Pathophysiology of encapsulating peritoneal sclerosis: lessons from findings of the past three decades in Japan.

Encapsulating peritoneal sclerosis (EPS), a condition with a high mortality rate, is a serious complication of peritoneal dialysis (PD). In Japan, EPS became a central issue in the clinical setting during the mid-90s and the beginning of this century. However, following the introduction of biocompatible neutral PD solutions containing lower levels of glucose degradation products, the incidence and clinical severity of EPS has been greatly lessened. During the past three decades, the etiology of EPS has been elucidated by findings obtained by peritoneal biopsy, laparoscopy, and surgical intervention. Accumulating findings suggest the need for a paradigm change on the nature of EPS pathophysiology; notably, EPS appears not to reflect peritoneal sclerosis per se, but rather the formation of a neo-membrane as a biological reaction to peritoneal injury. This narrative review looks back on the history of EPS in Japan, and discusses EPS pathophysiology, the impact of neutral PD solution on peritoneal protection, and a future novel diagnostic approach, ultra-fine endoscope, for the identification of patients at high risk of EPS.

Relationship between laparoscopic and microscopic findings of peritoneum in peritoneal dialysis patients.

Long-term exposure to the peritoneal dialysis solution (PDS) causes functional and morphological alterations that diminish the efficacy of peritoneal dialysis (PD). Macroscopic and microscopic findings, submesothelial compact zone (SMC) thickness and vascular patency, were associated with PD duration. The relationship between microscopic and laparoscopic morphological findings in PD patients was determined. A total of 78 laparoscopic intraperitoneal findings were recorded during PD catheter removal and 45 peritoneal tissues were obtained from the anterior parietal peritoneum. We examined macroscopic morphological findings in both parietal and visceral peritoneums and bowel movement and assessed the score semiquantitatively. SMC thickness and vascular patency were examined as microscopic findings. Total laparoscopic finding's score (LFS) and microscopic findings, SMC thickness and vascular patency, were associated with PD duration. Total LFS was related to SMC thickness in both visceral and parietal peritoneum, whereas it was related to vascular patency in parietal but not in visceral peritoneum. There was no relationship between microscopic findings and peritoneal surface color, properties, vasculopathy, and adhesion. Total LFS in patients with newly formed membrane and omentum atrophy was higher than in those without. There was a significant relationship between microscopic and laparoscopic findings in PD patients. It is important to evaluate laparoscopic findings in more PD patients to find the predictive findings of encapsulating peritoneal sclerosis development.

A nationwide observational study of locomotive syndrome in Japan using the ResearchKit: The Locomonitor study.

BACKGROUND: We developed the Locomonitor application (app), the world's first iOS app to study locomotive syndrome, using the ResearchKit and examined the prevalence and risk factors for locomotive syndrome in Japanese general individuals 20-69 years old in a nationwide cross-sectional observational study. METHODS: The participants were recruited from February to August 2016. The outcome measures for the locomotive function were evaluated by locomotive syndrome risk tests (LSRTs) using the Locomonitor app. The chi-squared test, a linear-by-linear association trend analysis, and Spearman's correlation test were performed as statistical analyses. RESULTS: A total of 2177 subjects from all prefectures in Japan were included (average 42.2 years old). The Locomo25 and Stand-Up test scores in female participants and the Two-Step test scores in male participants showed age-dependent deterioration. In the overall population, the incidence of Locomo stage 1 and 2, as evaluated by the Locomo25, Stand-Up test or Two-Step test, was 30.2% and 29.2%, respectively. In subjects without locomotive syndrome (40.5%), LSRT scores showed age-dependent deterioration in both sexes. Locomotive syndrome in participants with a body mass index (BMI) of ≥25 kg/m2 was more frequent than in those with a BMI of <25 kg/m2 (age- and gender-adjusted odds ratio [OR] 1.344 [95% confidence interval {CI} 1.03-1.75, p = 0.027]). Locomotive syndrome in participants with an exercise habit was less frequent than in those without an exercise habit (age- and gender-adjusted OR 0.499 [95% CI 0.33-0.755, p < 0.0001]). CONCLUSIONS: The Locomonitor app, a newly developed remote platform, revealed that approximately 20%-30% of Japanese individuals 20-69 years old in the general population met the definition of locomotive syndrome. Locomotive syndrome in participants with obesity was more frequent than those without obesity, while locomotive syndrome in participants with an exercise habit was less frequent than those without an exercise habit.

Effects of long-term treatment with low-GDP, pH-neutral solutions on peritoneal membranes in peritoneal dialysis patients.

BACKGROUND: The morphological changes induced by bio-incompatible peritoneal dialysis (PD) solutions are well known. However, the morphological damage induced by long-term low-glucose degradation product (GDP), pH-neutral solutions has not been reported in detail. The aim of this study was to investigate the long-term effects of pH-neutral PD solutions on morphological and functional changes in the peritoneal membrane. METHODS: We assessed peritoneal membrane biopsy samples from PD patients treated with acidic (Conventional group) or pH-neutral solutions (pH-neutral group) using pathology and immunopathology techniques. RESULTS: Analyses of 54 Conventional and 73 pH-neutral group samples showed that the peritoneal membrane was thicker (P < 0.001), the ratio of luminal diameter to vessel diameter (L/V ratio) was significantly smaller (P < 0.001), and advanced glycation end-product (AGE) accumulation was higher in the Conventional than in the pH-neutral group (P < 0.001). Comparison of samples from patients in the Conventional (n = 33) and pH-neutral groups (n = 33) who were treated for 4-10 years also showed significant differences in peritoneal thickness, L/V ratio and AGE score. Furthermore, the L/V ratio in the Conventional group significantly decreased over time (P < 0.01); however, no such change was seen in the pH-neutral group. Peritoneal membrane thickness was not associated with PD duration in both groups. Dialysate-to-plasma ratio of creatinine and L/V ratio negatively correlated with PD treatment duration in the Conventional group, but not in the pH-neutral group. CONCLUSIONS: These findings suggest that pH-neutral solutions prevent the morphological and functional peritoneal changes induced by long-term PD treatment.

Scavenging of reactive oxygen species by astaxanthin inhibits epithelial-mesenchymal transition in high glucose-stimulated mesothelial cells.

BACKGROUND: High glucose concentrations influence the functional and structural development of the peritoneal membrane. We previously reported that the oral administration of astaxanthin (AST) suppressed peritoneal fibrosis (PF) as well as inhibited oxidative stress, inflammation, and epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in a chlorhexidine-induced PF rat model. This suggests that oxidative stress induction of EMT is a key event during peritoneal damage. The present study evaluated the therapeutic effect of AST in suppressing EMT, in response to glucose-induced oxidative stress. METHODS: Temperature-sensitive mesothelial cells (TSMCs) were cultured in the presence or absence of AST and then treated with 140 mM glucose for 3 or 12 hours. Expression levels of TNF-α, TGF-ß, and VEGF were determined at the mRNA and protein levels, and nuclear factor kappa B (NF-κB) activity was evaluated. We measured NO2-/NO3- concentrations in cellular supernatants and determined 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in mitochondrial and nuclear DNA. The expressions of E-cadherin and alpha-smooth muscle actin (α-SMA) were evaluated by double immunofluorescence and protein levels. RESULTS: High glucose concentrations induced overproduction of reactive oxidative species (ROS), increasing 8-OHdG mitochondrial DNA and cytokine levels. The NF-κB pathway was activated in response to high glucose concentrations, whereas de novo α-SMA expression was observed with decreased E-cadherin expression. AST treatment attenuated ROS production, inflammatory cytokine production, NF-κB activation, and EMT. CONCLUSION: The findings of the present study indicate that AST may have an anti-EMT effect due to anti-oxidative and anti-inflammatory activities by scavenging glucose-induced ROS from mitochondria in PMCs. AST may be an efficacious treatment for PF.

Recommendations for pathological diagnosis on biopsy samples from peritoneal dialysis patients.

Peritoneal dialysis (PD) has been established as an essential renal replacement therapy for patients with end stage renal disease during the past half century. Histological evaluation of the peritoneal membrane has contributed to the pathophysiological understanding of PD-related peritoneal injury such as peritonitis, fibrosis, and encapsulating peritoneal sclerosis (EPS). Hyalinizing peritoneal sclerosis (HPS), also known as simple sclerosis, is observed in almost all of PD patients. HPS is morphologically characterized by fibrosis of the submesothelial interstitium and hyalinizing vascular wall, particularly of the post-capillary venule (PCV). Two histological factors, the thickness of submesothelial compact zone (SMC) and the lumen/vessel ratio (L/V) at the PCV, have been used for the quantitative evaluation of HPS. The measuring system on SMC thickness and L/V ratio is easy and useful for evaluating the severity of HPS. On the other hand, EPS is characterized by unique encapsulation of the intestines by an "encapsulating membrane". This newly formed membranous structure covers the visceral peritoneum of the intestines, which contains fibrin deposition, angiogenesis, and proliferation of fibroblast-like cells and other inflammatory cells. This review will cover the common understandings of PD-related peritoneal alterations and provide a basic platform for clinical applications and future studies in this field.

Pancreatic lipase activity in overnight effluent predicts high transport status in peritoneal dialysis patients.

BACKGROUND: Long-term peritoneal dialysis (PD) causes peritoneal morphological and functional changes, resulting in high transport status featuring increased peritoneal permeability. High transport status is diagnosed by peritoneal equilibration test (PET), a reliable but time-consuming method. We identifed a reliable biomarker in peritoneal effluent to predict high transport status in PD patients. METHODS: We collected peritoneal effluent and serum from 33 PD patients and measured common laboratory test parameters. High transport status was determined by PET if the dialysate/plasma ratio of creatinine at 4h dwell (D/P Cr 4h) was ≥0.81. RESULTS: There were significant correlations between D/P Cr 4h and some laboratory parameters in overnight effluent (pancreatic lipase activity, r=0.65, p<0.001; ß2-microglobulin concentration, r=0.59, p<0.001; IL-6 concentration, r=0.53, p<0.001; and CA125 concentration, r=0.29, p=0.027). In a multivariate logistic regression analysis, the pancreatic lipase activity in overnight effluent was identified as an independent predictor of high transport status even after adjusting for age, PD duration, and glomerular filtration rate [OR=1.43 (95% CI: 1.11-1.83), p=0.005]. CONCLUSIONS: The pancreatic lipase activity in overnight effluent is an independent predictor of high transport status in PD patients.

Determinants of coronary artery calcification in maintenance hemodialysis patients.

In addition to the well-known traditional risk factors, uremia-related so-called novel risk factors and medications appear to affect coronary artery calcification in hemodialysis patients. This study was performed to evaluate coronary artery calcification score (CACS) in maintenance hemodialysis (MHD) patients, and to identify significantly related factors. We assessed CACS using Agatston Score by MDCT, sex, age, dialysis vintage, presence of diabetes mellitus, smoking history, presence of ≥100 ml urine volume/day, normalized protein catabolic rate, geriatric nutritional risk index, administration of active vitamin D3, cinacalcet, phosphate binders or antihypertensive agents, and circulation parameters including creatinine, albumin, corrected calcium and phosphate in 207 MHD patients. Coronary artery calcifications were observed in 192 patients (92.8%). In multivariate analysis, CACS showed direct associations with age (p < 0.001), dialysis vintage (p < 0.001) and presence of diabetes mellitus (p < 0.01), and an inverse association only with active vitamin D3 administration (p < 0.001) in MHD patients. Patients with active vitamin D3 showed significantly lower CACS than in those without it (1349.6 ± 1635.0 vs. 2475.6 ± 2646.6 H, p < 0.05). Older age, longer duration of dialysis and diabetes mellitus are risk factors and administration of active vitamin D3 is protective factor for coronary artery calcification in MHD patients.

Morphological characteristics in peritoneum in patients with neutral peritoneal dialysis solution.

Peritoneal dialysis solution (PDS) plays a role in functional and morphological damage to the peritoneum. This study aimed to clarify the effect of neutral PDS in preventing morphological changes by assessing peritoneal damage and comparing morphological alterations between PD patients treated with neutral PDS and acidic PDS. Sixty-one patients participated from seven hospitals. All patients were treated with neutral PDS excluding icodextrin, during their entire PD treatment, and experienced no episode of peritonitis. The thickness of submesothelial compact (SMC) zone and the presence of vasculopathy in the anterior parietal abdominal peritoneum were assessed. The impact of icodextrin, hybrid therapy, and peritoneal rest and lavage in morphological alterations were determined. There was no significant difference in the average SMC thickness between neutral and acidic PDS. The vessel patency in patients using neutral PDS was significantly higher compared to that in acidic PDS at any time during PD. There were no significant suppressive effects from interventions or use of icodextrin with respect to peritoneal morphological injury. A monolayer of mesothelial cell was observed in approximately half the patients, especially in their receiving lavage patients. Neutral PDS, accompanied by other preventive approaches against peritoneal injury, might suppress the development of peritoneal morphological alterations.

Long-term outcome of encapsulating peritoneal sclerosis (EPS) patients in a single center.

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is the most serious complication of peritoneal dialysis (PD) with a high mortality rate. The objective of the present study was to determine the clinical characteristics, the incidence rate, and the long-term outcome of EPS patients compared with control patients. METHODS: Two hundred and seventy patients with end-stage kidney disease were started on PD from 1987 to 2013 in the Juntendo University Hospital. EPS was diagnosed by clinical findings, radiological findings, and macroscopic inspection at the time of laparoscopy or surgical operation. Patient medical records were analyzed retrospectively, including clinical characteristics, laboratory findings, treatment modality, and outcomes. Using a Kaplan-Meier analysis, we compared the survival rate between EPS patients and control PD patients, matched for age, gender, diabetes, and duration of PD. RESULTS: Among 270 PD patients, 13 patients (4.8 %) developed EPS. The mean duration of PD was 120.5 ± 42.8 months. There were no significant difference in demographic findings between EPS and control PD patients. Among the EPS patients, seven patients died, of which four deaths were directly attributed to EPS. All four patients that had had surgical enterolysis were doing well and had no recurrences. No significant difference in the survival rate between EPS and control PD patients was observed in the Kaplan-Meier analysis. CONCLUSIONS: There was no significant difference in the survival rate between EPS patients and control PD patients. It appears that an early diagnosis by laparoscopy and accurate treatment, including surgical enterolysis, might improve mortality.

Oral Astaxanthin Supplementation Prevents Peritoneal Fibrosis in Rats.

BACKGROUND: Preventing peritoneal damage during peritoneal dialysis is critical. Reactive oxygen species (ROS) have an important role in peritoneal damage; however, few studies have investigated this. We aimed to determine the effects of oral astaxanthin (AST) supplementation in a peritoneal fibrosis (PF) rat model. METHODS: Thirty-seven Sprague-Dawley rats were divided into 5 groups: Control 1 (fed a normal diet without stimulation), Control 2 (fed an AST-supplemented diet without stimulation), Group 1 (fed a normal diet with 8% chlorhexidine gluconate [CG] stimulation for 3 weeks), Group 2 (fed a 0.06% AST-supplemented diet with CG stimulation), and Group 3 (fed a 0.06% AST-supplemented diet that was initiated 4 weeks before CG stimulation). Peritoneal fibrosis, vascular proliferation, and fibrosis-related factor expression were examined. RESULTS: Peritoneal thickness was significantly suppressed by AST supplementation. Astaxanthin diminished the number of CD68-, 8-hydroxy-2'-deoxyguanosine (8-OHdG)-, and monocyte chemoattractant protein-1 (MCP-1)-positive cells. Type 3 collagen, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and MCP-1 mRNA expression was significantly lower in Group 3 than in Group 1. Increased transforming growth factor-ß (TGF-ß) and Snail mRNA expression, vascular density, and the number of α-smooth muscle actin (α-SMA)-positive cells were also decreased in Group 3. CONCLUSION: Astaxanthin suppressed PF development through the inhibition of inflammation and oxidation in PF rats. It appears that the anti-oxidative agent AST may be useful for the prevention of peritoneal damage.

Adipose-derived mesenchymal stem cells transplantation facilitate experimental peritoneal fibrosis repair by suppressing epithelial-mesenchymal transition.

BACKGROUND: Prevention or reversal of peritoneal damage is critical in peritoneal dialysis. Although autologous cell transplantation has beneficial effects on tissue repair in various organs, few studies have investigated the effects of transplantation of adipose-derived mesenchymal stem cells (ASCs) on peritoneal fibrosis (PF). Thus, we examined the mechanism of facilitated peritoneal reconstruction induced by ASC transplantation on chlorhexidine gluconate (CG)-induced PF in rats. METHODS: To induce PF in rats, continuous-infusion pumps containing 8 % CG were placed in the abdominal cavity for 21 days. The pumps were removed on day 22 and ASCs were immediately injected into the peritoneal cavity. Morphological alterations and mRNA expression levels of fibrosis-related factors were examined on days 29 and 35. RESULTS: ASC transplantation significantly facilitated peritoneal repair. mRNA expression of tumor necrosis factor-α, interleukin-1ß, monocyte chemotactic protein-1, and epithelial-mesenchymal transition (EMT) markers such as Snail and α-smooth muscle actin were suppressed, whereas that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB) were overexpressed after ASC transplantation. Immunofluorescence indicated that some transplanted ASCs expressed VEGF and PDGF-BB and differentiated into vascular cells. CONCLUSIONS: ASC transplantation facilitates peritoneal repair by suppressing EMT and modulating inflammation and angiogenesis during the early phase of tissue repair in experimental PF.

Suppressive effects of iron overloading on vascular calcification in uremic rats.

BACKGROUND: Medial vascular calcification is a specific complication in chronic kidney disease (CKD) patients although its pathogenesis is poorly understood. The administration of iron (Fe), generally used for the treatment of anemia in CKD patients, induces oxidative stress. Fe loading possibly affects the progress of vascular calcification in uremia. We investigated the effect of Fe on vascular calcification and its mechanism in uremic rats. METHOD: Thirty-two rats were divided into four groups: untreated rats (controls), rats fed a standard diet with Fe administration (Fe group), rats fed an adenine-enriched diet (uremic group), and rats fed an adenine-enriched diet with Fe administration (uremic + Fe group). Iron dextran was administered once a week for 5 weeks intraperitoneally. Morphological alterations and vascular calcification-associated factors in the aortic wall were evaluated. RESULTS: No aortic calcification was observed in the control group although uremic rats developed severe vascular calcification. Fe loading suppressed vascular calcification in the uremic groups. Expressions of runt-related transcription factor 2 (Runx2), single-strand (ss)DNA and phosphate transporter (Pit)-1 were increased in the uremic rats compared to the control rats. In the uremic group, Fe administration did not show any effect on ssDNA expression, but reduced Runx2 and Pit-1 expressions. CONCLUSION: Fe suppressed the development of vascular calcification through the prevention of Pit-1 and vascular smooth muscle cell osteoblastic transdifferentiation.

Paracrine effects of transplanted mesothelial cells isolated from temperature-sensitive SV40 large T-antigen gene transgenic rats during peritoneal repair.

BACKGROUND: The prevention and restoration of peritoneal damage is a critical mission in peritoneal dialysis (PD). Transplantation of mesothelial cells has been suggested to suppress peritoneal injury during PD. Few studies have examined the efficacy and safety of cell transplantation. We evaluated the paracrine effects of mesothelial transplantation during peritoneal repair using immortalized temperature-sensitive mesothelial cells (TSMCs) in chlorhexidine gluconate (CG)-induced peritoneal fibrosis rats. METHODS: Continuous-infusion pumps containing 8% CG were placed into the abdominal cavity for 21 days. After the removal of the pumps, the TSMCs were injected into the peritoneal cavity at Day 22 (Tx-1 group) or 29 (Tx-2 group). Morphological findings and mRNA expressions of regeneration-related factors were examined at Days 22, 29 and 35. RESULTS: Peritoneal thickness was aggravated in the Tx-1 group. Levels of transforming growth factor (TGF)-ß, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 mRNA in the Tx-1 group at Day 35 were comparable with those at Day 22. The levels of Snail, B-Raf and ERK-1, markers of epithelial to mesenchymal transition and of the RAS/MAPK pathway in the Tx-1 group, were significantly higher than those in the Tx-2 group. TGF-ß and VEGF were produced from the transplanted mesothelial cells and the surrounding cells in the Tx-1 group. CONCLUSION: It appears that the paracrine effect of transplanted mesothelial cells during peritoneal repair is associated with its surrounding condition. It is important to determine the most appropriate time for developing peritoneal repair through mesothelial transplantation.

Comparison between the fixation of peritoneal dialysis catheters to the peritoneal wall and the conventional placement technique: clinical experience and follow-up of a new implant technique for peritoneal dialysis catheters.

Peritoneal dialysis (PD) catheters often become severely dislocated, which may lead to malfunction. With the aim of preventing this complication, we have developed a simple method of fixing the catheter downwards in the peritoneal cavity (fixation technique), a technique that does not require a laparoscope. Sixteen patients were implanted using the conventional placement technique and 25 patients were implanted using the fixation technique. The location of the catheter tip was classified from grade 1 (downward, normal) to 5 (dislocated). The frequency of dislocation (defined as the extended time and/or decrease in volume when draining the PD solution) was measured for both the fixation technique and conventional placement technique. There was a significant difference in grade between the fixation technique (2.72 ± 1.01) and conventional technique (3.92 ± 1.31). The time until first dislocation was significantly different between the fixation technique (59.3 ± 48.1 days) and conventional technique (8.8 ± 14.6 days). The time until any dislocation was significantly different between the fixation technique (69.2 ± 41.9 days) and conventional technique (12.9 ± 13.7 days). Complications were not significantly different between the fixation technique and conventional technique. The fixation technique appears to be simple, safe, and useful for preventing severe dislocation and for lengthening the time until dislocation in PD patients.

Campylobacter bacteremia in hemodialysis patients by eating raw meat - the importance of sanitary education.

In 2011, simultaneous, widespread outbreaks of food poisoning by contaminated enterohemorrhagic Escherichia coli in beef, which killed four and hospitalized more than 30 people, occurred in Japan. While the press was widely reporting this disaster, two maintenance hemodialysis patients were suffering from Campylobacter bacteremia by eating undercooked meat. One patient was infected with C. upsaliensis and the other with C. fetus. Although these patients could be successfully treated, they led us to consider the characteristics of C. upsaliensis and C. fetus as opportunistic pathogens, as well as changes in dietary behaviors and food markets. Moreover, they emphasized the need for hemodialysis patients to be not only educated in that they should restrict potassium, phosphate and water intake, but also that they should take care of food sanitation.

Risk of overestimation of kidney function using GFR-estimating equations in patients with low inulin clearance.

BACKGROUND: Accurate estimation of the glomerular filtration rate (GFR) is very important in clinical practice. Although renal inulin clearance (Cin) is the gold standard for measuring GFR, the procedure for Cin measurement is complicated. Use of GFR-estimating equations has been increasing recently due to their simplicity. The objectives of the present study are to analyze the correlation between Cin and other GFR-estimating parameters and to investigate their clinical usefulness and limitation. METHODS: Seventy-two Japanese patients were enrolled in this study. Cin was measured by the continuous infusion method. Serum creatinine (s-Cr), cystatin C, uric acid (UA), and hemoglobin (Hb) were measured. The Japanese formula of estimated GFR (eGFR) was as follows: eGFR (ml/min/1.73m(2) ) = 194 × s-Cr(-1.094) × Age(-0.287) × 0.739 (if female). The endogenous creatinine clearance test was also performed. RESULTS: Levels of Cin were highly correlated with those of endogenous creatinine clearance (Ccr) (R(2) = 0.7585) and eGFR (R(2) = 0.5659). However, patients with lower Cin showed unexpectedly elevated levels of endogenous Ccr and eGFR. Moreover, the levels of eGFR tended to be unexpectedly increased in patients with low body surface area. CONCLUSION: Although GFR-estimating equations are useful for estimating GFR accurately, they pose a risk of overestimation of kidney function in patients with decreased GFRor a poor physique.

Differentiation of bone marrow-derived cells into regenerated mesothelial cells in peritoneal remodeling using a peritoneal fibrosis mouse model.

Marked thickening of the peritoneum and vasculopathy in the submesothelial compact zone have been reported in long-term peritoneal dialysis patients. Bone marrow (BM)-derived cell lines are considered to be useful tools for therapy of various diseases. To clarify the role of BM-derived cells in the peritoneal fibrosis (PF) model, we analyzed several lineages of cells in the peritoneum. BM cells from green fluorescent protein (GFP) transgenic mice were transplanted into naïve C57Bl/6 mice. Chlorhexidine gluconate (CG) was injected intraperitoneally to induce PF. Immunohistochemical analysis was performed with parietal peritoneum using anti-Sca-1 or -c-Kit and -GFP antibodies. Isolated BM cells were also transplanted into the CG-stimulated peritoneum. BM-derived cells from GFP transgenic mice appeared in the submesothelium from days 14 to 42. Both GFP- and stem cell marker-positive cells were observed in the submesothelium and on the surface. Isolated c-Kit-positive cells, transplanted into the peritoneal cavity, differentiated into mesothelial cells. In this study, we investigated whether or not BM-derived cells play a role in the repair of PF and immature cells have the potential of inducing repair of the peritoneum. The findings of this study suggest a new concept for therapy of PF.

Rapamycin inhibits transforming growth factor ß-induced peritoneal angiogenesis by blocking the secondary hypoxic response.

Patients with end-stage kidney disease on peritoneal dialysis often develop progressive scarring of the peritoneal tissues. This manifests as submesothelial thickening and is associated with increased vascularization that leads to ultrafiltration dysfunction. Hypoxia induces a characteristic series of responses including angiogenesis and fibrosis. We investigated the role of hypoxia in peritoneal membrane damage. An adenovirus expressing transforming growth factor (TGF) ß was used to induce peritoneal fibrosis. We evaluated the effect of the mTOR inhibitor rapamycin, which has been previously shown to block hypoxia-inducible factor (HIF) 1α. We also assessed the effect of HIF1α independently using an adenovirus expressing active HIF1α. To identify the TGFß1-independent effects of HIF1α, we expressed HIF1α in the peritoneum of mice lacking the TGFß signalling molecule Smad3. We demonstrate that TGFß-induced fibroproliferative tissue is hypoxic. Rapamycin did not affect the early angiogenic response, but inhibited angiogenesis and submesothelial thickening 21 days after induction of fibrosis. In primary mesothelial cell culture, rapamycin had no effect on TGFß-induced vascular endothelial growth factor (VEGF) but did suppress hypoxia-induced VEGF. HIF1α induced submesothelial thickening and angiogenesis in peritoneal tissue. The fibrogenic effects of HIF1α were Smad3 dependent. In summary, submesothelial hypoxia may be an important secondary factor, which augments TGFß-induced peritoneal injury. The hypoxic response is mediated partly through HIF1α and the mTOR inhibitor rapamycin blocks the hypoxic-induced angiogenic effects but does not affect the direct TGFß-mediated fibrosis and angiogenesis.

Establishment of a peritoneal mesothelial cell line from a transgenic rat harbouring the temperature-sensitive simian virus 40 large T-antigen gene.

BACKGROUND: Establishing a peritoneal mesothelial cell (MC) line in which the native characteristics of primary MCs are constantly maintained in vivo is of great significance for investigating their morphological and functional changes in peritoneal dialysis. We established transgenic (Tg) rats that expressed the temperature-sensitive tsA58 mutant of the simian virus 40 large T-antigen (tsSV40T), which served as a source of immortalized rat cell lines. The cells were immortalized at a permissive temperature of 33 degrees C, although they were differentiated at a non-permissive temperature of 38 degrees C. In this study, we established a novel MC line from tsSV40T Tg rats and evaluated its characteristics. METHODS: MCs were isolated from 8-week-old tsSV40T Tg rats and cloned. MCs from 8-week-old Wistar rats were used as controls. These cells were immunohistochemically and phenotypically evaluated by immunofluorescence, phase contrast and electron microscopy. The production of plasminogen activator inhibitor 1 (PAI-1) from MCs stimulated by tumour necrosis factor-alpha (TNF-alpha) was measured. RESULTS: The tsSV40T MCs showed a cobblestone-like appearance at 33 and 38 degrees C, which was similar to normal primary cultured MCs. Microvilli-like structures were observed on the cell surface by a scanning electron microscope at 33 and 38 degrees C. Wilms tumour-1 and pancytokeratin, as MC markers, were expressed at 33 and 38 degrees C. Following TNF-alpha stimulation, PAI-1 production of tsSV40T MCs was similar to that of normal primary cultured MCs. CONCLUSION: We established a novel, conditionally immortalized MC line that continuously retained the characteristics of primary cultured peritoneal MCs. This cell line might be a useful tool for various types of in vitro biological research on peritoneal dialysis.