Kidney Week

Abstract: TH-OR107

A Central Role of the Enzyme Asparagine Synthetase (ASNS) in Driving Glutamine Anaeplerosis in the Metabolic Rewiring of ADPKD

Session Information

• PKD: Genetic, Mechanistic, and Clinical
  October 25, 2018 | Location: 25A, San Diego Convention Center
  Abstract Time: 05:42 PM - 05:54 PM

Category: Genetic Diseases of the Kidney

• 1001 Genetic Diseases of the Kidney: Cystic

Authors

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

Christine Podrini, PhD

• Rowe, Isaline, San Raffaele Scientific Institute, Milan, Italy

Isaline Rowe,

• Pagliarini, Roberto, San Raffaele Scientific Institute, Milan, Italy

Roberto Pagliarini,

• Henriques da Costa, Ana Sofia, MRC Cancer Unit, University of Cambridge, Cambridge, United Kingdom

Ana Sofia Henriques da Costa,

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

Marco Chiaravalli,

• Kim, Hyunho, Center for Medical Innovation, Seoul National University Hospital, Seoul, Korea (the Republic of)

Hyunho Kim,

• Qian, Feng, University of Maryland School of Medicine, Baltimore, Maryland, United States

Feng Qian,

• Frezza, Christian, MRC Cancer Unit, University of Cambridge, Cambridge, United Kingdom

Christian Frezza,

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

Alessandra Boletta,

Background

We showed that the Warburg effect is a feature of PKD. However, additional pathways involved in the metabolic deregulation of ADPKD are likely present. Here, we explore global metabolomics and isotope tracing experiments to identify new targetable metabolic pathways in PKD.

Methods

16 newborn (P4) kidneys of Ksp-Cre;Pkd1^flox/− mice and their respective littermate controls were collected. Metabolomics was performed with LC-mass spectrometry (MS). Tracing studies with¹³C-labeled carbons from glucose or glutamine were performed in Pkd1-mutant cells, using LC-MS.

Results

Metabolomics screen identified 488 significantly altered metabolites. PCA and HCA showed a clear separation between cystic and control samples. The alterations included glycolysis, fatty acid oxidation and biosynthesis, and massive TCA cycle. To test which energy source was fuelling the accumulation of TCA cycle metabolites, alternate labelling with [¹³C₆] glucose or [¹³C₅,¹⁵N₂] glutamine was performed in-vitro. Pkd1^-/- cells had an enhanced glucose uptake, mostly used to produce lactate and minimally funneled into the TCA cycle. To compensate, Pkd1^-/- cells uptake more glutamine and use it both oxidatively and reductively in the TCA cycle to drive fatty acids biosynthesis. Following the fate of the ¹⁵N₂-glutamine, we found a significant increase of ¹⁵N-labelled asparagine in Pkd1^-/- cells suggesting that they exhibit increase asparagine synthesis from glutamine. This reaction is catalyzed by Asparagine synthetase (ASNS). Indeed, Asns was upregulated in Pkd1^-/- cells and KspCre;Pkd1 kidneys. Tracing with ¹⁵N₂ and ¹³C₅-glutamine showed that silencing of Asns reduced significantly the levels of labelled asparagine and glutamine-derived α-KG. Importantly, the down-regulation of Asns reduced growth in Pkd1^-/- cells, but not in controls. Combination with glucose starvation was lethal in Pkd1^-/- cells. Microarray analysis on Pkd1 mutant kidneys (Pkd1^V/V) and human PKD1 samples confirmed upregulation of ASNS and further validated the global metabolic perturbations observed.

Conclusion

Our data show that increased glutaminolysis, interlinked with asparagine metabolism is an important feature of PKD and targeting ASNS might offer a novel therapeutic opportunity.