An integrated, functional, molecular, and metabolomic approach to understand Oxalobacter-induced elimination of oxalate

一种了解草酸杆菌诱导的草酸盐消除的综合、功能、分子和代谢组学方法

• 批准号: 9136604
• 负责人: Marguerite Hatch
• 金额: $ 9.75万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9136604
• 关键词: APPBP2 gene; Address; Alanine-glyoxylate aminotransferase; American; Animal Model; Animals; Anions; Apical; Bacteria; Blood; Calcium Oxalate; Calculi; Carrier Proteins; Cessation of life; Chloride Ion; Chlorides; Clinical Management; Collection; Colon; Data; Deposition; Development; Disease; Distal; Enteral; Enterocytes; Enzymes; Excretory function; Gastric Bypass; Gene Family; Goals; Health; Hereditary Disease; Human; Human Genetics; Hyperoxaluria; In Vitro; Individual; Inorganic Sulfates; Intestines; Kidney; Kidney Calculi; Kidney Failure; Kidney Transplantation; Knock-out; Knockout Mice; Large Intestine; Lead; Libraries; Life; Liquid Chromatography; Liquid substance; Liver; Mass Fragmentography; Mass Spectrum Analysis; Measures; Membrane Transport Proteins; Modeling; Molecular; Movement; Mus; Nature; Nuclear Magnetic Resonance; Oxalates; Oxalobacter; Oxalobacter formigenes; Pathway interactions; Patients; Pattern; Physiological; Plasma; Population; Potassium; Primary Hyperoxaluria; Probiotics; Process; Production; Rattus; Resolution; Role; SLC26A3 gene; Signal Pathway; Signal Transduction; Small Intestines; Sodium; Sodium-Potassium-Chloride Symporters; System; Technology; Testing; Therapeutic; Time; Tissues; Unspecified or Sulfate Ion Sulfates; Vitamin B6; Xenopus oocyte; bariatric surgery; base; basolateral membrane; clinically significant; extracellular; ileum; intestinal epithelium; mRNA Expression; metabolomics; microbial host; mouse model; novel; oxalosis; patient population; protein expression; putative anion transporter 1; response; urinary

 DESCRIPTION (provided by applicant): In this application we are proposing to examine the mechanistic basis of how the non-pathogenic bacteria, Oxalobacter formigenes (Oxf), promotes intestinal elimination of oxalate leading to the beneficial effect of normalizing both plasma and urinary oxalate in an otherwise hyperoxalemic and hyperoxaluric animal model of the human genetic disease of Primary Hyperoxaluria, type 1 (PH1). In the genetic disease of PH1, an increased endogenous production of oxalate, due to a deficiency of the liver enzyme alanine-glyoxylate aminotransferase (AGT), results in hyperoxaluria and calcium oxalate kidney stone formation in addition to tissue deposition of oxalate (oxalosis), renal failure and death unless early aggressive clinical management is instigated. Unfortunately, the only known cure for PH1 is a liver or liver-kidney transplant. Thus, the potential impact of this probiotic/therapeutic approach may be highly clinically significant and could also extend to a much larger population of idiopathic calcium oxalate stone formers who comprise ~12% of Americans, individuals with Enteric Hyperoxaluria, and an emerging population of hyperoxaluric patients who have undergone bariatric surgery and develop kidney stone disease. Numerous key pieces of information that emerged from our studies of intestinal oxalate transport in rats and mice have provided the direction for the studies proposed here. First, we have consistently shown that both a wild rat and a human strain of Oxf can colonize the entire mouse intestine for varying periods of time where it induces segment-specific oxalate secretion/excretion in both the small and large intestine. Second, results from our earlier studies in rats indicate that Oxf potentially elaborate a soluble compound/secretagogue that promotes enteric oxalate elimination correlating with a reduced renal excretion of oxalate. Third, our studies examining the role of the two major apical oxalate transporters in the Slc26a gene family appear to indicate that neither one of these is required for Oxf-induced enteric oxalate elimination. These unexpected latter results suggest we should now focus on the major basolateral pathway which is the first rate-limiting step in moving oxalate from the blood across the intestinal wall and into the lumen. In this regard, we will focus on SAT1 (Sulfate Anion Transporter 1, Slc26a1) in addition to a prominent basolateral sodium/potassium/2 chloride transporter, NKCC1. Recently, we acquired compelling new evidence that NKCC1 may be either directly or indirectly involved with translocation of oxalate across the basolateral membrane. Thus, in Aim 1 we will test the hypothesis that the Oxf-induced enteric elimination of oxalate necessarily requires the functional presence of SAT1 and/or NKCC1 in the setting of PH1 as well as in healthy control mice. We will directly measure bi-directional oxalate movements across various intestinal segments removed from mice that are colonized compared to non-colonized mice. In parallel with these studies on the native living tissues, we will directly determine whether oxalate is a substrate for NKCC1 by expressing NKCC1 in Xenopus oocytes and measuring oxalate transport rates. Aim #2 is focused on identifying the compound/secretagogue produced by Oxf that promotes enteric elimination of oxalate by using the emerging metabolomics technologies. The plan will include an analysis of the mucosal metabolome in order to reveal segment-specific tissue responses to colonization by Oxf. Combined with the metabolomics studies of Oxf, tissue-specific metabolomics should reveal novel mechanistic information about the bacteria-host physiological interaction and possible bi- directional signaling pathways between Oxf and specific intestinal segments. The results from these studies should reveal a novel direction leading to the development of a probiotic system based upon bacteria/bacterial products/supplements that promote both enteric oxalate excretion and intestinal oxalate degradation thereby normalizing urinary oxalate excretion in potentially numerous patient populations.

 描述（由申请人提供）：在本申请中，我们提出检查非致病性细菌产草酸杆菌（Oxf）如何促进草酸盐的肠消除，从而在原发性高尿酸血症1型（PH 1）的人类遗传疾病的其他高血容量和高尿酸动物模型中产生使血浆和尿草酸盐正常化的有益效果的机制基础。在PH 1遗传性疾病中，由于肝酶丙氨酸-乙醛酸转氨酶（AGT）缺乏，草酸盐的内源性产生增加，除了草酸盐的组织沉积（草酸盐症）、肾衰竭和死亡之外，还导致高尿酸和草酸钙肾结石形成，除非早期积极的临床管理被激发。不幸的是，PH 1唯一已知的治疗方法是肝脏或肝肾移植。因此，这种益生菌/治疗方法的潜在影响可能具有高度临床意义，并且还可能扩展到更大的特发性草酸钙结石形成者群体，包括约12%的美国人、肠源性高尿酸个体和接受减肥手术并发展为肾结石疾病的高尿酸患者的新兴群体。从我们对大鼠和小鼠肠道草酸盐转运的研究中出现的许多关键信息为本文提出的研究提供了方向。首先，我们一直表明，无论是野生大鼠和人类菌株的Oxf可以殖民整个小鼠肠道不同的时间段，它诱导段特异性草酸分泌/排泄在小肠和大肠。第二，我们早期在大鼠中的研究结果表明，Oxf可能是一种可溶性化合物/促分泌素，可促进肠道草酸盐的消除，并减少草酸盐的肾脏排泄。第三，我们的研究中的两个主要的顶端草酸盐转运蛋白的Slc 26 a基因家族的作用似乎表明，这两个都不是必需的Oxf诱导的肠道草酸盐消除。这些意想不到的结果表明，我们现在应该把重点放在主要的基底外侧通路，这是第一个限速步骤移动草酸盐从血液中穿过肠壁，并进入内腔。在这方面，我们将重点 在SAT 1（硫酸盐阴离子转运蛋白1，Slc 26 a1）上，除了一个突出的基底外侧钠/钾/2氯转运蛋白，NKCC 1。最近，我们获得了令人信服的新证据，NKCC 1可能直接或间接参与草酸盐跨基底外侧膜的转运。因此，在目的1中，我们将检验以下假设：Oxf诱导的草酸盐的肠消除必然需要在PH 1的设置中以及在健康对照小鼠中SAT 1和/或NKCC 1的功能性存在。我们将直接测量与非定殖小鼠相比，从定殖小鼠中取出的各种肠段的双向草酸盐运动。在与这些研究的本地活组织，我们将直接确定草酸是否是NKCC 1的底物表达NKCC 1在非洲爪蟾卵母细胞和测量草酸的运输速率。目的#2专注于通过使用新兴的代谢组学技术来鉴定由Oxf产生的促进草酸盐的肠消除的化合物/促分泌素。该计划将包括粘膜代谢组学的分析，以揭示节段特异性组织对Oxf定植的反应。结合Oxf的代谢组学研究，组织特异性代谢组学将揭示关于细菌-宿主生理相互作用的新机制信息以及Oxf与特定肠段之间可能的双向信号传导途径。这些研究的结果应该揭示了一个新的方向，导致基于细菌/细菌产品/补充剂的益生菌系统的发展，促进肠道草酸盐排泄和肠道草酸盐降解，从而使潜在的许多患者群体的尿草酸盐排泄正常化。