SK was obtained from Xi'an Shiji Shengkang Pharmaceutical Industry Co., Ltd., (Xi'an, China). Enalapril (EN) was purchased from Merck Millipore (Billerica, MA, USA). Anti-extracellular signal-regulated kinase (ERK; #9102), anti-phosphorylated ERK (p-ERK; #4370), anti-p38 (#9212), and anti-phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK; #9216) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-c-Jun N-terminal kinase (JNK; sc-571), anti-phosphorylated JNK (p-JNK; sc-6254), anti-poly(ADP-ribose) polymerase (PARP; sc-8007) and anti-actin (sc-47778) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Merck Millipore. Hydrogen peroxide (H₂O₂) was purchased from Samchun Chemical Co. Ltd. (Seoul, Korea).

Six-week-old male Sprague-Dawley (SD) rats weighing 200±20 g were obtained from the Fourth Military Medical University (Xi'an, China). The rats were maintained under a regular 12 h light/dark cycle at stable room temperature for 1 week prior to the commencement of the experiments. The rats were fed standard rodent chow and had free access to tap water. All experimental procedures were carried out according to the protocols approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Each rat was anesthetized with chloral hydrate solution (33 mg/100 g) via intraperitoneal injection. A total of 18 rats was subjected to 5/6 nephrectomy (5/6 Nx) in which, the upper and lower poles of the left kidney and the entire right kidney were removed, as previousy described (5,34,35). A sham operation was performed on 6 additional rats as a non-Nx control (sham-operated) group. The nephrectomized rats were randomly divided into 3 groups as follows: i) no treatment (5/6 Nx, n=6); ii) treatment with SK (5/6 Nx + SK, n=6); and iii) treatment with EN (5/6 Nx + EN, n=6, positive control). The rats in the treatment groups received either SK (450 mg/kg/day; via tail vein injection) or EN (5 mg/kg/day; via intraperitoneal injection) daily for 1 week following surgery, whereas the rats in the sham-operated group and 5/6 Nx (no treatment) group received the vehicle (distilled water, 5 ml/kg/day) only. The animals were sacrificed by exsanguination at day 30 post-surgery.

Blood samples were collected from the orbital venous plexus on days 0, 7 and 20 post-surgery. At the end of the experiment, blood samples were obtained from the abdominal aorta, immediately following sacrifice. The serum concentrations of blood urea nitrogen (BUN) and serum creatinine (SCr) were determined using standard laboratory procedures, as previously described (36).

After the rats were sacrificed, the kidney tissue was removed from the abdominal cavity of the rats by surgical methods (isolated kidney tissue from adipose tissue, renal vasculature cut with scissors to obtain kidney tissue). Tissue was fixed in formalin, dehydrated with ethanol, rendered transparent with xylene, embedded with liquid paraffin and sliced with an automatic slicing machine. Slices of renal tissue fixed in 10% neutral-buffered formalin were embedded in paraffin, and 2-μm-thick sections were cut for morphological anlaysis. These sections were stained with hematoxylin and eosin (H&E), as previously descrbied (37). All tissue samples were evaluated by an independent investigator without prior knowledge of the group to which the rat belonged. All glomeruli and the entire microscopic area of each specimen were examined.

Renal proximal tubule epithelial cells (HK-2 cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HK-2 cells were passaged every 2–3 days in 100-mm dishes containing combined Dulbecco's modified Eagle's medium/F-12 [DMEM/F12(1:1)] supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Grand Island, NY, USA). The cells were grown at 37°C in a humidified 5% CO₂ atmosphere. These cells were treated with H₂O₂ in the presence or absence of SK at the indicated concentrations. For experimental use, the cells were harvested at the end of the treatment period for further analysis.

Cell viability was measured using the EZ-Cytox Cell Viability Assay kit (MTT) assay (Itsbio, Seoul, Korea). MTT assay is based on the cleavage of the tetrazolium salt, MTT, to the water-insoluble formazan (38,39). The formazan dye produced by viable cells can be quantified by measuring the absorbance of the dye solution at 460 nm. The HK-2 cells were seeded in 96-well plates (5×10³ cells/well) at 37°C in a 5% CO₂ incubator in DMEM/F12. Following an overnight incubation, the cells were incubated for 24 h in the presence or absence of SK (300, 600 and 900 μg/ml) for 1 h prior to exposure to H₂O₂ (200, 300, 400 and 500 μM). The final incubation of the cells with 10 μl of kit reagent was performed for 45 min at 37°C. The absorbance was measured at 460 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was calculated and averaged. The cells from the control group were treated in the same manner without H₂O₂, and cell viability was expressed as a percentage of the untreated controls.

Early apoptosis and late apoptosis/necrosis induced by H₂O₂ were detected quantitatively, using an Annexin V-FITC apoptosis detection kit (Invitrogen, Grand Island, NY, USA), as previously described (31). Briefly, the cells were treated with H₂O₂ and/or SK, and then harvested by centrifugation, washed with PBS, re-suspended in a Ca²⁺-enriched binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl₂), incubated with 10 μM fluorescein isothiocyanate (FITC)-conjugated Annexin V protein and propidium iodide (PI) for 15 min in the dark and analyzed by two-color flow cytometry. The cell samples were detected immediately in the FL1-H and FL2-H channels of a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), which measured the fluorescence at 488 nm excitation and 530 nm emission. The number of apoptotic cells was quantified and the percentage of apoptotic cells was calculated.

Changes in the nuclear morphology of apoptotic cells were detected using the DNA-specific fluorescent dye, DAPI (Vectashield; Vector Laboratories, Burlingame, CA, USA), as previously described (31). The HK-2 cells were grown on glass coverslips and treated with H₂O₂ and/or SK for 24 h. The treated cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, permeablized with 0.2% Triton X-100 for 10 min at room temperature, washed with PBS again and 20 μl of mounting medium (DAPI) was then added to the fixed cells for 5 min. The finalized slides were stored at 4°C, protected from light and examined under a fluorescence microscope (Nikon, Tokyo, Japan) in order to assess chromatin condensation and fragmentation of the nuclei. Cells that exhibited a reduced nuclear size, chromatin condensation, intense fluorescence and nuclear fragmentation were considered apoptotic.

The generation of intracellular ROS was measured with dihydroethidium (DHE; Invitrogen) (a ROS fluorescent probe), as previously described (1). Briefly, the HK-2 cells were seeded onto 6 cm plates and incubated with 500 μM H₂O₂ for 6 h in the presence or absence of 300 μg/ml SK. At the end of the experimental period, the cells were incubated with 5 μM DHE for 30 min at 37°C, washed and then collected by centrifugation (600 × g for 5 min at room temperature), and resuspended in PBS. The fluorescence intensity was measured using a FACSCalibur™ flow cytometer (BD Biosciences). An OxiSelect™ Total Antioxidant Capacity (TAC) assay kit (Cell Biolabs, San Diego, CA, USA) was employed to measure TAC, as previously described (40) and to estimate the reductive or antioxidant capacity of biomolecules. Briefly, HK-2 cells were incubated with 500 μM H₂O₂ for 6 h in the presence or absence of 300 μg/ml SK, washed 3 times with cold PBS, homogenized in cold PBS, and then centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was stored at −80°C and measured according to the manufacturer's instructions and with the appropriate controls.

Following treatment with H₂O₂ and/or SK, the HK-2 cells were placed on ice, washed twice in ice-cold PBS and lysed at 4°C for 30 min in cell lysis buffer containing 50 mmol/l Tris-HCl, pH 7.5, 1% (v/v) Nonidet P-40, 250 mmol/l NaCl, 0.1 mmol/l phenylmethylsulfonyl fluoride, 0.1 mmol/l sodium vanadate, 20 mmol/l β-glycerol phosphate, 2 mmol/l DTT, 1 mmol/l leupeptin and 10 mmol/l PNPP, as previously described (31). The lysate was then centrifuged at 16,000 × g for 20 min at 4°C. The supernatant was collected for use in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the protein content was estimated using a bovine serum albumin protein assay. Proteins were mixed with sample buffer containing β-mercaptoethanol and heated at 100°C for 2 min. A total of 40 μg of each cell lysate was fractionated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline (TBS) containing 0.02% Tween-20 at room temperature for 1 h, the membranes were then incubated with primary antibodies (diluted 1:1,000) overnight at 4°C. Actin (diluted 1:5,000) was used as a loading control. Following incubation with the primary antibodies, the blots were washed 4 times in TBS/Tween-20 prior to incubation for 1 h at room temperature in goat anti-mouse or anti-rabbit HRP-conjugate antibody at 1:2,000 dilution in TBS/Tween-20 containing 5% skim milk. Following extensive washing in TBS/Tween-20, the blots were processed for the detection of antigens using the enhanced chemiluminescence system. Proteins were visualized with the ECL-chemiluminescence kit (GE Healthcare Life Sciences, Logan, UT, USA).

The quantification of the results of western blot analysis was carried out using the ImageJ (1.47) software (version 1.47). Data are expressed as the means ± SEM of the 3 independent experiments and analyzed by the Student's unpaired t-test (SPSS version 17.0 software; SPSS Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the putative effects of SK on renal function, histopathological changes in the renal sections of 5/6 nephrectomized rats were examined (Fig. 1). In the control (sham-operated) group, proximal and distal tubules exhibited a normal structure (no histological lesions; Fig. 1). A significantly greater number of renal histological abnormalities (glomerular sclerosis, tubular vacuoles, interstitial fibrosis and inflammatory cell infiltration) was observed in the 5/6 Nx vs. the sham-operated group. These abnormalities were markedly decreased in the 5/6 Nx + SK group compared with the 5/6 Nx group, and similar effects were also observed in the 5/6 Nx + EN-treated group (Fig. 1). EN, an effective and widely used drug in the treatment of CKD (41–47), was used as a positive control treatment. These results indicate that SK may be beneficial in the treatment of CKD.

To evaluate renal function, SCr and BUN levels were measured. The SCr and BUN levels significantly increased in the 5/6 Nx group vs. the sham-operated group (Fig. 2), indicating that the 5/6 nephrectomy model, reflecting an impairment in renal function, had been successfully established. Notably, the SCr and BUN levels were significantly decreased in the 5/6 Nx + SK and 5/6 Nx + EN groups compared with the 5/6 Nx group (Fig. 2), which suggests that the administration of SK improves renal function in a similar manner to EN.

To elucidate the molecular mechanisms underlying the effects of SK, HK-2 cells were employed to examine the effects of SK on the apoptosis of renal proximal tubule epithelial cells induced by H₂O₂. The HK-2 cells were exposed to H₂O₂ at various concentrations at 37°C for 24 h. Cell viability was then measured by MTT assay. As shown in Fig. 3A, cell viability was decreased in the H₂O₂-treated cells in a dose-dependent manner (200–500 μM). Notably, H₂O₂-induced cell death was inhibited by treatment with SK (300, 600 and 900 μg/ml) (Fig. 3B) prior to exposure to H₂O₂ (500 μM), suggesting that treatment with SK effectively protects HK-2 cells against H₂O₂-induced cell death.

To further investigate the effects of SK on H₂O₂-mediated cell death, Annexin V and PI staining were employed to evaluate the events leading to cell death. FACS analysis with Annexin V and PI staining revealed that the apoptotic cell population, induced by exposure to H₂O₂ for 24 h, was elevated from 3.4±0.1 to 32.1±0.4% (Fig. 4A); this increase in the apoptotic cell population was prevented by treatment with SK prior to exposure to H₂O₂ (apoptotic cell population decreased to 19.1±0.3%). Statistical analysis further confirmed that treatment with SK protected the HK-2 cells against apoptosis (induced by H₂O₂; Fig. 4A, lower panel). To further evaluate the effects of SK, morphological changes in the nuclei were observed using DAPI staining with fluorescence microscopy. Normal nuclei were characterized by homogeneous staining, and regular oval and rounded shapes (Fig. 4B, left panel). Apoptotic nuclei, indicated by condensed nuclei and nuclear fragmentation, were apparent following exposure to 500 μM H₂O₂ for 24 h (Fig. 4B, center panel). However, these changes in nuclear characteristics were ameliorated by pre-treatment with SK in the HK-2 cells (Fig. 4B, right panel). This was confirmed by western blot analysis of PARP, which is cleaved under apoptotic conditions (48,49). As illustrated in Fig. 4C, the cleaved form of PARP significantly increased following exposure to H₂O₂ for 24 h. The cleavage of PARP was inhibited by pretreatment of the cells with SK (Fig. 4C), suggesting that SK prevents the molecular events involved in apoptotic signaling.

It is well-established that oxidative stress contributes to apoptosis (31–33). Therefore, the intracellular ROS levels, and TAC, were measured in the HK-2 cells. ROS production, measured by DHE staining, was elevated following exposure to H₂O₂; treatment with SK significantly reduced ROS production (Fig. 5A). In addition, TAC was also enhanced by treatment with SK (Fig. 5B). Taken together, our findings suggest that the SK-induced inhibition of ROS production is mediated by enhanced TAC in HK-2 cells.

To determine whether SK regulates the signaling mechanisms responsible for H₂O₂-induced apoptosis, the activation of different MAPK signaling pathways, including ERK, JNK and p38 MAPK, was monitored by western blot analysis. Notably, the H₂O₂-mediated activation of ERK and p38 was observed, but JNK activation was not affected by exposure to H₂O₂ (Fig. 6A). Furthermore, pre-treatment of the HK-2 cells with SK inhibited the phosphorylation of ERK and p38, but not that of JNK.