Summary of Study ST003113

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001933. The data can be accessed directly via it's Project DOI: 10.21228/M8VM8W This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003113
Study TitleInhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney Disease Progression, investigated with targeted tracing metabolomics analysis in MEF cells using 15N2-glutamine.
Study SummaryPolycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing studies we could confirm that the pyrimidine biosynthesis pathway is increased and rescued by silencing of Asns.
Institute
San Raffaele University
Last NameStefanoni
First NameDavide
AddressVia Olgettina 58, Milan, Milan, 20132, Italy
Emailstefanoni.davide@hsr.it
Phone+393337686005
Submit Date2024-02-28
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-04-30
Release Version1
Davide Stefanoni Davide Stefanoni
https://dx.doi.org/10.21228/M8VM8W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001933
Project DOI:doi: 10.21228/M8VM8W
Project Title:Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney Disease Progression.
Project Summary:Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against ASNS in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD control kidneys treated with ASNS-ASOScr-ASO revealed major changes in the mutants, several of which are rescued by ASNS silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by ASNS-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with ASNS-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD. Altogether, targeted metabolomics analysis performed in Tam-Cre;Pkd1ΔC/flox mouse model kidneys corroborates the central role of ASNS in the metabolic rewiring occurring in PKD, highlighting the therapeutic potential of its inhibition.
Institute:San Raffaele University
Last Name:Stefanoni
First Name:Davide
Address:Via Olgettina 58, Milan, Milan, 20132, Italy
Email:stefanoni.davide@hsr.it
Phone:+393337686005

Subject:

Subject ID:SU003228
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA3373182DG_1Brain 2DG
SA3373192DG_5Brain 2DG
SA3373202DG_4Brain 2DG
SA3373212DG_2Brain 2DG
SA3373222DG_3Brain 2DG
SA337323Saline_5Brain Saline
SA337324Saline_4Brain Saline
SA337325Saline_1Brain Saline
SA337326Saline_2Brain Saline
SA337327Saline_3Brain Saline
SA33734037MEF cells Labeled_siASNS_11_1
SA33734138MEF cells Labeled_siASNS_11_2
SA33734239MEF cells Labeled_siASNS_11_3
SA33734340MEF cells Labeled_siASNS_11_4
SA33734441MEF cells Labeled_siASNS_11_5
SA33734542MEF cells Labeled_siASNS_11_6
SA33734643MEF cells Labeled_siASNS_14_1
SA33734744MEF cells Labeled_siASNS_14_2
SA33734845MEF cells Labeled_siASNS_14_3
SA33734946MEF cells Labeled_siASNS_14_4
SA33735047MEF cells Labeled_siASNS_14_5
SA33735148MEF cells Labeled_siASNS_14_6
SA33732813MEF cells Labeled_Untreated_11_1
SA33732914MEF cells Labeled_Untreated_11_2
SA33733015MEF cells Labeled_Untreated_11_3
SA33733116MEF cells Labeled_Untreated_11_4
SA33733217MEF cells Labeled_Untreated_11_5
SA33733318MEF cells Labeled_Untreated_11_6
SA33733419MEF cells Labeled_Untreated_14_1
SA33733520MEF cells Labeled_Untreated_14_2
SA33733621MEF cells Labeled_Untreated_14_3
SA33733722MEF cells Labeled_Untreated_14_4
SA33733823MEF cells Labeled_Untreated_14_5
SA33733924MEF cells Labeled_Untreated_14_6
SA3373521MEF cells Unlabeled_Untreated_11_1
SA3373532MEF cells Unlabeled_Untreated_11_2
SA3373543MEF cells Unlabeled_Untreated_11_3
SA3373554MEF cells Unlabeled_Untreated_11_4
SA3373565MEF cells Unlabeled_Untreated_11_5
SA3373576MEF cells Unlabeled_Untreated_11_6
SA3373587MEF cells Unlabeled_Untreated_14_1
SA3373598MEF cells Unlabeled_Untreated_14_2
SA3373609MEF cells Unlabeled_Untreated_14_3
SA33736110MEF cells Unlabeled_Untreated_14_4
SA33736211MEF cells Unlabeled_Untreated_14_5
SA33736312MEF cells Unlabeled_Untreated_14_6
Showing results 1 to 46 of 46

Collection:

Collection ID:CO003221
Collection Summary:Animals were euthanized by CO2 inhalation 16 hours following the final injection and transcardially perfused with ice-cold PBS. Whole brains and spinal cords were then excised, flash frozen in liquid N2, and stored at –80°C for biochemical analysis.
Sample Type:Brain
  
Collection ID:CO003222
Collection Summary:Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media enriched with and without 15N2-glutamine. Following 24h were collected, pellet and instantly extracted with metabolomics lysis buffer.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003237
Treatment Summary:Mice were administered i.p. injections of saline (vehicle) or 4 g/kg 2DG (Cayman Chemical) twice weekly for six weeks.
  
Treatment ID:TR003238
Treatment Summary:Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media enriched or not with 15N2-glutamine.

Sample Preparation:

Sampleprep ID:SP003235
Sampleprep Summary:Metabolites from equal amounts of frontal cortex tissue were rapidly extracted in 80% ice-cold methanol. Extracted samples were vortexed twice, cleared by centrifugation at 14,000 x g for 20 minutes at 4°C, and stored at -80°C.
  
Sampleprep ID:SP003236
Sampleprep Summary:MEF cells samples were extracted in a ratio of 1milion/1mL of ice cold extraction solution (methanol:acetonitrile:water 5:3:2 v/v/v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble material was removed by centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for metabolomics analysis by UHPLC-MS.

Combined analysis:

Analysis ID AN005098 AN005099 AN005100 AN005101
Analysis type MS MS MS MS
Chromatography type HILIC HILIC Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column SeQuant ZIC- pHILIC (150 x 2.1mm,5um) SeQuant ZIC- pHILIC (150 x 2.1mm,5um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units peak intensity peak intensity Peak Area Peak Area

Chromatography:

Chromatography ID:CH003854
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:30
Flow Gradient:85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A
Flow Rate:150 μL/min
Solvent A:100% acetonitrile
Solvent B:100% acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH003855
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:30
Flow Gradient:85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A
Flow Rate:150 μL/min
Solvent A:100% acetonitrile
Solvent B:100% acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH003856
Chromatography Summary:5MM_POS_ESI
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B.
Flow Rate:0.450ml/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH003857
Chromatography Summary:5MM_NEG_ESI
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B.
Flow Rate:0.450ml/min
Solvent A:5% acetonitrile/95% water; 1mM ammonium acetate
Solvent B:95% acetonitrile/5% water; 1mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS004835
Analysis ID:AN005098
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:1. A Sequant ZIC-pHILIC column (2.1 mm i.d. × 150 mm, particle size of 5 µm, Millipore Sigma) was used for separation of metabolites. A 2.1 × 20 mm guard column with the same packing material was used for protection of the analytical column. Flow rate was set at 150 μL/min. Buffers consisted of 100% acetonitrile for mobile phase A, and 100% acetonitrile for mobile phase B. The chromatographic gradient ran from 85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A. Column temperature was 30 C.
Ion Mode:POSITIVE
  
MS ID:MS004836
Analysis ID:AN005099
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:1. A Sequant ZIC-pHILIC column (2.1 mm i.d. × 150 mm, particle size of 5 µm, Millipore Sigma) was used for separation of metabolites. A 2.1 × 20 mm guard column with the same packing material was used for protection of the analytical column. Flow rate was set at 150 μL/min. Buffers consisted of 100% acetonitrile for mobile phase A, and 100% acetonitrile for mobile phase B. The chromatographic gradient ran from 85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A. Column temperature was 30 C.
Ion Mode:NEGATIVE
  
MS ID:MS004837
Analysis ID:AN005100
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control. Metabolite assignments and correction for expected natural abundances of 13C isotopes were performed using MAVEN.
Ion Mode:POSITIVE
  
MS ID:MS004838
Analysis ID:AN005101
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control. Metabolite assignments and correction for expected natural abundances of 13C isotopes were performed using MAVEN.
Ion Mode:NEGATIVE
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