Abstract
Influence of the Status of Uremia on Peritoneal Microvessel Density
Objectives: Uremia in patients (pts) with end-stage renal disease (ESRD) may lead to peritoneal membrane structure change, which could affect transport after peritoneal dialysis (PD). However, the influence of uremia on the peritoneal vascular bed, which is regarded as an important barrier, is not very clear yet. In this study, we investigated the effect of uremia on the peritoneal vascular bed, and we also explored possible mechanisms.
Methods: Our single-center cross-sectional study included 36 nondiabetic ESRD pts (20 men, 16 women; mean age: 59.5 ± 9.8 years; no previous history of abdominal surgery or infection) selected from the new cases at the PD center in our hospital from December 2005 to January 2007. The study also included, as controls, 10 pts with normal renal function (7 men, 3 women; mean age: 55.7 ± 2.3 years) who were going to have abdominal surgery. General information was collected for all subjects, and residual renal function [RRF, as creatine clearance (CCr) in mL/min] was determined. Venous blood was obtained to evaluate C-reactive protein [CRP (mg/L)] on the day that the PD catheter was inserted. Specimens of parietal peritoneum 0.3×0.3 cm, obtained during the operations, were stained for CD34 (marker of vascular endothelium), and microvessel density [MVD (excluding vessels larger in diameter than 8 red blood cells or with muscular layer)] was counted. The MVD value of the specimen was taken as the mean of all selected high-power fields (HPFs). The MVD in ESRD pts was compared with that in pts with normal renal function, and the correlation between RRF and MVD in ESRD pts was studied.
Results: The difference in peritoneal MVD value between pts with ESRD and those with normal renal function was significantly different (525 ± 2.84/HPF vs. 3.10 ± 0.96/HPF, p = 0.034). In ESRD pts, CCr was 5.26 ± 1.70 mL/min, which was significantly negatively correlated with peritoneal MVD (p = 0.046) and serum CRP (24.82 ± 26.04 mg/L, p = 0.002).
Conclusions: Uremia in pts with ESRD may affect peritoneal MVD, and the worse the RRF, the higher the peritoneal MVD. Poor RRF in pts with a higher inflammatory status, which makes the peritoneal capillary bed more open or hyperplastic, may be the mechanism.
Peritoneal Membrane Morphology of 10 Japanese Patients who Underwent Long-Term Peritoneal Dialysis with Biocompatible Dialysis Fluid
Objectives: The morphology changes in peritoneal membrane after long-term peritoneal dialysis (PD), which consist of interstitial fibrosis and hyalinizing vasculopathy, are considered to be the result of long-term exposure to bioincompatible conventional dialysate. Although biocompatible dialysate has been used worldwide in this decade, its impact on peritoneal morphology has not been well reported. This report aimed to estimate the long-term effect of biocompatible fluid on membrane morphology.
Methods: Peritoneal biopsy specimens were obtained from 10 Japanese patients who underwent PD with biocompatible dialysate fluid over 3 years. Average thickness of the submesothelial compact zone at 5 randomly selected points and lumen/vessel diameter ratio at postcapillary venules were evaluated. Demographic data was obtained for each patient at baseline and with each peritoneal equilibration test (PET).
Results: The mean age of PD induction was 54.4 ± 12.4 years, and the mean PD duration was 59 ± 9.3 months. Only 1 patient had diabetes mellitus. Seven patients received hybrid therapy, and 8 patients experienced peritonitis (0.375 episode/patient–year) during their course of PD therapy. The average time to peritoneal biopsy was 55 ± 13.1 months. The median of average peritoneal thickness was 176 mm (range: 96 – 1424 mm), and the mean lumen/vessel diameter ratio was 0.62 ± 0.10. Average peritoneal thickness and lumen/vessel diameter ratio were both unchanged with duration of PD therapy. At the first and last PET, the mean dialysate-to-plasma ratio (D/P) of creatinine was 0.60 ± 0.07 and 0.64 ± 0.10 respectively. The average gradient of D/P creatinine was 0.0001 ± 0.0040/year.
Conclusions: The use of biocompatible dialysate might slow damage to peritoneal morphology. Patients who underwent long-term PD with biocompatible fluid kept favorable peritoneal solute transport.
Aliskiren Ameliorates Chlorhexidine Digluconate-Induced Peritoneal Sclerosis in Rats
Objectives: Peritoneal sclerosis (PS) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The renin–angiotensin system may have fibrotic effects on the peritoneum. The present study was designed to investigate the protective effects of aliskiren on chlorhexidine digluconate (CHD)–induced PS in rats.
Methods: PS was induced by daily administration of 0.5 mL 0.1% CHD in normal saline for 1 week via PD catheter in Sprague–Dawley rats. Rats received daily intravenous injections of low dose aliskiren (1 mg/kg) or high-dose aliskiren (10 mg/kg) for 1 week after CHD administration. After 7 days, conventional 4.25% glucose dialysate 30 mL was administered, and mean arterial pressure (MAP) and heart rate (HR) were monitored for 4 hours. Dialysate samples (0.5 mL) and blood samples (0.5 mL) were taken at 0 and 4 hours after the dialysate had been infused for measurements of blood urea nitrogen (BUN) and glucose. Transforming growth factor β1 (TGFβ1) was measured in serum at 4 hours of PD. At the end of dialysis, the rats were euthanized, and parietal peritonea at liver and anterior abdominal wall were harvested. Peritoneum was analyzed by microscopic examination and immunohistochemistry for TGFβ1, α–smooth muscle actin (αSMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF).
Results: There was no significant differences between the groups in MAP and HR. In the PS group as compared with the vehicle group, 4-hour dialysate-to-plasma (D4/P4) BUN, D4/P4 glucose, and end dialysate–to–initial dialysate (D4/D0) glucose were decreased; serum TGFβ1 was increased; the submesothelial compact zone in liver and muscle peritoneum was markedly thicker; and expression of TGFβ1, αSMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum was increased. Aliskiren decreased serum TGFβ1, decreased the thickness of the submesothelial compact zone in liver and muscle peritoneum, and decreased the expression of TGFβ1, αSMA, collagen, and VEGF-positive cells in liver peritoneum. Moreover, D4/P4 BUN, D4/P4 glucose, and D4/D0 glucose were more increased, serum TGFβ1 was more decreased, the submesothelial compact zone in liver and muscle peritoneum was more decreased, and expression of TGFβ1, αSMA, fibronectin, collagen, and VEGF-positive cells was more decreased in the high-dose aliskiren group than in the low-dose aliskiren group.
Conclusions: Aliskiren protected against CHD–induced PS in rats.
Attenuation of Acute Peritonitis-Induced Functional and Structural Peritoneal Membrane Changes in Cav1-Deficient Mice
Objectives: Previous studies have demonstrated that in caveolin-1 (Cav-1)–dependent caveolae, invaginations of the plasma membrane do not influence baseline small molecular transport (SMT) and ultrafiltration (UF) in mice. However, we hypothesized that Cav-1 may play a role in acute peritonitis-induced changes of SMT and UF in mice.
Methods: Two groups of age- and sex-matched mice (Cav1–/– vs. Cav1+/+, n = 12×2) were investigated at baseline (n = 12) and 18 hours after induction of acute peritonitis (n = 12) using intraperitoneal lipopolysaccharide (LPS). Basic SMT [dialysate-to-plasma (D/P) Na, urea, and glucose] and UF were assessed using a 2-hour peritoneal equilibration test. We then measured NOx and white blood cells (WBCs) in 2-hour dialysate effluent. Visceral peritoneum was sampled for immunoblotting (AQP-1, Cav1), hematoxylin and eosin staining, and immunohistochemistry (ICAM-1).
Results: Expression of Cav-1 in peritoneum confirmed the model. Cav1–/– and Cav1+/+ mice had similar baseline SMT, UF, tissue staining, and AQP-1 expression. After acute peritonitis, Cav1+/+ mice, as compared with Cav1–/– mice, had a significantly greater increase in SMT [D/P urea 0.71 ± 0.03 vs. 0.58 ± 0.03, and end dialysate–to–initial dialysate (D/D0) glucose 0.47 ± 0.02 vs. 0.53 ± 0.02; both p < 0.05] in the early 30 minutes and a greater loss of Na sieving (–10.9% ± 1.1% vs. –6.1% ± 1.1%, p < 0.05) and UF (28 ± 1.4 μL/g vs. 45±2.1 μL/g, p < 0.05). Post-peritonitis dialysate NOx did not differ between the groups. Membrane and dialysate WBCs both were lower in Cav1–/– mice (tissue: 3.89 ± 0.50 vs. 8.94 ± 1.71 per vessel; dialysate: 23.8 ± 6.2 vs. 79.2 ± 15.2; p < 0.05 for both). Finally, peritoneal AQP-1 expression was higher and ICAM-1 protein levels were significantly lower in Cav1–/– mice.
Conclusions: We present novel data suggesting a detrimental role for Cav-1 in peritoneal morphologic and functional changes following acute peritonitis in mice.
A New Rat Model of Peritoneal Fibrosis by Erythromycin Lactobionate
Objectives: Peritoneal fibrosis (PF) is one of the most serious complications in patients on long-term peritoneal dialysis (PD). The causes of PF are not very clear, but evidence shows that bacterial peritonitis and incompatible dialysate both correlate with it. However, because of improvements in catheter implantation technique, the incidence of bacterial PF has decreased. Meanwhile, nonbacterial PF has recently increased. Therefore, a kind of animal model of non-bacterial PF in PD needs to be constructed to obtain more meaningful research results on nonbacterial PF. The aim of the present study was to establish a nonbacterial PF model in rats.
Methods: We divided 34 Sprague–Dawley (SD) rats into five groups: group 1, control (n = 7); group 2, normal saline (n = 7); group 3, 4.25% dextrose solution (n = 7); group 4, 4.25% dextrose solution with lipopolysaccharide (n = 6); group 5, 4.25% dextrose solution with lactobionate erythromycin (n = 7). Over a period of 5 weeks, the animals in each group received a daily infusion of the applicable dialysate. Rats were weighed at 0, 7, 14, 21, 28, 35 days before food intake and infusion. Animals were humanely killed at 5 weeks. A 2-hour peritoneal equilibration test (PET) was performed with 20 mL 2.5% dextrose dialysate, and dialysate-to-plasma ratio (D/P) of urea, glucose reabsorption ratio (D2/D0), net ultrafiltration (UF) volume, and levels of hydroxyproline and fibronectin were determined. Peritoneal membrane histology was evaluated by light microscopy.
Results: The D2/D0 ratio and net UF volume were significantly lower in group 5 than in groups 1 and 2 (p < 0.05), and the level of hydroxyproline and fibronectin were significantly higher in group 5 than in groups 1 and 2 (p < 0.05). In comparison with groups 1, 2, and 3, D/P urea and thickness of the peritoneal membrane in group 5 were significantly higher (p < 0.05). Moreover, the quantity of peritoneal vessels was significantly greater in group 5 than in groups 1 and 2 (p < 0.05).
Conclusions: These data suggest that 4.25% dextrose solution with lactobionate erythromycin can induce changes in peritoneal transport and morphology, establishing a new type of nonbacterial PF model in rats.
Effect of Various Concentrations of Peritoneal Dialysis Fluid on MCP-1 Expression by Peritoneal Mesothelial Cells in Continuous Ambulatory Peritoneal Dialysis Rats
Objectives: Monocyte chemotactic protein 1 (MCP-1) is a chemokine associated with increased synthesis of extracellular matrix (ECM) and accumulation of fibronectin (FN) that is involved in the process of inflammation. In vitro experiments have shown that high glucose (glu) induces MCP-1 and transforming growth factor β (TGFβ) secretion by peritoneal mesothelial cells (PMCs). Our study investigated the effects of various glu concentrations in peritoneal dialysis fluids (PDFs) on MCP-1 expression by PMCs and accumulation of FN in parietal peritoneum during short-term continuous ambulatory PD (CAPD) in rats.
Methods: We randomly divided 32 male Sprague–Dawley rats weighing 250 – 300 g into 4 groups (gps), with 8 rats in each gp. The rats were injected intraperitoneally with normal saline (NS) or with 1.5%, 2.5%, or 4.25% PDF daily for 2 weeks. On the day 5, after a 4-hour dwell, dialysate samples and serum were collected to measure MCP-1 protein (ELISA kit), and parietal and visceral peritoneum samples were taken to investigate mRNA expression of MCP-1 (in situ hybridization) and FN accumulation (immunohistochemistry).
Results: In PDF, MCP-1 protein was increased in the 4.25% gp as compared with the 1.5% and 2.5% gps (statistically nonsignificant); in the NS gp, it was significantly increased compared with the 3 experimental (exp) gps (F = 4.890, p = 0.007). Serum MCP-1 protein was not significantly different in any of the 4 gps (F = 0.763, p = 0.524). In the NS gp as compared with the 4.25% gp, mRNA expression of MCP-1 by PMCs was increased, and expression was higher in the 4.25% gp than in the 1.5% and 2.5% gps (statistically significant: F = 8.564, p = 0.001). In parietal peritoneal tissue, FN was also higher in the NS gp than in any experimental gp; second-highest was the 4.25% gp, in which FN was higher than in the 1.5% and 2.5% gps (statistically nonsignificant: F = 0.640, p = 0.590). The FN in PDF was positively correlated with expression of MCP-1 in PDF (r = 0.613, p = 0.002), and FN and MCP-1 mRNA also showed the same correlation (r = 0.438, p = 0.042).
Conclusions: Expression of MCP-1 in rat PMCs after short-term exposure to PDf increases as the glu concentration increases; NS could accelerate the synthesis of MCP-1. In short-term CAPD, high glu PDF (4.25%) has no effect on the components of ECM, such as FN. Accumulation of FN is associated with increased expression of MCP-1. The increases expression of MCP-1 may be one contributor to CAPD-related peritoneal sclerosis.
In Vitro and in Vivo Effects of Calcium on Human Peritoneal Mesothelial Cells
Objectives: Ca is an important solute in peritoneal dialysis fluid (PDF) and its effect on human peritoneal mesothelial cells (HPMCs) has not been investigated so far. This study tested the influence of Ca at various concentrations on HPMC proliferation, damnification, and interstitial fibrosis under in vitro and in vivo conditions.
Methods: HPMCs were cultured ex vivo and treated with Ca at various concentrations (2.0, 1.75, 1.25, 1.0, or 0 mmol/L). Proliferation of HPMCs was assessed by tetrazolium salt colorimetric assay, and lactate dehydrogenase (LDH) in the supernatant was detected to evaluate damage to HPMCs. Expression of fibronectin (FN) in cytoplasm was detected by immunohistochemistry. The study recruited 30 patients who had undergone peritoneal dialysis (PD) with conventional PDFs (1.25 mmol/L Ca) during the past 19.3 ± 10.35 months. Each patient's baseline at study entry was used as a control in comparison with the experimental groups (self and pre–post control). Subsequently, patients were treated with PDF containing 1.75 mmol/L Ca for 4 weeks (other treatments remained unchanged). At the end of each week of treatment, the patients were considered as a group (group I, II, III, IV respectively). ELISA was used to measure FN and LDH in peritoneal effluent. Cancer antigen 125 (CA125) was detected by electrochemiluminescence assay. Serum phosphate, Ca, and parathyroid hormone (PTH) were simultaneously determined.
Results: In vitro, Ca enhanced HPMC proliferation in a time-dependent manner, especially at 1.25 mmol/L and 1.0 mmol/L. Ca at various concentrations significantly increased LDH in HPMCs, also in a time-dependent manner, with the effect of Ca 1.25 mmol/L being lightest (p < 0.01). Ca at 2.0 mmol/L and 1.75 mmol/L significantly upregulated FN expression (p < 0.01). In vivo, differences between the 4 groups in LDH concentration in peritoneal effluent were all statistically significant and increased in a time-dependent manner. In peritoneal effluent, FN and LDH were notably higher in group IV than in other groups (p < 0.01), and CA125 was markedly lower (p < 0.01). Serum Ca in group IV declined markedly, and the differences of serum phosphonium and PTH were not statistically significant between groups.
Conclusions: In vivo and in vitro, high Ca (1.75 mmol/L) damaged HPMCs, inhibited their proliferation, and upregulated FN synthesized by HPMCs; a physiologic concentration of Ca (1.25 mmol/L) protected peritoneum and prevented peritoneal fibrosis.
Effect of Peritoneal Dialysis Fluids on ET-1 and ETR Expression in Continuous Ambulatory Peritoneal Dialysis Rats
Objectives: Endothelin-1 (ET-1), a 21-aminopeptide hormone, has potent vasoconstrictor activity that also induces production of extracellular matrix proteins and is involved in fibrotic processes in numerous organs. These ET-1 effects are elicited through the activation of two receptor subtypes, endothelin receptor A (ETAR) and endothelin receptor B (ETBR). The present study investigated the effect of peritoneal dialysis fluids (PDFs) on ET-1, ETAR, ETBR, and fibronectin (FN), and analyzed the relationships between those molecules in non infection situations to gain a better understanding of the mechanism of continuous ambulatory peritoneal dialysis (CAPD)–related peritoneal fibrosis.
Methods: We randomly divided 40 adult male Sprague–Dawley rats weighing 280 – 350 g into 4 groups that received intraperitoneal injections of 20 mL normal saline (NS) and 1.5%, 2.5%, and 4.25% PDF daily for 4 weeks. On the 29th day, after a 4-hour dwell, parietal peritoneal tissue was taken to determine the ET-1, ETAR, ETBR, and FN protein expression by immunohistochemistry.
Results: Expression of ET-1, ETAR, ETBR, and FN was higher in the experimental groups than in the control group, and the differences were significant. The ET-1, ETAR, ETBR, and FN protein expression were all highest in the 4.25% group, and the differences between the 4.25% group and the 2.5% and 1.5% groups were statistically significant (p < 0.05). Values were higher in the 2.5% group than in the 1.5% group, but the differences had no statistical significance (p > 0.05). FN protein expression was higher in the 4.25% group than in the 2.5% group, and expression was higher in the 2.5% group than in the 1.5% group; the differences between all 3 groups were statistically significant (p < 0.05). Expression of ET-1, ETAR, and ETBR were positively correlated with expression of FN (r = 0.960, p < 0.01; r = 0.908, p < 0.01; r = 0.934, p < 0.01).
Conclusions: Expression of ET-1, ETAR, ETBR, and FN in peritoneum of CAPD rats increase with the increase of glucose concentration in PDF. The change of ET-1, ETAR, and ETBR may cause accumulation of FN and induce CAPD-related peritoneal fibrosis.
Effects of Peritoneal Dialysis Fluids on VEGF Expression and Neoangiogenesis in Continuous Ambulatory Peritoneal Dialysis Rats
Objectives: Vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) play important roles in the process of angiogenesis. This study investigated the effects of PD fluids (PDFs) on production by peritoneal mesothelial cells (PMCs) of MCP-1 and VEGF in non infection situations and the relationship of those molecules with continuous ambulatory PD (CAPD)–related neoangiogenesis.
Methods: We randomly divided 50 adult male Sprague–Dawley rats weighing 280 – 350 g into 5 groups (gps) and dialyzed them with various solutions: control gp, no solution (n = 10); 1.5% gp, 1.5% glucose (glu) PDF (n = 10); 2.5% gp, 2.5% glu PDF (n = 10); 4.25% gp, 4.25% glu PDF (n = 10); and normal saline (NS) gp, 0.9% saline solution (n = 10). On day 28, after a 4hour dwell, dialysate samples and serum were collected to measure protein levels of MCP-1 and VEGF (ELISA kit) and, samples of parietal peritoneum were taken to determine MCP-1 and VEGF expression (immunohistochemistry).
Results: In parietal peritoneum, expression of MCP-1 and VEGF protein was significantly increased in the 4.25%, 1.5%, 2.5%, and NS gps as compared with the control gp (p < 0.05). Expression of MCP-1 and VEGF protein was higher in the 4.25% gp than in the 1.5% and 2.5% gps (statistically nonsignificant (p > 0.05). Protein levels of MCP-1 and VEGF in dialysate were increased in the 4.25% gp as compared with the control gp and the 1.5% and 2.5% gps (statistically significant (p < 0.05). Values were significantly increased in the NS, 1.5%, and 2.5% gps as compared with the control gp (p < 0.05), but the difference between the 1.5% and 2.5% gps was statistically nonsignificant (p > 0.05). Protein levels of MCP-1and VEGF in serum was not significantly different between any of the 5 gps (p > 0.05). Neovascularization was more prominent in the 4.25% gp than in the control, 1.5%, and 2.5% gps; levels in the NS gp were significantly increased as compared with the control gp (p < 0.05). Neovascularization was significantly increased in the 2.5% and 1.5% gps as compared with the control gp (p < 0.05). But the differences between the 1.5% and 2.5% gps had no statistical significance (p > 0.05). Protein expression of MCP-1 and VEGF in parietal peritoneum were directly correlated (r = 0.850, p < 0.01), and VEGF was positively correlated with MCP-1 in PDF (r = 0.810, p < 0.01).
Conclusions: PDF could induce increased protein expression of MCP-1 and VEGF by PMCs in CAPD rats. High glu dialysate in CAPD rats may lead to neoangiogenesis and ultrafiltration failure by promoting expression of MCP-1 and VEGF. Accumulation of VEGF is associated with increased expression of MCP-1. But how these molecules affect each other still need further study.
Expression of bFGF in Rat Peritoneum and Its Effect on Peritoneal Neoangiogenesis
Objectives: To investigate the effect of peritoneal dialysis (PD) solution on the expression of basic fibroblast growth factor (bFGF) in rat peritoneum and the relationship between bFGF and peritoneal neoangiogenesis.
Methods: Male Sprague–Dawley rats were divided into four groups in this study: normal rats (group 1, n = 8), uremia rats (group 2, n = 8), and dialyzed rats exposed either to 1.5% glucose PD solution (group 3, n = 8) or to 4.25% glucose PD solution (group 4, n = 8). The catheters implanted in the dialysis rats were tunneled subcutaneously to the neck and PD solution exchanges (20 mL per rat) were kept in the abdominal cavity for 2 hours each day. After regular PD for 28 days, expression of bFGF mRNA and protein in the peritoneal tissue were detected in each group by RT-PCR and immunohistochemistry respectively. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody.
Results: Expression of bFGF in peritoneal tissue was constituent in normal rats. Compared with group 1, bFGF mRNA expression and protein expression were both upregulated in groups 2, 3, and 4 (0.59 ± 0.02 vs. 0.70 ± 0.03, 0.59 ± 0.02 vs. 0.85 ± 0.03, 0.59 ± 0.02 vs. 0.91 ± 0.04 respectively, all p < 0.05). MVD was upregulated in groups 2, 3, and 4 as compared with group 1 (p < 0.05).
Conclusions: Uremia and unphysiologic PD fluid could increase bFGF mRNA and protein expression and MVD, which might participate in and have some effect on the course of peritoneal neoangiogenesis.
Expression of Endostatin in Rat Peritoneum and Its Effect on Peritoneal Neoangiogenesis
Objectives: To investigate the expression of endostatin (ES) in rat peritoneum and its possible effect on peritoneal neoangiogenesis.
Methods: Male Sprague–Dawley rats were divided into 4 groups: normal rats (group 1, n = 8), uremia rats (group 2, n = 8), and dialyzed rats exposed to 1.5% glucose PD solution (group 3, n = 8) or to 4.25% glucose PD solution (group 4, n = 8). The catheters implanted in dialysis rats were tunneled subcutaneously to the neck and PD solution exchanges, 20 mL per rat, were kept in the abdominal cavity for 2 hours daily. After regularly peritoneal dialysis for 28 days, expression of ES mRNA and protein in the peritoneal tissues in each group was detected by immunohistochemistry and RT-PCR respectively. Microvessel density (MVD) of the peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody.
Results: Expression of ES in peritoneal tissue was constituent in normal rats. Compared with group 1, ES protein expression was upregulated in groups 2, 3, and 4 (3.13 ± 1.13 vs. 5.13 ± 1.14, 3.13 ± 1.13 vs. 9.00 ± 1.51, 3.13 ± 1.13 vs. 10.75 ± 1.83 respectively, all p < 0.05), but ES mRNA expression was not significantly different between the various groups (p > 0.05). MVD was upregulated in groups 2, 3, and 4 as compared with group 1 (p < 0.05).
Conclusions: Uremia and unphysiologic peritoneal dialysis fluide increases ES protein expression and MVD, which might participate in and have some effect on the course of peritoneal neoangiogenesis.
Effect of Losartan on Extracellular Matrix Accumulation in Rats
Objectives: To investigate the protective effect of losartan on extracellular matrix accumulation and peritoneal function in a rat peritoneal fibrosis (PF) model induced by 4.25% peritoneal dialysis solution (PDS) and lipopolysaccharide (LPS).
Methods: We randomly divided 24 Wistar rats into three groups: a control group (n = 6), a PF group (n = 12), and losartan-treated (LT) group (n = 12). Rats in the PF and LT groups were injected intraperitoneally with 4.25% glucose PDS and LPS for 28 days. Rats in the LT group received losartan (20 mg kg–1 d–1) in addition to the 4.25% glucose PDS and LPS. At day 28, peritoneal tissue was dissected out, an accurate ultrafiltration volume was measured, and mass transfer of glucose out of the peritoneum was calculated. The thickness of peritoneal membrane stained with the Masson trichrome was measured in sections from the abdominal wall. The expression patterns of collagen I, collagen IV, and plasminogen activator inhibitor 1 (PAI-1) were examined by immunofluorescence and RT-PCR.
Results: In the control group, the peritoneal tissue consisted of a peritoneal mesothelial monolayer and exiguity of connective tissues. In the PF and LT groups, thickening of the peritoneum and accumulation of collagen fiber at day 28 was attenuated by losartan. Dialysate also played a role in influencing peritoneal ultrafiltration by increasing the glucose transport. Attenuation of ultrafiltration dysfunction was investigated in LT rats and was confirmed by decreased glucose transport in that group. Expression of collagen I, collagen IV and PAI-1 were markedly upregulated in the PF group, but similar results were not obtained in the LT group or the control group.
Conclusions: Losartan inhibits accumulation of extracellular matrix induced by 4.25% glucose PDS and LPS, and protects the peritoneum from ultrafiltration dysfunction.
Improved Model of Peritoneal Dialysis in Uremic Animal and Observation of Lymphatic Changes
Objectives: To develop an improved model of peritoneal dialysis (PD) in a uremic animal that mimics human PD, and to investigate abnormal changes of lymphatic vessels.
Methods: Male Sprague–Dawley rats were divided into four groups: normal rats (group 1), 5/6 nephrectomy uremic rats (group 2), and dialyzed uremic rats exposed to 4.25% glucose PD solution (group 3) or to 1.5% glucose PD solution (group 4). The catheters implanted in dialysis rats were tunneled subcutaneously to the back. Each rat underwent PD solution influx (30 mL) every 2 hours. In the first and fourth weeks of the study, serum creatinine, urea nitrogen, and albumin were detected in each group, and weight and ultrafiltration volume were recorded. All of the rats were humanely killed and their diaphragms taken to investigate ultrastructural changes of stomata by electronic scanning microscope. The diameters of the stomata and lymphatic vessels in the diaphragms were analyzed using the ImageTool software application (Roswell, GA, U.S.A.).
Results: All of the uremic rats undergoing PD could receive exchanges of PD solution repeatedly, complications being catheter obstruction or dislocation. The drainage volume in weeks 1 and 4 was similar for groups 3 and 4 (17.66 ± 21.81 mL vs. 12.33 ± 8.31 mL, 18 ± 13.84 mL vs. 17.3 ± 12.18 mL), and levels of serum creatinine, urea nitrogen, and albumin were similar in the two groups. The diameters of stomata were remarkably dilated in groups 2 – 4 as compared with group 1 (p < 0.05). The dilated lymphatic and sprouting lymphatic vessels found in groups 3 and 4 indicated lymphangiogenesis in the diaphragm in uremic rats undergoing PD.
Conclusion: This reliable model mimics human PD, with the catheter tunneled subcutaneously to the back to achieve daily PD solution drainage in a conscious uremic rat. Lymphangiogenesis was investigated in uremic rats undergoing PD, which may correlate with peritoneal membrane dysfunction in PD.
Valsartan and Spironolactone Inhibit Peritoneal Fibrosis Induced by High Glucose Dialysate in Mice
Objectives: The mechanism of peritoneal fibrosis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) is poorly elucidated. Transforming growth factor β (TGFβ) may be a key regulator in the progression of peritoneal fibrosis. This study investigated the mechanism of high glucose–induced TGFβ overexpression and the inhibitory effect of valsartan and spironolactone.
Methods: Glucose dialysis solution (4.25%) was intraperitoneally injected into mice with or without valsartan or spironolactone for 8 weeks. Expression of fibronectin and Smad7 were detected by Western blot analysis. Expression of TGFβ1 was analyzed by reverse transcription polymerase chain reaction and ELISA. DNA-binding activity of nuclear factor (NF) κB was detected by ELISA.
Results: TGFβ1 mRNA and protein expression were significantly upregulated by high glucose and suppressed by valsartan and spironolactone. Upregulated fibronectin and downregulated expression of Smad7 mRNA and protein were investigated in the 4.25% glucose group. NF-κB activity was similarly changed in the 4.25% glucose group. Valsartan and spironolactone increased expression of Smad7 and inhibited high glucose–induced NF-κB DNA binding and fibronectin expression.
Conclusions: These results suggest that high glucose induces peritoneal fibrosis mainly through TGFβ1/Smad7 by activating NF-κB, which was inhibited by valsartan and spironolactone. The renin–angiotensin–aldosterone system (RAAS) may be a target in preventing peritoneal fibrosis in CAPD.
Effect of Astragalus Injection on Aquaporin-1 Expression in Peritoneal Mesothelial Cells of Peritoneal Dialysis Rat
Objectives: To study the effect of Astragalus injection on Aquaporin-1 (AQP-1) expression in peritoneal mesothelial cells of peritoneal dialysis rat.
Methods: We divided 27 male Sprague–Dawley rats into three groups: normal control group, model group, and Astragalus group. The model and Astragalus groups received daily intraperitoneal injections of 25 mL 4.25% glucose peritoneal dialysate and 4.25% glucose peritoneal dialysate plus 20 mg/mL Astragalus respectively. After 10 days, the changes of dialysis ultrafiltration volume (UF, net UF), peritoneal transport of sodium (D/P Na), peritoneal pathology, and aquaporin-1 (AQP-1) immunohistochemistry under optical microscopy were recorded.
Results: The dialysis ultrafiltration volume increased significantly in the Astragalus group as compared with the model group (p < 0.05). When dialysate was left in the abdomen at 30 minutes and 60 minutes, the D/P Na declined in the Astragalus group as compared with the model group (p < 0.05). The peritoneal layer was thicker in the model group than in the Astragalus group, and the situation of detached mesothelial cells was worse. Expression of AQP-1 in peritoneal mesothelial cells increased as compare with that in the model group (p < 0.05).
Conclusions: Astragalus injection may reduce damage from high glucose peritoneal dialysis solution on mesothelial cells, may enhance AQP-1 expression in peritoneal mesothelial cells, and may improve the early inter-cell water transport during a dialysis exchange, thereby enhancing the peritoneal removal of water to increase the dialysis ultrafiltration volume.
Advanced Oxidation Protein Products Induced the Expression of Transforming Growth Factor β1 in Human Peritoneal Mesothelial Cells via Reactive Oxygen Species Generation
Objectives: To investigate the effect of advanced oxidation protein products (AOPPs) on the expression of transforming growth factor β1 (TGFβ1) in human peritoneal mesothelial cells (HPMCs) cultured in vitro, and the regulatory mechanism of endogenous reactive oxygen species (ROS) in this course.
Methods: The model of AOPP–human serum albumin (HAS) was prepared in vitro. The HPMCs were separated from peritoneal dialysis effluent and were primarily cultured. HPMCs were divided into time-dependence groups, dose-dependence groups, and drug groups. Protein levels of TGFβ1 were measured by ELISA. The mRNA expression of TGFβ1 was determined by RT-PCR. The intracellular generation of ROS was assessed using flow cytometry. HPMCs were pretreated with three different concentrations of vitamin E and N-acetylcysteine (NAC) to investigate the effect of antioxidants on the expression of TGFβ1 in HPMCs and the regulatory mechanism of endogenous ROS in this course.
Results: AOPP–HSA can significantly induce secretion and gene expression of TGFβ1 and also significantly increase intracellular ROS production in cultured HPMCs in a time- and dose-dependent manner (p < 0.01). The pretreatment of HPMCs with antioxidants vitamin E and NAC significantly reduced AOPP-induced ROS production and secretion and gene expression of TGFβ1 in cultured HPMCs in a dose-dependent manner (p < 0.01). The 50 μmol/L vitamin E groups and 10 mmol/L NAC groups had the strongest inhibition.
Conclusions: AOPP induces expression of TGFβ1 in HPMCs, which may be partially mediated by the ROS-dependent pathway. Antioxidant vitamin E and NAC may significantly inhibit gene expression and secretion of TGFβ1 by scavenging ROS in HPMCs. This study may provide a potential therapeutic strategy to prevent peritoneal fibrosis.
Influence of Peroxisome Proliferator–Activated Receptor γ Ligand on the Expression of Connective Tissue Growth Factor and Plasminogen Activator Inhibitor 1 in Rat Peritoneal Mesothelial Cells by High Glucose
Objectives: To investigate the influence of high glucose (glu) on the expression of peroxisome proliferator–activated receptor γ (PPARG) in rat peritoneal mesothelial cells (RPMCs) and the effect of PPARG ligands, pioglitazone, and 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2) on the production of connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1) induced by high glu.
Methods: RPMCs were divided into 4 groups: (1) treated with 0.1%, 1.5%, 2.5%, and 4.25% glu for 24 hours; (2) treated with 2.5% glu for 0, 6, 12, 24, 36, 48, and 72 hours; (3) treated with 0.1%, 1.5%, 2.5%, and 4.25% mannitol for 24 hours; (4) pretreated with pioglitazone (5 or 15 μmol/L) or 15d-PGJ2 (5 or 15 μmol/L) for 2 hours, and then treated with 2.5% glu for 24 hours. Expression of CTGF and PAI-1 mRNA was detected by RT-PCR. Expression of PPARG, CTGF and PAI-1 protein was detected by Western blot analysis.
Results: In normal conditions, RPMCs expressed PPARG. Glucose reduced expression of PPARG protein (p < 0.05 and p < 0.01) dose- and time-dependently The same concentration of mannitol had no effect on expression of PPARG protein (p > 0.05). Expression of CTGF mRNA and protein was significantly upregulated after stimulation with 2.5% glu (p < 0.01). Both 15d-PGJ2 and pioglitazone (5 and 15 μmol/L) reduced expression of CTGF mRNA and protein in a concentration-dependent manner (p < 0.05 and p < 0.01). Glucose (2.5%) increased expression of PAI-1 mRNA and protein (p < 0.01). 15d-PGJ2 (5 μmol/L) decreased expression of PAI-1 mRNA, but not protein. 15d-PGJ2 (15 μmol/L) decreased expression of PAI-1 mRNA and protein (p < 0.05 and p < 0.01). Pioglitazone (5 and 15 μmol/L) resulted in a concentration-dependent decrease in expression of PAI-1 mRNA and protein (p < 0.05 and p < 0.01).
Conclusions: High glu can regulate the expression of PPARG in RPMCs, which is not correlated with hyperosmosis. PPARG ligands can strongly inhibit the expression of CTGF and PAI-1 production in RPMCs and may prevent peritoneal fibrosis related to peritoneal dialysis.
Crosstalk between Smad and JNK Signaling Pathways in Transforming Growth Factor β Signal Transduction in Rat Peritoneal Mesothelial Cells
Objectives: Our previous results showed that JNK signaling may play an important role in epithelial–mesenchymal transition (EMT) induced by transforming growth factor β1 (TGFβ1) in rat peritoneal mesothelial cells (RPMCs). However, the interrelationship between the JNK and Smad pathways in the fibrotic response remains unknown. The goal of the present study was to examine the potential crosstalk between the Smad and JNK signaling pathways during TGFβ1-induced EMT in primary cultures of RPMCs.
Methods: RPMCs harvested from male Sprague–Dawley rats were cultured in DMEM/F12 medium and transiently transfected with a pcDNA3 construct expressing the dominant–negative Smad3 sequence (pcDNA3-Smad3M) or dominant-negative JNK1 gene (Ad-DN-JNK1) for 24 hours and then stimulated with addition of TGFβ1 at 10 ng/mL for different time intervals. The protein expression of phosphorylated JNK and Smad3 were detected by Western blot analysis. The intracellular distribution and expression of phosphorylated Smad3 was determined by immunofluorescence.
Results: TGFβ1 was able to activate the JNK pathway with the first peak at 10 minutes and the second peak at 8–16 hours. Interestingly, overexpression of dominant-negative Smad3 produced no effect on TGFβ–induced JNK activation at 10 minutes, but significantly suppressed the secondary peak at 12 hours by 72% ± 5%. In contrast, RPMCs infected with Ad-DN-JNK1 attenuated TGFβ–induced phosphorylation of Smad3 at 30 minutes by 55% ± 3%.
Conclusions: We conclude that there is a crosstalk between the Smad and JNK signaling pathways in TGFβ–induced response in RPMCs: TGFβ is able to induce an early and late activation of the JNK pathway; TGFβ–induced late activation of JNK is Smad3-dependent; Smad activation in response to TGFβ is partially mediated by JNK1.
Effects of High Glucose on the Expression of VEGF and Its Receptors in Rat Peritoneal Mesothelial Cells
Objectives: Vascular endothelial growth factor (VEGF) is a central mediator of vascular hyperpermeability and angiogenesis. it binds and activates two tyrosine kinase receptors: VEGFR1 and 2. High glucose in peritoneal dialysate may contribute to dysfunction of peritoneal membrane through an influence on angiogenesis. The present study investigated the effects of high glucose on VEGF and its receptors VEGFR1, VEGFR2, sVEGFR1 synthesis in rat peritoneal mesothelial cells (RPMCs).
Methods: Primary rat mesothelial cells were exposed to normal medium (control) or the same medium with glucose at 90 mmol/L (HG), 60 mmol/L (MG), or 30 mmol/L (LG) or mannitol at 90 mmol/L (HM). The mRNA expressions of VEGF, VEGFR1, VEGFR2, and sVEGFR1 were detected by RT-PCR. Protein expressions of VEGFR1 and VEGFR2 were detected by immunoblotting. The release of VEGF and sVEGFR1 was detected by ELISA.
Results: RPMCs in culture expressed VEGFR1, VEGFR2, and sVEGFR1. Following HG treatment, mRNA expressions of VEGF and VEGFR2 decreased, reaching the lowest level at 24 hours, in contrast to upregulation of VEGFR1 and sVEGFR1 that peaked at the same time point. After glucose stimulation, the mRNA expressions of VEGFR1 and sVEGFR1 were significantly higher in the MG and HG groups as compared with the control group (p < 0.01 and p < 0.05 respectively), but VEGFR2 mRNA was significantly lower in the MG and HG groups as compared with the control group (p < 0.01 respectively). Expression of VEGF mRNA was higher in the LG group, and significantly lower in the HG group (p < 0.05), than in the control group. The changes in VEGFR1 and VEGFR2 protein expression assessed by immunoblotting were consistent with the results generated with RT-PCR. Similarly, the changes in VEGF and sVEGFR1 protein were confirmed by ELISA.
Conclusions: RPMCs expressed VEGFR1, VEGFR2 and sVEGFR1. HG upregulated the expressions of VEGFR1 and sVEGFR1 but downregulated the expressions of VEGF and VEGFR2 at both the mRNA and protein levels. Therefore, HG distinctively affects the VEGF system in RPMCs.
Effects of p53 on High Glucose–Induced Apoptosis in Mouse Peritoneal Mesothelial Cells
Objectives: To investigate the effect of p53 on apoptosis and proliferation in mouse peritoneal mesothelial cells (MPMCs) caused by high glucose in vitro.
Methods: MPMCs obtained from p53+/+ and p53–/– mouse omental tissue were cultured in RPMI 1640 medium. The cells were exposed for 3 days to RPMI 1640 medium containing: (1) 4.25% glucose (high glucose); (2) pifithrin-α (PFTα), a specific p53 inhibitor (50 μmol/L) in p53+/+ cells; (3) no additions (control). Apoptosis and proliferative cell nuclear antigen (PCNA) were measured by terminal deoxynucleotidyl transferase (TdT)–mediated in situ end-labeling and immunohistochemistry. Protein expression of p53 was measured by flow cytometry.
Results: Apoptotic cells were significantly increased in the high glucose group as compared with control (103 ± 62 cells per field unit vs. 57 ± 28 cells per field unit in p53+/+ cells, 56 ± 25 cells per field unit vs. 17 ± 9 cells per field unit in p53–/– cells, p < 0.01), and apoptotic cells were distinctly increased in p53+/+ cells as compared with p53–/– cells in high glucose (p < 0.01). After treatment with PFTα in p53+/+ cells, the apoptotic cells were distinctly decreased in the high glucose and control groups (62 ± 24 cells per field unit and 21 ± 7 cells per field unit respectively, p < 0.01). Cells with positive PCNA expression were fewer in the high glucose group than in the control group (69 ± 38 cells per field unit vs. 105 ± 50 cells per field unit in p53+/+ cells, 102 ± 56 cells per field unit vs. 166 ± 55 cells per field unit in p53–/– cells, p < 0.01), and the PCNA-positive cells were distinctly decreased in p53+/+ cells as compared with p53–/– cells in high glucose (p < 0.01). After treatment with PFTα in p53+/+ cells, PCNA-positive cells were distinctly increased in the high glucose and control groups (121 ± 62 cells per field unit and 153 ± 77 cells per field unit respectively, p < 0.01). In p53+/+ cells, expression of p53 was stimulated by high glucose (51% ± 22% vs. 23% ± 13% in the control group, p < 0.01), and PFTα could significantly inhibit expression of p53 (11% ± 7% in the high glucose group vs. 6% ± 3% in the control group, p < 0.01). Apoptotic cells were positively correlated with expression of p53 (r = 0.71, p < 0.01), and PCNA-positive cells were negatively correlated with expression of p53 (r = –0.68, p < 0.01).
Conclusions: Our data suggested that high glucose induced apoptosis and inhibited proliferation of MPMCs and stimulated the expression of p53 in MPMCs in vitro. p53 plays an important role in triggering apoptosis and inhibiting proliferation of MPMCs.
High Glucose and Glucose Degradation Products Inhibit Proliferation and Downregulate Angiopoietin 1 Expression in Vascular Pericytes
Objectives: To investigate the effect of high glucose and glucose degradation product (GDP) on proliferation and angiopoietin 1 (Ang1) expression in cultured vascular pericytes (VPs), thus helping to understand the potential mechanisms of peritoneal angiogenesis in patients undergoing long-term peritoneal dialysis.
Methods: Cultured VPs were incubated with serum-free media (control), media containing 1.5%, 2.5%, and 4.25% glucose (G groups), media containing mannitol at the same concentrations (M groups), or media containing 400 μmol/L or 800 μmol/L methylglyoxal (MGO groups). Proliferation of VPs was assessed by MTT assay. Expression of Ang1 mRNA and protein in VPs was detected by real-time RT-PCR amplification and ELISA respectively.
Results: After stimulation for 12 hours, 24 hours, 48 hours, and 72 hours, proliferation of VPs was significantly inhibited in 4.25% G, 4.25% M, and 800 μmol/L MGO as compared with control (p < 0.01). A trend of inhibition occurred with 2.5% G, but did not reach statistical significance. Lower concentrations of G, M, and MGO did not result in inhibition. Inhibition effect of 4.25% G was stronger than that of 4.25% M (p < 0.05). As compared with control, 2.5% G and M, 4.25% G and M, and 400 μmol/L and 800 μmol/L MGO downregulated Ang1 mRNA expression in VPs (p < 0.01). After incubation for 12 hours (as compared with 6 hours), Ang1 mRNA expression in each group showed a trend of upregulation that did not reach statistical significance. Also, as compared with control, either 2.5% or 4.25% G or M, and 400 μmol/L or 800 μmol/L MGO, resulted in a significantly decreased Ang1 protein level (p < 0.01). After incubation for 12 hours (as compared with 6 hours), Ang1 protein expression was significantly increased (p < 0.01). G showed stronger effect on decrease of Ang1 expression in cultured VPs than did M at the same concentrations (p < 0.05).
Conclusions: Both high G and high GDP concentrations reduce VP proliferation and Ang1 expression, and the inhibition effect of high G occurs partially through its hyperosmolarity. The results suggested that bioincompatible factors of conventional PD solution such as high G, high GDP, and hyperosmolarity may contribute to peritoneal angiogenesis by inhibiting proliferation of VPs and downregulating Ang1 expression in pericytes.
Effect of Glucose on Intercellular Adherens Junctions of Human Peritoneal Mesothelial Cells
Objectives: The mechanism of peritoneal mesothelial cell exfoliation after exposure to dialysis solution has not been thoroughly investigated. This study describes the effect of high glucose on intercellular adherens junctions of cultured human peritoneal mesothelial cells (HPMCs) to clarify the cause of decreased dialysis efficacy during prolonged peritoneal dialysis.
Methods: All tests were performed on the HPMC line HMrSV5. The cells were divided into 3 groups: normal glucose group (5.5 mmol/L d-glucose); (2) high glucose group (140 mmol/L d-glucose); and (3) high osmolality group (140 mmol/L mannitol). All groups were observed at 6, 12, 24, and 48 hours. Expression of E-cadherin and β–catenin were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), Western Blot analysis, and immunofluorescence. High glucose–induced actin disassembly was studied by a fluorescence labeling technique.
Results: High glucose caused a time-dependent increase in cell detachment. The mRNA and protein levels of intercellular adherens junctional proteins (E-cadherin and β–catenin) decreased, and immunostaining by anti–E-cadherin and anti–β–catenin antibodies became weak and often discontinuous along the cell contour. The result also showed that, in HPMCs incubated in high glucose, F-actin bundles appeared less prominent and changed to an irregular network. At the same osmolality, mannitol had no effect on E-cadherin/β–catenin/actin complex.
Conclusions: These findings suggest that the peritoneal mesothelium is damaged by prolonged peritoneal dialysis using high glucose dialysate and that impairment of the intercellular adherens junction of HPMCs by high glucose plays an important role in cell detachment and progressive reduction in dialysis efficacy.
Effects of TNFα on Expression of Matrix Metalloproteinases and Their Inhibitors in Human Peritoneal Mesothelial Cells
Objectives: Our previous study found that tumor necrosis factorα (TNFα) could induce epithelial–mesenchymal transition of human peritoneal mesothelial cells (HPMCs). This study investigated the effects of TNFα on the expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMP) in HPMCs, and also investigated the effects of treatment of TNFα+transforming growth factor β1 (TGFβ1), TNFα+TGFβ1+interleukin 1 (IL-1), or TNFα alone on MMP-9 activity in HPMCs.
Methods: After 24-hour stimulation by various concentrations of cytokines (TNFα, TGFβ1, IL-1), serum-free conditioned HPMC culture supernatants and the HPMCs were collected. The mRNA expression of MMP-2, MMP-9, TIMP-2, and TIMP-1 was detected by semi-quantitative RT-PCR. The activity of active and pro–MMP-9 in the conditioned media was quantitatively measured by Biotrak MMP-9 activity assay system (GE Healthcare, Piscataway, NJ, U.S.A.). Collagen I protein level was examined by ELISA.
Results: In vitro with treatment of TNFα, expression of MMP-9 mRNA in HPMCs was significantly upregulated in a dose- and time-dependent manner (2.3 – 4.9 times the base value, p < 0.05), and expression of TIMP-1 mRNA and TIMP-2 mRNA was slightly downregulated (77.2% and 61.3% of base value, p < 0.05); however, expression of MMP-2 mRNA did not change obviously. Activity and secretion of MMP-9 in the conditioned cell supernatant increased significantly with treatment of TNFα+TGFβ, TNFα+TGFβ+IL-1 or TNFα alone. Collagen I protein expression of HPMCs increased significantly with 24-hour stimulation of TNFα.
Conclusions: The increase of MMP-9 activity and expression induced by TNFα alone and TNFα with other cytokines may play a role in peritoneal fibrogenesis.
Tgfβ1 and Epithelial–Mesenchymal Transition of Human Peritoneal Mesothelial Cells
Objectives: Many studies showed that transforming growth factor β1 (TGFβ1) is a pleiotropic cytokine, possessing not only inflammatory activity, but also fibrogenetic activity. It can be produced by peritoneal mesothelial cells and resident peritoneal macrophages when peritoneal inflammation occurs. The present study investigated the effects of TGFβ1 on epithelial–mesenchymal transition in human peritoneal mesothelial cells (HPMCs).
Methods: All tests were performed on the HPMC line HMrSV5. Treatment groups were stimulated with TGFβ1 (1 – 10 ng/mL, separately). Morphology changes in HPMCs were observed by phase-contrast microscopy. The expression of cytokeratin 8, cytokeratin 18, and fibroblast-specific protein 1 (FSP-1) was detected by immunocytochemistry. Meanwhile, mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was detected by semiquantitative RT-PCR. Production of MMP-9 and type I collagen was measured by enzyme-linked immunosorbent assays.
Results: TGFβ1 (10 ng/mL) induced morphology change in about 50% – 60% of HPMCs. The original polygonal cobblestone monolayer was changed into the elongated spindle shape characteristic of fibroblasts. The original strong positive expression of cytokeratin 8 and cytokeratin 18 changed to negative in all morphologically changed HPMCs, but expression of FSP-1 was positive. After 48 hours of treatment with TGFβ1 (10 ng/mL), mRNA expression of MMP9 was significantly upregulated, and mRNA expression of TIMP-1 and TIMP-2 was downregulated. We also observed that production of MMP-9 and type I collagen increased significantly after 48 hours of treatment with TGFβ1 (10 ng/mL).
Conclusions: TGFβ1 induced epithelial–mesenchymal transition of HPMCs and upregulated the synthesis of MMP-9 and type I collagen, which may facilitate peritoneal extracellular matrix remodeling and fibrogenesis.
A Selective Cyclooxygenase-2 Inhibitor Decreases Connective Tissue Growth Factor Synthesis and Matrix Production in Human Peritoneal Mesothelial Cells
Objectives: To investigate the effects of sc58236, a highly selective cyclooxygenase-2 (COX-2) inhibitor, on connective tissue growth factor (CTGF) expression and matrix production in human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs were incubated in medium F12 with a high concentration of d-glucose (4.25%) in the presence and absence of 20 μmol/L sc58236. The mRNA expression of COX-2, CTGF, and fibronectin (FN) were determined by semi-quantitative RT-PCR. Prostaglandin E2 (PGE2) concentration in the culture medium was measured by ELISA. Expression of CTGF, COX-2, and fibronectin (FN) protein were determined by Western blot analysis.
Results: Expression of PGE2 was obviously upregulated after stimulation with 4.25% d-glucose (p < 0.01). Addition of 20 μmol/L sc58236 significantly inhibited PGE2 release into the culture medium (p < 0.05). Expression of CTGF and FN mRNA and protein were significantly increased in HPMCs stimulated with 4.25% d-glucose as compared with controls in F12 medium (p < 0.01), which was reversed in the presence of sc58236 (20 μmol/L). Obvious decrease of CTGF mRNA expression and protein were found in sc58236-treated groups as compared with the group stimulated with 4.25% d-glucose (p < 0.05). Exposure of HPMCs to sc58236 reduced FN secretion (p < 0.05).
Conclusions: Sc58236 reduces the expression of CTGF in HPMCs stimulated with 4.25% d-glucose and reduces ECM production through PGE2 production. This study suggests that sc58236, a highly selective cyclooxygenase-2 (COX-2) inhibitor, may have a specific role in ameliorating the course of progressive peritoneal fibrosis under long-term peritoneal dialysis.
Astragalus mongholicus Altered the Secretion and Expression of Transforming Growth Factor β1 and Basic Fibroblast Growth Factor in Cultured Human Peritoneal Mesothelial Cells
Objectives: To investigate the effect of Astragalus mongholicus (AM) injection on the secretion and mRNA expression of transforming growth factor β1 (TGFβ1) and basic fibroblast growth factor (bFGF) in cultured human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs obtained from patients undergoing surgery were cultured in vitro. Cells of the third passage were incubated with RPM I1640 culture medium containing 0.1% fetal bovine serum (FBS) for 24 hours and then divided into 5 groups: control group, peritoneal dialysis solution (PDS) group, AM group 1, AM group 2, and AM group 3. Each group was supplemented with an equal volume of RPM I1640 culture medium containing 20% FBS. After 24 hours of incubation, mitochondrial dehydrogenase activity (MTT assay), levels of TGFβ1 and bFGF in cell culture supernatant, and mRNA expression of TGFβ1 and bFGF were detected.
Results: Significant decreased mitochondrial dehydrogenase activity was observed in the PDS group as compared with the control and AM groups (p < 0.05). No significant difference in mitochondrial dehydrogenases activity was found among the AM groups (p > 0.05). The levels of TGFβ1 and bFGF in cell culture supernatant were significantly lower in the control group than in the PDS group. Markedly lower levels of TGFβ1 and bFGF were found in AM group 1, AM group 2, and AM group 3 than in the PDS group (p < 0.05). The level of TGFβ1 and bFGF were significantly higher in AM group 1 than in AM groups 2 and 3 (p < 0.05). The mRNA expressions of TGFβ1 and bFGF in the PDS group increased significantly as compared with those in the control group. And significant mRNA expression of TGFβ1 and bFGF was found in the PDS group as compared with the AM groups (p < 0.05).
Conclusions: The secretion and mRNA expression of TGFβ1 and bFGF induced by commercial PDS can be reduced by AM in cultured HPMCs.
Ctgf Knockdown Attenuated Matrix Protein Production and Vegf Expression Induced by Tgfβ1 in Cultured Human Peritoneal Mesothelial Cells
Objectives: Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor β1 (TGFβ1), has recently been implicated in peritoneal fibrosis. Extracellular matrix (ECM) accumulation and angiogenesis are characteristic changes in peritoneal fibrosis. In this study, we investigated the effect of CTGF knockdown by RNA interference (RNAi) on ECM production and VEGF expression induced by TGFβ1 in human peritoneal mesothelial cells (HPMCs).
Methods: Four CTGF short hairpinRNA (shRNA) expression constructs were generated using pRetroSuper(PRS) vector, and infectious retroviral particles were prepared for infection of HPMCs. Expression levels of CTGF, fibronectin (FN), collagen I (Col I), laminin, and vascular endothelial growth factor (VEGF) mRNA and protein were measured by semi-quantitative RT-PCR and Western blot assay respectively.
Results: CTGF expression was increased after stimulation with TGFβ1, but was inhibited by each of the four independent CTGF shRNA constructs (p < 0.01). Moreover, expression of ECM proteins (FN, Col I, laminin) and VEGF were upregulated after incubation with TGFβ1, but elevated levels of ECM and VEGF induced by TGFβ1 were significantly inhibited by RNAi (p < 0.01), although not by the empty retroviral vector (p > 0.05).
Conclusions: From these results, we concluded that retrovirus-mediated CTGF siRNA can effectively inhibit ECM production and VEGF expression induced by TGFβ1 in HPMCs. By decreasing ECM accumulation and angiogenesis, downregulation of CTGF may represent a potential therapeutic approach for peritoneal fibrosis.
Effect of Indomethacin, Sodium Ferulate, and Glucocorticoid on Expression of Fibronectin and Plasminogen Activator Inhibitor 1 in Cultured Human Peritoneal Mesothelial Cells
Objectives: To investigate the mechanism of peritoneal fibrosis induced by high glucose, and the effect of indomethacin, sodium ferulate, and glucocorticoid on high glucose–induced fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) secretion in cultured human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs were cultured in vitro and incubated in 2.5% glucose with various concentrations of indomethacin, sodium ferulate, and dexamethasone. The levels of FN and PAI-1 protein in the supernatant were determined by enzyme-linked immunosorbent assay. RT-PCR was used to detect expression of FN and PAI-1 mRNA in HPMCs.
Results: FN and PAI–1 protein was significantly increased in supernatant of HPMCs exposed to high glucose as compared with controls (p < 0.01). Expression of FN and PAI-1 mRNA was also upregulated in the high glucose group. The various concentrations of indomethacin, sodium ferulate, and dexamethasone inhibited FN and PAI-1 secretion and expression in a dose-dependent manner (p < 0.05).
Conclusions: High glucose increased secretion of FN and PAI-1 and upregulated expression of FN and PAI-1 mRNA in HPMCs. However, indomethacin, sodium ferulate, and dexamethasone protected HPMCs by reducing secretion and expression of FN and PAI-1 induced by high glucose.
Effect of Peritoneal Dialysis Solution on Connective Tissue Growth Factor in the Peritoneum of Peritoneal Dialysis Rats
Objectives: To investigate the effect of peritoneal dialysis fluid (PDF) on connective tissue growth factor (CTGF) expression and extracellular matrix (ECM) synthesis in the peritoneum of peritoneal dialysis rats.
Methods: Sprague–Dawley rats were randomized into four groups: control group, 0.9% saline solution (NS) group, 1.5% dextrose group, and 4.25% dextrose group. Peritoneal equilibration tests (PETs) were performed and ultrafiltration (UF) was measured 4 weeks later. Dialysate-to-plasma ratio (D/P) of creatinine (Cr), glucose reabsorption (D/D0 glucose), UF, and peritoneal thickness were measured. Expression of transforming growth factor β1 (TGFβ1), connective tissue growth factor (CTGF), and fibronectin (FN) mRNA and protein were determined by semi-quantitative RT-PCR and immunohistochemistry respectively.
Results: The UF volume was lower in the 1.5% and 4.25% dextrose groups than in the control and NS groups (p < 0.05), and lower in the 4.25% dextrose group than in the 1.5% dextrose group (p < 0.05). The D/P Cr was higher in the 4.25% dextrose group than in the control and NS groups (p < 0.05), but D/D0 glucose was lower in the 1.5% and 4.25% dextrose groups than in the control and NS groups (p < 0.05). Total collagen content in peritoneal membrane increased after stimulation with glucose (p < 0.05). Expression of TGFβ1, CTGF, and FN mRNA and protein were also upregulated by glucose in a dose-dependent manner.
Conclusions: Glucose-containing PDF can upregulate expression of TGFβ1, CTGF, and FN mRNA and protein, suggesting that high glucose PDF contributes to UF failure and peritoneal fibrosis.
Effect of TGFβ1 Antisense Oligodeoxynucleotides on Proliferation and Fibronectin Expression in Human Peritoneal Mesothelial Cells
Objectives: To investigate the effect of transforming growth factor α (TGFβ1) antisense oligonucleotides (ASON) on proliferation of human peritoneal mesothelial cells (HPMCs) and expression of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) in HPMCs.
Methods: HPMCs were isolated from human omenta by the trypsinization method and cultured with F12 medium. HPMCs were identified by invert, transmission electron, and scanning electron microscope and by immunohistochemistry. Cultured HPMCs were treated with F12/serum-free (control group), TGFβ1 sense oligodeoxynucleotides (sense group), TGFβ1 ASONs (antisense group), and TGFβ1 missense oligonucleotides (missense group). Lipofectamine 2000 was used for transfection. The levels of FN, PAI-1, and basic fibroblast growth factor (bFGF) mRNA expression were measured by RT-PCR. ELISA was used to detect expression of FN and PAI-1 protein
Results: At 24 and 48 hours, mRNA expression of FN, PAI-1, and bFGF was significantly increased in the TGFβ1 sense group as compared with control (p < 0.05). However, FN expression was significantly downregulated in the antisense group as compared with the other groups (p < 0.05). Proliferation of HPMCs was also inhibited in the TGFβ1 ASON group at 24 and 48 hours.
Conclusions: TGFβ1 ASONs can inhibit HPMC proliferation and overexpression of FN and PAI–1.
Effects of High Glucose on Human Peritoneal Mesothelial Cells: A Proteomic Analysis
Objectives: Ultrafiltration failure continues to be a major complication of long-term peritoneal dialysis (PD). Continuous exposure to bioincompatible PD solutions causes inflammation of the peritoneal membrane, which progressively undergoes fibrosis (PF) and angiogenesis, ultimately leading to ultrafiltration failure. The underlying mechanism is not completely understood, however. Proteomic tools offer a high-throughput analysis of protein expression in biologic samples. The global analysis of protein in human peritoneal mesothelial cells (HPMCs) cultured with glucose-containing PD fluid (PDF) will provide a better understanding of the effects of biocompatible PDF on HPMCs.
Methods: To study the effect of glucose-containing PDF on proteins secreted by HPMCs. HPMCs were isolated from human omenta by the trypsinization method and were grown in DMEM/F12 medium containing 4.25% glucose for 24 and 48 hours. HPMCs treated with DMEM/F12 served as a control. The proteomic analysis used two-dimensional gel electrophoresis and PDquest picture analysis software (Bio-Rad Laboratories, Hercules, CA, U.S.A.). Matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) identifies the differential expression of proteins.
Results: As compared with controls, HPMCs incubated with high glucose (HG) for 24 and 48 hours showed differential expression of 27 and 50 protein spots respectively (p < 0.05). Upregulated expression was indicated by 22 protein spots, and downregulated expression, by 50 protein spots. Of the differential protein spots, 23 analyzed by peptide mass finger (PMF), including cofilin 1, aldose reductase, glyceraldehyde-3-phosphate dehydrogenase, interleukin 1 family number 8 (IL-1F8), galectin 1, manganese superoxide dismutase (Mn-SOD), and surfactant, among others. These were involved in the regulation of cytoskeletal, signal transduction, glucose metabolism, cytokine expression, adhesion, surfactant, and antioxidative pathway, among other functions.
Conclusions: Our data suggest that HG may induce PF by altering various types of protein expression and may provide a better understanding of the mechanisms involved in PF. Moreover, these observations might be novel findings leading to the prediction of PF.
Effects of Simvastatin on High Glucose–Induced Type I Collagen, MMP-1 and TIMP-1 Expression in Cultured Human Peritoneal Mesothelial Cells
Objectives: In long-term continuous ambulatory peritoneal dialysis (CAPD) patients, peritoneal fibrosis (PF) is a serious complication characterized by extracellular matrix (ECM) accumulation in peritoneal mesothelial cells. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by human peritoneal mesothelial cells (HPMCs) play important roles in the process of balance between synthesis and degradation of ECM components. Previous studies have demonstrated that high glucose (HG) not only stimulates the synthesis of ECM, but also attenuates its degradation by decreasing MMP expression and increasing TIMP expression in HPMCs. This study investigated the effects of simvastatin on type I collagen, one of the major ECM components in PF, together with expression of MMP-1 and TIMP-1 in HPMCs cultured under HG conditions.
Methods: HPMCs were isolated from human omenta by the trypsin digestion method and subcultured. After synchronization of cell growth, HPMCs were divided into a normal control group, a HG group, and HG plus low-, medium-, and high-dose simvastatin (2.5 μmol/L, 5 μmol/L, and 10 μmol/L respectively) groups. Semi-quantitative RT-PCR was used to detect the expression of type I collagen, MMP-1, and TIMP-1 mRNA in HPMCs. The protein levels of type I collagen, MMP-1, and TIMP-1 in culture supernatants were determined by ELISA. Protein concentration in cells was measured by the trace bicinchoninic acid method to correct the ELISA results.
Results: Simvastatin significantly attenuated HG-induced type I collagen and TIMP-1 expressions in a dose-dependent manner at both the protein and gene levels (p < 0.01). In addition, high-dose treatment with simvastatin significantly increased MMP-1 expression that was inhibited by HG (p < 0.01).
Conclusions: These data demonstrate that simvastatin, an HMG–coenzyme A reductase inhibitor, not only decreases HG-induced type I collagen synthesis, but also promotes its degradation by modulating unbalanced MMP-1/TIMP-1 expression in HPMCs cultured in HG.
High Glucose Induces TGFβ1 Secretion by Human Peritoneal Mesothelial Cells via Reactive Oxygen Species Signaling Pathway
Objectives: This study investigated the effect of high glucose (HG) on generation of reactive oxygen species (ROS) and secretion of transforming growth factor β1 (TGFβ1) in cultured human peritoneal mesothelial cells (HPMCs). We applied various concentrations of antioxidant N-acetylcysteine (NAC) to pretreat HPMCs before addition of HG to explore whether HG induces TGFβ1 secretion by HPMCs via the ROS signaling pathway.
Methods: HPMCs were isolated from omental specimens by trypsin disaggregation, and a stable culture model was established. The cells were divided into dose-dependent groups (control and 1.5%, 2.5%, and 4.25% glucose), each cultured for 24 hours; time-dependent groups (control group, and 1.5% glucose) cultured for 12, 24, and 48 hours; pretreated groups (control and 0.1 mmol/L, 1.0 mmol/L, and 10 mmol/L NAC), pretreated for 1 hour followed by incubation in 1.5% d-glucose with DMEM for 24 hours. In each sample, TGFβ1 in supernatant was measured by ELISA. Intracellular ROS of each sample was measured using fluorescence microscopy and a multifunctional microplate reader to detect relative fluorescence intensity of dichlorofluorescein.
Results: HG significantly induced secretion of TGFβ1 in both a dose- and time-dependent manner in cultured HPMCs (p < 0.05), with the 4.25% glucose and 48-hour incubation groups being the most significant. HG also significantly stimulated ROS generation in a dose- and time-dependent manner (p < 0.05), with the 4.25% glucose and 48-hour incubation being the most significant. Compared with control, pretreatment of HPMCs at three different NAC concentrations inhibited HG-induced ROS generation and TGFβ1 secretion in vitro (p < 0.05). The most significant effect was observed in the 10 mmol/L NAC group.
Conclusions: HG induced increased TGFβ1 secretion in cultured HPMCs, which could be inhibited by antioxidant. In addition, HG stimulated ROS generation, resulting in oxidative damage in cultured HPMCs, an effect that could also be inhibited by antioxidant. Our findings suggested that HG induces TGFβ1 secretion by HPMCs partly via the ROS signaling pathway, which could be inhibited by antioxidant.
Inhibition of CTGF Expression in Human Peritoneal Mesothelial Cells by pcDU6 Vector–Mediated CTGF shRNA
Objectives: To investigate the effect of connective tissue growth factor (CTGF) short hairpin RNA (shRNA) mediated by pcDU6 plasmid on CTGF expression in human peritoneal mesothelial cells (HPMCs).
Methods: Using 2 selected fragments of a coding sequence containing 21 nt of the CTGF sequence starting with GGCC, two pairs of oligos were designed for the 2 fragments. After annealing, double-stranded DNA was formed and ligated separately to plasmid pcDU6 [pcDNA3.1(–) with U6 promoter]. The inverted motif contained 6 spacers and four Ts, which made it possible to form shRNAs called CTGF shRNA1 and CTGF shRNA2. HPMCs were isolated from human greater omentum by pancreatin disaggregation to establish a stable cell culture model. Plasmid pcDU6 mediating the expression of CTGF was transfected with lipofectamine 2000 into the third-passage HPMCs and the resulting transfected cells were stimulated with 4.25% d-glucose and 10 μg/mL lipopolysaccharide (GS+LPS). Semi-quantification RT-PCR was performed to detect the expression of CTGF. Protein levels of CTGF in the culture supernatant were measured with a sandwich ELISA.
Results: Expression of CTGF was significantly upregulated in HPMCs stimulated with GS+LPS (p < 0.01). Expression of CTGF was obviously downregulated in the pcDU6 plasmid vector–mediated CTGF shRNA groups as compared with the GS+LPS group and the pcDU6 void vector group (p < 0.01), with no significant difference among pcDU6 plasmid vector–mediated CTGF shRNA groups (p > 0.05). No significant difference was found between pcDNA3.1(–) vector plasmid–mediated antisense RNA group and pcDU6 void vector group (p > 0.05). The expression of CTGF was obviously downregulated in pcDU6 plasmid vector–mediated CTGF shRNA groups as compared with the pcDNA3.1(–) plasmid vector–mediated antisense RNA group (p < 0.01).
Conclusions: PcDU6 plasmid vector–mediated shRNA can significantly inhibit the expression of CTGF induced by GS+LPS in HPMCs. These results suggest the possible application of pcDU6 plasmid vector–mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.
Inhibition of CTGF Expression in Mouse Peritoneal Mesothelial Cells by Nano-Carrier PAMAM–Mediated pCTGF–shRNA
Objectives: To investigate the effects of connective tissue growth factor (CTGF) short hairpin RNA (shRNA) mediated by nano-carrier G9 polyamidoamine (PAMAM) on the expression of fibronectin (FN) and transforming growth factor β1 (TGFβ1) in primary mouse peritoneal mesothelial cells (MPMCs).
Methods: The CTGF shRNA1, CTGF shRNA2, and negative control shRNA were designed and ligated into pGenesil-1 plasmid. Transfection of these plasmids to MPMCs was mediated by G9 PAMAM nano-carrier. Then, 4.25% glucose (GS) was used as a stimulant to transfected MPMCs with CTGF shRNA1, CTGF shRNA2, and negative control shRNA mediated by nano-carrier. Untransfected MPMCs served as a control. The levels of CTGF, FN, and TGFβ1 mRNA and protein were detected by RT-PCR and Western blot analysis respectively.
Results: Expression of CTGF, FN, and TGFβ1 in MPMCs was significantly upregulated with GS incubation. Introduction of pCTGF–shRNA1 or pCTGF–shRNA2 could significantly inhibit high levels of CTGF and FN expression induced by GS (p < 0.05), but had no effect on TGFβ1 expression. Inhibition was more effective in the pCTGF2-shRNA group; pHK–shRNA did not have this effect.
Conclusions: Nano-carrier G9 PAMAM has the ability to efficiently transfect pCTGF–shRNA into primary MPMCs, which leads to a prominent inhibition effect on increased expression of CTGF and FN induced by high glucose. PAMAM-mediated pCTGF–shRNA could therefore be used as an efficient strategy in gene therapy of peritoneal fibrosis.
Protective Effect of Ginsenosides, Shen Mai, and Huang Qi Injection on Human Peritoneal Mesothelial Cells against Lactate-based Peritoneal Dialysis Solution
Objectives: To investigate the role of lactate-buffered peritoneal dialysis solution (L-PDS) on the function of human peritoneal mesothelial cells (HPMCs), and to study the protective effect of ginsenosides, shen mai, and huang qi injection on the cytotoxicity of L-PDS to HPMCs.
Methods: Isolated HPMCs were cultured in vitro and stimulated by various concentrations of ginsenosides, shen mai, and huang qi injection. The viability of HPMCs was assessed by lactate dehydrogenase (LDH) release; capacity of proliferation was examined by MTT assay.
Results: Levels of LDH in supernatant of cultured HPMCs increased and proliferation of HPMCs was inhibited by L-PDS in a time-dependent manner (p < 0.01). But there was no difference in HPMCs treated with various concentrations of L-PDS. Ginsenosides, shen mai, and huang qi injection decreased release of LDH, increased cell viability, and enhanced proliferation of HPMCs (p < 0.01).
Conclusions: Ginsenosides, shen mai, and huang qi injection protected HPMCs from toxicity of L-PDS, thereby enhancing cell viability and proliferation.
Relationship between MicroRNA194 Expression and Epithelial-to-Mesenchymal Transition in Human Peritoneal Mesothelial Cells
Objectives: Determine microRNA194 expression in human peritoneal mesothelial cells (HPMCs) isolated from effluents in dialysis fluid and demonstrate the relationship between microRNA194 expression and epithelial-to-mesenchymal transition (EMT) and ultrafiltration failure (UFF) in peritoneal dialysis (PD) patients.
Methods: HPMCs were isolated and randomly cultured from effluents of patients undergoing continuous ambulatory PD (CAPD). The cultured cells were identified by invert microscopy and immunohistochemistry. Patients were randomly divided into two groups: PD start (group A), and PD for more than 1 year (group B). Western blot analysis and quantitative real-time PCR were used to detect protein or mRNA expression of E-cadherin and α–smooth muscle actin (αSMA). MicroRNA194 expression in the samples was analyzed by real-time PCR using a TaqMan miRNA194 probe (ABI) based on the manufacturer's protocols.
Results: There was a markedly lower level of E-cadherin expression in HPMCs isolated from effluents of patients at CAPD start than of patients on CAPD for more than 1 year. Levels of αSMA were upregulated in group B as compared with group A. Expression of miRNA194 was upregulated in HPMCs from PD patients and was related to expression of E-cadherin and αSMA.
Conclusions: With prolongation of PD, EMT deepens in HPMCs cultured from effluents of patients undergoing CAPD. Our finding suggests that miRNA194 is expressed in HPMCs cultured from effluents of patients undergoing CAPD and that miRNA194 expression is related to EMT of HPMCs.
Tgfβ1-Induced Peritoneal Fibrosis through Smad and Mapk Signal Pathways
Objectives: To investigate the roles of the Smad and MAPK signal pathways on transforming growth factor β1 (TGFβ1)–induced peritoneal fibrosis.
Methods: Isolated human peritoneal mesothelia cells (HPMCs) were cultured in vitro and divided into 5 groups: control, TGFβ1 group, TGFβ1 missense oligonucleotides group, ERK inhibitor group, and p38 inhibitor group. Proliferation of HPMCs was detected by MTT assay. Expression of connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1), and type I collagen (Col I) protein and mRNA were detected by immunofluorescence, ELISA, Western blot analysis, and RT-PCR respectively. Expression of p-Smad2/3 and Smad7 protein was detected by immunofluorescence.
Results: TGFβ1 induced proliferation of HPMCs and increased expression of CTGF, αSMA, FN, PAI-1, and Col I mRNA and protein. p-Smad2/3 protein was mainly distributed in cytoplasm at 15 minutes and concentrated in nucleus and peri-nucleus from 30 minutes to 1 hour. It was then distributed in cytoplasm again at 2 hours. Its expression was induced by TGFβ1 at 15 minutes, peaked from 30 minutes to 1 hour, then gradually dropped until 2 hours. Expression of Smad7 protein time-dependently increased at 24 hours induced by TGFβb1, peaking at 48 hours. However, ERK inhibitor and p38 inhibitor suppressed proliferation of HPMCs and expression of FN.
Conclusions: Expression of CTGF, αSMA, FN, PAI-1, and Col I in HPMCs is induced by TGFβ1 via both the Smad and MAPK signal transduction pathways.
Effect of Glucose Peritoneal Dialysates on Permeability of Cultured Human Peritoneal Mesothelial Cells
Objectives: Transmesothelial electrical resistance (TER) is a measurement of paracellular solute permeability in various types of cells. To clarify the cause of peritoneal hyperpermeability, TER was used to evaluate the effect of various concentrations of glucose in peritoneal dialysis solutions (PDSs) on the permeability of human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs were cultured in the two-chamber culture system. TER was measured daily until a steady state was reached. Then HPMCs were treated with a 50:50 mix (by volume) of DMEM and PDS containing 1.5%, 2.5%, or 4.25% glucose. Serum-free DMEM was used as a control. TER measurement was taken at hours 0, 3, 6, 12, and 24 of incubation.
Results: TER reached a stable state at 34.7 ± 1.5 Ω·cm2 on the second day and then declined gradually in a time- and concentration-dependent manner after culture with various concentrations of glucose PDS. TER values in the 4.25% glucose PDS group declined gradually to 4.3 ± 1.5 Ω·cm2 within 24 hours (p < 0.01 vs. 2.5% glucose PDS group). TER values in lower concentrations of glucose PDS (1.5%, 2.5%) also showed a tendency to decline, but that decline was less pronounced (p < 0.01 vs. 1.5% glucose PDS group and control group respectively).
Conclusions: High glucose in peritoneal dialysate increased paracellular permeability of HPMCs, which may affect the progression of peritoneal hyperpermeability and ultrafiltration failure.
The Effects of Hepatocyte Growth Factor on Peritoneal Fibrosis Induced by High Glucose
Objectives: Peritoneal dialysis (PD) is a form of renal replacement based on the use of the peritoneum as a semipermeable membrane. Continuous exposure to bioincompatible PD solutions (PDSs) causes injury to the peritoneal membrane, which progressively undergoes fibrosis and, ultimately, ultrafiltration failure. Epithelial–mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) induced by transforming growth factor β1 (TGFβ1), which is upregulated by high glucose (HG) PDS, is a crucial event in peritoneal fibrosis. Endogenous hepatocyte growth factor (HGF) plays a role in antifibrosis. It has been reported that the homeostasis between TGFβ1 and HGF is closely involved in fibrosis. In this study, we investigated the effect of exogenous HGF on the expression of TGFβ1 induced by HG in HPMCs.
Methods: Omental HPMCs were obtained by digestion of samples of omentum from nonuremic patients. All the HPMCs were identified by phase-contrast microscopy and immunohistochemistry. Cells of the third passage were used in this study. After subconfluent culture, HPMCs were incubated in serum-free medium for 24 hours and then exposed to various concentrations of glucose (5.5, 30, 60 mmol/L d-glucose and 60 mmol/L mannitol). Recombinant human HGF (25, 50, 100 ng/mL) was added to HPMCs cultured with HG (60 mmol/L d-glucose) for 48 hours. TGFβ1 and FN protein in the supernatant were detected by ELISA. In HPMCs, α smooth muscle actin (αSMA) was detected by immunocytochemistry. Expression of TGFβ1, FN, and αSMA mRNA in HPMCs was detected by real-time PCR.
Results: Expression of αSMA, TGFβ1, and FN mRNA and protein increased significantly in HPMCs cultured in HG (p < 0.05) in a concentration-dependent manner (p < 0.01). A similar change was not revealed in HPMCs cultured with mannitol. Upregulation of αSMA, TGFβ1, and FN mRNA expression and protein induced by HG was attenuated by HGF in a concentration-dependent manner.
Conclusions: Overexpression of TGFβ1, EMT, and ECM synthesis by HPMCs are induced by HG and are inhibited by HGF.
The Role of Smad Signaling Pathway Mediated by TGFβ1 on Extracellular Matrix of Human Peritoneal Mesothelial Cells
Objectives: To investigate the effect of the Smad signaling pathway, mediated by TGFβ1, on expression of extracellular matrix (ECM) in human peritoneal mesothelial cells (HPMCs).
Methods: HPMCs were isolated from human omenta by the trypsinization method, and were grown in DMEM/F12 medium with or without 5 ng/mL TGFβ1 during incubation. Western blot analysis and immunohistochemistry were used to detect expression of p-Smad2/3 protein and its migration into HPMCs incubated with TGFβ1 at 0, 15, and 30 minutes and 1 and 2 hours. After incubation with TGFβ1 for 24, 48, and 72 hours, expression of Smad7, connective tissue growth factor (CTGF), α smooth muscle actin (αSMA) and type I collagen (Col I) protein were determined by Western blot analysis. Moreover, plasminogen activator inhibitor 1 (PAI-1) and fibronectin (FN) in the supernatant were detected using ELISA. Semi-quantification RT-PCR was used to detect mRNA expression of Smad7, CTGF, αSMA, PAI-1, FN, and Col I in HPMCs.
Results: Expression of p-Smad2/3 protein in HPMCs was remarkably increased at 15 minutes after incubation with TGFβ1, peaking from 30 minutes (81%) to 1 hour (84%), and dropping after 2 hours (37%, p < 0.01); Meanwhile, immunohistochemistry showed that p-Smad2/3 was mainly distributed in cytoplasm at 15 minutes, concentrated in cell nuclei and peri-nuclei from 30 minutes to 1 hour, and distributed in cytoplasm again at 2 hours. Smad7, CTGF, αSMA, Col I, PAI-1, and FN expression were obviously increased in HPMCs stimulated by TGFβ1, in a time-dependent manner (p < 0.01).
Conclusions: Extrinsic TGFβ1 can specifically activate the Smad signaling pathway in HPMCs and can induce transcription and expression of many downstream target genes such as Smad7, CTGF, αSMA, PAI-1, FN, and Col I mediated by Smad signaling pathway.
Transforming Growth Factor β1–Induced Epithelial–Mesenchymal Transition of Rat Peritoneal Mesothelial Cells via RhoA–ROCK Signaling Pathway
Objectives: To investigate the role of the RhoA–ROCK signaling pathway in epithelial–mesenchymal transition (EMT) induced by transforming growth factor β1 (TGFβ1) in rat peritoneal mesothelial cells (RPMCs).
Methods: Primary RPMCs were cultured in vitro. After synchronization for 24 hours, RPMCs were randomly assigned to 4 groups: group A (control), group B (10 μg/L TGFβ1), group C (pretreated for 2 hours with 10 μmol/L Y-27632, an inhibitor of ROCK, before stimulation with 10 μg/L TGFβ1), and group D (10 μmol/L Y-27632 alone). Growth-arrested and synchronized RPMCs were stimulated with 10 μg /L TGFβ1 for various times. Levels of E-cadherin, α smooth muscle actin (αSMA), and collagen I mRNA and protein expression were measured by RT-PCR and Western blot analysis respectively. Protein expression of vimentin was measured by Western blot analysis. Active RhoA was extracted using a plasma membrane protein extraction kit, and was then assessed by Western blot analysis.
Results: Stimulation with TGFβ1 elicited a robust and time-dependent increase in RhoA activity by a factor of 2.57 ± 0.52 as compared with the control group (p < 0.05) after 10 minutes. RhoA activity peaked at 1 hour, at a factor of 4.35 ± 0.41 times that in the control group (p < 0.05). TGFβ1 upregulated mRNA or protein expression (or both) of αSMA, vimentin, and collagen I, and downregulated mRNA and protein expression of E-cadherin in RPMCs. The ROCK inhibitor Y-27632 effectively reversed TGFβ1-induced expression of αSMA, collagen I, and vimentin: as compared with TGFβ1-stimulated groups, mRNA levels of αSMA and collagen I decreased by 53.8% and 55.7%, and protein levels of αSMA, vimentin, and collagen I decreased by 42.6%, 60.1%, and 58.1% (p < 0.05). However no effect on the level of E-cadherin was observed.
Conclusions: The RhoA–ROCK signaling pathway may mediate the EMT induced by TGFβ1 in RPMCs. The RhoA–Rock pathway may be a potential therapeutic target in the progress of peritoneal fibrosis.
Troglitazone Inhibits Synthesis of CTGF and Reduces Matrix Production in Human Peritoneal Mesothelial Cells
Objectives: Inhibition of connective tissue growth factor (CTGF) expression in human peritoneal mesothelial cells (HPMCs) may provide a potential treatment for peritoneal fibrosis. How troglitazone, one of the peroxisome proliferator–activated receptor γ (PPARG) agonists, effects extracellular matrix (ECM) turnover is unknown. This study investigated the effect of the PPARG agonist troglitazone on CTGF expression and ECM production in HPMCs.
Methods: HPMCs from human omentum were cultured in medium with or without 30 mmol/L d-glucose by an enzyme digestion method. Expression of CTGF and production and turnover of ECM were measured in the presence and absence of 15 μmol/L troglitazone. Expression of CTGF, collagen I (Col I), and fibronectin (FN) mRNA was determined by semi-quantification RT-PCR. The level of CTGF protein was determined by ELISA and Western blot analysis; expression of Col I and FN protein was determined by Western blot analysis.
Results: Expression of CTGF, Col I, and FN mRNA and protein were significantly increased in HPMCs stimulated with 30 mmol/L d-glucose (GS) as compared with a control group in F12 medium (p < 0.01), which was reversed in the presence of troglitazone (15 μmol/L). An obvious decrease in CTGF expression was found in troglitazone-treated groups as compared with GS-stimulated groups (p < 0.05). HPMCs exposed to troglitazone also showed reduced Col I and FN secretion (p < 0.05).
Conclusions: This study suggests that the PPARG agonists may have a specific role in ameliorating the course of progressive peritoneal fibrosis under long-term peritoneal dialysis.
Establishment of a Peritoneal Dialysis Barrier and Influence of Uremic Serum on Permeability of the Barrier by Transwell
Objectives: Transwell membrane (Sigma–Aldrich, St. Louis, MO, U.S.A.) is an experimental device/technique for establishing (among other things) a biologic barrier. We used Transwell with human umbilical vascular endothelial cells (HUVECs) and human peritoneal mesothelial cells (HPMCs) to construct a peritoneal dialysis (PD) barrier and to test its permeability in the uremic milieu.
Methods: We first cultured HUVECs and HPMCs, and then inverted a Transwell membrane (polyester, 3.0-mm pore size, Millicell) into a 12-well plate, and placed HPMCs on top of the membrane. After 5 days’ culture, the HPMCs had reproduced and were adhered to the membrane. The membrane was then turned over (with the HPMCs now upside-down) in 24-well culture plates, and HUVECs were subcultured on the top of the membrane. The cells were co-cultured for another 4 days, after which their morphology and distribution on both sides of the membrane were observed using a transmission electron microscope. Creatinine [Cr, low molecular weight (MW)], β2-microglobulin (β2M, intermediate MW), and human serum albumin (hAlb, high MW) were then used to test the permeability of the barrier at 6 hours and 24 hours. Uremic serum was added to the upper and lower wells in 24-well plates to stimulate the cells for 16 hours. Wells were washed from inside to outside, and 0.05 mg/mL fluorescein isothiocyanate (FITC)–hAlb was added to the upper wells. After 6 hours, the concentration of hAlb in the upper and lower wells was tested.
Results: A PD barrier was successfully established. Levels of Cr and β2M were not balanced until 24 hours; their concentration was higher in the upper wells than in the lower wells by a factor of approximately 4 at 6 hours. But the concentration of hAlb was greater by a factor of approximately 8 in upper wells at 6 hours. After uremic serum was added to the upper wells for 24 hours and FITC-hAlb for 6 hours, the concentration of fluorescence intensity in the upper and lower wells was statistically significantly different between uremic serum and controls (p < 0.05).
Conclusions: Uremic serum affected the permeability of the constructed PD barrier. Besides establishing a PD barrier, we also explore its permeability to substances of various MWs, finding that 6 hours is an appropriate interval for studying permeability changes.
Characterization of MicroRNA Expression Associated with Peritoneal Fibrosis
Objectives: Peritoneal fibrosis (PF) is an important long-term complication in peritoneal dialysis. MicroRNAs (miRNAs) constitute a highly conserved class of noncoding RNAs that control many biologic processes by sequence-specific inhibition of gene expression. Here, we preliminarily investigated the miRNA expression associated with PF in rat mesothelial cells (RMCs).
Methods: Primary RMCs were isolated from omentum majus of Sprague–Dawley rats and cultured in DMEM/F12 medium with 15% fetal bovine serum. The RMCs then were stimulated with 2.5% d-glucose for 24 hours. Expression of extracellular matrix [fibronectin (FN) and collagen I (Col I)] was assessed using real-time RT-PCR. Detection was normalized to expression of the endogenous control, α–tubulin. Expression of miRNAs in induced cells were further explored by stem–loop real-time RT-PCR using the endogenous control U6. The relative quantification of target genes (including miRNAs, FN, and Col I) in induced cells was calculated using the equation
Results: Increased FN and Col I mRNA after treatment with 2.5% d-glucose for 24 hours indicated that RMCs had begun the course of fibrosis. Expression of FN and Col I were 1.86 and 2.43 times higher respectively in induced cells than in untreated controls. Fifteen miRNAs (miR132, miR133a, miR141, miR145, miR155, miR192, miR194, miR203, miR21, miR212, miR29c, miR30c, miR429, miR451, and miR497) were selected for further study. Among them, miR155, miR192, and miR21 showed significantly more expression in induced cells than in untreated cells. The averages of the relative expressions of miR155, miR192, and miR21 were 3.26, 2.36, and 1.84 respectively. At the same time, expressions of miR29c, miR30c, and miR203 were significantly less in induced cells than in controls. The averages of the relative expressions of miR29c, miR30c, and miR203 were 0.35, 0.26, and 0.41 respectively.
Conclusions: Stimulation of peritoneal mesothelial cells by high glucose can result in different patterns of miRNA expression. The differential expression of miRNAs may play a role in PF.
Impairment of the Peritoneal Charge Barrier May Promote Protein Loss in Continuous Ambulatory Peritoneal Dialysis Patients
Objectives: To evaluate the role of the peritoneal charge barrier in protein loss in continuous ambulatory peritoneal dialysis (CAPD) patients.
Methods: We allocated 32 CAPD patients to either a diabetic nephropathy (DN) group (n = 14) or a chronic glomerulonephritis (CGN) group (n = 18). Blood samples and peritoneal fluid were collected; the peritoneal charge barrier was determined by differential clearance of isoamylase (CPAm/CSAm); and protein in peritoneal fluid was also measured. The relationship between CPAm/CSAm and protein loss was analyzed.
Results: Peritoneal CPAm/CSAm ratio in these patients was 6.296 ± 21.514, and loss of peritoneal protein was 4.14 ± 1.91 g. Loss of peritoneal protein was significantly higher in the DN group than in the CGN group (4.35 ± 1.88 g vs. 5.61 ± 0.86 g, p = 0.011). CPAm/CSAm ratio was significantly lower in the DN group than in the CGN group (9.94 ± 28.35 vs. 0.68 ± 0.86, p = 0.017). CPAm/CSAm ratio was negatively correlated with protein loss (r = –0.584, p < 0.01), and the natural logarithm of the CPAm/CSAm ratio was also linearly correlated with protein loss.
Conclusions: Loss of the peritoneal charge barrier may play a considerable role in loss of peritoneal protein in CAPD patients. Improving charge barrier status may lower the protein losses in CAPD patients.
Intraperitoneal Hepatocyte Growth Factor Reduces Peritoneal Micro-inflammatory State and Increases Ultrafiltration in Peritoneal Dialysis Patients
Objectives: To investigate the influence of hepatocyte growth factor (HGF) on peritoneal micro-inflammatory state and ultrafiltration in peritoneal dialysis (PD) patients.
Methods: During 2 treatment periods of 3 months each, separated by a wash-out period of 1 month, 20 PD patients were randomized to received either normal saline or HGF intraperitoneally. The HGF was added to dialysate to observe the change of inflammatory factors [C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and transforming growth factor β(TGFβ)] and peritoneal ultrafiltration.
Results: Treatment with HGF was accompanied by reduction in dialysate concentrations of CRP, IL-6, TNFα, and TGFβ (p < 0.05). Intraperitoneal HGF significantly reduced the dialysate-to-plasma ratios (D/P) of creatinine, urea, and albumin (p < 0.05). In addition, the end dialysate–to–initial dialysate ratio (D4/D0) of glucose was increased (p < 0.05), as was the ultrafiltration volume (p < 0.05). No significant change was found in the weekly total Kt/V urea or weekly creatinine clearance before and after treatment with HGF.
Conclusions: Intraperitoneal HGF reduces the peritoneal micro-inflammatory state, reduces peritoneal permeability to small solutes, and increases ultrafiltration in PD patients.