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132 protocols using creatinine

1

Isolated Organ Perfusion for Renal Assessment

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In the CI experiment, the isolated organ perfusion system was also used to assess renal function and injury during simulated reperfusion.
The reperfusion model differs from NMP in that whole autologous blood is used (containing all blood components), and certain protective ingredients are omitted (Table 1). The system has no ability to produce creatinine metabolically; therefore, 1,000 mol creatinine (Sigma-Aldrich, Steinheim, Germany) were added to the circuit to permit creatinine clearance measurement. After 1 h of NMP, the kidneys were removed from the system and flushed with 100 ml of Ringers lactate solution at 4°C to remove blood and cool the organ. Kidneys were then placed on the circuit and reperfused at a near-tonormal (11) RPP of 85 mmHg for 3 h at 38°C. All kidneys were reperfused with a 95% O 2-5% CO2 gas mixture.
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2

Linearity Evaluation of NMR Metabolites

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Linearity of creatinine, valine and myo-inositol NMR measurements were determined according to CLSI guideline EP6-A [33 ]. To generate a low-level fraction, an aliquot of this serum was cleared of small metabolites by dialysis against 1× PBS supplemented with 10 mg/dL of sodium-(L) Lactate as described above. To generate a very high-level fraction, another aliquot of the serum was supplemented with creatinine (Sigma Aldrich, St. Louis, MO, USA), valine (Sigma Aldrich) and myo-inositol (Sigma Aldrich) to final concentrations >1 mmol/L for creatinine and valine, and >0.4 mmol/L for myo-inositol. A total of 11 equidistant concentration levels were prepared by linear intermixture of the high- and low-level fractions ranging from 100% high level to 0% low level, as recommended [33 ].
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3

Biochemical Analysis of Animal Samples

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Blood samples were collected from animals at sacrifice. BUN was measured using a QuantiChrom assay kit (QuantiChrom™ Urea Assay Kit, BioAssay Systems, Hayward, Calif.) following the manufacturer's instructions. Serum creatinine was measured by high-performance liquid chromatography (HLPC) using creatinine (Sigma, Munich, Germany) as standard. Plasma tunicamycin levels were determined by HPLC using tunicamycin (Sigma, Munich Germany) as standard.
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4

Normothermic Machine Perfusion of Donor Kidneys

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The NMP system was assembled as previously described; a schematic diagram of the perfusion set-up can also be found in this reference.21 (link) A continuous supply of oxygen, delivered as 95% oxygen/5% carbon dioxide, was delivered to the oxygenator at a flow rate of 1.5 L/min. Creatinine was added to the circuit (Merck, Darmstadt, Germany) to enable subsequent quantification of Creatinine clearance (CrCl). The kidney was placed in a customized, 3D-printed perfusion chamber, with the renal vein left open.22 (link) Urine output (UO) was replaced with Hartmann’s solution. NMP was undertaken at a temperature of 37°C, with flow rates adjusted to maintain at a mean arterial pressure (MAP) of 75–85 mm Hg.
To provide a direct comparison between CS and NMP in the absence of the ability to transplant these kidneys, ex vivo reperfusion with whole blood was undertaken in paired kidneys to simulate transplantation (MAP 85–95 mm Hg and temperature 37°C).15 (link),23 (link),24 (link) Perfusion parameters, additives, and constituents in both the NMP and ex vivo whole blood reperfusion system are detailed in Methods S1 (SDC, http://links.lww.com/TXD/A226). Perfusion parameters (pressure and flow) and UO were sequentially recorded during NMP and whole blood reperfusion.
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5

Quantitative Analysis of Metabolites

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Uric acid, uricase, urea, glucose, creatinine, dopamine, and glutathione were purchased from Sigma-Aldrich (St. Louis, MO), while o-phenylenediamine (OPD), histidine, and phenylalanine were obtained from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Diethylenetriamine (DETA) was obtained from TCI Shanghai Chemical Industry Development Co., Ltd. (Shanghai, China). All chemicals used were of analytical grade. The ultrapure water (18.25 MΩ cm−1) was used in all experiments and was prepared from a Milli-Q water purification system (Millipore, USA).
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6

Intestinal Permeability Assay in Mice

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The intestinal permeability assay was performed as described before by Edelblum KL et al., 2017 (35 (link)). 14-week old Zbtb20-cKO and WT mice were fasted for 2 hours and then gavaged with a mixture of 100 mg/mL creatinine (Sigma C4255), 80 mg/mL FITC-dextran 4kDa (FD4) (Sigma 46944), and 20 mg/mL Rhodamine B-dextran 70 kDa (Sigma R9379). After 5h the 250–300μL of blood was collected by the retroorbital bleed. Samples were spun down at 1000 RPMs and ∼100μL of serum was collected for the analysis. Fluorescence intensity for FD4 and RD70 was determined by using a plate reader at 495 nm excitation/525 nm emission and 555 nm excitation/585 nm emission, respectively. The concentration of creatinine was measured using the Sciteck SVT creatinine kit (Sciteck 139–30) according to the manufacturer’s protocol.
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7

Fluorescent Probes for PS Microplastics

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Mice were administered with 0.2 mL saline containing 12 mg fluorescein isothiocyanate-4 kDa dextran, 8 mg rhodamine B isothiocyanate-70 kDa dextran, and 20 mg creatinine (Sigma) 3 h prior to the end of 24 h and 28 days PS micro- and nanoplastics exposure. Serum was harvested after 24 h of PS micro- and nanoplastics exposure. creatinine was measured using a kit from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Recovery of creatinine and fluorescent probes was measured in a Synergy HT plate reader (BioTek, Winooski, VT, USA) using freshly prepared standards. Fluorescein and rhodamine B fluorescence were measured using excitation wavelengths of 495 nm and 555 nm, and emission wavelengths of 525 nm and 585 nm, respectively.
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8

Rat Kidney Normothermic Machine Perfusion

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The rat NMP method has been described extensively earlier.18 In brief, NMP was performed for 90 minutes using a roller pump (Ismatec ISM404, Zürich, Switzerland). The perfusion pressure was set at 102 mmHg, controlled at the renal artery. In the control group, the perfusion fluid consisted of 100 mL William’s Medium E supplemented with 30 mmol/L HEPES, 50 g/L albumin, and 7 mmol/L creatinine (all Sigma‐Aldrich). In the experimental groups, either 30 or 300 mg/L metformin was added to the perfusate. The perfusion fluid was oxygenated with 95% oxygen and 5% carbon dioxide with a flow of 0.5 L/min. The temperature of the perfusion fluid was maintained at 37°C using a water bath and heat exchanger (Julabo, Seelbach, Germany). The flow was recorded every 10 minutes, using a calibrated flow sensor (ME1PXN Inline, Transonic Systems, Ithaca, NY). After NMP, biopsies of the kidneys were submerged immediately in 4% formaldehyde or snap‐frozen in liquid nitrogen and subsequently stored at −80°C.
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9

Determination of Heterocyclic Amines in TRE

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All of the chemicals and solvents were of HPLC or analytical grade. Analytical standards of 12 Has—IQ (2-amino-3-methyl-imidazo[4,5-f]quinoline), MeIQ (2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline), MeIQx (2-amino-3,8-dimethyl-imidazo [4,5-f]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8-trimethyl-imidazo[4,5-f]quinoxaline), 7,8-DiMeIQx (2-amino-3,7,8-trimethyl-imidazo[4,5-f]quinoxaline), PhIP (2-amino-1- methyl-6-phenylimidazo[4,5-f]pyridine), Harman (1-methyl-9H-pyrido[3,4-b]indole), Norharman (9H-pyrido[3,4-b]indole), Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b] indole), Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), AαC (2-amino-9H- pyrido[2,3-b]indole), and MeAαC (2-amino-3-methyl-9H-pyrido[2,3-b]indole)—were supplied by Toronto Research Chemicals (Downsview, ON, Canada). The standards of free amino acids, creatine, and creatinine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oasis MCX cartridges (60 mg, 3 mL) were supplied by Waters (Milford, MA, USA). The TRE was obtained followed our previous described [16 (link)] with the extraction conditions of extraction solvent of 60% ethanol, ultrasonic-assisted extraction power of 600 W for 40 min. The vacuum freeze dryer was employed to obtain the extract powders, which were stored at −20 °C until use.
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10

Creatine and Creatinine Riboside Analysis

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Reference standards of creatine (≥98.0 %), anhydrous grade, creatinine (≥98.0 %), anhydrous grade, ammonium acetate, LC–MS grade and ACS grade ammonium hydroxide were purchased from Sigma-Aldrich (St Louis, MO, USA). Synthesized creatine riboside (CR, ≥99.0 %), creatinine riboside (CNR, ≥99.0 %), creatine riboside-13C,15N2 (CR-13C,15N2, ISTD, ≥ 99.0 %) were synthesized in the Chemical Biology Laboratory, CCR, NCI (Frederick. MD). LC–MS grade acetonitrile, methanol and water were purchased from Fisher Scientific Chemicals (Fairlawn, NJ, USA).
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