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Abstract: TH-OR102

Polymyxin-B Induces Fas-Mediated Apoptosis in a Human Kidney Proximal Tubule Microphysiological System (Organ-on-a-Chip)

Session Information

  • Predicting AKI
    November 02, 2017 | Location: Room 282, Morial Convention Center
    Abstract Time: 05:18 PM - 05:30 PM

Category: Acute Kidney Injury

  • 003 AKI: Clinical and Translational

Authors

  • Bhatraju, Pavan K., University of Washington, Seattle, Washington, United States
  • Weber, Elijah, University of Washington, Seattle, Washington, United States
  • Himmelfarb, Jonathan, Kidney Research Institute, Seattle, Washington, United States
  • Kelly, Edward J., University of Washington, Seattle, Washington, United States
Background

In response to the emergence of multi-drug resistant gram negative infections, polymyxin-B (PMB) use has increased despite the clinical observations of severe nephrotoxicity. Our lab is modeling PMB-induced nephrotoxicity in 2D proximal tubule epithelial cells (PTECs) and in a 3D microphysiological system (MPS) to identify biological pathways in the development of renal epithelial cell injury.

Methods

PTECs were treated in 2D with escalating doses of PMB (0 uM to 800 uM). Cellular viability (cell cytotoxicity assay) and caspase 3, 7 activation (fluorescent caspase detection) were measured. The minimal concentration that led to cell death in 2D cultured PTECs was used in the MPS. The MPS system was treated with 50 uM for 48 hours. Effluent was collected at 24 hours and analyzed for caspase cleaved cytokeratin 18 (CK-18). Additionally, transcriptional response was analyzed via RNA-sequencing (RNA-seq) of PMB treated MPS.

Results

In two separate donors, PMB-induced toxicity was observed in 2D in a concentration dependent manner with decreasing cellular viability with increasing PMB concentrations. The EC50 for PMB was 130 uM (SD +/- 12.3) and the minimal concentration that led to cell death was 50 uM. Additionally, we observed increasing caspase activation with decreasing cellular viability (one way ANOVA p-value <0.0015). In the 3D MPS, PMB significantly increased CK-18 effluent levels (control 70 +/- 11 U/l versus PMB 207 +/- 24 U/l, p < 0.001). RNA-seq demonstrated increased transcription of Fas/FasL related genes including Fas cell surface receptor (FAS), Fas associated death domain (FADD) as well as others. The RNA seq response was distinct compared to MPS treated with cadmium, another known nephrotoxicant.

Conclusion

We have demonstrated that in 2D and in 3D, PMB induced injury is mediated through apoptosis. Furthermore, transcriptional response data demonstrates upregulation of Fas pathways in human PTECs after PMB exposure. This study supports continued study of apoptosis and the Fas-pathway to potentially develop therapies that ameliorate PMB induced nephrotoxicity. Clinically, CK-18 may be useful as a biomarker of PMB induced-AKI.This research was supported by an unrestricted gift from the Northwest Kidney Centers to the Kidney Research Institute, F32DK112532, UH3TR000504.

Funding

  • NIDDK Support