1. DEFINITION AND NEW INSIGHTS IN UREMIC TOXINS R Vanholder, University Hospital, Gent, Belgium

2. UREMIC TOXICITY: DEFINITIONS The uremic syndrome is a clinical condition, developing during the progression of renal failure, characterized by the loss of numerous biochemical and physiologic functions, and attributed to the retention of various solutes. Uremic toxins are uremic retention solutes which affect specific physiologic and biochemical functions. All these disturbed functions together are responsible for the uremic syndrome.

3. DEFINITION TOXIN (1) Compound should be chemically identified and accurate quantitative analysis in biological fluids should be possible The plasma level should be higher in uremic than in non-uremic subjects High concentrations should be related to specific uremic symptoms that decrease or disappear when concentration is reduced Concentrations in in vivo or in vitro studies should conform to those found in body fluids or tissue of uremic patients Vanholder et al, IJAO, 24, 695-725, 2001

4. DEFINITION TOXIN (2) Only a few solutes conform more or less with this strict defintion: –H 2 O? –Phosphate? –Potassium? –ß 2 -microglobulin? –……?

5. UREMIC RETENTION SOLUTES 1) adrenomedullin; 2) advanced glycation end products (AGE); 3) advanced oxidation protein products (AOPP); 4) angiogenin; 5) atrial natriuretic peptide; 6) ß 2 -microglobulin; 7) 3-carboxy-4-methyl-5-propyl-2- furanpropionic acid (CMPF); 8) chloramines; 9) Clara cell protein (CC16); 10) complement factor D; 11) cystatine C; 12) creatinine; 13) dimethylarginine (ADMA); 14) ß-endorphin; 15) glomerulopressin; 16) granulocyte inhibiting protein I (GIP I); 17) granulocyte inhibiting protein II; 18) guanidines; 19) hippuric acid; 20) homocysteine; 21) hyaluronic acid; 22) hypoxanthine; 23) indoles; 24) indoxyl sulfate; 25) leptin; 26) ß- lipotropin; 27) melatonin; 28) methionine-enkephalin; 29) methylamines; 30) myoinositol; 31) neuropeptide Y; 32) nitric oxide; 33) o- hydroxyhippuric acid; 34) oxalate; 35) parathyroid hormone; 36) p-cresol; 37) p-hydroxyhippuric acid; 38) peptides; 39) phenols; 40) phenylacetylglutamine; 41) phosphorus; 42) polyamines; 43) pseudouridine; 44) retinol binding protein; 45) trace elements; 46) trihalomethanes; 47) tryptophan; 48) urea; 49) uric acid; 50) xanthine; 51) xanthopterin

6. REVIEW ON UREMIC TOXINS: CLASSIFICATION, CONCENTRATION AND INTERINDIVIDUAL VARIABILITY EUToX: European Uremic Toxin Work Group within the context of ESAO Aim: to discuss and analyse matters related to the identification, characterization, analytical determination and evaluation of biological activity of uremic retention solutes Website: http://www.uremic-toxins.org

7. EUROPEAN UREMIC TOXIN WORK GROUP (EUToX) A Argiles P Brunet G Cohen PP De Deyn B Descamps-Latscha T Henle A Jörres ZA Massy M Rodriguez B Stegmayr P Stenvinkel R Vanholder C Wanner W Zidek U Baurmeister W Clark R Deppisch H Lemke J Passlick-Deetjen C Tetta

8. RESULTS MAPPING ANALYSIS Literature search of 857 publications 141 dealth with concentration (1968-2002) Data retained from 55 publications 90 solutes 68 with MW < 500 D, 22 middle molecules 12 > 12,000 D 25 solutes protein bound (mostly small compounds but also leptin and retinol binding protein) Concentrations range from ng/L (methionine- enkephalin) up to g/L (urea) Vanholder et al

9. CONCLUSIONS Concentrations of retention solutes in uremia vary over a broad range, from ng/L to g/L Low concntrations rae found especially for the MM A substantial number of molecules are protein bound and/or MM and hence difficult to remove Uremic retention is a complex problem which concerns much more solutes than the current markers urea and creatinine

10. SMALL WATER SOLUBLE COMPOUNDS

11. 0 100 200 300 400 500 UREA AND CELLULAR K- INFLUX. Lim et al, JCI, 96, 2126-2132, 1995. ControlUrea 45mM Bumetanide sensitive K influx µmol/(loc.h)

12. EFFECT OF INCREASING PLASMA UREA. Johnson et al, Mayo Clin. Proc., 47, 21-29, 1972. 600 500 400 300 200 150 100 50 Blood urea (mg/100 mL) 350 340 330 320 310 280 Serum (mOsm/Kg) 400 200 0 Dialysate urea (mg/100 ml) 5 10 15 20 30 40 50 60 70 80 90 100 110 Days 25 20 15 10 5 0 Plasma creatinine (mg/100mL) 286 596 Lethargy + + 0 0 0 0 0 0 + + + 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Headache 3+ 3+ 0 0 0 1+ 1+ 2+ 0 1+ 0 2+ 0 0 2+ 1+ 1+ 2+ 1+ 1+ 1+ 1+ 1+1+ 0 3+ 1+ 1+ 0 0 Emesis 0 0 0 0 1+ 0 2+ 1+1+1+ 0 2+ 0 0 2+1+ 2+ 2+ 2+ 2+ 2+ 0 0 0 0 2+ 2+ 1+ 0 0 Bleeding 0 2+ 2+ 2+ 0 1+ 1+ 1+1+1+ 2+ 2+ 1 + 0 0 1+ 1+ 1+ 1+ 0 0 0 0 0 0 0 1+ 0 0 0 0 Cramps 0 0 0 0 0 0 0 0 0 1+ 0 0 0 0 0 0 0 1+ 1+ 1+ 0 1+ 0 0 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2+ 0 0 0

13. UREA: CORRELATION OF PERCENTAGE REMOVAL Significant correlation –pseudouridine: 0.62 –uric acid: 0.73 –peak 4: 0.54 –peak 5: 0.46 –creatinine: 0.66 –pOHhippurate: 0.47 –hippuric acid: 0.46 –potassium: 0.26 No correlation –hypoxanthine: NS –xanthine: NS –indoxyl sulfate: NS –phosphate: NS Vanholder et al, Clin Chem, 1992.

14. SMALL WATER SOLUBLE COMPOUNDS

15. PHOSPHATE AND MORTALITY RISK. Block et al, AJKD, 31, 607-617, 1998. 2.0 1.5 1.0 0.5 RR 1.1-4.5 4.4-5.5 5.6-6.5 6.6-7.8 7.9-16.9 reference * p=0.03 ** p<0.0001 1.0 1.02 1.18* 1.39** Serum phosphorus Quintile (mg/dL)

16. PROTEIN BOUND COMPOUNDS

17. ** P-CRESOL : PHAGOCYTE FUNCTION. TIME (min) 0102030405060 0 20 40 60 80 100 0 µg cresol/ml 10 µg cresol/ml 30 µg cresol/ml 50 µg cresol/ml (CPM) Counts (x10 3 ) Vanholder et al, KI, 47, 510-517, 1995. CHEMILUMINESCENCE FACSCAN : PHAGOTEST/BURSTTEST Cell number 10 1 10 2 10 3 10 4 0 µg/mL PC 30µg/mL PC 0 µg/mL PC 30µg/mL PC Burst Phago Log fluorescence units 0 20 40 60 80 100 C pCSpCpCS+pC CO 2 production (DPMx10³) P-CRESOL vs. P-CRESYLSULPHATE PCS : p-cresylsulphate pC : p-cresol

18. P-CRESOL decrease oxygen uptake rat cerebral cortex slices (Lascelles 1968) increase free warfarin and diazepam (MacNamara 1981) change cell membrane permeability (Keweloh 1991) growth retardation in the weanling pig (Yokoyama 1992) LDH-leakage from liver cell slices (Thompson 1994) susceptibility to epilepsy (Yehuda 1994) inhibition phagocytic destruction of invading germs (Vanholder 1995) blockage cell K + channels (Elliott 1997) increase cellular toxicity aluminum (Abreo 1997) inhibition PAF-synthesis (Wratten 1999)

19. HOSPITALIZATION RATE In vivo longitudinal study (n=44) < 1 week > 1 week < 1 month > 1 month mg/dL * *: p<0.05 vs hospitalization < 1 week free p-cresol

20. MIDDLE MOLECULES

21.  2-M AMYLOID DEPOSITION. van Ypersele, KI, 39, 1012-1019, 1991.

22. WESTERN BLOTTING EXPERIMENTS. Niwa et al, KI, 50, 1303-1309, 1996 Lanes 2 :  2M-dimer

23. SERUM ß2-MICROGLOBULIN AND TREATMENT MODALITY Locatelli et al. Kidney Int 50: 1293-1302, 1996

24. RELATIVE RISK OF CARPAL TUNNEL SYNDROME Conventional High FluxAge Age + 1 Membrane Koda et al. Kidney Int 52: 1096-1101, 1997

25. SERUM LEPTIN vs. EVOLUTION LEAN BODY MASS. Stenvinkel et al, JASN, 11, 1303-1309, 2000 60 50 40 30 20 10 0 Serum leptin (ng/mL) Gained LBM Lost LBM Initial examinationFollow-up ***

26. EFFECT OF AGE AND AOPP ON LEUKOCYTE RESPONSE Witko-Sarsat J Immunol 161 : 2524, 1998 Stimulatory effect : increased oxidative stress HBBS Opsonized Zymosan PMA Control HSA AGE-HSA Control HSA AOPP -HSA Glycosylation products Oxidation products * ** 0 200 400 Lucigenin CL (counts/20min/monocyte)

27. GRANULOCYTE INHIBITORY PROTEINS ISOLATED FROM HEMO- AND/OR PERITONEAL DIALYSIS PATIENTS. Granulocyte inhibitory protein I: Immunoglobulin light chains : Granulocyte inhibitory protein II : Degranulation inhibiting protein I : Degranulation inhibiting protein II : Chemotaxis inhibiting protein: MW 28kDa, 80% homology to  and 40% homology to light chains MW 25kDa for monomers, MW 50kDa for dimers MW 9.5kDa, homology for  2- microglobulin MW 14.4kDa, identical to angiotensin MW 24kDa, identical to complement factor D MW 8.5kDa, homology to ubiquitin Cohen et al, KI, 52, S62, S79-S82, 1997.

28. MM WITH BIOLOGICAL POTENTIAL Adrenomedullin AGE Angiogenin AOPP Atrial natriuretic peptide Cholecystokin Clara cell protein Complement factor D Cystatin C Cytokines Delta sleep inducing protein Endothelin  -Endorphin Glomerulopressin GIP I GIP II Leptin  -Lipotropin Methionine-enkephalin ß 2 -Microglobulin Neuropeptide Y Retinol binding protein

29. CONCLUSIONS (1) Small water soluble compounds do not exert much toxicity. The most toxic ones show a kinetic behavior that is different from that of urea. Several protein bound compounds exert toxic effects. Their removal pattern is different from that of urea. Adding flux has no effect on removal of most of these molecules. Alternative removal concepts will have to be developed.

30. CONCLUSIONS (2) Several middle molecules exert toxic effects, especially in the area of inflammation/atherogenesis. Increasing membrane pore size and flux has improved their removal, but concentrations remain far above normal. More removal will have to be persued by changing concepts, although multicompartmental behavior might limit adequacy of removal.

31. STRATEGIC MODIFICATIONS Extracorporeal adsorption Intestinal adsorption Changes in dietary habits Prokinetics, probiotics Modification of metabolism, drug therapy Neutralisation of biochemical impact (e.g. scavengers) Modification protein binding in the devices Regenerative medicine –Artificial liver –Artificial tubule