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To evaluate the effect of cyclosporine A (CyA) at high concentrations (10(-4) and 10(-5) M) and the influence of endothelin-1 (ET-1) at physiological and pharmacological concentrations (10(-14) to 10(-6) M) on epithelial cell function, LLC-PK1 cells were studied as a model of the proximal tubule and MDCK cells as a model of the distal tubule/collecting duct. CyA caused time- and concentration-dependent acute toxicity. In LLC-PK1 cells, CyA caused a decrease in transepithelial resistance, indicating a loss of cell contacts, a release of lactate dehydrogenase (LDH) and villin into the supernatant, suggesting destruction of the apical membrane with loss of brush border, and finally release of uvomorulin, suggesting a disruption of the cell-cell adhesion, the zonula adherens. DNA synthesis, as evaluated by bromodeoxyuridine (BrdU) incorporation, was significantly affected at > or = 10(-5) M CyA. The toxicity of CyA was higher when given from the apical rather than the basolateral compartment. ET-1 alone was without effect, but in combination with CyA, ET-1 significantly enhanced toxicity. The ET-1 effect was partially inhibitable by an ET(B), but not an ET(A), antagonist. Immunofluorescence for alpha-catenin, another protein of the zonula adherens, demonstrated no change in polarity for this protein, and immunoprecipitation of the complex indicated relative stability of the zonula adherens despite loss of cadherin into the supernatant. In MDCK cells the effects were different. CyA was not associated with LDH release, but with an increase in transepithelial resistance, indicating increased paracellular resistance. Morphological alterations were significantly less, but BrdU incorporation was decreased. This pattern of toxicity is compatible with a direct toxic effect of CyA on cells of the proximal tubule, with predominant morphological destruction of the cells, with concomitant proximal tubular dysfunction, and a functional alteration in cells of the distal tubule associated with increased paracellular resistance, which may lead to solute and water loss.

Published high-performance liquid chromatographic (HPLC) methods for the determination of tetracyclines are modified to determine low levels of each of 15 tetracycline derivatives in small blood samples. The rapid and accurate methods are applicable to pharmacokinetic studies in small experimental animals. The chromatographic procedures allow run times ranging from 2.1-7.3 min using different reversed-phase materials as supports and eluents based on acetonitrile or isopropanol-diethanolamine-EDTA. Detection limits are estimated at 10-500 ng/mL according to the derivative analyzed. The extraction from plasma is based on acidic precipitation of the plasma proteins using trifluoroacetic acid (TFA). The recovery of 13 derivatives from spiked plasma samples (100 micrograms/mL) reached 85-94% (n = 5). The precision data, expressed as coefficient of variation, ranged between 2.1 to 9.1% within run (n = 10) and 4.3 to 11.0% between day (n = 8) respectively.