Aquaporins in Peritoneal Dialysis - HÔPITAL NECKER · Aquaporins in Peritoneal Dialysis. Zurich,...

O. Devuyst, MD, PhD

Necker Seminars in Nephrology

Paris, April 20th, 2011

Aquaporins in Peritoneal Dialysis

Zurich, 1890

Overton CE (1899)

On the general osmotic properties of the cell, their probable origin,

and their significance for physiology. Vierteljahrsschr Naturforsch Ges Zurich 44: 88–135

Charles Ernest Overton (1865–1933)In 1890, Overton wurde er «Privatdocent der Biologie» an der Universität Zürich,

wo er über anderthalb Jahrzehnte lang wirkte.

The permeation coefficient (diffusion, P) of a molecule

across a membrane:

is directly function of its lipophilicity (K, partition coefficient between the oily and aqueous

phases), with D is the diffusion coefficient of the molecule in the membrane

and l, the membrane thickness.

P =K D

l

The higher is the lipophilicity, the faster is the cell permeation rate

The Overton Rule

• The boundary of cells is mostly constituted of a lipidic material

• Water and ions (non-lipophilic substances) must use different pathways,

as the lipids prevent solvation of charged particles

Science 175: 720-731, 1972

Other Suggestions from Overton

Vehicle

• Hypo-osmolar swelling

• HgCl2 inhibited, no currents

AQP1

Dendrogram of Human Aquaporins

Aquaporins

Aquaglyceroporins

AQP4

AQP12

AQP9

AQP10

AQP3

AQP7

AQP11

AQP0

(MIP)

AQP8

AQP2

AQP6

AQP5

AQP1

Wang et al. Structure 13, 2005

General Structure of Aquaporins

Törnroth-Horsefield S et al. FEBS Lett 584, 2010

Two essential motifs:

• NPA: asparagine-proline-alanine

• ar/R: aromatic/arginine

General Structure of Aquaporins

Water transport – relevance for dialysis ?

• Mode of renal replacement therapy (>1980’s)

• >300,000 patients worldwide (40% UK, 90% Hong-Kong)

• Advantages : Simplicity, home dialysis, lower cost

• Positive choice for autonomous patients (waiting pre-TP)

• Preferred in developing countries

• Continuous dialysis through natural membrane

Peritoneal Dialysis

Comparison of HD and PD patient numbers in the 15 countries with the

highest dialysis patient populations.

Grassmann A et al. Nephrol. Dial. Transplant.

2005;20:2587-2593

Structure of the Peritoneal Membrane

mm

• Mesothelium

• Interstitium - MPS hydrogel + collagen

• Capillary network - endothelial barrier

Dialysate

Fact. VIII

m

Water and toxins

Glucose

n

n

n

n

n

n n n n n n n n n n

800

600

400

200

0

- 200

30 60 120 180 240 300 360 420 480

Time (min)

ΔV

olu

me (

ml)

from Krediet Kluwer 2000

3.86 % glucose (486 mOsm)

+12

+93

Net Crystalloid Osmotic Pressure

(mmHg)

1.36 % glucose (347 mOsm)

Water Removal : Glucose as an Osmotic Agent

The Endothelium as a Functional Barrier in PD

Ultrasmall pores : predict to account for 50% of water removal (UF)

Ultrasmall Pores across the Endothelium

Osmotic

Gradient

Hypothesis : Water Channels

Simple Diffusion

Facilitated Diffusion

Distribution of Aquaporin-1 in the

Endothelium Lining Peritoneal Capillaries

AQP1 -

GlyAQP1 -

-28

-35

-50

kD

m

lumen

rbc

Am J Physiol 275, 1998 ; Kidney Int 67, 2005

Hu. Perit.

Carotid artery Jugular vein

PUMP

Saline

BP TRANSDUCER

Peritoneal cavityPD catheter

Ni et al. Kidney Int 67; 2005

2-hour PET

2.5 ml dialysate

Sampling T0, T30, T60, T120

Synchron CX5 Beckman

Peritoneal Dialysis in Mouse

Structure of the Mouse Peritoneal Membrane


A

B

C

D

E

*

*

AQP1

eNOS

Water and Solute Transport in Mouse

0 30 60 90 1200.0

0.5

1.0

1.36% (n=6)

3.86% (n=6)

7% (n=6)

Dwell Time (min)

D/D

0 G

luco

se

*

*

*

Net U

F/B

W (

µl/g)

1.36%

0

20

40

60

3.86% 7%

*

*§

*

*

*

0 30 60 90 1200.0

0.5

1.0

Dwell Time (min)

D/P

Ure

a

0 30 60 90 1200.7

0.8

0.9

1.0

Dwell Time (min)

D/P

So

diu

m

*§

*§

** *

*


Species Dialysate volume

(ml)

Net UF

( l/g BW)

BW

(g)

BSA

(cm2)

Mouse 2.5 ml 29 25 70

Rat 15 ml 25 300 425

Man 2,000 ml 30 65,000 17,300

• Similar ultrafiltration across species

• Small solute transport scaled to BSA


Peritoneal Transport : Mouse vs. Rat vs. Man

Phenotype of the AQP1 KO Mouse

• Impaired urine concentrating ability : NDI

• Reduced water transport across descending vasa recta

• Reduced water permeability across lung capillaries

• Reduced corneal water permeability

• Reduced cerebrospinal fluid production

Transport across the peritoneal membrane ?

A.S. Verkman et al.

Aqp1+/+

Aqp1-/-

Positive

controls

Threshold (Ct) mRNA

Aqp1+/+ Aqp1-/-

GAPDH 17.2 0.1 17.2 0.2

AQP0 ND ND

AQP1 21.8 0.2 ND

AQP2 ND ND

AQP3 30.8 0.8 ND

AQP4 ND ND

AQP5 27.9 0.4 26.2 0.4

AQP6 ND ND

AQP7 22.9 0.2 23.4 0.2

AQP8 ND ND

AQP9 27.8 0.3 29.6 0.4

AQP10 ND 30.9 0.3

AQP Gene Family in the Mouse Peritoneum

• AQP1 : endothelium

• AQP7 (adipocytes), AQP9 (leukocytes), AQP3/5 (dendritic cells)

Real-time RT-PCR

Vascular Density in Aqp1 Mice

Aqp1 (+/+) Aqp1 (-/-)Vascular density (N/mm2)

0

20

40

60

80

100

120

AQP1 (+/+) (n=4)

AQP1 (-/-) (n=4)

Endothelial area (%)

0

1

2

3

4

5

6

7

AQP1

CD31

Ni et al. Kidney Int 69, 2006

Slope of volume curves

(125I-albumin)

0

10

20

30

40

Aqp1 Mice

+/+ +/- -/-

Init

ial

UF

rate

(µ

l/m

in)


Initial Water Flow in Aqp1 Mice

Intraperitoneal volume during PD


Ultrafiltration in Aqp1 KO Mice

Aqp1 (+/+) (n=6)

Aqp1 (-/-) (n=6)

0

10

20

30

40

50

60

70U

F/B

W(µ

l/g)

583.1

*#

271.8

PET: 2ml, 7% glucose dialysate, 2 hours

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6

Aqp1 (-/-)

Aqp1 (+/-)

Aqp1 (+/+)

D/P Osmolality at T30

Aqp1 (-/-) (n=6)

Aqp1 (+/+) (n=6)

Aqp1 (+/-) (n=6)

0 30 60 90 120

0.0

0.5

1.0

Dwell Time (min)

D/D

0 G

lucose

Unchanged Osmotic Gradient in Aqp1 KO Mice

• Studies in Aqp1 mice validate the 3-pore model :

AQP1 is the ultrasmall pore in endothelial cells

AQP1 mediates 50% of the water transport

AQP1 deletion does not affect solute transport

AQP1 in the Peritoneal Membrane

• Regulation of AQP1 : expression level and gating

0 6 12 18 24

Low UF

Low Average

High Average

High UF

40

100

90

80

70

60

50

Time in Months

Pe

rce

nt S

urv

ivin

g

Churchill et al. JASN 9: 1285-92, 1998

Survival According to PD Transport Characteristic

CANUSA

606 patients - initial PET

AQP1 in Capillary Endothelium : Lung

J Clin Invest 97, 1996

saline or betamethasone (0.35 mg/kg)

E15

P1

AQP1 Expression : Glucocorticoid Induction

Moon C. et al. AJP 273: C1562, 1997

4.4-kb mouse Aqp1

MEL cells

1 M Dexa

Dexa mg/Kg RU-486

0.04 1 4 Dexa +0

1

2

3

Sham

AQ

P1/

-acti

n r

ela

tiv

e

to s

ham

(fo

ld in

cre

ase

)

Expression of GR and Steroid Induction of AQP1

MW Control PM

- 534

bp

Anti-GR Pre-ad

GR-

- 150

- 100

- 75

- 50

kDVP PP VP PP

Stoenoiu et al. JASN 14: 555-65, 2003

lumen

lumen

Dexa 1mg/kg

Sham

E

E

Stoenoiu et al. JASN 14: 555-65, 2003

Glucocorticoids upregulate AQP1

in peritoneal capillaries

Sham

Dexa 1mg/kg

Water transport

(ml/kg)

*

38

52

10

20

30

40

50

60

± 3

± 1

Parameter Before → After

Sodium sieving (D/P sodium, T30-T0) -8 → -16

Ultrafiltration USP (mL) 108 → 190

D/P creatinine 0.53 → 0.53

Steroid Induction of AQP1: PET after Living Donor Transplantation

Coll. Javier Arteaga, Cordoba

• N=3, living related kidney transplantation (F11y, M52y, M5y)

• Methylprednisolone, 1mg/m2

• MiniPET, before and 22 days after transplant

Gating aquaporin-1 to improve UF ?

Aquaporins in Plants: Arabidopsis

AQP Kdown AQP WT

Plants counteract fluctuations in water supply by regulating their aquaporins:

→ Drought stress: dephosphorylation and closure

→ Flooding: anoxia – cytosolic acidification: protonation and closure

Yu et al. Structure 14, 2006

• Loop D contains a polyarginine motif

• cGMP bound to arginines

•E ffect on central pore or monomer

Gating of AQP1: Cytosolic Loop D Interacts with cGMP

Migliati et al. Mol Pharmacol 76, 2009

• Inhibition of AQP1 and AQP4

• Swelling assay, oocytes

• IC50 20mM, extracellular

Gating of AQP1: Interaction with B013 (Bumetanide)

• Chemical library screen: aryl sulfonamides

• Related to loop diuretic bumetanide

• Membrane permeable compounds → Ongoing tests in mouse

Devuyst & Yool, PDI 30, 2010

Gating of AQP1: Possibility for Agonists ?

• Studies in Aqp1 mice validate the 3-pore model :

AQP1 is the ultrasmall pore in endothelial cells

AQP1 mediates 50% of the water transport

AQP1 deletion does not affect solute transport

AQP1 in the Peritoneal Membrane

• Regulation of AQP1 : expression level and gating

• Genetic variation in AQP1 : individual variability

Distribution of Water Transport in PD Patients

Amsterdam data – SPA test

60 min – hypertonic dwell

Incident PD patients, n=211

Clinical variables account for only ~ 20% of the variability

Histogram of Data 1:Freq. dist. (histogram)

-31.5

9-1

1.5

98.4

128.4

148.4

168.4

188.4

1108.4

1128.4

1148.4

1168.4

1188.4

1208.4

1228.4

1248.4

1268.4

1288.4

1308.4

1328.4

1348.4

1

0

5

10

15

20

25

30

35

FWT at 60 min the dwell (mL)

Nu

mb

ers

Pati

en

ts

• Multicentric association study

•UCL – KUL – CHC Liège: N=215

• NECOSAD – AMC Amsterdam: N=500

• Initial water flow, small solute transport

• Survival : technique and patient

Influence of variants in AQP1 on Water transport and PD Outcome

rs283

62687

rs147

6597

rs207

5574

rs102

53374

rs104

9305

rs283

62687

rs147

6597

rs207

5574

rs102

53374

rs104

9305

AQP1 Haplotype

Exon1 Exon2 Exon3 Exon4

Promoter Intron 1Intron 2 Intron 3 3’UTR

Exon1 Exon2 Exon3 Exon4

Promoter Intron 1Intron 2 Intron 3 3’UTR

Blood 2003; 101: 1596-1602

CTCTG, core binding motif of a regulatory element for PKLR

0% 50% 100% 150%

CTCTT

Wt

Basic

Relative luciferase activity (percent)

Mutations in AQP1 promoter Down-regulate Promoter Activity

0% 50% 100% 150%

CTCTT

Wt

BasicpGL3-Basic

pGL3-Wt: CTGTC

pGL3-M3-CTCTT

Human Embryonic Kidney 293 cells (Hek293) (n=8)

Luciferase reporter constructs for human AQP1 promoter.

*, p < 0.001 vs. pGL3-Basic

#, p<0.001 vs. pGL3-Wt: CTGTC

6 ± 1

100 ± 5

45 ± 3

#

#

*

*


pGL3-Basic

pGL3-Wt: CTGTC

pGL3-M3-CTCTT


Human erythromyeloblastoid leukemia cell line (K562) (n=8)

Luciferase reporter constructs for human AQP1 promoter.

*, p < 0.001 vs. pGL3-Basic

#, p<0.001 vs. pGL3-Wt: CTGTC

4 ± 0

100 ± 11

39 ± 1 # *

*

#

Maréchal C et al. ASN 2010

Patient – rs2075574 CC

Patient – rs2075574 TT

Time (s)

Scat

tere

d li

ght

inte

nsi

ty

(vo

lt)

Measurements of water transport in human erythrocytes by stopped-flow light scattering

Coll. P. Ripoche, Paris

TT = 17 % TT = 4 %

P25: 431 ml P75: 842 ml

Ultrafiltration volume (ml)


rs2075574 CCN = 94

CTN =94

TTN = 23

P-value

Transport parameters

UF Mean (Std) 588 (338) 673 (309) 483 (266) 0.02

Seaving Na Mean (Std) (n = 155) 0.05 (0.03) 0.05 (0.02) 0.04 (0.03) 0.046

D/P creat 4 hours Mean (Std) 0.72 (0.12) 0.73 (0.11) 0.76 (0.12) NS

Promoter Variant of AQP1: Influence on Survival

UCL - KUL

0

0,5

1

1,5

2

2,5

HR for death HR for TF HR for death and TF

CC

CT

TTHR

*

*

R R R

(n=195)

(n=199)

(n=64)

NECOSAD -AMC

0

0,5

1

1,5

2

2,5

3

3,5

HR for death HR for TF HR for death and TF

CC

CT

TT

HR

*

* *

R R R

(n=85)

(n=82)

(n=20)


Conclusions

1. The peritoneal membrane is a useful model to investigate AQP1:

highly expressed in endothelium

baseline / inflammation

correlation with transport parameters

3. Pharmacoregulation of AQP1 : benefit for peritoneal dialysis

4. Individual variability in water transport : AQP1 genotype ?

2. Multiple defects related to AQP1 in endothelial cells:

defective water transport during peritoneal dialysis

defective vascular proliferation

defective leukocyte recruitment and macrophage activation

UCL Brussels - Nephrology

J. Ni

M. Stoenoiu S. Combet

Y. Cnops H. Debaix

E. Goffin O. Devuyst

UCL Brussels

FATH : J-L. Balligand, O. Feron

ANPS : P. Moulin, J-P. Cosyns

CEA Saclay, France

J-M. Verbavatz

FNRS, FRSM, UCL-FSR

CF Belgium - ARC

FP6 – EC

Cardiff, Wales

N. Topley

ULB Brussels, Belgium

C. Delporte

Acknowledgements

UCSF, San Francisco, USA

A. Verkman

Univ. of Lund, Lund, Sweden

A. Rippe, B. Rippe

Peter Agre

Soren Nielsen

Univ. of Nagasaki, Japan

T. Nishino, M. Miyazaki

Thank you for your attention

Wang et al. Structure 13, 2005

What Makes an Aquaporin a Glycerol Channel ?

Fasted state

Bloodstream

G3P

TG

Glycerol

ADIPOCYTEGlucose

HSL

AQP7

Glycerol

G3P Glucose

LIVER

AQP9

Bloodstream

G3P: glycerol-3-phosphate

TG: triglycerides

HSL: hormone-sensitive lipase

FFA: free fatty acid

IRE: negative insulin response

element

fatty acid binding protein

fatty acid translocase

fatty acid transporter protein

Modified from FEBS Letters 580 (2006), 4771-76

Insulin

Nucleus

FFAGlycerol

FFA

IRE

Glucose

During fasting, AQP7 and AQP9 expressions are elevated through the negative IRE of their promoter regions.

Triglycerides are hydrolysed to glycerol and FFA by HSL and released into the bloodstream. Glycerol is secreted

from adipocytes through AQP7. Plasma glycerol is introduced into hepatocytes through AQP9. In the liver, glycerol

is one of substrates for glucogenesis.

kD

AQP7

GlyAQP7

-25

-35

Expression of AQP7 in the Peritoneum

Visceral peritoneum

Testis

T. Nishino, unpublished data

Fasting Increases AQP7 and AQP1

Expression

-Actin-

AQP7-

AQP1-

Fed Fasted

-25

-25

kD

-37

Relative optical density

(% of Fed level)

Fed

Fasted

0

100

200

300

AQP1 AQP7

*

*

b

d

a

c

T. Nishino, unpublished data

Clinical Factors Influencing Baseline Transport

Churchill et al. JASN 1998

Davies SJ. Kidney Int 2004

Rumpsfeld et al. AJKD 2004

• AUSTRALIA – NEW-ZEALAND (Registry, N = 3188) :

MV : Age – cerebrovascular disease - BSA - ethnicity

The clinical variables account for only ~ 20% of the variability

of solute & water transport at the start of PD :

Room for variables that are not routinely measured …

Induction of iNOS and Release of NOx

iNOS

AQP1

-Actin

Control AQP1 WT-p AQP1 KO-pkD

-130

-25

-45

Immunoblotting: iNOS in catheter-model

Dialysate level of NOx

0

50

100

150

200

250

300

350

WT KO WT KO WT KO

Baseline Catheter LPS

(mM)

* #

* #

* # §

* # §

qPCR: iNOS in LPS-model

0

200

400

600

800

1000

1200

Baseline

WT WT KO

LPS

(%)

*

#

Defective Cell Margination in AQP1 KO

Aqp1 WT Aqp1 KONumber of marginating cells/vessel

[6-day catheter-model]

0

5

10

15

20

* #

§

WT KO WT KO

Baseline Peritonitis

Number of marginating cells/vessel

[18-h LPS-model]

0

10

20

30

* #

* # §

WT KO WT KO



[6-day catheter-model]

0

5

10

15

20

* #

§

WT KO WT KO



[18-h LPS-model]

0

10

20

30

* #

* # §

WT KO WT KO


J Biol Chem. 1998 Feb 20;273(8):4296-9.

Severely impaired urinary concentrating ability in transgenic mice

lacking aquaporin-1 water channels.

Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS.

Peritoneal Structure in Aqp1 Mice: Morphology

Aqp1 (+/+) Aqp1 (-/-)

PP

VP

The Nobel Prize in Chemistry 2003

Peter Agre Roderick MacKinnon

« for discoveries concerning channels in cell membranes »

Capillary Ultrastructure in Aqp1 Mice

Aqp1+/+

Aqp1-/-

rbc

rbc

! Descending vasa recta : vasodilation – higher blood flow

2.9 0.2 m (N=11)

2.9 0.4 m (N=9)

A

C D

B

L

L

(RBC)

N

N

L

EE

E

m

m

m

M

c

c

c

E

m

Visceral peritoneum Parietal peritoneum

AQP1 staining

Nature 434:786-792, 2005

Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene

disruption.

Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS.

« Our findings support a fundamental role of water channels in cell migration,

which is central to angiogenesis and wound healing. »

Acute Peritonitis : Structural Changes

Combet et al. JASN 10: 2185-96, 1999

Inflammatory infiltrate Vascular proliferation

Control

Peritonitis

Control

Peritonitis

Rat model

Catheter-induced peritonitis

5 days

Decreased Angiogenesis in AQP1 KO Mice

AQP1 KO-p (n=6)

(n=6)AQP1 WT

(n=6)AQP1 WT-p

(n=6)AQP1 WT

Number of vessels positive for CD31

0

50

100

150

200

250

(Nu

mb

er

of ve

sse

ls /m

m2)

* #

§

WT KO WT KO


*

**

#*

*

*

0 30 60 90 120

0.0

0.5

1.0

Dwell Time (min)D

/P U

rea

Small solute transport

Nishino et al. Unpublished data

Catheter model

6-day peritonitis

Impaired VCAM-1 Induction in AQP1 KO

Immunostaining for VCAM-1

Control AQP1 WT-p AQP1 KO-p

Immunoblotting: VCAM-1

Control AQP1 WT-p AQP1 KO-p

VCAM1

-Actin

kD

-45

-100

qPCR: VCAM-1

0

50

100

150

200

250

300

350

(%)

WT KO

LPS

WT KO

Baseline

* #

* # §

Nishino et al. Unpublished data

Heterozygous mutations in the c-kit proto-oncogene :

→ Pattern of hypopigmentation in (a) a patient with the piebald trait

(b) a mouse with dominant spotting

Comparative Genomics: Humans vs Rodents

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