Summary of Study ST002458
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 PR001585. The data can be accessed directly via it's Project DOI: 10.21228/M8TD8H 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 | ST002458 |
| Study Title | Mouse kidney metabolomics (Proximal tubular cells) |
| Study Type | MS quantitative analysis |
| Study Summary | Here, we reveal for the first time the misregulated metabolic pathways in TSC kidneys and their relevance to TSC-associated cytogenesis. To this end, we have analyzed the metabolic profile of the whole kidney as well as sorted proximal tubule cell (PTCs) extracts. The metabolomics data show that Tsc1 deletion in nephron progenitor cells changes the arginine biosynthesis pathway as well as a substantial number of metabolic pathways. |
| Institute | Hadassah Medical Center |
| Last Name | Ben-Dov |
| First Name | Iddo |
| Address | Hadassah Medical Center, Jerusalem, Israel 91120 |
| iddo@hadassah.org.il | |
| Phone | +97226776881 |
| Submit Date | 2023-01-31 |
| Num Groups | 3 |
| Total Subjects | 26 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-02-26 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001585 |
| Project DOI: | doi: 10.21228/M8TD8H |
| Project Title: | Mouse kidney metabolomic studies |
| Project Type: | MS quantitative analysis |
| Project Summary: | Chronic kidney disease secondary to cystic kidney disease is a leading cause of mortality in patients with tuberous sclerosis complex (TSC) disease. The mechanisms underlying TSC cystic kidney disease are obscure, with no interventions available to prevent cyst formation. Here, we reveal for the first time the misregulated metabolic pathways in TSC kidneys and their relevance to TSC-associated cytogenesis. To this end, we have analyzed the metabolic profile of the whole kidney as well as sorted proximal tubule cell (PTCs) extracts. The metabolomics data show that Tsc1 deletion in nephron progenitor cells changes the arginine biosynthesis pathway as well as a substantial number of metabolic pathways. These changes were associated with overexpression of argininosuccinate synthetase 1 (ASS1), a rate-limiting enzyme in the arginine biosynthetic pathway in TSC KO mice and human kidneys. The rise in ASS1 expression is dependent on mTORC1 activity. Arginine depletion in vitro and in vivo prevented the rise in mTORC1 activity and cell cycle progression and averted the overexpression of previously described cytogenetic signaling proteins, including c-Myc and P65. Accordingly, an arginine-depleted diet substantially reduced the TSC cystic load, indicating the potential therapeutic effects of arginine deprivation as a treatment of TSC-associated kidney disease. |
| Institute: | Hadassah Medical Center |
| Department: | Pediatric Nephrology |
| Laboratory: | Wohl Institute for Translational Medicine |
| Last Name: | Ben-Dov |
| First Name: | Iddo |
| Address: | Ein Karem Campus, Jerusalem, NA, 9211001, Israel |
| Email: | iddo@hadassah.org.il |
| Phone: | +97226776881 |
| Funding Source: | This work was supported by grants from the Israel Science Foundation (MN 2030/21 and OV 2358/18), the TSC alliance research grant (OV), the research authority of Hadassah Hebrew University Medical Center (OV). OV and MN are Wohl’s Translation Research Institute research associates at Hadassah-Hebrew University Medical Center |
| Contributors: | Athar Amleh, Lana Watad, Ifat Abramovich, Bella Agranovich, Eyal Gottlieb, Iddo Z. Ben-Dov, Morris Nechama and Oded Volovelsky |
Subject:
| Subject ID: | SU002548 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA246071 | 06_KO_3 | Tsc1-knockout | none |
| SA246072 | 04_KO_1 | Tsc1-knockout | none |
| SA246073 | 05_KO_2 | Tsc1-knockout | none |
| SA246074 | 09_KO_Rapha3 | Tsc1-knockout | rapamycin |
| SA246075 | 08_KO_Rapha2 | Tsc1-knockout | rapamycin |
| SA246076 | 07_KO_Rapha1 | Tsc1-knockout | rapamycin |
| SA246077 | 02_WT_2 | Wild-type | none |
| SA246078 | 03_WT_3 | Wild-type | none |
| SA246079 | 01_WT_1 | Wild-type | none |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO002541 |
| Collection Summary: | PTCs were isolated as previously described(PMID: 32484794). Briefly, kidneys were excised in ice-cold HBSS buffer. The kidneys were sliced and chopped into pieces (~0.5–1 mm) on ice using a surgical scalpel. The chopped kidneys were transferred into an HBSS solution containing 1 μg/μL collagenase/dispase (Sigma Aldrich, Cat. # 10269638001) and incubated for 25 minutes at 370 C. The cells were filtered through a 40-μm nylon cell strainer (Corning, Cat. # 431750) and washed twice with cold HBSS. For PTC isolation, the cells were stained with PE-conjugated antiCD133/prominin-1 antibody (Invitrogen, Cat. # 12-1331-82) according to the manufacturer's instructions. PE+ cells were isolated by Aria III flow cytometry-based cell sorting. |
| Sample Type: | Proximal tubular cells |
Treatment:
| Treatment ID: | TR002560 |
| Treatment Summary: | A mixture of six labeled internal standards was added to the extraction solution for quality control (13C6-Glucose, 13C5-Glutamine, 13C5-Glutamate, 13C1-Alanine, 13C3-Pyruvate and 13C3-Lactate). The exact volume at each tube was adjusted according to tissue weight (average volume of 200 µl). The samples were homogenized using Precellys 24 tissue homogenizer (Bertin Technologies) precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a 30 s gap between each cycle) and later centrifuged at 18,000 g for 15 min at 4°C. The supernatants were transferred into HPLC glass vials and kept at −80°C prior to LC-MS analysis. |
Sample Preparation:
| Sampleprep ID: | SP002554 |
| Sampleprep Summary: | The samples were homogenized using Precellys 24 tissue homogenizer (Bertin Technologies) precooled to 4°C (3 cycles × 30 s at 6000 rpm, with a 30 s gap between each cycle) and later centrifuged at 18,000 g for 15 min at 4°C. The supernatants were transferred into HPLC glass vials and kept at −80°C prior to LC-MS analysis. |
Combined analysis:
| Analysis ID | AN004010 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | concentration |
Chromatography:
| Chromatography ID: | CH002961 |
| Chromatography Summary: | UPLC setup consisted a ZIC-pHILIC column (SeQuant; 150 mm × 2.1 mm, 5 μm; Merck). 5 µl of cells or kidney extracts were injected using an autosampler. Compounds were separated using a 15 minutes gradient, starting at 20% aqueous (20 mM ammonium carbonate adjusted to pH 9.2 with 0.1% of 25% ammonium hydroxide) and 80% organic (acetonitrile), terminated with 20% acetonitrile. Flow rate and column temperature were kept at 0.2 ml/min and 45°C, respectively for a total run time of 27 min |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 45 |
| Flow Gradient: | 15 min terminated with 20% acetonitrile |
| Flow Rate: | 0.2 ml/min |
| Solvent A: | 20% water/80% acetonitrile; 20 mM ammonium carbonate; 0.1% of 25% ammonium hydroxide (adjusted to pH 9.2) |
| Solvent B: | 80%water/20% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003757 |
| Analysis ID: | AN004010 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Briefly, Dionex Ultimate ultra-high-performance liquid chromatography (UPLC) system coupled to Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific) was used. The resolution was set to 35,000 at 200 mass/charge ratio (m/z) with electrospray ionization and polarity switching mode to enable both positive and negative ions across a mass range of 67–1000 m/z. |
| Ion Mode: | UNSPECIFIED |
