Regulation of renal drug transport under physiological and pathological conditions
Rosalinde Masereeuw
Radboud University Nijmegen Medical Centre
Nijmegen Centre for Molecular Life Sciences
October 13, 2010
Drug transport in the kidney
nephron
Drug transport in the kidney
nephron
glomerulus
tubulus
Glomerular filtration
Reabsorption
Secretion
Drug transport in the kidney
nephron
glomerulus
tubulus
bloed urine
Tubule cell
Drug transport in the kidney
Renal drug handling
Two major pathways:
• Organic anion system (eg.
PAH, MTX, penicillines)
• Organic cation system (eg.
choline, dopamine, cisplatin)
Multiple transporters identified :
• Solute Carriers (SLCO,
SLC22, SLC47)
• ATP Binding Cassette
transporters (ABC)
Transport mechanismTransporter
The organic anion system
Substrates (examples)
blood urine
Renal tubular cell
antiport (dicarboxylates)OAT1/3 PAH, methotrexate (MTX), hippurate, indoxyl sulfate, etc.
OAT1/3
αKG2-
OA-
URAT1 antiport ((organic )anions) urate
OAT4/URAT1OA-
OAT4 OTA, DHEA-sulfate, estrone-sulfate, urate, MTX
antiport (dicarboxylates)
MRP2/4 (ABCC2/4)
MRP2/ABCC2 glutathione, glucuronide/sulfate conjugates, PAH, GSH, GSSG, MTX
pump (ATP)
cidofovir, PMEA, MTX, PAH, cAMP, cGMP, prostaglandins, urate
MRP4/ABCC4 pump (ATP)
BCRP (ABCG2)
BCRP/ABCG2 pump (ATP) dimer MTX, urate, sulfates
OATP4C1?
OA-
αKG2-
Organic cation transport system
blood urine
Renal tubular cellP-gp (ABCB1)
BCRP (ABCG2)
Transport mechanismTransporter Substrates (examples)
potential driven, electrogenic
OCT2 TEA, histamine, dopamine, norepinephrine, cisplatin,cimetidine, etc.
?OATP4C1 digoxin, ouabain, thyroxine, T3, cAMP, MTX
antiport (protons)
MATE1/2-K TEA, cimetidine, creatinine, guanidine, cisplatin, oxaliplatin
BCRP/ABCG2 pump (ATP) dimer chemotherapeutics
P-gp/ABCB1 pump (ATP) chemotherapeutics, antivirals, antibiotics, cimetidine
?
OATP4C1OC+
OCTN1/2
antiport (protons)
TEA, verapamil, carnitine
MATE1/2-KH+
OC+
OCTN1/2
OCT2OC+
OC+
Drug transporters and the kidney
• Many drugs and their metabolites leave the human body via the urine
• Nephrotoxic injury contributes to 30-40% of all acute renal failures, which complicates 5% of all hospital admissions
• In chronic renal failure, increased levels of uremic toxins cause severe problems, including cardiovascular injury
Human urate homeostasis
Humans excrete uric acid as the final breakdown product of unwanted purinenucleotides.
Urate scavenges potential harmful radicals in our body, but may cause gout, nephrolitiasis, hypertension, and vascular disease.
Blood levels of urate are maintained by the balance between generation and excretion by specialized transporters located in renal proximal tubule cells.
Glomerular filtration 100%
Secretion 50%
Renal urate handling
Post-secretory reabsorption 40%
Excretion 10%
Reabsorption 99-100%
URAT1
Renal urate handling: URAT1
Enomoto et al. Nature, 2002
Renal urate handling: MRP4
MRP4 apicalbasolateral
MRP4 MRP2 merge
Van Aubel et al. JASN, 2002
Renal urate handling: MRP4
Van Aubel et al. AJP Renal, 2005
MRP4 apicalbasolateral
MRP4 MRP2 merge
human rat
BCRP KO mouse
BCRP in renal proximal tubule
M. Huls, C.D. Brown et al. Kidney Int. 2008
Renal urate handling: BCRP
Woodward et al. PNAS. 2009
blood urine
OAT1/3
αKG2-
urateURAT1
OA-
MRP4 (ABCC4)
BCRP (ABCG2)
?
Renal urate handling
Multiple transporters responsible for physiological urate balance; multiple sites for drug interaction(s)!
blood urine
ATP Mrp2Multidrug Resistence
Protein 2 (MRP2)
Regulation of Mrp2 in renal proximal tubules
Mrp2 and the kidney
• Killifish renal proximal tubules
• MDCKII-MRP2 cells
• Rat in vivo
Mrp2 in killifish renal proximal tubules
Masereeuw et al., Mol. Pharmacol. 2000
Transmitted light
Confocal microscopy
FL-MTX
Mrp2
Contr
ol
10 M
Gen
t0
1000
2000
3000LumenCell
**
Fluo
resc
ence
inte
nsity
Terlouw et al., Mol. Pharmacol. 2001
FL-MTX +gentamicin
Mrp2 in killifish renal proximal tubules
Mrp2 in killifish renal proximal tubules
ET-1
Ca2+ET-1
ET-1
ATP
Mrp2
(-)
ETB(+)
NOS
PKCcGMP(+)
(+)NO
sGC
Notenboom et al., AJP Renal Physiol. 2004
aminoglycosides
radiocontrast agents
heavy metals
X
Killifish model, gentamicin exposure
• Reduction of ATP-consuming processes in order to conserve ATP for more important cellular functions
• A reduction of transport activity may contribute to the progression of cellular damage
Function of inhibition of Mrp2-mediated efflux?
Contr
ol
Gent
(1.5h
)
Gent
(3h)
Gent
(12h)
Gent
(24h)
0
25
50
75
100
125
150
175 LumenCell
*
*Fl
uore
scen
ce in
tens
ity(%
of c
ontr
ol)
Killifish model, gentamicin exposure
Notenboom et al., J Pharmacol Exp Ther. 2005
20 m
A
20 m
B
Increase of Mrp2 after 24h recovery
Notenboom et al., 2004
Contr
ol
M Ge
nt
10
M L-N
MMA
50
Gent+
L-NMM
A0
20
40
60
80
100
120
140 **
Fluo
resc
ence
inte
nsity
Control Preconditioned
0
50
100
150
200
250Cell
*
***
Fluo
resc
ence
inte
nsity
Preconditioning of cellular function measured using Mito Tracker Red CM-H2XRos
Notenboom et al., 2004
ATP
Mrp2
ATP
Mrp2
Short-termgentamicin (-)
gentamicinLong-term (+)
Killifish model, gentamicin exposure
• Killifish renal proximal tubules
• MDCKII transfected with MRP2
• Rat in vivo
Mrp2 and the kidney
MDCKII transfected with MRP2
1=Esterase2=Glutathione-S-transferase
CMFDA
CMFDA GSH
CMF
GSMFDA
1
1
2
2
GS-MF efflux from MDCKII cells(mean ± SD)
0 10 20 300
25
50
75
100
Time (min)
Rel
ativ
e flu
ores
cenc
ein
tens
ity (%
)
wild type
MRP2+MK571+CDNB
MDCKII transfected with MRP2
1h exposure+24h recovery
M 0
M
100
M
200
M
500
M
1000
0
50
100
150
200
*** ***
Gentamicin
% G
S-M
F flu
ores
cenc
e(e
xtra
cellu
lar/
intr
acel
lula
r)
Notenboom et al., J Pharmacol Exp Ther. 2006
MDCKII transfected with MRP2
250 kD
150 kD
Total membrane
Apical membrane
Con
trol
1h 24h
1h +
24h
reco
very
250 kD
150 kD
250 kD
150 kD
250 kD
150 kD
250 kD
150 kD
Con
trol
1h 24h
1h +
24h
reco
very
250 kD
150 kD
Con
trol
1h 24h
1h +
24h
reco
very
250 kD
150 kD
250 kD
150 kD- Mrp2
- Mrp2
0.0
0.5
1.0
1.5
2.0
2.5 Apical membrane
***
1h e
xpos
ure+
24h
reco
very
24h
expo
sure
1h e
xpos
ure
Con
trol
Rel
ativ
e pi
xel i
nten
sity
0.0
0.5
1.0
1.5
2.0
2.5 Total membrane
Rel
ativ
e pi
xel i
nten
sity
MDCKII transfected with MRP2
• Killifish renal proximal tubules
• Rat in vivo
Mrp2 regulation in rat kidney
computer
T
37.5°C
O2 pO2
pressure
Vit B12
urine flow
computer
T
37.5°C
O2 pO2
pressure
Vit B12
urine flow
Calcein-AM
Calcein-AM
Mrp2 regulation in rat kidney
Masereeuw et al., J Am Soc Nephrol. 2003
0 25 50 75 100 125 150
0
20
40
60
80
100
120 WH
Mrp2-/-
**
Time (min)
Excr
etio
nrat
e/G
FR (p
mol
/ml)
Mrp2 regulation in rat kidney
0 25 50 75 100 125 150
0
20
40
60
80
100
120 WH
siRNA Mrp2Mrp2-/-
**
*
Time (min)
Excr
etio
nrat
e/G
FR (p
mol
/ml)
Van de Water et al., Drug Metab Dispos. 2006
Mrp2 regulation in rat kidney
Mrp2 in killifish renal proximal tubules
ET-1
Ca2+ET-1
ET-1
gentamicin
Mrp2ETB
ATP(-)
(+)NOS
PKCcGMP(+)
(+)NO
sGC
0 25 50 75 100 125 1500
25
50
75
100
125 Control100 mg/kg Gent ip 7dg
**
Time (min)
Excr
etio
nrat
e/G
FR (p
mol
/ml)
Mrp2 regulation in rat kidney
Gen
t+B
os
Con
trol
Gen
t
Mrp2-
Mrp2 gene expression
Untre
ated
gent
gent
+ bos
entan
0
1
2
3
4
5
Xn v
alue
s
0 25 50 75 100 125 1500
25
50
75
100
125 Control100 mg/kg Gent ip 7dg
**
100 mg Gent+Bosentan ip 7dg
Time (min)
Excr
etio
nrat
e/G
FR (p
mol
/ml)
Mrp2 regulation in rat kidney
Notenboom et al., J Pharmacol Exp Ther. 2006
Gentamicin
exposure period
Killifish
MDCKII-
MRP2
Rat
Short = =
Short + recovery
Long
Regulation of Mrp2 from fish to mammal
Long-term effect of ET-signaling is nephroprotective
ATPMrp2iNOS
(+)NO
PKCcGMP(+)
sGC
Mrp2 regulation in rat kidney
LPS
A: iNOS
B: Mrp2
C: both
D + E:
nitrotyrosine
adducts
F + G:
tubule injury
F G
Heemskerk et al., Pflügers Arch. EJP. 2007
kDa
150
100
dLPS 3 6 12 24 48
iNOS --
Mrp2 --
Mrp2 regulation in rat kidney
Rat endotoxemia: effect on other transporters
P-gpATPADP
B: NO-independent
TNF-a
TIRLRRMD-2
TLR4
+
+ TRAF2
+
TRAF2
NF-kB
+
IKKb
NIK
+
+
+
+
TIRLRR
CD14
MD-2
P-gpATPADP
A: NO-dependent
iNOS NO+
?
+
++
TLR4
?
150
kDa
100
3 6 12 24 LPS-
150
kDa
100
3 6 12 24 LPS-
P-gp
Heemskerk et al., J. Biomed. Biotech. 2010
Contr
olg/m
l
LPS 1
0 10 ng
/ml
a
TNF-
a
LPS +
TNF-
0
50
100
150 *** ***
*
Rel
ativ
e P-
gp a
ctiv
ity (1
00%
)
Rat endotoxemia: effect on influx transporters
Protein (Oct-2)
50
75
150100
kDa LPS- 3 6 12 24 Am 6 Am 12
Heemskerk et al., Eur J Pharmacol. 2008 and unpublished
-2
-1
0
1Oat1Oat3
dLPS 3 6 12 24 48
mR
NA
exp
ress
ion
(rel
ativ
e Xn
)
-7-6-5-4-3-2-1012
dLPSLPS
Oatp1a1 Oatp1a3
mR
NA
exp
ress
ion
(rela
tive
Xn)
iNOS
NO
Heemskerk et al., 2007, 2008, unpublished
Mrp2
(+)
P-gp
OC+
OC+
a-KG
OA-
(-)
LPS
injuryMrp2
P-gp
OC+
OC+
a-KG
OA-
(-)
Regulation of drug transporters during endotoxemia
0
10
20
30
40
ControlsLPS
LPS + aminoguanidine
3 6 12 24Time after LPS injection (h)
cum
ulat
ive
GST
- a (µ
mol
)
LPS
basa
l
endo
toxem
ia
+ ami
nogu
anidi
ne0
20
40
60
80*
iNOS-gene expression
indu
ctio
n (X
n)
0 500 1000 1500 2000 25000
1020304050607080
r2 = 0.6688
p = 0.0131Controls
LPS
r2 = 0.0608p = 0.4922
CUM NOx (mol)
CU
M G
ST- a
( m
ol)
Kidney failure in human experimental endotoxemia
Conclusions
• A down-regulation of influx carriers and an up-regulation
of efflux pumps diminishes the accumulation of toxic
compounds and attenuates further proximal tubular
damage during inflammation
• Important consequences for pharmacotherapy
Regulation of drug transporters in kidney injury
Acknowledgements
Dept. of Intensive CarePeter PickkersHans van der Hoeven
Radboud University Nijmegen
Medical Centre
Inst. for Cell and Molecular BiosciencesColin D.A. Brown
Newcastle University
NIEHS/NIH; MDIBL
Lab. Pharmacology and ToxicologyDavid S. Miller
Science FacultyLab. Organismal Animal PhysiologyGert Flik
Dept. Pharmacology and ToxicologyFrans G.M. RusselJeroen van den HeuvelMiriam HulsSuzanne HeemskerkSylvia NotenboomSylvie TerlouwFemke van de Water