Perit Dial Int
29(Supplement_2):
32-35
2009
© 2009 International Society for Peritoneal Dialysis
Part 2: Cellular and Molecular Biology of the Peritoneum
and Peritoneal Dialysis |
HOW TO ASSESS TRANSPORT IN ANIMALS?
Bengt Rippe
Department of Nephrology, University Hospital of Lund, Sweden
Correspondence to: B. Rippe, Department of Nephrology, Lund University,
University Hospital of Lund, S-211 85 Lund, Sweden.
Bengt.Rippe{at}med.lu.se
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ABSTRACT
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The general principles for assessing solute and fluid transport across
the peritoneum in animal models are not different from those in human studies.
Animal models allow for extensive standardization of experimental conditions
and also for sampling of peritoneal tissues for analysis. The present review
will focus on (1) the scaling issue between various species, (2) how to
measure intraperitoneal volume in animal models, (3) the impact of an
indwelling catheter, (4) the difference between acute and chronic experiments,
and (5) the particular problems associated with transport measurements in
mice. If done correctly and after proper scaling, mass transfer area
coefficients and clearance measurements show marked similarity among different
species. Although animal models only partly mimic human peritoneal dialysis,
they are valuable tools for understanding the basic physiology and biology of
peritoneal dialysis.
KEY WORDS: Capillary permeability; mouse; rat; transcytosis; aquaporin-1.
The use of animal models for peritoneal research can be seen as an
intermediate step between in vitro (cell biology) studies and
clinical studies. The general principles for assessing solute and fluid
transport across the peritoneum in animal models are not different from those
in human studies. The advantages of using animal models are the possibility of
standardizing experimental conditions and the ability to take peritoneal
tissues for analysis. For example, the old enigma that a macromolecular tracer
injected into peritoneal dialysis (PD) fluid disappears from the fluid to the
peritoneal tissues at a much higher rate than it appears in plasma (direct
lymphatic absorption) was resolved only after animal experiments, since the
peritoneal tissues could be sampled for analysis in the animal model
(1). Trapping of tracer in the
interstitium was revealed. The great disadvantage of using animal models,
rodent models for example, is their small size. Furthermore, rodent models in
particular show very dramatic changes in peritoneal morphology and physiology
in response to exposure to PD fluids and the presence of an intraperitoneal
(IP) catheter. Even though the time scale for such changes is only a few weeks
in rats and mice, rodent models have been frequently utilized to test
strategies for preventing the long-term morphological and pathophysiological
changes that can occur in the peritoneum after years of PD in humans. One may,
however, question the representativity of such animal models in this
context.
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THE SCALING ISSUE
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The major problem with animal studies is the scaling problem. How can
peritoneal exchange parameters be scaled from mouse, rat, rabbit,
etc. to man? Whereas body weight (BW; cf. volume of the
body) is related to the third power of the length scale (L3), body
surface area (BSA) is related to the second power of the length scale
(L2). Mathematically, for a sphere, the scaling exponent of volume
to surface area is (volume)2/3. Since the metabolic rate is
approximately related to BSA, it would thus be logical to use 0.67 (2/3) as a
scaling exponent of metabolic rate-related parameters (such as glomerular
filtration rate or cardiac output) to BW. However, in a number of previous
studies, an equation having a scaling exponent of 0.7 (–0.75) was found
to be more appropriate
(1,2),
which is a compromise between scaling for BSA, with the theoretical scaling
factor (BW)0.67, and for BW, that is, (BW)1. In a recent
mouse study we were able to scale parameters, such as the
permeability–surface area products [PS; mass transfer area coefficient
(MTAC)] for glucose and 51Cr-EDTA, from mouse to man to obtain an
almost exact data match between measured mouse and human data
(3). Also, the clearance of
125I-albumin (RISA) from peritoneum to plasma (lymph flow) was
identical in mice compared to man. The only parameter that differed was the
clearance of RISA from plasma to peritoneum, which, after scaling, was only
approximately 20% of that in humans. Otherwise, our data were in general
agreement with those obtained by Flessner and co-workers
(4), who studied the transport
of fluid and solutes into and out of small "cups" glued to the
surface of the peritoneum in rats and mice.
The implication of scaling for BSA is that BSA (and peritoneal surface
area) increases relative to BW in small animals.
Table 1 shows a comparison of
IP area/volume ratios for mouse, rat, rabbit, and man. The consequence of a
high area/volume ratio in small animals is the more rapid solute and fluid
kinetics present in the latter. For example, the time to reach a
dialysate/plasma ratio for creatinine of 0.7 in mouse PD is only 55 minutes;
in rats it is approximately 80 minutes, in rabbits approximately 100 minutes,
and in man approximately 4 hours. In a similar fashion, volume kinetics is
different between the species. The time to reach the maximum ultrafiltered
volume for a 4% (3.86%) glucose solution is about 55 minutes in mouse,
approximately 100 minutes in rat, and approximately 4 hours (240 minutes) in
man. Without considering the impact of different area/volume ratios, it may be
difficult to interpret data from peritoneal equilibration tests (PET). The
same situation prevails for infants versus adults. Infants have much more
rapid PET kinetics than adults. Note that this is not related to permeability;
it is a matter of the increased surface area of the peritoneum
relative to BW in the infant.
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INTRAPERITONEAL VOLUME MEASUREMENTS IN ANIMAL MODELS
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Animal models offer the unique option of measuring the actual
(real) IP volume by direct volume recovery techniques through complete fluid
sampling (using syringes and gauze tissues) and weighing the collected fluid.
For many purposes, however, it is more practical to use a volume marker to
assess IP volume versus time. It is crucial for any volume marker technique in
conjunction with PD to know the exact pattern of marker disappearance from the
peritoneal fluid. By comparing directly (volumetrically) assessed IP volume
with that determined using a volume marker, we were able to assess the actual
disappearance of RISA from the peritoneal cavity in rats
(5). First, we found some (4%)
binding of the peritoneal marker (RISA) to the peritoneal linings, followed by
a biexponential, not a monoexponential, disappearance of the marker. The
initial (0 – 30 minutes) marker disappearance rate was much higher than
that occurring later in the dwell. These findings were later corroborated by
Van Biesen et al. in a similar rat study
(6). We noted that only
approximately 20% of the total RISA disappearance could be accounted for by
"true" lymphatic absorption. A major portion of the disappearance
of tracer (KE) occurred into the peritoneal tissues, coupled to net
volume reabsorption to the capillaries. In this process, fluid but not
macromolecular tracer is reabsorbed to plasma, and thus the tracer is trapped
interstitially. Another portion of KE evidently also reaches the
interstitium by hydrostatic pressure-driven transport. This represents net
transport into tissue without any net water transport, so-called
"fluid recirculation" between the peritoneal fluid and the
interstitium, which is explained in detail elsewhere
(5,7).
It should thus be noted that only a small portion of the disappearance of
tracer is associated with net fluid transport out of the peritoneal cavity,
that is, to true lymphatic reabsorption. The major portion of marker clearance
represents trapping of tracer within the interstitial tissues without any
volume change in the tissue. Therefore, it is not correct to denote the
clearance of a macromolecular marker out of the peritoneal cavity lymphatic
absorption or to use the apparent change in distribution volume for
the tracer as a reflection of true changes in IP volume. However, it is
correct to use the KE to correct the apparent volume to
obtain a real (measured) volume. Furthermore, it is not correct,
according to the three-pore model, to add KE to net ultrafiltration
during the dwell to establish a transcapillary ultrafiltration, since only
part of the KE is related to net fluid transport across the
peritoneum.
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IMPACT OF AN INDWELLING CATHETER
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Chronic animal models in which there is use of a subcutaneous injection
port (e.g., Rat-o-Port; Access Technologies, Skokie, IL, USA)
connected to a silicone catheter tunneled to the peritoneal cavity demonstrate
within weeks a marked thickening of the submesothelial compact zone combined
with angiogenesis (8). In an
attempt to avoid the use of a catheter, we performed daily IP needle
injections of dialysis fluids in rats
(9), and in this model both
fibrosis and angiogenesis were much less pronounced than in catheter-bearing
rats. This was recently corroborated in a study in which catheter-bearing
animals were compared with needle-injected animals in a longitudinal follow-up
fluid study in rats (10).
Furthermore, catheter-bearing animals treated with conventional solutions
usually show marked fibrosis and angiogenesis in response to high versus low
glucose degradation product solutions. This difference could not, however, be
detected in animals subjected to daily IP injections of dialysis fluid
(9). It was speculated that, as
a foreign body, a catheter could host bacteria that grow biofilm, which coats
the catheter (10). It was
therefore hypothesized that the catheter may have had a significant effect on
the inflammatory process in the peritoneum, and also on the process of
epithelial-to-mesenchymal transition of cells in the peritoneal membrane. A
peritoneal catheter may thus be a potent enhancer of peritoneal inflammatory
processes and represent an accelerated animal model for peritoneal fibrosis
and angiogenesis. On the other hand, a catheter-bearing animal may not be a
model that is representative of human chronic PD, in which peritoneal
fibrosis and angiogenesis are usually not problems until after 3 – 4
years of treatment.
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ACUTE VERSUS CHRONIC EXPERIMENTS
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Peritoneal exchange characteristics may be different in the acute compared
to the chronic setting. When the peritoneum is exposed to PD fluid for the
first time, there seem to be large changes in the peritoneal tissues, with
expansion of interstitial volume and partial washout of the hyaluronan content
(11). In only a few hours the
interstitial colloid osmotic pressure has been shown to drop from
approximately 12 mmHg to approximately 7 mmHg
(12). Acute exposure of the
peritoneum to peritoneal fluids raises the IP hydrostatic pressure and leads
to overhydration of the tissues surrounding the peritoneal cavity. This is due
to increases in the local pressure gradient between cavity and tissue.
However, the situation in long-term PD (cf. the situation in humans)
is different, with much less peritoneal tissue volume expansion. For example,
we noted in long-term PD (3 months in rats) a reduced degree of acute tissue
swelling after acute PD dwells, compared to rats that had just started PD
(13). Whereas these acute
interstitial changes may affect the measured fluid and macromolecule transport
out of the peritoneal cavity in acute experiments, alterations of
this kind will still be of subordinate importance with respect to
blood-to-tissue transport (of small solutes). This is because the interstitium
offers only a very small resistance to transport across the entire
blood–peritoneum barrier.
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ASSESSMENTS OF PERITONEAL TRANSPORT IN SMALL ANIMALS
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Compared to larger animals, mice offer a number of advantages, including
low cost, fast turnover, easy breeding, and, above all, the possibility of
genetic manipulation. Great varieties of knockout mice are available
commercially. One of the disadvantages of the mouse model is the small size.
For example, a 25-g mouse has a plasma volume of only approximately 1.5 mL.
Thus, sampling and injections should be downscaled to the microliter range.
Injections should not exceed 50 µL at a time and sampling of blood or PD
fluid should utilize sampling volumes on the order of 10 µL (max 20 µL).
Despite using microdissection techniques, it is not possible to cannulate more
than a limited number of vessels in a mouse. It is almost impossible to
cannulate lymphatics or ureters. In addition, because mice are sensitive to
temperature changes, a rigorous temperature control system is needed.
Ni et al. were the first to show that a mouse model of PD is
feasible for physiological experimentation
(14). In later studies using
an IP volume marker (RISA), it was possible to measure IP volume as a function
of time and, hence, to more exactly characterize the peritoneal membrane with
respect to MTACs (PS) for small solutes and clearance of macromolecules
(3). Knockout mice have been
used to demonstrate the importance of aquaporin-1 for fluid transport during
the first hour of a PD dwell, and that aquaporin-1 seems to be the molecular
correlate to the transcellular water pathway of the three-pore model
(15). Furthermore, knockout
mice deficient in endothelial caveolae demonstrated that mice completely
lacking the capacity for endothelial transcytosis actually show a moderately
increased transfer of RISA, radiolabeled IgM, and FITC-Ficoll 70/400
(molecular weights 70000 Da and 400000 Da) across the peritoneal capillaries
compared to wild-type mice
(16). This strongly suggests
that transcytosis is not the major mode of macromolecule transport between
plasma and interstitium.
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CONCLUSIONS
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Animal models are valuable tools for understanding the basic physiology and
biology of peritoneal exchange and the pathophysiological changes in long-term
PD. It is worth noting, however, that animal models do not entirely mimic
human PD. Furthermore, acute animal PD experiments are different from those
following chronic exposure of the peritoneal membrane to PD. In addition, an
indwelling catheter can markedly influence the pathophysiological alterations
occurring in animal PD. With respect to transport measurements, PET results
are size dependent and simple PET experiments (without further mathematical
interpretation) should be avoided. The measurements of PS (MTAC) and clearance
values require that IP volume curves be measured using a volume marker. To
interpret volume marker data, one has to have an exact knowledge of the
pattern of marker disappearance, which is not a linear process. If done
correctly, and after proper scaling, PS (MTAC) and clearance measurements show
a marked similarity among different species. Although animal models only
partly mimic human PD, they have greatly contributed to the understanding of
peritoneal biology and they will continue to do so in the future.
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ACKNOWLEDGMENTS
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This study was supported by the Swedish Research Council, grant no. 08285,
and the Swedish Heart and Lung Foundation.
The expert secretarial assistance by Kerstin Wihlborg is gratefully
acknowledged.
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ABSTRACT
THE SCALING ISSUE
