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

DEFINITION AND NEW INSIGHTS IN UREMIC

TOXINS

R Vanholder, University Hospital, Gent, Belgium

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.

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

DEFINITION TOXIN (2)

• Only a few solutes conform more or less with this strict defintion:– H2O?

– Phosphate?– Potassium?

– ß2-microglobulin?

– ……?

UREMIC RETENTION SOLUTES

1) adrenomedullin; 2) advanced glycation end products (AGE); 3) advanced oxidation protein products (AOPP); 4) angiogenin; 5) atrial natriuretic peptide; 6) ß2-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

REVIEW ON UREMIC TOXINS: CLASSIFICATION, CONCENTRATION AND INTERINDIVIDUAL

VARIABILITY

EUToX: European Uremic Toxin Work Groupwithin the context of ESAO

Aim: to discuss and analyse matters related to theidentification, characterization, analytical determination and evaluation of biological activityof uremic retention solutes

Website: http://www.uremic-toxins.org

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

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

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

SMALL WATER SOLUBLE COMPOUNDS

0

100

200

300

400

500

UREA AND CELLULAR K-

INFLUX.

Lim et al, JCI, 96, 2126-2132, 1995.Control Urea 45mM

Bu

met

an

ide

sen

sit

ive

K in

flu

x µ

mo

l/(lo

c.h

)

EFFECT OF INCREASING

PLASMA UREA.

Johnson et al, Mayo Clin. Proc., 47, 21-29, 1972.

600

500

400

300

20015010050B

loo

d u

rea

(mg

/100

mL

)

350

340

330

320

310

280

Ser

um

(m

Osm

/Kg

)

400200

0

Dia

lys

ate

ure

a(m

g/1

00

ml)

5 10 15 20 30 40 50 60 70 80 90 100 110

Days

2520151050

Plasm

a crea

tinin

e (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 0Headache 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 0Bleeding 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+ 01+ 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

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.

SMALL WATER SOLUBLE COMPOUNDS

Guanidines Neurotoxicity Inhibition NO-synthesis (?)

Oxalate Tissue deposition

Phosphate Vascular damage Hyperparathyroidism

Purines Resistance to vitamin D

Urea Isolated biochemical functions

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 1.01.02

1.18*1.39**

Serum phosphorus Quintile (mg/dL)

PROTEIN BOUND COMPOUNDS

****

P-CRESOL : PHAGOCYTE FUNCTION.

TIME (min)

0 10 20 30 40 50 60

0

20

40

60

80

1000 µg cresol/ml

10 µg cresol/ml

30 µg cresol/ml

50 µg cresol/ml

(CPM)Counts (x103 )

Vanholder et al, KI, 47, 510-517, 1995.

CHEMILUMINESCENCEFACSCAN :

PHAGOTEST/BURSTTEST

Cel

l nu

mb

er

101

102103

1040 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 pCS pC pCS+pC

CO

2 p

rod

uct

ion

(D

PM

x10³

)

P-CRESOL vs. P-CRESYLSULPHATE

PCS : p-cresylsulphatepC : p-cresol

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)

HOSPITALIZATION RATEIn vivo longitudinal study (n=44)

< 1 week > 1 week< 1 month

> 1 month

mg/

dL *

*: p<0.05 vs hospitalization < 1 week

0.00

0.05

0.10

0.15

0.20

0.25 free p-cresol

MIDDLE MOLECULES

2-M AMYLOID DEPOSITION.

van Ypersele, KI, 39, 1012-1019, 1991.

WESTERN BLOTTING EXPERIMENTS.

Niwa et al, KI, 50, 1303-1309, 1996Lanes 2 : 2M-dimer

SERUM ß2-MICROGLOBULIN AND TREATMENT MODALITY

25

30

35

40

45

50

0 6 12 18 24

Time (months)

2-m

icro

glo

bu

lin

(m

g/d

l)

Cu-HD

LfPS-HD

HfPS-HD

HfPS-HDF

Locatelli et al. Kidney Int 50: 1293-1302, 1996

RELATIVE RISK OF CARPAL TUNNEL SYNDROME

1,00

0,50

1,001,06

0,00

0,20

0,40

0,60

0,80

1,00

1,20

Rel

ativ

e R

isk

Conventional High Flux Age Age + 1 Membrane

Koda et al. Kidney Int 52: 1096-1101, 1997

SERUM LEPTIN vs. EVOLUTION LEAN BODY MASS.

Stenvinkel et al, JASN, 11, 1303-1309, 2000

60

50

40

30

20

10

0

Se

rum

lep

tin

(n

g/m

L)

Gained LBMLost LBM

Initial examination Follow-up

***

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

Glycosylationproducts

Oxidationproducts

*

**

0 200 400

Lucigenin CL(counts/20min/monocyte)

GRANULOCYTE INHIBITORY PROTEINSISOLATED 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.

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

• ß2-Microglobulin

• Neuropeptide Y • Retinol binding protein

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.

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.

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