Abstract
Autophagy plays an important role in the maintenance of podocyte homeostasis. Reduced autophagy may result in limited renal cell function during exposure to high glucose conditions. In this study we investigated the effects of ursolic acid (UA) on autophagy and podocyte injury, which were induced by high glucose.
Conditionally immortalized murine podocytes were cultured in media supplemented with high glucose and the effects of the PI3K inhibitor LY294002 and UA on protein expression were determined. miR-21 expression was detected by real-time RT-PCR. Activation of the PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related proteins and expression of podocyte marker proteins were determined by western blot. Immunofluorescence was used to monitor the accumulation of LC3 puncta. Autophagosomes were also observed by transmission electron microscopy.
During exposure to high glucose conditions, the normal level of autophagy was reduced in podocytes, and this defective autophagy induced podocyte injury. Increased miR-21 expression, decreased PTEN expression and abnormal activation of the PI3K/Akt/mTOR pathway were observed in cells that were cultured in high glucose conditions. UA and LY294002 reduced podocyte injury through the restoration of defective autophagy. Our data suggest that UA inhibits miR-21 expression and increases PTEN expression, which in turn inhibits Akt and mTOR and restores normal levels of autophagy.
Our data suggest that podocyte injury is associated with reduced levels of autophagy during exposure to high glucose conditions, UA attenuated podocyte injury via an increase in autophagy through miR-21 inhibition and PTEN expression, which inhibit the abnormal activation of the PI3K/Akt/mTOR pathway.
INTRODUCTION
Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes that contributes to end-stage renal disease (ESRD). The pathogenesis of DN is not fully understood, and currently available therapeutics do not effectively reverse the progression of DN. An in-depth study of DN may provide valuable information that is needed to develop targeted therapies. Recent studies have suggested that the impairment of podocytes induces proteinuria in cases of DN, early glomerular sclerosis and ESRD [1]. Podocytes are terminally differentiated cells that, upon injury, form lesions that are difficult to reverse. One major question is how to program podocytes to maintain their ability to respond under stressful circumstances such as organ dysfunction and hypoxia [2].
Autophagy is a process by which overexpressed or damaged proteins are cleared by lysosomes. Autophagy is an important process that maintains cellular integrity and intracellular homeostasis during conditions that lead to metabolic stress. Recent studies have shown that relatively high levels of autophagy occur in podocytes in physiological conditions [3, 4]. One central hypothesis is that altered autophagy in podocytes contributes to the development and progression of DN, which implicates autophagy as a potential therapeutic target [5, 6]. However, the role of autophagy in DN has yet to be studied and defined. The PI3K/Akt/mTOR signaling pathway is known to negatively regulate autophagy during times of cellular stress or nutritional deficiency. Alternatively, PTEN can up-regulate autophagy through negative regulation of the activity of type I PI3K kinase. Studies have also suggested that miR-21 is involved in the pathogenesis of DN via a decrease in PTEN expression, which leads to activation of the Akt/mTOR pathway. Therefore, miR-21 is one of the potential therapeutic targets of DN [7–9].
Currently available renoprotective drugs have safety concerns and/or limited efficacy. Therefore, the identification of new medicinal agents, especially those that are extracted from natural products, offers exciting possibilities for the future development of successful therapeutic agents. Pentacyclic triterpenes are abundant in herbal medicines. Ursolic acid (UA) is a pentacyclic triterpene compound that is present in many plants and has several advantageous properties, including glucose-reduction, anti-inflammatory activity and anti-tumor activity. Recent studies of various animal models have suggested that UA reduces blood glucose levels and oxidative stress in diabetic animals and inhibits the expression of inflammatory cytokines; these activities then exert a protective effect on the renal system [10]. Studies have also suggested that UA inhibits the proliferation of human glioblastoma cell lines and promotes apoptosis via the inhibition of miR-21 expression [11]. However, studies have yet to address whether UA protects renal cells, including podocytes, through the inhibition of miR-21 expression. In this study we investigate whether UA can lead to the recovery of autophagic function in podocytes through the inhibition of miR-21 expression, which leads to up-regulated PTEN expression and decreased abnormal activation of the PI3K/Akt/mTOR pathway. The objective of this study is to elucidate the protective mechanism of UA in podocytes.
MATERIALS AND METHODS
Cell culture
The conditionally immortalized murine podocytes cell (MPC) lines were kindly provided by Dr Perter Mundel (Mount Sinai School of Medicine, New York, NY, USA). Podocytes were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, USA) in the presence of 5% CO2 atmosphere. To sustain podocytes proliferation, the culture medium was supplemented with 10 U/mL recombinant murine interferon-γ (PeproTech, Rocky Hill, NJ, USA) and the cells were cultured at 33°C. To induce differentiation, cells were shifted to 37°C in interferon-γ-free medium for 7–14 days. All experiments were performed in differentiated podocytes.
Western blot analysis
After treatment, the cells were extracted with lysis buffer containing protease inhibitors (150 mM NaCl, 1% NP-40, 0.1% SDS, 2 μg/mL aprotinin, 1 mM phenylmethanesulfonyl fluoride) for 30 min at 4°C. The supernatants were centrifuged at 12 000 g for 15 min at 4°C. The supernatant containing total protein was harvested. The protein concentration was measured using the BCA Protein Kit (CWBiotech, Beijing, China). Aliquots containing 20 μg of proteins were separated by a 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes. The membranes were soaked in blocking buffer (5% skimmed milk) for 2 h. Subsequently, proteins were detected using different primary antibodies overnight at 4°C, then visualized using anti-goat or anti-rabbit IgG conjugated with peroxidase (horseradish peroxidase) (1:10 000; Abcam, Cambridge, MA, USA) for 2 h at room temperature. The primary antibodies were: anti-PTEN (1:300), anti-p85-PI3K (1:800), anti-p-Akt (ser473, 1:800), anti-t-Akt (1:800), anti-p-mTOR (ser2448, 1:500), anti-t-mTOR(1:500), anti-Beclin1 (1:500) and anti-p62/SQSTMI (1:500) were purchased from Cell Signaling technology (Danvers, MA, USA); anti-LC3 (1:800) and anti-synaptopodin (1:800) were purchased from Abcam; anti-podocin (1:300) and anti-nephrin (1:300) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and GAPDH (1:2000, Proteintech, Chicago, IL, USA) was used as an internal control. The EC3 Imaging System (UVP Inc., Upland, CA, USA) was used to catch up the specific bands, and the optical density of each band was measured using Image J software (National Institutes of Health, Bethesda, MD, USA).
Immunofluorescence
Cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After washing with phosphate buffer saline (PBS), cells were permeabilized with 0.2% Triton X-100 for 5 min. After washing with PBS, sections were incubated in a blocking buffer containing 5% bovine serum albumin for 30 min at room temperature, followed by incubation with anti-LC3 (1:200) antibody overnight at 4°C. Secondary antibodies labeled with fluorescein (Alexa Fluor®488, 1:1000, Abcam) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides. Microphotographs of LC3 fluorescence were captured on a wide-field fluorescent microscope (Olympus, Tokyo, Japan). The detection of punctated staining of LC3 from the diffuse staining indicated the formation of autophagosomes.
Transmission electron microscopy
The cells were harvested gently using trypsin–EDTA (Gibco) after 24 h of culture, followed by centrifugation, and the floating cells were collected. The cells were washed twice with cold PBS and fixed in 2% glutaraldehyde. After fixation, the cells were conventionally dehydrated, embedded, sectioned and stained and the formation of autophagosomes was observed using transmission electron microscopy (TEM). During TEM study, 10 random fields were captured in a grid (top left, middle left, bottom left, center, top right, middle right and bottom right) and the numbers of autophagosomes and cells were counted per field.
Real-time RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Waltham, MA, USA). The PCR reverse primer and real-time PCR reactions of mature miR-21 were performed using Hairpin-it™ miRNAs RT-PCR quantitation kit (GenePharma, Shanghai, China) according to the manufacture's protocols. The specific forward primer is 5′-TCGCCCGTAGCTTATCAG ACT-3′, reverse primer is 5′-CAGAGCAGGGTCCGAGGTA-3′ and probe sequence is 5′-CGCTCTGGACCCGACTCAACA-3′. The following reaction conditions were used: pre-denaturation at 95°C for 3 min, followed by 40 cycles, 95°C for 12 s and 62°C for 40 s. The miRNA expression data were normalized to U6 snRNA.
The PCR reverse primer for PTEN was performed using a PrimerScript™RT reagent kit (Takara Bio, Shiga, Japan) according to the manufacturer's protocols. The sequences of primer pairs were as follows: PTEN (forward) 5′-TGGATTCGACTTAGACTTGACCT-3′ and (reverse) 5′-GCGGTGTCATAATGTCTCTCAG-3′, GAPDH (forward) 5′-AGGTCGGTGTGAACGGATTTG-3′ and (reverse) 5′-GGGGTCGTTGATGGCAACA-3′. Real-time PCR was performed using SYBR Premix Ex Taq™ II (Takara Bio). Amplifications were carried out in a total volume of 20 μL and cycled 40 times after initial denaturation (95°C for 30 s) with the following parameters: 95°C for 5 s and 60°C for 30 s. The relative mRNA expression was quantified through a comparison of the cycle threshold (Ct) values. The experimental data were processed using the 2–ΔΔCt method: ΔΔCt = (Cttarget − Ctinternal control) experiment group − (Cttarget − Ctinternal control) normal control group. Each experimental group was repeated three times.
Statistical analysis
All experiments were repeated at least three times independently. The quantitative data are presented as mean ± SEM. Statistical analysis was performed using the Statistical Package for Social Science (SPSS) 17.0 software (SPSS, Chicago, IL, USA). Comparisons between multiple groups were made using one-way analysis of variance (ANOVA). Pair-wise comparisons were performed using the t-test. A P-value <0.05 was considered significant.
RESULTS
UA treatment ameliorates podocyte injury induced by high glucose
Podocytes were cultured in medium with a high concentration of glucose (25 mmol/L) and were treated with UA at different concentrations (1, 2.5, 5 and 7.5 μmol/L) for 24 h. To evaluate the effect of UA treatment on podocyte injury caused by high glucose, we investigated the expression of the podocyte markers synaptopodin, podocin and nephrin in the absence or presence of UA under high glucose conditions. The expression of all three proteins was decreased in podocytes that were exposed to high glucose (P < 0.01). Protein expression was not significantly altered in podocytes under conditions of high osmotic pressure, which suggested that podocyte injury induced by exposure to high glucose was not due to increased osmotic pressure. Importantly, UA treatment increased the expression of podocyte marker proteins in a dose-dependent manner (P < 0.05). We chose 5 μmol/L as the most appropriate concentration of UA to use in the remainder of the study because the most significant change in protein expression was observed at this concentration (Figure 1A and C). We also investigated the protein expression in cultured podocytes that were treated with UA over 48 h. Significant decreases in the expression of these proteins were observed in podocytes under high glucose conditions for 12 h (P < 0.01). UA treatment up-regulated the expression of podocyte marker proteins at 24 h, and no additional increase in the expression of these proteins was observed after 48 h of treatment; thus, all further experiments were conducted for 24 h (P < 0.01) (Figure 1B and D).
UA attenuated podocyte injury induced by high glucose. (A) Podocytes were cultured in normal glucose (NG, 5.5 mmol/L glucose), mannitol (MA, 5.5 mmol/L glucose + 19.5 mmol/L mannitol), high glucose (HG, 25 mmol/L glucose) or high glucose with ursolic acid (HG + UA, 25 mmol/L glucose + 1.0, 2.5, 5 and 7.5 μmol/L UA) for 24 h. (B) Podocytes were cultured in high glucose (25 mmol/L glucose) with 5 μmol/L ursolic acid for 0, 12, 24 and 48 h. Western blot analysis of synaptopodin, podocin, nephrin and GAPDH protein expression. (C and D) Quantitative analysis of synaptopodin, podocin and nephrin protein expression. Data are presented as the mean ± SEM of three experiments. #P < 0.01 versus NG, *P < 0.05 versus HG, **P < 0.01 versus HG.
High glucose inhibits autophagy, which can be restored with UA treatment
LC3II and Beclin1 are known biomarkers that are expressed during the formation of autophagosomes. Their expression is positively correlated with the level of autophagy. In addition, p62/SQSTMI is a biomarker of the degradation of autolysosomes. Impaired autophagy is often accompanied by the accumulation of p62/SQSTMI, as its expression is negatively correlated with the level of autophagy. To investigate autophagy in podocytes cultured under high glucose conditions, we investigated the expression of LC3II/LCI, Beclin1 and p62/SQSTMI. In podocytes that were cultured under high glucose conditions, the expression of LC3II/LCI and Beclin1 was significantly decreased compared with that in cells that were cultured with normal glucose. Additionally, the expression of p62/SQSTMI increased significantly (P < 0.01). No significant difference was noted in the expression of LC3II/LCI, Beclin1 or p62/SQSTMI between cells that were cultured in normal glucose and cells that were exposed to high osmotic pressure. UA treatment significantly up-regulated the expression of LC3II/LCI and Beclin1 and conversely down-regulated the expression of p62/SQSTMI (P < 0.01) in podocytes that were cultured under high glucose conditions (Figure 2A and B).
UA restored defective autophagy and attenuated podocyte injury induced by high glucose. (A and C) Podocytes were cultured in NG (5.5 mmol/L glucose), MA (5.5 mmol/L glucose + 19.5 mmol/L mannitol), HG (25 mmol/L glucose), 3-MA (2 mmol/L 3-MA), HG + UA (25 mmol/L glucose + 5 μmol/L UA) or HG + UA + 3-MA (25 mmol/L glucose + 5 μmol/L UA + 2 mmol/L) for 24 h. Western blot analysis of LC3II/LCI, Beclin1, p62, synaptopodin, podocin, nephrin and GAPDH protein expression in each group. (B and D) Quantitative analysis of LC3II/LCI, Beclin1, p62, synaptopodin, podocin and nephrin protein expression. Data are presented as the mean ± SEM of three experiments. #P < 0.01 versus NG, **P < 0.01 versus HG.
As a classic inhibitor of autophagy, 3-MA can effectively inhibit this process. In podocytes that were treated with 3-MA, LC3II/LCI and Beclin1 expression decreased and p62/SQSTMI expression increased, these differences were statistically significant (P < 0.01). In podocytes that were pretreated with 3-MA under high glucose conditions, autophagy could not be recovered as a result of UA treatment (P < 0.01) (Figure 2A and B). Additionally, UA could not induce the up-regulation of synaptopodin, podocin or nephrin expression in 3-MA-treated podocytes (P < 0.01) (Figure 2C and D).
The number of endogenous LC3 puncta increases in UA-treated podocytes
To further evaluate autophagy in podocytes under high glucose conditions, we measured LC3 puncta by fluorescence microscopy as a positive indicator of autophagy. In podocytes that were cultured with normal glucose, LC3 (green) was localized in a diffuse pattern in the cytoplasm. No significant difference was observed in the number of LC3 puncta in cells that were cultured in normal glucose, cells that were cultured under high osmotic conditions or in podocytes that were treated with UA. Compared with cells that were cultured with normal glucose, cells that were cultured in either high glucose conditions or were treated with 3-MA demonstrated decreased numbers of LC3 puncta. UA treatment resulted in increased numbers of intracellular LC3 puncta in podocytes that were cultured under high glucose conditions. Pretreatment with 3-MA significantly inhibited the effect of UA on increasing LC3 puncta in podocytes (Figure 3).
UA increased the number of endogenous LC3 puncta in podocytes cultured with high glucose. Podocytes were cultured in NG (5.5 mmol/L glucose), MA (5.5 mmol/L glucose + 19.5 mmol/L mannitol), HG (25 mmol/L glucose), 3-MA (2 mmol/L 3-MA), HG + UA (25 mmol/L glucose + 5 μmol/L UA) or HG + UA + 3-MA (25 mmol/L glucose + 5 μmol/L UA + 2 mmol/L) for 24 h. The bright fluorescent particles (LC3) were visualized by indirect immunofluorescence staining. Magnification, ×400. Bar = 50 μm.
Formation of autophagosomes increases in UA-treated podocytes under high glucose conditions
To further evaluate autophagy, we observed autophagic vacuolization by TEM. The number of autophagosomes (also referred to as initial autophagic vacuoles), which were identified by a double-layer membrane structure containing large cargo such as damaged organelles (e.g. mitochondria, endoplasmic reticulum), in 10 visual fields was counted. As shown in Figure 4, a large number of autophagosomes were detected in normal glucose conditions and high glucose-inhibited podocytes by TEM (P < 0.01). However, UA induced the number of autophagosomes in podocytes that were cultured with high glucose (P < 0.01). 3-MA pretreatment abolished the facilitative effect of UA on autophagosome formation in podocytes (P < 0.01) (Figure 4).
UA increased the number of autophagosomes in podocytes cultured with high glucose. (A) Podocytes were cultured in NG (5.5 mmol/L glucose), HG (25 mmol/L glucose), HG + UA (25 mmol/L glucose + 5 μmol/L UA) or HG + UA + 3-MA (25 mmol/L glucose + 5 μmol/L UA + 2 mmol/L) for 24 h. TEM micrographs demonstrated the presence of autophagosomal structures in podocytes. Autophagosomes with a typical double-layer membrane structure containing undigested organelle remnants are indicated by the arrows. Magnification, ×4000. Bar = 2 μm. (B) The number of autophagosomes was indicated in 10 randomly selected cells. #P < 0.01 versus NG, **P < 0.01 versus HG, †P < 0.01 versus HG + UA.
miR-21 expression is reduced by UA treatment
Compared with cells that were cultured with normal glucose, cells that were maintained under high glucose conditions demonstrated increased miR-21 expression and decreased PTEN expression. Similarly, the phosphorylation of PI3K, Akt and mTOR was increased. Likewise, LC3II/LCI and Beclin1 expression was decreased and p62/SQSTMI expression was increased. All differences in expression were statistically significant (P < 0.01). However, high osmotic conditions did not have a significant effect on the expression of any of these proteins. UA treatment resulted in a significant down-regulation of miR-21 expression and up-regulation of PTEN expression. The phosphorylation of PI3K, Akt and mTOR was also decreased in podocytes that were treated with UA. Similarly, LC3II/LCI and Beclin1 expression increased and p62/SQSTMI expression was decreased in UA-treated cells (P < 0.01 for each protein investigated). As predicted, the PI3K inhibitor LY294002 only inhibited the phosphorylation of PI3K, Akt and mTOR, thus LC3II/LC3I expression increased and p62/SQSTMI expression decreased, but did not affect the expression of miR-21 or PTEN (Figures 5 and 6).
UA inhibited the HG-induced up-regulation of miRNA-21 mRNA expression and down-regulation of PTEN mRNA expression. (A and B) Podocytes were cultured in NG (5.5 mmol/L glucose), MA (5.5 mmol/L glucose + 19.5 mmol/L mannitol), HG (25 mmol/L glucose), HG + LY (25 mmol/L glucose + 2 mmol/L LY294002) and HG + UA (25 mmol/L glucose + 5 μmol/L UA) for 24 h. Real-time RT-PCR analysis for miRNA-21 and PTEN mRNA expression in each group. #P < 0.01 versus NG, **P < 0.01 versus HG.
UA regulated PTEN expression and inhibited PI3K/Akt/mTOR activation in podocytes cultured with high glucose. (A and C) Podocytes were cultured in NG (5.5 mmol/L glucose), MA (5.5 mmol/L glucose + 19.5 mmol/L mannitol), HG (25 mmol/L glucose), HG + LY (25 mmol/L glucose + 2 mmol/L LY294002) or HG + UA (25 mmol/L glucose + 5 μmol/L UA) for 24 h. Western blot analysis of PTEN, p85-PI3K, p-Akt, t-Akt, p-mTOR, t-mTOR, LC3II/LCI, Beclin1, p62 and GAPDH protein expression in each group. (B and D) Quantitative analysis of PTEN, p85-PI3K, p-Akt, t-Akt, p-mTOR, t-mTOR, LC3II/LCI, Beclin1 and p62 protein expression. Data are presented as the mean ± SEM of three experiments. #P < 0.01 versus NG, **P < 0.01 versus HG.
DISCUSSION
UA has received much attention due to its benefits, which include anti-hyperglycemic, anti-hyperlipidemic, anti-inflammatory and anti-oxidative properties, and its potential application for the treatment of type 2 diabetes and associated complications [10, 12–16]. UA treatment can inhibit the STAT3-ERK1/2-JNK pathway in rats with DN and can reduce extracellular matrix deposition and glomerular hypertrophy, which are induced by streptozocin [13]. However, whether UA treatment can reduce the injury on podocytes that are cultured under high glucose conditions has not yet been reported. Our data suggest that high glucose conditions induced podocyte injury with a diminution in the expression of podocyte marker proteins. UA was able to impede the progression of high glucose-induced podocyte injury after 24 h of treatment.
Several studies have suggested that autophagy is a key pro-survival protective mechanism that maintains podocyte homeostasis [17–21]. Both in vitro and in vivo studies have suggested that the extent of autophagy decreases in podocytes in cases of DN, which prevents the clearance of damaged intracellular proteins. Additionally, ER stress and altered mitochondrial function increase when autophagy decreases. Collectively, decreased autophagy aggravates kidney damage and increases urinary albumin excretion [3, 22]. The recovery of autophagy restores mitochondrial morphology, significantly reduces albumin excretion and attenuates DN-associated damage to podocytes and renal tissue [19, 23, 24]. Taken together, these preclinical studies indicate an impairment of autophagy under conditions of experimental DN and that recovery of autophagy mitigates renal damage. Moreover, evidence of impaired autophagy has also been observed in kidney biopsy samples from obese patients with type 2 diabetes, who exhibit accumulation of p62/SQSTM1 protein in their proximal tubular cells. This suggests that an obesity-mediated deficiency in autophagy also occurs in human type 2 diabetes [25]. However, whether UA ameliorates podocyte injury induced by high glucose in an autophagy-dependent manner has not been addressed. Because autophagy is essential for the maintenance of the integrity of podocytes, our study suggests that the defective basal level of autophagy caused by high glucose seems to be involved in high glucose-induced podocyte injury. Importantly, UA treatment attenuates defective autophagy in podocytes under conditions of high glucose, and the expression of podocyte maker proteins was restored. We observed the inhibition of autophagy by an inhibitor (3-MA), the beneficial effect of UA on the restoration of autophagy and that podocyte injury was reversed. Our study suggests that UA ameliorates podocyte injury caused by high glucose through the restoration of autophagy.
The PI3K/Akt/mTOR signaling pathway negatively regulates autophagy. In contrast, PTEN increases autophagy via the inhibition of PI3K activation. PTEN dephosphorylates the 3′-carboxyl group of PIP3, which leads to the formation of PIP2 [26–28]. The inhibition of the PTEN/Akt/mTOR signaling pathway in tumor cells results in an increased level of autophagy, reduced cell growth and an induction of apoptosis, which provides an effective treatment modality for tumors [29–31]. However, the role of the PTEN/Akt/mTOR signaling pathway in the regulation of autophagy in renal cells after UA treatment has not been evaluated. Our study suggests that high glucose down-regulated PTEN expression, which might be associated with the activation of PI3K/Akt/mTOR signaling and reduced autophagy. Importantly, UA treatment restored PTEN expression, which thus inhibits the abnormal activation of the PI3K/Akt/mTOR signaling pathway and attenuates the autophagic deficiency caused by high glucose. Additionally, LY294002, which inhibits abnormal activation of the PI3K/Akt/mTOR signaling pathway, restored autophagy to basal levels but did not alter PTEN expression; this suggests that UA increases autophagy in podocytes under high glucose conditions through a PTEN/Akt/mTOR-dependent mechanism.
During the development of DN, decreased PTEN expression results in activation of the PI3K/Akt/mTOR signaling pathway and induces a morphological change in podocytes [32]. Cytoskeletal proteins rearrange and urinary albumin excretion increases [33]. Our study supports these observations, and the result may be related to autophagic deficiency caused by high glucose, but a specific mechanism has not yet been identified. miRNAs negatively regulate the expression of target proteins at the transcriptional level by binding to the 3′-UTR of the target mRNA. Several miRNAs have been implicated in the progression of DN. miRNA-26a, miRNA-216, miRNA-217 and miRNA-21 activate the Akt signaling pathway via the inhibition of PTEN, which results in a decrease in the survival of glomerular mesangial cells and the induction of mesangial cell hypertrophy [7, 34–36]. In addition, deposition of the matrix proteins fibronectin and type I collagen also increases [7, 35], as does urinary albumin excretion [36]. Previous studies have suggested that UA induces apoptosis of tumor cells through inhibition of miR-21 expression in human glioblastoma cell lines [11]. Additional studies have suggested that UA ameliorates fibrosis and hypertrophy induced by transverse aortic constriction in cardiac muscle cells through inhibition of miR-21 expression [37]. However, whether miR-21 is involved in activation of the PTEN/Akt/mTOR signaling pathway in DN and whether UA affects miR-21 expression in renal cells have not yet been addressed. In this study, miR-21 expression was elevated in podocytes that were cultured under high glucose conditions. Likewise, we observed a decrease in PTEN expression and abnormal activation of the PI3K/Akt/mTOR pathway. UA treatment inhibited miR-21 expression, which resulted in increased PTEN expression and decreased activation of the PI3K/Akt/mTOR pathway. Treatment with LY294002 inhibited activation of the PI3K/Akt/mTOR pathway but did not affect the expression of miR-21 or PTEN, which suggests that UA may regulate the PTEN/Akt/mTOR pathway via inhibition of miR-21 expression (Figure 7).
A schematic model of the protective effect of UA on podocytes cultured under high glucose conditions. HG stimulates miR-21 expression, which resulted in the down-regulation of PTEN and activation of the PI3K/Akt/mTOR pathway. UA may inhibit HG-induced cell injury through inhibition of this pathway.
Collectively, our data suggest that high glucose results in increased miR-21 expression, decreased PTEN expression and activation of the PI3K/Akt/mTOR signaling pathway, which may inhibit autophagy and induce podocyte injury. Our suggestion that UA reduces podocyte injury and restores defective autophagy through an miR-21- and PTEN-dependent mechanism may hold promise as a novel avenue for the treatment of DN.
ACKNOWLEDGEMENTS
This work is supported by National Natural Science Foundation of China (81270808), Science and Technology Plan of Liaoning Provincial Technology Department (2012225019) and Innovation Research Foundation of Immune Dermatology Key Laboratory in Higher Education Institution Grand Science and Technology Program of Liaoning Province (201303). The authors thank Hongzheng Meng for his skillful technical support.
AUTHORS’ CONTRIBUTIONS
Q.F. conceived and designed the experiments; L.X., L.L., Y.Y. and X.L. performed the experiments; L.X., X.W., X.L. and L.W. analyzed the data; X.C., J.L., X.Z. and L.W. contributed reagents/materials/analysis tools and L.X. wrote the paper. All the authors declared no competing interests.
CONFLICT OF INTEREST STATEMENT
None declared.
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