Abstract: FR-PO157

PKC-a Inhibition Normalizes Nephrotoxic Serum Induced Disruption of Mitochondrial Membrane Potential and Morphology in Glomerular Endothelial Cells

Session Information

  • Mitochondriacs and More
    November 03, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Kvirkvelia, Nino, Augusta University, Augusta, Georgia, United States
  • McMenamin, Malgorzata, Augusta University, Augusta, Georgia, United States
  • Warren, Marie, Augusta University, Augusta, Georgia, United States
  • Raju, Raghavan, Augusta University, Augusta, Georgia, United States
  • Lucas, Rudolf, Medical College of Georgia, Augusta University, Augusta, Georgia, United States
  • Madaio, Michael P., Augusta University, Augusta, Georgia, United States
Background

Nephrotoxic serum nephritis (NTN), an inflammatory model of antibody mediated nephritis caused by single injection of nephrotoxic serum (NTS), proceeds to end stage kidney disease. Earlier studies have shown that NTS treatment damages cultured glomerular endothelial cells by disrupting mitochondrial respiration, and that PKC-a Inhibition normalized these perturbations. The goal of this study was to determine the mitochronical pathways involved in this process.

Methods

Murine glomerular endothelial cell (GEC) viability was evaluated by LDH release following NTS exposure. PKC-α was pharmacologically inhibited with Ro-320432 (EMD Millipore, Billerica, MA). Changes in mitochondrial membrane potential (Δψm) were measured using cationic carbocyanine dye, which changes fluorescence spectrum depending on mitochondrial potential status. Mitochondrial morphology and distribution were examined using Mitotracker–mitochondrion selective probes.



Results

Following NTS exposure, LDH release, measured as per cent of LDH cytotoxicity, was dramatically increased in GEC up to 50%, whereas PKC-α inhibition (50 nM Ro-320432) reduced cytotoxicity significantly (12%), indicating that PKC-α is involved in NTS-induced endothelial cell cytotoxicity. NTS treatment of GEC resulted in reduction of Δψ and in an increase of cells with depolarized mitochondria by 73%, PKC-α inhibition of NTS-treated cells reduced the number of cells with depolarized mitochondria to 15%. Furthermore, NTS reduced the fraction of cells with healthy mitochondria by 34%, whereas PKC-α inhibition increased that fraction to 79%. Severe mitochondrial swelling, documented by the appearance or large and round shaped mitochondria, was observed in endothelial cells after NTS treatment using Mitotracker–mitochondrion selective probes, which coincided with a loss of Δψm. PKC-α inhibition in NTS-treated GEC restored the rod-type appearance in the majority of mitochondria.

Conclusion

PKC-α participates in NTS induced mitochondrial dysfunction in GEC cells and inhibition of PKC-α significantly improves endothelial cell viability by normalizing mitochondrial membrane potential and morphology. These results may foster the design of novel therapeutic approaches that preserve mitochondrial function during kidney injury.

Funding

  • NIDDK Support