IS was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary antibodies against CYP1B1 (cat no. ab185954) and AhR (cat no. ab2769) were obtained from Abcam (Cambridge, MA, USA). The antibodies anti-Histone H3 (cat no. 4499) and anti-β-actin (cat no. 3700) were obtained from Cell Signaling Technology, Inc. Small interfering RNA (siRNA) of CYP1B1 and AhR were obtained from Shanghai GenePharma Co., Ltd. and the sequences of these siRNA are presented in Table SI. Transfection reagent Lipofectamine^® 2000 was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The chromatin immunoprecipitation (ChIP) kit EZ-ChIP was obtained from EMD Millipore. All primers were synthesized by Shanghai Sangong Pharmaceutical Co., Ltd.

A total of 15 patients with primary CKD at stage V (age range 55–64 years, mean age 59.4, 8 male and 7 female, Chinese and ethnically Han, and 15 sex- and age-matched controls were enrolled from the Department of Nephrology of Xinqiao Hospital (Chongqing, China) between July and October 2018. The protocol of the present study was ethically approved by the Ethics Committee of Xinqiao Hospital (approval no. 2018-006-01), and was performed in accordance with the Declaration of Helsinki. All patients and healthy controls provided written informed consent as a form provided by the Ethical Committee of Xinqiao Hospital (Third Military Medical University) prior to the start of the study to confirm the collection and use of the samples in the present study.

Male C57BL/6J mice were obtained from Beijing HFK Bioscience Co., Ltd. Mice (n=18) at 8 weeks of age were housed under controlled temperature (25°C), humidity (50–70%) and 12-h light/dark cycle with free access to food and water. Mice were divided into 3 groups. The first group (n=6) received dimethyl sulfoxide (DMSO; 50% in saline, v/v) intraperitoneally. The second group (n=6) was treated with a single daily dose of 100 mg/kg IS for 8 weeks intraperitoneally. The third group (n=6) was administered 300 µg/kg TMS (MedChemExpress LLC) in DMSO every third day plus a daily dose of 100 mg/kg IS for 8 weeks intraperitoneally. Subsequently, the mice were euthanized by cervical dislocation under isoflurane (2%)-inhaled anesthesia 24 h following the final injection. Hearts were immediately frozen in liquid nitrogen and stored at −80°C. The parameters of heart function were monitored by micro-computed tomography. All animal studies were ethically approved by the Institutional Animal Care and Use Committee of Third Military Medical University, and were performed in accordance with the animal care guidelines of Third Military Medical University.

Rat embryonic cardiomyoblast H9c2 cell line was purchased from Shanghai Cell Bank (http://www.cellbank.org.cn). Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, at 37°C in a humidified incubator containing 5% CO₂. For the IS treatment, increasing concentrations of IS (0, 0.1, 0.25 and 0.5 mM) were added to the medium. Cells were treated with IS for the indicated periods (0, 24, 48 and 72 h). For treatment with human serum, individual serum samples were diluted at room temperature with the complete culture media (1:4, v/v), and the cells were treated with diluted serum for 48 h.

Total RNA was isolated from the cell culture and heart tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was reverse transcribed into cDNA and RT-qPCR was performed as previously described (9). Briefly, a reaction volume of 10 µl containing primers (1.0 µM), cDNA (100 ng) and 1XPCR mixture with SYBR I Green (Takara Biotechnology Co., Ltd.) was amplified on an iCycler iQ (Bio-Rad Laboratories, Inc.), with the thermocycling conditions of one cycle of 95°C for 5 min, 36 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 60 sec, then one cycle of 72°C for 5 min. Expression levels of CYP1B1, AhR, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were calculated relative to GAPDH expression, using the 2^−ΔΔCq method (23). All primers are listed in Table SII.

Protein samples were prepared from the cell culture (H9c2, 1×10⁶) and heart tissues using RIPA buffer containing protease inhibitors (Beyotime Institute of Biotechnology, Haimen, China). Protein expression levels were measured by assessing band thickness in the gel-phase of the western blot analysis as previously described (9). The following antibodies were used for the western blot analysis: Anti-CYP1B1 (1:1,000), anti-AhR (1:1,000), anti-Histone H3 (1:1,000) and anti-β-Actin (1:1,000). The blots were then incubated with the corresponding secondary antibodies (1:2,000) conjugated with horseradish peroxidase (cat nos. ZDR-5306 or ZDR-5307, OriGene Technologies, Inc.). The immunoreactive proteins were detected using a ECL Detection System (Thermo Fisher Scientific, Inc.). Finally, the membranes were scanned and the densitometry analysis was performed using an image analysis system (Image Lab, v.3.0; Bio-Rad Laboratories Inc.). To validate the data, the western blot analysis was repeated at least 3 times.

The immunofluorescence assay was performed on H9c2 cells (2×10⁵) fixed in 4% paraformaldehyde for 15 min at room temperature as previously described (9). The following antibodies were used: Anti-AhR (1:100) and anti-α-actinin (1:200, ab137346; Abcam). The nucleus was labeled with 1 mg/ml 4′,6-Diamidino-2-phenylindole, dihydrochloride in phosphate buffered saline (PBS; Roche Diagnostics) for 5 min at room temperature. Images were captured using a Leica TCS-SP5 laser-scanning confocal microscope (magnification, ×400; Leica Microsystems, Inc.).

The hearts were fixed in buffered formalin for 24 h at room temperature and dehydrated with increasing concentrations of ethanol (70, 80, 95 and 100% for 2 h, respectively) and subsequently with xylene (30 min, 3 times). Finally, the hearts were embedded in paraffin. The embedded tissues were cut into 3 µm sections and were processed with xylene (10 min, twice) and rehydrated with decreasing concentrations of ethanol (100, 95, 90, 80 and 70% for 5 min, respectively). Then the sections were stained with hematoxylin-eosin (HE) with a commercial kit (C0105, Beyotime Institute of Biotechnology) in accordance with the manufacturer's protocol and were observed under a light microscope (magnification, ×200, Olympus CX22; Olympus Corporation).

The paraffin sections (3 µm) of the hearts were analyzed via immunohistochemistry using antibodies against CYP1B1 or AhR using standard methods. Following rehydration, nonenzymatic antigen retrieval was performed by heating the sections to 95–100°C in citrate buffer (10 mM, pH 6.0) for 15 min and washed three times with phosphate buffered saline (PBS) for 5 min. The sections were immersed in 3% (v/v) H₂O₂ for 5 min and washed three times with PBS for 5 min prior to using QuickBlock Blocking Buffer (P0231, Beyotime Institute of Biotechnology) for 10 min. Following incubation with primary antibodies (CYP1B1 or AhR, 1:100) at 4°C overnight, the slides were then incubated with enzyme-conjugated secondary antibodies (PV9001 or PV9002, 1:200, OriGene Technologies, Inc.) and visualized using 3,3′-diaminobenzidine. Slides were then observed using an inverted microscope (magnification, ×400, Olympus BX63; Olympus Corporation). The negative controls used PBS in place of the primary antibody. Images were randomly captured using the microscope.

ChIP was performed using a commercial EZ-ChIP kit as previously described (24). Briefly, IS-treated H9c2 cells or normal controls (1×10⁷) were harvested and washed in cold PBS. Cells were then immediately fixed in 1% formaldehyde for 10 min at room temperature and halted using 0.125 M glycine. Cell lysates were sonicated, and the sheared cross-linked chromatin was immunoprecipitated with antibodies against CYP1B1 (target), histone H3 (positive control) or normal immunoglobulin G (IgG; negative control) overnight. Chromatin was then collected, washed and decross-linked at 65°C. DNA was purified using spin columns and detected with regular and quantitative PCR. Primers are listed in Table SIII.

H9c2 cells (2×10⁴) in 6-well plate were transfected with 50 µM of siRNA against CYP1B1 or AhR or negative control siRNA using Lipofectamine^® 2,000 according to the manufacturer's protocol. After incubating with siRNA for 4 h, the media were refreshed. Following 2 days, the efficiency of the knockdown of the target was evaluated by analyzing the protein level of CYP1B1 or AhR. In order to analyze the biological functions of the knockdown, H9c2 cells were further treated with IS as aforementioned.

All data are expressed as the mean ± standard error of the mean. Unpaired, two-tailed t-tests and one-way analysis of variance followed by Tukey's multiple comparison test were used. The statistical analysis was performed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

H9c2 cells were treated with serum from patients with CKD in the present study. The effect of the serum on cardiac hypertrophy markers, including ANF, BNP and β-MHC, was detected using RT-qPCR. As presented in Fig. 1A, all these markers were significantly induced by CKD serum when compared with that of the healthy controls (P<0.01). The protein levels of CYP1B1 were then detected via western blot analysis, and the results revealed that the CYP1B1 protein was substantially upregulated by CKD serum compared with the control (Fig. 1B). In order to further determine whether the induction of CYP1B1 was at the translational or transcriptional level, the mRNA level of CYP1B1 was detected via RT-PCR. As presented in Fig. 1C, the CYP1B1 mRNA levels in H9c2 cells treated with CKD serum were significantly increased compared with the controls (P<0.01).

IS is the most abundant uremic toxin in CKD serum. The present study further investigated its effect on gene expression of CYP1B1. As the IS concentration in patients with CKD was reported to vary from 7.6–656.0 µM (25), 500 µM IS was selected as the maximal dose in the present study. Consistent with previous results, all markers of myocardial hypertrophy were significantly increased at an IS concentration of 0.25 or 0.50 mM compared with the control (P<0.05), as determined by RT-PCR (Fig. S1A-B). In order to assess whether IS may induce the gene expression of CYP1B1, H9c2 cells were also treated with an increasing dose of IS. The results from the present study revealed that the protein levels of CYP1B1 were significantly upregulated by IS in a dose- and time-dependent manner (P<0.05; Fig. 1D and E). In addition, the mRNA levels of CYP1B1 were detected using RT-PCR. As presented in Fig. 1F and G, the CYP1B1 mRNA levels in IS-treated H9c2 cells were significantly increased compared with the control (P<0.05), while also revealing a similar dose- and time-dependent manner. These data indicated that the gene expression of CYP1B1 in H9c2 cells was induced by the uremic toxin.

In order to further investigate the effect of the uremic toxin on the gene expression of CYP1B1 in vivo, two mouse models were applied. As the IS concentration in CKD mice has previously been reported to be increased (26), a CKD model was established by 5/6 electrocoagulation and was applied in the present study. The other model was established by direct intraperitoneal injections of IS. The pathological changes of myocardial hypertrophy of mouse models were confirmed by HE staining (Fig. 2A). In order to investigate the CYP1B1 mRNA level, total RNA was isolated from the heart tissues and detected using RT-PCR. As presented in Fig. 2B, the CYP1B1 mRNA levels were significantly increased in the CKD and IS groups compared with the control (P<0.05). The heart tissues from these groups were then assessed via western blot analysis and the results revealed that the CYP1B1 protein levels were significantly increased in the CKD and IS groups when compared with those in the normal control group (P<0.05; Fig. 2C). Similarly, the mRNA levels of ANF, BNF and β-MHC were also significantly increased in these two groups, compared with that in controls (P<0.05; Fig. 2D). To further confirm the gene expression of CYP1B1 in vivo, sections from the heart tissues were detected using immunochemistry. As presented in Fig. 2E, CYP1B1 was substantially increased in the CKD and IS groups compared with the control, which was consistent with the results of the western blot analysis. These data clearly demonstrated that the expression of CYP1B1 was induced by IS in vivo.

The present study then investigated whether the induction of CYP1B1 by IS is dependent on AhR. Thus, the effect of IS on the protein expression of AhR in H9c2 cells was examined. However, the total protein of AhR was not significantly altered by IS treatment, even at the maximal dose of IS (Fig. 3A). Similarly, the total protein and mRNA levels of AhR remained unchanged when the cells were treated with CKD serum (Fig. S2). The protein samples were further assessed to isolate the nuclear and cytosol proportions. Notably, the cytosol AhR level was significantly decreased compared with the control (P<0.05) while the nuclear AhR level was significantly increased compared with the control (P<0.05), indicating that nuclear translocation of AhR occurs in a dose-dependent manner (Fig. 3B and C). In order to further confirm this result, the cellular distribution of AhR was detected using immunofluorescence. As presented in Fig. 3D, AhR was detected as smeared in the cytosol in control cells. However, following the treatment of IS, AhR was visible as dotted foci in the nucleus of the H9c2 cells, which indicated the marked nuclear translocation of AhR induced by IS.

AhR is able to bind with the ARNT to form a heterodimeric transcription factor, which is responsible for the induction of a number of different targets (15). It was further investigated whether AhR may directly bind to the promoter region of CYP1B1. Therefore, ChIP-PCR was performed in order to analyze the location of AhR in the promoter region (P1) and the intron 2 (I2) of CYP1B1 (Fig. 4A). Following IS treatment, H9c2 cells were immunoprecipitated with antibodies against AhR, IgG (negative control) and histone H3 (positive control). The immunoprecipitated DNA was detected via PCR and positive bands of primer pair P1 were detected in the immunoprecipitated samples of AhR and histone H3, but not in IgG (Fig. 4B, lower panel). Of note, the band of primer pair I2 was only visible in immunoprecipitated samples of histone H3, indicating the specific binding of AhR in the promoter region (Fig. 4B, upper panel). RT-qPCR was further applied to detect the enrichment of AhR. As presented in Fig. 4C and D, significantly increased amounts of AhR-binding DNA were detected in IS-treated cells compared with the negative control (P<0.001). Overall, the data in the present study clearly demonstrated that AhR was able to bind with the promoter region of CYP1B1 and this binding was enhanced by IS treatment.

To further prove that AhR is crucial for the induction of CYP1B1 by IS, siRNAs of AhR were used to treat H9c2 cells. Compared with NC-siRNA, si-AhR-2 had the greatest significant efficiency when inhibiting the expression of AhR and was used in the subsequent experiments (P<0.001; Fig. 5A). H9c2 cells were pre-treated with si-AhR or its control prior to IS treatment, and the gene expression of CYP1B1 was detected. It was revealed that the pretreatment of si-AhR significantly reversed the induction of CYP1B1 protein and mRNA induced by IS (P<0.01; Fig. 5B and C). Accordingly, the transcription of cardiac hypertrophy markers including ANF, BNF and β-MHC was also reversed by si-AhR (P<0.05; Fig. 5D). Thus, AhR knockdown attenuated the induction of CYP1B1 by IS, and prevented cardiac hypertrophy.

In order to elucidate the association between CYP1B1 expression and cardiac hypertrophy, siRNAs of CYP1B1 were synthesized and used to knockdown the expression of CYP1B1 in H9c2 cells. It was revealed that si-CYP1B1-2 was the optimum siRNA for inhibiting the expression of CYP1B1, and was therefore used in subsequent experiments (Fig. 6A). Furthermore, the pretreatment of si-CYP1B1 significantly reversed the induction of CYP1B1 protein induced by IS (P<0.01; Fig. 6B). In order to gain an improved understanding of this association, H9c2 cells were pretreated with si-CYP1B1 prior to IS treatment, and the gene expression of ANF, BNF and β-MHC was detected via RT-PCR. As presented in Fig. 6C, the induction of all these cardiac hypertrophy markers was significantly reversed by pretreatment with si-CYP1B1 (P<0.01; Fig. 6C). In addition, the cell size was determined by immunofluorescence staining using α-actinin antibodies. The results revealed that the cell size induced by IS treatment was significantly inhibited by si-CYP1B1 (P<0.01; Fig. 6D). This suggested that CYP1B1 knockdown inhibited cardiac hypertrophy.

Finally, in order to investigate the function of CYP1B1 inhibition in vivo, a specific inhibitor of CYP1B1 was combined with IS for use in the mouse model. Previous studies (21,27) have reported TMS as a selective inhibitor of CYP1B1, and the dosage in vivo was selected according to these studies. Following 2 months of treatment, the thickness of the ventricular wall and the relative heart weight in the IS group were significantly increased, compared with that in the controls (P<0.01). However, these parameters were significantly ameliorated by TMS treatment (P<0.05; Fig. 7A and B). Furthermore, the results of the HE staining revealed that the decreased length of the left ventricular midchamber and the increased cell size induced by IS were also substantially ameliorated by TMS treatment (Fig. 7C). In addition, the increased mRNA levels of cardiac hypertrophy markers induced by IS were significantly reversed by TMS treatment (P<0.001; Fig. 7D). These results clearly indicate that the inhibition of CYP1B1 ameliorated cardiac hypertrophy induced by IS.