Emodin Ameliorates High Glucose-Induced Podocyte Apoptosis via Regulating AMPK/mTOR-Mediated Autophagy Signaling Pathway

To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro. MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation. Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.

Research has shown that the basic level of autophagy in podocytes is significantly higher than that in other intrinsic glomerular cells, and high level of autophagy is necessary to maintain the normal physiological function of podocytes. (6) The high level of autophagy activity of podocytes is conducive to the degradation or removal of damaged proteins and aging organelles for maintaining cell homeostasis. Apoptosis is a programmed method of gene regulation and biological autonomy for maintaining a constant number of cells. Studies have demonstrated that autophagy is closely related with apoptosis in the development and progression of DN. (7,8) Apoptosis of podocytes is present in early stage of DN, and autophagy activity is signifi cantly increased when the podocytes are damaged. (9) Therefore, exploring the relationship between podocyte autophagy and apoptosis may provide an important therapeutic strategy for drug treatment of DN.
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active anthraquinone constituent that extracted from the rhizome of rhubarb Rheum offi cinale Baill. (10) It has been shown that emodin possesses various pharmacological properties, (11) including anti-bacterial, anti-infl ammation, immunosuppressive, antiproliferation, anticancer and antioxidant activities, etc. Rhubarb preparations have been widely used in clinical treatment of DN. Previous studies have shown that the treatment mechanism of emodin in DN may be related to the inhibition of cell proliferation and inflammatory response. (12,13) Recent study has shown that emodin can improve the damage of DN by regulating autophagy signaling pathway. (14) The pathogenesis of DN is related to nutrition sensitive pathways such as AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), (15) autophagy is positively regulated by AMPK, but negatively regulated by mTOR. (16) AMPK activation leads to the phosphorylation and activation of tuberous sclerosis complex 1/2 (TSC1/2) complex, which can indirectly inhibit the activity of mTOR by suppressing the activity of Rheb enzyme. It can also directly phosphorylate a subunit of mTORC1, raptor, to inhibit mTOR and enhance autophagy. It has been reported that emodin is an effective AMPK activator, (17) and can also regulate mTOR pathway. (18) Our in vivo study shows that emodin ameliorates podocyte injury in DN rats by regulating AMPK/mTORmediated autophagy signaling pathway. (19) To further verify the protective effect of emodin on podocytes of DN, we conducted experiments in vitro and the effects and molecular mechanisms of emodin on podocyte injury induced by high glucose was investigated.

Cell Culture
The conditioned immortalized mouse podocytes (MPC5) cells were kindly provided by Prof. YUAN Jun, Department of Nephrology, the Affiliated Hospital of Hubei University of Chinese Medicine, which were obtained from Professor Peter Mundel Laboratory (Mount Sinai Medical Center, New York, USA). Undifferentiated podocytes were cultured in RPMI 1640 medium (Hyclone, Thermo Fisher, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) containing 10 U/mL IFN-γ, 100 U/mL penicillin G and 100 μg/mL streptomycin (Gibco USA) at 33 ℃ under an atmosphere of 5% CO 2 , and induced to differentiate supplemented with 10% FBS without IFN-γ at 37 ℃ and 5% CO 2 for 10-14 days in RPMI-1640 medium. The differentiated podocytes were used in subsequent experiments.

Evaluation of Viable Cells
The number of viable cells was assessed by trypan blue exclusion. MPC5 cells were incubated in medium containing normal glucose (5.5 mmol/L) and different concentrations of HG (20, 40, 80, 160 mmol/L), or 40 mmol/L HG with emodin (2, 4, 8 μmol/L) for 48 h and were subjected to phase-contrast microscopy (magnification, ×200), then the viable cells were collected and resuspended in the trypan blue solution (0.4%), fi nally the number of viable cells were counted under a light microscope with a hemacytometer. At least 3 independent experiments were conducted.

Cell Proliferation Assay
The cell counting kit-8 (CCK-8, C0037, Beyotime Biotechnology, Shanghai, China) was used to detect cell viability according to the manufacturer's instructions. The differentiated MPC5 cells (5×10 3 cells per well) were seeded into 96-well plates and incubated with 5% CO 2 at 37 ℃. After the cells proliferated to 70%-80% fusion in the plate, subsequently, the cells were divided into several groups and treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L) medium respectively. After 48 h treatment, CCK-8 and serumfree RPMI 1640 medium were mixed at a ratio of 1:10, and then the cells were incubated for 2 h. The absorbance values of each well were measured at 450 nm using a microplate reader (SpectraMax i3x, Molecular Devices, Shanghai, China)，the relative ratio was used to refl ect the proliferation rate.

Flow Cytometric Analysis of Apoptosis
The apoptosis of MPC5 cells was assessed by flow cytometry (Becton Dickinson, USA) using the Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, KGA108, Nanjing, China) according to the manufacturer's protocol. Cells were incubated in medium containing normal glucose (5.5 mmol/L) and 40 mmol/L HG with or without 100 nmol/L Rap for 48 h, and then MPC5 cells were collected and resuspended in binding buffer. Subsequently, cells were incubated with Annexin V-FITC (5 μL) and PI (5 μL) for 15 min. (20) The percentage of Annexin V-FITC and PI-stained cells was calculated using Accuri C6 software (Becton Dickinson, USA).

Western Blot Analysis
Podocytes were collected and lysed with RIPA lysis buffer (Beyotime, Hainan, China). Samples were obtained via centrifugation at 13,000×g and 4 ℃ for 5 min. The supernatants were boiled at 100 ℃ for 5 min in loading buffer. Lysate protein concentrations were determined by bicinchoninic acid (BCA) protein concentration assay kit (Beyotime Biotechnology, P0012, Shanghai, China). Equal amounts of protein were separated using 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then was electrophoretically transferred to polyvinylidene fluoride membrane (Millipore, Bedford, USA). The membranes were blocked with 5% skimmed milk at room temperature for 1 h, and then incubated overnight at 4 ℃ with primary antibody as follows: antirat AMPK (Cat. No. ab80039), anti-rat p-AMPK (Cat. No. ab23875), anti-rat β-actin (Cat. No. ab227387) antibodies were purchased from Abcam Ltd, HKSP, New Territories, HK, China; anti-rat LC3 Ⅰ/Ⅱ (Cat. No. 12741), anti-rat mTOR (Cat. No. 2983), anti-rat p-mTOR (Cat. No. 5536), anti-rat cleaved caspase-3 (Cat. No. 9661) antibodies were obtained from Cell Signaling Technology Company, Beverly, MA, USA; anti-rat caspase-3 (Cat. No. BM4620) antibodies were purchased from Boster Biological Technology Co., Ltd., Wuhan, China; Horseradish peroxidase (HRP)conjugated anti-rabbit immunoglobulins and anti-mouse immunoglobulins (KPL Company, USA) were used as the secondary antibody. The membranes were coated using HRP-labeled chemiluminescent substrates (Millipore, Bedford, USA), eventually exposed, and fixed in the dark box. This procedure was carried out 3 times. The results were quantified using Image-Pro Plus 6.0 software (Media Cybernetic, Washington, USA), which were contrasted with densitometric signal of β-actin, respectively, and the ratios were expressed as the relative protein contents.

Statistical Analysis
The data were analyzed using statistical software SPSS 24.0 (SPSS Inc., Chicago, Illinois, USA). Significant differences were evaluated using oneway ANOVA with Bonferroni post-hoc test (GraphPad Prism 6.0, La Jolla, CA, USA). A P-value less than 0.05 was statistically signifi cant.

HG Induces Apoptosis in Podocytes
Results of CCK-8 analysis revealed that the proliferation rate of cells was gradually decreased when HG concentration is higher than 5 mmol/L ( Figure 1A). Additionally, HG resulted in concentration-dependent cell apoptosis, and cells treated with 40 mmol/L HG showed obviously apoptosis (P<0.01, Figure 1B).
Western blot analysis showed that after 12-h treatment with 40 mmol/L HG, the expression of cleaved caspase-3 increased (P<0.01, Figure 1C). After 48 h of treatment, the results of flow cytometry revealed that the proportion of apoptotic cells increased after 40 mmol/L HG treatment (11.4% early apoptotic cells and 1.69% late apoptotic cells; P<0.01, Figure 1D).

Figure 1. High Glucose Induces Apoptosis in Podocytes
Notes: A: MPC5 cells were treated with HG (2.5-160 mmol/L) for 48 h, cell proliferation rate was detected by CCK-8 assay. B: MPC5 cells were treated with HG (20-160 mmol/L) for 48 h and were subjected to phase-contrast microscopy (magnifi cation, ×200). C: MPC5 cells were treated with 40 mmol/L HG at different time points and subjected to Western blot analysis of caspase-3 and cleaved caspase-3 expressions. D: MPC5 cells were exposed to 40 mmol/L HG for 48 h and Annexin V and PI assay was used to assess apoptosis. P<0.01

Effect of Emodin on Podocyte Apoptosis Induced by HG
The results indicate that 40 mmol/L HG-treated cells for 48 h exhibited abundant cellular apoptosis that was markedly attenuated by 4 μmol/L emodin treatment (P<0.01, Figure 2A). Under phase-contrast microscopy, 40 mmol/L HG treatment decreased the number of viable cells, which was obviously reversed by 4 μmol/L emodin ( Figure 2B). Additionally,

Emodin Induces Autophagic Activity in MPC5 Cells
As shown in Figure 3A, when MPC5 cells were exposed to 40 mmol/L HG at 0, 1, 2, 3, 6, 12 h, LC3-Ⅱ markedly increased and reached a maximum level at 1 h (P<0.01). Similarly, MPC5 cells were treated with 4 μmol/L emodin, the level of LC3-Ⅱ was also increased at 1 h (P<0.01, Figure 3B). Under the fl uorescence microscope, when MPC5 cells were treated with 4 μmol/L emodin for 1 h, the bright fluorescent particles increased significantly, whereas cells without emodin treatment showed a diffuse distribution of red fl uorescence ( Figure 3C), which indicated an increase in the formation of autophagosomes.

Autophagy Protects Podocytes from Apoptosis Induced by HG
Western blot analysis showed that autophagic  Figure 4A). Subsequently, cells were exposed to 40 mmol/L HG in the absence or presence of 100 nmol/L Rap for 48 h and were subjected to morphological observation, the results showed that Rap treatment can signifi cantly attenuate the apoptosis induced by HG ( Figure 4B). Additionally, cells were similarly exposed to 40 mmol/L HG with or without 100 nmol/L Rap for 48 h. Flow cytometric analysis revealed that when Rap was added, the percentages of early and late apoptotic cells was all decreased (P<0.01, Figure 4C). Finally, Western blot analysis showed that similar to the effects of 100 nmol/L Rap, 4 μmol/L

Emodin Induces Autophagy by Regulating the AMPK/mTOR Signaling Pathways
When MPC5 cells were exposed to 4 μmol/L emodin at 0, 1, 2, 3, 6 h, the phosphorylation of AMPK was signifi cantly increased, especially at 1 h, while the phosphorylation of mTOR was markedly suppressed, which also most obviously at 1 h (P<0.01, Figure 5A). As shown in Figure 5B, when MPC5 cells were exposed to 4 μmol/L emodin for 1 h, pmRFP-tagged LC3-transfected cells exhibited increased punctate have suggested that HG could induce apoptosis in glomerular podocytes, (22,23) in this experiment, apoptosis of podocyte and expression of pro-apoptotic protein cleaved caspase-3 increased signifi cantly with 40 mmol/L HG. After emodin intervention, all these indice were signifi cantly reversed, which indicates that emodin has protective effect on podocyte injury.
Autophagy participates in organelle metabolism and bioenergy supply through degrades long-lived proteins and organelles, which can maintain the stability of the cell environment. (24) Under normal physiological conditions, the basic level of autophagy exists in almost all cells and plays an important role in cell growth, proliferation, and death. Studies have reported that in the pathophysiological process of the kidney, autophagy is closely related to the intrinsic cells of the kidney, such as podocytes and renal tubular epithelial cells. (25,26) There are also studies reported that autophagy has appeared in diabetic kidney injury, (27) renal ischemia-reperfusion injury, (28) and toxic kidney injury, (29) indicating that autophagy may be involved in a variety of kidney diseases. Under normal conditions, podocytes maintain a certain level of autophagy; in our experimental results, a few autophagosomes were found in podocytes cultured with basic concentration of HG (5.5 mmol/L). LC-3, known as microtubule associated protein light structures, while these number of punctate structures was significantly decreased when 10 μmol/L compound C was added (P <0.01). Western blot showed that 4 μmol/L emodin treated cells for 48 h signifi cantly increased the ratio of LC3-Ⅱ/LC3-Ⅰ, while the ratio was signifi cantly decreased when 10 μmol/L compound C was added (P<0.01, Figure 5C).

DISCUSSION
DN has now gradually become a major cause of end stage renal disease. However, the completely effective treatment is still limited. Increasingly studies show that Chinese medicine treatment can delay the progression of DN through variety mechanisms, (21) including antioxidation, anti-infl ammation, immune regulation, anti-fi brosis, etc. In the present investigation, emodin, a bioactive substance found in rhubarb, which increased autophagy and suppressed HG-induced podocyte apoptosis, might exert protective effects via inhibiting podocyte apoptosis and promoting cell autophagy in DN.
Podocytes were highly differentiated glomerular epithelial cells and located on the surface of GBM, which play a key role in maintaining the structure and function of the glomerular fi ltration barrier. The loss and impairment of podocytes is a major cause of nephrotic proteinuria and glomerular sclerosis, which lead to the initiation and progression of DN. (4) Previous studies  (30) is synthesized in cells and located in the cytoplasm. In the process of autophagy, LC-3 type Ⅰ is modifi ed by ubiquitin-like system, covalently combined with phosphatidylethanolamine, and located on the autophagosome membrane to form LC-3 type Ⅱ. The inversion of the relative expression ratio of type Ⅰ and type Ⅱ can be used to indicate the activity of autophagy. (31,32) In this experiment, the results of Western blot showed that the ratio of LC3-Ⅱ/LC3-Ⅰ was signifi cantly increased at 1 and 6 h after HG and emodin treatment, indicating that autophagy activity was enhanced. The results indicated that autophagy has a self-stabilizing effect and plays a protective role in podocyte damage, and emodin may reduce HG-induced podocyte apoptosis by enhancing autophagy.
Autophagy is regulated by two main nutrientsensing pathways, ie., mTOR and AMPK. (33) Rapamycin, the activator of autophagy, which has been reported to activate autophagy by inhibiting mTOR signaling pathway, (16) was used to verify the protective effect of autophagy on HG-induced podocyte apoptosis. mTOR is a target protein of rapamycin, which can regulate cell growth and autophagy. In the condition of adequate nutrition or without stress, mTOR is activated and autophagy is inhibited; however, mTOR activity is inhibited and autophagy pathway is activated when the cells in a stress state or starvation environment under nutritional deficiency. (34) Under stress conditions, rapamycin can specifically bind to mTOR and inhibit the protein kinase activity of mTOR, thus inducing autophagy. (15) In this study, Rap was used to interfere with MPC5 cells. It was found that rapamycin treatment could increase the ratio of LC3-Ⅱ/LC3-Ⅰ. Our previous studies have shown that emodin can regulate mTOR pathway, (19,20) in the present experiment, the results showed that the ratio of LC3-Ⅱ/LC3-Ⅰ also increased significantly, which indicated that emodin can induce autophagy analogue to rapamycin. Emodin treatment also increased the autophagy fluorescence granules, which further confi rmed that emodin can induce autophagy.
AMPK pathway is one of the upstream pathways of mTOR. Activation of AMPK can inhibit mTOR and enhance autophagy. It has been reported that emodin is an effective AMPK activator, (17) our results showed that the expression of p-mTOR protein was signifi cantly down-regulated and the expression of p-AMPK was upregulated with the prolongation of emodin intervention time, which most obvious at 1 h. Therefore, emodin may induce autophagy in MPC5 cells by regulating AMPK/mTOR signaling pathway. Compound C is a well-known AMPK inhibitor, (35) when cells were treated with emodin alone or in combination with compound C, autophagy fl uorescence granules increased by emodin was obviously suppressed when added compound C. Similarly, Western blot results showed that emodin can increase the ratio of LC3-Ⅱ/LC3-Ⅰ, which reversed by compound C. It was further confi rmed that emodin might regulate AMPK/mTOR signaling pathway.
In conclusion, experimental results indicated that emodin could induce autophagy in HG-treated MPC5 cells and revealed the underlying mechanism of emodin against HG-induced podocyte apoptosis, that involved induction of autophagy through the AMPK/ mTOR pathway. This study confirmed that emodin ameliorates HG-induced podocyte apoptosis and provides additional evidence in support of the clinical usage of emodin in the treatment of DN.