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J Am Soc Nephrol 14:1296-1306, 2003
© 2003 American Society of Nephrology

Effects of Conventional and New Peritoneal Dialysis Fluids on Leukocyte Recruitment in the Rat Peritoneal Membrane

Siska Mortier*, An S. de Vriese*, Rachel M. McLoughlin{dagger}, Nicholas Topley{dagger}, Thomas P. Schaub{dagger}, Jutta Passlick-Deetjen{dagger} and Norbert H. Lameire*

*Renal Division, University Hospital, Gent, Belgium; {dagger}Institute of Nephrology, University of Wales College of Medicine, Cardiff, United Kingdom; and {ddagger}Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.

Correspondence to Dr. Siska Mortier, Renal Division, University Hospital, OK12, De Pintelaan 185, B-9000 Gent, Belgium. Phone: 32-9-2405301; Fax: 32-9-2404599;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Peritonitis remains an important cause of morbidity and technique failure in peritoneal dialysis (PD). Conventional peritoneal dialysate fluids (PDF) inhibit peritoneal leukocyte function in vitro and may thus adversely affect the immune response to peritonitis. New PDF have been designed with neutral pH, low glucose degradation product (GDP) contents, and bicarbonate as buffer. The present intravital microscopy study examined the effects of conventional and new PDF on leukocyte behavior in the peritoneal microcirculation of Wistar rats. The visceral peritoneum was superfused by a control solution (EBSS), a conventional (CAPD), or a new bicarbonate-buffered PDF with neutral pH and low GDP content (CAPD BicaVera). In addition, spent conventional and new PDF were tested. The number of rolling, adhering, and extravasated leukocytes and leukocyte rolling velocity were assessed at different time intervals after exposure to lipopolysaccharide (LPS) or cell-free supernatants of coagulase-negative staphylococci (CNS-CFS). Exposure to LPS or CNS-CFS dissolved in EBSS dramatically increased the number of rolling, adhering and extravasated leukocytes and decreased leukocyte rolling velocity. Superfusion by CAPD abolished the LPS- or CNS-CFS-induced leukocyte recruitment, whereas CAPD BicaVera had significantly fewer depressant effect. Spent PDF affected the leukocyte response in a similar way as fresh PDF. High lactate concentrations, GDP, and hypertonicity appeared to be mainly responsible for the inhibition of leukocyte recruitment. In conclusion, conventional PDF abolish in vivo leukocyte recruitment in reponse to potent inflammatory stimuli. Bicarbonate-buffered pH-neutral PDF with low GDP contents have fewer depressant effects and may therefore contribute to a better preservation of peritoneal host defense. E-mail: siska.mortier@rug.ac.be


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite substantial improvements in bag connection technology, peritonitis remains an important cause of morbidity and technique failure in patients undergoing peritoneal dialysis (PD). Gram-positive organisms are the most common pathogens, with coagulase-negative staphylococci being responsible for 30 to 40% of peritonitis episodes (1). However, the relative contribution of Gram-negative organisms to PD-related peritonitis has risen considerably in the past few years (1).

The peritoneal immune system plays a central role in the prevention and clearance of peritonitis in PD. The chief components of this system are resident macrophages, neutrophils that are recruited from the systemic circulation, mesothelial cells, and fibroblasts. Shortly after peritoneal infection, local macrophages and mesothelial cells secrete inflammatory cytokines and chemoattractants, resulting in emigration of neutrophils from the bloodstream to the site of inflammation (2). Leukocyte recruitment is a multi-step process, directed by specific adhesive interactions between the leukocyte and the endothelium. Selectins and their carbohydrate-containing ligands mediate the initial and transient contact between the circulating leukocyte and the vascular endothelium, the so-called "rolling." The leukocyte becomes thus exposed to tissue-derived chemokines and other activating stimuli. For a rolling cell to subsequently adhere, a reduction in leukocyte rolling velocity must occur. The molecular mechanisms for slow rolling are incompletely understood, but they may include an increased expression of selectins at the surface of the leukocyte (3). Finally, firm adherence and transendothelial migration takes place, mediated by interaction of integrins with their Ig-like receptors (4).

A large body of evidence indicates that conventional peritoneal dialysate fluids (PDF) cause a functional impairment of peritoneal host defense mechanisms. Viability, bactericidal activity, and chemokine production of different leukocyte populations, mesothelial cells, and fibroblasts are substantially inhibited after exposure to PDF (5). Conventional PDF are unphysiologic due to their acidic pH, hypertonicity, high lactate and high glucose concentrations, and the formation of glucose degradation products (GDP) during heat-sterilization and storage of the dialysate. In an attempt to improve these biocompatibility aspects, new PDF have been developed with pH-adjustment to physiologic values, use of bicarbonate as buffer system, and markedly reduced GDP levels through the use of double-chamber bags (6). Several authors have reported that the new PDF may exert less suppressive effects on peritoneal leukocyte functions (5). The large majority of these studies have, however, been performed in vitro. As in vivo data on PDF biocompatibility are scarce, the potential clinical relevance of the reported perturbations of various cell functions remains unclear.

Against this background, we evaluated the effects of conventional and new PDF on the recruitment of circulating leukocytes in response to different inflammatory stimuli in the rat peritoneal membrane, using a well-standardized intravital microscopy model (7–9). The consecutive leukocyte-endothelial interactions, i.e., leukocyte rolling, adhesion, and emigration, were evaluated and quantified in venules of the rat peritoneal membrane. The potential contribution of different dialysate components in mediating changes in peritoneal leukocyte recruitment was assessed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Laboratory Animals and Dialysate Solutions
The studies were performed in 102 female Wistar rats (Iffa Credo, Brussels, Belgium) that received care in accordance with the national guidelines for animal protection. The following PDF (Fresenius Medical Care, Bad Homburg, Germany) and self-generated solutions were evaluated (Table 1):

  1. Earle’s Balanced Salt Solution (EBSS; Life Technologies Ltd., Paisley, Scotland), containing 5.6 mmol/L glucose, 26 mmol/L NaHCO3, 117 mmol/L NaCl, 1.8 mmol/L CaCl2, 5.3 mmol/L KCl, 0.8 mmol/L MgSO4, and 1 mmol/L NaH2PO4 (n = 6)
  2. A conventional, single-chamber bag, acidic pH, L-lactate-buffered PDF with 1.5% (83 mmol/L) D-glucose and an osmolarity of 358 mOsm/L (CAPD2) (n = 6) or with 4.25% (236 mmol/L) D-glucose and an osmolarity of 511 mOsm/L (CAPD3) (n = 6)
  3. A conventional, single-chamber bag, acidic pH, lactate-buffered PDF adjusted to pH 7.4 with NaOH, containing 4.25% glucose (CAPD3-NaOH) (n = 6)
  4. A new, double-chamber bag, pH-neutral, bicarbonate-buffered PDF with 1.5% D-glucose (CAPD20 BicaVera) (n = 6) or with 4.25% D-glucose (CAPD30 BicaVera) (n = 6)
  5. A new, double-chamber bag, pH-neutral, bicarbonate-buffered PDF, resterilized (second steam sterilization process) (10) to increase GDP content, containing 4.25% glucose (CAPD30 Bica Vera-R) (n = 6)
  6. EBSS with addition of D-glucose to achieve a final glucose concentration of 236 mmol/L and a final osmolarity of 511 mOsm/L (n = 6)
  7. EBSS with addition of D-mannitol to achieve a final osmolarity of 511 mOsm/L (n = 6)
  8. Sterile water with addition of electrolytes (117 mmol/L NaCl, 1.8 mmol/L CaCl2, 5.3 mmol/L KCl, 0.8 mmol/L MgSO4), a D-glucose concentration of 5.6 mmol/L, and a L-lactate concentration of 35 mmol/L (n = 6)
  9. Sterile water with addition of electrolytes (117 mmol/L NaCl, 1.8 mmol/L CaCl2, 5.3 mmol/L KCl, 0.8 mmol/L MgSO4), a D-glucose concentration of 236 mmol/L, and a L-lactate concentration of 35 mmol/L (n = 6)
  10. Spent CAPD2 (n = 6) and spent CAPD20 BicaVera (n = 6) obtained after a 6-h dwell of a single patient using 4 x 2 dwells per day


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  		Table 1. Differences in pH, osmolality, glucose concentration, buffer, and glucose degradation product (GDP) content of the peritoneal dialysate fluids (PDF) and homemade solutions
 
Inflammatory Stimuli
Lipopolysaccharide (LPS) (Escherichia coli serotype 0127:B8; Sigma, St. Louis, MO) was added to the different solutions at a concentration of 0.1 µg/ml. In pilot experiments, this concentration was found to induce substantial leukocyte recruitment in the absence of systemic BP effects.

Additional experiments were conducted utilizing lyophilized cell free supernatants (CFS) from a strain of coagulase-negative staphylococci (CNS), which had previously been obtained from a PD patient with peritonitis (11). In brief, bacteria were isolated from a stationary phase culture by centrifugation (1800 x g at 20°C for 20 min) and resuspended in Tyrode’s salt solution without gelatin (Sigma) to an absorbance of 0.5 at 560 nm. Previous serial plate count analysis had established that an optical density of 0.5 at 560 nm is equivalent of 5 x 108 colony forming units/ml (12). This solution was incubated at 37°C for 24 h. Suspensions were centrifuged (1800 x g at 20°C for 20 min) to remove the remaining bacteria particles. Thereafter, the supernatants were filter-sterilized through 0.2-µm filters (Millipore, Bedford, MA) and dialysed against distilled water at 4°C through size 5 dialysis tubings (Medicell International Ltd., London, UK). Fractions were freeze-dried, and aliquots were stored at -70°C until use. In pilot experiments, a dose of 30 x 109 colony forming units was found to induce substantial leukocyte recruitment in the absence of systemic BP effects.

Intravital Microscopy
Rats were anesthetized with thiobutabarbital (Inactin, RBI; 100 mg/kg subcutaneously). The trachea was intubated to facilitate breathing, a jugular vein was cannulated for continuous infusion of isotonic saline, and a carotid artery was cannulated for continuous monitoring of arterial BP. Cromoglycate (cromolyn sodium salt; 10 mg/kg intavenously) was administered 15 min before surgery to block degranulation of mast cells induced by the surgical manipulation. A small midline abdominal incision was made, and a short segment of the small bowel was exteriorized, carefully avoiding stretching, spread over a plexiglass plate, and superfused continuously with EBSS maintained at 37°C (Figure 1). The preparation was allowed to stabilize for 30 min after completion of surgery. Observations were made with an Axiotech Vario 100 HD microscope (Zeiss, Jena, Germany) using a water immersion objective (Achroplan 40x). The tissue was transilluminated via a fiberoptic using a light source (KL 1500; Schott, Wiesbaden, Germany) equipped with a 150-W halogen lamp. The resulting image was displayed on a television monitor by a TK-1281 camera (Victor Company of Japan LTD-JVC, Tokyo, Japan) or a high-speed video camera (Kodak Motioncorder Analyser, Eastman Kodak Company, San Diego, CA) and recorded by a videorecorder (S-VHS Panasonic AG-7355, Matsushita, Japan) for off-line analysis. The video images were digitized with an IP-8/AT Matrox image processing board and analyzed with image analysis software (Cap-Image, Ingenieurbüro Zeintl, Heidelberg, Germany), as described previously (7–9).



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  		Figure 1. Intravital microscopy. A segment of the small bowel is exteriorized, spread over a plexiglass plate, and superfused continuously with Earle’s Balanced Salt Solution (EBSS) or peritoneal dialysate fluids (PDF). Observations are made with an Axiotech Vario 100 HD microscope, using a water immersion objective and transillumination.

 
Study of Leukocyte Recruitment
In each experimental animal, a single unbranched venule with a diameter of 20 to 30 µm and a length of 150 to 200 µm was selected. Rolling leukocytes were defined as those that moved at a velocity lower than that of the red blood cells and were in contact with the endothelial surface. The flux of rolling leukocytes was determined by counting the number of rollers crossing an imaginary line perpendicular to the axis of the venule per minute. The number of leukocytes adhering to the venular endothelial lining and not moving during a 30-s period was counted and expressed as the number per 100-µm length of venule. The number of extravasated leukocytes was determined as the number counted within a predefined area of perivenular tissue. To minimize the influence of preactivation of the tissue, only vessels in which baseline leukocyte rolling was <30 cells/min and baseline adhesion was <3 cells/100 µm of vascular endothelium were considered for further analysis.

After stabilization, the peritoneal membrane was superfused with one of the above-mentioned solutions. The number of rolling, adhering, and extravasated leukocytes, leukocyte rolling velocity, red blood cell velocity (vRBC), and vessel diameter (D) were measured twice with an interval of 10 min. Venular wall shear rate ({gamma}w) was calculated as {gamma}w = 8 x vRBC/D. The measurements were repeated at 30, 60, 90, 120, and 150 min after exposure to LPS or CNS-CFS. Solutions containing bicarbonate were bubbled continuously with CO2 to maintain the pH neutral and the pCO2 and HCO3- concentrations stable throughout the entire experiment.

Circulating Leukocytes
A 25-µl sample of arterial blood was added to 475 µl of 2% orthophosphoric acid (VWR International, Leuven, Belgium) to lyse the red blood cells. The total number of peripheral leukocytes was thereafter counted in a Bürker chamber and expressed as number/mm3.

Statistical Analyses
The results are expressed as mean ± SEM. Statistical analyses were performed using ANOVA, and the Tukey test was used as post hoc test where appropriate. An a-priori level of alpha = 0.05 was used to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leukocyte Recruitment in Response to LPS and CNS-CFS
Exposure to both LPS and CNS-CFS caused a dramatic rise in the number of rolling, adhering, and extravasated leukocytes in the peritoneal microcirculation, as compared with superfusion with EBSS alone (Figure 2; Figure 3, A through C). In addition, the velocity of the rolling leukocytes decreased after exposure to the infectious stimulus (Figure 3D). Leukocyte parameters were similar after LPS and CNS-CFS stimulation (Figure 3, A through D).



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  		Figure 2. Leukocyte rolling (open arrow), adhesion (closed arrow), and extravasation in response to LPS dissolved in EBSS after t = 0 min (A) and t = 150 min (B) and to LPS dissolved in CAPD3 after t = 0 min (C) and t = 150 min (D).

 


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  		Figure 3. The number of rolling (A), adhering (B), and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points during superfusion by EBSS ({blacksquare}, n = 6), EBSS with LPS ({blacktriangleup}, n = 6) and EBSS with cell free supernatants (CFS; •, n = 6). (A) *P < 0.005 versus EBSS; (B) *P < 0.05 versus EBSS; (C) *P < 0.001 versus EBSS; (D) *P < 0.01 versus EBSS.

 
The Effect of Conventional and New PDF on LPS-Induced Leukocyte Recruitment
Superfusion of the peritoneal membrane with CAPD3 abolished leukocyte recruitment in response to LPS (Figure 2; Figure 4, A through D). The number of rolling leukocytes decreased over time, indicating that baseline rolling was also impaired (Figure 4A). The number of adhering and extravasated leukocytes did not increase after stimulation with LPS (Figure 4, B and C). Leukocyte rolling velocity remained stable throughout the experiment (Figure 4D).



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  		Figure 4. The number of rolling (A), adhering (B), and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points before and after addition of LPS during superfusion by EBSS ({blacktriangleup}, n = 6), CAPD3 (•, n = 6), CAPD30 BicaVera ({blacksquare}, n = 6). (A) *P < 0.005 versus EBSS, # P < 0.01 versus CAPD3; (B) *P < 0.05 versus EBSS; (C) *P < 0.05 versus EBSS, # P < 0.005 versus CAPD3; (D) *P < 0.05 versus EBSS.

 
In contrast, the effects of CAPD30 BicaVera on LPS-induced leukocyte recruitment were much milder. The number of rolling leukocytes increased initially to the same extent as in the EBSS-exposed peritoneal venules but decreased thereafter (Figure 4A). The number of adhering and extravasated leukocytes increased during the first 60 min after LPS exposure and remained stable thereafter (Figure 4, B and C). Leukocyte rolling velocity fell initially, but it returned to baseline values thereafter (Figure 4D).

CAPD2 and CAPD20 BicaVera had similar effects on LPS-induced leukocyte recruitment as CAPD3 and CAPD30 BicaVera, respectively (data not shown).

The Effect of Conventional and New PDF on CNS-CFS-Induced Leukocyte Recruitment
Exposure to CAPD3 abolished the leukocyte response to CNS-CFS. The number of rolling, adhering, and extravasated leukocytes, as well as leukocyte rolling velocity, did not change over time (Figure 5, A through D). CAPD30 BicaVera partially inhibited leukocyte recruitment after CNS-CFS stimulation, in a similar way as during LPS stimulation (Figure 5, A through D).



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  		Figure 5. The number of rolling (A), adhering (B), and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points before and after addition of CFS during superfusion by EBSS ({blacktriangleup}, n = 6), CAPD3 (•, n = 6), and CAPD30 BicaVera ({blacksquare}, n = 6). (A) *P < 0.005 versus EBSS, # P < 0.01 versus CAPD3; (B) *P < 0.01 versus EBSS, # P < 0.05 versus CAPD3; (C) *P < 0.01 versus EBSS, # P < 0.005 versus CAPD3; (D) *P < 0.005 versus EBSS, # P < 0.005 versus CAPD3.

 
The Effect of Spent Dialysate on LPS-Induced Leukocyte Recruitment
Spent CAPD2 inhibited the rise in rolling, adhering, and extravasated leukocytes and the fall in leukocyte rolling velocity after LPS exposure, although to a somewhat lesser extent than fresh dialysate (Figure 6, A through D). Spent CAPD20 BicaVera had similar effects on LPS-induced leukocyte recruitment as fresh CAPD BicaVera (Figure 6, A through D).



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  		Figure 6. The number of rolling (A), adhering (B) and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points before and after addition of LPS during superfusion by EBSS ({blacktriangleup}, n = 6), spent CAPD2 (•, n = 6) and spent CAPD20 BicaVera ({blacksquare} n = 6). (A) *P < 0.005 versus EBSS, # P < 0.005 versus spent CAPD2; (B) *P < 0.05 versus EBSS; (C) *P < 0.05 versus EBSS, # P < 0.0005 versus spent CAPD2; (D) *P < 0.01 versus EBSS, # P < 0.05 versus spent CAPD2.

 
The Effect of Lactate and Hyperosmolarity on LPS-Induced Leukocyte Recruitment
Addition of D-glucose to EBSS to obtain the same concentration as present in CAPD3 and CAPD30 BicaVera did not impair LPS-induced leukocyte rolling during the initial phase of the experiment, but it decreased the number of rolling leukocytes thereafter (Figure 7A). The number of adhering and extravasated leukocytes increased initially but remained unaltered thereafter (Figure 7, B and C). Leukocyte rolling velocity only decreased during the first 30 min (Figure 7D). D-Mannitol, added to EBSS to obtain the same osmolarity as present in CAPD3 and CAPD30 BicaVera, had virtually identical effects on LPS-induced leukocyte recruitment as D-glucose. Addition of L-lactate in a concentration of 35 mmol/L substantially impaired leukocyte rolling, adhesion, and extravasation and prevented the decrease of leukocyte rolling velocity. Finally, a combination of high L-lactate and high D-glucose concentrations abolished LPS-induced leukocyte recruitment (Figure 7, A through D).



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  		Figure 7. The number of rolling (A), adhering (B), and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points before and after addition of LPS during superfusion by EBSS ({blacktriangleup}, n = 6), EBSS with 236 mmol/L glucose ({circ}, n = 6), EBSS with 511 mosm/L mannitol (•, n = 6), sterile water with 35 mmol/L lactate ({square}, n = 6), and sterile water with 35 mmol/L lactate and 236 mmol/L glucose ({blacksquare}, n = 6). (A) *P < 0.005 versus EBSS, # P < 0.05 versus sterile water with 35 mmol/L lactate and 236 mmol/L glucose; (B) *P < 0.05 versus EBSS, # P < 0.05 versus sterile water with 35 mmol/L lactate and 236 mmol/L glucose; (C) *P < 0.05 versus EBSS, # P < 0.05 versus sterile water with 35 mmol/L lactate and 236 mmol/L glucose; (D) *P < 0.05 versus EBSS.

 
The Effect of pH and GDP on LPS-Induced Leukocyte Recruitment
Addition of NaOH to CAPD3 to adjust the pH to 7.4 did not alter the effects on leukocyte recruitment. Leukocyte rolling, adhesion, and extravasation, as well as leukocyte rolling velocity, were affected to the same extent as by CAPD3 (Figure 8, A through D).



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  		Figure 8. The number of rolling (A), adhering (B), and extravasated (C) leukocytes and the velocity of the rolling leukocytes (D) at different time points before and after addition of LPS during superfusion by EBSS ({blacktriangleup}, n = 6), CAPD3 (•, n = 6), pH-neutralized CAPD3-NaOH ({circ}, n = 6), and resterilized CAPD30 BicaVera-R ({blacksquare}, n = 6). (A) *P < 0.005 versus EBSS; (B) *P < 0.05 versus EBSS; (C) *P < 0.001 versus EBSS; (D) *P < 0.05 versus EBSS, # P < 0.05 versus CAPD3.

 
Resterilization of CAPD BicaVera increased GDP levels without otherwise altering the chemical composition of the PDF (10). CAPD30 BicaVera-R inhibited leukocyte recruitment in a similar way as CAPD3 (Figure 8, A through D).

BP, Circulating Leukocytes, Hematocrit, and Baseline Leukocyte Parameters
BP was not different between the experimental groups and did not change throughout the experiments (Table 2). The number of circulating leukocytes did not change significantly during the experiments. There were no differences in the number of circulating leukocytes among the different experimental groups at any of the time points (Table 2). Hematocrit values were stable during the experiments and not different among the groups (Table 2).


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  		Table 2. BP, the number of circulating leukocytes, and hematocrit in the experimental groups at different time points after the exposure to the inflammatory stimulia
 
The number of rolling, adhering, and extravasated leukocytes, leukocyte rolling velocity, and venular shear rate at baseline were not different between the experimental groups (Table 3). There was no correlation between the number of rolling leukocytes and venular shear rate at any of the time points (data not shown).


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  		Table 3. Baseline leukocyte characteristics in the different experimental groupsa
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Exposure of the rat peritoneal membrane to LPS derived from Escherichia coli causes an impressive increase in the number of rolling, adhering, and extravasated leukocytes in the venules. In addition, leukocyte rolling velocity decreased substantially, allowing for intense signaling between the leukocyte and the vascular endothelium. These phenomena represent a physiologic host response to infection (3,4). To determine whether the changes in leukocyte-endothelial interactions observed with LPS could be extrapolated to other inflammatory stimuli of relevance to PD-related peritonitis, additional experiments were conducted with supernatants of a strain of coagulase-negative staphylococci previously isolated from a PD patient with peritonitis (11). The resultant leukocyte recruitment appeared similar to that induced by LPS.

The leukocyte response to LPS and CNS-CFS was dramatically affected by concomitant exposure to conventional dialysate. In contrast, superfusion with a pH-neutral, bicarbonate-buffered PDF with a low GDP content had much less depressant effects on leukocyte recruitment. The differences could not be attributed to variability of systemic BP, circulating leukocyte numbers, or baseline levels of rolling, adhesion, extravasation, leukocyte rolling velocity, or venular shear rate, as these parameters were not significantly different among the groups. In addition, no correlation was found between the number of rolling leukocytes and venular wall shear rate at any time point, indicating that potential dialysate-induced variations in blood flow (9) were not responsible for the observed effects. Spent PDF obtained from a patient after a 6-h dwell affected leukocyte kinetics to a similar extent as fresh PDF. Taken together, the results indicate that the presence of conventional PDF in the peritoneal cavity has important and persistent deleterious effects on the host response to peritonitis.

Additional experiments were conducted to identify the causative PDF-components in greater detail. Addition of D-glucose to EBSS in identical concentrations as found in conventional and new PDF resulted in a partial inhibition of leukocyte-endothelial interaction that was similar to that caused by CAPD30 BicaVera. Addition of D-mannitol, an osmotic agent that is not transported into the cell, yielded virtually identical results. These observations emphasize the importance of hyperosmolarity rather than glucose per se in mediating inhibitory effects on leukocyte recruitment. The results are in line with previous in vitro studies, showing that inhibition of phagocytosis and leukotriene generation by polymorphonuclear leukocytes (PMN) is related to the osmolarity but not to the glucose content of the fluid (13). An amino acid and glucose solution with similar osmolarity exerted comparable effects on monocyte cytokine release and cytotoxicity (14). In contrast, PMN cytokine release and cytotoxicity were found to be at least partly dependent on the glucose content of the solution (13,15). Finally, PMN respiratory burst activation remained unaffected by the hyperosmolarity and high glucose concentration of the solution (13). Taken together, the results indicate that the various leukocyte functions are differentially affected by glucose and hyperosmolarity. However, as efficient recruitment of leukocytes to the area of infection is a prerequisite for the effector functions to be meaningful, the effects of hyperosmolarity will be predominant.

While superfusion of the peritoneum with a pH-neutral solution containing high lactate concentrations and physiologic glucose levels caused a partial inhibition of leukocyte recruitment, a pH-neutral solution with both high lactate and high glucose concentrations abolished the leukocyte response similarly to conventional PDF, suggesting additive effects of lactate and hyperosmolarity on leukocyte kinetics. Impairment of leukocyte recruitment by conventional PDF persisted after pH-adjustment to 7.4, indicating that, although low pH has well-documented inhibitory effects on various leukocyte effector functions in vitro (5), it does not appear to be essential for the observed inhibition in vivo. After resterilization, CAPD BicaVera inhibited leukocyte recruitment to a similar extent as CAPD3. Resterilization is expected to increase GDP levels without otherwise altering the chemical composition of the PDF; therefore, the results support an inhibitory effect of GDP on leukocyte recruitment, as suggested by in vitro experiments (16). However, as the combination of lactate and hyperosmolarity already caused a maximal suppression of leukocyte recruitment, lowering the GDP content of PDF alone may not be sufficient to improve peritoneal host defense. The subordinate effect of GDP on leukocyte recruitment is supported by previous observations of a lower influx of neutrophils in the peritoneal cavity of rats infected with Staphylococcus aureus after previous exposure to both a pH-neutral lactate-buffered PDF with low GDP content and a conventional dialysate (17).

Whereas lactate and GDP caused an immediate suppression of leukocyte recruitment, the effects of hyperosmolarity were delayed. After 60-min exposure, leukocyte rolling and adhesion decreased, leukocyte rolling velocity increased, and no further leukocytes extravasated. These results suggest that the underlying pathophysiologic mechanisms of inhibition by lactate, GDP, and hyperosmolarity are different. Further work is required to clarify this issue.

The nonphysiologic composition of PDF disappears progressively during the dwell time. Osmolarity decreases due to glucose absorption and water ultrafiltration, although it never reaches physiologic values. Lactate concentration also diminishes rapidly during the dwell. We therefore determined the effect of spent dialysates on leukocyte recruitment. Results were very similar to those obtained with fresh dialysates, suggesting that osmolarity and lactate concentration remain sufficiently elevated to profoundly inhibit leukocyte recruitment. Alternatively, uremic toxins (18) and reactive carbonyl compounds accumulating in the dialysate during the dwell may have affected peritoneal leukocyte behavior. Taken together, the results indicate that the inhibition of leukocyte recruitment by conventional dialysate will persist throughout the entire PD cycle.

The molecular mechanisms of the impaired leukocyte response were not investigated in the present study. Several possibilities may be advanced, including changes in the expression of adhesion molecules on the leukocyte membrane such as increased L-selectin shedding (19) or decreased CD11b/18 upregulation (20), competition by soluble adhesion molecules such as soluble P-selectin released from activated platelets (21), defective generation of chemokines by mesothelial cells or resident macrophages, or alterations of the adhesion molecules at the vascular endothelial cell surface. Additional experiments need to be conducted to elucidate this issue.

In conclusion, both fresh and spent conventional PDF abolish leukocyte recruitment in response to LPS or CNS-CFS exposure, while a bicarbonate-buffered PDF exhibited less severe inhibitory effects. The depressant action largely results from a combination of high lactate concentrations, hyperosmolarity, and GDP. Glucose per se and acidity do not appear to be essential for the inhibitory effects on leukocyte recruitment. Whether the use of bicarbonate-buffered PDF with low GDP content may portend an improvement in peritonitis rates in PD patients remains to be determined in long-term prospective clinical trials.


   Acknowledgments
 
We thank Julien Dupont and Mieke Van Landschoot for their expert technical assistance and Wim Van Biesen for his kind cooperation. SM is supported by a grant from Fresenius Medical Care-Germany.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gokal R: Peritoneal dialysis. Prevention and control of infection. Drugs Aging 17: 269–282, 2000[CrossRef][Medline]
  2. Topley N: Biocompatibility of peritoneal dialysis solutions and host defense. Adv Ren Replac Therapy 3: 309–311, 1996
  3. Kubes P, Kerfoot SM: Leukocyte recruitment in the microcirculation: the rolling paradigm revisited. News Physiol Sci 16: 76–80, 2001[Abstract/Free Full Text]
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Received for publication October 2, 2002. Accepted for publication January 17, 2003.




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