Differential Toxicity of Parenteral Antibiotic Drugs in Renal Cells (LLC-PK₁) Grown on Permeable Membrane Filters

Abstract

Growth of renal epithelial cells on permeable membrane niters promotes the expression of polarized function and facilitates the study of directional aspects of exposure of kidney cells to nephrotoxins (apical versus basolateral). Monolayer cultures of LLC-PK₁ cells were grown on membrane filter inserts coated with a collagen-laminin matrix and incubated with three parenteral antibiotic drugs (vancomycin, 2.5–15 mg/ml; tobramycin, 0.5–10 mg/ml; and cephaloridine, 0.05–0.5 mg/ml) at 37d`C for 48 h. Growth medium containing the experimental antibiotic drugs was placed in the upper or lower chamber for apical or basolateral exposure, respectively. After incubation, cellular viability was determined by nigrosin dye exclusion and by the in situ uptake of the fluorescent dye, propidium iodide, which is increased in dead cells. Cytotoxicity was seen with increasing concentrations of each antibiotic drug after basolateral or apical exposure. Profound basolateral and apical differences, expressed as a percent of control viability, were observed with all three antibiotic drugs when viability was assessed by nigrosin dye exclusion. Basolateral versus apical percent viabilities were 0 versus 67% with vancomycin (15 mg/ml), 38 versus 76% with tobramycin (5 mg/ml), and 4 versus 89% with cephaloridine (0.2 mg/ml). No significant differences in toxicity were observed after basolateral and apical exposure to vancomycin and tobramycin when propidium iodide was used as the viability endpoint. Cytotoxicity due to cephaloridine, on the other hand, was greater after basolateral exposure by propidium iodide monitoring (e.g., 4,046 versus 2,628 relative fluorescence units after basolateral versus apical exposure, respectively, at 0.25 mg/ml). These results demonstrate enhanced sensitivity of LLC-PK₁ cells to nephrotoxins from the basolateral surface and also identify differences between cellular viability end-points.

Key Words: :

• LLC-PK
• Vancomycin
• Cephaloridine
• Tobramycin
• Propidium iodide
• BCECF-AM
• Nigrosin dye