Molecular Mechanisms Regulating Ammonia Metabolism

调节氨代谢的分子机制

• 批准号: 10366279
• 负责人: I. David Weiner
• 金额: $ 33.55万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366279
• 关键词: Acids; Acute; Address; Alkalosis; Ammonia; Anions; Apical; Atrophic; Bicarbonates; CXCL12 gene; Cardiovascular system; Cell membrane; Cell physiology; Characteristics; Chronic Kidney Failure; Clinical Medicine; Coupled; DNA Sequence Alteration; Disease; Disease Progression; Duct (organ) structure; Electron Microscopy; Enzymes; Event; Excretory function; GDF15 gene; GPR4 gene; Gene Combinations; Gene Deletion; Generations; Genes; Glycoproteins; Health; Homeostasis; Hypokalemia; Impairment; In Vitro; Intercalated Cell; Intercalated Duct; Intervention Trial; Kidney; Knockout Mice; Lead; Limb structure; Maintenance; Mediating; Metabolic acidosis; Metabolism; Modeling; Molecular; Mus; Pathway interactions; Pattern; Phenotype; Physical Function; Play; Potassium; Procedures; Process; Proteins; Proton-Translocating ATPases; Protons; Publishing; RNA Splicing; Regulation; Renal Replacement Therapy; Role; Series; Serum; Signal Pathway; Signal Transduction; Site; Techniques; Thick; Thyroid Function Tests; Urine; Variant; Vesicle; Work; base; bone metabolism; collecting tubule structure; extracellular; glucose metabolism; hensin; hyperkalemia; improved; insulin sensitivity; kidney interstitial tissue; mortality; muscle form; muscle strength; response; transcription activator-like effector nucleases

Project Summary/Abstract The renal maintenance of acid-base homeostasis is critical for optimal health. Collecting duct intercalated cells play a central role in this process through adaptive changes in proton secretion, bicarbonate secretion, and Rh glycoproteins-mediated ammonia transport. Our current paradigm is that the primary determinant of this response involves direct effects of extracellular pH on intercalated cells. We suggest a new paradigm. Deletion of the proximal tubule-specific basolateral bicarbonate transporter, NBCe1-A, causes severe metabolic acidosis, yet inhibits intercalated cell phenotypic characteristics of acid secretion and inhibits the intercalated cell plasticity response to acid-loading. This is not an off-target effect of the TALEN gene-editing procedure; identical effects were seen with NBCe1-A/B deletion generated using Cre-lox techniques. Published work shows that the K+ disorders, hypokalemia and hyperkalemia, alter intercalated cell phenotype and plasticity in a pattern which cannot be explained by extracellular pH. The effects of NBCe1-A or NBCe1-A/B deletion, or of K+ disorders likely involve ammonia. Each alters proximal tubule- derived ammonia generation in a pattern which parallels the observed intercalated cell responses. Isolated perfused collecting duct studies show ammonia acutely and directly regulates intercalated cell H⁺ and bicarbonate transport. Thus, we propose a new paradigm, that proximal tubule-derived ammonia, which is concentrated in the renal interstitium by the TAL, is a primary determinant of intercalated cell phenotypic characteristics and plasticity response. Our proposed studies investigate this new paradigm in detail. Specific Aim 1 will determine the effect of gene deletion maneuvers which directly and specifically alter proximal tubule and thick ascending limb ammonia metabolism on intercalated cell phenotype and plasticity. We will use a combination of gene deletion approaches, including proximal tubule-specific deletion of PDG, the initial and the rate-limiting enzyme in ammoniagenesis, and TAL NHE4 deletion, which directly impacts ammonia concentration into the renal interstitium where we propose it regulates intercalated cells. We will study the effect of these gene deletion so in a variety of conditions, including basal state, acid-loading, and abnormal potassium homeostasis, both hypokalemia and hyperkalemia. Specific Aim 2 will determine the signaling mechanisms through which proximal tubule regulates intercalated cell phenotypic characteristics and plasticity. We will identify whether this is direct stimulator of pathways or acts parallel to and independent of signaling pathways known to alter intercalated cells, including GDF15, hensin, GPR4, and SDF1. These studies will substantially advance our understanding of the molecular mechanisms regulating thereby acid-base homeostasis.

项目摘要/摘要 肾脏维持酸碱平衡对最佳健康至关重要。集热管插层 在这一过程中，细胞通过质子分泌、碳酸氢盐分泌和 Rh糖蛋白介导的氨转运。我们目前的模式是，这一点的主要决定因素 反应涉及细胞外pH对嵌入细胞的直接影响。 我们提出了一种新的范式。近端小管特异的碱侧重碳酸盐的缺失 转运蛋白NBCe1-A导致严重代谢性酸中毒，但抑制嵌入细胞的表型特征 抑制酸分泌，抑制细胞对酸负荷的可塑性反应。这不是偏离目标 TALEN基因编辑程序的效果；在产生NBCe1-A/B缺失的情况下看到相同的效果 使用Cre-lox技术。已发表的工作表明，K+紊乱、低钾血症和高钾血症、 插层细胞的表型和可塑性不能用细胞外pH来解释。其影响 NBCe1-A或NBCe1-A/B缺失，或K+紊乱可能与氨有关。每一种都改变了近端小管- 衍生氨的产生模式与观察到的嵌入细胞的反应平行。孤立 灌流集合管研究表明，氨能敏锐而直接地调节嵌入细胞H⁺和 小苏打运输。因此，我们提出了一种新的范式，即近端小管衍生的氨，这是 集中在肾间质的TAL，是插层细胞表型的主要决定因素 特性和塑性反应。我们提出的研究对这一新范式进行了详细的研究。 具体目标1将确定基因缺失操作的效果，这些操作直接和特定地改变 近端小管和粗大升肢氨代谢对间质细胞表型和可塑性的影响。我们 将使用基因缺失的组合方法，包括近端小管特异的PDG缺失，最初 氨化作用中的限速酶，以及直接影响氨的TAL NHE4缺失 浓度进入肾间质，我们认为在那里它调节着嵌入的细胞。我们将研究这一影响 这些基因的缺失在各种条件下都是如此，包括基础状态、酸负荷和异常的钾 动态平衡，包括低钾血症和高钾血症。 特定目标2将确定近端小管调节信号的信号机制 嵌合细胞的表型特征和可塑性。我们将确定这是否是直接刺激因素 与已知的改变插入细胞的信号通路平行或独立的通路或作用，包括 GDF15、Hensin、GPR4和SDF1。 这些研究将极大地促进我们对调控分子机制的理解 从而实现酸碱平衡。