WNK & SPAK/OSR1 regulation of SLC12 cotransporters in Drosophila

温克

• 批准号: 8235050
• 负责人: AYLIN RACHEL RODAN
• 金额: $ 14.53万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235050
• 关键词: Academic Medical Centers; Acid-Base Equilibrium; Acids; Address; Adult; Advisory Committees; Affect; Basic Science; Biochemistry; Biological Assay; Blood Pressure; Cardiovascular system; Cations; Cell Culture Techniques; Chloride Ion; Chlorides; Committee Members; Data; Development; Diet; Disease; Distal renal tubular acidosis Type 1; Diuretics; Doctor of Philosophy; Drosophila genus; Drosophila melanogaster; End stage renal failure; Epithelial; Epithelium; Event; Family; Fellowship; Financial compensation; Fluids and Secretions; Food; Future; Gene Family; Gene Transfer Techniques; Genes; Genetic; Goals; Hematological Disease; Homologous Gene; Human; Hypertension; Hypotension; Immersion Investigative Technique; In Vitro; Ion Transport; Ions; K-Series Research Career Programs; Kidney; Knock-out; Laboratories; Lead; Life Cycle Stages; Malpighian Tubules; Measurement; Measures; Medical center; Mentors; Modeling; Molecular; Mus; Mutate; Mutation; Oocytes; Pathogenesis; Pathway interactions; Performance; Phosphorylation; Phosphotransferases; Physicians; Physiology; Play; Population; Potassium; Protein Isoforms; Publishing; Regulation; Renal function; Renal tubular acidosis; Renal tubule structure; Research; Research Personnel; Resources; Risk Factors; Role; Scientist; Serine; Sodium; Sodium Chloride; Syndrome; System; Testing; Texas; Threonine; Tissues; Training Programs; United States; United States National Institutes of Health; Universities; Urine; Work; Xenopus oocyte; base; career; career development; chloride-cotransporter potassium; combinatorial; experience; extracellular; familial hypertension; field study; fly; gain of function mutation; hyperkalemia; improved; in vitro activity; in vivo; insight; loss of function; loss of function mutation; mouse model; mutant; protein protein interaction; public health relevance; reconstitution; research and development; research study; skills; sodium-chloride cotransporter; sodium-potassium chloride cotransporter 2 protein; thiazide; wasting

DESCRIPTION (provided by applicant): This NIH mentored Career Development Award proposal describes a five year training program for the candidate, a physician scientist with the long-term goal of becoming an independent academic investigator with a research focus on epithelial ion transport related to kidney physiology and diseases. The candidate proposes to build on a background in basic research developed during undergraduate, Ph.D., and fellowship studies, and in particular previous experience studying Drosophila melanogaster, by developing new scientific skills in physiology and biochemistry. These will be applied to the immediate goal of understanding the molecular mechanisms of regulation of SLC12 cation-chloride cotransporters by the WNK and SPAK/OSR1 kinases, which play essential roles in epithelial ion transport in the kidney. The candidate will develop these skills with the support of a primary mentor, a co-mentor, and advisory committee members with extensive experience in fields related to the candidate's proposed field of study. The candidate and her advisors are located at the University of Texas Southwestern Medical Center, a leading academic medical center with the substantial physical and intellectual resources necessary for the career development of young investigators and the performance of cutting-edge research. In addition to the intensive immersion in research in the laboratory, the candidate will take advantage of the numerous research and career development seminars and courses available at UT Southwestern to further develop her career. Project Description: Epithelial ion transport underlies essential kidney functions, such as the regulation of extracellular volume and blood pressure and acid-base regulation. The SLC12 family of cation-chloride cotransporters, which includes sodium-chloride, sodium-potassium-two-chloride, and potassium-chloride cotransporters (NCC, NKCC, and KCC), plays a key role in renal epithelial ion transport. Loss-of-function mutations in human NCC and NKCC2 result in hypotension due to renal sodium wasting, and these transporters are the targets of thiazide and loop diuretics, respectively. Knockout of the KCC4 gene in mice results in distal renal tubular acidosis. Regulation of these cotransporters remains incompletely understood, but recent evidence suggests that the WNK and SPAK/OSR1 kinases are involved. Human WNK gain-of-function mutations lead to hypertension and hyperkalemia, and loss-of-function mutations in WNK and SPAK lead to renal salt wasting and hypotension in the mouse. Data from in vitro, cell culture, and Xenopus oocyte models suggest that WNKs phosphorylate and activate SPAK and OSR1, which then phosphorylate and activate NCC and NKCCs, and oocyte data also suggest regulation of KCCs by WNKs. However, many open questions remain. The immediate goal of this project is to use the fruitfly Drosophila melanogaster to better understand the molecular mechanisms of SLC12 regulation by WNKs and SPAK/OSR1. The sophisticated genetics of the fly, its rapid life cycle, and the well-characterized physiology of its renal tubule allows for efficient, yet detailed, study of the molecular mechanisms of epithelial ion transport in vivo. Moreover, the predominant single gene representation of most mammalian gene families decreases combinatorial complexity and gene compensation as encountered in mammalian models. Preliminary and published data suggest a role for SLC12 cotransporters in the fly renal tubule. The aims of this proposal are to assess whether WNK and Fray (the Drosophila SPAK/OSR1 homolog) regulate epithelial ion transport; if they do so by regulation of NKCC and/or KCC; and the detailed mechanisms by which WNK and Fray regulate NKCC versus KCC. This will be tested by assaying tubule physiology, including measurement of transepithelial potential and potassium flux, rates of urine secretion, and lethality of adult flies on high-potassium food, as well as in vitro and cell culture assays of protein-protein interactions and kinase activity. The differences between the four mammalian WNK isoforms will be explored by their introduction into flies lacking endogenous WNK. Finally, the importance of specific phospho-serines and phospho-threonines on the in vivo functioning of fly and mammalian NKCC and KCC will be tested by assaying tubule physiology in flies expressing mutant transporters. Insights gained from these studies can in future be directly tested in mouse models, with the long-term goal of better understanding mammalian renal physiology and human disorders such as hypertension and distal renal tubular acidosis. PUBLIC HEALTH RELEVANCE: SLC12 cotransporters are important in the kidney's ability to regulate blood pressure and acid-base balance in the body. How these cotransporters are regulated is not well understood. This project seeks to better understand the molecular mechanisms by which these cotransporters work and how they are regulated, to improve understanding and treatment of high blood pressure and disorders of acid-base balance.

描述（由申请人提供）：这份由NIH指导的职业发展奖提案描述了一个为期五年的培训计划，该候选人是一名内科科学家，其长期目标是成为一名独立的学术研究者，研究重点是与肾脏生理学和疾病相关的上皮离子运输。候选人建议在本科，博士和奖学金研究期间发展的基础研究背景，特别是以前研究果蝇的经验，通过发展生理学和生物化学方面的新科学技能。这些将用于理解WNK和SPAK/OSR1激酶调控SLC12阳离子-氯共转运体的分子机制，它们在肾脏上皮离子运输中起重要作用。候选人将在主要导师、共同导师和咨询委员会成员的支持下发展这些技能，这些成员在候选人提议的研究领域相关的领域具有丰富的经验。候选人和她的导师位于德克萨斯大学西南医学中心，这是一个领先的学术医学中心，为年轻研究者的职业发展和前沿研究的表现提供了必要的物质和智力资源。除了在实验室进行深入的研究外，候选人还将利用德克萨斯大学西南分校提供的众多研究和职业发展研讨会和课程来进一步发展她的职业生涯。项目描述：上皮离子转运是肾脏基本功能的基础，如细胞外体积和血压的调节以及酸碱调节。SLC12家族的阳离子-氯共转运蛋白，包括氯化钠、二氯化钠-钾和氯化钾共转运蛋白（NCC、NKCC和KCC），在肾上皮离子转运中起关键作用。人类NCC和NKCC2的功能缺失突变可导致由于肾脏钠消耗而导致的低血压，而这两种转运蛋白分别是噻嗪类药物和环状利尿剂的靶点。敲除小鼠KCC4基因可导致远端肾小管酸中毒。这些共转运体的调控尚不完全清楚，但最近的证据表明WNK和SPAK/OSR1激酶参与其中。人类WNK功能获得突变导致高血压和高钾血症，WNK和SPAK的功能丧失突变导致小鼠肾盐消耗和低血压。来自体外、细胞培养和爪蟾卵母细胞模型的数据表明，WNKs磷酸化并激活SPAK和OSR1，然后SPAK和OSR1磷酸化并激活NCC和nkcc，卵母细胞数据也表明WNKs调节kcc。然而，仍有许多悬而未决的问题。本项目的近期目标是利用果蝇来更好地了解WNKs和SPAK/OSR1调控SLC12的分子机制。果蝇复杂的遗传学，其快速的生命周期，以及其肾小管的生理特征，使得对体内上皮离子运输的分子机制进行有效而详细的研究成为可能。此外，大多数哺乳动物基因家族的优势单基因代表降低了在哺乳动物模型中遇到的组合复杂性和基因补偿。初步和已发表的数据表明SLC12共转运蛋白在果蝇肾小管中的作用。本研究的目的是评估WNK和Fray（果蝇SPAK/OSR1同源物）是否调节上皮离子运输；如果他们这样做是根据NKCC和/或KCC的规定；以及WNK和Fray调节NKCC与KCC的详细机制。这将通过分析小管生理学来测试，包括测量经上皮电位和钾通量，尿液分泌率，以及高钾食物对成年苍蝇的致死率，以及体外和细胞培养的蛋白质-蛋白质相互作用和激酶活性分析。四种哺乳动物WNK亚型之间的差异将通过将它们引入缺乏内源性WNK的果蝇来探索。最后，我们将通过分析表达突变转运体的果蝇的小管生理学来测试特异性磷酸化丝氨酸和磷酸化苏氨酸对果蝇和哺乳动物NKCC和KCC体内功能的重要性。从这些研究中获得的见解可以在未来直接在小鼠模型中进行测试，其长期目标是更好地了解哺乳动物肾脏生理学和人类疾病，如高血压和远端肾小管酸中毒。