Kidney Week

Abstract: SA-PO481

Investigating the Centrality of the Asparagine Synthetase Enzyme in the Metabolic Reprogramming and Glutamine Usage in ADPKD

Session Information

• Cystic Kidney Diseases: Basic/Translational
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Podrini, Christine, San Raffaele Scientific Institute, Milan, Milano, Italy

Christine Podrini,

• Chiaravalli, Marco, San Raffaele Scientific Institute, Milan, Milano, Italy

Marco Chiaravalli,

• Boletta, Alessandra, San Raffaele Scientific Institute, Milan, Milano, Italy

Alessandra Boletta,

Background

We showed that metabolic alterations in the TCA cycle and increased glutaminolysis interlinked with asparagine metabolism are important features of ADPKD (Podrini et al. Commun Biol, 2018). Tracing studies with labelled [¹³C₅,¹⁵N₂] glutamine showed that Pkd1^-/- cells increased glutamine uptake and ¹⁵N-labelled asparagine, indicating a central role for asparagine synthase (ASNS). Indeed, siAsns in vitro abrogated glutamine contribution to the TCA cycle. Here, we explore ASNS as an ER-bound protein activator of the integrated stress response (ISR) sitting at the intercross of metabolic and ER stress response. Further, we propose ASNS as a novel therapeutic target for ADPKD.

Methods

Protein expression for ISR targets, metabolomics, tracing with siAsns in Pkd1-mutant cells. Given the absence of inhibitors for ASNS we developed a strategy using Antisense LNA GapmeRs to achieve stable downregulation in vitro and in vivo.

Results

Central to the ISR is the eIF2α/ATF4 axis which drive ASNS transcription. Elevated expression of ASNS was associated with increased ATF4 protein expression in cystic kidneys (KspCre;Pkd1) compared to controls. p-eIF2α, which promotes ATF4 synthesis, was increased in Pkd1-mutant cells. However, when Pkd1-mutant cells were deprived from glucose, p-eIF2α was decreased, suggesting the induction of a pathway involved in ROS production. This was supported by increased GSH (reduced glutathione) in metabolomics studies in cystic kidneys (KspCre;Pkd1) and Pkd1-mutant cells compared to respective controls. Further, amino acids availability for ROS production was significantly decreased, suggesting that eIF2α/ATF4 axis might be responsive to amino acids depletion. Importantly, downregulation of Asns in Pkd1-mutant cells causes cell death and rescues the accumulation of metabolites of the TCA cycle, suggesting a potential dual role for ASNS in glutaminolysis and cross-talk with ROS production. To validate ASNS as a therapeutic target, we have designed specifically the silencing of ASNS by LNA GapmeR. Initial testing in vitro was done in Pkd1-mutant cells on five different GapmeRs. The most effective is currently being tested in vivo in an inducible Pkd1 KO mouse model.

Conclusion

Our data raises the possibility that asparagine metabolism interlinked with the ISR might offer a novel therapeutic intervention in PKD.

Funding

• Government Support - Non-U.S.