Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that is considered to be a promising target for the treatment of patients with type 2 diabetes. However, the mechanisms underlying the protective effects of GLP-1 on diabetic nephropathy are yet to be fully elucidated. Sirtuin (SIRT)1 encodes a member of the SIRT family of proteins that serves an important role in mitochondrial function and is reported to be associated with the pathogenesis of chronic kidney disease. The present study treated mouse podocytes with various concentrations of D-glucose to establish a high glucose (HG)-induced model of renal injury. The results of a 2′,7′-dichlorodihydrofluorescein diacetate assay, Annexin V/propidium iodide staining and ELISA demonstrated that treatment of podocytes with HG significantly enhanced the production of reactive oxygen species (ROS), promoted cell apoptosis and increased the secretion of proinflammatory cytokines, respectively. The cytokines increased following HG treatment included tumor necrosis factor-α, interleukin (IL)-1β and IL-6. Notably, treatment with GLP-1 attenuated HG-induced increases in ROS production and podocyte apoptosis, which may occur via downregulation of the expression of caspase-3 and caspase-9, and increased expression of nephrin, podocin and SIRT1, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Treatment with GLP-1 led to protective effects in podocytes that were similar to those of resveratrol. Furthermore, SIRT1 knockdown using short hairpin RNA significantly enhanced the expression of caspase-3 and caspase-9 in mouse podocytes, compared with normal mouse podocytes. SIRT1 knockdown with or without GLP-1 administration significantly decreased the expression of caspase-3 and caspase-9 in mouse podocytes, compared with SIRT1 knockdown mouse podocytes. In conclusion, the results of the present study indicated that GLP-1 may be a promising target for the development of novel therapeutic strategies for HG-induced nephropathy, and may function through the activation of SIRT1.
Diabetic nephropathy (DN) is among the most common microvascular complications of type 1 and type 2 diabetes mellitus, and is the major cause of end-stage renal disease globally (
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is secreted by intestinal L-cells, which are enteroendocrine cells present throughout the gastrointestinal tract, from the duodenum to the rectum. GLP-1 has been previously associated with various biological functions (
Sirtuins (SIRTs) are proteins that are members of the silent information regulation-2 family and possess nicotinamide adenine dinucleotide (NAD)-dependent deacetylase activity. Sirtuin (SIRT)1 is involved in the deacetylation of histones and numerous transcriptional regulators, thus regulating diverse biological processes (
In the present study, treatment of podocytes with high glucose (HG) was demonstrated to induce the generation of ROS, promote podocyte apoptosis and enhance the secretion of proinflammatory cytokines, thus indicating that podocyte apoptosis or depletion may represent a novel early mechanism implicated in the pathogenesis of DN. SIRT1 may act as a key mediator of podocyte apoptosis
MPC-5 mouse podocytes were obtained from BeNa Culture Collection (Kunshan, China) and were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100X penicillin-streptomycin solution (100 U/ml penicillin and 100 mg/l streptomycin) and 10 U/ml interferon (IFN)-γ (ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA). Cells were maintained in a humidified atmosphere at 33°C with 5% CO2. Following proliferation to 80% confluence, podocytes were cultured in the aforementioned medium without 10 U/ml IFN-γ and were incubated in a humidified atmosphere at 37°C with 5% CO2 for 10–14 days.
To establish the diabetic injury model, mouse podocytes (1×105 cells/well) were exposed to normal glucose (5.5 mM) or HG (15, 30 and 50 mM) for 48 h. Cells maintained in normal glucose (5.5 mM) were used as the control group. In addition to HG induction, mouse podocytes were simultaneously treated with GLP-1 (10, 100 and 500 nM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or 10 µM resveratrol (RSV; Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China) for 48 h.
A 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe combined with flow cytometric analysis was used to detect alterations in the ROS levels generated in podocytes 48 h after the various treatments, as described previously (
Cell apoptosis was analyzed 48 h after the various treatments using flow cytometry and an Annexin V-FITC Apoptosis Detection kit (C1062; Beyotime Institute of Biotechnology). Briefly, mouse podocytes were plated in 6-well plates at a density of 1×105 cells/well and incubated with 195 µl Annexin V-fluorescein isothiocyanate (FITC) and 5 µl propidium iodide (PI) for 15 min in the dark at 4°C. Analysis of cell apoptosis was subsequently performed using a flow cytometer. The early apoptotic cells are presented in the lower right quadrant of the fluorescence-activated cell sorting (FACS) histograms, and the late apoptotic cells, which were stained with FITC and PI, emitted red-green fluorescence and are presented in the upper right quadrant of the FACS histograms. FACS was performed on an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (version 9.0.2; FlowJo LLC, Ashland, OR, USA).
TNF-α, IL-6 and IL-1β levels present in the culture supernatants of mouse podocytes 48 h after the various treatments were determined using Mouse Interleukin 1β (IL-1β) ELISA kit (PI301; Beyotime Institute of Biotechnology), Mouse Interleukin 6 (IL-6) ELISA kit (KMC0061; Thermo Fisher Scientific, Inc.), Mouse Tumor necrosis factor α (TNF-α) ELISA kit (PT512; Beyotime Institute of Biotechnology), respectively, according to the manufacturer's protocol.
The activity of caspase-3 and caspase-9 was analyzed using Caspase-3 and Caspase-9 Colorimetric Assay kits (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. Briefly, mouse podocytes (1×105 cells/well) 48 h after the various treatments were collected, resuspended in 50 µl chilled cell lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) and incubated on ice for 10 min. Following centrifugation for 1 min at 400 × g at 4°C, the supernatants were transferred to a fresh tube and the protein concentration was assessed using a bicinchoninic acid protein assay kit (PICPI23223; Thermo Fisher Scientific, Inc.). 100 µg protein was diluted in 50 ml cell lysis buffer for each assay. The absorbance of each sample was measured at 405 nm using a Multiskan EX microplate reader (Thermo Fisher Scientific, Inc.).
The pLKO.1 lentiviral vector, psPAX2 packaging plasmid and pMD2G envelope plasmid were obtained from Addgene, Inc. (Cambridge, MA, USA). The SIRT1-targeting short hairpin (sh)RNA (position 516–534: GCG GAT AGG TCC ATA TAC T; forward: CCG GGC GGA TAG GTC CAT ATA CTT TCT CGA GGA ATA CCT CAT CTT TCC TCT TTT TTT C; reverse: AAT TGA AAA AAA GAG GAA AGA TGA GGT ATT CCT CGA GAA AGT ATA TGG ACC TAT CCG CGG CCT) or a scramble shRNA sequence (AGA GCT ATC GGC ATC ATG T; forward: CCG GAG AGC TAT CGG CAT CAT GTT TCT CGA GGA ATA CCT CAT CTT TCC TCT TTT TTT C; reverse: AAT TGA AAA AAA GAG GAA AGA TGA GGT ATT CCT CGA GAA ACA TGA TGC CGA TAG CTC TGG CCT; Sangon Biotech Co., Ltd., Shanghai, China) was cloned into the pLKO.1 lentiviral vector using Agel I and Ecol I restriction enzymes. The pLKO.1 lentiviral vector with scramble shRNA was used as the negative control (NC). 293T cells (American Type Culture Collection, Manassas, VA, USA) at the density of 1×105 cells/well cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) were seeded in 60-mm culture dishes 37°C and, after 24 h, they were co-transfected with 1 µg pLKO.1-SIRT1-shRNA or pLKO.1-NC-shRNA, and 0.1 µg psPAX2 and 0.9 µg pMD2G, using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent, according to the manufacturer's protocol. The recombinant lentiviral particles were collected 48 h post-transfection and were used to infect mouse podocytes. Mouse podocytes (1×105 cells/well) cultured in RPMI-1640 medium 10% fetal bovine serum were infected with 2 µl lentivirus at a multiplicity of infection of 20 in the presence of 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 4–6 h at 37°C. Sequencing was performed by Shanghai Majorbio Pharmaceutical Technology Co., Ltd. (Shanghai, China) to confirm the recombinant virus.
Total RNA was extracted from podocytes 48 h after the various treatments using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using an AMV reverse transcriptase kit (Fermentas; Thermo Fisher Scientific, Inc.) for 60 min at 37°C, 5 min at 85°C and 5 min at 4°C. qPCR was performed on cDNA using SYBR-Green 10X Supermix (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 45 sec, and a final extension step of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. The sequences of the primers that were used in the present study were: SIRT1 forward, 5′-TGACGCTGTGGCAGATTG-3′ and reverse, 5′-CAAGGCGAGCATAGATACCG-3′; nephrin forward, 5′-GGACCCACACTACTACTC-3′ and reverse, 5′-CTCTCCACCTCGTCATAC-3′; podocin forward, 5′-TTGTTTCCTGGCTCCTTC-3′ and reverse, 5′-TGCCTTGGGACTACTTTC-3′; caspase-3 forward, 5′-CTGACTGGAAAGCCGAAAC-3′ and reverse, 5′-GCAAAGGGACTGGATGAAC-3′; caspase-9 forward, 5′-GTGAAGAACGACCTGACTG-3′ and reverse, 5′-GCATCCATCTGTCCCATAG-3′; and GAPDH forward, 5′-ATCACTGCCACCCAGAAG-3′ and reverse, 5′-TCCACGACGGACACATTG-3′. GAPDH was used the internal control for normalization. Relative gene expression was calculated using the 2−ΔΔCq method (
Total proteins were isolated from mouse podocytes 48 h after the various treatments in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing 0.01% protease and phosphatase inhibitor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The protein concentration was assessed using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Proteins (20–30 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics GmbH, Mannheim, Germany). Following blocking with 5% skimmed milk at 4°C overnight, the membranes were incubated at 4°C overnight with the following primary antibodies: Anti-SIRT1 (ab28170; 1:1,000), anti-nephrin (ab58968; 1:500), anti-podocin (ab181143; 1:2,000), anti-caspase-3 (ab44976; 1:500), anti-caspase-9 (ab2013; 1:1,000) and anti-GAPDH (5174; 1:2,000). All primary antibodies were purchased from Abcam (Cambridge, MA, USA). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (A0208, A0181, A0216; 1:1,000; Beyotime Institute of Biotechnology) for 1 h at 37°C. Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA) and blots were semi-quantified by densitometry using Quantity One software version 4.5.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by Tukey's post-hoc test was performed to statistically analyze the differences among groups. P<0.05 was considered to indicate a statistically significant difference.
It is established that ROS are implicated in DN (
To determine the effect of GLP-1 on HG-induced apoptosis in mouse podocytes, flow cytometry was performed. Treatment with HG (15, 30 and 50 mM) induced a significant dose-dependent increase in apoptotic cells (
To investigate whether GLP-1 may counteract HG-induced alterations in SIRT1 expression in mouse podocytes, RT-qPCR and western blot analysis were performed. The results demonstrated that 30 mM glucose induced a significant decrease in the mRNA and protein expression of SIRT1, compared with cells maintained under normal glucose conditions (
To determine whether HG treatment may induce podocyte depletion, the expression of podocyte-specific markers, including nephrin and podocin, was assessed using RT-qPCR and western blot analysis. As presented in
RT-qPCR and western blot analysis were performed to assess the mRNA and protein expression of caspase-3 and caspase-9 in podocytes. The results demonstrated that 30 mM glucose significantly enhanced the mRNA and protein expression of caspase-3 and caspase-9, compared with cells maintained under normal glucose (
To determine the effect of GLP-1 on HG-induced inflammatory responses in mouse podocytes, ELISA assays were performed to detect the levels of proinflammatory cytokines secreted by podocytes. The results demonstrated that treatment with 30 mM glucose induced a significant increase in the levels of TNF-α, IL-1β and IL-6 secreted by mouse podocytes (
To investigate the roles of SIRT1 in the protective effects of GLP-1 in mouse podocytes, SIRT1 expression was silenced in mouse podocytes following infection with a pLKO.1-SIRT1-shRNA recombinant lentiviral vector. The results demonstrated that the mRNA and protein expression of SIRT1 in mouse podocytes following transduction with the pLKO.1-SIRT1-shRNA vector was significantly decreased by 81.1±0.03 and 60.3±0.03%, respectively (
In the present study, the protective effects of GLP-1 against cellular damage were investigated in HG-treated mouse podocytes
One of the major biomarkers for the prediction of DN progression is a reduced density of podocytes (
Based on
Inflammatory responses are an important factor contributing to renal injury in HG-induced DN (
The present study demonstrated that SIRT1 expression was downregulated in HG-treated mouse podocytes, while it was upregulated following GLP-1 treatment in a dose-dependent manner. RSV has been reported to exert protective effects against numerous diseases, including diabetes, neurodegenerative disorders, cognitive disorders, cancer, kidney diseases and cardiovascular diseases, through the activation of SIRT1 (
In conclusion, to the best of our knowledge, the present study demonstrated for the first time that GLP-1 may exert protective effects against HG-induced damage in mouse podocytes, as indicated by the suppression of HG-induced ROS production, apoptosis and inflammatory responses, through the activation of SIRT1
The present study was supported by the Youth Scientific Research Funds of Changhai Hospital in 2014 (grant no. CH 201508).
Effects of GLP-1 on oxidative stress in HG-treated mouse podocytes. (A) Mouse podocytes were treated with various concentrations of glucose and the ROS levels were measured using the DCFH-DA fluorescent probe combined with flow cytometry. (B) Mouse podocytes were treated with 30 mM glucose in the absence or presence of various concentrations of GLP-1 and the ROS levels were measured using the DCFH-DA fluorescent probe combined with flow cytometry. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal group; ##P<0.01 vs. HG group. GLP, glucagon-like peptide; HG, high glucose; ROS, reactive oxygen species; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate.
Effects of GLP-1 on HG-induced apoptosis in mouse podocytes. (A) Mouse podocytes were treated with various concentrations of glucose and cell apoptosis was assessed using Annexin V/PI staining followed by flow cytometry. (B) Mouse podocytes were treated with 30 mM glucose in the absence or presence of various concentrations of GLP-1 and cell apoptosis was assessed using Annexin V/PI staining followed by flow cytometry. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal group; ##P<0.01 vs. HG group. GLP, glucagon-like peptide; HG, high glucose; PI, propidium iodide.
Effects of GLP-1 on SIRT1 expression in HG-treated mouse podocytes. Mouse podocytes were treated with 30 mM glucose in the absence or presence of various concentrations of GLP-1 and the (A) mRNA and (B) protein expression levels of SIRT1 were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal group; ##P<0.01 vs. HG group. GLP, glucagon-like peptide; SIRT, sirtuin; HG, high glucose.
Effects of GLP-1 on the expression of podocyte-specific markers and apoptosis-associated proteins in HG-treated mouse podocytes. (A) Mouse podocytes were treated with 30 mM HG in the absence or presence of 500 nM GLP-1 or 10 µM RSV, and the mRNA expression of SIRT1, nephrin and podocin was determined by RT-qPCR. (B) Protein expression levels of SIRT1, nephrin and podocin were quantified by densitometric analysis following treatment with 30 mM HG in the absence or presence of 500 nM GLP-1 or 10 µM RSV. (C) Representative western blot bands for SIRT1, nephrin, podocin, caspase-3 and caspase-9 are presented. GAPDH was employed as the loading control. (D) Protein expression levels of caspase-3 and caspase-9 were quantified by densitometric analysis. (E) RT-qPCR results for caspase-3 and caspase-9 mRNA expression following treatment with 30 mM HG in the absence or presence of 500 nM GLP-1 or 10 µM RSV. The activity of (F) caspase-3 and (G) caspase-9 in HG-treated mouse podocytes was also measured using colorimetric biochemical assays. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal group; ##P<0.01 vs. HG group. GLP, glucagon-like peptide; HG, high glucose; RSV, resveratrol; SIRT, sirtuin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Effects of GLP-1 on inflammatory responses in HG-treated mouse podocytes. Mouse podocytes were treated with 30 mM glucose in the absence or presence of 500 nM GLP-1 or 10 µM RSV, and the levels of (A) TNF-α, (B) IL-1β and (C) IL-6 secreted by mouse podocytes were measured using ELISA. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal group; ##P<0.01 vs. HG group. GLP, glucagon-like peptide; HG, high glucose; RSV, resveratrol; TNF, tumor necrosis factor; IL, interleukin.
Effects of GLP-1 on caspase-3 and caspase-9 expression in mouse podocytes following SIRT1 knockdown. (A) SIRT1 mRNA expression in mouse podocytes transduced with pLKO.1-SIRT1-shRNA lentiviral vectors was determined by RT-qPCR. (B) Protein levels of SIRT1 in mouse podocytes transduced with pLKO.1-SIRT1-shRNA lentiviral vectors were determined by western blot analysis and quantified by densitometric software. (C) Representative western blot bands for SIRT1 protein expression in control cells and mouse podocytes transduced with NC- or SIRT1-shRNA. (D) Western blot analysis was performed to measure the protein expression of SIRT1, caspase-3 and caspase-9 following transduction of mouse podocytes with NC- or SIRT1-shRNA lentiviral vectors with or without 500 nM GLP-1. (E) Densitometric analysis was performed to quantify the protein expression in different groups. (F) RT-qPCR was performed to measure the mRNA expression of SIRT1, caspase-3 and caspase-9 in NC- or SIRT1-shRNA-transduced mouse podocytes with or without treatment with 500 nM GLP-1. Data are presented as the mean ± standard deviation. For parts A and B, **P<0.01 vs. NC group; for parts E and F, **P<0.01 vs. normal group, ##P<0.01 vs. NC group and ΔΔP<0.01 vs. SIRT1-shRNA group. GLP, glucagon-like peptide; SIRT, sirtuin; shRNA, short hairpin RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Control, untransduced cells; NC, negative control; HG, high glucose; NC, NC-shRNA-transduced cells.