Animals.

The animal experiments in this study were approved by the Animal Care and Ethics Committee of Fourth Military Medical University. Male adult Sprague-Dawley rats (Department of Laboratory Animal Science, Fourth Military Medical University, Xi'an, China), weighing 250–300 g, were used in the present study. All animal welfare and experimental procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23, National Academy Press, Washington, DC, rev. 1996). All experimental rats were kept in an environmentally controlled breeding room for 5 days before experiments and fed with standard laboratory food and water.

Chemicals.

Sal A (97599) (≥98% by HPLC) and 4′,6-diamidino-2-phenylindole (DAPI) (9542) were obtained from Sigma (St. Louis, MO). CM-H2DCFDA (C6827) and the MTT assay kit were procured from Thermo Fisher Scientific (Waltham, MA). Malondialdehyde (MDA) (S0131) and superoxide dismutase (SOD) (S0060) kits were obtained from Beyotime Bio-technology (Jiangsu, China). A terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kit (ApoBrdu DNA fragmentation assay kit) (K403-50) was procured from Biovision (Milpitas, CA). Rapamycin (no. 9904), LY294002 (no. 9901), and monoclonal antibodies specific for phosphorylated-Akt (p-Akt, Ser-473; no. 4060), Akt (no. 4685), phosphorylated-mTOR (p-mTOR, Ser-2448; no. 5536), mTOR (no. 2983), eukaryotic initiation factor 4E binding protein 1 (4EBP1; no. 9644), phosphorylated-4EBP1 (p-4EBP1, Thr-70; no. 9455), and GAPDH (no. 5174) were obtained from Cell Signaling Technology (Danvers, MA). Bcl-2 (sc-7382) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), while anti-active caspase-3 (ab2302) was purchased from Abcam (Cambridge, UK). Secondary antibodies goat anti-rabbit (621140380011730) and goat anti-mouse (621140680011730) were procured from Merck Genei (Karnataka, India). HK-2 cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). DMEM and other cell culture supplies were purchased from Gibco (Grand Island, NY). All materials for SDS-PAGE were obtained from Bio-Rad Laboratories (Hercules, CA). Hypoxic/ischemic chambers were obtained from Billups-Rothenberg (San Diego, CA). All other chemicals used were of analytical grade and were purchased from Sigma.

Rat model of renal I/R injury.

All rats were anesthetized with sodium chloral hydrate (85 mg/kg ip) and placed in a prone position on a warming pad at 37°C to perform surgical procedures. The sham-operated groups (sham group) were only subjected to the removal of the right kidney, whereas rats in I/R, I/R+Sal A, and I/R+Rapa+Sal A groups were also subjected to acute I/R injury to the left kidney, which was induced by clamping the renal artery for 45 min using nontraumatic vascular clips. The clamp was then removed for reperfusion. Blood was collected from the eye socket at 4, 10, and 24 h postreperfusion, and the left kidney was removed at 24 h. The rats were euthanized by decapitation and exsanguination at 24 h after the I/R procedure. The kidney and blood were collected for further analyses.

Rat experimental design.

Forty rats were randomly divided into four groups: 1) a sham-operated group (sham group: n = 10) treated with tail vein injection of saline; 2) an I/R group treated with tail vein injection of saline (I/R+Vehicle group, n = 10); 3) an I/R group treated with tail vein injection of Sal A (I/R+Sal A group, n = 10); and 4) an I/R group (I/R+Rapa+Sal A group: n = 10) were administered intraperitoneally with rapamycin (Rapa: 1.5 mg/kg body wt) 24 h before the I/R procedure. Sal A was administered to animals in the same manner as the I/R+Sal A group. Sal A (40 mg/kg, dissolved in normal saline) or the same volume of saline was injected intravenously into the tail vein 30 min before surgery. The rat model of renal I/R injury and the surgical procedures involved were similar to those previously described (1, 14).

To clarify the effect of Sal A on non-I/R injury rats, we performed an experiment in which Sal A was injected into the tail vein of the sham group (n = 10). And the protein expression (Akt and mTOR) in the renal tissue was compared with the sham group (n = 10) treated with saline at 24 h after injection.

In addition, we performed another experiment in which Sal A was injected into the tail vein after I/R (n = 10), and renal function (plasma creatinine and urea nitrogen) was assessed and compared with the I/R group (n = 10) at 24 h of reperfusion.

Cell culture and treatment.

HK-2 cells, a human proximal tubular cell line, were cultured in DMEM/F12 (1:1) and were passaged every 2–3 days in 100-mm dishes supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were grown at 37°C in a humidified 5% CO₂ atmosphere. To mimic ischemia-like conditions in vitro, HK-2 cells were exposed to ischemia by replacing the medium with an “ischemic buffer” (5 mM HEPES, 137 mM NaCl, 4 mM KCl, 1 mM MgCl₂, and 1.5 mM CaCl₂, pH 7.0). The cells were then incubated in a hypoxic/ischemic chamber at 37°C for 60 min in a humidified atmosphere of 5% CO₂ and 95% N₂. Finally, the cells were incubated again in the culture medium in an incubator with 95% air and 5% CO₂ for an additional 24 h, as previously described (14, 18, 20, 21). For all experiments, cells were plated at an appropriate density, according to the experimental design and grown for 24 h before treatment. Five sets of experiments were performed: 1) for the Control Group, cells were treated with PBS; 2) for the Hypoxia/Reoxygenation (H/R) Group, cells were treated as described above to mimic ischemia-like conditions; 3) for the Sal A+H/R Group, cells were treated with 40 μM Sal A for 1 h and then treated with the H/R condition; 4) for the RA+Sal A+H/R group, cells were treated with rapamycin (100 nM) for 1 h and 40 μM Sal A for 1 h and then treated with the H/R condition; and 5) for the LY+Sal A+H/R group, cells were treated with an inhibiter LY294002 (20 μM) for 1 h and 40 μM Sal A for 1 h, and then treated with the H/R condition.

Assessments of biochemical parameters.

Blood was collected using retro-orbital puncture at 4, 10, and 24 h following reperfusion. Serum was separated by centrifugation at 2,700 g and at 4°C, and serum creatinine (Scr) and blood urea nitrogen (BUN) levels were determined by staff at the Clinical Laboratory of Xijng Hospital, who were blinded to the treatments given. Blood samples were stored at −80°C for analyses.

Histological analyses.

Staff at the Pathology Department of Xijng Hospital, who were blinded to the treatments given, performed the morphological assessments. The left kidney was fixed and embedded, and each paraffin-embedded kidney was cut into 3-μm sections and subjected to hematoxylin-and-eosin staining, as previously reported (14). The histopathological changes in the cortex and medulla were evaluated by a pathologist in a blinded fashion on a scale from 0 to 5: 0, none; 1: 0–10%; 2: 11–25%; 3: 26–45%; 4: 46– 75%; and 5: 76–100%, as previously described (35).

Determination of apoptosis.

Renal cell apoptosis was detected using a TUNEL assay with tissue paraffin blocks. Renal slides were incubated with TUNEL reaction mixture in a humidified chamber for 60 min at 37°C in the dark. The renal sections were then rinsed three times in PBS, and the nuclei were mounted with DAPI (300 nM). Apoptosis was quantified by calculating the percentage of TUNEL-positive nuclei out of the total nuclei in an average of 20 high-power fields for each section in a blinded manner.

Measurements of oxidative stress markers.

Dissected kidneys were immediately rinsed in ice-cold PBS. Tissues were homogenized in 10% 150 mM phosphate buffer (pH 7.4) (1/10 wt/vol). The homogenate was centrifuged at 6,000 g for 10 min at 4°C. The total protein level in the supernatant was measured using a BCA protein assay reagent kit. The concentrations of MDA and SOD in renal tissue were measured using commercial kits, according to the manufacturer’s instruction. The final levels of MDA and SOD were normalized to the protein concentration of kidney tissue homogenate, as previously described (34).

Assessment of cell viability.

Cell viability was measured using the cell viability assay kit (MTT) assay. 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 overnight incubation, the cells were incubated in the presence or absence of rapamycin or LY294002 and subsequently treated Sal A for 1 h before exposure to the H/R condition. 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 Laboratories). Cell viability was calculated and averaged. The cells from the control group were treated in the same manner without exposure to the H/R condition, and cell viability was expressed as a percentage of the untreated controls.

Assessment of reactive oxygen species production in HK2 cell.

CM-H₂DCFCA, a reactive oxygen species (ROS)-sensitive fluorescent dye, was used to measure ROS levels. To evaluate the effect of Sal A on I/R injury-induced ROS production, HK-2 cells were cultured in 96-well plates. Cells were washed twice with PBS, incubated with 5 mM CM-H₂DCFCA for 30 min in Dulbecco’s PBS, and again washed three times with PBS. Fluorescence intensities were measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microplate fluorescence reader.

Western blot analysis.

Protein extraction and Western blot analysis were done as previously described (14). Cell or renal lysates (50 μg) were separated on polyacrylamide SDS gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with antibodies with optimal dilutions against Bcl-2, cleaved caspase-3, p-Akt (Ser-308), Akt, p-mTOR (Ser-2448), mTOR, p-4EBP1 (Thr-70), or 4EBP1 overnight at 4°C. After six washes with PBS-Tween (each for 5 min), the membranes were probed with an horseradish peroxidase-conjugated secondary antibody. Blots were developed using enhanced chemiluminescent reagents (Thermo Fisher Scientific), and blotting signals were visualized by a ChemiDoc MP System (Bio-Rad Laboratories). GAPDH was used for normalization, and the intensity of each band was analyzed using the densitometry plugin on ImageJ (https://imagej.nih.gov/ij/) as instructed.

Statistical analysis.

Data are expressed as means ± SD. Numerical data are presented as means ± SD from at least three individual experiments. All statistical analyses were performed using the SPSS 16.0 software package. Comparisons between the two groups were performed using an independent-sample t-test. Experiments with more than two groups were compared by ANOVA followed by the Tukey post-hoc test. P values <0.05 were considered statistically significant.

Sal A provides protection against renal I/R injury in vivo.

To assess the effect of Sal A pretreatment on renal I/R injury, rats were administered with Sal A, followed by I/R insult (I/R+Sal A Group). In addition, rats were pretreated with vehicle (I/R Group) or had undergone similar surgical processes absent of I/R insult (sham group). I/R insult led to a significant impairment of renal function. Renal I/R injury led to a significant increase in the levels of Scr and BUN in a time-dependent manner following reperfusion (Fig. 1, B and C). Sal A-pretreated rats displayed well-preserved renal function, as manifested by significantly lower levels of serum BUN (Fig. 1B) and Scr (Fig. 1C), as compared with rats from the I/R+Vehicle Group. Twenty-four hours after renal reperfusion, rats in the I/R+Vehicle group developed significant renal dysfunction indicated by an increase in Scr (343.45 ± 47.08) and BUN (60.85 ± 4.73) levels. Rats pretreated with Sal A did not exhibit such a significant increase in Scr (161.36 ± 11.90) and BUN (24.21 ± 1.58) levels.

In addition, to confirm these observations, histological analyses of renal sections were performed. Compared with the sham group, rats in the I/R group showed significant pathological changes, as evidenced by severe tubular necrosis, dilation, and cast formation (Fig. 2A). Meanwhile, the pathological score of histological lesions was significantly lower (P < 0.05) in Sal A-treated I/R rats (2.53 ± 0.68) than untreated I/R rats (3.87 ± 0.54) at 24 h after reperfusion. These histopathological alterations were moderately reduced in I/R+Rapa+Sal A group. The renal histopathological score of all the rats are presented in Fig. 2B.

In the Sal A treatment after I/R experiment, the Sal A-treated groups also showed lower Scr (I/R: 343.3 ± 47.1; Sal A after I/R: 285.6 ± 76.8 μmol/l), and BUN (I/R: 60.3 ± 4.5; Sal A after I/R: 45.6 ± 9.7 mmol/l).

HK-2 cells were also pretreated with Sal A (10, 20, and 40 µM) for 1 h, after which, they were exposed to the H/R condition to mimic ischemic-like conditions in vitro. Exposure to the H/R condition resulted in 43.03 ± 4.21% cell death. However, cell viability was significantly increased (85.12 ± 5.37%) when cells were pretreated with Sal A (40 µM) (Fig. 2C). Altogether, the data support that administration of Sal A may attenuate I/R-induced renal damage.

Sal A prevents I/R-induced tubular apoptosis.

Tubular apoptosis is a crucial event in I/R-induced renal injury and drives the severity of tissue damage (6, 31). To determine whether the beneficial effects of Sal A on renal I/R injury were associated with the inhibition of apoptosis, TUNEL assays were performed on kidney sections. Similar to immunohistochemistry results, TUNEL-positive cells were almost absent in the sections of mice from the sham group, while a substantial increase of apoptotic cells was noted in I/R+Vehicle rats (Fig. 3A). However, I/R+Sal A rats manifested a threefold reduction of apoptotic cells as compared with I/R+Vehicle rats (Fig. 3B), indicating that Sal A pretreatment prevented I/R-induced tubular apoptosis. Furthermore, the expression of Bcl-2 and cleaved caspase-3 was also examined. Similarly, I/R led to a substantial decrease in the expression of Bcl-2 and an increase in active caspase-3 activity (Fig. 3C). Our data show that Bcl-2 expression was restored to nearly normal levels by Sal A preconditioning, while cleaved caspase-3 expression was dramatically suppressed. In vitro, Sal A pretreatment in HK-2 also suppressed H/R-induced activation of caspase-3 (Fig. 3D). Taken together, Sal A administration mitigated renal cell apoptosis in I/R injury rats.

Sal A reduces I/R-induced ROS accumulation.

To explore whether the renoprotection conferred by Sal A in I/R injury was associated with oxidative stress, related markers of oxidative stress were examined. MDA is a naturally occurring product of lipid peroxidation and an indicator of ROS production. MDA levels were increased in I/R injury rats compared with the sham group. Pretreatment with Sal A decreased renal MDA levels. The activity of SOD, a critical enzyme responsible for the detoxification of ROS, was then analyzed. Surprisingly, I/R injury resulted in a significant reduction of SOD activity, and pretreatment with Sal A restored renal SOD levels (Fig. 4B). To determine whether Sal A reduces I/R-induced ROS production, HK-2 cells were incubated with increasing concentrations of Sal A in the presence or absence of the H/R condition. Subsequently, ROS were detected quantitatively using a fluorescence microplate reader. The H/R condition resulted in a significant increase in intracellular ROS production, which was reduced by Sal A pretreatment (Fig. 4C), suggesting that Sal A can attenuate oxidative stress from two directions in rats with I/R injury.

Sal A activates the Akt/mTOR/4EBP1 pathway in renal I/R injury.

Cellular proliferation and survival require the involvement of mTOR and phosphoinositide 3-kinase (PI3-K)/Akt pathways (30). Therefore, whether the Akt/mTOR pathway is involved in the renoprotection provided by Sal A was investigated. Interestingly, I/R injury significantly suppressed p-Akt and p-mTOR proteins, while Sal A pretreatment significantly attenuated this phenomenon. The higher levels of p-Akt and p-mTOR (Fig. 5A) were also noted in Sal A-pretreated rats compared with I/R+Vehicle rats.

In the presence of Rapa (an inhibitor of mTORC1), phosphorylation of mTOR, 4EBP1 was significantly lower compared with I/R+Sal A group. (Fig. 5B). In addition, Sal A did not affect significantly the protein expression of p-Akt and p-mTOR on non-I/R-injured rats (data not shown).

To confirm the above results, HK-2 cells were cultured under the H/R condition in the presence or absence of Sal A, as described earlier. As is known, mTOR phosphorylates and activates 4EBP1, which serves as a marker for mTOR activity. The expression of phosphorylated mTOR and its downstream target p-4EBP1 were assessed. Induction of the H/R condition almost blocked the expression of p-mTOR and p-4EBP1, while Sal A (40 μM) significantly restored the expression of these proteins in the presence of the H/R condition (Fig. 5C). We hypothesized that Akt/mTOR/4EBP1 activation is responsible for the increased cell survival in response to the H/R condition and, thus, performed inhibitory experiments using rapamycin. Rapamycin completely reversed Sal A-mediated mTOR/4EBP1 phosphorylation in response to the H/R condition (Fig. 5C). Furthermore, increased expression of p-mTOR and p-4EBP1 by Sal A during exposure to the H/R condition was blocked following the application of the inhibitor LY294002 (20 μM) (Fig. 5D). In addition, a considerable decrease in cell survival was found when rapamycin (77.8% of Sal A+H/R group, P < 0.01) or LY294002 (75.6% of Sal A+H/R group, P < 0.01) was applied in combination with Sal A (Fig. 5E). These data indicate that Sal A protects the kidney from I/R-induced injury, at least partially via activation of the Akt/mTOR/4EBP1 pathway.