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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Expression of Glutathione-Dependent Enzymes and Cytochrome P450s in Freshly Isolated and Primary Cultures of Proximal Tubular Cells from Human Kidney

Brian S. Cummings, Jerome M. Lasker and Lawrence H. Lash
Journal of Pharmacology and Experimental Therapeutics May 2000, 293 (2) 677-685;
Brian S. Cummings
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Jerome M. Lasker
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Lawrence H. Lash
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Abstract

The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of γ-glutamyltransferase, γ-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.

Footnotes

  • Send reprint requests to: Dr. Lawrence H. Lash, Department of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield Ave., Detroit, MI 48201-1928. E-mail: l.h.lash{at}wayne.edu

  • ↵1 This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK40725 and National Institute of Environmental Health Sciences Grant R01-ES08828 (to L.H.L.) and National Institute of Alcohol Abuse and Alcoholism Grant R01-AA07842 (to J.M.L.).

  • ↵2 Current address: Division of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 638, Little Rock, AR 72205-9985.

  • ↵3 The standard nomenclature for P450 isoenzymes described in Nelson et al. (1996) is used in this study.

  • Abbreviations:
    P450
    cytochrome P450
    GST
    glutathioneS-transferase
    GSH
    glutathione
    hPT
    human proximal tubular
    GPX
    glutathione peroxidase
    GRD
    glutathione disulfide reductase
    GCS
    γ-glutamylcysteine synthetase
    GGT
    γ-glutamyltransferase
    PAGE
    polyacrylamide gel electrophoresis
    FITC
    fluorescein isothiocyanate
    • Received October 8, 1999.
    • Accepted January 7, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 293 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 293, Issue 2
1 May 2000
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Expression of Glutathione-Dependent Enzymes and Cytochrome P450s in Freshly Isolated and Primary Cultures of Proximal Tubular Cells from Human Kidney

Brian S. Cummings, Jerome M. Lasker and Lawrence H. Lash
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 677-685;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Expression of Glutathione-Dependent Enzymes and Cytochrome P450s in Freshly Isolated and Primary Cultures of Proximal Tubular Cells from Human Kidney

Brian S. Cummings, Jerome M. Lasker and Lawrence H. Lash
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 677-685;
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