Using Drosophila to understand WNK and SPAK/OSR1 regulation of SLC12 cotransporte

利用果蝇了解 WNK 和 SPAK/OSR1 对 SLC12 共转运蛋白的调控

• 批准号: 8636462
• 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/8636462
• 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）：这项NIH指导的职业发展奖提案描述了候选人为期五年的培训计划，该计划是一名医师科学家，其长期目标是成为独立的学术研究员，研究重点是与肾脏有关生理和疾病。该候选人建议通过开发新的生理学和生物化学方面的新科学技能，以研究果蝇Melanogaster在本科，博士学位和研究金研究期间开发的基础研究的背景。这些将应用于理解WNK和SPAK/OSR1激酶对SLC12阳离子 - 氯化物共转运蛋白调节的分子机制的直接目标，该机制在肾脏中上皮离子转运中起着重要作用。候选人将在主要导师，联合委员会和咨询委员会成员的支持下发展这些技能，并在与候选人提议的研究领域有关的领域经验丰富。候选人及其顾问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，拥有年轻研究人员的职业发展所需的大量物理和知识分子，以及尖端研究的表现。除了沉浸在实验室研究中，候选人还将利用UT Southwestern提供的众多研究和职业发展研讨会和课程，以进一步发展她的职业生涯。项目描述：上皮离子转运是必需肾功能的基础，例如细胞外体积和血压调节以及酸碱调节。 SLC12阳离子 - 氯化物共转运蛋白，其中包括氯化钠，钠 - 二氯化钠和氯化钾 - 氯化钾共转运蛋白（NCC，NCC，NKCC和KCC）在肾上皮离子离子转运中起关键作用。人NCC和NKCC2的功能丧失突变导致肾钠浪费引起的低血压，这些转运蛋白分别是噻嗪类和环二尿的靶标。小鼠中KCC4基因的敲除导致远端肾小管酸中毒。这些共转运蛋白的调节仍然不完全理解，但最近的证据表明，WNK和SPAK/OSR1激酶涉及。人类WNK功能收益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的肾脏盐浪费和低血压。来自体外，细胞培养和爪蟾卵母细胞模型的数据表明，WNK磷酸化并激活SPAK和OSR1，然后磷酸化并激活NCC和NKCC，并且卵母细胞数据也表明WNKS对KCC进行了调节。但是，仍然存在许多公开问题。该项目的直接目的是使用果蝇果蝇melanogaster更好地了解WNK和SPAK/OSR1 SLC12调节的分子机制。苍蝇的复杂遗传学，其快速生命周期以及其肾小管的特征性生理学，可以对体内上皮离子转运的分子机制进行有效但详细的研究。此外，大多数哺乳动物基因家族的主要单基因表示会降低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共转运蛋白在蝇肾小管中的作用。该提案的目的是评估WNK和Fray（果蝇SPAK/OSR1同源物）是否调节上皮离子运输；如果他们这样做是通过NKCC和/或KCC的规定； WNK和Fray对NKCC与KCC的调节的详细机制。这将通过测定小管生理学来测试，包括测量跨跨剂量和钾通量，尿液分泌速率以及成年蝇对高磷酸食品的致命性，以及蛋白质 - 蛋白质相互作用和激酶的体外和细胞培养试验活动。四种哺乳动物WNK同工型之间的差异将通过引入缺乏内源性WNK的苍蝇进行探索。最后，通过在表达突变体转运蛋白的苍蝇中测定小管生理学来测试特定磷酸链和磷酸硫代硫酸盐对苍蝇和哺乳动物NKCC和KCC体内功能的重要性。从这些研究中获得的洞察力可以在小鼠模型中直接测试，其长期目标是更好地了解哺乳动物的肾脏生理和人类疾病，例如高血压和肾小管远端管状酸中毒。 公共卫生相关性：SLC12共转运蛋白对肾脏调节体内血压和酸碱平衡的能力很重要。这些共转运蛋白的调节方式尚不清楚。该项目旨在更好地了解这些共转运蛋白起作用的分子机制及其调节方式，以提高对高血压和酸碱平衡疾病的理解和治疗。