Male Sprague-Dawley rats (weight, 180–200 g; age, 6–8 weeks; n=32) were purchased from the Experimental Animal Center of Wenzhou Medical University (Wenzhou, China). Rats were housed under a controlled temperature (22–25°C), humidity (40–60%) and light environment (12-h dark/light), and fed with standard rat chow (10–15 g twice a day) and water (20–45 ml a day), and this access was controlled, except for one day of fasting prior to the operation. The weight-matched rats were randomly assigned to one of four groups: Sham-operated, treated with vehicle (saline, n=8) or SSBE (100 mg/kg/day, n=8), and unilateral ureteral obstruction (UUO) treated with vehicle (n=8) or SSBE (100 mg/kg/day, n=8). UUO surgery was performed as previously described (11). All rats were sacrificed by cervical dislocation and were anesthetized by 0.2% pentobarbital natrium (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Kidneys were excised at day 8 for rats in the UUO SSBE and vehicle control groups as previously described (11). SSBE (cat. no. 20101017; Xuancheng Baicao Plant Industry and Trade Co., Ltd., Anhui, China) was extracted according to the standard protocol (10). The animal study protocols were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.

The paraffin-embedded kidney sections were stained using standard histology procedures as previously described (11), including hematoxylin and eosin (H&E) and Masson's trichrome staining (both from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China). Immunohistochemical analysis was performed on 4-µm-thick kidney sections using an automatic slicing machine (YD-335; Wuxiang Instrument, Shanghai, China) that had been dewaxed with xylene and rehydrated using sequential ethanol (100, 95, 85 and 75%) and distilled water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed by heating the sections in 0.1% sodium citrate buffer (pH 6.0). Immunohistochemical analysis was performed using anti-TGF-β1 (dilution 1:800, cat. no. bs0103R; BIOSS, Beijing, China), anti-type III collagen (Col3α1; dilution 1:800, cat. no. bs-0549R; BIOSS) and anti-proliferating cell nuclear antigen (PCNA; dilution 1:1,000, cat. no. sc-9857; Santa Cruz Biotechnology, Dallas, TX, USA) primary antibodies at 4°C overnight and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (dilution 1:10,000, cat. no. P0211; Beyotime Institute of Biotechnology) at 37°C for 30 min. The integrated optical density was measured using Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). All samples were semi-quantitatively or quantitatively assessed by two blind independent investigators.

The NRK-52E renal epithelial cell line was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and was maintained in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen). NRK-52E cells were seeded into 6-well culture plates at a density of 3×10⁵ cell/well to confluence in complete medium containing 5% FBS for 24 h, and then changed to serum-free medium for 24 h before treatment with 5 ng/ml TGF-β1 (cat. no. 0312209-1; PeproTech, Inc., Rocky Hill, NJ, USA), 10 µg/ml AA (cat. no. A5512; Sigma-Aldrich) or 10-1,000 µg/ml SSBE.

NRK-52E cells were cultured with TGF-β1, AA, and/or SSBE in 6-well plates at a seeding density of 3×10⁵ cells/well containing glass slides. Cells were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (Sigma-Aldrich) at 4°C for 30 min. Following permeabilization with 0.1% Triton X-100 for 10 min, specimens were washed with PBS, and the substrate was blocked with 10% FBS to eliminate nonspecific fluorescence. Immunofluorescence staining was performed using anti-Col3α1 (dilution 1:200), anti-E-cadherin (cat. no. ab53033, dilution 1:400; Abcam, Cambridge, MA, USA), and anti-α-smooth muscle actin (α-SMA; dilution 1:400; cat. no. sc-32251), anti-protein patched homolog 1 (Ptch1; dilution 1:400; cat. no. sc-9016), anti-smoothened (Smo; dilution 1:400; cat. no. sc-13943) and anti-Gli family zinc finger 1 (Gli1; dilution 1:100; sc-6153), purchased from Santa Cruz Biotechnology, primary antibodies at 4°C overnight. Following washing with PBS three times, the cell preparations were incubated with fluorescein isothiocyanate (green)/tetramethylrhodamine-(red) labeled secondary antibodies (dilution 1:2,000; Sigma-Aldrich) for 1 h at room temperature. Following washing with PBS, cell preparations were placed in acacia and covered with a slide. Immunofluorescence studies were semi-quantitatively or quantitatively assessed by two blind independent investigators.

Total RNA was extracted from NRK-52E cells or kidney tissues using TRIzol^® reagent (Invitrogen). Reverse transcription into cDNA templates were performed using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). qPCR was performed using a SYBR-Green Real-Time PCR Master Mix Plus (Toyobo). Quality was analyzed on agarose gels, and quantities were measured using Varioskan Flash (Thermo Fisher). Sequence-specific primers of α-SMA, tight junction protein 1 (ZO-1), type I collagen (Col1α1), Col3α1, sonic hedgehog (Shh), Ptch1, Smo, Gli1, TGF-β1 and TGF-β1 receptor (TGFβ1R), all listed in Table I, were synthesized by Invitrogen; Thermo Fisher Scientific, Inc., and β-actin served as an endogenous reference gene. Samples were analyzed in triplicate. The melting curve was examined to verify that a single product was amplified. For quantitative analysis, all samples were analyzed using the 2^−ΔΔCq value method (14). For semi-quantitative analysis, all samples were analyzed using gel electrophoresis.

Whole proteins from NRK-52E cells were collected using RIPA lysis buffer (Beyotime Institute of Biotechnology) by centrifugation at 12,900 × g for 10 min, and protein concentrations were determined using a Bicinchoninic Acid protein assay kit (Beyotime). Whole proteins (30 µg) from each sample were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Beijing Solarbio Science & Technology, Beijing, China). Following blocking with 5% skimmed milk at 37°C for 1.5 h, membranes were incubated with anti-Col3α1 (dilution 1:1,000), anti-α-SMA (dilution 1:1,000) and anti-Smo (dilution 1:1,000) primary antibodies at 4°C overnight, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (dilution 1:5,000; Beyotime Institute of Biotechnology) at 37°C for 2 h. Bound antibodies were visualized using chemiluminescence detection (ECL, cat. no. 32109; Thermo Fisher Scientific, Inc.) on autoradiographic film. Quantification was performed by measuring the intensity of signals using Image-Pro Plus version 6.0 software (Media Cybernetics), and normalized to that for the anti-GAPDH antibody (dilution 1:2,000; cat. no. AP0063; Bioworld Technology).

All results are presented as mean ± standard error. Statistical analyses were performed using a Statistical Package for Social Sciences version 16.0 software (SPSS, Inc., Chicago, IL, USA). Student's t-test was used to analyze differences between the two groups, and one-way analysis of variance followed by least significant difference post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Evidence from H&E staining revealed marked tubular dilation and atrophy associated with interstitial fibrosis in the obstructed kidney tissues (Fig. 1A). The total collagen deposition determined by Masson trichrome staining was more severe as obstructive time progressed (Fig. 1A). SSBE administration significantly alleviated renal tubular injury and reduced total collagen deposition (Fig. 1A). These findings suggested that SSBE alleviated UUO-induced interstitial fibrosis in rats.

Compared with those in the sham-operated group, the mRNA expression levels of Col3α1 (Fig. 1B), and the protein expression levels of Col1α1 and Col3α1 (Fig. 1C) in UUO kidneys were significantly increased. These results supported that UUO induced excessive ECM deposition and interstitial fibrosis in kidney tissues. The fibrotic alterations in UUO kidneys were associated with enhanced gene (Fig. 1B) and protein (Fig. 1C) expression of the profibrotic factor TGF-β1, and mRNA expression of its receptor TGF-β1R (Fig. 1B). However, the upregulated expression of TGF-β1, TGF-β1R and ECM components were inhibited by the treatment of SSBE. Furthermore, SSBE suppressed cellular proliferation in the tubules and interstitium by reducing the numbers of PCNA-positive cells (Fig. 1D). Therefore, the inhibitory effect of SSBE on cellular proliferation indirectly regulates the tubular epithelial cell phenotype and myofibroblast accumulation, resulting in the reduction of interstitial fibrogenesis.

UUO has been demonstrated to induce cell proliferation in kidney tissues, which may occur via a feedback model, and is accompanied with activation of proliferation-associated signaling, including the hedgehog signaling pathway (15). Therefore, the present study examined the gene and protein expression levels of key molecules involved in the hedgehog signaling pathway, in the obstructed kidney. As presented in Fig. 1E, UUO induced the synthesis and secretion of Smo, and inhibited the expression of Ptch1, a hedgehog inhibitor by targeting Smo. In addition, upregulated mRNA expression levels of Gli1 and downregulated expression levels of Ptch1 were observed in UUO kidneys (Fig. 1F). These findings suggested that UUO induced the activation of hedgehog signaling. Previous studies have demonstrated that in UUO rats, hedgehog signaling is activated by a paracrine signaling loop and mediates epithelial-mesenchymal communication and promotes renal fibrosis (3,4). Blockade of hedgehog signaling may alleviate the extent of fibrosis (3,16). In the present study, the activity of hedgehog signaling in UUO rats was decreased following SSBE treatment. Thus, it was hypothesized that SSBE exerts renal anti-fibrotic effects via suppressing the hedgehog signaling pathway.

In UUO kidneys, upregulated expression levels of molecules involved in the activation of hedgehog signaling are primarily located around the tubules, which are rich in tubular epithelial cells (17). However, whether activation of hedgehog signaling is responsible for the proliferation of epithelial cells remains unknown. The present study investigated the effects of SSBE on EMT induction and ECM deposition in RTECs (NRK-52E cells) treated with TGF-β1, an important inducer triggering EMT and ECM deposition. As expected, in TGF-β1-treated NRK-52E cells, upregulated expression of Col3α1 and the myofibroblast marker α-SMA, and downregulated expression of the epithelial marker E-cadherin, were observed (Fig. 2A). In addition, TGF-β1 decreased the mRNA expression levels of ZO-1, and increased the expression of α-SMA, Col1α1 and Col3α1, compared with the control group (Fig. 2B). These TGF-β1-mediated fibrotic alterations, including EMT induction and ECM accumulation, were inhibited by SSBE treatment in a dose-dependent manner; SSBE at the higher concentration (100 µg/ml) had a stronger anti-fibrotic activity. However, SSBE at too high concentrations (>1,000 µg/ml) inhibited cellular proliferation and induced apoptosis (data not shown), suggesting that an overdose of SSBE may have a cytotoxic effect.

In addition to the induction of EMT and deposition of ECM, TGF-β1 enhanced the activity of hedgehog signaling in NRK-52E cells. As presented in Fig. 3, upregulated expression of PCNA in association with activated hedgehog signaling was observed in TGF-β1-treated NRK-52E cells. However, SSBE treatment effectively inhibited PCNA expression. Furthermore, SSBE downregulated the mRNA expression levels of Shh and Gli1 in NRK-52E cells after TGF-β1 treatment (Fig. 3B and C), although the changes of Smo expression levels were not significant. Thus, this in vitro experiment reconfirmed that inhibiting hedgehog activity may be an important molecular mechanism for the anti-fibrotic effect of SSBE on renal tissues in vivo.

AA is regarded as a potent mutagen that induces significant cytotoxic effects on RTECs. In vivo, AA may cause a devastating renal disease called AA nephropathy, and the histopathology features interstitial matrix deposition and fibrosis (18). Therefore, the present study evaluated the effect of SSBE on RTECs following AA injury. The results demonstrated that SSBE treatment inhibited AA-induced overexpression of α-SMA and Col3α1 protein (Fig. 4A), but also decreased mRNA expression levels of TGF-β1, α-SMA and Col1α1 (Fig. 4B). Furthermore, SSBE reduced the overactivation of hedgehog signaling in RTECs following AA injury by upregulating the mRNA expression of Ptch1 and downregulating mRNA and protein expression of Smo (Fig. 4A and C). Therefore, in an injured micro-environment, SSBE exhibits inhibitory activities on hedgehog signaling, resulting in the reduction of ECM deposition and a reduction in fibrosis.