Amphotericin B-induced renal tubular cell injury is mediated by Na+ influx through ion-permeable pores and subsequent activation of mitogen-activated protein kinases and elevation of intracellular Ca2+ concentration

Abstract

Amphotericin B (AMB) is one of the most effective antifungal agents; however, its use is often limited by the occurrence of adverse events, especially nephrotoxicity. The present study was designed to determine the possible mechanisms underlying the nephrotoxic action of AMB. The exposure of a porcine proximal renal tubular cell line (LLC-PKl cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propldium iodide uptake was enhanced, while terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was not affected by AMB, suggesting a lack of involvement of apoptosis in AMB-induced cell injury. The cell injury was inhibited by the depletion of membrane cholesterol with methyl-ß-cyclodextrin, which lowered the extracellular Na+ concentration or the chelation of intracellular Ca. The rise in the intracellular Ca2+ concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na+-Ca2+ exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na+-Ca exchanger inhibitor). Moreover, AMB-induced cell injury was reversed by PD169316 (a p38 mitogen-activated protein [MAP] kinase inhibitor), c-Jun N-terminal kinase inhibitor II, and PD98059 (a MEK1/2 inhibitor). The phosphorylations of these MAP kinases were enhanced by AMB in a calcium-independent manner, suggesting the involvement of MAP kinases in AMB-induced cell injury. These findings suggest that Na+ entry through membrane pores formed by the association of AMB with membrane cholesterol leads to the activation of MAP kinases and the elevation of the intracellular Ca2+concentration, leading to renal tubular cell injury. Copyright © 2009, American Society for Microbiology. All Rights Reserved.