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NDT Advance Access originally published online on July 29, 2007
Nephrology Dialysis Transplantation 2007 22(12):3593-3600; doi:10.1093/ndt/gfm497
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


The cellular contribution to effluent potassium and its relation to free water transport during peritoneal dialysis

Annemieke M. Coester1, Dirk G. Struijk1,2, Watske Smit1,2, Dirk R. de Waart3 and Raymond T. Krediet1

1Division of Nephrology, Department of Internal Medicine, Academic Medical Center, University of Amsterdam, 2Dianet Foundation Amsterdam-Utrecht and 3Department of Experimental Hepatology, Academic Medical Center, University of Amsterdam, The Netherlands

Correspondence to: Annemieke Marcella Coester, MD, Academic Medical Center, Department of Internal Medicine, Division of Nephrology, A01-114, PO Box 22700, 1100 DE Amsterdam, The Netherlands. Email: a.m.coester{at}amc.uva.nl



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Aquaporin-1 (AQP-1) dysfunction is one of the valid theories for decreased free water transport (FWT) in long-term peritoneal dialysis (PD) ultrafiltration failure (UFF). We questioned whether apoptosis of peritoneal cells could be reflected in an increased release of cellular (CR) K+ and explain AQP-1 dysfunction. If so, negative relationships between CR-K+ and FWT would be expected. Therefore, we analysed CR-K+ to total peritoneal K+ removal, for possible relationships with FWT, the duration of PD, the presence of late UFF and effluent cancer antigen (CA) 125.

Methods. Standard peritoneal permeability analyses done with 3.86% glucose were investigated cross-sectionally in three extreme groups: group I: 19 patients <1year on PD; group II: 20 patients >4 years on PD without UFF; group III: 19 patients >4 years on PD with UFF.

Results. Group III had the lowest values of FWT and CR-K+ (P < 0.01). CR-K+ had a positive correlation with FWT in groups I and II, but not in group III. These correlations were also present using much simpler methodologies: replacement of CR-K+ by mass transfer area coefficient (MTAC)-K+/MTAC-creatinine ratio or dialysate over plasma (D/P)-K+/D/P-creatinine ratio and replacement of FWT by Na+-sieving. No relationship with CA125 was present.

Conclusions. This study shows that other than diffusive and convectional, K+ transport is not excluded in patients treated with conventional glucose-based PD solutions. We found evidence for release of K+ from cells. In general, CR-K+ was related to parameters of FWT, except for long-term patients with UFF. This suggests glucose-induced hypertonic cell shrinkage as a basic physiological phenomenon during PD. The absence of this relationship in long-term PD patients with UFF either suggests a reduction or inhibition of K+-channels and may be due to another mechanism than AQP-1 dysfunction. Most likely, CR-K+ in UFF does not reflect apoptosis. However, the D/P-K+/D/P-creatinine ratio may be useful in detecting peritoneal changes.

Keywords: fluid kinetics; peritoneal dialysis; peritoneal transport; potassium; ultrafiltration failure; water channels



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Long-term peritoneal dialysis (PD) can lead to ultrafiltration failure (UFF) [1]. The International Society of Peritoneal Dialysis defined UFF as net ultrafiltration (NUF) <400 ml after a 4 h dwell using a modified peritoneal equilibration test with a 3.86% glucose solution [2]. It is mostly associated with high transport rates of small solutes, such as creatinine and glucose, the latter leading to a fast disappearance of the crystalloid osmotic gradient [3]. This suggests a large peritoneal vascular surface area, which is reflected by a high mass transfer area coefficient (MTAC) of creatinine. In long-term PD this has been morphologically demonstrated as neoangiogenesis, a more permanent peritoneal membrane alteration [4]. Long-term PD UFF is also associated with a decreased osmotic conductance to glucose, suggesting decreased free water transport (FWT). This was present in 43% of patients with UFF, who had PD for more than 4 years [5]. There is evidence that aquaporin-1 (AQP-1) represents the most important route for FWT across the peritoneal membrane [6,7]. However, decreased FWT can be present without reduced expression of AQP-1, suggesting functional impairment [8]. Another equally valid theory of reduced AQP-1-mediated water transport in late UFF is the presence of a thickened interstitium or interstitial fibrosis, hampering FWT in late UFF [9]. At present, the exact pathogenetic mechanism is unclear, but this study focuses on AQP-1 dysfunction as a possible explanation for late UFF.

Recently Jablonski et al. [10] reported that thymocytes and granulocytic ovarian cells, going into apoptosis, had a decreased AQP-1 function despite a normal AQP-1 expression. In vitro studies demonstrated that exposure to glucose and/or glucose degradation products can lead to apoptosis of several cultured cells, including endothelial [11] and mesothelial cells [12]. The apoptotic process plays a critical role in tissue homoeostasis: a balance between proliferation and apoptosis. K+-ions are important in the maintenance of the so-called ‘basic’ cell physiology. Regulated K+-transmembrane transport is a major determinant of cell volume regulation, electropotential control, and the balance of cell proliferation and apoptosis [13]. Excessive K+-efflux and intracellular K+-depletion are key early steps in apoptosis [10]. In PD, peritoneal K+-transport is probably not only determined by diffusion, but also by K+-release from cells [14,15].

We questioned whether apoptosis of peritoneal cells could be reflected in an increased release of cellular (CR) K+ and explain AQP-1 dysfunction. If so, negative relationships between CR-K+ and FWT would be expected. Therefore, the aim of the study was to analyse cellular contribution of K+ to total peritoneal K+ removal, for possible relationships with FWT, the duration of PD, and the presence of late UFF. Because mesothelial cells form the first layer of cells in the peritoneal cavity, cellular contribution of K+ was also analysed for possible relationships with effluent cancer antigen (CA) 125, which reflects mesothelial cell mass [16]. We found positive correlations between CR-K+ and FWT in patients starting PD and in long-term PD patients without UFF. These results made apoptosis of peritoneal cells unlikely as an explanation of AQP-1 dysfunction. The phenomenon of hypertonic cell shrinkage is suggested as possible explanation for these findings.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Standard peritoneal permeability analyses of clinically stable patients, available from two earlier studies [17,18], were investigated cross-sectionally. The patients were divided into three groups: group I consisted of 19 patients who had PD treatment less than 1 year. Group II consisted of 20 patients without UFF and group III of 19 patients with UFF. Both groups had more than 4 years of PD treatment.

Subjects
In group I, median age was 52 years, range 24–73. Median PD duration was 4 months, range 1–8. Median NUF was 568 ml/4 h, range –295 to 1169. In group II, median age was 53 years, range 26–75. Median PD duration was 57 months, range 48–144. Median NUF was 671 ml/4 h, range 429–1115. In group III, median age was 46 years, range 20–75. Median PD duration was 82 months, range 48–158. Median NUF was 120 ml/4 h, range –659 to 390. In group I, 6 patients had UFF. These patients had very high effective lymphatic absorption (ELA) (median 788 ml/4 h, range 327–1297) in contrast to those in group III (median 451 ml/4 h, range 104–1583). They were not excluded from group I, because UFF accompanied by a high ELA is considered a different mechanism than long-term PD UFF. NUF is given in Table 1. All patients used commercially available glucose-based solutions (Dianeal®, Baxter Healthcare Ltd., IRL).


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  		Table 1. A comparison of peritoneal fluid kinetics between the three groups

 
Procedure
The standard peritoneal permeability analyses were performed during 4 h dwells, as described previously [18]. Briefly, after a rinsing procedure, a fresh 3.86% glucose-based dialysis solution (Dianeal®, Baxter Healthcare Ltd., IRL) was instilled for a test dwell. Dialysate samples were taken at 0, 10, 20, 30, 60, 120, 180 and 240 min. Blood samples were taken at the beginning and the end of the test. To calculate peritoneal fluid kinetics, dextran 70 (Hyskon®, Medisan Pharmaceuticals AB, Uppsala, Sweden) 1 g/l was added. Dextran 1 (Promiten®, NPBI, Emmercompascuum, The Netherlands) was given intravenously before instillation of the test solution to prevent possible anaphylactic reactions to dextran 70.

Measurements
All measurements were done as described previously [18]. High-performance liquid chromatography was used to determine total dextran concentration in effluent. Ion selective electrodes were used to measure Na+ and K+. Creatinine, urea and urate in plasma and effluent were measured by enzymatic methods (Hitachi, Boehringer Mannheim, Germany). β2-Microglobulin was determined with an IMx system, applying a micro-particle enzyme immuno assay (MEIA) (Abbott Laboratories, North Chicago, IL, USA). Total plasma protein was determined by biuret methodology (Roche, Almere, The Netherlands), also with the use of an automated analyser (Hitachi 747, Boehringer Mannheim, Germany). Dialysate CA125 was determined in the 4 h effluent by a MEIA (Abbott Laboratories, North Chicago, IL, USA) using a commercially available monoclonal antibody OC125 (Fujirebio Diagnostics, Inc, Malvern, PA, USA) on a IMx auto-analyser. This assay has a lower detection limit of 0.4 U/ml.

Calculations of solute and fluid transport
Solute and fluid transport parameters were calculated as described previously [18]. Transport of small solutes was calculated as MTAC and dialysate over plasma (D/P) ratios. MTACs were calculated according to the model of Waniewski et al. [19]. Solute concentrations in serum were corrected for plasma water. Pathways of fluid transport during PD are shown in Figure 1. An explanation of the model is given in the legend of Figure 1.


Figure 1
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  		Fig. 1. Schematic overview of the pathways of fluid transport in peritoneal dialysis. TCUF is induced by the crystalloid osmotic gradient across the peritoneal membrane. It comprises water transport trough small interendothelial pores (SPT) and ultrasmall transendothelial pores, so-called FWT. The amount of transported water across the large pores (LPT) is considered negligible. Changes in intraperitoneal volume ({Delta}IPV) result from TCUF and fluid reabsorption. Fluid reabsorption includes lymphatic absorption, disappearance to the interstitial tissues (together with ELA) and backfiltration into the capillaries. {Delta}IPV during the dwell was assessed with the intraperitoneal administered volume marker dextran 70. Its clearance from the peritoneal cavity underestimates the amount of backfiltration into the capillaries, first, because it is unlikely that it accompanies colloid osmosis-induced backfiltration across small pores. Secondly, backfiltration through the large pores is also unlikely due to the hydrostatic pressure in the capillaries. Therefore, in our model NUF or {Delta}IPV is TCUF minus ELA. NUF was calculated as the difference between the IPV at the end and the initial IPV. TCUF was calculated from the dilution of the volume marker, by subtracting the initial IPV from the theoretical IPV (when both fluid absorption and sampling would not be present).

 
Calculation of free water transport
Along with the transport of water solutes are transported, so-called solvent drag or convective transport. As sodium (Na+) has an Einstein–Stokes radius in vivo not exceeding 3 Å, its sieving coefficient across the small pores should approach zero. This implies that small pore water transport (SPT) is always associated with convective transport of Na+, so-called sieving of Na+. According to the current understanding of the pore theory of transcapillary transport during PD, the dissociation in the transport of water induced by crystalloid osmotic pressure and that of Na+ can only be caused by transcellular FWT. Therefore, FWT was calculated as described previously by Smit et al. [20] by substracting transcapillary ultrafiltration (TCUF) coupled to Na+-transport, which is fluid transport through small pores (SPT), from TCUF. The contributions of FWT and SPT were examined after 60 and 240 min and expressed in absolute values and as a percentage of TCUF. Additionally, the maximal dip in the D/P of Na+, which is the difference between the initial D/P Na+ and the lowest D/P Na+ (usually after 1–2 h), was determined. Most often the dialysate Na+ concentration before instillation is lower than the plasma Na+ concentration, resulting in some diffusion of Na+ from the extracellular volume (the circulation). For that reason, a diffusion correction for Na+ sieving was performed using the MTAC of urate [21].

Calculations on cellular release of K+
For each individual patient, diffusion/transport lines were calculated, based on the least squares regression analysis of the D/P ratios of the small solutes and their free diffusion coefficients in water at 20°C (D20,w in cm2/s/107) when plotted on a double logarithmic scale. The slope of this line represents the size selectivity of the peritoneal membrane. D20,ws of urea (130), creatinine (83.3), urate (76.4) and β2-microglobulin (13.3) were used (22]. The regression coefficients all exceeded 0.97 (P < 0.01). In the formed diffusion/transport lines, interpolation of the D20,w of a solute results in its transport by means of diffusion only [22,23]. Interpolating the D20,w of K+ (93.0), obtained from an in vitro PD model, resulted in the expected D/P ratio. Effluent K+ due to cellular release was defined as the difference between the measured and expected D/P ratio. The measured D/P–K+ exceeded diffusion in 18 of 19 patients in group I, all patients in group II and 17 of 19 patients in group III. D/P ratios at the different time points (10, 20, 30, 60, 120, 180 min and at the end of the dwell) were used to calculate CR-K+ as the absolute amount of K+ released by cells. This was done by multiplying the difference between measured and expected D/P–K+ by the mean plasma concentration, corrected for plasma water and Gibbs–Donnan equilibrium, at the beginning and the end of the dwell and multiplying this by the intraperitoneal volume (IPV) at the appropriate time point during the dwell.

In vitro PD model
References for the D20, w of K+ are sparse [24]. Therefore, the D20, w of K+ was estimated first by interpolation of the permeability coefficient for diffusion across a lipid bilayer using those of urea and urate [25] in the same system. The D20, w of K+ was estimated as 80.0 cm2/s/107, as the D20, w of urea and urate are available from literature. An in vitro PD model, described earlier by McGary et al. [26], was used to verify the estimated D20, w of K+. The model consisted of a rustproof steel cavity designed to allow a hollow fibre dialyser (Hemoflow F5 High Performance Steam, Fresenius Medical Care, Germany), with an effective surface area of 1.0 m2 comparable to that of an adult, with the outer shell removed to be submerged in 2 l of 1.36% commercially available glucose solution (Physioneal®, Baxter Healthcare Ltd., IRL). Glucose of about 1.36% was used to reduce the effects of crystalloid osmotic pressure. Perfusate consisting of bovine serum albumin (BSA, 30 g/l) to mimic colloid osmotic pressure, KCl (6 mmol/l), urea (25 mmol/l) and creatinine (500 µmol/l) dissolved in 0.9% NaCl, was pumped single-pass by a haemodialysis monitor (Gambro AK 200 Ultra S) through the hollow fibre dialyser. Perfusate flow was set as low as possible at 80 ml/min, to reduce effects of hydrostatic pressure. At 0, 1, 2, 3, 4, 5, 10, 15, 20 and 30 min perfusate in- and outflow and dialysate samples were taken. To reach near equilibrium of K+ between perfusate and dialysate 30 min was considered sufficient. The D20,w of K+ was calculated from the power relation of the MTACs of urea and creatinine and their D20,w by interpolating the MTAC of K+ obtained from this experiment. The experiment was performed in triplicate, from which the mean value was 93.0 cm2/s/107.

Statistical analysis
Mean values and SDs are presented, unless stated otherwise, as most of the data were normally distributed. For Figures 2–4GoGo means and SEMs are given. Analysis of variance (ANOVA) with Bonferroni correction was used to compare the three different groups. Pearson correlation analysis and Spearman rank correlation were performed to examine possible relationships. Regression analysis was done to analyse differences in relationships between the two groups. This was done by analysing whether the interaction between the group and the predictive parameters had a significant influence on the dependent variable in the regression model.


Figure 2
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  		Fig. 2. FWT profiles of the three groups during the dwell. During the initial phase of the dwell FWT increases and levels off after 120 min of the dwell. The time course during the dwell was significantly different among the groups (P < 0.01). Highest values for FWT during the dwell were present in group II. Lowest, but rather stable values were present in group III.

 

Figure 3
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  		Fig. 3. This figure shows small pore water transport profiles of the three groups during the dwell. The time course during the dwell was significantly different among the groups (P < 0.01). Group I had the highest values during the dwell, but not significantly different at the end of the dwell compared to group II. Group III had the lowest values.

 

Figure 4
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  		Fig. 4. The time course of the amount of potassium additional to diffusion is shown in this figure. For all the groups an initial increase, steepest for groups I and II, was followed by a subsequent decrease. Maximal values were present at 60 min of the dwell. Significant differences were present among the groups (P < 0.01) during the dwell. During the dwell highest values were present for group II, lowest for group III. The time course of the three different groups followed the profile of FWT.

 


   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
Peritoneal transport characteristics and fluid kinetics
Transport characteristics of small solutes and effluent CA125 are summarized in Table 2. Patients from group III had the highest values for MTAC-creatinine and MTAC-urate. The D/P creatinine and urate were also highest in group III. Patients from group III had the lowest values for CA125. Figure 2 shows the FWT profile of the three groups during the dwell. The FWT contribution to TCUF was significantly less in group III compared to group II (Table 1). Figure 3 shows SPT profiles of the three different groups. The contribution of SPT to TCUF at 60 min was not significantly different among the groups (Table 1). Values for K+-transport and CR-K+ are given in Table 3. CR-K+, the MTAC-K+/MTAC-creatinine and D/P-K+/D/P-creatinine ratio were lowest in group III. The profile of CR-K+ during the dwell is shown in Figure 4.


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  		Table 2. A comparison of peritoneal solute transport and CA125 between the three groups

 

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  		Table 3. Transport and cellular release of K+ of the three groups during a 4 h dwell SPA

 
Relationships between K+-transport, parameters of FWT and CA125
Positive relationships were present between CR-K+ and FWT at either 60 and 240 min of the dwell in group I and group II (Table 4). This was also the case in group I and II for the relationships between the max dip D/P Na+ and either the MTAC-K+/MTAC-creatinine (group I: r = 0.50, P < 0.05, group II; r = 0.66, P < 0.01) or D/P-K+/D/P-creatinine ratio (group I: r = 0.69, P < 0.01, group II; r = 0.73, P < 0.01) There was no relationship between CR-K+ and FWT in group III (Table 4). Likewise, there were no relationships in this group between the max dip D/P Na+ and either the MTAC-K+/MTAC-creatinine (r = –0.16) or D/P-K+/D/P-creatinine ratio (r = 0.36). The associations in group III were significantly different from those in the other groups (all P ≤ 0.05). In none of the groups associations between CR-K+ and SPT were present (Table 4). There were no relationships for CA125 and either CR-K+, the MTAC-K+/MTAC-creatinine or D/P-K+/D/P-creatinine ratio when examined in the whole population (Figure 5). This was also the case when groups were analysed separately (data not shown).


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  		Table 4. Spearman rank correlation coefficients of cellular release of K+ in relation to FWT and SPT at either 60 min or 240 min of the dwell

 

Figure 5
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  		Fig. 5. Using Pearson correlation analysis, no relationships were present between CR-K+ and CA125 (r = –0.04, P > 0.5) (left panel), the MTAC-K+/MTAC-creatinine ratio and CA125 (r = –0.07, P > 0.5) (middle panel) and the D/P-K+/D/P-creatinine ratio and CA125 (r = 0.06, P > 0.5) (right panel).

 
For calculation of CR-K+ the D20, w of 93.0 cm2/s/107 was used, as derived by us. Using the estimated value of 80.0 cm2/s/107 instead, did not influence the results significantly (data not shown).



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, it was demonstrated that total effluent K+ during the entire period of the dwell was higher than could be expected, based on transport from the circulation. Highest values were present at 60 min of the dwell. Our results support findings of earlier experiments by Waniewski et al. (15,27). These studies suggested additional release of K+ from cells, because sieving coefficients for K+ were substantially higher than 1.0. During PD, transport of small solutes is mainly determined by diffusion and to a lesser extent by convection. The studies of Waniewski et al. [15,27] and Imholz et al. [14] reported that with a hypertonic PD solution convective transport of urea, creatinine and K+ was substantial during the first hour of an exchange. Especially higher values were found for urea and K+. In contrast, sieving coefficients for K+ substantially lower than 1.0 have been reported as well [28,29]. However, the direct calculated sieving coefficients of these isochratic studies, done in intermittent PD patients, were calculated from 70 min exchanges with 30 min dwells. Also, the glucose concentration used was 7% and the diffusion coefficient of K+ was as assumed 0.0. Waniewski et al. [30] reported that values of sieving coefficients for urea and K+ are time dependent, i.e. lower values are present when the dwell time is shorter. It is conceivable that differences in methodology explain the different results. The findings of our study, using 4 h dwells, are in accordance with those obtained with 4–6 h exchanges [14,15,27]. Mixing of solutes within the membrane also remains a possibility for these different results [31]. As the peritoneal vessels are distributed discretely within the membrane, regional variability in convective water flow is possible. This can, when substantially increased during hypertonic exchanges, enhance small solute mixing. We are also aware of the fact that there is much discussion among the experts about the interpretation of fluid transport and its approximation [32]. The present study has used the models for fluid transport which are in line with previous studies from our research group and depicted in the schematic diagram Figure 1.

Because of these anomalous findings of sieving coefficients for K+ in earlier studies, it is obvious that it is difficult to distinguish K+ sieving from other mechanisms, like transport of K+ from and to cells, which are most certainly present in a biological membrane. The present models used to estimate diffusive and convective transport do not include cellular phenomena. The possibility that convection itself was responsible for the whole amount of K+ additional to diffusion in our study was less likely, whereas it had no relationship with SPT, or convective water flow. Therefore, we had reasonable evidence that the main source of effluent K+ additional to diffusion, albeit the result of calculations for which approximations have been used, was cellular and we could term it CR-K+.

The objective of our study was to investigate whether CR-K+ could be used as a reflection of apoptosis of peritoneal cells. If so, negative relationships between CR-K+ and FWT would be expected. We found the opposite: there were positive correlations between CR-K+ and FWT in patients starting PD and in long-term PD patients without UFF. These results either mean that our initial hypothesis was incorrect, or that apoptosis of peritoneal cells is not an important mechanism. Our results most likely indicate the basic physiological phenomenon of hypertonic cell shrinkage. Hypertonicity can directly impinge on the apoptotic machinery [33]. However, as the effect of osmotic shrinkage during apoptosis seems to be most prominent in cells that are incapable of performing regulatory volume increase, it does not necessarily lead to apoptosis. The osmotic pressure gradient is very important for AQP-1-mediated water transport [6] and can also up-regulate AQP-1 epxression [33]. This can be reflected by our findings showing highest values for FWT during the dwell in the long-term PD group without UFF. In hypertonic cell shrinkage, the loss of water raises the cell K+-concentration, thereby promoting passive K+-diffusion through K+-channels [34]. Passive influx of Na+ occurs simultaneously [34]. In the calculation of FWT, which is calculated from Na+ kinetics, the correction of Na+ sieving can therefore cause a slight overestimation of FWT. It can be questioned to which extent passive influx of Na+ can influence the relationship between CR-K+ and FWT. Other reasons for redistribution of K+ are the rate of cell breakdown, as was our initial hypothesis, and a low extracellular pH. However, it has been demonstrated that using conventional PD solutions in vivo pH is in the physiological range after only a few minutes [35]. Additionally, a low extracellular pH caused by lactate is less likely to elevate extracellular K+ [36].

Given the correlation coefficient performed with a Spearman rank correlation of –0.02 in the association between CR-K+ and FWT at 240 min in the long-term PD UFF group, only 0.04% can explain the association. This is not clinically relevant. Based on a sample size calculation with 80% power with a 0.05 two-sided significance level, 19 620 patients are necessary to make this association of –0.02 significant. As this is not realistic in a PD population, it provides us the second reason why at 240 min and also at 60 min the association can be considered absent. This was due to both a lower FWT and CR-K+. It can be suggested that these results represent less apoptosis. We could have verified this by determining effluent lactate dehydrogenase, however, there were no useful samples for analysis. But, although not examined histologically in our study, it is very likely that the peritoneum of the patients from this group shows characteristics of cell proliferation: basement membrane reduplications of peritoneal capillaries, expansion of extracellular matrix with extensive deposition of collagen IV and neoangiogenesis, as these are the morphological alterations present during long-term PD UFF [4]. In addition to AQP-1 dysfunction, a thickened interstitium or interstitial fibrosis is also considered an equally valid theory for reduced AQP-1-mediated FWT in late UFF [9], which could explain decreased FWT in our group III. The theories do not exclude each other. Both decreased apoptosis and increased proliferation of cells represent a status of disturbed tissue homoeostasis [13], following from a rise in survival signals and a reduction in death signals. Vascular endothelial growth factor and transforming growth factor-β are important survival signals and involved in the peritoneal membrane alterations in long-term PD UFF [37]. Hypoxia can interfere in the balance between apoptosis and cell proliferation. This was shown for primary pulmonary hypertension [13]. In this condition, hypoxia reduces the expression or inhibits K+-channels, leading to a reduced K+-efflux and maintenance of the intracellular K+-concentration. This attenuates apoptotic volume decrease and also suppresses the activation of apoptotic enzymes. It may result in less apoptosis and subsequently less CR-K+. As a consequence fibrosis develops, leading to pulmonary hypertension. In PD with glucose-based/lactate-buffered solutions a situation of pseudohypoxia is likely to exist [38]. In the presence of a high intracellular glucose concentration, its break-down is not only by glycolysis, but also by the sorbitol pathway. In both pathways NADH is formed from NAD+. The resulting increase in NADH/NAD+ ratio is similar to what happens during true hypoxia. The increased NADH/NAD+ ratio is reversed by the conversion of pyruvate to lactate, in which NADH is regenerated to NAD+. This process may be inhibited by high lactate concentrations as present in PD solutions. It is conceivable that pseudohypoxia, like true hypoxia, can reduce the expression of K+-channels or inhibit their function in peritoneal tissue. This could trigger cell proliferation and thereby contribute to the development of peritoneal fibrosis. AQP-1 dysfunction is most likely due to another mechanism.

The relationships between CR-K+ and FWT in group I and II were also present using much simpler methodologies, that is, replacement of CR-K+ by MTAC-K+/MTAC-creatinine or D/P-K+/D/P-creatinine ratio and replacement of FWT by Na+-sieving. These parameters can be calculated from the well-established peritoneal equilibration test, when performed with a 3.86% glucose solution. The absent relationships between CR-K+ and CA125 in any of the groups suggest that mesothelial cells are not likely or not only involved. Using functional parameters, it remains speculative to address release of K+ to a particular type of peritoneal cell. It was also not the scope of this functional study. The endothelial cells are, however, the most likely candidates, because AQP-1 is specifically located in venule and capillary endothelium [33].

In conclusion, this study shows that other than diffusive and convectional, K+ transport is not excluded in patients treated with conventional glucose-based PD solutions. We found evidence for the release of K+ from cells. Our results showed that, in general, CR-K+ was related to parameters of FWT, suggesting glucose-induced osmotic cell shrinkage as a basic physiological phenomenon during PD. The absence of this relationship in the long-term patients with UFF either suggests a reduction or inhibition of K+-channels and may be due to a mechanism other than AQP-1 dysfunction. Most likely, effluent K+ in UFF does not reflect apoptosis and it may not be causative for AQP-1 dysfunction. The extent to which in vitro data can be extrapolated to the in vivo situation remains uncertain. Furthermore, the identity and interplay of pathways during cell shrinkage in hypertonic stress during PD are poorly understood and need to be elucidated in the development of more biocompatible PD solutions. As the D/P-K+/D/P-creatinine ratio is a simple method, it may be promising in detecting peritoneal membrane alterations.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors acknowledge Joke Korevaar for her statistical advice and thank the reviewers for their valuable criticisms and comments. We highly appreciate the assistance of Gerrit Burger and Bert Land for constructing the in vitro PD model. This study was supported by a grant of the Dutch Kidney Foundation—C06.2186.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 7.12.06
Accepted in revised form: 29. 6.07


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A. M. Coester, W. Smit, D. G. Struijk, and R. T. Krediet
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A. M. Coester, M. M. Zweers, D. R. de Waart, and R. T. Krediet
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