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Effects of Inosine Monophosphate Dehydrogenase Inhibition on High Glucose-Induced Cellular Reactive Oxygen Species in Mesangial Cells
In vitro
Effects of Inosine Monophosphate Dehydrogenase Inhibition on High Glucose-Induced Cellular Reactive Oxygen Species in Mesangial Cells J.S. Song, K.H. Huh, J. Park, M.K. Ju, M.S. Kim, and Y.S. Kim ABSTRACT Mesangial cell extracellular matrix (ECM) synthesis plays an important role in chronic renal diseases including chronic renal allograft dysfunction and diabetic nephropathy. Although inosine monophosphate dehydrogenase 2 (IMPDH2), as a target of mycophenolic acid (MPA), is important for de novo guanosine synthesis in lymphocytes, mesenchymal cells are not wholly dependent on it. To explore the importance of IMPDH2 on the inhibitory effects of MPA in mesangial cells (MC), we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular reactive oxygen species (ROS). Mouse mesangial cells (MMC) were stimulated with HG (30 mmol/L D-glucose) in the presence or absence of MPA pretreatment or IMPDH2 siRNA transfection. Fibronectin secretion was measured by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS assessed by flow cytometry. HG increased fibronectin secretion by 1.8-fold at 24 hours and DCF-sensitive cellular ROS by 1.5-fold at 1 hour. MPA at 10 mol/L totally inhibited HG-induced fibronectin secretion and cellular ROS in MMC. However, IMPDH2 siRNA only partially suppressed HG-induced fibronectin secretion and cellular ROS. These results suggested that MPA may inhibit HG-induced fibronectin secretion partially through inhibiting cellular ROS and the inhibition of IMPDH2 may be partially involved in the mechanism of MPA.
From the Research Institute for Transplantation (J.S.S., K.H.H., J.P., M.S.K., Y.S.K.) and Department of Surgery (K.H.H., M.K.J., M.S.K., Y.S.K.), Yonsei University College of Medicine, Seoul, Korea, and BK21 Team of Nanobiomaterials for Cell-Based Implants, Yonsei University, Seoul, Korea (Y.S.K.). J.S. Song and K.H. Huh contributed equally to this work. 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.016 464
This work was supported by a grant from Korea Science and Engineering Foundation (KOSEF R01-2006-000-10829-0). Address reprint requests to Yu Seun Kim, MD, PhD, Department of Surgery, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Ku, Seoul 120-752, South Korea. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 464 – 466 (2008)
EFFECTS OF IMPDH2 INHIBITION
D
ESPITE THE RECENT improvement of short-term renal allograft survival, chronic renal allograft dysfunction remains the leading cause of long-term graft attrition. Extracellular matrix (ECM) accumulation in mesangial cells (MC) is considered a key feature in chronic renal allograft dysfunction as it is in various chronic renal diseases including diabetic nephropathy.1 Mycophenolic acid (MPA), a potent immunosuppressive agent, has the potential to prevent and treat chronic renal allograft dysfunction because it can inhibit mesenchymal cell and lymphocyte activation by several stimuli. MPA is a selective, noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), which is the rate-limiting enzyme in the de novo biosynthesis of guanosine nucleotide. Although guanosine depletion has been, in fact, reported to be a possible mechanism that MPA inhibits MC proliferation, the role of IMPDH in mesenchymal cells including MC is limited.2 In this connection, reactive oxygen species (ROS) have been proposed to be important signaling molecules in ECM synthesis.3,4 We previously reported that MPA inhibits platelet-derived growth factor (PDGF)-induced cellular ROS and subsequent activation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activation in vascular smooth muscle cells (VSMC)5 and in MC.6 Diabetes, whether it occurs before or after transplantation, is dependent on hyperglycemia and causes glomerulosclerosis. Plasma glucose concentration is the most important factor for diabetes. After renal transplantation, diabetes can be caused by various factors including suboptimal immunosuppression.7 Although MPA can reduce the toxicity of calcineurin inhibitors and the glomerulosclerosis of diabetic animal models, the mechanisms of MPA have not been clearly understood. In the present study, to explore the importance of IMPDH2 on the inhibitory effects of MPA in MCs, we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular ROS.
465 Tokyo, Japan) was used for the detection of fibronectin secretion into the media.
Flow Cytometry 5 (and 6)-Chloromethyl-2=,7=-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, Ore, United States)sensitive cellular ROS were measured as previously described.5
Statistical Analysis Results are expressed as mean values ⫾ standard errors. Statistical comparisons were performed by ANOVA followed by the Fisher least significant difference method. The level of significance was set at P ⬍ .05.
RESULTS Effects of MPA and IMPDH2 siRNA on HG-Induced Fibronectin Secretion
Fibronectin secretion by MMC was measured at 24 hours after the addition of HG. HG increased fibronectin secretion by 1.8-fold compared with control. MPA at 1 and 10 mol/L totally abrogated HG-induced fibronectin secretion (Fig 1A), and IMPDH2 siRNA at 10 nmol/L effectively
MATERIALS AND METHODS Cell Culture Mouse mesangial cells (MMC) from an SV40 transgenic mouse (MES-13) were purchased from American Type Culture Collection (Manassas, Va, United States) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Gaithersburg, Md, United States) containing 5% fetal bovine serum (FBS; Invitrogen). Predesigned mouse IMPDH2 siRNA was purchased from Ambion (Austin, Tex, United States). IMPDH2 siRNA was transfected by the lipofectamine method (Invitrogen) for 24 hours. MPA at 0.1, 1, and 10 mol/L was administered 1 hour before the addition of 30 mmol/L D-glucose (HG). Control groups were maintained in DMEM containing 5.6 mmol/L D-glucose (LG).
Western Blot Analysis Western blot analysis was performed as previously described.5 The peroxidase-conjugated fibronectin antibody (DAKO Japan Co,
Fig 1. (A) Effects of MPA on HG-induced fibronectin secretion. (B) Effects of IMPDH2 siRNA on HG-induced fibronectin secretion. The experimental protocol is detailed in Materials and Methods. Data are presented as the representative Western blot and mean values ⫾ SE of 5 experiments. *P ⬍ .05 vs LG; †P ⬍ .05 vs HG.
466
SONG, HUH, PARK ET AL
DISCUSSION
MPA totally blocked HG-induced fibronectin secretion in MMC. These results agreed with the previous results, which were the effects of MPA on TGF-1- and PDGF-induced fibronectin synthesis in human and rat MCs, respectively.6,8 The dose of MPA that inhibited HG-induced fibronectin secretion in this study is clinically attainable. IMPDH2 siRNA also partly, but not totally, suppressed HG-induced fibronectin secretion. These observations suggested that MPA may inhibit HG-induced ECM synthesis partly through inhibiting IMPDH2 in MC. HG-induced cellular ROS were increased and maintained from 1 hour to 48 hours. MPA wholly suppressed HG-induced cellular ROS in MMC, but IMPDH2 siRNA also partially down-regulated HG-induced cellular ROS. Thus, it appears that IMPDH2 is an important mediator for HG-induced cellular ROS, but HG-induced cellular ROS up-regulation is not totally dependent on IMPDH2. In conclusion, we demonstrated that MPA and IMPDH2 siRNA inhibited HG-induced fibronectin secretion partly through IMPDH2-dependent cellular ROS and that MPA has other mechanisms for the inhibition of MC activation including ECM synthesis and cellular ROS. REFERENCES Fig 2. (A) Effects of MPA on HG-induced cellular ROS. (B) Effects of IMPDH2 siRNA on HG-induced cellular ROS. The experimental protocol is detailed in Materials and Methods. Data are presented as the mean values ⫾ SE of 5 experiments. *P ⬍ .05 vs LG; †P ⬍ .05 vs HG.
transfected into MMC and then suppressed IMPDH2 mRNA expression (data not shown). The reduction of fibronectin secretion by IMPDH2 siRNA at 10 nmol/L was 1.3-fold of control (Fig 1B).
Effects of MPA and IMPDH2 siRNA on HG-Induced Cellular ROS
HG increased DCF-sensitive cellular ROS by 1.6-fold at 1 hour and this increment was maintained at 48 hours. HG-induced cellular ROS was also blocked by MPA (Fig 2A), but partially ameliorated by IMPDH2 siRNA (Fig 2B).
1. Nankivell BJ, Borrows RJ, Fung CL, et al: The natural history of chronic allograft nephropathy. N Engl J Med 349:2326, 2003 2. Allison AC, Eugui EM: Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 80:S181, 2005 3. Ha H, Lee HB: Reactive oxygen species and matrix remodeling in diabetic kidney. J Am Soc Nephrol 14:S246, 2003 4. Ha H, Park J, Kim YS, et al: Oxidative stress and chronic allograft nephropathy. Yonsei Med J 45:1049, 2004 5. Park J, Ha H, Seo J, et al: Mycophenolic acid inhibits platelet-derived growth factor-induced reactive oxygen species and mitogen-activated protein kinase activation in rat vascular smooth muscle cells. Am J Transplant 4:1982, 2004 6. Ha H, Kim MS, Park J, et al: Mycophenolic acid inhibits mesangial cell activation through p38 MAPK inhibition. Life Sci 79:1561, 2006 7. van Hooff JP, Christiaans MH, van Duijnhoven EM: Tacrolimus and posttransplant diabetes mellitus in renal transplantation. Transplantation 79:1465, 2005 8. Dubus I, Vendrely B, Christophe I, et al: Mycophenolic acid antagonizes the activation of cultured human mesangial cells. Kidney Int 62:857, 2002
Effects of Inosine Monophosphate Dehydrogenase Inhibition on High Glucose-Induced Cellular Reactive Oxygen Species in Mesangial Cells J.S. Song, K.H. Huh, J. Park, M.K. Ju, M.S. Kim, and Y.S. Kim ABSTRACT Mesangial cell extracellular matrix (ECM) synthesis plays an important role in chronic renal diseases including chronic renal allograft dysfunction and diabetic nephropathy. Although inosine monophosphate dehydrogenase 2 (IMPDH2), as a target of mycophenolic acid (MPA), is important for de novo guanosine synthesis in lymphocytes, mesenchymal cells are not wholly dependent on it. To explore the importance of IMPDH2 on the inhibitory effects of MPA in mesangial cells (MC), we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular reactive oxygen species (ROS). Mouse mesangial cells (MMC) were stimulated with HG (30 mmol/L D-glucose) in the presence or absence of MPA pretreatment or IMPDH2 siRNA transfection. Fibronectin secretion was measured by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS assessed by flow cytometry. HG increased fibronectin secretion by 1.8-fold at 24 hours and DCF-sensitive cellular ROS by 1.5-fold at 1 hour. MPA at 10 mol/L totally inhibited HG-induced fibronectin secretion and cellular ROS in MMC. However, IMPDH2 siRNA only partially suppressed HG-induced fibronectin secretion and cellular ROS. These results suggested that MPA may inhibit HG-induced fibronectin secretion partially through inhibiting cellular ROS and the inhibition of IMPDH2 may be partially involved in the mechanism of MPA.
From the Research Institute for Transplantation (J.S.S., K.H.H., J.P., M.S.K., Y.S.K.) and Department of Surgery (K.H.H., M.K.J., M.S.K., Y.S.K.), Yonsei University College of Medicine, Seoul, Korea, and BK21 Team of Nanobiomaterials for Cell-Based Implants, Yonsei University, Seoul, Korea (Y.S.K.). J.S. Song and K.H. Huh contributed equally to this work. 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.016 464
This work was supported by a grant from Korea Science and Engineering Foundation (KOSEF R01-2006-000-10829-0). Address reprint requests to Yu Seun Kim, MD, PhD, Department of Surgery, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Ku, Seoul 120-752, South Korea. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 464 – 466 (2008)
EFFECTS OF IMPDH2 INHIBITION
D
ESPITE THE RECENT improvement of short-term renal allograft survival, chronic renal allograft dysfunction remains the leading cause of long-term graft attrition. Extracellular matrix (ECM) accumulation in mesangial cells (MC) is considered a key feature in chronic renal allograft dysfunction as it is in various chronic renal diseases including diabetic nephropathy.1 Mycophenolic acid (MPA), a potent immunosuppressive agent, has the potential to prevent and treat chronic renal allograft dysfunction because it can inhibit mesenchymal cell and lymphocyte activation by several stimuli. MPA is a selective, noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), which is the rate-limiting enzyme in the de novo biosynthesis of guanosine nucleotide. Although guanosine depletion has been, in fact, reported to be a possible mechanism that MPA inhibits MC proliferation, the role of IMPDH in mesenchymal cells including MC is limited.2 In this connection, reactive oxygen species (ROS) have been proposed to be important signaling molecules in ECM synthesis.3,4 We previously reported that MPA inhibits platelet-derived growth factor (PDGF)-induced cellular ROS and subsequent activation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activation in vascular smooth muscle cells (VSMC)5 and in MC.6 Diabetes, whether it occurs before or after transplantation, is dependent on hyperglycemia and causes glomerulosclerosis. Plasma glucose concentration is the most important factor for diabetes. After renal transplantation, diabetes can be caused by various factors including suboptimal immunosuppression.7 Although MPA can reduce the toxicity of calcineurin inhibitors and the glomerulosclerosis of diabetic animal models, the mechanisms of MPA have not been clearly understood. In the present study, to explore the importance of IMPDH2 on the inhibitory effects of MPA in MCs, we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular ROS.
465 Tokyo, Japan) was used for the detection of fibronectin secretion into the media.
Flow Cytometry 5 (and 6)-Chloromethyl-2=,7=-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, Ore, United States)sensitive cellular ROS were measured as previously described.5
Statistical Analysis Results are expressed as mean values ⫾ standard errors. Statistical comparisons were performed by ANOVA followed by the Fisher least significant difference method. The level of significance was set at P ⬍ .05.
RESULTS Effects of MPA and IMPDH2 siRNA on HG-Induced Fibronectin Secretion
Fibronectin secretion by MMC was measured at 24 hours after the addition of HG. HG increased fibronectin secretion by 1.8-fold compared with control. MPA at 1 and 10 mol/L totally abrogated HG-induced fibronectin secretion (Fig 1A), and IMPDH2 siRNA at 10 nmol/L effectively
MATERIALS AND METHODS Cell Culture Mouse mesangial cells (MMC) from an SV40 transgenic mouse (MES-13) were purchased from American Type Culture Collection (Manassas, Va, United States) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Gaithersburg, Md, United States) containing 5% fetal bovine serum (FBS; Invitrogen). Predesigned mouse IMPDH2 siRNA was purchased from Ambion (Austin, Tex, United States). IMPDH2 siRNA was transfected by the lipofectamine method (Invitrogen) for 24 hours. MPA at 0.1, 1, and 10 mol/L was administered 1 hour before the addition of 30 mmol/L D-glucose (HG). Control groups were maintained in DMEM containing 5.6 mmol/L D-glucose (LG).
Western Blot Analysis Western blot analysis was performed as previously described.5 The peroxidase-conjugated fibronectin antibody (DAKO Japan Co,
Fig 1. (A) Effects of MPA on HG-induced fibronectin secretion. (B) Effects of IMPDH2 siRNA on HG-induced fibronectin secretion. The experimental protocol is detailed in Materials and Methods. Data are presented as the representative Western blot and mean values ⫾ SE of 5 experiments. *P ⬍ .05 vs LG; †P ⬍ .05 vs HG.
466
SONG, HUH, PARK ET AL
DISCUSSION
MPA totally blocked HG-induced fibronectin secretion in MMC. These results agreed with the previous results, which were the effects of MPA on TGF-1- and PDGF-induced fibronectin synthesis in human and rat MCs, respectively.6,8 The dose of MPA that inhibited HG-induced fibronectin secretion in this study is clinically attainable. IMPDH2 siRNA also partly, but not totally, suppressed HG-induced fibronectin secretion. These observations suggested that MPA may inhibit HG-induced ECM synthesis partly through inhibiting IMPDH2 in MC. HG-induced cellular ROS were increased and maintained from 1 hour to 48 hours. MPA wholly suppressed HG-induced cellular ROS in MMC, but IMPDH2 siRNA also partially down-regulated HG-induced cellular ROS. Thus, it appears that IMPDH2 is an important mediator for HG-induced cellular ROS, but HG-induced cellular ROS up-regulation is not totally dependent on IMPDH2. In conclusion, we demonstrated that MPA and IMPDH2 siRNA inhibited HG-induced fibronectin secretion partly through IMPDH2-dependent cellular ROS and that MPA has other mechanisms for the inhibition of MC activation including ECM synthesis and cellular ROS. REFERENCES Fig 2. (A) Effects of MPA on HG-induced cellular ROS. (B) Effects of IMPDH2 siRNA on HG-induced cellular ROS. The experimental protocol is detailed in Materials and Methods. Data are presented as the mean values ⫾ SE of 5 experiments. *P ⬍ .05 vs LG; †P ⬍ .05 vs HG.
transfected into MMC and then suppressed IMPDH2 mRNA expression (data not shown). The reduction of fibronectin secretion by IMPDH2 siRNA at 10 nmol/L was 1.3-fold of control (Fig 1B).
Effects of MPA and IMPDH2 siRNA on HG-Induced Cellular ROS
HG increased DCF-sensitive cellular ROS by 1.6-fold at 1 hour and this increment was maintained at 48 hours. HG-induced cellular ROS was also blocked by MPA (Fig 2A), but partially ameliorated by IMPDH2 siRNA (Fig 2B).
1. Nankivell BJ, Borrows RJ, Fung CL, et al: The natural history of chronic allograft nephropathy. N Engl J Med 349:2326, 2003 2. Allison AC, Eugui EM: Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 80:S181, 2005 3. Ha H, Lee HB: Reactive oxygen species and matrix remodeling in diabetic kidney. J Am Soc Nephrol 14:S246, 2003 4. Ha H, Park J, Kim YS, et al: Oxidative stress and chronic allograft nephropathy. Yonsei Med J 45:1049, 2004 5. Park J, Ha H, Seo J, et al: Mycophenolic acid inhibits platelet-derived growth factor-induced reactive oxygen species and mitogen-activated protein kinase activation in rat vascular smooth muscle cells. Am J Transplant 4:1982, 2004 6. Ha H, Kim MS, Park J, et al: Mycophenolic acid inhibits mesangial cell activation through p38 MAPK inhibition. Life Sci 79:1561, 2006 7. van Hooff JP, Christiaans MH, van Duijnhoven EM: Tacrolimus and posttransplant diabetes mellitus in renal transplantation. Transplantation 79:1465, 2005 8. Dubus I, Vendrely B, Christophe I, et al: Mycophenolic acid antagonizes the activation of cultured human mesangial cells. Kidney Int 62:857, 2002