Renal Transporters in Drug Disposition, Drug‐Drug Interactions, and Nephrotoxicity

Bo Feng1, Ayman F. El‐Kattan1, Zaher A. Radi2

1 Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Groton, Connecticut, 2 Drug Safety Research and Development, Pfizer Worldwide Research and Development, Cambridge, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 23.3
DOI:  10.1002/0471140856.tx2303s53
Online Posting Date:  August, 2012
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Abstract

This unit describes in detail the in vitro methods for measuring the interaction of new chemical entities (NCEs) with human renal transporters (hOAT1, hOAT2, and hOCT2) as both a substrate and inhibitor. Renal transporter substrate assays help in the identification of renal secretion mechanisms and assessment of the potential renal drug‐drug interactions (DDIs) for NCE as a target, as well as to predict its renal clearance in humans. Human renal transporter (hOAT1, hOAT2, and hOCT2) inhibition assays characterize the inhibition potency of NCE and predict the potential for renal DDIs as a perpetrator with xenobiotics and drugs that are mainly renally cleared. In addition, such inhibition assays enable a better assessment of the potential for renal transporter‐mediated nephrotoxicity and pathology. Therefore, renal transporter substrate and inhibition assays are pivotal in drug discovery and development for renally cleared drugs and those that are co‐administered with marketed compounds mainly eliminated via the kidney. Curr. Protoc. Toxicol. 53:23.3.1‐23.3.15. © 2012 by John Wiley & Sons, Inc.

Keywords: renal transporters; nephrotoxicity; drug‐drug interaction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Organic Cation Transporter 2 (OCT2), Organic Anion Transporter 1 (OAT1), and Organic Anion Transporter 3 (OAT3)–Mediated Uptake Assay
  • Basic Protocol 2: OAT1, OAT3, and OCT2 Renal Transporter–Mediated Inhibition (IC50) Assay
  • Support Protocol 1: Transfecting, Selecting, and Culturing HEK293 Cells for Transporter Assays
  • Support Protocol 2: Sample Processing and Quantification for Both Scintillation Counting and LC‐MS/MS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Organic Cation Transporter 2 (OCT2), Organic Anion Transporter 1 (OAT1), and Organic Anion Transporter 3 (OAT3)–Mediated Uptake Assay

  Materials
  • HEK293 Flp‐In cells (Invitrogen, cat. no. R750‐07)
  • Growth medium 1 (see recipe)
  • HEK293 Flp‐In cell lines (see protocol 3)
  • HEK293 Flp‐In cells transfected with hOCT2
  • HEK293 Flp‐In cells transfected with hOAT1
  • HEK293 Flp‐In cells transfected with hOAT3
  • Growth medium 2 (see recipe)
  • Radiolabeled probe substrates:
    • [14C]Tetraethyl ammonium (TEA) (ARC, cat. no. 0577; 50 µCi, 55 mCi/mmol, 0.1 mCi/ml); [14C] TEA at 50 µM final concentration as a positive control substrate for OCT2 transporter assay
    • [3H]p‐Aminohippuric acid (PAH) (Perkin Elmer, cat. no. NET053001MC; 4.54 Ci/mmol, 1 mCi/ml); [3H] PAH at 2 µM final concentration as a positive control substrate for OAT1 transporter assay
    • [3H]Estrone‐3‐sulfate (E‐3‐S) (NEN Life Science Products, cat. no. NET203250UC; 46 Ci/mmol, 1 mCi/ml); [3H]E‐3‐S at 100 nM final concentration as a positive control substrate for OAT1 transporter assay
  • Transport buffer (see recipe)
  • DPBS buffer (e.g., Invitrogen), ice cold
  • Pierce BCA Protein Assay kit (Thermo Scientific)
  • 1% SDS with 0.1% (v/v) 1 M NaOH in DPBS buffer
  • Scintillation fluid
  • 24‐well poly‐D‐lysine‐coated plates with covers (Biocoat)
  • 37°C heating block
  • Platform shaker
  • Scintillation vials
  • Scintillation counter
  • Statistical analysis program (e.g., GraphPad PRISM)

Basic Protocol 2: OAT1, OAT3, and OCT2 Renal Transporter–Mediated Inhibition (IC50) Assay

  Materials
  • NCEs to be evaluated as inhibitors for OAT1, OCT2, or OAT3 transporters
  • Appropriate positive control inhibitors
  • 37°C heated orbital shaker
  • PRISM 5.0 (GraphPad)
  • Additional reagents and equipment for HEK293 Flp‐In cells expressing the desired transporters (see protocol 1)

Support Protocol 1: Transfecting, Selecting, and Culturing HEK293 Cells for Transporter Assays

  Materials
  • Appropriate cDNAs for transfection (varies depending on the target protein to be expressed, e.g., hOCT2, NM_003058; hOAT1, AF097490; hOAT3, NM_004254)
  • pcDNA5/FRT vector
  • HEK293 Flp‐In cells (Invitrogen)
  • LipofectAMINE 2000 (Invitrogen)
  • Hygromycin
  • Specified media (see recipe)

Support Protocol 2: Sample Processing and Quantification for Both Scintillation Counting and LC‐MS/MS

  Materials
  • Samples in plates ready for processing (see Basic Protocols protocol 11 and protocol 22)
  • 100% methanol
  • Internal standard for LC/MS‐MS (IS and source for IS vary depending on compound analyzed)
  • Nitrogen stream
  • Platform shaker
  • 96‐well, deep‐well plate
  • LC‐MS/MS
  • Curve‐fitting statistical software (e.g., PRISM 5.0, GraphPad)
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Figures

Videos

Literature Cited

Literature Cited
   Cha, S.H., Sekine, T., Fukushima, J.I., Kanai, Y., Kobayashi, Y., Goya, T., and Endou, H. 2001. Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol. 59:1277‐1286.
   Feng, B., LaPerle, J.L., Chang, G., and Varma, M.V. 2010a. Renal clearance in drug discovery and development: Molecular descriptors, drug transporters and disease state. Expert Opin. Drug Metab. Toxicol. 6:939‐952.
   Feng, B., LaPerle, J.L., Chang, G., and Varma, M.V. 2010b. Renal clearance in drug discovery and development: Molecular descriptors, drug transporters and disease state. Expert Opin. Drug Metab. Toxicol. 6:939‐952.
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   Matsuzaki, T., Morisaki, T., Sugimoto, W., Yokoo, K., Sato, D., Nonoguchi, H., Tomita, K., Terada, T., Inui, K., Hamada, A., and Saito, H. 2008. Altered pharmacokinetics of cationic drugs caused by down‐regulation of renal rat organic cation transporter 2 (Slc22a2) and rat multidrug and toxin extrusion 1 (Slc47a1) in ischemia/reperfusion‐induced acute kidney injury. Drug Metab. Dispos. 36:649‐654.
   Miyazaki, H., Sekine, T., and Endou, H. 2004. The multispecific organic anion transporter family: Properties and pharmacological significance. Trends Pharmacol. Sci. 25:654‐662.
   Mulato, A.S., Ho, E.S., and Cihlar, T. 2000. Nonsteroidal anti‐inflammatory drugs efficiently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1. J. Pharmacol. Exp. Ther. 295:10‐15.
   Okuda, M., Saito, H., Urakami, Y., Takano, M., and Inui, K. 1996. cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2. Biochem. Biophys. Res. Commun. 224:500‐507.
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