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

点击翻译按钮获取中文摘要

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.