BACKGROUND: Toll like receptor (TLR), an element of innate immunity, is upregulated by Ischemia/reperfusion (IR) injury and may be involved in adaptive immune response. Immunosuppressive agents may increase or attenuate IR injury and TLR expression. To explore the involvement of TLRs in hypoxic tubular injury and modification by mycophenolic acid (MPA) rapamycin (RAP), this study examined TLR expression in hypoxia-induced human renal proximal tubular epithelial cells (HK-2). METHODS: HK-2 cells were cultured in keratinocyte-SFM media supplemented with epidermal growth factor and bovine pituitary extract. The Induction of hypoxia was achieved using GasPak pouch system. TLR 2, 3, and 4 mRNA expression was analyzed by real time RT-PCR using SYBR green and TLR 4 protein expression was evaluated by Western blot analysis. MPA at concentration of 100 nM and 1uM and RAP at concentration of 20, 50, and 100 nM were added to culture medium. RESULTS: TLR4 but noTLR2 or TLR3 mRNA expressions increased in hypoxic HK-2 cells at 24 and 48 hrs. TLR4 protein expression also increased in hypoxic HK-2 cells at 24 and 48 hrs. MPA (100 nM and 1uM) and RAP (20, 50, and 100 nM) decreased hypoxia-induced TLR4 mRNA expression in HK-2 cells compared to normoxia at 24 hrs. However, TLR4 protein expression was decreased only by RAP at 20 and 50 nM. CONCLUSIONS: The results suggest that RAP may modify hypoxic renal tubular damage by decreasing TLR4-mediated inflammatory and immune reactions.
Adaptive Immunity ; Anoxia ; Blotting, Western ; Epidermal Growth Factor ; Epithelial Cells ; Humans ; Immunity, Innate ; Immunosuppressive Agents ; Mycophenolic Acid ; RNA, Messenger ; Sirolimus ; Toll-Like Receptors

Oxidative stress defined as outbalanced generation of reactive oxygen species (ROS) than the existing antioxidative defense mechanisms plays an important role in tissue injury. Ischemia/reperfusion accompanied during organ transplantation is well- established oxidative stress-induced tissue injury. We hypothesized that oxidative stress may also play a role in the development and progression of chronic allograft nephropathy (CAN), since that ROS are major signaling molecules of growth factors and cytokines [platelet-derived growth factors, transforming growth factor-beta1 (TGF-beta1) ] upregulated in the kidney of CAN, that ROS in turn upregulate TGF-beta1, and that mycophenolic acid may inhibit features of CAN [proliferation and extracellular matrix (ECM) accumulation in vascular smooth muscle cells and glomerular mesangial cells] through inhibiting cellular ROS. Cellular ROS activate signal transduction cascade (protein kinase C, mitogen-activated protein kinases, and janus kinases) and transcription factors (nuclear factor-kappa B, activated protein-1, specificity protein 1, and signal transducers and activators of transcription) leading to regulation of genes and proteins involved in cellular proliferation, ECM remodeling, and apoptosis accompanied in CAN. This review is intended to provide an overview of oxidative stress in renal allograft nephropathy.
Chronic Disease ; Humans ; Kidney Diseases/*etiology ; Kidney Transplantation/*adverse effects ; *Oxidative Stress ; Research Support, Non-U.S. Gov't

PURPOSE: Proliferation, migration, and the accumulation of extracellular matrix (ECM) protein of vascular smooth muscle cells (VSMC) play roles for transplant arteriosclerosis. We have previously reported that carvedilol (CA) inhibits the proliferation and the migration of VSMCs. The present study examined the effects of CA on platelet-derived growth factor (PDGF)-induced collagen synthesis in VSMC and the roles of reactive oxygen species (ROS), extracellular signal- regulated protein kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). METHODS: Primary cultured rat VSMCs were obtained from aorta of Sprague-Dawley rats. Growth arrested and synchronized cells were pretreated with CA (10 nM~10micrometer) at 1 hour before the addition of PDGF 10 ng/ml. Collagen synthesis was measured by 3[H]-proline incorporation, ROS by flow cytometry using ROS-sensitivedichlorofluorescein (DCF) dye, and the activation of ERK andp38 MAPK by Western blot analysis. RESULTS: PDGF significantly increased collagen synthesis by 2.0-fold, intracellular ROS by 1.6-fold, the activation of ERK 1/2 and p38 MAPK by 4.2-fold and 3.9-fold compared to control, respectively. CA above 1micrometer inhibited PDGF-induced collagen synthesis. CA also inhibited DCF-sensitive ROS and the activation of ERK and p38 MAPK. All pharmacological inhibitors of ROS, ERK, and p38 MAPK effectively inhibited PDGF-induced collagen synthesis. CONCLUSION: These data suggest that CA inhibit PDGF-induced collagen synthesis possibly through inhibiting intracellular ROS and ERK 1/2 and p38 MAPK activation.
Animals ; Aorta ; Arteriosclerosis ; Blotting, Western ; Collagen* ; Extracellular Matrix ; Flow Cytometry ; Muscle, Smooth, Vascular* ; p38 Mitogen-Activated Protein Kinases ; Platelet-Derived Growth Factor ; Protein Kinases ; Rats* ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; Signal Transduction*

PURPOSE: Typical pathologic lesions of chronic allograft rejection or transplant vascular sclerosis are similar to arteriosclerotic vascular lesion of non-transplant patients, or vascular remodeling process after vascular injury. Abnormal and excess proliferation of vascular smooth muscle cells (VSMC) which are triggered by endothelium-derived cytokines or growth factors, play a major role during these process. Effective prophylactic or therapeutic strategies against chronic rejection or transplant vasculopathy is not yet clearly established. Recent in vitro cell culture study showed that carvedilol, a novel antihypertensive agent has the significant inhibitory activities against the proliferation of VSMC. METHODS: Using in vitro VSMC culture techniques, we measured anti-proliferative activity of carvedilol alone, or in combination of cyclosporine, a basic immunosuppressive agent for transplantation. Growth-arrested early passage (3-5) cultured VSMC from the aorta of rat (Sprague-Dawley) were exposed to platelet derived growth factor (PDGF), endothelin-l, or antiotensin-ll, respectively. Carvedilol and/or cyclosporine was added as inhibitors. Proliferation was assessed by incorporated [(3)H]-thymidine activity. RESULTS: PDGF stimulated mitogenesis most effectively. Carvedilol inhibited mitogenesis in dose-dependent manner in the presence of PDGF(10ng/ml). Compared to control, proliferation was significantly decreased to 60.3 (+/-10.4)% and 18.3 (+/- 5.9)% in the presence of 1 micro M and 10 micro M of carvidilol, respectively (p<0.05, each). Carvedilol also produced significant concentration-dependent inhibitory activities against VSMC stimulated by endothelin-1 (10 nM) and angiotensin-II (100 nM). The IC50 of carvedilol in PDGF-, endothelin-l, and angiotensin-ll-stimulated VSMC were 1-10 micro M. Cyclosporine (100 nM) did not show significant inhibition of VSMC regardless of the kinds of cytokines. However, combined addition of carvedilol and cyclosporine produced significant VSMC inhibition. The pattern of inhibition in c very similar with that of carvedilol alone group regardless of the kinds of cytokines. CONCLUSION: We demonstrated that carvedilol significantly inhibited the proliferation of VSMC regardless of the kind of cytokines, and even under the presence of cyclosporine in VSMC cultures. These indicated that carvedilol has the unique potential to reduce the development of transplant vasculopathy when used with cyclosporine in hypertensive renal transplant recipients.
Allografts ; Animals ; Aorta ; Cell Culture Techniques ; Culture Techniques ; Cyclosporine* ; Cytokines ; Endothelin-1 ; Humans ; Inhibitory Concentration 50 ; Intercellular Signaling Peptides and Proteins ; Muscle, Smooth, Vascular* ; Platelet-Derived Growth Factor ; Rats* ; Sclerosis ; Transplantation ; Vascular System Injuries

PURPOSE: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which are triggered by endothelium-derived cytokines or growth factors, play a major role in the chronic transplant vasculopathy or vascular remodeling process after vascular injury. We have reported that carvedilol, a new anti-hypertensive agent, inhibits cytokine-triggered proliferation of cultured rat VSMCs. In this study, we investigate the effect of carvedilol on the migration of rat VSMCs. METHODS: Growth-arrested cultured VSMCs (passage 8-11) from the aorta of rat (Sprague-Dawley) were used. Migration was measured using a microchemotaxis chamber with a polycarbonate membrane. Platelet derived growth factor (PDGF) or angiotensin-II (ANG-II) was used as a stimulator and was added into the lower well of the chamber. A density of 1X104 cells per well with carvedilol and/or cyclosporine A (CsA) was seeded into the upper well of chamber. Degree of migration was assessed by using the number of migrated cells per high power field of light microscopy. RESULTS: PDGF and ANG-II stimulated VSMC chemotaxis effectively. Carvedilol decreased PDGF-induced migration to 88.9 (+/-16.0)% and 37.4 (+/-10.5)% at 1 microM and 10 microM, respectively. Carvedilol inhibited both PDGF and ANG-IIinduced chemotaxis in a concentration-dependent manner. The IC50 of carvedilol in PDGF and ANG-ll-induced VSMC migration was around 10 microM. CsA (100 nM) neither significantly inhibited the migration of VSMC, regardless of the kinds of cytokines, nor affected the inhibitory activities of carvedilol. The pattern of inhibition in the group with a combined addition of carvedilol and CsA was very similar to that of carvedilol alone group, regardless of the kinds of cytokines. CONCLUSION: We demonstrated that carvedilol alone or in the presence of CsA significantly inhibited the cytokine- induced migration of VSMC. These data indicate that carvedilol has a unique potential to reduce the development of chronic transplant vasculopathy when used with CsA in hypertensive renal transplant recipients.
Animals ; Aorta ; Chemotaxis ; Cyclosporine* ; Cytokines ; Inhibitory Concentration 50 ; Intercellular Signaling Peptides and Proteins ; Membranes ; Microscopy ; Muscle, Smooth, Vascular* ; Platelet-Derived Growth Factor ; Rats* ; Transplantation ; Vascular System Injuries

Rapamycin, a macrocyclic lactone, is effective in reducing the incidence of acute rejection after renal transplantation. The inhibitory effects of rapamycin on lymphocyte proliferation and the molecular mechanisms that were involved have been described. However, its effects on glomerular mesangial cells have not been clearly understood, and here, we examined the effect of rapamycin on platelet-derived growth factor (PDGF) - induced extracellular matrix synthesis as well as cell proliferation in mesangial cells. Rat mesangial cells were isolated from the glomeruli of Sprague-Dawley rats and cultured with Dulbecco's modified Eagles medium containing 20% fetal bovine serum. Different concentrations of rapamycin were administered 1 hour before the addition of 10 ng/ml of PDGF into growth arrested and synchronized cells. Cell proliferation was assessed by [3H]thymidine incorporation, total collagen synthesis by [3H]proline incorporation, and fibronectin secretion into the medium by Western blot analysis. In the mesangial cells, PDGF increased cell proliferation by 4.6-fold, total collagen synthesis by 1.8-fold, and fibronectin secretion by 3.2-fold. Rapamycin above 10 nM significantly inhibited PDGF-induced proliferation and collagen synthesis, but the treatment of rapamycin up to 1micrometer did not show any significant effects on PDGF-induced fibronectin secretion. These inhibitory effects of rapamycin on PDGF-induced mesangial cell proliferation and collagen synthesis reflect the potential value of rapamycin in the prevention and treatment of glomerulosclerosis in patients with chronic allograft nephropathy.
Animals ; Cells, Cultured ; Collagen/*antagonists & inhibitors/biosynthesis ; Fibronectins/*biosynthesis ; Glomerular Mesangium/cytology/drug effects/*metabolism ; Immunosuppressive Agents/*pharmacology ; Male ; Platelet-Derived Growth Factor/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Sirolimus/*pharmacology

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of chronic allograft vasculopathy as in atherosclerosis. We already reported that mycophenolic acid (MPA) inhibited VSMC proliferation, cellular reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) in human VSMCs. In this study, we examined further molecular mechanisms involved in the anti-proliferative effect of MPA in rat VSMCs. METHODS: Primary rat VSMCs were stimulated with PDGF-BB 10 ng/mL in the presence or absence of MPA and various kinds of cell signaling inhibitors. Cell proliferation was assessed by [H3]- thymidine incorporation, NAD(P)H oxidase subunits mRNA expression by RT-PCR, dichlorofluorescein- sensitive cellular ROS by FACS, and the activation of PDGF receptor-beta (Tyr 751), rac1, and MAPK by Western blot analysis. RESULTS: PDGF increased cell proliferation and cellular ROS, activation of PDGF receptor-beta (Tyr 751), rac1, expression of p22phox and MOX1 mRNA, ERK 1/2, and p38 MAPK, compared to control. MPA inhibited up-regulation of rac1 phosphorylation, p22phox and MOX1 mRNA expression, cellular ROS, and phosphorylation of ERK 1/2 and p38 MAPK. However, MPA did not affect PDGF receptor-beta (Tyr 751) activation. Wortmannin, diphenyleniodonium (DPI), trolox, and NAC, each inhibited PDGF- induced ERK 1/2 and p38 MAPK activation. PD98059 and p38 MAPK inhibitor also inhibited PDGF-induced cell proliferation. CONCLUSION: These results suggest that MPA inhibits PDGF-induced VSMC proliferation through inhibiting NAD(P)H oxidase-dependent cellular ROS leading to ERK 1/2 and p38 MAPK activation.
Allografts ; Animals ; Atherosclerosis ; Blotting, Western ; Cell Proliferation ; Humans ; Mitogen-Activated Protein Kinases ; Muscle, Smooth, Vascular* ; Mycophenolic Acid* ; NADPH Oxidase ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Rats ; Reactive Oxygen Species ; RNA, Messenger ; Thymidine ; Up-Regulation

PURPOSE: Increased oxidative stress and chronic inflammatory process have been substantially linked with the development and progression of chronic renal failure. However, the roles of oxidative stress and inflammation in chronic renal allograft dysfunction are not yet clear. The present study examined the plasma levels of lipid peroxides (LPO), C-reactive protein (CRP), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), transforming growth factor-beta1 (TGF-beta1), and heat shock protein 70 (Hsp 70) as the representative markers of oxidative stress, the inflammatory process and fibrosis among healthy control subjects (kidney donors, n=24), end-stage renal disease (ESRD) patients (n=28), transplant recipients with a serum creatinine (Scr) less than 1.5 mg% (n=30), and transplant recipients with Scr between 1.5 and 5.0 mg% (n=32) during the time at least one year after renal transplantation. METHODS: The plasma LPO concentration was measured by a thiobarbituric acid (TBA) reaction, CRP was measured by latex-enhanced immunonephelometric assay, and TGF-beta1, IL-6, sIL-6R, and HSP 70 were measured by enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: The Plasma concentrations of LPO, TGF-beta1, Hsp 70, and IL-6 were significantly higher in both the ESRD and renal transplant recipients with Scr levels between 1.5 and 5.0 mg% than in both the healthy controls and transplant patients with Scr levels below 1.5 mg%. Plasma sIL-6R was significantly increased only in the ESRD patients. Among the other 3 groups, the concentrations of sIL-6R were not different between the groups. The plasma CRP concentrations were not different among the 4 study groups. Upon regression analysis, the plasma concentrations of LPO, TGF-beta1, Hsp 70, IL-6 and sIL-6R were positively correlated with the Scr level, respectively. CONCLUSION: Our results demonstrate that the markers of oxidative stress, inflammation and fibrosis were correlated with the Scr level which represents the graft (and native) renal function.
Allografts ; C-Reactive Protein ; Creatinine* ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Fibrosis ; HSP70 Heat-Shock Proteins ; Humans ; Inflammation ; Interleukin-6 ; Kidney Failure, Chronic ; Kidney Transplantation* ; Lipid Peroxides ; Living Donors* ; Oxidative Stress* ; Plasma* ; Receptors, Interleukin-6 ; Tissue Donors ; Transforming Growth Factor beta1 ; Transplantation ; Transplants

PURPOSE: Proliferation and extracellular matrix (ECM) accumulation in the vascular smooth muscle cell (VSMC) and glomerular mesangial cell (MC) play key roles in the development and the progression of transplant glomerulosclerosis and chronic allograft nephropathy. Tautomycetin (TMC), a newly developed immunosuppressive agent, induces T-lymphocyte apoptosis through the inhibition of tyrosine kinase and protein phosphatase 1. We examined the effects of TMC on platelet-derived growth factor (PDGF)-induced proliferation and ECM synthesis in cultured VSMCs and MCs of Sprague- Dawley rats, and investigated the molecular mechanisms that are involved. METHODS: Different concentrations of TMC were administered 1 hour before the addition of PDGF 10 ng/mL into the growth-arrested and synchronized cells. Cell proliferation was assessed by methylthiazoletetrazolium (MTT) assay. Caspase-3 cleavage, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK were assessed by Western blot analysis, respectively. RESULTS: PDGF 10 ng/ mL increased cell proliferation, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK in both VSMCs and MCs. In both cultured cells, TMC at above 1 microgram/mL significantly reduced basal MTT and increased cleavage caspase-3 in a dose-dependent manner. TMC at 100 ng/mL decreased the PDGF-induced VSMC and MC proliferation without cytotoxicity. However, fibronectin secretion and the activation of Akt, ERK, and p38 MAPK were not affected at this low concentration of TMC, respectively. CONCLUSION: The present data demonstrated that low-dose TMC reduced PDGF-induced VSMC and MC proliferation without affecting the fibronectin secretion and cellular kinase activation.
Allografts ; Animals ; Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Proliferation ; Cells, Cultured ; Extracellular Matrix ; Fibronectins* ; Mesangial Cells* ; Muscle, Smooth, Vascular* ; p38 Mitogen-Activated Protein Kinases ; Phosphotransferases ; Platelet-Derived Growth Factor ; Protein Phosphatase 1 ; Protein-Tyrosine Kinases ; Rats ; T-Lymphocytes

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-beta1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 microM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-beta1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-beta1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-beta1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.
Animals ; Blotting, Western ; Diabetic Nephropathies ; Extracellular Matrix ; Fibrinolysin ; Fibronectins ; Fibrosis ; Glucose ; Liposomes ; Mesangial Cells ; Oligodeoxyribonucleotides ; Plasminogen ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Rats ; Renal Insufficiency, Chronic ; Transfection ; Transforming Growth Factor beta1 ; Transforming Growth Factors ; Up-Regulation