Epithelial-to-mesenchymal transition of mesothelial cells is an early event during peritoneal dialysis and is associated with high peritoneal transport.

Ultrafiltration (UF) failure is a consequence of long-term peritoneal dialysis (PD). Fibrosis, angiogenesis, and vasculopathy are causes of this functional disorder after 3-8 years on PD. Epithelial-to-mesenchymal transition (EMT) of mesothelial cell (MC) is a key process leading to peritoneal fibrosis with functional deterioration. Our purpose was to study the peritoneal anatomical changes during the first months on PD, and to correlate them with peritoneal functional parameters. We studied 35 stable PD patients for up to 2 years on PD, with a mean age of 45.3+/-14.5 years. Seventy-four percent of patients presented loss of the mesothelial layer, 46% fibrosis (>150 microm) and 17% in situ evidence of EMT (submesothelial cytokeratin staining), which increased over time. All patients with EMT showed myofibroblasts, while only 36% of patients without EMT had myofibroblasts. The number of peritoneal vessels did not vary when we compared different times on PD. Vasculopathy was present in 17% of the samples. Functional studies were used to define the peritoneal transport status. Patients in the highest quartile of mass transfer area coefficient of creatinine (Cr-MTAC) (>11.8 ml min(-1)) showed significantly higher EMT prevalence (P=0.016) but similar number of peritoneal vessels. In the multivariate analysis, the highest quartile of Cr-MTAC remained as an independent factor predicting the presence of EMT (odds ratio 12.4; confidence interval: 1.6-92; P=0.013) after adjusting for fibrosis (P=0.018). We concluded that, during the first 2 PD years, EMT of MCs is a frequent morphological change in the peritoneal membrane. High solute transport status is associated with its presence but not with increased number of peritoneal vessels.

Effects of rapamycin on the epithelial-to-mesenchymal transition of human peritoneal mesothelial cells.

The preservation of the peritoneal membrane is crucial for long-term survival in peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) is a process demonstrated in mesothelial cells (MC), responsible for negative peritoneal changes and directly related to PD. EMT enables neovascularization and fibrogenic capabilities in MC. Vascular endothelial growth factor (VEGF) is the mediator for neo-vascularization. Rapamycin is a potent immunosuppressor with antifibrotic action in renal allografts and has a demonstrated anti-VEGF effect. We performed this study with the hypothesis that rapamycin may regulate the EMT of MC. MC from human omentum were cultured. When mesothelial cells reached confluence, some of them were stimulated with r-TGF-beta (1 ng/mL) to induce EMT, co-administered with rapamycin (0.2, 2, 4, 20 and 40 nM). Other groups of cells received similar doses of rapamycin or r-TGF-beta, separately. Cells were analyzed at 6, 24, 48 hours and 7 days. As markers of EMT we included alfa -SMA, E-cadherin and snail nuclear factor by quantitative RT-PCR. EMT markers and regulators demonstrated the following changes with rapamycin: E-cadherin (a protective gene for EMT) increased 2.5-fold relative to controls under 40 nM, at 24h. Importantly, rapamycin inhibited snail expression induced by TGF-beta at 6h, whereas TGF-beta increased snail 10-fold. At day 7, rapamycin showed no anti-EMT properties. An important decrease in alfa -SMA expression by MC after rapamycin addition was observed. In conclusion, rapamycin shows a mild protective effect on EMT, as it increases E-cadherin and decreases alfa -SMA expression. Consequently, rapamycin might partially regulate the epithelial-to-mesenchymal transition of mesothelial cells.

Tissue models of peritoneal fibrosis.

OBJECTIVE: To evaluate the utility of peritoneal pathologic samples, unrelated to peritoneal dialysis (PD) treatment, for the study of peritoneal fibrosis and inflammation. METHODS: Comparative morphologic and immunohistochemical study of peritoneal pathologic samples unrelated to PD with peritoneal biopsies from PD patients with special emphasis on the expression of myofibroblastic and epithelial-to-mesenchymal transition markers. RESULTS: Regarding morphology, PD-related simple fibrosis was less cellular, with greater stromal hyalinization, determining a homogeneous, hypocellular aspect of the submesothelium. In contrast, non-PD fibrosis was more cellular with an extracellular matrix showing a dense and fibrillar quality with wide bundles of collagen. Hylinazing vasculopathy was only present in PD samples. Myofibroblastic differentiation and epithelial-to-mesenchymal transition were common findings in all situations of peritoneal fibrosis. Calponin and calretinin are useful cellular markers to study such fibrogenic mechanisms and correlate with other well-known markers such as a -SMA and cytokeratins. Their expression was much more intense in those samples showing acute inflammation (peritonitis). CONCLUSIONS: Non-PD models of peritoneal fibrosis seem very useful to evaluate important features of human peritoneal pathology such us fibrogenesis, and inflammation. Fibrogenic events such as myofibroblastic differentiation and epithelial-to-mesenchymal transition are evident in these tissue samples allowing us to use them as an accessible source for in vivo and ex vivo studies. Both events show their maximal expression in situations of acute inflammation supporting the important role that peritonitis episodes play in the progression of fibrosis.

[Influence of different pharmacological agents in the ex vivo proliferation of mesothelial cells obtained from the peritoneal effluent of patients treated with peritoneal dialysis]. / Influencia de factores exógenos en la capacidad de proliferación ex vivo de las células mesoteliales obtenidas de efluente de pacientes tratados con diálisis peritoneal crónica.

UNLABELLED: Mesothelial cells (MC) are the first peritoneal membrane barrier in contact with dialysate. The aim of this study was to analyze the in vitro capacity of different pharmacological agents to modify the ex vivo proliferation of MC obtained from the peritoneal effluent of patients treated with peritoneal dialysis (PD). MATERIAL AND METHODS: Thirty cultures of MC taken from nocturnal peritoneal effluent were performed. After identification, MC are subcultured in 24 multi-well plates, adding the different exogenous agents. Proliferative capacity and cell morphology were estimated on day 16th of culture. The agents evaluated were insulin, IGF-1, tamoxifen, labetalol, carvedilol, enalapril and losartan. RESULTS: Insulin shows a dose-dependent effect on MC growth, with a limit that is stimulated by the addition of fetal bovine serum (FBS). Concentrations higher than 100 micrograms/ml, are not associated with further growth, even with cell damage. In contrast, the wide range of IGF-1 dose used did not affect to MC proliferation. Tamoxifen causes negative effects on MC growth just a very high doses, not resembling doses in clinical practice. Labetalol does not modify MC proliferation used under therapeutic calculated range. However, concentrations higher than 40 micrograms/ml showed a negative influence on growth, behaving as lethal doses that over 100 micrograms/ml. The addition of FBS attenuates this effect. These effects were very similar to that caused by carvedilol addition. Enalapril and losartan act as antiproliferative agents for MC. This effect is potentiated with angiotensin II, reaching lethal concentrations increasing the dose. In conclusion, mesothelial cell growth ex vivo taken from nocturnal peritoneal effluent on PD patients is an useful tool to explore the effects of any pharmacological agent on the biology of the cell of the peritoneum. The agents used had any influence in the proliferation capacity of mesothelial cells.

Hemodialysis vascular assessment by an ultrasound dilution method (transonic) in patients older than 65 years.

UNLABELLED: Malfunction of vascular access is one of the most frequent causes of morbidity and mortality in hemodialysis patients (HD). Early diagnosis makes possible the most frequent vascular access (VA) used in HD patients. The arteriovenous fistula (AVF), both autologous or heterologous, is the appropriate correction by an interventional radiologist or by surgery, before thrombosis appears. For this purpose, a follow-up of VA is mandatory. New technologies offer non-invasive methods for this purpose. In HD sessions ultrasound 'on line' and ultrasound-dilution techniques permit us to monitor vascular access in HD patients. Also transonic technology has been validated for this purpose, although the limitations of its use among elderly patients is unknown. Using the Transonic HD01 monitor, we studied vascular access in 45 patients in HD older than 65 years, and compared them with 47 patients who were younger than 65 years. The parameters analyzed were: effective flow Qt, recirculation, venous pressure and access flow. We found no significant differences between these parameters but in both groups found that the effective flow measure by Transonic was lower than that measured by a blood pump. Both groups contained patients who had no recirculation but had an access flow that was lower than expected. To rule out stenosis of VA in those patients, we performed an Eco Doppler confirming that all patients had stenosis. With this method, one can determines the access flow and thus predicts the possibility of future thrombosis. CONCLUSION: Our data confirm that one can evaluate VA in patients older than 65 years with Transonic HD01 monitor, and also in patients younger than 65 years. Due to the special characteristics of the vessels in elderly patients, Transonic HD01 monitor is a good method by which to monitor VA in them.

Vascular endothelial growth factor (VEGF) levels in peritoneal dialysis effluent.

BACKGROUND: Long-term peritoneal dialysis (PD) patients who develop peritoneal ultrafiltration failure have an abnormally large number of capillaries and sclerotic changes in peritoneal biopsy. Peritoneal vascular endothelial growth factor (VEGF) production has been suggested to explain the higher levels in peritoneal effluent than in plasma. The high effluent VEGF levels have been related to peritoneal changes consisting of increased permeability to small molecules. To further analyze the relationship between peritoneal neoangiogenesis induced by VEGF and peritoneal transport, we studied peritoneal effluent VEGF levels in active PD patients. METHODS: VEGF levels were determined in serum and plasma, and in peritoneal effluent (PE) after 4, 8 and 15 h dwell times. RESULTS: PE VEGF levels were 58.6+/-33.7 pg/mL, with a mean VEGF D/P ratio of 0.45+/-0.29 (range 0.06-0.93). In low-transport patients (n = 7) this ratio did not differ from high-average ones (n=5) (0.48+/-0.3 and 0.41+/-0.1, NS). In multivariate analysis, the VEGF D/P ratio showed no correlation with the independent variables included in this study. VEGF levels were higher in 15 h than in 8 h effluent; so the VEGF D/P ratios were higher as well. Regression analysis showed a direct correlation between PEVEGF levels and dwell time (r: 0.57, p = 0.03), but not between VEGF D/P ratio and dwell time. PEVEGF levels directly correlated with effluent protein content. Regression analysis showed no correlation between PEVEGF levels and age, time on PD, days of peritonitis, urea and creatinine-mass transfer coefficients, ultrafiltration capacity, and accumulated glucose dose. Multivariate regression analysis showed correlation only between PEVEGF levels and dwell time, but not with the other independent variables. CONCLUSIONS: This study confirms that VEGF is present in fresh PE from PD patients at levels that suggest local production and filtration from plasma. Peritoneal effluent VEGF levels are not significantly associated with peritoneal functional parameters and background, and seem to be influenced by ultrafiltration in a dilution process. We believe that the role of VEGF in peritoneal pathophysiology is part of a complex relationship involving multiple peritoneal structures and other growth factors, including local counteracting factors for VEGF that regulate neoangiogenesis.

Helicobacter pylori infection: a new cause of anorexia in peritoneal dialysis patients.

OBJECTIVE: Helicobacter pylori (HP) infection has frequently been found in dialysis patients. Chronic infections induce overproduction of pro-inflammatory substances. Inflammation has been associated with cachexia and anorexia. We explored the relationship between HP infection, anorexia, and malnutrition in peritoneal dialysis (PD) patients. PATIENTS AND METHODS: The study included 48 clinically stable PD patients divided into four groups: HP+ with anorexia (group I, n = 12); HP+ without anorexia (group II, n = 4); HP- with anorexia (group III, n = 5); and HP- without anorexia (group IV, n = 27). Infection with HP was diagnosed by breath test. Anorexia was evaluated using a personal interview and an eating motivation scale (VAS). The VAS included five questions that are answered before and after eating. The questions concern desire to eat, hunger, feeling of fullness, prospective consumption, and palatability. Biochemical markers of nutrition and inflammation were also determined. RESULTS: At baseline, group I showed lower scores for desire to eat, hunger sensation, prospective consumption, and palatability. They also showed lower lymphocyte counts, prealbumin, transferrin, serum albumin, normalized equivalent of protein-nitrogen appearance (nPNA), and residual renal function (RRF). In addition, the same group showed higher levels of C-reactive protein (CRP) and more sensation of fullness than the remaining groups. In the entire series, we found significant linear correlations between the following markers of nutrition and certain questions on the VAS: albumin with before-lunch desire to eat (r = 0.38, p < 0.05), and prealbumin with before-lunch hunger (r = 0.41, p < 0.05) and after-lunch hunger (r = -0.35, p < 0.05). Negative linear correlations were found between albumin and fullness before lunch (r = -0.45, p < 0.01), and between prealbumin and before-lunch desire to eat (r = -0.39, p < 0.05). Negative linear correlations were also seen between CRP and albumin (r = -0.35, p < 0.05) and between CRP and prealbumin (r = -0.36, p < 0.05). Similarly, CRP showed a negative correlation with before-lunch desire to eat (r = -0.38, p < 0.05) and afterlunch desire to eat (r = -0.45, p < 0.01). After HP eradication, group I showed a significant increase in markers of nutrition and in VAS scores for almost all questions. Simultaneously, they showed a decrease in CRP level. Significant differences were also found in lymphocyte count (1105 +/- 259.4 cells/mm3 vs 1330.8 +/- 316 cells/mm3, p < 0.05), nPNA (0.9 +/- 0.16 g/kg/day vs 1.07 +/- 0.3 g/kg/day, p < 0.05), prealbumin (26.7 +/- 6.5 mg/dL vs 33.9 +/- 56.6 mg/dL, p < 0.01), albumin (3.48 +/- 0.3 g/dL vs 3.67 +/- 0.35 g/dL, p < 0.05), CRP (1.16 +/- 1.14 mg/dL vs 0.88 +/- 1.2 mg/dL, p < 0.054), before-lunch desire to eat (56.6 +/- 6.8 vs 72.2 +/- 4, p < 0.001), after-lunch desire to eat (5.4 +/- 2.6 vs 12.3 +/- 2, p < 0.01), hunger before lunch (55.4 +/- 5.4 vs 73.1 +/- 4.6, p < 0.001), hunger after lunch (5.8 +/- 2.9 vs 11 +/- 4, p < 0.01), fullness before lunch (36.6 +/- 10.3 vs 18.7 +/- 8.8, p < 0.001), consumption after lunch (5 +/- 4.7 vs 17.5 +/- 18, p < 0.05), and palatability (61 +/- 5.3 vs 74.1 +/- 4.1, p < 0.001). CONCLUSION: Infection with HP is associated with anorexia, inflammation, and malnutrition in PD patients. Eradication of HP significantly improves this syndrome. Residual renal function seem to have a protective effect on appetite preservation. The present study supports the hypothesis of the involvement of inflammation in the pathogenesis of malnutrition in PD patients.

Comparisons of hemodialysis and CAPD in patients over 65 years of age: a meta-analysis.

This meta-analysis had the aim of studying the available studies on comparison between Hemodialysis and Peritoneal Dialysis in the elderly. The final objective was to reach, if possible, evidence for potential differences. In the case that no differences could be demonstrated, contribute to accept that HD and PD are similar techniques to be offered to elderly people requiring dialysis. The question formulated was this: Do we have adequately contrasted data on results for survival, hospitalization rate, quality of life and morbidity on hemodialysis and peritoneal dialysis in the elderly (more than 65 years old)? As data sources we selected eight papers that compared the general results of these two dialysis techniques. Different elements were considered in this selection because none reached the two first levels in the hierarchy of sources of evidence, and only two reached the third level--that of prospective studies; this is because an oral presentation of data has been included in a meta-analysis. Another four papers--uni- or multicenter retrospective studies compared the results obtained with PD and HD. The remaining two papers--reports from nationwide registries that compare of mortality rates, adjusted for co-morbid conditions and age, present specific results on groups of elderly patients. Three papers compare particular aspects of the two techniques, including nutritional status, psychiatric and psychosocial aspects and rehabilitation, in this case comparing PD with home hemodialysis patients. Finally, we have included the opinions of healthy elderly people on dialysis issues. This meta-analysis of these different studies suggests that the mortality and hospitalization rate of elderly people treated by PD is similar to that of similar people treated by HD. In consequence, we have no reasons to select either therapy on behalf of the patient. The nephrologist should consider and inform the patient and family about the relative advantages and disadvantages of both techniques and tailor dialysis technique choice to the specific individual to assure the best results. Local circumstances should also be considered.

Serum level of vascular endothelial growth factor is influenced by erythropoietin treatment in peritoneal dialysis patients. (Grupo de Estudios Peritoneales de Madrid).

Some patients on long-term peritoneal dialysis (PD) develop a hyperpermeability state, owing to peritoneal neoangiogenesis. Vascular endothelial growth factor (VEGF), a potent mitogen for endothelial cells, has been implicated in most diseases characterized by microvascular neoformation. Erythropoietin (EPO) is able to induce endothelial proliferation in vitro. Our aim was to elucidate whether VEGF serum levels are influenced by EPO treatment, and whether VEGF serum level maintains a relationship with peritoneal transport data. We analyzed serum levels of VEGF in 35 PD patients (18 males, 17 females). Mean age was 58 years, with a mean time on PD of 98 +/- 75 months. Of the 35 patients, 19 were on automated peritoneal dialysis, and 16 were on continuous ambulatory peritoneal dialysis. Seven patients had diabetes. Peritoneal transport parameters were: urea mass transfer coefficient (MTC), 19.5 +/- 6.6 mL/min; creatinine MTC, 9.9 +/- 4.7 mL/min; net ultrafiltration, 491 +/- 166 mL per 4-hour dwell. Twenty seven patients were under therapy with recombinant human erythropoietin (rHuEPO). Mean serum VEGF levels were 347 +/- 203 pg/mL (range 66-857 pg/mL), with most patients in the normal range (60-700 pg/mL). VEGF levels did not correlate with age, sex, primary renal disease, diabetes, type of PD, time on PD, peritonitis, and cumulative glucose load. We found no correlation with urea MTC, creatinine MTC, ultrafiltration rate, or protein effluent levels. However, a significant negative correlation with residual renal function was seen (r = -0.39, p < 0.05). Patients treated with rHuEPO showed significantly higher serum levels of VEGF than non treated patients (375 +/- 220 pg/mL vs 251 +/- 75 pg/mL, p < 0.05), although they had similar residual renal function. We conclude that increased serum VEGF levels are associated with EPO treatment. Consequently, VEGF might have a role in the EPO effects found in PD patients. Whether both agents are related to peritoneal neoangiogenesis requires further research.

Catheter malfunction due to spontaneous permcath displacement into medium suprahepatic vein.

One of the last options, when the other possibilities of vascular access present malfunction, is the insertion of a permanent catheter in a central vein, preferentially internal jugular vein. This option is considered when arteriovenous access is impossible. We report a case of malfunction due to a permanent catheter dis-placement solved by vascular interventional radiology.

Risk factors responsible for ultrafiltration failure in early stages of peritoneal dialysis.

OBJECTIVE: To define risk factors for ultrafiltration failure (UFF) during early stages of peritoneal dialysis (PD). DESIGN: Retrospective analysis of a group of patients whose peritoneal function was prospectively followed. SETTING: A tertiary-care public university hospital. PATIENTS: Nineteen of 90 long-term PD patients required a peritoneal resting period to recover UF capacity: 8 had this requirement before the third year on PD (early, EUFF group) and 11 had a late requirement (LUFF group). The remaining 71 patients, those with stable peritoneal function over time, constituted the control group. MAIN OUTCOME MEASURES: Peritoneal UF capacity under standard conditions (monthly) and small solute peritoneal transport (yearly). RESULTS: None of the conditions appearing at the start of PD or during the observation period could be definitely identified as the cause of UFF. There were no differences in characteristics between the EUFF group and the other two groups, except for the higher prevalence of diabetes in the EUFF group. Residual renal function (RRF) declined in all three groups during the first 2 years, with rapid loss during the third year in the EUFF group. This rapid loss in RRF was coincident with UFF. Peritoneal solute and water transport at baseline was similar in the three groups. After 2 years on PD, individuals in the EUFF group showed a significantly lower UF and higher creatinine mass transfer coefficient values than those in the LUFF group. Diabetic patients in the control group showed remarkable stability in UF capacity over time. During the second year on PD, requirement for increases in dialysate glucose concentration was 3.4 +/- 0.5% in the LUFF group, but as high as 25.5 +/- 24.2% in the EUFF group. The accumulated days of active peritonitis (APID, days with cloudy effluent) were similar for the three groups after 1, 2, and 3 years on PD. Interestingly, diabetic patients in the control group showed an APID index significantly lower than the overall EUFF group. Diabetics in the control group also had significantly lower APID versus nondiabetics in the control group (p = 0.016). CONCLUSIONS: Our findings suggest that certain patients develop early UFF type I. Diabetic state and a higher glucose requirement to obtain adequate UF suggest that glucose on both sides of the peritoneal membrane could be responsible. The mechanisms for this higher requirement remain to be elucidated. The identification of a larger cohort of these early UFF patients should lead to a better exploration of the primary pathogenic mechanisms.

Tumor necrosis factor alpha as a uremic toxin: correlation with neuropathy, left ventricular hypertrophy, anemia, and hypertriglyceridemia in peritoneal dialysis patients.

Tumor necrosis factor alpha (TNF alpha) is usually excreted by the kidney. In dialysis patients, it accumulates. TNF alpha has been implicated in the pathogenesis of malnutrition, diabetic neuropathy, and erythropoietin resistance. We studied TNF alpha plasma levels in 49 stable peritoneal dialysis (PD) patients, with the aim of correlating those levels with the presence and severity of peripheral neuropathy, hypertrophic cardiomyopathy, and anemia. Kt/Vurea' residual renal creatinine clearance (CrC), nutritional markers, and general biochemistry were also determined. The average plasma level of TNF alpha was 67 +/- 32 pg/mL (range: 18.1-156.3 pg/mL; normal value 3-20 pg/mL). No correlation was observed between TNF alpha and KT/Vurea' but a negative correlation with CrC was seen (r: -0.37, p < 0.05). TNF alpha levels were higher in patients with neuropathy as compared to patients with normal results (72.5 +/- 32 pg/mL vs 44 +/- 22 pg/mL, p < 0.05). Patients with neuropathy also showed a lower CrC value (1.5 +/- 1.7 mL/min vs 3.9 +/- 2.6 mL/min, p < 0.01). TNF alpha levels were higher in patients with left ventricular hypertrophy (LVH) with respect to normal individuals (70.4 +/- 32 pg/mL vs 38.5 +/- 20.8 pg/mL, p < 0.05). Patients with severe LVH showed the lowest CrC value. A direct, significant relationship was found between TNF alpha levels and weekly erythropoietin dose (r: 0.41, p < 0.05). Patients with hypertriglyceridemia or taking lipid-lowering agents showed a positive linear correlation between TNF alpha and triglycerides (r = 0.7, n = 14, p < 0.05). These data suggest that accumulation of TNF alpha may contribute to the development or maintenance of some neurologic, hematologic, and cardiac complications of uremic syndrome. Loss of residual renal function conditions an increment in TNF alpha levels. These data continue to add support to the idea that TNF alpha may be considered a uremic toxin.