Introduction
Diabetic nephropathy (DN) is a common and debilitating complication of diabetes (1). According to the United States renal data system, DN is the leading cause of end-stage renal disease and a major contributor to morbidity and mortality in patients with diabetes worldwide (2). Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are prominent features of DN (3).
Under physiological conditions, MCs are located between glomerular capillary loops and secrete extracellular matrix, produce cytokines, support the glomerular capillary plexus, phagocytize and clear macromolecular substances and contract (4). In the presence of hyperglycemia and other stimulating factors, MCs proliferate abnormally and secrete a large amount of extracellular matrix, which is deposited in the mesangial area (5). MCs can migrate to the gap between glomerular capillary endothelial cells and the basement membrane and even protrude into the surrounding capillary cavity, resulting in capillary occlusion and ultimately leading to glomerulosclerosis (6). MC proliferation serves a key role in the process of DN, but the mechanism of MC proliferation in diabetic patients is complex. It is generally believed that the kidneys of diabetic patients produce too much reactive oxygen species (ROS) due to stimulation of the mitochondrial respiratory chain by long-term hyperglycemia (7). Low levels of ROS serve roles in signal transduction and proliferation, which may be one of the important mechanisms of the abnormal proliferation of MCs. In addition, transforming growth factor β, connective tissue growth factor, angiotensin II and other cellular growth factors are also involved in the abnormal proliferation of MCs (8,9). Studies have also shown that a decrease in the level of hydrogen sulfide (H2S) serves an important role in the proliferation of MCs (10).
H2S has been traditionally viewed as a toxic gas. However, few individuals realize that H2S is also a biological gas that is endogenously produced by the cytosolic enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), which use homocysteine and L-cysteine as substrates (11). H2S can not only regulate inflammation, oxygen sensing, cell growth and ageing but also protect a variety of organs from ischemia-reperfusion injury, serving an important biological role in vivo (12). There is abundant endogenous H2S in kidney tissues (13). H2S can increase the glomerular filtration rate, inhibit the reabsorption of sodium by the renal tubules and inhibit the proliferation of glomerular MCs (10,14).
Dopamine receptors (DRs) have seven transmembrane domains and belong to the G protein coupled receptor superfamily (15). According to their different effects on adenylate cyclase, DRs are divided into two subfamilies: DR1 (D1, D5) and DR2 (D2, D3, D4) (16). DR1 binds to Gs protein to activate adenylate cyclase and stimulate phospholipase C, resulting in an increase in the intracellular free calcium concentration [(Ca2+)i]; DR2 inhibits adenylate cyclase and calcium channels (17). DRs are mainly distributed in the central nervous system and in the cardiovascular system, kidneys and adrenal glands. DR1 is also expressed and serves an important role in glomerular MCs (18).
It has been reported that H2S can inhibit the proliferation of MCs (10). Yang et al (19) found that CSE is physiologically activated by calcium-calmodulin, which increases the production of endogenous H2S in vascular endothelial cells. Our research group reported that DR1 activation increases the (Ca2+)i in myocardial ischemia-reperfusion injury (20). However, it is not clear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. To examine this possibility, the present study performed animal and cellular experiments and measured changes in related factors and signaling pathways.
Materials and methods
Materials and drugs
Mouse renal MC lines (SV40-MES13) were purchased from Shanghai Cell Bank of Chinese Academy of Science. SKF38393 (DR1 agonist), sodium hydrogen sulfide (NaHS), PPG (a CSE inhibitor), PD98059 (an ERK1/2 inhibitor), streptozocin (STZ) and 7-Azido-4-Methylcoumarin (H2S probe) were purchased from Sigma-Aldrich (Merck KGaA). The primary antibodies for anti-CSE, cyclin D1, PCNA, P21, COL1, α-smooth muscle actin (α-SMA), and MMP9 were from ProteinTech Group, Inc. DR1 antibody was purchased from GeneTex, Inc. The total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies were obtained from Cell Signaling Technology, Inc. The Cell Counting Kit-8 (CCK-8) kit and the primary antibody for β-actin were obtained from Wuhan Boster Biological Technology, Ltd. The EdU kit was obtained from Guangzhou RiboBio Co., Ltd. The BCA Protein Assay kit and Enhanced Chemiluminescence (ECL) reagent were purchased from Beyotime Institute of Biotechnology.
Animal use and experimental design
At total of 48 C57BL/6J (male:female 50:50; 6-weeks-old; 20-22 g) mice were provided by the Experimental Animal Center of Second Affiliated Hospital in Harbin Medical University. All experiments were approved by the Animal Care Committee of Harbin Medical University for the use of experimental animals (approval no. SCXK2013-001). All animals were given free access to normal chow and water and were housed in cages at room temperature with 40-70% humidity and a fixed 12-h light/dark cycle.
After animal adaptation to the environment for 1 week, male and female C57BL/6J (6-weeks-old, 20-22g) mice were made diabetic by a single injection of streptozotocin (STZ; 150 mg/kg, intraperitoneally). The STZ was dissolved in 100 µM citrate buffer (citric acid: sodium citrate=1:1.32). After 3 days, the mice with glucose levels >16.67 mmol/l were considered hyperglycemic (diabetic) (21). The daily water intake and food intake of these diabetic mice were simultaneously recorded and the weekly weight change monitored. At the same time, blood glucose was monitored weekly to eliminate mice whose blood glucose had returned to <16.67 mmol/l.
Briefly, the experimental groups were as follows (n=8): i) Control group (Control); the mice were injected with the same volume of physiological saline solution daily. ii) Diabetes group (T1D); the diabetic mice were injected with the same volume of physiological saline solution daily. iii) Diabetes + SKF38393 group (T1D + SKF38393); the diabetic mice were injected with SKF38393 (50 µg/kg) daily. iv) Diabetes + NaHS group (T1D + NaHS); the diabetic mice were injected with NaHS (100 µM/kg) daily. According to the results of pre-experiment and published papers, a dose of 100 µM/kg/day NaHS was used in present study (11,22). All drugs were dissolved in physiological saline solution and all the above mice were injected intraperitoneally. After the treatment reached 4, 8 and 12 weeks, the mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and then the chest was opened and the kidneys, hearts, livers and arteries of mice were removed for related experimental research.
Renal morphology assessment
Kidneys were dehydrated in a series of alcohols and then embedded. Tissue sections from paraffin-embedded kidneys with a 4 µm thickness were stained with hematoxylin and eosin and Masson's trichrome at room temperature for 3 min following deparaffinization. Staining was conducted to assess renal morphology and collagen accumulation to estimate the progression of diabetic nephropathy. Images of random fields under a microscope (Olympus Corporation) were captured by an individual blinded to the grouping.
Transmission electron microscopy
Renal tissues (1 mm3) were immersed immediately in fixative (2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate, pH 7.2). Following 2-3 days of storage at 4°C, specimens were rinsed in PBS, postfixed in cacodylate-buffered 1% osmium tetroxide, dehydrated in an ethanol series, and embedded in polybed 812. Ultra-thin (90 nm) sections were made with microtome, and ultrastructural changes of glomerular basement membrane were observed under an electron microscope. The thickness of glomerular basement membrane was quantified using ImageJ software (National Institutes of Health; version 1.48).
Measurement of H<sub>2</sub>S levels in the kidney
H2S production rate was measured as described previously (23). In brief, following different treatments, the kidney tissues were collected and homogenized in 50 mM ice-cold potassium phosphate buffer (pH 6.8). The flasks containing the reaction mixture (100 mM potassium phosphate buffer, 10 mM l-cysteine, 2 mM pyridoxal 5-phosphate and 10% cell homogenates) and center wells containing 0.5 ml 1% zinc acetate and a piece of filter paper (2×2.5 cm) were flushed with N2 gas and incubated at 37°C for 90 min. The reaction was stopped by adding 0.5 ml of 50% trichloroacetic acid and the flasks were incubated at 37°C for another 60 min. The contents of the center wells were transferred to test tubes, each containing 3.5 ml of water. Then 0.5 ml of 20 mM N, N-dimethyl-p-phenylenediamine sulfate in 7.2 M HCl and 0.5 ml 30 mM FeCl3 in 1.2 M HCl were added. The absorbance of the resulting solution at 670 nm was measured 20 min later with an ultraviolet spectrophotometer.
Cell culture and treatment
The SV40-MES13 were cultured in DMEM/F-12 medium (3:1) (cat. no. PM150323; Procell Life Science & Technology Co., Ltd., and cat. no. 31600; Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 14 mM HEPES, 5% fetal bovine serum, 1% penicillin and streptomycin in a 5% CO2 humidified atmosphere at 37°C. For the following experiments, the SV40-MES13 were incubated with serum-free DMEM/F-12 for 12-16 h (the cells reached 70-80% confluence) and then the cells were randomly divided into six groups: i) normal-glucose control group (Control); incubated in DMEM/F-12 medium containing 5.6 mM glucose. ii) high glucose group (HG); incubated in DMEM/F-12 medium containing 30 mM glucose for 48 h. iii) SKF38393 treated group (HG + SKF38393); 10 µM SKF38393 was added to the medium 1 h before HG induction. iv) NaHS treated group (HG + NaHS): 100 µM NaHS was added to the medium 1 h before HG condition. v) PPG and SKF38393 treatment group (HG + PPG + SKF38393); 2 mM PPG and 10 µM SKF38393 were added to the medium 1 h before HG condition. vi) PD98059 treatment group (HG + PD98059): 10 µM PD98059 (an ERK1/2 inhibitor) was added to the medium 1 h before HG condition.
Cell viability assay
Cell viability was measured by Cell Counting Kit-8. Cells were seeded in 96-well plates at a concentration of 1×103 cells/well. Prior to different treatments, the cells were serum starved for 12 h. Subsequently, they were incubated in 10 µl of CCK-8 reagent at 37°C for 1 h. A microplate reader was used to determine the optical density at 450 nm.
Measurement of cell proliferation
5-Ethynyl-20-Deoxyuridine (EdU) incorporation assay was performed with the Cell-Light EdU In Vitro Imaging kit to detect the proliferation rates of SV40-MES13 (21). Briefly, cells subjected to different treatment were incubated with 20 mM EdU in complete growth medium at 37°C for 2 h before fixation in 4% paraformaldehyde at room temperature for 30 min. After EdU staining, cell nuclei were stained with Hoechst 33342 at room temperature for 30 min in a darkroom and visualized under fluorescence microscopy (magnification, ×200). The percentage of EdU-positive cells was calculated from six random fields in three wells.
Detection of H<sub>2</sub>S by 7-Azido-4-Methylcoumarin
The fluorescence response of H2S in MCs was detected using 7-Azido-4-Methylcoumarin (C-7Az; Sigma-Aldrich; Merck KGaA), as described previously (24,25). MCs were incubated with 1 ml PBS (containing 12 µl C-7Az) for 30 min at room temperature and then the cells were washed with PBS. The fluorescence activation response of C-7Az to H2S in MCs were visualized using a microscope (Olympus Corporation) and six fields (magnification, ×200) were randomly selected for statistical analysis.
Western blotting analysis
Western blotting assay was conducted as described previously (12). In brief, cells were lysed and extracted total protein with RIPA lysis buffer pre-mixed with 1 mM PMSF. The protein concentration in each sample was determined using the BCA Protein Assay kit. In each western blot analysis, the same amount of total protein (25-50 µg) from each group was separated by 8-12% SDS-PAGE and electro-transferred to PVDF membranes. The membranes were blocked for 1.5 h at room temperature in TBST (Tris-buffered saline with 0.05% Tween-20, pH 7.4) plus 5% non-fat milk and incubated overnight at 4°C with diluted primary antibodies against DR1 (cat. no. GTX100354, 1:1,000; Gene Tex, Inc.), CSE (cat. no. 12217-1-AP, 1:1,000; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig, 1:5,000; ProteinTech Group, Inc.), PCNA (cat. no. 10205-2-AP, 1:5,000; ProteinTech Group, Inc.), P21 (cat. no. 27296-1-AP, 1:500; ProteinTech Group, Inc.), α-SMA (cat. no. 14395-1-AP, 1:1,000; ProteinTech Group, Inc.), COL1 (cat. no. 14695-1-AP, 1:1,000; ProteinTech Group, Inc.), MMP9 (cat. no. 10375-2-AP, 1:1,1000; ProteinTech Group, Inc.), total (t)-ERK1/2 (cat. no. 4695, 1:5,000; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (cat. no. 4370, 1:5,000; Cell Signaling Technology, Inc.), or β-actin (cat. no. BM0627, 1:1,000; Boster Biological Technology, Ltd.). Secondary goat anti-rabbit antibody and goat anti-mouse antibody were used respectively (diluted 1:5,000, cat. no. BA1054 and cat. no. BA1050; Boster Biological Technology, Ltd.). The secondary antibodies were incubated for 1.5 h at room temperature (25°C). The signals were detected by an ECL kit and a multiplex fluorescent imaging system (ProteinSimple). The integrated optical density of the analyzed bands on the film was quantified using ImageJ software (National Institutes of Health; version 1.48). The levels of analyzed proteins were normalized to the internal control (β-actin).
Statistical analysis
All data are expressed as the mean ± standard error of the mean and represent at least three independent experiments. Statistical comparisons were made using one-way ANOVA followed by a post hoc analysis (Tukey test) where applicable. P<0.05 was considered to indicate a statistically significant difference.
Results
DR1 activation upregulates the CSE/H<sub>2</sub>S pathway in the kidneys of T1D mice
As shown in Fig. 1, hyperglycemia downregulated DR1 and CSE expression and H2S production in the T1D-4 W, T1D-8 W and T1D-12 W groups compared with the control group. Compared with that in the control group, this effect was most evident in the T1D-12 W group. Compared with the T1D-12 W group, SKF38393 (DR1 agonist) treatment significantly increased the expression of DR1 and CSE and the production of H2S. NaHS (an exogenous H2S donor) treatment only increased the expression of CSE and the production of H2S but had no significant effect on the expression of DR1.
DR1 activation inhibits cell proliferation and collagen deposition by upregulating the CSE/H<sub>2</sub>S pathway in the kidneys of T1D mice
The data showed that compared with those in the control group, renal injury was aggravated, including glomerular hypertrophy, collagen deposition in interstitial kidney tissue and the thickness of the glomerular basement membrane were increased in the T1D-4 W, T1D-8 W and T1D-12 W groups. These changes were most significant in the T1D-12 W group. Pretreatment with SKF38393 or NaHS significantly reduced these pathological changes in the T1D-12 W group (Fig. 2).
DR1 activation increases the CSE/H<sub>2</sub>S pathway in HG-induced MCs
As shown in Fig. 3, the levels of DR1, CSE and H2S were lower in the HG group compared with the control group. Compared with those in the HG group, the levels of DR1, CSE and H2S were significantly increased in cells treated with HG + SKF38393. Treatment with HG + NaHS only increased the levels of CSE and H2S but had no significant effect on DR1 levels. These results were consistent with the results of the animal experiments.
Activation of the DR1-CSE/H<sub>2</sub>S pathway attenuates HG-induced MC proliferation
Cell viability, the proliferation rate and the expression of cyclin D1 and PCNA were increased and the expression of P21 was decreased in the HG group compared with the control group. Compared with the HG group, HG + SKF38393 and HG + NaHS reduced cell viability, the proliferation rate and the expression of cyclin D1 and PCNA and increased the expression of P21. PPG (an inhibitor of CSE) abrogated the inhibitory effect of SKF38393 on MC proliferation. In addition, the effect of HG + SKF38393 on these indicators was similar to that of HG + PD98059 (an ERK1/2 inhibitor; Fig. 4).
Activation of the DR1-CSE/H<sub>2</sub>S pathway inhibits HG-induced collagen deposition in MCs
α-smooth muscle actin (α-SMA) and collagen 1 (COL1) expression was significantly upregulated and MMP9 expression was downregulated in the HG group compared with the control group. Compared with that in the HG group, the expression of α-SMA and COL1 was markedly decreased and the expression of MMP9 was significantly increased in the HG + SKF38393 and HG + NaHS groups. These beneficial effects of SKF38393 were blocked by PPG. The effect of SKF38393 on collagen deposition was similar to that of PD98059 (Fig. 5).
Activation of the DR1-CSE/H<sub>2</sub>S pathway downregulates the ERK1/2 pathway in HG-induced MCs
The activity of p-ERK1/2 was significantly higher in the HG group than in the control group. HG + SKF38393 or HG + NaHS significantly decreased p-ERK1/2 activity and similar suppression was observed in the HG + PD98059 group. The inhibitory effect of SKF38393 on p-ERK1/2 activity was abolished by PPG. The activity of t-ERK1/2 remained unchanged in different treatments (Fig. 6).
Discussion
Diabetic nephropathy is one of the leading causes of mortality in individuals with diabetes and is characterized by diffuse or nodular glomerulosclerosis (26). MC proliferation and extracellular matrix deposition are the main causes of glomerulosclerosis (3). Proliferating MCs secrete a large amount of extracellular matrix, resulting in glomerular basement membrane thickening and mesangial expansion (27). Inhibiting the abnormal proliferation of MCs is critical for diabetic patients. The results of the present study showed that the level of H2S and the expression of DR1 and CSE were decreased in vivo and in vitro (Figs. 1 and 3). This finding suggests that the proliferation of MCs is associated with a decrease in DR1 expression and the downregulation of the CSE/H2S pathway. To test this hypothesis and examine the relationship between these factors, SKF38393 (a DR1 agonist) and NaHS (an exogenous H2S donor) were administered. Our previous research showed that SKF38393 could increase the expression and activity of DR1 (20). It has been reported that 120 µM exogenous H2S increased the transcription and expression of CSE, while at concentrations >160 µM, the transcription and expression of CSE were completely inhibited, suggesting that higher levels of H2S may be toxic (28,29). The experimental results of the present study showed that SKF38393 increased the expression of DR1 and CSE/H2S pathway factors, but NaHS only increased CSE/H2S pathway factors and did not affect the expression of DR1. In addition, it was also found that PPG (a CSE inhibitor) abolished SKF38393-mediated enhancement of the CSE/H2S pathway in the pre-experiment (data not shown). These results indicated that DR1 activation inhibited the proliferation of MCs by upregulating the CSE/H2S pathway and that DR1 is an upstream regulatory factor of the CSE/H2S pathway (Figs. 1 and 3).
How can DR1 regulate the CSE/H2S pathway to inhibit MC proliferation? DR1, with seven transmembrane domains, belongs to the G protein-coupled receptor superfamily, which can increase (Ca2+)i (17). A previous study by our research group has also shown that DR1 activation promotes myocardial ischemia-reperfusion injury by increasing (Ca2+)i (20). It has been reported that an increase in (Ca2+)i can activate calmodulin (CaM), upregulate the expression of CSE and promote the production of endogenous H2S (19). Therefore, DR1 increases endogenous H2S production by increasing (Ca2+)i, which inhibits MC proliferation.
The cell cycle is precisely controlled by cell cycle regulatory proteins. Cell cycle regulatory proteins can be divided into positive and negative regulatory proteins according to their effects on the cell cycle. Positive regulatory proteins include cyclins and cyclin-dependent kinases; the negative regulatory proteins are mainly cyclin-dependent kinase inhibitors (30). Cyclins bind with CDK to form cyclin/CDK active complexes, which regulate cell cycle initiation and phase transition. CKIs include the INK4 family and CIP/Kip family. The former includes p16INK4a, p15INK4B, p18INK4C and p19INK4D, which can inhibit the activity of cyclin/CDK complexes in G1 and S phases; the latter includes p21WAF1/CIP1, p27kip1 and p57kip2, which inhibit most cyclin/CDK complexes (31-33). Proliferation-related proteins were measured to explore what caused the inhibition of MC proliferation. The data showed that HG increased the thickness of the glomerular basement membrane, cell viability, proliferation and cyclin D1 and PCNA expression and decreased P21 expression. SKF38393 reduced the thickness of the glomerular basement membrane, cell viability, proliferation and cyclin D1 and PCNA expression and increased P21 expression. This was the same as the effect of directly using NaHS to supplement exogenous H2S in MCs. PPG (a CSE inhibitor) abolished the effect of SKF38393 (Figs. 2 and 4). These results suggested that the DR1-CSE/H2S pathway suppresses cell proliferation by affecting the expression of cell proliferation-related proteins (cyclin D1, PCNA and P21).
Renal fibrosis is the common pathway of chronic kidney disease that leads to end-stage renal failure (26). The deposition of extracellular matrix is the main cause of renal fibrosis. Collagen is the most abundant protein in the extracellular matrix and provides structural support to cells (34). α-SMA is an important marker of the phenotypic transformation of MCs. Positive α-SMA expression in diabetic glomeruli is a sign of impaired MC activation (35). Under physiological conditions, MCs have a contractile phenotype, maintaining a balance between the synthesis and degradation of mesangial matrix (36). However, under continuous hyperglycemia, MCs transform to a synthetic phenotype, secrete a large amount of mesangial matrix such as COL1 to resist traumatic stimulation and reduce the expression of MMP9 (37). To examine the protective effect of the DR1-CSE/H2S pathway on renal fibrosis, changes in the relevant indicators (COL1, α-SMA and MMP9) of renal fibrosis were measured and it was found that HG increased the expression of COL1 and α-SMA and decreased the expression of MMP9. SKF38393 and NaHS reduced COL1 and α-SMA expression and increased the expression of MMP9. PPG abrogated the effect of SKF38393 (Fig. 5). The results of the present study demonstrated that the DR1-CSE/H2S pathway improved renal fibrosis by inhibiting the deposition of collagen and increasing the degradation of extracellular matrix. MMP2 and membrane-type matrix metalloproteinase-1 (MT1-MMP) are also fibrosis markers of diabetic nephropathy (34-37) and these two indicators will be detected in future studies.
A number of signaling pathways can regulate the proliferation of MCs, such as ERK1/2, TGF-β, Wnt/β-Catenin and PI3K-AKT (38-40). ERK1/2 is a major member of the MAPK family that is involved in a number of physiological processes, such as gene expression, mitosis, cell metabolism, survival, apoptosis and differentiation (41). The ERK1/2 pathway activates the cyclin D1 gene, which leads to cell growth, proliferation and differentiation (42). The ERK1/2 pathway becomes activated in MCs cultured under HG conditions (38). The results of the present study showed that HG increased the activity of p-ERK1/2. SKF38393 and that NaHS reduced the activity of p-ERK1/2. PPG reversed the effect of SKF38393 (Fig. 6). The effect of SKF38393 on cell viability, proliferation, proliferation-associated protein expression, fibrosis-associated protein expression and p-ERK1/2 activity was similar to that of PD98059 (Figs. 1-6). This finding suggested that the DR1-CSE/H2S pathway inhibited MC dysfunction by inhibiting ERK1/2 activation. However, it remains to be explored whether the DR1-CSE/H2S pathway also affects other signaling pathways, such as Wnt/β-Catenin, PI3K-AKT, TGF-β and others.
Notably, the roles of SKF38393 (a DR1 agonist), NaHS (an exogenous H2S donor) and PD98059 (an ERK1/2 inhibitor) in MC proliferation were similar, suggesting that these factors inhibit MC proliferation by regulating the ERK1/2 pathway. Whether there are other mechanisms involved will be explored in the future.
In conclusion (Fig. 7), the findings of the present study suggested that: i) MC proliferation and extracellular matrix deposition were associated with decreased DR1 expression and the CSE/H2S pathway. ii) DR1 activation upregulated the CSE/H2S pathway in HG-induced kidney tissues and MCs. iii) The DR1-CSE/H2S pathway inhibited MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway. The present study explored the potential protective mechanism of the DR1-CSE/H2S pathway in diabetic nephropathy and provided a new therapeutic target for antifibrosis strategies against diabetic nephropathy.