Male ATF4+/− heterozygous and ATF4+/+ wild-type (WT) mice in the C57BL/6J background were obtained from GemPharmatech Co., Ltd. All mice were raised in the environment of 21 ± 3 ° C and light / dark cycle for 12 hours. Before the experiment, the mice need to adapt to the environment for at least one week, In the meantime, they are free to get water and food. All experiments were approved by the Ethics Committee on Animal Experiments of Jinan University (Approval No.2019022501). Fourteen WT and fourteen ATF4+/− mice were randomly divided into control group (n = 7) and streptozotocin (STZ) group (n = 7) respectively. 150 mg/kg STZ (Sigma-Aldrich, St. Louis, USA) dissolved in 0.1 mM citrate buffer at pH 4.5 was administered to mice via an intraperitoneal injection to induce DN. Control group were injected with an equivalent volume of citrate buffer. When the blood glucose level is higher than 16.5 mmol/l, it is considered as a successful DN model. Ten weeks later, pentobarbital sodium (60 mg/kg iP) was used to anesthetize mice, and blood was collected from eyeballs to measure biochemical parameters. Renal tissues were collected for further study.
Cell Culture And Transfections
NRK-52E cells were purchased from The Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM containing 5% fetal bovine serum with 5% CO2. The temperature of the incubator is 37℃. To mimic normal and diabetic pathological conditions, cells were cultured in DMEM containing 5.5 mmol/L glucose (normal glucose, NG) or 30 mmol/L glucose (high glucose HG). pcDNA3.1A-ATF4 plasmid and vector were obtained from Hanbio Biotechnology Co., Ltd. (Shanghai, China). ATF4 siRNA and siRNA-NC vector were synthesized by Guangzhou Ribobio Co., Ltd. (GuangDong, China) In the experiment, Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection.
Western blot analysis
After the total protein was extracted from renal tissue by RIPA method, BCA protein detection kit was used to determine the total protein content. Aliquots of proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were blotted overnight with primary antibodies: anti-Col-IV (1:1000, Sigma), anti-LC3A/B (1:1000, CST), anti-P62 (1:2000, CST), anti-ATF4 (1:1000, CST), anti-GAPDH (1:1000, CST). After incubation with secondary antibodies (1:10000 dilution), chemiluminescence was performed with Pierce® ECL western blotting substrate. The gray value of bands was calculated by Gel-pro analyzer software.
Real-time Quantitative RT-PCR
Trizol kit (Invitrogen, Carlsbad, CA) were used to extract total RNA from collected samples. Then Invitrogen (Carlsbad, CA) kit were used to reverse transcribe RNA into cDNA for later PCR reaction. Quantitative RT-PCR was performed by bio rad 96fx circulatory (bio rad, USA) with SYBR Green Master Mix. The primer sequences of ATF4 gene and GAPDH reference gene are as follows: ATF4: forward 5’-CGACTTTTATTACACTTTCTGGGAG-3’ and reverse 5’-GGGACAGATTGGATGTTGGAG-3’; GAPDH: forward 5’-TCTCTGCTCCTCCCTGTTC-3’ and reverse 5’-ACACCGACCTTCACCATCT-3’. The results were analyzed by 2-ΔΔCq method.
Transmission electron microscopy
Firstly, the samples were fixed with 2.5% glutaraldehyde and then fixed with 1% osmium tetroxide, embedded in the Durcupan ACM after dehydrated. Then uranyl acetate and lead citrate were used to stain ultrathin sections of samples. Finally, transmission electron microscopy (JEOL-100CXII, JEOL, Japan) was used to observe autophagosomes. Ten fields were randomly extracted from each group, and the number of autophagosomes was analyzed.
Tandem mRFP-GFP-LC3 Fluorescence Microscopy
The autophagy flow was observed by tandem mRFP-GFP-LC3 fluorescence microscopy. After 24 hours transfection of NRK-52E cells with adenovirus expressing mRFP-GFP-LC3 (Hanbio Co., Ltd., Shanghai, China), images were observed and collected by confocal laser scanning microscope (LSM 510; Zeiss, Oberkochen, Germany).
Histology and immunofluorescence
Harvested kidney samples were conventionally immersed in 4% paraformaldehyde overnight under ordinary temperature. 4 µm thick paraffin embedded mouse kidney sections were prepared. Hematoxylin and eosin (HE), Masson and Periodic acid-Schiff (PAS) staining were carried out and morphologic analysis was performed by light microscopy.
The expression of renal light chain 3 (LC3) was detected by immunofluorescence staining. The tissue sections were soaked with anti LC3B (1:200, CST) and incubated at 4 ℃ for about 12 hours, and then incubated with alexa Fluor 594-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG and cy3-conjugated goat anti-rabbit IgG at 37˚C for 1 h. Finally, the nuclei were labeled with DAPI, and the images were observed and collected by laser scanning confocal fluorescence microscope (UltraVIEW VoX; PerkinElmer, Inc.).
Detection of blood glucose in mice was performed with Glucometer (Roche Diabetes Care GmbH, UK). Serum creatinine and blood urea nitrogen was analyzed by an automatic chemistry analyzer (AU480; Beckman Coulter Inc; Kraemer Boulevard Brea, CA). Mice stayed in a metabolic cage and urine was collected for 24 hours. The level of urinary albumin was determined by ELISA Kit (Bethel laboratory, Montgomery, TX).
All experimental data are shown as mean ± SE. SPSS software 22.0 is used for statistical analysis (SPSS, Inc.). When two groups are compared, Student’s t-test is selected for analysis. One-way ANOVA with Tukey post hoc test was performed to analysis when comparing three or more groups. The results were considered to be significant with values of 𝑃 < 0.05.