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