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Molecular Mechanisms Regulating Ammonia Metabolism

调节氨代谢的分子机制

基本信息

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
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/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

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.

项目总结/摘要肾脏维持酸碱平衡对最佳健康至关重要。集合管闰细胞通过质子分泌、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，反应涉及细胞外pH对嵌入细胞的直接影响。我们提出了一个新的范例。近端小管特异性基底外侧重碳酸盐缺失转运蛋白NBCe 1-A可导致严重的代谢性酸中毒，但抑制了嵌入细胞的表型特征的酸分泌和抑制嵌入细胞可塑性响应酸负荷。这不是脱靶 TALEN基因编辑程序的效果;在产生NBCe 1-A/B缺失的情况下观察到相同的效果。使用Cre-lox技术。已发表的研究表明，K+障碍，低钾血症和高钾血症，改变插入细胞表型和可塑性的模式，不能解释细胞外pH值。 NBCe 1-A或NBCe 1-A/B缺失或K+障碍可能涉及氨。每一个都改变了近端小管- 衍生的氨产生的模式，平行于观察到的嵌入细胞的反应。分离灌注集合管研究表明氨急性和直接调节闰细胞H β，碳酸氢盐运输因此，我们提出了一个新的范例，即近端小管来源的氨，这是通过TAL在肾间质中浓缩，是闰细胞表型的主要决定因素。特征和可塑性反应。我们提出的研究详细调查了这种新的模式。具体目标1将决定基因删除策略的效果，这些策略直接和特异性地改变近端小管和粗升支氨代谢对闰细胞表型和可塑性的影响。我们将使用基因缺失方法的组合，包括近端小管特异性PDG缺失，最初的氨合成中的限速酶，以及直接影响氨的TAL NHE 4缺失浓度进入肾间质，我们建议它调节闰细胞。我们将研究这些基因的缺失，所以在各种条件下，包括基础状态，酸负荷，和异常钾体内平衡，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，闰细胞表型特征和可塑性。我们将确定这是否是与已知改变闰细胞的信号传导途径平行且独立的途径或行为，包括 GDF 15、hensin、GPR 4和SDF 1。这些研究将极大地推进我们对调节细胞凋亡的分子机制的理解。从而达到酸碱平衡