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sonicated with Bioruptor (CosmoBio Co. Ltd, Tokyo, Japan) for 4 min (30-s ON/30-s
OFF) in ice-water.
For frozen sections, the kidneys were fixed with 4% paraformaldehyde (PFA; Wako
Chemical Co.) for 2 h on ice, incubated overnight in 30% (vol/vol) sucrose in
phosphate-buffered saline (PBS) at 4 °C and embedded in optimum cutting
temperature compound (Sakura FineTek Japan Co., Ltd, Tokyo, Japan).
Subsequently, 5-
μ
m thick sections were cut.
Oil Red O staining and area calculation. Oil Red O powder (75 mg) was
dissolved in 25 ml of 100% isopropyl alcohol and the solution was filtered to remove
undissolved powder. PFA-fixed samples were washed with PBS and 60% (v/v)
isopropyl alcohol. The samples were stained with 60% (v/v) Oil Red O solution for
15 min. Fat droplets in adipocytes were stained. Oil Red O-stained cells and frozen
section samples were observed and images were captured with an IX71 inverted
microscope (Olympus, Tokyo, Japan) or a BZ-X700 digital microscope (Keyence,
Osaka, Japan). The percentage of total cell culture area positive for Oil Red O
staining was calculated using ImageJ software (National Institutes of Health,
Bethesda, MD, USA). At least three different wells were measured for each
condition. For frozen sections, Mayer's hematoxylin was used as a counter-stain.
Hierarchical clustering, PC and GO analyses. Gene expression
analysis was performed using a SurePrint G3 Mouse GE Microarray Kit 8 × 60 K
(Agilent Technologies, Santa Clara, CA, USA). Raw data were normalized and
analyzed using GeneSpring GX11 software (Agilent Technologies). These
normalized data were analyzed using the NIA (National Institute on Aging) Array
Analysis website (http://lgsun. grc.nia.nih.gov/ANOVA/),
22
a web-based tool for
microarray data analysis using hierarchical clustering of averages and PCA. A
hierarchical clustering analysis was performed using a minimum distance value of
0.001, a separation ratio of 0.5, and the standard definition of the correlation
distance. GO and KEGG pathway enrichments were evaluated statistically following
the instructions provided by the Database for Annotation, Visualization and
Integrated Discovery (DAVID) 6.7.
57
The gene expression microarray data have
been submitted to the GEO (Gene Expression Omnibus) online database (http://
www.ncbi.nlm.nih.gov/geo/) under accession number GSE70088.
In vitro
and
in vivo
IRI models. For the in vitro IRI model, CMPs were
grown to 80% confluence and incubated in PBS for 3 h under hypoxic (1% O
2
, 5%
CO
2
, balanced N
2
) conditions at 37 °C in a hypoxic chamber (ASTEC, Fukuoka,
Japan), and subsequently incubated in growth media for 21 h under normoxic
conditions (21% O
2
, 5% CO
2
). CMPs were collected at various time points (0, 3, 4,
6 and 24 h).
58
Ten- to 12-week-old C57BL/6 mice were anesthetized by
intraperitoneal injection of pentobarbital (50 mg/kg body weight) (Kyoritsu Seiyaku,
Tokyo, Japan), and were intubated and ventilated under a respirator (SN-480-7,
Shinano Manufacturing, Tokyo, Japan). General anesthesia was maintained by
isoflurane. Following left thoracotomy, 7-0 Prolene suture thread was passed
beneath the LAD just distal to the main trunk. The threads were tied transiently over
a polyethylene tube for 60 min for ischemia, and were thereafter released for
reperfusion. The LVs, including the areas at risk of IRI, were collected at various
time points (0, 1, 1.5, 2 and 24 h) to examine gene profiles. At the end of the 24- h
reperfusion period, Evans blue and TTC double staining was performed to verify
IRI.
59
Western blotting. Samples (50
μ
g) were mixed with bromophenol blue and
2-mercaptoethanol, boiled for 10 min, electrophoresed on 10% SDS polyacrylamide
gel and electroblotted onto a PVDF transfer membrane (Millipore, Billerica, MA,
USA). The membrane was blocked with PBS containing 5% skimmed milk, 0.05%
Tween 20 and then incubated for 1 h with rabbit polyclonal antibodies to PPAR-
γ
(sc-7196; Santa Cruz Biotechnologies, Inc., Dallas, TX, USA), and mouse
monoclonal antibodies to GAPDH (MAB374; Millipore), which were diluted to
1 : 500 with blocking buffer. After washing, the membrane was incubated with
1 : 5000 dilution of horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG
or HRP-conjugated donkey anti-mouse IgG (GE Healthcare, Little Chalfont, UK) in
blocking buffer. Subsequently, the blots were developed using the ECL detection kit
(GE Healthcare) and protein bands were visualized using the VersaDoc system
(Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Statistical analysis. Results are expressed as mean values
±
S.E. The
statistical significance of differences between groups was evaluated using Student
’
s
t -test, and P -values
o
0.05 were considered significant.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements . We would like to express our sincere thanks to Toyoda
Masashi. (Tokyo Metropolitan Institute of Gerontology) for helpful discussions
regarding the results presented in the manuscript. This study was supported by a
Grant-in-Aid for Exploratory Research from JSPS KAKENHI 24659594.
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Cell Death and Disease