ies have demonstrated that in oivo compound potency differences are reflected in vitro, and rat hepatocyte cultures have been used to investigate structure-activity relationships for peroxisome proliferation, 6°-62 which presumably also holds for P450IVA induction. Another valuable use of hepatocytes is to investigate species differences in response. Studies with primary hepatocyte cultures from the rat, mouse, Syrian hamster, Chinese hamster, dog, guinea pig, and primates have demonstrated good in vivo/ in vitro correlations in induction. 24'46'52,63-68Human hepatocyte cultures have been reported not to respond to these compounds.46,63,6s Acknowledgments We thank the S.E.R.C., M.R.C., Wellcome Trust, and the U.K. Ministry of Agriculture, Fisheries, and Food for financial support of our studies. 61 B. G. Lake, T. J. B. Gray, D. F. V. Lewis, J. A. Beamand, K. D. Hodder, R. Purchase, and S. D. Gangolli, Toxicol. Ind. Health 3, 165 (1987). 62 p. I. Eacho, P. S. Foxworthy, R. D. Dillard, C. A. Whitesitt, D. K. Herron, and W. S. Marshall, Toxicol. Appl. Pharmacol. 100, 177 (1989). 63 C. R. Elcombe, Arch. Toxicol. Suppl. 8, 6 (1985). 64 B. G. Lake, T. J. B. Gray, P. Sjoberg, K. D. Hodder, J. A. Beamand, C. R. Stubberfield, and S. D. Gangolli, Food Chem. Toxicol. 24, 573 (1986). F. Bieri, W. Staubli, F. Waechter, S. Muakkassah-Kelly, and P. Bentley, Cell Biol. Int. Rep. 12, 1077 (1988). 66 E. G. Butler, P. J. England, and G. M. Williams, Carcinogenesis 9, 1459 (1988). 67 p. S. Foxworthy, S. L. White, D. M. Hoover, and P. I. Eacho, Toxicol. Appl. Pharmacol. 104, 386 (1990). 6s B. J. Blaauboer, C. W. M. Van Holsteijn, R. Bleumink, W. C, Mennes, F. N. A. M. Van Pelt, S. H. Yap, J. F. Van Pelt, A. A. J. Van Iersel, A. Timmerman, and B. P. Schmid, Biochem. Pharmacol. 40, 521 (1990).

[35] Cytochrome P450 Expression and Metabolism in Isolated Rabbit Renal Epithelium By D E N N I S R . KOOP, R O N A L D M . L A E T H E M , A R T H U R L . GOLDNER, a n d JANICE G . DOUGLAS

Introduction The kidney is a heterogeneous organ with respect to transport function, bioelectric properties, distribution of hormone receptors, and enzymes, including the cytochrome P450-dependent mixed-function oxidase system. The cytochromes P450 present in the kidney include isozymes that may METHODSIN ENZYMOLOGY,VOL. 206

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be categorized as being primarily involved in the biotransformation of xenobiotics. In many instances, the potential toxicity of a compound is dependent on the isozymes of P450 present in a particular cell.1 The second class of isozymes present in the kidney are those involved in biosynthetic reactions such as the hydroxylation of arachidonic acid and 25-hydroxycholecalciferol. Both general classes of isozymes have a significant role in the function of the kidney. Although the expression of both classes of P450 is not clearly linked, it is possible that the expression and induction of the biotransformation class of isozymes by environmental exposures may affect the expression and/or activity of all the biosynthetic isozymes. Since chemical toxicity is region specific within the nephron (i.e., acetaminophen exhibits toxicity in the proximal tubule2), it is important to identify the distribution and activity of distinct P450 isozymes across the nephron. An uneven distribution of total spectrally determined P450 was reported in 1978. 3 The direct analysis for distinct isozymes involved in the biotransformation of xenobiotics in rabbit renal tissue by immunohistochemical methods was reported by Dees e t al. 4 Antibodies to P450IAl and P450IA2, P450IIC3 (subsequently identified as P450K or P450IIC25), and P450IIB4 were used. The distribution and inducibility of the enzymes were distinct; immunoreactive material was identified in the $2 and S 3 portions of the proximal nephron. There was no staining observed in regions identified as the S 1 portion by antibodies to the various P450 isozymes, but there was significant staining observed with antibody to cytochrome-P450 reductase.4 The failure to observe P450 in the S~ segment of early proximal tubule may have been due to the relative concentration of specific isozymes in the various regions of the nephron. The oxidation of arachidonic acid leads to the formation of a variety of products which exhibit unique pharmacological properties 6 as described in more detail elsewhere in this volume. 7 The cytochrome P450 system transforms arachidonic acid into a variety of oxygenated products including monohydroxyeicosatrienoic acids, epoxyeicosatrienoic acids, and the 1 F. P. Guengerich, Crit. Rev. Biochem. Mol. Biol. 25, 97 (1990). 2 j. Mohandas, G. G. Duggin, J. S. Horvath, and D. J. Tiller, Toxicol. Appl. Pharmacol. 61, 252 0981). 3 T. V. Zenser, M. B. Mattammal, and B. B. Davis, J. Pharmacol. Exp. Ther. 207, 719 (1978). 4 j. H. Dees, B. S. S. Masters, U. Muller-Eberhard, and E. F. Johnson, Cancer Res. 42, 1423 (1982). 5 M. J. Finlayson, B. Kemper, N. Browne, and E. F. Johnson, Biochem. Biophys, Res. Commun. 141, 728 (1986). 6 p. Needleman, J. Turk, B. A. Jakschik, A. R. Morrison, and J. B. Lefkowith, Annu. Rev. Biochem. 55, 69 (1986). 7 j. H. Capdevila, E. Dishman, A. Karara, and J. R. Falck, this volume [42].




to- and to-l-hydroxyeicosatrienoic acids. 6,7 The formation of the products is regiospecific in the kidney, and thus isozymes catalyzing the hydroxylations are unevenly distributed in the tubular epithelium. For example, arachidonic acid is metabolized predominately to epoxides in the cortex, whereas in the medulla the toko-1- hydroxylated metabolites are the principal products. 8 As a result of the importance of cell-specific expression of P450, it is important to be able to perform analysis on cells from specific regions of the nephron. A primary goal in our laboratories is to isolate cells that retain the biological and physiological properties exhibited in vivo. The use of isolated cells permits both the identification of P450s immunochemically and the measurement of enzyme activity. The purpose of this chapter is to describe the isolation of renal cortical epithelial cells by Percoll density gradient centrifugation into fractions enriched in proximal epithelial cells and more distal segments including cortical ascending limb cells and collecting duct cells. We also describe characterization of the cells, detection of P450 isozymes by immunoblot analysis of whole cell lysates, and the use of the isolated cells for the metabolism of arachidonic acid. Isolation of Cortical Epithelial Cells Epithelial cells are routinely prepared from 2-kg New Zealand White male rabbits (Hazelton, Denver, PA, barrier sustained). The animals are placed in quarantine and acclimated to the environment for at least I week prior to the cell isolation procedure. The animals are left untreated or treated with inducers such as Aroclor 1254 (generous gift of Dr. Elena McCoy, Case Western Reserve University) or acetone by reported procedures prior to cell isolation. 9 Pretreatment does not affect the isolation procedure as described below. Rabbits are injected with 12,000 units of heparin via the marginal ear vein followed in 15 min by a lethal dose of Nembutal. The kidneys are removed as rapidly as possible and placed in oxygenated Dulbecco's modified Eagle's medium (DMEM) from GIBCO (Grand Island, NY) at 4° during transport. Each kidney is perfused to remove blood with 20 ml of Collins' solution (140 mM dextrose, 15 mM potassium chloride, 15 mM potassium phosphate, monobasic, 19 mM sodium bisulfite, 61 mM magnesium chloride, and 10 mM sodium bicarbonate; the preparation is described in detail in Ref. I0) containing 10% (v/v) fetal calf serum (FCS), 8 K. Takahashi, J. Capdevila, A. Karara, J. R. Falck, H. R. Jacobson, and K. F. Badr, Am. J. Physiol. 258, F781 (1990). 9 G. G. Schnier, C. L. Laethem, and D. R. Koop, J. Pharmacol. Exp. Ther. 251, 790 (1989). 10 C. M. Nagineni, P. J. Leveille, D. B. Lee, and N. Yanagawa, Biochem. J. 223, 353 (1984).





Cell Type f cell debris collecting duct


proximal tubule

ascen ascending limb

~_/) RBCs FIG. 1. Schematic representation of the pattern of resolution of cortical epithelial cells following separation on a Percoll density gradient.

5 /zg/ml insulin and transferrin, and 26/zg/ml DNase employing an 18gauge angiocath. Kidneys are perfused a second time with an equal volume of the same solution containing 5 mM EGTA. The cortex is then removed with a Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro, N J). Cortex remaining around the medulla is carefully removed with a scalpel and placed in ice-cold Collins' solution without EGTA. The cortex is minced with a scalpel into coarse pieces and gently homogenized in 40 ml of Collins' solution with a Dounce homogenizer with the B pestle (Wheaton, Millville, NJ) for 4 strokes. The homogenate is filtered through a series of Nitex filters (250, 53, and 25/zm, Tetko, Elmsford, NY) on ice to remove glomeruli and intact tubules. Cells which pass through the 25-/zm filter are washed twice with 10 ml of DMEM with DNase in sterile 50-ml Falcon tubes (Becton Dickinson Labware, Lincoln Park, N J). Yields are in the range of 25-40 x 107 cells/kidney. The cells from both kidneys are resuspended in 6 ml of DMEM, and 3 ml is layered on each of two discontinuous Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradients: 30% 10 ml, 40% 10 ml, 45% 8 ml, 50% 3 ml, and 60% 1 ml. The gradients are centrifuged at 1500 g for 15 min at 4° in a Du Pont Sorvall RC-5B (DuPont Co., Wilmington, DE) employing an HS-4 rotor. It is critical that the centrifugation step be performed reproducibly in order to obtain consistent cell fractions. A typical separation that is obtained is schematically shown in Fig. 1. Fraction 1, between 3 and 7 ml, is primarily collecting duct cells. Fraction 2, from 9.5 to 17 mi, is primarily proximal tubular in origin, and fraction 3, from 20 to 31 ml, is primarily ascending limb cells. The average cell yield for fraction 1 is 58.8 x 10 6 cells; fraction 2, 73.0 × 106 cells; and fraction 3, 18.3 x 10 6 cells.




Characterization of Cell Fractions

Electron micrographs of the epithelial cells were published previously and substantiate the sites of origin. 1~ Earlier studies documented that cyclooxygenase is localized to the distal nephron segment.~2 Consistent with this observation, we reported prostaglandin E2 (PGE2) production by collecting duct epithelial cells only. H By contrast, proximal tubular cells metabolized arachidonic acid to both epoxides and co/~-I metabolites. ~3 We also documented that ascending limb cells display P450-dependent arachidonic acid metabolism consistent with studies reported by Schwartzman et al. ~4Additional characterization of these epithelial cells demonstrated that vasopressin stimulated cAMP production in collecting duct cells but not in proximal tubule cells, consistent with the known distribution of receptors. ~1

Immunoblot Analysis of Isolated Cells The isolated cells are pelleted in a 1.5-ml microcentrffuge tube at 500 g for 5 rain at 4 °. The supernatant is removed by careful aspiration; if the cell pellet is not used immediately for sodium dodecyl sulfate (SDS)polyacrylamide gel electrophorcsis 15or metabolic assays, it can be stored at - 7 0 ° for several months. The cell pellet is dissolved in SDS gel sample buffer which contains 70 mM Tris-acetate, pH 6.8, 4% S DS (w/v), 0.001% (w/v) pyronin Y, 25% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, and 5 /~g/ml each of leupeptin and aprotinin. The final concentration of cells can vary, but we usually do not exceed a final concentration of l0 × 106 cells/ml. The sample is placed in a boiling water bath for 2 rain, then cooled at room temperature. It is often impossible to pipette the samples at this point as a result of the high viscosity of the DNA. The DNA is sheared by sonication in a Heat Systems sonicator with a water-cooled cup horn probe (Heat Systems-Ultrasonics, Plainview, NY) at a power setting of 5 for 2-3 rain. The DNA can also be sheared by passing the sample through an 18-gauge then a 25-gauge, needle 3 times cach. The latter procedure is more time consuming because only one sample can be manipulated at a time. It is possible to sonicate four samples in the cup horn simultaneously. It C. Welsh, G. Dubyak, and J. G. Douglas, J. Clin. Invest. 81, 710 (1988). 12 M. Irnbert-Teboul, S. Siaume, and F. Morel, MoL Cell. Endocrinol. 45, 1 (1986). 13R. M. Laethem, C. L. Laethem, and D. R. Koop, unpublished observations. 14 M. Schwartzman, N. R. Ferreri, M. A. Carrol, E. Songu-Mize, and J. C. McGiff, Nature (London) 314, 620 (1985). 15 U. K. Laemrnli, Nature (London) 227, 680 (1970).




The sheared samples are diluted as necessary in sample diluting buffer prior to application to a 7.5% polyacrylamide gel. For minigels the samples are adjusted to yield from 5000 to 100,000 cells/20/xl of sample to be applied to the gel. Serial dilutions are made from the original stock solution, and the same volume of sample is applied to each well to ensure consistent lane width for accurate quantification. After electrophoresis, the samples are transferred to nitrocellulose filters and immunochemically stained with the desired antibody. 16,17We have screened the cell fractions with a variety of polyclonal and monoclonal antibodies and have not found it necessary to change the protocol for immunoblot analysis from that used for microsomal samples. 16.17The number of solubilized cells needed for detectable signal depends on many factors including the antibody, the detection system, and the pretreatment of the rabbit from which the cells were isolated. For each sample, the optimal concentrations must be determined. Figure 2 shows the results of a typical analysis in which ceils from the Percoll gradient fraction 3 from untreated or Aroclor 1254-treated rabbits were screened for P450IIE1 and P450IA1. P450IIE1 was detectable in as few as 30,000 ceils from untreated rabbits, whereas P450IA1 was readily detectable in as few as 5000 cells from Aroclor 1254-treated rabbits. Catalytic Activity of Isolated Cells The metabolism of arachidonic acid in the isolated cells is determined in an effort to correlate the metabolic activity with pharmacological parameters such as Ca 2+ and Na + transport, lsA9 Acutely isolated cells (5-20 x 106) are homogenized in 0.5 ml of 50 mM H E P E S buffer, pH 7.6, containing 5/zg/ml each of leupetin and aprotinin with 35 strokes of a 1-ml PotterElvehjem (0.004-0.006 inch clearance) homogenizer. The homogenate is kept ice cold during the procedure by placing the homogenizer in a 16 × 125 mm tube filled with a salt water-ice solution. A portion of the cell homogenate is then added to a 0.5 ml reaction mixture containing 50 mM HEPES, pH 7.6, 7.6/zM [14C]arachidonic acid (0.2/zCi, 52.8 mCi/mmol), 0.5 mM NADP, 10 mM glucose 6-phosphate, 10 mM MgCI2, and 1 unit of glucose-6-phosphate dehydrogenase, which is added to initiate the reaction. After 30 rain at 37°, the reactions are quenched by acidification to 16 D. R. Koop, B. L. Crump, G. D. Nordblom, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 82, 4065 (1985). 17 X. X. Ding, D. R. Koop, B. L. Crump, and M. J. Coon, Mol. Pharmacol. 30, 370 (1986). 18 M. F. Romero, U. Hopfer, Z. T. Madhan, W. Zhou, and J. G. Douglas, Renal Physiol. Biochem. 14, 199 (1991). 19M. Laniado-Schwartzman, K. L. Davis, J. C. McGiff, R. D. Levere, and N. G. Abraham, J. Biol. Chem. 263, 2536 (1988).




6 3a










FIG. 2. Immunoblot analysis of P450IIEI and P450IA1 in cells from the PercoU gradient fraction 3. Cells were submitted to immunoblot analysis as described in the text. Lanes a-d were loaded with cells from Arocolor 1254-treated rabbits (a, 20,000 cells; b, 15,000 cells; c, 10,000 cells; d, 5,000 cells). Lanes e - h were loaded with cells from untreated rabbits (e, 80,000 cells; f, 60,000 cells; g, 40,000 cells; h, 30,000 cells). Lane i was loaded with hepatic microsomes (I/xg) from acetone-treated rabbits. The positions of P450IIE 1 and P450IA 1 are indicated by 3a and 6, respectively. (From D. R. Koop, R. M. Laethem, and J. G. Douglas, unpublished results.)

pH 4.5 with 1 M citric acid. Arachidonic acid and metabolites are extracted from the mixture with 1 ml of ethyl acetate 3 times. The pooled organic layers are dried under argon and the residue dissolved in 100/zl of ethanol. The metaborites are separated by reversed-phase HPLC using an Ultrasphere C]s column (Beckman, San Ramon, CA) (4.6 x 250 ram) using a linear gradient of 1.23%/min from acetonitrile-water-acetic acid (49:51:0.1) to acetonitrile-acetic acid (100:0.1) at a flow rate of 1.0 ml/min. Radioactivity is monitored with a Radiomatic flow scintillation detector (Radiomatic Instrument and Chemical Co., Inc., Meriden, CT). Depending on the cell fraction and the pretreatment of the rabbit, metaborites are observed which correspond to the to/to-l-monohydroxylated metabolites as well as the monoepoxides and the corresponding diols. 6'7




Conclusions The identity of the isozymes catalyzing the epoxidation and the alkyl and aUylic hydroxylation of arachidonic acid in various regions of the nephron has not been determined, although purified hepatic and renal P450 isozymes from rats and rabbits can metabolize arachidonic acid.~-2~ We are currently isolating arachidonic acid monooxygenases from cortical microsomes and are using the isolated cell preparations to determine the distribution of these isozymes across the nephron. The catalytic activity of the isozymes in isolated cells is also being examined. We feel that through the use of the isolated cell preparations we will be able to characterize the role specific P450 isozymes have in arachidonic acid-mediated signal transduction in renal epithelial cells. Similarly, the isolated cell preparations provide a valuable system to monitor the activity of the biotransformation isozymes and more carefully examine their role in regiospecific toxicity along the nephron. Acknowledgments We would like to acknowledge the technical assistance of Carmen Laethem, Carson White, and Chris Erhart in many aspects of these studies. Research performed in our laboratories was supported by U.S. Public Health Service Grants HL22990, HL39012, and HL41618 (J.G.D.) and AA07219 (DRK). 2o S. Tanaka, S. Imaoka, E. Kusunose, M. Kusunose, M. Maekawa, and Y. Funae, Biochim. Biophys. Acta 1043, 177 (1990). 21 R. K. Sharma, M. V. Doig, D. F. V. Lewis, and G. G. Gibson, Biochem. Pharmacol. 38, 3621 (1989).

[36] C u l t u r i n g S t e r o i d o g e n i c Cells

By PETER J. HORNSBY and JAN M. MCALLISTER Introduction Cultures of steroidogenic cells have been invaluable in many studies of the molecular biology, cell biology, and physiology of steroidogenic tissues. Here, procedures for preparation, growth, and storage of steroidogenic cells from the adrenal cortex, ovary, and testis are described. Procedures for culturing cells from tumor tissue are not described; the culture and use of the Y1 adrenocortical cell line was described in an earlier METHODSIN ENZYMOLOGY,VOL. 206

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Cytochrome P450 expression and metabolism in isolated rabbit renal epithelium.

364 CELL CULTURE SYSTEMS [35] ies have demonstrated that in oivo compound potency differences are reflected in vitro, and rat hepatocyte cultures h...
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