Oxalate exposure impacted host hepatic activity in a microbiota dependent fashion. A) Swiss Webster mice were given neomycin, followed by an allograft (SWM) or xenograft (NALB) fecal transplant, then maintained on a 0% or 1.5% oxalate diet prior to necropsy for fecal metabolomics (Fig. 2) and hepatic transcriptomics. B) PCoA of normalized, whole-transcriptome data. p=0.02 for microbiome composition, p=0.07 for dietary oxalate content, p=0.3 in 2-way analysis; 2-way PERMANOVA. C) Total number of hepatic genes significantly stimulated or inhibited by dietary oxalate. Significant genes are plotted by Log2FoldChange. Positive values reflect genes increased with oxalate exposure and negative values are genes decreased with exposure. FDR < 0.05, Wald test. Hepatic genes are annotated to pathway (Kegg, Uniprot, PubChem, Metacyc) and the total number of genes that exhibit a positive or negative shift with oxalate exposure are listed in the legend. Complete gene list is in Table S2.

Oxalate exposure impacted microbial metabolic activity in a microbiota dependent fashion. A) PCoA of protein normalized, log-transformed metabolomic data. p=0.001 for microbiome composition, p=0.1 for dietary oxalate content, p=0.3 in 2-way analysis; 2-way PERMANOVA. B) Total number of fecal metabolites significantly stimulated or inhibited by dietary oxalate. Significant metabolites are plotted by Log2FoldChange. Positive values reflect metabolites increased with oxalate exposure and negative values are metabolites decreased with exposure. FDR < 0.05, Mann- Whitney U or Fisher’s exact test. Metabolites are annotated to pathway (Kegg, Uniprot, PubChem, Metacyc) the total number of metabolites that exhibit a positive or negative shift with oxalate exposure are listed in the legend. Complete list is in Table S3. C) Change in the host-microbe interaction network upon exposure to oxalate, quantified as hepatic gene-microbe metabolite correlations R > +/- 0.3 and FDR < 0.05 with SparCC, visualized in Cytoscape. All host-microbe interactions are listed in Table S4.

Oxalate exposure stimulates taxonomically diverse microorganisms, with a few strains that dominate the response. A) Neotoma albigula with native microbiota were given increasing amounts of dietary oxalate up to 6% w/w. *indicates samplingtimepoints. B) PCoA of normalized metagenomic data. p=0.02; PERMANOVA. C) Total number of microbial genes significantly stimulated or inhibited by dietary oxalate, annotated to pathway (Kegg, UniProt, PubChem, Metacyc) and listed by Log2FoldChange. The total number of genes stimulated or inhibited by oxalate are listed in the legend. Genes with unknown annotation are not listed. The complete list of annotated genes is listed in Table S5. D) Number of significantly differentiated genes involved in oxalate degradation, sulfate reduction, acetogenic, methanogenic, or sugar metabolic pathways or utilization of by-products of those pathways stimulated (positive) or inhibited (negative) by dietary oxalate. Genes are listed by their Log2FoldChange between no and high oxalate diets. FDR < 0.05, Wald test. E) The number of genes/genome significantly altered by oxalate, mapped to microbial genomes extracted from N. albigula. Number of genes/genomes are log10-transformed to show the distribution more clearly. A total of 92% of genomes had at least one significantly altered gene population mapped to them.

Genes related to the metabolism or handling of oxalate are present in >50% of 248 full length NALB microbial genomes from the gut. A) Proportion of the genomes extracted from N. albigula that had at least one oxalate-related gene. B) Relative distribution of oxalate-handling genes by gene function. C) Proportion of genomes that have a complete pathway for oxalate degradation, specifically. oxdd=oxalate oxidoreductase; acec=acetyl-CoA:oxalate CoA-transferase; oxc=Oxalyl-CoA decarboxylase; succ=succinyl-CoA:coenzyme A transferase; frc=Formyl-CoA transferase; D) Number of oxalate-handling genes by genome.

Microbial community composition and available substrates impact oxalate metabolism and the impact of oxalate on growth. A) Substrates associated with metabolic pathways enriched by exposure to dietary oxalate in vivo differentially impact oxalate metabolism. p<0.001 in one-way and two-way ANOVA against bacterial group and substrate; +/- reflects p<0.05, Holm’s-corrected, pairwise t-test compared to base media for an increase (+) or decrease (-) in oxalate degradation. B) Substrates differentially impact the influence of oxalate on microbial growth; p<0.001 in two-way ANOVA against substrate and bacterial group, and one-way analysis against substrate; +/- reflects p<0.05, Holm’s-corrected, one-sample t-test compared 0 (no impact of oxalate) for an increase (+) or decrease (-) in growth due to oxalate exposure. The impact of oxalate on growth was not calculated for cellulose or Media B. C) Culture- based means to quantify proportion of the NALB community that can use substrates identified through shotgun metagenomics as sole carbon and energy sources. D) Defined microbial communities to assess oxalate metabolism in vitro and in vivo. Listed are the microbial consortia, which substrate the microbes utilize that corresponds to the shotgun metagenomic data, taxonomic classification of microorganisms used in the two cohorts, and the proportion of studies in which microorganisms in the taxonomic cohort were stimulated by oxalate exposure. E,F) Oxalate metabolism in minimal media with 20mM oxalate from the microbial communities listed in 5D, in comparison to the NALB community. p<0.001, ANOVA comparing microbial groups. *p<0.05, **p<0.01; ***p<0.001; Holm’s corrected, one sample t-tests against 0 (no oxalate metabolism). Blue letters reflect statistical groups between microbial groups for oxalate metabolism.

Microbial community composition (taxonomic cohort) impacted the effect of exogenous oxalate on host health. A) Swiss Webster mice were given neomycin, followed by inoculation of microbial consortia that included either no bacteria or the taxonomic cohort listed in Figure 5C. B,C) The effect of microbial transplants on urinary (B) or fecal (C) oxalate levels over the course of the diet trial, compared to baseline. ANOVA p<0.001 for microbial group, but was not significant by timeperiod or 2-way analyses for both B & C. D) The effect of microbial transplants on urinary formate levels over the course of the diet trial, compared to baseline. ANOVA was not significant in one-way and two-way analyses. E) Renal calcium oxalate deposition. Arrows show stained calcium deposits, which were quantified through an automated algorithm in QuPath. F) Quantification of renal calcium oxalate deposition by group. p<0.001, ANOVA. Blue letters reflect statistical groups between microbial groups for renal calcification by Holm’s corrected paired t-tests. G) Pearson correlation between urinary oxalate and renal calcium oxalate deposition. R=0.22, p=0.32 with NALB samples included (blue circle); R=0.7, p=0.001 excluding the NALB group. H) Representative colon tissues from the No_bact (Group 1) and All (Group 4) groups, exhibiting high and low colitis severity scores, respectively. Tissues were stained with hematoxylin and eosin and scored based on standardized, multifactorial metrics. I) Quantification of colitis severity by group. p<0.001, ANOVA. Blue letters reflect statistical groups between microbial groups for renal calcification by Holm’s corrected paired t-tests. J) Pearson correlation between colitis severity and renal calcium oxalate deposition. R= 0.7, p=0.002. Colitis severity was not quantified for the NALB group.

Phylogeny of genomes extracted from the N. albigula metagenome, in comparison to >3000 full length microbial genomes. Stars indicate taxonomic placement of extracted genomes. All other points indicate reference genomes.

Representation of formate metabolism genes in the NALB metagenome derived from 248 full length genomes. A) Proportion of the genomes extracted from N. albigula that had at least one formate metabolism gene. B) Representation of formate metabolism genes among the metagenome, broken down by gene function and proportion of total formate metabolism genes. C) Representation of formate metabolism genes broken down by taxon.

Representation of acetogenic genes in the NALB metagenome derived from 248 full length genomes. A) Proportion of the genomes extracted from N. albigula that had at least one acetogenic gene. B) Representation of acetogenic genes among the metagenome, broken down by gene function and proportion of total acetogenic genes. C) Representation of acetogenic genes broken down by taxon.

Representation of methanogenic genes in the NALB metagenome derived from 248 full length genomes. A) Proportion of the genomes extracted from N. albigula that had at least one methanogenic gene. B) Representation of methanogenic genes among the metagenome, broken down by gene function and proportion of total methanogenic genes. C) Representation of methanogenic genes broken down by taxon.

Representation of sulfate-reducing genes in the NALB metagenome derived from 248 full length genomes. A) Proportion of the genomes extracted from N. albigula that had at least one sulfate-reducing gene. B) Representation of sulfate-reducing genes among the metagenome, broken down by gene function and proportion of total sulfate-reducing genes. C) Representation of sulfate-reducing genes broken down by taxon.

Microbial transplant composition impact on urinary inflammation, renal health, and overall mouse health for the taxonomic cohort. A-G) The effect of microbial transplants on urinary IL-6 (A), IL-18 (B), and creatinine (C) levels, along with water intake (D), urine output (E), food intake (F) and body mass (G). H) Masson’s Trichrome staining of heart tissue reveals fibrosis. Shown are representative images from the No_bact (Group 1) and All (Group 4) groups. I) Quantification of cardiac fibrosis from different microbial transplant groups. Statistical significance - 2-way ANOVA (shown on charts) and post-hoc, Holm’s corrected, paired t-tests. Blue letters reflect statistical groups between microbial transplants and *p<0.05, **p<0.01; ***p<0.001 for longitudinal comparisons within each microbial transplant group.

Effect of microbial transplants on microbial community composition for the taxonomic cohort. A,B) Beta-diversity analysis based on a weighted UniFrac dissimilarity matrix of colon feces collected at the end of the study period vs. stool (A; . p=0.006, PERMANOVA) or in the endpoint stool samples by group (B; no significant differences). Similar results for (B) were obtained with colon feces. Statistical differences shown by blue letters in legend. C) Within group beta-diversity distance of colon feces. p=0.006, one-way ANOVA. Blue letters represent statistical groups, based on post-hoc, Holm’s corrected, paired t-tests. Similar results were found with endpoint stool samples. D) Change in phylogenetic diversity of stool for each microbial transplant group. Stats, shown on graph, are based on Pearson correlations. E) Normalized counts of transplanted microorganisms in the stool across timepoints and in the colon feces at the end of the diet trial.

Microbial community composition (metabolic cohort) impacted the effect of exogenous oxalate on kidney health. A) Experimental design. Swiss Webster mice were given neomycin, followed by inoculation of one of five microbial consortia that included either no bacteria, the NALB community, or the metabolic cohort listed in Figure 5C. Animals were maintained on a 3% oxalate diet throughout the trial except for a 0% oxalate washout period after the microbial transplant period. B,C) The effect of microbial transplants on urinary (B) or fecal (C) oxalate levels over the course of the diet trial, compared to baseline. ANOVA p<0.001 for microbial group (B&C), and p<0.01 for both timeperiod and in 2-way analysis for (B only). D) The effect of microbial transplants on urinary formate levels over the course of the diet trial. ANOVA p<0.001 for microbial group and timeperiod. E) Renal calcium oxalate deposition based on Von Kossa staining of renal tissue sections. Arrows show calcium deposits stained black, which were quantified through an automated algorithm in QuPath. F) Quantification of renal calcium oxalate deposition by group. p=0.014; ANOVA. G,H) Pearson correlation between urinary oxalate (R=0.7, p<0.001) (G) or formate (R=0.026, p=0.9) (H) and renal calcium oxalate deposition. Where applicable, statistical significance was assessed through an ANOVA and post-hoc, Holm’s corrected, paired t-tests. Statistical groups shown by blue letters.

Microbial transplant composition impact on urinary inflammation, renal health, and overall mouse health for the metabolic cohort. A-G) The effect of microbial transplants on urinary IL-6 (A), IL-18 (B), and creatinine (C) levels, along with water intake (D), urine output (E), food intake (F) and body mass (G). Statistical significance - 2-way ANOVA (shown on charts) and post-hoc, Holm’s corrected, paired t-tests. Blue letters reflect statistical groups between microbial transplants and *p<0.05, **p<0.01; ***p<0.001 for longitudinal comparisons within each microbial transplant group.

Effect of microbial transplants on microbial community composition for the metabolic cohort. A,B) Beta-diversity analysis based on a weighted UniFrac dissimilarity matrix of colon feces collected at the end of the study period vs. stool (A; . p=0.006, PERMANOVA) or in the endpoint stool samples by group (B; no significant differences). Similar results for (B) were obtained with colon feces. Statistical differences shown by blue letters in legend. C) Within group beta-diversity distance of colon feces. p=0.006, one-way ANOVA. Blue letters represent statistical groups, based on post-hoc, Holm’s corrected, paired t-tests. Similar results were found with endpoint stool samples. D) Change in phylogenetic diversity of stool for each microbial transplant group. Stats, shown on graph, are based on Pearson correlations. E) Normalized counts of transplanted microorganisms in the stool across timepoints and in the colon feces at the end of the diet trial.

Effect of oxalate and formate on oxalate quantification using an enzymatic, ELISA-based assay. A) Correlation between oxalate or oxalate + formate, added to water, on oxalate concentration quantified through the enzymatic kit. B) Ratio of the calculated oxalate vs. the added concentration of oxalate and formate. C) The effect of adding oxalate or formate to urine, or of extracting oxalate from urine. R values and p- values (A) or p-values from one-way ANOVA (B,C) shown on chart. Blue letters represent statistical groups, based on pairwise t-tests.

Composition of the diets used in the diet trials.

Media recipe used to test the proportion of the NALB community that can utilize substrates as sole carbon and energy sources.

Microorganisms used in the taxonomic cohort.