NDT Advance Access originally published online on July 28, 2006
Nephrology Dialysis Transplantation 2006 21(10):2874-2880; doi:10.1093/ndt/gfl368
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Effects of anaesthesia on fluid and solute transport in a C57BL6 mouse model of peritoneal dialysis
1Research Service, VA Salt Lake City Health Care System 2Department of Medicine and 3Department of Bioengineering, University of Utah, Salt Lake City, UT, USA
Correspondence and offprint requests to: John (Ken) Leypoldt, PhD, Dialysis Program, University of Utah, 85 N. Medical Drive East Room 201, Salt Lake City, UT 84112-5350, USA. Email: ken.leypoldt{at}hsc.utah.edu
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Abstract |
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Background. Genetically modified mice show promise as animal models for studying the physiology and pathophysiology of the peritoneum during peritoneal dialysis (PD). Methods for evaluation of the functional characteristics of the mouse peritoneum have not been studied extensively, and the effects of anaesthesia on fluid and solute transport in mouse models of PD are unknown.
Methods. A single exchange of dialysis solution was performed in C57BL6 mice by injecting fluid into the peritoneal cavity using a 27-gauge needle and allowing fluid to dwell for 30, 60 or 120 min. Experiments evaluated the effect of ketamine (plus xylazine) anaesthesia on fluid and solute transport; these effects were examined in separate experiments using glucose and mannitol as the osmotic agent added to the injected dialysis solution. After euthanasia, blood was collected, the remaining dialysis solution was drained and their contents analysed for concentrations of the osmotic solute (glucose or mannitol), urea nitrogen (UN), sodium (Na) and a volume marker (fluorescein-labelled albumin) added to the initial, injected dialysis solution. Determined parameters included final volume of dialysis solution (drained plus residual fluid volume), dialysate concentration (D/D0) of glucose (or D/D0 mannitol), dialysate-to-plasma concentration ratio for (D/P) UN and D/P Na and the apparent dialysis solution volume by indicator dilution. Peritoneal permeability-area (PA) values or mass transfer-area coefficients were also calculated for the osmotic solutes.
Results. Final volumes of dialysis solution were higher when mice were anaesthetized with ketamine than in unanaesthetized mice, independent of whether glucose or mannitol was used as the osmotic agent. The increases in final volume were paralleled by higher dialysate concentrations (D/D0 values) and lower calculated PA values for both glucose and mannitol. When using either osmotic agent, anaesthesia also increased plasma glucose concentrations, suggesting that ketamine altered glucose metabolism.
Conclusions. Ketamine anaesthesia in the mouse decreases PA values for glucose and mannitol when used as osmotic agents in PD solutions. The decrease in transperitoneal transport for these osmotic agents increases the final volume of fluid which can be obtained from the peritoneal cavity.
Keywords: D/D0 glucose; D/P sodium; D/P urea; final drained volume; mass transfer-area coefficient; peritoneal permeability
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Introduction |
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Animal models for the study of peritoneal dialysis (PD) have been developed to achieve various objectives since this therapy was first demonstrated to remove a number of uraemic solutes from the cat [1]. Practical considerations have often constrained the choice of animals for these studies, and previous studies have commonly employed either dogs, rabbits or rats. During the past two decades, the rabbit and rat have most often been used [2], and the advantages of rat models of PD have recently been reviewed [3].
Genetically modified mice have proven to be a powerful tool for the study of animal physiology and pathophysiology, and mouse models for evaluating the functional properties of the peritoneum have been recently developed [4,5]. Those studies evaluated rates of fluid and solute transport across the peritoneum in anaesthetized mice, and others have also used a similar experimental approach [6]. The current experiments were performed to evaluate whether a mouse model of PD could be developed without the need for anaesthesia and to study the effect of ketamine anaesthesia on peritoneal fluid and solute transport. The results showed that ketamine anaesthesia significantly alters certain parameters related to peritoneal fluid and solute transport; thus, the method of anaesthesia should be considered when evaluating mouse models of PD. The results from these studies also imply that the method of anaesthesia should be considered in experimental studies of chronic exposure to PD fluids in mouse models.
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Methods |
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Experimental methods
Acute experiments were performed on 96 male C57BL6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) with initial body weights averaging 22.1 g (range 19.2–25.6 g). In each mouse, 2 ml of hypertonic PD solution with the addition of 5 µg/ml of fluorescein isothiocyanate (FITC)-labelled albumin (Sigma Chemical, St Louis, MO, USA) was injected into the peritoneal cavity using a 27-gauge needle. The FITC-labelled albumin was dialysed at 4°C overnight to remove any free FITC before use. After the needle was removed, a small quantity of cyanoacrylate cement (Superglue, Superglue Corporation, Rancho Cucamonga, CA, USA) was spread across the exit-site to prevent fluid from leaking out of the peritoneal cavity. At the end of the dwell period the mouse was euthanized, a blood sample of 0.5–1.0 ml was obtained and a 1–2 cm midline incision was made to surgically open the abdominal cavity. The mouse was then inverted and the fluid within the peritoneal cavity was drained as completely as possible. The drained fluid was weighed using a digital scale (XE series, Model 400, Denver Instruments, Denver, CO, USA). The residual fluid volume remaining in the peritoneal cavity after drainage was measured by swabbing all peritoneal tissues with sterile gauze and determined by the difference in weight, before and after swabbing. All fluid weights were then converted to volumes, assuming a solution density of 1.0 g/ml. The final volume of fluid in the peritoneal cavity at the end of the dwell was calculated as the sum of that drained and the residual fluid volume. The residual fluid volume averaged 0.12 ml for all experiments combined and was frequently only a small fraction of the volume drained. The blood sample was obtained either by cardiac puncture or by exsanguination after decapitation. Measurements of blood pressure or heart rate were not attempted in these studies.
Experiments were performed for dwell periods of 30, 60 and 120 min for dialysis solutions containing two different osmotic agents. Half of the experiments were performed using commercial PD solution containing glucose at a concentration of 3.86% (4.25% monohydrate glucose) as the osmotic agent (Delflex, Fresenius Medical Care North America, Ogden, UT, USA). The other half were performed using a PD solution prepared in the laboratory to be otherwise identical except that it contained mannitol (Sigma Chemical) at a concentration of 3.86% instead of glucose as the osmotic agent. Mannitol was chosen for comparison with glucose because it is the same molecular size as glucose but is not readily metabolized. After preparation, the latter solution was sterilized by filtration through a microporous membrane filter (0.2 µm, Nalgene, Rochester, NY, USA). The experiments at all dwell periods and both osmotic agents were performed without anaesthesia and also using ketamine anaesthesia (100 µg/g of ketamine and 10 µg/g of xylazine as an initial intramuscular injection followed by additional doses as necessary). All experiments were repeated under each set of conditions eight times.
Analytical assays
The blood sample was collected in a tube containing heparin and was centrifuged to obtain plasma for further analysis. Glucose and urea nitrogen (UN) were measured in plasma and dialysate samples using an automated analyser (Dade-Behring, Deerfield, IL, USA). Mannitol was measured in plasma and dialysate using a commercial assay kit (Megazyme, Wicklow, Ireland). To assess transperitoneal ultrafiltration into the peritoneal cavity during each dwell, the concentration of FITC-labelled albumin in the effluent dialysate was measured by first diluting the sample 1:10 in a 1.0 M Tris buffer (pH 8.0) with the absorbance measured at 460 nm using a spectrophotometer (Cecil 1021, Cambridge, UK).
Calculations and data analysis
To evaluate transperitoneal ultrafiltration during the dwell, the apparent (indicator dilution) volume of fluid within the peritoneal cavity at the end of the dwell (IDV(T)) was first calculated by the change in the concentration of FITC-labelled albumin by the following equation [7]
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The disappearance rate of glucose (or mannitol) out of the peritoneal cavity and the appearance rate of UN into the peritoneal cavity were first inspected by calculating the dialysate concentration relative to its concentration in the initial infused dialysis solution (D/D0) for glucose (or mannitol) and the dialysate-to-plasma concentration ratio (D/P) for UN, respectively. In addition, transperitoneal transport of sodium (Na) was also assessed by calculating D/P for Na. No corrections for differences in ultrafiltration were made to D/D0 or D/P values. In all experiments, permeability-area (PA) values (or mass transfer-area coefficients) for glucose or mannitol were calculated from the change in the dialysate concentration, the plasma concentration and the indicator dilution volume with time as described previously [8,9]. This calculation assumes that the glucose (or mannitol) sieving coefficient across the peritoneal membrane is zero; however, the calculated PA values are relatively insensitive to the assumed sieving coefficient.
All empirical and calculated values are reported as mean ± SE of the mean or SEM. Differences in measured and calculated parameters as a function of time and whether anaesthesia was used were analysed using two-way analysis of variance (ANOVA). All data presented graphically use bar graphs instead of x–y plots with connecting lines to emphasize that all results were obtained at only one dwell time for each mouse.
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Results |
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Experiments using glucose as osmotic agent
The results for experiments using glucose as the osmotic agent are shown in Figure 1. Figure 1A shows the measured final volumes of fluid remaining in the peritoneal cavity after dwell periods of 30, 60 and 120 min. The final volumes remained at approximately 2 ml after all dwell periods, but the volumes were higher when mice were anaesthetized with ketamine than in unanaesthetized mice. The higher final volumes of fluid with ketamine anaesthesia were accompanied by higher calculated indicator dilution volumes, indicating higher transperitoneal ultrafiltration (Table 1). These differences in transperitoneal ultrafiltration were not, however, statistically significant. Further, calculated fluid absorption rates were lower when mice were anaesthetized with ketamine (Table 2).
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The disappearance of glucose from the peritoneal cavity as assessed by D/D0 glucose in mice anaesthetized with ketamine and unanaesthetized mice is shown in Figure 1B. As expected, D/D0 glucose was lower with longer dwell periods indicating additional loss of glucose from peritoneal dialysate. The disappearance of glucose from the peritoneal cavity was less in mice anaesthetized with ketamine than in unanaesthetized mice. Figure 1C shows plasma glucose concentrations after different dwell periods in mice anaesthetized with ketamine and in unanaesthetized mice; the concentrations were >2-fold higher in anaesthetized mice at all dwell periods.
Figure 1D and E shows D/P for UN and Na at different dwell periods in mice anaesthetized with ketamine and in unanaesthetized mice. These D/P values were higher with longer dwells and were lower for anaesthetized mice. These data demonstrate significant Na sieving, especially in mice anaesthetized with ketamine, since D/P for Na measured in mice after a very short (<1 min) peritoneal dwell using hypertonic glucose dialysis solution was 0.925 ± 0.002 (N = 3, unpublished observations). Calculated PA values for glucose are shown in Figure 1F. PA values for glucose were time-independent and were lower in mice anaesthetized with ketamine than in unanaesthetized mice. The overall PA value for glucose in unanaesthetized mice was 57 ± 5 µl/min and in mice anesthetized with ketamine was 34 ± 4 µl/min.
Experiments using mannitol as osmotic agent
The results for experiments using mannitol as the osmotic agent are shown in Figure 2. In general, the results parallel those when using glucose as the osmotic agent with few exceptions. For example, final volumes of fluid remaining in the peritoneal cavity at the end of the dwell period were higher (Figure 2A), calculated indicator dilution volumes were higher (Table 1) and fluid absorption rates from the peritoneal cavity were lower (Table 2) in mice anaesthetized with ketamine than in unanaesthetized mice. Further, D/D0 mannitol was higher (Figure 2B), but D/P for UN (Figure 2D) and Na (Figure 2E) were lower in anaesthetized than in unanaesthetized mice. Plasma mannitol concentrations were slightly lower with longer dwell periods (data not shown), but were relatively low and were 102 ± 7 and 98 ± 9 mg/dl in mice anaesthetized with ketamine and in unanaesthetized mice, respectively. In contrast, plasma glucose concentrations were dependent on dwell time when mice were anaesthetized with ketamine (Figure 2C); the plasma glucose concentration was higher for longer dwell periods in these experiments even in the absence of glucose in the initial PD solution. Calculated PA values for mannitol were independent of the length of the dwell period and substantially lower when mice were anaesthetized with ketamine (Figure 2F). The overall PA value for mannitol in unanaesthetized mice was 46 ± 4 µl/min and in mice anaesthetized with ketamine was 28 ± 5 µl/min.
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Discussion |
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Mouse models of PD have been recently developed, largely to preferentially use knockout mice and more specifically examine molecular mechanisms in understanding peritoneal physiology and pathophysiology [4,5]. Similar to other animal models of PD, mouse models often require anaesthesia because surgical procedures are necessary to implant catheters for measuring blood pressure and to sample blood frequently for chemical analyses. In addition, anaesthetics act to sedate and immobilize animals so that PD experiments can be readily performed. The results from models of PD which use anaesthetized animals during an experimental dwell provide fluid and solute transport characteristics which are reasonable and often directly comparable with those for PD patients.
On the other hand, it is well known that the use of anaesthetics alters numerous physiological functions [10], especially those related to the cardiovascular system [11,12]. Although the action of numerous drugs has been shown to alter peritoneal membrane transport properties [13], the effects of anaesthetics on peritoneal fluid and solute transport have not been extensively studied. Studies by Breborowicz and Knapowski [14] more than 20 years ago showed that intraperitoneal administration of the local anaesthetic procaine increased urea and inulin clearances in a rabbit model of PD. More recent studies have examined the effect of anaesthesia on lymphatic drainage and fluid absorption rates from the peritoneal cavity. By directly cannulating lymphatic vessels in a sheep model, Tran et al. [15] showed that halothane anesthesia profoundly decreased flow rates from lymph vessels draining fluid from the peritoneal cavity by 
80%. These same investigators also showed that halothane anaesthesia decreased fluid absorption rates from the peritoneal cavity by 30% [16].
Our initial, preliminary studies in an unanaesthetized mouse model of PD [17] demonstrated low final volumes of fluid remaining in the peritoneal cavity after a 2 h dwell. The current studies therefore directly compared peritoneal fluid and solute transport in mice anaesthetized with ketamine (and xylazine) with that in unanaesthetized mice. The results from our study demonstrate that ketamine anaesthesia results in higher final volumes of fluid at the end of the study dwells (Figures 1A and 2A). These higher final fluid volumes were due to both enhanced transperitoneal ultrafiltration (Table 1) and lower fluid absorption rates from the peritoneal cavity (Table 2). Thus, anaesthesia decreases fluid absorption rates from the peritoneal cavity in the mouse, similar to that observed in the sheep [16]. It is also possible that anaesthesia could decrease other physiological parameters affecting fluid absorption, rates such as capillary surface area. Similar results were obtained when using either glucose or mannitol as the osmotic agent.
The higher rates of transperitoneal ultrafiltration in mice anaesthetized with ketamine are caused, at least in part, by the slower loss of the osmotic agent from the peritoneal cavity (Figures 1B and 2B). This is likely caused by a generalized decrease in transperitoneal transport since it was accompanied by lower rates of equilibration for both urea and Na (Figures 1D and E and 2D and E). A decrease in the calculated PA values for both osmotic agents, glucose and mannitol, in mice anaesthetized with ketamine was also observed (Figure 1F and 2F). These results suggest that ketamine anaesthesia decreases transperitoneal transport for small solutes. It is possible that such general decreases in transperitoneal transport are the result of a decrease in peritoneal blood flow [18,19], although a general decrease in the permeability or effective surface area of peritoneal capillaries or decreases in interstitial tissue permeability cannot be excluded as alternative explanations of these data.
The current studies also show that ketamine anaesthesia can significantly increase plasma concentrations of glucose in the C57BL6 mouse, especially when using glucose as the osmotic agent (Figure 1C). The increase in plasma concentration of glucose when using mannitol as the osmotic agent is more gradual (Figure 2C) and suggests that ketamine anaesthesia decreases glucose clearance from the blood, perhaps by decreasing glucose metabolism. Indeed, others [20] have recently shown that plasma glucose concentrations and glucose metabolism can be altered significantly in C57BL6 mice not undergoing PD when using ketamine anaesthesia. Those investigators also noted that glucose concentrations and metabolism were also altered by isoflurane anaesthesia, but these effects were less significant than during ketamine anaesthesia. The effects of ketamine anaesthesia on plasma glucose concentrations are likely amplified in the C57BL6 mouse when a large glucose load is administered intraperitoneally, such as during PD.
Ni et al. [4] have recently characterized fluid and solute transport in the C57BL/6J mouse anaesthetized with ketamine (and xylazine). Those investigators demonstrated that PD using 3.86% glucose dialysate yielded equilibration curves for urea and glucose, Na sieving and net ultrafiltration that were similar to those obtained in their rat model. Further, an increase in dialysate glucose concentration resulted in higher net ultrafiltration and additional Na sieving. Interestingly, these investigators also observed that female mice had lower Na sieving. The results from our study are consistent, in general, with those findings. Our equilibration curves are qualitatively similar to those reported by Ni et al.; however, the rates of equilibration we observed were more rapid for all solutes evaluated. For example, we attempted to calculate PA values for urea from our experiments but the rate of equilibration was too high to obtain very accurate estimates. Limiting our analyses to the experiments using a 30 min dwell, we could estimate (using the same methods described above for glucose) PA values for urea of 236 ± 26 µl/min in unanaesthetized mice and 140 ± 27 µl/min in mice anaesthetized with ketamine when using glucose as the osmotic agent and 273 ± 36 µl/min and 110 ± 18 µl/min, respectively, when using mannitol as the osmotic agent. These estimates are considerably greater than the PA value reported for urea of 23 ± 2 µl/min by Ni et al. [5]. In contrast, our calculated PA values for glucose and mannitol are comparable with that reported for glucose of 49.6 ± 3.1 µl/min in another mouse model of PD [6].
Significant challenges when using mice as experimental models include their large ratio of body surface area to body weight and their high rates of metabolism. The large surface area-to-body weight ratio creates difficulty in determining the most relevant fluid volume to mimic the physiology during PD in humans. Assuming that peritoneal membrane surface area scales with body surface area [21], it would be necessary to scale the volume of the initial dialysis solution fluid by mouse body weight to the two-thirds power in order to maintain the same peritoneal membrane surface area to dialysis solution volume as in human PD patients. This would suggest that the initial volume of dialysis solution used should be (25 g/70 000 g)2/3 x 2000 ml or 10 ml. Such a large volume of PD solution would not likely appropriately scale a number of other physiological parameters, however. Thus, the use of anaesthesia to lower peritoneal permeability may be beneficial in creating equilibration curves similar to those in other animal models and human PD patients, without the need to use such large volumes of PD solution. As can be observed from the current study, a large surface area-to-body weight ratio in unanaesthetized mice results in the rapid absorption of glucose from the peritoneal cavity, and accordingly, low net ultrafiltration and final volume remaining in the peritoneal cavity after a 2 h dwell.
An additional implication from the findings of this study is that the use of anaesthesia in PD models using the C57BL6 mouse may directly alter plasma and dialysate glucose concentrations. Because high concentrations of glucose may be detrimental to the longevity of the peritoneum as a dialysing membrane [22], chronic exposure models of PD must account for their use of anaesthetic agents.
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This material is based upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
Conflict of interest statement. None declared.
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[Abstract/Free Full Text]
Accepted in revised form: 26. 5.06
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