Materials
Streptozotocin (STZ, S0130) was obtained from Sigma Chemical company (Louis, MO, USA). GMCs (147589) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). BBR (181013) was got from BeNa Culture Collection (Beijing, China). MTT (M8180) and 0.4% Trypan blue solution (ST798) were obtained from Solarbio (Beijing, China). Membrane and Cytosol Protein Extraction Kit (P0033), EdU (5-ethynyl-2-deoxyuridine) (C0071S) and Cell cycle detection kit (BB-4104) were acquired from Beyotime Biotechnology (Shanghai, China). 2-NBDG (N13195) was obtained from Thermo Fisher Scientific (Carlsbad, USA). Rabbit anti-PI3K-p85 (60225-1-lg) was got from Proteintech (60225-1-lg, Wuhan, China), while rabbit anti-AKT (4691S), anti-phospho-AKT (ser473), anti-GAPDH (5174), anti-AS160 (2670), anti-phospho-AS160 (8619) antibodies were obtained from Cell Signaling Biotechnology (Boston, MA, USA). Rabbit anti-GLUT4 antibody (48547) was bought from Abcam Biotechnology (Cambridge, Cambridge shire, UK). All other chemicals applied in these experiments were belong to analytical grade and were acquired from commercial sources.
Animals
C57BL/6 male mice were selected as subjects. Mice (n=50) were fed with a standard diet and water ad libitum and adapted to the experimental conditions (20 ± 2°C, humidity of 60 ± 5%) for 7 days. Then, they were randomly divided into five groups: normal group (n=10, NC), DN model group (n=10, DN), DN mice treated with BBR (n=10, 90 mg/kg) group, BBR (n=10, 180 mg/kg) group, and Metformin (n=10, 200 mg/kg) group. Before the start of the experiment, 10 mice were assigned to the NC group with only normal diet. Other mice were fed with high glucose/ lipid diet for 6 months and received intraperitoneal injections of streptozotocin (50 mg/kg/day) for 5 consecutive days to build type II diabetic model. The development of hyperglycemia (type II diabetes) in mice was confirmed by estimating the fasting blood glucose (FBG) level (≥16.7 mmol/L) after 72 h. Control mice given vehicle alone showed a normal range of 5-6 mmol/L. Mice in the NC and DN groups were given an equal volume of vehicle (CMC-Na), while the BBR treatment groups were received BBR dissolved in 0.5% carboxymethyl cellulose at doses of 90 mg/kg, 180 mg/kg per day for 10 weeks intragastrically. After 10 weeks, kidney samples were rapidly and frozen in liquid nitrogen or fixed in 10% formaldehyde buffer. The animal experiment has been ethically acceptable, and where relevant conforms to national guidelines for animal usage in research.
Histopathology and immunohistochemistry
After dissection of the animals, one entire kidney was placed in 10% neutral formalin solution. Sections (3-5 μm) were taken, processed and stained with HE staining to detect the structure of kidney and periodic acid-Schiff (PAS) reagent to detect glycogen. For immunohistochemistry, renal sections were dewaxed in xylene and dehydrated with an ethanol gradient. After antigen retrieval by microwaving in o.1 M citric saline at 95℃ for 3 min, sections were incubated with peroxide blocking reagent for 10 min, rinsed with phosphate buffer and again incubated with power block solution for 10 min. Non-specific binding was minimized by leaving the sections in 3% BSA in phosphate buffered saline for 30 min. Sections were incubated overnight with a 1:200 dilution of GLUT4 antibody. Finally, they were rinsed well with phosphate buffer and incubated in a supersensitive polymer-horseradish peroxidase immunohistochemistry detection system. Then, the sections were washed with buffer and incubated with diaminobenzidine substrate solution for 5 min, and subsequently were counterstained with hematoxylin and detected using Leica Biosystems.
Transmission electron microscopy (TEM)
Kidney tissues were fixed in 2.5% glutaraldehyde and then osmic acid for 12 h. The sections were dehydrated in alcohol (from 50% to 90%) and araldite sequentially. Finally, the tissues sections were embedded in pure Araldite. Ultrathin sections (70 nm) were cut with ultra-microtome (UC-7, Leica) and mounted on a copper grid (200 mesh). The sections were stained with uranyl acetate and lead citrate. The transmission electron microscope (JEM1400, Japan) was used for viewing and morada G3 was used for photographing.
Cell culture
GMCs were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin at 37℃ in a humidified atmosphere containing 5% CO2. Serial passaging was performed when the cells reached 80% confluence.
MTT assay and cell survival
The viability of GMCs was measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Briefly, cells (1.0×104 cells per well) were seeded into 96-well plates and incubated at 37°C overnight. After that, cells were pre-treated with serum-free medium for 12 h and subsequently cultured with additional D-glucose (30 mmol/L) or serum-free normal glucose medium for different hours (12, 24, 36, 48 h). The absorbance was measured by a microplate reader (SpectraMax i3x, Molecular Devices, Shanghai, China) at 490 nm according to the operations manual. Cell viability was expressed as the percentage of viable cells relative to that of the non-treated control cells. All results were repeated for three times.
Trypan blue
GMCs were cultured under different concertations of high glucose condition and treated with BBR at different times. After treatment, the cell suspension mixed with 0.4% trypan blue solution at volume ratio 9:1. Then, countess® cell counting chamber slides were inserted into the instrument. The dead cells were stained blue, and the live cells were colorless and transparent.
Flow cytometry analysis the cell cycle
GMCs were washed twice with cold PBS and fixed in cold 70% ethanol overnight at 4°C. Fixed cells were washed with cool PBS and stained using the cell cycle assay kit. The experiment was repeated three times. Finally, the samples were analyzed on a Beckman flow cytometer, and data were collected for 10,000 single cell events. The percentage of cells in the G1, S and G2 phases of the cell cycle was determined by CytExpert software. Finally, data are presented as histograms.
Fluorescent EdU analysis
GMCs with a density of 1×104 cells/well were cultured in six-well plate for 24 h, then EDU assay was performed using fluorescence microscopy (DM2000, Leica, Germany) after different treatment in different groups. Briefly, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, and then permeabilized by 0.3% Triton-X100 for 15 min, following the click reaction solution was added and incubated at room temperature for 30 min in the dark. Following this, samples were washed with PBS. Then, cells were stained with Hoechst33342 for 10 min at room temperature. Finally, the images were obtained with a microscope and were analyzed with Image-Pro plus. The EDU incorporation rate was calculated as the ratio of EDU positive cells (green cells) to total Hoechst positive cells (blue cells).
Western blotting analysis
After treated with indicated drugs, the cells were washed with 1ml ice-cold PBS. To examine the translocation of GLUT4, we extracted and isolated the cytoplasmic and membrane protein were performed according to the Membrane and Cytosol Protein Extraction Kit(Beyotime, Jiangsu, China).Cell and tissue total proteins were extracted or isolated according to the manufacturer protocols. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore Corporation, Bedford, MA). The PVDF membranes were blocked with 5% nonfat milk powder for 2 h at room temperature and then incubated with the appropriate primary and secondary antibodies. Anti-PI3K-p85 antibody (1: 1000), anti-AKT antibody (1: 1000), anti-p-AKT antibody (1: 1000),anti-AS160 antibody (1: 1000), anti-p-AS160 antibody (1: 1000) and anti-GLUT4 antibody (1: 1000) were used as primary antibodies. All of the immunoblots were detected with HRP western blotting detection reagents (Millipore Corporation. Equal loading was confirmed using an anti-GADPH antibody (1: 1000).
RT-qPCR analysis
Total RNA was extracted from GMCs with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc). RNA concentrations were measured using analytikjena (Germany, Jena). RNA samples with an A260/A280 ratio of 1.8-2.0 were used for next steps. To detect the relative mRNA level, a Transcription First Strand cDNA Synthesis Kit (Vazyme Biotech, China) was used to perform the reverse transcription. Reaction conditions were as follows: 50˚C for 15 min, 85˚C for 5 seconds. A PCR reaction system was prepared using SYBR®-Green Real‑Time PCR Master mix (Thermo Fisher Science ABI7500). PCR reaction conditions were as follows: 95˚C for 10 sec, followed by 40 cycles of 10 sec at 95˚C and 30 sec at 60˚C. The GAPDH was served as an internal control to normalize the relative expression of PI3K, AKT, GLUT4 and AS160. All data were quantified using the 2‑∆∆Ct method and run in triplicate for each sample. Primer sequences used in RT-qPCR are listed in Table 2.
2-NBDG assay for glucose uptake
2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose), a fluorescent glucose analog that has been used to monitor glucose uptake in live cells, as an indicator of cell viability. The GMCs were seeded at a concentration of 1 × 105 cells/mL/well in duplicate in a six wells plate and incubated overnight at 37℃. The cells were washed twice with cold KBR after high glucose treatment, then were cultured with 2-NBDG for 30 min at 37 °C. The last, cells were washed twice with cold KBR buffer to halt glucose uptake, and resuspend with KBR to fluorescence detection using a flow cytometer at a fluorescence excitation of 488 nm and emission of 520 nm. The experiment was performed in triplicate.
Statistical analysis
Data were assessed using SPSS 24.0 (IBM Corporation, Armonk, NY, USA). Significant differences were evaluated using one-way ANOVA using a post hoc Bonferroni correction (GraphPad Prism 5.0; GraphPad Software, La Jolla, CA, USA). A two-sided p<0.05 was considered significant. The data are presented as the mean ± SD (n ≥ 3).