LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity

Abstract

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects 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 release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were 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 already investigated.

Delayed Apoptosis Post-Cadmium Injury in Renal Proximal Tubule Epithelial Cells

“…With the advent of bettercharacterized cell lines and culture techniques, in vitro models are becoming more useful in toxicological research [14,41]. Pointedly, most of the previous work on Cd nephrotoxicity in vitro has focused on the acute event [20,43].…”

Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK 1 Cells Subjected to Electroporation

“…These, however, must be exposed to high concentrations of gentamicin because of poorly efficient drug uptake (12). Using LLC-PK 1 cells, an accepted model of renal proximal tubular cells for the study of aminoglycoside toxicity (24,64,66,67,72), we observed that gentamicin causes an early permeabilization of lysosomes before activation of the mitochondrial pathway of apoptosis can be detected (61). In parallel, recent data indicate that a minor but probably significant proportion of the gentamicin accumulated by cells can reach the cytosol and the mitochondria by trafficking through the Golgi apparatus and the endoplasmic reticulum (56).…”

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for organ toxicity.

“…Epithelial leakiness has been shown to be more sensitive than biochemical measures (enzyme leakage) for renal epithelial cell injury (46).…”