Standard Peritoneal Dialysis Fluid Research Articles

MiR-122-5p promotes peritoneal fibrosis in a rat model of peritoneal dialysis by targeting Smad5 to activate Wnt/β-catenin pathway

Peritoneal fibrosis (PF) is the main reason leading to declining efficiency and ultrafiltration failure of peritoneum, which restricts the application of peritoneal dialysis (PD). We aimed to investigate the effects and mechanisms of miR-122-5p on the PF. Sprague-Dawley (SD) rats were infused with glucose-based standard PD fluid to establish PF model. HE staining was performed to evaluate the extent of PF. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were performed to measure the expression level of miR-122-5p. Western blot was used to test the expression of transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF)-A, Fibronectin 1 (FN1), extracellular matrix protein 1 (ECM1), Smad5, α-smooth muscle actin (SMA), collagen type 1(COL-1), Vimentin, E-Cadherin, Wnt1, β-catenin, p-β-catenin, c-Myc, c-Jun, and Cyclin D1. Immunohistochemistry (IHC) staining was used to detect type I collagen alpha 1 (Col1α1), α-SMA, and E-Cadherin expression. We found PF was glucose concentration-dependently enhanced in peritoneum of PD rat. The PD rats showed increased miR-122-5p and decreased Smad5 expression. MiR-122-5p silencing improved PF and epithelial–mesenchymal transition (EMT) process in PD rats. MiR-122-5p silencing attenuated the activity of the Wnt/β-catenin signaling pathway. Importantly, dual-luciferase reporter assay showed Smad5 was a target gene of miR-122-5p. Smad5 overexpression significantly reversed the increases of PF and EMT progression induced by miR-122-5p overexpression. Moreover, miR-122-5p mimic activated Wnt/β-catenin activity, which was blocked by Smad5 overexpression. Overall, present results demonstrated that miR-122-5p overexpression showed a deterioration effect on PD-related PF by targeting Smad5 to activate Wnt/β-catenin pathway.

Curcumin ameliorates peritoneal fibrosis via inhibition of transforming growth factor-activated kinase 1 (TAK1) pathway in a rat model of peritoneal dialysis

BackgroundPeritoneal fibrosis (PF) remains a serious complication of long-term peritoneal dialysis (PD). The goal of this study was to investigate the anti-fibrotic effects of curcumin on the PF response to PD and its’ mechanism.MethodsMale Sprague–Dawley rats were infused with 20 mL of 4.25% glucose-based standard PD fluid for 8 consecutive weeks to establish PF model and then divided into five groups: Control, received sham operation and 0.9% physiological saline; PD, received 4.25% standard PD fluid; Curcumin, PD rats injected intraperitoeally with curcumin for 8 weeks at doses of 10, 20 or 40 mg/kg. Masson’s staining was performed to evaluate the extent of PF. Peritoneal Equilibration Test (PET) was conducted to assess ultrafiltration volume (UFV) and mass transfer of glucose (MTG), quantitative RT-PCR, and immunohistochemistry or western blotting were performed to measure the expression levels of inflammation and fibrosis-associated factors. We also detected the TGF-β1 in peritoneal fluid by ELISA.ResultsCompared with the control group, the PD rats showed decreased UFV (2.54 ± 0.48 to 9.87 ± 0.78 mL, p < 0.05] and increased MTG (18.99 ± 0.86 to 10.85 ± 0.65 mmol/kg, p < 0.05) as well as obvious fibroproliferative response, with markedly increased peritoneal thickness (178.33 ± 4.42 to 25.26 ± 0.32um, p < 0.05) and higher expression of a-SMA, collagen I and TGF-β1. Treatment with curcumin significantly increased UFV, reduced MTG and peritoneal thickness of PD rats. The elevated TGF-β1 in peritoneal fluid of PD rats was significantly decreased by curcumin. It attenuated the increase in protein and mRNA of TGF-β1, α-SMA and collagen I in peritoneum of PD rats. The mRNA expressions of TAK1, JNK and p38, as well as the protein expressions of p-TAK1, p-JNK and p-p38 in peritoneum of PD rats were reduced by curcumin.ConclusionsPresent results demonstrate that curcumin showed a protective effect on PD-related PF and suggest an implication of TAK1, p38 and JNK pathway in mediating the benefical effects of curcumin.

A randomized controlled trial of alanyl-glutamine supplementation in peritoneal dialysis fluid to assess impact on biomarkers of peritoneal health

In early clinical testing, acute addition of alanyl-glutamine (AlaGln) to glucose-based peritoneal dialysis (PD) fluids restored peritoneal cellular stress responses and leukocyte function. This study was designed to test the effect of extended treatment with AlaGln-supplemented PD fluid on biomarkers of peritoneal health. In a double-blinded, randomized crossover design, stable PD patients were treated with AlaGln (8 mM) or placebo added to PD fluid for eight weeks. As primary outcome measures, dialysate cancer-antigen 125 (CA-125) appearance rate and exvivo stimulated interleukin-6 (IL-6) release were assessed in peritoneal equilibration tests. In 8 Austrian centers, 54 patients were screened, 50 randomized, and 41 included in the full analysis set. AlaGln supplementation significantly increased CA-125 appearance rate and exvivo stimulated IL-6 release. AlaGln supplementation also reduced peritoneal protein loss, increased exvivo stimulated tumor necrosis factor (TNF)-α release, and reduced systemic IL-8 levels. No adverse safety signals were observed. All 4 peritonitis episodes occurred during standard PD fluid treatment. A novel AlaGln-supplemented PD fluid improves biomarkers of peritoneal membrane integrity, immune competence, and systemic inflammation compared to unsupplemented PD fluid with neutral pH and low-glucose degradation. A phase 3 trial is needed to determine the impact of AlaGln supplementation on hard clinical outcomes.

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD.We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln).With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense.Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

Functional and Transcriptomic Characterization of Peritoneal Immune-Modulation by Addition of Alanyl-Glutamine to Dialysis Fluid

Peritonitis remains a major cause of morbidity and mortality during chronic peritoneal dialysis (PD). Glucose-based PD fluids reduce immunological defenses in the peritoneal cavity. Low concentrations of peritoneal extracellular glutamine during PD may contribute to this immune deficit. For these reasons we have developed a clinical assay to measure the function of the immune-competent cells in PD effluent from PD patients. We then applied this assay to test the impact on peritoneal immune-competence of PD fluid supplementation with alanyl-glutamine (AlaGln) in 6 patients in an open-label, randomized, crossover pilot trial (EudraCT 2012-004004-36), and related the functional results to transcriptome changes in PD effluent cells. Ex-vivo stimulation of PD effluent peritoneal cells increased release of interleukin (IL) 6 and tumor necrosis factor (TNF) α. Both IL-6 and TNF-α were lower at 1 h than at 4 h of the peritoneal equilibration test but the reductions in cytokine release were attenuated in AlaGln-supplemented samples. AlaGln-supplemented samples exhibited priming of IL-6-related pathways and downregulation of TNF-α upstream elements. Results from measurement of cytokine release and transcriptome analysis in this pilot clinical study support the conclusion that suppression of PD effluent cell immune function in human subjects by standard PD fluid is attenuated by AlaGln supplementation.

The effects of Panax notoginseng saponins on the cytokines and peritoneal function in rats with peritoneal fibrosis

Background: Due to the long-term and chronic exposure to the peritoneal dialysis fluid, patients could develop peritoneal fibrosis and ultrafiltration failure which compromises treatment efficacy and outcome, and fibrosis is the major cause of peritoneal dialysis (PD) withdraw among patients. Methods: Twenty-one male WISTAR rats were randomly assigned to three groups, namely saline group, standard peritoneal dialysis fluid (PDF) group, and panax notoginseng saponins (PNS) group. Peritoneal fibrosis was induced by daily injection of PDF for 4 weeks. After execution, multiple histological techniques including HE and Masson's trichrome staining and transmission electron microscopy (TEM) were applied to observe the pathological changes and concentrations of multiple cytokines may involve in the process of fibrosis were determined by enzyme-linked immune sorbent assay (ELISA). Biochemistry parameters were determined by automated chemistry analyzer. Results: PNS can significantly inhibit the expression of transforming growth factor beta (TGF-β1), connective tissue growth factor (CTGF), and monocyte chemoattractant protein (MCP-1) in the peritoneum of rats. Furthermore, pathological damages, including extracellular matrix deposition, vascularization, and fibroblast, were ameliorated in PNS group when being compared with standard PDF group. Peritoneal functions were improved by regular PNS treatment with significantly elevated ultrafiltration. Conclusion: PNS is capable of improving peritoneal function in subjects with PDF exposure and can possibly applied in patients with PD after further verification.

Peritoneal Fibrosis Induced by Intraperitoneal Methylglyoxal Injection: The Role of Concurrent Renal Dysfunction

Background: Peritoneal fibrosis (PF) is a serious pathophysiology of peritoneal dialysis (PD). An ongoing focus of research is the potential fibrogenic nature of methylglyoxal (MGO) in conventional PD fluid (PDF). The aim of the current study was to explore the effects of the uremic milieu on the promotion of PF by MGO using rats with adenine-induced renal failure (RF). Methods: Adenine-treated Sprague-Dawley rats were randomly assigned to receive continuous peritoneal injections of PDF with or without MGO for three weeks or were left untreated for the same duration. Rats without RF were also assigned to three groups. The peritoneal histology and expression levels of type I collagen, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (αSMA), Snail, matrix metalloproteinase-2 (MMP-2), advanced glycation end-products (AGEs) and the receptor for AGE (RAGE) were then analyzed. Results: Peritoneal treatment with 5 m<smlcap>M</smlcap> MGO accelerated the fibrous peritoneal thickening progression promoted by exposure to standard PDF in the rats with RF, but not in the rats with a normal renal function. Treatment with MGO significantly augmented the proliferation of mesenchymal-like mesothelial cells, accumulation of AGE, de novo expression of αSMA and RAGE and gene expression of type I collagen, TGF-β1, Snail and MMP-2, whereas both MGO and RF alone had, at most, marginal effects on the changes in these biological parameters. Conclusions: In the present study, the adverse effects of MGO on the peritoneum became more prominent under conditions of a uremic milieu. These findings imply that MGO and uremia act cooperatively to induce PF.

Astragalus membranaceus inhibits peritoneal fibrosis via monocyte chemoattractant protein (MCP)-1 and the transforming growth factor-β1 (TGF-β1) pathway in rats submitted to peritoneal dialysis.

Inflammation and transforming growth factor-β1 (TGF-β1) contribute to the development of peritoneal fibrosis (PF), which is associated with peritoneal dialysis (PD). Astragalus membranaceus (Astragalus) has anti-inflammatory and anti-fibrotic effects in many diseases. The goal of this study was to determine the anti-fibrotic effects of Astragalus on the PF response to PD. A rat model of PD was induced using standard PD fluid, and PF was verified by HE and Masson’s staining, as well as through the expression of fibroblast surface protein (FSP) and collagen III. The expression levels of monocyte chemoattractant protein (MCP)-1, F4/80 (macrophage/monocyte marker in rat), TGF-β1 and the downstream proteins phospho-SMAD 2/3 in dialyzed peritoneal tissue treated with or without Astragalus was evaluated using immunohistochemistry analysis. Overall correlations between MCP-1 and TGF-β1 staining were analyzed using both the Spearman and Pearson methods. The results showed that Astragalus could inhibit the recruitment and activation of monocytes/macrophages, thereby reducing the production of TGF-β1 in the dialyzed peritoneal membrane. PF was also significantly decreased following treatment with Astragalus. MCP-1 expression had a strong positive correlation with TGF-β1 sensitivity, suggesting that the anti-fibrotic function of Astragalus was mediated by MCP-1 and the TGF-β1 pathway. Our results indicate that Astragalus could be a useful agent against PD-induced PF.

Influence of Bicarbonate/Low-GDP Peritoneal Dialysis Fluid (Bicavera) on in Vitro and Ex Vivo Epithelial-to-Mesenchymal Transition of Mesothelial Cells

Peritoneal membrane damage induced by peritoneal dialysis (PD) is largely associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), which is believed to be a result mainly of the glucose degradation products (GDPs) present in PD solutions. This study investigated the impact of bicarbonate-buffered, low-GDP PD solution (BicaVera: Fresenius Medical Care, Bad Homburg, Germany) on EMT of MCs in vitro and ex vivo. Omentum-derived MCs were incubated with lactate-buffered standard PD fluid or BicaVera fluid diluted 1:1 with culture medium. Ex vivo studies: From 31 patients randomly distributed to either standard or BicaVera solution and followed for 24 months, effluents were collected every 6 months for determination of EMT markers in effluent MCs. Culturing of MCs with standard fluid in vitro resulted in morphology change to a non-epithelioid shape, with downregulation of E-cadherin (indicative of EMT) and strong induction of vascular endothelial growth factor (VEGF) expression. By contrast, in vitro exposure of MCs to bicarbonate/low-GDP solution had less impact on both EMT parameters. Ex vivo studies partially confirmed the foregoing results. The BicaVera group, with a higher prevalence of the non-epithelioid MC phenotype at baseline (for unknown reasons), showed a clear and significant trend to gain and maintain an epithelioid phenotype at medium- and longer-term and to show fewer fibrogenic characteristics. By contrast, the standard solution group demonstrated a progressive and significantly higher presence of the non-epithelioid phenotype. Compared with effluent MCs having an epithelioid phenotype, MCs with non-epithelioid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin and VEGF. In comparing the BicaVera and standard solution groups, MCs from the standard solution group showed significantly higher secretion of interleukin 8 and lower secretion of collagen I, but no differences in the levels of other EMT-associated molecules, including fibronectin, VEGF, E-cadherin, and transforming growth factor β1. Peritonitis incidence was similar in both groups. Functionally, the use of BicaVera fluid was associated with higher transport of small molecules and lower ultrafiltration capacity. Effluent MCs grown ex vivo from patients treated with bicarbonate/low-GDP BicaVera fluid showed a trend to acquire an epithelial phenotype, with lower production of proinflammatory cytokines and chemokines (such as interleukin 8) than was seen with MCs from patients treated with a lactate-buffered standard PD solution.

A Study of the Clinical and Biochemical Profile of Peritoneal Dialysis Fluid Low in Glucose Degradation Products

Although peritoneal dialysis (PD) is a widely accepted form of renal replacement therapy, concerns remain regarding the bioincompatible nature of standard PD fluid (PDF). Short-term studies of new biocompatible PDFs low in glucose degradation products (GDPs) reveal divergent results with respect to peritoneal integrity. We studied 125 patients on maintenance PD who were assigned, by simple randomization, to receive either conventional or low-GDP PDF at PD initiation. Parameters of dialysis adequacy and peritoneal transport of small solutes were determined at initiation and after a period of maintenance PD at the time when serum and overnight effluent dialysate were simultaneously collected and assayed for various cytokines, chemokines, adipokines, and cardiac biomarkers. All patients were further followed prospectively for an average of 15 months from the day of serum and effluent collection to determine patient survival and cardiovascular events. Patients treated with conventional or low-GDP PDF were matched for sex, age, duration of dialysis, dialysis adequacy, and incidence of cardiovascular disease or diabetes. After an average of 2.3 years of PD treatment, the weekly total and peritoneal creatinine clearance, and the total and peritoneal Kt/V were comparable in the groups. However, urine output was higher in patients using low-GDP PDF despite there having been no difference between the groups at PD initiation. Patients using low-GDP PDF also experienced a slower rate of decline of residual glomerular filtration and urine output than did patients on conventional PDF. Compared with serum concentrations, effluent concentrations of tumor necrosis factor α, hepatocyte growth factor, macrophage migration inhibitory factor, interleukins 8 and 6, C-reactive protein, and leptin were found to be higher in both groups of patients after long-term PD, suggesting that the peritoneal cavity was the major source of those mediators. Compared with patients on low-GDP PDF, patients on conventional fluid showed elevated leptin and reduced adiponectin levels in serum and effluent. The effluent concentration of interleukin 8 was significantly lower in patients using low-GDP PDF. The survival rate and incidence of cardiovascular complications did not differ between these groups after maintenance PD for an average of 3.6 years. It appears that low-GDP PDF results in an improvement of local peritoneal homeostasis through a reduction of chronic inflammatory status in the peritoneum.

PPAR-γ agonist rosiglitazone protects peritoneal membrane from dialysis fluid-induced damage

Exposure to non-physiological solutions during peritoneal dialysis (PD) produces structural alterations to the peritoneal membrane and ultrafiltration dysfunction. The high concentration of glucose and glucose degradation products in standard PD fluids induce a local diabetic environment, which leads to the formation of advanced glycation end products (AGEs) that have an important role in peritoneal membrane deterioration. Peroxisome proliferator-activated receptor γ (PPAR-γ) agonists are used to treat type II diabetes and they have beneficial effects on inflammation, fibrosis, and angiogenesis. Hence, we evaluated the efficacy of the PPAR-γ agonist rosiglitazone (RSG) in ameliorating peritoneal membrane damage in a mouse PD model, and we analyzed the mechanisms underlying the protection offered by RSG. Exposure of the peritoneum to PD fluid resulted in AGEs accumulation, an inflammatory response, the loss of mesothelial cell monolayer and invasion of the compact zone by mesothelial cells, fibrosis, angiogenesis, and functional impairment of the peritoneum. Administration of RSG diminished the accumulation of AGEs, preserved the mesothelial monolayer, decreased the number of invading mesothelial cells, reduced fibrosis and angiogenesis, and improved peritoneal function. Interestingly, instead of reducing the leukocyte recruitment, RSG administration enhanced this process and specifically, the recruitment of CD3+ lymphocytes. Furthermore, RSG treatment augmented the levels of the anti-inflammatory cytokine interleukin (IL)-10 and increased the recruitment of CD4+ CD25+ FoxP3+ cells, suggesting that regulatory T cells mediated the protection of the peritoneal membrane. In cell-culture experiments, RSG did not prevent or reverse the mesothelial to mesenchymal transition, although it decreased mesothelial cells apoptosis. Accordingly, RSG appears to produce pleiotropic protective effects on the peritoneal membrane by reducing the accumulation of AGEs and inflammation, and by preserving the mesothelial cells monolayer, highlighting the potential of PPAR-γ activation to ameliorate peritoneal deterioration in PD patients.

Low-GDP peritoneal dialysis fluid ('balance') has less impact in vitro and ex vivo on epithelial-to-mesenchymal transition (EMT) of mesothelial cells than a standard fluid

Peritoneal membrane deterioration during peritoneal dialysis (PD) is associated with epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MC), which is believed to be mainly due to glucose degradation products (GDPs) present in PD solutions. Here we investigate the impact of GDPs in PD solutions on the EMT of MC in vitro and ex vivo. For in vitro studies, omentum-derived MC were incubated with standard PD fluid or low-GDP solution diluted 1:1 with culture medium. For ex vivo studies, 33 patients, who were distributed at random to either the 'standard' or the 'low GDP' groups, were followed over 24 months. Effluents were collected every 6 months to determine EMT markers in effluent MC. Exposure of MC to standard fluid in vitro resulted in morphological change into a non-epitheloid shape, down-regulation of E-cadherin, indicative of EMT, and in a strong induction of vascular endothelial growth factor (VEGF) expression. In contrast, in vitro exposure of MC to low-GDP solution did not lead to these phenotype changes. This could be confirmed ex vivo, as the prevalence of non-epitheloid phenotype of MC in the standard group was significantly higher with increasing PD duration and MC isolated from this group showed significantly higher levels of EMT-associated molecules including fibronectin, collagen I, VEGF, IL-8 and TGF-β levels when compared with the low-GDP group. Over time, the expression of E-cadherin also decreased in the standard but increased in the low-GDP group. In addition, the levels of EMT-associated molecules (fibronectin, VEGF and IL-8) increased in the standard but decreased in the low-GDP group. A similar trend was also observed for collagen I and for TGF-β (for the first year), but did not reach global statistical significance. Accordingly, effluent MC with non-epitheloid morphology showed significantly lower levels of E-cadherin and greater levels of fibronectin, collagen I, VEGF and IL 8 when compared with MC with epitheloid phenotype. The incidence of peritonitis did not significantly influence these results. Drop-out due to technique failure was less in the 'balance' group. The functional, renal and peritoneal evaluation of patients being treated with either standard or 'balance' fluid did not show any significant difference over time. MC from PD effluent of patients treated with a PD fluid containing low GDP levels show fewer signs of EMT and the respective molecules than MC from patients treated with standard fluid, indicating a better preservation of the peritoneal membrane structure and a favourable outcome in patients using low-GDP fluid. It also confirms the hypothesis that the protection of EMT by GDP-reduced fluids is also present in vivo.

Low-GDP fluid (Gambrosol trio(R)) attenuates decline of residual renal function in PD patients: a prospective randomized study

Residual renal function (RRF) impacts outcome of peritoneal dialysis (PD) patients. Some PD fluids contain glucose degradation products (GDPs) which have been shown to affect cell systems and tissues. They may also act as precursors of advanced glycosylation endproducts (AGEs) both locally and systemically, potentially inflicting damage to the kidney as the major organ for AGE elimination. We conducted a clinical study in PD patients to see if the content of GDP in the PD fluid has any influence on the decline of the residual renal function. In a multicentre approach, 80 patients (GFF > or = 3 mL/min/1.732 or creatinine clearance > or =3 mL/min/1.73 m(2)) were randomized to treatment with a PD fluid containing low levels of GDP or standard PD fluid for 18 months. RRF was assessed every 4-6 weeks. Fluid balance, mesothelial cell mass marker CA125, peritoneal membrane characteristics, C-reactive protein (CRP), total protein, albumin, electrolytes and phosphate were measured repeatedly. Data from 69 patients revealed a significant difference in monthly RRF change: -1.5% (95% CI = -3.07% to +0.03%) with low GDP (43 patients) vs -4.3% (95% CI = -6.8% to -2.06%) with standard fluids (26 patients) (P = 0.0437), independent of angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker medication. Twenty-four-hour urine volume declined more slowly with low-GDP fluid compared to standard fluids (12 vs 38 mL/month, P = 0.0241), and monthly change of phosphate level was smaller (+0.013 vs +0.061 mg/dL, P = 0.0381). Our prospective study demonstrates for the first time a significant benefit concerning preservation of RRF and urine volume of using a PD fluid with low GDP levels. These findings suggest that GDPs might affect patient outcome related to RRF.

Cellular stress-response modulators in the acute rat model of peritoneal dialysis

Cytotoxicity of peritoneal dialysis fluids (PDF) not only results in cellular injury, but also induces heat-shock proteins (HSP), the main effectors of the cellular stress response. This study investigated effects of modulation of mesothelial HSP expression on peritoneal membrane integrity during acute PDF exposure. In the acute in vivo rat model of peritoneal dialysis (PD), either the HSP coinducer indomethacin or the HSP suppressor quercetin was added to standard PDF (CAPD 3, Fresenius, Germany). HSP-72 expression, number of detached mesothelial cells, and peritoneal protein loss were evaluated at the end of a 4-h dwell time. Compared with pure PDF exposure, addition of indomethacin resulted in increased expression of mesothelial HSP-72, reduced mesothelial cell exfoliation, and reduced peritoneal protein loss. Addition of quercetin resulted in decreased expression of HSP-72, increased mesothelial cell exfoliation, and higher peritoneal protein loss. Differences were statistically significant between indomethacin-treated and quercetin-treated rats. Mesothelial HSP expression was related to markers of peritoneal membrane integrity upon in vivo PDF exposure, consistent with HSP-mediated cytoprotection. These data clearly demonstrate the potential for clinically feasible pharmacologic interventions with the cellular stress response as a novel therapeutic approach to improve PD outcome.

Nephrology literature watch

Nephrology literature watch

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

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. 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). 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. 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.

Citrate supplementation of PD fluid: effects on net ultrafiltration and clearance of small solutes in single dwells

Inflammatory reactions affect the general performance as well as the technique survival of peritoneal dialysis (PD). Anti-inflammatory additives like heparin and sodium citrate have shown favourable results in these respects. The present study is the first to evaluate citrate-supplemented PD fluids (PDFs) in humans. Crossover design was used to evaluate sodium citrate and heparin-supplemented Gambrosol Trio (2.5% glucose) in 28 stable outpatients from the PD unit. Comparisons were made between single dwells of each fluid. Citrate supplementation at 5 mM/L was compared with standard PDF, and citrate supplementation at 10 mM/L was compared with low-molecular-weight heparin (4500 units of tinzaparin) supplementation and standard PDF. The initial osmolarity of the fluids was equalized by adding sodium chloride. Citrate supplementation at 5 mM/L significantly increased net ultrafiltration, measured as drained volume gain, by 126 mL. Creatinine and phosphate clearance, but not glucose clearance, was significantly improved by supplementation with citrate or heparin. Heparin supplementation created an insignificant trend towards an increased ultrafiltration (P = 0.08). No negative side effects were reported for any of the treatments; however, citrate supplementation led to a small calcium loss by the drained PD fluid (0.4 mmol) and a transient fall in the plasma concentration (0.04 mM/L) of free calcium ions at 5 mM/L citrate. Effects on plasma bicarbonate concentration were insignificant. Citrate supplementation of PD fluid improved ultrafiltration and clearance of small solutes with only minor effects on calcium turnover. The mechanism is unknown and, according to the results, not related to complement inhibition.

Peritoneal Dialysis Solutions Low in Glucose Degradation Products—evidence for Clinical Benefits

In Japan, two types of new peritoneal dialysis fluid (PDF) are ordinarily used: two-chambered PDF, and icodextrin PDF. Two-chambered PDF has several biocompatible characteristics, one being low glucose degradation products (GDPs). Of the several GDPs in PDF, 3,4-dideoxyglucosone-3-ene (3,4-DGE) is thought to be strongly associated with the cytotoxicity of standard PDF. Using a PDF low in GDPs may reduce exposure of the peritoneum to 3,4-DGE, helping to preserve peritoneal function in PD patients. Additionally, use of a PDF low in GDPs may reduce plasma levels of advanced glycosylation end-products in PD patients, a change that may help to preserve vascular function in PD patients. Peritoneal rest for 24 hours after exposure to a PDF with low GDPs improves the activity of human peritoneal mesothelial cells. As compared with the use of standard PDF, the use of low-GDP PDF in combination therapy (peritoneal dialysis plus hemodialysis) may more effectively preserve peritoneal function. The new PDF low in GDPs has biocompatible characteristics relative to peritoneum and system that may help to preserve peritoneal function or reduce complications such as atherosclerosis or dialysis-related amyloidosis in dialysis patients.

Quercetin protects human mesothelial cells against exposure to peritoneal dialysis fluid

During peritoneal dialysis, mesothelial cells have been shown to undergo severe damage due to continuous exposure to peritoneal dialysis fluid (PDF) with cytotoxic physicochemical properties. In this study, we investigated the cytoprotective role of the bioflavonoid Quercetin in the in vitro model of peritoneal dialysis. Immortalized human mesothelial cells (Met5A) were exposed either to regular growth medium or to standard acidic lactate-buffered PDF (Dianeal PD4) or to a more biocompatible lactate-bicarbonate-buffered PDF (Physioneal 40). Parallel cell cultures were supplemented with 200 microM Quercetin. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by the release of cytoplasmic lactate dehydrogenase and fluorescence-activated cell sorting (FACS). PDF exposure with bioincompatible Dianeal PD4 resulted in severe disruption of cell cultures and in significantly increased lactate dehydrogenase (LDH) release (p=0.0007 vs. control). Addition of 200 microM Quercetin significantly decreased the LDH release (p=0.04 vs. "pure" Dianeal PD4 exposure), comparable to control exposure and to more biocompatible Physioneal 40 exposure (p=0.37) and resulted in marked preservation of cell culture monolayers and cellular viability as assessed by FACS. Introduction of cytoprotective agents such as Quercetin may represent an alternate approach to protect mesothelial cells from cytotoxicity of frequently used PDFs, comparably effective to the introduction of novel, more biocompatible, PDFs.

ImprovedIn VitroBiocompatibility of Bicarbonate-Buffered Peritoneal Dialysis Fluid

Conventional peritoneal dialysis fluids (PDFs) have been shown to damage the mesothelial layer and are associated with the development of peritoneal fibrosis and neoangiogenesis. New-generation PDFs have therefore been developed with physiological pH and reduced levels of glucose degradation products (GDPs), precursors of advanced glycation end products (AGEs). In this work, we evaluated and compared the improved biocompatibility of two new-generation PDFs (Balance and bicaVera) using mesothelial cell biology; we also compared them to a standard PDF (stay.safe) (all PDFs by Fresenius Medical Care, Fresnes, France). stay.safe, Balance, and bicaVera were tested for their effect on human peritoneal mesothelial cell (HPMC) viability by measuring cell proliferation and apoptosis, and oncosis induction. The formation of AGEs was evaluated by immunoassay. Transforming growth factor beta-1 and vascular endothelial growth factor (VEGF) were immunoassayed in HPMC supernatants exposed to the above PDFs. At 15 g/L glucose concentration, HPMC exposure to bicaVera resulted in higher cell proliferation compared to Balance (p < 0.001) and stay.safe (p < 0.001). Compared to the lactate-buffered PDFs (Balance and stay.safe), oncosis was significantly lower in cells exposed to bicaVera (p < 0.05). bicaVera, containing lower amounts of GDPs, generated less AGE formation (p < 0.05) and VEGF production (p < 0.05) than either Balance or stay.safe. New-generation PDFs with physiological pH and lower GDP levels, especially if bicarbonate-buffered (bicaVera), have fewer in vitro toxic effects on mesothelial cells and may contribute to peritoneal preservation, thus improving long-term treatment of PD patients.