Renal tubular cell apoptosis and tubular dysfunction is an important process underlying diabetic nephropathy (DN). Understanding the mechanisms underlying renal tubular epithelial cell survival is important for the prevention of kidney damage associated with glucotoxicity. Curcumin has been demonstrated to possess potent anti-apoptotic properties. However, the roles of curcumin in renal epithelial cells are yet to be defined. The present study investigated advanced glycation or glycoxidation end-product (AGE)-induced toxicity in renal tubular epithelial cells via several complementary assays, including cell viability, cell apoptosis and cell autophagy in the NRK-52E rat kidney tubular epithelial cell line. The extent of apoptosis was significantly increased in the NRK-52E cells following treatment with AGEs. The results also indicated that curcumin reversed this effect by promoting autophagy through the phosphoinositide 3-kinase/AKT serine/threonine kinase signaling pathway. These conclusions suggested that curcumin exerts a renoprotective effect in the presence of AGEs, at least in part by activating autophagy in NRK-52E cells. Collectively, these findings indicate that curcumin not only exerts renoprotective effects, however may also act as a novel therapeutic strategy for the treatment of diabetic nephropathy.
Diabetic nephropathy (DN) previously known as idiopathic nodular glomerulosclerosis is the leading cause of end-stage renal disease. It is characterized by increased blood pressure, increased urinary albumin and glomerular lesions, leading to the loss of glomerular filtration is the main microvascular complications of diabetes (DM) (
Previous study found that AGEs could induce apoptosis and dysfunction of tubular cells may contribute in part to glomerular hyperfiltration, an early renal dysfunction in diabetes, so that late glomerulosclerosis (
Curcumi, AGEs, Triton X-100, DMSO, LY294002 and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DMEM and fetal bovine serum (FBS) were both obtained from HyClone (Thermo Fisher Scientific, Inc., Logan, UT, USA). Anti-GAPDH, anti-bax, anti-AIF, anti-caspase-3 and anti-p-AKT were obtained (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Anti-Beclin 1 and anti-LC3 were both obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). ECL kit was purchased (Pierce; Thermo Fisher Scientific, Inc.). Flow cytometer (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA). All reagents used were trace element analysis grade. All water used was glass distilled.
Rat kidney tubular epithelial cell line NRK-52E was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM; low glucose), supplemented with 10% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin at a density of 6×103 cells/well in six-well culture plates.
We use 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect cell viability. Briefly, the sample medium were added to 10 µl MTT (500 µg/ml) and incubated for 3 h at 37°C following treatment. Subsequently, the MTT solution was removed and 100 µl dimethyl sulfoxide (DMSO) was added to dissolve the colored formazan crystals. The absorbance of each aliquot at 490 nm was measured using a Sunrise microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The cell viability was determined as the ratio of the signal.
We detected apoptosis by Annexin V labeled with FITC. Propidium iodide was used to determine cell necrosis. After exposure to various experimental conditions, cells were trypsinized and labeled with fluorochromes at 37°C, and then cytofluorometric analysis was performed with a FACS can (Becton-Dickinson).
Apoptosis was also evaluated by the TUNEL method. For the TUNEL assays, cells grown on a coverslip were pretreated with various experimental conditions. The TUNEL assay kit was used to detect apoptotic cells under a fluorescence microscope (Leica TCS SPE; Leica Microsystems GmbH, Wetzlar, Germany). After treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized with 0.2% Triton X-100 in citrate buffer. Samples were incubated with TdT and fluorescein-labeled dUTP, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and then observed under a fluorescence microscope (Leica TCS SPE). Percentages of apoptotic cells were estimated by counting 300 cells in random fields.
NRK-52E cells were fixed with 4% paraformaldehyde at 4°C for 30 min, incubated with 0.2% Triton X-100 for 10 min. The cells were blocked at non-specific antibody binding sites by incubating with 10% goat serum in PBS containing 0.3% Triton X-100 and 0.5% bovine serum albumin (BSA) for 30 min at room temperature, followed by incubation with a mouse monoclonal antibody against Beclin 1 and LC3 (1:400 in PBS; Cell Signaling Technology, Inc.) overnight. Then TRITC and FITC-conjugated goat anti-mouse IgG (1:100 in PBS) was used to incubate for 0.5 h at room temperature. Heochst 33342 was added to the cells for 15 min. After washing three times with PBS, cells were visualized under fluorescence microscopy.
The NRK-52E cells were lysed in protein extraction reagent (Tissue Protein Extraction kit; Pierce, Rockford, IL, USA) and the lysates were extracted by centrifugation. Equal amounts of protein were loaded per sample in each experiment, separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. The protein-bound membranes were blocked and washed in Tris-buffered saline (TBS)-Tween-20. The membranes were incubated overnight with primary antibodies. The primary antibodies used in the present study were as follows: anti-GAPDH, anti-bax, anti-AIF, anti-caspase-3 and anti-p-AKT (Santa Cruz Biotechnology, Inc.). Anti-Beclin 1 and anti-LC3 (Cell Signaling Technology, Inc.). After washing in TBS-0.1% Tween-20, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies overnight at 4°C. Finally, the blots were developed using an enhanced chemiluminescence system (Pierce). To compare the levels of proteins, the density of each signal was evaluated by image analysis software (CS Analyzer; ATTO, Tokyo, Japan).
Statistical comparison was made on the differences in mean values among multiple groups by using one-way analysis of variance with post hoc Scheffe's test. Statistical significance was achieved if P-values were <0.05.
The cell viability of NRK-52E cells under AGEs conditions were assessed by MTT. As shown in (
Curcumin, the active ingredient from the spice turmeric (Curcuma longa L.), has been demonstrated recently to possess anti-apoptosis effects (
Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival under various cellular conditions. In this text, we explored the role of autophagy in the cytoprotective effect of curcumin. As above, the NRK-52E cells were treated with 700 µg/ml AGEs with or without 10 µM curcumin for 48 h. After both curcumin and AGEs treatment, the number of LC3 and Beclin 1 fluorescent dots dramatically increased (
To investigate whether the mechanism of curcumin anti-apoptotic effects was the induction of chondrocyte autophagy, 3-Methyladenine (3-MA), a type of potent pharmacological inhibitors on autophagy, was used to suppress the autophagy. As (
Given that PI3K/AKT pathway activation promotes autophagy in different cell lines (
Curcumin as a kind of plant polyphenol extracted from turmeric is the most important active ingredient of turmeric to play the pharmacological effect (
The relationship between autophagy and apoptosis is complex and can be induced by both processes in response to similar stimuli, through which superfluous, damaged, or aged cells or organelles are eliminated (
In conclusion, we have shown the protective potential of curcumin for AGEs-mediated apoptosis. Autophagy caused by the activation of PI3K/AKT pathway plays a significant role in this process. Although the present study is limited to
This study was supported by The International Science and Technology Cooperation Program of China (2010DFB33260; 2010DFB33260).
normal rat kidney tubular epithelial cells
epithelial-to-mesenchymal transition
3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide
Dulbecco's modified Eagle's medium
dimethyl sulfoxide
bovine serum albumin
phosphate-buffered saline
Tris-buffered saline
diabetic nephropathy
advanced glycation or glycoxidation end-products
diabetes mellitus
3-methyladenine
fetal bovine serum
the terminal uridine nick 3′ end labeling
AGEs decreased the cell viability in NRK-52E cells. (A) Cells were treated with 0–2,000 µg/ml AGEs for 24, 48 and 72 h, and the cell viability were detected by MTT. (B) Cells were treated with 300, 700 and 1,000 µg/ml AGEs for 0–72 h, and the cell viability were detected by MTT.
The effect of curcumin on cell apoptosis induced by AGEs. (A) Cells were treated with 700 µg/ml AGEs with or without 10 µM curcumin for 48 h, and the apoptosis was determined by TUNEL analysis. (B) Cells were treated with above, and the apoptosis was determined by flow cytometry followed by Annexin V/PI double staining. Each value represented mean ± SEM (n=6). (C-E) Cells were treated with above, and the expression of bax, AIF and caspase-3 were detected by western blot analysis. Corresponding protein levels were assessed using densitometry and are expressed as relative intensities. GAPDH was used as loading control. Each value represents the mean ± SEM (n=6) (**P<0.01 vs. control; #P<0.01 vs. AGEs group).
Curcumin promoted autophagy in NRK-52E cells. (A) Cells were treated with 700 µg/ml AGEs with or without 10 µM curcumin for 48 h, and the Beclin 1 protein was stained and observed under a fluorescence microscope as described in Materials and methods. (B) Cells were treated with above, and the LC3 protein was stained and observed under a fluorescence microscope as described in Materials and methods. (C and D) Cells were treated with above, and the expression of LC3 and Beclin 1 were detected by western blot analysis. The results were representatives of three independent experiments. GAPDH was used as loading control (**P<0.01 vs. control).
The role of autophagy in the protective effect of curcumin. (A) Cells were treated with 700 µg/ml AGEs, 10 µM curcumin, as well as 2 mM 3-MA for 48 h or a combined treatment of 700 µg/ml AGEs and 10 µM curcumin or 2 mM 3-MA, 700 µg/ml AGEs and 10 µM curcumin for 48 h, and apoptosis was determined by flow cytometry followed by Annexin V-PI double staining. (B and C) Cells were treated aboved, and the expression of Bax and Beclin 1 were detected by western blot analysis. The results were representatives of three independent experiments. GAPDH was used as loading control (**P<0.01 vs. control; #P<0.05 vs. AGEs group; ▲P>0.05 vs. AGEs group).
The role of PI3K/AKT signaling pathway in authphagy. (A) Cells were treated with 700 µg/ml AGEs, 10 µM curcumin, as well as 10 mM LY294002 for 48 h or a combined treatment of 700 µg/ml AGEs and 10 µM curcumin or 10 mM LY294002, 700 µg/ml AGEs and 10 µM curcumin for 48 h, and the expression of p-AKT, Beclin 1 were determined by western blot analysis. (B) The results were representatives of three independent experiments. GAPDH was used as loading control (**P<0.01 vs. control; #P<0.05 vs. AGEs group).