2.1. Animal Experimental Design
All experiments described were
approved by the Animal Ethical and Experimental Committee of the Tianjin
Medical University and performed in accordance with their guideline.
Animal studies are reported in compliance with the ARRIVE guidelines
(Kilkenny, Browne, Cuthill, Emerson & Altman, 2010; McGrath & Lilley,
2015) and with the recommendations made by the British Journal of
Pharmacology. All study animals were purchased from
HFK Bioscience Co. Ltd, Beijing.
Fifteen 8-week-old female
C57BL/6J wild-type mice
(average weight 19.4 ± 3.2 g) and
30 spontaneous diabetic KK-Ay mice
(average
weight 25.6 ± 3.6 g) were used in this study.
Lot of studies have confirmed that
KK-Ay mice developed proteinuria, mesangial matrix accumulation and GBM
thickening, which validated this strain as a good model for studying DKD
(Ito et al., 2006).
Animals
were housed in Specific Pathogen Free facilities with maintained
temperature (23 ± 1°C) and humidity under 12/12-h light-dark cycle. Mice
were maintained in cages with a maximum of four mice per cage. The
C57BL/6J mice were fed with regular chow that comprises of 5% fat, 53%
carbohydrate and 23% protein, and the KK-Ay mice were fed with high-fat
diet that consists of 17.9% fat, 48% carbohydrate and 17.5% protein.
All the mice could drink water freely.
Mice were given analgesia
immediately preceding surgery with buprenorphine (0.1 mg·kg−1
intraperitoneally). The Schematic diagram of the experimental procedure
was shown in Figure 1A. Meanwhile, in order to testify if the renal
positive effect of Empa was independent of its
glycemia controlling function, we
introduced another group of KK-Ay mice which accepted insulin therapy
(INS group n=15) to our study.
2.2. Cellular Experiments
HK2 cells (purchased from Chinese Academy
of Sciences Shanghai Cell Bank, Shanghai, China) were cultured in
Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12)
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100
ug/ml streptomycin in an atmosphere containing 5% CO2 at 37℃. HK2 cells
were trypsinized and seeded into 6-well culture plates at a density of
1*106/ well and grown to over 80% confluence.
Serum-free medium containing 0.2% BSA was cultured overnight to
synchronize cells. HK2 cells were treated with normal glucose (NG, 17.5
mmol/l D-glucose), or high glucose (HG, 33.3 mmol/l D-glucose), or high
glucose plus Empa (HG +EMPA, 33.3 mmol/l D-glucose+ 8 μg/ml Empa) or
high osmotic pressure solution (MA, 17.5 mmol/l D-glucose + 15.8 mmol/l
D-mannitol).
2.3. Morphological Analysis of Kidney
Unilateral renal tissue was fixed with 10%
phosphate-buffered saline (PBS)-buffered formalin and embedded in
paraffin. Tissue sections with 5 µm thickness were prepared and stained
with hematoxylin-eosin (HE) stain. The changes of tissue morphology were
observed with an inverted microscope (magnification, 40x; BX51; Olympus
Corp., Tokyo, Japan) and captured by the attached camera. All HE stains
were performed using five independent histological
slides.
2.4. Western Blotting
The frozen kidney tissues of mice were lysed with RIPA buffer (Beyotime,
Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF,
Thermo Fisher Scientific, Inc.,
cat.No.36978B). Cell lysate of HK2 was treated with RIPA buffer
(Beyotime, Shanghai, China) and cocktail (Roche Diagnostics, Mannheim,
Germany). The extractions of mitochondrial fractions were obtained by
Mitochondria/Cytosol Fractionation Kit (KeyGEN BioTECH, cat.No. KGP8100)
according to the manufacturer’s protocol. The primary antibodies used in
the present study included: AMPK (1:1, 000; Proteintech, cat.No.
10929-2-AP), p-AMPK (1:1, 000; Cell Signaling
Technology, #8208),
DRP1 (Cell Signaling Technology,
#8570), DRP1S637 (Abcam, cat. No.
ab193216), DRP1S616
(Cell
Signaling Technology, #3455), MFN 2 (Cell Signaling Technology,
#9482), BAX (Proteintech, cat.No.
50599-2-Ig), BCL-2 (Proteintech, cat.No. 12789-1-APS),
Cleaved-CASPASE 3 (1:1, 000;
Abcam; #ab2302), Cytochrome C (Proteintech, cat.No.10993-1-AP), Tomm20
homolog (1:1000; Abcam, cat. No. ab56783), SP1
(Proteintech, cat.No.21962-1-AP),
PGAM5 (Abcam, cat. No. ab126534), GAPDH (Abcam, cat. No. ab181602).
All immunoblot assays were
performed using five independent samples.
2.5. Terminal transferase dUTP
nick end labelling assay
The apoptotic terminal transferase dUTP nick end labelling (TUNEL) assay
was performed to detect the DNA fragmentation in the cell nuclei which
is a marker of apoptosis. The TUNEL Apoptosis Assay Kit (Beyotime,
Shanghai, China) was applied according to the manufacturer’s protocol.
Each treatment was performed in six replicates.
2.6. JC-1 Staining
The mitochondrial potential was tested by a JC-1 probe according to the
manufacture’s protocol. HK2 Cells
were incubated with 10 mg/ml JC-1 for 10 min at 37°C in the dark and
monitored with a fluorescence microscope (magnification, 150x; BX51;
Olympus Corp., Tokyo, Japan). The Red-orange fluorescence reflects the
potential-dependent dye aggregation in the mitochondria and the green
fluorescence represents the monomeric form of JC-1, which suggests the
depolarization of the mitochondrial membrane (Reers, Smiley,
Mottola-Hartshorn, Chen, Lin & Chen, 1995).
Each treatment was performed in
five replicates.
2.7. Mitotracker Experiments
The Mitotracker assay (Cell Signaling Technology, #9082P) was used to
label the mitochondria in HK2 cells. HK2 Cells were treated respectively
and incubated with Mitotracker (200 nM) for 45 minutes at 37 ℃.
Fluorescence signals were detected by confocal fluorescence microscopy
(Leica Microsystems, Germany). The data from five independent
experiments are presented.
2.8. RNA Interference
Gene silencing was performed by
infecting HK2 cells with siRNA oligonucleotides (GenePharma, Shanghai,
China). The cells were seeded (5 × 104 cells per well)
in six-well plates and incubated for 24h. The sequences of PGAM5 siRNA
(sense 5’-CUG UGC AGU AUU ACG AAG ATT-3’, antisense 5’-UCU UCG UAA UAC
UGC ACA GTT-3’), SP1 siRNA (sense
5’-CAG CUU GGU AUC AUC ACA ATT-3’, antisense 5’-UUG UGA UGA UAC CAA GCU
GTT-3’) and negative control (sense 5’-UUC UCC GAA CGU GUC ACG UTT-3’,
antisense 5’-ACG UGA CAC GUU CGG AGA ATT-3’) were transfected with
lipo2000 Transfection Agent (Invitrogen, USA) according to the
manufacturer’s protocol. After 48h of the transfection, cells were
harvested for next experiments. Each treatment was performed in six
replicates.
2.9. Luciferase Reporter Assay
A wild-type mouse PGAM5 promoter fragment was amplified by PCR using the
primers listed in Figure 5B and cloned into the promoterless pEZX-PG02
(FulenGen Co., Ltd, Guangzhou,China). The online program, JASPAR was
used to predict the putative binding sequences of sp1 within the
promoter. Deletion mutations in the SP1 binding sequence at -713, -539
and -320 nt were made using the Quikchange II XL kit (Stratagene, La
Jolla, CA, USA). To study the involvement of SP1 in the regulation, the
wild-type or mutant PGAM5 promoter constructs were cotransfected with
the SP1 expression plasmid, or the empty vector into HK2 cells for 36 h
by using X-tremeGENE transfection reagent. The Renilla luciferase
reporter plasmid pRL-SV40 (10 ng) was included in the cotransfection
mixture. The luciferase assay was performed with a dual-luciferase
reporter assay kit (Promega). The result from five independent
experiments are presented.
2.10. Chromatin immunoprecipitation assay
We conducted the ChIP experiment using ChIP assay Kit (22188S, Cell
Signalling Technology, America) according to the manufacturer’s
protocol. Cell lysates containing soluble chromatin were incubated
overnight with anti-SP1 antibody (Proteintech, cat.No.21962-1-AP) ,
Histone H3 (4620, Cell Signaling
Technology, America, provided in the ChIP Assay Kit) and normal mouse
IgG (2729, Cell Signaling Technology, America, provided in the ChIP
Assay Kit) respectively. Then the
immunoprecipitant were purified using spin columns (Cell Signaling
Technology) and the de-cross-linked DNA was adopted as templates to PCR
amplify mouse PGAM5-specific sequences. The data from five independent
experiments are presented.
2.11. Randomization and blinding
In the in vivo experiments, the KK-Ay mice were randomly assigned to
either Empa group (n=15) or diabetic model group (n=15). All the urinal
and serum samples, the created
histological slides and tissue specimen were numbered, and the analysis
was performed under blinded conditions. In vitro experiments the cells
were harvested and numbered, tests were also conducted under blind
conditions to reduce the possible operator bias.
2.12. Statistical Analysis
The data and statistical analysis comply with the recommendations of the
British Journal of Pharmacology on experimental design and analysis in
pharmacology (Curtis et al., 2018). Statistical analysis was performed
using GraphPad prism7.00 software (GraphPad, RRID:SCR_000306). The
normal probability plot was used to examine data distributions. Results
are expressed as the mean ± standard deviation. Statistical comparison
of two groups was performed by Student’s t test and three or more
conditions were conducted by one-way
ANOVA followed by Tukey’s or Sidak’s multiple comparison correction,P <0.05 was
considered as a statistically significant difference.
3. Results