Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Virginia Vaenuku - University Of Washington
Co-Author(s): Elijah Weber, University of Washington, WA;Pavan Bhatraju, University of Washington, WA
Aminoglycosides, such as Gentamicin and Tobramycin, are commonly used to treat bacterial infections, but in some instances, have been observed to cause acute kidney injury (AKI). Oftentimes, AKI is poorly predicted by preclinical models of drug development. To bridge the gap between in vitro and in vivo systems, the Kelly lab has utilized a 3D microphysiological system (MPS) that mimics the proximal tubule of the kidney 1. Primary human proximal tubule epithelial cells (PTECs) cultured within the 3D kidney MPS demonstrate transport and metabolic characteristics of the proximal tubule and is also sensitive to cadmium chloride, a known heavy metal nephrotoxin2. Our goal is to model aminoglycoside nephrotoxicity in the 3D kidney MPS using Gentamicin and Tobramycin. Gentamicin and Tobramycin have limited toxicity in in vitro studies and we hypothesize that aminoglycoside-induced nephrotoxicity will be observed to a greater magnitude in the 3D kidney MPS compared to 2D culture. Gentamicin dosimetry was performed in 2D culture to define the concentration responsible for reducing cell viability by 50 percent (EC50). Polymyxin B was used as a positive control for injury and cell medium was used as a negative control. After 72 hours of exposure, cell viability was not affected at concentrations tested (max: 10mM). For 3D kidney MPS treatments, a concentration of 1mM was used. Exposure to 1mM Gentamicin for 72 hours resulted in morphological changes in the tubule structure and cell sloughing. Additionally, the loss of fluorescence of a cellular biosensor indicated gentamicin-induced cytotoxicity. These results show increased sensitivity of the 3D kidney MPS to gentamicin-induced cytotoxicity compared to traditional 2D cultures. Future efforts will include quantification of renal injury biomarkers from device effluents, including kidney injury molecule-1 (KIM-1) and Cytokeratin-18 (CK-18). Furthermore, RNA was isolated from gentamicin-treated cells and will be used for transcriptional profiling to better understand the mechanisms of toxicity. Tobramycin will also be evaluated to construct a robust injury profile of aminoglycosides.
(1) Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., . Kelly, E. J. (2016). Development of a microphysiological model of human kidney proximal tubule function. Kidney International,90(3), 627-637.
(2) Adler, M., Ramm, S., Hafner, M., Muhlich, J. L., Gottwald, E. M., Weber, E., . Vaidya, A. S. (2015). A Quantitative Approach to Screen for Nephrotoxic Compounds in Vitro . American Society of Nephrology,27, 1015-1028.
(3) J. X. Huang, G. Kaeslin, M. V. Ranall, M. A. Blaskovich, B. Becker, M. S. Butler, M. H. Little, L. H. Lash, M. A. Cooper. Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells, Pharma Res Per, 3(3), 2015, e00148.Abstract_ ERN conference.docx
Funder Acknowledgement(s): Special regards to the University of Washington Office of Research for funding and supporting the UW GenOM Project (NIH 5R25HG007153-05). Greatest appreciation goes to principal investigator of the lab, Dr. Edward J Kelly, and my mentor, Elijah Weber. I would also like to thank Kevin Lidberg, Dr. Ranita Patel, and Dr. Pavan Bhatraju for their help.
Faculty Advisor: Edward J Kelly, email@example.com
Role: With close guidance from my mentor, I was able to extract epithelial cells from the cortex of donated human kidney samples that would later be used in our experiments. I maintained cultured cell growth by exchanging PTEC media solution accordingly (every 3-4 days). I performed 2D gentamicin dosimetry by using a multichannel 1000 ul pipette to sequentially dilute concentrations at a 1:1 ratio. I assisted in making gentamicin concentrated solutions for the 3D model experiment. I also analyzed gentamicin toxicity affects in 3D model using kidney Injury bio-markers and abstract cell staining.