philippe rieu service de néphrologie, chu reims umr cnrs...
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Les toxines urémiques
Philippe RieuService de Néphrologie, CHU Reims
UMR CNRS 7369
Uremic toxicity: Definition
The uremic syndrome
a clinical condition developing during the progress ion of renal failure
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Uremic toxicity: Definition
The uremic syndrome
a clinical condition developing during the progress ion of renal failure
Encéphalopathiepéricardite
neuropathieostéodystrophie
athérome accélérécardiopathie
thrombopathieimmuno-dépression
etc....
The uremic syndrome
a clinical condition developing during the progress ion of renal failure
Uremic toxins
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Uremic toxin: Definition
The high concentration of the compound should be related to a specific uremic dysfonction and/or symptom that decrease or disappear when the concentration is reduced
The total body and plasma level of the compound should be higher in uremic than in non uremic subjects
1.
2.
Uremic toxins: Classification
1. Inorganic molecules: H 2O, ions
2. Free water-soluble low-molecular weight solutes (< 500 D)
3. Middle molecules (500 – 60000 D)
4. Protein-bound solutes
4
JASN p1258-70, 2012
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Free water-soluble low-molecular weight uremic solutes (N=45)
Vanholder et al.KI, 2003, 63:1934EUTox (European Uremic Toxin Work Group )
Free water-soluble low-molecular weight uremic solutes (N=45)
Vanholder et al.KI, 2003, 63:1934EUTox
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Uremic toxin: Definition
The high concentration of the compound should be related to a specific uremic dysfonction and/or symptom that decrease or disappear when the concentration is reduced
The total body and plasma level of the compound should be higher in uremic than in non uremic subjects
1.
2.
Uremic toxins: Definition
Only a few solutes conform more or less with this strict definition:– H2O?– Phosphate?– Potassium?– ß2-microglobulin?– ……?
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Uremic toxins: Classification
1. Inorganic molecules: H 2O, ions
2. Free water-soluble low-molecular weight solutes (< 500 D)
3. Middle molecules (500 – 60000 D)
4. Protein-bound solutes
H2O
Na+
K+
PO42-
Intracellular hyperhydration
Extracellular hyperhydration
Cardiac arrhythmias
HyperparathyroidismVascular calcifications
Inorganic molecules
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H2O
Na+
K+
PO42-
Intracellular hyperhydration
Extracellular hyperhydration
Cardiac arrhythmias
HyperparathyroidismVascular calcifications
Inorganic molecules
Conventional Hemodialysis
Phosphate MassRemoval Rate
mmol/h
Time (hours)
2
4
6
8
10
1 2 3 4 5
2
1
1,5
2,5
0,5
Phosphoremiammol/L
Phosphate removal during HD
Man et al. ASAIO 1991, 37:4639 15 20 24 mmol
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Phosphate MassRemoval Rate
mmol/h
Time (hours)
2
4
6
8
10
1 2 3 4 5
2
1
1,5
2,5
0,5
Phosphoremiammol/L
Inaccesibility of phosphate during HD
Man et al. ASAIO 1991, 37:4639 15 20 24 mmol
SerumP
93 mg
Intracellular P
63 g
2.5
3.5
4.5
5.5
6.5
7.5
PreDialyse
T0 T30 T60 T90 T120
time
Phosphatemg/dL
30
45
60
75
90
105
15
BUN
mg/dL
PreDialyse
T0 T30 T60 T90 T120
time
Postdialytic Rebound of Serum Phosphorus
Minutolo et al. 2002 JASN 13: 1046
10
Phosphate MassRemoval Rate
mmol/h
Time (hours)
2
4
6
8
10
1 2 3 4 5
2
1
1,5
2,5
0,5
Phosphoremiammol/L
Phosphate removal during HD
Man et al. ASAIO 1991, 37:463
9 15 20 24 mmol
Phosphate MassRemoval Rate
mmol/h
Time (hours)
2
4
6
8
10
1 2 3 4 5
2
1
1,5
2,5
0,5
Phosphoremiammol/L
Short daily dialysis
Man et al. ASAIO 1991, 37:463
9 15 20 24 mmol2 hours daily dialysis Conventional hemodialysis
15 x 6 = 90 mmol/week 24 x 3 = 72 mmol/week
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Uremic toxins: Classification
1. Inorganic molecules: H 2O, ions
2. Free water-soluble low-molecular weight solutes (< 500 D)
3. Middle molecules (500 – 60000 D)
4. Protein-bound solutes
Free water-soluble low-molecular weight uremic solutes (N=45)
Vanholder et al.KI, 2003, 63:1934EUTox work group
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Guanidines NeurotoxicityInhibition NO-synthesis (?)
Oxalate Tissue deposition
Purines Resistance to vitamin D
Urea ?
Free water-soluble low-molecular weight uremic solutes
Free water-soluble low-molecular weight uremic solutes (N=45)
Vanholder et al.KI, 2003, 63:1934EUTox work group
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Johnson et al, Mayo Clin. Proc.,47, 21-29, 1972.
Blo
od u
rea
(mg/
100
mL)
350
340
330
320
310Ser
um (
mO
sm/K
g)
400200
0
Dia
lysa
te u
rea
(mg/
100
ml)
5 10 15 20 30 40 50 60 70 80 90 100 Days
Plasm
a creatinine(m
g/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 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 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 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
2520151050
600
500
400
300
200
100
Plasma Urea
Effect ofIncreasing
plasma urea
Carbamylation
C NH2
O
NH2 NH4+ NCO-+
Urea Cyanate
R-NH2 C O
NH+ R-N
H
C NH2
O
Cyanic acide Carbamylated proteinProtein
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Carbamylation
• Blood proteins are carbamylated in vivo in kidney failure. • Carbamylation alters enzyme and hormone activity in vitro• Role of carbamylation in uremic toxicity ?
P Gillery Nephrol Ther 2015
C NH2
O
NH2 NH4+ NCO-+
Urea Cyanate
R-NH2 C O
NH+ R-N
H
C NH2
O
Cyanic acide Carbamylated proteinProtein
Free water-soluble low-molecular weight uremic solutes (N=45)
Vanholder et al.KI, 2003, 63:1934
Conventional Hemodialysis
EUTox work group
15
HEMO-Study
Kt/Vsp = 1.32 ± 0.09(1.16±0.08)
Kt/Vsp = 1.71 ± 0.11(1.53±0.09)
Free water-soluble low-molecular weight uremic solutes
Eknoyan G et al, N eng J Med (2002); 347: 2010
Uremic toxins: Classification
1. Inorganic molecules: H 2O, ions
2. Free water-soluble low-molecular weight solutes (< 500 D)
3. Middle molecules (500 – 60000 D)
4. Protein-bound solutes
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Middle molecules (500 – 60000 D)
Vanholder et al.KI, 2003, 63:1934EUTox work group
PTH
IL-1IL-6
TNF-ααααFacteur D
Leptin
ββββ2-microglobuline
Hyperparathyroïdie
Anorexie ?
Amylose
Inflammation ?
Middle molecules (500 – 60000 D)
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Middle molecules (500 – 60000 D)
Vanholder et al.KI, 2003, 63:1934EUTox work group
ββββ2-microglobuline
• Chaîne légère HLA classe I• 99 AA, 11 800 D• Exprimée par toute les cellules
nuclées de l ’organisme• Taux sérique = 1,5-3mg/L, • Taux x 10-60 en cas d ’IRC
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Amylose ββββ2-microglobuline
• Dépôts amyloïdes dans les tissus articulaires et pé riarticulaires :
– Syndrome du canal carpien +++– Ténosynovite des fléchisseurs de la main– Arthralgies chroniques (épaules, autres articulatio ns)– Arthropathies destructrices, hémarthroses, fracture s– Spondyloarthropathies destructrices (cervicale) ave c
compression radiculaire ou médullaire.• Dépôts extra-osseux
– Cœur, tube digestif, vaisseaux, poumons... – Le plus souvent asymptomatique
Drüeke, KI, 1999, 56:S89Floege, KI, 2001, 59:164
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van Ypersele, KI, 39, 1012-1019, 1991.
Floege, KI, 2001, 59:164
Carpal tunnel syndromeand ββββ2m amyloidosis
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ββββ bulge
HLA ββββ 2m
M ββββ 2m
Trinh et al, PNAS, 2002, 99:9775
HLA ββββ 2m
M ββββ 2m
Trinh et al, PNAS, 2002, 99:9775
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Middle molecules (500 – 60000 D)
Vanholder et al.KI, 2003, 63:1934
HF- Hemodialysis
EUTox work group
Middle molecules removal in HF-HDversus LF-HD
Maduell F et al, Am J Kidney Dis 2002; 40: 582-589
81
69
40
-4
82
70
5461
25
0
20
40
60
80
100
Urea60d
Creat113d
Osteoc5.8kd
ββββ2M11.8kd
Myogl16kd
LF-HD
HF-HD
Per
cent
age
of r
educ
tion
22
Actuarial occurrence of carpal tunnel syndrome
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mantel-Cox, p=0.0118
100
80
60
40
20
J. Chanard BMJ 1989, 298: 867-868
Years of haemodialysis
%tage of patients without
carpal tunel syndromeLF-HD (Cuprophane)
HF-HD AN69
1,0
1,61,6
1,4
1,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
<27.5 27.5-35 35-42.5 42.5-50 >50
Relative Risk of Death
Cheung AK et al, HEMO-Study, JASN (ePub Dec2005)
[ß2M], mg/l
ββββ2 microglobuline and risk of death
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MPO-Study Locatelli et al, JASN (2009); 20: 645
Middle molecules (500 – 60000 D)
LF-HD HF-HD
Middle molecules (500 – 60000 D)
Vanholder et al.KI, 2003, 63:1934
ConvectiveHD
EUTox work group
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Middle molecules removal in HDFversus LF-HD and HF-HD
Maduell F et al, Am J Kidney Dis 2002; 40: 582-589
HDF post 26.8l/s81
69
40
-4
82
70
5461
25
83
72
64
75
63
0
20
40
60
80
100
Urea60d
Creat113d
Osteoc5.8kd
ββββ2M11.8kd
Myogl16kd
LF-HD
HF-HD
HF-HDFPer
cent
age
of r
educ
tion
p<0,01 p<0,01 p<0,01
ß2-MICROGLOBULIN REMOVAL
Lornoy et al, NDT, 15 (Suppl 2), 49-54, 2000
HDF (mL/min) post dilution
0 20 40 60 80 100 120
60
80
100
120
140
160
180 PS 1,8M2, F80QB= 300 ml/minQD = 600ml/min
Clairanceml/min
25
High-efficiency HDF reduces all-cause mortality compared vith conventional HD
Maduell, JASN, 02 2013
HDF 82%
HD 73%
30% risk reductionLog-rank p-value : 0.01
Uremic toxins: Classification
1. Inorganic molecules: H 2O, ions
2. Free water-soluble low-molecular weight solutes (< 500 D)
3. Middle molecules (500 – 60000 D)
4. Protein-bound solutes
26
Protein-bound solutes (N=25)
Vanholder et al.KI, 2003, 63:1934EUTox work group
Protein-bound solutes
Leptin
Homocystein
p-cresol
AGE AOPPCarbamylated protein
16 000 D
108 D
150 AA
1 AA 135 D
1 AA to > 1000 AA
100 D to > 100 kD
27
Protein-bound solutes
Leptin
Homocystein
p-cresol
AGE AOPPCarbamylated protein
Covalent binding
Non covalent binding
Protein-bound solutes
Leptin
Homocystein
p-cresol
Indole
16 000 D
108 D
10-50%
75 % 135 D
94 %
Protein boundfraction
MW
Kraus, KI, 2001, 59: S102Kraus, KI, 2001, 59: S102
117 D70-75 %
28
Protein-bound solutes
Leptin
Homocystein
p-cresol
Indole
16 000 D
108 D
10-50%
75 % 135 D
94 %
Protein boundfraction
MW
Kraus, KI, 2001, 59: S102Kraus, KI, 2001, 59: S102
117 D70-75 %
Tyrosine Tryptophan
Indole
Indoxyl sulfate
P-cresol P-cresol sulfate P-cresol P-cresol sulfate
Uremic solutes from colon microbes
29
Organic anion transporters
OAT
Miyamoto Y, NDT 2011 26: 2498Enomoto JASN 2002 13:1711
Aryl Hydrocarbon Receptor
Indoxyl sulfate
Sallée M. Toxins 2014 6:934
récepteur aux hydrocarbures aromatiques
30
The AhR/p38MAPK/NF- κB pathway is involved in COX-2 protein induction.
Laetitia Dou et al. JASN 2015;26:876-887
Indole-3 acetic acid
Cumulative survival (A) and major cardiovascular events (B) to serum IAA
Laetitia Dou et al. JASN 2015;26:876- 887
Indole-3 acetic acid (IAA)
31
In Vitro: p-cresylsulfate
Increases leukocyte free radical production(Schepers NDT 2007)
synergistic effect of pCS and pCG(Meert NDT 2011)
Induction of endothelial microparticle release(Meijers AJKD 2008)
Induction of insulin resistance (Koppe JASN 2013)
Renal tubular damage by inducing oxidative stress(Watanabe KI 2013)
Liabeuf et al, NDT, 25: 1183-1191; 2010
Free p-cresylsulfate predictor of overall mortality and CV death in CKD patients
32
Protein-bound solutes (N=25)
Vanholder et al.KI, 2003, 63:1934EUTox work group
uf
Adsorption
Dialysate in
Dialysate out
Hemodiafiltration with reinfusion
(HFR)
ultrafiltrate
Convection(through protein
leaking membrane)
Diffusion
Adsorption
Dialysate in
Dialysate out
Hemodiafiltration with endogenous reinfusion
Alb
Alb
Alb
resin
33
uf
Convection
Diffusion
Adsorption
Dialysate in
Dialysate out
Hemodiafiltration with endogenous reinfusion
11.6 µM
2.5
[Hcy] t10
Splendiani, Artif Org2004 28: 592
Protein-bound solutes
Leptin
Homocystein
Phenol et indoles (p-cresol)
AGE AOPPCarbamylated protein
Covalent binding
Non covalent binding
34
Early Glycosylation Products
CC
(CHOH)(CHOH)33
CHCH 22OHOH
OO
HH NHNH 22 NHNH++
CCHH
NHNH
CHCH 22
(CHOH)(CHOH)33
CHCH 22OHOH
CC OO
Amadori ProductAmadori ProductSchiff BaseSchiff BaseProteinProteinGlucoseGlucose
++
CHOHCHOH
(CHOH)(CHOH)33
CHCH 22OHOH
CHOHCHOH
LysLys
Amadori ProductAmadori Product
Advanced Glycation EndAdvanced Glycation End--ProductsProductsAGEsAGEs
O2OXIDATION Dehydratationcondensationfragmentationcyclisation ...
Schiff BaseSchiff Base
GlucoseGlucose ++ NHNH--ProteinProtein
35
Amadori ProductAmadori Product
O2
Dehydratationcondensationfragmentationcyclisation ...
Schiff BaseSchiff Base
NHNH--ProteinProteinGlucoseGlucose ++O2
Arabinose Glyoxal
O2
NHNH--ProteinProtein
CarbonylStress
Advanced Glycation EndAdvanced Glycation End--ProductsProductsAGEsAGEs
CarboxymethyllysinePyrralinePentosidineGlyoxal-lysine-dimerMethyl-glyoxal-lysine-dimer
LysylpyrropyrineVesperlysineDehydrofuroimidazole
CrosslineImidazoloneGlucosepanCrosspy
CrosslineImidazoloneGlucosepanCrosspy
36
NHNH
AGEsAGEs
NHNH
NHNH
AGEsAGEs
Protein cross linkingProtein cross linking
NHNH
AGEsAGEs
RAGE
AGE receptorsAGE receptors
Effects of AGEs on End-Organ Damage in ESRD patients
Mallipattu S et al. Seminars in Dialysis 2012;25:5 29
- Cardiovascular morbidity
- Dysregulation of the immune system
- Dialysis-related amyloidosis
- Progression of chronic renal failure
- Uremic Neuropathy
- Peritoneal damage
37
NHNH
AGEsAGEs
NHNH
NHNH
AGEsAGEs
Protein cross linkingProtein cross linking
NHNH
AGEsAGEs
RAGE
AGE receptorsAGE receptors
Normal
Mesure des Calcifications in vitro par Rouge Alazarin
Transformation ostéoblatique Co-transporteurs du phosphate
In Vitro
RAGE KO
WT
7 j 14 j 21 j 28 jPi ou Ligand de RAGE
0
P- AKT
Runx2
Pit -1
ß Actine
WT RAGE KO
+ Stim + Stim 0
Belmokhtar K et al, NDT 2019
Effects of AGEs/RAGE on vascular calcifications
38
Témoin IRC0
1
2
3
4
5
ApoE -/-
Exp
ress
ion
rela
tive
**
CML Aortique qPCR RAGE
Témoin IRC0.0
0.5
1.0
1.5
ApoE -/-
CML/lysine (
µM/M
)
*
ApoE-/- ApoE-/-RAGE-/-
In Vitro
Effects of AGEs/RAGE on vascular calcifications
Belmokhtar K et al, NDT 2019
NHNH
AGEsAGEs
NHNH
NHNH
AGEsAGEs
Protein cross linkingProtein cross linking
NHNH
AGEsAGEs
RAGE
AGE receptorsAGE receptors
39
Mediators Gradient
Blood FluxPost-capillary veinules
Activated endothelial cells
Red blood cells
Collagen
Mediators Gradient
Blood FluxPost-capillary veinules
Activated endothelial cells
Red blood cells
Effect of AGE-Collagen on
neutrophils migration ?
AGE-Collagen
40
Non stimulated FMLP -8
Native Collagen
AGE-Collagen
Migration Rate ( µm/min )
*
0
5
10
15 *
0
5
10
15
Non stimulated Stimulated FMLP-8
Touré et al. ASN 2003
Migration
En conclusion…
41
Toxine urémique
Défaut d’éliminationrénale de la toxine
Modification du métabolisme
au cours de l’urémie
b2-microglobuline HomocystéineAGE
Toxine urémique A Symptôme urémique A
42
Toxine urémique A Symptôme urémique A
Toxine urémique I
Toxine urémique J
Toxine urémique K
Toxine urémique L
Toxine urémique M
Toxine urémique …
Toxine urémique B
Toxine urémique C
Toxine urémique D
Toxine urémique E
Toxine urémique F
Toxine urémique G
Toxine urémique A Symptôme urémique A
Symptôme urémique B
Symptôme urémique C
Symptôme urémique E
Symptôme urémique F
Symptôme urémique G
Symptôme urémique …
Symptôme urémique H
Symptôme urémique I
Symptôme urémique J
Symptôme urémique K
Symptôme urémique L
Symptôme urémique L
43
Symptôme urémique A
Symptôme urémique B
Symptôme urémique C
Symptôme urémique E
Symptôme urémique F
Symptôme urémique G
Symptôme urémique …
Symptôme urémique H
Symptôme urémique I
Symptôme urémique J
Symptôme urémique K
Symptôme urémique L
Symptôme urémique L
Toxine urémique A
Toxine urémique I
Toxine urémique J
Toxine urémique K
Toxine urémique L
Toxine urémique M
Toxine urémique …
Toxine urémique B
Toxine urémique C
Toxine urémique D
Toxine urémique E
Toxine urémique F
Toxine urémique G