Molecular Mechanisms Regulating Ammonia Metabolism

调节氨代谢的分子机制

基本信息

批准号：
10491245
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
项目类别：
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/10491245
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 Persons 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

项目摘要

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.

项目概要/摘要肾脏维持酸碱平衡对于插入集合管的最佳健康至关重要。细胞通过质子分泌、碳酸氢盐分泌和代谢的适应性变化在此过程中发挥核心作用。 Rh 糖蛋白介导的氨转运是这一现象的主要决定因素。反应涉及细胞外 pH 对闰细胞的直接影响。我们提出了一种新的范例，即删除近端小管特异性基底外侧碳酸氢盐。转运蛋白 NBCe1-A 会导致严重的代谢性酸中毒，但会抑制闰细胞表型特征酸分泌并抑制插层细胞对酸负荷的可塑性反应，这不是脱靶。 TALEN 基因编辑程序的效果；与 NBCe1-A/B 删除产生的效果相同使用 Cre-lox 技术已发表的研究表明 K+ 疾病、低钾血症和高钾血症会改变。插层细胞表型和可塑性的模式无法用细胞外 pH 值来解释。 NBCe1-A 或 NBCe1-A/B 缺失，或 K+ 疾病可能涉及氨。衍生的氨生成模式与观察到的孤立细胞反应平行。灌注集合管研究表明，氨能敏锐地直接调节嵌入细胞 H⁺ 和因此，我们提出了一种新的范例，即近端小管衍生的氨，即 TAL 集中在肾间质，是闰细胞表型的主要决定因素我们提出的研究详细研究了这种新范式。具体目标 1 将确定基因删除操作的效果，该操作直接且具体地改变近曲小管和粗升肢氨代谢对闰细胞表型和可塑性的影响。将使用基因删除方法的组合，包括近端小管特异性的 PDG 删除，最初的以及氨生成中的限速酶，以及直接影响氨的 TAL NHE4 缺失浓度进入间质，我们建议它调节嵌入细胞，我们将研究其效果。这些基因缺失在多种条件下都会发生，包括基础状态、酸负荷和钾异常体内平衡，低钾血症和高钾血症。具体目标 2 将确定近端小管调节的信号传导机制我们将确定这是否是插入细胞表型特征和可塑性的直接刺激物。与已知改变嵌入细胞的信号通路平行且独立的通路或作用，包括 GDF15、hensin、GPR4 和 SDF1。这些研究将极大地增进我们对调节分子机制的理解从而实现酸基稳态。