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Abstract: TH-OR018

Organelle-Specific Oxidant Stress and CORE Disruption Mediate Proximal Tubule Cell Injury During Gentamicin Exposure

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Igwebuike, Chinaemere, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
  • Pimentel, David, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
  • Feng, Hui, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
  • Campbell, Joshua, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
  • Sherman, Michael, Ariel University, Jerusalem, Israel
  • Yaglom, Julia, Ariel University, Jerusalem, Israel
  • Wang, Zhiyong, Boston Medical Center, Boston, Massachusetts, United States
  • Havasi, Andrea, Boston Medical Center, Boston, Massachusetts, United States
  • Borkan, Steven C., Boston Medical Center, Boston, Massachusetts, United States
Background

The Cross-Organelle Stress Response (CORE) is an adaptive mechanism that maintains mitochondrial and endoplasmic reticulum (ER) proteostasis. We hypothesized that gentamicin causes nephrotoxic acute kidney injury (AKI) by causing both mitochondrial specific oxidative stress and fragmentation resulting in CORE disruption before activating the lethal unfolded protein response (UPR).

Methods

Mito HyPer, a mitochondrial-specific H2O2 probe, was used to detect early mitochondrial ROS accumulation in human proximal tubule epithelial cells (HK2) during gentamicin exposure. Mitochondria and ER were stained with MitoTracker and ER Tracker, respectively, and time course experiments were performed using a Nikon Super Resolution microscope. Mitochondrial-ER disassociation, mitochondrial morphology and immunoblots of CORE-associated mitochondrial pro-fission proteins (Total DRP1 and pDRP1) were used as surrogates of CORE function. Misfolded protein stains (Thioflavin T), protein ubiquitination, and immunoblots for whole cell oxidative stress (4HNE) were measured to assess proteotoxicity. The efficacy of preserving CORE on protein misfolding, lethal UPR activation (CHOP), and cell survival was assessed using geranylgeranylacetone (GGA), a protein chaperone inducer, prior to the introduction of gentamicin.

Results

Gentamicin exposure caused characteristic features of disrupted CORE, including mitochondrial-specific H2O2 accumulation, DRP-1 activation and organelle fragmentation, followed by mitochondrial-ER dissociation. Importantly, CORE disruption occurred before detectable changes in whole cell oxidative stress, protein ubiquitination, protein misfolding or lethal UPR activation (CHOP) were observed. GGA significantly decreased mitochondrial-specific oxidative stress, prevented fragmentation, preserved mitochondrial-ER association and ameliorated lethal UPR activation.

Conclusion

Gentamicin exposure causes early mitochondrial H2O2 accumulation and disrupts the CORE. These untoward events contribute to gentamicin-induced proteotoxicity and lethal UPR activation. GGA preserves the CORE and decreases subsequent lethal UPR activation that contributes to the proximal tubule cell injury caused by gentamicin.

Funding

  • NIDDK Support