Paulina Nguyen Mentor: Dr. Ganesh Cherala OSU/OHSU College of Pharmacy

1. 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

2. 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%

3. 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

4. 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

5. Blood Proximal Tubule PROXIMAL TUBULE OCT2 LUMEN OCTN1 OCTN2 Basolateral Membrane • Transporters • OCT

6. Data from study of Summer’2010

7. 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.

8. 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

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

10. 2 1 4 3 5 Infared Dye

11. Data/Results

12. OCTN2 Molecular marker Plasma Membrane Cytosol Microsome

13. 2 1 4 3 5 Infared Dye Blocking Peptide

14. 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

15. Endogenous control • Used to account for • Technician error (pipetting) • Error during transfer onto membrane • Loading control

16. 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

17. Positive Control OCTN2 Molecular marker

18. Westerns Data – Day 150

19. SummarY • Found fold increases in mRNA expression of OCTN2 • Prominent changes in females • No changes in western blotting protein expression.

20. 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

21. Carnitine Assay Carnitine + Acetyl-CoA Carnitineacetyltransferase Acetylcarnitine + CoASH DTNB TNB (λ412nm) + CoAS-SNB

22. Carnitine Assay Procedure • Urine • Kidney or Liver Tissue • Plasma KHCO3 Perchloric Acid Removal of protein Total Carnitine Free Carnitine KOH H2O HCl Hydrolyze acylcarnitine

23. 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)

24. Collected Data

25. Future Studies • Determine the functional status of OCTN2 • Measure carnitine levels in: • Urine • Blood Plasma • Various organ tissues

26. 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