Supplementary Material for: Uremic Toxin-Producing Gut Microbiota in Rats with Chronic Kidney Disease

Background: In patients with chronic kidney disease (CKD), many metabolites of gut microbiota retain in the body as uremic toxins (UTs). However, the kinds of bacteria producing UTs are rarely discussed. Methods: We analyzed UT production and the composition of gut microbiota in CKD rats and cecectomized rats. AST-120, a spherical carbon adsorbent, was administrated to evaluate how the precursors of UT affect gut microbiota. Serum and urine levels of UTs were quantified by liquid chromatography/electrospray ionization-tandem mass spectrometry. Gut microbiota were analyzed using 454-pyrosequencing of the 16S rRNA gene. Operational taxonomic unit (OTU) clustering and UniFrac analysis were performed to compare gut microbiota among the groups. Results: Serum and urine levels of indoxyl sulfate and phenyl sulfate were higher in CKD versus control rats (p < 0.05). AST-120 administration decreased UT production (p < 0.01) and changed overall gut microbiota composition in CKD rats. UT urinary excretion and gut microbiota composition changed in cecectomized rats, with the relative abundance of Clostridia- and Bacteroidia-affiliated species being significantly reduced (p < 0.01). We identified candidate indole- and phenol-producing intestinal microbiota, 3 Clostridia, and 2 Bacteroidia. These OTUs have a tryptophanase/tyrosine phenol-lyase gene in the closest sequenced genome out of the OTUs declined following cecectomy. Conclusion: Our data suggest that UT production is correlated with a subset of indigenous gut microbiota. However, UT may be induced by other non-symbiotic microbiota that are influenced by factors other than microbiota populations. The relationship between specific microbiota and UTs in patients requires further clarification.