To evaluate the function of sinigrin in the modulation of hypertension-induced kidney damage, a hypertensive rat model was constructed using Ang II. Briefly, Sprague-Dawley rats (n=10 per group, weighing 200-225 g, male, SiPeiFu Biotechnology Co., Ltd.) were subcutaneously administered Ang II (Sigma-Aldrich; Merck KGaA) for 28 days via low-dose Ang II infusion from osmotic minipumps (model 2002, ALZET, DURECT Corporation) in the dorsum of the neck under anesthesia (pentobarbital sodium, 50 mg/kg i.p.). Beginning from the first day of Ang II pump implantation, sinigrin (#85440, Sigma-Aldrich; Merck KGaA) was administered at the indicated doses to the rats once daily via oral gavage and continued for 28 days. The rats were assigned to 5 groups as follows: The sham-operated (sham) group (n=10, infused with normal saline and orally administered normal saline); Ang II group (n=10, infused with Ang II and orally administered normal saline); Ang II + sinigrin (10 mg/kg) group (n=10, infused with Ang II and orally administered 10 mg/kg sinigrin); Ang II + sinigrin (20 mg/kg) group (n=10, infused with Ang II and orally administered 20 mg/kg sinigrin); Ang II + sinigrin (40 mg/kg) group (n=10, infused with Ang II and orally administered 40 mg/kg sinigrin). At the end of the treatment period, the rats were housed in individual metabolic cages for 24 h, and provided with water and food ad libitum, and urine samples were obtained from the rats. Prior to sacrifice by cervical dislocation, the rats were anesthetized by pentobarbital sodium (50 mg/kg i.p.) and blood samples (5 ml) were harvested from the abdominal aorta for one time. Subsequently, the kidneys were excised and collected; one part was placed in 10% formalin and embedded in paraffin for histopathological analysis, and the other was snap-frozen in liquid nitrogen for protein evaluation. All procedures and experimental protocols were approved by the Animal Ethics Committee of the Third Affiliated Hospital of Shandong First Medical University (no. LL202001003).

SBP and DBP were recorded and analyzed using the CODA Monitor (Kent Scientific) with a tail-cuff non-invasive method according to the instructions of the manufacturer. Prior to the measurement, the rats were placed in the holder for 5 min, and the SBP and DBP were recorded three times for each rat at 0, 7, 14, 21 and 28 days of Ang II infusion.

The levels of serum creatinine (SCR) and blood urea nitrogen (BUN) were measured using an automatic biochemical analyzer (Beckman Coulter, Inc.) as described in a previous study (16). The levels of urinary protein were examined using a BCA Protein Assay kit (#P0010, Beyotime Institute of Biotechnology).

For the histological analysis of kidney tissues, the kidneys were perfused with physiological salt solution, treated with formalin (10%) and longitudinally sectioned, followed by fixation in formalin (10%) overnight. Subsequently, the kidney tissues were paraffin-embedded and sectioned as previously described (17). Glomerular basement membrane thickness was analyzed by periodic acid-Schiff (PAS) staining (Beyotime Institute of Biotechnology). Briefly, the sections were treated with periodic acid at room temperature for 5 min, and then incubated with Schiff at room temperature for 15 min.

Human HK-2 proximal tubule epithelial cell lines were maintained at the Central Laboratory of the Third Affiliated Hospital of Shandong First Medical University and incubated at 37°C with 5% CO₂ in Dulbecco's modified Eagle medium (Cytiva) containing fetal bovine serum (15%, Gibco; Thermo Fisher Scientific, Inc.), streptomycin (0.1 mg/ml; Beijing Solarbio Science & Technology Co., Ltd.) and penicillin (100 units/ml; Beijing Solarbio Science & Technology Co., Ltd.). To induce HK-2 cell injury, the cells were exposed to Ang II (Sigma-Aldrich; Merck KGaA) at a concentration of 1 µM. Subsequently, the cells were treated with sinigrin at1, 10 and 100 µg/ml.

The viability of the HK-2 cells was evaluated by MTT assays. In brief, ~2×10⁴ cells were plated in 96-well plates and incubated for 12 h at room temperature. Following the indicated treatments, the cells were mixed with MTT solution (5 mg/ml, 10 µl) and incubated for 4 h at room temperature. The medium was then removed, and DMSO (150 µl) was added to the cells. Cell viability was determined by measuring the absorbance at 570 nm using an ELISA browser (Bio-Tek EL 800; BioTek Instruments, Inc.).

HK-2 cells (~2×10⁵) were plated in 6-well plates. Cell apoptosis was assessed using the Annexin V-FITC Apoptosis Detection kit (#6592, Cell Signaling Technology, Inc.) according to the manufacturer's instructions. In brief, ~2×10⁶ cells collected and washed cells were resuspended in binding buffer, dyed with propidium iodide at 25°C, and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences).

Oxidative stress-related enzymes, including superoxide dismutase (SOD), malondialdehyde (MDA) and catalase (CAT), were analyzed in vivo and in vitro. The activity of SOD was measured using a SOD assay kit (#706003; Cayman Chemical Company). The activity of SOD was analyzed using a microplate reader (BioTek Instruments, Inc.) at 450 nm. The activity of MDA was measured using a MDA assay kit (#700870; Cayman Chemical Company). The activity of MDA was analyzed using a microplate reader (BioTek Instruments, Inc.) at 405±414 nm. The activity of CAT was measured using a CAT assay kit (#707002, Cayman Chemical Company). The activity of CAT was analyzed using a microplate reader (BioTek Instruments, Inc.) at 340 nm.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was manufactured using the PrimeScript™ II 1st Strand cDNA Synthesis kit (#6210A; Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The qPCR assays were performed using Premix Ex Taq SYBR-Green (#RR820A; Takara Biotechnology Co., Ltd.). The primer sequences were as follows: TNF-α forward, 5′-CCC AGG CAG TCA GAT CAT CTT C-3′ and reverse, 5′-GCT TGA GGG TTT GCT ACA ACA TG-3′; IL-6 forward, 5′-TCA GGA AAT TTG CCT ATT GAA AAT TT-3′ and reverse, 5′-GCT TTG TCT TTC TTG TTA TCT TTT AAG TTG T-3′; monocyte chemoattractant protein-1 (MCP-1) forward, 5′-CAT AGC AGC CAC CTT CAT TCC -3′ and reverse, 5′-TCT CCT TGG CCA CAA TGG TC-3′; GAPDH forward, 5′-AAC GGA TTT GGT CGT ATT GGG -3′ and reverse, 5′-CCT GGA AGA TGG TGA TGG GAT -3′. The RT-qPCR reaction conditions were as follows: 95°C, 10 sec (denaturation); 55°C, 30 sec (annealing); 72°C, 30 sec (extension) for 40 cycles. The relative expression levels were calculated using the 2^−ΔΔCq method (18).

Total protein was isolated from the cells using RIPA buffer (Cell Signaling Technology, Inc.) and quantified using the BCA Protein Quantification kit (Abbkine Scientific Co., Ltd.). Protein samples were subjected to 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore), followed by blocking with 5% skimmed milk at room temperature and incubating with primary antibodies at 4°C overnight. The horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000, cat. no. BA1039; Wuhan Boster Biological Technology, Ltd.) were used to incubate the membranes for 1 h at room temperature, followed by visualization by using a chemiluminescence detection kit (Beyotime Institute of Biotechnology, Inc.). The primary antibodies used in the present study were the following: Collagen I (#ab260043, 1:500; Abcam), collagen IV (1:500, cat. no. ab236640; Abcam), fibronectin (1:1,000, cat. no. ab268020; Abcam), ERK (1:500, cat. no. 4695; Cell Signaling Technology, Inc.), p65 (1:1,000, cat. no. 8242; Cell Signaling Technology, Inc.), IκB α (1:500, cat. no. 8412; Cell Signaling Technology, Inc.), p-ERK (1:300, cat. no. 4370; Cell Signaling Technology, Inc.), p-p65 (1:500, cat. no. 3033; Cell Signaling Technology, Inc.), p-IκBα (1:300, cat. no. 2859; Cell Signaling Technology, Inc.) and GAPDH (1:2,000, cat. no. ab181602; Abcam). The density of the protein bands was quantified using ImageJ software (version V1.8.0; National Institutes of Health).

Data are expressed as the mean ± SD, and statistical analysis was conducted using GraphPad Prism 7 (GraphPad Software, Inc.). One-way ANOVA followed by Tukey's post hoc test was used to assess the differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of sinigrin in the modulation of hypertension-induced kidney damage, a spontaneously hypertensive rat model was constructed using Ang II. It was observed that SBP and DBP were increased in the Ang II-challenged rats, and sinigrin treatment attenuated this increase in a dose-dependent manner (Fig. 1A and B). The levels of BUN and SCR were enhanced by Ang II in the rats, and sinigrin reversed this effect in a dose-dependent manner (Fig. 1C and D). Moreover, the Ang II-induced increase in urinary protein levels was inhibited by sinigrin treatment in the rats (Fig. 1E). These results suggest that sinigrin may alleviate Ang II-induced kidney dysfunction in vivo.

The present study then explored the influence of sinigrin on Ang II-induced kidney injury in a rat model. The results revealed that renal glomerular basement membrane (GBM) thickness was increased in the Ang II-challenged rats, which was reduced by treatment with sinigrin in a dose-dependent manner (Fig. 2A). Moreover, the expression of fibronectin, collagen IV and I was increased by Ang II in the rats, and sinigrin reversed this effect in a dose-dependent manner (Fig. 2B-E). These results indicate that sinigrin may alleviate Ang II-induced kidney injury in vivo.

Given that oxidative stress and inflammatory processes are involved in hypertension-related kidney damage, the present study further explored the effects of sinigrin on associated markers in the rat model. It was found that the expression of TNF-α, IL-6 and MCP-1 was upregulated by Ang II and treatment with sinigrin attenuated this upregulation in the rats (Fig. 3A-D). Moreover, the levels of SOD and CAT were decreased, whereas the levels of MDA were increased in the Ang II-challenged rats; these effects were reversed by treatment with sinigrin (Fig. 3E-G). Taken together, the results suggest that sinigrin may reduce Ang II-induced inflammation and oxidative stress in vivo.

The present study then investigated the underlying mechanisms of sinigrin-mediated hypertensive kidney damage. Notably, the phosphorylation of ERK, p65 and IκBα was stimulated in the Ang II-challenged rats; however, this was reduced by sinigrin treatment (Fig. 4), indicating that sinigrin may attenuate hypertension-induced kidney damage by inactivating ERK and NF-κB signaling in vivo.

Ang II is a vasoconstrictive peptide that modulates blood pressure homeostasis and can cause hypertensive renal inflammation in renal tubular epithelial cells (19,20). Therefore, in the present study, the Ang II-induced injury model using HK-2 cells, a human renal tubular epithelial cell line, was used to determine the function of sinigrin in vitro. HK-2 cells were treated with sinigrin at the indicated concentrations, which did not exhibit cytotoxicity to the cells (Fig. 5A). The viability of the Ang II-exposed HK-2 cells was reduced however, and this was attenuated by by sinigrin treatment in a concentration-dependent manner (Fig. 5B). Sinigrin also reversed the Ang II-induced apoptosis of HK-2 cells in a concentration-dependent manner (Fig. 5C). In addition, the levels of SOD and CAT were reduced, whereas the levels of MDA were enhanced the Ang II-exposed HK-2 cells; these effects reversed by treatment with sinigrin (Fig. 5D-F). Taken together, these results suggest that sinigrin attenuates Ang II-induced HK-2 cell injury in vitro.

It was demonstrated that the expression of TNF-α, IL-6 and MCP-1 was upregulated by Ang II in the HK-2 cells, and treatment with sinigrin abrogated this effect (Fig. 6A-C). Furthermore, the expression of fibronectin, collagen IV and I was elevated by Ang II in the cells, and this increase was reversed by sinigrin treatment in a concentration-dependent manner (Fig. 6D-G). These results indicate that sinigrin may alleviate Ang II-induced inflammation and ECM degradation in HK-2 cells.

The results revealed that the phosphorylation of ERK, p65 and IκBα was enhanced in Ang II-exposed HK-2 cells, and sinigrin treatment reduced this effect in a concentration-dependent manner (Fig. 7). Therefore, sinigrin may attenuate hypertension-induced kidney damage by inactivating ERK and NF-κB signaling in vitro.