The protocols described were undertaken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the University of Liverpool Animal Ethics Committee. Male Sprague Dawley rats received daily intraperitoneal (IP) dosing with gentamicin (200mg/kg/day), rosuvastatin (40mg/kg/day), combined gentamicin and rosuvastatin, or 0.9% saline (2ml/kg, control group) for 9 days at 10am each day. Four animals were included in each group. On day 9, animals were placed into metabolic cages for 2 hours each for collection of urine samples. Urine samples were transferred to 1.5ml eppendorfs and frozen at -80˚C.
Animals were sacrificed at 10am on day 10, 24 hours after the final treatment. Animals were euthanized in their groups, using a rising concentration of carbon dioxide. Blood was obtained from each animal by cardiac puncture. Blood was allowed to clot in the refrigerator for 24 hours. Serum was prepared by the removal of red blood cells by centrifugation at 3000rpm for 1 minute, the supernatant was removed and then frozen at - 80˚C for later analysis.
The liver and both kidneys were removed from euthanized animals. Half of the liver and one kidney were placed in 10% formalin. The other half of the liver, and the other kidney were divided into three and immediately frozen in liquid nitrogen, before being transferred to -80˚C.
152 6.2.1.1 Measurement of urinary NAG
A colorimetric assay was used for the determination of urinary N-Acetyl-β-D- glucosaminidase (NAG) (Roche, Germany). The assay reagent, 3-cresolsulfonphthaleinyl-N- acetyl-β-D-glucosaminide, sodium salt, is hydolysed by NAG, with the release of 3- cresolsulfonphthalein, sodium salt (3-cresol purple) which can be measured photometrically. Prior to running the assay, the reagents were prepared following the manufacturer’s instructions.
NAG standard (Roche, Germany) was prepared by dissolving the contents of one bottle in 3ml sterile distilled water to give an initial concentration of 25.40mU/ml. A calibration curve was first prepared from this standard using serial dilutions of 25µl of standard in 25µl distilled water to give further standard concentrations of 12.70, 6.35, 3.18, 1.59, 0.79 and 0.40 mU/ml. A final control calibration standard of distilled water was prepared. Urine samples were removed from the freezer and allowed to thaw at room temperature.
Calibrators and samples were run in triplicate on the same plate. First 100µl of the assay substrate solution (3-cresolsulfonphthaleinyl-N-acetyl-β-D-glucosaminide, sodium salt) was added to each well using a reagent reservoir and a multichannel pipette. The plate was covered and incubated at 37˚C for 5 minutes. Next, 5µl of calibration standard or urine sample was added to each well. The plate was sealed and incubated at 37˚C for 45 minutes, after which 200µl of stop reagent (sodium carbonate) was added to each well. The plate was covered and placed onto a plate shaker at 300rpm for 10 minutes at room temperature. The optical density was then read at 570nm. Sample concentration was calculated using the following formula:
153 6.2.1.2 Measurement of urinary creatinine
A colorimetric assay was used for the determination of urinary creatinine based on the Jaffe reaction first established in 1886. Prior to running the assay, the assay reagents were prepared as per table 6.1. Reagent A was then wrapped in tin foil to protect it from light.
Table 6.1 Creatinine assay reagents
Reagent Compound Preparation Volume
Reagent A
Sodium hydroxide 1g in 50ml dH2O (0.5M) 50.0ml Sodium Phosphate, dibasic 0.7098g in 50ml dH2O (0.1M) 25.0ml
Sodium borax 1.06g in 50ml dH2O 29.5ml
Sodium dodecyl sulphate 1.06g in 50ml dH2O 25.0ml
Picric Acid 45.0ml
Dimethyl sulphoxide 5.0ml
Reagent B
Acetic Acid 5.0ml
Concentrated sulphuric acid 1.0ml
Distilled water 44.0ml
Creatinine standard was prepared by dissolving 50mg creatinine (anhydrous) in 5ml distilled water to give an initial concentration of 10mg/ml (1000mg/dl). A calibration curve was prepared from this standard. 25µl of 1000mg/dl was diluted with 225µl distilled water to give a concentration of 100mg/dl. 100µl of 100mg/dl was added to 150µl distilled water to give a concentration of 40mg/dl. This was the maximum concentration standard for the calibration curve. Serial dilutions were then made of 100µl of the previous standard in 100µl distilled water to give further standard concentrations of 20, 10, 5, 2.5, 1.25 and 0.625mg/dl. A final control calibration standard of distilled water only was prepared. Urine samples were thawed, mixed and centrifuged (3000rpm, 5min). The resulting supernatant was prepared for analysis by ten-fold dilution (20µl sample in 180µl distilled water).
The assay was run in a standard 96 well plate with calibrators and samples run in duplicate on the same plate. First 25µl of standard or sample was added to each well. Next, 125µl of Reagent A was added to each well using a reagent reservoir and multichannel pipette and
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incubated at room temperature for 2 minutes. 5µl of Reagent B was added to each well using a reagent reservoir and multichannel pipette. The plate was covered and incubated on a plate shaker at 300rpm for 10 minutes at room temperature. After 10 minutes the optical density was read using a microplate reader at 490nm. A calibration curve was constructed using the measured optical densities for the standard concentrations. The equation for the best fit line was then used to calculate a creatinine concentration from the measured optical density for each sample.
6.2.1.3 Measurement of urinary Kim-1
Urinary Kim-1 was measured using a Rat TIM-1/KIM-1/HAVCR assay kit (Meso Scale Discovery (MSD), US). This assay is a commercially available electrochemiluminescent sandwich immunoassay run on a 96-well plate, and works in the same way as the MSD assays for human urinary KIM-1 and NGAL described in chapter 3.
The assay was run as below, according to the manufacturer’s protocol with some slight modification. The assay plate was brought to room temperature. MSD Diluent 5, MSD stock calibrator and urine samples were thawed. 5% MSD Blocker A was prepared by dissolving 1.25g of blocker A powder in 20ml of deionised water, and then adding 5ml of MSD phosphate buffer (5X). Phosphate buffered saline plus 0.05% Tween-20 (PBS-T) was prepared for plate washing. 150µl of Blocker A was added to each well of the plate using a multichannel pipette and reservoir. A reverse pipetting technique was used at all stages in order to avoid the introduction of bubbles. The plate was sealed using an adhesive plate seal, and incubated on a plate shaker at 300rpm at room temperature for 1 hour.
Seven standard concentrations were prepared from the stock calibrator for a standard curve by serial dilutions of the stock calibrator in MSD Diluent 5. The highest calibrator was prepared by adding 10µl of the stock calibrator to 190µl of MSD Diluent 5 to give a
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concentration of 10000pg/ml. Six further calibrator concentrations were prepared by serial three-fold dilutions of the previous calibrator (80µl of calibrator in 160µl MSD Diluent 5), to give concentrations of 3333, 1111, 370, 123, 41.2 and 13.7pg/ml. The final calibrator was MSD Diluent 5 only.
Urine samples were thawed, mixed and centrifuged (3000rpm, 5min). The resulting supernatant was prepared for analysis by five-fold dilution (20µl of sample in 80µl MSD Diluent 5).
After 1 hour, the plate was removed from the plate shaker. The plate was inverted over a sink to remove Blocker A, and washed four times with 150µl of PBS-T per well. Following the fourth wash, excess PBS-T was removed from the wells by inverting the plate and banging sharply onto absorbent paper towels on the bench. 25µl of MSD Diluent 5 was added to each well of the plate using a multichannel pipette and reservoir. 25µl of sample or calibrator was then added to each well of the plate. The plate was sealed and incubated on the plate shaker at 300rpm at room temperature for 2 hours.
After 2 hours the plate was removed and washed as before. 25µl of MSD detection antibody solution was added to each well of the plate. This was prepared within 15 minutes prior to use by dilution of 60µl of the 50X stock detection antibody solution in 2.94ml of MSD Diluent 5. The plate was sealed and incubated on the plate shaker at 300rpm at room temperature for 2 hours.
After 2 hours, the plate was removed and washed as before. MSD read buffer T (1X) was prepared during the previous incubation by dilution of 5ml MSD read buffer T (4X) with 15ml deionised water. 150µl of MSD read buffer T (1X) was added to each well of the plate with a reverse pipetting technique using a multichannel pipette and reservoir. The plate
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was then immediately read using an MSD SECTOR imager 6000. The plate data was analysed using the MSD DISCOVERY WORKBENCH software (version 4.0), which uses 4- parameter logistic curve fitting to generate the calibration curve.