db/db mice and healthy control db/m mice were housed and maintained under a 12-h light/12-h dark cycle, with ad libitum access to water and standard mouse chow at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China). Animals were maintained under specific pathogen-free conditions and received humane care according to the criteria outlined in the National Institutes of Health (NIH) Guide 21. The animal study was approved by the Institutional Animal Care and Use Committee of Zhengzhou University (2020-KY-273).
According to the manufacturer's instructions (Genechem Co., LTD.,Shanghai,China ), an adeno-associated virus (AAV) vector that interferes with Risa. Risa was amplified by PCR using specific primers (forward: 5′-TCTGGAGAGCCCAACCT-3′, reverse: 5′-TCCTTCAA ACGCGAGAGAG-3′). qRT-PCR was used to detect the virus titer. Mice were divided into four groups: db/m, db/db, db/db+Risa-vehicle, and db/db+Risa-AAV. 10-weeks-old db/db mice were injected with Risa-AAV and the empty virus control group by tail vein injection under the red light vein imaging instrument. Blood, urine, kidney tissue, and clinical data (blood glucose, body weight, serum creatinine, and urinary albumin- creatinine ratio) were collected at the 6th, 10th, 14th, 18th and 22th weeks of age, while quantitative real-time polymerase chain reaction (qRT-PCR), Western blot analysis, transmission electron microscope (TEM), PAS and confocal immunofluorescence staining and analysis were used to observe the relevant indicators.
Cell Culture and Transfection
The immortalized mouse podocyte cell (MPC) was donated by academician Liu Zhihong of Nanjing University School of Medicine (Nanjing, China). MPCs were immediately placed into a water bath at 37 °C with gentle shaking after the liquid nitrogen had evaporated. When completely melted, the cryopreservation-containing cells were transferred to a sterilized 5 mL centrifuge tube and centrifuged for 10 min at 1000 × g. After the supernatant was removed, the cells were resuspended in complete culture solution and inoculated in a culture dish with complete culture medium containing 45 mL of Royal Park Memorial Institute (RPMI)-1640 medium, 5 mL of fetal bovine serum (FBS), 50 μL of a mixture of penicillin/streptomycin at a ratio of 1:1000 and 1000 U γ-interferon (γ-IFN). The cells were evenly distributed through gentle shaking and then placed in the culture incubator. The culture conditions were set at 33 °C with 5% CO2 under saturated humidity. The medium was changed the next day, and cell growths were observed. When cell confluence reached 70–80%, the cells were subcultured at a ratio of 1:3. The cells that were cultured at 33 °C were inoculated into a fresh, precoated culture bottle with γ-IFN-free 1640 medium added. Then, the culture bottle was placed into an incubator at 37 °C with 5% CO2. Microscopically, the cells proliferated slowly, the cytoplasm extended to the periphery, and a large number of secondary protrusions emerged from the cell body. With 10–14 days of differentiation and maturation, the cells could be used in related experiments. Grouping was conducted as follows: i) NG (5.6 mM glucose); ii) HM (5.6 mM glucose + 44.4 mM mannitol); iii) HG (30 mM glucose); iv) HG + Lithium chloride (Licl) (30 mM); v) HG+Risa-LV; vi) HG+Risa-vehicle; vii) HG+Risa-LV+ EX-527 (Sirt1 inhibitor). After 0-96 h, the podocytes were collected to be used for various assays. Licl was purchased from Beijing DingGuo Changsheng Biotechnology (Beijing, China) and EX-527 was purchased from Beijing Bioss Biotechnology (Beijing, China).
Stable transfection of MPC cells with LncRNA AK044604 Lentivirus (Risa-LV) (Genechem, Shanghai, China) and empty vector (Risa-vehicle) were conducted referring to the instructions of the Reliable partner in gene function & drug target validation (Genechem, Shanghai, China).
RNA Extraction and qRT-PCR
Tissue and cell total RNA were extracted using TRIZOL reagent (Thermo Fisher Scientific, Shanghai, China). RNA was used to perform reverse transcription using First Strand cDNA Synthesis Kit and polymerase chain reaction using SYBR Green qPCR kit (Thermo Fisher Scientific). Relative gene expression data were calculated using the comparative threshold cycle method with GAPDH or β-actin (Servicebio Co., LTD.,Wuhan,China) as housekeeping genes. All assays were run in triplicate. The primers for each gene are as follows: Beclin-1, 5′-GAGTGGAATGA AATCAATGCTGC-3′ and 5′-TTTCCACCTCTTC TTTGAACTGC-3′; LC3B, 5'-CCGTCCGAGAAGACCTTCAA-3' and 5'-TCTTGCGGCAG GAGAACCTA-3'; GAPDH, 5'-CCTCGTCCCGTAGACAAAATG-3' and 5'-TGAGGT CAA TGAAGGGGTC GT-3'.
Western Blot Assay
Total protein lysates were extracted from mouse renal cortex tissue or cells using RIPA lysis buffer (Solarbio Co., LTD.,Beijing,China). Protein samples were separated by 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride. After being blocked with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1 h, the membranes were incubated with a primary antibody at 4°C overnight and then with a secondary antibody at room temperature for 1 h. High sig ECL Western blotting substrate development kit was used to develop the imprint. The relative gray intensity of each target protein expression band was quantitatively analyzed by Image J, and GAPDH was set as an internal reference. The ratio of each target protein to GAPDH was the relative expression level of the target protein. All measurements were repeated at least three times. Primary antibodies against SYSTM1/P62, Beclin-1, GSK3β, LC3B, Desmin, and WT-1 were purchased from Servicebio Biotechnology (Wuhan,China), GSK3β (phospho Ser9) was gained from Cell Signaling Technology (Shanghai,China), Nephrin and NPHS2 were purchased from Abcam (Shanghai, China), Sirt1 and phospho Sirt1 (Ser 27) were obtained from Bioss Biotechnology (Beijing, China) , and GAPDH was acquired from Good Here (Hangzhou, China).
Transmission electron microscopy
The kidney specimens were fixed in glutaraldeyde immediately after biopsy or nephrectomy. Ultrathin sections were cut with an ultramicrotome, stained with 2% uranyl acetate and lead citrate, and examined with a JEOL JEM-1400 Plus transmission electron microscope. Autophagosomes in podocytes were identified [7,8] under 5000× and 25,000× high-power field. Each tissue sample was examined; ten podocytes were randomly selected to count the number of autophagosomes and autophagolysosomes in podocytes. After counting, statistical analysis were performed by an experienced pathologist and nephrologist.
Formalin-fixed and paraffin-embedded kidney tissues were prepared as 4μm sections and deparaffinized and hydrated. Sections were processed for periodic acid-Schiff staining. Slides were rinsed in distilled water after oxidizing in 0.5% periodic acid for 5 min, incubated in Schiff reagent for 15 min, washed in lukewarm tap water for 5 min, and counterstained in Mayer’s hematoxylin.
Confocal immunofluorescence staining and analysis
Kidney tissue sections or cells were fixed using cold methanol for 40 min and incubated overnight at 4 °C with primary antibodies. Secondary antibodies conjugated with Alexa Fluor® 488 or 594 were incubated at 25 °C for 1 h. Finally, sections were counterstained with Propidium Iodide and visualized by the Zeiss LSM 880 confocal microscope. Primary antibodies against SQSTM1/P62 and Nephrin were purchased from Servicebio Biotechnology (Wuhan,China), LC3B was obtained from Sigma-Aldrich (Shanghai, China), GSK3β (Phospho Ser9) was gained from Cell Signaling Technology (Shanghai, China), WT-1 was acquired from Santa Cruz Biotechnology (Shanghai, China), and phospho Sirt1 (Ser 27) was gained from Bioss Biotechnology (Beijing, China).
Statistical analyses were performed using SPSS or Prism software. Data were expressed as means ± standard deviation (Unless otherwise stated). Continuous variables were compared using ANOVA and independent t-test. P-values < 0.05 in two-tailed tests were considered statistically significant in all analyses.