Abstract
Peritoneal Morphologic and Functional Changes in Uremic Rats Dialyzed with Icodextrin-Based Solution
Objectives: To investigate the effect of icodextrin-based peritoneal dialysis (PD) solution on peritoneal morphology and transport function in uremic rats.
Methods: A uremic PD rat model was induced by subtotal nephrectomy and catheter insertion. The rats were then dialyzed with either conventional PD fluid [group G: 4.25% Dianeal (Baxter Healthcare Corporation, Deerfield, IL, U.S.A.),n = 12] or icodextrin-based PD fluid [group I: 7.5% Extraneal (Baxter Healthcare Corporation),n=12] daily for 8 weeks. Sham operation rats served as controls (group C). After 4 or 8 weeks, rats underwent a 120-minute peritoneal equilibration test (PET) before being euthanized. Peritoneal biopsies were obtained to assess peritoneal morphology.
Results: Compared with control rats at 4 weeks, rats in groups G and I both showed significantly mesothelial hypercellularity (p = 0.002, p = 0.006) and increased thickness of the submesothelial compact zone (SCZ: 18.0 μm vs. 4.2 μm, p = 0.004; 14.3 μm vs. 4.2 μm, p = 0.004) and vessel density (106.2 ± 30.2/mm2 vs. 22.3 ± 13.4/mm2, p < 0.001; 72.7 ± 20.3/mm2 vs. 22.3 ± 13.4/mm2, p < 0.001). Compared with group G rats, group I rats had fewer vessels (p = 0.048). After 8 weeks on PD, rats in groups G and I both showed marked mesothelial cell loss (p = 0.002, p = 0.02), a thickened SCZ (24.5 μm vs. 4.3 μm, p = 0.001; 12.7 μm vs. 4.3 μm, p = 0.004), and higher vessel density (122.2 ± 64.3/mm2 vs. 29.5 ± 11.9/mm2, p < 0.001; 81.3 ± 22.1/mm2 vs. 29.5 ± 11.9/mm2, p < 0.001). Thickness of the SCZ was significantly higher in group G than in group I (p = 0.005). The PET showed that, at 4 weeks, dialysate-to-plasma creatinine (D/P Cr) of rats in group G was significantly higher than that in groups C and I (0.89 ± 0.49 vs. 0.74 ± 0.11, p = 0.001; 0.89 ± 0.49 vs. 0.74 ± 0.10, p = 0.007), and net ultrafiltration (UF) was less than in groups C and I (1.13 ± 2.86 mL vs. 7.23 ± 1.56 mL, p = 0.001; 1.13 ± 2.86 mL vs. 7.07 ± 2.44 mL, p = 0.01). Similarly, at 8 weeks, rats in group G had a higher D/P Cr than that in groups C and I (0.90 ± 0.06 vs. 0.69 ± 0.11, p = 0.002; 0.90 ± 0.06 vs. 0.65 ± 0.06, p < 0.001) and less net UF than that in group C (1.57 ± 3.97 mL vs. 6.4 ± 1.92 mL, p = 0.023). As compared with group C rats, group I rats showed similar D/P Cr and UF (p > 0.05).
Conclusions: Compared with conventional PD solution, icodextrin-based solution could induce less morphologic deterioration and preserve peritoneal transport.
Genetic Effect of Proinflammatory Cytokine Polymorphism on Patients Undergoing Peritoneal Dialysis
Aim: Proinflammatory cytokines (PCs) regulate the inflammatory reaction that plays a pivotal role in many pathophysiologic processes. Uremia is frequently complicated with chronic inflammation which substantially influences outcome. For peritoneal dialysis (PD) patients (pts), exposure to glucose-based PD solution elicits local and systemic reactions; both can lead to chronic inflammation. Polymorphism (polym) of PCs has rarely been investigated in PD pts, specifically regarding inflammatory status.
Methods: Stable chronic PD pts were enrolled for genetic analysis, and their demographic and clinical data were reviewed and recorded. The PD therapy was evaluated using weekly creatinine clearance and Kt/V; residual kidney function (RKF) was recorded as daily urine volume. Peritoneal permeability was assessed by a standardized peritoneal equilibration test. Vascular calcification (VC) was interpreted by the presence of aortic arch calcification in plain posterior–anterior chest radiographs. Restriction fragment length polym analysis was performed, and the PCs interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) were analyzed.
Results: The IL-6 polym was found in promoter region –572 (A→G), but no polym was found in –174 (G→C) and –597 (G→A). High producer IL-6 (GG) was associated with increased peritoneal permeability [dialysate-to-plasma creatinine (D/P Cr): 0.64 ± 0.11 vs. 0.60 ± 0.10,p < 0.05], higher IL-18 (463.9 ± 227.2 pg/mL vs. 399.9 ± 168.2 pg/mL, p < 0.05), and lower adiponectin (19.7 ± 12.9 μg/mL vs. 24.8 ± 14.8 μg/mL, p < 0.05). With regard to VC, the distribution of genetic background was uneven: more IL-6 –572 (GG) was observed in pts with VC (70.2% vs. 51.1%, p < 0.05). There was no significant difference in RKF albumin, and high-sensitivity C-reactive protein (hs-CRP). Initial peritoneal transport was not influenced by IL-6 polym. Genetic analysis of TNFα found that polym at promoter region –308 (G→A) constituted 18.5% of study population, and most of the high producer was heterozygotic. There was no significant difference in albumin, hs-CRP and IL-6, IL-18, and adipokines (adiponectin, leptin, resistin). The presence of VC was not affected by the genetics of TNFα.
Conclusions: The IL-6 promoter polym in Chinese PD pts is different from that in Caucasian populations. The IL-6 –572 (A→G) is associated with alterations in the characteristics of peritoneal transport. Pts with IL-6 –572 polym had higher IL-18 and lower adiponectin levels. This genetic factor also influenced VC in PD pts. No significant effect was found in TNFα –308 (G→A) polym.
Serum Human Decoy Receptor 3 (DcR3) is a Surrogate of Malnutrition–Inflammation in Peritoneal Dialysis Patients
Aim: Malnutrition–inflammation complex (MIC) is frequently encountered in chronic peritoneal dialysis (PD) patients. Serum human decoy receptor 3 (DcR3) is a novel member of the tumor necrosis factor receptor family. Recent studies have shown that serum DcR3 is elevated in patients with autoimmune disorders and malignancy and serves as a predictor of poor prognosis. However, the role of DcR3 in the MIC status of PD patients is uncertain. We therefore assessed whether serum DcR3 could be a marker of inflammation and malnutrition in chronic PD patients.
Methods: The study enrolled 74 PD patients (38 men, 36 women; mean age: 57 years) with a dialysis duration of more than 3 months (mean PD vintage: 11 months). Those having infections or cardiovascular events within 1 month of enrollment were excluded. A standard peritoneal equilibration test was performed. Serum levels of DcR3, tumor necrosis factor α (TNFα) and interleukin 6 (IL-6) were measured by ELISA.
Results: Serum DcR3 was significantly negatively correlated with serum albumin (r = –0.50, p < 0.001) and transferrin (r = –0.27, p < 0.05). Serum DcR3 was positively correlated with serum IL-6 (r = 0.51, p < 0.001), TNFα (r = 0.43, p < 0.05), and C-reactive protein (r = 0.33, p < 0.05). On evaluation of peritoneal function, serum DcR3 was positively correlated with dialysate-to-plasma ratio of creatinine at 4 hours (r = 0.30, p < 0.05).
Conclusions: Serum level of DcR3 is a surrogate marker of MIC in patients undergoing chronic PD. The clinical and pathologic role of increased serum DcR3 in MIC-related outcomes in PD patients remains to be studied.
Association between Protein-Bound Uremic Toxin and Proinflammatory Cytokines in Patients on Peritoneal Dialysis
Aim: Patients with chronic kidney disease accumulate uremic toxins as renal function deteriorates. These retention molecules can cause both functional and structural alterations in individual cells and eventually lead to dysfunction of organs and even elicit clinical manifestations constituting the uremic syndrome. The relationship between protein-bound uremic toxins and inflammation has not yet been explored.
Methods: Blood samples were collected from stable peritoneal dialysis (PD) patients. Demographic and biochemical data, residual kidney function (RKF), dialysis dosage [weekly creatinine clearance (wCCr), Kt/V], and markers of nutrition were reviewed and recorded. Levels of proinflammatory cytokines [interleukins 6 (IL-6) and 10 (IL-10)] were measured. The concentration of the uremic toxin indoxyl sulfate (IS), total and free form, was determined by ultra-performance liquid chromatography assay.
Results: The study enrolled 157 stable PD patients (mean age: 49.1 ± 13.8 years; mean duration of PD therapy: 42.3 ± 34.2 months). Their blood concentration of total and free IS was 41.1 ± 16.3 mg/L and 4.0 ± 2.6 mg/L respectively. The presence of RKF affected the levels of IL-6 and total and free IS (4.5 ± 0.5 pg/mL vs. 6.7 ± 0.9 pg/mL; 38.3 ± 16.4 mg/L vs. 45.1 ± 15.3 mg/L, and 3.7 ± 2.4 mg/L vs. 4.6 ± 2.8 mg/L; all p < 0.05). Peritoneal permeability did not influence levels of total and free IS. No significant difference was noted between PD modalities (continuous ambulatory PD vs. automated PD). Univariate analysis revealed a reverse association between total IS level and urine volume, Kt/V, and wCCr. Total IS was directly associated with blood urea nitrogen, creatinine, albumin, and IL-6. Free IS was negatively correlated with urine volume, Kt/V, and wCCr. A significant positive relationship was noted for BMI, creatinine, IL-6, and total IS. Multivariate regression analysis found that creatinine and IL-6 were independently correlated with total IS; age, IL-6, and total IS predicted free IS level.
Conclusions: The presence of RKF influences the concentration of total and free IS. There was a strong association between the proinflammatory cytokine IL-6 and the protein-bound uremic toxin IS, both total and free form.
Interleukin-1β –Induced Cyclooxygenase-2 Gene Expression via Atypical Protein Kinase C in Cultured Human Peritoneal Mesothelial Cells
Background: In a recent study, cyclooxygenase-2 (COX2) inhibition resulted in reduced fibrosis of peritoneum and in partial recovery of ultrafiltration, and thus it was suggested that COX2 inhibition can be a potential strategy to ameliorate peritoneal deterioration in peritoneal dialysis patients. Interleukin-1β (IL-1β) can be elevated in the peritoneal fluid because of bioincompatible dialysate or during peritonitis. Protein kinase C (PKC) is one of the important mediators of IL-1β–induced signal transduction. PKCs consist of three groups of isoenzymes: conventional, novel, and atypical. Atypical PKCs include PKCζ and PKCι. In this study, we investigated whether IL-1β increases COX2 messenger RNA (mRNA) expression, and whether PKC is involved in this process.
Methods: The COX2 mRNA level and phosphorylated PKCζ/ι in total cell lysate were measured by Northern blot and Western blot analysis respectively.
Results: IL-1β (1 ng/mL) increased COX2 mRNA expression in a dose- and time-dependent manner. Pretreatment of human peritoneal mesothelial cells (HPMCs) with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1000 nmol/L caused a dose-dependent inhibition of IL-1β–induced COX2 mRNA expression. GF109203X, another pan-PKC inhibitor, at doses of 1, 5, and 10 μmol/L also downregulated IL-1β–induced COX2 mRNA expression in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated COX2 mRNA expression. After prolonged treatment of the cells for 72 hours with PMA, another dose of PMA did not increase COX2 mRNA expression, but IL-1β still stimulated it. IL-1β increased phospho-PKCζ/ι, an active form of the enzyme. Transfection of the cells with either pcDNA3 containing dominant negative PKCζ or pcDNA3 containing dominant negative PKCι attenuated IL-1β–induced COX2 mRNA expression.
Conclusions: The results suggest that IL-1β–induced COX2 gene expression in HPMCs occurs through the activation of atypical PKCs (PKCζ/ι). Thus, atypical PKCs could be a target to prevent overproduction of COX2.
Long-Term Effect of Peritoneal Dialysis Fluids with Neutral pH and Low Glucose Degradation Product Concentrations: A 5-Year Retrospective Cohort Study
Long-term peritoneal dialysis (PD) has shown that peritoneal membrane performance and anatomy deteriorates with duration of exposure to peritoneal dialysis fluids (PDFs). In this retrospective cohort study, we compared the long-term clinical impact of PD treatment with either conventional PDFs or PDFs low in glucose degradation products (GDPs).
From January 2003, 178 PD patients treated with either conventional PDFs (C-PDF group, n = 66) or low-GDP PDFs (L-PDF group, n = 112) in a single Korean center were included in the study. We investigated clinical and biochemical parameters simultaneously with nutrition status as assessed by subjective global assessment (SGA), dialysis adequacy, and peritoneal equilibration test (PET) in two groups (baseline vs. 5-year values).
Baseline clinical parameters including SGA, BMI, albumin, CRP, solute clearance, and urea kinetics were not significantly different between the two groups. At 5 years, daily ultrafiltration in the L-PDF group was greater than that in the C-PDF group (775.0 ± 260.9 mL vs. 502.6 ± 261.8 mL, p < 0.05), and CRP levels were lower in the L-PDF group (0.36 ± 0.29 mg/dL vs. 2.02 ± 3.58 mg/dL, p < 0.05). Actual dialysate-to-plasma creatinine (D/P Cr) was not significantly different between the L-PDF and C-PDF groups. Total weekly Kt/V urea and creatinine clearance at 5 years were not significantly different between the two groups.
Our data show that PD patients on low-GDP PDFs can maintain the long-term preservative capacity of ultrafiltration and less systemic inflammation than are seen in PD patients on conventional PDFs.
High Dose of Intravenous Iron Aggravates the Peripheral T-Lymphocyte Imbalance in Peritoneal Dialysis Patients
Objectives: Uremia and iron deficiency can both cause inflammation and immune dysfunction, impair the balance of T-cell subsets, resulting increased infection susceptibility. The aim of this study was to evaluate the impact of high-dose intravenous iron supplementation on peripheral blood T-helper lymphocyte balance in peritoneal dialysis (PD) patients with iron deficiency anemia (IDA).
Methods: In 19 PD patients with IDA, low molecular weight iron dextran (Cosmofer: Schwarz Pharma) was administered in a single dose of 1000 mg. Subsets of T lymphocytes and T helper lymphocyte subpopulations, including natural killer (NK) cells, helper T lymphocytes (Th), cytotoxic T lymphocytes (Ts), helpwe T lymphocyte types 1, 2, and 17 (Th1, 2, 17), and regulatory T cells (Tregs) were measured by flow cytometry 12 hours before and after the infusion. Serum levels of interferon γ (IFN-γ) and of interleukins 4,6, 10, and 17A (IL-4, -6, -10, -17A), and expression of T-helper lymphocyte differentiation-related genes were assessed by ELISA and by real-time quantitative polymerase chain reaction respectively.
Results: Proportions of NK cells, Th cells, and Tc cells were higher after the iron than before, but not significantly so. Nevertheless, the percentages of Th1, Th2, and Th17 increased after iron infusion, and Tregs decreased significantly. Meanwhile, the levels of cytokines corresponding to Th cells also rose after iron transfusion (IL-4: 57.2 ± 28.7 pg/mL vs. 102.3 ± 69.5 pg/mL, p = 0.011; IL-10: 32.6 ± 20.3 pg/mL vs. 59.2 ± 31.4 pg/mL, p = 0.064; IL-17A: 12.6 ± 5.3 pg/mL vs. 19.4 ± 9.4 pg/mL, p = 0.059; IFN-γ: 32.5 ± 28.6 pg/mL vs. 57.2 ± 36.0 pg/mL, p = 0.154; IFN-γ/IL-4: 0.91 ± 0.59 vs. 0.49 ± 0.17, p = 0.068). Consistent with the cytokine profile, expression of both T-bet and GATA3 were markedly higher after the iron than before (T-bet: p = 0.087; GATA3: p = 0.012), with a dramatic reduction of Foxp3 expression and the ratio T-bet/GATA3 (Foxp3: p = 0.024; T-bet/GATA3: p = 0.043).
Conclusions: Comprehensively elevated T-cell subsets, along with decreased Tregs, could be induced by a high dose of intravenous iron dextran. Furthermore, the ratios in the cellular and cytokine profiles indicated that an imbalance of helper T cells had switched to Th2. And the shift of immuno-imbalance should be connected with clinical episodes in the future.
Role of Poly (ADP-ribose) Polymerase-1 in Peritoneal Fibrosis Induced by High Glucose in Peritoneal Mesothelial Cells
Objectives: To investigate the role of poly (ADP-ribose) polymerase-1 (PARP-1) in peritoneal fibrosis induced by high glucose (HG) in peritoneal mesothelial cells (PMCs).
Methods: Rat PMCs (RPMCs) were harvested and maintained under defined in vitro conditions. Human PMC (HPMC) lines (HMrSv5) were incubated with serum-free media for 24 hours to arrest and synchronize growth. The cells were then separated into groups that were stimulated with or without HG for up to 24 hours. Some groups were treated with PARP-1 inhibitor PJ34 (3×10–6 mol/L) or PARP-1 small interfering RNA (siRNA—5×10–8 mol/L). PARP-1, α–smooth muscle actin (αSMA), E-cadherin, fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1), vimentin, and collagen-1 (COL1) messenger RNA (mRNA) and protein (prot) expression were examined.
Results: Glucose at 2.27% (126 mmol/L) significantly increased PARP-1 mRNA and prot expression in RPMCs. PJ34 significantly inhibited, by 45.0% and 51.1% respectively (p < 0.05), PARP-1 mRNA and prot overexpression induced by HG. HG was also associated with elevated levels of α–SMA, E-cadherin, PAI-1, and FN mRNA, and with increased RPMC prot expression of α–SMA, E-cadherin, PAI-1, and COL-1. These effects were suppressed by PJ34 (p < 0.05). PARP-1, α–SMA, and PAI-1 mRNA and prot expression were all significantly upregulated by HG (2.27%) in HPMCs. PARP-1 siRNA significantly inhibited the upregulation of PARP-1, α–SMA, and PAI-1 expression induced by HG. Compared with the HG stimulation group, the PARP-1 siRNA–treated group showed mRNA expression inhibition rates for PARP-1, α–SMA, and PAI-1 of 34.3%, 52.8%, and 62.1% respectively (p < 0.05). Comparison of prot expression in the same groups showed inhibition rates of 33.3%, 30.6%, and 68.3% respectively (p < 0.05). In addition, HG significantly downregulated both E-cadherin mRNA and prot expression in HPMCs (p < 0.05). PARP-1 siRNA effectively reversed HG-induced E-cadherin mRNA and prot expression by 81.0% and 37.0% respectively (p < 0.05). HG levels were associated with upregulated FN mRNA expression in HPMCs. Treatment with PARP-1 siRNA effectively downregulated HG-induced FN mRNA overexpression by 50.9% (p < 0.05). Vimentin prot expression was upregulated by HG 24 hours after stimulation, and PARP-1 siRNA inhibited this effect by 42.3% (p < 0.05).
Conclusions: HG stimulates PARP-1 overexpression in PMCs. PARP-1 mediates the endothelial mesenchymal transformation and the accumulation of extracellular matrix in PMCs subjected to HG.
Effect of Glucose-Based and Icodextrin-Based Peritoneal Dialysis Fluid on the Expression of TLR-2 and -4 and the Activation of MAPK and NF-κB Signaling in Human P eritoneal Mesothelial Cells
Objectives: Recent studies have shown that toll-like receptors (TLRs) play a critical role in innate immunity. Our study investigated the effect of glucose (GLU)–based and icodextrin (ICO)–based peritoneal dialysis solution (PDF) on the expression of TLR-2 and -4 and on the response of TLR signaling molecules [mitogen-activated protein kinase (MAPK), nuclear factor κB (NF-κB)] and on TLR-mediated proinflammatory cytokines [interleukin 1β (IL-1β)] to pathogen-associated molecules in human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs were separately treated with GLU-based PDFs (1.5%, 2.5%, 4.25%) and ICO-based PDF (7.5%) for 48 hours and 72 hours respectively. After pre-treatment with these fluids for 48 hours, HPMCs were separately incubated with TLR-2 ligand (Pam3CSK4, 250 ng/mL) and TLR-4 ligand [lipopolysaccharide (LPS),1000 ng/mL]. The phosphorylation levels of ERK1/2, JNK, p38 MAPK, and NF-κB p65 at 1 hour and the expression of IL-1β messenger RNA (mRNA) at 2 hours were measured.
Results: Compared with standard medium (StdM), treatment with 1.5%, 2.5%, and 4.25% PDF for 48 hours decreased HPMC expression of TLR-2 by 5.5% ± 2.8%, 31.4% ± 7.5% (p < 0.05), and 54.9% ± 1.9% (p < 0.05). Treatment also decreased expression of TLR-4 by 32.9% ± 17.6%, 47.7 ± 13.5%, and 66.4 ± 13.5% respectively (each p < 0.01). In addition, significantly decreased cytoplasmic staining of TLR-2 and TLR-4 was observed in HPMCs. After extending treatment to 72 hours, expression of TLR-2 and -4 was also decreased (TLR-2: 29.4% ± 14.7%, 38.9% ± 9.9%, 63.5% ± 16.5%, p < 0.05; TLR-4: 59.5% ± 16.8%, 63.1% ± 9.5%, 79.2% ± 14.0%, p < 0.01). In contrast, no significant changes in TLR-2 and -4 protein expression were seen in cells treated with ICO for the same period. Pre-incubation of HPMCs with GLU-based PDF, as compared with StdM, significantly decreased Pam3CSK4 or LPS-induced phospho-ERK1/2, phospho-JNK, phospho-p38 MAPK, and NF-κB phospho-p65 protein levels, and IL-1β mRNA expression. However, treatment with ICO, as compared with StdM, did not significantly decrease levels of phospho-ERK1/2, phospho-JNK, phospho-p38 MAPK, and NF-κB phospho-p65 proteins or the expression of IL-1β mRNA in HPMCs.
Conclusions: Reduced expression of TLR-2 and -4 in HPMCs exposed to GLU-based PDF may be associated with compromised function of the innate immune system within the peritoneal cavity. The absence of similar findings in cells treated with ICO suggests that icodextrose may be superior for preserving peritoneal defense.