aquaporins in peritoneal dialysis - hÔpital necker · aquaporins in peritoneal dialysis. zurich,...
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O. Devuyst, MD, PhD
Necker Seminars in Nephrology
Paris, April 20th, 2011
Aquaporins in Peritoneal Dialysis
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Zurich, 1890
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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.
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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
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• 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
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Vehicle
• Hypo-osmolar swelling
• HgCl2 inhibited, no currents
AQP1
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Dendrogram of Human Aquaporins
Aquaporins
Aquaglyceroporins
AQP4
AQP12
AQP9
AQP10
AQP3
AQP7
AQP11
AQP0
(MIP)
AQP8
AQP2
AQP6
AQP5
AQP1
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Wang et al. Structure 13, 2005
General Structure of Aquaporins
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Törnroth-Horsefield S et al. FEBS Lett 584, 2010
Two essential motifs:
• NPA: asparagine-proline-alanine
• ar/R: aromatic/arginine
General Structure of Aquaporins
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Water transport – relevance for dialysis ?
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• 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
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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
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Structure of the Peritoneal Membrane
mm
• Mesothelium
• Interstitium - MPS hydrogel + collagen
• Capillary network - endothelial barrier
Dialysate
Fact. VIII
m
Water and toxins
Glucose
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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
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The Endothelium as a Functional Barrier in PD
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Ultrasmall pores : predict to account for 50% of water removal (UF)
Ultrasmall Pores across the Endothelium
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Osmotic
Gradient
Hypothesis : Water Channels
Simple Diffusion
Facilitated Diffusion
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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.
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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
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Structure of the Mouse Peritoneal Membrane
Ni et al. Kidney Int 67; 2005
A
B
C
D
E
*
*
AQP1
eNOS
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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
*§
*§
** *
*
Ni et al. Kidney Int 67; 2005
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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
Ni et al. Kidney Int 67; 2005
Peritoneal Transport : Mouse vs. Rat vs. Man
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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.
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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
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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
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Slope of volume curves
(125I-albumin)
0
10
20
30
40
Aqp1 Mice
+/+ +/- -/-
Init
ial
UF
rate
(µ
l/m
in)
Ni et al. Kidney Int 69, 2006
Initial Water Flow in Aqp1 Mice
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Intraperitoneal volume during PD
Ni et al. Kidney Int 69, 2006
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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
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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
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• 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
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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
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AQP1 in Capillary Endothelium : Lung
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J Clin Invest 97, 1996
saline or betamethasone (0.35 mg/kg)
E15
P1
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AQP1 Expression : Glucocorticoid Induction
Moon C. et al. AJP 273: C1562, 1997
4.4-kb mouse Aqp1
MEL cells
1 M Dexa
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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
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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
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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
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Gating aquaporin-1 to improve UF ?
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Aquaporins in Plants: Arabidopsis
AQP Kdown AQP WT
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Plants counteract fluctuations in water supply by regulating their aquaporins:
→ Drought stress: dephosphorylation and closure
→ Flooding: anoxia – cytosolic acidification: protonation and closure
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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
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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)
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• 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 ?
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• 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
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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
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• 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
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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
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Blood 2003; 101: 1596-1602
CTCTG, core binding motif of a regulatory element for PKLR
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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
#
#
*
*
Relative luciferase activity (percent)
pGL3-Basic
pGL3-Wt: CTGTC
pGL3-M3-CTCTT
Relative luciferase activity (percent)
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
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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
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TT = 17 % TT = 4 %
P25: 431 ml P75: 842 ml
Ultrafiltration volume (ml)
Maréchal C et al. ASN 2010
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
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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)
Maréchal C et al. ASN 2010
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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
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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
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Thank you for your attention
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Wang et al. Structure 13, 2005
What Makes an Aquaporin a Glycerol Channel ?
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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.
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kD
AQP7
GlyAQP7
-25
-35
Expression of AQP7 in the Peritoneum
Visceral peritoneum
Testis
T. Nishino, unpublished data
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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
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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 …
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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
(%)
*
#
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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
Baseline Peritonitis
Number 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
Baseline Peritonitis
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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.
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Peritoneal Structure in Aqp1 Mice: Morphology
Aqp1 (+/+) Aqp1 (-/-)
PP
VP
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The Nobel Prize in Chemistry 2003
Peter Agre Roderick MacKinnon
« for discoveries concerning channels in cell membranes »
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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)
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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
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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. »
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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
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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
Baseline Peritonitis
*
**
#*
*
*
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
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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
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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