Abstract: TH-PO240
Genetic Screening Reveals Potential Therapeutic Targets in Gentamicin-Induced AKI
Session Information
- AKI Basic: Cell Death and Biomarkers
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Acute Kidney Injury
- 001 AKI: Basic
Authors
- Igwebuike, Chinaemere, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
- Wang, Zhiyong, BOSTON MEDICAL CENTER, BOSTON, Massachusetts, United States
- Havasi, Andrea, Boston University Medical Center, Boston, Massachusetts, United States
- Sherman, Michael Y, Boston University School of Medicine, Boston, Massachusetts, United States
- Yaglom, Julia, Boston University School of Medicine, Boston, Massachusetts, United States
- Wang, Yongmei, Boston University School of Medicine, Boston, Massachusetts, United States
- Schwartz, John H., Boston Medical Center-Evans Biomedical Center, Boston, Massachusetts, United States
- Borkan, Steven C., Boston Medical Center, Boston, Massachusetts, United States
Background
Gentamicin is a highly effective, nephrotoxic antibiotic that accounts for >20% of AKI, a complication that increases morbidity and mortality but lacks effective therapy. We propose that high throughput genetic screening can characterize the signal pathways that mediate gentamicin-induced proximal tubule epithelial cell (PTEC) injury and that once identified, pharmaceuticals that favorably alter these signal pathways will ameliorate gentamicin-induced AKI.
Methods
Human proximal tubule cells (HK2) were exposed to gentamicin sulfate under conditions that replicate the human disease. An shRNA library directed against 5,000 signal genes was introduced into human PTEC exposed to gentamicin for 10 (“low stringency”) or 11 days (“high stringency”). shRNA that were over-represented vs. luciferase control were considered “protective” and under-represented genes were considered “sensitizing” to gentamicin-induced death measured by image cytometry of Hoechst and propidium iodide stained cells. Signal pathways that mediate gentamicin toxicity were identified using Ingenuity Pathway Analysis based on genes with a similar change in expression under both low and high stringency conditions. PTECs were then exposed to drugs that either stimulate protective or inhibit sensitizing pathways during gentamicin exposure.
Results
Genetic screening detected 406 genes in gentamicin exposed vs. control PTEC with at least a 1.8-fold change in expression. Pathways analysis showed that these genes were involved in cell metabolism, survival and inflammatory pathways. Three distinct pathway modifying drugs, including metformin, an FDA approved agent, were found to be non-toxic to normal PTEC. Approximately 59% and 88% of PTEC died after 10 or 11 days exposure to 26 mM gentamicin, respectively. In contrast, pre-incubation with metformin, a CAMP and AMP kinase mediator, significantly prevented PTEC death (P<0.05).
Conclusion
Genetic screening successfully identifies modifiable pathways responsible for gentamicin-induced nephrotoxicity that can be selectively targeted using existing pharmaceuticals. Together, studies define an effective strategy for gaining mechanistic insight into the pathogenesis of nephrotoxic AKI, facilitating the selection of agents to prevent and treat its devastating consequences.
Funding
- NIDDK Support