A High‐Throughput Screening Assay to Identify Kidney Toxic Compounds

Susanne Ramm1, Melanie Adler1, Vishal S. Vaidya2

1 Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, 2 Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.10
DOI:  10.1002/cptx.12
Online Posting Date:  August, 2016
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Abstract

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high‐throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO‐1) protein expression is upregulated as a protective mechanism during kidney toxicant‐induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO‐1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO‐1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.

Keywords: biomarker; in vitro; heme oxygenase 1; high‐throughput; HTRF; primary human proximal tubular epithelial cells; screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Throughput HTRF Assay for HO‐1 in Kidney Cells
  • Basic Protocol 2: An Immunofluorescent‐Based Screen to Identify Toxic Compounds
  • Support Protocol 1: Thawing, Expansion, and Seeding of Kidney Cells
  • Support Protocol 2: Addition of Compound Libraries for Dose‐Response Curves
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: High‐Throughput HTRF Assay for HO‐1 in Kidney Cells

  Materials
  • HTRF lysis buffer (LB1; Cisbio Bioassays)
  • 100× protease inhibitor cocktail (Cell Signaling Technology)
  • HTRF detection buffer (Cisbio Bioassays)
  • Europium cryptate (k)‐labeled rabbit monoclonal HO‐1‐antibody (CST #5853‐k) (Cell Signaling Technology)
  • D2‐conjugated rabbit polyclonal anti HO‐1‐antibody (CST #5061‐d2) (Cell Signaling Technology)
  • Cadmium chloride (Sigma Aldrich)
  • Microlab STARlet liquid handling system (Hamilton)
  • Pin transfer robot equipped with 384 pipetting heads (Seiko)
  • EL 406 washer dispenser & Biostack 3 microplate stacker (Bio‐Tek)
  • White low volume 384‐well plates (Greiner Bio One)
  • 1‐µl cassette with 8‐tips and plastic tubes for peristaltic pump of EL406 plate washer and dispenser
  • 8‐channel pipets, optional
  • Microseal Foil (BioRad)
  • Titer plate shaker (Lab Line Instruments)
  • Allegra X‐14R Centrifuge (Beckmann Coulter)
  • SpectraMax Paradigm (Molecular Devices) equipped with:
  • Paradigm Cisbio HTRF cartridge

Basic Protocol 2: An Immunofluorescent‐Based Screen to Identify Toxic Compounds

  Materials
  • 37% Paraformaldehyde (PFA; Sigma)
  • Phosphate‐buffered saline (PBS; Gibco, cat. no. 10010049), pH 7.4
  • Methanol (Sigma)
  • Odyssey blocking buffer (LiCor)
  • Mouse monoclonal anti‐HO‐1 antibody (Santa Cruz)
  • Goat anti‐mouse antibody (Alexa 555) (Life Technologies)
  • Mouse IgG Isotype control (Thermo Fisher Scientific)
  • Hoechst 33342
  • EL 406 washer dispenser & Biostack 3 microplate stacker (Bio‐Tek)
  • Straight 24‐place stainless steel Manifold for Microtest Plates for 384‐well plates (Drummond)
  • Microseal Foil for IF (BioRad)
  • Operetta high‐content microscope with plate handler (Perkin Elmer)

Support Protocol 1: Thawing, Expansion, and Seeding of Kidney Cells

  Materials
  • Collagen IV solution (see recipe)
  • Sterile‐distilled water
  • Primary human proximal tubule epithelial cells (HPTECs) (Biopredic) (alternative: Lonza, ATCC)
  • FBS medium based on DMEM/hAM F12 with GlutaMAX (Invitrogen; see recipe)
  • Growth medium based on DMEM/hAM F12 with GlutaMAX (Invitrogen; see recipe)
  • 1× PBS (phosphate buffered saline) (‐/‐) (Corning)
  • 0.25% Trypsin/EDTA (1 ×) (Gibco)
  • 25‐cm2 and 75‐cm2 flasks (Sigma)
  • 37°C incubator
  • Water bath
  • Centrifuge
  • Hemacytometer or automated cell counter (TC20) (Bio‐Rad)
  • 384‐well plates (Poly‐D‐lysine‐coated, black, clear bottom) (Corning)
  • Multidrop Combi Reagent Dispenser (Thermo Scientific)
  • 50‐ml sterile reagent reservoirs (Corning)
  • 10‐ to 100‐µl 12‐channel Multi‐Channel Pipets (Eppendorf)
  • Standard Tube Dispensing Cassette (Thermo Scientific)
  • Straight 24‐place stainless steel Manifold for Microtest Plates for 384‐well plates (Drummond)

Support Protocol 2: Addition of Compound Libraries for Dose‐Response Curves

  Materials
  • 68 compounds from SCREEN‐WELL Nephrotoxicity library (Enzo, cat. no. ENZ‐LIB100‐0100)
  • 100% dimethyl sulfoxide (DMSO; Sigma)
  • 96‐well library screening plates (ABGene)
  • Microlab STARlet liquid handling system (Hamilton)
  • 8‐Channel Transferpette, 0.5 to 10 µl (BrandTech)
  • 384‐well pin transfer plates (V&P Scientific)
  • Pin transfer robot equipped with 384 pipetting heads (Seiko)
  • Humidity‐controlled incubator
NOTE: The 68 compounds include mainly nephrotoxic compounds (Cadmium chloride was considered a positive control), but also a limited number of heptotoxic compounds (e.g., Troglitazone), and ten nontoxic controls (e.g., Dexamethasone, Carboplatin, Methylparaben, Atenolol, Aspirin).
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
  http://iccb.med.harvard.edu
  Homepage of the ICCB‐L screening facility at the Harvard Medical School with available compound libraries.
  http://www.enzolifesciences.com/ENZ‐LIB100/screen‐well‐nephrotoxicity‐library/
  Focused nephrotoxicity library used for 8‐point dose‐response curves of 65 compounds.
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