Avaliação das linhagens Vero e MDCK na sinalização celular e como alternativas para estudo da nefrotoxicidade utilizando Anfotericina B

Abstract

Due to intense pressure from society and scientific communities to reduce, refine and replace (3R´s) the use of animals in toxicity tests, alternative methods (in vitro) have been developed and recognized by various institutions worldwide. Cell lines are now widely available in cell banks and cellular cultivating techniques have become the most used in vitro methods within the fields of pharmacology and toxicology for their enormous research potential. One of the main toxic effects of medicines is nephrotoxicity and this is why the use of nephrotoxic cells and drugs is of crucial importance to validate methods which enable the study of toxiceffects. Amphotericin B is an antifungus agent widely used to treat systemic fungus infections; however, nephrotoxicity is a commonside-effect. Some studies have suggested that Amphotericin B exerts its toxic effect by means of the phosphorylation of Protein kinase C (PKC), an important protein involved in cellular signaling. The primary objective of our study is to use two different renal cell lines VERO (monkey) and MDCK (dog) to study nephrotoxicity caused by Amphotericin B. Both lines were exposed to eight different concentrations of this drug (2, 4, 6, 8, 10, 15, 20, and 30 g/mL) for 1, 6, 18, 24 and 48 hours with Red Neutral in order to determine citotoxicity, a dye that evaluates lisossomal integrity. They both presented a significant reduction (p<0.05) of cellular viability after exposure to concentrations of 15, 20 and 30 g/mL in all five rounds of the test. No fundamental difference was found between the three doses when the two lines were exposed for both one and 6 hours. By contrast, the doses presented significant difference (p<0,05) after 18, 24 and 48-hour-exposures. In other words, the MDCK and the VERO lines showed different profiles. More importantly, for both lines, toxicity in the sequent time-spans could be predicted after only one hour of exposure to the drug. In order to examine cellular signaling by PKC, both lines were exposed to Amphotericin B (30 g/mL) for one and 18 hours. The PKCsignaling pathway was observed in both lines. However, in the VERO line, Amphotericin B did not use it as the main pathway to exert its citotoxic effect. In the MDCK, the inhibition of the PKC pathway increased the citotoxic effect of the Amphotericin B. Inshort, our findings demonstrated that both lines were effective for studying nephrotoxicity caused by Amphotericin B. They also proved adequate for investigating cellular signaling, given that we could enhance the citotoxic effect of Amphotericin B by inhibiting the PKC signaling pathway