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Abstract: FR-PO085

NAD+ Augmentation Improves Cell Survival Against Cisplatin via Enhanced Mitophagy

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms


  • Lynch, Matthew R., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Tran, Mei T., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Parikh, Samir M., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States

Cisplatin nephrotoxicity is a common cause of acute kidney injury. Activation of autophagy in the renal tubule may be protective against cisplatin. We previously reported that nicotinamide adenine dinucleotide (NAD+) precursor treatment ameliorated cisplatin nephrotoxicity in mice. Given the role of NAD+ in mitochondrial function and homeostasis, we investigated if the protection afforded by NAD+ augmentation against cisplatin involved the induction of mitochondrial autophagy, i.e., mitophagy.

Mitophagy has been traditionally difficult to quantify, requiring co-staining of cells with mitochondrial and lysosomal dyes which can be cytotoxic, non-specific, or both. Recently, a pH-sensitive non-toxic biosensor protein, mtKeima, has been developed to track the fraction of mitochondria that undergo autophagy and breakdown in the acidic lysosome.


We developed a stable renal tubular cell line expressing mtKeima, which we treated with cisplatin (10 mM) or vehicle. Another set of cells were pre-treated with the NAD+ precursor nicotinamide mononucleotide (NMN, 1 mM) 30m prior to cisplatin or vehicle. General autophagy was assessed by Western Blot for LC3 and p62. Cells were imaged 24h after treatment on a confocal microscope with two sequential excitations 440 nm and 559 nm and a 570 to 695 nm emission range. A mitophagy index was calculated by determining the ratio between the area of the red (acidic) and green (basic) emission.


LC3 and p62 revealed no differences in response to cisplatin or NMN. However, cisplatin markedly inhibited mitophagy (p<0.05, 2-fold change). Conversely, NMN enhanced basal mitophagy (p<0.001, 4-fold change). Pre-treatment of cisplatin-exposed cells with NMN abrogated the decline in mitophagy induced by cisplatin (p<0.001, 6-fold change). Finally, application of the lysosome inhibitor, chloroquine, enhanced cisplatin toxicity.


Whereas traditional measurements of autophagy were uninformative, use of mtKeima illuminated a specific reduction in mitophagy triggered by cisplatin. This effect was abrogated with the addition of NMN. Our results indicate that preservation of mitophagy with its safe mitochondrial disposal, rather than generalized autophagy, may be critical for renal tubular cells to resist cisplatin. Further, NAD+ supplementation may represent a therapeutic option for the treatment of cisplatin nephrotoxicity.


  • NIDDK Support