Summary of Study ST002784

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 PR001736. The data can be accessed directly via it's Project DOI: 10.21228/M89Q7K 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.

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Study IDST002784
Study TitleMetabolic changes of the pig kidney during isolated normothermic perfusion with red blood cell based perfusate - perfusate
Study TypeExperimental
Study SummaryThis study investigates how glucose, lactate and 20 amino acids in the perfusate of normothermically perfused pig kidneys changes over time. It also studies how type and severity of ischemia before perfusion (cold or warm ischemia) change this profile.
Institute
KU Leuven
DepartmentMicrobiology, Immunology and Transplantation
LaboratoryLab of Abdominal Transplantation
Last NameJochmans
First NameIna
AddressHerestraat 49, Leuven, 3000, Belgium
Emailina.jochmans@kuleuven.be
PhoneNone
Submit Date2023-07-17
Num Groups3
Total Subjects10 kidneys
Num Males5 male pigs
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-08-07
Release Version1
Ina Jochmans Ina Jochmans
https://dx.doi.org/10.21228/M89Q7K
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001736
Project DOI:doi: 10.21228/M89Q7K
Project Title:Metabolic changes during normothermic isolated kidney perfusion
Project Type:Experimental
Project Summary:Normothermic isolated kidney perfusion is being developed as a method to preserve donor kidneys and to assess their future function before they are transplanted. Donor kidneys can be exposed to different types and degrees of ischemic injury before they are preserved. This project studies how the metabolome changes in relation to different types and degrees of ischemia.
Institute:KU Leuven
Department:Microbiology, Immunology and Transplantation
Laboratory:Lab of Abdominal Transplantation
Last Name:Jochmans
First Name:Ina
Address:Herestraat 49, Leuven, 3000, Belgium
Email:ina.jochmans@kuleuven.be
Phone:None
Funding Source:FTBO, KOOR, FWO
Contributors:Julie De Beule, Bart Ghesquière, Ina Jochmans

Subject:

Subject ID:SU002891
Subject Type:Mammal
Subject Species:Sus scrofa
Taxonomy ID:9823
Age Or Age Range:~4 months
Weight Or Weight Range:35-45 kg
Gender:Male
Animal Animal Supplier:TOPIGS TN70, Tojapigs, the Netherlands
Animal Light Cycle:12 hours
Animal Water:ad libitum

Factors:

Subject type: Mammal; Subject species: Sus scrofa (Factor headings shown in green)

mb_sample_id local_sample_id Group Timepoint
SA29881152_T0CI 0
SA29881254_T0CI 0
SA29881348_T0CI 0
SA29881450_T0CI 0
SA29881548_T2CI 120
SA29881650_T2CI 120
SA29881754_T2CI 120
SA29881852_T2CI 120
SA29881950_T0.25CI 15
SA29882048_T0.25CI 15
SA29882152_T0.25CI 15
SA29882254_T0.25CI 15
SA29882350_T3CI 180
SA29882452_T3CI 180
SA29882548_T3CI 180
SA29882654_T3CI 180
SA29882748_T4CI 240
SA29882850_T4CI 240
SA29882954_T4CI 240
SA29883052_T4CI 240
SA29883148_T0.50CI 30
SA29883252_T0.50CI 30
SA29883354_T0.50CI 30
SA29883450_T0.50CI 30
SA29883550_T0.75CI 45
SA29883652_T0.75CI 45
SA29883748_T0.75CI 45
SA29883854_T0.75CI 45
SA29883950_T1CI 60
SA29884052_T1CI 60
SA29884154_T1CI 60
SA29884248_T1CI 60
SA29884351_T0Control 0
SA29884449_T0Control 0
SA29884547_T0Control 0
SA29884649_T2Control 120
SA29884751_T2Control 120
SA29884847_T2Control 120
SA29884951_T0.25Control 15
SA29885049_T0.25Control 15
SA29885147_T0.25Control 15
SA29885249_T3Control 180
SA29885351_T3Control 180
SA29885447_T3Control 180
SA29885551_T4Control 240
SA29885649_T4Control 240
SA29885747_T4Control 240
SA29885849_T0.50Control 30
SA29885947_T0.50Control 30
SA29886051_T0.50Control 30
SA29886147_T0.75Control 45
SA29886249_T0.75Control 45
SA29886351_T0.75Control 45
SA29886451_T1Control 60
SA29886549_T1Control 60
SA29886647_T1Control 60
SA29886745_T0WI 0
SA29886846_T0WI 0
SA29886953_T0WI 0
SA29887045_T2WI 120
SA29887153_T2WI 120
SA29887246_T2WI 120
SA29887353_T0.25WI 15
SA29887446_T0.25WI 15
SA29887545_T0.25WI 15
SA29887646_T3WI 180
SA29887745_T3WI 180
SA29887853_T3WI 180
SA29887953_T4WI 240
SA29888046_T4WI 240
SA29888145_T4WI 240
SA29888246_T0.50WI 30
SA29888345_T0.50WI 30
SA29888453_T0.50WI 30
SA29888553_T0.75WI 45
SA29888645_T0.75WI 45
SA29888746_T0.75WI 45
SA29888845_T1WI 60
SA29888946_T1WI 60
SA29889053_T1WI 60
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Collection:

Collection ID:CO002884
Collection Summary:Perfusate samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes, at baseline (before the kidney was mounted on the normothermic perfusion device), every 15 minutes during the first hour and hourly thereafter. One milliliter perfusate aliquots (centrifuged 1000g, 10 minutes, 4°C) were snap frozen in liquid nitrogen and stored at -80°C until analysis.
Sample Type:Perfusate
Collection Frequency:Baseline (T0) and during perfusion at 15 min (T0.25), 30 min (T0.50), 45 min (T0.75), 1 hour (T1), 2 hours (T2), 3 hours (T3), 4 hours (T4)
Storage Conditions:-80℃
Additives:EDTA

Treatment:

Treatment ID:TR002900
Treatment Summary:Three experimental conditions were investigated (n=3/group for Control and WI, n=4/group for CI): (a) Controls; (b) warm ischemia (WI) simulating hypoxic acute kidney injury; (c) cold ischemia (CI) replicating clinical cold storage. Control kidneys were retrieved, flushed with cold IGL-1, and immediately reperfused. CI group, kidneys were exposed to 22h of CI by submerging them in IGL- 1 (a clinical preservation solution for cold storage) and storing them on ice. In WI, the renal artery and vein were clamped for 60 min before retrieval. All kidneys were flushed with 200 ml of Ringer’s solution before mounting them on the ex situ circuit to wash out IGL-1.
Animal Anesthesia:Pigs were sedated by an intramuscular injection of Tiletamine/Zolazepam (8 mg/kg, Zoletil®, Virbac, Belgium) and Xylazine (2 mg/kg, Xylazine®, VMD pharma, Belgium) to allow orotracheal intubation. Anesthesia was maintained by inhalation of isoflurane (1% Isovet®, Piramal Critical Care B.V., Belgium) and continuous infusion of fentanyl (8 µg/kg, Fentanyl®, Janssen Pharmaceutica, Belgium).
Animal Acclimation Duration:Minimum of 2 days
Animal Fasting:Overnight
Animal Endp Euthanasia:Non-recovery study

Sample Preparation:

Sampleprep ID:SP002897
Sampleprep Summary:Samples were extracted in an 80% methanol (80:20 methanol:water) (Methanol ≥99.9%, HiPerSolv CHROMANORM®, ULTRA for LC-MS, suitable for UPLC/UHPLC-MS instruments, VWR, Belgium) extraction buffer containing 1 µM of deuterated D27 myristic acid, 5 µM D12 glucose, 3 µM 13C5-D5-15N Glutamic acid and 3 µM D7-15N4-Arginine as internal standards. 10 µl of sample was added to 990 µl of the extraction buffer and stored overnight at -80 °C. Insolubilities and precipitated proteins were removed by centrifugation at 20.000 g, for 15 min at 4 °C. 200 µL of the supernatant was transferred to an appropriate mass-spectrometry vial. 10 µl of sample was added to 990 µl of the extraction buffer and stored overnight at -80 °C.
Extract Storage:-80℃

Combined analysis:

Analysis ID AN004531
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 1290 Infinity
Column Agilent InfinityLab Poroshell 120 (2.1 x 150 mm, 2.7 um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Focus
Ion Mode NEGATIVE
Units Peak area

Chromatography:

Chromatography ID:CH003404
Chromatography Summary:10 µl was loaded onto an Ultra Performance Liquid Chromatograph (UPLC) equipped with a Hydrophilic Interaction Liquid Chromatography (HILIC) column (InfinityLab Poroshell 120 HILIC-Z PEEK-lined 2.1 x 150 mm, 2.7 µm column; Agilent, Santa Clara, USA)) and connected in-line to a Q-exactive Orbitrap Focus (Thermo Fisher Scientific, Massachusetts, USA) mass spectrometer. A linear gradient was built up starting with 90% solvent A (LC-MS grade acetonitrile, Acetonitrile hypergrade for LC-MS LiChrosolv, Supelco (Merck), Germany) and 10% solvent B (10 mM ammonium acetate (LiChropur™, eluent additive for LC-MS, (Merck), Germany), pH 9.3). At 2 min the gradient increased to 60% of solvent B and maintained at 60% until 15 min. The gradient returned to 10% solvent B at 16 min and remained until 25 min. The flow rate was 250 µl/min and the column was kept at 25°C throughout the analysis. The MS operated in negative ion mode, with a spray voltage of 2.9 kV and a temperature of the capillary of 325 °C. Gas settings were as follows: sheath gas 40 and auxiliary gas 15. The vaporizer temperature was set at 300 °C. A full scan (resolution of 70.000 and scan range of m/z 70-1050) was applied. XCalibur version to operate the LC-MS was 4.2.47.
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent InfinityLab Poroshell 120 (2.1 x 150 mm, 2.7 um)
Column Temperature:25°C
Flow Gradient:A linear gradient was built up starting with 90% solvent A and 10% solvent B. At 2 min the gradient increased to 60% of solvent B and maintained at 60% until 15 min.
Flow Rate:250 µl/min
Solvent A:100% acetonitrile
Solvent B:100% water; 10 mM ammonium acetate, pH 9.3
Chromatography Type:HILIC

MS:

MS ID:MS004278
Analysis ID:AN004531
Instrument Name:Thermo Q Exactive Focus
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Raw MS data were converted into mzML using the MSConvert tool of Proteowizard (version 3.0.20247). Peak picking was performed with El_Maven (El_Maven, Elucidata, Massachusetts, USA, version 0.12.0). Metabolites were identified using an in-house library containing exact mass and retention time. The mass accuracy during data processing in El Maven was set at 10 ppm. Calculation of abundances was done in the LC-MS Workflow of El_Maven. Raw abundances (peak area values) for each metabolite were corrected for internal standard (Myristic acid d27).
Ion Mode:NEGATIVE
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