The animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the First People Hospital of Foshan. Male BALB/c mice at 8–12 weeks of age, weighing 20–30 g, were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Kidneys were exposed through flank incision and subjected to ischemia by clamping of the renal pedicles with non-traumatic microaneurism clamps. The time of ischemia was chosen to obtain a reversible model of ischemic acute renal failure (IARF) and to avoid animal mortality. After 30 min, the clamps were removed and blood reflow was confirmed. Body temperature was maintained at 36.5–37.5°C throughout the procedure. Mice were randomly allocated into the following groups: (1) NC+sham group (n = 8); (2) NC+I/R group (n = 8); (3) Curcumin+sham group (n = 8); and (4) Curcumin+I/R group (n = 8). Sham control mice underwent an identical surgical procedure but without pedicle clamping. Mice were administered curcumin (100 mg/kg) 0.5 h before I/R induction. All animals were sacrificed at 24 h after reperfusion. Kidneys were perfused and harvested.

Blood samples were obtained from the inferior vena cava 24 h after reperfusion. Serum creatinine was measured using a creatinine assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer's instructions. Blood urea nitrogen was determined fluorometrically as described (Ramesh et al., 2007).

Kidney tissue was fixed in 10% buffered formalin, embedded in paraffin and then cut at 4 μm thickness. After deparaffinization and rehydration, sections were stained with H&E. Tissue damage was examined in a blinded manner and scored according to the percentage of damaged tubules as previously reported (Chen et al., 2011): 0, no damage; 1, <25% damage; 2, 25%−50% damage; 3, 50%−75% damage; and 4, >75% damage.

Mouse renal tubular epithelial cells (EpiCM-a) (Sciencell, San Diego, California, US) were thawed in a 38°C water bath and then centrifuged at 1,000 rpm for 5 min. The supernatant was discarded and the cells were cultured in DMEM medium in an incubator containing 5% CO₂ at 37°C. The medium was replaced when cells adhered to the bottle wall. The cells were subcultured until 80% of the bottle bottom was covered with cells. To induce hypoxia, mouse renal tubular epithelial cells were incubated in serum-free DMEM in a hypoxia chamber containing 95% N2 and 5% CO₂ at 37°C. Following exposure to hypoxic conditions, cell medium was replaced with fresh oxygenated DMEM and cells were reoxygenated for 24 h in normoxic conditions (5% CO², 21% O₂ and 74% N₂) at 37°C.The cells with traces of APPL1 siRNA treatment were divided into six groups: Sham group, HR group, HR+Curcumin (25 μM) group, HR+Curcumin+APPL1 siRNA group, HR+Curcumin+NC siRNA group, and HR+Curcumin+APPL1 siRNA+AZD5363 (30 μM) group. HR treatment consisted of hypoxia for 3 h and then reoxygenation for 24 h. Curcumin and AZD5363 were administered at the beginning of HR treatment.

APPL1 siRNA was manufactured by Sigma (US). For each well that was transfected, RNAi duplex-Lipofectamine™ RNAiMAX complexes were prepared according to the following protocol: 6 pmol RNAi duplex in 100 μl Opti-MEM® I Medium was diluted without serum in the well of the tissue culture plate. Lipofectamine™ RNAiMAX was mixed gently before use, and then 1 μl Lipofectamine™ RNAiMAX was added to each well containing the diluted RNAi molecules. Cells were mixed gently and incubated for 10–20 min at room temperature. Cells were diluted in complete growth medium without antibiotics 24 h after plating. For suspension cells, 20,000–50,000 cells/well were used. In each well, 500 μl diluted cells were added with RNAi duplex—Lipofectamine™ RNAiMAX complexes. The final volume was 600 μl and the final RNA concentration was 10 nM. Cells were incubated for 24–72 h at 37°C in a CO₂ incubator.

NC siRNA is transfected with non-characteristic negative siRNA cells by transfection reagent.

Immunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories) or proteinase K. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 min. After blocking with 5% normal serum, slides were incubated with primary antibodies in a humidified chamber overnight. After washing, slides were incubated with appropriate secondary antibodies and ABC solution sequentially according to the ABC kit (Vector Laboratories). Slides were then visualized by incubation in diaminobenzidine solution for an appropriate duration of time. Nuclear staining was performed with hematoxylin. The slides were dehydrated, cleared, and mounted. The images from these slides were obtained and analyzed by NIS Element software (Nikon Instruments) with the Nikon microscope image system (Nikon Instruments).

Apoptotic cell death was determined with terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining with the DeadEnd Colorimetric Apoptosis Detection System (Millipore, Billerica, MA) according to the manufacturer's instructions. The number of TUNEL-positive cells per high-power field was counted and analyzed in a blinded fashion.

Cells were added to 96-well plates (100 μL/well; 2 × 10⁴ cells/well) and incubated overnight, followed by cell transfection. After cells were treated with HR or sham, the medium was discarded and cells were fixed with 4% paraformaldehyde. Cells were incubated at room temperature for 30 min and washed with PBS. Permeabilization solution (0.1% Triton X-100 dissolved in 0.1% sodium citrate solution) was added to plates for a 2-min incubation in an ice-bath. With the addition of 50 μL TUNEL reagent, cells were cultured in a humidified incubator at 37°C for 60 min. After three PBS washes, 50 μL of DAPI was added to the cells, followed by incubation at 37°C in the dark and another three PBS washes. Cells were observed under a fluorescence microscope and images were captured.

An Annexin V-PE(V-phycoerythrin) and 7-AAD (7-amino-actinomycin D) double-staining Apoptosis Detection kit (KeyGEN, Nanjing, China) was used to detect apoptotic activity according to the manufacturer's instructions. Cells were harvested, washed, and incubated for 15 min with annexin V-FITC and PI. The cells were washed and transferred into 12 × 75-mm Falcon 2052 FACS tubes (Becton Dickinson, San Jose, CA), and data from cells were collected on FACS with a Becton Dickinson Biosciences FACScan and CellQuest software version 3.3. This combination allows for the differentiation between early apoptotic cells (annexin V-FITC positive, PI negative), late apoptotic and/or necrotic cells (annexin V and PI positive), and viable cells (unstained).

Cells were washed with ice-cold PBS and lysed in ice-cold modified RIPA lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 0.5% deoxycholic acid, 1% NP-40, 1 mM sodium orthovana-date, 0.1% SDS). Insoluble material was removed by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentrations were measured using a bicinchoninic acid assay (Perbio Science, Cramlington, UK) following the manufacturer's instructions. Protein was extracted using RIPA buffer containing a cocktail of proteinase inhibitors and quantified with a Bio-Rad protein assay. An equal amount of protein was separated on SDS-polyacrylamide gels in a Tris/SDS buffer system and then transferred onto nitrocellulose membranes. Blotting was performed according to standard procedures with primary antibodies overnight, followed by incubation with appropriate fluorescence-conjugated secondary antibodies. The proteins of interest were analyzed using an Odyssey IR scanner (LI-COR Biosciences). Signal intensities were quantified using NIH Image/J software (National Institutes of Health).

The data were presented as the means ± SEM. Comparisons of multiple groups were performed by ANOVA followed by the Bonferroni post-hoc test. A P < 0.05 was considered statistically significant.

We first determined if curcumin has nephroprotective action in a mouse model of kidney ischemia-reperfusion injury (IRI). BALB/c mice were treated i.p. with curcumin or equivalent normal saline (NS). Then, we detected the damage in the kidneys by assessing serum creatinine, urea nitrogen and histological injury. Renal dysfunction was induced as evidenced by a marked elevation of serum creatinine and urea nitrogen at 24 h after IR treatment. Curcumin significantly alleviated renal damage, as serum creatinine and urea nitrogen were markedly lower than those observed in mice in the NC group (Figures 1A,B). Consistent with the serum creatinine and urea nitrogen results, there was a substantial change in the level of histological injury in the IR treated kidneys, with less injury of the tubular epithelial cell in the curcumin group compared with the NC group (Figure 1C). The tubular damage score was markedly decreased in IR-treated kidneys of the curcumin group compared to the NC group (Figure 1D). These data indicate that curcumin may have a nephroprotective effect in mice with IR-induced AKI.

A host of evidence has indicated that apoptosis of renal tubular epithelial cells is an important manifestation of IR-induced AKI (Gao et al., 2012). Thus, the extent of apoptosis in IR-treated tubular epithelial cells was detected in both curcumin and NC treated mice. Figure 2A provides the amount of apoptotic tubular cells in kidneys via terminal transferase dUTP nick end labeling (TUNEL) assay. The results showed that IR treatment induced a large number of apoptotic cells in kidneys. TUNEL cells were markedly decreased in IR-treated kidneys of the curcumin group (Figure 2B). Bax and C-caspase-3 have been used to evaluate the level of apoptosis in a number of studies because the two proteins are sensitive to apoptosis. They are the most commonly used indices of apoptosis. Thus, we evaluated apoptosis in kidney tubules cells by Bax and C-caspase-3 protein expression using Western blotting (Figures 2C–E). The results showed that IR treatment led to upregulation of Bax and C-caspase-3 protein expression in the kidneys of mice, while the expression of Bax and C-caspase-3 were markedly decreased in kidneys of mice receiving i.p. curcumin compared to NC treatment. These data indicated that curcumin could reduce apoptosis in kidneys of mice with IR-induced AKI.

The role of adiponectin (APN) in ischemia of some important organs has been reported, and the Akt signaling pathway was found to be a downstream signaling pathway of APN involved in a variety of pathological processes (Cheng et al., 2013). Considering that the adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) is an adapter protein that positively mediates adiponectin signaling, we examined the expression of APPL1 and phosphorylated Akt (p-Akt) in kidneys of mice via immunohistochemical staining. Significantly increased APPL1 and p-Akt positive cells were observed in the kidneys of IR-treated mice compared to sham controls. Curcumin treatment significantly increased the expression of APPL1 and inhibited the activation of Akt in the kidneys after IR treatment (Figure 3). These results suggested that APPL1 and the Akt signaling pathway could play an important role in the pathogenesis of IR-induced AKI.

To further confirm whether curcumin mediated APPL1/Akt protects against IR-induced AKI by inhibiting apoptosis, the effect on the apoptosis of renal tubular epithelial cell treated with HR in vitro with curcumin and the role of the APPL1/Akt signaling pathway were verified. First, we observed the expression of APPL1 and phosphorylated Akt via Western blot in our curcumin treated HR model of renal tubular epithelial cells (Figures 4A,C). The results showed that HR induced increased expression of APPL1 and activation of Akt; curcumin treatment significantly upregulated the expression of APPL1 and inhibited the activation of Akt. Downregulation of APPL1 gene expression could directly result in the activation of Akt, while AZD5363 could specifically inhibit Akt phosphorylation (Figures 4B,C).

The effect on TUNEL cells treated with HR with curcumin is presented in Figure 5A. Our results indicated that TUNEL cells induced by HR were significantly increased and that curcumin treatment can significantly alleviate TUNEL cell induction by HR. In addition, APPL1 siRNA could weaken the inhibition of apoptosis induced by curcumin, while inhibition of Akt could decrease the effect of APPL1 siRNA (Figure 5B). The effect on HR-treated renal tubular epithelial cells administered curcumin using flow cytometry analysis was demonstrated in Figure 6A. The apoptosis rates during the early period (Figure 6B), later period (Figure 6C), and the total period are shown (Figure 6D). In agreement with the TUNEL staining, the results of flow cytometry analysis indicated that curcumin augmented APPL1/Akt signaling to inhibit the apoptosis of renal tubular epithelial cells treated with HR.

To provide additional supporting evidence, we also evaluated the effect on the apoptosis of renal tubular epithelial cells treated with IR and curcumin by assessing Bax and C-caspase-3 protein expression via Western blot (Figures 7A,C). The results showed that Bax and C-caspase-3 protein expression were elevated in renal tubular epithelial cells treated with HR, while Bax and C-caspase-3 protein expression were markedly decreased in renal tubular epithelial cells treated with curcumin. Downregulation of APPL1 expression could reduce the inhibition of apoptosis with curcumin treatment, and inhibition of Akt could weaken the effect of APPL1 siRNA (Figures 7B,C). All the original pictures can be seen in the Supplementary Material.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewers AS, VP and handling Editor declared their shared affiliation.