29 research outputs found

    Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis

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    Background. Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. Methods. We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. Results. Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). Conclusions. EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EP

    Immunohistochemical findings concerning the pathogenesis of encapsulating peritoneal fibrosis (EPS) in peritoneal dialysis patients

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    Immunhistochemische Untersuchungen ĂŒber die Pathogenese der verkapselnden Peritonealsklerose (EPS) bei Peritonealdialyse-Patienten Hintergrund Die verkapselnde Peritonealsklerose (EPS) stellt eine schwerwiegende und teilweise lebensbedrohliche Erkrankung bei Peritonealdialyse-Patienten dar, die in vielen FĂ€llen zum Abbruch der Peritonealdialyse fĂŒhrt. Die Pathogenese der EPS ist nicht endgĂŒltig geklĂ€rt. Es gibt mehrere AnsĂ€tze, die ĂŒber den Einfluss von Dialysaten, einer sukzessiven EntzĂŒndung und einer Hochregulierung von Transkriptionsfaktoren, wie NF-kappa B versuchen, die Ätiologie zu erklĂ€ren. In dieser Arbeit haben wir den Einfluss verschiedener Transkriptionsfaktoren und anderer Proteine auf die Entwicklung der EPS untersucht. Methoden Es wurden histologische Proben von Peritonealdialyse-Patienten mit EPS [n=9] verwendet. Diese wurden mit verschiedenen histologischen und immunhistologischen FĂ€rbungen behandelt. Neben einer HĂ€matoxylin-Eosin-FĂ€rbung (HE) wurden Antikörper gegen NF-kappa B, TGF-beta 1, TGF-beta 2, TGF-beta-Rezeptor I, TGF-beta-Rezeptor II, FGF-BP, CTGF und VEGF angewandt. Die Arbeit umfasste weiterhin vier verschiedene Kontrollgruppen: eine Gruppe mit Peritonealdialyse-Patienten (CAPD) ohne EPS [n=10], eine Gruppe mit Patienten mit Peritonealfibrose [n=11], eine Gruppe mit chronischer Appendizitis [n=10] und eine Gruppe mit Peritonea, die histologisch als Normalbefund gewertet worden war [n=10]. Die Proben wurde verblindet untersucht und mittels semiquantitativer Graduierung wurden fĂŒr HE Mittelwerte und fĂŒr die immunhistochemischen FĂ€rbungen ein Immunreaktiver Score (IRS) bestimmt. Weiterhin wurden fĂŒr alle Patienten im Labor Blutkonzentrationen fĂŒr Kreatinin, Calcium, HĂ€moglobin und Leukozyten bestimmt. Ergebnisse Bei den Blutwerten gab es signifikant höhere Leukozyten-Konzentrationen bei den Patienten mit der Appendizitis. Die HĂ€moglobin-Konzentrationen waren bei den Gruppen mit EPS und CAPD signifikant niedriger als in den Vergleichsgruppen, gleichzeitig war die Kreatinin-Konzetration in den gleichen Gruppen signifikant höher. Bei den Calcium-Konzentrationen gab es keine signifikanten Unterschiede. Bei der HE-FĂ€rbung zeigte sich bei der Gruppe der Appendizitiden eine signifikant stĂ€rkere AnfĂ€rbung. Bei den immunhistochemischen AnfĂ€rbungen gegen NF-kappa B und FGF-BP ergaben sich nur bei den Gruppen der CAPD und Appendizitis signifikant höhere Werte. Bei den Untersuchungen der TGF-beta-Familie zeigte sich, dass vor allem die Gruppen Peritonealfibrose, CAPD und Appendizitis höhere Werte erzielten. Die Gruppe der EPS erzielte nie höhere Werte als die Gruppe und CAPD. In den meisten FĂ€llen sich eine deutlich geringere AnfĂ€rbung. Bei der Untersuchung von CTGF erreichten wieder nur die Peritonealfibrose und die CAPD signifikant höhere Werte. Die Untersuchung von VEGF zeigte als einzige eine signifikant stĂ€rkere AnfĂ€rbung bei der EPS. Diskussion Bisher war man von einer Pathogenese ausgegangen, die ĂŒber mehrere Schritte ablĂ€uft. Durch jahrelange Exposition mit sauren oder stark glucosehaltigen Dialysaten kommt es zu einer chronischen EntzĂŒndung. Durch die Wirkung proinflammatorischen und profibrotischen Faktoren, die bei der Reizung und EntzĂŒndung des Peritoneum ausgeschĂŒttet werden, kommt es zu einer starken Fibrosierung des Peritoneum. Die angesprochenen proinflammatorischen und profibrotischen Proteine wie TGF-beta, NF-kappa B, FGF-BP und CTGF, die wir bei der Studie untersucht haben, waren jedoch nicht hochreguliert, sondern in allen FĂ€llen sogar schwĂ€cher exprimiert als bei der Gruppe der CAPD, die im Hinblick auf die Pathogenese der EPS am Anfang stehen mĂŒsste. Auch in der Gruppe der Peritonealfibrose waren die entsprechenden Faktoren deutlich stĂ€rker exprimiert, obwohl man bei der EPS histologisch von einer starken Fibrosierung spricht. Der einzige untersuchte, subzellulĂ€re Faktor, der bei der EPS erhöht und im Vergleich zum normalen Gewebe sogar signifikant erhöht war, war VEGF. VEGF ist ein starker angiogenetischer Faktor, der viele neue, aber brĂŒchige und durchlĂ€ssigere GefĂ€ĂŸe wachsen lĂ€sst. Dieses Ergebnis stĂŒtzt ein weiteres ErklĂ€rungsmodell fĂŒr die EPS: die sogenannte „two-hit-Theorie“. Nach dieser Theorie besteht der erste „hit“ in der langjĂ€hrigen SchĂ€digung durch die Exposition mit Dialysaten. Es kommt durch die Wirkung von VEGF zur Neoangiogenese, wie oben beschrieben. In einem zweiten „Schlag“ kommt es in einem als „Plasmaleak“ bezeichneten Prozess zum vermehrten Austritt von Fibrin. Fibrin wird unter anderem von der Mastzell-Tryptase abgebaut. Die Mastzellen und damit auch die Tryptase ist bei der EPS jedoch erniedrigt und somit wird die Akkumulation von Fibrin nicht gehindert und das Gewebe fibrosiert.Immunohistochemical findings concerning the pathogenesis of encapsulating peritoneal fibrosis (EPS) in peritoneal dialysis patients Background Encapsulating peritoneal fibrosis (EPS) is a severe and partially life-threatening disease in peritoneal dialysis patients, which in many cases leads to withdrawal from peritoneal dialysis. The pathogenesis of EPS has not been solved definitively yet. Many authors explain its etiology through the influence of certain dialysis solution leading to a consecutive inflammation and up regulation of transcription factors, such as NF-kappa B eventually causing severe fibrosis. The goal of this dissertation is to determine the influence of certain transcription factors and other proteins on the development of EPS. Methods Histological samples of patients with EPS [n=9] were stained with different histological and immunohistochemical staining, such as hematoxylin-eosin (HE) and antibodies against NF-kappa B, TGF-beta 1, TGF-beta 2, TGF-beta-receptor I, TGF-beta-receptor II, FGF-BP, CTGF and VEGF. We included samples of four control groups in the study that underwent the same stainings: one group of peritoneal dialysis patients without EPS [n=10], one group of patients with histologically confirmed peritoneal fibrosis which could not be linked to peritoneal dialysis (CAPD) [n=11], one group of patients with histologically confirmed chronic appendicitis [n=10] and one group of patients whose peritonea were found normal in histological examinations. The stains were graded through blinded semiquantitative examination leading to a mean value in HE stains and to an immunoreactive score (IRS) in the antibody stains. Finally we collected laboratory values for creatinine, calcium, hemoglobin and complete WBC for the above mentioned groups. Results Concerning the WBC we found significantly higher values in patients with chronic appendicitis compared to the other groups. Hemoglobin levels were significantly lower in patients with EPS and peritoneal dialysis without EPS, while also having significantly higher creatinine levels, compared with the other groups. The evaluation of the calcium levels showed no significant differences. In samples of chronic appendicitis we found a significantly more intense staining compared to the other groups. Immunohistochemical stains with antibodies against NF-kappa B and FGF-BP lead to a significantly higher IRS in the groups of peritoneal fibrosis without CAPD and chronic appendicitis. Examining the stains of the TGF-beta family we found significantly higher IRS in the groups of chronic appendicitis, peritoneal dialysis without EPS and peritoneal fibrosis without CAPD. The EPS group never showed higher values in the IRS in these stains compared to the group of peritoneal fibrosis without CAPD. The stains with antibodies against CTGF showed a significantly higher IRS in the groups of peritoneal fibrosis and peritoneal dialysis without EPS. Finally we examined the stains with antibodies against VEGF, showing significantly higher levels in the IRS compared to the control groups. Discussion Long lasting exposition with acidic dialysis solutions or solutions with high concentrations of glucose leads to chronic inflammation of the peritoneum. The development of severe fibrosis is promoted by profibrotic cytokines and transcription factors which are expressed in higher levels in tissues with high inflammatory activity. Assuming a development of EPS as a result of long lasting CAPD, many authors expect higher levels of NF-kappa B, TGF-beta, FGF-BP and CTGF in peritonea of EPS patients. We showed that those factors were not up-regulated in the group of patients with EPS and had in some cases even lower IRS than the group of peritoneal dialysis without EPS. VEGF is the only subcellular factor reached by our investigations, which was significantly up-regulated in patients with EPS. VEGF is a potent angiogenetic factor leading to the growth of many new, but very fragile vessels especially in malignancies. Our findings support another explanatory model for the development of EPS: the “two-hit theory”. According to this theory, the first “hit” is represented by the long lasting exposition to dialysis solutions and the consecutive impairment of the peritoneum. This impairment leads to an increased expression of VEGF and consecutively to neoangiogenesis. The second “hit” marks a process known as the plasma leak, which leads to an increased discharge of fibrin through these vessels. Fibrin is usually degraded by the mast cell tryptase. In EPS patients the activitiy of the mast cell tryptase in the peritonea is reduced and therefore an accumulation of further fibrin cannot be prevented and the tissue becomes fibrotic

    Classification of EPS and non-EPS cases by using random forests.

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    <p>The first column shows a scaled measure of the relative importance of each predictor variable. The second column lists the p-values related to one-sided z-tests. The appearance of the predictor variables was ordered according to decreasing importance. The estimated misclassification error rate for new cases is 14%.</p

    Histopathological findings in EPS compared to simple sclerosis.

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    <p><b>A</b> D2–40 stained section showing podoplanin positive cells not associated to vessels (arrows). EPS, original magnification×400; <b>B</b> HE staining showing acute and chronic inflammation with round cells and neutrophils (arrows). EPS, original magnification×400; <b>C</b> HE staining showing fibroblast like cells, eosinophils, plasma cells and round cells (arrows). EPS, original magnification×400; <b>D</b> HE staining showing vasculitis, round cells and calcium deposits (arrows). EPS, original magnification×100.</p

    Histopathological findings in EPS compared to simple sclerosis.

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    <p><b>A</b> HE staining showing an increased cellularity, round cells and fibroblast like cells (arrows). EPS, original magnification×400; <b>B</b> HE staining showing a decreased cellularity, fibrin deposits and a complete denudation of the mesothelial cell layer with fibrin exudations (arrows). EPS, original magnification×100; <b>C</b> HE staining showing a decreased cellularity with intracellular matrix (arrows), complete mesothelial denudation with fibrin exudations. EPS, original magnification×200; <b>D</b> HE staining showing an increased cellularity, hemorrhage, round cells, fibroblast like cells and fibrin deposits (arrows). EPS, original magnification×400; <b>E</b> Fe staining showing vessels, intraluminal erythrocytes and Fe deposits (arrows). EPS, original magnification×400; <b>F</b> D2–40 stained section showing podoplanin positive cells associated to vessels (arrows). EPS, original magnification×400.</p

    Clinical data of study patients.

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    <p>PD, peritoneal dialysis; EPS, encapsulating peritoneal sclerosis; PDF, peritoneal dialysis fluid; Hb, haemoglobin; N.D., not determined; PTH, parathyroid hormone,</p>***<p>p<0.001.</p>**<p>p<0.05.</p>*<p>p<0.1.</p
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