Introduction

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The mammalian kidney is a complex organ composed of numerous different cell types that function together to facilitate the filtering of blood and the regulation of systemic blood pressure (Guyton, 1991; Tisher and Madsen, 1996). The mammalian kidney also can oxidize and conjugate drugs because xenobiotic-metabolizing enzyme expression and/or activity have been reported in this tissue (for review, see Lohr et al., 1998). Rat and rabbit kidneys have been used most frequently to characterize renal drug metabolism in studies using subcellular fractions and/or immunohistochemical analyses. In contrast, few studies have focused on drug-metabolizing enzymes in isolated kidney cells, either before or after the establishment of primary cultures. We recently determined P450 hemoprotein and glutathione S-transferase (GST) isoform expression in freshly isolated renal proximal tubular cells from male Fischer 344 rats (Cummings et al., 1999, 2000) and found that these cells contained CYP2B1/2,³ CYP2C11, CYP2E1, CYP4A2/3, and GST but not CYP3A1/2, GSTµ, or GST. After primary culture, expression of the P450 enzymes markedly decreased, whereas expression of GST was maintained at levels close to or only modestly lower than those found in freshly isolated cells (Lash et al., 1995; Cummings et al., 1997).

There have been several reports on select aspects of renal drug metabolism in humans (Hayes and Pulford, 1995; Amet et al., 1997; Rodilla et al., 1998), and primary cultures of human kidney cells originating mainly from the proximal tubules have been used for a number of years in both physiological and toxicological studies (Detrisac et al., 1984; Trifillis et al., 1985; Kempson et al., 1989; Chen et al., 1990; Garrett et al., 1998). However, the composition of P450 hemoproteins and glutathione (GSH)-dependent enzymes in the human kidney, freshly isolated human proximal tubular (hPT) cells, and/or cultures of hPT cells have been poorly characterized. A description of the drug-metabolizing properties of human kidney cells could provide not only a better explanation of their biochemical and physiological responses to xenobiotics but also could decrease the uncertainty involved in extrapolating such data obtained in animals to humans. Development of an in vitro model that mimics human kidney cell function in vivo would have obvious benefits in pharmacology and the study of toxicological risk assessment. Although cell lines are available and human kidney cells can be immortalized (Taub, 1996), these various models use transformed cell lines that, at least with regard to drug metabolism, may no longer be suitable for comparison to the in vivo state.

The goals of this study were to determine the expression of exemplary drug-metabolizing enzymes in hPT cells isolated from kidney samples and to assess the effects of primary culture on these enzymes. Freshly isolated hPT cells were found to express high levels of certain P450s and GSH-dependent enzymes. Although primary hPT cell cultures exhibited a decrease in activity and/or content of most of these enzymes, enhanced expression of one enzyme, namely CYP4A11, was observed in cultures exposed to ethanol and/or dexamethasone. Our data indicate that hPT cells can serve as a suitable model for studies of human renal drug metabolism, and that primary cultures of these cells can be used to study factors regulating renal P450 enzyme expression.

	Materials and Methods

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Chemicals. Unless otherwise stated, all chemicals used were purchased from Sigma Chemical Co. (St Louis, MO). 1-[¹⁴C]Lauric acid (specific activity = 40 mCi/mmol) was purchased from ICN Pharmaceuticals, Inc. (Irvine, CA) and exhibited radiochemical purity >99%, as assessed by thin-layer chromatography. Antibodies for CYP2E1, GSTA, GSTP, and GSTM were purchased from Oxford Biomedical (Oxford, MI). The antibody for GSTT was purchased from Biotrin International (Newton, MA). Note that the convention used for naming GST isoforms is that Greek letters are used for rat enzymes, whereas Arabic letters are used for the analogous human enzymes.

Isolation of Microsomes and Cytosol from Human Kidney Homogenates. Freshly isolated slices of human kidney cortex were obtained from the Human Tissue Resources Core of the Department of Pathology, Harper Hospital (Detroit, MI). Human kidney samples were obtained from 15 female (age = 60.9 ± 11.8; mean ± S.D.; range = 45-77) and 11 male (age = 63.6 ± 9.5; mean ± S.D.; range = 44-77) patients, each of whom underwent a unilateral nephrectomy. Each slice (generally 1-4 g of tissue) was weighed, rinsed with buffer (250 mM sucrose, 10 mM triethanolamine/HCl, 1 mM EDTA·Na₂, pH 7.6), and homogenized in 3 ml of buffer/g tissue with a Teflon-glass device. Homogenates were initially centrifuged at 9000g for 20 min. The supernatant was filtered through cheesecloth and then centrifuged for 60 min at 105,000g. The resulting supernatant (cytosolic fraction) was used for enzyme assays and was stored at 80°C until use. The resulting pellets were resuspended in buffer and were centrifuged an additional 60 min at 105,000g to produce "washed" microsomes. The microsomal pellets were resuspended in buffer containing 10% (v/v) glycerol and were stored at 80°C until used. Enzyme activities were normalized to protein concentrations, which were measured with the BCA protein kit from Sigma Chemical Co. according to the manufacturer's instructions.

Isolation of hPT Cells from Renal Cortical Slices. hPT cells were derived from human kidney cortical slices obtained from the source described above after scoring by a pathologist as normal (i.e., derived from noncancerous, nondiseased tissue). The fibrous renal capsule was removed from the slice, and the slice was then weighed. The slices were washed with sterile PBS, minced, and the pieces were placed in a trypsinization flask filled with 30 ml of Hanks' buffer containing 25 mM NaHCO₃; 25 mM HEPES, pH 7.4; 0.5 mM EGTA; 0.2% (w/v) BSA; 50 µg/ml gentimicin; 1.3 mg/ml collagenase; and 0.59 mg/ml CaCl₂, which was filtered before use. All buffers were continuously bubbled with 95% O₂, 5% CO₂ and were maintained at 37°C. Minced cortical pieces were subjected to collagenase digestion for 15 min, after which the supernatant was filtered through a 70-µm mesh filter to remove tissue fragments, centrifuged at 150g for 7 min, and the pellet resuspended in Krebs-Henseleit buffer I [118 mM NaCl, 4.8 mM KCl, 0.96 mM KH₂PO₄, 0.12 mM MgSO₄ · 7H₂O, 25 mM NaHCO₃, 25 mM HEPES, and 2% BSA (w/v)]. These steps were repeated until complete digestion of the tissue was achieved (usually four or five cycles). Resuspended cells were combined and centrifuged at 150g for 7 min, pellets were washed with Krebs-Henseleit buffer I, centrifuged at 150g for 7 min, and the final pellet (hPT cells) was resuspended in Krebs-Henseleit Buffer II (same as Krebs-Henseleit buffer I except no BSA was added). Approximately 50 to 70 × 10⁶ cells were obtained from 1 g of human kidney cortical tissue.

Culturing of hPT Cells. Isolation of hPT cells was achieved as explained above, except sterile conditions were used (i.e., all instruments and glassware were autoclaved and all buffers were filtered through a 0.2-µm pore-size filter). After isolation, cells were resuspended in 2 ml of Krebs-Henseleit Buffer II and diluted to 30 ml with cell culture media. Basal medium was a 1:1 mixture of Dulbecco's modified Eagle's medium:Ham's F12. Standard supplementation included 15 mM HEPES, pH 7.4; 20 mM NaHCO₃; antibiotics for day 0 through day 3 only (192 I.U. penicillin G/ml + 200 µg of streptomycin sulfate/ml) to inhibit bacterial growth; 2.5 µg of amphotericin B/ml to inhibit fungal growth; 5 µg of bovine insulin/ml (= 0.87 µM); 5 µg of human transferrin/ml (= 66 nM); 30 nM sodium selenite; 100 ng of hydrocortisone/ml (= 0.28 µM); 100 ng of epidermal growth factor/ml (= 17 nM); and 7.5 pg of 3,3',5-triiodo-DL-thyronine/ml (= 111 nM) (Lash et al., 1995). Cells were seeded at densities of 50 to 100 µg of protein/cm² (0.51.0 × 10⁶ cells/ml) in polystyrene culture dishes. Cultures were grown at 37°C in a humidified incubator under an atmosphere of 95% air, 5% CO₂ at pH 7.4. Cultures were allowed to attach and grow for at least 24 h before treatment with any agent. Cells were harvested from the dishes by either scraping the plates with a Teflon scraper or by brief incubation with 0.05% (w/v) trypsin/0.2% (w/v) EDTA (in Ca²⁺- and Mg²⁺-free Hanks' buffer).

Isolation of Microsomes and Cytosol from hPT Cells. Microsomal and cytosolic fractions were prepared from both freshly isolated and primary cultures of hPT cells by homogenization of the cells with a Polytron ultrasonic device (Brinkmann Instruments, Westbury, NY), followed by centrifugation at 11,000g for 20 min to pellet nuclei, mitochondria, and cellular debris. The supernatant from this step was centrifuged in a tabletop ultracentrifuge at 105,000g for 90 min at 4°C to separate the cytosolic fraction from the microsomal pellet.

Enzyme Assays. Activities of glutathione peroxidase (GPX) with 0.25 mM H₂O₂ as substrate; GST with 1-chloro-2,4-dinitrobenzene as substrate; glutathione disulfide reductase (GRD), -glutamylcysteine synthetase (GCS), and -glutamyltransferase (GGT) with -glutamyl-p-nitroanilide and glycylglycine as substrates; and hexokinase were determined by spectrophotometric assays as described previously (Lash and Tokarz, 1989; Lash et al., 1998 and references therein). Total extracts of cells were used as the source of enzymes for each assay.

Flow Cytometry Analysis of DNA. Cell cultures were washed twice with sample buffer [PBS plus glucose (1 g/l) filtered through a 0.22-µm filter], dislodged by trypsin/EDTA (0.1% w/v) incubation, centrifuged at 400g for 10 min, and resuspended in sample buffer. Cell concentrations were adjusted to 1 to 3 × 10⁶ cells/ml with sample buffer and 1 ml of the cell suspension was centrifuged at 400g for 10 min. All of the supernatant except 0.1 ml/10⁶ cells was removed and the remaining cells were mixed on a vortex mixer in the remaining fluid for 10 s. Next, 1 ml of ice-cold ethanol (70%, v/v) was added to the sample drop by drop, with samples being mixed for 10 s between drops. The tubes were capped and fixed in ethanol at 4°C. After fixing, the cells were stained in propidium iodide (50 µg/ml) containing RNase A (100 U/ml). Samples were then mixed, centrifuged at 1000g for 5 min, and all the ethanol except 0.1 ml was removed. Cells were mixed in the residual ethanol and 1 ml of the propidium iodide staining solution was added to each tube. After mixing again, cells were incubated at room temperature for at least 30 min. Samples were analyzed within 24 h by flow cytometry with a Becton Dickinson FACSCalibur flow cytometer.

Western Blot Analysis of Individual P450 and GST Isoforms. Individual P450 and GST enzymes were detected by subjecting samples derived from kidney cells (microsomes or cytosolic fractions) to SDS-polyacrylamide gel electrophoresis (PAGE) on 7 to 10% slab gels, followed by electrophoretic transfer of the gels to nitrocellulose membranes. The membranes were then immunostained with polyclonal antibodies to CYP4A11 (Powell et al., 1996), CYP2E1, GSTA, GSTP, GSTM, or GSTT (Cummings et al., 1999, 2000). Alkaline phosphatase staining intensity was determined by scanning laser densitometry.

Immunohistochemical Staining for Cytokeratins and Vimentin. Cultures of hPT cells were grown on 35-mm polystyrene dishes. Cytokeratins were monitored as an epithelial cell marker by indirect immunofluorescent staining as described by Chopra et al. (1987). Vimentin was monitored as an endothelial cell marker and should not be present in control cells (Vamvakas et al., 1988). After fixation with 3.7% (v/v) formalin in PBS, cells were washed several times with PBS containing saponin (0.1% w/v), then incubated with -keratin conjugated to fluorescein isothiocyanate (FITC) antibody from guinea pig or a mouse anti-donkey vimentin antibody (Sigma Chemical Co.). After 1 h, cultures were washed with PBS and viewed under a Carl Zeiss laser microscope at the Confocal Imaging Core Facility in the School of Medicine at Wayne State University (Detroit, MI). For vimentin, cultures were incubated with a secondary antibody solution conjugated to Texas Red.

Data Analysis. All values are means ± S.D. of measurements made on the indicated number of separate preparations. Significant differences between means for data were first assessed by a one-way ANOVA. When significant F values were obtained, the Fisher's protected least significance t test was performed to determine which means were significantly different from one another, with two-tail probabilities <.05 considered significant.

	Results

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Morphology and Expression of Cytokeratins and Vimentin in Primary Cultures of hPT Cells. Primary cultures of hPT cells grown for 4 days, at which time the cultures reach or approach confluence, exhibited characteristic epithelial morphology (Fig. 1A). Expression of cytokeratins, a marker for epithelial cells, and of vimentin, a marker for endothelial cells, was assessed in two separate confluent primary cultures of hPT cells by immunohistochemical staining with monoclonal FITC-conjugated mouse antibody to cytokeratins and a monoclonal Texas Red-conjugated mouse antibody to vimentin. Primary cultures of hPT cells expressed high levels of cytokeratins after 4 days of cell culture (Fig. 1B). In contrast, vimentin staining was not detected in these cells (data not shown).

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Photomicrograph and expression of cytokeratins in primary cultures of hPT cells. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow to confluency (~4 to 5 days). Cells were incubated with a monoclonal FITC-conjugated mouse cytokeratin antibody and cytokeratin staining was visualized with laser scanning microscopy at 340 nm. Original magnification, 100×.

Flow Cytometry Analysis of Primary Cultures of hPT Cells Flow cytometry analysis of primary cultures of hPT cells was performed to assess the proportion of these cells in different phases of the cell cycle (Fig. 2). This method also can detect cells undergoing apoptosis, which would appear to the left of the G_o/G₁ peak in the subdiploid region. The confluent hPT cells (4 days of cell culture) were all diploid, viable, and predominantly in the G_o/G₁ phase of the cell cycle, with <10% of the cells in the S phase and no apoptotic cells.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Flow cytometry analysis of primary cultures of hPT cells. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow to confluency (~4 to 5 days). Cells were then harvested by trypsin-EDTA digestion, washed in sterile PBS, and fixed overnight in ethanol. Cells were stained with propidium iodide and analyzed by flow cytometry with a Becton Dickinson FACSCalibur flow cytometer.

Activities of GSH-Dependent Enzymes in Freshly Isolated hPT Cells. Activities of GGT, GCS, GST, GRD, GPX, and hexokinase were measured in freshly isolated hPT cells from three individual donors (Fig. 3). Viability of these cells after isolation was typically ~90%, as determined by trypan blue exclusion and lactate dehydrogenase release (data not shown). Freshly isolated hPT cells had ~10 times higher levels of GGT, a proximal tubular cell marker enzyme on the brush-border membrane, than hexokinase, a distal tubular cell marker enzyme, consistent with these cells being derived from the proximal tubular region of the nephron (Lash and Tokarz, 1989). Specific activities of the other four GSH-dependent enzymes varied somewhat between individual donors, but were all within a factor of 5 of each other.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Activity of GSH-dependent enzymes in freshly isolated hPT cells. Freshly isolated hPT cells were obtained from human renal cortical slices from three patients with enzymatic digestion in the presence of collagenase followed by differential centrifugation. Cells were used immediately for enzymatic assays. Typically, 1 to 2 g of tissue yielded 50 to 80 × 106 cells. Typical viability was ~90% as based on trypan blue exclusion and lactate dehydrogenase release assays. Data represent the mean ± S.D. of at least three separate measurements.

Expression of CYP4A11 and CYP2E1 in Freshly Isolated hPT Cells. Freshly isolated hPT cells from five male and three female patients ranging in age from 44 to 77 years were analyzed for the expression of CYP4A11 with a polyclonal rabbit anti-human antibody (Fig. 4, A and B). All of the patients tested expressed CYP4A11 and there was no significant difference in expression levels among patients, with the exception of one patient, or among male and female patients, as determined by densitometric analysis. CYP2E1 expression was not detected in either freshly isolated hPT cells or in microsomes isolated from human renal cortical slices in any patient tested (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of CYP4A11 expression in freshly isolated hPT cells. A, purified human liver CYP4A11 (1 µg) and freshly isolated hPT cells (60 µg) were subjected to SDS-PAGE and then transferred to a nitrocellulose blot that was then incubated with a polyclonal rabbit anti-human antibody to CYP4A11. Numbers refer to individual patients' samples. B, densitometric analysis of CYP4A11 expression from samples represented in A. Densitometry was performed by scanning blots into a Power Macintosh 8100/100 computer with a Microtek ScanMaker IIsp scanner at a resolution of 150 lines per inch, with Adobe Photoshop 5.0 and NIH Image 1.6.1 software.

Expression of GST Isoforms in Freshly Isolated hPT Cells. Freshly isolated hPT cells from two male and three female patients, ranging in age from 44 to 63 years, were analyzed for expression of GSTA with a polyclonal goat anti-rat GST antibody that also recognizes human GSTA (Fig. 5, A and B). All of the patients tested expressed GSTA. There was no major difference in GSTA expression among any of the patients tested, based on densitometric analysis.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of GST isoforms in freshly isolated hPT cells. A, human kidney cytosol (5 µg) and hPT cells (60 µg) were subjected to SDS-PAGE and then transferred to a nitrocellulose blot that was then incubated with a polyclonal goat anti-rat antibody to GST, which recognizes human GSTA. B, densitometric analysis of GSTA expression from samples represented in A. C, human kidney cytosol (5 µg) and freshly isolated hPT cells (60 µg) were subjected to SDS-PAGE and then transferred to a nitrocellulose blot that was then incubated with a polyclonal rabbit anti-human antibody to GSTP. D, densitometric analysis of GSTP expression from samples represented in C. E, freshly isolated hPT cells (60 µg) were subjected to SDS-PAGE and then transferred to a nitrocellulose blot that was then incubated with a polyclonal rabbit anti-human antibody to GSTT. F, densitometric analysis of GSTT expression from samples represented in E. Densitometry was performed and analyzed as described in the legend to Fig. 4. Numbers refer to individual patient samples.

Effect of Duration of Cell Culture on Activity of GSH-Dependent Enzymes. Freshly isolated hPT cells were seeded at 0.5 to 1.0 × 10⁶ cells/ml in the supplemented cell culture media. Cells were grown to confluency (~4 to 5 days) and isolated from culture dishes by trypsin-EDTA treatment. At each passage, one-half the cells was harvested and the other half was subcultured. Figure 6 displays the activity of GSH-dependent enzymes and hexokinase in freshly isolated hPT cells (passage 0) and after four subsequent passages. The ratio of GGT to hexokinase decreased slightly compared with that in freshly isolated cells, but GGT activity was still >5 times higher than hexokinase activity at passage 4. GGT activity remained relatively constant through passage 4. GRD activity significantly increased in passage 1 but decreased significantly in passages 2 to 4. The activities of GST and GPX remained unchanged until passage 2, after which significant decreases were observed. GCS activity remained unchanged throughout the four passages.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effect of subculturing hPT cells on GSH-dependent enzymes. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow to confluency (~4 to 5 days). At each time point, half the cells were harvested and the other half was passaged. Activities of GSH-dependent enzymes and hexokinase were determined with spectrophotometric methods. Passage 0 represents freshly isolated hPT cells. Data are the mean ± S.D. of at least three separate experiments with material derived from three separate donors.

Effect of Duration of Cell Culture on Expression of CYP4A11 in hPT Cells. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 10⁶ cells/ml. After 24 h, media were removed and media containing either solvent control ([ethanol] = 0.1%, v/v) or ciprofibrate (100 µM), a peroxisome-proliferating agent similar to clofibrate, were added. Cells were allowed to grow for an additional 24 or 48 h, after which microsomes were isolated from these cells and analyzed for the expression of CYP4A11 by Western blot analysis with a polyclonal rabbit anti-human CYP4A11 antibody (Fig. 7). CYP4A11 expression was maintained in primary cultures of hPT cells after both 48 and 72 h of culture (Fig. 7). Ciprofibrate had no effect on CYP4A11 expression at any time point tested (Fig. 7). Interestingly, the levels of CYP4A11 expression in primary cultures of hPT cells were slightly higher than those in freshly isolated hPT cells. This was not due to loading differences because equal amounts of protein were loaded onto the gel.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Effect of primary culture and ciprofibrate on CYP4A11 expression in hPT cells. Freshly isolated hPT cells (99-026) were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow for 24 h before treatment with ciprofibrate or solvent control (ethanol = 0.1%, v/v), and then allowed to grow for an additional 24 or 48 h. At the indicated time, cells were harvested and microsomes were isolated. CYP4A11 expression was measured by Western blot analysis as described in Fig. 4. Densitometric analysis of CYP4A11 expression in primary cultures of hPT cells is shown. Densitometry was performed and analyzed as described in the legend to Fig. 4.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of ethanol and dexamethasone on CYP4A11 expression in primary cultures of hPT cells. Freshly isolated hPT cells (98-415, 99-026, and 99-223) were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow for 24 h before treatment with either 25 or 50 mM ethanol (EtOH), or dexamethasone (7 nM) and then allowed to grow for an additional 24 or 48 h. At the indicated time, cells were harvested and microsomes were isolated. CYP4A11 expression was measured by Western blot analysis as described in Fig. 4. Densitometric analysis of blots is shown. Densitometry was performed and analyzed as described in the legend to Fig. 4. A, effect of ethanol on CYP4A11 expression. B, effect of ethanol and dexamethasone on CYP4A11 expression.

Expression of GSTA, GSTP, and GSTT in Primary Cultures of hPT Cells. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 10⁶ cells/ml and allowed to grow to confluency (~4 to 5 days). Cells were then harvested, cytosol was isolated, and expression of GSTA, GSTP, and GSTT determined by immunoblot analysis. GSTA was detected in 30 µg of cytosolic protein isolated from six separate cultures, representing two different patients, after 4 days of culture [Fig. 9A, sample 98-415 (lanes 2-4) and sample 99-026 (lanes 5-7)]. Although a direct comparison of expression in freshly isolated cells and cell cultures is not possible due to different amounts of protein loading, GSTA expression appeared to be well maintained during the course of primary culture (Fig. 9A, lane 1). The expression of GSTP and GSTT in cytosol isolated from hPT cells after 4 days of culture also was determined by Western blot analysis [Fig. 9, B (lanes 2-4) and C (lanes 2-5)]. Unlike GSTA, the expression of both GSTP and GSTT appeared to decrease significantly from levels seen in freshly isolated hPT cells (Fig. 9, B and C, lane 1).

View larger version (72K): [in this window] [in a new window]   Fig. 9.   Expression of GSTA, GSTP, and GSTT in cytosol isolated from primary cultures of hPT cells. Freshly isolated hPT cells were seeded at a density of 0.5 to 1.0 × 106 cells/ml and allowed to grow to confluency (~4 to 5 days). GSTA, GSTP, and GSTT expression was measured by Western blot analysis with a polyclonal goat anti-rat GST antibody, which also recognizes human GSTA, a polyclonal rabbit anti-human GSTP antibody, and a polyclonal rabbit anti-human GSTT antibody. A, GSTA expression. Lane 1 is cytosol (5 µg) from freshly isolated hPT cells, whereas lanes 2 to 7 are cytosol (30 µg) from six separate preparations from two different patients (98-415 (lanes 2-4) and 99-026 (lanes 5-7). B, GSTP expression. Lane 1 is cytosol (5 µg) from freshly isolated hPT cells, whereas lanes 2 to 4 are cytosol (30 µg) isolated from two separate preparations from two different patients [98-415 (lane 2) and 99-026 (lanes 3 and 4)]. C, GSTT expression. Lane 1 is cytosol (5 µg) from freshly isolated hPT cells, whereas lanes 3 through 5 are cytosol (30 µg) isolated from two separate preparations from two different patients [99-026 (lanes 2 and 3) and 99-015 (lanes 4 and 5)].

	Discussion

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The present results describe the use of freshly isolated and primary cultures of hPT cells as models to study drug metabolism mediated by P450 and GSH-dependent enzymes in the human kidney. Confluent primary cultures of hPT cells maintained their epithelial properties, as demonstrated by cytokeratin and vimentin expression, and remained viable and diploid, as shown by flow cytometry. Freshly isolated hPT cells exhibited high levels of GSH-dependent enzyme activities, and subcellular fractions prepared from these cells expressed specific P450 and GST isoforms. We showed that GSTT is expressed in human kidney and that CYP4A11 is present in the proximal tubules. In other studies (Lasker et al., 2000), we found renal CYP4A11 to be localized exclusively in the S2 and S3 segments of the proximal tubules. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of most GSH-dependent enzyme activities and no longer expressed P450s or GSTs. Nevertheless, steady-state levels of CYP4A11 in hPT cell primary cultures could be enhanced by treatment with specific xenobiotics. Primary cultures of hPT cells thus appear to be a suitable in vitro model for studying regulation of the renal expression of CYP4A11 and specific GST isoforms.

Activity and Expression of Renal P450 Enzymes. In contrast to human liver, human kidney appears to express few P450 enzymes. In fact, the only P450 enzymes found at significant levels in human renal microsomes are CYP4A11 and CYP4F2 (Lasker et al., 2000) and CYP3A isoforms (Schuetz et al., 1992; Kharasch et al., 1995). Herein, we also were able to detect CYP4A11 but not CYP2E1 in microsomes from freshly isolated hPT cells. This agrees with a study by Amet et al. (1997), which reported the presence of a single "CYP4A" immunoreactive protein in human kidney microsomes with a polyclonal sheep anti-rat CYP4A1 antibody and that CYP2E1 was undetectable in renal microsomes from 18 different subjects. The catalytic activity of renal CYP4A11, as measured by lauric acid hydroxylation, was significantly lower than the mean value reported by Amet et al. (1997). However, the lauric acid -hydroxylase activities reported by Amet et al. (1997) varied by >10-fold and the activities reported herein are at the lower end of that range. Differences in assay methods also may have contributed to these differences in rates.

Activity and Expression of GSH-Dependent Enzymes. Freshly isolated hPT cells expressed high levels of several GSH-dependent enzymes. This report describes activities of enzymes that are critical to second-phase drug metabolism and redox status in hPT cells. Certain of these GSH-dependent enzymes (GCS, GPX, and GRD) exhibited activities that were higher than those measured in rat kidney PT cells, whereas other enzyme activities (GST, GGT, and hexokinase) were lower (Lash et al., 1995). Whereas the overall activity of GST (with 1-chloro-2,4-dinitrobenzene as substrate) was lower in hPT cells than in rat kidney PT cells, GSTA, GSTP, and GSTT were expressed in hPT cells, whereas only GST and GSTµ could be detected in rat kidney cells (Fig. 5; Cummings et al., 2000). Such interspecies differences in GST activity and in GST isoform expression may be explained by the markedly different affinities of the various GST isoforms for 1-chloro-2,4-dinitrobenzene (Mannervik, 1985).

Comparisons of P450 and GST Isoform Expression between Rat and Human Kidney. From the data presented herein and our previous work (Cummings et al., 1999, 2000), it is obvious that rat and human kidneys differ significantly in the expression of GST and P450 enzymes. This information bears directly on the utility of data from rat studies for extrapolation to humans for risk assessment. For example, chemicals such as acetaminophen, various chloroethylenes, and other low-molecular-weight hydrocarbons are metabolized by CYP2E1 (Guengerich, 1991). Although these compounds are metabolized primarily in the liver, it is possible, especially in cases of exposure to high chemical concentrations (e.g., suicide attempts), that the kidneys also are exposed to significant levels of these compounds. In such cases, the differences observed in renal CYP2E1 expression between rats and humans may play a pivotal role in the development of nephrotoxicity. Although the majority of studies on xenobiotic metabolism and kidney toxicity have been performed in rats, the results obtained with nephrotoxins activated by CYP2E1 would not be applicable to humans due to the lack of expression of this P450 enzyme in the human kidney. However, both rat and human kidney exhibit extensive expression of CYP4A enzymes as well as substantial lauric acid -hydroxylase activity. However, the rat kidney may only be somewhat applicable for the study of renal CYP4A function and/or expression in humans because four CYP4A subfamily proteins are found in the rat kidney (CYP4A1, CYP4A2, CYP4A3, and CYP4A8) (Ito et al., 1998; Nguyen et al., 1999), whereas the human kidney expresses only a single CYP4A enzyme, namely, CYP4A11 (Powell et al., 1996, 1998).

Influence of Primary Culture and Subculturing on Enzyme Expression and Cellular Function. Much of the previous work with primary cultures of hPT cells has focused on nondrug metabolism issues. Data from this study describe for the first time changes that occur in the drug-metabolizing capacity of primary and subcultures of hPT cells. The cytokeratin and vimentin expression data and the flow cytometry analysis presented herein indicate that the confluent, primary cultures of hPT cells are normal, diploid epithelial cells. As would be expected for hPT cells in the intact kidney, primary cultures of hPT cells are mostly in the resting phase and are not undergoing cell death by either necrosis or apoptosis. Thus, these cells may provide a useful tool for studying the injurious effects of chemicals on the hPT cell cycle. Importantly, activities of GSH redox cycle enzymes as well as those of GSH synthesis and degradation enzymes were maintained in primary cultures of hPT cells (Fig. 6). The expression of GSTA also was retained in primary hPT cell cultures, whereas that of GSTP and GSTT decreased. Both GSTP and GSTT were still detected after 4 days of culture but at levels that were apparently much lower than those of freshly isolated hPT cells. The differences observed in GST enzyme expression in these cultures may reflect the role of each individual isoform in kidney function or may stem from differences in the manner via which expression of each isoform is regulated. Interestingly, GSH-dependent enzyme activities were generally not well maintained after subculturing. This suggests that use of the hPT cells after passage should be done with caution, particularly if the chemical being studied is metabolized by any of the enzymes whose expression changes under these conditions.

	Summary and Conclusions

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The expression and activity of several drug-metabolizing enzymes were determined in freshly isolated hPT cells and in primary cultures thereof. The drug-metabolizing enzymes detected in freshly isolated hPT cells were all found in primary hPT cell cultures but at lesser levels. These data provide validation for the use of primary cultures of hPT cells to study chemical-induced injury and physiological functions of the human kidney.