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

摘要 在这项应用中，我们建议检查非致病细菌如何， 草酸杆菌，促进草酸在肠道中的排出，从而产生有益的效果 在高草酸血症和高草酸尿症动物模型中血浆和尿草酸的正常化 人类遗传性高草酸尿症，1型(PH1)。在PH1的遗传病中，一种 由于肝酶丙氨酸-乙二醇酸缺乏，内源性草酸产量增加 氨基转移酶(AGT)导致高草酸尿和草酸钙肾结石形成，此外 草酸盐组织沉积(草酸中毒)，肾功能衰竭和死亡，除非早期积极的临床治疗 被煽动的。不幸的是，唯一已知的治疗PH1的方法是肝脏或肝肾移植。因此，潜在的 这种益生菌/治疗方法的影响可能在临床上具有非常重要的意义，也可能延伸到 占美国人约12%的特发性草酸钙结石患者的数量要多得多， 肠源性高草酸尿症患者和新出现的高草酸尿症患者 接受了减肥手术，并患上了肾结石疾病。 大量关键信息来自我们对大鼠肠道草酸转运的研究，以及 老鼠为这里提出的研究提供了方向。首先，我们一直表明，两者都是 野鼠和人类OXF菌株可以在整个小鼠肠道内定居不同的时间段，其中 在小肠和大肠中诱导特定节段的草酸分泌/排泄。第二，结果 从我们早期对大鼠的研究表明，Oxf潜在地阐述了一种可溶化合物/促分泌剂， 促进草酸在肠道中的排出，与减少肾脏草酸排泄有关。第三，我们的学习 研究SLC26a基因家族中两个主要的顶端草酸转运体的作用似乎表明 OXF诱导的肠道草酸消除不需要这两种物质。这些意想不到的后者 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。 草酸从血液中穿过肠壁进入肠腔。在这方面，我们将重点关注 SAT1(硫酸盐阴离子转运体1，SLc26a1)除了突出的基侧钠/钾/2 氯离子转运体，NKCC1。最近，我们获得了令人信服的新证据，NKCC1可能是 直接或间接地参与了草酸跨基底膜的转运。因此，在目标1中，我们 将检验这一假设，即Oxf诱导的草酸在肠道中的消除必然需要功能性的 在PH1和健康对照小鼠中存在SAT1和/或NKCC1。我们会直接 测量从小鼠身上取出的不同肠段之间的双向草酸运动 与非殖民地小鼠相比，被殖民的小鼠。在对本土生物组织进行这些研究的同时，我们将 通过在非洲爪哇卵母细胞中表达NKCC1，直接确定草酸是否为NKCC1的底物 测量草酸盐的传输速率。目标2的重点是鉴定由 OXF通过使用新兴的代谢组学技术促进草酸的肠道消除。这个计划 将包括对粘膜代谢组的分析，以揭示节段特异性组织对 被牛津饥荒救济委员会殖民。结合OXF的代谢组学研究，组织特异性代谢组学应该 揭示了细菌-宿主生理相互作用和可能的双向作用的新机制信息。 OXF和特定肠段之间的定向信号通路。这些研究的结果 应该揭示一个新的方向，导致基于细菌/细菌的益生菌系统的发展 促进肠道草酸排泄和肠道草酸降解的产品/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.