philippe rieu service de néphrologie, chu reims umr cnrs...

1

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

2

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

3

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

5

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

6

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?– ……?

7

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

8

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

9

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

11

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

12

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

13

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

14

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

16

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)

17

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

18

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

19

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

Floege, KI, 2001, 59:164

Carpal tunnel syndromeand ββββ2m amyloidosis

20

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

21

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

23

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

24

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