Nephrology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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A major factor
that affects solute and water transport through tissue is the state of
tissue hydration. The amount of interstitial water directly affects the
transport coefficients for both diffusion and convection. To
investigate the effect of simultaneous exposure of tissue to
hydrostatic and osmotic pressures on the state of tissue hydration and
the pattern of distribution of tissue water, we dialyzed rats with
isotonic (290 mosmol/kg) or hypertonic (510 mosmol/kg) solution at
intraperitoneal pressures (Pip) between 0 and 6 mmHg, and
we infused isotopic markers intravenously and determined their
equilibrium distribution volumes (VD) in the anterior
abdominal muscle (AAM) by quantitative autoradiography. Total tissue
water volume (TW) was determined from dry-to-wet weight
ratios.
urea, the VD of
[14C]urea, equals the sum of the extracellular
water volume (
EC, VD of
[14C]mannitol) and intracellular water volume
(
IC =
urea
EC). If
if = interstitial water volume and
IV = vascular water volume (VD of 131I-labeled IgG),
then
EC =
if +
IV. AAM
hydrostatic pressure profiles were measured by a
micropipette/servo-null system and demonstrated that elevation of
Pip above 3 mmHg significantly (P < 0.05)
increases mean tissue pressure (PT) to the same level regardless of intraperitoneal osmolality. The increase in
PT resulted in a nonlinear tissue expansion primarily in
the interstitium regardless of osmolality. From 0 to 6 mmHg,
if (in ml/g dry tissue) increased from 0.59 ± 0.02 to
1.7 ± 0.05 and to 1.5 ± 0.05 after isotonic and hypertonic
dialysis, respectively, whereas
IC increased from 2.8 ± 0.08 to 3.0 ± 0.1 after isotonic dialysis and decreased to 2.6 ± 0.1 after hypertonic dialysis. After dialysis at 6 mmHg with
isotonic or hypertonic solutions,
IV increased from
0.034 ± 0.001 to 0.049 ± 0.001 and 0.042 ± 0.002, respectively.
urea during hypertonic dialysis at
Pip between 0 and 6 mmHg increased in a nonlinear
fashion (F = 26.3, P < 0.001), whereas
IC invariably decreased (F = 11.1, P < 0.001) and
if doubled from its control value at low
Pip. In conclusion, elevation of intraperitoneal hydrostatic pressure causes tissue expansion, primarily in
interstitium, irrespective of osmolality of the bathing solution.
Tissue hydrostatic pressure is therefore the primary determinant of
tissue properties with respect to hydration, which in turn affects
diffusive and convective transport.
interstitium; peritoneal dialysis; hydraulic conductivity; transport; convection
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INTRODUCTION |
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THE SIMULTANEOUS STRESSES of hydrostatic and osmotic pressures on tissue occur in peritoneal dialysis, a technique that relies on solute and water exchange between a hypertonic solution in the peritoneal cavity and the blood circulating in the surrounding tissues. Typically 2-3 liters of a hyperosmolar dialysis solution containing glucose as an osmotic agent are repeatedly instilled into and drained from the peritoneal cavity to remove excess solutes and body water. Because of the relatively large fill volume, the intraperitoneal hydrostatic pressure (Pip) in peritoneal dialysis patients increases to between 2 and 10 mmHg (10, 18), whereas the initial osmolality of the fluid depends on the concentration of dextrose in the solution and varies between 330 and 510 mosmol/kg. Despite the widespread use of peritoneal dialysis, the details of the mechanisms underlying the complex process of fluid and mass transport between the blood and dialysate are still to be defined.
The general equation (Darcy's Law, see Ref. 12) that governs fluid
flow through tissue is
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(1) |
In experiments with isotonic solutions, we have shown that the
dependency of the flow on Pip is due to both a marked
increase in the interstitial hydraulic conductivity (K) and a
moderate increase in the interstitial hydrostatic pressure gradient
(dPT/dx), when Pip exceeds a threshold
pressure of 1.5 mmHg (23). Theoretically, K is a complex
function of the tissue structure and the interstitial water volume,
if (12). In a subsequent study (24), we reported that
the increase in K with Pip is due to the combined
effects of expansion of the interstitium, dilution of the interstitial macromolecules from the influx of water, and a washout of mobile interstitial hyaluronan in the direction of flow. These
prior results have indicated that the dynamics of the volume change in
whole tissue and interstitium are important determinants of tissue
hydration and the resistance to hydraulic fluid flow across tissue. Our
previous study (24) of the expansion of the subperitoneal tissue space
was carried out with isotonic solution. Although clinical dialysis
routinely employs hypertonic solution, the effect of combined
hydrostatic and osmotic pressure of the peritoneal solution on the
surrounding tissue space is unknown. Our hypothesis is that the
expansion of the interstitium as well as the total tissue water space
will be altered by the hyperosmotic solution.
To test this hypothesis, we determined the in vivo effect of
hydrostatic and osmotic pressures on the tissue water of the anterior
abdominal muscle. The experiments were designed to measure the
components of tissue (ml/g of dry or wet weight tissue) making up the
total tissue water. These are as follows: the extracellular fluid
volume (EC), the vascular volume (
IV),
the intracellular volume (
IC), and the interstitial
volume (
if =
EC
IV). Our model tissue is the rat anterior abdominal
muscle (AAM), when it is exposed to isotonic or hypertonic solution in
the peritoneal cavity, which is maintained at hydrostatic pressures
between 0 and 6 mmHg.
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METHODS |
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Animals
All experiments were performed in 250-350 g female Sprague-Dawley rats (Charles River Laboratories). Animals had free access to water and standard rat chow until the morning of the experiment. At least three animals were used for each pressure level investigated. All procedures were approved by the University of Rochester Committee on Animal Resources.Materials
Tracers.
Immunoglobulin G (anti-rabbit IgG, no. IM-134; Amersham Life Science,
Arlington, IL) was used as a marker of the local vascular volume
IV. Prior to the experiment, the isotope was checked for degradation and for free 125I by precipitation with
trichloroacetic acid (TCA). If free 125I was >1%, then
the solution was purified further by mixing it with saline and
concentrating the mixture with a Centricon 30 microconcentrator
(Millipore, Bedford, MA) by centrifugation (IEC Centra CL2). Dilution
and concentration were repeated until the free 125I was
<1% by TCA precipitation. [14C]mannitol was
purchased from Moravek Biochemicals (Brea, CA). The volume of
distribution and half-life of the product in the rat have previously
been determined to be 0.174 ± 0.006 l/kg and 13 min, respectively
(4). With these values, an infusion rate of labeled mannitol was chosen
to maintain a constant plasma concentration during the course of the
experiment. 3H2O and
[14C]urea were purchased from Moravek
Biochemicals and used as supplied.
Dialysis solutions. The dialysis solutions employed in the present experiments were made from an isotonic Krebs-Ringer bicarbonate solution (containing, in mol/l, 0.12 NaCl, 0.01 KCl, 0.0021 CaCl2 · 2H2O, 0.025 NaHCO3, 0.00028 KH2PO4, and 1.18 ml of 1 M MgSO4 · 7H2O). All solutions were filtered with a 0.45-µm pore size membrane (Nalgene) and stored at 4°C. The hypertonic solution was made by adding mannitol to the base solution to a concentration of 4%; the initial osmolality of the hypertonic solution was 510 ± 5 mosmol/kg. Bovine serum albumin (5%) was added to all solutions before each experiment.
Surgery
Anesthesia was induced by an intramuscular injection of pentobarbital sodium (60 mg/kg) to the hind leg and maintained with subsequent intravenous injections. Surgery was initiated on loss of the blink reflex. A tracheostomy was performed to reduce airway resistance. Two arterial lines were established using PE-50 catheters. The left carotid artery was cannulated to allow for continuous blood pressure measurements on a pressure measurement system (model PE-10z Statham pressure transducer; Window Graf, Gould Valley Instruments, OH), and a tail artery catheter was used for blood sampling. A venous catheter was secured into the left external jugular vein for continuous infusion of [14C]mannitol from an infusion pump (model 22; Harvard Apparatus, Holliston, MA). The animal's rectal temperature was continuously monitored and maintained between 35.5 and 38.5 with a servo-controlled warming blanket (Harvard Apparatus) and an overhead heating lamp. The peritoneal cavity was exposed through a midline abdominal incision ~1.5 cm, and the hollow viscera (duodenum to rectum) were removed using the technique as described in our previous publication (26). The slitlike abdominal incision was closed using a continuous suture after careful inspection to ensure there was no bleeding. This maneuver was necessary to ensure that fluids in the cavity have access to the entire abdominal wall. With the aid of a trocar, a multihole catheter was placed through the abdominal wall into the peritoneal cavity and secured with a purse stitch. A three-way valve was connected to the multihole catheter to administer and sample the dialysate and to continually measure the Pip with a glass capillary manometer. A urethral catheter was inserted for collection of urine during the experiment.Measurements
Radioactive tracer detection. 14C- and 3H-labeled tracers were quantified with a scintillation counter (model LS6000IC; Beckman, Fullerton, CA). 125I-labeled tracers were quantified with a counter (Gamma 4000; Beckman, Irvine, CA).
Interstitial hydrostatic pressure. The hydrostatic pressure profile within the AAM was measured by the technique of Wiig and colleagues (22) as later modified by Flessner (3) to allow for in vivo measurements of the interstitial pressure profile in the rat anterior abdominal wall. Details of the procedure are found in our previous publication (3).
Quantitative autoradiography.
Quantitative autoradiography (QAR) was used to determine the local
concentration of each tracer in the tissue at the time of animal death.
The general assumption for the determination of a particular space
within the tissue was that the tracer was equilibrated between the
plasma and the volume of distribution of the tracer within the tissue.
The volume of tissue, i, equals the ratio of the
tissue concentration to the plasma concentration, Ctissuei/Cplasmai.
Briefly, tissue tracer concentrations were determined from the thin
tissue sections from the frozen carcass at the end of each experiment (see Dialysis procedure in Experimental Protocols,
below). The sections were placed with standards (tissues with known
isotopic concentration) against X-ray film (Biomax MR; Kodak,
Rochester, NY) to produce autoradiograms. After developing the films,
the tissue slides were stained with hematoxylin and eosin. Each slide was examined by light microscopy to determine the mesothelial layer and
the skin side. This procedure is important in tissue samples where
tracer concentration profiles are to be determined. The films were
analyzed with a computerized densitometer (model MCID; Imaging
Research, St. Catherines, Ontario, Canada), which measures optical
density (OD) vs. position in the tissue. The isotopic standards are
used to construct a calibration curve (concentration vs. OD) to convert
the unknown OD values from the tissue samples to concentration. By
superimposing the tissue histology over the autoradiogram, the location
of the reading was carefully determined, and a concentration vs.
position curve was obtained (concentration profile data) or mean
concentration in a large area of the tissue. These concentrations
divided by the plasma concentration provided an estimate of the volume
that was marked by the specific tracer. The term "local" refers
to
i at a particular location in the tissue or
within the
i profile that has been obtained from
QAR. The use of this macro-QAR technique is detailed in our previous
publications (5, 24).
Extracellular volume.
The extracellular volume, EC, is defined as a unit of
volume within the AAM tissue that is not occupied by cells or solid material.
EC was determined by an intravenous infusion
of [14C]mannitol at a rate to compensate for
renal excretion and to maintain a constant concentration of the labeled
mannitol. The tracer equilibrates with the extracellular space, and its
tissue concentration divided by the plasma concentration provides the estimate of
EC. After surgical preparation, 5 µCi of
[14C]mannitol was given as an intravenous
injection followed by a continuous infusion at a rate of 0.25-0.5
µCi/min for 90 min. Thirty minutes after commencing the infusion, the
dialysis fluid was injected into the peritoneal cavity in an amount
sufficient to reach the designated Pip. Attempts were made
to obtain at least one measurement of the interstitial hydrostatic
pressure profile across the AAM. The Pip tested were 0, 1.5, 3, 4.4, and 6 mmHg. A minimum of three animals were used for each
pressure level. Blood and peritoneal fluid were sampled at 15-min
intervals. Tissue samples from the AAM were processed for single-label
([14C]mannitol) QAR.
Intravascular volume.
Intravascular volume, IV, is defined as the volume
within the blood vessels that is not occupied by cells. After surgical preparation, a 40-min dwell time was initiated with the hypertonic dialysis solution at Pip of 0, 3, and 6 mmHg. Ten minutes
before termination of the experiment, a bolus injection of
125I-IgG (50-100 µCi) was given intravenously to
mark the plasma space,
IV. Blood was sampled just before
the death of the animal for both 125I and osmolality. The
assumption in this series of experiments is that the overall
transcapillary escape rate of the labeled tracer in 10 min is
negligible. Tissue concentrations were determined by single-label
(125I-IgG) QAR.
IV was calculated from the
ratio of the tissue concentration and the plasma concentration. The
interstitial volume,
if, in the AAM was calculated from
the difference between the extracellular volume and the intravascular
volume (
if =
EC
IV).
Total tissue water volume.
The total tissue water volume, TW, is equal to the sum
of the volumes of water in the extracellular space (
EC)
and the intracellular space (
IC) and is calculated from:
TW = (tissue wet weight
tissue dry
weight)/tissue dry weight (ml/g dry tissue, where 1 g water = 1 ml).
Total tissue water in the AAM was also obtained from the equilibrium
distribution volume of [14C]urea
(
urea) at Pip of 1.5, 3, 4.4, and 6 mmHg.
For each pressure level, three animals were used. The intracellular
volume (
IC) in the AAM was obtained from the difference
between the total tissue water and the extracellular volume as
(
IC =
TW
EC) ml/g
dry tissue, or (
IC =
urea
EC) ml/g wet tissue. Values for tissue volumes were
converted from a wet weight basis to a dry weight by multiplying by the
wet-to-dry weight ratio of the tissue.
Experimental Protocols
Dialysis procedure.
An experiment to determine i was initiated with
a bolus injection of tracer "i" via a short jugular
venous catheter, followed by continuous tracer infusion. Thirty minutes
after commencing the infusion, the dialysis fluid [warmed to
37°C; in some cases containing the labeled tracer in a
concentration estimated to equal that in the plasma (see below)]
was injected through a three-way valve connected to the peritoneal
catheter in an amount sufficient to raise Pip to a value
slightly below the desired pressure. A reservoir containing the rest of
the dialysis fluid was then connected to the three-way valve attached
to the intraperitoneal catheter. The reservoir was maintained at the
exact level above the right atrium to produce the desired
Pip, which was determined every 15 min with a water
manometer. Attempts were made to obtain at least one pressure profile
measurement in the AAM during the 1-h dwell time. At the end of the
experiment the following steps were taken in rapid succession as
follows: the animal was euthanized by an anesthetic overdose followed
by decapitation. The fluid was drained from the cavity, and the animal
was rapidly frozen using chlorodifluoromethane (Dust-off; Falcon
Safety Products, Branchburg, NJ) precooled to
75°C. The
abdominal wall was carefully cut from the carcass with an autopsy saw.
Four small portions of muscle (50-100 mg each) were taken from the
abdominal wall. Each piece was thawed, gently blotted to remove any
residual fluid, and placed in a previously weighed vial to determine
its weight. The tissue was then solubilized and counted for either
125I-IgG or
[14C]mannitol. From the frozen
abdominal wall, thin sections (20 µm) were taken horizontally with a
cryomicrotome (model OTF; Bright-Hacker, Fairfield, NJ) and dried on a
slide warmer. Sections were used for single-label (125I or
14C) QAR and for histology after staining with hematoxylin
and eosin.
Bidirectional [14C]mannitol transport
studies.
To test the effects of dwell time and the decrease in osmolality on the
estimation of VD of [14C]mannitol
in the AAM, we designed a series of experiments in which the dwell time
was set at 120 min with the dialysis fluid exchanged at 60 min, to keep
osmolality within 90% of its initial value. A total of three rats (280 ± 21 g) were used in this series. The animals were surgically
prepared (see Surgery, above) and were rendered anephric by
bilateral ligation of the renal pedicle; this eliminated the need to
continuously infuse the tracer to make up for renal clearance. Fifteen
microcuries of [14C]mannitol were given as an
intravenous bolus injection. For each animal, the dialysis solution was
prepared as 150 ml of a 4% mannitol, 5% bovine serum albumin in Krebs
ringer solution containing 40 µCi of
[14C]mannitol. Initial osmolality of the
solution was (511 ± 1.2 mosmol/kg). The dialysis solution was
instilled into the peritoneal cavity in an amount sufficient to raise
Pip to ~3 mmHg. Approximately 20 ml of the dialysis
solution was added to the reservoir connected to the
peritoneal cavity. The rest of the fluid was kept in a water bath at
37°C to be used for the second exchange. At 60 min, the peritoneal
fluid was aspirated in a 60-ml syringe within 1 min. The rest of the
dialysis solution from the water bath was injected into the peritoneal
cavity to raise Pip to ~3 mmHg. The reservoir was
reconnected. The fresh solution was allowed to dwell for another 60 min. Blood and peritoneal fluid were sampled 15 min after
initiation of the experiment and every 30 min thereafter for a
total dwell of 120 min. At 120 min, final blood and peritoneal fluid
samples were obtained; the animal was then euthanized, and the
abdominal muscle was harvested (see Dialysis procedure, above) and prepared for QAR. If EC obtained from these 120-min
dwell studies equaled
EC obtained from 60-min dwell
times, then our assumption that 60 min equilibration for a
small-molecular-weight tracer to reach a steady state of diffusion
equilibrium in the AAM will be justified.
Bidirectional [14C]urea transport studies.
The purpose of these studies was the determination of VD, a
surrogate for the volume of tissue that was made up of water. Since the
technique of QAR requires dehydration of the frozen tissue slice prior
to placement against the X-ray film, 3H2O
cannot be used as a marker for tissue water content. To ensure that the tracer urea is equivalent to tritiated water in terms of
volume of distribution, six animals were surgically prepared (see
Surgery, above). A 5% bovine serum albumin in Krebs-Ringer solution was instilled into the peritoneal cavity to produce
Pip of 1.5 mmHg, and 150 µCi of either
3H2O or [14C]urea was
given as an intravenous bolus injection. The total bolus dose was
carefully determined from the initial concentration and the weight of
the syringe before and after the injection. Blood and peritoneal fluid
were sampled every 15 min for a total equilibration time of 180 min.
Final peritoneal, blood sample, and the total urine volume were
collected, and their specific activities were determined in a beta
counter. The volume of distribution was calculated as the given dose of
3H2O or [14C]urea
divided by the tracer concentration at time 0, which was found
by extrapolating the plasma concentration vs. time curve to t = 0 on a log-log plot. The plasma curve was fitted to
C(t)/C0 = e(kt),
where C(t) is the plasma tracer concentration at time
t, C0 is the tracer concentration at time 0,
and k is the decay constant. In these control experiments we
determined VD of both 3H2O (240 ± 2 g rat, mean ± SE, n = 3) and
[14C]urea (267 ± 5 g rat, mean ± SE,
n = 3), as well as the decay constants. The VD
[equivalent to total body water (TBW)] determined by
3H2O averaged 658 ± 14 ml/kg rat
(n = 3), which is comparable to 626 ± 8 ml/kg rat as obtained
using [14C]urea as a marker, whereas the
half-lives (T1/2) were 545 ± 21 min and
317 ± 47 min, respectively. Since k = 0.693/T1/2, the corresponding values for k
were calculated to be 1.27 × 10
3
min
1 and 2.19 × 10
3 min
1.
From these parameters, the bolus injection and infusion rate were
calculated to attain a concentration of labeled urea in the plasma
equal to that in the peritoneal cavity.
Statistics
All data are presented as means ± SE unless stated otherwise. One-way ANOVA was used to analyze the effect of a single factor (i.e., Pip) on measured ![]() |
RESULTS |
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Hydrostatic Pressure Profiles
Figure 1 displays the measured hydrostatic pressure profiles (means ± SE) for Pip elevations during hypertonic dialysis or isotonic dialysis (isotonic data from Ref. 24 was plotted for comparison). The hypertonic profiles were similar to our previous measurements at the corresponding Pip using isotonic dialysis solution in the peritoneal cavity (3, 24). A one-way ANOVA demonstrated that elevation of Pip > 3 mmHg significantly (F = 4.15, P < 0.05) increased the mean tissue pressure as calculated from the distance-averaged hydrostatic pressure (PT within the first 600 µm from peritoneal edge) regardless of the osmolality of the dialysis solution. A multiple comparison Bonferroni t-test at each Pip demonstrated that the numerical difference between the calculated PT for the two solutions was not statistically significant (all Bonferroni P > 0.05). The mean PT values are listed in Table 1.
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Pressure-Volume Curves
The change in the interstitial fluid volume (
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Extracellular Volume Profiles
Figure 3 displays the results designed to measure
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Total Tissue Water and Its Distribution During Hypertonic Dialysis
Figure 4 displays the pattern of distribution of total tissue water (
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As seen in Table 1, total body water normalized to kilogram body weight
tends to be numerically lower with gradual elevation of
Pip, but the difference among the group means was not
significant (n = 12, F = 1.42, P > 0.1).
if and
IV were determined from a separate
set of experiments, whereas total tissue water fraction was either
determined from dry-to-wet weight ratio from AAM tissue (
TW) or obtained from the normalized tissue
[14C]urea concentration (
urea).
Elevation of Pip from 1.5 to 6 mmHg caused
urea to increase from 0.78 ± 0.01 to 0.85 ± 0.01 ml/g wet tissue (F = 29.7, P < 0.001). This increase
in
urea is primarily elicited in
if,
which accounted for 20% of
urea at Pip = 1.5 mmHg, but significantly increased (F = 135.4, P < 0.001) to 40% of
urea after Pip elevation
to 6 mmHg.
IV was not affected by this pressure change.
However,
IC was significantly reduced (F = 21.2, P < 0.001) from 0.57 ml/g wet tissue (73% of
urea) at Pip = 1.5 mmHg, to 0.50 (59% of
urea) upon elevation of Pip to 6 mmHg.
Tissue Profiles of urea
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In Fig. 6, the fractions making up the
total tissue water (urea) in the AAM are shown for
control nondialyzed rats and for animals that underwent hypertonic
dialysis at Pip elevations between 1.5 and 6 mmHg. A
significant increase in
urea is only observed in animals
dialyzed at Pip > 1.5 mmHg. This is consistent with our
finding that 1.5 mmHg is a threshold for a hydrostatic-driven convection during peritoneal dialysis in rats. The increase in
urea is primarily in the interstitium,
if, which doubled its volume upon elevation of
Pip
3 mmHg. There was no significant change in
IV; however,
IC invariably decreased in
all animals dialyzed at Pip > 1.5 mmHg.
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DISCUSSION |
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The present study investigated the changes in total tissue water and
its distribution in AAM upon simultaneous exposure to changes in
hydrostatic and osmotic pressures. As demonstrated in the present study
(see Fig. 6) and in our previous publications (9, 24), an
intraperitoneal threshold pressure must be reached for changes in
tissue water to occur. The threshold pressure in the rat peritoneal
cavity is ~1.5 mmHg, which results in an average tissue pressure
(PT) of 1.2 mmHg in the abdominal wall. Below this
threshold pressure, there is neither a significant change in tissue
water content nor a significant change in the pattern of distribution
of tissue water. Above the threshold pressure, a hydrostatic pressure
profile is set up across the abdominal wall, as seen in Fig. 1, which
illustrates the profiles for both the hypertonic dialysis of this study
and the isotonic solution of our previous study (24). As stated in the
results, the hydrostatic pressure profiles varied with the
Pip and had no dependency on the tonicity of the solution
in the peritoneal cavity; this observation matches our previous
pressure profile measurements in the anterior abdominal wall (3). Since
the slope of the tissue pressure profile (dPT/dx)
defines the driving force for hydrostatic pressure-driven convection
from the cavity into the interstitial space of the anterior abdominal
wall muscle during dialysis (see Eq. 1), it is not
surprising that a significant increase in tissue water is observed
regardless of osmolality of peritoneal fluid. This tissue expansion is
primarily elicited in the interstitium (if) (see Fig.
4), which doubles from its control value irrespective of dialysis fluid
osmolality when Pip is raised from 1.7 to 4 mmHg (Fig. 2).
Observations of tissue hydration depend to a great extent on the tissue
model employed, and a comparison to those used in other physiological
studies will be made below. In addition, these observations allow us to
predict the specific alterations to the transport coefficients for
diffusion and convection through the tissue space when it is expanded
by fluid in the cavity during peritoneal dialysis.
The Model Tissue and the Assumptions
The model tissue employed in this study is rat AAM simultaneously exposed to changes in hydrostatic and osmotic pressures. The model allows for measurement of tissue hydrostatic pressure and tracer concentration profiles for any Pip and peritoneal fluid osmolality. The total tissue water and its distribution in AAM were measured indirectly using dry-to-wet weight ratios or radioactive tracers. Since there is no specific marker for the interstitium, it must be determined from the difference between extracellular fluid volume (Studies of the Interstitium by Others
Levick and McDonald (13) have demonstrated simultaneous flow into and flow out from the rabbit knee after increases in both intra-articular pressure and albumin concentration in the infusate, suggesting an internal circulation of fluid. However, the rabbit knee synovium is only 20 µm thick, with little "tissue" surrounding the blood capillaries. It therefore resembles a capillary surrounded by a minimum of interstitial space. The rat AAM is ~2,000 µm thick with the nearest blood capillaries typically 40 µm from the peritoneum. Thus the mechanisms operating in the rabbit synovial model may be different from those of our model system.Wiig and Reed (20) dialyzed cats with 20% glucose in saline solutions
in the peritoneal cavity and measured EC,
PT, and
TW in gracilis and sartorius
muscles, which are anatomically separate from the tissue adjacent to
the cavity. The high glucose concentration in the peritoneal cavity
causes a rapid and large flux of fluid into the peritoneal cavity. The
immediate effect of this large flux is an increase in the effective
transcapillary oncotic pressure in favor of capillary absorption. This
resulted in the decrease in all volumetric parameters in tissues
distant from the peritoneal cavity (20, 21). Because of a greater absolute reduction of
TW than in
if, the
authors concluded that
IC was also reduced after
hypertonic dialysis. The authors extended their experiments in dogs
with the same measurements in gracilis and sartorius muscles (21).
if was reduced by 43% with reduction in
TW greater than the fall in
if. In both
species, a remarkable reduction in PT was observed after
hypertonic dialysis: from
0.8 ± 0.9 to
4.0 ± 1.1 mmHg
in cats and from
0.1 ± 0.8 to
4.2 ± 1.7 mmHg in dogs.
However, blood glucose had increased 5- to 10-fold from a control value
of 4-5.5 mmol/l, despite insulin injection.
The results and conclusions from these studies in cats and dogs cannot
be compared with our studies because of the different experimental
designs. First, by varying the volume and composition of the
intraperitoneal solution, we have exposed the AAM directly and
simultaneously to increases in osmolality and hydrostatic pressure,
which results in effects on if and
TW. In
the cat and dog studies,
if was manipulated by
dehydration alone. Second, in none of the animals dialyzed at
Pip > 0 mmHg with the hypertonic solution were negative
pressures in the tissue recorded, whereas all PT values
were negative in the cat and dog studies. Third, the net change in
TW in this study involves more complex mechanisms of
fluid shift consisting of an osmotic flux into the cavity with simultaneous hydrostatic-driven fluid flux into the AAM in situations where PT reaches a threshold of ~1.2 mmHg. In contrast,
in the cat and dog studies, there was likely little hydrostatic-driven fluid flow, and the use of a 20% glucose-based solution caused depletion of the plasma volume by 30% and increased the effective colloid osmotic pressure by 76% compared with baseline values. This
may in part explain the lower
if obtained after
hypertonic dialysis (20, 21). In summary, our results are derived from manipulation of the interstitial side of the Starling forces, whereas
previous studies were based on changes in the plasma pressures.
Pressure-Volume Curve
The pressure-volume curve assessed in this study (Fig. 2) revealed the classic nonlinear form reported in literature (16, 19, 21, 24), in which the tissue pressure (PT) must reach a pressure threshold to elicit a change inEffects of Hypertonic Dialysis on Local Water Content Profiles
Under normal physiological conditions in the intact rat, the total water content in the rat abdominal muscle accounts for ~75% of the total tissue volume.The tissue profile for urea, a surrogate for total
tissue water, illustrates some of the effects of hypertonicity in the tissue space closest to the peritoneum. All of the curves decrease in
magnitude in the 200 µm closest to the peritoneum, with the lowest
value measured at the peritoneum where the osmolality is highest. Part
of this decrease in local concentration of urea may be due to the
dilution of the tracer in the cavity caused by the influx of
solute-free water due to the osmotically driven flow. Evidence for the
dilution is provided in Fig. 5B with values of the ratio of
dialysate urea concentration to that in plasma concentration
[(D/P)urea] less than 1 for some of the
Pip values. When the urea tracer concentration in the
cavity drops below the plasma concentration, urea from the tissue will
diffuse from the tissue toward the dialysis fluid; this might cause a
slight decrease in the local concentration of urea in the vicinity of
the peritoneum. However, a clear pattern is present in all curves of
Fig. 5C, including those curves with (D/P)urea
equal to one. The decrease in total tissue water appears to coincide
with the region of highest hypertonicity (see mannitol concentration
profile in Fig. 5). Although we have no independent measure of the
intracellular space adjacent to the peritoneum, we can surmise from the
relationship
IC =
urea
EC and from the relatively flat profiles of
EC (Fig. 3; also see figure 2 of Ref. 24)
that the hyperosmolality that is present in the vicinity of the
peritoneum results in the decrease of the intracellular space. The
fluid that transports from
IC may contribute to the
local tissue expansion or to the osmotically induced flow from the
tissue to the cavity.
Hyperosmolality in the peritoneal cavity produces a quantitatively
lower TW at Pip > 1.5 mmHg than does
isotonic dialysis due to a decrease in
IC (see Figs. 4
and 6 and Table 1). As discussed in the introduction, we have
previously observed that hypertonic dialysis produces an osmotically
driven volume flux from tissue into the cavity (2, 6, 9) and a
hydrostatic pressure-driven flux into surrounding tissue and that these
events appear to occur at the same time (7-9). Whereas the
underlying mechanisms of these complex flow phenomena are not
completely clear at this time,
TW (or
urea) measured during hypertonic dialysis results from
this combination of osmotic and hydrostatic forces and the complex flow
into and out of the tissue space.
Effects of Hydrostatic and Osmotic Pressures on Transport During Peritoneal Dialysis
The general expansion of the tissue space surrounding the peritoneal cavity increases the rates of diffusion of solutes such as urea and creatinine through the tissue. The effective diffusion coefficient in tissue (Deff) equals the product of the diffusivity of the solute within the tissue space (DT) and the volume of the tissue available to the solute (Expansion of the extracellular volume in the tissue surrounding the
peritoneal cavity increases the rate of convection. This follows from
the dependency of the hydraulic conductivity of the tissue space on the
interstitial volume. One theoretical expression that is used to relate
the hydraulic conductivity of a porous bed to its structural properties
is the Carmen-Kozeny equation (12): K = (if)3/(GS2),
where G is Kozeny factor (a dimensionless proportionality
factor), and S is the wetted surface area within the porous
bed. As shown in Figs. 2 and 6 and in Ref. 24,
if
doubles once Pip increases from 1.5 to 4.4 mmHg; this
expansion would theoretically increase K by a factor of 8 (K1/K0 = 23), if
S is constant. However, the expansion of the interstitium may
increase the wetted surface area of interstitial matrix molecules, such
as hyaluronan (12), and the prediction of in vivo hydraulic conductivity cannot be easily made independently from experiment. We
have previously demonstrated that K in Eq. 1 varies
directly as the Pip is raised above 1.5 mmHg and increases
by a factor of 5 between Pip of 1.5 and 8 mmHg (23). An
increase in tissue hydraulic conductivity will increase the rate of
fluid flow through the tissue. Thus the raised Pip expands
the tissue, decreases its resistance to flow, and results in higher
rates of convection through the tissue than if it were in a nonexpanded
state. However, the direction of net flow will depend on the complex
relationship between hydrostatic and osmotic pressure forces within the
tissue space. Although the theory of hydrostatic pressure-driven flow is well established (Eq. 1), the effects of osmotic gradients are still controversial.
In conclusion, instillation of a dialysis solution in the peritoneal
cavity results in a rise in the intraperitoneal hydrostatic pressure,
Pip, that is maximally exerted across the AAM causing elevation of the local interstitial pressure, PT. It is not
until PT reaches a threshold pressure of ~1.2 mmHg that a
significant tissue expansion is observed. This tissue expansion is
nonlinear, occurs primarily in the interstitium (if),
and is unaffected by the osmolality of the bathing solution. Since the
interstitial space expands to the same degree under isotonic or
hypertonic conditions, the primary determinant of the resistance to
passive transport through the extracellular space in tissue surrounding the peritoneal cavity is the hydrostatic pressure exerted by the fluid
in the cavity and the corresponding changes in the adjacent tissue
space. The relatively small pressures seen in the cavity (2-20
mmHg) during dialysis are more than sufficient to cause interstitial
expansion. Thus transport through the subperitoneal interstitium under clinical dialytic conditions occurs at an
accelerated rate compared with movement through normal, nonexpanded tissue.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Whitaker Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant R29-DK-48479, and by a grant-in-aid from the American Heart Association.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. F. Flessner, Box 675, 601 Elmwood Ave., Univ. of Rochester Medical Center, Rochester, NY 14642 (E-mail: Michael_Flessner{at}URMC.Rochester.edu).
Received 23 April 1999; accepted in final form 16 December 1999.
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