Cephaloridine toxicity in primary cultures of rat renal cortical epithelial cells

Toxic. in Vitro Vol. 1, No. I, pp. 23-29, 1987 Printed in Great Britain

0887-233/87 $3.00+0.00 Pergamon Journals Ltd

C E P H A L O R I D I N E T O X I C I T Y IN P R I M A R Y C U L T U R E S OF RAT R E N A L C O R T I C A L E P I T H E L I A L CELLS M. A. SMITH* and D. ACOSTAt Department of Pharmacology and Toxicology, College of Pharmacy, The University of Texas, Austin, TX 78712 and J. V. BRUCKNER School of Pharmacy, University of Georgia, Athens, GA 30602, USA (Received 4 August 1986)

Abstract--Primary cultures of rat renal cortical epithelial cells were used to assess the in vitro nephrotoxicity of cephaloridine (Cph). Several different indices were used to follow the course of Cph-induced nephrotoxicity in the cultures. Plasma membrane' integrity was determined by the effect of Cph on lactate dehydrogenase (LDH) leakage, cellular K + content and plasma membrane Na+/K ÷ ATPase activity. Significant LDH leakage and decreased cellular K ÷ content occurred after 8 hr of exposure to Cph. Na+/K + ATPase activity was depressed as early as 4 hr after Cph treatment. Brush border integrity was assessed by the effect of Cph on the activity of the brush border enzyme alkaline phosphatase, which was significantly decreased following 12 hr of exposure to Cph. Treatment with Cph resulted in an initial elevation of cellular glutathione (GSH)--as indicated by cellular non-protein sulphydryl content--followed by a decrease in GSH content 16 hr later. The mitochondrial response to Cph was assessed by determining mitochondrial succinate dehydrogenase (SDH) activity and cellular ATP content. SDH activity was significantly depressed after 4 hr of Cph exposure; ATP content was not significantly depressed until 12 hr after treatment. The time course of Cph-induced injury in our culture system suggests that early injury involves alterations at the level of the mitochondrial membrane and the plasma membrane. The most sensitive indicators of Cph-induced toxicity in this system were mitochondrial SDH activity and plasma membrane Na+/K ÷ ATPase activity.

INTRODUCTION During recent years, there has been an increased interest in the development of in vitro systems for toxicity evaluation. This has been partly due to the trend towards limiting the number of animals used in toxicity testing and to the need to screen large numbers of chemical entities for toxicity. Interest in observing the direct cellular effects of toxicants on target organ tissue, without the influence of the compensatory hormonal and vascular mechanisms of the intact animal, has also increased the demand for in vitro methods. The development of such systems requires that sensitive, reproducible indices of cell damage be used to assess xenobiotic-induced toxicity in vitro. In addition, toxicity manifested in vitro should correlate with the degree of functional impairment observed following in vivo exposure to a toxicant. With these goals in mind, primary cultures of a variety of tissues such as rat liver, heart, nerve and kidney have been developed for use in evaluating

the target organ toxicity of various compounds in vitro (Acosta et al. 1985).

The kidneys are the target for a wide variety of chemical agents, including heavy metals, haloalkanes, analgesics and antibiotics (Hook, 1980). The complexity of renal structure and function results in the kidneys being particularly susceptible to xenobioticinduced toxic injury. Factors such as the high rate of renal blood flow, renal concentrating mechanisms, transport systems and a capacity for xenobiotic biotransformation may predispose the kidneys to toxicant-induced injury. The structural and functional heterogeneity of the kidneys complicates the development of methods to evaluate renal damage. Evaluation of renal function by traditional methods such as determining blood urea nitrogen (BUN) concentrations and creatinine clearance provides an index of renal function following toxic insult. However, these methods are not capable of discerning the effects of a nephrotoxic agent at the cellular level. In addition, these indices may not be altered, even in the presence of significant cellular damage. Our laboratory has developed a primary cell culture system of rat renal cortical epithelial cells to evaluate nephrotoxic agents in vitro (Smith et al. 1986). To characterize our system further, we have chosen to evaluate the toxicity of a well-known nephrotoxic agent cephaloridine (Cph). Cph, a broad-spectrum antibiotic, produces nephrotoxicity characterized by acute proximal tubular necrosis,

*Current address: Smith, Kline and French Laboratories, 1500 Spring Garden Street, P.O. Box 7929, Mail Code L-66, Philadelphia, PA 19101. tTo whom reprint requests should be addressed. Burroughs Wellcome Scholar in Toxicology. Abbreviations: AP = alkaline phosphatase; BSS = balanced salt solution; Cph = cephaloridine: DMEM = DulbeccoVogt modification of Eagle's minimum essential medium; GSH = glutathione; LDH = lactate dehydrogenase; SDH = succinate dehydrogenase. 23

M. A. SMITHet al.

24

which correlates with the extent of its renal cortical accumulation and subsequent intracellular concentration (Atkinson et al. 1966; Tune, 1975). The mechanisms of Cph-induced renal damage are still unclear, but peroxidative injury has been suggested as a possible biochemical mechanism for Cph-induced nephrotoxicity (Cojocel et al. 1985; Goldstein et al. 1986; Kuo et al. 1983). The purpose of this study was to characterize further the utility of primary cultures of rat renal cortical epithelial cells for assessing nephrotoxicity in vitro, by evaluating the toxicity of a nephrotoxic agent that has been studied in detail using other systems. MATERIALS AND METHODS

Materials. The cell culture medium and newborn calf serum were obtained from the Grand Island Biological Co. (Grand Island, NY). Potassium penicillin G and amphotericin B were purchased from E. R. Squibb & Sons, Inc. (Princetown, N J) and streptomycin sulphate was from Pfizer Laboratories (New York). Disposable plastic culture dishes were purchased from Falcon Labware (Oxnard, CA), and sterilizing filters from Gelman Instrument Co. (Ann Arbor, MI). All other chemicals and reagents were purchased from Fisher Scientific Co. (Fairlawn, N J) or Sigma Chemical Co. (St Louis, MO). Cell culture procedure Primary cultures of renal cortical epithelial cells were prepared from the kidneys of 12-14-day-old Sprague--Dawley rats. The culture method was a modification of the procedure described by Belleman (1980) for adult rat kidney culture and the method used by Acosta et al. (1980) for liver cell culture. Rats were decapitated, and the renal artery was exposed. The kidneys were perfused through the renal artery via a 22-gauge, 1.5in. needle with approximately 0.75 ml of dissociation medium. The complete dissociation medium was made from Hank's Ca + +-free balanced salt solution (BSS), which contained Sigma type IV collagenase (100 U/ml), Sigma type II hyaluronidase (60 U/ml) and Sigma bovine serum albumin (1 mg/ml). After perfusion, all the kidneys were collected in a small beaker containing dissociation medium. The cortex was removed from each kidney and collected in a 10-ml beaker and the tissue was minced into l-ram fragments with small scissors. These tissue fragments were transferred to a 30-ml dissociation flask containing 15 ml of dissociation medium. The dissociation flask, containing a magnetic stirrer, was kept in a 37°C water-bath. After an initial 10-min period of gentle mixing, the supernatant was discarded and replaced with 15ml of fresh dissociation medium. After a 15-20-min dissociation, a Pasteur pipette was used to transfer the supernatant to a serum tube. The cell suspension was centrifuged at 900 rpm in a tabletop clinical centrifuge for 5 min. The dissociation flask was refilled with 15 ml of fresh dissociation medium and placed into the water-bath for the second 15-20-min dissociation period. After the 5-min centrifugation, the pellet was resuspended in 2.5ml of arginine-deficient, ornithine-supplemented Dulbecco-Vogt's modification of Eagle's

minimum essential medium (DMEM) which contained D-valine (0.094g/litre), insulin (0.01 g/litre), hydrocortisone (0.05 g/litre), niacinamide (0.3 g/litre), transferrin (0.005 g/litre), bovine calf serum (10%, v/v) and bovine serum albumin (0.1 g/litre). This cell suspension was centrifuged at 1500 rpm for 5 min. The pellet was resuspended in 2.5 ml of complete D M E M and filtered through a stainless-steel 25-mm screen mesh. The cell suspension was collected in a 25-ml graduated cylinder. The second and third dissociation periods were conducted in the same manner as the first. The kidney cell suspensions from the three dissociation periods were pooled and diluted with complete D M E M plus serum and antibiotics (potassium penicillin G, 200 U/ml, streptomycin sulphate, 200/~g/ml, and amphotericin B, 4/tg/ml). The volume of D M E M added was such that each 35-ram culture dish was plated with 0.1-0.5 mg cell protein in 2 ml culture medium. The cultures were grown in a humidified environment of 5% CO2/95% air at 37°C to maintain a pH of 7.2-7.4. Renal epithelial cells were separated from other cell types by using a differential pour-off technique based on the rate of cell attachment to the culture dishes (Waymouth, 1978). The cell suspension was swirled and transferred to fresh culture dishes 3 hr after the initial plating. Fibroblasts and debris had attached at the end of the first 3 hr and the epithelial cells were still in suspension when they were transferred to the fresh culture dishes. The culture medium was replaced with fresh medium 48 hr after initial plating; the timing of the medium change was such that the glomerular cells had not yet attached (Glasgow et al. 1981). This ensured that the cultures were made up mostly of cortical tubular epithelial cells. Two other steps taken to suppress fibroblastic overgrowth and ensure that the cultures were primarily cortical tubular cells were the use of arginine-free, ornithinesupplemented medium (Leffert & Paul, 1973) and the substitution of L-valine with D-valine (Gilbert & Migeon, 1975). Cephaloridine treatment. All experiments were conducted 3 4 days after initial plating of the cells to ensure that a complete monolayer had formed prior to drug treatment. Cph was dissolved in plain D M E M without serum prior to addition to the cultures. The concentrations of Cph tested were 2 x 10 5, 2 x 10 4 and 1 × 10 -3 M. Stock solutions of Cph were freshly prepared for each experiment. Analytical procedures Lactate dehydrogenase ( L D H ) activity. Leakage of LDH into the culture medium was measured according to the method described by Mitchell et al. (1980). The rate of disappearance of N A D H in the presence of sodium pyruvate was measured spectrophotometrically at 340 nm. The rate of change in absorbance is directly proportional to the amount of LDH leaked from the cells into the medium. LDH leakage was expressed as units/mg cell protein. Renal cell potassium content. Flameless atomic absorption spectroscopy was used to measure intracellular potassium concentrations (Sanui & Rubin, 1979). The culture medium was aspirated from the dishes and the cells were rinsed twice with 10 mM-Tris HCI buffer (pH 7.4, 37°C). One ml of 1.5 N-nitric acid

Cephaloridine toxicity in renal cells was added to each dish to digest the cell monolayer. The cells were scraped and transferred to plastic vials for storage at - 2 0 ° C . The samples were thawed at room temperature prior to assay. For the assay, samples were dried at 100°C for 20sec, ashed at 500°C for 15 sec and atomized at 2000°C for 2 sec. Potassium was analysed at 7690 A,. Sample potassium values were determined by comparing them to a standard curve generated from samples of known KC1 concentration in 1,5 N-nitric acid. Results were expressed a s / t m o l potassium/g cell protein. Alkaline phosphatase ( A P ) activity. AP activity was determined spectrophotometrically by a procedure modified from that described by Kyle et al. (1983). The assay is based on the reaction between AP and the substrate p-nitrophenyl phosphate. The change in absorbance at 400 nm was recorded over a 3-min period and a molar extinction coefficient of 17.52 was used to calculate AP activity, which was expressed as units/mg cell protein. Glutathione (GSH). The concentration of cellular non-protein sulphydryls (indicative of cellular GSH content) was determined using Ellman's reagent (Beutler et al. 1963). The supernatant resulting from precipitation of cell protein with metaphosphoric acid was mixed with Eilman's reagent and the absorbance was read at 412nm. Absorbance values were compared to a standard curve generated from samples of known GSH concentration. GSH content was expressed as #g/mg cell protein. M itochondrial succinate dehydrogenase ( SD H ) activity. Following isolation of mitochondria from control and Cph-treated cell cultures, SDH activity was determined according to the method described by Singer (1974). An aliquot of a mitochondrial suspension was incubated with sodium succinate, sodium deoxycholate and potassium cyanide in a phosphate buffer for 15 min at 37°C. At the end of the incubation period, potassium ferricyanide was added to the reaction mixture and the blanching of ferricyanide was followed spectrophotometrically at 450 nm over a 10-rain period. A millimolar extinction coefficient of 0.262 was used to calculate SDH activity, which was expressed as pmol/mg protein/min. Cellular A TP content. ATP concentration in cultured renal cells was determined according to a method modified from Debetto et al. (1982) and George et al. (1982). The luciferin-luciferase reaction was used to determine ATP content and was quantitated with a New Brunswick ATP.integrating photometer. ATP content was expressed as nmol ATP/mg cell protein. N a ÷ / K ÷ ATPase. The activity of renal plasma membrane Na+/K + ATPase was determined according to the method of Sulakhe et al. (1976). ATPase activity in plasma membranes isolated from cultured cells was quantitated by assaying for the release of inorganic phosphate, which was determined spectrophotometrically at 720 nm using an acid molybdate reagent (Rockstein & Herron, 1951). Na+/K + ATPase activity was expressed as #mol/mg protein/min. Morphology Cell morphology was monitored using an inverted

25

phase-contrast microscope (Leitz Diavert, magnification).

x400

Statistical analysis The data were analysed by ANOVA. The Scheffe's post-hoc test was used to assess the differences (P ~<0.05 and P ~<0.01). Values were expressed as the mean ___SEM. RESULTS

There was no statistically significant LDH leakage into the culture medium for the first 4 h r after addition of Cph to the cultures (Table 1), but LDH leakage was significantly increased after Cph treatment for 8 hr at all concentrations tested. Table 2 illustrates the cellular potassium content in cultures treated with Cph. As with LDH leakage, cellular potassium content was not significantly decreased until the cells had been exposed to Cph for 8 hr. Cellular potassium was significantly decreased by 8-hr exposure to 2 x 10 4 or to 1 x 10 -3 M-Cph. By 12hr, both the LDH and potassium levels had returned to control levels. Treatment with the highest concentration of Cph resulted in significant decreases in plasma membrane Na+/K ÷ ATPase activity following 4 and 8 hr of exposure (Fig. 1). ATPase activity was 23% of the control value at 4 h r and dropped to zero by 8 hr. No significant decreases in AP activity were evident after 8 hr of Cph exposure (Fig. 2a). By 12 hr there was a 40% decline in AP activity following treatment with 1 x 10 -3 M-fph. Treatment with the two lower concentrations of Cph resulted in a significant increase in GSH concentrations after 8 hr (Fig. 2b). These values were 25 and 50% greater than the controls for the 2 x 10 -4 and 2 x 10-SM concentrations, respectively. GSH depletion was not observed until 16 hr after the beginning of treatment, by which time exposure to

Table 1. Leakage of lactate dehydrogenase (LDH) into the culture medium following exposure of cultures of rat renal cortical epithelial cells to cephaloridine (Cph) for 4 - 1 2 h r L D H activity (U/mg cell protein) after: Cph concn (M) 0 (control) 2 x 10 -5 2 x 10 -4 1 x 10 -3

4 hr

8 hr

12 hr

359.2 + 12.5 359.8 + 12.7 352.3-+9.9 349.7_+5.6

312.8 _+ 7.6 390.8 + 2.8* 352.0-+7.0* 350.7_+4.3*

359.5 _+ 10.0 375.7 -+ 5.2 375.7-+7.3 391.5_+2.3

Values are means_+ SEM (n = 6). An asterisk indicates those differing significantly (P < 0.05) from the corresponding control value. Table 2. Cellular potassium content following exposure of cultures of rat renal cortical epithelial cells to cephaloridine (Cph) for 4--I 2 hr K + content (/amol/g cell protein) after: Cph concn (M) 0 (control) 2 × 1 0 -5 2x10 4 1 × 10 -3

4 hr

8 hr

12 hr

171.5 + 4.3 150.3+_3.6 186.3_+14.1 181.3_+ 11.0

247.5 +_ 17.7 227.8_+5.5 145.3_+8.6" 192.0_+5.6"

196.0 + 3.5 191.8_+4.7 190.5_+2.9 177.8_+7.2

Values are m e a n s + SEM (n =4). An asterisk indicates those differing significantly (P ~< 0.05) from the corresponding control value.

26

M.A. SMITHet al. 100

has been implicated as a possible mechanism of Cph-induced nephrotoxicity (Cojocel et al. 1985; Goldstein et al. 1986; Kuo et al. 1983). Cph-induced lipid peroxidation in renal cortical slices preceded the effects of Cph on organic ion accumulation by the 8 75 slices (Goldstein et al. 1986). Co-incubation of the 'S slices with Cph and antioxidants prevented lipid .< peroxidation and the subsequent changes in organic >, ion accumulation. In this study, a variety of indices was used to trace > 50 the course of Cph-induced nephrotoxicity in primary cultures of rat renal cortical epithelial cells. LDH leakage, cellular K ÷ content and plasma membrane g_ FNa+/K + ATPase activity were used as indicators of %,. 25 plasma membrane integrity. Slight increases in LDH leakage and small decreases in cellular K ÷ content +o Z were observed after 8 hr of Cph exposure. These observations suggested that Cph did not severely impair renal cell membrane integrity. Morphological examination revealed only scattered areas of cell 2 x 1 0 -5 2 x 1 0 -4 lx10 -3 membrane disruption after 8-hr Cph exposure. This correlates well with the findings of Silverblatt et al. Cph conch (M) (1970) who found that disruption of the plasma Fig. 1. Plasma membrane Na+/K + ATPase activity follow- membrane did not occur until 16 hr after Cph treating exposure of rat renal cortical epithelial cells in culture ment. LDH leakage is a gross indicator of membrane to cephaloridine (Cph) for 4 ( t ) and 8 (l~) hr. Values damage and one would not expect to see substantial repre~nt the mean + SEM (n = 4) and an asterisk marks those significantly(P ~<0.01) different from the correspond- LDH leakage until the plasma membrane has been disrupted. Slight alterations in the configuration of ing control. the plasma membrane would not be expected to result in significant LDH leakage. Brush border damage 1 x 10-3 M-Cph resulted in a 35% drop in the GSH was assessed by determining AP activity following concentration. treatment with Cph. Although Cph did inhibit AP Mitochondrial SDH activity was significantly deactivity, this effect did not appear until after exposure creased by all three concentrations of Cph after 4 and to Cph for 12 hr. Hsu et al. (1981) have suggested that 8 hr of exposure (Fig. 2c). These values were 60, 68 AP is embedded in the lipid matrix of the brush and 46% of control values for cells treated for 4 hr border membrane and is not as vulnerable as other with 2 x 10 -5, 2 × 1 0 - 4 and 1 x 10 3M_Cph, rebrush border enzymes to damage by nephrotoxic spectively. No effect on cellular ATP content was agents. Plasma membrane Na+/K ÷ ATPase activity observed following treatment with Cph for 4 or 8 hr declined as early as 4 hr after addition of Cph. (Fig. 2d). ATP content was significantly decreased Changes in Na+/K ÷ ATPase activity preceded alteronly after exposure to 2 × 10 -4 or 1 × 10 -3 M-Cph for ations in LDH leakage, cellular K ÷ content and AP 12 hr. activity. Renal Na+/K ÷ ATPase is located in the Cellular morphology was not visibly altered at the basal infoldings of the plasma membranes of cortical light microscopic level after 4 hr of Cph exposure. By cells. The composition of plasma membranes in the 8 hr, there were scattered areas of plasma membrane apical pole or brush border of the cortical cell is disruption at all the Cph concentrations tested, but thought to differ from that of the membranes that the damage was not diffuse. After 12 hr of exposure make up the basal infoldings of the cells (Kinne et al. to 1 x 10-3M-Cph, there was an increase in the 1971). Differences in membrane composition could number of vacuoles and in plasma membrane disaccount for the increased sensitivity of plasma memruption. brane Na+/K ÷ ATPase to Cph toxicity. Cph may be metabolized to an as yet unidentified reactive metabolite prior to causing cell injury (McDISCUSSION Murtry & Mitchell, 1977). We monitored cellular The nephrotoxicity of Cph has been well estabGSH content after exposure to Cph as an index of the lished. Cph has been shown to cause tubular necrosis, potential of the cultured cells to produce a reactive enzymuria, mitochondrial swelling and changes in intermediate. The cells initially responded to the tubular membrane structure (Atkinson et al. 1966; presence of Cph by an increase in GSH content. Perkins et al. 1968; Silverblatt et al. 1970). Electron There was an inverse relationship between the conmicroscopic evaluation of Cph-induced nephcentration of Cph and the extent of the increase in rotoxicity in rabbits revealed early mitochondrial and GSH. A significant decrease did not appear until Cph plasma membrane alterations between 1 and 5 hr exposure had continued for 16 hr. GSH depletion has after Cph treatment (Silverblatt et al. 1970). Depresbeen reported to occur within 1 hr of Cph treatment sion of mitochondrial respiration has been demonin rabbits (Kuo & Hook, 1982). This depletion of strated as early as 15 min after in vitro incubation GSH was followed by a return to control GSH with Cph and 2 hr after in vivo exposure (Tune & concentrations by 4 hr. Kidney cells have a high Fravert, 1981; Tune et al. 1979). Peroxidative injury turnover rate of GSH and are able to synthesize GSH

Cephaloridine toxicity in renal cells

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Fig. 2. (a) Alkaline phosphatase (AP) activity, (b) cellular glutathione (GSH) content, (c) mitochondrial succinate dehydrogenase (SDH) activity and (d) cellular ATP content, following exposure of cultures of rat renal cortical epithelial cells to 0 (Fq; control), 2 x 10 -5 (m), 2 x 10 -4 (A) or 1 × 10 -3 (O) ra-cephaloridine for periods between 2 and 16 hr. Values are means + SEM for (a) six or (b
significantly until after 12 hr of treatment. S D H is an inner mitochondrial membrane enzyme; inhibition of S D H in our system indicates that the cultured cells were able to concentrate Cph intracellularly. The time course of Cph-induced injury in our culture system suggests that early injury involves alterations at the level of the mitochondrial membrane and the plasma membrane, Overt toxicity was not manifested until the cells had been exposed to Cph for several hours. The most sensitive indicators of Cph toxicity in this system were mitochondrial S D H activity and plasma membrane N a + / K ÷ ATPase activity. The concomitant inhibition of plasma membrane N a + / K + ATPase and mitochondrial S D H

28

M . A . SMITH et al.

activity may suggest some similarities in the susceptibilities of mitochondrial membranes and plasma membranes to Cph-induced toxicity. The maintenance of phospholipid structure is necessary for N a + / K + ATPase activity (Katz, 1982). Disruption of this membrane structure by peroxidative injury could result in loss of enzyme activity. Alterations in mitochondrial S D H activity could adversely affect mitochondrial electron transport functions and this would result in deficient cellular energy production. Cellular transport functions and cellular protective mechanisms, such as G S H , would then suffer. Membranes of subcellular organelles, such as mitochondria, are composed of large amounts of polyunsaturated lipids and are highly susceptible to peroxidative injury (Tappel, 1970). Mitochondria can generate free radicals during oxygen reduction and are equipped with protective mechanisms against free radical-induced damage (Neal, 1980). Cph may directly alter mitochondrial lipids or inactivate the enzymes involved in protecting the mitochondria from free radicalinduced damage. Data from this study provide evidence that primary cultures of rat renal cortical epithelial cells may be useful for the in vitro evaluation of nephrotoxic agents. Primary renal cell culture offers several advantages over other methods for assessing the effects of toxic agents on renal cells. Defined populations of cells from specific regions of the kidney can be isolated and grown in culture. Toxicant-induced alterations in cellular biochemistry and morphology can easily be observed within the same sample. Unlike renal cell suspensions, cultured renal cells have had the opportunity to recover from the isolation process and maintain their cell-to-cell interactions and the polarity that is observed in vivo (Ojakian & Herzlinger, 1984). Use of a cell culture system results in fewer animals being used per experiment. C o m p a r e d with other in vitro systems, cell cultures have a greater duration of viability; thus, it is possible to conduct toxicity evaluations over several hours or days. There are, however, limitations to the use of cultured renal cells in toxicity evaluation. In contrast to slice or tubule systems, cell culture is an expensive endeavour. It is more demanding in terms of reagent and equipment expense, in the time involved and in the training of personnel in tissue culture. Moreover, the decline in renal drug metabolizing enzyme activities over time in culture could pose potential problems when evaluating compounds that are enzymatically detoxicated or activated. If the limitations of cell culture systems are taken into consideration prior to designing experiments, however, primary cultures of renal cells can provide a unique way of observing direct renal cellular responses to toxicants. Acknowledgements--This research was supported in part by

grants from the Upjohn Company and EPA Cooperative Agreement, CR-811215. REFERENCES

Acosta D., Anuforo D. C. & Smith R. V. (1980). Preparation of primary monolayer cultures of postnatal rat liver cells. J. Tiss. Cult. Meth. 6, 35 37.

Acosta D., Sorenson E. M. B., Anuforo D. C., Mitchell D. B., Ramos K., Santone K. S. & Smith M. A. (1985). An in vitro approach to the study of target organ toxicity of drugs and chemicals. In Vitro Cell Dev. Biol. 21, 495 504. Atkinson R. M., Caisey J. D., Currie J. P., Middleton T. R., Pratt D. A. H., Sharpe H. M. & Tomich E. G. (1966). Subacute toxicity of cephaloridine to various species. Toxic. appl. Pharmac. 8, 407-428. Belleman P. (1980). Primary monolayer culture of liver parenchymal cells and kidney cortical tubules as a useful new model for biochemical pharmacology and experimental toxicology. Archs Toxicol. 44, 63-84. Beutler G. C., Duron O. & Kelley B. M. (1963). Improved method for the determination of blood glutathione. J. Lab. clin. Med. 61, 882 888. Cojocel C., Hannemann J. & Baumann K. (1985). Cephaloridine-induced lipid peroxidation initiated by reactive oxygen species as a possible mechanism of cephaloridine nephrotoxicity. Biochim. Biophys. Acta 834, 402-4 10. Debetto P., Dal Toso R., Varotto R., Bianchi V. & Luciani S. (1982). Effects of potassium dichromate on ATP content of mammalian cells cultured in vitro. ChemicoBiol. Interactions 41, 15 24. George M., Chenery R. J. & Krishna G. (1982). The effect of ionophore A 23181 and 2,4-dinitrophenol on the structure and function of cultured liver cells. Toxic. appl. Pharmac. 66, 349 360. Gilbert S. F. & Migeon B. R. (1975) D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 5, 11 17. Glasgow E. F., Hancock W. W. & Atkins R. C. (1981). The technique of glomerular culture. In Techniques for Nephropathology. Edited by D. E. Allen & J. P. Dowling. pp. 87-103. CRC Press, Boca Raton, FL. Goldstein R. S., Pasino D. A., Hewitt W. R. & Hook J. B. (1986). Biochemical mechanisms of cephaloridine nephrotoxicity: Time and concentration dependence of peroxidative injury. Toxic. appl. Pharmac. 83, 261-270. Hook J. B. (1980). Toxic responses of the kidney. In Toxicology: The Basic Science o f Poisons. Edited by J. Doull, C. D. Klaassen & M. O. Amdur. pp. 232-245. Macmillan, New york. Hsu B. Y., McNamara P. D., Schlesinger H., Pepe L. M., Marshall C. M. & Segal S. (1981). Ease of solubilization of five marker enzymes in three preparations of rat renal brush border membranes. Enzyme 25, 170-181. Katz A. (1982). Renal Na+/K + ATPase: Its role in tubular sodium and potassium transport. Am. J. PhysioL 242, F207 F219. Kinne R., Schmitz J. E. & Kinne-Saffran E. (1971). The localization of the Na÷/K ÷ ATPase in the cells of the rat kidney cortex. Pflugers Arch. 329, 191-206. Kuo C.-H. & Hook J. B. (1982). Depletion of renal glutathione content and nephrotoxicity of cephaloridine in rabbits, rats and mice. Toxic. appl. Pharmac. 63, 292-302. Kuo C.-H., Maita K. Sleight S. D. & Hook J. B. (1983). Lipid peroxidation: a possible mechanism of cephaloridine-induced nephrotoxicity. Toxic. appl. Pharmac. 67, 78-88. Kyle, G. M., Luthra, R., Bruckner, J. V., MacKenzie, W. F. & Acosta, D. (1983). Assessment of functional, morphological and enzymatic tests for acute nephrotoxicity induced by mercuric chloride. J. ToxicoL envir. Hlth 12, 99-117. Leffert H. & Paul D. (1973). Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine-deficient medium. J. cell. comp. Physiol. 81, 113-124.

McMurtry R. J. & Mitchell J. R. (1977). Renal and hepatic necrosis after metabolic activation of 2-substituted furans

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