Paulina Nguyen HHMI Recipient Mentor: Dr. Ganesh Cherala Lab: OSU-College of Pharmacy

Effect of Perinatal Low Protein Diet on the Expression of Renal Organic Cation Transporters in Adult Rats

P A U L I N A N G U Y E NH H M I R E C I P I E N T

M E N T O R : D R . G A N E S H C H E R A L A

L A B : O S U - C O L L E G E O F P H A R M A C Y

Low Birth Weight (LBW) About 1 in every 12

babies in the United States is born with low birth weight

Increased risk for serious health problems such as lasting disabilities and even death.

Stunted growth Higher incidence of

diseases

Poor conditions in the womb • Greater susceptibility to diseases in adult life

(Barker’s Hypothesis)

Low protein diet (LPD)–Rodent model for LBW• Similar to human epidemiological findings• LPD in rodent model can be manipulated in

laboratory conditions

Fetal Programming and Low Birth Weight (LBW)

Altered tissue development leads to altered development of organs such as in kidney and liver

◦ Affect renal organic transporter functionality

◦ Affects drug transport and excretion in and out of the body

LBW individuals will need different drug dosages

Consequences of LBW

Morphological Physiological

Low Birth Weight

LPD

Various Organs

Kidney

Liver

Heart, etc.

Drug Transport & PharmacokineticsPharmacokinetics (PK)

Study of how drug travels through the bodyoAbsorptionoDistributionoMetabolismoExcretion

Drug Metabolism and TransportRenal Excretion

ADME

Transporters• OAT• OCT• PGP• PEPT• MRP

PROXIMAL TUBULE

Blood

PROXIMAL TUBULE

LUMEN

Basolateral Membrane

5 Organic Cation Transporters found in rat models

1. OCT12. OCT23. OCT34. OCTN15. OCTN2

Renal Organic Cation Transporters (OCT)

OCT2

OCTN1

OCTN2

Hypothesis • Low birth weight, as a result of altered

perinatal environment, leads to permanent alterations in the expression of renal OCT transporters.

Specific Aims To quantify mRNA expression of renal OCT2,

OCTN1, and OCTN2 at various ages To quantify protein expression of renal OCT2 at

various ages

Study Design

Gestation3 weeks

Lactation4 weeks

Day 65 Day 150

Weaned onto regular diet

Birth weights noted; litters culled

Day 28

ControlLow Protein (LP)

Measure the mRNA level of OCT2, OCTN1, and OCTN2 expression in male and female offspring in both LP and Control groups using RT-PCR

Measure the protein expression of OCT2 in male and female offspring in both LP and Control groups using western blotting

Sample Analysis

RESULTS

OCT2 OCTN1 OCTN2

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

Day 28 mRNA levels for males and females

Males Females

Transporters

Fold

Diff

eren

ce (r

elat

ive

to c

ontro

l)RT-PCR Day 28

OCT2 OCTN1 OCTN2

-4

-3

-2

-1

0

1

2

3

Day 65 mRNA levels for males and females

Males Females

Transporters

Fold

Diff

eren

ce (r

elat

ive

to c

ontro

l)

RT-PCR Day 65

OCT2 OCTN1 OCTN2

-40

-30

-20

-10

0

10

20

30

40

50

Day 150 mRNA levels for males and females

Males FemalesTransporters

Fold

diff

eren

ce (r

elat

ive

to c

ontro

l)

* Statistically Significant

RT-PCR Day 150

*

*

*

OCT2 OCTN1 OCTN2 OCT2 OCTN1 OCTN2 OCT2 OCTN1 OCTN2

-40

-30

-20

-10

0

10

20

30

40

50Fold difference in Level of OCT Expression

Males FemalesTransporters

Fold

diff

eren

ce (r

elat

ive

to c

ontro

l)

Day 28Day 150

Day 65

RT-PCR Summary

*

*

*

* Statistically Significant

Day 28 Day 65 Day 1500

0.0020.0040.0060.0080.01

0.0120.0140.0160.018

Control LBW

Den

sity

of

OCT

2 Ba

nd (

norm

aliz

e to

β-

acti

n)OCT2 Western Blotting – Males

Day 28 Day65 Day1500

0.020.040.060.080.1

0.120.140.16

Control LBW

Den

sity

of

OCT

2 Ba

nd (

norm

aliz

e to

β-

acti

n)

OCT2 Western Blotting - Females

Homogenize

1000 G

Tissue

Pellet- Nuclei

- Cell Debris

Tissue Homogenate

35000 G

Pellet- Mitochondria

- Plasma Membrane

100000 G

Pellet- Microsomes

Supernatant

- Cytosol

Supernatant

Supernatant

27 fold decrease in expression of OCT2 in Day 150 LP females relative to control (regular diet) females

Effect of LP diet seen in later life

Results – Discussion

Sex hormone regulation of OCT2 a possible reason for 27 fold difference in LP Day 150 females

Sex hormone regulation of OCT2• Testosterone up-regulates expression of OCT2• Estrogen down-regulates expression of OCT2

Discussion - Regulation of OCT2?

Howard Hughes Medical Institute

Cripps Funding

Cherala Lab◦ Dr. Ganesh Cherala◦ Barent DuBois◦ Jacob Pearson◦ Dr. Shobana Ganesan◦ Tahir Mahmood◦ Shawn Mahmood◦ Alexandru Cioara

OSU-College of Pharmacy

Dr. Kevin Ahern

Acknowledgements

Cherala G, Shapiro BH, and D'mello AP (2006) Two low protein diets differentially affect food consumption and reproductive performance in pregnant and lactating rats and long-term growth in their offspring. J Nutr 136: 2827–2833

Kathleen M. Giacomini, et al. (2010) Membrane transporters in drug development Nature Reviews Drug Discovery , 215-236

Hagenbuch, B (2010) Drug Uptake Systems in Liver and Kidney: A Historic Perspective Clinical Pharmacology & Therapeutics 87 1, 39–47

Works Cited