title: 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-PK1 cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propidium 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-beta-cyclodextrin, which lowered the extracellular Na(+) concentration or the chelation of intracellular Ca(2+). The rise in the intracellular Ca(2+) concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na(+)-Ca(2+) exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na(+)-Ca(2+) 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 Ca(2+) concentration, leading to renal tubular cell injury.
authors.affiliation: Department of Pharmacy, Kyushu University Hospital, Fukuoka, Japan. tyano@pharm.med.kyushu-u.ac.jp
authors.forename: Takahisa
authors.initials: T
authors.lastname: Yano
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Itoh
authors.forename: Eiko
authors.initials: E
authors.lastname: Kawamura
authors.forename: Asuka
authors.initials: A
authors.lastname: Maeda
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Egashira
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nishida
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurose
authors.forename: Ryozo
authors.initials: R
authors.lastname: Oishi
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D000666:Amphotericin B / Q000633:toxicity*; D000818:Animals; D000935:Antifungal Agents / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D007684:Kidney Tubules / Q000187:drug effects* / Q000473:pathology; D018374:LLC-PK1 Cells; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D013552:Swine
chemical_list: D000935:Antifungal Agents; D000666:Amphotericin B; D000255:Adenosine Triphosphate; D002784:Cholesterol; D012964:Sodium; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium