Poster Presentation Lorne Infection and Immunity 2014

Quantitative mapping of polymyxin in rat and human kidney cells using X-ray fluorescence microscopy (#106)

Mohammad A. K. Azad 1 , Kade D. Roberts 1 , Heidi H. Yu 1 , Boyin Liu 2 , Alice V. Schofield 1 , Simon James 3 , Daryl L. Howard 3 , Roger L. Nation 1 , Kelly Rogers 4 , Martin de Jonge 3 , Philip E. Thompson 1 , Jing Fu 2 , Tony Velkov 1 , Jian Li 1
  1. Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette , Indiana, USA
  2. Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC, Australia
  3. X-ray fluorescence microscopy Beamline Team, Australian Synchrotron, Clayton, VIC, Australia
  4. Centre for Dynamic Imaging, Walter and Eliza Hall Institute, Parkville, VIC, Australia

Objective: Polymyxins, a last-line therapy against multidrug-resistant Gram-negative ‘superbugs’, induce nephrotoxicity in up to 60% of patients after intravenous administration. Quantification of the polymyxin concentration in kidney tubular cells is important for understanding its nephrotoxicity, albeit the concentration has yet to be determined due to the lack of an appropriate experimental technique. This study aimed to design, synthesize and evaluate a novel fluorescent polymyxin probe (FADDI-096) for quantitative mapping of polymyxin in single rat (NRK-52E) and human (HK-2) kidney proximal tubular cells.
Methods: A 3D molecular model in complex with LPS from Escherichia coli was constructed to design the probe labelled with a dansyl and an iodine. Fmoc solid-phase synthesis was employed to prepare FADDI-096. Minimum inhibitory concentration (MIC) of FADDI-096 against Pseudomonas aeruginosa was determined. Intra-cellular accumulation and localisation of FADDI-096 was investigated at single-cell level using correlative fluorescence microscopy (FM), synchrotron-based X-ray fluorescence microscopy (XFM) and scanning electron microscopy (SEM).
Results: FADDI-096 had an MIC of 4 mg/L against P. aeruginosa ATCC 27853. FM and XFM images revealed that FADDI-096 was highly localised in the nuclear and perinuclear area. Correlation between FM, XFM and SEM images demonstrated that the fluorescent signal originated intra-cellularly. FADDI-096 concentrations in cells were approximately 2,000- to 4,500-fold higher than extra-cellular concentrations. The uptake of FADDI-096 was higher in NRK-52E cells treated for 4 h with 50 µM (110.1 ± 28.2 mM) compared to those treated with 5 µM FADDI-096 (23.8 ± 6.6 mM), indicating a concentration-dependent but less than dose-proportional accumulation. The intra-cellular concentration of the probe in HK-2 cells was 31.0 ± 5.7 mM after 4-h treatment with 10 µM FADDI-096.
Conclusions: Our study is the first to quantitate polymyxin accumulation in kidney cells and it provides key information for the understanding of polymyxin-induced nephrotoxicity.