Kidney SLC Transporter Toxicity Models: RPTEC/TERT1-OAT1 and OCT2

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Days in culture

Growth curve comparasion

RPTEC/TERT1-OAT1

RPTEC/TERT1-OCT2

RPTEC/TERT1

Growth Curve and Morphology

Kidney transporter over-expressing cell lines compared with parental RPTEC/TERT1. OAT1 and OCT2 clones display the same renal epithelial growth pattern as parental RPTEC/TERT1 cells.

OAT1, OCT2 western blot, presence of OAT1, OCT2 when compared to parental line

75 kDa

50 kDa

50 kDa anti-Tubulin

Western Blot

100 kDa

75 kDa

50 kDa

50 kDa

Milliporeanti-OCT21:1,000

anti-Tubulin

Abcamanti-OAT11:1,000

Molecular characterization of the RPTEC SLC transporter cells. Left) Immunoblot demonstrates OAT1 protein expression levels inRPTEC/TERT1 parental and OAT1 cells. Right) Immunoblot demonstrates OCT2 protein expression levels RPTEC/TERT1 parental and OCT2cells.

RT-PCR

RT-PCR

500 GAPDH

1000

1500 hOAT1

OAT1, and OCT2 presence detected in RT-PCR early and late passage

MCB, DI and MCB+15 passages

Molecular characterization of the RPTEC SLC transporter cells. Top) RT-PCR demonstrates the presence of OAT1 mRNA in RPTEC/TERT1OAT1 cells. Bottom) RT-PCR demonstrates the presence of OCT2 mRNA in RPTEC/TERT1 OCT2 cells.

RP

TE

C/T

ER

T1 O

CT

2R

PT

EC

/TE

RT

1

OCT2 Merged with DAPIOAT1 Merged with DAPI RP

TE

C/T

ER

T1-O

AT

1R

PT

EC

/TE

RT

1

Scale bar: 100µm

Surface markers and method of detection: OAT1 and OCT2

ICC

Molecular characterization of the RPTEC SLC transporter cells. Left) IF/ICC demonstrates OAT1 expression and localization. Right) IF/ICCdemonstrates OCT2 expression and localization.

Merged with DAPIE-cadherinR

PT

EC

/TE

RT

1 O

CT

2R

PT

EC

/TE

RT

1R

PT

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/TE

RT

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Scale bar: 100µm

CD31 Merged with DAPI

Surface markers and method of detection: OAT1 and OCT2

ICC

Kidney transporter over-expressingcell lines compared with parentalRPTEC/TERT1. RPTEC/ TERT1 SLCtransporter cells were subjected toimmunostaining and dome formationassay. OAT1 and OCT2 clones displaythe same renal epithelial growthpattern as parental RPTEC/TERT1cells. The renal epithelial markersCD13 and E-cadherin are expressed inboth parental RPTEC/TERT1 cells andin the OAT1 and OCT2 lines.

Dome formation

RPTEC/TERT1 RPTEC/TERT1-OAT1 RPTEC/TERT1-OCT2

Scale bar: 100µm

Figure 3. Kidney transporter over-expressing cell lines compared with parental RPTEC/TERT1. RPTEC/ TERT1 SLC transporter cells weresubjected to dome formation assay. OAT1 and OCT2 clones display the same renal epithelial growth pattern as parental RPTEC/TERT1cells.

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

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5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

3 0 0 0 0

3 5 0 0 0

4 0 0 0 0

R P T E C /T E R T 1 -O A T 1 5 -C F u p ta k e

5 -C F c o n c e n tra t io n , M

up

tak

e(R

FU

)

R P T E C /T E R T 1

R P T E C /T E R T 1 -O A T 1

K m = 2 9 4 .7 M

5-CF con(µm) 0 0.5 2 5 10 25 75 150 300 500

uptake ratio1.061636 1.512075 2.323561 3.1003 2.633725 6.718272 5.00061 6.100167 6.350512 6.641002

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

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

7 0 0 0 0

R P T E C /T E R T 1 -O A T 1 (M C B ) 6 -C F u p ta k e

6 -C F c o n c e n tra t io n , M

RF

U

R P T E C /T E R T 1

R P T E C /T E R T 1 -O A T 1

K m = 1 4 .1 7 M

6-CF conc. 0µM 0.5µM 1.0µM 2.0µM 3.0µM 5.0µM 10.0µM 25.0µM 50.0µM 100.0µM

uptake ratio1.160812 169.6708 226.9443 239.1342 260.7467 274.1321 287.706 198.1971 146.0713 84.16204

Evidence of transporter functionality- OAT1 transporter assay, 5-CF (6-CF)

Use 5-CF as uptake substrate

Use 6-CF as uptake substrate

Drug Kinetic Profiles of RPTEC/TERT1OAT1 Transporter Cells. Solute uptakeactivity of RPTEC/TERT1 OAT1 cells wasassessed using 5-CF or 6-CF as substrate. Asexpected, uptake increases with increasing5-CF or 6-CF concentration in OAT1-expressing cells but not in parentalRPTEC/TERT1 cells (n=3), indicating thatthe observed transport is due to OAT1expression.

-7 -6 -5 -4 -3 -2

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

N o v o b io c in In h ib its R P T E C /T E R T 1 O A T 1 6 -C F u p ta k e

N o v o b io c in C o n c e n tra tio n , L o g (M )R

FU

IC 5 0 = 5 9 .1 7 M

-8 -7 -6 -5 -4 -3 -2

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

P ro b e n e c id In h ib its R P T E C /T E R T 1 O A T 1 6 -C F u p ta k e

P ro b e n e c id c o n c e n tra t io n , L o g (M )

RF

U

IC 5 0 = 1 6 .9 1 M

-8 -7 -6 -5 -4 -3 -2

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

P r o b e n e c id In h ib its R P T E C /T E R T 1 O A T 1 5 -C F u p ta k e

P ro b e n e c id c o n c e n tra t io n , L o g (M )

RF

U

IC 5 0 = 4 .4 7 M

-7 -6 -5 -4 -3 -2

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

N o v o b io c in In h ib its R P T E C /T E R T 1 O A T 1 5 -C F u p ta k e

N o v o b io c in C o n c e n tra tio n , L o g (M )

RF

U

IC 5 0 = 7 7 .6 5 MUse 5-CF as uptake substrate

Use 6-CF as uptake substrate

Evidence of transporter functionality- OAT1 transporter assay, 5-CF (6-CF)-uptake inhibition

Transport inhibition kinetics ofRPTEC/TERT1 OAT1-expressingcell lines. OAT1- expressing cellswere exposed to increasingconcentrations of the knownOAT1 inhibitors probenecid andnovobiocin while 5-CF or 6-CFconcentration and uptake timewere held constant at 100 µMand 20 minutes, respectively.The resulting inhibition curvesindicate that OAT1 hasphysiologically relevanttransport activity whenoverexpressed in RPTEC/TERT1cells (n=3).

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0

2 0 0

4 0 0

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R P T E C /T E R T 1 -O C T 2 c lo n e 3 2 u p ta k e a s s a y

A S P+

c o n c e n tra tio n , M

up

tak

e(R

FU

)

R P T E C /T E R T 1

R P T E C /T E R T 1 -O C T 2

Asp con 0µM 0.5µM 2µM 5µM 10µM 25µM 75µM 150µM 300µM 500µM

ratio 1.017543 1.523934 2.033221 2.989123 3.455444 4.540586 4.776906 4.883032 4.38785 4.050386

K m = 2 0 .3 9 M

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R P T E C -O C T 2 E A M -1 u p ta k e

E A M -1 c o n c e n tra tr io n , M

RF

U

R P T E C /T E R T 1

R P T E C /T E R T 1 -O C T 2

K m = 3 5 .3 7 M

EAM-1 conc0µM 0.5µM 1µM 2µM 3µM 5µM 10µM 25µM 50µM 100µM

uptake ratio2.267334 21.56116 23.0057 22.65206 22.73024 28.13118 29.92067 23.72269 21.58114 21.21723

Use ASP+ as uptake substrate

Use EAM-1 as uptake substrate

Evidence of transporter functionality- OCT2 transporter assay, Asp+(EAM-1)

Drug Kinetic Profiles of RPTEC/TERT1 OCT2 Transporter Cells. Solute uptake activity of RPTEC/TERT1 OCT2 cells was assessed using ASP+ or EAM-1 as substrate. As expected, uptake increases with increasing amounts of ASP+ and EAM-1 in OCT2-expressing cells but not in parental RPTEC/TERT1 cells (n=3), indicating that the observed solute transport is due to OCT2 expression.

-7 -6 -5 -4 -3 -2

2 0 0

3 0 0

4 0 0

5 0 0

Q u in it in in h ib i ts R P T E C /T E R T 1 O C T 2 A S P+

u p t a k e

Q u in it in c o n c e n tra t io n , lo g (M )

RF

UIC 5 0 = 5 5 .1 4 M

-7 -6 -5 -4 -3 -2

1 0 0

2 0 0

3 0 0

4 0 0

C im e t id in e in h ib its R P T E C /T E R T 1 O C T 2 A s p+

u p t a k e

C im e tid in e c o n c e n tra tio n , L o g (M )

RF

U

IC 5 0 = 2 9 8 .5 M

-7 -6 -5 -4 -3

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2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

Q u in it in in h ib its O C T 2 E A M -1 u p ta k e

Q u in it in c o n c e n tra t io n , lo g (M )

RF

U(u

pta

ke

)

IC 5 0 = 5 9 .4 9 M

-7 -6 -5 -4 -3 -2

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

C im e tid in e in h ib its O C T 2 E A M -1 u p ta k e

C im e tid in e c o n c e n tra tio n , L o g (M )

RF

U(u

pta

ke

)

IC 5 0 = 9 3 .5 M

Use ASP+ as uptake substrate

Use EAM-1 as uptake substrate

Evidence of transporter functionality- OCT2 transporter assay, Asp+(EAM-1)-uptake inhibition

Transport inhibition kinetics ofRPTEC/TERT1 OCT2-expressingcell lines. OCT2 expressing cellswere exposed to increasingconcentrations of the knownOCT2 inhibitors cimetidine andquinitin while ASP+ concentrationand uptake time were heldconstant at 100 µM and 20minutes, respectively. Theresulting inhibition curves indicatethat OCT2 has physiologicallyrelevant transport activity whenoverexpressed in RPTEC/TERT1cells (n=3).