Animals
All animal experiments in the present study were reviewed and approved by the Institutional
Animal Care and Use Committee of Shionogi Research Laboratories (Osaka, Japan). Male
C57BL/6 J mice, weighing 18.2–24.6 g, were purchased from CLEA Japan, Inc. (Tokyo,
Japan), housed in a temperature-controlled room maintained on a 12-h light/dark cycle
with lights on at 8:00 am, and allowed free access to chow and tap water. At 8 weeks
of age, mice of the cisplatin group were intraperitoneally administered with a single
dose of cisplatin (30-mg/kg body weight, Wako Pure Chemical Industries Ltd., Osaka,
Japan) dissolved in saline at 3 mg/mL. This dose of cisplatin was selected based on
previous studies using mice with cisplatin-induced nephrotoxicity 10], 23]. Mice of the cisplatin + DMTU group were intraperitoneally administered with DMTU
(100-mg/kg body weight, Sigma-Aldrich, St. Louis, MO, USA) dissolved in saline at
10 mg/mL, 30 min prior to the cisplatin administration (30 mg/kg). The control + DMTU
mice were administered with DMTU (100 mg/kg) alone. For the histopathological examination,
DMTU was injected into mice 30 min prior to the cisplatin administration and then
injected once a day until 72 h after cisplatin administration, as described in a previous
report 10]. The groups of mice at 5, 10, or 18 h after cisplatin administration were used for
autoradiographic study ( = 4–6). The groups of mice at 24 h after cisplatin administration were used for assessment
of renal function and lipid peroxidation ( = 6). The groups of mice at 72 h after cisplatin administration were used for histopathological
examination ( = 6–10).
Synthesis of [
3
H]hydromethidine
[
3
H]hydromethidine (specific activity; 74 GBq/mmol, radiochemical purity; 98.2 %) was
synthesized by N-methylation using [methyl-
3
H]Methyl nosylate as described previously 22]. [
3
H]hydromethidine was diluted with distilled water containing 5 % DMSO (v/v), giving
97.8 % of radiochemical purity.
Autoradiographic study with kidney sections from mice injected with [
3
H]hydromethidine
An aqueous solution (5 % DMSO, v/v) of [
3
H]hydromethidine (185 kBq) was injected intravenously into the tail vein of mice at
5, 10, or 18 h after cisplatin administration. The mice were sacrificed by decapitation
at post-injection time points of 1 or 60 min under deep anesthesia with isoflurane.
The kidney was rapidly removed and frozen, and sections (20-?m thick) were prepared
using a cryostat. The sections were exposed to an imaging plate (BAS-TR, GE Healthcare,
Buckinghamshire, UK) for 14 days. After exposure, the plates were read with a FLA-3000
(Fujifilm Corp., Tokyo, Japan). Regions of interest (ROIs) were drawn on the corticomedullary
junction 24], and the photo-stimulated luminescence value for each ROI (PSL/mm
2
) was determined using Multi Gauge version 2.3 (Fujifilm Corp.). The radioactivity
concentrations in each ROI were expressed as (PSL ? background)/area (mm
2
) [(PSL ? BG)/mm
2
].
Sample collection for assessment of renal function, lipid peroxidation, and histopathology
Apart from the autoradiographic study, mice from each group were euthanized by isoflurane
anesthesia and exsanguinated via the inferior vena cava at 24 or 72 h after cisplatin
administration. Blood samples were collected for assessment of renal function. Kidneys
were stored at ?80 °C until measurement of lipid peroxidation or fixed with 10 % neutral
buffered formalin for histopathological examination.
Assessment of renal function
The blood samples were centrifuged at 1000×g for 20 min at 4 °C to obtain serum. Blood urea nitrogen (BUN) and creatinine levels
in the serum were measured using the urea nitrogen B-test kit (Wako Pure Chemical
Industries Ltd.) and creatinine colorimetric/fluorometric assay kit (BioVision, Inc.,
Milpitas, CA, USA), respectively.
Histopathological examination
The formalin-fixed kidney samples were embedded in paraffin. Paraffin sections were
stained with hematoxylin and eosin using standard methodologies. The tissues were
examined under a light microscope.
Measurement of lipid peroxidation
The kidney samples were homogenized in ice-cold phosphate buffered saline (0.01 M,
pH 7.4) containing 1-mM ethylenediaminetetraacetic acid to obtain 10 % (w/v) homogenate.
The homogenate was centrifuged at 10,000×g for 5 min at 4 °C, and the supernatant was used for the determination of the malondialdehyde
(MDA) level, an index of lipid peroxidation, using an MDA assay kit based on reaction
with thiobarbituric acid (Northwest Life Science Specialties LLC, Vancouver, WA, USA).
Statistical analysis
Data were expressed as mean ± SD. Statistical differences between the two groups were
determined by an unpaired t-test. P 0.05 was considered statistically significant.