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

摘要 在本申请中，我们提出检查非致病性细菌， 产草酸杆菌（Oxf）促进草酸盐的肠消除，从而产生以下有益效果： 在另外的高血压和高尿酸动物模型中使血浆和尿草酸盐正常化， 人类遗传性疾病原发性高尿酸血症，1型（PH 1）。在PH 1遗传病中， 由于肝酶丙氨酸乙醛酸缺乏，草酸的内源性产生增加 氨基转移酶（AGT），除了导致高尿酸和草酸钙肾结石形成外， 草酸盐组织沉积（草酸盐中毒），肾衰竭和死亡，除非早期积极的临床治疗， 煽动。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/治疗方法影响可能具有高度临床意义， 占美国人约12%的特发性草酸钙结石形成者的人数要多得多， 肠型高尿症患者，以及新出现的高尿症患者人群， 接受减肥手术并患上肾结石。 从我们对大鼠肠道草酸盐转运的研究中出现的许多关键信息， 小鼠为本文提出的研究提供了方向。首先，我们一直表明， 野生大鼠和人类的Oxf菌株可以在不同的时间段内定殖整个小鼠肠道， 诱导小肠和大肠中的节段特异性草酸盐分泌/排泄。二、成果 从我们早期的大鼠研究表明，Oxf可能会产生一种可溶性化合物/促分泌素， 促进肠内草酸盐消除，这与草酸盐的肾排泄减少有关。第三，我们的研究 研究Slc 26 a基因家族中两种主要顶端草酸转运蛋白的作用似乎表明， 这些都不是Oxf诱导的肠道草酸盐消除所必需的。这些意想不到的 结果表明，我们现在应该关注主要的基底外侧通路，这是第一个限速步骤。 草酸盐从血液中穿过肠壁进入肠腔。在这方面，我们将重点关注 SAT 1（硫酸盐阴离子转运蛋白1，Slc 26 a1）除了显著的基底外侧钠/钾/2 氯化物转运蛋白NKCC 1。最近，我们获得了令人信服的新证据，表明NKCC 1可能是 直接或间接参与草酸盐穿过基底外侧膜的转运。因此，在目标1中， 将测试的假设，Oxf诱导的肠道消除草酸盐必然需要功能 在PH 1环境中以及在健康对照小鼠中存在SAT 1和/或NKCC 1。我们会直接 测量从小鼠中取出的各种肠段的双向草酸盐运动， 与未定植的小鼠相比。在对天然活组织进行这些研究的同时，我们将 通过在非洲爪蟾卵母细胞中表达NKCC 1直接确定草酸盐是否是NKCC 1的底物， 测量草酸盐转运速率。目的#2集中于鉴定由以下物质产生的化合物/促分泌素： 利用新兴的代谢组学技术促进草酸盐在肠道中的消除。该计划 将包括粘膜代谢组的分析，以揭示节段特异性组织反应， 结合Oxf的代谢组学研究，组织特异性代谢组学应 揭示了细菌-宿主生理相互作用和可能的双- Oxf和特定肠段之间的定向信号通路。这些研究的结果 应该揭示了一个新的方向，导致基于细菌/细菌代谢的益生菌系统的发展。 从而促进肠草酸盐排泄和肠草酸盐降解的产品/补充剂 在潜在的众多患者群体中使尿草酸排泄正常化。

Abstract 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 elaborates 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.