Polymyxin B induces apoptosis in kidney proximal tubular cells

Mohammad Abul Kalam Azad, Benjamin Arthur Llewellyn Finnin, Anima Poudyal, Kathryn Erin Davis, Jinhua Li, Prudence A Hill, Roger Leigh Nation, Tony Velkov, Jian Li

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53 Citations (Scopus)

Abstract

The nephrotoxicity of polymyxins is a major dose-limiting factor for treatment of infections caused by multidrug-resistant Gram-negative pathogens. The mechanism(s) of polymyxin-induced nephrotoxicity is not clear. This study aimed to investigate polymyxin B-induced apoptosis in kidney proximal tubular cells. Polymyxin B-induced apoptosis in NRK-52E cells was examined by caspase activation, DNA breakage, and translocation of membrane phosphatidylserine using Red-VAD-FMK [Val-Ala-Asp(O-Me) fluoromethyl ketone] staining, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and double staining with annexin V-propidium iodide (PI). The concentration dependence (50 effective concentration [EC50]) and time course for polymyxin B-induced apoptosis were measured in NRK-52E and HK-2 cells by fluorescence-activated cell sorting (FACS) with annexin V and PI. Polymyxin B-induced apoptosis in NRK-52E cells was confirmed by positive labeling from Red-VAD-FMK staining, TUNEL assay, and annexin V-PI double staining. The EC50 (95 confidence interval [CI]) of polymyxin B for the NRK-52E cells was 1.05 (0.91 to 1.22) mM and was 0.35 (0.29 to 0.42) mM for HK-2 cells. At lower concentrations of polymyxin B, minimal apoptosis was observed, followed by a sharp rise in the apoptotic index at higher concentrations in both cell lines. After treatment of NRK-52E cells with 2.0 mM polymyxin B, the percentage of apoptotic cells (mean +/- standard deviation [SD]) was 10.9 +/- 4.69 at 6 h and reached plateau (>80 ) at 24 h, whereas treatment with 0.5 mM polymyxin B for 24 h led to 93.6 +/- 5.57 of HK-2 cells in apoptosis. Understanding the mechanism of polymyxin B-induced apoptosis will provide important information for discovering less nephrotoxic polymyxin-like lipopeptides.
Original languageEnglish
Pages (from-to)4329 - 4335
Number of pages7
JournalAntimicrobial Agents and Chemotherapy
Volume57
Issue number9
DOIs
Publication statusPublished - 2013

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