All procedures involving animals were approved by the Ethics Committee for Animal Research of Wuhan University (Wuhan, China). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and the National Research Council (15). A total of 24 male C57BL6 mice (age, 8 weeks; weight, 20–25 g) were purchased from the Animal Center of Wuhan University; these mice were randomly divided into 3 groups (n=8): Sham operation, UUO + vehicle (0.5% carboxymethylcellulose, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and UUO + 60 mg/kg/day HES (Sigma-Aldrich; Merck KGaA). The mice were maintained at 25°C under a 12-h light/dark cycle and provided ad libitum access to food and water. UUO was performed in accordance with an established procedure (16). The mice in the UUO + HES group were orally treated with 60 mg/kg/day HES from 7 days prior to surgery to 7 days post-surgery. The mice in the UUO + vehicle group were treated with the same volume of vehicle solution (0.5% carboxymethylcellulose). The mice were sacrificed on day 7 post-surgery for renal tissue and blood sampling. Blood samples were harvested from the left renal vein when the mice were sacrificed, and immediately centrifuged at 13,000 × g for 5 min at 4°C. Sera were then collected and stored at −20°C. Blood urea nitrogen (BUN) and serum creatinine levels were determined using commercially available kits (BUN, 48T/96T, Biofine, Blaine, WA, USA; creatinine, 48T/96T, Shanghai Yanjin Bioscience Company, Shanghai, China) using an AU5800 automatic biochemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA).

NRK-52E cells were purchased from Wuhan Boster Bioengineering Co., Ltd. (Wuhan, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences; Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO₂. NRK-52E cells were seeded onto a 6-well dish (3×10⁶ cells/well) and starved in serum-free medium for 24 h at 37°C, and subsequently treated with 5 ng/ml recombinant human transforming growth factor (TGF)-β1 and 50 or 100 µM HES for 24 h at 37°C. The cells were subsequently assigned into 4 treatment groups as follows: Control, 5 ng/ml transforming growth factor (TGF)-β1 (positive control; R&D Systems, Inc., Minneapolis, MN, USA); 5 ng/ml TGF-β1 + 50 µM HES; and 5 ng/ml TGF-β1 + 100 µM HES.

Renal tissues extracted from C57BL6 mice in each group were placed in 10% buffered formalin for 24 h at room temperature, embedded in paraffin, and sliced into 4-µm thick sections. The sections were stained with H&E at room temperature for 5 min, and then visualized by light microscopy to assess the extent of renal injury. Interstitial damage was graded as previously described (17).

Immunohistochemical analysis was performed in accordance with an established procedure (18). Kidney tissues were fixed in 4% paraformaldehyde at room temperature overnight, embedded in paraffin and sliced into 4-µm thick sections. Sections were dewaxed and hydrated in a decreasing series of ethanol, and antigens were retrieved in 0.01% sodium citrate buffer at 100°C for 1 h. Endogenous peroxidase activity was reduced using 3% H₂O₂ at room temperature for 10 min and 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) was used to block nonspecific binding at room temperature for up to 1 h. The sections were incubated with the following primary antibodies at 37°C for 1 h: Anti-α-smooth muscle actin (SMA, 1:100; BM0002, Wuhan Boster Bioengineering Co., Ltd.), anti-E-cadherin (1:200; 610181; BD Biosciences, Franklin Lakes, NJ, USA), anti-collagen I (1:100; PB0981; Wuhan Boster Bioengineering Co., Ltd) and anti-fibronectin (FN, 1:200; Ab2413; Abcam, Cambridge, UK). Goat anti-rabbit secondary antibodies labeled with horseradish peroxidase (1:200; BA1080; Wuhan Boster Bioengineering Co., Ltd.) were then added to the tissues and incubated at room temperature for 1 h. Diaminobenzidine (Sigma-Aldrich; Merck KGaA) was added to detect positive signals at room temperature for 10 sec. A total of 10 random fields were selected to capture images using an inverted light microscope (Olympus Corporation, Tokyo, Japan). Images were subsequently quantified with Image Pro-Plus 6.0 software (Rockville, MD, USA).

Total RNA was extracted from renal tissues and NRK-52E cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse-transcribed into complementary DNA from 2 ug total RNA using oligo (dT) primers with a Prime Script™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. PCR amplifications were conducted with SYBR Premix Ex Taq II (Takara Bio, Inc.) according to the manufacturer's instructions in an AB7500 real-time PCR system (Thermo Fisher Scientific, Inc.) and normalized to GAPDH. The PCR primers used are presented in Table I. The qPCR thermocycling conditions were as follows: 95°C for 10 min, followed by by 40 cycles of 5 sec at 95°C, 30 sec at 60°C and 1 min at 72°C. The Cq values of each samples were calculated using the 2^−∆∆Cq method (19).

Renal tissues and NRK-52E cells were lysed with radioimmunoprecipitation assay lysis buffer (Biyuntian, Haimen, China) on ice and the lysates were centrifuged for 15 min (12,000 × g, 4°C). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Sigma-Aldrich; Merck KGaA). Proteins (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore; Billerica, MA, USA). Following blocking with 5% non-fat milk at room temperature for 2 h, the membranes were probed with the primary antibodies at 4°C overnight. The antibodies used were as follows: Anti-β-actin (1:400; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), anti-α-SMA (1:1,000; BM0002; Wuhan Boster Bioengineering Co., Ltd.), anti-E-cadherin (1:1,000; 610181; BD Biosciences). Membranes were subsequently incubated with a secondary IRDye^® 800CW-conjugated antibody (1:1,000; 925-32211; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The signals were visualized with an Odyssey Imaging System (Odyssey; LI-COR Biosciences, USA) and the target protein expression levels were normalized to those of β-actin. Protein levels were assessed using ImageJ 1.46 software (National Institutes of Health, Bethesda, MD, USA).

All experiments were repeated at least three times independently Statistical analyses were performed with SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Inter-group comparisons were analyzed by one-way analysis of variance followed by Student-Newman-Keuls' method. Data were expressed as the mean + standard deviation. All P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.

Renal histology and BUN and creatinine levels in the blood were assessed to determine the effect of HES on renal function. H&E staining identified epithelial cell necrosis, inflammatory cell infiltration and tubular dilation in the renal interstitium of the UUO kidneys (Fig. 1A). Furthermore, results suggested that the levels of BUN and creatinine in the UUO + vehicle group were significantly elevated when compared with those in the sham group (P<0.05; Fig. 1B and C). Following treatment with HES (60 mg/kg/day), the BUN and creatinine levels significantly declined in UUO mice (P<0.05; Fig. 1B and C, respectively). Additionally, the significant damage in the renal interstitium induced by UUO (P<0.05) was significantly alleviated by treatment with HES (P<0.05; Fig. 1A and D). Therefore, HES may prevent renal dysfunction induced by ureteral obstruction.

UUO mice are commonly used animal models of renal interstitial fibrosis (20). The primary feature of renal interstitial fibrosis is the accumulation of ECM components, including FN and collagen (21). As depicted in Fig. 2A, immunohistochemistry staining indicated that HES treatment significantly reversed the accumulation of FN and collagen I protein in UUO mice (P<0.05; Fig. 2B and C). Similarly, RT-qPCR analysis demonstrated that the elevated mRNA expression levels of FN and collagen I in UUO mice (P<0.05) were significantly reduced by HES treatment (P<0.05; Fig. 2D and E). Myofibroblasts are considered to be a primary renal interstitial cell responsible for the production of ECM during fibrosis (22). Furthermore, EMT activation has been associated with myofibroblast production (23). In the present study, the protein expression levels of α-SMA were upregulated in UUO mice, while those of E-cadherin were downregulated when compared with the sham group; however, these changes were significantly reversed following treatment with HES (Fig. 3A-C). Similarly, the mRNA expression levels of α-SMA were significantly upregulated and those of E-cadherin were significantly downregulated in UUO mice (P<0.05), and these alterations were significantly reversed by HES treatment (P<0.05; Fig. 3D and E).

Although the in vivo studies indicated that HES may inhibit EMT, in vitro experiments were performed to further investigate the effects of HES. Western blot analysis demonstrated that 24-h treatment with TGF-β1 altered the protein expression levels of α-SMA and E-cadherin in NRK-52E cells (Fig. 4A). Quantification of these results (Fig. 4B and C) indicated that TGF-β1 significantly increased the protein expression levels of α-SMA and significantly decreased those of E-cadherin in NRK-52E cells when compared with the control group (P<0.05). These changes were significantly reversed by HES in a dose-dependent manner (P<0.05; Fig. 4B and C). Thus, a greater dose of HES (100 µM) elicited a stronger inhibitory effect on EMT. These results were consistent with the RT-qPCR data (Fig. 4D and E); however, the mRNA expression levels of α-SMA between the 5 ng/ml TGF-β1 + 50 µM HES and 5 ng/ml TGF-β1 + 100 µM HES groups did not differ significantly. Though notably, TGF-β1 significantly increased the mRNA expression levels of α-SMA and significantly decreased those of E-cadherin in NRK-52E cells (P<0.05), and these changes were significantly reversed following HES intervention (P<0.05; Fig. 4D and E).

In vivo experimental results indicated that the mRNA expression levels of Shh and Gli1 were significantly increased in the UUO group when compared with the sham group (P<0.05). These increased levels were significantly reduced by HES treatment (P<0.05; Fig. 5A and B, respectively), thus suggesting that HES inhibited hedgehog signaling in UUO mice.

Consistent with these in vivo data, the results of in vitro experiments indicated that the mRNA expression levels of Shh and Gli1 were upregulated by 24-h treatment with TGF-β1 in NRK-52E cells, and this effect was significantly reversed by HES treatment (P<0.05; Fig. 5C and D). Additionally, the higher dose of HES (100 µM) inhibited hedgehog signaling to a greater extent.