Pergamon 0887-2333(94)E0051-T

Toxic. in Vitro Vol. 8, No. 4, pp. 601-603, 1994 Copyright 6) 1994 Elsevier Science Ltd

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USE OF S C A N N I N G ELECTRON MICROSCOPY IN THE S T U D Y OF N E P H R O T O X I N - I N D U C E D RENAL PROXIMAL

T U B U L A R D A M A G E IN VITRO

J. A. REES, J. G. EVANS, M. BLACKMORE, A. J. SPENCER and T. J. B. GRAY Sterling Winthrop Pharmaceuticals Research Division, Willowburn Avenue, Alnwick,

Northumberland NE66 2JH, UK

Abstract--Scanning electron microscopy was used to study cephaloridine and gentamicin-induced renal cell injury in vitro. Exposure of renal proximal tubular cells to these toxicants produced changes in cell surface morphology that occurred at concentrations similar to (or below) those affecting neutral red uptake or monolayer permeability.

INTRODUCTION

The use of renal cell lines in in vitro nephrotoxicity studies is well documented, and a common approach is the measurement of cell survival or proliferation after toxic insult (Linseman et al., 1990; Williams et al., 1988). We have assessed the toxicity of cephaloridine and gentamicin to LLC-PKj cells by scanning electron microscopy (SEM; for surface morphology), measurement of the flux of sodium fluorescein (for monolayer permeability) and neutral red (NR) uptake (for cytotoxicity).

MATERIALS AND METHODS

LLC-PK l cells were maintained and seeded onto culture inserts as described by Blackmore et al. (1994) and exposed (72 hr post seeding) to cephaloridine or gentamicin for 24 hr by both apical and basolateral routes.

SEM. Inserts were rinsed in phosphate buffered saline containing Ca 2÷ and Mg 2+ (PBS), fixed for 1 hr in modified Karnovsky's fixative, and a semicircle of insert membrane was carefully excised and pinned to a piece of flat cork under alcohol. Conventional SEM preparation and observation techniques were followed.

Sodium fluorescein exclusion assay. Inserts were washed with PBS and placed into the wells of a six-well plate containing 2ml PBS. 2ml of a 0.1 mg/ml solution of sodium fluorescein (in PBS) was then added to the apical surface of each mono- layer. The basolateral compartment was sampled after 40 rain at 37°C and analysed for the presence of fluorescein using an autoanalyser.

Neutral red uptake. This was determined as described by Biackmore et al. (1994). Briefly, cells were incubated in a medium containing NR

Abbreviations: NR --- neutral red; PBS = phosphate buffered saline; SEM = scanning electron microscopy.

(50 mg/ml) for 1 hr at 37°C and washed in PBS. NR was extracted by a 1-hr incubation with 1% acetic acid-50% ethanol. Optical absorbance was read at 540 nm.

RESULTS

Cephaloridine treatment

The NR assay showed cytotoxicity at cephaloridine concentrations of 1-2 mM and above (IC50 4.1 mM); monolayer permeability was increased at 2-3 mM, as determined by sodium fluorescein exclusion. SEM of cultures treated with 1 mM increments of cephaloridine showed altered surface morphology at concentrations of 2 mM and above. A typical control culture is illustrated in Plate 1 (p. 603). At 2 mM microvilli were fattened onto the cell surface or clumped together in tufts (Plate 2, p. 603). At 3-5 mM there was necrosis and a loss of cell-cell contact. At concentrations between 1 and 3 mM (intervals of 0.1 mM) little effect on monolayer permeability was evident until 2.3 mM, but altered morphology was observed with SEM at the 1.5 mM level and above. In addition to the above changes, blebbing was observed at concentrations from 1.5 to 2.2 mM. At concen- trations from 2.2 to 3 mM, flattened microvilli and surface protrusions were in abundance, with the frequency of occurrence increasing with dose.

Gentamiein treatment

Concentrations of gentamicin up to and including 30mM had no effect on NR uptake by LLC-PK~ cells. Monolayer permeability was unaffected at 10mM but increased with 20mM and 30mM genta- micin (120 and 140% of basal fluorescence compared with controls). SEM revealed changes in morphology at 20 mM, showing small blebs and the clumping of microvilli into tufts. At 30 mM, there were more blebs, which were often attached to the cells with 'stalks' (Plate 3, p. 603).

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DISCUSSION

SEM examination of LLC-PK~ cells appeared to be as sensitive as assessment of N R uptake for detecting cephaloridine-induced damage, and more sensitive than measurement of monolayer permeability. With gentamicin, SEM and monolayer permeability ap- peared to be more sensitive for detecting damage than the assessment of N R uptake.

SEM, compared with transmission electron microscopy, allows a large number of cells to be examined in a short time and in greater detail than with light microscopy. SEM can be performed on cultures previously tested with viability assays and, unlike a biochemical approach, there is retention of the spatial relationships of cells allowing possible heterogeneity in response to be investigated.

To our knowledge, there are no reported SEM investigations of the effects of cephaloridine and gentamicin treatment on proximal tubular cell sur- face morphology in vivo.

REFERENCES

Blackmore M., Richardson J. C., Costello S., Harpur E. S. and Gray T. J. B. (1994) In vitro toxicity of cephalosporin and aminoglycoside antibiotics to LLC-PK k cells assessed by changes in glucose uptake. Toxicology in Vitro 8, 771 773.

Linseman D. A., Raczniak T. J., Aaron C. S. and Bacon J. A. (1990) Comparative toxicity rankings of four amino- glycoside antibiotics in Chang, SIRC and LLC-PKI cell lines. A TLA 18, 283-290.

Williams P. D., Laska D. A., Tay L. K. and Hottendorf G. H. (1988) Comparative toxicities of cephalosporin antibiotics in a rabbit kidney cell line (LLC-RKI). Antimicrobial Agents and Chemotherapy 32, 314 318.

(Plates 1-3 opposite)

Plate I. SEM micrograph of a control monolayer of LLC-PK~ cells. Note tightly packed, flattened cells with numerous microvilli.

Plate 2. SEM micrograph of LLC-PK, cells treated with 2 mM cephaloridine showing flattened microvilli.

Plate 3. SEM micrograph of LLC-PK~ cells treated with 30 mM gentamicin showing large blebs attached to the monolayer surface.