Comparative in Vitro Nephrotoxicity of a Novel Antifungal Agent

Objectives: Candida albicans is the major human fungal pathogen that causes superficial mucosal infections such as oral candidiasis and systemic mycoses with high mortality. Nephrotoxicity is the major treatment complication associated with antifungal agents. Recently, we discovered a novel antifungal small molecule, SM21, with promising antifungal activity. The aim of the present study was to comparatively evaluate the in vitro nephrotoxicity of SM21 with current antifungal agents.
Methods: The present work evaluated the effects of SM21 and its active analogue CID2147408 (SM21-A) comparatively with amphotericin B(AmB), lyposomal amphotericin B as positive controls and Voriconazole, Caspofungin as negative controls on immortalized (HK-2) cells. The cells were exposed to five different concentrations (0.1, 1, 3, 5, 10μg/mL) of drugs for 24 hours. A panel of sensitive and specific nephrotoxic and apoptotic markers was selected based on earlier in vivo studies. Protein expression of well accepted pre-clinical renal proximal tubule injury marker HAVCR1(KIM1) and apoptotic markers such as CASP3 and BCL were analyzed by Western blotting and cell viability was determined by CellTiter-Glo® Luminescent assay. The total RNA was extracted from the cells, and RT-PCR was performed to evaluate differential gene expression profiles of the selected genes.
Results: Our data indicated that cytotoxic impact of AmB and lypososmal AmB on HK-2 viability and gene expression is stronger than that of SM21 and SM21-A in vitro. High concentration (3-6 μM) of SM21 and SM21-A had no significant influence on viability or protein and mRNA expression in HK-2. In contrast AmB (0.1-3 μM) decreased HK-2 viability and highest decrease of expression include HAVCR1(KIM1) and CASP3. Increased expression of HAVCR1(KIM1) and CASP3 at protein and mRNA level in HK-2 cells was correlated well with voriconazole and caspofungin in a dose dependent manner showing comparatively lower nephrotoxicity than AMB.
Conclusions: The present study demonstrated that SM21 and its analogues have less acute nephrotoxic effect than AMB and liposomal AMB, providing essential safety aspect, before commencing human trials in future.