Male Wistar rats (age, 8 weeks old; weight, 200-250 g) were purchased from the animal care facility at the Second Affiliated Hospital of Harbin Medical University (Harbin, China). All animal experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (13). The present study was approved by the Institutional Animal Research Committee of Harbin Medical University. All animals were housed at the animal care facility of Harbin Medical University at 25°C with a 12/12 h light/dark cycle in a vivarium with humidified airflow, and were allowed free access to normal rat chow and water throughout the study period.

Diabetes can be induced by an intraperitoneal of streptozotocin (STZ). A total of 45 rats were randomly divided into three groups: i) Control (n=15), 0.1 M sterile citrate buffer (pH 4.5) was injected intraperitoneally as a control at the same volume as that in the model group; ii) T1D (n=15), a single intraperitoneal injection of STZ (60 mg/kg, dissolved in 0.1 M, pH 4.5 citric acid-citrate sodium buffer) was used to establish the T1D model. When the concentration of serum glucose was >16.7 mmol/l, the T1D model was considered to be established successfully (14); and iii) T1D + Sp (n=15), spermine solution (2.5 mg/kg/day, dissolved in normal saline) was injected intraperitoneally every day for 2 weeks before STZ injection, followed by an injection of spermine (2.5 mg/kg/day) every other day for 12 weeks. Rats in each group were euthanized at week 12 of modeling. Under anesthesia with intraperitoneal 2% pentobarbital (50 mg/kg, dissolved in normal sodium), the kidneys were removed by opening the abdomen and 0.1% lidocaine (2 mg/kg, dissolved in normal saline) was used to infiltrate the incision wound for analgesia. The rats were sacrificed after surgery with an overdose of 2% pentobarbital (100 mg/kg injected intravenously) (15).

Blood glucose and body weight were measured at weeks 0, 4, 8 and 12 in each group. Urine samples were collected for 24 h on week 12, while blood was collected from the retro-orbital vein by removing the eyeballs. Subsequently, the rats were euthanized, and the kidneys were then removed and weighed. The levels of urinary albumin excretion (UAE; cat. no. ZK-H1678), serum creatine (Scr; cat. no. GMS70021.7) and urea (cat. no. GMS70022.1) were measured using appropriate ELISA kits (Shenzhen Ziker Biological Technology Co., Ltd.).

Sections of kidney tissues were fixed with 4% paraformaldehyde at room temperature for 48 h, embedded in paraffin and sectioned into 4-µm sections. Renal sections were stained at room temperature with hematoxylin and eosin (H&E) for 3 min, and with periodic acid-Schiff staining (PAS) for 30 sec for 5 min, and then sections were assessed by light microscopy. Images of 20 glomeruli per rat were obtained, and the PAS-positive (purple) area per glomeruli was quantified using ImageJ v1.8.0 software (National Institutes of Health).

For immunohistochemistry, paraffin-embedded sections were blocked with 5% BSA (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature and stained with an antibody against podocin (1:50; cat. no. ab50339; Abcam) at 4°C overnight, and then washed with PBS. Next, the mixture was incubated with the secondary antibody (goat anti-rabbit IgG; 1:500; cat. no. ab6721; Abcam) for 1 h at room temperature and washed with PBS. Finally, the sections were stained with a solution of diaminobenzidine for 2 min at room temperature, washed with PBS, dehydrated and permeabilized with xylene. Stained images (10 glomeruli per kidney) were visualized by light microscopy and all the images were analyzed with ImageJ software.

Rat podocytes (cat. no. BNCC338697; BeiNa Culture Collection; Beijing Beina Chunglian Biotechnology Research Institute) were cultured at 37°C in a 5% CO₂ humidified incubator in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin. Podocytes were cultured for 6-12 h in DMEM containing 5.5 mM D-glucose (Sigma-Aldrich; Merck KGaA) without FBS before exposure to various experimental conditions. The cells were randomly divided into six groups: i) Normal-glucose group (NG), where the podocytes were incubated in DMEM containing 5.5 mM glucose for 48 h; ii) mannitol group (M), where the podocytes were incubated in DMEM containing 40 mM mannitol and 5.5 mM glucose for 48 h; iii) high-glucose group (HG), where the podocytes were incubated in DMEM containing 40 mM glucose for 48 h; iv) spermine-treated group (HG + Sp), where the podocytes were pretreated with 1, 5, 10, 20 and 50 µM spermine (Sigma-Aldrich; Merck KGaA) for 2 h before being subjected to HG conditions; v) rapamycin-treated group (HG + Rap), where the podocytes were pretreated with 10 mM rapamycin (MCE) for 2 h before being subjected to HG conditions; and vi) Compound C treatment group (HG + Sp + Compound C), where 10 mM Compound C (Selleck Chemicals), an AMPK inhibitor, was added to the medium 1 h before HG and spermine addition.

Quantitative evaluation of cell viability was performed using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay according to the manufacturer's instructions. Cells were seeded in 96-well plates at a concentration of 1×10³ cells/well. After 24 h of treatment in each group of cells, 10 µl CCK-8 was added to each well and incubated for 2 h at 37°C. Next, the absorbance at 570 nm was determined using a microplate spectrophotometer reader.

Hoechst 33342 is a blue, fluorescent dye that can penetrate the cell membrane and is used for apoptosis detection. Podocytes were plated into 24-well dishes (2×10⁵ cells/well) and were cultured under the aforementioned conditions. Then, cells were rinsed with PBS and incubated for 10 min at room temperature using 5 mg/ml Hoechst 33342. Staining was observed by fluorescence microscopy (Nikon Corporation) at ×400 magnification. The excitation wavelength was maintained at 380 nm. When apoptosis occurred, the nuclei appeared as dense or fragmented.

The ultrastructure of the kidney was detected by electron microscopy. Samples of renal sections were cut into pieces (<1 mm) and fixed in 2.5% glutaraldehyde for 4 h at room temperature. Tissues were post-fixed in 1% osmium tetroxide with 0.1 M sodium cacodylate, dehydrated through graded concentrations of ethanol and propylene oxide, and subsequently embedded in Epon 812 for 24 h at 60°C. Ultrathin sections were cut from blocks and mounted on copper grids for counterstaining with lead citrate and uranium acetate for 7 min each at 75°C. The sections were observed under a Philips CM 120 electron microscope (Philips Medical Systems, Inc.).

Rat renal tissues and rat podocytes were homogenized in 0.5 ml RIPA buffer prior to being transferred to small tubes and rotated 20 sec per 5 min six times. Solubilized proteins were collected after centrifugation at 10,000 × g at 4°C for 30 min. The supernatant was collected and stored at −80°C. The protein concentration of each sample was quantified using an enhanced BCA protein assay kit. For detection of protein levels, protein lysates (40 µg protein loaded per lane) from each group of cells and tissues were separated via SDS-PAGE on a 10% gel, and subsequently separated proteins were electrotransferred onto a PVDF membrane (EMD Millipore). After blocking with TBS with 0.1% Tween-20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C, the membrane was incubated overnight at 4°C with antibodies against ODC (1:1,000; cat. no. ab97395; Abcam), SSAT (1:1,000; cat. no. ab105220; Abcam), nephrin (1:400; cat. no. BA1669; Boster Biological Technology), podocin (1:400; cat. no. BA3416; Boster Biological Technology), CD2-associated protein (CD-2AP; 1:1,000; cat. no. bs-0512R; BIOSS), microtube-associated proteins 1A/1B light chain 3 (LC3; 1:1,000; cat. no. 3868T; Cell Signaling Technology, Inc.), Beclin 1 (1:1,000; cat. no. 3495T; Cell Signaling Technology, Inc.), P62 (1:1,000; cat. no. 8025T; Cell Signaling Technology, Inc.), autophagy protein 5 (Atg5; 1:1,000; cat. no. 12994T; Cell Signaling Technology, Inc.), cleaved caspase-3 (1:1,000; cat. no. 9661T; Cell Signaling Technology, Inc.), total (t)-mTOR (1:1,000; cat. no. A00003-2; Boster Biological Technology), phosphorylated (p)-mTOR (1:600; cat. no. BM4840; Boster Biological Technology), t-AMPK (1:1,000; cat. no. A00994-6; Boster Biological Technology), and p-AMPK (1:1,000; cat. no. P00994; Boster Biological Technology). The membrane blots were washed and incubated at 4°C with anti-rabbit (cat. no. ab6721) or anti-mouse (cat. no. ab6728) IgG antibodies (1:10,000; Abcam) for 1.5 h. The signals were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and the Multiplex Fluorescence Imaging System (ProteinSimple). The intensities of the protein bands were semi-quantified by AlphaView SA 3.0 software (ProteinSimple) and normalized to β-actin.

Podocytes were plated into 24-well dishes (2×10⁵ cells/well) and cultured under the indicated conditions. Cells were fixed with 4% paraformaldehyde at room temperature for 24 h, permeabilized in 0.5% Triton X-100 for 30 min and blocked with 1% BSA for 1 h at 4°C. Next, cells were washed and incubated with anti-nephrin antibodies (1:1,000; cat. no. ab216341; Abcam) overnight at 4°C. Finally, the cells were incubated with a secondary anti-body (goat anti-rabbit IgG; 1:1,000; cat. no. ab6721; Abcam) for 2 h at room temperature, and observed with a fluorescence microscope at ×400 magnification (Nikon Corporation).

Data are expressed as the mean ± standard error of the mean. Each measurement was obtained by performing ≥3 independent experiments. Statistical comparisons were conducted using paired or unpaired t-tests or one-way ANOVA followed by Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study established a rat model of intraperitoneally injected STZ-induced diabetes. The results showed that, compared with those of the control group, the blood glucose and the kidney weight/body weight ratio of the T1D rats were increased (Fig. 1A and C), while their body weight was mildly decreased (Fig. 1B). The animals also developed common signs of T1D, including polydipsia, polyuria, and noticeable hypoactivity and weakness (data not shown). Renal function parameters were evaluated, and it was found that Scr, serum urea and UAE in the T1D group were significantly increased (Fig. 1D-F).

Next, the expression of ODC and SSAT was detected by immunoblotting. The results showed that the expression of ODC was decreased, while that of SSAT was increased, in the T1D group (Fig. 1G), indicating that the metabolism of polyamines was altered and the content of endogenous spermine was decreased. Therefore, exogenous spermine pretreatment was added in subsequent experiments. The results revealed that exogenous spermine could improve the general condition and renal function-related indicators in T1D rats, which suggested that spermine may exert a protective effect on diabetic renal function.

PAS and H&E staining were used to observe the morphological changes of the kidney. The results revealed moderate mesangial proliferation, matrix accumulation and parietal endothelial cell proliferation in the T1D group, which further verified renal injury. These pathological changes were attenuated in the presence of spermine (Fig. 2A, B and D). Electron microscopic examination revealed that podocytes were disorganized with obvious loss and effacement of foot processes (FPs) and thickening of the glomerular basement membrane (GBM) in the T1D group, but these abnormalities were alleviated in the T1D + Sp group (Fig. 2C and E). Taken together, these results suggested that spermine may protect against diabetic kidney damage.

In vivo, the expression levels of podocyte-specific proteins, including nephrin, CD-2AP and podocin, were detected by immunoblotting and immunohistochemistry. The expression levels of podocyte-related proteins were significantly reduced in the T1D group (Fig. 3A). Immunohistochemical staining of podocin further supported the aforementioned results (Fig. 3B).

Next, the expression of autophagy-related proteins were examined. In comparison with the control group, the expression levels of Atg5, Beclin 1 and LC3II/LC3I were decreased and P62 was increased in the T1D group (Fig. 3C). Immunohistochemical staining revealed a mildly decreased expression of LC3 localized to glomeruli in T1D rats (Fig. 3D), which confirmed autophagy insufficiency in DN rats. These changes were obviously improved by exogenous spermine pretreatment. Collectively, these results indicated that the effect of spermine in DN was associated with activation of autophagy.

A CCK-8 assay was used in vitro to detect the activity of podocytes subjected to different glucose concentrations. Previous studies reported that podocytes exhibited lower activity under HG stimulation for 48 h (16). The present study found that, when podocytes were treated with glucose at a concentration of >40 mM for 48 h, cell viability was decreased significantly (Fig. 4A). LC3 is a soluble protein, which can be hydrolyzed to LC3I to form LC3II, thus reflecting the change in autophagy flow (17). Immunoblotting was used to observe the protein expression of podocin and LC3II/LC3I at different time points in order to determine the association between podocyte injury and autophagy. As shown in Fig. 4B, under HG conditions, podocin expression was decreased in a time-dependent manner, while LC3II/LC3I expression was decreased gradually after 12 h, which indicated that podocyte injury may be associated with a reduction of autophagy.

To observe whether podocyte injury was associated with polyamine metabolism, the present study detected changes in polyamine metabolizing enzymes. As shown in Fig. 4C, immunoblotting revealed decreased ODC expression and increased SSAT expression in HG-induced podocytes. Podocytes were treated with different concentrations of spermine (1, 5, 10, 20 and 50 µM) in HG medium. The results indicated that 5 and 10 µM spermine treatment of HG-induced podocytes increased cell viability. However, when the concentration of spermine was >10 µM, cell activity was decreased (Fig. 4D). The aforementioned results validated the conditions used in subsequent experiments.

To determine the role of autophagy during spermine treatment of podocytes, the effect of rapamycin, which is known to induce autophagy, on podocyte injury was examined. The podocytes were pretreated with rapamycin for 2 h before HG treatment. In accordance with the results of spermine pretreatment, rapamycin also partially reversed the effects of HG on cell viability (Fig. 5A), indicating that spermine can also act as an autophagic agonist.

Cell apoptosis was quantified by Hoechst 33342 staining and immunoblotting. It was observed that HG-induced podocytes showed markedly increased nuclear aggregation, DNA fragmentation and caspase-3 cleavage activity (Fig. 5B and E), indicating that HG treatment induced podocytes apoptosis. Next, podocyte injury was evaluated by immunoblotting and immunofluorescence with podocyte-specific markers. There was a significant reduction in nephrin, podocin and CD-2AP expression in the HG group (Fig. 5C). In agreement with the results of immunoblotting, the data were confirmed by measuring the nephrin-positive area by immunofluorescence staining (Fig. 5D). Finally, to further observe the autophagy of podocytes in vitro, autophagy-related proteins were detected. Immunoblotting showed that Beclin 1 and LC3II/LC3I were decreased, while P62 was increased (Fig. 5E), which was consistent with the in vivo results. Taken together, the aforementioned findings indicated that DN changes were improved by spermine and rapamycin treatment, which suggested that the protective effect of spermine on podocyte injury may be due to a reduction in autophagy.

mTOR kinase is a key molecule in autophagy induction that can be inhibited by activated AMPK. In the present study, p-AMPK and p-mTOR expression were determined by immunoblotting. The results revealed that p-AMPK was decreased and p-mTOR was increased in the HG group. However, spermine and rapamycin reversed the inhibitory effect of HG on AMPK and mTOR activity. The addition of Compound C, an AMPK inhibitor, further confirmed the autophagy-promoting effect of spermine by increasing AMPK phosphorylation and decreasing mTOR phosphorylation (Fig. 6).