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 1

 ~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% 2

Maternal Low Protein Diet Full-term Low Birth Weight babies Fetal Programming Altered excretion of xenobiotic substances out of body Require altered optimal drug dosage for patient Altered PhysiologyAltered Morphology Kidney; Liver Hypertension Hyperlipidemia Diabetes Mellitus Obesity Altered Pharmacokinetics 3

 Pharmacokinetics (PK) Study of how drug travels through the body o Absorption o Distribution o Metabolism o Excretion  Drug Metabolism and Transport Renal Excretion Organic Cation Transporters (OCTs) ADMEADME 4

Blood Proximal Tubule LUMEN BasolateralMembrane Transporters OCT OCT2 OCTN1 OCTN2 5

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 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

Birth weights noted; litters culled Control Low Protein (LP) 8

 To quantify protein expression of renal OCTN2 – Western blotting OCTN2 (Day 150 males and females) 9

Infared Dye

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Molecular marker OCTN2 Cytosol Microsome Plasma Membrane 12

Infared Dye Blocking Peptide 13

1° Ab + 2° Ab 1° Ab + B. peptide + 2° Ab Molecular marker OCTN2 Β-actin 2° Ab Only Molecular marker B. peptide + 2° Ab 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 Pipetting error Loading error  %CV is <5%  Dilute samples to same protein concentration for loading equal sample volume 16

OCTN2 Positive Control Molecular marker 17

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 Found fold increases in mRNA expression of OCTN 2 – Prominent changes in females  No changes in western blotting protein expression. 19

 Establish an assay to assess functionality of OCTN 2 Carnitine – reabsorbed by OCTN 2  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 Acetylcarnitine + CoASH TNB (λ412nm) + CoAS-SNB DTNB Carnitine acetyltransferase 21

Urine Kidney or Liver Tissue Plasma Perchloric Acid KHCO 3 Free Carnitine Total Carnitine H2OH2O KOH HCl Removal of protein Hydrolyze acylcarnitine 22

Free Carnitine Total Carnitine Reagent solution o DTNB o Acetyl CoA o DI H 2 O Carnitine Acetyl Transferase (CAT) Reagent solution o DTNB o Acetyl CoA o DI H 2 O Carnitine Acetyl Transferase (CAT) 23

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 Determine the functional status of OCTN 2 – 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