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1 2 3 498 4 Supplementary Figure 1| Overview of the workflow for the multi-omics strategy 5 499 used in this study. Host phenome, serum and faecal metabolomes, and gut 6 500 microbiome of 235 ESRD patients and 81 healthy controls were characterised and 7 501 integrated into a multi-omics dataset. (A) Data types; (B) Differential filtering refers 8 9 502 to statistical significance computation based on differential abundance of features in 10 503 the patient and control groups; (C) Correlation and effect size analyses of multi-omics 11 504 dataset; (D) Predictive model building and clustering of metagenomics species with 12 Confidential: For Review Only 505 13 metabolites into co-abundance based networks; (E) Animal experiments testing the 14 506 effect of ESRD microbiota (left panel), ESRD-associated species (centre panel) and 15 507 probiotics (left panel). 16 508 17 18 509 Supplementary Figure 2| Differences in levels of serum metabolites between 19 510 ESRD patients and healthy controls. (A) Boxplot shows the serum metabolites that 20 511 differ significantly between ESRD patients and healthy controls. Top panel, ESRD 21 22 512 patient-enriched metabolites. Bottom panel, healthy control-enriched metabolites. The 23 513 uremic toxins that are enriched in ESRD patients are highlighted in red. Boxes 24 514 represent the interquartile range between the first and third quartiles and median 25 515 26 (internal line). Whiskers denote the lowest and highest values within 1.5 times the 27 516 range of the first and third quartiles, respectively; dots represent outliers beyond the 28 517 whiskers. (B) Changes in the composition of bile acids in two groups. Average 29 518 relative abundance (normalized from their raw abundance) of 16 bile acids of ESRD 30 31 519 patients and healthy controls are shown. Bottom panel: the ratios of unconjugated vs. 32 520 conjugated bile acids, unconjugated PBA/SBA and conjugated PBA are calculated for 33 521 each individual and compared between patients and controls. Abbreviations of the bile 34 35 522 acids are listed at Online Methods. (C) Correlation analysis of the quantified 36 523 concentrations of five targeted serum uremic toxins and non-targeted abundance in 60 37 524 randomly chosen serum samples (ESRD patients, n=40; and healthy controls, n=20). 38 525 39 40 526 Supplementary Figure 3| Schematic representation of the metabolic pathways of 41 527 the serum uremic toxins and bile acids associated with gut microbiota. (A) 42 528 Production of serum uremic toxins by gut microbiota via degrading diet-originated 43 44 529 aromatic amino acids, polyphenols and choline. (B) Metabolism of bile acids. The key 45 530 enzymes encoded by gut microbes are highlighted by orange boxes, and details are 46 531 shown in online supplementary table 14. 47 48 532 49 533 Supplementary Figure 4| Concentrations of serum uremic toxins and bile acids 50 534 are highly correlated with ESRD-associated clinical parameters. The heatmap 51 535 52 panels show the Spearman correlation coefficient between serum uremic toxins or bile 53 536 acids and ESRD-associated clinical parameters, for which significance levels in 54 537 correlation tests are denoted: +, q < 0.05; *, q < 0.01; **, q < 0.001. Top: all cohort; 55 538 bottom left and right: patients and controls, respectively. Information on metabolites 56 57 539 included in clusters S10, S19, S20, S24, S37, and S38 are provided in online 58 540 supplementary table 5. 59 541 60

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1 2 3 542 4 Supplementary Figure 5| Differences in faecal metabolite levels between ESRD 5 543 patients and healthy controls. (A) Boxplot shows the faecal metabolites that differ 6 544 significantly between ESRD patients and healthy controls. The uremic toxins that are 7 545 enriched in ESRD patients and the SCFAs that are prominently enriched in healthy 8 9 546 controls are highlighted in red. (B) Targeted metabolomic profiling of 16 faecal bile 10 547 acids of ESRD patients and healthy controls. Bottom panel: the abundance of total 11 548 PBAs, total SBAs, and the ratio of PBA/SBA are calculated for each individual and 12 Confidential: For Review Only 549 q q 13 compared between patients and controls. Significance levels: ‘*’, < 0.05, ‘+’, < 14 550 0.10. (C) Effect size of faecal metabolites that drive the variance of faecal 15 551 metabolome. 16 552 17 18 553 Supplementary Figure 6| Correlation analysis between serum uremic toxins and 19 554 their faecal precursors. Fitted lines and 95% confidence intervals are shown. 20 555 21 22 556 Supplementary Figure 7| Validation of the ESRD-associated serum (A) and 23 557 faecal metabolites (B) in an independent cohort. Boxplot shows the comparison of 24 558 serum uremic toxins (left panel) and faecal uremic toxin precursors and SCFAs (right 25 559 26 panel). Red boxes, ESRD patients (n = 12); blue boxes, healthy controls (n = 12). 27 560 Significance levels: ‘***’, q < 0.001, ‘**’, q < 0.01, ‘*’, q < 0.05, ‘+’, q < 0.10. 28 561 29 562 30 Supplementary Figure 8| Alteration of microbial taxonomic composition and 31 563 functions in ESRD patients. (A) Shannon’s diversity index of ESRD patients and 32 564 healthy controls. (B-C) Distance-based redundancy analysis (dbRDA) reveals a 33 565 significant difference of gut microbial taxonomic composition (B) and functional 34 35 566 profiles (C) in ESRD patients (n = 223) and healthy controls (n = 69). KEGG 36 567 orthologue (KO)-based and MGS-based Bray-Curtis dissimilarity was used to assess 37 568 the functional and taxonomic difference, respectively. The display is based on sample 38 569 39 scores on the primary constrained axis (CAP1) and primary multidimensional scaling 40 570 (MDS1), see Online Methods for details. 41 571 42 572 43 Supplementary Figure 9| Differences in bacterial species between ESRD patients 44 573 and healthy controls. (A) List of species that were significantly enriched in ESRD 45 574 patients or healthy controls. (B-C) Stratification of patients and healthy controls. The 46 575 20 most discriminant species enriched in ESRD patients (B) or healthy controls (C). 47 48 576 The receiver operating characteristic (ROC) curves for classification of ESRD 49 577 patients and healthy controls are shown; values of the area under the ROC curve 50 578 (AUC) are listed in the right panels. (D-F) Distribution of MGS encoding the key 51 579 52 acetate (D), propionate (E) and butyrate (F) synthesis genes in ESRD patients and 53 580 healthy controls. MGS were grouped in genera. Red colour denotes statistically 54 581 significant abundance difference (q < 0.05). 55 582 56 57 583 Supplementary Figure 10| Differences in microbial functions between ESRD 58 584 patients and healthy controls (A-C) Alterations in microbial pathways and 59 60

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1 2 3 585 functional modules. ( Alterations in gut functional modules involved in amino acid 4 D) 5 586 degradation and bile acid metabolism. Red, ESRD-enriched; cyan, control-enriched. 6 587 7 588 Supplementary Figure 11| The correlation networks in ESRD patients and 8 9 589 healthy controls. (A-C) ESRD-specific (A), control-specific (B) and shared (C) 10 590 correlation networks of the gut MGS, serum and faecal metabolomes. Vertices 11 591 indicate variables and lines indicate significant Spearman’s correlations (|ρ| > 0.35 12 Confidential: For Review Only 592 q 13 and < 0.01). The serum and faecal variables were clustered to simplify the networks. 14 593 (D-E) Number of correlations between serum metabolite clusters and the gut 15 594 microbiota (D) or faecal metabolite clusters (E). The serum metabolite clusters of 16 595 uremic toxins and bile acids are highlighted in red. ( ) The clusters of serum uremic 17 F 18 596 toxins and bile acids correlated to MGSs (listed on the right; unclassified MGS are 19 597 omitted) and faecal metabolites. (G) Conservation of MGS-uremic toxins/SBAs 20 598 correlations in the ESRD and control networks. Correlations that were significant 21 22 599 (|ρ| > 0.35 and q < 0.01) in at least one network (ESRD or control) are shown. ‘**’, P 23 600 < 0.01; ‘***’, P < 0.001. 24 601 25 602 26 Supplementary Figure 12| The effect size of gut microbiota on the host serum 27 603 metabolome in different studies. The approach described in the Methods section 28 604 was used for all studies. 29 605 30 31 606 Supplementary Figure 13| Number of medications used for ESRD patients. 32 607 33 608 Supplementary Figure 14| Distribution of the key synthetases involved in the 34 35 609 biosynthesis of uremic toxins and SBAs. (A) Relative abundance of key synthetase- 36 610 encoding genes in ESRD patients and healthy controls (left panels) and in MGS- 37 611 assigned genes (right panels). The number of synthetase-encoding genes in each MGS 38 612 39 is shown in brackets. Boxes represent the interquartile range between the first and 40 613 third quartiles and median (internal line). Whiskers denote the lowest and highest 41 614 values within 1.5 times the range of the first and third quartiles, respectively, and 42 615 circles represent outliers beyond the whiskers. ( ) Correlations between the 43 B 44 616 abundance of key synthetase-encoding genes and faecal concentrations of 45 617 corresponding metabolites. Best-fit lines and 95% confidence intervals (CI) are 46 618 indicated. 47 619 48 49 620 Supplementary Figure 15| Network view of the uremic toxins/SBAs and the 50 621 MGSs used to construct predictive models. Central boxes represent p-cresol, indole, 51 622 52 phenol and SBAs in faeces, and TMAO, hippuric acid and phenylacetylglutamine in 53 623 serum. The surrounding circles and squares connected to the central boxes by grey 54 624 lines represent the correlated species with or without key synthetase-encoding genes, 55 625 respectively. ESRD- and control-enriched MGSs are highlighted in red and green, 56 57 626 respectively. MGSs that are not significantly different by abundance in the two groups 58 627 are indicated in grey. The MGSs without taxonomic annotation are not included in the 59 628 lists. 60

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1 2 3 629 4 5 630 Supplementary Figure 16| Relative abundances of the MGSs associated to the 6 631 toxins or SBAs in the predictive models are highly correlated to ESRD-associated 7 632 clinical parameters. (A) Spearman correlation coefficients between the MGSs and 8 9 633 the ESRD-associated clinical parameters across the whole cohort are displayed as 10 634 heat-maps. ‘+’, q < 0.05; ‘*’, q < 0.01; ‘**’, q < 0.001. Overall effect size of microbes 11 635 on each clinical parameter is shown on the right. The MGSs are listed at the bottom 12 Confidential: For Review Only 636 13 (red, ESRD-enriched; cyan, control-enriched; black, not significant between ESRD 14 637 patients and healthy controls). (B) Spearman correlation coefficients between the 15 638 MGSs and the ESRD-associated clinical parameters in ESRD patients only are 16 639 displayed as heat-maps. 17 18 640 19 641 Supplementary Figure 17| Classification of ESRD clinical status by toxin/SBA- 20 642 linked microbes. (A) Receiver-operating characteristic (ROC) analysis of the 21 22 643 classification of ESRD status using the random forest model trained on the 23 644 toxin/SBA-correlated microbes. The classification performance of the model was 24 645 assessed by the area under the ROC curve (AUC). Black bars denote 95% confidence 25 646 26 intervals (CI), and the area between the two outside curves represents the 95% CI 27 647 shape. (B) The 20 species for which the highest mean decrease accuracy (indicated by 28 648 the bars) in the model distinguishing ESRD patients from healthy controls are shown. 29 649 30 31 650 Supplementary Figure 18| Faecal microbiota transplantation experiment in germ 32 651 free mice. (A) Experimental design. (B) Comparison of gut microbial taxa of donors 33 652 at the time of first sampling and new visit sampling. Microbial compositions were 34 35 653 determined by 16S rRNA sequencing among ESRD (pool of n = 13) and control 36 654 donors (pool of n = 13). Upper panel: principal component analysis of the gut 37 655 microbiota. Samples on the first and second principal components (PC1 and PC2) are 38 656 39 plotted. Bottom panel: Bray-Curtis dissimilarity between the samples that at the time 40 657 of first sampling and new visit sampling. (C) Comparison of gut microbial taxa 41 658 among donors and recipients. (D) Immunohistochemistry of TGF-β and quantification 42 659 of the TGF-β+ area in the renal cortex. PAS staining and quantification of the 43 (E) 44 660 glomerulosclerotic index in renal glomeruli. (F) Immunohistochemistry for 8-OH-dG 45 661 and quantification of 8-OH-dG+ cells in the renal cortex. (G-H) Serum levels of urea 46 662 (G) and creatinine (H) in recipient mice after faecal transplantation for two weeks. 47 48 663 Data are shown as mean ± s.e.m. ‘*’, P < 0.05; ‘**’, P < 0.0 49 664 50 665 Supplementary Figure 19| Faecal microbiota transplantation experiment in 51 666 52 antibiotics-treated rats. (A) Experimental design. (B) Bray-Curtis dissimilarity 53 667 between recipients and donors. Compositions of gut microbial taxa of ESRD donors 54 668 (pool of n = 13), control donors (pool of n = 13), ESRD-microbiota recipients 55 669 (abbreviated as E recipients, n = 9) and control-microbiota recipients (abbreviated as 56 57 670 C recipients, n = 9) were determined by 16S rRNA sequencing. (C-D) Serum levels of 58 671 urea (C) and creatinine (D) in recipient rats before and after faecal transplantation for 59 672 three weeks. (E) Changes of serum uremic toxin levels in CKD rats after faecal 60

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1 2 3 673 transplantation for three weeks. ( Masson’s trichrome staining and quantification of 4 F) 5 674 the proportion of the fibrotic area in the renal cortex. (G) Immunofluorescence of α- 6 675 SMA and quantification of the relative fluorescence intensity of the α-SMA+ area in 7 676 renal tubular. (H) Immunohistochemistry of TGF-β and quantification of the TGF-β+ 8 9 677 area in the renal cortex. (I) PAS staining and quantification of the glomerulosclerotic 10 678 index in renal glomeruli. (J) Immunohistochemistry for 8-OH-dG and Quantification 11 679 of 8-OH-dG+ cells in the renal cortex. Data are shown as mean ± s.e.m. ‘*’, P < 0.05; 12 Confidential: For Review Only 680 P 13 ‘**’, < 0.01. 14 681 15 682 Supplementary Figure 20| Effects of E. lenta and F. nucleatum supplementation 16 683 ) 17 on serum levels of creatinine and urea, and renal pathophysiological changes. (A 18 684 Experimental design. (B-C) Serum levels of urea (B) and creatinine (C) in CKD rats 19 685 before and after eight-week gavage-feeding E. lenta or F. nucleatum (D) 20 686 Immunohistochemistry of TGF-β and quantification of the TGF-β+ area in the renal 21 22 687 cortex. (E) PAS staining and quantification of the glomerulosclerotic index in renal 23 688 glomeruli. (F) Immunohistochemistry for 8-OH-dG and Quantification of 8-OH-dG+ 24 689 cells in the renal cortex. Data are shown as mean ± s.e.m. ‘*’, P < 0.05; ‘**’, P < 25 690 26 0.01. 27 691 28 692 Supplementary Figure 21| Alteration of gut microbiota in bacterial- and sham- 29 693 The abundance of E. lenta and F. nucleatum in bacterial- 30 gavaged CKD rats. (A) 31 694 gavaged and sham-gavaged CKD rats following eight weeks of gavage, as quantified 32 695 by qPCR analysis. Data are shown as mean ± s.e.m. ‘*’, P < 0.05; ‘**’, P < 0.01. (B- 33 696 C) PCA analysis (B) and comparison of taxa (C) revealed no significant alterations of 34 35 697 the microbial composition of E. lenta- and F. nucleatum-gavaged rats compared with 36 698 sham-gavaged rats. Gut microbial abundances of rat faecal samples were quantified 37 699 by 16S rRNA amplicon sequencing analysis (see Online Methods). Significance levels 38 700 q 39 ( value) of the comparison of taxa between bacterial-gavaged and sham-gavaged 40 701 CKD rats are shown in the bottom of (C). (D-E) Correlation between the change of 41 702 faecal E. lenta (D) or Fusobacterium spp. (E) abundance (after – before gavage, as 42 703 quantified by qPCR analysis) and the change in concentration of serum uremic toxins 43 44 704 (after – before gavage) in both bacterial- and sham-gavaged rats. 45 705 46 706 Supplementary Figure 22| Alteration of the gut microbiota in probiotic- and 47 48 707 sham-gavaged CKD rats. (A) Experimental design. (B-C) PCA analysis (B) and 49 708 comparison of taxa (C) between probiotic- and sham-gavaged rats. Gut microbial 50 709 abundances of rat faecal samples were quantified based on 16S rDNA amplicon 51 710 q 52 analysis (see Online Methods). Significance levels ( value) of the comparison of taxa 53 711 between probiotic-gavaged and sham-gavaged CKD rats are shown. (D) The 54 712 abundance of E. lenta and Fusobacterium spp. in probiotics-gavaged and sham- 55 713 gavaged rats as quantified by qPCR analysis. Data are shown as mean ± s.e.m. ‘*’, P 56 57 714 < 0.05; ‘**’, P < 0.01. 58 715 59 716 Supplementary Figure 23| Effects of probiotic supplementation on serum 60

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1 2 3 717 4 metabolic parameters and renal pathophysiological changes in CKD rats. (A) 5 718 Changes in rat serum uremic toxin levels after eight weeks of probiotics gavage. (B- 6 719 C) Changes in rat serum creatinine (C) and urea (D) levels after eight weeks of 7 720 probiotics gavage. (D) Masson’s trichrome staining and quantification of the 8 9 721 proportion of the fibrotic area in the renal cortex. (E) Immunofluorescence of α-SMA 10 722 and quantification of the relative fluorescence intensity of the α-SMA+ area in renal 11 723 tubular. (F) Immunohistochemistry of TGF-β and quantification of the TGF-β+ area in 12 Confidential: For Review Only 724 13 the renal cortex. (G) PAS staining and quantification of the glomerulosclerotic index 14 725 in renal glomeruli. (H) Immunohistochemistry for 8-OH-dG and quantification of 8- 15 726 OH-dG+ cells in the renal cortex. Data are shown as mean ± s.e.m. ‘*’, P < 0.05; ‘**’, 16 727 P < 0.01. 17 18 728 19 729 20 730 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Supplementary material Gut online supplementary figure 1 validation cohort: 12 ESRD Page 61 of 338 223 ESRD patientsGut and 69 healthy controls Confidential: For Review patients and 12 Onlyhealthy controls

A Serum metabolites Faecal metabolites Gut microbiome Phenome 1 180 detatonna 11.4 million non-redundant genes 25 host properties metabolites 2 255 detatonna 16 bile acids 69 clinical indexes metabolites 3 900 metagenomic 11,867 functional 4 species categories

5 730 significant metabolites KEGG functional modules 6 AAAs degradation B 134 significant 98+2 significant 457 significant MGS 7 AAAs biosynthesis 8 Differential filtering Bile acid metabolism 9 37 clusters 45 clusters and 43 singletons SCFA biosynthesis 10 and 135 singletons ESRD- Control- 11 enriched enriched C 12 Correlation and effect size analyses 13 correlations 14 15D Multi-omic correlation analysis 16 building predictive models based on quantification 17 of genes encoding key synthetases 18 correlations 19 20E Animal expriments increase aggravate increase aggravate decrease alleviate 21ESRD microbiome uremic toxins ESRD Toxin species uremic toxins ESRD Probiotics uremic toxins ESRD

22 E. lenta-gavage Germ-free FMT from healthy donors CKD+healthy (n=6) SPF CKD CKD+E. lenta (n=8) SPF CKD probiotics-gavage CKD+probioticis (n=13) CKD mice FMT from ESRD donors F. nucleatum-gavage rats 23 CKD+ESRD (n=6) rats CKD+F. nucleatum (n=10) sham-gavage CKD control (n=12) sham-gavage CKD control (n=12) 24 uremic toxins renal disease severity 25 uremic toxins renal disease severity uremic toxins renal disease severity 26 27

28Antibiotics-treated FMT from healthy donors CKD+healthy (n=9) CKD rats 29 FMT from ESRD donors CKD+ESRD (n=9) 30 uremic toxins renal disease severity 31 https://mc.manuscriptcentral.com/gut 32 33 34 35 36 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 37 online supplementary figure 2 Gut Gut Page 62 of 338 A ESRD patient-enriched serum metabolites (wilcoxon q < 0.05, fold change ≥ 1.2) B ESRD Healthy C 1.0e6 patients controls 7.5e6 r = 0.9833 1 7.5e5 5e6 17.5e6 ESRD patients unconjugated 2 Healthy controls 2.5e6 5.0e5 UDCA 3 SBA DCA -Cresol sulphate P = 4.6e-11 2.5e5 p 0 ESRD-vs-healthy Confidential: For Review0.8 OnlyNon-targeted abundance 45.0e6 CDCA 0 200 600 PBA doi: 10.1136/gutjnl-2019-319766 400 800 5 0 CA Targeted concentration (ng/μL) –12. :1 6 TCA 1.0e7 0 TDCA GDCS Proline rnithine urenine TUDCA GCDCS GUDCS A C22:1 conjugated O ylhistidine ylxanthine Creatinine

7 FF Methionine h Kyn 2.5e6 h

Normalized abundance r 0.6 TUDCA 7.5e6 = 0.9791 Carnitine C5:1 Carnitine C4:1 ylacetylglycine 2020; Carnitine C16:2 Carnitine C14:0

8 D-citramalic acid GCDCA−3−glucu 3−Met TDCA Phen Gut

1,7−Dimet 5.0e6 9 GUDCS SBA 10 GUDCA 2.5e6

0 , et al. GDCS 11 P = 4.1e-9 0.4 ESRD-vs-healthy GDCA Non-targeted abundance 0

12 GCA

Phenylacetylglutamine 0 50 200

Wang X Wang 100 150 TMAO TCDCA Choline TCDCA GCDCA yl sulfate ylalanine 13 Carnitine Targeted concentration (ng/μL) TCA Carnitine C4 Carnitine C5 Hippuric acid Relative abundance of serum bile acids Phen Phen Homocysteine Carnitine C8:1 Carnitine C8:0 Carnitine C3:0 Carnitine C2:0 Carnitine C6:0 Indoxyl sulfateIndoxyl LPE 18:2 sn−1 LPE 16:0 sn−1 LPE 20:4 sn−1 LPE 18:1 sn−1 LPE 22:6 sn−1 LPE 18:2 sn−2 LPE 20:4 sn−2 LPE 18:1 sn−2 LPE 22:6 sn−2 LPE 16:0 sn−2 LPC 22:5 sn−1 14 −Cresol sulfate G S Carnitine C18:1 Carnitine C10:1 Carnitine C10:0 Carnitine C16:0 Carnitine C14:2 Carnitine C14:1 Carnitine C18:0 Carnitine C12:0 Carnitine C12:1 Carnitine C16:1 CDC Indolelactic acid p LPE 18:0−1 sn−1 LPE 18:0−2 sn−2 Cinnamoylglycine PBA Carnitine C10:2 *2 Carnitine C10:2 *1 0.2 GCDCA r 15 −Cresyl glucuronide 2e5 = 0.9212 p Phenylacetylglutamine Androsterone sulfate−1 16 Androsterone sulfate−2 GCDCA 1-Amlnocydohexanecarboxylic acid 17 GCA Healthy control-enriched serum metabolites (wilcoxon q < 0.05, fold change ≤ 0.8) 1e5 18 TMAO 8e+6 0 P 19 ESRD-vs-healthy = 7.7e-11

Non-targeted abundance 0

206e7 ESRD patients 0 10 20 30 ESRD Healthy 21 Healthy controls 6e+6 P-value Targeted concentration (ng/μL) patients controls 3e6 22 unconjugated / 4e7 0.35 1.11 <2e-16 23 conjugated BA r = 0.9832 4e+6 2e6 24 unconjugated 2.44 2.33 0.277 25 PBA/SBA 2e7 1e6 2e+6 Normalized abundance conjugated 26 1.26 1.85 4e-11 PBA/SBA Hippuric acid P ESRD-vs-healthy = 1.6e-14 27 Non-targeted abundance 0 0 60 28 0 0 30 90 29 Targeted concentration (ng/μL) DCA CDCA 30 UDCA A C24:1 A C12:0 A C17:1 A C16:2 A C19:1 A C10:0 A C18:1 A C18:2 A C22:5 A C22:2 A C20:4 A C16:1 A C22:6 A C20:2 A C20:3 r Phe−Phe = 0.9615 ryptophan xadecanol 3e6 A C20:5−1 A C14:1−1 A C14:1−2 A C20:5−2 FF FF FF FF FF FF FF FF FF FF FF FF FF FF FF Cholic acid T 31 e LPC O−16:0 LPC O−16:1 LPC O−18:1 LPC O−18:0 FF FF FF FF ylisoquinoline h arnesylacetone 1−H

32 F 2e6 Carnitine C4−OH xadecatrienoic acid 4−Hydroxyquinoline

33 3−Met He 1e6

34 Dehydroepiandrosterone sulfate https://mc.manuscriptcentral.com/gut

lndoxyl sulphate P 0 ESRD-vs-healthy = 9.6e-19 35 Non-targeted abundance 0 200 600 36 ESRD patients (n = 40) 400 Healthy controls (n = 20) Targeted concentration (ng/μL) 37 38

39 Supplementary material 40 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Confidential: For Review Only online supplementary figure 4 Supplementary material All individuals (n = 292) Gut Page 64 of 338Gut S20: Serum uremia toxins ****************** * ********** * ** * S19: Serum uremia toxins ****************** + + + ** + ****** + ** 1 S10: Serum uremia toxins ************** * * + ************ + + ** 2 S37: Serum uremia toxins ****************** * + * ************ + ++ 3 S24: Conjuncted SBA + 4 ** * * ****** ** UDCA + + ****** * ** * 5 Spearman correlation coefficient 6S38: Primary bile acids ************** ** * ******** * DCA ************** * ** * ****** 7 −0.5 0 0.5 TG UA Age ALT LBP ALB AST SBP HCT GLU DBP CRP

8 HGB KT/V eGFR Leptin UREA CREA TCHO LDL_C HDL_C 9 Gender Fecal_pH 10 Dialysis period

11 Dialysis frequency ESRD (n = 223) Healthy control (n = 69) 12 S20: Serum uremia toxins + ****** * ** 13 ** S19: Serum uremia toxins + + + + + 14 + + + ** + ** ** * S10: Serum uremia toxins 15 * + * + + + S37:16 Serum uremia toxins ****** * ** * + + + + + + 17S24: Conjuncted SBA + + * + + * + + 18 UDCA ** + * + * 19S38: Primary bile acids + + + + + ** + + + + 20 DCA ** + * + *** + TG TG UA UA Age 21 Age ALT ALT ALB LBP LBP AST AST SBP SBP GLU DBP GLU DBP HCT HCT CRP HGB HGB KT/V Leptin Leptin eGFR eGFR UREA CREA UREA CREA TCHO TCHO LDL_C LDL_C HDL_C HDL_C 22 https://mc.manuscriptcentral.com/gutGender Gender Fecal_pH 23 Fecal_pH Dialysis period

24 Dialysis frequency 25 26 27 28 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 online supplementary figure 5 Page 65 of 338Gut Gut ESRD patient-enriched faecal metabolites (wilcoxon q < 0.05, fold change ≥ 1.2) A B Faecal bile acids (*: q < 0.05; +: q < 0.1) 60 300 ESRD patients ESRD patients 200 1 Healthy controls Healthy controls Confidential: For Review40 6000 Only 2 3 200 100 4 20 4000

550 6 100

2000 doi: 10.1136/gutjnl-2019-319766 7 Normalized abundance Normalized abundance 8 –12. :1 0 9 0 0 10 +** + 2020; CA hyl hyl nyl TCA DCA LCA GCA *M1 TLCA TDCA 11 GLCA CDCA UDCA rneol GDCA hydro exane Indole TUDCA TCDCA GCDCS GUDCA GCDCA Phenol Gut Acetoin hydroxy Pentanol p−Cresol Mequinol Quinoline

12 xy−phe Dodecane , 5−met Label colors: PBAs SBAs 3− o exadecanol hylpentanol e Isopentanol 272_Xylene 269_Xylene (−)−Bo 2−Butanone Tridecanone 2−Octanone Octadecanol Tridecadiene Pentanedione 2−Oxoh (−)−Menthone 2− Carbon dioxide 2−Undecanone 13 2−Dodecanone 1−H Pentadecanone 1−Heptadecyne yl−3−heptanone xy−2,7−dimet , 5−heptyldi h Dimethyl trisulfide Dimethyl , et al. 2,3− 2− Tributyl phosphate Tributyl ESRD Healthy Phenylethyl alcohol Z−1,6− 14 Phenylacetaldehyde Benzyl methyl ketone Benzyl methyl

others 36 metabolites P-value hydro 2−Met

2−Heptanon patients controls ranone entenoic acid, 4− Oxime−, meth , 5− (S)−3,4−Dimet 15 P e unconjugated 2− 323 1012 0.006 16 PBAs X Wang 17 2(3H)−Fu unconjugated

4−Octanon 2603 0.041 SBAs 2108 18 Healthy control-enriched faecal metabolites (wilcoxon q < 0.05, fold change ≤ 0.8) unconjugated 0.22 0.61 0.028 19 20 PBA/SBA ESRD patients 10020 21 Healthy controls 10 22 50 C

23 ) 2 0.15 2425 2 25 0.10 Combined R = 49.6% Normalized abundance 26 0.05 27 28 0.00 Explained Variance (R hyl hyl oxy *M2 xy−4

29 o CA hydride Octanal Toluene butyrate Hexanal LCA nyl ester nyl DCA Benzene Terpinene 1−Butanol Indole 3−Octanol CDCA UDCA Mesitylene hyl alcohol hyl hyl acetate hyl 1−Decanol Acetic acid Acetonitrile Phenol hyl valerate hyl hyl butyrate hyl 1−Propanol 30 2−Heptenal nylpropanol Butyric acid 309_Xylene yl−4−met yanatoethyl) Ethyl acetate Ethyl h Ethylbenzene Pentadecanal butoxyethoxy) p−Cresol yl−, an Hexanoic acid Hexanoic hyl propionate hyl Propionic acid hylpropanoate nylpropanoate hyl isobutyrate hyl Met Met Met h Met 4,5−Nonadiene Acetic acid Ethyl propionate Ethyl

31 Butyric acid yl−1,6−octadiene Met Ethanol, 2−eth Met 3−Phe h hyl tetradecanoate hyl , 1−et Isobutyl isovalerate Isobutyl Propionic acid hydroxyphenylglycol 1,1−Dichloropentane Ethyl methyl disulfide methyl Ethyl hyl hexadecanoic acid hexadecanoic hyl ans−2−Dodecen−1−ol 32 Met yl 2−met yl 3−phe oxane, tetradecamethyl oxane, tr Butanoic acid, 3−methyl h , (2−isothioc 1,4−p−Menthadien−7−al , 3−bromo−5−meth Butanoic acid, butyl ester Butanoic acid, butyl Propanoic acid, 2−met Met 3,4−Di Phenylacetaldehyde Hexanoic acid, methyl ester acid, methyl Hexanoic Cyclohexanecarboxylic acid Cyclohexanecarboxylic Met 33 Benzene Propanoic acid, propyl ester Propanoic acid, propyl Prop Ethanol, 1−(2− Heptasiloxane, hexadecamethyl Heptasiloxane, yl−1,3−pentanediol diiso R(−)3,7−Dimet Butanoic acid, butyl ester

34 Benzene Butanoic acid, 3−methyl−

https://mc.manuscriptcentral.com/gutoic acid, 3,4−dichlorophe Cyclohexasiloxane, dodecamethyl Cyclohexasiloxane, Propanoic acid, propyl ester Cycloheptasil Propanoic acid, 2−met Propanoic acid, propyl ester.1

35 Benz Benzaldehyde Trimeth Propanoic acid, 2−methyl−, anhydride Cyclohexanecarboxylic acid, methyl ester acid, methyl Cyclohexanecarboxylic 36 Benzoic acid, 3,4−dichlorophenyl ester 37 2,2,4− 38

39 Supplementary material 40 41 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Confidential: For Review Only online supplementary figure 7 Page 67 ofSerum 338 metabolites (uremic toxins)Gut Faecal metabolites (uremic toxin precursors and SCFAs) SupplementaryA material B Gut

1 2 3 4 5 6 7

8 Normalized abundance Normalized abundance 9 10 11 0 20 40 60 12 0 4e8 8e8 12e8 Significant 13level (q): *** *** *** *** *** *** *** * * ++ + + + + 14 TMAO -Cresol Indole Phenol 15 p Acetic acid Hippuric acid Butanoic acid 16 https://mc.manuscriptcentral.com/gut Propanoic acid -Cresol sulphatePhenyl sulphateIndoxyl sulphate Phenol, 4-ethyl- p Indole, 3-methyl- -Cresyl glucuronide 17 p Phenylacetaldehyde Phenylacetylglutamine Trimethylamine (TMA) ButanoicButanoic acid, 3-methyl- acid, 2-methyl- 18 Propanoic acid, 2-methyl- 19 20 21 22 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Gut Page 68 of 338 SupplementaryA material B Metagenomic species profiles C KEGG orthologue (KO) profiles Gut Confidential:P = 0.002 For Review Only ESRD patients ESRD patients 13.5 −1.5 Healthy controls Healthy controls 1 2 13.0 −1.0 1.0 3 12.5 4 −0.5 0.5 5 12.0 6 0.0 11.5 7 0.5 8 0.0 11.0 Shannon’s diversity index MDS1 9 1.0 MDS1

10 10.5 https://mc.manuscriptcentral.com/gut CAP1 CAP1 11 ESRD Healthy −0.5 −2 −1 0 12 patients controls 2 1 0 −1 −2 13 14 15 16 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Supplementary material Gut onlinePage 69 supplementary of 338 figure 10 Gut

A KEGG pathway level C B ESRD-enriched pathways/KOs

Cell growth and death K00428 cytochrome c peroxidase 1 Confidential:Cell motility For ReviewK00430 peroxidase Only Cellular community - eukaryotes K00432 glutathione peroxidase Cellular community - prokaryotes Cellular Processes K00433 non-heme chloroperoxidase 2 Transport and catabolism K00435 peroxiredoxin Membrane transport K03386 peroxiredoxin Oxidative stress Signal transduction K03564 peroxiredoxin Q/BCP 3 Signaling molecules and interaction Environmental Information resistance Folding, sorting and degradation Processing K03781 catalase 4 Replication and repair K03782 catalase-peroxidase Transcription Genetic Information K04565 superoxide dismutase 5 Translation Processing K05919 superoxide reductase Amino acid metabolism K11065 thiol peroxidase, peroxiredoxin Biosynthesis of other secondary metabolites K16301 deferrochelatase/peroxidase 6 Carbohydrate metabolism Basal transcription factors Energy metabolism RNA polymerase Transcription 7 Glycan biosynthesis and metabolism Spliceosome Lipid metabolism Metabolism Purine metabolism Nucleotide 8 Metabolism of cofactors and vitamins Pyrimidine metabolism metabolism Metabolism of other amino acids 9 Metabolism of terpenoids and polyketides -1 10 2 Nucleotide metabolism 10 Xenobiotics biodegradation and metabolism Report Z-score -2 -1 0 1 2 enriched in ESRD patients 11 Report Z-score enriched in healthy controls 12 C13 D 14 Control-enriched pathways/modules Amino acid degradation 15 Melatonin biosynthesis, tryptophan => serotonin => melatonin M00015 Proline biosynthesis, glutamate => proline Tryptophan metabolism, tryptophan => kynurenine => 2-aminomuconate 16 M00016 Lysine biosynthesis, succinyl-DAP pathway, aspartate => lysine Capsaicin biosynthesis, L-Phenylalanine => Capsaicin M00017 Methionine biosynthesis, apartate => homoserine => methionine Monolignol biosynthesis, phenylalanine/tyrosine => monolignol M00018 Threonine biosynthesis, aspartate => homoserine => threonine Flavanone biosynthesis, phenylalanine => naringenin 17 M00019 Valine/isoleucine biosynthesis, pyruvate => isoleucine Catecholamine biosynthesis, tyrosine => dopamine => noradrenaline => adrenaline M00020 Serine biosynthesis, glycerate-3P => serine Tyrosine degradation, tyrosine => homogentisate 18 M00021 Cysteine biosynthesis, serine => cysteine Homoprotocatechuate degradation, homoprotocatechuate => 2-oxohept-3-enedioate Mooo22 Shikimate pathway, phosphoenolpyruvate + erythrose-4P => chorismate Leucine degradation, leucine => acetoacetate + acetyl-CoA M00023 Tryptophan biosynthesis, chorismate => tryptophan 19 Amino acid Pantothenate biosynthesis, valine/L-aspartate => pantothenate M00024 Phenylalanine biosynthesis, chorismate => phenylalanine Cephamycin C biosynthesis, aminoadipate + cycteine + valine => cephamycin C 20 M00025 Tyrosine biosynthesis, chorismate => tyrosine biosynthesis Methionine degradation M00026 Histidine biosynthesis, PRPP => histidine Ethylene biosynthesis, methionine => ethylene M00030 Lysine biosynthesis, AAA pathway, 2-oxoglutarate => lysine Polyamine biosynthesis, arginine => ornithine => putrescine 21 M00031 Lysine biosynthesis, 2-aminoadipate => lysine Histidine degradation, histidine => N-formiminoglutamate => glutamate M00034 Methionine salvage pathway Inosine monophosphate biosynthesis, PRPP + glutamine => IMP 22 M00040 Tyrosine biosynthesis, prephanate => pretyrosine => tyrosine Uridine monophosphate biosynthesis, glutamine (+ PRPP) => UMP M00047 Creatine pathway Ectoine biosynthesis, aspartate => ectoine 23 M00136 GABA biosynthesis, prokaryotes, putrescine => GABA NAD biosynthesis, aspartate => NAD M00338 Cysteine biosynthesis, homocysteine + serine => cysteine Glutathione biosynthesis, glutamate => glutathione M00432 Leucine biosynthesis, 2-oxoisovalerate => 2-oxoisocaproate Heme biosynthesis, glutamate => protoheme/siroheme 24 M00433 Lysine biosynthesis, 2-oxoglutarate => 2-oxoadipate Lysine degradation, lysine => saccharopine => acetoacetyl-CoA M00525 Lysine biosynthesis, acetyl-DAP pathway, aspartate => lysine 25 M00526 Lysine biosynthesis, DAP dehydrogenase pathway, aspartate => lysine M00527 Lysine biosynthesis, DAP aminotransferase pathway, aspartate => lysine 26 M00535 Isoleucine biosynthesis, pyruvate => 2-oxobutanoate Bile acid metabolism M00570 Isoleucine biosynthesis, threonine => 2-oxobutanoate => isoleucine 27 M00609 Cysteine biosynthesis, methionine => cysteine Bile acid biosynthesis, cholesterol => cholate ABC transporters Membrane Conjugated bile acid biosynthesis, cholate => taurocholate/glycocholate Bacterial secretion system Multiple steps 7α/β-dehydroxylation (secondary bile acids biosynthesis) 28 Phosphotransferase system (PTS) transport Bacterial chemotaxis 29 Flagellar assembly https://mc.manuscriptcentral.com/gutCell motility -2 -1 0 1 2 Regulation of actin cytoskeleton Report Z-score 30 -2 -1 0 1 31 Report Z-score 32 33 34 35 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 36 Supplementary material Gut

online supplementary figure 9 Gut Page 70 of 338 A B Totle number # ESRD patients # healthy control- Taxonimic name Top 20 discriminatory power species (ESRD patient-enriched) of MGSs -enriched MGSs enriched MGSs Akkermansia muciniphila 2 1 1.00 AUC 1Alistipes 6 1 Alistipes finegoldii 3 2 Anaerostipes caccae_M535 0.818 Alistipes shahii 11 5 2Anaerostipes caccae 2 2 Eggerthella lenta_M388 0.803 Bacteroides cellulosilyticus 13 2 Bacteroides fragilis 2 1 Flavonifractor_M648 0.797 3Bifidobacterium dentium 1 1 Intestinimonas_M541 0.794 Bilophila wadsworthia 2 2 Blautia 14 6 2 0.75 Clostridium_M074 0.788 4Blautia hansenii 3 3 Blautia sp. YL58Confidential: 2 2 For Review Only Flavonifractor_M413 0.785 Clostridiales bacterium 1_7_47FAA 1 1 5Clostridioides difficile 1 1 Flavonifractor_M390 0.785 Clostridium leptum 1 1 6Clostridium ramosum 1 1 Flavonifractor_M641 0.771 Clostridium saccharolyticum 1 1 Clostridium_M790 0.767 Clostridium scindens 2 2 7Clostridium spiroforme 2 2 0.50 Flavonifractor_M543 0.760 Clostridium symbiosum 1 1 Desulfovibrio 6 4 Blautia sp. YL58_M796 0.744 8Desulfovibrio fairfieldensis 1 1 Sensitivity Desulfovibrio piger 1 1 Clostridioides_M822 0.737 Eggerthella lenta 2 2 9Enterococcus faecalis 1 1 Flavonifractor plautii_M389 0.736 Enterococcus faecium 2 1 Flavonifractor_M051 0.735 Erysipelotrichaceae bacterium 5_2_54FAA 1 1 0.25 10Erysipelotrichaceae bacterium I46 2 2 Ruminiclostridium_M216 0.733 Faecalibacterium 14 6 3 Flavonifractor 21 17 1 Lachnoclostridium_M873 0.733 11Flavonifractor plautii 2 2 Fusobacterium nucleatum 1 1 Ruminiclostridium_M217 0.729 Fusobacterium ulcerans 1 1 12Holdemania filiformis 2 1 Oscillibacter_M391 0.725 Intestinimonas 2 2 Clostridioides difficile_M685 0.718 Intestinimonas butyriciproducens 1 1 0 13Lachnoclostridium 8 4 2 Blautia_M323 0.722 Lachnoclostridium sp. YL32 6 6 Lachnospiraceae bacterium 1_4_56FAA 1 1 0 0.25 0.50 0.75 1.00 14Lachnospiraceae bacterium 3_1_57FAA_CT1 1 1 Lactobacillus amylovorus 1 1 Lactobacillus casei 2 2 1 - Specificity 15Lactobacillus fermentum 1 1 Lactobacillus plantarum 1 1 Lactobacillus vaginalis 1 1 16Parabacteroides 10 5 Parabacteroides merdae 4 2 17Paraprevotella xylaniphila 1 1 Prevotella buccae 1 1 Ruminiclostridium 2 2 18Ruminococcaceae bacterium D16 1 1 C Ruminococcus 30 13 4 Ruminococcus gnavus 1 1 Top 20 discriminatory power species (healthy control-enriched) 19Ruminococcus sp. 5_1_39BFAA 3 3 Ruminococcus sp. SR1/5 6 1 Streptococcus infantarius 1 1 AUC 20Streptococcus thermophilus 1 1 1.00 Turicibacter sp. H121 1 1 Eubacterium_M652 0.799 Weissella 1 1 21Weissella cibaria 2 2 Clostridium_M349 0.784 Bacteroides 11 1 2 Burkholderiales 3 3 Roseburia_M328 0.775 22butyrate-producing bacterium SS3/4 2 2 Clostridium bartlettii 1 1 Clostridium_M300 0.767 Clostridium sp. L2-50 1 1 Prevotella copri_M186 0.765 23Coprobacillus sp. 29_1 2 1 0.75 Coprococcus 4 4 Prevotella copri_M107 0.765 Coprococcus comes 1 1 24Coprococcus sp. ART55/1 1 1 butyrate producing Dorea formicigenerans 1 1 bacterium SS3/4_M295 0.761 Dorea longicatena 3 3 25Eubacterium 15 15 Prevotella_M898 0.760 Eubacterium biforme 4 1 Prevotella copri_M891 0.759 26Eubacterium rectale 1 1 0.50 Eubacterium ventriosum 1 1 Prevotella copri_M233 0.758 Faecalibacterium prausnitzii 17 9

27Fusobacterium 1 1 Sensitivity Clostridium_M366 0.757 Lachnospiraceae bacterium 2_1_46FAA 1 1 Mitsuokella multacida 1 1 Lachnoclostridium_M294 0.757 28Oxalobacter formigenes 2 1 Parasutterella excrementihominis 1 1 Eubacterium_M664 0.750 Phascolarctobacterium succinatutens 1 1 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 29Prevotella 16 3 11 0.25 Clostridioides_M501 0.748 Prevotella copri 4 4 Roseburia 22 2 17 Prevotella_M184 0.746 30Roseburia intestinalis 2 2 Prevotella_M146 0.745 Ruminococcus bicirculans 2 2 Ruminococcus obeum 1 1 Eubacterium_M315 0.743 31Ruminococcus torques 3 2 Sutterella wadsworthensis 1 1 Prevotella_M743 0.726 Clostridioides 10 4 3 32Clostridium 47 17 14 0 Clostridium_M365 0.737 Coprococcus catus 2 1 1 Eubacterium eligens 2 1 1 Oscillibacter_M052 0.736 33Oscillibacter 14 7 5 0 0.25 0.50 0.75 1.00 34Overall 419 172 129 1 - Specificity 35 Acetate biosynthesis Propionate biosynthesis Butyrate biosynthesis D (acetyl-CoA decarbonylase/synthase) E (methylmalonyl-CoA decarboxylase; F (butyryl-CoA:acetate CoA-transferase; 36 lactoyl-CoA dehydratase; butyrate kinase) 37 propionaldehyde dehydrogenase) Bacteroides Faecalibacterium Faecalibacterium 38

39 Roseburia 40 Ruminococcus 41 Prevotella 42 unclassified_MGS unclassified_MGS 43 Prevotella unclassified_MGS Eubacterium 44 1e−05 2e−05 3e−05 Blautia Clostridium Megamonas Clostridium 45 2e−05 4e−05 6e−05 8e−05 other_taxa other_taxa Clostridioides 1e−05 2e−05 3e−05 4e−05 Clostridium Ruminococcus Roseburia Blautia Coprococcus Escherichia Average abundance in healthy controls (n=223) in healthy abundance Average Average abundance in healthy controls (n=223) 46 other_taxa Bifidobacterium Average abundance in healthy controls (n=223) Escherichia Flavonifractor Lachnoclostridium Flavonifractor https://mc.manuscriptcentral.com/gutMegasphaera Anaerostipes 0 47 Bifidobacterium 0 0 2e−05 4e−05 6e−05 8e−05 1e−05 2e−05 3e−05 4e−05 0 1e−05 2e−05 3e−05 48 Average abundance in ESRD patients (n=69) Average abundance in ESRD patients (n=69) Average abundance in ESRD patients (n=69) 49 50 51 52 53 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Confidential: For Review Only online supplementary figure 12 Supplementary material Gut Page 72 of 338Gut 0.5

1 2 3 4 0.4 5 6 7 8 0.3 9 10 11 12 13 0.2 14

15fcet size on serum metabolome 16Ef 17 18 0.1 19 20 21 22 ACVD patients (n = 59) ESRD patients (n = 292) Healthy controls (n = 69) Healthy controls (n = 43) 23 0.0 https://mc.manuscriptcentral.com/gutObese individuals (n = 72) Healthy controls (n = 79) Diabetic patients (n = 80) Non-diabetic controls (n = 291) 24 ESRD ACVD Obese Diabetes 25 This study Jie et al. 2017 Liu et al. 2017 Pedersen et al. 2017 26 27 28 29 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Confidential: For Review Only

Pageonline 73 ofsupplementary 338 Gut figure 13 Supplementary material Gut 60 1 2 3 4 5 6 7 Number of patients 8

9 0 10 20 30 40 50

10 0 2 4 6 8 10 11 Number of medications 12 13 https://mc.manuscriptcentral.com/gut 14 15 16 17 18 Wang X, et al. 19Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 online supplementary figure 14 Gut Page 74 of 338 A p−Cresol synthesis: 4-hydroxyphenylacetate decarboxylase Indole synthesis: tryptophanase Phenol synthesis: tyrosine phenol-lyase 1.2e−05

4e−06 P = 2.4e-5 6e−05 1 P = 5.2e-06 9.0e−06 P = 1.6e-05 2 3e−06 4e−05 2.0e−05 6.0e−06 3 2e−04 2e−06 2e−05 1.5e−05 2e−05 4 1e−06 3.0e−06 1.0e−05 5 1e−04 0 0 1e−05 5.0e−06 6 0 Total relative abundance (5) (1) (4) Gut

Total relative abundance (3) 3 (1) (1) (1) (1) (1) 09 (1) 02 (1) 6 (1) (2) 58 0 52 (1) 66 6 0 7 (2) 4 M045 (3) other (1)

Total relative abundance 7 2 71 (2) 72 (1) 7 10 6 4 83 (1) 0 M5 M562 (1) 6 _M3 _M4 _M4 other (2) A M0 M030 (2) M0 M209 (1) sedioidirtsolC M5 M_eliciffid ESRD Healthy ens_M134 (1) ESRD Healthy um ella_M5 0 FA 8 io_M545 (2)a_M5 patients controls 7 er atum hia_M4 patients controls ausnitzii_M014 (1) le rt ichia coli_M095 (6) ESRD Healthy rtif ridium_M0ridium_M0 wo la lent Bacteroides_M115 Prevot patients controls mo 9 1_7_4 Confidential:ramosum_M3 For Review Only Roseburia_M354 (1) ds hel Alistipes shahii_M161 (5) rium pr Alistipes shahii_M169 (1) m m nuc Clost Clost Escher Escherichia coli_M099 (1) m m wa Desulfovibr Desulfovibrio_M567 ert Alistipes finegoldii_M162 eriu Ruminococcus_M443a (1) Bacteroides ovatus_M118 (5) Lachnoclostridium_M293 (5) Lachnoclostridium_M294 (1) eriu Escherichia coli_M095 (1) Citrobacter freundii_M090 (1) eriu gg ibacter splanchnicus_M150 (3) ct 10 ridiu Bacteroides vulgatus_M140 (1) ct Bacteroides stercoris_M110 (9) E Bacteroides uniformis_M176 (8) ct

ost ba oba oba ilophil Odor Fusobacterium ulcerans_M168 (10) Bacteroides xylanisolvBacteroides xylanisolvens_M133 (5) s Cl B Phascolarctobacterium_M613 (1) BacteroidesBacteroides cellulosilyticus_M119 cellulosilyticus_M120Bacteroides (2) cellulosilyticus_M121 (2) 11 Fus Faecalibacte Fus Bacteroides thetaiotaomicron_M123 (1) Phascolarctobacterium faecium M414 (7) Phenylacetylglutamine synthesis: phenylacetaldehyde dehydrogenase, Benzoic acid synthesis: fldH, fldBC, acdA, PAL ridiale 12 st

Clo phenylpyruvate decarboxylase 0.00020 13 P = 2.6e-4 2e-4 P = 0.0011 144e−04 0.00025 0.00015 doi: 10.1136/gutjnl-2019-319766 15 1.5e-4 0.00020

3e−04 –12. 16 0.00010 0.00015 :1 17 1e-4 0 2e−04 0.00010 0.00005 18 5e-5 2020; 1e−04 0.00005

19 Total relative abundance

0.00000 Gut Total relative abundance 0 20 ESRD Healthy ) ) ) ) ) ) ) 4 4 4 0 (3) (9) (3) (1) (3) (1) (1) 6 6 4 4 patients controls 0 ( 6 7 (2) 6 4 2 (1) 0 ( 8 (2) 1 (2) 8 (1) 6 6 4 (5) (3) 03 (1) 0 (8) (17) 7 (3) 9 (5) (11)9 (1) 4 4 4 6 4 4 44 6 6 44 3 4 223 (1) 21 ESRD Healthy 4 4 29 ( 11 (1) 6 9 others 523 (3) 259 (8) 393 ( M374 (5) M292 (5) M M2 4 4 46 44 46 6 , et al. patients controls M598 (2) M M207 (1) M M 22 others (388) hia coli_M09 ic

Sutterella_M Sutterella_M Bacteroides_M1 Clostridium_M 23 Eubacterium_M319 (1) Eubacterium_M31 Ruminococcus_M322 (2) Flavonifractor_M EscherichiaEscher coli_M099 (1) EscherichiaEscherichia coli_M09Escherichia coli_M098Escherichia coli_M097 (2) coli_M099 (1) (1) Faecalibacterium_M00Bacteroides dorei_M1 X Wang Escherichia coli_M095 (13) Eggerthella lenta_M388 ( Alistipes putredinis_M1 Bacteroides dorei_M137 (1) Bacteroides fragilis_M1 Bacteroides caccae_M132 (3) Lachnoclostridium_M293 (5) Lachnoclostridium_M301 (1) Eubacterium eligens_M318 (7) 24 tococcus salivarius_M Bacteroides vulgatus_M1 AnaerostipesBacteroides hadrus_M351 eggerthii_M131 (1) (1) Bacteroides uniformis_M17 Ruminococcus bromii_M225Clostridium (1) ramosum_M310 (2) Bacteroides vulgatus_M1 Enterobacter cloacae_M875 ( Ruminococcus gnavus_M321 (3) Megasphaera elsdenii_M373 (2) Clostridium sp. L2-50_M399 (1) Clostridium sporogenes_M Ruminococcus gnavus_M321 (1) LactobacillusAkkermansia rhamnosus_M100 muciniphila_M38 ( Klebsiella pneumoniae_M09 ParabacteroidesStrep merdae_M170 (3) MegamonasRuminococcus hypermegale_M bicirculans_M22 Enterobacter hormaechei_ Sutterella wadsworthensis_M Raoultella ornithinolytica_M88 Faecalibacterium prausnitzii_M007 (3) 25 Parabacteroides distasonis_M172 (3) Dialister sp. Marseille-P5638_M Ruminococcus sp. 5_1_39BFAA_M291 (5) Trimethylamine26 (TMA) synthesis: choline trimethylamine-lyase Secondary bile acid synthesis: 7α/β-dehydroxylation ESRD patients 2.0e−05 Healthy controls 27 6e−05 28 P = 1.9e-6 1.5e−05 P = 6.0e-4 4e−05 29 1.0e−05 2e−04 301e−04 31 5.0e−06 2e−05 32 0 1e−04 5e−05 0 ) 225M_ )1( 6 (3) (2) ) 225M_ )1( 125M_ ) )1( 8 (3) 9 (1) 0 ( 4 4 6 4 33 72 (3) 9 (10) 0 2 6 7 71 (2) 10 (3) Total relative abundance 6

Total relative abundance 4

M040 (1) M0 M0 88 ( 22 (5) 10 ( _M1 21 (11) 64 0 M045 (3) M022 (1) M025 (1) M030 (1) or_M2 34 0 M044 (19) _M3 I muiretcab eaecahcirtolepisyrE hia_M4 or_M4 muiretcab I a_M3 64 64 rt ESRD Healthy I muiretcab eaecahcirtolepisyrE eaecahcirtolepisyrE muiretcab I muiretcab I ESRD Healthy ridium_M0 us_M3 um_M3 wo 35 s patients controls av os patients controls la lent Clostridium_M349 (4) Clostridium_M053 (1) vonifract Clostridium_M3 Clost gn wad la Desulfovibrio_M232 (4) nococcus ram Flavonifractor_M028 (1) s hel F des distasonis a Flavonifract Ruminococcus_M3 m 36 cu ert Alistipes shahii_M161 (1) Lachnoclostridium_M301 (8) Escherichia Rumicoli_M095 (2) eroi gg ridiu Faecalibacterium_M003 (2) E Bacteroides dorei_M137 (1) ilophil ococ Dorea longicatena_M337 (1) st B bact Klebsiella pneumoniae_M094 (1) abacteroides distasonis_M174 (1) 37 Klebsiella michiganensis_M092 (1) Clo Ruminococcus torques_M320 (9) ara Par Rumin P

Faecalibacterium prausnitzii_M010Faecalibacterium (1) prausnitzii_M013 (1) 38 Phascolarctobacterium faecium_M414 (1) 39 Phascolarctobacterium faecium_M414 (2) B p−Cresol Indole Phenol Phenylacetylglutamine 40 Pearson r P Pearson r = 0.316, P = 5.6e-8 Pearson r = 0.307, P = 0.143, = 0.016 Pearson r = 0.205, P = 5.2e-4 8 = 1.4e-7 yde) Spearman ρ = 0.378, P = 1.0e-10 ) Spearman ρ = 0.146, P = 0.014 3 Spearman ρ = 0.230, P = 9.5e-15 41 Spearman ρ = 0.257, P = 1.3e-5 ol

40 le) 100 aldeh

(phen 6

42 (indo ylacet 30 ce 43 ce 2

4 (phen

44 20 50 abundan abundan ance ance 1 lized lized 2

45 lized 10 abund ed ed 46 Norma Norma 0 0 Normalized abundance (p-Cresol) 0 0 47 0 1e−04 2e−04 0 5.0e−06 1.0e−05 1.5e−05 2.0e−05 2.5e−05 0 1e−05 2e−05 1e−04 2e−04 3e−04 4e−04 5e−04 Total relative abundance in gut microbiome Total relative abundance in gut microbiome Normaliz 48 Total relative abundance in gut microbiome Total relative abundance in gut microbiome

49 Benzoic acid Trimethylamine (TMA) Secondary bile acids Supplementary material

50 ） 0.015 Pearson r = 0.118, P = 0.047 Pearson r = 0.129, P = 0.0307 Pearson r = 0.212, P = 0.0036 P = 0.159 Spearman ρ = 0.084, 3e+05 Spearman ρ = 0.234, P = 7.4e-5 Spearman ρ = 0.182, P = 0.013 51 um) 15000 er

52 0.010 2e+05

53 (DCA+LCA) ce 10000

54 s in (TMAO ance 0.005 abundan 55 1e+05 5000 abund https://mc.manuscriptcentral.com/gut lized 56 ed

0.000 Norma 57 Normalized abundance (Hippuric acid 0 0

Normaliz 0 1e−04 2e−04 0 1e-04 2e-04 3e-04 0 5e−05 1e−04 58 Total relative abundance in gut microbiome Total relative abundance in gut microbiome Total relative abundance in gut microbiome 59 60 online supplementary figure 15 SupplementaryPage 75 of material 338 Gut Gut M047 Oscillibacter M278 Ruminococcus obeum M802 M325 M650 M45 M737 Alistipes shahii M852 M424 M416 M452 Fusobacterium mortiferum M77 M650 Lachnoclostridium M109 M770 M743 Prevotella Confidential:M59 For ReviewM470 Only M95 M541 Intestinimonas M301 M555 M53 M656 Clostridioides M301 Lachnoclostridium M443 M873 Lachnoclostridium M836 M153 M656 M562 M095 Escherichia coli M847 M278 Blautia M466 Fusobacterium nucleatum M567 M388 Eggerthella lenta 1 M679 M200 M502 M790 Clostridium M140 M881 M019 Faecalibacterium prausnitzii M323 Blautia M370 Cellulosilyticum M616 p-Cresol M685 Indole M47 M873 M1402 Bacteroides vulgatus M44 M743 Phenol M472 Blautia hansenii M847 Citrobacter freundii M583 M863 M460 M33 Fusobacterium nucleatum_M466 M7703 Flavonifractor M795 M864 M452 M712 Lactobacillus crispatus M26 M712 M388 Eggerthella lenta M337 M31 M228 M19 4 M761 M427M766 M222 M572 M585 M547 Eggerthella lenta M547 M323 M278 M370 M212 M790 M737 M523 Blautia hansenii_M472 M4245 Clostridium leptum M554 M324 M541 M355 M266 M258 M163 M399 M163 Alistipes finegoldii M570 M488 Roseburia Clostridium scindens_M494 M464 M3376 Dorea longicatena Desulfovibrio_M827 M293 Alistipes finegoldii_M164 M679 Eubacterium dolichum M488 M641 M213 M257 M4437 Ruminococcus M195 M578 M059 Alistipes shahii M207 M571 M8638 Bacteroides Phenylacetylglutamine M758 M210 M449 M460 Bilophila wadsworthia Citrobacter freundii_M744 Mitsuokella multacida_M800 M6859 Clostridioides difficile Eggerthella lenta_M388 M823 M413 M802 Citrobacter freundii M064 M109 Bacteroides eggerthii M592 M544 10 Flavonifractor/Intestinimonas_M449 Flavonifractor_M543 M836 Lactobacillus vaginalis M426 M675 M226 M503 M35 M670 M547 Eggerthella lenta M868 M773 M410 M091 11 M320 M689 M779 Eubacterium_M652 M644 M870 M613 M819 M695 M673 M727 M322 Ruminococcus M483 M886 M532 M854M122 M312 M116 Faecalibacterium_M708 M31712 Eubacterium M420 M361 M453 M341 M830 M805 Fusobacterium M295 M489 M288 M413 Flavonifractor M40113 Coprococcus sp. ART55/1 M772 M542 M706 M488 Roseburia M682 Clostridium perfringens M191 M601 M644 M652 Eubacterium M320 Ruminococcus torques M805 14 M340 M389 M543 Flavonifractor M388 Eggerthella lenta SBAs M839 TMAO M374 Hippuric acid M667 M608 M509 M464 Bacteroides M50315 Clostridium M401 M052 M734 Holdemania filiformis M102 M71 M708 Faecalibacterium M317 M210 Flavonifractor 16 M234 M895 M735 M270 M517 M705 M823 Flavonifractor M689 Faecalibacterium prausnitzii M206 M843 M69 M877 M735 Flavonifractor M274 M399 Clostridiumsp.L2-50 M335 M309 M889 M53517 Anaerostipes caccae M682 M298 M730 M266 Faecalibacterium prausnitzii M734 M322 M40 M535M312 M231 M112 M361 Clostridium M640 M585 Desulfovibrio fairfieldensis M89518 Clostridium saccharolyticum M309 Clostridiales bacterium 1_7_47FAA M172 Parabacteroides distasonis M295 butyrate-producing bacterium SS3/4 M052 Oscillibacter M116 Bacteroides finegoldii M712 Lactobacillus crispatus M88619 Raoultella ornithinolytica M877 Oxalobacter formigenes M517 Anaerostipe shadrus M426 Clostridium M453 Clostridium bartlettii M830 Roseburia M312 Clostridiums piroforme M410 Bifidobacter iumlongum M819 Burkholderiales M389 Flavonifractor plautii M509 Coprococcus catus 20 M673 Clostridium M839 Parabacteroides acrEF (indole exporter) M288 Ruminococcus M390 Flavonifractor M779 Cloacibacillus synthetase-encoding species M644 Parabacteroides merdae M112 Bacteroidesc ellulosilyticus M449 Flavonifractor M10221 Ruminococcus phenylethylamine permease M295 butyrate-producing bacteriumSS3/4 M427 Ruminococcus M670 Bacteroides synthetase-lacking species pmtBCD (phenol export transporter) M355 Roseburia M31222 Clostridium spiroforme ESRD patients-enriched https://mc.manuscriptcentral.com/gut pcmK (p-cresol transporter) M293 Lachnoclostridium healthy control-enriched M641 Flavonifractor trimethylamine permease 23 not signficant M122 Bacteroides cellulosilyticus baiG (bile acid transporter) M091 Enterococcus gallinarum 24 M675 Prevotellabuccae 25 26 27 28 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 online supplementary figure 16 Gut Page 76 of 338

A Effect size (Q2 ) 0.2 0.4 0.6 ** ** ** ** ** ** ** ** **** * ** ** ** ** ** ** ** ** ** ** ** **** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** * * ** ** ** ** ** ** ** * ** ** ** *** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **** * ** ** ** ** ** ** ** ** ** ** ** ** ** *** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** *** ** * ** ** ** *** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** *** * ** ** ** *** ** ** ** ** ** ** * ** ** ** ** ** ** *** ** ** ** ** ** ** ** ** ** * * * ** ** ** ** ** ** ** ** ** ** ** *** ** ** * ** ** ** ** ** ** ** * ** ** *** ** *** ** ** ** ** ** ** ** ** ** **** * ** ** ** *** ** **** *** * ** ** * * ** ** **** * ** ** ** ** *** ** * ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** *** ** ** ** * * ** ** ** ** ** ** ** ** ** ** *** ** ** ** ** ** ** ** ** ** **** * ** ** ** ** **** * ** ** ** ** **** * ** ** ** ** **** * ** ** ** ** ** ** ** *** *** * ** * ** ** * ** ** **** * ** ** ** + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1 + ** * * * * ** * * * * * *** ** * * * * * * * * * * * ** ** * * * * * ** * *** * ** * ** * * * * * * * * * * * * ** HGB ** * * + + + + + + + + + + + + + * * * 2 HCT ** * ** ** ** ** ** ** ** ** ** ** + + + + + + + + + + + + + + + + + + + * * * * * ** * * * * * * * * 3 eGFR * *** ** *** ** *** ** ** ** + + + + + + + + * * * * * * * * * * * ALT * * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + * * * * * Gut 4 AST ** + + + + + + + + + + + + + + + + + + + + + + * Dialysis5 frequency * * ** + + + + + + + + + + + + + + + + + *** *** ** * * ** * ** * * * * * * * * KT/V * + + + + 6 + DBP + + + + + + + + + + + 7 HDL_C * Gender +

8 + Confidential: For Review Only* BMI 9 LDL_C + + + + + + + + + + ** *** ** * ** ** * * ** * 10 TCHO * + + + + + + + + * * PTH * + + + + + + + + + ** * ** **** * ** * * ** ** ** * * * ** * 11 ALB ** * + 12Dialysis period * ** + + CRP + + doi: 10.1136/gutjnl-2019-319766 ****** * * * * ** + + + + + + + + ** * 13 ** * Leptin *** * ** * ** *** ** ** *** ** ** + + + + + + + + + + + *** * ** * * * * * ** ** ** ** ** * * ** ** * * ** * * * * * ** * * * * *

14 Age –12. ** ** ** ** **** ** ** * * ** **** * * ** ** ** ** ** ** * ** ** ** ** **** * ** ** ** ** ** ** ** ** ** * ** ** ** ***** ** ** ** ** ** * * * ** ** * ** ** + + + + + + + + + + + + + + + + ** * * * * * * * **** *** ** ** * * ** * ** ** * * ** * * * * * * * * * * Fecal_pH :1 0 ** **** ** ** * * ** *** ** ** * ** * ** ** *** ** * ** ** ** ** ** ** ** *** ** ** ** ** ** *** ** * ** ** ** ** *** ** + + + + + + + + + + + + + + + + + + + + + + + + 15 + ** *** ** ** ** * * * *** *** ** ** * ** * * * ** * *** ** * * * SBP * ** ** + + + + + + + + + + + + + + + + + + + + + + + + * * * * * * * * * ** *** ** ** ** * 16 GLU * *** ** ** * ** ** *** *** ** ** * ** ** ** ** ** ** ** + + + + + + + + + + + + + + + + + + + + + + + + + ** *** ** ** ** * ** * * ** ** * * *** ** ** * ** * * ** * *** ** ** * ** *

UA 2020; ** ** *** * ** * ** ** *** ** ** ** ** ** ** 17 ** ** * ** + + + + + + + + + + + + + + + + + + + + * * * ** ** * * ** * * * * * * * LBP * ** ** ** ** ** ** ** ** ** ** ** ** Gut + + + + + + + + + + + + + + + + + + + + + ** * * * * * * ** * * * * * * 18 TG ** ** * *** ** *** ** *** ** *** ** *** ** *** ** ** ** *** ** *** ** ** ** ** ** *** ** ** ** *** ** *** ** *** ** *** ** *** ** *** ** *** ** *** ** + + + + + + + + + + + + ** * * * ** * * * * * * ** * ** * * * * * ** * * 19 UREA ** * ** + + + + + + + + + + + * * * CREA * 20 , et al. M044 M033 M761 M502 M228 M864 M889 M489 M494 M424 M881 M542 M064 M222 M578 M572 M071 M616 M213 M069 M601 M608 M298 M040 M583 M795 M554 M705 M312 M730 M766 M592 M026 M206 21 M727

22 idium_M650 Wang X Wang Blautia_M323 Blautia_M278 Blautia_M472 Olsenella_M374 Sutterella_M570 Prevotella_M743 Roseburia_M355 Clostridium_M503 23 Clostridium_M426 Eggerthella_M532 Clostridium_M361 Clostridium_M341 Clostridium_M673 Clostridium_M758 Clostridium_M420 Clostridium_M270 Clostridium_M870 Clostridium_M790 Oscillibacter_M854 Oscillibacter_M052 Oscillibacter_M047 Eubacterium_M571 Eubacterium_M652 Eubacterium_M317 Desulfovibrio_M460 Desulfovibrio_M827 Lactobacillus_M836 Clostridioides_M656 Megasphaera_M772 Anaerostipes_M535 Coprococcus_M544 Flavonifractor_M449 Flavonifractor_M770 Flavonifractor_M543 Flavonifractor_M641 Flavonifractor_M210 Flavonifractor_M413 Flavonifractor_M390 Flavonifractor_M735 Clostridioides_M453 Selenomonas_M800 Fusobacterium_M452 Intestinimonas_M541 Anaerotruncus_M212 Fusobacterium_M466 Fusobacterium_M805 Ruminococcus_M324 Ruminococcus_M322 Alistipesshahii_M737 Ruminococcus_M337 Ruminococcus_M427 rococcus catus_M509 Burkholderiales_M819 Eggerthella lenta_M388Eggerthella Eggerthella lenta_M547Eggerthella

24 Faecalibacterium_M708 Lachnoclostridium_M301 Citrobacter freundii_M744 Citrobacter Citrobacter freundii_M847 Citrobacter Lachnoclostridium_M309 Lachnoclostridium_M873 Lachnoclostridium_M293 Lachnoclostr Cop Raoultella planticola_M886 Anaerostipes hadrus_M517 Anaerostipes Flavonifractor plautii_M389 Flavonifractor Clostridioidesdifficile_M685 25 Clostridiumsp. L2-50_M399 Clostridiumsporogenes_M340 Enterococcus gallinarum_M091 Parabacteroides merdae_M644 Parabacteroides Desulfovibrio fairfieldensis_M585 Desulfovibrio Parabacteroides distasonis_M172 Parabacteroides Coprococcus sp. ART55/1_M401 Faecalibacterium prausnitzii_M019Faecalibacterium T: ESRD patients enriched Faecalibacterium prausnitzii_M266Faecalibacterium CandidatusMelainabacteria_M843 Clostridiumsaccharolyticum_M895

26 sp. Marseille-P5638_M523 Dialister Spearman correlation coefficient 27 T: Healthy controls enriched Lachnospiraceae bacterium GAM79_M335 bacterium Lachnospiraceae 28 butyrate-producing bacteriumSS3/4_M295 B ESRD patients only −0.4 0 0.4 ** ** ** ** ** + + + + + + + + + + + + + + + + + * * * * 29 * HGB + + + + + + + + + 30 HCT * + + + + + + + + + + + + + + + + + + + + + + + * * * * eGFR * + + + + + + + + + + + + + 31 + ALT + 32 * AST * ** + + 33 CRP + ** *** ** + + + + + + + + + + + + + + + + + + + + + + + + + * * * Dialysis frequency * *** * ** + + + + + + + + ** ** * * * * * * * * * 34 KT/V * * + + + + + * 35 Leptin * + + + + + + + + + + + * Gender *

36 * DBP + + + + + 37 BMI + + + + + + + + + LDL_C * * + + 38 + + TCHO ** + + + + + + 39 HDL_C * + + + + + + + + + + + + + + * * * * 40 PTH * + + + + + + ** * ** * * * * * ALB * * ** + + 41Dialysis period * ** ** + + + + + + * ** * * * 42 Age * * + + + + * * * * Fecal_pH * + + + + + + + * * * * * * * * 43 * GLU ** ** 44 SBP + ** ** + + + + + + + + + * * * * UA *

45 + LBP + * 46 TG * ** + + + + + + * * * * 47 CREA * + + + + + * 48 UREA M044 M033 M761 M502 M228 M864 M889 M489 M064 M424 M069 M542 M222 M312 M578 M583 M601 M572 M616 M295 M298 M554 M040 M795 M766 M730 M026 M494 M206 M705 M881 M592 M071 M213 M608 49 M727 Supplementary material ia_M323 50 um_M790 densis_M585 Blaut Blautia_M472 Blautia_M278 Sutterella_M570 Olsenella_M374 Prevotella_M743 Roseburia_M355 Clostridium_M503 Clostridium_M426 Eggerthella_M532 Clostridium_M341 Clostridium_M361 Clostridium_M673 Clostridium_M758 Clostridium_M420 Clostridium_M870 Clostridium_M270 Clostridi 51 Oscillibacter_M854 Oscillibacter_M052 Oscillibacter_M047 Eubacterium_M571 Eubacterium_M652 Eubacterium_M317 Desulfovibrio_M460 Desulfovibrio_M827 Lactobacillus_M836 Clostridioides_M656 Megasphaera_M772 Anaerostipes_M535 Coprococcus_M544 Flavonifractor_M449 Flavonifractor_M543 Flavonifractor_M641 Flavonifractor_M770 Flavonifractor_M413 Flavonifractor_M210 Flavonifractor_M735 Flavonifractor_M390 Clostridioides_M453 Selenomonas_M800 Fusobacterium_M452 Intestinimonas_M541 Anaerotruncus_M212 Fusobacterium_M805 Ruminococcus_M324 Fusobacterium_M466 Ruminococcus_M322 Alistipesshahii_M737 Ruminococcus_M427 Ruminococcus_M337 Burkholderiales_M819 Eggerthella lenta_M388Eggerthella Eggerthella lenta_M547Eggerthella Faecalibacterium_M708

52 Lachnoclostridium_M301 Citrobacter freundii_M744 Citrobacter Citrobacter freundii_M847 Citrobacter Lachnoclostridium_M293 Lachnoclostridium_M873 Lachnoclostridium_M309 Lachnoclostridium_M650 Coprococcus catus_M509 Raoultella planticola_M886 Anaerostipes hadrus_M517 Anaerostipes Flavonifractor plautii_M389 Flavonifractor Clostridioidesdifficile_M685 53 Clostridiumsp. L2-50_M399 https://mc.manuscriptcentral.com/gut Clostridiumsporogenes_M340 Enterococcus gallinarum_M091 Parabacteroides merdae_M644 Parabacteroides Desulfovibrio fairfiel Desulfovibrio Parabacteroides distasonis_M172 Parabacteroides Coprococcus sp. ART55/1_M401 Faecalibacterium prausnitzii_M019Faecalibacterium Faecalibacterium prausnitzii_M266Faecalibacterium CandidatusMelainabacteria_M843 Clostridiumsaccharolyticum_M895 54 sp. Marseille-P5638_M523 Dialister

55 GAM79_M335 bacterium Lachnospiraceae butyrate-producing bacteriumSS3/4_ 56 57 58 59 Confidential:Page 77 of 338 ForGut Review Only

A Gut B 30

1 20 2 T : ESRD-enriched sepcies 3 T : Control-enriched species 10 doi: 10.1136/gutjnl-2019-319766 4 –12. :1 5 0 Mean Decrease Accuracy 2020;

6 0 Gut , et al. 7 Sensitivity (%) M601 M044 M795 M705

8 X Wang ii_M453 da_M800 um_M466

9 tlett 10 ar Roseburia_M611 AUC= 97.0% Clostridium_M366 Eubacterium_M652 idium b Flavonifractor_M251

11 Megasphaera_M772 ium nucleat tr Alistipes shahii_M737 Alistipes

0 20 40 60 8095% 100 CI: 95.1%− 98.9% er Eggerthellalenta_M388 12 los https://mc.manuscriptcentral.com/gut C Clostridium scindens_M494 Clostridium spiroforme_M312 Butyricimonas faecalis_M077 13 100 50 0 Mitsuokella multaci usobact 14 Specificity (%) F Dialister sp. Marseille-P5638_M523 Flavonifractor/Intestinimonas_M413 15 Victivallales bacterium CCUG 44730_M191 16 17

18 Supplementary material 19 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Supplementaryonline supplementary material figure 20 Gut Gavage (every other day) Gut Page 80 of 338 A Finish 5/6 B C nephrectomy 0.08 Confidential: For Review) Only 1.2 60 )

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4 erum S erum

5 F ecesa F ecesa F ecesa Sacrifice 0.0 S 0 Serum Serum Serum Tissues week 0 week 8 week 0 week 8 6 CKD 7 CKD+F.nucleatum D CKD controlCKD + F. nucleatum CKD + E. lentan CKD+E.lenta 8 9 CKD control **

10 ** PAS CKD + F. nucleatum 11 (x20) 12 CKD + E. lenta 13 0 0.5 1.0 1.5 2.0 2.5 Glomerulosclerotic index E14 15 CKD control 16 * CKD + F. nucleatum * 17 TGF-β1 18 (x20) CKD + E. lenta 19 0 0.5 1.0 1.5 2.0 2.5 3.0 20 Relative mean density of TGF-β1 F21 22 CKD control ** ** 23 CKD + F. nucleatum 8-OHdG 24 CKD + E. lenta (x20) https://mc.manuscriptcentral.com/gut 25 0 10 20 30 40 26 8-OHdG posltive cells(%) 27 28 29 30 Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 31 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766 Supplementary material Gut

Wang X, et al. Gut 2020;0:1–12. doi: 10.1136/gutjnl-2019-319766