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Abstract: PO1506

Defective NAD+ Homeostasis in ADPKD and the Effects of PC1CTT on Redox Modulation and Disease Progression

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Onuchic, Laura, Yale University School of Medicine, New Haven, Connecticut, United States
  • Padovano, Valeria, Broad Institute, Cambridge, Massachusetts, United States
  • Gresko, Nikolay P., Yale University School of Medicine, New Haven, Connecticut, United States
  • Schena, Giorgia, Yale University School of Medicine, New Haven, Connecticut, United States
  • Rajendran, Vanathy, Yale University School of Medicine, New Haven, Connecticut, United States
  • Caplan, Michael J., Yale University School of Medicine, New Haven, Connecticut, United States
Background

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the genes encoding polycystin-1 or 2 (PC1 or PC2). Defective metabolism is a hallmark of ADPKD. The C-terminal cleavage product of PC1 (PC1CTT) can translocate to mitochondria and its expression in Pkd1 KO cells may rescue defective mitochondrial phenotypes. Altered oxidoreductase activity is detected in an ADPKD in vitro model. Here we assess the degree of redox imbalance between NAD/NADH in in vitro and animal ADPKD models. Moreover, we use an in vitro model to express variations of PC1CTT that localize to different subcellular compartments and reveal an association between PC1CTT localization and Redox modulation. Finally, we show that expression of a PC1CTT variant with predominant nuclear as opposed to mitochondrial localization exacerbates the cystic phenotype in a PKD1 KO mouse model.

Methods

We quantified the ratio between NAD+ and NADH in lysates from Pkd1+/- and Pkd1-/- mouse renal epithelial cells. We also assessed the NAD/NADH ratio in kidney lysates from ADPKD mouse models and WT controls. We generated 3 variations of PC1CTT with either an N-terminal HA-tag (2HA-PC1CTT), a C-terminal HA-tag (PC1CTT-HA) or no additional tag, and applied the assay to lysates from HEK cells transfected with these constructs. Finally, we expressed 2HA-PC1CTT in a PKD1 KO mouse model and observed phenotypic differences.

Results

The NAD/NADH ratio was 80% higher in Pkd1+/- cells in comparison to Pkd1-/- cells. Kidney lysates from WT mice had double the NAD/NADH ratio compared to that observed in cystic and pre-cystic animals. Transfection of HEK cells with the PC1CTT constructs revealed distinct patterns: 2HA-PC1CTT localized to nuclei, PC1CTT-HA localized to mitochondria and PC1CTT was found in both. PC1CTT and PC1CTT-HA expressing HEK cells exhibited 40% and 60% higher NAD/NADH ratios than those measured in 2HA-PC1CTT-expressing cells. ADPKD mice expressing 2HA-PC1CTT exhibited a 3-fold increase in kidney-weight/body-weight in comparison to control ADPKD mice.

Conclusion

ADPKD is characterized by defective NAD+ homeostasis. Mitochondrial localization of PC1CTT can potentially rescue this redox imbalance, while nuclear localization of PC1CTT appears to aggravate this imbalance and exacerbate the cystic phenotype in animal models.

Funding

  • Other NIH Support