Kidney Week

Abstract: TH-PO103

Hsp70 Induction by GGA Treatment Attenuates the Proapoptotic Unfolded Protein Response and Inhibits Gentamicin-Induced Renal Cell Death

Session Information

• AKI: Inflammation, New Technologies, Omics
  October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Igwebuike, Chinaemere, Boston Univerisity School of Medicine , Boston, Massachusetts, United States

Chinaemere Igwebuike,

• Huiting, Leah, Boston Univerisity School of Medicine , Boston, Massachusetts, United States

Leah Huiting,

• Feng, Hui, Boston Univerisity School of Medicine , Boston, Massachusetts, United States

Hui Feng,

• Wang, Zhiyong, Boston Medical Center, Boston, Massachusetts, United States

Zhiyong Wang,

• Havasi, Andrea, Boston Medical Center, Boston, Massachusetts, United States

Andrea Havasi,

• Schwartz, John H., Boston Medical Center, Boston, Massachusetts, United States

John H. Schwartz,

• Sherman, Michael, Ariel University, Jerusalem, Israel

Michael Sherman,

• Borkan, Steven C., Boston Medical Center, Boston, Massachusetts, United States

Steven C. Borkan,

Background

Exposure to nephrotoxic antibiotics damages the proximal tubule epithelial cells and causes substantial morbidity and mortality. Gentamicin is a common nephrotoxic antibiotic that promotes proximal tubule damage and acute kidney injury (AKI). However, the lack of a unifying mechanism for gentamicin-induced renal cell injury limits AKI therapy. We hypothesize that computational analysis of an unbiased, broad-based shRNA cell signal screen will identify a “best fit” mechanism(s) of renal cell injury as well as targeted drugs to ameliorate gentamicin-induced AKI.

Methods

Dual shRNA screens of 25,000 individually barcoded signal pathway genes were performed in gentamicin-exposed human proximal tubule cells. Differentially expressed shRNAs were analyzed by Ingenuity Pathways Analysis (IPA) software. Predictions based on computational data were then tested using Unfolded Protein Response (UPR) markers: ATF6, CHOP, BiP and XBP1. Additionally, protein ubiquitination, protein misfolding (thioflavin T staining), oxidative stress (4HNE), mitochondrial integrity (MitoTracker), endoplasmic reticulum (ER)-mitochondrial association (ER Tracker), and cell death (cleaved PARP and propidium iodide staining) were measured.

Results

Interference RNA screening detected 226 differentially expressed shRNAs. Pathway analysis of these shRNAs identified the UPR as the “best fit” mechanism of cell injury and suggested Hsp70 as a potential cytoprotectant. Gentamicin exposure increased protein ubiquitination, protein misfolding, oxidative stress, activated all 3 UPR arms, caused mitochondrial fragmentation followed by mitochondria-ER dissociation, and finally, induced cell death. Chemical induction of Hsp70, a protein that regulates several UPR steps, markedly reduced protein ubiquitination, misfolding, oxidative stress, suppressed both mitochondrial fragmentation and dissociation from ER, and dramatically improved renal cell survival.

Conclusion

This study suggests that the proapoptotic UPR contributes to gentamicin-induced renal cell injury. Hsp70 induction by chemical treatment ameliorates the proapoptotic UPR events and significantly improves renal cell survival during gentamicin exposure.

Funding

• NIDDK Support