Abstract
Aim:
To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.
Methods:
Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.
Results:
Treatment of the cells with HG, H2O2, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002.
Conclusion:
The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.
Similar content being viewed by others
Introduction
Diabetic nephropathy (DN) is characterized by glomerular hypertrophy, excessive accumulation of extracellular matrix (ECM) and glomerulosclerosis, which ultimately lead to a progressive loss of renal function1. Hyperglycemia is considered to be the first step in initiating DN, in which high glucose (HG) activates multiple intracellular signaling factors and causes target organ injury2. Over-production of reactive oxygen species (ROS) and oxidative stress are the direct consequences of hyperglycemia3,4, and excessive mitochondrial ROS serve as a common upstream mediator of diabetic complications5. Our previous research demonstrated that extracts from herbal medicines could prevent DN progression by scavenging ROS6,7,8.
Nitric oxide (NO) plays an important role in DN progression. There have been reports suggesting that the physiological and morphological changes in DN are mediated by the NO level in the kidneys9,10,11,12. However, there is still disagreement over the effect of hyperglycemia on NO production in renal cells13,14. Mesangial cells (MCs) provide structural support for capillary loops and modulate glomerular filtration, playing a crucial role in maintaining the structure and function of the renal glomerular tufts. Lipopolysaccharide increases the enzymatic activity and protein and mRNA levels of inducible nitric oxide synthase (iNOS) in MCs, suggesting that MCs are a potential source of NO production15,16. Reports from animal experiments show that increased renal NO production is one of the major pathogenic features of early DN17,18,19. However, the molecular mechanism for the effects of hyperglycemia on renal NO production remains unclear.
Hyperglycemia can be mediated through the induction of various growth factors and cytokines. Data from other laboratories20,21,22 and our team6,7,8,23,24,25 have demonstrated that transforming growth factor β1 (TGF-β1) is the core factor that contributes to DN pathogenesis. Most of the studies on the pathologic effects of TGF-β1 on DN simply focus on its profibrotic effect, while other potential mechanisms remain unclear. Studies have shown that TGF-β1 expression in MCs is increased under high glucose conditions20,21,22,24,25 and that TGF-β1 participates in activation of the PI3K/Akt pathway26,27,28,29. PI3K/Akt pathway activation by TGF-β1 induces the phosphorylation and inactivation of FoxO3a and decreases expression of its downstream target genes, Bim and MnSOD30,31,32,33,34,35,36. Consequently, both the cell proliferation rate and ROS production increase37. Furthermore, Bim deficiency in Birt-Hogg-Dubé (−/−) cells can be attributed to reduced Bim transcription, which is associated with a loss of TGF-β-mediated transcription and chromatin modifications38. Autocrine TGF-β protects breast cancer cells from apoptosis through Bim reduction39. Additionally, iNOS is a major enzyme for NO over-production after stress or under pathological conditions, but whether increased TGF-β1 expression can promote iNOS protein expression is not well understood. Therefore, investigating the relationships among high glucose-induced TGF-β1, NO production and the downstream targets of the PI3K/Akt pathway in rat MCs under high glucose conditions is necessary.
Based on the above findings, we investigated whether NO over-production in rat mesangial cells cultured in HG is related to activation of the TGF-β1/PI3K/Akt pathway.
Materials and methods
Mesangial cell culture and treatment
A rat mesangial cell line (HBZT-1) was purchased from the China Center for Type Culture Collection (Wuhan University, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C. The cells were cultured under normal glucose conditions (NS, 5.56 mmol/L) for 24 h and treated with mannitol (MA, 24.44 mmol/L), high glucose (HG, 24.44 mmol/L), high glucose+dimethyl sulfoxide (HG+DMSO, with 20 μmol/L DMSO), high glucose+the TGF-β1 inhibitor SB43154 (HG+SB431542, with 10 μmol/L SB431542), high glucose+the PI3K inhibitor LY294002 (HG+LY294002, with 20 μmol/L LY294002), high glucose+the NADPH oxidase inhibitor diphenylene iodonium (HG+DPI, with 10 μmol/L DPI), hydrogen peroxide (H2O2, 10 μmol/L), hydrogen peroxide+the TGF-β1 inhibitor SB431542 (H2O2+SB431542, with 10 μmol/L SB431542), H2O2+the PI3K inhibitor LY294002 (H2O2+LY294002, with 20 μmol/L LY294002), or TGF-β1 (5 ng/mL). All of the experiments were performed with cells between passages 5 and 8. After treatment with the different agents for 16 h, the cells were harvested for analysis.
Spectrophotometric determination of total nitric oxide level
The MCs were treated with different inhibitors and then cultured for 16 h before supernatant collection for the NO assay. The total NO level was estimated using the Griess method, which determined the levels of nitrate and nitrite to reflect the total NO level. The Griess kit was purchased from the Beyotime Institute of Biotechnology (Haimen, China), and the measurements were performed according to the manufacturer's instructions.
ELISA assay for TGF-β1 measurement
The MCs were seeded in 12-well plates at approximately 105 cell/well with DMEM medium containing 10% FBS. After 24 h, the medium was replaced with serum-free DMEM containing penicillin/streptomycin. The conditioned medium was collected after 16 h of treatment and centrifuged at 10 000×g for 15 min. The resulting supernatant was subjected to TGF-β1 measurement using a rat enzyme-linked immunosorbent assay (ELISA) kit (Boster Bio-engineering Co, Ltd, Wuhan, China), according to the manufacturer's instructions. The colorimetric reaction was measured at 450 nm. Each experiment was repeated in triplicate.
Western blotting analysis for Akt, iNOS and Bim protein expression
The cell samples were separated by SDS-PAGE under reducing conditions and then transferred onto nitrocellulose membranes. The membranes were blocked with western blocking buffer and then incubated with different antibodies, including rabbit polyclonal antibodies against t-Akt and p-Akt (Beyotime Institute of Biotechnology, Haimen, China, both 1:1000 dilution), rabbit monoclonal antibody against Bim (Cell Signaling Technology, Beverly, MA, USA, 1:1000 dilution), rabbit polyclonal antibodies against iNOS and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, both 1:500 dilution), and goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution). Detection was performed by using Image J version 1.34s software (Wayne Rasband National Institutes of Health, Detroit, MI, USA) and normalized to β-actin expression. The statistical data for Bim protein expression were obtained by determining the average of band optical density of three different molecular masses.
Analysis of TGF-β1 and Bim by RT-PCR
The MCs were cultured for 16 h in the normal and experimental groups. Total RNA was extracted using TRIzol reagent (Tiangen Biotech, Co, Ltd, Beijing, China) according to the manufacturer's instructions and was then subjected to reverse transcription using a Quant One Step RT-PCR kit (Applied Biosystems, Carlsbad, CA, USA). Complementary DNA (cDNA) was amplified using the appropriate primers. The primer sequences used were as follows: TGF-β1, 5′-CCCGCATCCCAGGACCTCTCT-3′ (forward) and 5′-CGGGGGACTGGCGAGCCTTAG-3′ (reverse); Bim, 5′-GCCAAGCAACCTTCTGATGTA-3′ (forward) and 5′-CAGTGCCTTCTCCAGACCAG-3′ (reverse); and β-actin, 5′-GCTGCGTGTGGCCCCTGAG-3′ (forward) and 5′-ACGCAGGATGGCATGAGGGA-3′ (reverse). The resulting products were separated by 1.5% agarose electrophoresis, and the band densities were analyzed by using Image J version 1.34s software (Wayne Rasband National Institutes of Health, Detroit, MI, USA). The data for Bim mRNA expression were obtained by determining the average band optical density of three different molecular masses.
Cell proliferation determination using a BrdU incorporation assay
Cells (104/well) were seeded in Falcon 96-well plates for 24 h and then exposed to different agents. After incubation for 16 h, a cell proliferation assay was performed using a BrdU Cell Proliferation Assay kit (Roche, Berlin, Germany). The optical density was measured at 370 nm using an automatic microplate reader (ClinBio 128Ce, Asys Hitech Gmbh, Austria).
Statistical analysis
All of the data in the different experimental groups were expressed as the mean±SD. Differences between the groups were assessed using one-way ANOVA and the t-test. P<0.05 was considered to be statistically significant.
Results
Effects of osmotic pressure and solvent
Mannitol was used to mimic the osmotic pressure produced under high glucose conditions, and DMSO served as the solvent. We examined the effects of mannitol and DMSO on NO release from rat MCs; mRNA expression of TGF-β1 and Bim; the protein expression of Akt, Bim, and iNOS; as well as cell proliferation. The results in the mannitol group were almost identical to the NS group, indicating that the osmotic pressure induced by high glucose did not affect the experimental data. The results in the HG+DMSO group were similar to the HG group, demonstrating that the presence of DMSO had no effect on the experimental data. Therefore, neither the osmotic pressure induced by high glucose nor the presence of DMSO significantly affected the experimental results.
NO level in rat mesangial cells
The amount of NO released from rat MCs was examined by visible spectrophotometry. Compared to the NS group, NO release from the rat MCs significantly increased (P<0.01) in the HG, HG+DMSO, and H2O2 groups and was decreased in the HG+DPI group (P<0.01) (Figure 1). This result indicates that excessive ROS generation can stimulate NO over-production in MCs cultured in high glucose. NO release from the rat MCs also increased (P<0.01) in the TGF-β1 group compared to the NS group. Compared to the HG group, NO release was decreased (P<0.01) in the HG+SB431542 and HG+LY294002 groups. In conclusion, the TGF-β1/PI3K pathway may participate in NO over-production in MCs with high glucose.
TGF-β1 protein and mRNA expression in rat mesangial cells
Compared to the NS group, TGF-β1 expression in the rat MCs was significantly increased (P<0.05) in the HG group, but decreased (P<0.05) in the HG+DPI group. Moreover, TGF-β1 expression was also markedly elevated (P<0.01) in the rat MCs treated with H2O2 (Figure 2A). These results indicate that excessive ROS generation mediates increased TGF-β1 expression in MCs with high glucose.
To further evaluate the role of TGF-β1 in NO over-production in MCs under high glucose conditions, we examined the changes in TGF-β1 mRNA expression (Figure 2B). The results showed that TGF-β1 mRNA expression in the rat MCs was significantly increased in the HG group (P<0.05) but decreased in the HG+DPI group (P<0.01). Similarly, TGF-β1 mRNA expression was markedly elevated (P<0.01) in the rat MCs treated with H2O2. These results indicate that excessive ROS generation promotes TGF-β1 mRNA expression in the MCs under high glucose conditions.
Akt phosphorylation in rat mesangial cells
Both high glucose and H2O2 significantly increased (P<0.01) the phosphorylated Akt (p-Akt) level in rat MCs, which was reduced (P<0.01) by treatment with the TGF-β1 inhibitor SB431542 or the PI3K inhibitor LY294002 (Figure 3A). This finding indicates that high glucose activates the TGF-β1/PI3K/Akt pathway in rat MCs. Furthermore, the level of p-Akt in the rat MCs decreased after adding DPI (P<0.01). Therefore, these findings demonstrate that excessive ROS generation leads to activation of the TGF-β1/PI3K/Akt pathway.
iNOS protein expression in rat mesangial cells
Compared to the NS group, iNOS protein expression was significantly increased in the HG group (P<0.01) but decreased (P<0.01) in the HG+SB431542, HG+LY294002, and HG+DPI groups (Figure 3B). Moreover, iNOS protein expression was markedly elevated (P<0.01) in the rat MCs treated with H2O2 but reduced (P<0.01) in the H2O2+SB431542 and HG+LY294002 groups. These results indicate that excessive ROS generation increases iNOS expression in MCs under high glucose conditions, which may be mediated by the TGF-β1/PI3K/Akt pathway.
Bim protein and mRNA expression in rat mesangial cells
Compared to the NS group, Bim protein expression in the rat MCs was significantly decreased (P<0.01) in the HG group but increased (P<0.01) in the HG+SB431542, HG+LY294002, and HG+DPI groups (Figure 4A). Moreover, Bim protein expression was remarkably (P<0.01) decreased in the rat MCs treated with H2O2 but increased (P<0.01) in the H2O2+SB43154 and H2O2+LY294002 groups. These results indicate that excessive ROS generation decreases Bim expression in MCs under high glucose conditions, which may be mediated by the TGF-β1/PI3K/Akt pathway. The same effect can be seen for Bim mRNA expression (Figure 4B).
Cell proliferation in the rat mesangial cells
Compared to the NS group, the cell proliferation rate was significantly increased (P<0.01) in the HG group but decreased (P<0.01) in the HG+SB431542, HG+LY294002, and HG+DPI groups (Figure 5). Moreover, increased cell proliferation rate was observed (P<0.05) in rat MCs treated with H2O2, but the cell proliferation rate was lower (P<0.05) in the rat MCs treated with H2O2+SB431542 or H2O2+LY294002. These findings indicate that inhibiting the TGF-β1/PI3K/Akt pathway can suppress the proliferation of mesangial cells caused by hyperglycemia.
Discussion
DN is characterized by the excessive accumulation of ECM with thickened glomerular and tubular basement membranes and increased mesangial area1, and NO may play an important role in these changes13,14. However, the molecular mechanism for the effects of hyperglycemia on NO over-production in the kidneys remains unclear. In this study, we found that oxidative stress in response to hyperglycemia might promote NO over-production in rat MCs due to decreased Bim protein and mRNA expression and increased iNOS protein expression, and the iNOS over-production is at least partially a result of the TGF-β1-induced activation of the PI3K/Akt pathway.
The change in NO production is an important biochemical feature in DN. Several reports have shown that the NO level is significantly increased in the early stages of kidney disease in diabetic rats9,17,18,19 and rat MCs under high glucose conditions10. However, the molecular mechanism involved is not well understood. In the current study, NO release was significantly increased in the rat MCs treated with high glucose or H2O2 for 16 h and was then reduced by treatment with DPI (a NADPH oxidase inhibitor). These results indicate that excessive ROS generation stimulates NO over-production in MCs under high glucose conditions. Moreover, the NO level was substantially elevated in the MCs treated with TGF-β1, but was then reduced by SB431542 (a TGF-β1 receptor kinase inhibitor) or LY294002 (a PI3K inhibitor). The results suggest that the TGF-β1/PI3K/Akt pathway may mediate the effect of high glucose on NO release from MCs.
TGF-β1, a very important growth factor in DN pathogenesis, has been reported to be up-regulated in the kidney under diabetic conditions using in vitro20,21,22,23,25 and in vivo6,7,8,24 studies. In the present study, both high glucose and H2O2 led to increased TGF-β1 protein and mRNA expression in rat MCs, and these were then reduced by treatment with DPI, suggesting that ROS mediated high glucose-induced TGF-β1 production. Our previous reports have demonstrated that TGF-β1 protein and mRNA expression were increased in the renal cortices of diabetic rats6,7,8. These findings indicate that TGF-β1 plays an important role during hyperglycemia.
To investigate the mechanism associated with TGF-β1-induced NO production in MCs, we examined the expression of iNOS, a major enzyme for NO biosynthesis after stress. High glucose dramatically increased iNOS protein expression in MCs, which was then reduced by treatment with SB431542 or LY294002. This result suggests that the TGF-β1/PI3K pathway participates in iNOS generation in MCs under high glucose conditions. TGF-β1 is required for NF-kappaB-mediated modulation of iNOS activity when controlling the induction of the Epstein-Barr virus replication cycle40. A report showed that TGF-β1 stimulates NO production in astrocytes by recruiting distinct cell subpopulations41. Therefore, the iNOS expression induced by the TGF-β1/PI3K/Akt pathway may be a main reason for NO over-production in MCs cultured in high glucose (Figure 6). Our study is the first report on the mechanism of induced iNOS expression in MCs under high glucose conditions.
High glucose can stimulate the over-proliferation of MCs and ECM secretion in MCs, which can be reduced by hydrogen sulfide via decreased ROS and TGF-β1 levels42. In the current study, the rat MCs under high glucose conditions showed NO over-production, increased TGF-β1 protein and mRNA expression, and increased cell proliferation (one of the most important pathological manifestations of diabetic kidney disease). Treatment with SB431542, LY294002, or DPI inhibited the mesangial cell proliferation caused by high glucose. These results indicate that excessive ROS generation causes MC proliferation under a high glucose condition, which may be mediated by the TGF-β1/PI3K/Akt pathway.
Reduced expression of the pro-apoptotic protein Bim can result in cell proliferation37. The lack of Bim expression had a different impact on ECM production in renal endothelial and epithelial cell migration, adhesion, eNOS activity and NO bioavailability43. In the current study, Bim protein expression was remarkably reduced in the MCs cultured in high glucose and was associated with increased cell proliferation and NO, which were both significantly inhibited by treatment with SB431542, LY294002, or DPI. Moreover, both high glucose and H2O2 showed similar effects on MCs. These results demonstrate that TGF-β1/PI3K/Akt pathway activation by ROS contributes to reduced Bim protein expression in the MCs cultured in high glucose. We also found that the change in Bim mRNA expression in the rat MCs was consistent with Bim protein expression. Reports have indicated that the nuclear factor FoxO3a is controlled by the activation of the TGF-β1/PI3K/Akt pathway, and Bim and MnSOD, which are associated with oxidative stress, represent two of the important downstream target genes of FoxO3a30,31,32,33,34,35,36. Therefore, FoxO3a may regulate Bim mRNA in MCs cultured in high glucose. Moreover, our previous study showed that FoxO3a participate in the down-regulation of MnSOD at the protein and mRNA levels in MCs cultured in high glucose via activation of the TGF-β1/PI3K/Akt pathway44. Therefore, reduced Bim expression is one of the important factors for rat MC proliferation and subsequent NO over-production in diabetic kidneys (Figure 6). Additionally, a report has indicated that exposure of MCs for up to 24 h can increase the cell number; however, after that time, the cell number remained unchanged, and the cells underwent hypertrophy, reflecting the observations found in experimental and clinical studies in vivo45. Therefore, the pathways discussed in the present study could be targeted during the early stage of DN.
In conclusion, TGF-β1 up-regulation by oxidative stress in MCs under high glucose conditions leads to NO over-production, mediated by increased iNOS protein expression and decreased Bim expression, which are at least partially due to activation of the PI3K/Akt/FoxO3a pathway.
Author contribution
Xiao-xing YIN designed the study and revised the paper; Yun-peng ZHAI performed most of the experiments; Qian LU performed some of the experiments and wrote this paper; Yao-wu LIU analyzed part of the data and made the figures; Qian CHENG and Cheng-lin LI performed some of the experiments; Ya-Qin WEI and Fang ZHANG purchased the reagents.
References
Kanwar YS, Wada J, Sun L, Xie P, Wallner EI, Chen S, et al. Diabetic nephropathy: mechanisms of renal disease progression. Exp Biol Med (Maywood) 2008; 233: 4–11.
Ha H, Lee HB . Reactive oxygen species amplify glucose signalling in renal cells cultured under high glucose and in diabetic kidney. Nephrology (Carlton) 2005; Suppl: S7–10.
Sedeek M, Callera G, Montezano A, Gutsol A, Heitz F, Szyndralewiez C, et al. Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy. Am J Physiol Renal Physiol 2010; 299: F1348–58.
Jaimes EA, Hua P, Tian RX, Raij L . Human glomerular endothelium: interplay among glucose, free fatty acids, angiotensin II, and oxidative stress. Am J Physiol Renal Physiol 2009; 298: F125–32.
Brownlee M . The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005; 54: 1615–25.
Yin X, Zhang Y, Yu J, Zhang B, Shen J, Qiu J, et al. The antioxidative effects of astragalus saponin I protect against development of early diabetic nephropathy. J Pharmacol Sci 2006; 101: 166–73.
Yin X, Zhang Y, Yu J, Zhang B, Shen J, Qiu J . Protective effects of bendazac lysine against development of diabetic nephropathy. Chin J Clin Pharmacol Ther 2007; 12: 552–7.
Lu Q, Yin XX, Wang JY, Gao YY, Pan YM . Effects of Ginkgo biloba on prevention of development of experimental diabetic nephropathy in rats. Acta Pharmacol Sin 2007; 28: 818–28.
Levin-Iaina N, Iaina A, Raz I . The emerging role of NO and IGF-1 in early renal hypertrophy in STZ-induced diabetic rats. Diabetes Metab Res Rev 2011; 27: 235–43.
Noh H, Ha H, Yu MR, Kang SW, Choi KH, Han DS, et al. High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production. Nephron 2002; 90: 78–85.
Erdely A, Freshour G, Maddox DA, Olson JL, Samsell L, Baylis C . Renal disease in rats with type 2 diabetes is associated with decreased renal nitric oxide production. Diabetologia 2004; 47: 1672–6.
Keynan S, Hirshberg B, Levin-Iaina N, Wexler ID, Dahan R, Reinhartz E, et al. Renal nitric oxide production during the early phase of experimental diabetes mellitus. Kidney Int 2000; 58: 740–7.
Komers R, Anderson S . Paradoxes of nitric oxide in the diabetic kidney. Am J Physiol Renal Physiol 2003; 284: F1121–37.
Prabhakar SS . Role of nitric oxide in diabetic nephropathy. Semin Nephrol 2004; 24: 333–44.
Lee HY, Noh HJ, Gang JG, Xu ZG, Jeong HJ, Kang SW, et al. Inducible nitric oxide synthase (iNOS) expression is increased in lipopolysaccharide (LPS)-stimulated diabetic rat glomeruli: effect of ACE inhibitor and angiotensin II receptor blocker. Yonsei Med J 2002; 43: 183–92.
Shultz PJ, Archer SL, Rosenberg ME . Inducible nitric oxide synthase mRNA and activity in glomerular mesangial cells. Kidney Int 1994; 46: 683–9.
Sefi M, Fetoui H, Soudani N, Chtourou Y, Makni M, Zeghal N . Artemisia campestris leaf extract alleviates early diabetic nephropathy in rats by inhibiting protein oxidation and nitric oxide end products. Pathol Res Pract 2012; 208: 157–62.
Anjaneyulu M, Chopra K . Effect of irbesartan on the antioxidant defence system and nitric oxide release in diabetic rat kidney. Am J Nephrol 2004; 24: 488–96.
Levine DZ . Hyperfiltration, nitric oxide, and diabetic nephropathy. Curr Hypertens Rep 2006; 8: 153–7.
Ho C, Lee PH, Hsu YC, Wang FS, Huang YT, Lin CL . Sustained Wnt/β-Catenin signaling rescues high glucose induction of transforming growth factor-β1-mediated renal fibrosis. Am J Med Sci 2012; 344: 374–82.
Du J, Wang L, Liu L, Fan Q, Yao L, Cui Y, et al. IFN-γ suppresses the high glucose-induced increase in TGF-β1 and CTGF synthesis in mesangial cells. Pharmacol Rep 2011; 63: 1137–44.
Uttarwar L, Gao B, Ingram AJ, Krepinsky JC . SREBP-1 activation by glucose mediates TGF-β upregulation in mesangial cells. Am J Physiol Renal Physiol 2012; 302: F329–41.
Yin X, Zhang Y, Wu H, Zhu X, Zheng X, Jiang S, et al. Protective effects of Astragalus saponin I on early stage of diabetic nephropathy in rats. J Pharmacol Sci 2004; 95: 256–66.
Wang JY, Yin XX, Wu YM, Tang DQ, Gao YY, Wan MR, et al. Ginkgo biloba extract suppresses hypertrophy and extracellular matrix accumulation in rat mesangial cells. Acta Pharmacol Sin 2006; 27: 1222–30.
Ji L, Yin XX, Wu ZM, Wang JY, Lu Q, Gao YY . Ginkgo biloba extract prevents glucose-induced accumulation of ECM in rat mesangial cells. Phytother Res 2009; 23: 477–85.
Runyan CE, Schnaper HW, Poncelet AC . The phosphatidylinositol 3-kinase/Akt pathway enhances smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1. J Biol Chem 2004; 279: 2632–9.
Qureshi HY, Ahmad R, Sylvester J, Zafarullah M . Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-beta in human chondrocytes. Cell Signal 2007; 19: 1643–51.
Yi JY, Shin I, Arteaga CL . Type I transforming growth factor beta receptor binds to and activates phosphatidylinositol 3-kinase. J Biol Chem 2005; 280: 10870–6.
Ghosh Choudhury G, Abboud HE . Tyrosine phosphorylation-dependent PI3 kinase/Akt signal transduction regulates TGF beta-induced fibronectin expression in mesangial cells. Cell Signal 2004; 16: 31–41.
Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA, et al. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 2003; 278: 49795–805.
Essafi A, Fernandez de Mattos S, Hassen YA, Soeiro I, Mufti GJ, Thomas NS, et al. Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene 2005; 24: 2317–29.
Urbich C, Knau A, Fichtlscherer S, Walter DH, Brühl T, Potente M, et al. FOXO-dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells. FASEB J 2005; 19: 974–6.
Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 2002; 419: 316–21.
Kato M, Yuan H, Xu ZG, Lanting L, Li SL, Wang M, et al. Role of the Akt/FoxO3a pathway in TGF-β1-mediated mesangial cell dysfunction: A novel mechanism related to diabetic kidney disease. J Am Soc Nephrol 2006; 17: 3325–35.
Li M, Chiu JF, Mossman BT, Fukagawa NK . Down-regulation of manganese-superoxide dismutase through phosphorylation of FOXO3a by Akt in explanted vascular smooth muscle cells from old rats. J Biol Chem 2006; 281: 40429–39.
Liu H, Luo LL, Qian YS, Fu YC, Sui XX, Geng YJ, et al. FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries. Biochem Biophys Res Commun 2009; 381: 722–7.
Kozuma Y, Ninomiya H, Murata S, Kono T, Mukai HY, Kojima H . The pro-apoptotic BH3-only protein Bim regulates cell cycle progression of hematopoietic progenitors during megakaryopoiesis. J Thromb Haemost 2010; 8: 1088–97.
Cash TP, Gruber JJ, Hartman TR, Henske EP, Simon MC . Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene 2011; 30: 2534–46.
Hoshino Y, Katsuno Y, Ehata S, Miyazono K . Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim. J Biochem 2011; 149: 55–65.
Oussaief L, Ramírez V, Hippocrate A, Arbach H, Cochet C, Proust A, et al. NF-kappaB-mediated modulation of inducible nitric oxide synthase activity controls induction of the Epstein-Barr virus productive cycle by transforming growth factor beta 1. J Virol 2011; 85: 6502–12.
Hamby ME, Hewett JA, Hewett SJ . TGF-beta1 potentiates astrocytic nitric oxide production by expanding the population of astrocytes that express NOS-2. Glia 2006; 54: 566–77.
Yuan P, Xue H, Zhou L, Qu L, Li C, Wang Z, et al. Rescue of mesangial cells from high glucose-induced over-proliferation and extracellular matrix secretion by hydrogen sulfide. Nephrol Dial Transplant 2011; 26: 2119–26.
Sheibani N, Morrison ME, Gurel Z, Park S, Sorenson CM . BIM deficiency differentially impacts the function of kidney endothelial and epithelial cells through modulation of their local microenvironment. Am J Physiol Renal Physiol 2012; 302: F809–19.
Lu Q, Zhai Y, Cheng Q, Liu Y, Gao X, Zhang T, et al. The Akt-FoxO3a-manganese superoxide dismutase pathway is involved in the regulation of oxidative stress in diabetic nephropathy. Exp Physiol 2012. DOI: 10.1113/expphysiol.2012.068361.
Wolf G . Molecular mechanisms of diabetic mesangial cell hypertrophy: a proliferation of novel factors. J Am Soc Nephrol 2002; 13: 2611–3.
Acknowledgements
We thank Miss Hao GUO for help with the manuscript. The work was supported by the National Natural Science Foundation of China (No 81173104), the Jiangsu University Natural Science Foundation of China (No 11KJD310004), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhai, Yp., Lu, Q., Liu, Yw. et al. Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose. Acta Pharmacol Sin 34, 507–514 (2013). https://doi.org/10.1038/aps.2012.207
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/aps.2012.207
Keywords
This article is cited by
-
MiR-29b expression is altered in crescent formation of HSPN and accelerates Ang II-induced mesangial cell activation
World Journal of Pediatrics (2020)
-
Ameliorative Effect of Selenomethionine on Cadmium-Induced Hepatocyte Apoptosis via Regulating PI3K/AKT Pathway in Chickens
Biological Trace Element Research (2020)
-
Seismic rock physical modelling for gas hydrate-bearing sediments
Science China Earth Sciences (2018)
-
Therapeutic effects of DZ2002, a reversible SAHH inhibitor, on lupus-prone NZB×NZW F1 mice via interference with TLR-mediated APC response
Acta Pharmacologica Sinica (2014)
-
Selective α1B- and α1D-adrenoceptor antagonists suppress noradrenaline-induced activation, proliferation and ECM secretion of rat hepatic stellate cells in vitro
Acta Pharmacologica Sinica (2014)