A growing body of evidence indicates that epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMC) may play an important role in the development and progression of peritoneal fibrosis during long-term peritoneal dialysis (PD) leading to failure of peritoneal membrane function. Here, we review our own observations and those of others on the mechanisms of EMT of HPMC and suggest potential therapeutic strategies to prevent EMT and peritoneal fibrosis during long-term PD. We found that high glucose and H2O2 as well as transforming growth factor-beta1 (TGF-beta1) induced EMT in HPMC and that high glucoseinduced EMT was blocked not only by inhibition of TGF-beta1 but also by antioxidants or inhibitors of mitogen-activated protein kinases (MAPK). Since MAPKs are downstream target molecules of reactive oxygen species (ROS), these data suggest that high glucose-induced generation of ROS and subsequent MAPK activation mediate high glucose-induced EMT in HPMC. We and others also observed that bone morphogenetic protein-7 (BMP-7) prevented EMT in HPMC. Glucose degradation products (GDP) were shown to play a role in inducing EMT. Involvement of a mammalian target of rapamycin (mTOR) in TGF-beta1-induced EMT has also been proposed in cultured HPMC. A better understanding of the precise mechanisms involved in EMT of HPMC may provide new therapeutic strategies for inhibiting peritoneal fibrosis in long-term PD patients.
Epithelial Cells/*pathology ; Fibrosis ; Humans ; Mesoderm/*pathology ; Peritoneal Dialysis/*adverse effects ; Peritoneum/*pathology

PURPOSE: Bone morphogenic protein (BMP)-7, a member of TGF-beta1 superfamily, is an endogenous antifibrotic protein highly expressed in normal kidney. It is not known, however, whether human peritoneal mesothelial cells (HPMC) express BMP-7 or if BMP-7 protects against peritoneal fibrosis and by what mechanism. We examined the effect of BMP-7 overexpression in TGF-beta1-induced epithelial-mesenchymal transition (EMT) of HPMC and in TGF-beta1 signaling in HPMC to elucidate the mechanisms of antifibrotic effect of BMP-7. METHODS: Growth arrested and synchronized HPMC were stimulated with 2 ng/mL of TGF-beta1 to induce EMT. HPMC were transiently transfected with adenovirus-mediated human BMP-7 (AdBMP-7) or with GFP (AdGFP). EMT was defined as downregulation of E-cadherin and upregulation of alpha-smooth muscle actin (SMA). RESULTS: HPMC constitutively expressed BMP-7 mRNA and protein. BMP-7 mRNA and protein expression were significantly inhibited by 50 mM D-glucose, 2x diluted commercial peritoneal dialysis solution, and 2 ng/ml of TGF-beta1. Transfection of AdBMP-7 resulted in 2.5-fold increase in BMP-7 mRNA expression in HPMC. TGF-beta1 significantly decreased E-cadherin and increased alpha-SMA expression in GFP transfected cells. BMP-7 overexpression effectively reversed TGF-beta1-induced E-cadherin and alpha-SMA expression and significantly suppressed TGF-beta1-induced phosphorylation of Smad2/3, ERK1/2, JNK, and p38 MAPK in HPMC as compared to GFP transfected cells. CONCLUSION: BMP-7 is an endogenous antifibrotic protein and downregulation of BMP-7 in HPMC by high glucose, PD solution, and TGF-beta1 may permit the development of peritoneal fibrosis during long-term PD. Our data demonstrate that BMP-7 overexpression reverses TGF-beta1-induced EMT of HPMC and consequent peritoneal fibrosis possibly through inhibition of Smad2/3 and MAPK phosphorylation.
Actins ; Bone Morphogenetic Protein 7* ; Cadherins ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Glucose ; Humans ; Kidney ; p38 Mitogen-Activated Protein Kinases ; Peritoneal Dialysis ; Peritoneal Fibrosis ; Peritoneum* ; Phosphorylation ; RNA, Messenger ; Transfection ; Transforming Growth Factor beta1 ; Up-Regulation

Internal jugular vein catheter is frequently used for emergency hemodialysis. Various complications have been reported. Infection is one of the problem after long term use. There have been reports of osteomyelitis of clavicle secondary to subclavian catheterization but not osteomyelitis associated with internal jugular vein catheterization. There are two possible pathways of infection. One is hematogenous spread from another focus in the body or sepsis. The other is transmission of focal infection. Manipulation of the needle may perforate the vein and produce hematoma. The infected hematoma may have resulted in abscess formation around the rib and subsequent osteomyelitis. Herein we report a case of osteomyelitis of the rib complicating internal jugular vein catheterization with a review of the literature.
Abscess ; Catheterization ; Catheters* ; Clavicle ; Emergencies ; Focal Infection ; Hematoma ; Jugular Veins* ; Needles ; Osteomyelitis* ; Renal Dialysis ; Ribs* ; Sepsis ; Veins

BACKGROUND: The transforming growth factor-beta1 (TGF-beta1) plays a key role in lung fibrosis. However, the mole?cular mechanisms involved in TGF-beta1-induced lung fibrosis are unclear. TGF-beta1 is the key inducer of myofibroblast transdifferentiation via de novo synthesis of alphasmooth muscle actin (alpha-SMA). Since TGF-beta1 signals through reactive oxygen species (ROS) and ROS have been shown to induce accumulation of extracellular matrix (ECM) in various tissues, this study examined if ROS play a role in TGF-beta1-induced fibronectin secretion and alpha-SMA expression in human lung fibroblasts, MRC-5 cells. METHODS: Growth arrested and synchronized MRC-5 cells were stimulated with TGF-beta1 (0.2-10 ng/ml) in the presence or absence of N-acetylcysteine (NAC) or diphenyleneiodonium (DPI) for up to 96 hours. Dichlorofluorescein (DCF)- sensitive cellular ROS were measured by FACScan and secreted fibronectin and cellular alpha-SMA by Western blot analysis. RESULTS: TGF-beta1 increased the level of fibronectin secretion and alpha-SMA expression in MRC-5 cells in a dose- dependent manner. Both NAC (20 and 30 mM) and DPI (1 and 5 microM significantly inhibited TGF-beta1-induced fibronectin and alpha-SMA upregulation. The TGF-beta1-induced cellular ROS level was also significantly reduced by NAC and DPI. CONCLUSIONS: The results suggest that NADPH oxidase-dependent ROS play an important role in TGF-beta1-induced fibronectin secretion and alpha-SMA expression in MRC-5 cells, which leads to myofibroblast transdifferentiation and progressive lung fibrosis.
Acetylcysteine ; Actins* ; Blotting, Western ; Extracellular Matrix ; Fibroblasts* ; Fibronectins* ; Fibrosis ; Humans* ; Lung* ; Muscle, Smooth* ; Myofibroblasts ; NADP ; Pulmonary Fibrosis ; Reactive Oxygen Species* ; Transforming Growth Factor beta1 ; Transforming Growth Factors* ; Up-Regulation

PURPOSE: To compare, in terms of their feasibility and normal range, 99mTc-DTPA renal perfusion imaging and renal perfusion imaging using harmonic ultrasound (US) with a microbubble contrast agent for the evaluation of renal perfusion after renal transplantation. MATERIALS AND METHODS: During a six-month period, thirty patients who had received a renal transplant underwent both 99mTc-DTPA renal perfusion imaging and renal perfusion imaging using harmonic US with a microbubble contrast agent. Sonographic renal perfusion images were obtained before and after a bolus injection of the microbubble contrast agent LevovistTM (SH U 508A; Schering AG, Berlin, Germany) every 3 seconds for 3 minutes. Sonographic renal perfusion images were converted into a renal perfusion curve by a computer program and Tpeak of the curve thus obtained was compared with that of the 99mTc-DTPA curve. RESULTS: Average Tpeak of the 99mTc-DTPA renal perfusion curve was 16.2 seconds in the normal group and 39.6 seconds in the delayed perfusion group, while average Tpeak of the sonographic renal perfusion curve was 23.7 seconds and 46.2 seconds, respectively. Tpeak of the sonographic renal perfusion curve showed a good correlation with that of the 99mTc-DTPA curve (correlation coefficient=0.8209; p=0.0001). The cut-off value of Tpeak of the sonographic renal perfusion curve was 35 seconds (sensitivity=90%, specificity=95%). CONCLUSION: In patients who have received a renal transplant, the findings of renal perfusion imaging using harmonic US with a microbubble contrast agent show close correlation with those of 99mTc-DTPA renal perfusion imaging. The optimal cut-off value of Tpeak of the sonographic renal perfusion curve was 35 seconds.
Berlin ; Humans ; Kidney Transplantation ; Microbubbles ; Perfusion Imaging ; Perfusion* ; Reference Values ; Ultrasonography*

PURPOSE: Oxidative stress plays an important role in the development and progression of renal injury. However, the role of reactive oxygen species (ROS) in renal allograft dysfunction is not clear. The present study examined the level of intracellular ROS in healthy control (kidney donor, n=37), end-stage renal disease (ESRD) patients (n=36), transplant recipients with serum creatinine (Scr) less than 1.5 mg% (n=33), and recipients with Scr between 1.5 and 5.0 mg% (n=36) at least one year after renal transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient method. Dichlorofluorescein (DCF)-sensitive ROS was measured by flow cytometry and expressed as an arbitrary unit. RESULTS: Basal ROS production in PBMC was significantly increased in ESRD patients compared to healthy control. Basal ROS production in both transplant patient groups was not significantly different from healthy control. Phorbol-12-myristate-13- acetate (PMA) and hydrogen peroxide significantly enhanced intracellular ROS in all 4 groups. PMA- and hydrogen peroxide-induced cellular ROS was significantly higher in renal recipients with Scr between 1.5 and 5.0 mg% than in both healthy control and patients with Scr below 1.5 mg%. In regression analysis all, PMA- and hydrogen peroxide- induced as well as basal intracellular ROS in PBMC was correlated with Scr. CONCLUSION: Our results demonstrate that oxidative stress correlates with the declining of renal graft function.
Allografts ; Creatinine ; Flow Cytometry ; Humans ; Hydrogen ; Hydrogen Peroxide ; Kidney Failure, Chronic ; Kidney Transplantation ; Oxidative Stress ; Reactive Oxygen Species* ; Tissue Donors ; Transplantation* ; Transplants*

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.
Animals ; Blotting, Western ; Chemokine CCL2* ; Collagen ; Fibroblasts* ; Fibronectins* ; Fibrosis ; Glucose ; Hand ; Humans* ; Lung ; Mesangial Cells ; Mice ; Monocytes* ; Peritoneal Fibrosis ; Rats ; Transforming Growth Factor beta1

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.
Animals ; Blotting, Western ; Cell Proliferation ; Diabetic Nephropathies ; Enzyme-Linked Immunosorbent Assay ; Fibronectins ; Galectin 3 ; Glucose ; Glycosylation End Products, Advanced ; Kidney ; Mesangial Cells* ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Rage ; Rats ; Risk Factors ; Transforming Growth Factor beta ; Up-Regulation ; Advanced Glycosylation End Product-Specific Receptor

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.
Animals ; Blotting, Western ; Cell Survival ; Culture Media, Serum-Free ; Epithelial Cells* ; Fibronectins ; Fibrosis ; Hydrocarbons, Aromatic ; Lactic Acid ; LLC-PK1 Cells ; Madin Darby Canine Kidney Cells ; Mesangial Cells ; Swine ; Tetrachlorodibenzodioxin

High glucose activates protein kinase C, induces reactive oxygen species generation, and upregulates expression of transforming growth factor-beta1(TGF-beta1) and fibronectin by human peritoneal mesothelial cells(HPMC). High glucose also induces premature senescence in mesothelial cells. Mesothelial cells shrink after exposure to hypertonic medium and intracellular uptake of amino acids increase to ensure subsequent volume increase. Based on these observations, new and more biocompatible peritoneal dialysis solutions that are glucose free and/or iso-osmolar have been developed. We investigated the effects of different osmolality and different osmotic agents including glucose, mannitol, and icodextrin on viability and proliferation of HPMC. HPMC were obtained from the omental tissues of consenting patients undergoing Cesarean section or elective abdominal surgery. All experiments were performed using cells in the 2nd or 3rd passage. Near-confluent HPMC grown in culture dishes were incubated with serum-free medium for 48 hours to arrest and synchronize cell growth. Lactate dehydrogenase(LDH) release was measured for cell viability and [3H]-thymidine incorporation for proliferation of cultured HPMC, after exposing HPMC to different concentrations of glucose, mannitol, and icodextrin for up to 96 hours. High glucose and mannitol at concentrations up to 100 mM(375 mOsm) did not increase LDH release up to 96 hours compared to control M199. When HPMC were exposed to 2, 4, 7.5, and 9% of icodextrin for 24-96 hours, LDH release did not increase. Glucose at 30, 50, and 100 mM significantly inhibited [3H]-thymidine incorporation by HPMC at 24 and 48 hours. Mannitol at 30, 50, and 100 mM for 24 hours and at only 100 mM for 48 hours also significantly inhibited cell proliferation. Icodextrin 9% (305 mOsm) inhibited cell proliferation compared with control M-199 at 24 hours. In conclusion, high osmolality per se dose not appear to increase HPMC death. However, high osmolality appears to inhibit HPMC proliferation at early stage. In addition, high glucose appears to inhibit HPMC proliferation independent of osmolality since high glucose continues to inhibit cell proliferation at 48 and 72 hours when mannitol at the same concentration did not. Icodextrin 9% of which osmolality is 305 mOsm inhibits HPMC proliferation at early stage but does not appear to increase HPMC death.
Humans