Faculty Bibliography

PURPOSE OF REVIEW/OBJECTIVE:Enteric hyperoxaluria is commonly observed in malabsorptive conditions including Roux en Y gastric bypass (RYGB) and inflammatory bowel diseases (IBD). Its incidence is increasing secondary to an increased prevalence of both disorders. In this review, we summarize the evidence linking the gut microbiota to the risk of enteric hyperoxaluria. RECENT FINDINGS/RESULTS:In enteric hyperoxaluria, fat malabsorption leads to increased binding of calcium to free fatty acids resulting in more soluble oxalate in the intestinal lumen. Bile acids and free fatty acids in the lumen also cause increased gut permeability allowing more passive absorption of oxalate. In recent years, there is more interest in the role of the gut microbiota in modulating urinary oxalate excretion in enteric hyperoxaluria, stemming from our knowledge that microbiota in the intestines can degrade oxalate. Oxalobacter formigenes reduced urinary oxalate in animal models of RYGB. The contribution of other oxalate-degrading organisms and the microbiota community to the pathophysiology of enteric hyperoxaluria are also currently under investigation. SUMMARY/CONCLUSIONS:Gut microbiota might play a role in modulating the risk of enteric hyperoxaluria through oxalate degradation and bile acid metabolism. O. formigenes is a promising therapeutic target in this population; however, further studies in humans are needed to test its effectiveness.

There has been increasing interest in the human anaerobic colonic bacterium Oxalobacter formigenes because of its ability to metabolize oxalate, and its potential contribution to protection from calcium oxalate kidney stones. Prior studies examining the prevalence of this organism have focused on subjects in developed countries and on adults. Now using O. formigenes-specific PCR, we have compared the prevalence of these organisms among subjects in two remote areas in which modern medical practices have hardly been present with a USA group of mothers and their infants for the first three years of life. Among the Amerindians of the Yanomami-Sanema and Yekwana ethnic groups in Venezuela and the Hadza in Tanzania, O. formigenes was detected in 60-80% of the adult subjects, higher than found in adults from USA in this and prior studies. In young children, the prevalence was much lower in USA than in either tribal village. These data extend our understanding of the epidemiology of O. formigenes carriage, and are consistent with the hypothesis that the rising incidence of kidney stones is associated with the progressive loss of O. formigenes colonization in populations that have been highly impacted by modern medical practices.

Background: Kidney stones represent a disease of worldwide prevalence with significant public health implications. About 60-80% of stones are composed of calcium oxalate (CaOx); hyperoxaluria is a major risk factor for CaOx stones. Oxalate is an endproduct of mammalian digestion and as with urea, must be excreted. We obtain oxalate from diet, or from endogenous production. Certain intestinal bacteria have the ability to degrade oxalate, protecting against oxalate nephropathy, including nephrolithiasis. To understand the role of the gut microbiome in oxalate metabolism, we compared conventional mice with germ-free mice (that lack a microbiota). In addition to the stress of endogenous oxalate production, we challenged groups with dietary and metabolic (via hydroxyproline (Hyp) supplementation) oxalate loads.
Method(s): Conventional (CO) and germ-free (GF) mice were fed normal chow diets supplemented with either 1% Oxalate (Ox), 1% Hydroxyproline (Hyp) or were unsupplemented (NC) for 6 weeks (n=3-4/mice group). After 6 weeks, we obtained 48-hour urine collections for measurement of the oxalate/creatinine ratio (Uox/cr).
Result(s): In CO mice, Uox/cr increased with the Ox diet compared with NC (0.57 + 0.17 vs 0.16 + 0.05, p= 0.03 by Student's t test), but not with the Hyp diet (0.14 +0.03 vs 0.16 +0.05, p=ns). However, in germ-free mice, both dietary Hyp and Ox led to increased Uox/ cr compared to NC diet (0.50 +/- 0.04, 0.85 +/- 0.11, vs. 0.31+/- 0.06, p<0.05 by ANOVA, respectively). Uox/Cr was lower in CO mice than GF mice when receiving Hyp (p=0.01, by Student's t test), Ox (p=0.06), and NC diets (0.06).
Conclusion(s): In conclusion, oxalate excretion was higher in the germ-free than in the conventional mice under all three dietary conditions (Ox, Hyp, NC), providing direct evidence that the normal gut microbiome plays a protective (symbiotic) role in oxalate metabolism. With the metabolic stress of the Hyp diet, the CO mice but not the germfree mice could compensate. Since mice are not colonized with O. formigenes, this work indicates that other members of their microbiota have the functional capacity to alter oxalate metabolism

Background: About 60-80% of kidney stones are composed of calcium oxalate (CaOx); idiopathic CaOx kidney stones (CaOPx), primary hyperoxaluria (PH) and enteric hyperoxaluria (EH) are diseases predisposing to stones. Oxalobacter formigenes (Oxf) is a human gut commensal that depends on oxalate for its carbon and energy, and may be protective against CaOx stones. We hypothesize that the microbiome community structure differs between patients with CaOx, PH, EH and normal subjects (NS). We also expect that Oxf isolates from PH patients will result in further reduction in urinary oxalate when compared to Oxf reference strain CC13, in a germ-free (GF) mouse model.
Method(s): We collected fecal specimens from 34 subjects (mean age: 39.1 +/- 11.9 years) with PH (n=6), CaOPx (n=10), EH (n= 5) and NS (n=13) in a cross-sectional observational study, and tested fecal samples from the groups by: 1)16S rRNA sequencing to determine the microbiome community structure, 2)PCR and qPCR for Oxf colonization and, 3) culturing in high oxalate selective media for indication of Oxf presence and subsequent isolation. We isolated Oxf from 4 PH (Oxf PH) subjects. We gavaged a growing culture of PH Oxf (n=6), Oxf reference strain CC13 (Oxf CC13) (n=5), and sham (n=6) into adult C5B6 GF mice, observing them for 4 weeks. We collected urine from mice for 48 hours before sacrifice to be tested for oxalate and creatinine (Uox/cr).
Result(s): Oxf was detected in 6 (46%) of 13 NS, 1 (10%) of 10 CaOPx, 0 (0%)of 4 EH, and 5 (83%) of 6 PH. Microbiome analysis revealed that the 4 groups differed in beta diversity, based on Bray-Curtis dissimilarity (p=0.08). Alpha diversity analysis trended toward lower Shannon and phylogenetic diversity index in the CaOPx and EH subjects compared to PH and NS. Introducing the PH Oxf to GF mice led to lower Uox/cr than in uninoculated controls (0.68 +/- 0.14, and 2.26 +/-0.49, respectively, p=0.04 by Mann-Whitney U test), but not significantly different from the Oxf CC13-innoculated mice (0.68 +/- 0.14, and 0.91 +/-0.24, respectively, p=0.26 by Mann-Whitney U test).
Conclusion(s): These studies provide evidence of differences in Oxf colonization rates and in microbiome composition in patients with CaOx stones and show the functional capacity of a PH Oxf strain to ameliorate hyperoxaluria. Studies to expand these patient groups are on-going

Background: Many uremic retention solutes are products of gut bacterial metabolism. Protein-binding renders these solutes poorly dialyzable. In a prior study we observed that a single dose of 250 mg of vancomycin, given by mouth, resulted in a significant (40%) decrease in the plasma concentration of indoxyl sulfate and p-cresyl sulfate over a period of one week. In this study we compared the changes in plasma concentration of a panel of protein-bound uremic retention solutes in response to the once-weekly oral administration of 250 mg of vancomycin or placebo over a period of 8 weeks.
Method(s): Eight subjects with chronic, stable ESRD on thrice-weekly hemodialysis via AV fistula in the River Renal Dialysis Unit in Bellevue Hospital, were randomized to two groups, utilizing a single-blinded procedure. Baseline plasma samples were collected prior to the initial dose of vancomycin or placebo and at weeks one, two, three, four, and eight. Uremic retention solutes were measured by MS-HPLC.
Result(s): Six of the eight uremic retention solutes (Table 1) demonstrated a significant decline in concentration over the eight week period of once-weekly vancomycin administration. The magnitude of the decline makes it more likely that gut production was reduced rather than renal excretion increased. Solute concentrations remained unchanged over the same period of placebo administration.
Conclusion(s): The significant decline in the plasma concentrations of multiple uremic retention solutes provides evidence of the importance of the gut microbiome in the generation of these solutes. The reduction in concentrations of indoxyl sulfate, p-cresyl sulfate, and kynurenic acid, recognized as likely uremic toxins, suggests that altering the gut microbiome might provide a valuable therapeutic strategy in the management of ESRD

Background: Multiple bacterial species are capable of degrading oxalate in vitro. Certain taxa degrade oxalate as their sole source of energy and carbon (e.g. Oxalobacter formigenes), whereas others use oxalate as an auxiliary carbon source. For oxalate metabolism, it is not yet well-understood how genomic potential relates to transcriptional regulation. We asked whether the human gut could have a community of oxalatedegrading taxa working synergistically to diminish the effects of this toxic metabolite. Our hypothesis is that oxalate metabolism is regulated by a multi-organism oxalatedegrading community (oxalobiome) that is dominated by specialist oxalate degraders.
Method(s): We used data from 2 public databases: (i)8 healthy subjects in the USA; and (ii)471 healthy subjects in the Netherlands as part of the Human Functional Genomic Project (HFGP). Both collected fecal samples for metagenomic and/or metatranscriptomic high throughput sequencing. Using HUMAnN2 with customized settings, we profiled the metabolic activity of oxalate-degrading bacterial species. Output from these analyses was expressed as Reads per Kilobase per Million mapped reads (RPKM).
Result(s): We identified the oxalate degradation pathway (ODP) in the metagenome and metatranscriptome of all 8 subjects. Mean ODP is 35.3+/-28.1 and 90.1+/-43.5 RPKM in the metagenome and the metatranscriptome, respectively, indicating active expression. O. formigenes, E. coli, and unclassified bacteria were present in metagenomic and metatranscriptomic reads. B. dentium had detectable ODP in its genome but was not transcribing it. In the HFGP database, we identified ODP in 328 subjects of the 471 tested (70%) (Mean=18.1+/-2.1 RPKM). ODP was detected in B. animalis, B. dentium, B. pseudocatenulatum, E.coli/Shigella, L. acidophilus, L. gasseri, L. mucosae, O. formigenes and unclassified bacteria. ODP was examined in the metagenome of 265 females (Mean ODP= 21.7+/-3.3) and 200 males (Mean ODP=13.3+/-1.9 RPKM; p=0.04 by unpaired t test).
Conclusion(s): We have identified a community of bacteria with the potential to degrade oxalate in healthy humans and species actively transcribing ODP. These include E.coli, which might be a common contributor of oxalate degradation in humans. The sex differences in ODP is consistent with the ~ 2:1 male/female incidence and prevalence of calcium oxalate stones

Background: Oxalobacter formigenes (O.f.) are symbiotic bacteria in the human gut that degrade oxalate, a component of most kidney stones. Observational studies suggest that O.f. colonization reduces the risk for kidney stones. Given the importance of dietary oxalate and calcium levels, studies in mice are more practical than in humans; however, O.f. do not naturally colonize laboratory rodents. Our objective was to develop a humanized murine model to investigate the therapeutic potential of O. formigenes in its native microbiome.
Method(s): To humanize mice, we transplanted feces from a pool of healthy human donors who were O.f.-negative (confirmed by PCR, qPCR, and oxalate degradation assay), supplemented with a human O.f. strain: OXCC13 (108 CFU/mL). The inoculum was introduced to C57BL/6J mice via esophageal gavage three times over six days. We compared two methods of humanization, transplanting inocula into mice that were (i) germ free; or (ii) treated with high-dose, broad-spectrum antibiotics (0.5g/L vancomycin, 1g/L ampicillin, 1g/L neomycin, 1g/L metronidazole in drinking water for 6 days) to suppress their native microbiome. As controls, one group received humanization with no pre-treatment and another received a sham gavage.
Result(s): Based on oxc qPCR and 16S rRNA sequencing, all humanized groups were stably colonized with O.f. through 8 weeks post-gavage, whereas mice that received sham gavage remained uncolonized (p<0.001). Humanization significantly changed microbial community structure as measured by unweighted UniFrac distances (p<0.001) and humanized germ-free and antibiotic-treated groups were highly similar in beta-diversity. We also assessed humanization by the number of shared OTUs between treatment groups and donor inoculum over time. Both germ-free and antibiotic-treated mice had a significant increase in shared OTUs compared to sham (p=0.024, p=0.036). The number of shared OTUs was stable in each group through 8 weeks post-gavage without significant difference between germ-free and antibiotic-treated mice.
Conclusion(s): Our method of transplanting human feces and O.f. conferred a new microbial phenotype in mice that resembled a human microbiome and was stable over time. Antibiotic pre-treatment, a simpler alternative to germ-free mice, provided comparable results. This model may allow insights to O.f.'s role in preventing calcium oxalate stones

BACKGROUND: Increasing evidence shows the importance of the commensal microbe Oxalobacter formigenes in regulating host oxalate homeostasis, with effects against calcium oxalate kidney stone formation, and other oxalate-associated pathological conditions. However, limited understanding of O. formigenes in humans poses difficulties for designing targeted experiments to assess its definitive effects and sustainable interventions in clinical settings. We exploited the large-scale dataset from the American Gut Project (AGP) to study O. formigenes colonization in the human gastrointestinal (GI) tract and to explore O. formigenes-associated ecology and the underlying host-microbe relationships. RESULTS: In >8000 AGP samples, we detected two dominant, co-colonizing O. formigenes operational taxonomic units (OTUs) in fecal specimens. Multivariate analysis suggested that O. formigenes abundance was associated with particular host demographic and clinical features, including age, sex, race, geographical location, BMI, and antibiotic history. Furthermore, we found that O. formigenes presence was an indicator of altered host gut microbiota structure, including higher community diversity, global network connectivity, and stronger resilience to simulated disturbances. CONCLUSIONS: Through this study, we identified O. formigenes colonizing patterns in the human GI tract, potential underlying host-microbe relationships, and associated microbial community structures. These insights suggest hypotheses to be tested in future experiments. Additionally, we proposed a systematic framework to study any bacterial taxa of interest to computational biologists, using large-scale public data to yield novel biological insights.

Background.: Observational studies have suggested a relationship between the plasma concentration of indoxyl sulfate (IS) and p -cresyl sulfate (PCS), small gut-derived 'uremic solutes', and the high incidence of uremic cardiomyopathy in patients with end-stage renal disease (ESRD). IS and PCS are derived from the metabolism of dietary components (tryptophan and tyrosine) by gut bacteria. This pilot study was designed to examine the effects of a poorly absorbable antibiotic (vancomycin) on the plasma concentration of two gut-derived 'uremic solutes', IS and PCS, and on the composition of the gut microbiome. Methods.: Plasma concentrations of IS and PCS were measured by MS-HPLC. The gut microbiome was assessed in stool specimens sequenced for the 16S rRNA gene targeting the V4 region. Results.: The pre-dialysis mean plasma concentrations of both IS and PCS were markedly elevated. Following the administration of vancomycin (Day 0), the IS and PCS concentrations decreased at Day 2 or Day 5 and returned to baseline by Day 28. Following vancomycin administration, several changes in the gut microbiome were observed. Most striking was the decrease in diversity, a finding that was evident on Day 7 and was still evident at Day 28. There was little change at the phylum level but at the genus level, broad population changes were noted. Changes in the abundance of several genera appeared to parallel the concentration of IS and PCS. Conclusions.: These findings suggest that alteration of the gut microbiome, by an antibiotic, might provide an important strategy in reducing the levels of IS and PCS in ESRD.