Renal fibrosis is one of the main characteristics of diabetic nephropathy. TGF-β1/Smad3 pathway is expected to reveal the pathogenesis of renal fibrosis in diabetic nephropathy (DN). Pyridoxamine (PM), a natural form of vitamin B6, is a powerful inhibitor of advanced glycation end products (AGEs). PM plays an anti-apoptotic, anti-oxidative stress, and fibrosis role in DN. The purpose of this study was to assess whether PM has a protective effect in renal tubular epithelial and to investigate its possible mechanism.
The effects of PM were investigated in HK-2 cells induced by high glucose. HK-2 cells were administered with PM at a dose of 1 mmol/L. Western blot and Realtime PCR were used to detect the expression levels of renal fibrosis related proteins. The possible mechanism of PM was examined by expression of transforming growth factor-β1 (TGF-β1)/Smad3 pathway.
PM could reduce the expression of Fibronectin (FN) and α-smooth muscle actin (α-SMA) induced by high glucose. PM could also affect the activity of TGF-β1/Smad3 pathway in HK-2 cells. FN and α-SMA were up-regulated by overexpression of Smad3 for 48 h. After adding PM, the levels of FN and α-SMA are significantly decreased.
Our findings indicate that PM showed a protective effect in HK-2 cells through the inhibition of TGF-β1/Smad3 pathway.
- diabetic nephropathy
- renal fibrosis
- TGF-β1/Smad3 pathway
The pathological changes of diabetic nephropathy (DN) are mainly manifested as glomerular stromal hyperplasia, glomerular basement membrane thickening, glomerular sclerosis, renal tubular atrophy, and interstitial fibrosis. Tubulointerstitial fibrosis is the common outcome in chronic kidney diseases (CKD), including diabetic nephropathy. Renal fibrosis is a reliable predictor of prognosis and also a major determinant of renal insufficiency . Renal fibrosis is one of the main characteristics of diabetic nephropathy, which is manifested as glomerular mesangial cell hyperplasia hypertrophy, extra-cellular matrix (ECM) excessive deposition, and interstitial fibrosis. It leads to the formation of scar tissue and the production of proteinuria .
Transforming growth factor-β1 (TGF-β1) has various biological characteristics, including cell proliferation, differentiation, apoptosis, autophagy, and ECM production. Previous researches have shown that TGF-β1 is one of the key factors during the progress to renal fibrosis, activates the downstream ways Smad3, and then increases the secretion of ECM collagen . Similarly, the role of TGF-β1/Smad3 pathway has been concerned in diabetic nephropathy. Smad3 is an important factor which causes renal fibrosis in diabetic nephropathy. In diabetic nephropathy rat models, the progression of renal fibrosis was prevented by Smad3 knocked out . Smad3 directly binds to the collagen gene promoter region, and then promotes renal interstitial fibrosis, inhibits ECM degradation, and reduces the activity of MMP-1 in fibroblasts cells . Further studying the role of TGF-β1/Smad3 pathway is expected to reveal the pathogenesis of renal fibrosis in diabetic nephropathy.
The pathogenesis of diabetic nephropathy is very complex. To explore the effective drugs for prevention and treatment of DN have always been focused on. The production of advanced glycation end products (AGEs) is increased in diabetic nephropathy. The expression of AGEs receptor (RAGE) is up-regulated to activate AGE-RAGE, and then many pathways such as mitogen-activated protein kinase (MAPK) and Smad are activated to participate in the process of renal fibrosis . Pyridoxamine (PM), a natural component of vitamin B6, is an inhibitor of AGEs and removes carbonyl compounds directly . In diabetic nephropathy rat models, PM alleviated the progression of diabetic nephropathy by reacting with precursors of AGEs . PM can inhibit cell apoptosis and down-regulate the expression of fibrotic molecules by reducing levels of AGEs-RAGE . In summary, PM plays an anti-apoptotic, anti-oxidative stress, and fibrosis role in DN by reducing the level of AGE. Finally, PM improves renal function and reduces renal damage. But the specific mechanism needs to be further improved. In this study, we took HK-2 cells as the subjects. The changes of fibrosis-related proteins FN and TGF-β1/Smad3 pathway in HK-2 cells under a high glucose environment were observed. Then, Smad3 plasmid was further applied to explore whether PM could play a protective role by inhibiting the TGF-β1/Smad3 pathway in delaying renal fibrosis.
The HK-2 cells were purchased from American Type Culture Collection (Manassas, USA). Smad3 antibody was obtained from Abcam (Cambridge, UK). Antibodies for FN, α-SMA, TGF-β1, and β-actin were obtained from Proteintech (Chicago, USA). Reverse Transcription System and qPCR Master Mix were purchased from Vazyme (New Jersey, USA). TRIzol reagent was purchased from Thermo Fisher (Carlsbad, USA). d-glucose and PM were obtained from Sigma (St. Louis, USA).
The HK-2 cells were cultured in DMEM (Invitrogen, CA, USA) with 10% fetal bovine serum (FBS) under 5% CO2 and 95% air at 37°C. The HK-2 cells were stimulated with DMEM containing 5.5 mmol/L glucose (NG group) or 30 mmol/L glucose (HG group) and PM (1 mmol/L) plus HG medium for 48 h. After all treatments, the cells were harvested and then washed twice with PBS.
After treatments, HK-2 cells were washed twice with PBS. Total cell protein was extracted with RIPA lysate buffer. The RIPA lysate buffer was mixed with protease and phosphatase inhibitor. Then the lysate mixture was added to the cells at 4°C for 40 min. The cells were scraped off with a cell scraper. The cells were collected into a 1.5 mL centrifuge tube, incubated on ice for about 10 min, centrifuged at 12,000 rpm for 20 min, and centrifuged at 4°C. The protein content was determined according to the instructions of the BCA protein quantitative kit. Then protein samples were denatured in the SDS sample buffer at 100°C for 7 min and were stored at −80°C until further experimentation.
About 30 μg of total protein was separated on SDS-PAGE gels (10% or 12%) and transferred to PVDF membranes. Then the membranes were blocked with 5% skim milk for 1 h at 37°C and incubated with primary antibodies at 4°C overnight. Primary antibodies used in this study were: anti-FN (1: 1000), anti-TGF-β1 (1: 1000), anti-Smad3 (1: 1000), and anti-α-SMA (1: 1000). Next day, they were washed with TBST for 15 min three times and incubated with secondary antibodies (1: 2000). The membranes were detected by the Odyssey Fc System (LICOR, USA) with an ECL detection reagent.
Total cell RNA was isolated with TRIzol. The cDNA was synthesized with cDNA synthesis kit according to the manufacturer's protocol. The primers used in the study were as follows: TGF-β1 (forward: 5′-AGCGACTCGCCAGAGTGGTTA-3′ and reverse: 5′-GCAGTGTGTTATCCCTGCTGTCA-3′). FN (forward: 5′-CACCCTCACCAACCTCA-3′ and reverse: 5′-C CTCGGAACATCAGAAAC-3′), α-SMA (forward: 5′-CGGGAC ATCAAGGAGAAACT-3′ and reverse: 5′-CCATCAGGCAACTC GTAACTCT-3′), Smad3 (forward: 5′-GAGCCTGGTCAAGAAA CTCAA-3′ and reverse: 5′-CTCTGGTAGTGGTAGGGATTCA C-3′), and β-actin (forward: 5′-TGACGTGGACATCCGCAA AG-3′ and reverse: 5′-CTGGAAGGTGGACAGCGAGG-3′). β-actin was used as an internal control. The PCR cycling conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. PCR was performed on an Agilent Mx3000P QPCR Systems (Agilent, CA, USA).
The target nucleotide sequences of Smad3 plasmid are as follows: forward: 5′-TAATACGACTCACTATAGG-3′ and reverse: 5′-TAGAAGGCACAGTCGAGG-3′. Cells were seeded in 6-well plates for 12 h and then transfected with Smad3 plasmid for 12 h using transfection reagent Lipo3000 and P3000. Then the cells were cultured with different medium at indicated time points. The level of Smad3 post-transfection was assessed at 48 h by western blot or Realtime-PCR.
Data results were expressed as the mean ± standard deviation. Data were analyzed using SPSS software version 22.0. The differences among groups were analyzed using one-way ANOVA, followed by the Student–Newman–Keuls (SNK) test for multiple comparisons.
To study whether PM could protect HK-2 cells from fibrosis under a high glucose environment, we tested the protein and mRNA levels of FN and α-SMA via western blot and Realtime-PCR. Compared with the NG group, the protein and mRNA levels of FN and α-SMA sharply increased in the high glucose (HG) group. However, they were significantly restored by PM (1 mmol/L) (
TGF-β1/Smad3 is one of the important signal pathways for renal fibrosis. In order to observe whether PM could affect its activity, the levels of TGF-β1 and Smad3 were detected using Western blot and Realtime-PCR. Compared with the NG group, HG significantly increased the expression of TGF-β1 and Smad3. Compared with the HG group, the levels of TGF-β1 and Smad3 in the HG + PM group were decreased (
In order to investigate whether the TGF-β1/Smad3 pathway was involved in the protective effect of PM on renal tubular epithelial cells, Smad3 plasmids were transfected. After over-expression Smad3, the effection of PM on the expression of fibrosis involved protein in HK-2 cells was observed. As shown in
Renal fibrosis is a common pathological feature of CKD. It is mainly manifested by the structural alteration of renal tissue and the loss of renal function caused by excessive deposition of ECM . Fibrosis is the main characteristic of renal tubular interstitial damage in diabetic nephropathy. TGF-β1 is the main mediating factor of DN . TGF-β1 promotes the overproduction of ECM, and leads to tubulointerstitial fibrosis . In this study, the results suggest that PM has an effect against HG-induced fibrosis in HK-2 cells. PM could decrease the expression of FN and α-SMA in HK-2 cells under a high glucose environment. PM could also inhibit the activity of the TGF-β1/Smad3 signaling pathway. After adding PM, the expressions of TGF-β1 and Smad3 in HK-2 cells were significantly down-regulated. As overexpression Smad3, the expression of FN and α-SMA were significantly up-regulated. As adding PM, the upregulation of FN and α-SMA caused by overexpression Smad3 could be partially eliminated. This illustrates that PM achieves protective effects through inhibiting the TGF-β1/Smad3 signaling pathway.
Renal fibrosis is a common pathway for the development of CKD. TGF-β1 is an important fibrogenic factor, which is secreted by mesangial cells and renal tubular epithelial cells. Currently, Smads are believed to be the only kinase substrate of TGF-β1 receptor. They mediate the downstream intracellular signal transduction, thus induce renal cell hypertrophy, and promote the thickening of glomerular basement membrane. They promote the development of glomerulosclerosis and tubulointerstitial fibrosis [13, 14]. TGF-β1 promotes the transformation of epithelial and endothelial cells into myofibroblasts. Its inhibitors or antagonists can block the TGF-β1/Smad3 signal pathway, and alleviate or reverse the process of epithelial–mesenchymal transformation or endothelial–mesenchymal transformation . Previous studies have shown that Smad2 and Smad3 are extensively activated in renal tissues with CKD and in animal models . Smad2 knockout enhances Smad3-mediated fibrosis in renal tubular epithelial cells, which is associated with increased Smad3 phosphorylation and nuclear translocation . Diabetic nephropathy is a complication of diabetes, and its renal fibrosis is mainly mediated by TGF-β1 through Smad-dependent or non-dependent signal pathways .
PM, a natural form of vitamin B6, is a powerful inhibitor of AGEs. It inhibits the generation of AGEs by interfering with glycation reactions and directly scavenges oxygen free radicals, which plays an important role in antioxidant stress . It has attracted attention due to its therapeutic effect on diabetic complications [8, 18]. It has been shown that PM can reduce glomerular hypertrophy, mesangial matrix hyperplasia, and proteinuria in diabetic db/db maternal mice . PM activates the formation of autophagosome in podocytes by inhibiting mTOR and transcription factor EB (TFEB) nuclear translocation . PM also plays an important role in fighting against renal fibrosis. Previous studies have shown that, in IR-AKI mouse model, treatment with PM reduced the severity of renal injury, decreased postinjury fibrosis, and improved long-term functional recovery after AKI . In this study, we found that PM significantly down-regulated the expression of FN and α-SMA in HK-2 cells under high glucose. The activities of TGF-1/Smad3 pathway were also down-regulated. It suggested that PM had an inhibitory effect on the process of fibrosis in HK-2 cells induced by high glucose. In order to further confirm whether TGF-β1/Smad3 pathway was involved in the protective effects of PM, Smad3 plasmid was used. Here, we observed that FN and α-SMA were up-regulated after Smad3 overexpression for 48 h. After adding PM, the level of FN and α-SMA was significantly decreased. Our findings indicated that PM showed a protective effect in HK-2 cells through the inhibition of TGF-β1/Smad3 pathway.