Mechanisms of Oxalobacter-Induced Enteric Oxalate Excretion

描述（由申请人提供）：高草酸尿被认为是草酸钙结石疾病的主要危险因素，约12%的美国人口发生草酸钙结石，除了减少草酸盐的饮食来源外，目前没有可用的药物治疗。与高草酸尿和高草酸血症相关的更严重的并发症，如肾衰竭、结石和草酸盐组织沉积（草酸沉积）也发生在原发性高草酸尿症（PH）中，这是一种进行性遗传代谢疾病，由肝脏缺乏丙氨酸-乙醛酸氨基转移酶（AGT）引起，只能通过肝肾联合移植来治愈。显然，草酸相关疾病的替代治疗方案需要关注，我们建议研究促进肠道消除和草酸降解作为减少高草酸尿和高氧血症的一种方法。我们最近在大鼠/小鼠结肠草酸转运研究中获得的几个关键信息为本文提出的研究提供了方向。首先，我们已经证明，除了降解草酸的膳食来源外，只存在于大肠中的底物/草酸特异性微生物草酸杆菌（Oxalobacter sp.）可以通过诱导结肠草酸分泌/排泄来显著降低尿草酸的排泄量。我们也有令人信服的结果表明，这种厌氧细菌通过产生促分泌剂与运输粘膜相互作用，激活肠道草酸处理中这些有益的变化。其次，基于我们对野生型（WT）和敲除型（KO）小鼠的研究，我们确定了两个关键的阴离子交换体，即PAT1 （slc26a6）和DRA (slc26a3)，它们对草酸盐在肠道中的运动有重要贡献，我们假设草酸盐转运的变化取决于这些转运体的表达和功能。因此，我们将在PAT1和DRA单KO小鼠以及PAT1/AGT双KO小鼠模型和携带草酸杆菌的WT小鼠中研究肠道和肾脏对草酸盐的处理，并将其与相应的非定殖小鼠进行比较。了解细菌细胞调节肠道草酸处理的机制基础，对于未来确定哪些菌株和/或细菌产物将有效治疗原发性高草酸尿和草酸钙结石病至关重要。公共卫生相关性：提出的研究解决了草酸杆菌在大肠定植诱导肠内草酸消除和降低尿中草酸排泄的机制。此外，这些研究将通过靶向缺失slc26a3 （DRA）和slc26a6 （PAT1）的敲除小鼠模型，评估草酸杆菌是否调节两种已知重要草酸转运蛋白的功能。然而，与当前应用特别重要和相关的是，最近可获得的PAT1/AGT双KO小鼠模型为我们提供了一个独特的机会来评估原发性高草酸尿，1型（PH1）的PAT1功能/活性，有或没有草酸杆菌定植。了解草酸盐在PH1中的处理以及PAT1和草酸杆菌在调节草酸盐在这种遗传疾病中的过度身体负担中的潜在作用和相互作用现在是可能的。

DESCRIPTION (provided by applicant): Hyperoxaluria is considered to be a major risk factor in calcium oxalate stone disease which occurs in about 12% of the American population and, apart from reducing dietary sources of oxalate, there is currently no pharmacological treatment available. More serious complications associated with hyperoxaluria and hyperoxalemia, such as renal failure, stones, and tissue deposits of oxalate (oxalosis) also occur in Primary Hyperoxaluria (PH), a progressive inherited metabolic disorder caused by a liver deficiency of the enzyme alanine-glyoxylate aminotransferase (AGT) which is cured only by a combined liver-kidney transplant. Clearly, alternative treatment options for the oxalate-associated diseases require attention and we are proposing to examine promoting intestinal elimination and degradation of oxalate as a way of reducing both hyperoxaluria and hyperoxalemia. Several key pieces of information from our recent studies of colonic oxalate transport in rats/mice have provided the direction for the studies proposed here. First, we have shown that, in addition to degrading dietary sources of oxalate, the substrate/oxalate-specific microorganism, Oxalobacter sp., which resides exclusively in the large intestine, can significantly lower urinary oxalate excretion by inducing colonic oxalate secretion/excretion. We also have compelling results indicating this anaerobic bacterium interacts with the transporting mucosa by producing a secretagogue that activates these beneficial changes in intestinal oxalate handling. Second, based upon our studies using wild type (WT) and knockout (KO) mice, we have identified two key anion exchangers, namely PAT1 (slc26a6) and DRA (slc26a3), that contribute significantly to the movements of oxalate in the intestine and we hypothesize that the changes in oxalate transport induced by Oxalobacter are dependent upon the expression and function of these transporters. Thus, we will examine intestinal and renal handling of oxalate in PAT1 and DRA single KO mice as well as in a PAT1/AGT double KO mouse model and WT mice colonized with Oxalobacter and compared to their appropriate non-colonized counterparts. An understanding of the mechanistic basis for bacterial cell modulation of intestinal oxalate handling is fundamental to future efforts in identifying which strains of bacteria and/or bacterial products will be effective in the treatment of Primary Hyperoxaluria and calcium oxalate stone disease. PUBLIC HEALTH RELEVANCE: The studies proposed address the mechanisms by which Oxalobacter colonization of the large intestine induces enteric oxalate elimination and lowers urinary oxalate excretion. In addition, these studies will evaluate whether Oxalobacter modulates the function of two known important oxalate transporters using knockout mouse models with targeted deletion of slc26a3 (DRA) and slc26a6 (PAT1). Especially important and relevant to the present application, however, is the recent availability of a PAT1/AGT double KO mouse model which affords us a unique opportunity to evaluate PAT1 function/activity in the setting of Primary Hyperoxaluria, type1 (PH1), with and without Oxalobacter colonization. Understanding oxalate handling in PH1 and the potential roles and interactions of PAT1 and Oxalobacter in modulating the excessive body burden of oxalate in this genetic disease are now possible.