WNK kinase cascade in health and disease

WNK 激酶级联在健康和疾病中的作用

• 批准号: 10523732
• 负责人: Chou-Long Huang
• 金额: $ 44.06万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10523732
• 关键词: Action Potentials; Acute; Affect; Alanine; Animals; Axon; Blinded; Blood Circulation; Body Water; Brain; Cell Volumes; Cells; Central Diabetes Insipidus; Cultured Cells; Diabetes Insipidus; Disease; Electrolytes; Electrophysiology (science); Environment; Equilibrium; Exhibits; Experimental Water Deprivation; Family; Feedback; Frequencies; Genotype; Goals; Health; Homeostasis; Human; In Situ; Individual; Ion Channel; Kidney; Knockout Mice; Life; Liquid substance; Lysine; Measures; Mediating; Metabolic; Molecular; Molecular Target; Mus; Mutation; Neurons; Organ; Osmolalities; Osmoregulation; Oxidative Stress; Pathway interactions; Phenotype; Phosphotransferases; Physiology; Play; Polyuria; Posterior Pituitary Gland; Process; Proline; Protein Kinase; Recombinants; Recovery; Refractory; Regulation; Role; Serum; Signal Transduction; Single-Blind Study; Slice; Subfornical Organ; Testing; Thirst; Travel; Type II Pseudohypoaldosteronism; Urine; Vasopressins; Virus; Voltage-Gated Potassium Channel; Water; blood-brain barrier function; conditional knockout; drinking; drinking water; equilibration disorder; experimental study; extracellular; gain of function mutation; hypertensive; inhibitor; insight; knock-down; magnocellular; metabolic abnormality assessment; novel; organum vasculosum of the lamina terminalis; paraventricular nucleus; protein function; response; sensor; small hairpin RNA; supraoptic nucleus

Project Summary Maintaining internal environment constancy is essential for life. The circumventricular organs (CVO’s) of the brain including organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO) lack a blood-brain-barrier, function as central sensors to provide feedback regulation for maintaining osmotic equilibrium. Neurons in CVO’s detect changes in osmolality and transduce the signals into action potentials (AP’s) travelling down the axon projecting to magnocellular neurons in the paraventricular (PVN) and supraoptic nuclei (SON). Upon activation by AP’s, magnocellular neurons synthesize antidiuretic hormones (ADH), which is transported via axon process to the posterior pituitary gland and released into blood circulation herein. ADH acts on kidney to effect free water reabsorption. While hyperosmolality also stimulates thirst and drinking, separate neuronal networks are involved. Together, renal free water reclamation and drinking restore serum osmolality in response to water deprivation. The molecular identity of the osmolality sensor(s) in the OVLT/SFO neurons remains elusive. With-no-lysine [K] kinases WNK1-4 are protein kinases in which gain-of-function mutations of WNK1 and 4 in humans cause familial hypertensive and hyperkalemic disease called pseudohypoaldosteronism type II (PHA2). Our preliminary results strongly support the central hypothesis that WNK1 functions as a central osmosensor for osmolality regulation of ADH release. Mice with neuronal conditional knockout (cKO) of Wnk1 exhibit phenotypes of partial central diabetes insipidus (DI). WNK1 activate downstream oxidative-stress responsive-1 kinase (OSR1) and related SPAK (Ste20-related proline/alanine-rich kinase). WNK1-OSR1/SPAK kinase cascade regulates many ion channels and transporters. To support the hypothesis, Specific Aim-1 will test the hypothesis that OSR1 and/or SPAK acts downstream of WNK1 to regulate osmolality-induced ADH release. Control and mice with genetically altered WNK1 kinase cascade will be studied in metabolic cage under free water access and water restriction. Urine volume, urine and serum electrolyte, osmolality, ADH and copeptin levels will be measured. Specific Aim-2 will test the hypothesis that activation of Kv3.1b voltage-gated K+ channels by WNK1 increases AP firing in OVLT/SFO neurons leading to stimulation of ADH release by hyperosmolality. Electrophysiological recording of freshly isolated individual OVLT/SFO neurons, native neurons in situ in acute brain slice, and HEK cells expressing recombinant channels and WNK1 cascades will be performed. The proposed studies will reveal novel findings that an intracellular protein functions as a sensor for extracellular osmolality and provide fresh insights into how body maintains osmotic equilibrium in health and into disease processes that affect total body water homeostasis.

项目概要 维持内部环境的恒定对于生命至关重要。室周器官（CVO） 大脑，包括终板血管器 (OVLT) 和穹窿下器官 (SFO) 缺乏 血脑屏障，充当中央传感器，提供反馈调节以维持渗透压 平衡。 CVO 中的神经元检测渗透压的变化并将信号转换为动作电位 （AP）沿着轴突行进，投射到室旁（PVN）和视上的大细胞神经元 原子核（SON）。被 AP 激活后，大细胞神经元合成抗利尿激素 (ADH)， 经由轴突转运至垂体后叶并释放到此处的血液循环中。 ADH 作用 影响肾脏的自由水重吸收。虽然高渗透压也会刺激口渴和饮酒，但单独 神经网络参与其中。肾游离水回收和饮用共同恢复了患者的血清渗透压 对缺水的反应。 OVLT/SFO 神经元中渗透压传感器的分子特性 仍然难以捉摸。无赖氨酸 [K] 激酶 WNK1-4 是蛋白激酶，其中 WNK1 和 4 在人类中引起家族性高血压和高钾血症，称为假性醛固酮增多症 II 型（PHA2）。我们的初步结果有力地支持了中心假设，即 WNK1 发挥着中心作用 渗透压传感器用于 ADH 释放的渗透压调节。 Wnk1 神经元条件性敲除 (cKO) 小鼠 表现出部分中枢性尿崩症（DI）的表型。 WNK1 激活下游氧化应激 响应 1 激酶 (OSR1) 和相关 SPAK（Ste20 相关脯氨酸/富含丙氨酸激酶）。 WNK1-OSR1/SPAK 激酶级联调节许多离子通道和转运蛋白。为了支持这一假设，Specific Aim-1 将 检验 OSR1 和/或 SPAK 作用于 WNK1 下游以调节渗透压诱导的 ADH 的假设 发布。对照小鼠和具有基因改变的 WNK1 激酶级联的小鼠将在代谢笼中进行研究 免费取水和限水。尿量、尿液和血清电解质、渗透压、ADH 和和肽素 将测量水平。具体的 Aim-2 将测试 Kv3.1b 电压门控 K+ 激活的假设 WNK1 的通道增加了 OVLT/SFO 神经元中的 AP 放电，从而刺激 ADH 的释放 高渗透压。新鲜分离的单个 OVLT/SFO 神经元、天然神经元的电生理记录 在急性脑切片中原位，表达重组通道和 WNK1 级联的 HEK 细胞将被 执行。拟议的研究将揭示细胞内蛋白质作为传感器的新发现 细胞外渗透压，并为身体如何维持健康的渗透平衡提供新的见解 影响全身水稳态的疾病过程。