Adverse perinatal environment alters
the expression of renal organic cation
transporters in adult rats in a gender
specific fashion.
Paulina Nguyen
Mentor: Dr. Ganesh Cherala
1
OSU/OHSU College of Pharmacy

~1 in every 12 babies
(8.33%) in the United
States is born fullterm with low birth
weight (LBW)

Global incidence of
LBW ~17%
2
Maternal Low Protein
Diet
Full-term Low Birth
Weight babies
Fetal Programming
Altered Physiology
Altered Morphology
Kidney; Liver
Altered excretion of
xenobiotic substances
out of body
•Hypertension
•Hyperlipidemia
•Diabetes Mellitus
•Obesity
Require altered optimal
drug dosage for patient
Altered
Pharmacokinetics
3

Pharmacokinetics (PK)
 Study of how drug travels
through the body
A o Absorption
D o Distribution
M o Metabolism
E o Excretion

Drug Metabolism and
Transport
 Renal Excretion
 Organic Cation Transporters
(OCTs)
4
Proximal
Tubule
LUMEN
OCT2
OCTN1
OCTN2
Basolateral
Membrane
Transporters
• OCT5
Fold difference (relative to control)
Day 150 mRNA levels for males and females
60
50
40
30
20
10
0
-10
OCT2
OCTN1
OCTN2
-20
-30
-40
-50
Transporters
Males
Females
*
Statistically Significant
6
Adverse perinatal environment leaves a long
term permanent effect on the expression of
organic cation transporters in a gender
dependent fashion mediated by sex hormones.

7
Control
Low Protein (LP)
Weaned onto
regular diet
Birth weights noted;
litters culled
8
 To quantify protein expression of renal
OCTN2
– Western blotting
• OCTN2 (Day 150 males and females)
9
2
1
4
3
5
Infared
Dye
10
11
OCTN2
Molecular marker
Plasma Membrane
Cytosol Microsome
12
2
1
4
3
5
Infared
Dye
Blocking
Peptide
13
1° Ab + 2° Ab
1° Ab + B. peptide + 2° Ab
Molecular marker
Molecular marker
OCTN2
OCTN2
Β-actin
2° Ab Only
Β-actin
B. peptide + 2° Ab
Molecular marker
Molecular marker
14

Used to account for
– Technician error (pipetting)
– Error during transfer onto membrane
– Loading control
15

Accounts for errors during transfer onto
membrane

Doesn’t account for
• Pipetting error
• Loading error
 %CV is <5%
 Dilute samples to same
protein concentration for
loading equal sample volume
16
Positive Control
OCTN2
Molecular marker
17
OCTN2 - Western Blotting
Density of OCTN2 Band (relative to pos. control)
0.9
0.8
0.7
0.6
0.5
LP
0.4
control
0.3
0.2
0.1
0
Male
Female
18

Found fold increases in mRNA expression
of OCTN2
– Prominent changes in females

No changes in western blotting protein
expression.
19

Establish an assay to assess functionality
of OCTN2
• Carnitine – reabsorbed by OCTN2
 Carrier of activated fatty acids of variable
chain length, esterified to its hydroxyl group.
 Essential for the mitochondrial beta
oxidation of acyl-CoA esters for energy
generation
20
Carnitine + Acetyl-CoA
Carnitine acetyltransferase
Acetylcarnitine + CoASH
DTNB
TNB (λ412nm) + CoAS-SNB
21
• Urine
• Kidney or Liver Tissue
• Plasma
Perchloric Acid
KHCO3
Removal of protein
Free Carnitine
H2O
Total Carnitine
KOH
HCl
Hydrolyze acylcarnitine
22
Free Carnitine
•Reagent solution
o DTNB
o Acetyl CoA
o DI H2O
• Carnitine Acetyl Transferase
(CAT)
Total Carnitine
• Reagent solution
o DTNB
o Acetyl CoA
o DI H2O
• Carnitine Acetyl Transferase
(CAT)
23
Carnitine Assay Standard Curve
0.4
0.35
y = 0.0083x + 0.1868
R² = 0.8838
Absorbance
(Abs)
0.3
0.25
y = 0.0075x + 0.1758
R² = 0.9678
0.2
free carnitine
total carnitine
0.15
0.1
0.05
0
0
5
10
15
20
25
Carnitine Concentrations (mg/L)
24

Determine the functional status of
OCTN2
– Measure carnitine levels in:
• Urine
• Blood Plasma
• Various organ tissues
25

Howard Hughes Medical Institute

Cripps Funding

OSU-College of Pharmacy

Dr. Kevin Ahern
Cherala Lab
• Dr. Ganesh Cherala
• Dr. Shobana Ganesan
• Ali Aldakhil
• Barent DuBois
• Bonnie Hastings
• Tahir Mahmood
• Shawn Mahmood
•Jacob Pearson
26