Shinji Namoto

Synergistic Cytotoxicity of Acidity and 3,4‐Dideoxyglucosone‐3‐ene Under the Existence of Lactate in Peritoneal Dialysis Fluid

Abstract:  Of the non‐physiological compounds in glucose‐rich peritoneal dialysis fluid, we investigated the synergistic cytotoxicity of acidity and 3,4‐Dideoxyglucosone‐3‐ene(3,4‐DGE) under the existence of lactate using human peritoneal mesothelial cells (HPMC). The effect of pH on cell viability at various levels of pH (5.5, 6.7, 7.15), with or without lactate was examined by adding 1N‐HCl to phosphate buffer solution. We also examined the cytotoxic effects of 3,4‐DGE and pH (5.5, 6.7 or 7.15). Additionally, we compared the cytotoxic effects of 3,4‐DGE and pH (5.5, 6.7 or 7.15) under existence of lactate (40meq/L) or absence of lactate. The cells were exposed to these solutions for 2 or 4 h. Cell viability was determined by MTT (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenylterazolium bromide) assay. 3,4‐DGE or acidic solution alone had no significant effects on MTT viability under the absence of lactate. However, acidic solutions containing 3,4‐DGE significantly decreased MTT viability under the existence of lactate. The MTT viability of HPMC was not decreased by 3,4‐DGE or acidity alone under the absence of lactate. However, the combination of acidity and 3,4‐DGE markedly decreased MTT viability under the existence of lactate, strongly suggesting the synergistic cytotoxicity of 3,4‐DGE and acidity under the existence of lactate.

Peritoneal Fibrosis and High Transport Are Induced in Mildly Pre-injured Peritoneum by 3,4-Dideoxyglucosone-3-ene in Mice

Peritoneal dialysis (PD) solution contains high concentrations of glucose and glucose degradation products (GDPs). One of several GDPs—3,4-dideoxyglucosone-3-ene (3,4-DGE)—was recently identified as the most reactive and toxic GDP in PD fluids. In vitro, 3,4-DGE has been shown to induce mesothelial cell damage; however, its role in peritoneal fibrosis in vivo remains unclear. In the present study, we intraperitoneally administered chlorhexidine gluconate (CG) for mild peritoneal injury, and we then injected 3,4-DGE [38 μmol/L (low concentration) or 145 μmol/L (high concentration)] 5 times weekly for 4 weeks. Significant thickening of the parietal peritoneal membrane was observed only when treatment with low or high concentrations of 3,4-DGE occurred after CG administration, but not when either CG or 3,4-DGE alone was given. The combination of CG and 3,4-DGE also caused upregulation of messenger RNA expression of transforming growth factor β1, connective tissue growth factor, fibronectin, collagen type 1 α1 chain, alpha smooth muscle actin (α-SMA), vascular endothelial growth factor 164, NADPH oxidase 1 and 4, p22phox, p47phox, and gp91phox in peritoneal tissue. Treatment with CG alone was sufficient to cause significant F4/80-positive macrophage infiltration, appearance of α-SMA-positive cells, and vessel formation in the submesothelial layer. Addition of 3,4-DGE markedly enhanced those changes and induced apoptosis, mainly in leukocytes. The concentration of 3,4-DGE in the abdominal cavity declined more rapidly in CG-treated mice than in PBS-treated mice. Peritoneal membrane permeability determined by peritoneal equilibration test showed high transport conditions in peritoneum treated with both CG and 3,4-DGE. These results indicate that, when mild peritoneal damage is already present, 3,4-DGE causes peritoneal thickening and fibrosis, resulting in deterioration of peritoneal membrane function.

Glutathione depletion as a mechanism of 3,4-dideoxyglucosone-3-ene-induced cytotoxicity in human peritoneal mesothelial cells: role in biocompatibility of peritoneal dialysis fluids

BACKGROUND The potential detrimental effects of glucose degradation products (GDPs) contained in peritoneal dialysis fluids (PDFs) on peritoneal mesothelial cells (PMCs) may impair intraperitoneal homeostasis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A recent study showed that 3,4-dideoxyglucosone-3-ene (3,4-DGE) was the most strongly cytotoxic among all identified GDPs in PDFs. The present study examined the effects of clinically relevant concentrations of 3,4-DGE on the proliferative capacity of PMCs and oxidative injury to them. METHOD The concentrations of eight GDPs in commercially available PDFs were determined by HPLC. The effect of cell growth media spiked with GDPs on the proliferation capacity of PMCs was evaluated. As a marker of the cellular redox status, total cellular glutathione (tGSH) was determined in PMCs incubated with GDPs. The reaction of 3,4-DGE with GSH under nonenzymatic conditions was analysed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). RESULT The concentrations of 3,4-DGE in a heat-sterilized single-compartment standard-type PDF (S-PDF) and in a heat-sterilized dual-chamber-type PDF (N-PDF) were 16 microM and 1.7 microM, respectively. The most cytotoxic GDP was 3,4-DGE, and the concentration at which it causes 50% inhibition of cell growth was 35 microM. A significant decrease in the cellular tGSH levels was observed in the cells treated with 10 microM 3,4-DGE. 3,4-DGE disappeared on incubation with GSH under nonenzymatic conditions for 1 h, and the 3,4-DGE-GSH conjugate was confirmed by accurate mass measurement using LC-ESI-MS. These data demonstrated that the change in the cellular redox status by GSH depletion might be a contributory factor in 3,4-DGE-induced cytotoxicity. CONCLUSION 3,4-DGE is a highly reactive GDP and is responsible for the depletion of the total intracellular glutathione. 3,4-DGE has an intense impact on PMC growth at concentrations found in standard PDFs. It is desired that the amount of 3,4-DGE in PDFs should be minimized.

Impact of 3,4-dideoxyglucosone-3-ene (3,4-DGE) on cytotoxicity of acidic heat-sterilized peritoneal dialysis fluid

Of the glucose degradation products (GDPs) in glucose-rich peritoneal dialysate, we investigated the influence of 3,4-dideoxyglucosone-3-ene (3,4-DGE) on the cytotoxicity of acidic heat-sterilized peritoneal dialysis fluid (L-H PDF) using human peritoneal mesothelial cells (HPMC). We prepared acidified filtration-sterilized PDF (glucose concentration 3.86%) containing eight types of added GDP [3,4-DGE, glyoxal (GO), methylglyoxal (MGO), 3-deoxyglucosone (3-DG), formaldehyde (FA), acetaldehyde (AA), 5-hydroxymethyl-2-furaldehyde (5-HMF), and furfural (FF)] or seven types of GDP (GO, MGO, 3-DG, FA, AA, 5-HMF, and FF). HPMC were exposed to these two types of solution and acidic heat-sterilized PDF (glucose concentration 3.86%, L-H 3.86) for 4 h. Cell viability was determined by 3,(4,5-dimethythiazol-2-yl)2,5-diphenyl-terazolium bromide (MTT) assay. MTT viability was decreased significantly compared with the control when treated with L-H 3.86 or acidified neutral filtration-sterilized PDF (glucose concentration 3.86%) containing eight GDPs. However, no significant decrease in MTT viability was observed when HPMC were treated with acidified neutral filtration-sterilized PDF (glucose concentration 3.86%) containing seven GDPs. Thus, 3,4-DGE strongly affects the cytotoxicity of L-H PDF. It is suggested that the cytotoxicity of L-H PDF is based on the presence of 3,4-DGE.

Availability of a Novel Criterion for Peritoneal Diffusive Selectivity on Peritoneal Dialysis

In the peritoneal dialysis, it is well-known that the peritoneal permeability increases gradually over the duration of treatment. The enhancement of the peritoneal permeability causes an under-dialysis and ultrafiltration failure, which is one of the grave factors which disrupt the homeostasis of body fluid. Hence, the monitoring of the peritoneal permeability is very important in order to give each patient some better prescriptions. In this study, we designed a novel criterion which can evaluate quantitatively the peritoneal permeability by applying a kinetic model for the peritoneal dialysis. Moreover clinical implementations of the novel criterion were validated with using the clinical data. By employing PD NAVI (JMS Co.,Ltd., Hiroshima, Japan), 50 well-being peritoneal dialysis patients performed the peritoneal function test including the peritoneal equilibration test (PET), which measured both urea and creatinine concentrations in the dialysate and drainage volumes. The overall mass transfer area coefficient for urea (MTACu) and that for creatinine (MTACc) were determined from a peritoneal mass transfer model and the clinical data for each patient. An average of MTACu/c which is the ratio of MTACu to MTACc was 1.78 and a standard deviation of that was 0.32. MTACu/c correlated with the drainage volume of PET (r2>0.66). Moreover a decrease of MTACu/c showed the enhancement of the peritoneal permeability (p<0.0001). Furthermore, since MTACu/c implies a peritoneal diffusive selectivity, MTACu/c also correlated with dialysis outcomes such as Kt/V for urea and weekly creatinine clearance normalized to 1.73m2. Then, we could derive some recommendations for MTACu/c by applying the recommendations for the dialysis outcomes such as NKF-DOQI guideline. Thus, MTACu/c can evaluate not only the peritoneal permeability, but also the dialysis outcome. We proposed that MTACu/c is available as the criterion which manages the peritoneal permeability and the therapeutic efficiency of the peritoneal dialysis.

Clinical Application of Computer-Aided Diagnostic System for Harmonious Introduction of Complementary Dialysis Therapy

In chronic peritoneal dialysis (PD) therapy, peritoneal permeability is gradually enhanced over the duration of the therapeutic course, leading to a grave decline in the therapeutic efficiency. In recent years, a novel therapy (CD therapy), which integrates PD therapy with hemodialysis therapy, is being applied to end-stage PD patients to complement the decline of therapeutic efficiency caused by the grave degeneration of the peritoneal tissue. To realize a harmonious introduction of the CD therapy, this study developed a useful index (KAu/c), which evaluates both therapeutic efficiency and degeneration of peritoneal tissue. Using a mathematical model and KAu/c, we were able to validate the therapeutic efficiency in the PD patients, and, in one case, propose a better prescription for the patient by employing the CD therapy. The clinical implementation of this methodology is indispensable with regard to expanding the therapeutic monitoring system for renal replacement therapy.

Development of Computer-Aided Diagnostic System for Peritoneal Dialysis: NAVI LightTM

Peritoneal dialysis, which is well-known as the renal replacement therapy, has been widely prescribed for the end stage renal disease in the world. With peritoneal dialysis, most of the patients that have preservation of the residual renal function achieve a dialysis outcome and ultrafiltration volume that is necessary to maintain their homeostasis. However, the therapeutic efficiency of the peritoneal dialysis decreases gradually over the duration of peritoneal dialysis treatment. Therefore, a monitoring of both the peritoneal permeability and dialysis outcome is indispensable to explore an optimal prescription and implement a long-term peritoneal dialysis. We developed a novel computer-aided diagnostic system for the peritoneal dialysis (NAVI LightTM) with employing both a kinetic mathematical model for peritoneal mass transfer and clinical test (NAVI test) which accumulates required clinical data. A set of unknown kinetic parameters on each patient were optimized by applying the clinical data to NAVI LightTM. To evaluate a validity of the parameter optimization, we statis-tically analyzed a relationship between the ultrafiltration vol-ume and MTACu/c which was the ratio of MTAC for urea to that for creatinine. Since MTACu/c showed a positive correla-tion with the ultrafiltration volume, NAVI LightTM could ana-lyze the peritoneal permeability of various therapeutic stages. Using NAVI LightTM, we estimated both the dialysis outcome and the ultrafiltation volume on the NAVI test, and analyzed a discrepancy between the estimated and the meas-ured data on the test. The estimated creatinine clearance, Kt/V for urea and ultrafiltration volume showed little discrepancy with measured those data. Moreover, those data on the ordinary prescription was also predicted by NAVI LightTM, which were compared with observed data on the ordinary prescription. The predicted those data also agreed with observed data. Therefore, these findings demonstrated that NAVI LightTM is useful for analysis of the peritoneal permeability and exploration of better prescription.