LLC-PK1 EPITHELIA AS A MODEL FOR IN-VITRO ASSESSMENT OF PROXIMAL TUBULAR NEPHROTOXICITY

LLC-PK1 cells, an established epithelial cell line derived from pig ki dney, were used as a model system for assessment of nephrotoxic side e ffects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the relea se of cellular marker enzyme activities into the culture medium. In ad dition, LLC-PK1 cells were grown on microporous supports, and cephalos porin-induced alteration of epithelial functional integrity was monito red by a novel electrophysiologic approach. For this purpose, an Ussin g chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditio ns in which defined fractions of the apical culture medium NaCl conten ts were replaced iso-osmotically by mannitol. This method of determini ng the functional intactness of the epithelial barrier by measuring di lution potentials was found to be far more sensitive than monitoring c ell injury by means of morphology;or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption o f monolayer integrity was detected with ah three methodologies applied . Cephaloridine was found the most toxic compound followed by ceftazid ime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of c ephaloridine was needed to produce cell injury. Measurement of transep ithelial dilution potentials was more sensitive as compared to the rel ease of the apical plasma membrane marker enzyme activities alkaline p hosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate deh ydrogenase, or the mitochondrial glutamate dehydrogenase. The data wer e compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was alr eady investigated.