Amphotericin B inhibits EPO expression. (A) AmB inhibition of hypoxia-induced EPO production in rat kidneys. After rats were exposed to air or 10% O₂ at 1 atm for 16 hours, kidneys were quickly frozen and stored at -70°C. The AmB stock solution (20 mg/mL) was dissolved in 35% (wt/wt) sodium deoxycholate and sodium phosphate provided by the manufacturer (Sigma-Aldrich) and stored at -20°C. Sodium deoxycholate for untreated animals or AmB (4 mg/kg in 4 divided doses) was injected into rats before rats were exposed to air or hypoxic atmosphere. After 16 hours of exposure, kidneys were excised to obtain RNA and protein samples. Tissue levels of EPO mRNA and protein were analyzed by semiquantitative RT-PCR and Western blotting, respectively, as described in “Materials and methods.” Results presented in lanes were obtained from individual animals. (B) AmB inhibition of anemia-induced EPO production in rat kidneys. After anemia was induced by removing blood, AmB solution (1 mg/kg) was once injected through the femoral vein. After 16 hours of incubation, kidneys were excised and quickly frozen. EPO and β-actin mRNAs were analyzed by the semiquantitative RT-PCR. Sham operations were performed in control rats in an identical manner, except for blood aspiration. (C) Renal toxicity of AmB. Rats were treated with sodium deoxycholate (Control) or AmB (4 mg/kg), or with KBrO₃ (160 mg/kg) as a reference sample for renal injury. After 24 hours, the kidneys were excised and prepared for TUNEL staining, as described in “Materials and methods.” The stained slides were separately analyzed for the cortex (left panel) and medulla (right panel) parts. Arrows indicate examples of TUNEL-positive nuclei. Results are representative of 3 separate experiments. (D) Cytotoxicity of AmB. Cell viabilities were measured using an MTT-labeling method, as described in “Materials and methods.” Hep3B (•) and HEK293 (○) cells were treated with various concentrations of AmB for 16 hours. Points represent the means ± SD of 7 experiments. (E) AmB inhibition of HIF-1 target gene expression. RNAs were isolated from Hep3B cells subjected to normoxia (N) or 16 hours of hypoxia (H) with sodium deoxycholate (lanes 1-2) or AmB (lanes 3-7). The mRNAs of HIF-1 target genes and β-actin were analyzed by semiquantitative RT-PCR. Data are representative of 3 separate experiments. EPO indicates erythropoietin; VEGF, vascular endothelial growth factor; PGK 1, phosphoglycerate kinase 1; and Enol 1, enolase 1.