MECHANISM OF HYPOALBUMINEMIA
Mechanism of hypoalbuminemia in
rodents
Maria Koltun1, Julijana Nikolovski1, Kimberley Strong1, David
Nikolic-Paterson2 and Wayne D. Comper1
1Department of Biochemistry and Molecular Biology, Monash University,
Clayton, Victoria 3800, Australia
2Department of Nephrology and Monash University Department of
Medicine, Monash Medical Centre, Clayton, Victoria 3168, Australia
Running Head: Mechanism of Hypoalbuminemia
Corresponding author:
Wayne D. Comper, PhD, DSc
Department of Biochemistry and Molecular Biology, Monash University
Wellington Road, Clayton
Victoria, 3800
Australia
Phone: 61 3 9905 3774
Fax: 61 3 9905 1117
Email: [email protected]
Articles in PresS. Am J Physiol Heart Circ Physiol (November 11, 2004). doi:10.1152/ajpheart.00808.2004
Copyright © 2004 by the American Physiological Society.
MECHANISM OF HYPOALBUMINEMIA
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Mechanism of hypoalbuminemia in rodents.
Normal albumin loss from the plasma is thought to be minimized by a number of
mechanisms including charge repulsion with the capillary wall and an intracellular
rescue pathway involving the major histocompatibility compex (MHC)-related Fc
receptor (FcRn)-mediated mechanism. This study investigates how these factors may
influence the mechanism of hypoalbuminemia. Hypoalbuminemia in rats was induced
by treatment with puromycin aminonucleoside (PA). To test the effects of PA on
capillary wall permeability, plasma elimination rates were determined for tritium
labeled tracers of different sized Ficolls, negatively charged Ficolls and carbon-14
labeled tracer of albumin in control and PA-treated Sprague-Dawley rats. Urinary
excretion and tissue uptake were also measured. Hypoalbuminemia was also examined
in two strains of FcRn deficient mice, β2-microglobulin (b2m) knockout (KO) mice and
FcRn α-chain KO mice. The excretion rates of albumin and albumin-derived fragments
were measured. PA-induced hypoalbuminemia was associated with a 2.5-fold increase
in the plasma elimination rate of albumin. This increase could be completely accounted
for by the increase in urinary albumin excretion. Changes in the permeability of the
capillary wall were not apparent as there was no comparable increase in the plasma
elimination rate of Ficoll (hydrodynamic radius range 36-85 Å) or negatively charged
Ficoll (50-80 Å). In contrast, hypoalbuminemic states in b2m and FcRn KO mice were
associated with decreases in excretion of both albumin and albumin-derived fragments.
This demonstrates that the mechanism of hypoalbuminemia consists of at least of two
distinct forms: one specifically associated with the renal handling of albumin and the
other mediated by systemic processes.
MECHANISM OF HYPOALBUMINEMIA
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Key Words: albumin, capillary wall permeability, glomerular permeability,
macromolecular transport probes, plasma elimination rate, b2m, β2-microglobulin,
FcRn, Fc receptor
MECHANISM OF HYPOALBUMINEMIA
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Albumin a 66 kDa protein is a critical and major component in the circulation. It
provides the dominant colloid osmotic properties of blood but also acts as a carrier to
many different types of ligands (23). The steady state levels of albumin in the
circulation are governed by its synthesis rate in the liver and its elimination rate from
the plasma. The elimination rate of albumin in humans corresponds to approximately
0.233 g.kg-1day-1 or around 16-17 g.day-1 (27), whereas in rats values in the range of
4.5-7.9 mg per 100g body wt per hour have been obtained (12, 16, 17). Endogenous
albumin catabolism has been assumed to be responsible for plasma albumin elimination
once corrected for the portion of albumin that is eliminated in the urine. The major sites
of albumin catabolism have been demonstrated to be liver, kidney, muscle (29, 31, 33)
and skin where the fibroblast plays an active role (28). It has been recently shown that
in control rats urinary excretion of albumin-derived material can account for 20-30% of
the albumin eliminated from the plasma (3, 11, 13, 22).
Hypoalbuminemic states in plasma are often associated with liver and kidney
disease and these states may have a profound influence on albumin plasma elimination.
The basic mechanism is still unresolved (1, 15). Kaysen et al have demonstrated that it
may increase significantly in 7/8 nephrectomized rat (17) and in rats with Heymann
nephritis (16). Excess urinary excretion of albumin in nephrotic states is thought to arise
from structural changes in the glomerular capillary wall (GCW). It is expected that
similar changes in permeability would occur in the capillary wall of the general
circulation particularly those tissues with fenestrated or discontinuous capillary beds. In
order to investigate this we induced hypoalbuminemia with an intravenous bolus
injection of puromycin aminonucleoside (PA). This agent is well known to produce
biochemical and structural alterations to the capillary wall (4, 20).
MECHANISM OF HYPOALBUMINEMIA
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Recently it has been suggested that minimization of albumin loss from the
plasma occurs through a rescue pathway governed by the major histocompatibility
complex (MHC)-related Fc receptor (FcRn)-mediated mechanism (5). Albumin is
pinocytosed by many cells of the body and is transported to acidic endosomes, where it
may encounter FcRn. This receptor binds albumin and diverts it from degradation in the
lysosomes, and instead transports albumin back to the cell surface. Under the influence
of neutral pH, albumin dissociates from the receptor and is free to recycle. It was
demonstrated that the lifespan of albumin is shortened in FcRn-deficient mice and the
plasma albumin concentration of these mice is less than half that of wild-type mice (5).
β2-microglobulin (b2m) knockout (KO) mice are apparently not albuminuric as
determined by a dipstick that determines total protein (18, 19) suggesting that increased
albumin elimination is due to higher levels of albumin degradation in cells around the
body. This may involve increased excretion of albumin-derived material (not measured
in previous studies) and this may contribute significantly to hypoalbuminemia in these
KO mice.
Albumin is excreted in the urine as a mixture of intact albumin and albumin-
derived low molecular weight fragments (< 10,000 Da). We have found that
immunoassays do not detect the bulk of the fragments whereas when radiolabeled
albumin is used all of the albumin-derived material (intact albumin plus albumin-
derived fragments) is detected (13). We refer to albumin as native albumin meaning
detected by immunoassay and albumin-derived material as being measured by
radioactivity. The total protein assay (biuret) measures all protein-derived material
including protein fragments (11).
This study specifically examines the plasma elimination rate and clearance from
the circulation in normal and nephrotic states of tritium labeled polydisperse Ficoll.
MECHANISM OF HYPOALBUMINEMIA
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Ficoll is a spherical cross-linked polysucrose which has a globular structure similar to
albumin (2). Ficoll will model extracellular transport and non-specific intracellular
transport (pinocytosis) of albumin whereas albumin may undergo both extracellular
transport, non-specific and specific intracellular transport. The relative plasma
elimination rates of albumin and Ficoll are compared. We also examine the renal
processing of albumin in two strains of FcRn KO mice (b2m-deficient where b2m is a
subunit of the FcRn receptor and FcRn α-chain deficient) to determine whether
hypoalbuminemia in these mice was due to excessive excretion of albumin-derived
material.
METHODS
Experimental animals
All animal studies were approved by the Monash University Animal Ethics
Committee. Male Sprague-Dawley rats (350-450 g in weight, 10-12 weeks in age) were
obtained from the Monash University Central Animal House (Melbourne, Australia).
Throughout the experimental period, the rats were maintained under a 12-hour day/night
cycle with free access to standard rat food and water.
Two strains of FcRn-deficient male mice (6-8 weeks old) were used. B2m KO
mice (B6.129P2-B2mtml) and FcRn α-chain KO mice (B6.129X1/SvJ-FcgrtTmDcr(N6))
were obtained from Jackson Laboratories (USA). The relevant wild type (WT) control
strain (C57BL/6J) was obtained from Monash University Central Animal House
(Melbourne, Australia). The KO mice have been backcrossed onto the C57BL/6J
background for 6 generations (FcRn) or 12 generations (b2m). This provides
justification for using C57BL/6J as the control strain.
MECHANISM OF HYPOALBUMINEMIA
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Induction of hypoalbuminemia with puromycin aminonucleoside
Hypoalbuminemia was induced with PA as previously described (13, 22). Rats
were injected intravenously (via the tail vein) with PA (Sigma Chemical, St. Louis,
MO), as a 3.5% solution in phosphate buffered saline (PBS), pH 7.4, at a concentration
of 10 mg.100 g-1 body weight. Age- and weight-matched controls were injected with
equivalent volume of PBS. Rats were placed in metabolic cages and urine was collected
over a 24-hour period at baseline (day 0) and days 5 and 8 following PA or PBS
administration. Total urinary protein excretion was determined at each of these days to
ensure the onset of proteinuria. The experiments were performed on day 9 after
injection of PA or PBS.
Plasma elimination of albumin and Ficoll in hypoalbuminemic rats
Control and PA-treated rats were injected with a mixture of [14C]albumin and
[3H]Ficoll or [14C]albumin and [3H]CM-Ficoll by a bolus injection into the tail vein.
The amount of tracer injected into each rat was: 1-2 × 107 dpm [14C]albumin, 1 × 108
dpm [3H]Ficoll and 3 × 107 dpm [3H]CM-Ficoll.
Blood samples were taken from the tail vein at 3, 6 and 16 hours post-
administration of the radiolabeled tracers. At 24 hours, the rats were anaesthetized with
an intraperitoneal injection of pentobarbitone sodium (20 mg; Rhone Merieux Pty Ltd,
Pinkeba, QLD, Australia) and sacrificed by cardiac puncture. Urine was collected over
the 24-hour period.
The plasma elimination rate of each tracer was determined from the decrease in
log plasma radioactivity over the 3-16 hour period (linear regression coefficient > 0.98).
The plasma elimination rate was the gradient of this plot (24). Volume of distribution
MECHANISM OF HYPOALBUMINEMIA
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(the extent of material distribution in the body following intravenous administration)
was determined for each tracer as the dose administered (dpm)/plasma concentration of
the tracer (dpm.ml-1; after equilibration within the body has been achieved). Plasma
clearance from the circulation was calculated as the plasma elimination rate (h-1) ×
volume of distribution of each tracer (ml). Multiplying the plasma clearance of albumin
by the plasma albumin concentration (mg.ml-1) gives total albumin loss in mg.h-1.
Albumin catabolic rate (mg.h-1) is determined as the total albumin loss (mg.h-1) less
albumin urinary excretion rate (mg.h-1) (17).
Ficoll and CM-Ficoll were polydisperse mixtures, containing molecules in the
radius range 36-85 Å. Plasma elimination parameters were calculated for molecules of
individual radii by fractionating plasma samples taken at different time points on size
exclusion chromatography.
Steady state excretion rates of albumin-derived material in hypoalbuminemic mice
using the osmotic pump method
As previously described (3) Alzet osmotic pumps were filled with [14C]-MSA.
The Alzet osmotic pump model 1007D has a mean filling volume of 100 ± 4 µl,
pumping rate of 0.5 ± 0.02 µl.h-1 with a duration of 7 days. Osmotic pump has a length
1.5 cm, diameter 0.6 cm and unfilled weight 0.4 g. Once the pumps were filled, they
were incubated in PBS for 4 hours at 37°C. The amount of radioactivity initially in the
pumps was 1 × 106 dpm [14C]MSA.
The mice were anesthetized with Isoflurane Inhalation Anaesthetic (Abbott,
Australia) and the osmotic pumps filled with [14C]MSA were implanted subcutaneously
between the scapulae using sterile technique. The mice were maintained in mouse boxes
in groups of five with free access to food and water at all times and were placed in
MECHANISM OF HYPOALBUMINEMIA
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metabolic cages on days 2, 5 and 7 for 24 hours and urine collections and corresponding
plasma samples were taken at the end of the 24h period. The samples were then
analyzed for radioactivity. Samples were taken on days 2, 5 and 7 to ensure that the
steady state was reached on day 7. Urine flow rate (UFR) (ml.min-1) was determined by
measuring the volume of the 24 h urine collection and glomerular filtration rate (GFR)
(ml.min-1) was determined by the creatinine assay (6). Specific activity of albumin in
the plasma (calculated from the ratio of plasma dpm to plasma albumin concentration)
was used to calculate albumin-derived material excretion rate.
Radiolabeling
Rat serum albumin (RSA) and mouse serum albumin (MSA)(Sigma Chemical,
St. Louis, MO) were labeled with [14C]-formaldehyde (56 mCi.mmol-1; New England
Nuclear (NEN) Life Science Products, Boston, Massachusetts), using a reductive
methylation procedure described by Eng (7). The specific activity of the RSA was 6.9 ×
106 dpm.mg-1 and that of MSA was 7.9 × 106 dpm.mg-1.
Polydisperse Ficoll 70 (Mw = 70,000) (Sigma Chemical, St. Louis, MO) and
negatively charged carboxymethyl Ficoll 40 (CM-Ficoll 40; Mw = 40,000; TdB
Consultancy AB, Uppsala, Sweden) were tritiated with sodium boro-[3H]hydride
according to Van Damme and colleagues (30). The specific activity of [3H]Ficoll was
5.39 × 107 dpm.mg-1 and that of [3H]CM-Ficoll was 6.33 × 107 dpm.mg-1.
Characterization of carboxymethyl Ficoll
Measurement of the charge substitution has been previously described (14). The
degree of carboxyl group substitution per sucrose residue on the CM-Ficoll was in the
range of 0.34-0.54.
MECHANISM OF HYPOALBUMINEMIA
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Column chromatography
Plasma samples containing polydisperse Ficoll or CM-Ficoll were fractionated
on a Sephacryl S-300 (column dimensions 2 × 66 cm2) (Pharmacia Fine Chemicals,
Uppsala Sweden). Plasma samples were eluted with PBS containing 0.2% BSA (used to
prevent adsorption) and 0.02% sodium azide, at 4°C, at a rate of 20 ml.h-1. Ninety-five
fractions of approximately 1.7 ml were collected with recoveries greater than 90%. The
column was calibrated using blue dextran (2 mg.ml-1) and tritiated water (4 × 104
dpm.ml-1) to determine the void volume (Vo) and the total volume (Vt), respectively.
The available volume of material fractionated on the column, Kav, was determined by
the formula, Kav = (Ve – Vo)/(Vt-Vo), where Ve is elution volume of material.
A calibration curve was constructed for the column using Kav values for
molecules of known molecular weight and size – albumin (radius = 36 Å), transferrin
(radius = 48 Å), immunoglobulin G (radius = 55 Å) and glucose oxidase (radius = 70
Å). A linear relationship was obtained for the plot of radii versus Kav (r = 0.993). Other
radii estimates were obtained by both interpolation and extrapolation of this plot.
Radioimmunoassay for albumin
The concentration of albumin in urine and plasma samples was determined using
[125I]RSA or [125I]MSA, prepared using the Chloramine T method (10), along with
rabbit antiserum (polyclonal) to rat albumin (ICN Biomedicals Inc., Aurora, OH, USA)
and sheep anti-rabbit antibodies (generously supplied by David Casley of the
Department of Medicine, Austin and Repatriation Medical Center, Victoria, Australia).
The urinary albumin concentration measured by this double antibody
radioimmunoassay (RIA) had an interassay coefficient of variation of 7% at a
MECHANISM OF HYPOALBUMINEMIA
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concentration of 180 ng.ml-1. The detection limit of the assay was 31.2 ng.ml-1. The
standard curve was prepared using albumin standard (1 mg.ml-1), which was diluted to
give a range of 4000 to 31.2 ng.ml-1.
Total urinary protein
All collected blood and urine samples were centrifuged at 1600g for 10 minutes
in a KS-5200C Kubota bench top centrifuge (Kubota Corp., Tokyo, Japan) to obtain
plasma and sediment-free urine respectively. Total urinary protein was determined by
the biuret assay (8), using bovine serum albumin (BSA) as a standard.
Tissue uptake of [3H]Ficoll
To determine organ uptake of polydisperse Ficoll, organ tissues (kidneys,
spleen, liver, muscle) were excised from the rats following cardiac puncture 24 h after
being injected with [3H]Ficoll. The tissues were briefly washed in saline, weighed and
minced, and 1.4 M NaOH was added to make a final volume of 6 ml (kidney, spleen, 1-
2 g liver, 1-2 g muscle). The samples were suspended in boiling water for 15-30 min to
allow digestion to occur. Four sample aliquots of 100 µl each were taken, 50 µl of
hydrogen peroxide was added to decolorize the samples, and the volume made up to one
ml with 850 µl of water. Four ml of scintillation fluid was added to the samples and
they were rested overnight in the dark to reduce chemiluminescence. The samples were
counted for radioactivity and the presence of the tracer in the tissues, plasma and urine
determined as percentage of injected dose.
Counting of radioactivity
MECHANISM OF HYPOALBUMINEMIA
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Radioactivity from carbon-14 and tritium labeled material was determined by
beta scintillation counting in a LKB Wallac 1409 liquid scintillation analyzer (Wallac,
Finland), using 1:3 aqueous sample to Optiphase scintillation cocktail ratio.
Statistical analysis
All experimental data are expressed as mean ± standard deviation (SD). N
denotes the number of experiments performed. Statistical significance was determined
using unpaired, two-tailed student’s t-test. Statistical significance was accepted when
probability, P < 0.05. Linear regression analysis was performed using the computer
program Sigma Plot (Version 4 for Windows 98, Jandel Corporation, San Rafeal,
California) or Microsoft Excel.
RESULTS
Integrity of radiolabeled probes in the plasma
Carbon-14 labeled albumin as well as tritium labeled Ficoll used in the study
were not biochemically altered over the course of 24 hours in the circulation, as
determined by size-exclusion chromatography (Figure 1), which could otherwise affect
the determination of their elimination rate from the plasma. Chromatographic analysis
of plasma samples also demonstrated that there was no binding of any of the tracer
molecules to other plasma components to generate higher molecular weight material.
Characteristics of PA induced hypoalbuminemia
Table 1 summarizes the physiological parameters obtained in control and PA-
treated experimental groups. Treatment with PA induced significant hypoalbuminemia
MECHANISM OF HYPOALBUMINEMIA
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in rats. This was accompanied by nephrosis as suggested by the significant increase in
the urinary excretion of total protein and albumin and the increase in the urine flow rate,
similar to those observed previously (13, 22). The plasma elimination rate of albumin
increased significantly by 2.5-fold with an accompanying increase in albumin volume of
distribution and hence albumin clearance from the plasma. Volume of distribution is a
direct measure of the extent of distribution and will encompass albumin losses into the
urine. With increased volume of distribution and decreased plasma concentration of
albumin, total albumin clearance did not increase significantly in PA-treated rats. In the
case of control animals, albumin catabolic rate is similar to total albumin loss (less
small losses of albumin in the urine). However, in PA-treated rats, total albumin
clearance is predominantly accounted for by albumin urinary excretion as albumin
catabolism has been significantly reduced. Other studies had established reduced
albumin catabolism in PA-treated rats (13, 22).
Plasma elimination rate of Ficoll
Plasma elimination rate of polydisperse [3H]Ficoll mixture was 0.020 ± 0.002 h-1
(n = 5) in controls which did not change significantly in PA-treated rats, 0.021 ± 0.003
h-1 (n = 7). Elimination rates of Ficoll of individual radii were compared to the plasma
elimination rate of albumin in control and PA-treated groups as shown in Figure 2. This
figure clearly demonstrates a lack of significant difference in the plasma elimination
rate of [3H]Ficoll in PA-treated as compared to controls at any of the radii examined,
36-85 Å (n = 5).
In control rats the elimination rate of albumin was comparable to that of Ficoll
molecules with radii of 65 Å and greater. In PA-treated, the plasma elimination rate of
albumin was comparable to the elimination rate of a 36 Å Ficoll.
MECHANISM OF HYPOALBUMINEMIA
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Volume of distribution and total clearance of Ficoll
The volume of distribution of Ficoll was compared to that of albumin in control
and PAN experimental groups (Figure 3). Despite a significant increase in albumin
volume of distribution in PA treated rats, the volume of distribution did not change
significantly for Ficoll molecules across the examined radius range, 36-85 Å (n = 4-5).
Total clearances of Ficoll and albumin in control and PAN animals are shown in
Figure 4. The total clearance of Ficoll molecules of individual radii did not change
significantly across the examined radius range, 36-85 Å (n = 4-5), in the nephrotic
group, except for Ficoll with a radius of 55 Å (P < 0.05). This was contrary to the
significant increase in the total clearance of albumin in PA treated group.
Tissue accumulation of Ficoll
Table 2 shows the accumulation of radiolabeled material in the tissues as
compared to plasma and urine 24 hours after bolus injection of [3H]Ficoll (expressed as
a percentage of injected dose). The relative changes in uptake for each tissue were
generally moderate being within a factor of 2. The exception is the PA-treated kidney
where a significant increase in uptake was observed but in relative terms the PA-treated
kidney contained only small percent of the initial dose. These results are supported by
the lack of change in the volume of distribution of Ficoll across the examined radii.
Plasma elimination rate of carboxymethyl Ficoll
Control plasma elimination rate of albumin (hydrodynamic radius of 36 Å) was
comparable to that of a Ficoll molecule of 65 Å or greater. This suggests that albumin
elimination from the circulation is minimized by some mechanism. One mechanism
MECHANISM OF HYPOALBUMINEMIA
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proposed to account for this has been charge repulsion of the negatively charged
albumin by the fixed charges of the capillary wall. In order to test this possibility we
examined the elimination of CM-Ficoll (valence = -60). Results in Figure 5 show
plasma elimination rate of [3H]CM-Ficoll over the examined radius range, 50-80 Å (n =
5), decreased in PA-treated as compared to controls with statistical significance (P <
0.05) for radii 75 and 80 Å. The elimination rate of this negatively charged probe was
significantly higher (P < 0.001) than the elimination rate of uncharged Ficoll (both in
control and PAN rats) and also the elimination of albumin (in controls). These results
demonstrated that charge interactions associated with electrostatic repulsion were not
responsible for the minimization of albumin plasma elimination.
β2m and FcRn KO mice
Hypoalbuminemic states were apparent in both b2m and FcRn KO mice (Table 3) and
comparable to the levels seen in PA-treated rats (Table 1). Albumin excretion did not
increase in these knockout mice which is in agreement with that reported previously (5),
rather it appeared to decrease. Importantly we found no evidence of peptideuria or
excessive excretion of albumin-derived fragments that may not be detected by
immunoassay. Again we found that there was a decrease in the amount of albumin-
derived material being excreted in the FcRn deficient mice. We did observe,
particularly with the b2m mice a gradual increase in plasma albumin with age (Figure 6)
but were always lower than that of controls.
DISCUSSION
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The extent to which albumin is retained in the circulation as compared to other
molecules of equivalent size has hitherto been unmeasured. Previous studies had
established that the plasma elimination of certain molecular weight dextran fractions
(32, 34) was unexpectedly rapid given their high molecular weight. In this study we
demonstrate that the plasma elimination rate of albumin (hydrodynamic radius of 36 Å)
was comparable to that of Ficoll molecules with radii of ≥ 65 Å. In PA-treated rats, the
plasma elimination rate of albumin was comparable to the elimination rate of a 36 Å
Ficoll, which is the same as the hydrodynamic radius of albumin. This means that PA
specifically destroyed the mechanism of minimization of plasma albumin elimination.
PA is known to exert major effects on the synthesis on the components of the
capillary wall and its morphology (4, 20). Despite these changes we found no
significant change in capillary wall permeability with PA treatment as measured by the
plasma elimination of different sized Ficolls. There was also no major change in the
renal elimination of Ficoll (there was an increase in the percent excreted into the urine
(Table 2), which would be offset by the corresponding increase in UFR in PA-treated
rats). It would also seem unlikely that there was an heterogeneous effect associated with
the clearance of Ficoll i.e. an increase in uptake in some tissues and decrease in others,
as both elimination rate and volume of distribution did not change for any of the
hydrodynamic radii studied in PA-treated rats.
We found that the negative charge repulsion between albumin and the capillary
wall did not play a significant role in the minimization of albumin plasma clearance.
The changes observed with CM-Ficoll were opposite to those seen with albumin. This is
consistent too with other recent studies that have not been able to identify electrostatic
charge repulsion of albumin (13, 14, 25, 26).
MECHANISM OF HYPOALBUMINEMIA
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Measurements of the plasma elimination rate and clearance of albumin are a
function of albumin distribution from plasma into various non-renal tissues, as well as
urinary excretion. We have previously shown that after 24 hours, 10-20% of albumin
from the initial injected dose is found in the liver, muscle and urine (13). [125I]human
albumin clearance has been demonstrated to occur in a range of tissues including the
liver and bowel (21). Similarly, the primary sites of Ficoll distribution are liver and
urine (Table 2).
The changes in PA-treated rats however are quite specific. The profound
increase in the total intact albumin plasma clearance can be entirely accounted for by
the increase in its urinary excretion in PA-treated rats (Table 1). These conclusions are
in agreement with studies of Kaysen et al (16, 17) in hypoalbuminemic/nephrotic rats.
In 7/8-nephrectomized rats total albumin plasma clearance in the control was 4.5
mg.100g body wt-1.h-1 which increased to 5.99 mg.100g body wt-1.h-1 in nephrotic rats
where plasma albumin concentration was reduced by 19%. The increase was
accompanied by an increase in albumin excretion of 1.16 mg.100g body wt-1.h-1 which
would account for 78% in the increase in plasma clearance (17). In another study (16),
it was demonstrated in Heymann nephritis where plasma albumin concentrations were
reduced by up to 70%, and plasma elimination rates increased up to 2-fold, the albumin
urinary excretion could account for up to 100% of the increase in the plasma elimination
rate in nephrotic states. In a more recent study, Öqvist et al (21) very clearly
demonstrated that the kidney was primary organ responsible for albumin loss in PA-
treated rats. Importantly, they found no major change in the albumin
permeability/uptake by non-renal tissues. Although hypoalbuminemia in PA-treated rats
is due to increased albumin loss into the urine, it is not due to altered capillary wall
MECHANISM OF HYPOALBUMINEMIA
18
permeability as suggested by the lack of change in the plasma elimination rate of Ficoll
of different radii.
It has been recently suggested that albumin elimination from the circulation may
be governed by the major histocompatibility complex (MHC)-related Fc receptor
(FcRn) mediated mechanism (5). MHC molecules are involved in the development of
several autoimmune diseases of the kidney and it was found that the mice that lacked
FcRn receptor (b2m KO) failed to develop proteinuria/albuminuria as measured by
dipstick (18, 19), suggesting that increased albumin elimination was due to higher levels
of albumin degradation in cells around the body and/or increased excretion of albumin-
derived fragments in the urine that was not detected by dipstick. Studies in Table 3
demonstrated that hypoalbuminemia in the KO mice is also not due to increased
excretion of albumin-derived material. The results of this study would suggest that the
factors that control the increased plasma elimination in PAN are quite different to those
proposed for FcRn-deficient mice. In nephrosis it is essentially the increased urinary
excretion of albumin that accounts for the increase in the loss of albumin from the
plasma. It appears that no other organ offers significant opportunity for the loss of
albumin. Other factors must control plasma albumin levels in the FcRn deficient mice.
These factors might be associated with albumin synthesis as both b2m and FcRn KO
mice exhibit similar albumin excretion in spite of having considerably different plasma
concentrations (Table 3). The plasma concentrations are also age-dependent (Figure 6).
Chaudhury et al (5) reported that albumin biosynthetic rate was lower in FcRn-deficient
mice, implicating FcRn in the albumin biosynthetic pathway.
While the results of nephrotic studies focus on a renal centric mechanism that
confers major control of plasma albumin levels, this mechanism appears cellular-
mediated as capillary wall permeability seems unaltered. It had been suggested earlier
MECHANISM OF HYPOALBUMINEMIA
19
that a high capacity post-filtration retrieval pathway exists for albumin (8), which when
inhibited by PA treatment could account for the observed changes seen in this study.
ACKNOWLEDGEMENTS
We gratefully acknowledge Mrs. Lynette Pratt, Department of Biochemistry and
Molecular Biology, Monash University, Clayton, Victoria, Australia, for her excellent
technical assistance and Dr Tanya Osicka and Mr Steve Sastra, AusAm Biotechnologies
Inc., for their technical assistance with the radioimmunoassays.
MECHANISM OF HYPOALBUMINEMIA
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24
Table 1. Physiological parameters in the control and PAN experimental groups
Control PAN
Plasma albumin concentration (mg.ml-1) 23.94 ± 1.28 (n = 12) 7.63 ± 2.05† (n = 12)
Total protein excretion (mg.h-1) 2.60 ± 0.34 (n = 12) 21.78 ± 4.69† (n = 12)
Urine flow rate (ml.min-1) 0.007 ± 0.002 (n = 12) 0.015 ± 0.003† (n = 12)
Glomerular filtration rate (ml.min-1) 1.19 ± 0.29 (n = 5) 1.01 ± 0.5 (n = 5)
Albumin urinary excretion rate (mg.h-1) 0.025 ± 0.012 (n = 7) 13.42 ± 3.20† (n = 7)
Albumin plasma elimination rate (h-1) 0.019 ± 0.003 (n = 18) 0.050 ± 0.011† (n = 19)
Volume of distribution (ml) 24.2 ± 2.9 (n = 18) 32.7 ± 9.9* (n = 19)
Total albumin clearance (ml.h-1) 0.460 ± 0.065 (n = 18) 1.62 ± 0.48† (n = 12)
Total albumin loss (mg.h-1) 11.01 ± 0.08 (n = 18) 12.36 ± 0.98 (n = 19)
Albumin catabolic rate (mg.h-1) 10.98 ~0
Values are means ± SD; n, number of rats. Plasma albumin concentration was measured
by RIA. Total protein excretion was measured by the biuret assay. Albumin excretion
rate was determined by RIA. * P < 0.05; † P < 0.001 versus control.
MECHANISM OF HYPOALBUMINEMIA
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Table 2. Accumulation of [3H]Ficoll in tissues, plasma and urine as a percentage of
injected radioactivity 24 hours after intravenous administration of tracer
Control
(% of injected radioactivity)
PAN
(% of injected radioactivity)
Kidney 0.34 ± 0.06 (n = 5) 4.15 ± 1.12† (n = 7)
Spleen 0.65 ± 0.18 (n = 5) 0.50 ± 0.10 (n = 7)
Liver 17.68 ± 3.38 (n = 5) 31.15 ± 4.05† (n = 7)
Muscle 3.07 ± 0.51 (n = 5) 2.39 ± 0.68 (n = 7)
Plasma 7.80 ± 0.96 (n = 5) 4.82 ± 0.81† (n = 7)
Urine 4.18 ± 1.50 (n = 7) 8.00 ± 3.57* (n = 7)
Values are means ± SD; n, number of rats. The percentage of injected radioactivity was
estimated by assuming that a rat weighing ~400 g has, on average, 15 ml of blood (7 ml
of plasma), 10 g of liver and 100 g of muscle. * P < 0.05; † P < 0.001 versus control.
MECHANISM OF HYPOALBUMINEMIA
Table 3. Physiological parameters and albumin excretion in control and FcRn deficient
mice
Control b2m KO FcRn KO
Plasma albumin
concentration
(mg.ml-1)
25.02 ± 3.89
(n = 7)
7.39 ± 1.85†
(n = 4)
14.21 ± 3.73*
(n = 4)
Urine flow rate
(ml.min-1)
0.0011 ± 0.0004
(n = 7)
0.0007 ± 0.0003
(n = 4)
0.0012 ± 0.0006
(n = 4)
Glomerular filtration
rate (ml.min-1)
0.1061 ± 0.0411
(n = 7)
0.0527 ± 0.0257*
(n = 4)
0.0424 ± 0.0120*
(n = 4)
Total protein excretion
(mg.24h-1)
6.55 ± 3.69
(n = 5)
2.71 ± 1.61
(n = 4)
4.04 ± 1.62
(n = 4)
Albumin excretion
rate (mg.24h-1)
0.0154 ± 0.009
(n = 7)
0.0049 ± 0.0039*
(n = 4)
0.006 ± 0.0027*
(n = 4)
Albumin-derived
material excretion rate
(mg.24h-1)
4.93 ± 1.69
(n = 7)
1.09 ± 0.44*
(n = 4)
1.84 ± 1.10*
(n = 4)
Values are means ± SD; n, number of mice. Plasma albumin concentration was
measured by RIA. Total protein excretion was measured by the biuret assay. Albumin
excretion rate was measured by RIA and albumin-derived material was measured by
radioactivity. * P < 0.05; † P < 0.001 versus control.
MECHANISM OF HYPOALBUMINEMIA
27
FIGURE LEGENDS
Figure 1. Size exclusion chromatography profile of [3H]Ficoll integrity in the plasma 24
hours after administration in (A) control and (B) PA-treated rats.
Figure 2. The plasma elimination rates (h-1) of [14C]albumin in control (filled triangle)
and PAN (unfilled triangle) rats (n = 18-19). Plasma elimination rate of [3H]Ficoll as a
function of hydrodynamic radius (Å) in control (filled circles) and PA-treated rats
(unfilled circles) rats (n = 5 at each radius). *P < 0.05 versus control.
Figure 3. Volume of distribution (ml) of albumin in control (filled triangle) and PAN
(unfilled triangle) rats (n = 18-19) and [3H]Ficoll as a function of hydrodynamic radius
(Å) in control (filled circles) and PA-treated rats (unfilled circles) rats (n = 4-5 at each
radius). Volume of distribution = injected dose of tracer (dpm) / plasma concentration of
tracer (dpm.ml-1). *P < 0.05 versus control.
Figure 4. Plasma clearance (ml.h-1) of [14C]albumin in control (filled triangle) and PAN
(unfilled triangle) rats (n = 18-19) and [3H]Ficoll as a function of hydrodynamic radius
(Å) in control (filled circles) and PA-treated rats (unfilled circles) rats (n = 4-5 at each
radius). Total clearance = plasma elimination rate (h-1) × volume of distribution (ml). *P
< 0.05 versus control.
Figure 5. The plasma elimination rate (h-1) of [14C]albumin in control (filled triangle)
and PAN (unfilled triangle) rats (n = 18-19) and [3H]CM-Ficoll as a function of
hydrodynamic radius (Å) in control (filled circles) and PA-treated rats (unfilled circles)
rats (n = 4-5 at each radius). *P < 0.05 versus control.
MECHANISM OF HYPOALBUMINEMIA
28
Figure 6. The plasma albumin concentration (mg/ml) in β-2-microglobulin (filled
circle) and FcRn (unfilled circle) knockout mice (n = 2-9 at each timepoint) as a
function of age (days).
MECHANISM OF HYPOALBUMINEMIA
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Figure 1
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Figure 2
MECHANISM OF HYPOALBUMINEMIA
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Figure 3
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Figure 4
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Figure 5
MECHANISM OF HYPOALBUMINEMIA
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Figure 6