Avaliação da nefrotoxicidade causada pela anfotericina B utilizando linhagens celulares LLC-PK1 e MDCK.2

Abstract

Amphotericin B (AmB), a polyene macrolide antibiotic, is one of the most effective antifungal drug for the treatment of systemical fungal infecction. However, its use is often limited by the occurrence of adverse events, especially nephrotoxicity. Druginduced nephrotoxicity is one of the manly frequently observed effects in long-term pharmacotherapy. Such situations have been tardily discovered because of existing methods to determine its toxicity. The present study was designed to propose in vitro alternative methods for early identification of AmB cell toxicity. Therefore, we exposed two different renal cell lines, LLC-PK1 (proximal tubule) and MDCK.2 (distal tubule), to nine different concentrations (2, 4, 6, 8, 10, 12, 15, 20 and 30 ìg/mL) of AmB during 24 hours. Gene tests were carried out according to results from MTT assay. A panel of sensitive and specific nephrotoxic genes was selected based on earlier in vitro and in vivo studies. The search for sequences of mRNAs encoding proteins that had been previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information - NCBI (USA). RNA was extracted from the cells, and RT-PCR was performed to evaluate differential gene expression profiles of the selected genes. The genes with the highest fold change include HAVCR1 (KIM1), CASP3, ANXA5, and VDAC1.