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 N
H 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
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
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
Fo
ld D
iffe
ren
ce
(re
lati
ve
to
co
ntr
ol)
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
Fo
ld D
iffe
ren
ce
(re
lati
ve
to
co
ntr
ol)
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 Females
Transporters
Fo
ld d
iffe
ren
ce
(re
lati
ve
to
co
ntr
ol)
* 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 Females
Transporters
Fo
ld d
iffe
ren
ce
(re
lati
ve
to
co
ntr
ol)
Day 28Day 150
Day 65
RT-PCR Summary
*
*
*
* Statistically Significant
Day 28 Day 65 Day 1500
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
Control LBW
Densit
y o
f O
CT2 B
and (
norm
alize
to β
-act
in)
OCT2 Western Blotting – Males
Day 28 Day65 Day1500
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Control LBW
Densit
y o
f O
CT2 B
and (
norm
alize
to β
-act
in)
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