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Division of Nephrology and Blood Purification, Department of Internal Medicine, Tokyo Women's Medical University Medical Center East, Tokyo, Japan
Correspondence to: H. Nishimura, 2-1-10 Nishiogu, Arakawa-ku Tokyo, Japan. hidekigm{at}dnh.twmu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSUREREFERENCES |
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Objective: The development of a glucose-free peritoneal
dialysis (PD) solution is important because glucose has been associated with
functional and morphological damage to the peritoneal membrane. The
ultrafiltration (UF) and biocompatibility of new PD solutions containing
taurine (PD-taurine) instead of glucose as an osmolite were tested in a rat PD
model. Methods: To determine the solution's UF ability,
different concentrations of taurine in PD solutions were compared to
glucose-based PD solutions (PD-glucose) by giving single intraperitoneal
injections for 2, 4, and 6 hours. To examine the biocompatibility of
PD-taurine, the rats were divided into 3 groups: a 3.86% PD-glucose group, a
3.5% PD-taurine group and a not dialyzed group. The rats were given 10-mL
injections of PD fluids intraperitoneally 3 times daily for 7 days. A
peritoneal equilibration test (PET) was performed using a 1.9% xylitol
solution at the time the rats were sacrificed. Mesothelial cell monolayers
were obtained from the animals and studied based on a population
analysis. Results: The net UF of PD-taurine increased in a
dose-dependent manner; the 3.5% PD-taurine solution was equivalent to the
3.86% PD-glucose solution after 4 hours. The PET showed that the drainage
volume and the D4/D0 ratio for xylitol after 4 hours
with PD-taurine solution were significantly greater than with the PD-glucose
solution (p < 0.001 and p < 0.001 respectively).
Mesothelial and fibroblast-like cell proliferation was significantly less with
PD-taurine than with PD-glucose (p < 0.01). Conclusions: These results indicate that PD-taurine
resulted in net UF equivalent to that of PD-glucose and was more biocompatible
than PD-glucose with respect to the peritoneal membrane.
KEY WORDS: Biocompatibility; GDP; osmolite; peritoneal dialysis fluid; taurine.
Glucose is widely used as the osmotic agent for peritoneal dialysis (PD) solutions because it is effective as an osmolite, inexpensive, easily metabolized, and may serve as a natural source of energy. However, there are some disadvantages related to the use of glucose-based solutions. During PD, various metabolic problems, including hyperlipidemia, hyperglycemia, and obesity, may occur due to continuous absorption of glucose from the dialysate (1). Other problems that occur are due to the high glucose levels, which stimulate peritoneal mesothelial cells to produce extracellular matrix, upregulate transforming growth factor-beta, fibronectin, and plasminogen activator inhibitor-1 (2–4), and increase cellular reactive oxygen species through activation of protein kinase C, NADPH oxidase, and mitochondrial metabolism (5). In addition to these direct effects of glucose, glucose degradation products (GDPs) are generated during heat sterilization of PD solutions that contain glucose. Although the acute toxicity of GDPs contained in PD solutions is limited, large amounts of data demonstrate that exposure of the peritoneal membrane to a low level of GDPs over a long period results in structural and functional changes (6,7). Moreover, it has also been reported that GDPs in the peritoneum contribute to the formation of advanced glycation end products (AGEs), which affect the morphology and function of the peritoneum (8,9). The accumulation of AGEs accelerates peritoneal permeability and thus causes ultrafiltration (UF) failure. Of note, double chambered, neutral-pH PD solutions that have recently been developed contain levels of GDPs that are not negligible. Therefore, the development of a glucose-free PD fluid is important. One such solution is an amino acid-based PD solution. This solution is comparable to glucose-based PD solution for solute clearance and UF; it is especially efficient in patients that have a poor nutritional status. On the other hand, using amino acid solutions for PD in clinical practice increases blood urea nitrogen levels and plasma ornithine levels, which reflect increased nitrogen load and activation of the urea cycle and provoke a metabolic acidosis. The metabolism of amino acids results in increased production of H+ ions. Basic amino acids and sulfur-containing amino acids, methionine and cysteine in particular, generate protons (10,11).
Taurine (2-aminoethanesulfonic acid) is a sulfonic beta-amino acid found in high concentration in mammalian cells; it is a physiological substance that regulates osmotic balance and ion transport. The molecular weight of taurine is 125 Da, which is less than that of glucose (180 Da). Due to its zwitterionic nature, taurine has a high water solubility, a low lipophilicity, and a high dipole; that is, it keeps a neutral pH and can have a strong buffering effect. Taurine is known to be an extremely safe substance: animal studies have shown no toxic effects, even when taurine is given to rats at the maximum dose (12). The use of taurine supplementation is effective in controlling hypertension and hyperlipidemia (13). Taurine is relatively cheap and easily available. For all these many reasons, we identified taurine as a good candidate for use in PD solutions.
Thus, in this study, we evaluated peritoneal transport during dialysis using taurine as osmotic agent instead of glucose and tested the biocompatibility of the new PD solution in a rat PD model.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSUREREFERENCES |
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Animals: In experiment 1, 152 male Sprague–Dawley rats (Sankyo Lab Service, Tokyo, Japan), weighing 250 – 290 g, were used: 72 rats were allocated to the PD-glucose group, with 24 for each of the 3 different percentages of the PD glucose solution; 72 rats were allocated to the PD-taurine group, with 24 for each of the three different concentrations of taurine solution; and 8 rats were used as controls. None of the rats used in this study were lost. The protocols were carried out in strict accordance with the guidelines of the US National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Tokyo Women's Medical University. All the rats had normal kidney function.
The rats were divided into three groups: the PD-glucose group, which received 1.36%, 2.27%, or 3.86% glucose-based PD solutions; the PD-taurine group, which received PD solutions using 1.0%, 1.8%, or 2.8% taurine as osmotic agent; and a control group (Table 1). All solutions had a neutral pH. Except the control rats, all rats were injected with 30 mL PD fluid intraperitoneally using a 22-gauge needle and the solutions were allowed to be absorbed from the peritoneal cavity. The animals had access to a standard diet (Oriental Yeast Co., Tokyo, Japan) and water ad libitum until sacrifice. The animals were anesthetized with diethyl ether and blood samples were collected by direct cardiac puncture 2, 4, and 6 hours after the injection of the PD solution. To determine the validity of dialysate recovery, another 8 rats were injected with 30 mL PD solution and the peritoneal cavity was then immediately opened. The fluid was collected with a syringe and the recovery volume was measured. A mean of 29.5 ± 0.6 mL of fluid was recovered. Net UF was evaluated as follows: net UF (mL) = drainage volume (mL) after the 2-, 4-, and 6-hour dwells – 29.5 mL. Blood samples were obtained to measure the plasma taurine levels.
After the animals were sacrificed, a midline abdominal incision was made, the peritoneal fluid was completely collected with a syringe, and its volume was measured. The dialysate and blood samples were centrifuged and stored at –20°C until assayed. Glucose in the dialysate samples was measured with an automated analyzer (Cobas Mira Plus; Roche Diagnostics, Basel, Switzerland). Plasma and dialysate taurine concentrations were measured as follows using HPLC (Sugar CH-G guard column + Sugar CH-801 column, Showa Denko, Tokyo, Japan; Alliance 2690, Waters, Tokyo, Japan). The plasma taurine levels were measured using the Waters AccQ·Fluor Reagent Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Taurine concentration was calculated from its peak height based on the standard taurine height.
EXPERIMENT 2: EVALUATION OF THE TIME COURSE OF UF
Animals and Procedure: Twelve male Sprague–Dawley rats,
weighing 300 – 320 g, were used. Each rat was anesthetized using a
single intraperitoneal injection of pentobarbital (50 mg/kg body weight). The
use of pentobarbital at this dose has been reported to not alter peritoneal
fluid transport, such as the peritoneal fluid absorption rate and peritoneal
lymphatic flow, in rats (15).
After 2 hours, the rats were given 25 mg/kg pentobarbital sodium
subcutaneously every hour to maintain the depth of anesthesia during the
experiment. The fur over the abdominal wall was closely shaved. The animal was
placed in the supine position and kept at 37°C with a heating pad. An open
airway was established by tracheotomy. Two vascular access points were used
during the study: a femoral artery catheter (3 fr; Atome Medical, Tokyo,
Japan) for obtaining blood samples and a femoral vein catheter (3 fr) for
infusing fluid (isotonic saline, 2 mL/hour) to prevent hypovolemia. Using
scissors, multiple holes were made in a silicone tube (1.0-mm diameter; Kaneka
Medix, Osaka, Japan), which was sterilized before being used. The modified
silicone tube was inserted percutaneously below the xiphoid process on the
right side.
A standard peritoneal permeability analysis (SPA) in the rat was carried out using a modification of the human SPA described by Pannekeet et al. (16). The experiment was started with an intraperitoneal injection of 30 mL PD-taurine 2.5% (n = 6) or PD-glucose 2.27% (n = 6) that contained 1 g/dL dextran 70 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) to allow the calculation of fluid kinetics. The solution was administered via a 1.0-mm catheter over 1 minute and instilled in the peritoneal cavity for 4 hours. Dialysate samples (400 µL) were taken at 30, 60, 120, 180, and 240 minutes after the dialysis solution had been infused. Prior to each sampling, 1 mL dialysate was flushed back and forth five times through the catheter to ensure that the PD solutions were homogeneously distributed. Blood samples were drawn at 0, 60, 120, and 240 minutes to assay the glucose or taurine concentration. After 240 minutes, the dialysate was completely collected from the peritoneal cavity using a syringe and the volume was recorded. In the SPA, none of the animals were lost during the study period.
The dialysate and blood samples were centrifuged and stored at –20°C until assayed. Total dextran was determined using HPLC (17). Peritoneal fluid transport was calculated as previously described (16,18). In brief, the amount of net UF is the sum of transcapillary ultrafiltration (TCUF), which increases the peritoneal volume, uptake in the lymphatic system, and back filtration, which causes fluid loss from the peritoneal cavity. Transcapillary UF was calculated as the dilution of dextran 70, which is a volume marker. The TCUF rate was calculated by dividing TCUF by dwell time. The convective loss of volume marker from the peritoneal cavity during the dwell can be used as an indirect method to quantify the contribution of peritoneal lymphatic absorption (LA) (19). These calculations include all pathways of lymphatic system uptake, both interstitial and subdiaphragmatic. The LA rate was determined by dividing LA by dwell time. The change in the intraperitoneal volume during the dwell can be calculated from the dilution of volume marker after correcting for incomplete recovery. Net UF rate was defined as the change in the intraperitoneal volume divided by dwell time.
EXPERIMENT 3: EVALUATION OF BIOCOMPATIBILITY
Animals: Thirty male Sprague–Dawley rats were divided into
three groups: a control group (n = 10), which was not exposed to any
PD solutions (sham operated); a PD-glucose group (n = 10), which
received a 3.86% glucose-based PD solution (low GDP, neutral pH); and a
PD-taurine group (n = 10), which received a 3.5% taurine-based PD
solution. All injections were given intraperitoneally three times daily (10 mL
each) for 1 week. Based on experiment 1, we estimated that a 3.5% taurine
concentration was necessary to achieve the equivalent net UF seen with a 3.86%
glucose PD solution. We chose these high concentration solutions to maximize
the effects of glucose, thus hoping to more easily detect any differences
between the solutions. Dialysis solutions were supplemented with antibiotics
(tobramycin 0.12 mg/10 mL and cefazolin sodium hydrate 2.5 mg/10 mL). Body
weight was monitored at the beginning and the end of the experimental period.
A modification of the method of Irwin
(20) was used for evaluation
of acute behavioral, neurological, and autonomic effects in the rats. A
semiquantitative scoring scale was used. Briefly, while placed in an open
field, rats were assessed for transfer arousal, involuntary movements
(tremors, convulsions, etc.), gait abnormalities, mobility, arousal,
respiration, stereotypy, unusual behavior, rears (defined as any time both
front paws left the floor), defecation, urination, diarrhea, polyuria,
piloerection, and response to stimuli to assess reactivity (approach with
blunt object, tail pinch). Corneal reflex, righting reflex, and inverted grid
test were also assessed. For the assay of the plasma taurine concentrations,
blood samples were taken from the jugular vein before the daily injection on
experimental days 0, 1, 4, and 7. No rats were lost and all animals appeared
to be healthy during the study, which involved repeated infusions of PD
fluid.
Peritoneal Equilibration Test (PET) and Measurement of Hyaluronic Acid in the Effluent: At the end of the experimental period, 30 mL 1.9% xylitol solution was injected into the peritoneal cavity using a 22-gauge needle. Four hours later, the animals were anesthetized with diethyl ether and direct cardiac puncture was used to collect blood samples. A midline abdominal incision was then made, residual peritoneal fluid was collected with a syringe, and the total drained dialysate volume was measured. The concentration of xylitol in the dialysate samples was measured using HPLC. Since glucose or taurine solutions were used, xylitol was used for the PET to avoid contamination by residual PD solution (21). The peritoneal fluid was cultured on blood agar culture plates for several days (22) and stained with Samson's solution. Animals whose peritoneal fluid developed colonies on the culture plates and had colonies growing or developed adhesions in the peritoneal cavity upon incision of the abdomen were excluded. One rat in each group was excluded.
The level of dialysate hyaluronic acid was measured using a hyaluronic acid plate (Chugai Pharmaceutical, Tokyo, Japan).
Preparation for Imprints to Evaluate Morphology: After the rats were sacrificed, the mesothelial cell monolayer was immediately peeled off from the liver surface using a slide coated with 1% agar, as previously described (23). Two imprints were taken from each animal. The imprints were fixed with 70% ethanol for 2 minutes, washed with phosphate-buffered saline, and then stained with hematoxylin & eosin and 1% pyronine B. To evaluate the imprints, four microscope fields (the area of 1 microscope field was 131691 µm2; BX50; Olympus Optical, Tokyo, Japan) were randomly selected from one slide and scanned with a digital camera (DP50CU; Olympus Optical) at x200 magnification. The density of the mesothelial cells was measured by Image Pro Plus (Planetron, Tokyo, Japan). The number of fibroblast-like cells was also counted. For fibroblast-like cells, after endogenous peroxidase activity was blocked with 3% hydrogen peroxide, the imprints were incubated with the following primary antibodies for 60 minutes at room temperature: a monoclonal antibody against human cytokeratin (1A4; Dako Japan, Kyoto, Japan) and a monoclonal antibody against bovine vimentin (Vim 3B4; Dako Japan). All antibodies are known to have cross-reactivity with rat antigens. Peroxidase activation was detected with diaminobenzidine. The specimens were counterstained with hematoxylin, then dehydrated in a series of ethanol solutions, dipped in xylene, and mounted with a mounting medium (Daido Sangyo, Saitama, Japan). Cells that were vimentin positive and cytokeratin negative were considered fibroblast-like cells.
Measurement of Peritoneal Thickness: To measure the peritoneal thickness, additional groups of rats were studied using the same procedure described above except that a different tissue fixation method was used (controls, n = 8; PD-glucose 3.86%, n = 10; PD-taurine 3.5%, n = 10). After 1 week, the rats were deeply anesthetized with diethyl ether and blood samples were collected by direct cardiac puncture. The peritoneal cavity was then filled with 2.5% glutaraldehyde for 1 hour prior to the collection of tissues. After fixation, to avoid artifact, a section of the anterior peritoneal wall that was deliberately free of any injections was carefully collected; the rats had been injected solely on the right side of the peritoneal wall and only the left side was used for morphological study. The samples were immersed in 2.5% glutaraldehyde for 24 hours for additional fixation and then embedded in paraffin (24). The specimens were sectioned in 3-µm slices and stained with Masson's trichrome for morphometric analysis. The thickness of the peritoneal membrane, including the mesothelial cell layer and the submesothelial interstitium, was measured using the digital camera-fitted microscope. Ten microscope fields at a magnification of x400 were randomly chosen in each specimen. The area and length of the peritoneal membrane were measured using Image Pro Plus and then peritoneal thickness was calculated.
STATISTICAL ANALYSIS
All data are expressed as mean ± SD. Student's t-test for unpaired
data was used to assess the UF experiment data, while analysis of variance
(ANOVA) followed by Fisher's protected least significant difference (PLSD)
test were used to assess the data of the biocompatibility experiment. All
statistical calculations were performed using StatView software (Abacus
Concepts, Berkeley, CA, USA). p Values less than 0.05 were considered
significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSUREREFERENCES |
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EXPERIMENT 1: ASSAY OF UF ABILITY
Fluid Transport: Figure
1(a) shows net UF for each PD solution. Ultrafiltration of the
PD-taurine solution was less than that of the PD-glucose solution at an
equivalent osmolarity. A substantial increase in peritoneal dialysate volume
was seen with the 3.86% and 2.27% PD-glucose solutions: 6 hours after
injection, net UF was 19.1 ± 0.4 mL and 12.1 ± 0.5 mL
respectively. Using the 1.36% PD-glucose solution, peritoneal dialysate volume
increased only slightly over a 6-hour period (4.5 ± 0.6 mL). The 1.0%,
1.8%, and 2.8% PD-taurine solutions increased the PD volume
[Figure 1(a)]. The greatest
increase was observed in animals treated with the 2.8% PD-taurine solution.
The maximum UF for 1.8% and 2.8% PD-taurine solutions (8.2 ± 0.4 mL and
13.4 ± 0.4 mL respectively) was achieved after the 4-hour dwell and did
not increase further during the 6-hour dwell. The maximum UF for the 1.0%
PD-taurine solution was achieved with the 2-hour dwell time (0.29 ±
0.45 mL) and did not increase further with the 4-hour dwell.
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Plasma Taurine Concentration: The plasma taurine concentration in conscious rats fed standard chow was 160 ± 40 µmol/L. After the 2-hour dwell, plasma taurine concentration achieved its maximum value (1.0% PD-taurine: 2692 ± 290 µmol/L; 1.8%: 4791 ± 458 µmol/L; 2.8%: 9887 ± 1061 µmol/L) [Figure 1(d)], then gradually decreased but did not return to normal levels at 6 hours.
EXPERIMENT 2: EVALUATION OF THE TIME COURSE OF UF
Fluid Transport: To determine net UF of the PD-taurine solution in
detail, the SPA was done using the 2.5% PD-taurine solution and the 2.27%
PD-glucose solution, which had different osmolarities. In experiment 1, they
were found to have the same UF efficiency after a 4-hour dwell.
Figure 2(a) shows the time
course of TCUF, LA, and change in net UF. After 1 hour of the dwell, net UF of
the PD-taurine solution was significantly higher than that of the PD-glucose
solution [net UF: 8.3 ± 0.6 mL and 6.1 ± 1.5 mL, p <
0.05; TCUF: 9.6 ± 0.6 mL and 7.7 ± 1.4 mL, p < 0.05;
Figure 2(a)]. After the 4-hour
dwell, net UF, TCUF rate, and LA rate were not significantly different between
the PD-glucose and the PD-taurine solutions
(Table 3).
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Osmotic Agent Transport: The amount of glucose and taurine in the dialysate decreased exponentially with time [Figure 2(b)]. At all time points, the fractional absorption of taurine was significantly higher than that of glucose (PD-glucose solution at 30, 60, 120, 180, 240 minutes: 77.2% ± 4.3%, 63.7% ± 6.9%, 47.7% ± 6.6%, 34.2% ± 5.9%, 26.6% ± 5.9%, respectively; PD-taurine solution: 64.7% ± 3.1%, 49.8% ± 3.0%, 34.8% ± 3.7%, 24.5% ± 4.1%, 15.6% ± 5.0%; p < 0.001, p < 0.01, p < 0.01, p < 0.05, p < 0.01, respectively).
The baseline plasma taurine level of the unconscious rats fed standard chow was 355 ± 35 µmol/L. After 60 minutes of the dwell, plasma taurine concentration achieved its maximum value (15 000 ± 537 µmol/L). After 120 minutes, plasma taurine concentration was almost the same as that at 60 minutes. After 240 minutes, plasma taurine concentration gradually decreased but it did not return to normal.
EXPERIMENT 3: EVALUATION OF BIOCOMPATIBILITY
Peritoneal Equilibration Test: To determine the biocompatibility
of PD-taurine, the In vivo effect of PD-taurine on peritoneal
membrane was examined. A PD-glucose 3.86% solution and a PD-taurine 3.5%
solution were used to evaluate the biocompatibility of PD-taurine, since the
higher osmotic agent concentration allowed the evaluation to be done in a
short period of time. Change in body weight was not significantly different
between the two PD fluid groups. No gross abnormalities were detected in the
rats treated with PD-taurine. All the rats appeared healthy, active, and
well-groomed. No differences were observed between the PD-glucose and
PD-taurine groups in a functional observational battery. Gross alterations of
locomotor activity and reactivity to auditory and tactile stimulation were not
apparent in the rats treated with PD-taurine. No tremors or convulsions were
observed. All reflexes appeared normal. One day after PD treatment, plasma
taurine concentration increased three times the baseline value and then
reached a plateau until dialysis was terminated
[Figure 3(a)]. After 1 week, a
PET using a 1.9% xylitol PD solution was done to determine peritoneal
function. The drainage volume of the control group was significantly higher
than that of the PD-glucose and PD-taurine groups [40.7 ± 0.6 mL, 29.7
± 1.8 mL, 34.0 ± 2.3 mL; p < 0.0001 and p
< 0.001, respectively; Figure
3(b)]. The drainage volume of the PD-taurine group was
significantly larger than that of the PD-glucose group (p <
0.001). The D4/D0 xylitol ratio of the control group was
significantly higher than that of the PD-glucose and PD-taurine groups [0.28
± 0.02, 0.13 ± 0.04, 0.19 ± 0.03; p < 0.0001
and p < 0.001, respectively;
Figure 3(c)]. The
D4/D0 xylitol ratio of the PD-taurine group was
significantly higher than that of the PD-glucose group. Peritoneal function of
the PD-taurine group was more preserved than that of the PD-glucose group.
Hyaluronic acid concentration in the dialysate of the PD-glucose group (117.2
± 61.6 ng/mL) was significantly higher than that of the control group
(31.2 ± 4.9 ng/mL, p < 0.001) and that of the PD-taurine
group (59.2 ± 13.3 ng/mL, p < 0.05); the difference between
the control and the PD-taurine 3.5% group was not significant
[Figure 3(d)].
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Many of the cells seen in the PD-treated groups had a morphology that differed from mesothelial cells. Since these cells stained positive for vimentin and negative for cytokeratin they were identified as fibroblast-like cells (6). The number of fibroblast-like cells was evaluated using the imprint method. At 1 week, the number of fibroblast-like cells (x200, 131691 µm2) in the control, PD-glucose, and PD-taurine groups was 0.35 ± 0.19, 3.31 ± 1.44, and 1.63 ± 0.49 cells/HPF, respectively [Figure 4(e)]; the control group had significantly fewer cells than the PD-glucose and PD-taurine groups (p < 0.0001, p < 0.01, respectively). Interestingly, the PD-taurine group had significantly fewer cells than the PD-glucose group (p < 0.01).
Peritoneal Thickness: Figure 5 shows the morphometric analysis of the Masson's trichrome-stained sections. The thickness of the peritoneum was significantly less in the control group than in the PD-glucose and PD-taurine groups (control, 6.13 ± 0.73 µm; PD-glucose, 9.39 ± 1.83 µm; PD-taurine, 8.27 ± 1.06 µm; p < 0.001 and p < 0.01, respectively). Although the difference was not statistically significant, the peritoneum tended to be thicker in the PD-glucose group than in the PD-taurine group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSUREREFERENCES |
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The composition of the single-chambered PD-taurine solution was the same as the two-chambered PD-glucose solution, which was characterized by a neutral pH and a low GDP content. Taurine was added to the PD solution in an amount that would allow it to be equivalent to PD-glucose in terms of osmolarity and UF (Table 1). It was found that the PD-taurine solution was so stable that, even in a one-compartment system, there was little change in its composition, pH, and osmolarity after heat sterilization and a long storage time under severe conditions. Taurine has strong buffering capacity and resistance to heat. As we expected, the GDP level was below detectable levels in the PD-taurine solutions. It has been shown in vitro that GDPs damage mesothelial cell viability and function (25). Moreover, some GDPs accelerate the formation and accumulation of AGEs, leading to dysfunction of the peritoneal membrane during PD therapy (26,27). Since the GDP level in PD-taurine solutions is below the detectable level, the use of such solutions may result in less accumulation of AGEs during continuous ambulatory PD (CAPD) treatment than with glucose-based PD solutions.
Net UF of PD-taurine solutions was significantly lower than that of PD-glucose solutions of equivalent osmolarity, since the absorption rate of taurine from the peritoneal cavity is slightly faster than that of glucose. This difference appears to be related to the molecular weight of these two molecules. Since the net UF of PD-taurine solutions increased linearly in a dose-dependent fashion, the theoretical PD-taurine dose at which net UF was equivalent to each PD-glucose dose could be calculated. With respect to UF, the 1.5%, 2.5%, and 3.5% PD-taurine solutions were comparable to the 1.36%, 2.27%, and 3.86% PD-glucose solutions, respectively. However, the period of effective UF of the PD-taurine solution was slightly shorter than that of the PD-glucose solution.
The SPA, adjusted for rats, using 2.5% PD-taurine and 2.27% PD-glucose solutions was done. Net UF of the PD-taurine solution was significantly greater than that of the PD-glucose solution after a 1-hour dwell, since the fractional absorption of taurine from the peritoneal cavity was slightly faster than that of glucose. It has been reported that, at the same concentration, the maximum increase in the net UF of small molecular weight substances is faster and greater than that of larger molecular weight substances (28). However, net UF after a 4-hour dwell was not appreciably different between the PD-taurine and PD-glucose solutions; thus, by increasing the concentration, taurine had the same UF ability as glucose.
In experiment 2, pentobarbital was used because it can easily maintain anesthesia in rats without causing mortality. There has been one report that pentobarbital can affect peritoneal permeability (29); thus, the absolute value may have been affected because pentobarbital may affect peritoneal permeability. On the other hand, another report found that the dose of pentobarbital used in our study did not affect peritoneal permeability (15). The difference in the 2.27% PD-glucose net UF between experiments 1 and 2 might be accounted for by the use of pentobarbital or by the assay method. The former would have had a direct effect, while the latter would have had an indirect effect. It has been reported that change in intraperitoneal fluid volume evaluated by an indicator dilution method is not completely identical to that evaluated using a volumetric measurement (30).
In experiment 1, after a 2-hour dwell, the plasma taurine concentration increased 16, 30, and 60 times over baseline levels with 1.0%, 1.8%, and 2.8% PD-taurine solutions, respectively, and then decreased gradually. This observation is consistent with previously published data (31). In experiment 1, we wanted to show the time course of the plasma taurine concentration after a single taurine PD administration, whereas in experiment 3 we measured the trough level of taurine to assess its accumulation in the body. Therefore, plasma taurine concentration was much lower in experiment 3 than in experiment 1. The discrepancy in the taurine levels between the two experiments indicates that taurine absorbed into the body can be easily eliminated. The baseline plasma taurine concentration can be easily changed by certain factors such as diet. Furthermore, the blood sampling procedures could have affected the results. The time of the sampling could have affected the results due to the circadian rhythm of feeding behavior. As well, anesthesia may have affected the baseline plasma taurine concentration: in experiment 1, blood was drawn from conscious rats, while in experiment 2 it was collected from anesthetized rats that had surgery. In the literature, normal rat basal plasma taurine level varies from 150 to 400 µmol/L depending on study conditions (31,32); thus, our data were not inconsistent with these findings.
The biocompatibility of the PD-taurine solution was evaluated In vivo in the rat model by examining and comparing the morphological and histological effects of a 3.5% PD-taurine solution and a 3.86% PD-glucose solution. The UF of these two solutions was almost the same for a 4-hour dwell, but the osmolarity was higher for the PD-taurine solution than for the PD-glucose solution. In experiment 3, a multiple-injection design (three times per day) was used in order to replicate a clinical CAPD regimen, which usually involves several dwells per day. Furthermore, Jonasson and Braide (24) pointed out that once-per-day injection was insufficient to examine peritoneal morphological changes; for this, injections at least three times per day are necessary. We conducted a pilot study before the study design was finalized and we found that a 1-week time period was long enough to demonstrate the superior biocompatibility of taurine compared to glucose as long as a multi-injection design was used. Even in this short-term study, there were several obvious differences. Furthermore, a single-injection design with a longer duration (e.g., 10 – 15 weeks, which has been used by several investigators) is not always feasible. Since CAPD patients require renal replacement therapy for many years, it could be that even a 10- to 15-week study is too short to allow biocompatibility to be assessed. As well, once-per-day PD therapy may not be adequate, since most PD patients change their PD solutions several times per day.
The level of hyaluronic acid in the effluent reflects the inflammatory process that occurs in the peritoneal cavity. After peritoneal membrane injury (especially the mesothelial cell layer), a process of tissue repair commences. This process can be described as an inflammatory response that is characterized by remesothelialization of the wounded area, neovascularization, and fibrosis of the submesothelial cell extracellular matrix (33). In the present study, the level of hyaluronic acid in the effluent of the PD-taurine group was significantly lower; indicating that the injury and remesothelialization due to the PD-taurine solution was mild.
The imprint technique that was used in this study is the most suitable method for observing in situ changes that occur in peritoneal mesothelial cells during PD therapy (23). The density of mesothelial cells increased more in the PD groups than in the control group, which did not undergo PD. It would appear that, no matter which type of PD solution is used, it is in some way nonphysiological for peritoneal mesothelial cells. Some investigators have pointed out that mesothelial cell hyperplasia may be induced by the glucose present in PD-glucose solution (34). Of particular note, after 1 week, the density of the mesothelial cells with PD-taurine solution was less than that noted with PD-glucose solution. These observations suggest that mesothelial cell hyperplasia can be largely attributed to glucose. Although Hekking et al. observed fibroblast-like cells in imprint specimens (34), they did not verify that they were, in fact, fibroblasts. In the current study, since the fibroblast-like cells in question were immunochemically vimentin positive and cytokeratin negative, these cells were most likely fibroblasts. Exposure to the PD solutions increased the number of fibroblast-like cells and the density of mesothelial cells; the number of fibroblast-like cells was significantly increased at 1 week in the PD-glucose group. Peritoneal thickness showed a tendency to be thicker in the PD-glucose group than in the PD-taurine group, although the difference was not signif icant. This finding reinforces the notion that PD-taurine is more biocompatible than conventional glucose-based PD fluid.
To investigate the safety of the PD-taurine fluid, every day during the study period, the rats' body weights were followed and changes in their behavior were recorded: no side effects of taurine were evident. The results of routine blood tests, including liver and kidney function tests, as well as electrolyte levels and blood cell counts, were the same before and at the end of the study.
On the basis of these examinations, we conclude that the PD-taurine solution is relatively safe, despite the increase in the plasma taurine level that was noted. The relatively short duration of the study and the lack of a detailed toxicity profile for taurine are limitations of this study. After taurine is administered systemically, it undergoes very little metabolism; most of it is excreted directly into the urine. Since rats with normal kidney function were used in this study, the taurine was immediately excreted from the kidneys. However, a rapid increase in plasma taurine concentration was observed in the early phase after the injection of the taurine PD fluid into the peritoneal cavity. When taurine doses of up to 5000 mg/kg/day were given intravenously, which is a much higher dose than the dose used in the present study, no serious problems, including central nervous system disturbances, were noted (35).
In patients with renal failure, the absorption, distribution, metabolism, and excretion of taurine still need to be fully elucidated. The few papers that have dealt with this issue found that, although the excretion of taurine into the bile is substantially increased in renal failure patients, a large amount of the taurine is still eliminated in the urine (36). In this situation, accumulation of taurine in the body is expected; however, the plasma taurine level associated with toxicity has yet to be determined. Suliman et al. reported that a 100 mg/kg/day taurine intake for 10 weeks by patients with end-stage renal disease resulted in accumulation of taurine in the plasma and muscles; 2 of the 4 patients complained of dizziness (37). However, the side effects that they attributed to taurine may not have been caused by taurine but may have been the result of other factors unrelated to taurine. In fact, only 4 patients were enrolled in their study and the authors did not report their clinical characteristics. If the patients had liver dysfunction, then taurine side effects may easily have occurred. We have found that taurine is effective against the leg cramps that are often seen in hemodialysis patients due to taurine's stabilization of the membrane potential in skeletal muscle and the peripheral nervous system (38). In our experience, anuric end-stage renal failure patients given 3 g taurine per day developed no serious problems, such as dizziness, even though their plasma taurine levels increased 40 – 60 times above baseline (668 – 1328 µmol/L) 2 weeks after the start of treatment and then remained stable. Similar plasma taurine levels can occur without any evidence of side effects in individuals that consume seafood daily.
Furthermore, although the safety of a few grams of taurine intake per day may not be a problem, even in anuric CAPD patients, it remains to be determined whether a taurine intake that results in a couple of dozen times greater absorption of taurine is safe. We are currently continuing our investigation of the use of PD-taurine solution in an adenine-induced renal failure model (39), which still maintains an adequate urine volume. After 4 weeks on a 0.75% adenine diet, rats were injected with 10 mL of the 1.5% PD-taurine solution intraperitoneally, once daily, for 6 days. The peak plasma taurine level was about 8 times higher than the baseline level and no appreciable abnormalities were seen in those animals. This range of increase in plasma taurine levels is considered to be within a safe range. Thus, to date, we have found that low-concentration PD-taurine is safe when used once daily in animals with renal failure that are still producing residual urine. In CAPD patients with residual renal function, taurine can be excreted into the urine. In certain cases, the use of a glucose–taurine mixture PD solution may be appropriate. Further studies are needed to identify a safer clinical regimen using a taurine PD solution. In renal failure patients, residual renal function is a critical factor related to mortality (40) and, since it has been shown that taurine has a renal protective effect (41), a PD-taurine solution may protect the renal function of renal failure patients and thereby decrease their mortality.
In conclusion, with the use of taurine as osmotic agent, net UF increased in a dose-dependent manner. The PD-taurine solutions were found to be equivalent to the PD-glucose solutions in terms of UF. Furthermore, the PD-taurine solutions had neutral pH and undetectable levels of GDP. In the rat PD model used in the present study, PD-taurine solutions were found to be more biocompatible than conventional glucose-based PD solutions with respect to morphological and histological changes. Further studies are needed to assess the long-term biocompatibility and safety of taurine in patients with renal failure.
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Received 4 September 2006; accepted 15 July 2008.
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