Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure: Study design and DB289 administration
Procedure: Sample collection
Procedure: Clinical chemistry
Procedure: Measurement of biomarker kidney injury molecule-1 (KIM-1)
Procedure: Measurement of DB75 kidney tissue concentration
Steps Procedure Equipment Data collected 1 Mice acclimated to housing facility for 2 wks - - 2 Mice weighed before treatment and subsequently after each treatment balance scale body weights 3 Mice gavaged with DB289 or vehicle for 10 consecutive days gavaging set - 4 Urine collected 18 h overnight between study days 3-4, 7-8, and 10-11 metabolic cages total urine volume, urine CREA and KIM-1 concentration 5 Mice fasted for 18 h before necropsy - - 6 Mice euthanized, weighed, and terminal blood collected balance scale; blood collection kit body weights, serum ALT and BUN 7 Necropsy conducted, liver and kidney harvested and weighed balance scale kidney and liver weights 8 Kidney tissue samples processed sonic dismembrator - 9 Quantification of active metabolite DB75 from kidney tissue HPLC-MS/MS system DB75 concentration
- ProvantisTM 8 (Instem ProvantisTM; Conshohoken, PA) data collection system
- Balance scale
- Gavaging needles for mice
- Mouse metabolic cages (Hatteras Instruments, Inc., Cary, NC).
- Dissecting tools
- -80 Â°C Freezer
- Blood collection serum separation tubes
- Safe-lock tubes
- 96-well microtitre plates
- SpectraMax® microtitre plate reader (Molecular Devices, LLC)
- Sonic dismembrator (Model 120; Fisher Scientific Co.)
- HPLC-MS/MS system consisting of two Shimadzu solvent delivery pumps, a Valco (Valco Instruments Co. Inc.) switching valve, and a thermostatted (6°C) LEAP HTC autosampler (LEAP Technologies) connected to an Applied Biosystems API 4000 triple quadrupole mass spectrometer equipped with a heated nebulizer (APCI) ionization source
- Aquasil C18 HPLC column (50 Ã 2.1 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA)
- Analyst software (version 1.4.1; Applied Biosystems)
- CO2 gas euthanasia setup
- DB289, DB75 and deuterated standards (Consortium for Parasitic Drug Development (CPDD)
- Vehicle: 0.5% methyl 2-hydroxyethyl cellulose (Sigma-Aldrich)
- MaxDiscoveryâ¢ Blood Urea Nitrogen Enzymatic Kit (Bioo Scientific Corporation)
- MaxDiscoveryâ¢ Alanine Transaminase Enzymatic Kit (Bioo Scientific Corporation)
- Quantichrom™ Creatinine Assay Kit (BioAssay Systems)
- Liquid nitrogen
- Tissue fixative: 10% neutral phosphate buffered formalin (NBF)
- HPLC-grade water
- Mobile phase (A) consisting of HPLC-grade water with 35 mM formic acid and 15 mM ammonium acetate; Mobile phase (B) consisting of 80:20 (v/v) acetonitrile:water with 35 mM formic acid and 15 mM ammonium acetate.
- ELISA Kit for Kidney Injury Molecule 1 (KIM-1) (Uscn Life Science, Inc.)
Acclimation to test conditions
Mice are acclimated to housing facility for two weeks.
a. Mice are randomized to treatment groups by body weight using the Instem Provantis system.
b. Control group is weighed then dosed intragastrically (i.g.) with vehicle (0.5% methyl 2-hydroxyethyl cellulose) once daily for 10 days with a dosing volume of 10 mL/kg.
c. The experimental group is weighed first, before treatment regimen of 75 µmol/kg DB289 i.g. is administered once daily for 10 days with a dosing volume of 10 mL/kg. DB289 dosage selection is based on a pilot study in which CD-1 male and female mice were administered 0, 75, 125, or 250 µmol/kg of DB289 daily for 2, 4, or 14 days, wherein higher than 75 µmol/kg doses caused significant morbidity and mortality. Initial evaluation showed marked decrease in body weight in sensitive mice (BALB/cByJ and C3H/HeJ).
d. Dosing is performed at the same time of day throughout the study to avoid diurnal variability.
Oral gavaging technique
Gavaging is used to dose mice with a specified volume (10 mL/kg) of DB289 or vehicle directly into the stomach. Only specialized, commercially available gavage needles for mice are used. The syringe is first filled with the appropriate volume of material before the needle is attached.
a. A mouse is gently restrained by the scruff.
b. The tip or ball of the gavaging needle is placed into the mouse's mouth and then gently slid past the back of the tongue. Note: The tip of the needle should slide easily down the esophagus, otherwise, the needle is improperly placed and should never be forced. Any resistance encountered requires the removal of the needle and followed by reinsertion.
c. Further injury to surrounding tissues is carefully avoided by holding the syringe and preventing it from aspirating.
d. When the needle is properly placed, a dose of vehicle or DB289 is administered via the attachment of the syringe.
a. Urine is collected in chilled containers for 18 h overnight between study days 3â4, 7â8, and 10â11 using mouse metabolic cages.
b. Before necropsy/euthanasia, mice are fasted for 18 h.
c. Mice are fasted for 18 h and euthanized on day 11 by CO2 asphyxia and blood is collected via cardiac puncture. [Mice of strain P/J underwent necropsy 24 h ahead of schedule, on day 10, due to morbidity associated with treatment.]
d. The livers and kidneys are quickly excised and sections of the left liver lobe and left kidney are placed in 10% phosphate-buffered formalin for histological analyses.
e. The remaining tissue is snap-frozen in liquid nitrogen and stored at -80°C.
f. Blood samples are allowed to clot at room temperature for at least 1 h.
g. Serum is separated from blood cells using serum separation tubes and a table top centrifuge at a speed of 13,000 x g for 20 min.
h. The serum layer is pipetted carefully without disturbing the packed blood cell layer below and then transferred into a new pre-labeled tube and stored at -80°C until ready to be assayed.
a. Samples are assayed for serum biomarkers by standard enzymatic procedures using a SpectraMaxÂ® microtitre plate reader.
b. Blood urea nitrogen (BUN) is assayed using the MaxDiscoveryâ¢ Blood Urea Nitrogen Enzymatic Kit using the manufacturerâs recommendations.
c. Alanine transaminase (ALT) is assayed using the MaxDiscoveryâ¢ Alanine Transaminase Enzymatic Kit using the manufacturerâs instructions.
d. The Quantichromâ¢ Creatinine Assay Kit is utilized to measure creatinine (CREA) in the serum and urine using the manufacturerâs protocol.
a. KIM-1 is quantified in urine collected overnight prior to necropsy using the ELISA Kit for Kidney Injury Molecule 1 as detailed by the manufacturer.
b. Samples and standards are added to a microtitre plate pre-coated with biotin-conjugated polyclonal antibody preparation specific to KIM-1.
c. Avidin conjugated with horseradish peroxidase is then incubated in each well prior to the addition of a substrate solution that elicites a colorimetric change.
d. The reaction is halted with the addition of sulfuric acid solution.
e. KIM-1 is quantified spectrophotometrically at a wavelength of 450 nm.
f. Sample values are calculated using a standard curve and normalized to total urine volume collected over 18 h collection period.
a. Kidney specimens are processed for quantification of DB75 (active metabolite of DB289) by liquid chromatography/triple quadrupole mass spectrometry (HPLC/MS-MS) using a previously published method (Harrill et al., 2012) with modifications.
b. Kidney specimens are homogenized with 2 volumes of HPLC-grade water (1 g of wet tissue is considered equivalent to 1 mL in volume) using a sonic dismembrator.
c. Homogenates obtained from DB289-treated mice are diluted 1:10 (v/v) with blank rat kidney homogenate.
d. DB75 is extracted by mixing homogenates (25 µL) with 200 µL of 7:1 (v/v) methanol:water containing 0.1% (v/v) trifluoroacetic acid and 100 µM internal standard (DB75-d8).
e. Resulting supernatants are centrifuged and evaporated, and the remaining pellet is reconstituted with 100 µL of 15% (v/v) methanol containing 0.1% (v/v) trifluoroacetic acid.
f. DB75 quantification is measured using an HPLC-MS/MS system:
1. Following a 4 µL injection of reconstituted pellet, DB75 and DB75-d8 is eluted from an Aquasil C18 HPLC column using a reverse-phase gradient chromatography at a flow rate of 0.75 mL/min.
2. Initial gradient condition is 100% mobile phase (A), which is held for 0.5 min, then mobile phase (B) is increased linearly to 90% from 0.5 to 4 min.
3. The eluent is directed to waste for the first 0.6 min of each injection, then directed to the mass spectrometer.
4. At 4 min, the eluent is redirected to waste, and the column is washed with 90% mobile phase (B) for 1 min.
5. The system is re-equilibrated for 1 min with 100% mobile phase (A). Total run time is 6 min/injection.
6. Automated sample acquisition and data analysis are accomplished using Analyst software (version 1.4.1).
7. DB75 and DB75-d8 (internal standard) are analyzed in positive ion mode using the following transitions preset in multiple reaction monitoring scans: 305.1→288.1 (DB75) and 313.2→296.2 (DB75-d8).
8. Duplicate 11-point calibration curves for DB75 (100-250,000 nM), prepared in blank mouse kidney homogenate, are constructed using the peak area ratio of DB75 to DB75-d8 and fit with a quadratic equation using 1/x weighting.
9. Quality controls and dilution controls (1:10 in blank rat kidney homogenate) are prepared in triplicate. The lower limit of quantification (LLQ) is 100 nM. Accuracy and precision are at least 85% and 6%, respectively.
ALT alanine aminotransferase H&E hematoxylin and eosin Pafuramidine, treatment for trypanomiasis HPLC high performance liquid chromatography BUN blood urea nitrogen O.D. optical density in a colorimetric assay CREA creatinine enzyme-linked immunosorbent assay KIM-1 kidney injury molecule-1 NBF neutral buffered formalin
- Body weight
- ALT (serum)
- BUN (serum)
- Creatinine (serum, urine)
- Urine volume
- KIM-1 (urine)
- DB75 (DB289 metabolite; kidney)
- Kidney weight
- Liver weight
*MPD Note: "Difference" vectors were computed by MPD from the Harrill2 strain means (treated / control), and are available for download in a separate supplementary file.