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

 描述(由申请人提供)：在本申请中，我们建议检查非致病细菌草酸杆菌(Oxf)如何促进草酸在肠道中的消除，从而在另一种高草酸血症和高草酸尿的人类遗传性疾病1型(PH1)动物模型中实现血浆和尿草酸正常化的有益效果。在PH1遗传性疾病中，由于肝酶丙氨酸乙二酸氨基转移酶(AGT)缺乏，内源性草酸生成增加，导致高草酸尿和草酸钙肾结石形成，以及草酸组织沉积(草酸中毒)，肾功能衰竭和死亡，除非及早采取积极的临床治疗。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，这种益生菌/治疗方法的潜在影响可能具有非常重要的临床意义，也可能扩展到更多的特发性草酸钙结石患者，包括约12%的美国人，肠源性高草酸尿症患者，以及接受过减肥手术并发展为肾结石疾病的新出现的高草酸尿症患者。我们对大鼠和小鼠肠道草酸转运的研究中出现的大量关键信息为这里提出的研究提供了方向。首先，我们一直表明，野生大鼠和人OXF菌株都可以在整个小鼠肠道定居不同时间，在小肠和大肠中诱导特定节段的草酸分泌/排泄。其次，我们早期对大鼠的研究结果表明，Oxf潜在地开发了一种可溶性化合物/促分泌剂，促进了草酸在肠道中的消除，与减少肾脏草酸的排泄有关。第三，我们研究了SLC26a基因家族中两个主要的顶端草酸转运体的作用，似乎表明这两个转运体都不是Oxf诱导的肠道草酸消除所必需的。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，这是将草酸从血液穿过肠壁进入肠腔的第一个限速步骤。在这方面，我们将重点 在SAT1(硫酸盐阴离子转运体1，SLc26a1)上，除了突出的碱侧钠/钾/2氯化物转运体NKCC1。最近，我们获得了令人信服的新证据，NKCC1可能直接或间接参与了草酸通过基底膜的转运。因此，在目标1中，我们将测试这样的假设，即Oxf诱导的草酸盐在肠道中的消除必然需要SAT1和/或NKCC1在PH1设置以及健康对照组小鼠中的功能性存在。我们将直接测量与未定植的小鼠相比，从定植的小鼠身上取出的不同肠段之间的双向草酸移动。在对天然活体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并测量草酸转运速率来直接确定NKCC1是否为底物。目的#2重点是利用新兴的代谢组学技术鉴定Oxf产生的促进草酸在肠道中消除的化合物/促分泌剂。该计划将包括对粘膜代谢组的分析，以揭示特定节段的组织对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通路。这些研究的结果应该揭示出一个新的方向，导致基于细菌/细菌产品/补充剂的益生菌系统的开发，从而促进肠道草酸排泄和肠道草酸降解，从而使潜在众多患者群体的尿草酸排泄正常化。

 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.