型DCT特异性细胞质点，在常规饮食中没有看到的小鼠。多年来，这些冷凝水的结构和功能，我们称之为“ wnk尸体”，仍然是一个谜。博伊德·什瓦尔斯基（Boyd-Shiwarski）博士的最初工作已经确定这些DCT- 特定的点（i）取决于肾脏特异性（ks）-WNK1（ii）钾敏感的表达；（iii）无膜;; （iv）与常规细胞器无关；（v）与WNK-Spak蛋白有关。基于这些发现，我们假设WNK体是无膜微区域，可隔离 WNK-Spak途径在钾不平衡期间调节WNK信号传导。该假设将在评估WNK身体形式的生理意义和生物学基础的两个目标。该提议的基于生理学的AIM将为博伊德·什瓦尔斯基博士提供与动物模型合作的机会，（i）实施离体显微镜技术，（ii）量化尿液，血清和血压的变化，以及（iii）开发转基因小鼠模型。鉴于，该提案的基于生物学的部分将包括（i）分子克隆，（ii）蛋白质生物化学以及（iii）质谱和RNA SEQ的实现。这些技能将由导师，顾问，合作者和核心资源团队加强匹兹堡大学。主要导师Arohan Subramanya博士是NIH R01资助身体科学家拥有13年的WNK信号生物学经验和先前的心理经验 20名学员。联合学者汤姆·克莱曼（Tom Kleyman）博士是一位国际认可的物理科学家，他指导匹兹堡肾脏研究中心，并指导了九名职业发展获奖者和五个R01 在过去10年中的接收者。此外，由高血压，WNK-Spak信号和肾小管运输方面的专业知识将监测Boyd-Shiwarski博士通过两年一次的会议进行。博伊德·什瓦尔斯基博士将使用此建议来实现她的短期目标科学独立性以及她成为具有专业知识的任期医师科学家的长期目标在钾稳态，高血压和CKD中。该提案的结果将构成R01的巴西斯研究KS-WNK1依赖性WNK体在人类肾病模型中的翻译作用。