Animal preparation

Fifty Sprague–Dawley (SD) rats (female; 160–180 g; 10 weeks old) were collected from the Charles River, Beijing, China, and reared in the animal laboratory of our hospital. The rats were placed in a 12:12 h light/dark environment and provided with sufficient water and food. The rats were divided into control (C), IMN model, metformin, rapamycin, and metformin + rapamycin groups (n = 10 rats/group). The experimental protocol was approved by the Ethics Committee of Shengjing Hospital of China Medical University (2022PS092K).

Rat model establishment and treatment

The rats in the IMN model group received cationic bovine serum albumin (C-BSA; 9058; Chondrex, Redmond, USA) injected through the tail vein (6.5 mg·kg−1) 15 times in 30 days. Rats in the Control group were injected with sterile saline solution (6.5 mg·kg−1) according to the same schedule. To ensure the success of the IMN model rats, 24-h proteinuria was examined. The successful IMN rats were randomly allocated to four groups: IMN model, metformin, rapamycin, and metformin + rapamycin. Rapamycin and metformin groups were pretreated with C-BSA. Metformin was administered daily by oral gavage (200 mg/kg·d), and rapamycin was administered via intraperitoneal injection at 1.5 mg/kg·d. The IMN model groups were offered distilled water solutions. All rats were sacrificed after the four-week treatment, and blood samples were collected. In addition, kidney samples were removed and stored (Supplementary Fig. S1).

Serum and urine collection and analysis

Metabolic cages were used to collect urine samples at 24 h. Protein concentration was quantified using the Bradford method. Blood samples were collected from the abdominal aorta and centrifuged again. Serum samples were kept at − 80 °C. Urine protein levels were measured using an automatic biochemical analyzer.

qRT-PCR

RNA extraction kit procured from Biomed, China, was used to obtain total RNA samples from renal tissue, followed by quantification using Nanovue spectrophotometer. Further, reverse transcription of RNA for complementary DNA (cDNA) in qRT-PCR (make of Applied Biosystems, USA) with GAPDH as an internal control, was performed via First-Stand cDNA Synthesis SuperMix and Trans Start Green qPCR SuperMix procured from TransGen Biotech, China. Primers used are: TGF-β (F: 5′-AGCAACAATTCCTGGCGATACCTC-3′, R: 5′-TCTTCAGCTTTCCAGCGGAC-3′), α-SMA (F: 5′-TGCTGGACTCTGGAGATGGTGTG-3′, R: 5′-CGGCAGTAGTCACGAAGGAATAGC-3′), GAPDH (F: 5′-ACGGCAAGTTCAACGGCACAG-3′, R: 5′-CGACATACTCAGCACCAGCATCAC-3′). The 2−ΔΔCt method (Livak and Schmittgen 2001) was employed for estimating the expression levels of relative mRNA in all assays, which were done in triplicate.

ELISA

Blood samples were collected from five rats and serum samples were subsequently obtained. VEGF, TNF-α, and IL-6 levels in the serum and MPC-5 cells were determined using ELISA according to the manufacturer's protocol (BD Biosciences, USA).

Histological examination

The collected kidney specimens were fixed using buffered 4% paraformaldehyde, embedded in paraffin, sectioned into slices (4-μm thick), and stained with hematoxylin and eosin (HE) following the manufacturer’s instructions.

Transmission electron microscopy (TEM)

The samples (Renal tissues and podocytes cells) were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed in 2% osmium tetroxide. Subsequently, they were embedded in epoxy resin. Sections (0.1-μm thick) were stained with lead citrate and uranyl acetate. Representative autophagic vacuoles in podocytes observed using TEM. Transmission electron micrographs were obtained at 60 kV using a Zeiss EM-10 electron microscope (Zeiss, Gottingen, Germany).

Immunohistochemical assay

After deparaffinization and rehydration, the renal tissue slices were treated with H2O2 (3%) in methanol for 30 min and blocked with 5% fat-free milk for 1 h. The slices were then incubated with the primary antibody (anti-CD68, ab283654, 1:100, Abcam, UK) at 4 °C for 24 h. Bound antibodies were identified with a biotin-labeled secondary antibody using the ABC kit, visualized with diaminobenzidine, and analyzed using light microscopy after washing (Thermo Fisher Scientific, Waltham, USA). The immunoassayed slides were scored thrice and the mean values were calculated.

Western blot

Tissues were collected and the total protein concentration was examined using Coomassie brilliant blue. Total protein was separated using 10% SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane. PVDF membranes were blocked in skimmed milk (5%) in PBS at 25 °C for 2 h before incubation with primary antibodies (AMPK, mTOR, p-AMPK, LC3, p-mTOR, Beclin 1, ATG5, ATG7, ATG12, and β-actin) at 4 °C for 24 h. The PVDF membranes were incubated with anti-rabbit IgG (1:2000, A6154MSDS, Sigma-Aldrich, St. Louis, USA) for 2 h at 2 °C. Protein bands were visualized and analyzed using an ECL system (Thermo, Waltham, USA) and the ImageJ software, respectively.

Podocyte culture and treatment

The MPC-5 cell line (conditionally immortalized) was purchased from ATCC (Manassas, VA, USA). MPC-5 cells were kept in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Waltham, USA), 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin and grown at 33 °C for proliferation.

To establish the model of C5b-9 sub lysis of MPC-5 podocytes, 0.15 mol/L sodium chloride with 1% yeast polysaccharide was prepared, boiled in double-distilled water for 1 h, cooled to 25 °C, and centrifuged at 4000 rpm for 30 min. The supernatant was discarded and the precipitate was used as standby. As previously described (Ishikawa et al. 1993; Liu et al. 2017), cells were cultivated in medium containing 10% Yeast polysaccharide-activated serum (zymosan-activated serum; ZAS) at 37 °C for 60 min. The lactate dehydrogenase release rate of the culture medium supernatant was determined based on the manufacturer’s protocol to determine the optimal level of ZAS for the establishment of the podocyte C5b-9 sublysis model. MPC-5 cells were cultivated in 5% CO2 at 37 °C for 14 days in DMEM and then exposed to metformin (10 μmol/L) or rapamycin at a dose of 20 nmol/L for 24 h. The levels of TNF-a (A), IL-6 (B), and VEGF (C) in the MPC-5 cell supernatant measured using ELISA.

F-actin staining

Podocytes were fixed using 4% paraformaldehyde, permeabilized for 10 min on ice using TritonX-100 (0.1%) (T8787, Sigma, USA), and incubated with rhodamine-phalloidin (ab235138, Abcam, Cambridge, UK) (1:100) for 1 h at 25 °C. A confocal microscope (20 × , LSM 780, Zeiss) was used to examine the cells.

Statistical evaluation

Data were examined using SPSS 22.0 and GraphPadPrism7 and displayed as means ± SD (standard deviation). The experiment was repeated three times. Repeated-measures ANOVA (one-way analysis of variance) and Tukey’s test were used to identify significant differences. Differences were considered statistically significant at P < 0.05.