UREMIC TOXINS / MICROBIOME

UREMIC TOXINS / MICROBIOME R Vanholder University Hospital, Gent, Belgium

COMPLEX PATHO-PHYSIOLOGY Ikizler et al, KI, 84: 1096-1107; 2013 2 2

UREMIC TOXINS NUTRITION 3 3

THE IMPACT OF NUTRITION ON THE UREMIC SYNDROME

UREMIC TOXINS NUTRITION 5 5

ROLE INTESTINE IN GENERATION CRESOLS p-cresol thyrosine intestinal flora intestinal wall liver p-cresylsulfate p-cresylglucuronide Schepers et al, Blood Purif, 29: 130-136; 2010 6 6

Vanholder et al, JASN, 25:1897-1907; 2014 7 7

FLOW CHART REVIEW PROCEDURES Figure 1. Flowchart of the review procedure and quality scoring. Vanholder et al, JASN, 25:1897-1907; 2014 8 8

INDOXYLSULFATE AND P-CRESYLSULFATE HAVE BEEN LINKED TO A MYRIAD OF TOXIC EFFECTS: Leukocyte activation Endothelial morphological and functional changes Endothelial/leukocyte interaction Smooth muscle cell proliferation Whole vessel calcification & senescence Cardiac fibrosis and myohypertrophy Skeletal resistance to PTH Insulin resistance Renal tubular damage 9 9

COMMON PATHWAYS IN ENDOTHELIUM Vanholder et al, JASN, 25:1897-1907; 2014 10 10

TMAO ENHANCES ATHEROGENESIS Wang et al, Nature, 472: 57-63; 2011 11 11

TMAO ENHANCES ATHEROGENESIS Metabolomic analysis unraveled TMAO as linked to vascular disease Sources of TMAO: Choline, fosfatidyl choline, betaine Red meat, shellfish, eggs, dairy products Wang et al, Nature, 472: 57-63; 2011 12 12

TMAO ENHANCES ATHEROGENESIS Role of intestinal microbiota: effect precursors TMAO neutralized by antibiotics Wang et al, Nature, 472: 57-63; 2011 13 13

TMAO CLEARANCE BY DIALYSIS IS IN THE RANGE OF OTHER SMALL WATER SOLUBLE COMPOUNDS Hai et al, Plos One, e0143731,;2015 14 14

TMAO CLEARANCE BY DIALYSIS IS IN THE RANGE OF OTHER SMALL WATER SOLUBLE COMPOUNDS Urea Creatinine TMAO Normals* 55 ±14 119 ± 21 219 ±78 ESRD** 258 ±58 174 ± 52 165 ±72 *: renal clearance; **: dialyzer clearance Hai et al, Plos One, e0143731,;2015 15 15

INDOXYL SULFATE + P-CRESYL SULFATE: OUTCOME Clin J Am Soc Nephrol 4: 1551 1558; 2009 16 16

INDOXYL SULFATE AND SURVIVAL Kaplan-Meyer estimates of overall mortality for patients as a function of tertiles for serum IS levels Number of patients at risk Barreto et al, CJASN, 4: 1551-1558; 2009 17 17

PROOF OF CONCEPT

PLASMA PROTEIN BOUND SOLUTES ARE DECREASED IN GERMFREE MICE Wikoff et al, PNAS, 106: 3698-3703; 2009 19 19

PLASMA PROTEIN BOUND SOLUTES ARE DECREASED IN GERMFREE MICE Fig. 3. Sulfate profiling based on constant neutral loss scanning of 80 m/z in ESI mode for conv (Upper) and GF (Lower) pooled plasma samples. The m/z values for several species are listed above their respective peak. Identified sulfates include: phenyl sulfate (a), indoxyl sulfate (b), p-cresol sulfate (c), equol sulfate (d), and methyl equol sulfate (e). Wikoff et al, PNAS, 106: 3698-3703; 2009 20 20

THE COLON CONTRIBUTES TO UREMIC TOXIN GENERATION Aronov et al, JASN, 22:1769-1776; 2011 21 21

THE COLON CONTRIBUTES TO UREMIC TOXIN GENERATION Aronov et al, JASN, 22:1769-1776; 2011 22 22

THE COLON CONTRIBUTES TO UREMIC TOXIN GENERATION Aronov et al, JASN, 22:1769-1776; 2011 23 23

THE COLON CONTRIBUTES TO UREMIC TOXIN GENERATION Aronov et al, JASN, 22:1769-1776; 2011 24 24

BUT THERE IS MORE THAN THAT

INTESTINAL MICROBIOTA ARE MODIFIED IN CKD Vaziri et al, KI, 83: 308-315; 2013 26 26

Fig. 4 - Hierarchical clustering of probe sets representing significantly different operational taxonomic units (OTUs) between groups with a minimum two-fold change in untransformed intensities between samples to show relative abundance. Yellow indicates increased abundance and red indicates decreased abundance relative to the mean for each OTU. Columns represent rat fecal microbiota composition for control (CTL) and chronic renal failure (CRF) individual rats. Rows are OTUs with mean fold change among samples and cluster based on the similarity of their abundance profiles across the data set, with similar OTUs connected at the hierarchical tree on the left. Bars on the right represent (1) OTUs from the Firmicutes (especially Lactobacillaceae and a few Coprococcus within the Lachnospiraceae), Bacteroidetes (Prevotellaceae, two Rikenellaceae OTUs) that had higher abundances in CTL samples, or (2) OTUs from the Firmicutes (unclassified Lachnospiraceae) and other Rikenellaceae (in Bacteroides) that have higher abundances in CRF samples. Vaziri et al, KI, 83: 308-315; 2013 27 27

CHANGES IN ASSIMILATION MODIFY THE GENERATION OF P-CRESYLSUFATE Bammens et al, KI, 64: 2196-2203; 2003 28 28

CHANGES IN ASSIMILATION MODIFY THE GENERATION OF P-CRESYLSUFATE Group A: GFR > 60 Group B: GFR 30-60 Group C: GFR < 30 (A) The median values of urinary p-cresol output (mg/24 hours) in the three study groups. (B) The median values of the urinary p-cresol (mg/24 hours)/urinary urea nitrogen (g/24 hours) ratio. *P < 0.05 vs. group A. Bammens et al, KI, 64: 2196-2203; 2003 29 29

CKD DISTURBS INTEGRITY OF THE INTESTINAL EPITHELIUM Vaziri et al, NDT, 27: 2686-2693; 2011 30 30

CKD DISTURBS INTEGRITY OF THE INTESTINAL EPITHELIUM Vaziri et al, NDT, 27: 2686-2693; 2011 31 31

CKD IS ASSOCIATED WITH TRANSLOCATION OF GUT BACTERIAL DNA GUT GENUS DETECTED IN BLOOD (% OF TOTAL GUT GENUS) Wang et al, Nephrology, 17: 733-738; 2012 32 32

CKD IS ASSOCIATED WITH TRANSLOCATION OF GUT BACTERIAL DNA GUT GENUS DETECTED IN BLOOD (% OF TOTAL GUT GENUS) Wang et al, Nephrology, 17: 733-738; 2012 33 33

CKD IS ASSOCIATED WITH TRANSLOCATION OF GUT BACTERIAL DNA GUT GENUS DETECTED IN BLOOD (% OF TOTAL GUT GENUS) Controls (n=10) ESRD (n=30) Klebsiella spp 0.60 1.93 0.037 Proteus spp 0.12 10.61 0.011 Escherichia spp 0.42 4.33 0.045 Enterobacter spp 0 0.42 0.001 Pseudomonas spp 0.04 0.23 0.014 P Wang et al, Nephrology, 17: 733-736; 2012 34 34

THERAPEUTIC OPTIONS Diet Cave malnutrition Prebiotics Non-digestible compounds beneficially modifying composition and/or function of intestinal flora Probiotics Bacteria adminstered as food components or supplements providing specific benefits themselves Synbiotics Prebiotics + probiotics Sorbents Schepers et al, Blood Purif, 29: 130-136; 2010 35 35

Vanholder and Glorieux, Clin Kidney J, 8: 168-179; 2015 36 36

Unfortunately, the quality and the evidence base of many of these studies are debatable, especially in uraemia, and often results within one study or among studies are contradictory. Vanholder and Glorieux, Clin Kidney J, 8: 168-179; 2015 37 37

URINARY PROTEIN BOUND TOXINS ARE LOWER IN VEGETARIANS Patel et al, cjasn, 7: 982-988; 2012 38 38

URINARY PROTEIN BOUND TOXINS ARE LOWER IN VEGETARIANS Table 2. Urinary excretion of PCS and IS Vegetarian Unrestricted P Value (Vegetarian versus Unrestricted) Period 1 Period 2 Period 1 Period 2 Period 1 Period 2 PCS (mg/d per 1.73 m2) 30 (11 74) 23 (17 47) 100 (47 124) 95 (20 124) 0.03 0.03 IS (mg/d per 1.73 m2) 24 (12 42) 19 ( 17 26) 47 (40 59) 52 (39 58) 0.001 <0.001 Patel et al, cjasn, 7: 982-988; 2012 39 39

P-CRESYLSULFATE INDUCES INSULIN RESISTANCE Koppe et al, JASN, 24: 88-99; 2013 40 40

PREBIOTIC AXOS REDUCES P-CRESYLSULFATE AND CORRECTS UREMIC INSULIN RESISTANCE IN MICE Koppe et al, JASN, 24: 88-99; 2013 41 41

DECREASE UREMIC TOXINS WITH SYNBIOTICS: RCT Rossi et al, cjasn, 11: 223-231; 2016 42 42

DECREASE UREMIC TOXINS WITH SYNBIOTICS: RCT IS - AB FREE (N=21) IS -TOTAL (N=31) PCS - AB FREE (N=21) PCS - TOTAL (N=31) Figure 3. Treatment effect of synbiotics on serum uremic toxins in all completing patients (n=31) and patients who were antibiotic free (n=21). a Treatment effect (95% confidence interval) derived from regression modeling accounting for period effect. *P=0.03; **P=0.001. Rossi et al, cjasn, 11: 223-231; 2016 43 43

REMOVAL OF INDOXYLSULFATE BY AST-120 PROTECTS GLOMERULAR FILTRATION RATE Akizawa T. et al, AJKD, 54:459-467; 2009 44 44

egfr, relative to baseline REMOVAL OF INDOXYLSULFATE BY AST-120 PROTECTS GLOMERULAR FILTRATION RATE 1.0 0.9 AST-120 Control 0.8 0.7 0 8 16 24 32 40 48 56 Time (weeks) P<0.001 Akizawa T. et al, AJKD, 54:459-467; 2009 45 45

AST-120 HAS NO PROTECTIVE EFFECT ON KIDNEY FUNCTION Schulman et al, JASN, 26: 1732-1746; 2015 46 46

AST-120 HAS NO PROTECTIVE EFFECT ON KIDNEY FUNCTION Schulman et al, doi: 10.1681/ASN.2014010042 47 47

AST-120 DOES NOT IMPACT ON PROGRESSION OF CKD Cha et al, cjasn, 2016, doi: 10.2215/CJN.12011214 48 48

AST-120 DOES NOT IMPACT ON PROGRESSION OF CKD Progression (primary endpoint): - Screa x 2 - egfr / 2 - ESRD Figure 2. Occurrence of composite primary outcomes in each treatment arm. Log-rank P=0.45. Cha et al, cjasn, 2016, doi: 10.2215/CJN.12011214 49 49

NOT DISCUSSED Uric acid Urea Advanced glycation end products Guanidines Other protein bound solutes (apart from indoxyl sulfate: e.g. p- cresyl sulfate, p-oh-hippuric acid, phenylacetic acid) Phenylacetylglutamine 50 50

NOT DISCUSSED Uric acid Urea Advanced glycation end products Guanidines Other protein bound solutes (apart from indoxyl sulfate and p- cresyl sulfate: p-oh-hippuric acid, phenylacetic acid) Phenylacetylglutamine 51 51

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins 52 52

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins This effect is further enhanced by: 53 53

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins This effect is further enhanced by: Intestinal overgrowth of bacteria 54 54

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins This effect is further enhanced by: Intestinal overgrowth of bacteria Disturbances of the intestinal epithelial barrier 55 55

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins This effect is further enhanced by: Intestinal overgrowth of bacteria Disturbances of the intestinal epithelial barrier Translocation of intestinal microbiota 56 56

CONCLUSIONS The intestine plays a crucial role in the generation of uremic toxins This effect is further enhanced by: Intestinal overgrowth of bacteria Disturbances of the intestinal epithelial barrier Translocation of intestinal microbiota The results of therapeutic interventions to modify uremic toxin uptake via the intestine up to now have been deceiving but quality of most studies may be a matter of debate 57 57

Back-ups

MULTIFACTORIAL ANALYSIS: SOLUTE CONCENTRATION VS. VARIOUS FACTORS Eloot et al, Plos One, 8: e76838; 2013 59 59

MULTIFACTORIAL ANALYSIS: SOLUTE CONCENTRATION VS. VARIOUS FACTORS Solute Covariates / R² full model Urea Normalized protein catabolic rate / 0.850 Crea Normalized protein catabolic rate / 0.150 Asymmetric dimethylarginine Symmetric dimethylarginine Uric acid Normalized protein catabolic rate / 0.195 beta-2-microglobulin Residual renal function/ 0.172 Hippuric acid Residual renal function / 0.187 Normalized protein catabolic rate / 0.220 Indoxyl sulfate Residual renal function / 0.059 Indole acetic acid p-cresylsulfate Normalized protein catabolic rate / 0.268 p-cresylglucuronide Residual renal function / 0.134 Normalized protein catabolic rate / 0.151 3-carboxy-4-methyl-5-propyl-2- furanpropionic acid age / 0.120 Free hippuric acid Residual renal function / 0.206 Free indoxyl sulfate Residual renal function / 0.144 Free indole acetic acid Residual renal function / 0.166 Free p-cresylsulfate Normalized protein catabolic rate / 0.189 Residual renal function / 0.257 Free p-cresylglucuronide Residual renal function / 0.135 Normalized protein catabolic rate / 0.150 - Included: Age, gender, npcr, Kt/V, RRF, diabetes, body weight, vintage Eloot et al, Plos One, 8: e76838; 2013 60 60

MULTIFACTORIAL ANALYSIS: SOLUTE CONCENTRATION VS. VARIOUS FACTORS Solute Covariates / R² full model Urea Normalized protein catabolic rate / 0.850 Crea Normalized protein catabolic rate / 0.150 Asymmetric dimethylarginine Symmetric dimethylarginine Uric acid Normalized protein catabolic rate / 0.195 beta-2-microglobulin Residual renal function/ 0.172 Hippuric acid Residual renal function / 0.187 Normalized protein catabolic rate / 0.220 Indoxyl sulfate Residual renal function / 0.059 Indole acetic acid p-cresylsulfate Normalized protein catabolic rate / 0.268 p-cresylglucuronide Residual renal function / 0.134 Normalized protein catabolic rate / 0.151 3-carboxy-4-methyl-5-propyl- 2-furanpropionic acid age / 0.120 Free hippuric acid Residual renal function / 0.206 Free indoxyl sulfate Residual renal function / 0.144 Free indole acetic acid Residual renal function / 0.166 Free p-cresylsulfate Normalized protein catabolic rate / 0.189 Residual renal function / 0.257 Free p-cresylglucuronide Residual renal function / 0.135 Normalized protein catabolic rate / 0.150 - Included: Age, gender, npcr, Kt/V, RRF, diabetes, body weight, vintage Eloot et al, Plos One, 8: e76838; 2013 61 61

DIET: UNCOOKED VEGAN DECREASES PROTEIN BOUND SOLUTES Ling et al, J Nutr, 122: 924-930; 1992 62 62

Serum concentration Serum concentration DIET: UNCOOKED VEGAN DECREASES PROTEIN BOUND SOLUTES Test Test Ling et al, J Nutr, 122: 924-930; 1992 63 63

P-CRESYLSULFATE INDUCES INSULIN RESISTANCE Koppe et al, JASN, 24: 88-99; 2013 64 64

P-CRESYLSULFATE INDUCES INSULIN RESISTANCE Koppe et al, JASN, 24: 88-99; 2013 65 65

PREBIOTIC AXOS REDUCES P-CRESYLSULFATE AND CORRECTS UREMIC INSULIN RESISTANCE IN MICE Koppe et al, JASN, 24: 88-99; 2013 66 66

COMMON PATHWAYS IN ENDOTHELIUM Vanholder et al, JASN, 25:1897-1907; 2014 67 67

PLASMA PROTEIN BOUND SOLUTES ARE DECREASED IN GERMFREE MICE Fig. 3. Sulfate profiling based on constant neutral loss scanning of 80 m/z in ESI mode for conv (Upper) and GF (Lower) pooled plasma samples. The m/z values for several species are listed above their respective peak. Identified sulfates include: phenyl sulfate (a), indoxyl sulfate (b), p-cresol sulfate (c), equol sulfate (d), and methyl equol sulfate (e). Wikoff et al, PNAS, 106: 3698-3703; 2009 68 68

DECREASE UREMIC TOXINS WITH SYNBIOTICS: RCT N=31 N=21 N=31 N=21 Figure 3. Treatment effect of synbiotics on serum uremic toxins in all completing patients (n=31) and patients who were antibiotic free (n=21). a Treatment effect (95% confidence interval) derived from regression modeling accounting for period effect. *P=0.03; **P=0.001. Rossi et al, cjasn, 11: 223-231; 2016 69 69