Biotransformation Studies Using Rat Proximal Tubule Cells

G. J. Schaaf1, R. F. M. Maas1, J. Fink‐Gremmels1

1 Utrecht University, Utrecht
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.14
DOI:  10.1002/0471140856.tx0414s21
Online Posting Date:  September, 2004
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Abstract

The proximal tubule is the main target for nephrotoxic substances due to its specific properties, including efficient drug transport and biotransformation potential. The availability of a pure population of proximal tubule cells (PT cells) as a model to study a range of biological, pharmacological, and toxicological parameters is, therefore, of great value. A two‐step PT cell–isolation procedure, based on density‐gradient centrifugation, is described; this procedure can easily be introduced into each laboratory setting. The procedure routinely yields a highly pure PT cell population, comprising 20–40 × 106 cells, which can be used for preparation of subcellular fractions or brought into primary culture.

Keywords: Proximal tubule cell isolation; biotransformation; primary cultures

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Biotransformation Enzyme Activity in Primary Cultured PT Cells
  • Basic Protocol 2: Measuring CYP450 Activity using Sub‐Cellular PT Cell Fractions
  • Support Protocol 1: Isolation of Rat Proximal Tubule Cells
  • Support Protocol 2: Establishing Primary PT Cell Cultures
  • Support Protocol 3: Preparation of Microsomal and Cytosolic Fractions from PT Cells
  • Support Protocol 4: Determining Cell Viability and Number of Isolated PT Cells
  • Support Protocol 5: Determining the Purity of PT Cell Preparation by Selective Uptake of Fluorescein
  • Support Protocol 6: Coating Cell Culture Surfaces
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Biotransformation Enzyme Activity in Primary Cultured PT Cells

  Materials
  • Substrate stock solutions (Table 4.14.1)
  • Serum‐free culture medium (see recipe)
  • Primary cultured rat proximal tubule cells (plated at 5 × 104 cells/cm2 in. 60‐mm dishes; see protocol 4)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Rubber policeman

Basic Protocol 2: Measuring CYP450 Activity using Sub‐Cellular PT Cell Fractions

  Materials
  • 2× concentrated cofactor mixture (see recipe)
  • Subcellular fractions (microsomal suspension containing ∼100 µg protein; see Support Protocols protocol 31 and protocol 53)
  • Incubation buffer (see recipe)
  • Substrate stock solution
  • Internal standard (see Table 4.14.2)
  • Extraction fluid (see Table 4.14.2)
  • Nitrogen source
  • 10‐ml glass tubes with caps
  • 37°C water bath
NOTE: The substrate stock should preferably be prepared in incubation buffer. If the selected substrate is insoluble in buffer, DMSO, alcohol, or other solvents can be added. These solvents may have a direct effect on CYP450 activity (Hickman et al., ) so include solvent‐only controls when solvents are used.

Support Protocol 1: Isolation of Rat Proximal Tubule Cells

  Materials
  • 70% ethanol
  • 10× HH buffer (see recipe)
  • 50× EGTA stock solution (see recipe)
  • Collagenase B (Boehringer Mannheim)
  • Bovine serum albumin (BSA, fraction V; Sigma)
  • 0.5 M CaCl 2 (see recipe)
  • Wistar Hannover rats with an average body weight of 225 g (Charles River)
  • 100 mg/kg sodium pentobarbital solution, 60 mg/ml Nembutal, or any other anaesthetic drug
  • Heparin (5000 IE/ml)
  • 36% (w/v) Nycodenz stock solution (see recipe)
  • PBS (sterile, GIBCO)
  • Complete cell culture medium (see recipe)
  • Perfusion apparatus (see Fig. ) consisting of:
    • 37°C water bath
    • Electric pump
    • Bubble trap
    • Organ bath
    • Tubing
  • 100‐ml cylinder
  • 250‐ml bottles
  • 100‐ml bottles
  • 50‐ml bottle
  • 250‐ and 100‐ml beakers
  • 0.22‐µm sterile filters
  • Animal surgery table
  • Small set of surgical instruments including scissors, forceps, and clamps
  • 18 G, 51 mm Abbocath T canula (Venisystems)
  • 100‐mm petri dish
  • Nylon gauze filters (80‐µm)
  • 12‐ml tubes

Support Protocol 2: Establishing Primary PT Cell Cultures

  Materials
  • Cell suspension ( protocol 3)
  • Complete culture medium (see recipe)
  • Serum‐free DMEM/F12 medium (see recipe)
  • Collagen‐coated 60‐mm dishes (see protocol 8)
  • 37°C, 5% CO 2 incubator

Support Protocol 3: Preparation of Microsomal and Cytosolic Fractions from PT Cells

  Materials
  • Cell pellet, fresh or frozen
  • KCl/EDTA solution (see recipe)
  • Liquid nitrogen
  • Glycerol buffer (see recipe)
  • Refrigerated ultracentrifuge with fixed‐angle rotor
  • Potter‐Elvehjem homogenizer with PTFE pestle (5‐ml working capacity)
  • 8.5‐ml ultracentrige tubes (e.g., Nalgene UltraBottle Assemblies)
  • Ultra Turrax T8 homogenizer equipped with an 8‐mm diameter dispersing element

Support Protocol 4: Determining Cell Viability and Number of Isolated PT Cells

  Materials
  • PT cell suspension ( protocol 3)
  • 0.4% (w/v) trypan blue solution (Sigma) in PBS
  • 1.5‐ml microcentrifuge tubes
  • Hemacytometer with coverslips
  • Microscope

Support Protocol 5: Determining the Purity of PT Cell Preparation by Selective Uptake of Fluorescein

  Materials
  • Cell suspension ( protocol 3)
  • Serum‐free culture medium (see recipe)
  • 50 µM sodium fluorescein reagent (see recipe)
  • 200 mM sodium azide solution (see recipe)
  • PBS containing 5% (w/v) BSA
  • 37°C water bath
  • Fluorescence microscope: excitation wavelength between 470 and 490 nm, emission wavelength between 520 and 555 nm

Support Protocol 6: Coating Cell Culture Surfaces

  Materials
  • Collagen solution (see recipe)
  • PBS (GIBCO)
  • 60‐mm culture dishes, culture flasks, or culture plates
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Figures

Videos

Literature Cited

   Baer, P.C., Nockher, W.A., Haase, W., and Scherberich, J.E. 1997. Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. Kidney Int. 52:1321‐1331.
   Blackmon, D.L., Watson, A.J., and Montrose, M.H. 1992. Assay of apical membrane enzymes based on fluorogenic substrates. Anal. Biochem. 200:352‐358.
   Blackmore, M. 1996. Rat renal cortical slices: Maintenance of viability and use in in vitro nephrotoxicity testing. Toxicol. In Vitro. 11:723‐729.
   Boom, S.P., Gribnau, F.W., and Russel, F.G. 1992. Organic cation transport and cationic drug interactions in freshly isolated proximal tubular cells of the rat. J. Pharmacol. Exp. Ther. 263:445‐450.
   Burke, M.D. and Mayer, R.T. 1974. Ethoxyresorufin: Direct fluorimetric assay of a microsomal O‐dealkylation which is preferentially inducible by 3‐methylcholanthrene. Drug Metab. Dispos. 2:583‐588.
   Burke, M.D., Thompson, S., Weaver, R.J., Wolf, C.R., and Mayer, R.T. 1994. Cytochrome P450 specificities of alkoxyresorufin O‐dealkylation in human and rat liver. Biochem. Pharmacol. 48:923‐936.
   Butler, M.A., Iwasaki, M., Guengerich, F.P., and Kadlubar, F.F. 1989. Human cytochrome P‐450PA (P‐450IA2), the phenacetin O‐deethylase, is primarily responsible for the hepatic 3‐demethylation of caffeine and N‐oxidation of carcinogenic arylamines. Proc. Natl. Acad. Sci. U.S.A. 86:7696‐7700.
   Commandeur, J.N., Stijntjes, G.J., Wijngaard, J., and Vermeulen, N.P. 1991. Metabolism of L‐cysteine S‐conjugates and N‐(trideuteroacetyl)‐L‐cysteine S‐conjugates of four fluoroethylenes in the rat. Role of balance of deacetylation and acetylation in relation to the nephrotoxicity of mercapturic acids. Biochem. Pharmacol. 42:31‐38.
   Commandeur, J.N., Stijntjes, G.J., and Vermeulen, N.P. 1995. Enzymes and transport systems involved in the formation and disposition of glutathione S‐conjugates. Role in bioactivation and detoxication mechanisms of xenobiotics. Pharmacol. Rev. 47:271‐330.
   Cox, P.G.F., Van Os, C.H., and Russel, F.G.M. 1993. Accumulation of salicylic acid and indomethacin in isolated proximal tubular cells of the rat kidney. Pharmacol. Res. 27.
   Dayer, P., Leemann, T., and Striberni, R. 1989. Dextromethorphan O‐demethylation in liver microsomes as a prototype reaction to monitor cytochrome P‐450 db1 activity. Clin. Pharmacol. Ther. 45:34‐40.
   Habig, W.H., Pabst, M.J., and Jakoby, W.B. 1974. Glutathione S‐transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249:7130‐7139.
   Hickman, D., Wang, J.P., Wang, Y., and Unadkat, J.D. 1998. Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab. Dispos. 26:207‐215.
   Knodell, R.G., Hall, S.D., Wilkinson, G.R., and Guengerich, F.P. 1987. Hepatic metabolism of tolbutamide: Characterization of the form of cytochrome P‐450 involved in methyl hydroxylation and relationship to in vivo disposition. J. Pharmacol. Exp. Ther. 241:1112‐1119.
   Schaaf, G.J., de Groene, E.M., Maas, R.F., Commandeur, J.N., and Fink‐Gremmels, J. 2001. Characterization of biotransformation enzyme activities in primary rat proximal tubular cells. Chem. Biol. Interact. 134:167‐190.
   Seglen, P.O. 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29‐83.
   Stanton, R.C., Mendrick, D.L., Rennke, H.G., and Seifter, J.L. 1986. Use of monoclonal antibodies to culture rat proximal tubule cells. Am. J. Physiol. 251:780‐786.
   Takenaka, M., Imai, E., Kaneko, T., Ito, T., Moriyama, T., Yamauchi, A., Hori, M., Kawamoto, S., and Okubo, K. 1998. Isolation of genes identified in mouse renal proximal tubule by comparing different gene expression profiles. Kidney Int. 53:562‐572.
   Wortelboer, H.M., de Kruif, C.A., van Iersel, A.A., Falke, H.E., Noordhoek, J., and Blaauboer, B.J. 1990. The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochem. Pharmacol. 40:2525‐2534.
   Wortelboer, H.M., de Kruif, C.A., van Iersel, A.A., Noordhoek, J., Blaauboer, B.J., van Bladeren, P.J., and Falke, H.E. 1992. Effects of cooked brussels sprouts on cytochrome P‐450 profile and phase II enzymes in liver and small intestinal mucosa of the rat. Food Chem. Toxicol. 30:17‐27.
Key References
   Cummings, B.S., Zangar, R.C., Novak, R.F., and Lash, L.H. 1999. Cellular distribution of cytochromes P‐450 in the rat kidney. Drug Metab. Dispos. 27:542‐548.
  Provides the first overview of CYP450 (protein) expression in rat proximal and distal tubular cells determined in microsomal fractions.
   Masereeuw, R., van den Bergh, E.J., Bindels, R.J., and Russel, F.G. 1994. Characterization of fluorescein transport in isolated proximal tubular cells of the rat: Evidence for mitochondrial accumulation. J. Pharmacol. Exp. Ther. 269:1261‐1267.
  Describes the technique of isolation of proximal tubualr cells for short term use in suspension (ro measure cellular uptake/transport) in detail.
   Schaaf et al., 2001. See above.
  An overview of phase 1 and phase 2 biotransformation activities in primary cultured proximal tubule\ar cells.
Internet Resources
   www.gentest.com
  This site provides relevant examples of model substrates for the measurement of CYP450 acriviry.
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