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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 OSU/OHSU College of Pharmacy. Significance of Low Birth-Weight.

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slide1

Adverse perinatal environment alters the expression of renal organic cation transporters in adult rats in a gender specific fashion.

Paulina Nguyen

Mentor: Dr. GaneshCherala

OSU/OHSU College of Pharmacy

significance of low birth weight
Significance ofLowBirth-Weight
  • ~1 in every 12 babies (8.33%) in the United States is born full-term with low birth weight (LBW)
  • Global incidence of LBW ~17%
slide3

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

Altered

Pharmacokinetics

  • Require altered optimal drug dosage for patient
pharmacokinetics
Pharmacokinetics
  • Pharmacokinetics (PK)
    • Study of how drug travels through the body
      • Absorption
      • Distribution
      • Metabolism
      • Excretion
  • Drug Metabolism and Transport
    • Renal Excretion
      • Organic Cation Transporters (OCTs)

A

D

M

E

slide5

Blood

Proximal Tubule

PROXIMAL TUBULE

OCT2

LUMEN

OCTN1

OCTN2

Basolateral

Membrane

  • Transporters
    • OCT
hypothesis
Hypothesis
  • 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.
study design
Study Design

Control

Low Protein (LP)

Weaned onto regular diet

Birth weights noted;

litters culled

Day 150

Gestation

3 weeks

Day 28

Day 65

Lactation

4 weeks

methodology
Methodology
  • To quantify protein expression of renal OCTN2
    • Western blotting
      • OCTN2 (Day 150 males and females)
slide10

2

1

4

3

5

Infared Dye

slide12

OCTN2

Molecular marker

Plasma Membrane

Cytosol

Microsome

slide13

2

1

4

3

5

Infared Dye

Blocking Peptide

slide14

1° Ab + 2° Ab

1° Ab + B. peptide + 2° Ab

Molecular marker

Molecular marker

OCTN2

OCTN2

Β-actin

Β-actin

2° Ab Only

B. peptide + 2° Ab

Molecular marker

Molecular marker

endogenous control
Endogenous control
  • Used to account for
    • Technician error (pipetting)
    • Error during transfer onto membrane
    • Loading control
use of a positive control
Use of a Positive Control
  • Accounts for errors during transfer onto membrane
  • Doesn’t account for
  • Pipetting error
  • Loading error
  • Pipetting error
  • Loading error
  • %CV is <5%
  • Dilute samples to same protein concentration for loading equal sample volume
slide17

Positive Control

OCTN2

Molecular marker

summary
SummarY
  • Found fold increases in mRNA expression of OCTN2
    • Prominent changes in females
  • No changes in western blotting protein expression.
next step
NEXTSTEP
  • 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
carnitine assay
Carnitine Assay

Carnitine + Acetyl-CoA

Carnitineacetyltransferase

Acetylcarnitine + CoASH

DTNB

TNB (λ412nm) + CoAS-SNB

carnitine assay procedure
Carnitine Assay Procedure
  • Urine
  • Kidney or Liver Tissue
  • Plasma

KHCO3

Perchloric Acid

Removal of protein

Total Carnitine

Free Carnitine

KOH

H2O

HCl

Hydrolyze acylcarnitine

slide23

Total Carnitine

Free Carnitine

  • Reagent solution
        • DTNB
        • Acetyl CoA
        • DI H2O
  • Carnitine Acetyl Transferase (CAT)
  • Reagent solution
        • DTNB
        • Acetyl CoA
        • DI H2O
  • Carnitine Acetyl Transferase (CAT)
future studies
Future Studies
  • Determine the functional status of OCTN2
    • Measure carnitine levels in:
      • Urine
      • Blood Plasma
      • Various organ tissues
acknowledgements
Acknowledgements
  • Howard Hughes Medical Institute
  • Cripps Funding
  • OSU-College of Pharmacy
  • Dr. Kevin Ahern
  • Cherala Lab
    • Dr. GaneshCherala
    • Dr. ShobanaGanesan
    • Ali Aldakhil
    • BarentDuBois
  • Bonnie Hastings
  • TahirMahmood
  • Shawn Mahmood
  • Jacob Pearson