Effect of Mycophenolic Acid on Human Vascular Smooth Muscle Cell Proliferation and Its Signal Transduction

PURPOSE: Vascular smooth muscle cells (VSMCs) migration and proliferation play important roles in chronic allograft rejection. Mycophenolic acid (MPA) inhibits the proliferation of VSMCs, glomerular mesangial cells and fibroblasts as well as lymphocytes. Since reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) play important roles in the proliferation of VSMCs, the present study examined the effects of MPA on intracellular ROS generation, activation of ERK and p38 MAPK, and the proliferation of VSMCs cultured under platelet derived growth factor (PDGF). METHODS: Human VSMCs obtained from ATCC were cultured with RPMI-1640 containing 10% fetal bovine serum. Near confluent VSMCs were incubated with serum-free media for 48 hours to arrest and synchronize the cell growth. MPA was administered 1 hour before the addition of PDGF. 5-(and-6)- chloromethyl-2',7'-dichlorodihydrofluorescein (DCF)-sensitive intracellular ROS was detected by FACS. Activations of ERK1/ERK2 and p38 MAPK were measured by Western blot analysis. Proliferation of VSMC was assessed by [(3)]. RESULTS: PDGF administered at 10 ng/ml, which induced human VSMCs proliferation, rapidly increased intracellular ROS by 1.6-fold (P<0.05), ERK1/ERK2 activation by 2.1-fold, (P<0.05) and p38 MAPK activation by 1.9-fold (P<0.05), respectively, as compared to the control. MPA 1 and 10nM effectively inhibited PDGF-induced human VSMCs proliferation. MPA also effectively inhibited PDGF-induced intracellular ROS generation as well as ERK1/ERK2 and p38 MAPK activation. CONCLUSION: The present study suggests that MPA inhibits PDGF-induced human VSMCs proliferation, possibly by inhibiting intracellular ROS generation and the phosphorylation of ERK1/ERK2 and p38 MAPK. H]-thymidine incorporation.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin(TCDD) on Glomerular Mesangial and Tubular Epithelial Cell Activation / 대한신장학회잡지

BACKGROUND: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototype compound of polyhalogenated aromatic hydrocarbons, produces diverse biologic effects. Although nephrotoxicity of aromatic hydrocarbons such as benzo[a]pyrene(BP) is well known, little is known about the effects of TCDD on renal function. Thus, the present study examined the effects of TCDD on cell viability, proliferation, and extracellular matrix(ECM) synthesis by glomerular mesangial cells, LLC-PK1 cells representing proximal tubular epithelial cells, and MDCK cells representing distal epithelial cells and compared with the effects of BP. METHODS: Quiescent cells were incubated with serum free media containing different concentrations of TCDD(1-100 nM) and BP(3 and 30 micro M) for 24- 96 hours. Cell viability and proliferation were assessed by lactate dehydrogenase(LDH) release and [3H]-thymidine incorporation, respectively. Secreted fibronectin was measured by Western blot analysis. RESULTS: When cells were continuously exposed to TCDD, LDH release significantly increased in MMC, LLC-PK1, and MDCK in a dose- and a time- dependent manner. [3H]-Thymidine incorporation was increased in MMC and LLC-PK1 but decreased in MDCK by TCDD. Contrary to TCDD, 30 micro BP significantly inhibited [3H]-thymidine incorporation in MMC and MDCK but not in LLC-PK1. Both TCDD and BP increased fibronectin secretion by MMC, LLC-PK1, and MDCK cells, suggesting that TCDD and BP may cause renal fibrosis leading to loss of renal function. CONCLUSION: These data provide experimental evidence that TCDD can alter cell viability and proliferation and increase ECM synthesis by renal cells which may lead to renal injury.

N epsilon(Carboxymethyl)Lysine-Induced Mesangial Cell Activation / 대한신장학회잡지

BACKGROUND: Advanced glycation end products (AGE) are independent risk factors in the development and progression of diabetic nephropathy. Receptor for AGE(RAGE) is considered the main receptor involved in AGE-induced cell activation. Galectin-3, another AGE receptor, has recently been found upregulated in mesangial cells(MC) cultured under high glucose and in diabetic rat kidneys. N epsilon(carboxymethyl)lysine(CML) is a well characterized AGE but its role in MC activation is unknown. The present study examined the effects of CML on MC proliferation and extracellular matrix(ECM) secretion. METHODS: Synchronized rat MC were stimulated with different concentrations of CML-bovine serum albumin(BSA), control BSA, and transforming growth factor-beta(TGF-beta) for up to 72 hours. Cell proliferation was measured by [3H]-thymidine incorporation. Fibronectin, TGF-beta, plasminogen activator inhibitor(PAI)-1 secreted into the media and RAGE and galectin-3 expression in MC were measured by Western blot analysis and ELISA. RESULTS: 1,000 micro /mL of CML-BSA decreased [3H]-thymidine incorporation by MC at 48 hours and 10 ng/mL TGF-beta at 24 and 48 hours. CML-BSA 100 and 1,000 micro /mL, control BSA 1,000 micro /mL, and TGF-beta 10 ng/mL increased fibronectin secretion at 48 hours. CML-BSA up to 1,000 micro /mL did not affect TGF-beta or PAI-1 secretion. TGF-beta 10 ng/mL, however, significantly increased PAI-1 secretion. Cultured MC expressed both RAGE and galectin-3. CML-BSA 100 micro /mL upregulated galectin-3 expression. CONCLUSION: CML-BSA decreased MC proliferation and increased fibronectin secretion, suggesting that CML may lead to ECM accumulation and glomerulosclerosis in diabetic animals. MC express RAGE and galectin-3 constitutively and CML-induced galectin-3 upregulation may have a role in AGE-induced MC activation.

Monocyte Chemoattractant Protein-1 Upregulates Fibronectin Secretion by Human Peritoneal Fibroblasts / 대한신장학회잡지

BACKGROUND: High glucose upregulates MCP-1 expression in rat glomerular mesangial cells and in human peritoneal mesothelial cells. However, the role of high glucose-induced MCP-1 on the development and progression of diabetic renal injury and peritoneal injury during peritoneal dialysis(PD) using high glucose PD solutions are not clear. Since MCP-1 was shown to upregulate transforming growth factor-beta1(TGF-beta1) and collagen expression in lung fibroblasts, the present study investigated the effects of MCP-1 on fibronectin secretion by mouse mesangial cells(MMC), human peritoneal mesothelial cells (HPMC), and human peritoneal fibroblasts(HPFB). METHODS: Synchronized cells were stimulated by different concentrations of MCP-1(0.1-100 ng/mL) or TGF-beta1(0.1-10 ng/mL) for 48 hours. Fibronectin protein secreted into the media was analyzed by Western blot analysis. RESULTS: MCP-1 up to 100 ng/mL did not affect fibronectin secretion by MMC. TGF-beta1 10 ng/mL, however, increased fibronectin secretion by MMC 2.8 fold that of control. MCP-1 up to 100 ng/mL did not affect fibronectin secretion by HPMC. But, TGF-beta1 0.1 ng/mL increased fibronectin secretion by HPMC 1.8 fold compared to control. On the other hand, MCP-1 increased fibronectin secretion by HPFB in a dose-dependent manner. MCP-1 at 1-10 ng/mL significantly increased fibronectin when compared to M199 control. 100 ng/mL MCP-1 further increased fibronectin secretion by HPFB compared to 0.1-10 ng/mL MCP-1. CONCLUSION: These results suggest a possible role for MCP-1 in the development and progression of peritoneal fibrosis and support the view that in addition to recruiting inflammatory cells MCP-1 may play a role in tissue fibrosis in other organs.

The Effects of Lactate and pH on Human Peritoneal Mesothelial Cell Biology / 대한신장학회잡지

Preservation of peritoneal membrane function is important in the success of long-term peritoneal dialysis (PD). During PD, human peritoneal mesothelial cells (HPMC) are continuously exposed to unphysiological peritoneal dialysis solution(PDS) charaterized by high glucose and lactate concentrations, low pH, and hyperosmolality. Since few studies have examined the effects of lactate and pH on HPMC biology, the present study investigated the effects of lactate and pH on the viability and proliferation of cultured HPMC and on the production of TGF-beta1, a fibrogenic cytokine, and fibronectin by cultured HPMC. HPMC were obtained from the omental tissue of pregnant women who were undergoing Cesarean section. Cells at confluence were utilized to determine the viability(LDH release), proliferation([3H]-thymidine incorporation), and the production of fibronectin and TGF-beta1(ELISA) after synchronizing the cell growth by incubating with serum free media for 24 hours. After exposure to the media containing lactate and pH, LDH release increased in dose- and time-dependent manner. Both 1.5% and 4.25% commercial PD solutions were cytotoxic and induced more than 80% LDH release within 24 hours. LDH release decreased with increasing dilution of commercial peritoneal dialysate, but there was no significant difference in LDH release between 1.5% and 4.25% PDS. LDH release increased in response to pH 5.5. Thymidine incorporation assay revealed that lactate and low pH significantly inhibited proliferation of HPMC. ELISA revealed that exposure of HPMC to lactate and low pH decreased fibronectin protein synthesis, when compared to cell exposed to bicarbonate containing M199 media. Our results clearly show that lactate and low pH lead to dose- and time-dependent cell death and reduce proliferation of cultured HPMC. Lactate and low pH per se appear to decrease fibronectin production by HPMC but may set a stage for other factors to promote progressive fibrosis during the healing stage in long-term PD.

Effect of High Glucose on Nitric Oxide Production in Culteured Rat Mesangial Cells / 대한신장학회잡지

Diabetic nephopathy is one of the leading causes of end-stage renal disease and characterized pathologically by the glomerular mesangial expansion and increased extracellular matrix(ECM) formation. Glomerular hyper-filtration and increased vascular permeability observed in the early stage of diabetic nephropathy have been proposed to play a significant pathophysiologic role in the eventual development of glomerulosclerosis of dia-betic nephropathy. Some studies have suggested that this glomerular hyperfiltration is mediated by increased nitric oxide(NO) production via the constitutive nitric oxide synthase(cNOS) pathway present in endothelial cells under the high glucose environment. However, the exact role of the inducible NOS(iNOS) pathway present in mesangial cells in the pathogenesis of diabetic neph-ropathy is not clearly established. The present study was carried out to examine whether NO production via the iNOS pathway is mo-dulated in cultured rat mesangial cells exposed to the high glucose environment and underlying mechanism of this modulation. For this purpose, the production of the stable metabolite of NO(nitrite), intracellular cyclic gu-anosine monophosphate(cGMP), iNOS mRNA expression and iNOS protein synthesis were examined under different glucose concentrations. Rat mesangial cells cultured in high glucose concen- tration(30mM D-glucose) increased significantly nitrit#e/ nitrate production and intracellular cGMP levels upon stimulation with lipopolysaccharide(LPS) plus interfer-on-r (IFN-r ) compared with control glucose concen- tration(5.6mM D-glucose). Mesangial iNOS mRNA expression and protein synthesis also increased signifi- cantly in response to high glucose. This enhanced iNOS mRNA expression induced by high glucose concentration was significantly suppressed by protein kinase C(PKC) inhibitor, calphostin C, and the aldose reductase inhibitor, 6-bromo-l, 3-dioxo-1H- benz[d, elisoquinoline-2(3H)-acetic acid. These results indicate that high glucose in combination with stimulation by LPS plus IFN- r enhances NO production from mesangial cells by the iNOS pathway, and that the activation of PKC and the polyol pathway may play a role in this enhancement.

Effects of Graded Control of Blood Glucose with Insulin on the Progression of Experimental Diabetic Nephropathy / 대한신장학회잡지

Intensive insulin therapy effectively delays the onset and slows the progression of nephropathy in patients with IDDM. TGF- 0 has recently been implicated in the pathogenesis of diabetic nephropathy. We evaluated the effects of different level of glucose control with insulin therapy on the progression of diabetic nephropathy in age-matched control rats(C) and 3 groups of streptozotocininduced diabetic rats', high blood glucose diabetic rats without insulin therapy(HG), rnoderate glucose diabetic rats with insulin therapy(MG), and normal glucose diabetic rats with intensive insulin treatment (NG). Glomerular volume(VG) was measured using Image-Pro morphometric software, glomerular TGF- Bl mRNA expression by in situ hybridization, and glomerular expression of TGF-8 and type IV collagen proteins by immunohistochemical staining. VG was significantly higher in HG than in other groups in 12 weeks. Kidney weight(KW) was the highest while the body weight the lowest in HG of all groups in 12 weeks. Daily urine albumin excretion (UAE) increased with time in all groups but was significantly larger in HG than in all other groups in 12 weeks. MG also had significantly larger UAE than C in 12 weeks. There was no difference in VG, KW, and UAE between NG and C. Glomerular TGF-Bl mRNA expression was significantly higher in HG than in all the rest of the groups in 4 and 12 weeks. Glomerular expression of TGF-B and type IV collagen proteins was proportional to the levels of blood glucose, being the highest in HG in 12 weeks. There was little or no expression of TGF-0 1 mRNA and protein or type IV collagen protein in NG. Thus these results support the view that high blood glucose is the prerequisite for glomerular injury in diabetes mellitus and that the glomerular injury in diabetes mellitus is mediated, in part, by TGF-01 and suppressed by glucose control.