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ObjectiveTo demonstrate the presence of an independent renin–angiotensin system (RAS) in the peritoneum and to determine the role of locally produced angiotensin (Ang) II in high glucose-induced upregulation of transforming growth factor (TGF)-β1 and fibronectin by human peritoneal mesothelial cells (HPMC).MethodsIn cultured HPMC, the expression of mRNAs for angiotensinogen, angiotensin-converting enzyme (ACE), Ang II type 1 receptor (AT1), and TGF-β1 was evaluated by real-time polymerase chain reaction; ACE, AT1, and fibronectin proteins by Western blot analysis; and Ang I, Ang II, and TGF-β1 proteins by ELISA. Dichlorofluorescein (DCF)-sensitive cellular reactive oxygen species (ROS) were measured by fluorometry.ResultsHPMC constitutively expressed all the components of RAS, and 50 mmol/L D-glucose (high glucose) significantly increased angiotensinogen, ACE, and AT1 mRNAs and ACE, AT1, and Ang II proteins. Ang II increased TGF-β1 and fibronectin protein expression and DCF-sensitive cellular ROS. Losartan prevented Ang II-induced increase in cellular ROS. Both losartan and captopril inhibited high glucose-induced upregulation of TGF-β1 and fibronectin expression in HPMC in a dose-dependent manner. Antioxidant catalase and NADPH oxidase inhibitor diphenyleneiodinium effectively inhibited Ang II-induced TGF-β1 and fibronectin protein expression.ConclusionsThe present data demonstrate that HPMC constitutively express RAS, that Ang II produced by HPMC mediates high glucose-induced upregulation of TGF-β1 and fibronectin expression, and that Ang II-induced TGF-β1 and fibronectin expression in HPMC is mediated by NADPH oxidase-dependent ROS. These data suggest that locally produced Ang II and ROS in the peritoneum may be potential therapeutic targets in peritoneal fibrosis during long-term peritoneal dialysis.

Diabetes along with obesity has emerged as a silent epidemic worldwide owing to sedentary lifestyle and high calorie diets. This is certainly the case in Korea, where diabetes and pre-diabetes (impaired fasting glucose) accounted for 10.1 and 19.9 % of the population aged over 30 years or older, respectively, in 2010 (Diabetes Fact Sheet in Korea 2012). Likewise, in the United States, diabetes accounted for 25.8 million children and adults, 8.3 % of the population in 2010 (National Diabetes Fact Sheet 2011, USA). The physical and economic burden of diabetes to both the patient and society are enormous. Current anti-diabetic drugs with different mode of action have limited effectiveness and tolerability, and diabetic patients under current treatment modality will progress to microand macro-vascular complications. The prevalence of diabetes is predicted to rise continuously, and more people will experience diabetic complications unless new and more effective therapeutic and preventive measures become available. A large body of information has accumulated and important progress has been made in the last decade in our understanding of molecular mechanisms involved in the development and progression of insulin resistance, insulin secretion (b-cell function), and diabetic complications. These include autophagy, endoplasmic reticulum (ER) stress, metabolic sensors, abnormalities in metabolites formation and transcriptional machinery associated with glucose/lipid toxicity, dysregulation of adipokines, and chronic low grade inflammation. In addition, the discovery of microRNAs (miRNAs), short single-stranded RNA molecules comprising about 1 % of the genome and regulating target mRNA translational repression or cleavage, has increased our understanding on the pathogenesis of diabetes and its complications. Considering that diabetes has a strong genetic predisposition, the availability of genome-wide association (GWA) and exome sequencing studies will certainly improve our insights on genetics of diabetes and its complications. It is true, however, that effective treatment of diabetes and prevention of diabetic complications remain elusive goals. For future direction of research in diabetes to achieve a more effective correction of intracellular metabolic errors through exploration of new therapeutic targets, it is appropriate that we review current status of research in diabetic research. This special issue starts from the review by Cheon (2013), which provides the latest information about the novel molecular targets of type 2 diabetes mellitus (T2DM) pathogenesis and current development status of new T2DM therapeutics (Cheon 2013). Particularly, it highlights new therapeutic approaches, including incretin-based therapy, renal tubular glucose transporter inhibitors, b-hydroxysteroid dehydrogenase 1 inhibitors, peroxisome proliferator-activated receptor modulators, regulators of glucose/ lipid metabolism, and anti-inflammatory therapies. The next series of reviews deal with novel pathophysiological mechanisms of T2DM as an intension to seek for the novel therapeutic strategy. The second article by Lee (2013) and colleagues provides reviews on recent findings related to the roles of miRNAs in T2DM (Park et al. 2013a). The M.-K. Kwak College of Pharmacy, The Catholic University of Korea, Gyeonggi-do, Bucheon, South Korea e-mail: mkwak@catholic.ac.kr

Vascular smooth muscle cell (VSMC) proliferation is the major pathologic feature associated with chronic allograft nephropathy, and mycophenolic acid (MPA) inhibits VSMC proliferation. Since the role of inosine monophosphate dehydrogenase (IMPDH)-dependent de novo guanosine synthesis is limited in VSMCs, we examined the effects of MPA on platelet-derived growth factor (PDGF)-induced cellular ROS and mitogen-activated protein kinases (MAPK) activation in VSMCs. Primary cultured rat VSMCs were stimulated with PDGF-BB in the presence or absence of MPA. Cell proliferation was assessed by [3H]-thymidine incorporation, ROS by flow cytometry and MAPK activation by Western blot analysis. PDGF increased cell proliferation, cellular ROS and extracellular-regulated protein kinase (ERK) 1/2 and p38 MAPK activation by 3.4-, 1.6-, 3.3- and 3.9-fold, respectively. MPA at above 1 muM inhibited PDGF-induced cellular ROS and ERK 1/2 and p38 MAPK activation, as well as proliferation. Structurally different anti-oxidants and inhibitor of ERK or p38 MAPK blocked PDGF-induced proliferation. Anti-oxidants also inhibited ERK 1/2 and p38 MAPK activation. Exogenous guanosine partially recovered the inhibitory effect of MPA on VSMC proliferation. These results suggest that MPA may inhibit PDGF-induced VSMC proliferation partially through inhibiting cellular ROS, and subsequent ERK 1/2 and p38 MAPK activation in addition to inhibiting IMPDH.

Transforming growth factor-beta1 (TGF-beta1) promotes tissue fibrosis by upregulating genes encoding extracellular matrix proteins and by increasing the synthesis of plasminogen activator inhibitor-1 (PAI-1). TGF-beta1 induces cellular reactive oxygen species (ROS) and PAI-1 promoter region has binding sites for redox sensitive transcription factors. We, therefore, hypothesized that TGF-beta1-induced upregulation of PAI-1 is ROS-dependent. Using cultured glomerular mesangial cells, we confirmed that TGF-beta1 induces cellular ROS, upregulates PAI-1 mRNA and protein expression, and suppresses plasmin activity. We further demonstrated that H(2)O(2) stimulates PAI-1 expression and suppresses plasmin activity and that N-acetylcysteine effectively reverses TGF-beta1- and H(2)O(2)-induced changes in PAI-1 expression and plasmin activity. Basal as well as TGF-beta1- and H(2)O(2)-induced PAI-1 expression was upregulated by depletion of intracellular GSH. The present data demonstrate that TGF-beta1-induced PAI-1 in mesangial cells is ROS-dependent and imply that cellular ROS may be potential therapeutic targets in glomerular fibrosis.

The interaction of heme oxygenase-1 (HO-1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO-1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO-1 and caveolin-1 or caveolin-2. The experiments of immuno-precipitation showed complex formation between the HO-1 and caveolin-1 or caveolin-2 in the Cd treated MMCs. Confocal microscopic results also support co-localization of HO-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with HO-1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO-1.

Diabetic nephropathy is characterized by excessive deposition of extracellular matrix (ECM) in the kidney. TGF-beta1 has been identified as the key mediator of ECM accumulation in diabetic kidney. High glucose induces TGF-beta1 in glomerular mesangial and tubular epithelial cells and in diabetic kidney. Antioxidants inhibit high glucose-induced TGF-beta1 and ECM expression in glomerular mesangial and tubular epithelial cells and ameliorate features of diabetic nephropathy, suggesting that oxidative stress plays an important role in diabetic renal injury. High glucose induces intracellular reactive oxygen species (ROS) in mesangial and tubular epithelial cells. High glucose-induced ROS in mesangial cells can be effectively blocked by inhibition of protein kinase C (PKC), NADPH oxidase, and mitochondrial electron transfer chain complex I, suggesting that PKC, NADPH oxidase, and mitochondrial metabolism all play a role in high glucose-induced ROS generation. Advanced glycation end products, TGF-beta1, and angiotensin II can also induce ROS generation and may amplify high glucose-activated signaling in diabetic kidney. Both high glucose and ROS activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein 1) and upregulate TGF-beta1 and ECM genes and proteins. These observations suggest that ROS act as intracellular messengers and integral glucose signaling molecules in diabetic kidney. Future studies elucidating various other target molecules activated by ROS in renal cells cultured under high glucose or in diabetic kidney will allow a better understanding of the final cellular responses to high glucose.

Excessive deposition of extracellular matrix (ECM) in the kidney is the hallmark of diabetic nephropathy. Although the amount of ECM deposited in the kidney depends on the balance between the synthesis and degradation of ECM, the role of ECM degradation in matrix remodeling has been less well appreciated. High glucose, advanced glycation end products, angiotensin II, and TGF-beta1 all increase intracellular reactive oxygen species (ROS) in renal cells and contribute to the development and progression of diabetic renal injury. The role of ROS in increased ECM synthesis has been well documented. ROS may also play a critical role in decreased ECM degradation by mediating high glucose- and TGF-beta1-induced inhibition of the proteolytic system, plasmin, and matrix metalloproteinases in the glomeruli. A recent observation suggests that ROS play an important role in tubulointerstitial fibrosis by mediating TGF-beta1-induced epithelial-mesenchymal transition (EMT). Accelerated ECM degradation is required to disrupt tubular basement membrane and complete EMT. ROS thus seem to be involved in both decreased and increased ECM degradation. It is not clear how cells determine when and where to increase or decrease ECM degradation in response to ROS. Precise definition of ROS-activated signaling pathways leading to ECM remodeling in the kidney will provide new strategies to prevent or treat diabetic renal injury.

Extracellular matrix (ECM) accumulation in the glomerular mesangium is a characteristic feature of diabetic nephropathy. While transforming growth factor-beta1 (TGF-beta1) is the final mediator of ECM accumulation, reactive oxygen species (ROS) and protein kinase C (PKC) are the upstream signaling molecules that mediate hyperglycemia-induced ECM expansion. Magnesium lithospermate B (LAB) is an active component isolated from Salvia miltiorrhizae with known renoprotective properties due to its antioxidative effects. Thus, the present study examined the effects of LAB on renal injury in streptozotocin-induced diabetic rats (STZR) and on the activation of mesangial cells cultured under high glucose conditions. Ten micrtograms of LAB/kg per day was started 8 wk after streptozotocin injection and continued for a period of 8 wk. It significantly suppressed renal malondialdehyde (MDA), microalbuminuria, glomerular hypertrophy, mesangial expansion, and the upregulation of renal TGF-beta1, fibronectin, and collagen in STZR without significantly affecting plasma glucose. Both 30 mM of glucose and 100 uM of H(2)O(2) significantly increased TGF-beta1 and fibronectin protein secretion by mesangial cells. LAB at 10 micro g/ml inhibited high glucose- and H(2)O(2)-induced TGF-beta1 and fibronectin secretion. LAB also inhibited glucose-induced intracellular ROS generation and PKC activation in mesangial cells, but it did not directly inhibit PKC activity at dosages that inhibited ROS generation. The in vitro data of this study show that LAB inhibits ROS generation leading to PKC activation and TGF-beta1 and fibronectin upregulation in mesangial cells cultured under high glucose conditions. Moreover, delayed treatment with LAB was found to significantly suppress the progression of renal injury in STZR. LAB may become a new therapeutic agent for the treatment of diabetic nephropathy.

Fractalkine (CX3CL1) is a unique chemokine that functions not only as a chemokine but also as an adhesion molecule. Fractalkine plays an important role in the recruitment of macrophages into the kidneys by binding to its specific receptor CX3CR1, and renal fractalkine expression was shown to be increased in chronic renal allograft rejection. Considering that microcapillary inflammation is a key feature of chronic renal allograft rejection, the present study examined whether monocytes bind to mesangial cells cultured in the presence of lipopolysaccharide (LPS) through fractalkine/CX3CR1 in order to understand their regulation with respect to inflammation-induced renal allograft dysfunction. Mouse mesangial cells were stimulated with LPS in the presence or absence of fractalkine or CX3CR1 siRNA. Calcein-AM-labeled monocytes were used to evaluate monocyte binding. Fractalkine and CX3CR1 mRNA and protein expression were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. LPS at 100 ng/mL significantly increased monocyte binding to mesangial cells. Each siRNA against fractalkine or CX3CR1 effectively inhibited LPS-induced monocyte-mesangial cell binding. Fractalkine and CX3CR1 mRNA expression were enhanced in mesangial cells stimulated with LPS. Fractalkine protein synthesis in media and lysate of mesangial cells were also induced by LPS. These results demonstrated that LPS induces monocyte-mesangial cell binding through the fractalkine/CX3CR1 system and suggested that fractalkine/CX3CR1 system may contribute to renal inflammation leading to chronic renal allograft rejection.

Mesangial cell proliferation is one of the main features of chronic renal allograft rejection. One unique feature of fractalkine (CX3CL1) is its existence as both a membrane-tethered and a soluble form. Fractalkine expression is increased in acute and chronic allograft rejection. However, its role in mesangial cell proliferation has not yet been clearly explored. Thus, the present study examined whether fractalkine induced mesangial cell proliferation through production of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK), two known mediators of mesangial cell proliferation. Growth-arrested and synchronized mouse mesangial cells were stimulated with fractalkine in the presence versus absence of inhibitors against ROS, extracellular signal-regulated protein kinase (ERK), and p38 MAPK. Cell proliferation was assessed by methylthiazoletetrazolium assay, dichlorofluorescein (DCF)-sensitive cellular ROS production by a fluorometer, and MAPK activation by Western blot analysis. Fractalkine (10–50 ng/mL) significantly increased mesangial cell proliferation at 24 hours in a dose-dependent manner, an effect that was abrogated by the ROS and MAPK inhibitors. Fractalkine (50 ng/mL) also induced cellular ROS production and activation of ERK1/2 and p38 MAPK in mesangial cells. These results demonstrated that fractalkine can induce mesangial cell proliferation through production of cellular ROS and activation of MAPK.

SUMMARY: Progressive peritoneal membrane hyperpermeability, ultrafiltration failure, and peritoneal fibrosis have been observed in long-term peritoneal dialysis (PD) patients, and these alterations in peritoneal structure and function may be responsible for the poor technique survival in PD. While frequent and/or severe peritonitis can result in alterations of the peritoneum, continuous exposure of the peritoneum to PD solutions may also adversely affect peritoneal structure and function. Peritoneal mesothelial cells (PMC) are directly and continuously exposed to unphysiological components of PD solution. Low pH, lactate, hyperosmolality, and glucose degradation products (GDP) reduce PMC viability and proliferation. High glucose, GDP, and advanced glycation end products (AGE) upregulate vascular endothelial growth factor (VEGF), monocyte chemoattractant protein (MCP)-1, transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, and extracellular matrix protein expression by PMC, and may thus lead to peritoneal hyperpermeability, ultrafiltration failure, and peritoneal fibrosis, as observed in long-term PD. Activation of diacylglycerol (DAG)-protein kinase C (PKC) and generation of reactive oxygen species (ROS) are important upstream signalling events in high glucose-induced PMC activation. Thus, strategies to inhibit high glucose-induced PKC activation and ROS generation and the use of new PD solutions with non-glucose osmotic agents, pH neutral solutions, or solutions containing low GDP may allow better preservation of the structural and functional integrity of the peritoneal membrane during long-term PD.

PURPOSE There is still no reliable method of preventing or treating chronic rejection or transplant vascular sclerosis. A recent in vitro cell culture study showed that carvedilol significantly inhibited the proliferation of vascular smooth muscle cells (VSMCs); however, the effect of carvedilol in the presence of cyclosporine (CsA) has not yet been reported. Using in vitro cultured VSMCs, we measured the antiproliferative activity of carvedilol alone and in combination with CsA. METHODS Growth-arrested cultured VSMCs from the thoracic aorta of Sprague-Dawley rats were exposed to platelet-derived growth factor (PDGF)-BB, endothelin (ET)-I, and angiotensin (ANG)-II. Carvedilol (1 and 10 microM) and/or CsA (100 nM) were added as test drugs. DNA synthesis was assessed by measuring the incorporated [3H]thymidine activity, and the percentage of inhibition in the presence of the test drugs was determined. RESULTS Compared with the PDGF-stimulated control, DNA synthesis decreased significantly to 60.3% +/- 10.4% and 18.3% +/- 5.9% in the presence of 1 and 10 microM of carvedilol, respectively (P < 0.05, each). Carvedilol significantly inhibited the activity of VSMCs stimulated by ET-1 and ANG-II. The IC50 of carvedilol was 1-10 microM. CsA only inhibited VSMCs significantly in the PDGF-stimulated subgroup. The addition of CsA in the presence of carvedilol did not affect the inhibitory activity of carvedilol. The pattern of inhibition in the combined group was uniform and similar to that of the carvedilol alone group, regardless of the stimulator used. CONCLUSION Carvedilol significantly inhibited the DNA synthesis of VSMCs regardless of the kind of stimulators examined, even in the presence of CsA. These results indicate that carvedilol has the unique potential to inhibit the development of transplant vascular sclerosis in hypertensive transplant recipients under CsA-based immunosuppression.

♦ ObjectiveTo compare the effects of different peritoneal dialysis solutions (PDS) on secretion of vascular endothelial growth factor (VEGF), transforming growth factor-β1(TGFβ1), procollagen I C-terminal peptide (PICP), procollagen III N-terminal peptide (PIIINP), and fibronectin by cultured human peritoneal mesothelial cells (HPMC).♦ DesignUsing M199 culture medium as control, commercial PDS containing 1.5% or 4.25% glucose and 40 mmol/L lactate [Dianeal 1.5 (D 1.5) and Dianeal 4.25 (D 4.25), respectively; Baxter Healthcare, Deerfield, Illinois, USA]; PDS containing 1.5% or 4.25% glucose with 25 mmol/L bicarbonate and 15 mmol/L lactate [Physioneal 1.5 (P 1.5) and Physioneal 4.25 (P 4.25), respectively; Baxter]; and PDS containing 7.5% icodextrin [Extraneal (E); Baxter] were tested. Growth-arrested and synchronized HPMC were continuously stimulated for 48 hours by test PDS diluted twofold with M199, TGFβ11 ng/mL, or different concentrations of icodextrin. VEGF, TGFβ1, and fibronectin secreted into the media were analyzed by ELISA, and PICP and PIIINP by radioimmunoassay.♦ ResultsDianeal 1.5, D 4.25, and P 4.25, but not P 1.5 and E, significantly increased VEGF secretion compared with control M199. D 4.25- and P 4.25-induced VEGF secretion was significantly higher than induction by D 1.5 and P 1.5, respectively, suggesting that high glucose may be involved in the induction of VEGF. Physioneal 1.5- and P 4.25-induced VEGF secretion was significantly lower than induction by D 1.5 and D 4.25, respectively, suggesting a role for glucose degradation products (GDP) in VEGF production. TGFβ1secretion was significantly increased by D 4.25 and E. Icodextrin increased TGFβ1secretion in a dose-dependent manner. All PDS tested significantly increased secretion of PIIINP compared with control. D 1.5- and D 4.25-induced PIIINP secretion was significantly higher than P 1.5, P 4.25, and E. Physioneal 4.25-induced PIIINP secretion was significantly higher than P 1.5, again implicating high glucose and GDP in PIIINP secretion by HPMC. There was no significant increase in PICP or fibronectin secretion using any of the PDS tested. Addition of TGFβ11 ng/mL into M199 control significantly increased VEGF, PICP, PIIINP, and fibronectin secretion by HPMC.♦ ConclusionsThe present study provides direct evidence that HPMC can secrete VEGF, TGFβ1, and PIIINP in response to PDS, and that HPMC may be actively involved in the development and progression of the peritoneal membrane hyperpermeability and fibrosis observed in long-term PD patients. This study also suggests that both high glucose and GDP in PDS may play important roles in inducing VEGF and PIIINP production/secretion by HPMC.

We recently reported that autologous adipogenic differentiated adipose-derived stem cells (ASCs) can potentially be used as an effective and safe therapy for soft-tissue regeneration. In the present study, we investigated whether adipogenic differentiated ASCs can be used for allogenic applications to enlarge their therapeutic use. The allogenic immune response of adipogenic differentiated ASCs was investigated by flow cytometry and mixed lymphocyte culture. To determine whether adipogenic differentiated ASCs can form new adipose tissue without immune rejection, these cells were implanted subcutaneously into allo- or xenogenic recipient mice. In addition, the safety of the allogenic implantation of adipogenic differentiated ASCs was explored in a phase I clinical study. Adipogenic differentiated ASCs do not express major histocompatibility complex (MHC) class II molecules and costimulatory molecules, and the expression levels of MHC class I decreased after differentiation. In addition, these cells do not elicit an immune response against MHC-mismatched allogenic lymphocytes and formed new adipose tissue without immune rejection in the subcutaneous region of MHC-mismatched mice. Moreover, these cells did not induce clinically significant local and systemic immune responses or adverse events in the subcutaneous region of donor-independent healthy subjects. These results suggest that adipogenic differentiated ASCs can be used as a “universal donor” for soft-tissue engineering in MHC-mismatched recipients.

Introduction Residual renal function (RRF) plays an important role in outcome of peritoneal dialysis (PD) including mortality. It is, therefore, important to provide a strategy for the preservation of RRF. The objective of this study was to evaluate relative protective effects of new glucose-based multicompartmental PD solution (PDS), which is well known to be more biocompatible than glucose-based conventional PDS, on RRF compared to conventional PDS by performing a systematic review (SR) of randomized controlled trials. Methods We searched studies presented up to January 2014 in MEDLINE, EMBASE, the COCHRANE library, and local databases. Three independent reviewers reviewed and extracted prespecified data from each study. The random effects model, a more conservative analysis model, was used to combine trials and to perform stratified analyses based on the duration of follow-up. Study quality was assessed using the Cochrane Handbook for risk of bias. Eleven articles with 1,034 patients were identified for the SR. Results The heterogeneity of the studies under 12 months was very high, and the heterogeneity decreased substantially when we stratified studies by the duration of follow-up. The mean difference of the studies after 12 months was 0.46 mL/min/1.73 m2 (95% confidence interval = 0.25 to + 0.67). Conclusion New PDS showed the effect to preserve and improve RRF for long-term use compared to conventional PDS, even though it did not show a significant difference to preserve RRF for short-term use.

Background/Aim: Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells. Methods: NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined. Results: High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-γ compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression. Conclusions: These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition.

Hyperglycemia-induced oxidative stress and protein kinase C (PKC) activation are implicated in the development and progression of diabetic nephropathy. Although PKC activation under hyperglycemia largely is related to an increase in de novo synthesis of diacylglycerol (DAG), activation of PKC can be regulated sensitively by oxidative stress. We investigated the expression and translocation of PKC isoforms in streptozotocin (STZ)-induced diabetic rat glomeruli and tubules and the effect of an antioxidant taurine. Experimental diabetes was induced by intravenous injection of 50 mg/kg of STZ. Two days after STZ, diabetic rats were assigned to one of two groups: untreated or treated with taurine 1% in drinking water. Four weeks after STZ, PKC isoforms were measured by Western blot analysis in the isolated glomeruli and tubules. DAG-dependent PKC isoforms PKC-α, PKC-βI, PKC-βII, PKC-δ, and PKC-ϵ and DAG-independent PKC-ζ all were detected in control rat glomeruli and tubules. Streptozotocin increased plasma glucose from 167 ± 11 mg/dL to 575 ± 35 mg/dL (n = 9, P

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide. Although death rates of diabetic patients on hemodialysis and peritoneal dialysis (PD) have decreased substantially, they remain higher than rates in nondiabetics on both modalities. PD offers equal or better survival than hemodialysis for younger diabetic patients during early years of dialysis. PD technique survival does not appear different between diabetic and nondiabetic patients but is inferior to hemodialysis technique survival. PD may accelerate changes in peritoneal membrane structure and function in diabetics. Peritonitis and conventional PD solutions containing high glucose and glucose degradation products are implicated in PD technique failure. Increased peritoneal expression of vascular endothelial growth factor and transforming growth factor-beta1 and excessive accumulation of advanced glycosylation end products may be involved in the progressive increase in membrane permeability, loss of ultrafiltration, and peritoneal fibrosis. Nonglucose PD solutions or solutions containing low glucose degradation products may prevent or delay alterations in peritoneal membrane structure and function in diabetic as well as nondiabetic patients during long-term PD.

High glucose–induced PKC activation mediates TGF-β1 and fibronectin synthesis by peritoneal mesothelial cells. Background Progressive peritoneal fibrosis, membrane hyperpermeability, and ultrafiltration failure have been observed in long-term peritoneal dialysis (PD) using glucose as an osmotic agent. High glucose activates protein kinase C (PKC), which is one important signal pathway in the activation of transforming growth factor-β1 (TGF-β1) and fibronectin (FN). To gain a better understanding of mechanisms involved in peritoneal fibrosis, we examined the effects of high glucose on human peritoneal mesothelial cell (HPMC) TGF-β1 and FN mRNA expression and protein synthesis and determined the involvement of PKC in the high glucose–induced HPMC activation. Methods Synchronized confluent HPMC were incubated with different concentrations of glucose with and without inhibition of PKC. PKC activity and diacylglycerol (DAG) levels were measured. The expression of TGF-β1 and FN mRNAs by HPMC was measured by Northern blot analysis. TGF-β1 protein was measured by enzyme-linked immunosorbent assay (ELISA) and mink lung epithelial cell growth inhibition assay. FN protein was measured by Western blot analysis and ELISA. Results PKC activity and DAG levels in HPMC cultured under 50 mmol/L (high) glucose increased 2.3- and 2.0-fold, respectively, that of 5.6 mmol/L (control) glucose at 24 hours and this was sustained up to 72 hours. The expression of TGF-β1 and FN mRNA by HPMC cultured under high glucose increased 1.6- and 1.7-fold, respectively, that of control values at 24 hours. TGF-β bioactivity as well as protein content in heat-activated conditioned media from high glucose was significantly higher than that of control values at 24 and 48 hours. FN protein also increased in response to high glucose, as measured by Western blot analysis and ELISA. PKC activator phorbol 12-myristate 13-acetate (PMA) induced 2.2- and 1.4-fold increase in TGF-β1 and FN mRNA expression, respectively. Depletion of PKC and calphostin C, a PKC inhibitor, effectively prevented both PMA and high glucose–induced, but not constitutive, expression of TGF-β1 and FN. Conclusion The present data demonstrate that high glucose up-regulates TGF-β1 and FN synthesis by HPMC, and that this high glucose–induced up-regulation is largely mediated by PKC. These results suggest that activation of PKC by high glucose in conventional PD solutions may constitute an important signal for activation of HPMC, leading to progressive accumulation of extracellular matrix and eventual peritoneal fibrosis.

Objective To investigate the biocompatibility of “new” peritoneal dialysis (PD) solutions with bicarbonate/lactate buffer, non glucose osmotic agents (icodextrin or amino acids), neutral pH, and low levels of glucose degradation products (GDPs). Design Using M199 culture medium as a control, we compared conventional and new PD solutions with respect to their effects on the viability of human peritoneal mesothelial cells (HPMCs) [using lactate dehydrogenase (LDH) release], on DNA damage in HPMCs [using single-cell gel electrophoresis (Comet assay)], and on HPMC proliferation (using [3H]-thymidine incorporation). The experiments were performed after cell growth was synchronized by incubation with serum-free media for 24 hours. The PD solutions tested included commercial 1.5% glucose and 4.25% glucose solutions with 40 mmol/L lactate (D 1.5 and D 4.25, respectively), 7.5% icodextrin (E), 1.1% amino acid (N), 1.5% glucose solution in a triple-chambered bag (Bio 1.5), 1.5% glucose solution in a dual-chambered bag with neutral pH (Bal 1.5), and 1.5% glucose and 4.25% glucose solution containing 25 mmol/L bicarbonate and 15 mmol/L lactate (P 1.5 and P 4.25, respectively). Results When HPMCs were continuously exposed to undiluted PD solutions, D 1.5, D 4.25, P 4.25, and E increased LDH release by more than 60% at 24 hours. All PD solutions tested increased LDH release by more than 75% at 96 hours. With 2-fold diluted PD solutions, only D 4.25 significantly increased LDH release at 96 hours, though not at 24 hours. When cells were exposed to undiluted PD solutions for 60 min and allowed to recover in M199 for up to 96 hours, LDH release was significantly higher at 24 – 96 hours in E (55% – 69%) and D 1.5 (48% –72%) as compared with control [M199 (18%)]. Release of LDH was significantly lower with PD solutions containing lower levels of GDPs than those in D 1.5, suggesting that GDPs may have a role in cell viability. The D solutions (D 1.5 and D 4.25) and E solution also induced significant DNA damage. Both LDH release and DNA damage by D and E were significantly attenuated by adjusting the solution pH to 7.4, suggesting that low pH may be implicated in PD solution–induced DNA damage and cell death. When diluted 2-fold, D 1.5, D 4.25, and P 4.25 decreased [3H]-thymidine incorporation to 43%, 34%, and 41% of control, respectively, at 24 hours and to 45%, 26%, and 35% of control, respectively, at 96 hours. When cells were exposed to undiluted PD solutions for 5 minutes and allowed to recover in M199 for up to 96 hours, D 1.5 and P 4.25—but not D 4.25—significantly inhibited cell proliferation at 24 hours. This effect was sustained up to 96 hours. Conclusions The present in vitro data demonstrate that PD solutions with low pH, or high levels of GDPs, or both, promote HPMC death and DNA damage, and that PD solutions with high osmolality inhibit cell proliferation. Solutions with neutral pH, amino acids, and “low GDPs” appear to be more biocompatible than conventional PD solutions. These results require confirmation in in vivo animal and clinical studies.

Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Background Oxidative stress is one of the important mediators of vascular complications in diabetes including nephropathy. High glucose (HG) generates reactive oxygen species (ROS) as a result of glucose auto-oxidation, metabolism, and formation of advanced glycosylation end products. The concept of ROS-induced tissue injury has recently been revised with the appreciation of new roles for ROS in signaling pathways and gene expression. Methods and Results. High glucose rapidly generated dichlorofluorescein-sensitive cytosolic ROS in rat and mouse mesangial cells. Neither L-glucose nor 3-O-methyl-D-glucose increased cytosolic ROS and cytochalasin B, an inhibitor of glucose transporter, effectively inhibited HG-induced ROS generation, suggesting that glucose uptake and subsequent metabolism are required in HG-induced cytosolic ROS generation. H 2 O 2 up-regulated fibronectin mRNA expression and protein synthesis; this up-regulation was effectively inhibited by protein kinase C (PKC) inhibitor or by depletion of PKC. The HG-induced generation of ROS was, in turn, related to activation of PKC and transcription factors nuclear factor-κ;B (NF-κ;B) and activator protein-1 (AP-1) as well as to the up-regulation of transforming growth factor-β1 (TGF-β1), fibronectin mRNA expression and protein synthesis, because antioxidants effectively inhibited HG-induced PKC, NF-κ;B, AP-1 activation, and TGF-β1 and fibronectin expression in mesangial cells cultured under HG. Conclusions Although signal transduction pathways linking HG, ROS, PKC, transcription factors, and extracellular matrix (ECM) protein synthesis in mesangial cells have not been fully elucidated, the current data provide evidence that ROS generated by glucose metabolism may act as integral signaling molecules under HG as in other membrane receptor signaling.

Objective This study reviews evidence that implicates high glucose (HG) in the pathogenesis of peritoneal fibrosis and proposes mechanisms potentially involved in the HG-induced peritoneal fibrosis that is observed in long-term peritoneal dialysis (PD) patients. Design Selected Western literature is reviewed, examining the effect of HG on rat or human peritoneal mesothelial cell (HPMC) biology with particular reference to extracellular matrix (ECM) gene expression and protein synthesis. Results HG up-regulated the expression of monocyte chemotactic peptide–1 (MCP-1), transforming growth factor beta 1 (TGFβ1), and fibronectin messenger RNAs (mRNAs) and proteins. These HG-induced up-regulations were effectively blocked by the inhibition of protein kinase C (PKC). In addition, cytosolic reactive oxygen species (ROS) rapidly increased in HPMC cultured under HG, and treatment with antioxidant effectively inhibited HG-induced fibronectin protein synthesis by HPMC. Conclusion Continuous exposure of the peritoneal membrane to HG may induce changes in HPMC biology, leading to excessive deposition of ECM and peritoneal injury. HG-induced activation of diacylglycerol PKC (DAG–PKC) plays a major role in up-regulation of MCP-1, TGFβ1, and fibronectin synthesis by HPMC cultured under HG. In addition, ROS, recently recognized as signalling molecules, are rapidly generated in HPMC as a result of increased glucose metabolism and may prove to be an important mediator of HG-induced peritoneal injury.

Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea D iabetic nephropathy is one of the major long-term complications of diabetes mellitus and has emerged as a leading cause of end-stage renal disease in certain parts of the world (1) including Korea (2). Data from the Diabetes Control and Complications Trial (3) established hyperglycemia as the main determinant of initiation and progression of diabetic microvascular complications including nephropathy. However, strict control of blood glucose in diabetes is difficult and sometimes dangerous. Therefore, understanding the distal pathways involved in high glucose-induced renal injury will have clinical significance. Several lines of evidence suggest the central role of oxidative stress in the development of diabetic nephropathy and the beneficial effects of antioxidants in renal injury owing to diabetes.

The mechanism of glomerular infiltration of monocytes remains unknown in diabetic nephropathy. We examined the effect of a high glucose concentration on monocyte chemotactic peptide 1 (MCP-1) expression in human mesangial cells (MCs) by using enzyme-linked immunosorbent assay and reverse transcription coupled with polymerase chain reaction (PCR). More than a 50% increase in the MCP-1 protein production was observed in MCs cultured in high-glucose medium (450 mg/dl) as compared to normal glucose (100 mg/dl; 1,496 ± 75 vs. 966 ± 15 pg/ml after 24 h, 1,910 ± 93 vs. 1,250 ± 55 pg/ml after 48 h). Semiquantitative PCR showed that phorbol myristate acetate (100 nM) increased the ratio of PCR products for MCP-1 to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase on densitometric results at 24 h by 2.7-fold, which was prevented by calphostin C (200 nM) pretreatment. High glucose increased the ratio by 3-fold as compared to normal glucose at 24 h (0.72 ± 0.11 vs. 0.24 ± 0.01). This was also suppressed by calphostin C pretreatment. These findings demonstrate that high glucose can directly increase MCP-1 expression in MCs, which may contribute to monocyte infiltration in diabetic nephropathy, and this is regulated by protein kinase C.

Sequential effects of high glucose on mesangial cell transforming growth factor- β 1 and fibronectin synthesis. Background Transforming growth factor (TGF)- β is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF- β 1 synthesis and between TGF- β 1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF- β 1 and fibronectin (FN) mRNA expression and protein synthesis. Methods Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) m m glucose after synchronizing the growth by incubation with serum-free media for 48 hours. Results. Mesangial cell TGF- β 1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF- β 1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF- β activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF- β antibody completely abolished HG-induced FN synthesis. Conclusions The present findings demonstrate that HG stimulates TGF- β 1 very early and prior to FN production and that HG-induced FN production is mediated by TGF- β . This finding is consistent with the view that TGF- β mediates increased ECM accumulation by MC under high glucose conditions.

Aims and background Colorectal cancer is one of the commonest cancers. Chemoradiotherapy gives better results than radiotherapy or chemotherapy in colorectal cancer. To enhance radiosensitivity of tumor cells for chemoradiotherapy, targeted therapy drugs that act as radiosensitizers can be used. In the present study, we provide a scientific rationale for the clinical application of sorafenib as a radiosensitizer in colorectal cancer, without causing significant adverse effects on normal intestinal tissue. Methods Three human colorectal adenocarcinoma cell lines (HCT116, HT-29, and SW480) were treated with sorafenib alone, or radiation followed by sorafenib. In vitro tests were performed using colony forming assays, cell cycle analysis, and comet assays. In addition, the effects of sorafenib and radiation therapy on the inhibition HT-29 tumor growth and survival of intestinal jejunum crypts were examined in vivo. Results Sorafenib increased the radiosensitivity of tumor cells in human colon adenocarcinoma cell lines (HCT116, HT-29, and SW480), as well as in HT-29 xenograft animal models. Sorafenib, in combination with ionizing radiation, induced the accumulation of tumor cells in the G2-M phase and delayed the repair of DNA damage caused by ionizing radiation. The combination of sorafenib and ionizing radiation did not enhance the apoptosis of intestinal crypt cells, compared with the use of radiation alone. Conclusions We provide a scientific rationale for the use of sorafenib in combination with radiotherapy in colorectal cancer.

Monocyte chemotactic protein-1 (MCP-1), a specific chemoattractant for monocytes, has been thought to play an important role in the recruitment and accumulation of monocytes within the glomerulus seen in glomerular diseases. This study examined the role of tumor necrosis factor (TNF)-alpha-mediated cellular signal transduction pathways on mesangial cell MCP-1 gene expression and monocyte migration. Incubation of mesangial cells with TNF-alpha stimulated MCP-1 mRNA expression in a dose- and time-dependent manner. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, increased MCP-1 message by mesangial cells while depleting PKC decreased MCP-1 gene expression to control levels. Activation of PKC-depleted mesangial cells with PMA but not with TNF-alpha inhibited MCP-1 mRNA expression. Similarly, calphostin C, a PKC inhibitor, failed to inhibit TNF-alpha-induced MCP-1 expression. The incubation of mesangial cells with various protein tyrosine kinase inhibitors (PTK, e.g., herbimycin, tyrphostin, genistein) blocked TNF-alpha-induced MCP-1 mRNA message. Additional experiments examining the role of cAMP on MCP-1 expression indicated that the preincubation of mesangial cells with various cAMP generating substances (pertussis toxin, isoproterenol, dbcAMP) did not induce mesangial cell MCP-1 mRNA transcripts. However, the coincubation of mesangial cells with TNF-alpha and dbcAMP completely inhibited TNF-alpha-induced MCP-1 gene expression. Finally, TNF-alpha-activated mesangial cell media increased monocyte transmigration that could be blocked by neutralizing anti-MCP-1. These studies indicate that TNF-alpha facilitates monocyte transmigration into the glomerulus mediated by the increased expression of MCP-1 by mesangial cells. TNF-alpha-induced mesangial cell MCP-1 expression is regulated by signal transduction pathways involving PTK but not those dependent on PKC or cAMP.

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A 3 adenosine receptor (A 3 AR) is up-regulated under stress, but the therapeutic effects of A 3 AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2 R ,3 R ,4 S )-2-[2-chloro-6-(3-iodobenzylamino)-9 H -purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A 3 AR antagonist, on unilateral ureteral obstruction (UUO)–induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A 3 AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)–induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A 3 AR blockade. UUO- or TGF-β1–induced c-Jun N-terminal kinase and extracellular signal–regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A 3 AR blockade reduced UUO- or TGF-β1–induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A 3 AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

Aims The present study examined renoprotective effect of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) in diabetes. NRK-52E cells were utilized to determine the paracrine effect of hUCB-MSC. Methods hUCB was harvested with the mother's consent. MSC obtained from the hUCB were injected through the tail vein. Growth arrested and synchronized NRK-52E cells were stimulated with transforming growth factor-β1 (TGF-β1) in the presence of hUCB-MSC conditioned media. Results At 4 weeks after the streptozotocin (STZ) injection, diabetic rats showed significantly increased urinary protein excretion, renal and glomerular hypertrophy, fractional mesangial area, renal expression of TGF-β1 and α-smooth muscle actin, and collagen accumulation but decreased renal E-cadherin and bone morphogenic protein-7 expression, confirming diabetic renal injury. hUCB-MSC effectively prevented diabetic renal injury except renal and glomerular hypertrophy without a significant effect on blood glucose. CM-DiI-labeled hUCB-MSC and immunostaining of PKcs, a human nuclei antigen, confirmed a few engraftment of hUCB-MSC in diabetic kidneys. hUCB-MSC conditioned media inhibited TGF-β1-induced extracellular matrix upregulation and epithelial-to-mesenchymal transition in NRK-52E cells in a concentration-dependent manner. Conclusions These results demonstrate the renoprotective effect of hUCB-MSC in STZ-induced diabetic rats possibly through secretion of humoral factors and suggest hUCB-MSC as a possible treatment modality for diabetic renal injury.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide. Excess accumulation of extracellular matrix and the epithelial-to-mesenchymal transition contribute to renal fibrosis, which is associated with DKD. The present study examined whether delayed treatment with human umbilical cord blood–derived stem cells (hUCB-SC) showed a therapeutic effect on DKD progression. Experimental diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg) into 6-week-old male Sprague-Dawley rats. Age-matched control rats received an equivalent volume of sodium citrate buffer alone. At 4 weeks after the STZ injection when diabetic renal injury had developed, hUCB-SC were administered (1 × 106 cells/rat) through the tail vein. Four weeks after administering the hUCB-SC, rats were sacrificed and we measured indices of DKD, including urinary protein excretion as well as fibronectin, α-smooth muscle actin (α-SMA), and E-cadherin mRNA, and protein expression. Diabetic rats developed significantly increased urinary protein excretion and renal hypertrophy compared to those in control rats. Renal expression of fibronectin and α-SMA mRNA, and protein were increased significantly in diabetic rats compared to those in the controls. E-cadherin protein expression in diabetic kidneys decreased significantly. Intravenously administered hUCB-SC effectively reduced proteinuria, renal fibronectin, and α-SMA up-regulation, as well as renal E-cadherin down-regulation in diabetic rats without a significant effect on blood glucose. Engrafted hUCB-SC in diabetic kidneys were confirmed by human DNA PKcs. The results demonstrated that delayed treatment with hUCB-SC attenuated the progression of diabetic renal injury.

Diabetes, whether it occurs before or after transplantation, plays an important role to decrease graft function and survival. In addition renal lipid accumulation has been suggested to play a role in the development and progression of chronic renal allograft rejection. Intracellular lipid accumulation is governed by a balance between the influx and efflux of lipid. Cholesterol transporters, such as scavenger receptor (SR)-A1, CD36, and ATP binding cassette (ABC) A1 and G1 (ABCG1), coordinate to regulate cellular lipid status. Therefore, in the present study, we examined whether high glucose caused lipid accumulation in mesangial cells as a result of altered cholesterol transporters. Mouse mesangial cells were stimulated with 30 mmol/L D-glucose (high glucose); 100 μmol/L oleic acid (OA) used as a positive control. Cellular lipid accumulation was measured by Oil Red O staining. Protein and mRNA expression of cholesterol influx (SR-A1 and CD36) and efflux (ABCA1 and ABCG1) transporters were evaluated using Western blot analysis and real-time quantitative polymerase chain reaction, respectively. High glucose was shown to significantly increase lipid accumulation in mesangial cells at 24 hours as was observed for OA. SR-A1 and CD36 mRNA expression levels were 1.5-fold and 3.5-fold higher, respectively, in high glucose-stimulated than control mesangial cell, whereas ABCG1 mRNA expression decreased to 60% of controls; however, there was no decrease in ABCA1 mRNA. Altered protein expression of each transporter in mesangial cells cultured under conditions of high glucose concentrations was consistent with mRNA expression. Osmotic control using mannitol did not significantly affect any of the measured parameters in the present study. These results demonstrated that high glucose, in itself, can induce mesangial lipid accumulation; this effect may be associated with an impaired balance between the influx and efflux of cholesterol.

Despite recent improvements in immunosuppressive regimens, chronic renal allograft rejection remains a major problem. Tubulointerstitial fibrosis is one of the major histological features of chronic renal allograft rejection, but its exact pathogenic mechanisms are not fully understood. Adenosine present in the normal kidney is significantly elevated in response to cellular damage. The cellular effect of adenosine occurs through 4 known adenosine receptor (AR) subtypes; A 1 AR, A 2A AR, A 2B AR, and A 3 AR. All AR subtypes are expressed in the kidney, but the expression of each AR subtype has not been defined under fibrotic conditions. In the present study, we examined AR subtype expression in kidneys that underwent unilateral ureteral obstruction (UUO), a well-characterized model for tubulointerstitial fibrosis. At 5 days after the induction of UUO, we observed α-smooth muscle actin (α-SMA), fibronectin, and collagen I messenger RNA (mRNA) and protein expressions to be significantly up-regulated in the obstructed compared with the sham kidneys, confirming that fibrosis had occurred in the former organs. A 1 AR mRNA expression in the obstructed kidney cortex was 3.2-fold higher than an the sham kidney cortex. Relative to the sham kidney A 2A AR and A 2B AR mRNA expressions were also 2.6- and 2.0-fold increased, respectively. A 3 AR mRNA expression in the obstructed kidney cortex was also up-regulated by 3.3-fold. These data demonstrated that all 4 subtypes of AR were increased in the obstructed kidney, which was accompanied by tubulointerstitial fibrosis. Further studies are needed to determine which subtypes of AR play a protective or pathogenic role in tubulointerstitial fibrosis.

Tubulointerstitial fibrosis, which is characterized by the progressive accumulation of extracellular matrix (ECM), is the main feature of chronic renal allograft dysfunction. Transforming growth factor-β1 (TGF-β1) is the key inducer of tubulointerstitial fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of ECM degradation, is increasingly recognized to play an important role in renal fibrosis. ECM accumulation is the net result of ECM synthesis and degradation. We previously reported that reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) are required for the TGF-β1-induced epithelial-to-mesenchymal transition in renal proximal tubular epithelial cells. In the present study, we examined the role of the ROS-MAPK pathways in TGF-β1-induced fibronectin and PAI-1 up-regulation in renal tubular epithelial cells. Growth arrested, synchronized normal rat kidney epithelial (NRK-52E) cells were stimulated with TGF-β1 (0.2-20 ng/mL) or H(2)O(2) (1-500 μmol/L) in the presence or absence of inhibitors of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (diphenyleneiodonium [DPI] and apocynin [Apo]) and MAPK (PD98059, an MEK inhibitor, or a p38 MAPK inhibitor) for up to 48 hours. Both TGF-β1 and H(2)O(2) increased fibronectin and PAI-1 secretion in dose-dependent manners. Chemical inhibition of NADPH oxidase, extracellular signal-regulated kinase (ERK), or p38 MAPK all inhibited TGF-β1-induced and H(2)O(2)-induced fibronectin and PAI-1 up-regulation. These results suggested that NADPH oxidase-mediated ROS and subsequent ERK and p38 MAPK activation play important roles in ECM accumulation in the renal tubulointerstitium.

8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, has been recently rediscovered to inhibit Rac1 in neutrophils and macrophages, thereby inhibiting Rac1-linked functions of these cells, including reactive oxygen species production through NADPH oxidase activation, phagocytosis, chemotaxis, and cytokine release. In vascular smooth muscle cells (VSMCs), reactive oxygen species also induce abnormal proliferation and migration leading to progression of atherosclerosis. Based upon the involvement of reactive oxygen species in phagocytic cells and VSMCs during the atherosclerotic process, we hypothesized that 8-OHdG could have antiatherosclerotic action and tested this hypothesis in an experimentally induced atherosclerosis in mice. Partially ligated ApoE knockout mice, a more physiologically relevant model of low and oscillatory flow, developed an advanced lesion in 2 weeks, and orally administered 8-OHdG significantly reduced plaque formation along with reduced superoxide formation, monocyte/macrophage infiltration, and extracellular matrix (ECM) accumulation. The effects of 8-OHdG observed in primary VSMCs were consistent with the in vivo effects of 8-OHdG and were inhibitory to angiotensin II or platelet-derived growth factor-induced production of reactive oxygen species, proliferation, migration, and ECM production. Also, angiotensin II-induced Rac1 activity in VSMCs was significantly inhibited by 8-OHdG, and transfection of constitutively active Rac1 reversed the inhibitory effect of 8-OHdG on VSMC activation. Molecular docking study showed that 8-OHdG stabilizes Rac1-GEF complex, indicating the physical contact of 8-OHdG with Rac1. These findings highly suggest that the antiatherosclerotic effect of 8-OHdG is mediated by inhibition of Rac1 activity. In conclusion, our results show a novel action of orally active 8-OHdG in suppressing atherosclerotic plaque formation in vivo and VSMC activation in vitro through inhibition of Rac1, which emphasizes a new therapeutic avenue to benefit atherosclerosis.

Oxidative stress defined as an excessive production of reactive oxygen species (ROS) surpassing existing antioxidative defense mechanisms plays a critical role in the development and progression of diabetic vascular complications including nephropathy. Over production of ROS in diabetic milieu is both a direct consequence of hyperglycemia and an indirect consequence through advanced glycation end products (AGEs) or mediators of glucotoxicity such as cytokines and growth factors. Among many pathways, nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase and mitochondrial dysfunction have been recognized as two major sources of ROS generation in diabetic kidneys, and NADPH oxidase-derived ROS has been shown to facilitate renal mitochondrial superoxide production in hyperglycemia. Low antioxidant bioavailability promotes cellular oxidative stress leading to additional cellular damage. Although large-scale clinical trials using classical antioxidants have failed to show a significant effect on the development of vascular complications in diabetes, new strategies targeting NF-E2-related factor 2, the primary transcription factor that controls the antioxidant response, mitochondrial dysfunction, or NADPH oxidase might provide a potential approach for the prevention and treatment of diabetic nephropathy.

Oxidative stress defined as an excessive production of reactive oxygen species (ROS) surpassing existing antioxidative defense mechanisms plays a critical role in the development and progression of diabetic vascular complications including nephropathy. Over production of ROS in diabetic milieu is both a direct consequence of hyperglycemia and an indirect consequence through advanced glycation end products (AGEs) or mediators of glucotoxicity such as cytokines and growth factors. Among many pathways, nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase and mitochondrial dysfunction have been recognized as two major sources of ROS generation in diabetic kidneys, and NADPH oxidase-derived ROS has been shown to facilitate renal mitochondrial superoxide production in hyperglycemia. Low antioxidant bioavailability promotes cellular oxidative stress leading to additional cellular damage. Although large-scale clinical trials using classical antioxidants have failed to show a significant effect on the development of vascular complications in diabetes, new strategies targeting NF-E2-related factor 2, the primary transcription factor that controls the antioxidant response, mitochondrial dysfunction, or NADPH oxidase might provide a potential approach for the prevention and treatment of diabetic nephropathy.

Fibrosis of epithelial parenchymal organs and end-stage organ failure, the final common pathway of many progressive chronic diseases including chronic kidney disease, continue to increase worldwide and are a major determinant of morbidity and mortality. Fibrosis is an active biosynthetic healing response initiated to protect the tissue from injury through the timed release of proteins but leads to serious tissue damage when it becomes independent from the initiating stimulus. Massive deposition of extracellular matrix by accumulation of myofibroblasts and disruption of the normal tissue architecture are characteristic of tissue fibrosis. The highly conserved Wnt/beta-catenin signaling pathway is essential to embryonic development in general and kidney morphogenesis in particular by regulating the expression of target genes, most often through the transcription factor T cell factor (TCF) and/or lymphoid enhancer factor (LEF). Emerging evidence from studies of renal fibrosis suggests that altered Wnt/beta-catenin signaling is linked to the pathogenesis of renal fibrosis. The renoprotective properties of some currently available drugs might be attributable in part to inhibition of Wnt signaling. The development of orally active Wnt modulators will provide a potentially important pharmacological tool for further investigating the role of Wnt/beta-catenin signaling and might offer a novel therapeutic strategy in renal fibrosis.

Excessive accumulation of extracellular matrix (ECM) in the kidneys and epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells contributes to the renal fibrosis that is associated with diabetic nephropathy. Histone deacetylase (HDAC) determines the acetylation status of histones and thereby controls the regulation of gene expression. This study examined the effect of HDAC inhibition on renal fibrosis induced by diabetes or transforming growth factor (TGF)-beta1 and determined the role of reactive oxygen species (ROS) as mediators of HDAC activation. In streptozotocin (STZ)-induced diabetic kidneys and TGF-beta1-treated normal rat kidney tubular epithelial cells (NRK52-E), we found that trichostatin A, a nonselective HDAC inhibitor, decreased mRNA and protein expressions of ECM components and prevented EMT. Valproic acid and class I-selective HDAC inhibitor SK-7041 also showed similar effects in NRK52-E cells. Among the six HDACs tested (HDAC-1 through -5 and HDAC-8), HDAC-2 activity significantly increased in the kidneys of STZ-induced diabetic rats and db/db mice and TGF-beta1-treated NRK52-E cells. Levels of mRNA expression of fibronectin and alpha-smooth muscle actin were decreased, whereas E-cadherin mRNA was increased when HDAC-2 was knocked down using RNA interference in NRK52-E cells. Interestingly, hydrogen peroxide increased HDAC-2 activity, and the treatment with an antioxidant, N-acetylcysteine, almost completely reduced TGF-beta1-induced activation of HDAC-2. These findings suggest that HDAC-2 plays an important role in the development of ECM accumulation and EMT in diabetic kidney and that ROS mediate TGF-beta1-induced activation of HDAC-2.

The survival of patients with end-stage renal disease (ESRD) resulting from diabetes continues to improve, but the survival rate among diabetic ESRD patients remains the lowest among all primary diagnoses probably because of the higher prevalence of cardiovascular comorbidity associated with diabetes. Diabetes, age, and comorbidity all significantly modify the effect of treatment modality on patient survival. As compared with hemodialysis (HD), peritoneal dialysis (PD) offers an equal or lower risk of death across all subgroups during the first 1 – 2 years of dialysis. The association of PD with better outcomes than are seen with HD is probably a result of a lower prevalence of infections and congestive heart failure and better preservation of residual renal function (RRF) in PD patients. Use of angiotensin converting-enzyme inhibitor (ACEI) or angiotensin II receptor blocker (ARB) helps to preserve RRF in ESRD patients and to maintain peritoneal membrane integrity longer in PD patients. Antioxidants can also support preservation of peritoneal membrane function. Peritoneal dialysis should be the initial modality of dialysis in all ESRD patients. Older patients (age ≥ 45 years) with diabetes and patients without diabetes may switch to HD or receive a kidney graft in 1 – 2 years’ time; younger patients (age < 45 years) with diabetes may stay on PD longer. Use of ACEI and ARB or antioxidants can help to maintain peritoneal membrane function longer.

There is an increasing evidence that reactive oxygen species (ROS) play a major role in the development of diabetic complications. Oxidative stress is increased in diabetes and the overproduction of ROS in diabetes is a direct consequence of hyperglycemia. Various types of vascular cells including renal cells are able to produce ROS under hyperglycemic condition. Both NADPH oxidase and mitochondrial electron gradient play roles in hyperglycemia-induced ROS generation. In addition to their ability to directly inflict macromolecular damage, ROS can function as signaling molecules. ROS mediate hyperglycemia-induced activation of signal transduction cascades and transcription factors leading to transcriptional activation of profibrotic genes in the kidney. Furthermore, ROS-activated signaling molecules generate and signal through ROS and thus ROS act as a signal amplifier. Intensive glycemic control and inhibition of angiotensin II delay the onset and progression of diabetic nephropathy, in part, through prevention of overproduction of ROS. Conventional and catalytic antioxidants have been shown to prevent or delay the onset of diabetic nephropathy. Combination of strategies to prevent overproduction of ROS and to increase the removal of preformed ROS may prove to be effective in preventing the development and progression of diabetic nephropathy.

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.

Histone deacetylase (HDAC) inhibitors are currently being tested as anticancer agents in clinical trials. Chromatin remodeling, such as through histone acetylation, is a fundamental phenomenon in eukaryotic cell biology, bearing implications to numerous physiological and pathological phenomena. Here, we discuss recent data from our own laboratory and those of others demonstrating antifibrotic and renoprotective effect of HDAC inhibitors in diabetic kidneys, and the possible mechanisms including the role of reactive oxygen species. HDAC inhibitors may prove to be a novel class of multitarget agents in the treatment of diabetic nephropathy.

We evaluated the role of oxidative stress in diabetic nephropathy by measuring intracellular reactive oxygen species (ROS) and redox-sensitive transcription factors in isolated peripheral mononuclear cells (PBMC) in 66 diabetic patients with or without diabetic nephropathy (Groups III and II, respectively) and 49 normal controls (Group I). Stimulated ROS was significantly higher in Group III compared to Group II (increment of H(2)O(2)-induced ROS production: 21.8+/-2.2% vs. 11.1+/-2.0%; increment of PMA-induced ROS production 23.5+/-4.5% vs. 21.6+/-2.2%; both respectively), and the activity of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), but not specificity protein 1 (Sp1) was significantly higher in Group III than in Group II (2.53-fold vs. 2.0-fold vs. 1.43-fold, respectively). Both PBMC- and urinary TGF-beta1 levels were higher in Group III than Group II (3.23+/-0.39 ng/g vs. 1.99+/-0.68 ng/g in PBMCs, 16.88+/-6.84 (ng/g Cr) vs. 5.61+/-1.57 (ng/g Cr) in urine, both respectively), and they correlated with the activity of NF-kappaB and AP-1 and 24-h urine albumin excretion (UAE). Increased intracellular ROS generation in PBMCs of diabetic patients is involved in the pathogenesis of diabetic nephropathy via activation NF-kappaB and AP-1 and an increased expression of TGF-beta1.

Background Vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis are major pathologic features of chronic allograft vasculopathy. Carvedilol, an anti-hypertensive agent, might be an effective agent for preventing the development and progression of chronic allograft vasculopathy, since it can inhibit VSMC proliferation and migration. The present study was designed to examine the effect of carvedilol on platelet-derived growth factor (PDGF)-induced ECM synthesis in rat VSMCs. Furthermore, we evaluated whether carvedilol inhibits PDGF-induced cellular reactive oxygen species (ROS) and the activation of mitogen-activated protein kinase (MAPK). Methods Primary cultured rat VSMCs were stimulated with PDGF-BB (10 ng/ml) in the presence or absence of carvedilol, and the effects of carvedilol were compared with those of ROS or MAPK inhibitors. Fibronectin secretion, proliferating cell nuclear antigen (PCNA) expression, and each MAPK activation were determined by Western blot analysis, total collagen synthesis by [ 3 H]-proline incorporation, and cellular ROS by flow cytometry. Results PDGF significantly increased PCNA expression, fibronectin secretion, total collagen synthesis, cellular ROS, and MAPK activation in rat VSMCs. Carvedilol at doses that inhibited PDGF-induced cell proliferation, inhibited ECM synthesis, cellular ROS, or subsequent MAPK activation. Structurally different anti-oxidants and extracellular signal-regulated protein kinase or p38 MAPK inhibitor effectively inhibited PDGF-induced fibronectin secretion and total collagen synthesis. Conclusions These results suggest that carvedilol inhibits PDGF-induced VSMC proliferation and matrix protein synthesis by inhibiting cellular ROS and the subsequent activation of MAPK. Thus the targeted inhibition of cellular ROS and MAPK might provide an effective therapeutic strategy to treat chronic allograft vasculopathy.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Reactive oxygen species (ROS) play a central role in the ECM synthesis and degradation in the glomeruli and tubulointerstitium leading to renal fibrosis. High glucose (HG) induces cellular ROS through protein kinase C (PKC)-dependent activation of NADPH oxidase and through mitochondrial metabolism. ROS thus generated activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein-1), up-regulate transforming growth factor-beta1 (TGF-beta1), angiotensin II (Ang II), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) gene and protein expression, and promote formation of advanced glycation end-products (AGE). PKC, TGF-beta1, Ang II, and AGE also induce cellular ROS and signal through ROS leading to enhanced ECM synthesis. NF-kappaB-MCP-1 pathway is activated by ROS and promotes monocyte recruitment and profibrotic process in the kidney. HG- and TGF-beta1-induced PAI-1 up-regulation is mediated by ROS and contribute to ECM accumulation via suppression of plasmin ativity. TGF-beta1-induced myofibroblast transformation of renal tubular epithelial cells (epithelial-mesenchymal transition) is also mediated by ROS and contribute to tubulointerstitial fibrosis. In summary, ROS transduce and amplify glucose signalling in renal cells under high glucose environment and play a critical role in excessive ECM deposition in the diabetic kidney. A better understanding of ROS production and removal will allow more effective therapeutic strategies in diabetic renal and other vascular complications.

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 extracellular matrix synthesis in cultured vascular smooth muscle cells (VSMCs) and mesangial cells (MCs) of Sprague-Dawley rats, and investigated the molecular mechanisms involved. Different concentrations of TMC were administered 1 hour before the addition of 10 ng/mL PDGF into the growth-arrested and synchronized cells. Cell proliferation was assessed by methylthiazoletetrazolium (MTT) assay, fibronectin secretion, and the activation of Akt, ERK, and p38 MAPK by Western blot analysis. PDGF 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 >1 μg/mL significantly reduced basal MTT. TMC at 100 ng/mL significantly decreased the PDGF-induced VSMC and MC proliferation. However, fibronectin secretion and the activation of Akt, ERK, and p38 MAPK were not affected by this nontoxic concentration of TMC. The present data demonstrate that low-dose TMC reduced PDGF-induced VSMC and MC proliferation without affecting the fibronectin secretion and cellular kinase activation.