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MB Sample ID: SA298966
| Local Sample ID: | 33_T4 |
| Subject ID: | SU002893 |
| 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 |
| Species Group: | Mammals |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002893 |
| 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 |
| Species Group: | Mammals |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 33_T4 | SA298966 | FL036243 | 240 | Timepoint |
| 33_T4 | SA298966 | FL036243 | CI | Group |
| 33_T4 | SA298966 | FL036243 | 26 | Urine_volume |
Collection:
| Collection ID: | CO002886 |
| Collection Summary: | Urine production over 1 hour of perfusion was diverted to a collection bag. At the end of the hour, urine samples were taken in dry tubes from this collection bag. 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: | Urine |
| Collection Frequency: | Every hour during perfusion, so T1 a sample from urine produced during the first hour of perfusion, T2 for the second hour, T3 for the third hour, and T4 for the fourth hour |
| Storage Conditions: | -80℃ |
| Additives: | None |
Treatment:
| Treatment ID: | TR002902 |
| Treatment Summary: | Three experimental conditions were investigated (n=6/group): (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. In the All kidneys were flushed with 200 ml of Ringer’s solution before mounting them on the ex situ circuit to wash out IGL-1. Because little urine was produced by WI kidneys, too few samples were available for a meaningful analysis. |
| 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: | SP002899 |
| 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. 50 µl of samples was added to 950 µl of the extraction buffer and stored overnight at -80°C. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004533 |
|---|---|
| Chromatography ID | CH003406 |
| MS ID | MS004280 |
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters ACQUITY UPLC BEH C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Focus |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003406 |
| Chromatography Summary: | 10 µl of each sample was loaded into a Dionex UltiMate 3000 LC System (Thermo Scientific Bremen, Germany) equipped with a Waters ACQUITY UPLC BEH C18 (150 x 2.1mm,1.7um) column coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) operating in negative ion mode. A step gradient was carried out using solvent A (10 mM TBA and 15 mM acetic acid) and solvent B (100% methanol). The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. The flow was kept constant at 0.25 mL/min and the column was placed at 40°C throughout the analysis. The MS operated in full scan mode (m/z range: [70.0000-1050.0000]) using a spray voltage of 4.80 kV, capillary temperature of 300°C, sheath gas at 40.0, auxiliary gas at 10.0. The AGC target was set at 3.0E+006 using a resolution of 140000, with a maximum IT fill time of 512 ms. Data collection was performed using the Xcalibur software (Thermo Scientific). The data analyses were performed by integrating the peak areas (El-Maven - Polly - Elucidata). |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters ACQUITY UPLC BEH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 40°C |
| Flow Gradient: | The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. |
| Flow Rate: | 0.25 ml/min |
| Solvent A: | 100% water; 10 mM TBA; 15 mM acetic acid |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004280 |
| Analysis ID: | AN004533 |
| 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). These were corrected for urine volume. |
| Ion Mode: | NEGATIVE |
