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MB Sample ID: SA138002
| Local Sample ID: | CKD4Liver_Aq |
| Subject ID: | SU001705 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 13 months |
| Weight Or Weight Range: | (32.86±1.21 g (control mice) vs 23.57±1.27 g (CKD mice), P < 0.0001, N=7/group). |
| Gender: | Male |
| Animal Animal Supplier: | Jackson Labs (Stock # 000664) |
| Animal Housing: | Housed in a temperature of 22 oC |
| Animal Light Cycle: | 12-hour light/12-hour dark |
| Animal Feed: | Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) |
| Animal Water: | free access to food and water (3-5 animals per cage). |
| Animal Inclusion Criteria: | (3-5 animals per cage). |
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Subject:
| Subject ID: | SU001705 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 13 months |
| Weight Or Weight Range: | (32.86±1.21 g (control mice) vs 23.57±1.27 g (CKD mice), P < 0.0001, N=7/group). |
| Gender: | Male |
| Animal Animal Supplier: | Jackson Labs (Stock # 000664) |
| Animal Housing: | Housed in a temperature of 22 oC |
| Animal Light Cycle: | 12-hour light/12-hour dark |
| Animal Feed: | Ad libitum (Casein control diet vs. adenine-supplemented diet to induce CKD) |
| Animal Water: | free access to food and water (3-5 animals per cage). |
| Animal Inclusion Criteria: | (3-5 animals per cage). |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CKD4Liver_Aq | SA138002 | FL017557 | CKD | Group |
Collection:
| Collection ID: | CO001698 |
| Collection Summary: | While under isoflurane anesthesia, tissues were rapidly dissected and snap frozen in liquid nitrogen and stored at -80°C until metabolite extraction. The following tissues were used in this study: kidney, liver, heart (left ventricle), skeletal muscle (quadriceps). Euthanasia was carried out by thoracotomy followed by cervical dislocation. |
| Sample Type: | Liver |
| Collection Method: | While under isoflurane anesthesia, tissues were rapidly dissected and snap frozen in liquid nitrogen and stored at -80°C until metabolite extraction |
| Collection Location: | University of Florida, Applied Physiology and Kinesiology, 1864 stadium RD, Gainesville, FL 32611 |
| Storage Conditions: | -80℃ |
| Storage Vials: | cryovials |
Treatment:
| Treatment ID: | TR001718 |
| Treatment Summary: | To induce CKD, we utilized an established adenine-diet model. Briefly, mice were assigned to a casein-base chow for 7-days, followed by induction of renal tubular injury by supplementing the diet with 0.2% adenine for 7-days, and subsequently maintained on a 0.15% adenine diet for 5 months and two weeks. CKD mice were then placed back on casein control diet for 2-weeks prior to euthanasia and terminal experiments, allowing for a washout period of adenine based chow. Control mice received casein diet for the duration of the study. Total duration of CKD encompassed 6-months. |
| Animal Vet Treatments: | none |
| Animal Anesthesia: | isoflurane |
| Animal Fasting: | non-fasted |
| Animal Endp Euthanasia: | Euthanasia was carried out by thoracotomy followed by cervical dislocation. |
| Animal Endp Tissue Coll List: | kidney, liver, heart (left ventricle), skeletal muscle (quadriceps) |
Sample Preparation:
| Sampleprep ID: | SP001711 |
| Sampleprep Summary: | A modified form of FOLCH extraction protocol was used to extract metabolites from the tissues. Wet weights of all tissue samples were recorded prior to extraction. Tissue samples were immediately homogenized to prevent any possible enzymatic action using 1 mL of ice-cold methanol in a PowerLyzer 24 Homogenizer (QIAGEN Group, Hilden, Germany). The mixture was centrifuged using 13,200 rpm at 4oC for 30 minutes and the resulting supernatant was transferred to a new glass vial consisting 3 mL of ice cold chloroform:methanol (2:1, v/v) mixture. The homogenate was vortexed and left in an ice bath for 15 minutes to allow for phase separation. Next, 1 mL of 0.9% of saline was added, vortexed it for couple of minutes followed by a second incubation in an ice bath for 30-45 min for complete phase separation. The upper aqueous layer was transferred to a new falcon tube. To the remaining organic phase sample, 1 mL of 0.9% of saline was added again followed by vigorous mixing and letting it stand in ice bath (15 minutes) for a second phase separation. This second aqueous phase was combined with the first. The resulting aqueous and organic layers were dried separately. The aqueous layer was dried overnight with a Labconco freezer dryer (Labconco Corporation, MO, USA) and the organic layer was dried via inert nitrogen gas. These two dried powders (aqueous and organic phases) were stored at -80oC until performing NMR experiments. |
| Processing Method: | Lyophilization and Homogenization |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Modified FOLCH extraction |
| Extract Storage: | -80℃ |
| Sample Resuspension: | In 50 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS and 0.2% sodium azide for aqueous phase samples. |
| Sample Spiking: | 0.5 mM of DSS for aqueous phase samples |
Analysis:
| MB Sample ID: | SA138002 |
| Analysis ID: | AN002663 |
| Laboratory Name: | Terence lab, UF |
| Analysis Type: | NMR |
| Acquisition Date: | 09/08/2020 |
| Software Version: | Bruker Topspin |
| Operator Name: | Ram Khattri |
| Detector Type: | Bruker |
| Data Format: | fid, 1r |
| Num Factors: | 2 |
| Num Metabolites: | 60 |
| Units: | A.U. |
NMR:
| NMR ID: | NM000195 |
| Analysis ID: | AN002663 |
| Instrument Name: | Bruker Avance Neo 600 MHz/54mm console |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Field Frequency Lock: | Deuterium |
| Standard Concentration: | 0.5 mM DSS |
| Spectrometer Frequency: | 600.2328273 MHz |
| NMR Probe: | 1.7 mm TXI CryoProbe |
| NMR Solvent: | Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in deuterated environment |
| NMR Tube Size: | 1.7 mm O.D. |
| Shimming Method: | Topshim |
| Pulse Sequence: | noesypr1d |
| Water Suppression: | presat |
| Pulse Width: | 90-degree |
| Receiver Gain: | 101 |
| Offset Frequency: | 2827.31 Hz |
| Chemical Shift Ref Cpd: | DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) |
| Temperature: | 25 o C |
| Number Of Scans: | 128 scans |
| Dummy Scans: | 8 |
| Acquisition Time: | 4 s |
| Relaxation Delay: | 1 s |
| Spectral Width: | 7142.9 Hz |
| Num Data Points Acquired: | 28571 |
| Real Data Points: | 65536 |
| Line Broadening: | 0.22 Hz |
| Zero Filling: | 65,536 points |
| Apodization: | Exponential |
| Baseline Correction Method: | Spline |
| Chemical Shift Ref Std: | 0 ppm for DSS |
