Development of a primary culture system of rat kidney cortical cells to evaluate the nephrotoxicity of xenobiotics

Fd Chem. Toxic. Vol. 24. No. 6/7, pp. 551-556, 1986 Printed in Great Britain. All rights reserved

0278-6915/86 $3.00+ 0.00 Copyright © 1986 Pergamon Journals Ltd

DEVELOPMENT OF A PRIMARY CULTURE SYSTEM OF RAT K I D N E Y CORTICAL CELLS TO EVALUATE THE NEPHROTOXICITY OF XENOBIOTICS M. A. SMITH* and D. ACOSTAt College of Pharmacy, Department of Pharmacology and Toxicology, The University of Texas, Austin, TX 78712 and

J. V. BRUCKNER School of Pharmacy, Department of Pharmacology and Toxicology, University of Georgia, Athens, GA 30602, USA Abstraet--A method has been developed for preparing primary monolayer cultures of postnatal rat kidney cortical epithelial cells. These cultures maintained differentiated cell functions and epithelial-like morphology for several days in culture. The presence of alkaline phosphatase and maltase was used to confirm the presence of cells from the renal cortex. The concentrations of these enzymes were maintained in culture until day 3, but had declined significantly by day 5. Similar patterns were observed with cytochrome P-450 and glutathione content, although their concentrations remained stable from day 3 to day 5. Mercuric chloride, cadmium chloride and acetaminophen were evaluated for nephrotoxicity in this culture system. Treatment with these compounds resulted in dose-dependent changes in cell morphology and in biochemical parameters such as lactate dehydrogenase leakage, alkaline phosphatase activity and cellular glutathione content. With this culture system, it was possible to detect the acute toxicities of compounds that produce varying degrees of renal injury. Further development of this kidney culture system may have value in detecting potential nephrotoxins and in studying their mechanisms of toxicity.

Introduction Primary renal cell culture has not been widely explored as a tool for in vitro toxicity evaluation; hence, the literature concerning its use is sparse. Early studies with mercury and lead have used endpoints such as cell growth, morphological alterations and cell death as indicators of toxicity (Vickery & McCann, 1978; Walton & Buckley, 1977). More recent studies with cadmium have focused on biochemical parameters such as metallothionein synthesis, in addition to standard morphological evaluation of toxicity (Cherian, 1980). Belleman (1980) further explored the use of primary renal cell culture as a tool in toxicology by stressing the importance of biochemical markers of cell function, such as brush border enzyme activity. The purpose of this study was to evaluate certain differentiated functions of a primary culture system of kidney cortical cells over a period of 5 days. The ability of the culture system to retain these differentiated functions would then determine the potential suitability of the system as an in vitro model for assessing nephrotoxic agents. We chose to test three well-known nephrotoxic agents in our system. Mercuric chloride and cadmium chloride were selected as representative heavy metals, since they are known to *Current address: Smith, Kline & French Laboratories, 1500 Spring Garden Street, Philadelphia, PA 19101. fTo whom reprint requests should be addressed. Abbreviations: AP = alkaline phosphatase; DMEM = Dulbecco-Vogt modification of Eagle's minimum essential medium; GSH = glutathione; LDH = lactate dehydrogenase. 551

cause renal dysfunction in man (Buchet, Lauwerys, Roels & Bernard, 1980). Acetaminophen was chosen as a prototype of analgesic-induced nephrotoxicity, since metabolism of the compound has been implicated in its ability to cause damage (Duggin, 1980).

Experimental 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 Belleman's procedure for adult rat kidney culture (Belleman, 1980) and the method used by Acosta, Anuforo & Smith (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, 1.5 in. needle with approximately 0.75 ml of dissociation medium. The complete dissociation medium was made from Hank's Ca + +-free balanced salt solution, which contained type IV collagenase (100 units/ml), type II hyaluronidase (60units/ml) and bovine serum albumin (1 mg/ml), all from Sigma Chemical Co. (St Louis, MO). After perfusion, all of 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 1-mm fragments with small scissors. These tissue fragments were transferred to a 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-minute period of gentle mixing, the supernatant

552

M.A. SMITHet al.

was discarded and replaced with 15ml of fresh dissociation medium. After a 15-20-minute 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 minutes. The dissociation flask was refilled with 15 ml of fresh dissociation medium and placed into the water-bath for the second 15-20-minute dissociation period. After the 5-minute centrifugation, the pellet was resuspended in 2.5 ml of arginine-deficient, ornithine-supplemented DulbeccoVogt's modification of Eagle's minimum essential medium (DMEM) which contained (in g/litre) D-valine, (0.094), insulin (0.01), hydrocortisone (0.05), niacinamide (0.3), transferrin (0.005) and bovine serum albumin (0.1), in addition to bovine calf serum (10%,v/v). This cell suspension was centrifuged at 1500rpm for 5 minutes. The pellet was resuspended in 2.5ml 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. The volume of D M E M added was such that each 35-mm culture dish (Falcon) was plated with 0.1-0.5 mg cell protein in 2 ml of culture medium. The cultures were grown in a humidified environment of 5% COJ95% air at 37°C to maintain a pH of 7.2-7.4. Separation of endothelial cells and epithelial cells. Renal epithelial cells were separated from endothelial cells by a differential pour-off technique based on the rate of cell attachment to the culture dishes (Waymouth, 1978). Three hours after initial plating, the cell suspension was swirled and transferred to fresh culture dishes. Endothelial cells and debris had attached at the end of the first 3 hours. 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 hour after initial plating. The timing of the medium change was such that the glomerular cells had not yet attached (Glasgow, Hancock & Atkins, 1981). This ensured that the cultures consisted principally of cortical tubular epithelial cells. In addition, the lack of arginine in the medium helped to suppress fibroblastic overgrowth (Acosta et al. 1980). Determination of functional parameters. Alkaline phosphatase (AP) activity was determined spectrophotometrically by a procedure modified from that described by Wright, Leathwood & Plummer (1972). Maltase activity was determined spectrophotometrically by a procedure modified from that described by Kyle, Luthra, Bruckner et al. (1983). Microsomal cytochrome P-450 was assayed according to the method of Omura & Sato (1964) as modified for cultured cells by Nelson, Acosta & Bruckner (1982). A modified method of Beutler, Duron & Kelley (1963) was used to determine cellular glutathione (GSH) content by measuring non-protein sulphydryls with Ellman's reagent. Toxicity evaluation. Leakage of lactate dehydrogenase (LDH) into the culture medium was measured spectrophotometrically by following the disap-

pearance of N A D H at 340 nm (Mitchell, Santone & Acosta, 1980). To determine whether the agents tested were able to interact directly with LDH, a commercially-obtained LDH (no. 826-6, Sigma Chemical Co.) was incubated directly with each of the three test materials. Mercuric chloride was the only compound that inactivated the enzyme directly. Drug treatments. All experiments were conducted 3-4 days after initial plating of the cells. All of the compounds were dissolved in plain D M E M prior to their addition to the cultures. The stock solutions were freshly prepared for each experiment. Aliquots of the stock solutions were added to each culture dish so that the total volume of each dish contained the desired final concentration of the test compound. The concentrations of mercuric chloride tested were 1 x 10 5, 1 x 10 -4 and 1 x 10 -3 M, and the cells were exposed to mercuric chloride for 30 minutes, 1 hour and 4 hours. The concentrations of cadmium chloride were 50, 100, 200 and 400 pM and the exposures were for 2 and 4 hours. Acetaminophen was tested at concentrations of 1 x 10 -5, 1 x 1 0 - 4 and 1 x 10 - 3 M, with exposures lasting t'or 24, 36 and 48 hours. Statistical analysis. The statistical analysis was performed using analysis of variance and Scheffe's post hoc test for significance (P ~< 0.01 and P ~<0.05). Values always represent the mean + SEM. Results

Cell morphology Cell morphology was observed throughout the culture period with the aid of an inverted phasecontrast microscope. After 24 hours in culture, the cells were grouped in rounded clusters, which had not yet attached firmly to the culture dish. By 48 hours, these clusters of cells began to flatten out into small islands of cells. These islands of cells attained a more polygonal, cobblestone shape, typical of epitheliallike cells, by day 3 in culture. The cultures became confluent between day 3 and day 5.

Alkaline phosphatase and maltase activity AP activity was monitored on days 1, 3 and 5 after cell plating. The levels of AP on day 3 in culture were not significantly different from those observed on day 1. By day 5, AP levels had dropped significantly, to 39% of day 1 values (data not shown). Maltase activity was also monitored on days 1, 3 and 5 in culture. As with AP, maltase levels on day 3 were not significantly different from those observed on day 1. However, by day 5, maltase activity had dropped nearly to zero (data not shown). Cytochrome P-450 content Cytochrome P-450 content was monitored in microsomes isolated from cells grown for 1, 3, or 5 days. P-450 levels dropped by 35% after 3 days in culture. By day 5 these levels had dropped by 51%. The P-450 levels on days 3 and 5 were significantly lower than the levels seen on day 1 in culture (data not shown). Glutathione concentrations in culture GSH levels were measured in cells grown in regular D M E M (without insulin, hydrocortisone, transferrin

In vitro nephrotoxicity of xenobiotics

553

Table 1. Comparisonof functionalparameters in kidneypreparations Values for primary kidney cultures on Parameter

Day 1

Alkaline phosphatase (units/mg protein) Maltase (units/mg protein) Cytoehrome P-450 (nmol/mg protein) Glutathione (/ag/mg protein)

Day 3

0.022 0.03 0.93 11.91

Reported values*

0.020 0.025 0.60 5.69

0.100 isolated kidney tubules (a) 0.40 kidney homogenate (b) 0.49~.58 liver cell culture (c) 8.0 mouse kidney cell culture (d)

*References: (a) Belleman (1980), (b) Meister (1983), (c) Maack (1980) and (d) Reiss & Sacktor (1982).

and niacinamide) and in complex DMEM. The concentrations of GSH dropped by approximately 50% after 3 days in culture in both types of media. These concentrations remained stable through day 5. At each time point, GSH concentrations in cells grown in complex DMEM were not significantly different from those in the cells grown in regular DMEM (data not shown).

Comparisons of functional parameters in different kidney preparations Table 1 compares the values measured for each parameter in our primary kidney cell culture system with values reported in other types of kidney preparations. Whereas the activities of brush border enzymes are low, the levels of P-450 and GSH compare favourably with reported values for kidney preparations.

A E

Toxicity evaluations The leakage of LDH into the culture medium was used as an index of cytotoxicity following treatment with mercuric chloride, cadmium chloride or acetaminophen. AP activity was also monitored after treatment with mercuric chloride or cadmium chloride. GSH concentrations were determined following treatment with acetaminophen. Mercuric chloride. Exposure to mercuric chloride for 30 or 60 minutes caused a concentrationdependent decrease in LDH activity (Fig. 1). Inhibition of LDH activity, rather than LDH leakage, proved to be a reliable index of mercuric chloride toxicity. AP activity was not significantly decreased by mercuric chloride except in the 4-hour treatment (Fig. 2). Mercuric chloride treatment resulted in distinct changes in cellular morphology. The most striking alterations appeared after treatment for 1 hour with 1 x 10-3M-mercuric chloride. Plasma membrane destruction was evident with loss of intracellular constituents; in addition, there was nuclear condensation. Cells treated for 1 hour with 1 x 10 4 M-mercuric chloride appeared pyknotic with scattered plasma membrane degeneration. Early nuclear changes were also noticeable. Cadmium chloride. Treatment with cadmium chloride resulted in a dose- and time-dependent increase

200

004

--

Q.

E

%.

E

E 3

o T Q .j

0.03

N

100

'~

~N

Control

lo-s

10° 4

HgCl 2 concn (M)

i

10 -3

Fig. 1. Lactate dehydrogenase (LDH) activity after treatment for 30min ([~) or 1 hr ([]) with mercuric chloride. Values are means _+SEM (n = 4-8), with asterisks indicating those differing significantly from the corresponding control: *P ~<0.01.

0.02

- -

--

o cL 0.01

1 o

I 0

I

I 2

I 3

I 4

Duration of exposure (hr)

Fig. 2, Alkaline phosphatase (AP) activity after treatment for 0.5, l or 4 hr with 0 (I-l), 1 x I0 -5 (11), 1 x l0 -4 (A) or 1 × 10-3 (O) M-mercuricchloride. Values are means _+SEM (n = 4-6), w i t h asterisks indicating those differing significantly from the corresponding control: *P ~<0.05.

M . A . SMITH et al.

554 1000

--

900

--

800

-

Table 2. Leakage of LDH into culture medium following acetaminophen treatment Concn of LDH activity (units/rag protein) at: acetaminophen (M) 24 hr 48 hr 0 (control) 465 ± 14.1 734.3 + 77.9 ] x 10-5 485.3 _+8.3 822.0 + 24.0 l xlO 4 533.5_+31.9 764.3_+21.7 l x 10-3 550.8 _+20.1" 764.3 _+21.7

(a)

Values represent the mean _+SEM (n = 4-6), and that marked with an asterisk differs significantly from the corresponding control: P _<0.05.

E

700

-

600

-

500

-

400

-

E 'E >

,J'

"I/

500

T

T

-

Z_.

I 2

o Duration

I 4

of e x p o s u r e ( h r )

0.04 , (b)

in L D H leakage (Fig. 3a) a n d , with the 2- a n d 4 - h o u r e x p o s u r e s , in a d o s e - a n d t i m e - d e p e n d e n t decrease in A P activity (Fig. 3b). T r e a t m e n t o f the renal cells with c a d m i u m c h l o r i d e resulted in cell s h r i n k a g e a n d p l a s m a m e m b r a n e irregularities. A f t e r 2 0 0 p M c h l o r i d e e x p o s u r e , the cells s e p a r a t e d f r o m o n e ano t h e r a n d cell lysis was a p p a r e n t in s c a t t e r e d areas, while 400 p M - c a d m i u m c h l o r i d e t r e a t m e n t resulted in a l m o s t total cell lysis. Acetaminophen. L D H leakage into the m e d i u m w a s d e t e r m i n e d after e x p o s u r e to a c e t a m i n o p h e n for 24 a n d 48 h o u r s (Table 2). Significant leakage o f L D H was o b s e r v e d only after 2 4 - h o u r e x p o s u r e to 1 x 10 - 3 M - a c e t a m i n o p h e n . Cellular G S H c o n c e n t r a t i o n s were d e t e r m i n e d after e x p o s u r e for 24, 36 a n d 48 h o u r s (Fig. 4). T h e lowest c o n c e n t r a t i o n o f a c e t a m i n o p h e n resulted in G S H elevation. T h e interm e d i a t e a n d high c o n c e n t r a t i o n s c a u s e d G S H to decline at 36 h o u r s , with r e c o v e r y at 48 hours. M o r p h o l o g i c a l a l t e r a t i o n s were n o t e v i d e n t until 24 h o u r s after the a d d i t i o n o f 1 x 10 3M-acetaminop h e n . T h e c h a n g e s o b s e r v e d were m i n i m a l . T h e y 120

I

0.03

N z

110

, om

1oo

•~: 0.02

o

.f

1

c

°°

9o

z~

N

13_

T

=

8o

§

0.01

T

~

": \

-

i! ~

-

.KNI

.~'~

• . \

-

.~NI

.,,\

-

:K.'q

7o

60 0

/,I

I 2

Duration

of e x p o s u r e ( h r )

0

I 4

24

Fig. 3. (a) Lactate dehydrogenase (LDH) leakage into the medium and (b) alkaline phosphatase (AP) activity, after treatment for 2 or 4 hr with 0 (r-l), 50 (©), 100 (11), 200 (A) or 400 (@) #M-cadmium chloride. Values are means + SEM (for (a) n = 8 and for (b) n = 4), with asterisks indicating those differing significantly from the corresponding control: *P ~< 0.01.

Duration

36

48

of e x p o s u r e ( h r )

Fig. 4. Cellular glutathione (GSH) content following treatment for 24, 36 or 48 hr with 1 x 10 -5 (l~), I x 10 -4 ( i ) or 1 x 10 -3 ([]) u-acetaminophen. Values are m e a n s _ SEM (n = 4), with asterisks indicating those differing significantly from the corresponding control: *P ~<0.05.

In vitro nephrotoxicity of xenobiotics

included slight plasma membrane disruption and some vacuolization of the cells.

Discussion

We have developed a primary renal cell culture system capable of maintaining differentiated cell functions. The integrity of our primary kidney cultures was determined on the basis of cell morphology and function. Arginine-deficient and D-valine supplemented culture medium was used to enhance epithelial cell growth and to suppress fibroblast overgrowth (Gilbert & Migeon, 1975; Leffert & Paul, 1973). These cell cultures were observed through an inverted phase-contrast microscope to ensure that they maintained a characteristic epithelial-like cell morphology throughout the duration of the culture periods. The presence of marker enzymes, AP and maltase, indicated the proximal tubular nature of our cultured cells (Hsu, McNamara, Schlessinger et al. 1980). o u r cultures maintained a greater percentage of AP activity over the duration of the culture than that reported by Belleman (1980) for his system of cultured kidney cells. These observations indicate that alterations in AP activity in our cultured cells could serve as a useful index of xenobiotic-induced renal toxicity in vitro.

This cell culture system maintained stable concentrations of cytochrome P-450 and GSH for 3 days in culture. The P-450 concentrations in the kidney cultures were consistent with the values previously reported by our laboratory for liver cells cultured in complex D M E M (Nelson et al. 1982). Values for GSH content in our kidney cultures compared favourably with values reported for cultured mouse and hamster kidney cells (Summer & Wibel, 1981). As these primary cultures of renal cortical epithelial cells retained differentiated functions for an extended period, they should be useful tools for the in vitro evaluation of nephrotoxic agents. To establish the validity of primary renal cell culture as a toxicological tool, it was necessary to evaluate several compounds known to be nephrotoxic in vivo. Some heavy metals, analgesics and antibiotics are known to fall into this category (Hook, McCormack & Kluwe, 1979). The evaluation of known nephrotoxic agents in a cell culture system can help to establish guidelines for determining toxicity in vitro. Criteria delineated by changes in organ-specific functions, cellular organdie functions and cell morphology can then be applied to the evaluation of potential nephrotoxins in vitro. Exposure to mercuric chloride results in proximal tubular necrosis (Buchet et al. 1980; Fowler, 1983). In vitro studies have evaluated mercuric chloride nephrotoxicity on the basis of RNA, D N A and protein synthesis (Inamoto, Ino, Inamoto et al. 1976). The time frame of mercuric chloride toxicity studied by Inamoto et al. (1976) ranged from 1 to 6 days in culture. Vickery & McCann (1978) observed mercuric chloride alterations in cell growth over a period of 3 days. We were able to detect inhibition of LDH activity as early as 1 hour after mercuric chloride treatment and a decline in AP activity was seen after

555

4 hours. We saw morphological alterations due to mercuric chloride as early as l hour after treatment. Functional changes resulting from cadmium nephrotoxicity include proteinuria, enzymuria, aminoaciduria, glycosuria and polyuria ( C h a n & Rennert, 1981). Biochemical, histochemical and histological studies on AP activity in rats, after treatment with cadmium, indicated that AP activity decreased because of cadmium-induced cell degeneration (Peereboom-Stegeman, Melet, Peereboom & Hooghwinkel, 1979). Cherian evaluated cadmium toxicity in primary cultures of renal epithelial cells, using cell detachment and decreased colony size as indices of toxicity, rather than alterations in biochemical markers of renal function. We evaluated cadmium chloride toxicity in our primary culture system on the basis of changes in LDH leakage and AP activity. The increased LDH leakage correlated well with cadmium-induced enzymuria (Chan & Rennert, 1981). The decline observed in AP activity was in line with that observed by Peereboom-Stegeman et aL (1979). Large doses of acetaminophen can result in hepatotoxicity and nephrotoxicity (Boyd, Grygiel & Minchin, 1983; Hook et al. 1979). Covalent binding of a reactive acetaminophen metabolite, formed by cytochrome P-450, is thought to be responsible for the hepatotoxicity and nephrotoxicity (Hinson, 1980). McMurtry, Snograss & Mitchell (1978) reported early morphological alterations in proximal tubular cells 6 hours after acetaminophen exposure. They reported necrosis to be greatest at 24-48 hours, followed by regeneration at 72-96 hours. They also demonstrated that covalent binding to renal microsomal protein was greatest when cellular GSH stores were depleted. In addition, they found a rebound increase in renal GSH content following acetaminophen-induced GSH depletion. We observed the effect of acetaminophen on LDH leakage and cellular GSH content. LDH leakage increased at 24 hours, but was not significantly different from controls by 48 hours. It is possible that the concentrations of acetaminophen tested resulted in only slight membrane injury and the cells were able to recover. Also, the metabolism or concentrations of acetaminophen were such that an overt toxicity was not apparent. Our studies with GSH indicate a biphasic response of our system to cellular injury. GSH undergoes a rapid turnover in the kidneys and an injurious stimulus can result in a compensatory synthesis of GSH (Reed & Beatty, 1980). Our findings regarding LDH leakage correlated with the initial toxicity followed by regeneration seen by McMurtry et al. (1978). Similarly, the rebound increase in GSH we observed paralleled that observed by McMurtry et al. (1978). Our studies with mercuric chloride, cadmium chloride and acetaminophen have helped to establish the validity of our primary renal cell culture system as a tool for toxicity evaluation. Since our cultures can detect the gross toxicity induced by metals and the more subtle toxicity of acetaminophen, they should prove useful in assessing the toxicity of a variety of potential nephrotoxins. With the appropriate biochemical markers for renal cell and organelle function, further development of this culture system

M. A. SMITH et al.

556

should m a k e it a useful tool for studying the mechanisms o f nephrotoxicity. Acknowledgements--This research was supported in part by grants from the Upjohn Company and EPA Cooperative Agreement, CR-807499. 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. 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. Beutler G. L., Duron O. & Kelley B. M. (1963). Improved method for the determination of blood glutathione. J. Lab. clin. Med. 61, 882. Boyd M. R., Grygiel J. J. & Minchin R. F. (1983). Metabolic activation as a basis for organ selective toxicity. Clin. exp. Pharmac. 10, 87. Buchet J. P., Lauwerys R., Roels H. & Bernard A. (1980). Relationship between exposure to heavy metals and prevalence of renal dysfunction. Archs Toxicol. Suppl. 4, 215. Chan W. Y. & Rennert O. M. (1981). Cadmium nephropathy. Ann. clin. Sci. 11, 229. Cherian G. M. (1980). The synthesis of metallothionein and cellular adaptation to metal toxicity in primary rat kidney epithelial cell cultures. Toxicology 17, 225. Duggin G. G. (1980). Mechanisms in the development of analgesic nephropathy. Kidney Int. 15, 553. Fowler B. A. (1983). Role of ultrastructural techniques in understanding mechanisms of metal-induced nephrotoxicity. Fedn Proc. Fedn Am. Socs exp. Biol. 42, 2957. 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. Glasgow E. F., Hancock W. W. & Atkins R. C. (1981). The Technique of Glomerular Culture. Edited by D. E. Allen & J. P. Dowling, p. 87. CRC Press, Boca Raton, FL. Hinson J. A. (1980). Biochemical toxicology of acetaminophen. In Reviews in Biochemical Toxicology. Vol. 2. Edited by E. Hodgson, J. R. Bend & R. M. Philpot, p. 103. Elsevier, New York. Hook, J. B., McCormack, K. M. & Kluwe W. M. (1979). Biochemical mechanisms of nephrotoxicity. In Reviews in Biochemical Toxicology. Vol. 1. Edited by E. Hodgson, J. R. Bend & R. M. Philpot, p. 53. Elsevier, New York. Hsu B. Y., McNamara P. D., Schlesinger H., Pepe L. M., Marshall C. M. & Segal S. (1980). Ease of solubilization of five marker enzymes in three preparations of rat renal brush border membranes. Enzyme 25, 170. Inamoto H., Ino Y., Inamoto N., Wada T., Kihara H. K.,

Watanabe I. & Asano S, (1976). Effect of HgC12 on rat kidney cells in primary culture. Lab. Invest. 34, 489. 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. Leffert H. & Paul D. (1973). Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine-deficient medium. J. Cell Physiol. 81, 113. Maack T. (1980). Physiological evaluation of the isolated perfused rat kidney. Am. J. Physiol. 238, F71. McMurtry R. J., Snodgrass W. R. & Mitchell J. R. (1978). Renal necrosis, glutathione depletion, and covalent binding after acetaminophen. Toxic. appl. Pharmac. 46, 87. Meister A. (1983). Selective modification of glutathione metabolism. Science, N.Y. 220, 472. Mitchell D. B., Santone K. S. & Acosta D. (1980). Evaluation of cytotoxicity in cultured cells by enzyme leakage. J. Tiss. Cult. Meth. 6, 113. Nelson K. F., Acosta D. & Bruckner J. V. (1982). Longterm maintenance and induction of cytochrome P-450 in primary cultures of rat hepatocytes. Biochem. Pharmac. 31, 2211. Omura T. & Sato R. (1964). The carbon monoxide-binding pigment of liver microsomes. J. biol. Chem. 239, 2370. Peereboom-Stegeman J. H. J., Melet J., Peereboom J. W. C. & Hooghwinkel G. J. M. (1979). Influence of chronic cadmium intoxication on the alkaline phophatase activity in liver and kidney: biochemical, histochemical and histological investigations. Toxicology 14, 67. Reed D. J. & Beatty P. W. (1980). Biosynthesis and regulation of glutathione: Toxicological implications. In Reviews in Biochemical Toxicology. Vol. 2. Edited by E. Hodgson, J. R. Bend & R. M. Philpot, p. 2133. Elsevier, New York. Reiss U. & Sacktor B. (1982). Alteration of kidney brush border membrane maltase in aging rats. Biochem. biophys. Acta 7tl4, 422. Summer K. H. & Wiebel F. J. (1981). Glutathione and glutathione S-transferase activities of mammalian cells in culture. Toxicology Lett. 9, 409. Vickery H. M. & McCann D. S. (1978). Temperature and species differences in susceptibility of kidney cell cultures to mercury toxicity. In Vitro 14, 312. Walton J. & Buckley, I. K. (1977). The lead poisoned cell: A fine structural study using cultured kidney cells. Expl Molec. Path. 27, 167. Waymouth C. (1978). Studies on chemically defined media and nutritional requirements of cultures of epithel!al cells. In Nutritional Requirements o f Cultured Cells. Edited by H. Katsuta. p. 39. University Park Press, Baltimore. Wright P. J., Leathwood P. D. & Plummer D. T. (1972). Enzymes in rat urine: alkaline phosphatase. Enzymologia 42, 317.