Hyper-O-GlcNAcylation of AMP-Activated Protein Kinase (AMPK) and Acetylated α-Tubulin in ADPKD Pathogenesis

Background: Altered cell metabolism is an important component of autosomal dominant polycystic kidney disease (ADPKD) pathogenesis, but drivers of these alterations are not understood. The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) onto protein substrates by O-GlcNAc transferase (OGT) integrates multiple metabolic signals that are dysregulated in ADPKD. We have reported that protein O-GlcNAcylation is increased in kidneys of ADPKD patients and mouse models, and that genetic downregulation of O-GlcNAcylation markedly attenuates disease severity and extends mouse survival. To determine the underlying pathogenic mechanisms, we sought to identify proteins that are hyper-O-GlcNAcylated in ADPKD. Methods: We generated juvenile and adult Pkd1 conditional knockout (cko) and Pkd1;Ogt double knockout (dko) mice using the HoxB7-Cre and the doxycycline-inducible Pax8rtTA;LC1-Cre recombinases (induced from 4-6 weeks of age). Juvenile and adult mouse kidneys were analyzed on postnatal day (P)14 and at 4 months of age, respectively. Hyper-O-GlcNAcylated proteins were identified via immunoprecipitation and Western blot, after which sites were mapped via mass spectrometry with generation and analysis of relevant point mutants to follow. Results: In juvenile mice, Ogt deletion in Pkd1 cko mice reduced renal cystogenesis and kidney weight:body weight ratios (KW/BW); restrained renal cilia lengths; reduced inflammation and fibrosis; increased activation of the energy sensor AMPK; and improved kidney function. Further, while Pkd1 cko mice die between P14-P20, Pkd1;Ogt dko mice survive beyond one year. In adult mice, deletion of Ogt in Pkd1 cko mice reduced renal cystogenesis and KW/BW. In P14 kidney extracts, hyper-O-GlcNAcylation of metabolic regulator, AMPKα1/α2, as well as of acetylated α-tubulin, which comprises the ciliary axoneme, was observed. In AMPKα1, Ser184 was identified as an O-GlcNAc site, adjacent to the activating phosphorylation site. Conclusion: In PKD, protein O-GlcNAcylation, including of AMPK and acetylated α-tubulin, is increased, and deletion or inhibition of OGT reduces cyst growth and disease severity, demonstrating that O-GlcNAcylation is an important driver of PKD progression. We propose that targeting O-GlcNAcylation may have therapeutic potential in ADPKD. Funding: Other U.S. Government Support, Private Foundation Support