Regulation of renal drug transport under physiological and pathological conditions · 2013. 2. 14. · Urate scavenges potential harmful radicals in our body, but may cause gout,

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


nephron

glomerulus

tubulus

Glomerular filtration

Reabsorption

Secretion


nephron

glomerulus

tubulus

bloed urine

Tubule cell


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



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)


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 (+)



• 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


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)



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



• 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


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)


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



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)


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)



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


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 --


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

Documents

Regulation of renal drug transport under physiological and pathological conditions · 2013. 2. 14. · Urate scavenges potential harmful radicals in our body, but may cause gout,