http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2014; 36(4): 589–592 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.882713
LABORATORY STUDY
FK506 inhibits the mice glomerular mesangial cells proliferation by affecting the transforming growth factor-b and Smads signal pathways Ren Qi1*, Wen Li2*, and Shengyou Yu3* 1
Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, Guangdong Province, China, 2Department of Dermatologic Surgery, HaiNan Provincial Hospital of Skin Disease, HaiKou, HaiNan Provincial, China, and 3Guangzhou First People’s Hospital, Guangzhou Medical University, Guangdong Province, China Abstract
Keywords
TGF-b1 plays an important role in the pathogenesis of chronic renal diseases. Although the specific mechanism is unknown, a major factor is the potent fibrogenic activity of TGF-b1 in the chronic progression of renal diseases. TGF-b1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules. Recently it has been studied that, Smad proteins as intracellular mediators of TGF-b signaling pathways provide important insights into the mechanisms determining the specificity of TGF-b action in various renal cells. Some studies have proved that immunosuppressants can affect TGF-b expression, but the mechanisms are unclear. In this study, we investigated the effect of FK506 on mesangial cells via TGF-b and Smads signal pathways. Our results shows that FK506 effectively blocked the TGF-b/Smad signaling pathway by downregulation of TGF-b receptor, and played an important role in TGF-b1-induced Smad2 expression in mice mesangial cells. FK506 can inhibit the TGF-b1-stimulated cell proliferation via Smad-related pathways. And reduced the Smad2 protein and mRNA expression. Altogether, this study provided a theoretical proof for the protective and treating effect of FK506 on kidneys.
FK506, glomerular mesangial cells, proliferation, Smad, TGF-b1
Glomerular disease is one of the main causes of chronic kidney disease (CKD), and either primary or secondary glomerular disease, the common features were the proteinuria occurrence.1 The mice mesangial cells proliferation is one of the main organic pathological changes for kidney disease, and aggravated cell proliferation would finally lead to glomerular sclerosis. Lot of experiments confirmed that transforming growth factor b1 (TGF-b1) participates in glomerular sclerosis by regulating cell proliferation, differentiation, and apoptosis through the activation of Smad signaling pathways.2 FK506, a topical calcineurin inhibitor, is widely used in kidney disease in recent years and has been successfully used as an alternative to cyclosporine to prevent graft rejection and treat autoimmune diseases,3 such as lupus nephritis, systemic vasculitis, and primary nephrotic syndrome. The clinic study suggests FK506 can reduce the proteinuria in patients; however, it is still unclear that whether the pharmacological action has direct or indirect effects on the function of mesangial cells. The purpose of this study is to observe the *These authors contributed equally to this study. They are all the firstauthors. Address correspondence to Shengyou Yu, Guangzhou First People’s Hospital, Guangzhou Medical university, Guangdong Province, China. E-mail: [email protected]
Received 6 October 2013 Revised 30 November 2013 Accepted 4 December 2013 Published online 10 February 2014
effect of FK506 on Smad2 protein and mRNA expression induced by TGF-b1 and further confirms the role of TGF-b1/ Smad signaling pathways in glomerular sclerosis. We also investigate whether FK506 can delay renal fibrosis through regulating TGF-b1/Smad signaling pathways and play an important role in antiproteinuria.
Materials and methods Cell culture and treatments
20 14
Introduction
History
Mesangial cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), cultured at 37 C in RPMI-1640 (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum (Sigma Chemical Co, St Louis, MO), 100 U/mL penicillin/streptomycin (Gibco), the culture medium was changed every 2 d. FK506 (Calbiochem; San Diego, CA) used in this study was dissolved in dimethylsulphoxide (DMSO). Cells were treated with FK506 at doses of 10, 100, and 1000 ng/mL with or without human recombinant TGF-b1 8 ng/mL (Peprotech, Rocky Hill, NJ) during the indicated time points. All experiments were repeated thrice. Cell proliferation assay Cell proliferation was assessed by 5-bromo-20 -deoxyuridine (BrdU) cell proliferation kit (Roche, Basel, Switzerland).
590
R. Qi et al.
Ren Fail, 2014; 36(4): 589–592
Briefly, cells were seeded on 96-well plates at a density of 1 105 cells/well and incubated overnight. Then the cells were treated and incubated for 8 h, 24 h, and 48 h. The treated cells were fixed and stained according to the manufacturer’s instructions. The optical density was measured at 570 nm with an ELISA plate reader equipped with Microplate Manager software version 2.1 (Model 450; Bio-Rad, Hercules, CA).
experimental groups in the study. Values of p50.05 were considered as statistically significant. All the experiments in our study were performed at least thrice.
RT-PCR analysis
The results shown in Figure 1 revealed that FK506 at various concentrations exhibited no significant effect on cell proliferation. Treatment with TGF-b1 significantly increased the proliferation of Glomerular mesangial cells. The addition of FK506 significantly suppressed the enhancing effect of TGF-b1 on cell proliferation.
Smad2 mRNA was extracted from Cells using Trizol Reagent(Sigma Chemical Co) according to the manufacturer’s instruction. Isolated RNA (1 mg) of each sample was subjected to reverse transcription by using Rever Tra Ace (Sigma Chemical Co) according to the manufacture’s protocol. The resulting cDNA (3 mL) was used for PCR amplification. The sequence-specific primers were designed and synthesized by Shanghai Invitrogen Biotechnology Co, Ltd. (Shangai, China). Primers used were as follows: Smad2 upstream and downstream primers were the following forward: 50 -TTCCAGACTTTGTGCTGTCCAGTAA-30 ; reverse: 50 -AGGGCAGAGGCTCCACTGAGTA-30 , product length being 120 bp; GAPDH upstream and downstream primers were the following forward: 5-GGTGAAGGTCGGTGT GAACGGAT-3; reverse: 5-CCACTTTGCCACTGCAAATG GCAG-3, product length being 118 bp. Levels of gene expression were expressed as the integrated density value of PCR products normalized to that of the GAPDH in the same sample. Independent experiment was repeated thrice.
Results FK506 treatment alone showed no significant effect on cell proliferation but significantly suppressed the TGF-b1-induced proliferation
FK506 reduced the protein and mRNA expressions of phosphorylated Smad2 induced by TGF-b1 It was noticed that TGF-b1 significantly altered the protein and mRNA expressions of Smad2. However, FK506 treatment
Western blot analysis Cells were lysed in the buffer containing 1% Tritonx-100 (Sigma Chemical Co., St Louis, MO), 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.7), 1 mM NaF, 1 mM NaVO3, and a protease inhibitor cocktail (Sigma Chemical Co., St Louis, MO). Seventy-five micrograms of total protein was loaded to run 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was set up for transfer protein to nitrocellulose membranes (Sigma Chemical Co., St Louis, MO). Then, the membranes were incubated with rabbit anti-first antibody (Sigma Chemical Co, St Louis, MO); mouse anti-GAPDH antibody (Sigma Chemical Co, St Louis, MO). The membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Sigma Chemical Co., St Louis, MO) for 50 min at room temperature, and then developed using ECL chemiluminescence reagent (Sigma Chemical Co., St Louis, MO). The specific protein bands were scanned and quantitated using a densitometry in relation to the GAPDH. Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, UK). We repeated each Western blot analysis using protein from three different and separate experiments. Statistical analysis SPSS 17.0 (SPSS Inc., Chicago, IL) was used for the statistical analysis. The results were expressed as mean ± standard deviation (mean ± SD). Student’s t test was used for statistical evaluation between control and
Figure 1. The comparisons of cell proliferation between the FK506 group and the control. Notes: cells were seeded into 96-well plates at a density of 3 104 cells/well. After starving in the serum-free medium overnight, the cells were incubated in the medium for 8, 24, and 48 h. Cell proliferation was assessed by using MTT assay. Absorbance was measured by ELISA plate-reader at 570 nm. The values were expressed as a mean ± SD. The presented data are based on at least three independent experiments. *p50.05, compared to normal group. The proliferation of FK506 group treated with FK506 10, 30, and 60 ng/mL for 8, 24, and 48 h was assessed by MTT cell proliferation assay. The data shown here (mean± SD) were from one representative experiment performed in triplicate, repeated three times with similar results.
Role of FK506 in CKD
DOI: 10.3109/0886022X.2014.882713
591
Figure 2. FK506 reduced the expressions of Smad2 protein increased by TGF-b1 in mesangial cells. Notes: Mesangial cells were cultured and treated with TGF-b1 5 ng/mL with or without the addition of FK506 (10, 30, and 60 ng/mL) for 48 h. Total cell protein lysates were prepared and subjected to Western blotting analysis with antibody against Smad2 and GAPDH. Experiments were repeated thrice with similar results. *p50.05 as compared with control group; #p50.05 as compared with TGF-b1 stimulated group. Lane 1: control; Lane 2: TGF-b1 5 ng/mL treatment; Lane 3: TGF-b1 5 ng/ mL + FK506 10 ng/mL treatment; Lane 4: TGF-b1 5 ng/mL + FK506 30 ng/mL treatment; Lane 5: TGF-b1 5 ng/mL + FK506 60 ng/mL treatment.
Table 1. FK506 reduced the expressions of Smad2 mRNA increased by TGF-b1 in mesangial cells.
The control FK506 10 ng/mL FK506 30 ng/mL FK506 60 ng/mL TGF-b1 5 ng/mL TGF-b1 + FK506 10 ng/mL TGF-b1 + FK506 30 ng/mL TGF-b1 + FK506 60 ng/mL
8h
24 h
48 h
0.151 ± 0.002 0.150 ± 0.014 0.148 ± 0.020 0.149 ± 0.031 1.101 ± 0.020# 0.671 ± 0.010*
0.172 ± 0.014 0.171 ± 0.041 0.169 ± 0.037 0.168 ± 0.021 1.192 ± 0.047# 0.761 ± 0.014*
0.194 ± 0.004 0.191 ± 0.006 0.190 ± 0.010 0.187 ± 0.014 1.221 ± 0.010# 0.897 ± 0.011*
0.473 ± 0.022*
0.583 ± 0.031*
0.578 ± 0.006*
0.311 ± 0.012*
0.452 ± 0.016*
0.491 ± 0.015*
Notes: Cells were treated with TGF-b1 5 ng/mL followed by FK506 10, 30, and 60 ng/mL and then incubated at indicated time points for the determination of Smad2 mRNA. The data shown here (mean ± SD) are from one representative experiment performed in triplicate, repeated thrice with similar results. *p50.05, #p50.01 as compared with the TGF-b1 stimulated group.
significantly decreased the phosphorylated Smad2 protein and mRNA expressions that were enhanced by TGF-b1 treatment.
Discussion TGF-b is a multifunctional cytokine that plays an important role in regulating cell growth, differentiation, and biosynthesis of extracellular connective tissue.4–7 TGF-b1 can induce the expression and synthesis of fibronectin in human mesangial cells.8–10 The discovery of Smads further reveals the mechanism of action of TGF-b. Smads protein is the direct substrate of TGF-b superfamily and also serves as an intermediating molecular that transmits interaction signals between TGF-b and receptor from cytoplasm to cell nucleus.11–14 In the TGF-b1/Smad signaling pathways, TGF-b1 first interacts with II receptor in cell membranes, activates TGF-b1RI, and phosphorylates the downstream Smad2/3. The activated Smad2/3 interacts with Smad4 to form complex, transfers from cytoplasm to nucleus, interacts with DNA, and regulates corresponding cell functions.
Smads is expressed in various tissues and takes part in the regulation of multiple cell functions, but its expression and location in normal renal tissues are still unclear. TGF-b1 can induce the transposition of Smad2 phosphorylation to nucleus.15 Our observations partly confirm this view. Smad2-mediated TGF-b signal can induce the expression of connective tissue growth factor and possesses pro-fibrogenic effects,16 indicating that TGF-b/Smad signaling pathways play an important role in glomerular diseases. Blocking the Smad2 signals can inhibit the transformation of epithelial cells induced by TGF-b1, thus delaying renal fibrosis. Blocking the TGF-b/Smad signaling pathways has important implications in treating resistant nephrotic syndrome and preventing treatment of renal fibrosis. FK506 is a new generation of CNI used in the treatment of kidney diseases and graft rejection by inhibiting the activation of T cells, the proliferation of dependent B cells on Th helper cells, and the expression of lymphokines such as IL-2, IL-3, and g-interferon, and IL-2R, to reduce immune inhibitory effects.17 Our previous study found that dexamethasone can protect podocyte apoptosis induced by puromycin aminonucleoside.18 A large number of clinical studies demonstrated that FK506 significantly reduced renal tissue damage and proteinuria in various kidney diseases. Previous studies reported that FK506 is associated with less renal interstitial fibrosis than cyclosporine in de novo renal transplants,19,20 suggesting that FK506 exhibited an antifibrotic effect and may be used to treat hyper fibrotic disorders. However, there are less work on the effects of FK506 towards TGF-b1/Smad signaling pathways in vitro. Our results provided evidences indicating that FK506 inhibited mesangial cells’ proliferation in high TGF-b1 environment and suppression of TGF-b/Smad signaling pathway is the involved molecular mechanism. In this study, we use different concentrations of FK506 to precede intervention for Smad2 expression stimulated by TGF-b1 in mesangial cells. The expressions of mesangial cells Smad2 and mRNA are little normally, but significantly increased when stimulated suggesting transcription levels of
592
R. Qi et al.
Smad2 protein and mRNA are consistent. From this dynamic view, it is observed that FK506 can decrease the Smad2 protein and mRNA expressions induced by TGF-b1. The effects of high and low doses of FK506 intervention have little difference towards Smad2 protein and mRNA expressions and thus have no statistically significant. Both the Smad2 protein and mRNA expressions significantly decrease after FK506 intervention. The results also demonstrate that Smad2 is one of the important effectors in TGF-b1 signaling pathways, and both TGF-b1 and Smad2 take part in the occurrence and in the development of glomerular sclerosis. We get an important conclusion that FK506 can make influence to the effects of TGF-b1/Smad signaling pathways by restraining mesangial cells Smad2 expressions stimulated by TGF-b1 at mRNA and proteins levels, and then protect mesangial cells and treat glomerular diseases. This paper reveals that the protection of FK506 on kidney diseases may by suppressing transduction of TGF-b1/Smad signaling pathways, provides more theoretical and experimental basement for glomerular diseases and ways for treatment idea towards delaying and controlling glomerular sclerosis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References 1. Yu S Y, Qi R, Zhao H. Losartan reverses glomerular podocytes injury induced by AngII via stabilizing the expression of GLUT1. Mol Biol Rep. 2013;40(11):6295–6301. 2. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-b family signalling. Nature. 2003;425:577–584. 3. Faulds D, Goa KL, Benfield P. Cyclosporin: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs. 1993;45:953–1040. 4. Shehata M, Cope GH, Johnson TS. Cyclosporine enhances the expression of TGF-bold beta in the juxtaglomerular cells of the rat kidney. Kidney Int. 1995;48:1487–1496.
Ren Fail, 2014; 36(4): 589–592
5. Huse M, Chen YG, Massague´ J. Crystal structure of the cytoplasmic domain of the type I TGF b receptor in complex with FKBP12. Cell. 1999;96:425–436. 6. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791. 7. Piek E, Westermark U, Kastemar M. Expression of transforminggrowth-factor (TGF)-beta receptors and Smad proteins in glioblastoma cell lines with distinct responses to TGF-beta1. Int J Cancer. 1999;80:756–763. 8. Takakura K, Tahara A, Sanagi M. Antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells[J]. Renal Fail. 2012;34(10):1309–1316. 9. Saglam F, Cavdar Z, Sarioglu S. Pioglitazone reduces peritoneal fibrosis via inhibition of TGF-b, MMP-2, and MMP-9 in a model of encapsulating peritoneal sclerosis. Renal Fail. 2012;34(1):95–102. 10. Zhang Y, Huang P, Jiang T. Role of aldose reductase in TGF-b1induced fibronectin synthesis in human mesangial cells. Mol Biol Rep. 2010;37:2735–2742. 11. Firemann S, Feuring E, Padberg W. Improvement of nephrotoxicity, hypertension, and lipid metabolism after version of kidney transplant recipient from cyclosporine to tacrolimus. Transplant Proc. 1998;30:1240–1242. 12. Ikedo H, Tamaki K, Ueda S. Smad protein and TGF-b signaling in vascular smooth muscle cells. Int J Mol Med. 2003;11:645–650. 13. Schnaper HW, Hayashida T, Hubchak SC. TGF-beta signal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol. 2003;284:F243–F252. 14. Buranakarl C, Kitjtawonrat A, Pondeenana S. Comparison of dipyridamole and fosinopril on renal progression in nephrectomized rats. Nephrology (Carlton). 2003;8:80–91. 15. Lan HY. Tubular epithelial-myofibroblast transdifferentia-tionmechanisms in proximal tubule cells. Curr Opin Nephro Hypertens. 2003;12:25–29. 16. Coimbra TM, Janssen U, Grone HJ. Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int. 2000;57:167–182. 17. Aramburu J, Heitman J, Crabtree GR. Calcineurin: a central controller of signalling in eukaryotes. EMBO Rep. 2004;5: 343–348. 18. Yu SY, Qi R. Role of bad in podocyte apoptosis induced by puromycin aminonucleoside. Transplant Proc. 2013;45:569–573. 19. Murphy GJ, Waller JR, Sandford RS. Randomized clinical trial of the effect of microemulsion cyclosporin and tacrolimus on renal allograft fibrosis. Br J Surg. 2003;90:680–686. 20. Ji SM, Li LS, Sha GZ. Conversion from cyclosporine to tacrolimus for chronic allograft nephropathy. Transplant Proc. 2007;39: 1402–1405.
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LABORATORY STUDY
FK506 inhibits the mice glomerular mesangial cells proliferation by affecting the transforming growth factor-b and Smads signal pathways Ren Qi1*, Wen Li2*, and Shengyou Yu3* 1
Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, Guangdong Province, China, 2Department of Dermatologic Surgery, HaiNan Provincial Hospital of Skin Disease, HaiKou, HaiNan Provincial, China, and 3Guangzhou First People’s Hospital, Guangzhou Medical University, Guangdong Province, China Abstract
Keywords
TGF-b1 plays an important role in the pathogenesis of chronic renal diseases. Although the specific mechanism is unknown, a major factor is the potent fibrogenic activity of TGF-b1 in the chronic progression of renal diseases. TGF-b1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules. Recently it has been studied that, Smad proteins as intracellular mediators of TGF-b signaling pathways provide important insights into the mechanisms determining the specificity of TGF-b action in various renal cells. Some studies have proved that immunosuppressants can affect TGF-b expression, but the mechanisms are unclear. In this study, we investigated the effect of FK506 on mesangial cells via TGF-b and Smads signal pathways. Our results shows that FK506 effectively blocked the TGF-b/Smad signaling pathway by downregulation of TGF-b receptor, and played an important role in TGF-b1-induced Smad2 expression in mice mesangial cells. FK506 can inhibit the TGF-b1-stimulated cell proliferation via Smad-related pathways. And reduced the Smad2 protein and mRNA expression. Altogether, this study provided a theoretical proof for the protective and treating effect of FK506 on kidneys.
FK506, glomerular mesangial cells, proliferation, Smad, TGF-b1
Glomerular disease is one of the main causes of chronic kidney disease (CKD), and either primary or secondary glomerular disease, the common features were the proteinuria occurrence.1 The mice mesangial cells proliferation is one of the main organic pathological changes for kidney disease, and aggravated cell proliferation would finally lead to glomerular sclerosis. Lot of experiments confirmed that transforming growth factor b1 (TGF-b1) participates in glomerular sclerosis by regulating cell proliferation, differentiation, and apoptosis through the activation of Smad signaling pathways.2 FK506, a topical calcineurin inhibitor, is widely used in kidney disease in recent years and has been successfully used as an alternative to cyclosporine to prevent graft rejection and treat autoimmune diseases,3 such as lupus nephritis, systemic vasculitis, and primary nephrotic syndrome. The clinic study suggests FK506 can reduce the proteinuria in patients; however, it is still unclear that whether the pharmacological action has direct or indirect effects on the function of mesangial cells. The purpose of this study is to observe the *These authors contributed equally to this study. They are all the firstauthors. Address correspondence to Shengyou Yu, Guangzhou First People’s Hospital, Guangzhou Medical university, Guangdong Province, China. E-mail: [email protected]
Received 6 October 2013 Revised 30 November 2013 Accepted 4 December 2013 Published online 10 February 2014
effect of FK506 on Smad2 protein and mRNA expression induced by TGF-b1 and further confirms the role of TGF-b1/ Smad signaling pathways in glomerular sclerosis. We also investigate whether FK506 can delay renal fibrosis through regulating TGF-b1/Smad signaling pathways and play an important role in antiproteinuria.
Materials and methods Cell culture and treatments
20 14
Introduction
History
Mesangial cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), cultured at 37 C in RPMI-1640 (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum (Sigma Chemical Co, St Louis, MO), 100 U/mL penicillin/streptomycin (Gibco), the culture medium was changed every 2 d. FK506 (Calbiochem; San Diego, CA) used in this study was dissolved in dimethylsulphoxide (DMSO). Cells were treated with FK506 at doses of 10, 100, and 1000 ng/mL with or without human recombinant TGF-b1 8 ng/mL (Peprotech, Rocky Hill, NJ) during the indicated time points. All experiments were repeated thrice. Cell proliferation assay Cell proliferation was assessed by 5-bromo-20 -deoxyuridine (BrdU) cell proliferation kit (Roche, Basel, Switzerland).
590
R. Qi et al.
Ren Fail, 2014; 36(4): 589–592
Briefly, cells were seeded on 96-well plates at a density of 1 105 cells/well and incubated overnight. Then the cells were treated and incubated for 8 h, 24 h, and 48 h. The treated cells were fixed and stained according to the manufacturer’s instructions. The optical density was measured at 570 nm with an ELISA plate reader equipped with Microplate Manager software version 2.1 (Model 450; Bio-Rad, Hercules, CA).
experimental groups in the study. Values of p50.05 were considered as statistically significant. All the experiments in our study were performed at least thrice.
RT-PCR analysis
The results shown in Figure 1 revealed that FK506 at various concentrations exhibited no significant effect on cell proliferation. Treatment with TGF-b1 significantly increased the proliferation of Glomerular mesangial cells. The addition of FK506 significantly suppressed the enhancing effect of TGF-b1 on cell proliferation.
Smad2 mRNA was extracted from Cells using Trizol Reagent(Sigma Chemical Co) according to the manufacturer’s instruction. Isolated RNA (1 mg) of each sample was subjected to reverse transcription by using Rever Tra Ace (Sigma Chemical Co) according to the manufacture’s protocol. The resulting cDNA (3 mL) was used for PCR amplification. The sequence-specific primers were designed and synthesized by Shanghai Invitrogen Biotechnology Co, Ltd. (Shangai, China). Primers used were as follows: Smad2 upstream and downstream primers were the following forward: 50 -TTCCAGACTTTGTGCTGTCCAGTAA-30 ; reverse: 50 -AGGGCAGAGGCTCCACTGAGTA-30 , product length being 120 bp; GAPDH upstream and downstream primers were the following forward: 5-GGTGAAGGTCGGTGT GAACGGAT-3; reverse: 5-CCACTTTGCCACTGCAAATG GCAG-3, product length being 118 bp. Levels of gene expression were expressed as the integrated density value of PCR products normalized to that of the GAPDH in the same sample. Independent experiment was repeated thrice.
Results FK506 treatment alone showed no significant effect on cell proliferation but significantly suppressed the TGF-b1-induced proliferation
FK506 reduced the protein and mRNA expressions of phosphorylated Smad2 induced by TGF-b1 It was noticed that TGF-b1 significantly altered the protein and mRNA expressions of Smad2. However, FK506 treatment
Western blot analysis Cells were lysed in the buffer containing 1% Tritonx-100 (Sigma Chemical Co., St Louis, MO), 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.7), 1 mM NaF, 1 mM NaVO3, and a protease inhibitor cocktail (Sigma Chemical Co., St Louis, MO). Seventy-five micrograms of total protein was loaded to run 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was set up for transfer protein to nitrocellulose membranes (Sigma Chemical Co., St Louis, MO). Then, the membranes were incubated with rabbit anti-first antibody (Sigma Chemical Co, St Louis, MO); mouse anti-GAPDH antibody (Sigma Chemical Co, St Louis, MO). The membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Sigma Chemical Co., St Louis, MO) for 50 min at room temperature, and then developed using ECL chemiluminescence reagent (Sigma Chemical Co., St Louis, MO). The specific protein bands were scanned and quantitated using a densitometry in relation to the GAPDH. Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, UK). We repeated each Western blot analysis using protein from three different and separate experiments. Statistical analysis SPSS 17.0 (SPSS Inc., Chicago, IL) was used for the statistical analysis. The results were expressed as mean ± standard deviation (mean ± SD). Student’s t test was used for statistical evaluation between control and
Figure 1. The comparisons of cell proliferation between the FK506 group and the control. Notes: cells were seeded into 96-well plates at a density of 3 104 cells/well. After starving in the serum-free medium overnight, the cells were incubated in the medium for 8, 24, and 48 h. Cell proliferation was assessed by using MTT assay. Absorbance was measured by ELISA plate-reader at 570 nm. The values were expressed as a mean ± SD. The presented data are based on at least three independent experiments. *p50.05, compared to normal group. The proliferation of FK506 group treated with FK506 10, 30, and 60 ng/mL for 8, 24, and 48 h was assessed by MTT cell proliferation assay. The data shown here (mean± SD) were from one representative experiment performed in triplicate, repeated three times with similar results.
Role of FK506 in CKD
DOI: 10.3109/0886022X.2014.882713
591
Figure 2. FK506 reduced the expressions of Smad2 protein increased by TGF-b1 in mesangial cells. Notes: Mesangial cells were cultured and treated with TGF-b1 5 ng/mL with or without the addition of FK506 (10, 30, and 60 ng/mL) for 48 h. Total cell protein lysates were prepared and subjected to Western blotting analysis with antibody against Smad2 and GAPDH. Experiments were repeated thrice with similar results. *p50.05 as compared with control group; #p50.05 as compared with TGF-b1 stimulated group. Lane 1: control; Lane 2: TGF-b1 5 ng/mL treatment; Lane 3: TGF-b1 5 ng/ mL + FK506 10 ng/mL treatment; Lane 4: TGF-b1 5 ng/mL + FK506 30 ng/mL treatment; Lane 5: TGF-b1 5 ng/mL + FK506 60 ng/mL treatment.
Table 1. FK506 reduced the expressions of Smad2 mRNA increased by TGF-b1 in mesangial cells.
The control FK506 10 ng/mL FK506 30 ng/mL FK506 60 ng/mL TGF-b1 5 ng/mL TGF-b1 + FK506 10 ng/mL TGF-b1 + FK506 30 ng/mL TGF-b1 + FK506 60 ng/mL
8h
24 h
48 h
0.151 ± 0.002 0.150 ± 0.014 0.148 ± 0.020 0.149 ± 0.031 1.101 ± 0.020# 0.671 ± 0.010*
0.172 ± 0.014 0.171 ± 0.041 0.169 ± 0.037 0.168 ± 0.021 1.192 ± 0.047# 0.761 ± 0.014*
0.194 ± 0.004 0.191 ± 0.006 0.190 ± 0.010 0.187 ± 0.014 1.221 ± 0.010# 0.897 ± 0.011*
0.473 ± 0.022*
0.583 ± 0.031*
0.578 ± 0.006*
0.311 ± 0.012*
0.452 ± 0.016*
0.491 ± 0.015*
Notes: Cells were treated with TGF-b1 5 ng/mL followed by FK506 10, 30, and 60 ng/mL and then incubated at indicated time points for the determination of Smad2 mRNA. The data shown here (mean ± SD) are from one representative experiment performed in triplicate, repeated thrice with similar results. *p50.05, #p50.01 as compared with the TGF-b1 stimulated group.
significantly decreased the phosphorylated Smad2 protein and mRNA expressions that were enhanced by TGF-b1 treatment.
Discussion TGF-b is a multifunctional cytokine that plays an important role in regulating cell growth, differentiation, and biosynthesis of extracellular connective tissue.4–7 TGF-b1 can induce the expression and synthesis of fibronectin in human mesangial cells.8–10 The discovery of Smads further reveals the mechanism of action of TGF-b. Smads protein is the direct substrate of TGF-b superfamily and also serves as an intermediating molecular that transmits interaction signals between TGF-b and receptor from cytoplasm to cell nucleus.11–14 In the TGF-b1/Smad signaling pathways, TGF-b1 first interacts with II receptor in cell membranes, activates TGF-b1RI, and phosphorylates the downstream Smad2/3. The activated Smad2/3 interacts with Smad4 to form complex, transfers from cytoplasm to nucleus, interacts with DNA, and regulates corresponding cell functions.
Smads is expressed in various tissues and takes part in the regulation of multiple cell functions, but its expression and location in normal renal tissues are still unclear. TGF-b1 can induce the transposition of Smad2 phosphorylation to nucleus.15 Our observations partly confirm this view. Smad2-mediated TGF-b signal can induce the expression of connective tissue growth factor and possesses pro-fibrogenic effects,16 indicating that TGF-b/Smad signaling pathways play an important role in glomerular diseases. Blocking the Smad2 signals can inhibit the transformation of epithelial cells induced by TGF-b1, thus delaying renal fibrosis. Blocking the TGF-b/Smad signaling pathways has important implications in treating resistant nephrotic syndrome and preventing treatment of renal fibrosis. FK506 is a new generation of CNI used in the treatment of kidney diseases and graft rejection by inhibiting the activation of T cells, the proliferation of dependent B cells on Th helper cells, and the expression of lymphokines such as IL-2, IL-3, and g-interferon, and IL-2R, to reduce immune inhibitory effects.17 Our previous study found that dexamethasone can protect podocyte apoptosis induced by puromycin aminonucleoside.18 A large number of clinical studies demonstrated that FK506 significantly reduced renal tissue damage and proteinuria in various kidney diseases. Previous studies reported that FK506 is associated with less renal interstitial fibrosis than cyclosporine in de novo renal transplants,19,20 suggesting that FK506 exhibited an antifibrotic effect and may be used to treat hyper fibrotic disorders. However, there are less work on the effects of FK506 towards TGF-b1/Smad signaling pathways in vitro. Our results provided evidences indicating that FK506 inhibited mesangial cells’ proliferation in high TGF-b1 environment and suppression of TGF-b/Smad signaling pathway is the involved molecular mechanism. In this study, we use different concentrations of FK506 to precede intervention for Smad2 expression stimulated by TGF-b1 in mesangial cells. The expressions of mesangial cells Smad2 and mRNA are little normally, but significantly increased when stimulated suggesting transcription levels of
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Smad2 protein and mRNA are consistent. From this dynamic view, it is observed that FK506 can decrease the Smad2 protein and mRNA expressions induced by TGF-b1. The effects of high and low doses of FK506 intervention have little difference towards Smad2 protein and mRNA expressions and thus have no statistically significant. Both the Smad2 protein and mRNA expressions significantly decrease after FK506 intervention. The results also demonstrate that Smad2 is one of the important effectors in TGF-b1 signaling pathways, and both TGF-b1 and Smad2 take part in the occurrence and in the development of glomerular sclerosis. We get an important conclusion that FK506 can make influence to the effects of TGF-b1/Smad signaling pathways by restraining mesangial cells Smad2 expressions stimulated by TGF-b1 at mRNA and proteins levels, and then protect mesangial cells and treat glomerular diseases. This paper reveals that the protection of FK506 on kidney diseases may by suppressing transduction of TGF-b1/Smad signaling pathways, provides more theoretical and experimental basement for glomerular diseases and ways for treatment idea towards delaying and controlling glomerular sclerosis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References 1. Yu S Y, Qi R, Zhao H. Losartan reverses glomerular podocytes injury induced by AngII via stabilizing the expression of GLUT1. Mol Biol Rep. 2013;40(11):6295–6301. 2. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-b family signalling. Nature. 2003;425:577–584. 3. Faulds D, Goa KL, Benfield P. Cyclosporin: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs. 1993;45:953–1040. 4. Shehata M, Cope GH, Johnson TS. Cyclosporine enhances the expression of TGF-bold beta in the juxtaglomerular cells of the rat kidney. Kidney Int. 1995;48:1487–1496.
Ren Fail, 2014; 36(4): 589–592
5. Huse M, Chen YG, Massague´ J. Crystal structure of the cytoplasmic domain of the type I TGF b receptor in complex with FKBP12. Cell. 1999;96:425–436. 6. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791. 7. Piek E, Westermark U, Kastemar M. Expression of transforminggrowth-factor (TGF)-beta receptors and Smad proteins in glioblastoma cell lines with distinct responses to TGF-beta1. Int J Cancer. 1999;80:756–763. 8. Takakura K, Tahara A, Sanagi M. Antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells[J]. Renal Fail. 2012;34(10):1309–1316. 9. Saglam F, Cavdar Z, Sarioglu S. Pioglitazone reduces peritoneal fibrosis via inhibition of TGF-b, MMP-2, and MMP-9 in a model of encapsulating peritoneal sclerosis. Renal Fail. 2012;34(1):95–102. 10. Zhang Y, Huang P, Jiang T. Role of aldose reductase in TGF-b1induced fibronectin synthesis in human mesangial cells. Mol Biol Rep. 2010;37:2735–2742. 11. Firemann S, Feuring E, Padberg W. Improvement of nephrotoxicity, hypertension, and lipid metabolism after version of kidney transplant recipient from cyclosporine to tacrolimus. Transplant Proc. 1998;30:1240–1242. 12. Ikedo H, Tamaki K, Ueda S. Smad protein and TGF-b signaling in vascular smooth muscle cells. Int J Mol Med. 2003;11:645–650. 13. Schnaper HW, Hayashida T, Hubchak SC. TGF-beta signal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol. 2003;284:F243–F252. 14. Buranakarl C, Kitjtawonrat A, Pondeenana S. Comparison of dipyridamole and fosinopril on renal progression in nephrectomized rats. Nephrology (Carlton). 2003;8:80–91. 15. Lan HY. Tubular epithelial-myofibroblast transdifferentia-tionmechanisms in proximal tubule cells. Curr Opin Nephro Hypertens. 2003;12:25–29. 16. Coimbra TM, Janssen U, Grone HJ. Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int. 2000;57:167–182. 17. Aramburu J, Heitman J, Crabtree GR. Calcineurin: a central controller of signalling in eukaryotes. EMBO Rep. 2004;5: 343–348. 18. Yu SY, Qi R. Role of bad in podocyte apoptosis induced by puromycin aminonucleoside. Transplant Proc. 2013;45:569–573. 19. Murphy GJ, Waller JR, Sandford RS. Randomized clinical trial of the effect of microemulsion cyclosporin and tacrolimus on renal allograft fibrosis. Br J Surg. 2003;90:680–686. 20. Ji SM, Li LS, Sha GZ. Conversion from cyclosporine to tacrolimus for chronic allograft nephropathy. Transplant Proc. 2007;39: 1402–1405.
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