Summary of Study ST003111
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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST003111 |
| Study Title | Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney Disease Progression, investigated with targeted metabolomics in Tam-Cre;Pkd1ΔC/flox mouse model kidneys. |
| Study 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 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. 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 |
| stefanoni.davide@hsr.it | |
| Phone | +393337686005 |
| Submit Date | 2024-02-27 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-04-30 |
| Release Version | 1 |
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: | SU003226 |
| 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 | Factor |
|---|---|---|---|
| SA337280 | F6 - 3578 | Kidney | Ctrl-Asns-ASO |
| SA337281 | A4 - 2931 | Kidney | Ctrl-Asns-ASO |
| SA337282 | D3 - 3449 | Kidney | Ctrl-Asns-ASO |
| SA337283 | I2 - 4048 | Kidney | Ctrl-Scr-ASO |
| SA337284 | B2 - 3152 | Kidney | Ctrl-Scr-ASO |
| SA337285 | F4 - 3587 | Kidney | Ctrl-Scr-ASO |
| SA337286 | D1 - 3442 | Kidney | Ctrl-Scr-ASO |
| SA337287 | G4 - 3788 | Kidney | Ctrl-Scr-ASO |
| SA337288 | B4 - 3147 | Kidney | KO-Asns-ASO |
| SA337289 | I4 - 4047 | Kidney | KO-Asns-ASO |
| SA337290 | L6 - 4629 | Kidney | KO-Asns-ASO |
| SA337291 | F5 - 3580 | Kidney | KO-Asns-ASO |
| SA337292 | K5 - 4606 | Kidney | KO-Asns-ASO |
| SA337293 | C1 - 3365 | Kidney | KO-Scr-ASO |
| SA337294 | B1 - 3151 | Kidney | KO-Scr-ASO |
| SA337295 | L3 - 4630 | Kidney | KO-Scr-ASO |
| SA337296 | L4 - 4634 | Kidney | KO-Scr-ASO |
| SA337297 | N3 - 5560 | Kidney | KO-Scr-ASO |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO003219 |
| Collection Summary: | Kidney tissue samples were collected and instantly frozen in liquid nitrogen. Successively kidney tissue samples were grinded in dry ice and weighted. |
| Sample Type: | Kidney |
Treatment:
| Treatment ID: | TR003235 |
| Treatment Summary: | Tam-Cre;Pkd1ΔC/flox mice and relative controls were treated with antisense oligonucleotide targeting Asns (Asns-ASO). As the efficacy/tolerability was dosed at 50 mg/kg/week, ASOs were administered at the same dosage via weekly intraperitoneal injection for the first 2 months after Tamoxifen induction, and every second week after P100 till the end of the experiment. |
Sample Preparation:
| Sampleprep ID: | SP003232 |
| Sampleprep Summary: | Grinded kidney tissue samples were extracted in a ratio of 15mg/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 | AN005094 | AN005095 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak Area | Peak Area |
Chromatography:
| Chromatography ID: | CH003850 |
| 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: | CH003851 |
| 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: | MS004831 |
| Analysis ID: | AN005094 |
| 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: | MS004832 |
| Analysis ID: | AN005095 |
| 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 |
