Mechanism of hypoalbuminemia in rodentsMECHANISM OF HYPOALBUMINEMIA 4 Albumin a 66 kDa protein is a critical and major component in the circulation. It provides the dominant colloid

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.

mailto:[email protected]


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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.


3

Key Words: albumin, capillary wall permeability, glomerular permeability,

macromolecular transport probes, plasma elimination rate, b2m, β2-microglobulin,

FcRn, Fc receptor


4

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).


5

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.


6

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.


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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


8

(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


9

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.


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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


11

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


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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


13

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.


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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


15

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).


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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


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


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.


20

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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.


25

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.


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.


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.


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).


29

Figure 1


30

Figure 2


31

Figure 3


32

Figure 4


33

Figure 5


34

Figure 6

Documents

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