Recombinant Human Erythropoietin for Injection (rhEPO, S19991025, CHO Cell) was purchased from Shanghai Chemo Wanbang Biophama Co., Ltd. Recombinant Human Transforming Growth Factor β1 (rhTGF-β1, HY-P7118, CHO Cell) was purchased from MedChemExpress. Primary antibodies against α-SMA, collagen I, myeloperoxidase (MPO) and β-actin were purchased from Abcam (Cambridge, United States). Primary antibodies against SPRY1, ERK1/2, p-ERK1/2, NF-κB and p-NF-κB were purchased from Cell Signaling Technology (Massachusetts, United States). Primary antibodies against IL-6 and TNF-α were purchased from Proteintech (Wuhan, China). Horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody was purchased from Sangon Biotech (Shanghai, China). Other reagents are described below.

We purchased 24 8-week-old male C57BL/6 mice from the experimental animal center of the Medical College of Zhengzhou University, and they were housed at a 22 °C constant room temperature and 47% humidity with a 12-h light-dark cycle and free access to standard laboratory chow and tap water. All experimental procedures on mice were performed in accordance with the National Institutes of Health guidelines and were approved by the Ethical Committee, Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University (2020-KY-273).

We used a mouse UUO model with 7-day ureteral obstruction. All mice used in the present study were randomly divided into four groups: the sham group (n = 6), the UUO group (n = 6), the UUO + EPO group (n = 6) and the UUO + saline group (n = 6). We established UUO model according to the accepted procedures depicted in previous studies. Briefly, the mouse was anesthetized with inhaled isoflurane, and the left proximal ureter was exposed. Then ureter was ligated with 6-0 silk thread and severed. Mice in the sham group received the same surgery as mice in the UUO group, except that the ureters were not ligated and severed. For the mice in the UUO + EPO group, 100 U/kg body wt rhEPO was administered intraperitoneally every day after operation, which lasted 7 days. For the mice in the UUO + saline group, saline was administered intraperitoneally in a manner identical to the rhEPO treatment. All mice in the four groups were sacrificed at the 7th day after operation. The left kidney specimens were collected, and inferior vena cava blood samples were collected for renal function evaluation including blood urea nitrogen (BUN) and serum creatinine (SCr). Part of the kidney tissue was frozen in liquid nitrogen for total RNA and protein extraction, and the remainder was fixed with 4% paraformaldehyde.

The kidney specimens were fixed with 4% paraformaldehyde and embedded in paraffin. The rotary microtome (Leica, Heidelberg, Germany) was used to cut the specimens into 4-μm sections and subjected to hematoxylin and eosin (HE) staining and Masson’s trichrome. Then the sections were examined and taken pictures by Leica DM4B microscope equipped with Leica X software. At high magnification (×200), we selected six non-overlapping regions of the renal cortex in each section for scoring or image analysis. According to the scoring criteria reported in previous study (Chen and Li, 2018), the tubulointerstitial impairment was assessed, which included tubular atrophy, tubular necrosis, lymphocyte infiltration, and interstitial fibrosis. The scores for each criterion were as follows: 0 = none; 1 = mild or <25%, 2 = moderate or 25%–50%, and 3 = severe or >50%. The blue stained area stained by Masson was considered to be collagen deposition and was analyzed using Image-Pro plus 6.0 software and the results were expressed as the means ± SD.

Using a rotary microtome, the paraffin-embedded kidney specimens were cut into 4-μm sections, then deparaffinized with xylene, and rehydrated with graded ethanol (100%, 95%, 85%, and 75%) and distilled water. The sections were incubated with 3% hydrogen peroxide for 10 min to block the activity of endogenous peroxidase, and then microwaved in 0.01 mol/L citrate buffer (pH 6.0) for 25 min for antigen retrieval. We washed Sections 5 min using phosphate buffered saline (PBS), 3 times in total, and then incubated the sections with the primary antibodies, including anti-α-SMA (1:500), anti-collagen I (1:500), anti-TNF-α (1:300), anti-IL-6 (1:200), and anti-MPO (1:1000), overnight at 4 °C. Next day, the sections were washed with PBS, and then incubated with HRP-labeled goat anti-rabbit secondary antibody at room temperature for 1 h. Finally, dehydration, clearing, 3,3′-diaminobenzidine (DAB) staining and neutral resin sealing were performed in sequence. Then the sections were examined and taken pictures by Leica DM4B microscope equipped with Leica X software. At high magnification (×200), we selected six non-overlapping regions of the renal cortex in each section for image analysis. Image-Pro plus 6.0 software was used to evaluate the integral optical density (IOD) of the positive area, and the results were expressed as the means ± SD.

We purchased the human renal proximal tubular epithelial cell line HK-2 from Beina Chuanglian Biotechnology Institute (Beijing, China), and cultured the cells in a humidified atmosphere of 5% CO2 at 37 °C using Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12), which was supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin, and 10 mg/mL streptomycin. Cells were seeded in 6-well plate at a density of 1 × 10⁵ cells per well and cultured in 10% FBS complete medium for 24 h. The cells were then cultured in serum-free medium for 24 h, and then treated with rhTGF-β1 at 10 ng/mL in 1% FBS medium for 24 h to induce fibrosis. The concentration and time of rhEPO treatment for HK-2 cells were determined through establishing gradient, the fibrotic HK-2 cells induced by rhTGF-β1 were treated with rhEPO at concentration of 10 U/mL for 0 h, 12 h, 24 h, 48 h respectively, or treated with rhEPO for 24 h at concentration of 0 U/mL, 5 U/mL, 10 U/mL, 20 U/mL respectively.

Trizol (Sangon Biotech, B511311, Shanghai, China) was used to extract total RNA, which was stored at −80 °C and checked the concentration and quality via NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, United States). Then reverse transcription of total RNA was conducted using M-MuLV First-Strand cDNA Synthesis kit (Sangon Biotech, B532435, Shanghai, China). The 2×SG Fast qPCR Master Mix (Low Rox) kit (Sangon Biotech, B639272, Shanghai, China) was used to determine the levels of mRNA through the Applied Biosystems 7,500 Sequence Detection System. Each sample was analyzed in triplicate. The β-actin gene served as a control, and the data were analyzed through the 2^−(ΔΔCt) method. To detect the expression level of miR-21-5p, the microRNA First-Strand cDNA Synthesis kit (Sangon Biotech, B532453, Shanghai, China) was used to synthesize cDNA following the manufacturer’s protocol. A MicroRNAs Quantitation PCR Kit (Sangon Biotech, B532461, Shanghai, China) was used to conduct qRT-PCR, and an Applied Biosystems 7,500 Sequence Detection System was applied to detect the levels of miR-21-5p. Each sample was analyzed in triplicate. U6 small nuclear RNA served as a control, and the data were analyzed through the 2^−(ΔΔCt) method. The sequences of all primers are listed in Table 1.

According to the manufacturer’s instructions, the total protein was isolated from kidney tissues and HK-2 cells using efficient radioimmunoprecipitation assay (RIPA) tissue/cell lysis buffer (Solarbio, R0010, Beijing, China). Enhanced BCA Protein Assay Kit (Beyotime, P0009, Shanghai, China) was used to detect the concentration of total protein. The protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, loading amount was 30 µg per well. In transfer solution, the protein was transferred from the resolving gel to the PVDF membrane by electrophoresis. The membrane was blocked with 5% nonfat milk on a shaker for 2 h and then incubated with primary antibodies overnight at 4 °C. Primary antibodies included anti-collagen I (1:1000), anti-α-SMA (1:1000), anti-Spry1 (1:500), anti-ERK1/2 (1:1000), anti-p-ERK1/2 (1:1000), anti-NF-κB (1:500), anti-p-NF-κB (1:500), anti-IL-6 (1:500), anti-TNF-α (1:500), and anti-β-actin (1:2000). TBST solution was used to wash the membrane three times in 30 min. Then, at room temperature, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody on a shaker for 1 h. After washing the membrane three times again in TBST solution, the Omni-ECL™ Femto Light Chemiluminescence kit (EpiZyme, SQ201, Shanghai, China) was used to visualize the bands in the membrane through the Bio-Rad ChemiDoc™ MP Imaging System. ImageJ software was used to quantify the densities of the bands. The levels of β-actin served as controls, and the results were expressed as the means ± SD.

The pGV514-miR-21-5p-mimics plasmid for overexpressing miR-21-5p and the corresponding control plasmid were constructed and synthesized by GeneChem (Shanghai, China). The transfection was conducted using lipofectamine 3,000 reagent (Invitrogen) according to the manufacturer’s instructions. The cells were seeded in 6-well plate at a density of 1 × 10⁵ cells per well and transfected at 60%-70% confluence, and then harvested after 48 h of transfection for subsequent experiments.

Glass coverslips were placed in wells of 6-well plate in advance and HK-2 cells were seeded at 1 × 10⁵ per well. Then the cells adhered to the coverslips, which were taken out and washed with PBS. 4% formaldehyde solution was used to fix the cells for 30 min, and then the cells were permeabilized with 0.2% Triton X-100/PBS for 15 min. Thereafter, we blocked the cells with 2% bovine serum albumin for 30 min and then incubated the cells in primary antibodies overnight at 4 °C. Next day, the cells were washed with PBS, followed by incubation with FITC-/TRITC-conjugated secondary antibodies for 1 h at room temperature, and then DAPI staining was performed after washing with PBS. At last, the coverslips were checked and taken pictures under fluorescence microscope.

GraphPad Prism seven was used to determine statistically significant differences. All data are presented as the means ± SD and were analyzed using one-way analysis of variance with Tukey’s test. P < 0.05 was considered statistically significant.

HE staining showed that 7 days after the left ureters of mice were ligated, the proximal renal tubules in the cortex of UUO group were significantly dilated and the tubulointerstitial impairment score was increased obviously according to the score criterions (Figures 1A,B). Due to compensation by the right kidneys, the BUN and SCr levels of the UUO group and the UUO + saline group were roughly within the normal range (Table 2), but compared with Sham group, the upward trend within the normal range was still obvious (Figures 1D,E). The blue-stained area of Masson’s trichrome staining expanded significantly in UUO group (Figure 1C). However, comparing UUO + EPO group with UUO group or UUO + saline group showed that the tubulointerstitial impairment score, the renal function and the blue-stained area of Masson’s trichrome staining were decreased significantly.

The results of qRT-PCR showed that the expression levels of α-SMA and Collagen Ι were increased significantly after 7-day obstruction in kidneys of UUO group, but the elevations were inhibited by low dose rhEPO treatment in UUO + EPO group (Figures 2A,B). The similar results were also revealed by Western blotting (Figures 2C,D) and immunohistochemical staining (Figures 2E–G).

The levels of miR-21-5p were elevated obviously in kidneys of UUO group according to the results of qRT-PCR (Figure 3A). While the protein expression levels of SPRY1 decreased, the protein expression levels of p-ERK1/2, p-NF-κB, IL-6 and TNF-α increased, as shown by Western blotting in UUO group (Figures 3B–E). Impressively, the 7-day low dose rhEPO treatment in UUO + EPO group could reverse the results listed above according to the results of qRT-PCR and Western blotting.

The results of immunohistochemical staining showed that MPO-positive inflammatory cells were increased and the expression of IL-6 and TNF-α was enhanced significantly in the renal cortex after 7-day obstruction in kidneys of UUO group. In the UUO + EPO group, the intervention with 7-day low dose rhEPO treatment in mice could decrease the MPO-positive inflammatory cells and inhibit the expression of IL-6 and TNF-α (Figure 4).

The induction of HK-2 cells by rhTGF-β1 at 10 ng/mL for 24 h promoted the expression of α-SMA and Collagen Ι, and resulted in conversion of cell morphology to be spindle-shaped (Figures 5F,H). Results of the time gradient with 10 U/mL rhEPO in treatment of HK-2 cells revealed that the expression of α-SMA in HK-2 cells induced by rhTGF-β1 was decreased significantly after intervention for 24 h and 48 h (Figures 5A,C). And results of the concentration gradient with rhEPO intervention for 24 h showed that the rhEPO concentration at 10 U/mL and 20 U/mL inhibited the expression of α-SMA significantly (Figures 5B,D). So the concentration and time of rhEPO treatment for HK-2 cells were set to 10 U/mL for 24 h in the subsequent experiments. According to the results of Western blotting, the treatment with rhEPO could reverse the upregulation of α-SMA and Collagen Ι causing by rhTGF-β1 induction or transfection with miR-21-5p mimics in HK-2 cells (Figures 5F,G), and immunofluorescence staining showed the consistent results with Western blotting (Figures 5H, 6C). The rhTGF-β1 induction promoted the expression of miR-21-5p, but the upregulation was inhibit after treatment with rhEPO (Figure 5E).

The transfection with miR-21-5p mimics in HK-2 cells could increase the expression of α-SMA and Collagen Ι showing by the results of Western blotting and immunofluorescence staining, but the effect could be counteracted by rhEPO (Figures 5F,G, 6C). Moreover, the transfection with miR-21-5p mimics in HK-2 cells increased p-ERK1/2 and p-NF-κB protein levels, but decreased SPRY1 protein levels. However, the treatment with rhEPO reversed the SPRY1/ERK/NF-κB pathway activation, in which the protein level of SPRY1 was increased, but p-ERK1/2 and p-NF-κB were decreased (Figures 6A,B).

Taken together, our results indicate that the low-dose rhEPO treatment has obvious anti-inflammatory and anti-fibrotic effects on obstructive kidneys in mice, which may be achieved by inhibiting miR-21-5p/SPRY1/ERK/NF-κB signaling pathway (Figure 6D).