Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney.
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 gamma-glutamyltransferase, gamma-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.
Cummings
B S
BS
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201-1928, USA.
Lasker
J M
JM
Lash
L H
LH
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004798:Enzymes / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005978:Glutathione / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007633:Keratins / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008875:Middle Aged; D004364:Pharmaceutical Preparations / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D004798:Enzymes; D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D014746:Vimentin; D007633:Keratins; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione