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

2008; Elsevier BV; Volume: 73; Linguagem: Inglês

10.1038/sj.ki.5002598

1523-1755

Gloria del Peso, José A. Jiménez‐Heffernan, M. Auxiliadora Bajo, Luiz Stark Aroeira, A Aguilera, A Fernández-Perpén, Antonio Cirugeda, M J Castro, R. de Gracia, Rafael Sánchez, J.A. Sánchez-Tomero, Manuel López‐Cabrera, Rafael Selgas,

Muscle and Compartmental Disorders

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 μm) 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. 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 μm) 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. Chronic peritoneal dialysis (PD) for end-stage renal disease treatment has been used for more than 30 years.1.Gokal R. Khanna R. Krediet R. Nolph K. The Textbook of Peritoneal Dialysis. 2nd edn. Kluwer Academic Publishers, Dordrecht2000: 862Google Scholar Nowadays, the expansion of PD continues to be limited by the membrane incapacity to perform diffusive and/or convective transport over the long term.2.De Vriese A.S. Mortier S. Lameire N.H. What happens to the peritoneal membrane in long-term peritoneal dialysis?.Perit Dial Int. 2001; 21: S9-S18PubMed Google Scholar Water, sodium, and small solute transports can all be affected by this limitation.3.Selgas R. Fernández-Reyes M.J. Bosque E. et al.Functional longevity of the human peritoneum: how long is continuous peritoneal dialysis possible? Results of a prospective medium long-term study.Am J Kidney Dis. 1994; 23: 64-73Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 4.Selgas R. Bajo M.A. Del Peso G. et al.Preserving the peritoneal dialysis membrane in long-term peritoneal dialysis patients.Semin Dial. 1995; 8: 326-332Crossref Google Scholar, 5.Selgas R. Bajo M.A. Paiva A. et al.Stability of the peritoneal membrane in long-term peritoneal dialysis patients.Adv Ren Replace Ther. 1998; 5: 168-178PubMed Google Scholar, 6.Krediet R. The peritoneal membrane in chronic peritoneal dialysis patients.Kidney Int. 1999; 55: 341-356Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 7.Smit W. Van Dijk P. Lagedijk M.J. et al.Peritoneal function and assessment of reference values using a 3.86% glucose solution.Perit Dial Int. 2003; 23: 440-449Crossref PubMed Scopus (95) Google Scholar The worst functional consequence is UF failure, which results in extracellular volume overload, increased cardiovascular risk, and the restriction for technique continuity.8.Davies S.J. Phillips L. Russell G.I. Peritoneal solute transport predicts survival on CAPD independently of residual renal function.Nephrol Dial Transplant. 1998; 13: 962-968Crossref PubMed Scopus (201) Google Scholar, 9.Churchill D.N. Thorpe K.E. Nolph K.D. for CANUSA Study group et al.Increased peritoneal membrane transport is associated with decreased patient and technique survival for continuous peritoneal dialysis patients.J Am Soc Nephrol. 1998; 9: 1285-1292PubMed Google Scholar, 10.Díaz-Buxo J.A. Lowrie E.G. Lew N.L. et al.Associates of mortality among peritoneal dialysis patients with special references to peritoneal transport rates and solute clearance.Am J Kidney Dis. 1999; 33: 523-534Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 11.Ates K. Nergizoglu K. Keven K. et al.Effect of fluid and sodium removal on mortality in peritoneal dialysis patients.Kidney Int. 2001; 60: 767-776Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar Functional deterioration of the peritoneum is related to the damage induced by components of PD fluids, most likely pH, glucose, and glucose degradation products. Therefore, during the last decade many efforts have been made to improve the biocompatibility of fluids. Such an improvement of biocompatibility is expected to result in less peritoneal damage. Recently, important contributions from cell biology and histopathology studies have helped us to understand the pathophysiology of the peritoneal membrane response.12.Garosi G. Di Paolo N. Peritoneal sclerosis—an overview.Adv Perit Dial. 1999; 15: 185-192PubMed Google Scholar, 13.Krediet R.T. Lindholm B. Rippe B. Pathophysiology of peritoneal membrane failure.Perit Dial Int. 2000; 20: S22-S42PubMed Google Scholar, 14.Combet S. Miyata T. Moulin P. et al.Vascular proliferation and enhanced expression of endothelial nitric oxide synthase in human peritoneum exposed to long-term peritoneal dialysis.J Am Soc Nephrol. 2000; 11: 717-728Crossref PubMed Google Scholar, 15.Selgas R. Del Peso G. Bajo M.A. et al.Spontaneous VEGF production by cultured peritoneal mesothelial cells from patients on peritoneal dialysis.Perit Dial Int. 2000; 20: 798-801PubMed Google Scholar, 16.Selgas R. del Peso G. Bajo M.A. et al.Vascular endothelial growth factor (VEGF) levels in peritoneal dialysis effluent.J Nephrol. 2001; 14: 270-274PubMed Google Scholar, 17.Margetts P.J. Bonniaud P. Basic mechanisms and clinical implications of peritoneal fibrosis.Perit Dial Int. 2003; 23: 530-541Crossref PubMed Scopus (138) Google Scholar, 18.De Vriese A.S. Tilton R.G. Mortier S. et al.Myofibroblast transdifferentiation of mesothelial cells is mediated by RAGE and contributes to peritoneal fibrosis in uraemia.Nephrol Dial Transplant. 2006; 21: 2549-2555Crossref PubMed Scopus (107) Google Scholar Epithelial-to-mesenchymal transition (EMT) of MC has been identified as a key process leading to peritoneal fibrosis with functional deterioration.19.Yáñρez-Mó M. Lara-Pezzi E. Selgas R. et al.Peritoneal dialysis induces an epithelial–mesenchymal transition of mesothelial cells.N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (619) Google Scholar,20.Yang A.H. Chen J.Y. Lin J.K. Myofibroblastic conversion of mesothelial cells.Kidney Int. 2003; 63: 1530-1539Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Concerning human histopathology studies, it must be remarked that most peritoneal biopsy studies have been based on PD patients with long-term treatment and peritoneal functional problems, specifically UF failure.21.Mateijsen M.A.M. van der Wal A.C. Hendriks P.M.E.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525Crossref PubMed Scopus (292) Google Scholar, 22.Plum J. Hermann S. Fussholler A. et al.Peritoneal sclerosis in peritoneal dialysis patients related to dialysis settings and peritoneal transport properties.Kidney Int. 2001; 78: S424-S427Google Scholar, 23.Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479Crossref PubMed Google Scholar, 24.Bertoli S.V. Buzzi L. Ciurlino D. Morphofunctional study of peritoneum in peritoneal dialysis patients.J Nephrol. 2003; 16: 373-378PubMed Google Scholar As a consequence, advanced morphopathological changes such as fibrosis, angiogenesis, and vasculopathy are probably overrepresented. These lesions are the main cause of functional disorder after 3–8 years on PD.23.Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479Crossref PubMed Google Scholar,25.Krediet R.T. Zweers M.M. van der Wal A.C. et al.Neoangiogenesis in the peritoneal membrane.Perit Dial Int. 2000; 20: S19-S25PubMed Google Scholar Obtaining peritoneal biopsies from short- to medium-term PD patients with no functional anomalies is not easy. Except for renal transplantation, there are few other opportunities to have access to a noninjured peritoneal membrane. The understanding of pathologic processes leading to advanced peritoneal anatomical–functional disorders requires recognition of the earlier key points. Knowledge of premature peritoneal changes might reveal information sufficient to interpret the primary response to PD. The main objective of the present study was to examine such an initial phase of PD treatment. For this purpose, we have explored the peritoneal anatomical changes appearing during the first months on PD, and correlated these findings with peritoneal functional parameters determined in the same period. Since the low number of patients at first year (n=15) does not permit a deeper analysis, we performed the analysis of the overall group (35 patients) as a whole (Table 1).Table 1Peritoneal functional data of the whole seriesPatients35Peritonitis episodes7Days of peritonitis2.7±2Urea-MTAC (ml min−1)19.7±6Cr-MTAC (ml min−1)8.7±4.6UF (ml per 4 h)871±283D/P creatinine0.7±0.09D/P, dialysate/plasma; Cr-MTAC, mass transfer area coefficient of creatinine; Urea-MTAC, mass transfer area coefficient of urea; UF, ultrafiltration. Open table in a new tab D/P, dialysate/plasma; Cr-MTAC, mass transfer area coefficient of creatinine; Urea-MTAC, mass transfer area coefficient of urea; UF, ultrafiltration. Seventy-four percent of patients presented partial or total loss of the mesothelial layer. Forty percent of them showed no mesothelium at all. Sixteen patients (46%) showed some degree of submesothelial thickness (>150 μm) or fibrosis (the terms submesothelial thickness and fibrosis are used in this paper indistinctly) (Figure 1). Patients with submesothelial thickening had similar mean time on PD than patients without fibrosis (13.9±6.4 vs 13.8±7 months, P=0.97). The prevalence of submesothelial fibrosis did not vary during time on PD when we analyzed the four semesters (Figure 2). In situ evidence of EMT was present in six patients (17%). Mean time on PD was not statistically significantly different between patients with or without EMT (15±8.6 vs 13.6±6.3 months, P=0.56). There was a trend to a higher prevalence of EMT in the fourth semester on PD, but the low number of patients does not permit the statistical analysis to be performed (Figure 3). Submesothelial thickness was not associated with the presence of EMT: 83% patients with EMT had fibrosis, but 38% patients without EMT also showed fibrosis (P=0.07).Figure 2Prevalence of submesothelial fibrosis according to different semesters. The prevalence of submesothelial fibrosis was similar in the different semesters.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Prevalence of EMT according to different semesters. We found a trend to a higher prevalence of EMT in the fourth semester on PD, around 30%, when compared with the remaining, around 10–15%. The low number of patients does not permit to perform the statistical analysis between the four semesters.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Forty-seven percent of the biopsies showed myofibroblasts. All patients with EMT showed myofibroblasts, while only 36% of patients without EMT had myofibroblasts (P=0.006). One-third of patients with myofibroblasts (α-smooth-muscle actin+) also showed EMT. The number of peritoneal vessels did not vary when we compared different times on PD (Figure 4). Mild degree of vasculopathy was present in six patients (17% of the samples). Moderate degree was present only in 3% of patients, with no cases with severe vasculopathy. Patients with and without vasculopathy showed similar mean time on PD (13.6±4.6 vs 13.9±7 months, P=0.93). The prevalence of vasculopathy was similar in different semesters of treatment (Figure 5). There was no association between vasculopathy and fibrosis (83% patients with vasculopathy had fibrosis and 38% patients without vasculopathy had fibrosis; P=0.073) or EMT (33% of patients with vasculopathy had EMT and 14% patients without vasculopathy had EMT; P=0.26). Fifty percent of patients with vasculopathy had myofibroblasts, and 46% patients without vasculopathy had myofibroblasts; P=1.00. Table 2 summarizes the prevalence of different peritoneal lesions along the time observed.Table 2Prevalence of different peritoneal lesions along timeFirst semesterSecond semesterThird semesterFourth semesterSubmesothelial fibrosis33%44%54%44%EMT17%11%9%33%Vasculopathy033%9%22%Number of peritoneal vessels per field4.43.73.63.9EMT, epithelial-to-mesenchymal transition. Open table in a new tab EMT, epithelial-to-mesenchymal transition. The different peritoneal lesions were related to the peritoneal functional parameters divided into quartiles. Patients in the highest quartile of mass transfer area coefficient of creatinine (Cr-MTAC) and the median value of UF were considered the reference. Table 3 shows the prevalence of peritoneal lesions in the four quartiles of Cr-MTAC and UF values.Table 3Prevalence of peritoneal-specific lesions in different quartiles of solute and water transport for patients studied during the first year on PDCr-MTACUF capacityFirst quartileSecond quartileThird quartileFourth quartileFirst quartileSecond quartileThird quartileFourth quartileSubmesothelial fibrosis25%50%25%67%0100%25%33%EMT0025%33%25%0033%Vasculopathy25%25%033%025%25%33%Number of peritoneal vessels per field3.63.63.643.343.55.5Cr-MTAC, mass transfer area coefficient of creatinine; EMT, epithelial-to-mesenchymal transition; PD, peritoneal dialysis; UF, ultrafiltration. Open table in a new tab Cr-MTAC, mass transfer area coefficient of creatinine; EMT, epithelial-to-mesenchymal transition; PD, peritoneal dialysis; UF, ultrafiltration. Patients in the highest quartile of Cr-MTAC (>11.8 ml min−1) showed significantly higher prevalence of EMT (P=0.016) (Figure 6) and similar presence of myofibroblasts, fibrosis, and vasculopathy (P=1.00, NS) than the other quartiles. However, all showed a similar prevalence of fibrosis (first quartile, 30%; second quartile, 50%; third quartile, 56%; and fourth quartile, 50%), as well as a similar number of peritoneal vessels (first quartile: 3±1 vessels per field, n=8; second quartile: 4.7±2 vessels per field, n=2; third quartile: 4.4±1 vessels per field, n=2; and fourth quartile: 4.3±2 vessels per field, n=6) (NS). We found no relation between the presence of EMT and previous peritonitis, since one of the six patients with EMT (17%) had a previous episode of peritonitis in contrast to 6 of the 29 patients (21%) without EMT (P=0.82). Days of peritonitis did not influence these results either (2 days in EMT group vs 2.8 days in non-EMT, NS) No correlation was found between the peritoneal lesions evaluated and UF capacity. When we compared patients over and under the median value of UF (820 ml per 4 h), we found a trend to a higher prevalence of fibrosis in patients with lower UF capacity (over the median: 29.4% and under the median: 61.1%) (P=0.06). No statistical differences were found in the prevalence of EMT (> median: 5.9%, < median: 27.8%) (NS), myofibroblast presence (> median: 44%, < median: 50%) (NS), vasculopathy (> median: 18%, < median: 17%) (NS), and number of vessels (> median: 3.45±1.2 vessels per field, n=8; < median: 4.12±1.8, n=10) (NS). Table 4 shows the univariate analysis data (unadjusted odds ratio) for the presence of EMT in peritoneal biopsies. In the multivariate analysis, the highest quartile of Cr-MTAC (>11.8 ml min−1) 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). None of the variables included in our study significantly predicted the presence of submesothelial thickness or vasculopathy in peritoneal biopsies.Table 4Univariate analysisOR (95% confidence interval)PAge (years)1.05 (0.98–1.12)0.10Time on PD (months)1.03 (0.90–1.18)0.63β-blockers use (yes/no)0.81 (0.12–5.23)0.83ACEI use (yes/no)3.05 (0.31–29.7)0.33ARA-II use (yes/no)0.62 (0.06–6.32)0.69Previous peritonitis episodes (yes/no)0.76 (0.75–7.86)0.82Accumulated days of peritonitis0.72 (0.13–3.82)0.70Cr-MTAC>11.8 ml min−1 (fourth quartile)12.49 (1.69–92.23)0.01Median UF (ml per 4 h)0.16 (0.01–1.57)0.11Fibrosis (yes/no)8.17 (0.84–79.36)0.07Vasculopathy (yes/no)3.12 (0.42–23.06)0.26Number of vessels per field1.43 (0.68–2.98)0.33ACEI, angiotensin converting enzyme inhibitor; ARA-II, angiotensin II receptor antagonist; EMT, epithelial-to-mesenchymal transition; Cr-MTAC, mass transfer area coefficient of creatinine; OR, odds ratio; PD, peritoneal dialysis; UF, ultrafiltration.Unadjusted OR for the presence of EMT in peritoneal biopsies. Open table in a new tab ACEI, angiotensin converting enzyme inhibitor; ARA-II, angiotensin II receptor antagonist; EMT, epithelial-to-mesenchymal transition; Cr-MTAC, mass transfer area coefficient of creatinine; OR, odds ratio; PD, peritoneal dialysis; UF, ultrafiltration. Unadjusted OR for the presence of EMT in peritoneal biopsies. The six patients with biopsies obtained during the first 6 months on PD with no peritonitis showed two cases (33%) with submesothelial fibrosis, one (16%) with EMT tissue data, none with vasculopathy and a normal number of vessels (mean 4.45±1.2 vessels per field). In these patients, Cr-MTAC ranged from 8.7 to 16.6 ml min−1, and UF capacity ranged from 500 to 1800 ml per 4-h 3.86% glucose dwell time. Since this group consists of few patients, we examined the data from the 15 patients studied within the first year. The inclusion of two patients who have suffered peritonitis did not demonstrate significant differences when compared to the other 13. The quartile distribution of Cr-MTAC was marked by the following values: 6.26, 11.3, and 13 ml min−1. The corresponding quartile distribution for UF was 610, 800, and 1050 ml per 4-h dwell time. One case showed data of UF failure (UF=300 ml). The peritoneal biopsies showed six cases (40%) with submesothelial fibrosis, two (13%) with EMT tissue data, and three cases (20%) with vasculopathy and a normal number of vessels (mean 3.9±1.2 vessels per field). When compared to data from the first semester patients, an increased prevalence of vasculopathy was present. The remaining variables were similar. One of the two patients who had experienced peritonitis, showed fibrosis and vasculopathy. This study, based on peritoneal biopsies performed during the first 2 years on PD, showed that EMT of MCs is a frequent peritoneal morphological change more common in those patients with higher solute transport status, and that such higher transport status was not associated with an increase in the number of vessels. A new interpretation of the mechanisms associated with fast peritoneal solute transport at early PD stages arises from these data. Mesothelial-to-mesenchymal transition is defined, in vivo, by the presence of fibroblastic-like cells located in the submesothelium that express mesothelial markers such as cytokeratins. During their conversion into myofibroblasts, MCs gradually lose their location, morphology, and immunophenotype. What we detect using immunohistochemistry against cytokeratin is a subset of myofibroblasts or transitional cellular forms that still retain cytokeratin expression, reflecting their mesothelial origin. When final conversion has occurred, myofibroblasts will have lost the expression of cytokeratin and other mesothelial markers. Therefore, although specific, this detection method has low sensitivity since only a portion of transdifferentiated MCs will be detected. Mesothelial cell detachment was seen in 74% of the peritoneal biopsies. Almost half of the tissue samples showed some degree of submesothelial thickening. The two main features of EMT, that is cytokeratin and α-smooth-muscle actin+ submesothelial fibroblasts, were present in 17 and 47% of biopsies, respectively. All biopsies with cytokeratin+ fibroblasts also showed myofibroblasts, while 36% of patients with no cytokeratin+ fibroblasts had evidence of myofibroblastic differentiation (P=0.006). One-third of patients with myofibroblasts (α-smooth-muscle actin+) also showed EMT. A mild-to-moderate grade of vasculopathy was present in 17% of the series, and its association with submesothelial thickening and myofibroblast was sporadic. Longer time on PD was associated with submesothelial fibrosis only when EMT was also present. Analyzing the EMT findings over time, a remarkable higher prevalence of these findings at the fourth semester was demonstrated. It can be assumed that time on PD is a risk factor for submesothelial thickening associated with EMT after the first year. In fact, our previous findings in biopsies taken at longer PD periods26.Jiménez-Heffernan J.A. Aguilera A. Aroeira L.S. et al.Immunohistochemical characterization of fibroblast subpopulation in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis.Virchow Arch Pathol Anat Physiol Klin Med. 2004; 444: 247-256Crossref Scopus (100) Google Scholar confirmed a progressive increase in EMT incidence (up to 48%). Myofibroblast presence in biopsies was very erratic over the time examined. Although they are characteristics of EMT process, fibroblasts from other origins must be participating in the process as well.27.Bucala R. Spiegel L.A. Chesney J. et al.Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 28.Del Peso G. Jiménez-Heffernan J.A. Bajo M.A. et al.Myofibroblastic differentiation in simple peritoneal sclerosis.Int J Artif Organs. 2005; 28: 135-140PubMed Google Scholar, 29.Powell D.W. Mifflin R.C. Valentich J.D. et al.Myofibroblasts. I. Paracrine cells important in health and disease.Am J Physiol. 1999; 277: C1-C9Crossref PubMed Google Scholar Vasculopathy was absent during the first semester and present to a mild degree in the other periods examined. Its presence at these early stages reinforces the role of this lesion in the peritoneal changes secondary to PD. Vessel density (to be discussed also in transport discussion) remained stable over the periods examined. This observation suggests that vessel number remains similar during the first 2 years of a noncomplicated PD. In other words, the angiogenesis expected in later PD stages, has not yet started. One of the main concerns in peritoneal function is the status of the fast transporter. To avoid bias in the management of Cr-MTAC values in the study of its relationship with biopsy findings, we have divided this parameter into quartiles. In spite of the shortness of the series, we have had sufficient number of patients in each quartile to compare the biopsy findings. Figure 6 shows a remarkably different prevalence of EMT in fast transporters (the highest quartile of Cr-MTAC>11.8 ml min−1), approximately fivefold higher than in the other groups. The cut-off value of 11.8 ml min−1 determines a true relationship with the presence of EMT in the biopsy, confirmed in the univariate and multivariate analyses. In consequence, we can firmly corroborate the association between higher solute transport and EMT in the biopsy. This association did not exist with submesothelial thickness per se. This seems to indicate qualitative differences in the composition of the thickened submesothelial zone. To confirm this transport–anatomical relationship, it was necessary to know whether or not the vascular density was consistent with the expectation that the higher the transport, the larger the number of vessels.21.Mateijsen M.A.M. van der Wal A.C. Hendriks P.M.E.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525Crossref PubMed Scopus (292) Google Scholar,30.Numata M. Nakayama M. Nimura S. et al.Association between an increased surface area of peritoneal microvessels and a high peritoneal solute transport rate.Perit Dial Int. 2003; 23: 116-122PubMed Google Scholar Contrary to this paradigm, our patients have demonstrated that the sole presence of EMT and related fibrosis, with no increase in the number of capillaries, is sufficient to lead to a high transporter status. Other authors have found that angiogenesis is not necessarily associated with noncomplicated higher peritoneal transport at later PD stages (4–6 years).31.Sherif A.M. Nakayama M. Maruyama Y. et al.Quantitative assessment of the peritoneal vessel density and vasculopathy in CAPD patients.Nephrol Dial Transplant. 2006; 21: 1675-1678Crossref PubMed Scopus (34) Google Scholar Evidently, peritoneal lesions over PD time should be different in quality and quantity and probably are associated. The present data suggest that the early changes in response to PD are preceded by identifiable cell and extracellular matrix changes in the submesothelial compact zone. The increase in collagen and fibronectin,26.Jiménez-Heffernan J.A. Aguilera A. Aroeira L.S. et al.Immunohistochemical characterization of fibroblast subpopulation in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis.Virchow Arch Pathol Anat Physiol Klin Med. 2004; 444: 247-256Crossref Scopus (100) Google Scholar prior to the increase of vessel number, can be sufficient. In fact, submesothelial thickening is the more constant finding detected in other peritoneal biopsy studies.21.Mateijsen M.A.M. van der Wal A.C. Hendriks P.M.E.M. et al.Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis.Perit Dial Int. 1999; 19: 517-525Crossref PubMed Scopus (292) Google Scholar,23.Williams J.D. Craig K.J. Topley N. et al.Morphologic changes in the peritoneal membrane of patients with renal disease.J Am Soc Nephrol. 2002; 13: 470-479Crossref PubMed Google Scholar Animal models,32.Margetts P. Kolb M. Galt T. et al.Gene transfer of transforming growth factor-β1 to the rat peritoneum: effects on membrane function.J Am Soc Nephrol. 2001; 12: 2029-2039PubMed Google Scholar in which TGF-β transfection of MC is induced, reproduce the sequence of phenomena that seems to apparently occur in humans. These data have demonstrated that TGF-β transfection of rat MC causes peritoneal sclerosis, with previous development of angiogenesis, both processes anteceded by the EMT of MCs during the first 4 days after transfection.33.Margetts P.J. Bonniaud P. Liu L. et al.Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum.J Am Soc Nephrol. 2005; 16: 425-436Crossref PubMed Scopus (240) Google Scholar The process in humans, as it does in the transfection model, should start by the EMT-induced change of MCs secondary to TGF-β effects and continue with submesothelial zone modifications. The EMT process starts by the loss of tight junctions (E-cadherin) by MC with their subsequent detachment into effluent and migration toward the submesothelial compact zone.34.Kalluri R. Neilson E.G. Epithelial–mesen