Renal cell cultures: a tool for studying tubular function and nephrotoxicity

Toxicology Letters, 53 (1990) l-7 Elsevier

TOXLET 02376

Renal cell cultures: a tool for studying tubular function and nephrotoxicity

Gerhard Gstraunthaler, Institute oj Physialogy, University

Dieter Steinmassl and Walter Pfaller ofInnsbruck,Innsbruck

Key words: LLC-PI&ceI1 line, cell culture; Cephalosporins;

(Austria)

In vitro nephrotoxicity

INTRODUCTION

Investigation of normal as well as impaired renal function has traditionally been a ‘technique-oriented’ field. The methodological approaches developed in basic renaI physiological and nephrological research offer today a variety of in vitro model systems applicable in renal physiology, pharmacology and toxicology studies. The most widely used in vitro methods are listed in Table I. Out of these, renal epithelial cell and tissue cultures have emerged as a powerful tool to study renal growth and differentiation, epithelial transport and its regulation by metabolism, hormones or drugs VI. Modern cell culture techniques enable renal epithelial cells to grow and be maintained at a state of differentiation, comparable with the in vivo tissue [I]. Furthermore, homogenous populations of cells can be grown for biochemical analysis or toxicological studies [2-lo]. For investigating epithelial transport in vitro, cells can be grown on permeable supports, thus separating the apical from the basolateral compartment by the cultured epithelium [1 l-191. In recent years a number of continuous epithelial cell lines have been established, which are listed in Table II. The biochemical and physiological characteristics of these cell lines have been reviewed in detail [I]. Since continuous cell lines, by definition, are capable of indefinite growth, cells can be maintained for extended culture periods under defined and controlled environmental conditions. The use of cell biological, immunological and molecular biological methods has opened up new avenues in physiological and toxicologicaf research. Address for

correspondence: Gerhard Gstraunthaler, Ph.D., Institute Innsbruck, Fritz-PregI-Stmsse 3, A-6010 Innsbruck, Austria.

of Physiology,

0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

University

of

2 TABLE I IN VITRO MODEL SYSTEMS TO STUDY RENAL FUNCTION AND NEPHROTOXICITY Isolated perfused kidney Kidney slices Suspensions of tubular fragments Isolated perfused tubule Primary cultures of renal cells Continuous renal ceii lines Isolated kidney cell organelles Modified after Williams [20]. TABLE II CONTINUOUS

EPITHELIAL CELL LINES

Cell line

Species

Tissue origin

LLC-PK, OK JTC-I2 NRK MDCK A6 GRB-MAL TB-M, TB-6c HT-29 Caco-2 T 84

fig

Proximal tubule Proximal tubule Proximal tubule Proximal tubule Distal tubule/toll. duct Distal tubulefcofl. duct Medullary thick ascending limb Urinary bladder Colon adenocarcinoma Colon aden~arcinoma Colon adenocarcinoma

Opossum Monkey Rat Dog Xenopus laevis Rabbit Bujb marinus Human Human Human

Finally, the ability to modify, bi~hemically and genetically, cultured cells allows the selection and isolation of sublines, strains, clones and mutants out of a parental cell population. Thus the advantages of using cultured epithelial cells in studies of cell injury are obvious and manifold. Easy manipulation of the cells and the ability to change parameters individually, or in combination, singly or sequentially in shortor long-term application have provided cultured epithelia as valuable tools for investigating tubular cell damage at the cellular and subcellular level without inducing regulatory mechanisms as in complex organisms [20,21]. However, the use of cultured renal cells also bears specific limitations, as do other model systems including experimental animals. Although continuous epithelial cell lines retain a number of differentiated properties of their ancestor cells [l], cultured epithelia have lost some of the in vivo characteristics during adaptation to tissue culture. In addition, cell culture conditions, like culture media, serum and growth factors, culture substrata and the extracellular matrix, substantially influence the expression of specific morphological features and physiological functions and thus the degree of cell differentiation [22-

3

251. The advantages and disadvantages of the use of renal cell cultures in studies of tubular function and injury are summarized in Table III. The use of renal cell cultures in studies of drug-induced tubular cell injury or screening of new drugs for potential nephrotoxicity is still in the initial stages. Previously, on epithelial cultures (LLC-PK, and Caco-2) the nephrotropic action of the fungal toxin orellanine was studied in vitro [lo], and cultured bovine kidney cells were used to evaluate the cytotoxicity of aflatoxin Br [26]. In a series of papers [27321, the nephrotoxic action of gentamicin was studied on cultured renal cells. Since the primary target of aminoglycoside toxicity is the proximal tubular epithelium [33351, most of the studies were performed with LLC-PKi cells, which retain in tissue culture many properties of proximal tubular cells [l] and have been morphologically and biochemically described in detail in our laboratory [2,3-9,361. However, in the studies mentioned above, gentamicin-induced LLC-PKt cell injury was only monitored with respect to the release of cellular enzymes [28], and changes in cytosolic calcium content [20,27] and phospholipid composition [29,32]. We therefore tried, in a combined morphological, biochemical and electrophysiological study, to correlate gross morphological appearance and enzyme release of LLC-PKi cells with electrophysiological parameters as a measure of the integrity of the cultured epithelium. As nephrotoxic agents, the cephalosporins cephaloridine (CPH), ceftazidime (CTZ) and cefotaxime (CTX) were used, whose adverse side effects on renal tubular epithelium are well documented [35,37,38].

TABLE III ADVANTAGES AND DISADVANTAGES Advantages

Disadvantages

Extended viability, unlimited lifespan

Altered (reduced) functional and metabolic properties compared with cells in vivo

Long-term storage of specific cell types and strains

Technical limitations in cell culture

Study of cell-specific effects and responses

Possible morphologic and functional changes of cells during long-term culture

Separate, bidirectional exposure to drugs Bidirectional cellular and transepithelial transport Rapid screening Automation Modified after Williams [20].

4

EXPERIMENTAL

PROTOCOL

Stock cultures of LLC-PKt cells were routinely grown as described elsewhere [3, $9,361. Experiments were performed on LLC-PKt cells grown in plastic tissue culture dishes and on LLC-PKt epithelia cultured on collagen-coated cellulose ester filters (MillicellTM HA, Millipore) [13-17,24,39]. Culture dishes or filters grown 10-14 days to confluence with LLC-PKt monolayers were exposed to different concentrations of CPH, CTZ and CTX for 24 and 48 h, respectively. Toxic effects were checked by measuring the release of activities of the apical membrane enzymes alkaline phosphatase and y-glutamyltranspeptidase, cytosolic lactate dehydrogenase and mitochondrial glutamate dehydrogenase [40]. In parallel, culture gross morphology was monitored by phase-contrast microscopy. The functional integrity of filter-grown LLC-PKt epithelia was determined by measuring dilution potentials after imposing concentration gradients for both sodium and chloride over control and cephalosporin-treated epithelia, i.e. the anion-tocation permeability ratio [ 11,17,18]. The resulting transepithelial potential differences were recorded with a high-impedance microvoltmeter (Keithley 197 DMM) connected to the bathing solutions by calomel half cells with 3 M KC1/2% agar bridges [16,17,19]. RESULTS

AND DISCUSSION

The results of this series of experiments are summarized in Table IV. Treatment of LLC-PKt cells grown in plastic tissue culture dishes with CPH, CTZ or CTX caused a dose-dependent deterioration of cell monolayer integrity, changes in cell morphology, and release of cellular enzymes. Beginning disintegration of cell monolayer occurred after culture treatment for 48 h with 0.5 mg/ml CPH, 1.Omg/ml CTZ

TABLE

IV

SUMMARY

OF RESULTS

AND COMPARISON

WITH

Cephaloridine

IN VIVO DATA

FROM

Ceftazidime

RATS

(CTZ)

(CPH)

Cefotaxime (CTX)

(concentration:

mg/ml)

Morphological changes Enzyme release

0.5 -1 0.5 -1

0.5 -1 0.75-1.5

Epithelial

0.384.76

1.2 -2.4

3.8-7.6

1

1.5 -3

10

500

1000

5ooo

permeability

Factor Tubulotoxic

dose in rats (mg/kg/d

(Sack et al. [38])

KC.)

5-10 5-10

5

and 5.0 mg/ml CTX. The release of cellular marker enzymes displayed a similar pattern (Table IV). LLC-PKt epithelia grown on permeable filter supports were used to study transepithelial transport properties as a measure of epithelial integrity and function. The anion-to-cation ~rmeability ratio [ 11,17,18] was found to be the most valuable and sensitive parameter. Drastic decreases in LLC-PKt epithelial pe~sel~tivity were obtained at drug concentrations well below the doses where first increases in enzyme release were found (Table IV). Comparison of the concentrations of the 3 cephalosporins capable of eliciting cytotoxic effects on LLC-PKi cells show that the concentration of CTZ needed to be 2-3 times and that of CTX even 10 times higher than that of CPH. These differences in cytotoxic concentrations are closely comparable with the tubulotoxic threshold doses reported for rats [38] (Table IV). PERSPECTIVES

Since one aim of studying renal cell injury is to explore mechanistic aspects of xenobiotic toxicity and its prevention, renal epithelial cells in tissue culture may provide a powerful tool for this type of study at the cellular and subcellular level. Drug-induced impairment of renal function is mostty caused by tubular damage and loss of epithelial integrity resulting in a decrease of the reabsorptive capacity of the kidney [34,35,38]. As shown in the present report, this impairment of tubular integrity and function found in vivo can be well explored on cultured LLC-PK, cell sheets in vitro. Furthermore, as more data on basic metabolism [1,7-93 and specific detoxification pathways [41-43] of cultured renal cells become available, studies on renal biotransformation and detoxification of xenobiotics and drugs can be performed in vitro. Last, but not least, cultured epithelial cells may become valuable alternatives to animal experiments in nephrotoxicity screening. REFERENCES 1 Gstraunthaler, G,J.A. (1988) Epithelial cells in tissue culture (Review). Renal Physiol. B&hem. 1I, l-42. 2 Frick. H., Gstraunthaler, G. and Pfaller, W. (1989) Modification of membrane protein expression and protein secretion in LLC-PK, cultures grown on different carbohydrates. Renal Physiol. Biochem. 12, 393-399. 3 Gstraunthaler, G., Pfaller, W. and Kotanko P. (1985) Biochemical characterization of renal epithelial cell cultures (LLC-PK, and MDCK). Am. J. Physiol. 248, F536-F544. 4 Gstraunthaler, G., Pfaller, W. and Kotanko, P. (1985) Lack of f~cto~l,6-bisphospha~se activity in LLC-PK, cells. Am. J. Physiol. 248, C181-Cl83. 5 Gstraunthaler, G. and Handler, J.S. (1987) Isolation, growth and characterization of a gluconeogenic strain of renal cells. Am. J. Physiol. 252. C232-6238. 6 Gstraunthaler, G., Harris, H.W. and Handler, J.S. (1987) Precursors of ribose-S-phosphate suppress expression of glucose-regulated proteins in LLC-PK, cells. Am. J. Physiol. 252, C239-C243.

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7 Gstraunthaler, G. and Pfaller, W. (1989) The pathway of ammoniagenesis in LLC-PK, cells. Kidney lnt. 35,445. 8 Gstraunthaler, G. and Pfaller, W. (1989) ~oduiation of the a~oniagenic response of LLC-PK, cultured epithelia: the role of tran~mination. Miner. Electrolyte Metab. 15,371. 9 Gstraunthaler, G., Gersdorf, E., Fischer, W.M., Joannidis, M. and Pfaller, W. (1990) Morphological and biochemical changes of LLC-PK, cells during adaptation to glucose-free culture conditions. Renal Physiol, Biochem. 13, 137-153. 10 Rued& Ch., Gstraunthaler, G. and Moser, M. (1989) Differential inhibitory action of the fungal toxin orellanine on alkaline phosphatase isoenzymes. Biochim. Biophys. Acta 991,28&283. 11 Cereijido, M., Robbins, ES., Dolan, W.J., Rotunno, CA. and Sabatini, D.D. (1978) Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77,853-880. I2 Cho, M.J., Thompson, D.P., Cramer, CT., Vidmar, T.J. and Scieszka, J.F. (1989) The Madin Darby Canine Kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm. Res. 671-77. 13 Misfeldt, D.S., Hamamoto, ST. and Pitelka, D.R. (1976) Transepitheiial transport in cell culture. Proc. Natl. Acad. Sci. USA 73,1212-1216. 14 Misfeldt, D.S. and Sanders, M.J. (1981) Tran~pithelial transport in cell culture: n-glucose transport by a pig kidney cell line (LLC-PK,). J. Membrane Biol. 59, 13-18. 15 Mullin, J.S. and O’Brien, T.G. (1987) Spontaneous reversal of polarity of the voltage across LLC-PKr renal cell sheets. J. Cell. Physiol. 133, 515-522. 16 Pitt, A.M., Gabriels, J.E., Badmington, F., McDowell, J., Gonzales, L. and Waugh, M.E. (1987) Cell culture on a microscopically transparent microporous membrane. BioTechniques 5, 162-171. 17 Rabito, CA. (1986) Occluding junctions in a renal cell line (LLC-PK,) with characteristics of proximal tubular cells. Am. J. Physiol. 250, F734-F743. 18 Rabito, C.A., Tchao, R., Valentich, J. and Leighton, J. (1978) Distribution and characteristics of the occluding junctions in a monolayer of a call line (MDCK) derived from canine kidney. J. Membrane Biol. 43,351-365. 19 Simmons, N.L. (1981) Ion transport in ‘tight’ epithelial monolayers of MDCK cells. J. Membrane Biol. 59,105-l 14. 20 Williams, P.D. (1989) The application of renal calls in culture in studying drug-induced n~phrotoxicity. In Vitro Cell. Dev. Biol. 25,80&805. 21 Wilson, P.D., (1986) Use of cultured renal tubular cells in the study of cell injury. Miner. Electrolyte Metab. 12, 71-84. 22 Cook, J.R., Crute, B.E., Patrone, L.M., Gabriels, J., Lane, M.E. and Von Buskirk, R.G. (1989) Microporosity of the substratum regulates differentiation of MDCK cells in vitro. In Vitro Cell. Dev. Biol. 25,914-922. 23 Handler, J.S., Preston, A.S. and Steele, R.S. (1984) Factors affecting the diffe~ntiation of epithelial transport and responsiveness to hormones. Fed. Proc. 43,2221-2224. 24 Ip, T.K., Galletti, P.M. and Aebischer, P. (1990) Effects of attachment substrates on the growth and differentiation of LLC-PK, cells. In Vitro Cell. Dev. Biol. 26,162-168. 25 Leiderman, L.J., Tucker, J.A. and Dennis, V.W. (1989) Growth and differentiation of opossum kidney cells on microscopically transparent microporous membranes. Tissue Cell 21,355-360. 26 Yoneyama, M., Sharma, R.P. and Elsner, Y.Y. (1987) Effects of mycotoxins in cultured kidney cells: cytotoxicity of aflatoxin B, in Madin-Darby and primary fetal bovine kidney cells. Ecotoxicol. Environ. Safety 13, 174-184. 27 Holohan, P.D., Sokol, P.P., Ross, C.R., Coulson, R., Trimble, M.E., Laska, D.A. and Williams, P.D. (1988) Gentamicin-induced increases in cytosolic calcium in pig kidney cells (LLC-PK,), J. Pharmacol. Exp. Ther. 247,349-354. 28 Hori, R., Yamamoto, K., Saito, H., Kohno, M. and Inui, K.-I, (1984) Effect of aminoglycoside antibiotics on cellular functions of kidney epithelial cell line (LLC-PK,): a model system for aminoglycoside nephrotoxicity. J. Pharmacol. Exp. Ther. 230,742-748.

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29 Inui, K.-I., Saito, H., Iwata, T. and Hori, R. (1988) A~no~ycoside-i~du~ alterations in apical membranes of kidney epithelial cell line (LLC-PI&). Am. J. Physiol. 254, C25 1x257. 30 Ramsammy, L.S., Josepovitz, C., Lane, B. and Kaloyanides, G.J. (1989) Effect of gentamicin on phospholipid metabolism in cultured rabbit proximal tubular cells. Am. J. Physiol. 256, C204C213. 31 Saito, H., In& K.-I and Hori, R. (1986) Mechanisms of pntamicin transport in kidney epithelial cell line (LLC-PK,). J. Pharmacol. Exp. Ther. 238,1071-10’76. 32 Schwertz, D.W., Kreisberg, J.I. and Ventkatachalam, M.A. (1986) Gentamicin-indu~ alterations in pig kidney epithelial (LLC-PK,) cells in culture. J. Pharmacol. Exp. Ther. 236,254-262. 33 Matsuda, O., Beck, F.-X, Diirge, A. and Thurau, K. (1988) Electrolyte composition of renal tubular cells in gent~jcin nephrotoxicity. Kidney Int. 33,1107-l 112. 34 Tulkens, P.M. (1989) Nephrotoxicity of aminoglycoside antibiotics. Toxicol. Lett. 46,107-123. 35 Walker, R.J. and Duggin, G.G. (1988) Drug nephrotoxicity. Annu. Rev. Pharmacol. Toxicol. 28,33134.5. 36 Pfaller, W., Gstrauntbaler, G. and Loidl, P (1990) Morphology of the differentiation and maturation of LLC-PK, epithelia. J. Cell. Physiol. 142,247-254. 37 Goldstein, R.S., Smith, P.F., Tarloff, J.B., Contardi, L., Rush, G.F. and Hook, J.B. (1988) Biochemical mechanisms of cephaloridine nephrotoxicity. Life Sci. 42, 18091816. 38 Sack, K., Marre, R. and Schulz, E. (1985) Renale Nebenwirkungen von Beta-Lak~m-Antibiotika. Krankenhauspha~~e 6,415-418. 39 Steele, R.E., Preston, A.S., Johnson, J.P. and Handler, J.S. (1986) Porous-bottom dishes for culture of polarized cells. Am. J. Physiol. 251, C136-Cl39. 40 Pfaller, W., Joannidis, M., Gstraunthaler, G. and Kotanko, P. (1989) Quantitative morphologic changes of nephron structures and urinary enzyme activity pattern in sodium-maleate induced renal injury. Renal Physiol. Biochem. 12,5664. 41 Montine, T.J. and Borch, R.F. (1988) Quiescent LLC-PKi cells as a model for cis-diamminedichloroplatinum(H) nephrotoxicity and modulation by thiol rescue agents. Cancer Res. 48,6017-6024. 42 Schaeffer, V.H. and Stevens, J.L. (1987) The transport of S-cysteine conjugates in LLC-PK, cells and its role in toxicity. Mol. Pharmacol. 3 1, SW-512. 43 Stevens, J., Hayden, P. and Taylor, G. (1986) The role of glutathione conjugate metabolism and cysteine conjugate g-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK, cells. J. Biol. Chem. 261,3325-3332.