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Bench |
Department of Biomedicine,1 Göteborg University, Göteborg; Gambro AB,2 Lund, Sweden
Correspondence to: M. Braide, Department of Biomedicine, Göteborg University, Box 420, SE 405 30 Göteborg, Sweden. Magnus.Braide{at}gu.se
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Exposure to peritoneal dialysis (PD) fluid
induces an inflammatory response in the peritoneal cavity. Blockers of
complement and coagulation have improved ultrafiltration in animal models of
PD. Citrate is a clinically established anticoagulant that also blocks
complement activation. Objective: The aim of the present study was to evaluate
the effects on ultrafiltration of a gradual substitution of citrate for
lactate in an experimental model of PD. Methods: Fractions (0, 5, 10, and 15 mmol/L) of the 40
mmol/L lactate buffer of filter-sterilized 2.5% glucose PD fluid were replaced
by citrate. The modified fluids were compared in a rat model of single PD
fluid exposure through an indwelling catheter. The initial kinetics of citrate
and ionized calcium were evaluated in separate, single, short time dwell
experiments. Results: Replacing 10 and 15 mmol/L of the lactate
buffer by sodium citrate significantly increased osmotic ultrafiltration (by
24.7% ± 7.7% at 10 mmol/L), net ultrafiltration, and glucose retention
at 4 hours of dwell time in the rat model. Osmotic ultrafiltration was
significantly correlated to citrate concentration and glucose concentration.
Citrate was rapidly eliminated from the peritoneal cavity, concentrations
falling to less than half in 1 hour and concentrations of calcium ions
concomitantly normalized. Conclusions: Substituting citrate for lactate induced a
dose-dependent increase in ultrafiltration. Mechanisms probably involve the
relation between diffusion and ultrafiltration, leading to increased glucose
retention. The increase in ultrafiltration was quantitatively important at a
citrate concentration (10 mmol/L) that is compatible with clinical
applications of citrate.
KEY WORDS: Ultrafiltration; lactate; citrate; rats.
Peritoneal dialysis (PD) treatment has several advantages compared with hemodialysis but suffers from shorter technique survival and lower efficiency. Recent developments in PD have aimed at improving the biocompatibility of PD fluids by changing buffers, osmotic agents, and sterilization techniques, thereby reducing toxic effects on the immune system and functional deterioration of the peritoneal membrane. The use of pharmacological additives to PD fluids has been anticipated for a few years (1) but implementation has been slow. Recent efforts include supplementation with glycosaminoglycans such as heparin and sulodexide. It has been proposed that anti-inflammatory actions are responsible for the improved ultrafiltration (UF) associated with these compounds.
Peritoneal dialysis maintains a constant state of intraperitoneal inflammation characterized by recruitment of neutrophils (2), production of cytokines (3,4), and activation of the complement and coagulation cascades (5–7). Over time, the peritoneum changes in a characteristic way that is also suggestive of chronic inflammation (8–10). Consequently, in addition to its long-term effects on the peritoneal membrane, inflammation has the potential to affect the efficiency of each PD dwell.
In recent animal studies, we have shown that intraperitoneal blockade of the complement and coagulation systems improve UF, possibly by mechanisms that reduce glucose transport (11,12). If similar effects are present in clinical PD, it could be possible, using equivalent additives to PD fluids, to improve the efficiency of PD during the useful length of this treatment. Furthermore, if inflammation is involved in the development of UF failure following long-term PD, there is also a potential for improving the technique survival of PD.
Most macromolecular PD fluid additives do not resist heat sterilization. Consequently, those compounds are not suited for the manufacturing techniques used for commercial PD fluids. Low molecular weight compounds with anti-inflammatory effects are therefore interesting candidates for PD fluid supplementation. This group of drugs includes calcium chelators, which resist heat sterilization and have a range of anti-inflammatory effects. Ethylenediaminetetraacetic acid (EDTA) and sodium citrate are calcium chelators in clinical use, citrate being a standard anticoagulant in hemodialysis (13,14) and blood transfusion. There are data showing significant inhibition of the complement system (15) in the citrate concentration range used to obtain anticoagulation, although complement inhibition is not total (16,17). Calcium chelation also has the potential to block inflammatory cells such as mast cells (18). In addition, citrate acts a scavenger, reducing reactive oxygen species in vitro (19). In contrast to EDTA, citrate does not depend on renal excretion and is therefore suited for the treatment of patients with renal disease. A major part of the circulating citrate is metabolized to bicarbonate in the liver and bound calcium is thereby released (14). Thus, sodium citrate is a feasible additive to PD fluids in commercial production with a potential for complement and coagulation inhibition that might prove favorable to the biocompatibility of PD.
The present study was performed to evaluate the dose response of citrate-supplemented PD fluid on transperitoneal transport of water and small solutes in acute single dwells in catheterized rats. In order to establish possible mechanisms of action, the kinetics of citrate and free calcium ions were characterized in relation to intraperitoneal coagulation. In a standard 2.5% glucose, lactate-buffered PD fluid, citrate was substituted for lactate in concentrations ranging from 0 to 15 mmol/L.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In separate experiments based on 1-hour dwells, 10 mmol/L (n = 10) and 0 mmol/L (n = 10) citrate substitutions were further evaluated. Here, the transport of glucose and urea was compared between the fluids and the intraperitoneal kinetics of citrate and calcium were related to coagulation at 10 mmol/L citrate substitution. Sampling was performed at 0, 30, and 60 minutes.
ANIMALS AND ANESTHESIA
The study protocol was approved by the Göteborg Ethics Committee and
the NIH guide Humane Care and Use of Laboratory Animals was followed.
Male Sprague–Dawley rats weighing 300 – 400 g were used in the
experiments.
Catheter implantation was performed 1 week before the experiment. A 7-fr silicone catheter (Renasil SILO8O; Braintree Scientific, Braintree, MA, USA) was implanted under sterile conditions and general anesthesia. A mid-line incision was made through the abdominal skin, taking care not to cause any bleeding, and a hole was pierced through linea alba with a 3-mm diameter tapered needle. After inserting 2.5 cm of the tip through the hole, the catheter was sutured to the superficial abdominal muscle fascia and the rest of the catheter was tunneled subcutaneously to the neck region and mobilized through the skin. After injecting 5 mL of saline, a stainless clip was used to close the catheter and the wounds were closed with agrafes. No antibiotics were administered.
General anesthesia was induced and maintained by inhalation of isoflurane (Baxter Medical AB, Kista, Sweden) in room air. During catheter implantation, the duration of anesthesia was approximately 20 minutes. During PD fluid infusion and the sampling procedures included in the experiment, the duration of anesthesia was below 10 minutes each time. At the end of the experiment, the animals were sacrificed under general anesthesia.
FLUIDS AND ADDITIVES
The PD fluids used were laboratory made and filter sterilized (Nalgene 0.2
UM SFCA, 150 mL; Nalgene NUNC International, New York, NY, USA). Two stock
solutions, one buffered with 40 mmol/L citrate (11.76 g/L sodium citrate) and
the other with 40 mmol/L lactate (7.5 g/L 60% sodium lactate syrup) were used
to create the PD fluids for this study. Except for this difference, the
composition of both fluids was equal, including 25 g D-glucose
(2.5%), 5.4 g NaCl, 0.051 g MgCl2·6H2O, and 0.198
g CaCl·2H2O.
The stock solutions were mixed to obtain citrate concentrations of 0 mmol/L, 5 mmol/L, 10 mmol/L, and 15 mmol/L, corresponding to lactate concentrations of 40, 35, 30, and 25 mmol/L, respectively. Measurements of osmolarity (freezing point osmometer) showed only small differences between the fluids. Total osmolarity increased by 0.6 mOsm for each 1 mmol/L citrate substituted, an effect of different sodium-binding capacities (3:1) and degree of ionization.
PERITONEAL DIALYSIS AND SAMPLING
The rats were allowed to heal for a full week following catheter
implantation. After this, the experiment began with a single intraperitoneal
20-mL infusion of PD fluid. After 1 – 2 minutes of abdominal massage
(labeled 0 hours), a 1-mL fluid sample was drawn through the catheter and the
animal was allowed to wake up. Two and 4 hours later, the animal was
anaesthetized again and additional 1-mL fluid samples were taken. The PD fluid
samples and a blood sample taken at 4 hours were supplemented with sodium EDTA
at a final concentration of 10 mmol/L. Samples were used directly for cell
counting and, after centrifugation, the cell-free supernatant was stored
frozen for the remaining analyses.
In separate short dwell experiments, the corresponding sampling was performed at 0, 30, and 60 minutes of dwell time.
ANALYSIS OF COAGULATION
Coagulation was determined from measurements of the
thrombin–antithrombin (TAT) complex concentration (Enzygnost TAT micro
#OWMG 15; Dade Behring, Marburg GmbFl, Marburg, Germany). TAT levels are
expressed as total intraperitoneal quantities.
MEASUREMENTS OF GLUCOSE, UREA, AND CITRIC ACID
For glucose determinations, the samples were diluted 1:10 with sterile
water in order to obtain glucose concentrations within the measurement range.
The Glucose Hexokinase II reagent kit (ADVIA Chemistry, # 04903429; Bayer
Healthcare LLC, UK) was adapted for use with 96-well plates in a plate reader.
The method has a typical reproducibility of about ±5%.
For citric acid measurements, the samples were diluted 1:10 with sterile water in order to obtain concentrations within the kit's analytical range (UV method, #10139076035; R-Biopharm AG, Darmstadt, Germany).
Urea was measured using the QuantiChrom Urea Assay Kit (#DIUR-500; BioAssay Systems, Hayward, CA, USA).
MEASUREMENTS OF CALCIUM IONS
Free (ionized) calcium was determined from calibrated readings of a calcium
ion-selective electrode (Orion 97-20 ionplus; Thermo Electron, Waltham, MA,
USA).
DETERMINATION OF UF VOLUME
Radiolabeled albumin was used as intraperitoneal volume marker to allow
measurements of UF and PD fluid reabsorption. Thus, 10 kBq
125I-labeled human serum albumin (GE Healthcare, Kjeller, Norway),
in combination with 1 mg unlabeled bovine serum albumin, which blocked surface
adsorption, was added to the PD fluid. The concentration of this volume marker
was determined by gamma count.
At the end of the experiment, 10 mL PD fluid without additives was injected through the catheter after the 4-hour sample had been collected. The dilution of tracer induced by this injection was used to calculate the final intraperitoneal fluid volume. Osmotic UF, net UF, and fluid reabsorption were calculated by combining data from tracer dilution during the dwell with the measured final intraperitoneal fluid volume and the known total activity of tracer infused. Fluid reabsorption was assumed to occur at a constant volume flow rate during the dwell. Intraperitoneal volumes at 2 and 4 hours were calculated from the dilution of the amount of tracer remaining at those times. Net UF was defined as the net volume gain at 2 and 4 hours; osmotic UF was calculated as net UF + fluid reabsorption. Ultrafiltration data are expressed as milliliters per 100 g body weight.
STATISTICS
Nonparametric statistics were used to evaluate differences between
treatments (Mann–Whitney U test) and correlations between variables in
groups of animals (Spearman rank test). The chosen level of significance was
p = 5%. Measured variables are presented in the text as mean ±
SEM.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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There was also a strongly significant correlation between glucose concentration and osmotic UF at 2 and 4 hours in the material as a whole, suggesting that changes in UF may have been caused by changes in intraperitoneal glucose retention (Figure 4). Despite this, the positive correlation between citrate dose and glucose retention did not reach significance (p = 0.08 and p = 0.13 at 2 and 4 hours respectively).
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The osmotic UF increase induced by 10 mmol/L citrate substitution was quantitatively important, amounting to 24.7% ± 7.7% at 4 hours. Fluid loss from the peritoneal cavity did not differ significantly between the groups (25.7 ± 3.6 vs 32.9 ± 1.6 µL/minute at 10 and 0 mmol/L respectively, p = 0.11).
Coagulation was not significantly inhibited by citrate at 2 and 4 hours. At time 0, due to the sampling procedure corresponding to approximately 2 minutes of dwell time, there was a tendency toward lower TAT levels in the citrate-treated animals (71.4 ± 23.3 vs 131.0 ± 25.0 ng, p = 0.07).
In the separate short-dwell experiments comparing 10 mmol/L and 0 mmol/L citrate, there was a similar tendency toward lowered TAT levels at 0 minutes but not at 30 or 60 minutes. Citrate was rapidly eliminated from the peritoneal cavity and, at 30 minutes, citrate concentrations were approximately half those measured initially. Ionized calcium concentrations rose concomitantly and were 0.6 mmol/L at 30 minutes and 1.2 mmol/L at 60 minutes (Figure 5). The transperitoneal transport of glucose and urea was evaluated from calculated values for D/D0 glucose (dialysate concentration/initial dialysate concentration) and dialysate/plasma concentrations (D/P) of urea. Substitution of 10 mmol/L citrate significantly lowered D/P urea at 30 minutes (Table 1); otherwise there were no significant effects of citrate substitution.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Citrate substitution at 10 mmol/L significantly increased glucose retention (Figure 2) and glucose concentration was significantly correlated with osmotic UF (Figure 4). Taken together, this is strong evidence that the increase in UF was related to reduced glucose transport, caused by the citrate substitution. The fact that citrate concentration did not correlate significantly with glucose concentration (p = 0.08 and 0.13 at 2 and 4 hours respectively) suggests that either the dose response of this effect was not linear or that the statistical power of the study was insufficient.
The connection between known actions of citrate and the observed effect on glucose transport and UF, however, remains unclear. Data on urea transport showed that the D/P value was reduced by citrate (Table 1), suggesting that citrate interacts with the general relation between filtration and diffusion rather than specifically affecting glucose transport mechanisms. This assumption is further supported by a similar trend for all measurements of glucose and urea in the 1-hour dwell experiments (Table 1). Detailed characterization of how citrate interacts with the different transport processes involved in PD requires specifically designed experiments.
It could be argued that the improvement in UF was due to direct osmotic effects of sodium citrate; however, this is inconsistent with the small measured difference in osmolarity between the fluids. The concentration of free sodium ions was only 6 mmol/L higher in the 10 mmol/L citrate solution than in the pure lactate solution. The higher molecular weight of citrate, compared with lactate, also added a transient osmotic effect since it slowed down the out-diffusion of citrate from the intraperitoneal space. Based on known empirical relations between transperitoneal diffusion rates and molecular weights in humans (20), the mass transfer area coefficient (MTAC) of lactate should be approximately 70% that of citrate. Our unpublished measurements of citrate kinetics in humans confirm this relation. The intraperitoneal half-life of citrate in the rat model was approximately 40 minutes (Figure 5). If the same relation between molecular weight and MTAC were valid in rats and humans, there would be a maximum osmolarity increment of less than 2 mOsm at 10 mmol/L citrate substitution due to slower out-diffusion. Consequently, the osmotic effects of citrate substitution were effective only during the first hour of the dwell and amounted to a total of less than 2% of the osmolarity of the PD fluid. Therefore, the 25% improvement in UF by 10 mmol/L citrate substitution has to be explained on the basis of the pharmacological effects of citrate, and was most likely due to the calcium-chelating properties of this molecule.
There are a number of calcium-dependent biological systems present in the peritoneum. The intention was to block the cascade systems present in the peritoneal cavity, that is, the complement and coagulation systems. A total inhibition of the alternative pathway of the complement system, however, requires that magnesium is removed in addition to calcium (17) and this action is not provided by citrate. Nor did citrate induce any significant anticoagulation in terms of reduced thrombin formation. The measurements from the initiation of the dwell indicate that there might have been an anticoagulation effect that came and went during the first 30 minutes of the dwell.
The transient calcium elimination caused by citrate was not synchronized with the sustained effects on glucose transport or UF. This indicates an indirect mechanism of action, probably via cellular mechanisms that were initiated during the first 30 minutes of the dwell. Cellular targets for calcium chelation include mast cells (18), eosinophils (21), and sensory nerve endings (22) that may be triggered by hyperosmolar PD solutions. Citrate has also been shown to act as a direct scavenger of superoxide radicals (19) and it therefore has a potential to interact with the signaling pathways of inflammatory cells (23).
Clinical PD means repeating the acute exposure and thus risking cumulative effects. The low calcium levels created in the peritoneal cavity by citrate may be potentially harmful to cells, especially the mesothelial cell layer and intraperitoneal cells, mainly leukocytes. Neutrophils of blood anticoagulated by citrate seem to have normal responses in terms of chemotaxis and respiratory burst (24). Some lymphocyte responses may, however, be partly inhibited by citrate anticoagulation (25). The minimum intraperitoneal calcium concentration measured in the present study was approximately 150 µmol/L (Figure 5) and was compatible with the stability constant of 3.5 for the calcium–citrate complex. Severe calcium depletion might contribute to cell injury. Calcium levels below 4 µmol/L — obtainable in vitro using strong chelators such as EGTA — disrupt intercellular junction complexes and cytoskeletal structures (26). At calcium concentrations below the level of the cytoplasm (0.1 – 0.4 µmol/L), irreversible cell damage occurs.
In the clinical situation, the quick elimination of citrate prevents calcium loss by the drained dialysate but leads to a rapid uptake of citrate into the blood. Substitution of 10 mmol/L lactate by citrate will supply the PD patient with 20 mmol citrate per standard 2-L dwell. Assuming an elimination rate that is proportional to that of the rat (a similar intraperitoneal half-life), the maximum uptake of citrate during the first minute of the dwell would be 0.2 mmol/minute. Maximum infusion rates in clinical practice are in the range of 0.015 – 0.020 mmol/(minute x kg body weight), corresponding to 1 mmol/minute in a person with 50 – 70 kg body weight. This is probably an overestimation since citrate elimination is faster in rats than in humans.
Differences between man and rat also apply to the time history of the citrate effect. Net UF was significantly improved already at 2 hours of dwell time. Peritoneal dialysis in rats is generally faster (approximately twice as fast) than in humans due to, for example, the scaling down of size and the metabolic differences. The favorable effects also at 2 hours in the rat may thus translate to conditions during 4-hour CAPD dwells in humans. It is hard to speculate on the effects of citrate on automated PD in humans and we plan studies of citrate supplementation in this setting.
To summarize, intermittent intraperitoneal exposure to citrate at concentrations below 10 mmol/L over longer periods of time seems to be a safe treatment. Obviously, this assumption has to be confirmed by adequate long-term studies focusing on possible cytotoxic effects of calcium depletion.
We used filter-sterilized fluids in the present study in order to simulate PD fluids with low glucose degradation product (GDP) content. In this way, the obtained results are relevant to future development of modern biocompatible PD fluids. Any impact of citrate substitution on the old type of heat-sterilized PD fluids is hard to predict since the effects of GDPs are complex and include anti-inflammatory as well as proinflammatory actions.
In conclusion, partially substituting the lactate buffer of PD fluids with citrate significantly improves the osmotic UF and net volume gain of a single dwell in a dose-dependent way. A 10 mmol/L citrate substitution that improves UF by 25% in rats seems to be compatible with clinical applications to PD; however, the efficiency of this concept remains to be confirmed in humans. The mechanisms of action are not directly connected to anticoagulation but seem to involve the relation between diffusion and UF, thereby increasing glucose retention.
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ACKNOWLEDGMENTS |
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Received 28 June 2007; accepted 12 May 2008.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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