Shorter daily dwelling time in peritoneal dialysis attenuates the epithelial-to-mesenchymal transition of mesothelial cells
© Lee et al.; licensee BioMed Central Ltd. 2014
Received: 5 February 2013
Accepted: 14 February 2014
Published: 20 February 2014
Peritoneal dialysis (PD) therapy is known to induce morphological and functional changes in the peritoneal membrane. Long-term exposure to conventional bio-incompatible dialysate and peritonitis is the main etiology of inflammation. Consequently, the peritoneal membrane undergoes structural changes, including angiogenesis, fibrosis, and hyalinizing vasculopathy, which ultimately results in technique failure. The epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs) plays an important role during the above process; however, the clinical parameters associated with the EMT process of MCs remain to be explored.
To investigate the parameters impacting EMT during PD therapy, 53 clinical stable PD patients were enrolled. EMT assessments were conducted through human peritoneal MCs cultured from dialysate effluent with one consistent standard criterion (MC morphology and the expression of an epithelial marker, cytokeratin 18). The factors potentially associated with EMT were analyzed using logistic regression analysis. Primary MCs derived from the omentum were isolated for the in vitro study.
Forty-seven percent of the patients presented with EMT, 28% with non-EMT, and 15% with a mixed presentation. Logistic regression analysis showed that patients who received persistent PD therapy (dwelling time of 24 h/day) had significantly higher EMT tendency. These results were consistent in vitro.
Dwelling time had a significant effect on the occurrence of EMT on MCs.
KeywordsDwelling time Epithelial-to-mesenchymal transition Mesothelial cells Peritoneal dialysis
In recent decades, peritoneal dialysis (PD) has been used as a therapy for end-stage renal disease (ESRD) and has been shown to provide equivalent adequacy and similar mortality rates to those of hemodialysis (HD) [1, 2]. However, 5- and 10-year technique survival rates are only about 70% and 50%, respectively [3–5]. The main causes of treatment failure are the development of recurrent peritonitis, loss of residual renal function, inadequate solute clearance, and loss of peritoneal membrane function .
There have been many advances in PD technique; however, ways to prevent the loss of peritoneal membrane function are still not fully understood. Continuous exposure to conventional bio-incompatible dialysate, which is acidic and contains large amounts of glucose degradation products (GDPs), is now thought to be one of the main causes of peritoneal morphological and functional deterioration [6, 7]. After undergoing PD, patients present with a reduction of microvilli, epithelial-to-mesenchymal transition (EMT) of mesothelial cells (MCs), sub-mesothelial fibrosis, and microvasculature disorders [8–10]. Consequently, the efficiency of dialysis decreases and leads to PD failure .
The EMT of MCs plays an important role in this pathophysiology . After dialysis, peritoneal MCs initiate the EMT process via a progressive loss of the epithelial phenotype, and they acquire a myofibroblast-like morphology [10, 13]. Then, those MCs contribute to peritoneal angiogenesis and fibrosis, two main causes of PD failure [10, 12]. Therefore, the EMT may be a key factor in peritoneal alterations during PD. However, there is limited information and no consistent result regarding the clinical parameters associated with EMT in previous studies [10, 12, 14–18]. Moreover, classifying EMT only according to morphology is subjective and unreliable.
The aims of this study were to determine the clinical factors associated with the EMT in PD patients and then to explore potential therapeutic interventions.
Patients and mesothelial cells
Subgroup classifications of peritoneal dialysis patients
In vitrostudy design
Flow cytometry and immunofluorescence
The purity of the MC from effluents and omentum was determined by ICAM-1 expression (anti-ICAM-1; eBioscience, San Diego, CA), and subgroup classifications of MCs from effluents were determined by cytokeratin 18 expression (anti-cytokeratin 18, Santa Cruz Biotechnology, Santa Cruz, CA). Phalloidin-labeled staining was used to investigate the expression of MCs cytoskeletal actin as described previously .
Western blotting for E-cadherin
To validate the changes in EMT of MCs, the expression of the epithelial marker, E-cadherin (anti-E-cadherin; BD Bioscience, Bedford, MA), were identified . The blots were incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich, San Luis, MO) to each primary antibody and detected using enhanced chemiluminescence (Chemicon International, Billerica, MA).
Calculation of mesothelium cell count
The MC count was determined by repeated calculations in a counting chamber to identify the rest of the surviving cells.
Continuous data were expressed as mean ± S.D. P < 0.05 was considered to be statistically significant. The associations between patients’ clinical characteristics and factors associated with the EMT of the MCs were analyzed using a univariate logistic regression. Significant predictors (P < 0.2) in the univariate analyses and other possible factors that may influence EMT of the MCs were included in the multivariate analysis. Odds ratios (OR) with 95% confidence intervals (CI) for each variable were used to estimate the relative risk (RR) of EMT. Data of the in vitro study were analyzed using nonparametric statistics. All experimental data were performed at least in triplicate. All data were analyzed using SPSS statistical software package, version 18 (SPSS Inc., Chicago, IL, USA).
The study was approved by the ethics committee/institutional review board of E-Da Hospital, and written informed consent was obtained from all patients. (IRB number: EMRP18100N).
Effects of dwelling time on the EMT of MCs
Basic characteristics of patients, according to presence of epithelial-to-mesenchymal transitioning
Non-EMT group (N = 15)
Mixed group (N = 8)
EMT group (N = 25)
57.7 ± 14.7
57.4 ± 7.4
51.0 ± 9.1
Male, n (%)
23.0 ± 5.2
24.6 ± 3.6
24.5 ± 3.4
Diabetes mellitus (%)
3.7 ± 0.5
3.8 ± 0.4
3.8 ± 0.3
10.0 ± 1.5
10.8 ± 1.4
10.0 ± 1.5
Residual renal function
2.0 ± 0.4
2.4 ± 0.9
2.1 ± 0.5
Glucose load (kg)a
52.3 ± 49.4
103.8 ± 102.1
77.1 ± 95.2
PD duration (months)
15.9 ± 13.9
20.2 ± 23.0
15.9 ± 11.9
Total duration of dialysate exposure (months)b
12.4 ± 14.7
20.1 ± 23.1
14.3 ± 12.0
Group 1 (%)
Group 2 (%)
Group 3 (%)
0.8 ± 1.7
1.0 ± 1.5
0.4 ± 1.0
RAAS inhibitor (%)
Univariate and multivariate ordinal logistic regression analyses for determining the predictive factors of epithelial-to-mesenchymal transition
OR (95% CI)
OR (95% CI)
Diabetes Mellitus (yes/no)
Residual renal function
Glucose load (Kg)a
PD duration (months)
Total duration of dialysate exposure (months)b
Dwelling time (grouping)c
Group 2 to 1
Group 3 to 1
RAAS inhibitor (yes/no)
Cell survival was evaluated and determined indirectly by the cell counts, (Figure 5B). On day 3, the 24 h group demonstrated significantly lower cell counts compared to the PBS group (P < 0.05). On day 6, the 16 and 24 h groups demonstrated significantly lower cell counts compared to the PBS group (PBS vs. 16 h: P < 0.05; PBS vs. 24 h: P < 0.01). Additionally, the 24 h group also had a significantly lower cell count compared to the TGF-β1 group (P < 0.05). These indicate that a shorter dwelling time provides a better environment for MC survival, as it may induce less EMT-related stress.
The EMT of peritoneal MCs plays an important role in peritoneal membrane dysfunction [10, 13, 14]. However, there is limited information and no consistent result regarding the clinical parameters associated with EMT (Additional file 1: Table S1) [10, 12, 14–18]. This may be related to the fact that there are no standard and objective criteria for the classification of EMT of MCs from effluents because of classifying the EMT of MCs only according to morphology is both subjective and unreliable. In previous studies, only a type of dialysate, PD duration, and peritonitis/hemoperitoneum have been found to have a potential association with EMT [10, 15–17]. However, the results on PD duration and episodes of peritonitis/hemoperitoneum differed between studies, and only dialysates containing low GDPs concentrations have been shown to prevent EMT absolutely [15, 17, 18]. To our knowledge, no previous study has investigated whether dwelling time influences the EMT of MCs. In a present study, we established “standard and objective” criteria for the classification of EMT. In addition to morphology, we classified MCs from effluents also according to cytokeratin 18 expression because cytokeratin 18 is one of the most common EMT markers [11, 13, 22]. We believed this method to be more objective, and based on these criteria, we determined that the dwelling time per se affects peritoneal EMT. Patients who received continuous bio-incompatible PD therapy (i.e. dwelling time of 24 h/day) had greater EMT tendency, and this tendency was not suppressed by one exchange per day of icodextrin-containing solution. This result means that persistent contact with bio-incompatible dialysate leads to MCs suffering from continuous stress and as a result, causes EMT. On the other hand, intermittent contact with bio-incompatible dialysate (dwelling time ranging from 8 to 20 hours) may allow MCs to organize a repair process during the resting period. One concern is that RRF may be one factor which can influence EMT, so we also took it into analysis. The result showed that RRF was not a factor influence EMT.
Herein, we replaced the PD duration with the total duration of dialysate exposure because we thought that the latter may be more correlated with total dialysate contact time rather than the former. We also considered that the controversial findings on the effects of PD duration on EMT in previous studies may have been due to the above stated reason. Ultimately, we found there to be no correlation between the total duration of dialysate exposure and EMT.
It has been proven that patients who use more bio-compatible dialysate containing lower amounts of GDPs have a lower occurrence of peritoneal EMT [15, 17, 18]. On the contrary, the effect of icodextrin-containing solution on EMT still unknown. In our study, there was no difference in the occurrence of the EMT of MCs in either subjects who received continuous dialysis with a daily dwelling time of up to 24 h or in those who were interrupted with a daily exchange of icodextrin-containing solution. This may be related to a moderate amount of GDPs and the acidic feature of the icodextrin-containing solution, which may activate inflammatory cytokines, chemokines and promote EMT [23, 24]. In addition, glucose load also showed no correlation with the EMT process. Taken together, this may mean that although glucose may induce the EMT process , it may play an insignificant role.
There are still some limitations remaining that should be noted. First, there is still some controversy about whether the MCs collected from effluent can represent the MCs that are still attached at the peritoneum. It has been suggested that a peritoneal biopsy is a more reliable method for such assessments . However, the biopsy procedure is difficult and impractical in clinical practice for most clinically stable PD patients. Additionally, the tissues obtained from these biopsies represent only a very small part of the entire peritoneum. In contrast, the MCs from the effluent are easily collected, and there are no complications in practice. Furthermore, increasing data from in vivo and in vitro studies all indicate that MCs from the effluent are representative of the MC population remaining on the peritoneum [8, 10, 12]. The second limitation is that the culture failure rate from the effluent was quite high. We think this may be due to the fact that the number of cells collected from the dialysate effluent was always less than the cell culture requirement because when the MC culture was from omentum; our success rate was almost 100%. The third limitation is that few incident PD patients were enrolled in our study. This may suggest that we couldn’t completely exclude the effect of PD duration. Another limitation is that in our in vitro study model, persistent high concentration of glucose, pH and GDP in culture medium is different with markedly decreased concentration of glucose and increased pH with time during one dwell in PD patients. We think in vivo study in future will be a good solution to this problem and can prove more solid evidence . Finally, in our study, we didn’t provide the mechanisms to explain which the most important factor on EMT is in short dwelling PD including glucose, GDP, and dialysate pH. This indicates the necessary for further study in the future.
We provide evidence that daily dwelling time per se has a significant impact on the occurrence of EMT of MCs. Because there’s no evidence showing that continuous PD therapy provides better outcomes in people who don’t have ultrafiltration or clearance problems, perhaps these patients should avoid 24 h of continuous dialysis in the future.
α-smooth muscle actin
Body mass index
End-stage renal disease
Glucose degradation products
Intercellular adhesion molecule-1
Residual renal function
Transforming growth factor-β1.
We are indebted to Shin-Han Tseng, Chu-Hung Tsai and Yi-Chun Teng for critical discussion. This study was supported by EDAHP-102034, EDAHP-102026 & NCKUEDA10202 from Research Foundation of E-DA Hospital, and National Cheng Kung University, Taiwan.
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