AJP - Heart Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H1604-H1610, 2005. First published November 11, 2004; doi:10.1152/ajpheart.00808.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/H1604    most recent
00808.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koltun, M.
Right arrow Articles by Comper, W. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koltun, M.
Right arrow Articles by Comper, W. D.

Mechanism of hypoalbuminemia in rodents

Maria Koltun,1 Julijana Nikolovski,1 Kimberley Strong,1 David Nikolic-Paterson,2 and Wayne D. Comper1

1Department of Biochemistry and Molecular Biology, Monash University, and 2Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia

Submitted 9 August 2004 ; accepted in final form 10 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 complex-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 14C-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: {beta}2-microglobulin ({beta}2M) knockout (KO) mice and FcRn {alpha}-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, inasmuch as there was no comparable increase in the plasma elimination rate of 36- to 85-Å Ficoll or negatively charged 50- to 80-Å Ficoll. In contrast, hypoalbuminemic states in {beta}2M and FcRn KO mice were associated with decreases in excretion of albumin and albumin-derived fragments. This demonstrates that the mechanism of hypoalbuminemia consists of at least two distinct forms: one specifically associated with the renal handling of albumin and the other mediated by systemic processes.

albumin; capillary wall permeability; glomerular permeability; macromolecular transport probes; plasma elimination rate; {beta}2-microglobulin; Fc receptor

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 ~0.233 g·kg–1·day–1 or ~16–17 g/day (27); in rats, albumin elimination rates of 4.5–7.9 mg·100 g body wt–1·h–1 have been obtained (13, 16, 17). Endogenous albumin catabolism has been assumed to be responsible for plasma albumin elimination after correction for the portion of albumin that is eliminated in the urine. The major sites of albumin catabolism have been demonstrated to be the 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, 12, 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. demonstrated that it may increase significantly in seven-eights-nephrectomized rats (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. 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. We induced hypoalbuminemia with an intravenous bolus injection of puromycin aminonucleoside (PA), an agent that is well known to produce biochemical and structural alterations to the capillary wall (4, 20).

Recently, it has been suggested that minimization of albumin loss from the plasma occurs through a rescue pathway governed by the major histocompatibility complex-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). {beta}2-Microglobulin ({beta}2M) 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-mass (<10,000 Da) fragments. We have found that immunoassays do not detect the bulk of the fragments, whereas when radiolabeled albumin is used, all the albumin-derived material (intact albumin + albumin-derived fragments) is detected (12). 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. Ficoll is a spherical cross-linked polysucrose with a globular structure similar to that of albumin (2). Ficoll will model extracellular transport and nonspecific intracellular transport (pinocytosis) of albumin, whereas albumin may undergo extracellular transport and nonspecific 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 ({beta}2M deficient, where {beta}2M is a subunit of the FcRn receptor, and FcRn {alpha}-chain deficient) to determine whether hypoalbuminemia in these mice was due to excessive excretion of albumin-derived material.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. All animal studies were approved by the Monash University Animal Ethics Committee. Male Sprague-Dawley rats (350–450 g body wt, 10–12 wk of age) were obtained from the Monash University Central Animal House (Melbourne, Australia). Throughout the experimental period, the rats were maintained under a 12:12-h day-night cycle with free access to standard rat food and water.

Two strains of FcRn-deficient male mice (6–8 wk old) were used. {beta}2M KO mice (B6.129P2-B2mtml) and FcRn {alpha}-chain KO mice [B6.129X1/SvJ-FcgrtTmDcr(N6)] were obtained from Jackson Laboratories. The relevant wild-type control strain (C57BL/6J) was obtained from Monash University Central Animal House. The KO mice have been backcrossed onto the C57BL/6J background for 6 generations (FcRn) or 12 generations ({beta}2M). This provides justification for using C57BL/6J as the control strain.

Induction of hypoalbuminemia with PA. Hypoalbuminemia was induced with PA as previously described (12, 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 10 mg/100 g body wt. Age- and weight-matched controls were injected with an equivalent volume of PBS. Rats were placed in metabolic cages, and urine was collected over a 24-h period at baseline (day 0) and 5 and 8 days after 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 9 days 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]carboxymethyl (CM)-Ficoll by a bolus injection into the tail vein. The amount of tracer injected into each rat was 1–2 x 107 disintegrations/min (dpm) [14C]albumin, 1 x 108 dpm [3H]Ficoll, and 3 x 107 dpm [3H]CM-Ficoll.

Blood samples were taken from the tail vein at 3, 6, and 16 h after administration of the radiolabeled tracers. At 24 h, the rats were anesthetized with pentobarbitone sodium (20 mg ip; Rhone Merieux, Pinkeba, QLD, Australia) and killed by cardiac puncture. Urine was collected over the 24-h period.

The plasma elimination rate of each tracer was determined from the decrease in log plasma radioactivity over the 3- to 16-h period (linear regression coefficient >0.98). The plasma elimination rate was the gradient of this plot (24). Volume of distribution (the extent of material distribution in the body after intravenous administration) was determined for each tracer as the dose administered (dpm) ÷ plasma concentration of the tracer (dpm/ml) after equilibration within the body. Plasma clearance from the circulation was calculated as the plasma elimination rate (h–1) x volume of distribution of each tracer (ml). Multiplying the plasma clearance of albumin by the plasma albumin concentration (mg/ml) gives total albumin loss in milligrams per hour. Albumin catabolic rate (mg/h) is determined as the total albumin loss (mg/h) – albumin urinary excretion rate (mg/h) (17).

Ficoll and CM-Ficoll were polydisperse mixtures containing molecules in the radius range of 36–85 Å. Plasma elimination parameters were calculated for molecules of individual radii by fractionation of plasma samples taken at different time points on size exclusion chromatography.

Steady-state excretion rates of albumin-derived material in hypoalbuminemic mice with use of the osmotic pump method. As previously described (3), Alzet osmotic pumps were filled with 14C-labeled mouse serum albumin (MSA). The osmotic pump (model 1007D, Alzet) has a mean filling volume of 100 ± 4 µl and a pumping rate of 0.5 ± 0.02 µl/h with a duration of 7 days. The osmotic pump has a length of 1.5 cm, a diameter of 0.6 cm, and an unfilled weight of 0.4 g. After the pumps were filled, they were incubated in PBS for 4 h at 37°C. The amount of radioactivity initially in the pumps was 1 x 106 dpm [14C]MSA.

The mice were anesthetized with isoflurane inhalation anesthetic (Abbott), and the osmotic pumps filled with [14C]MSA were implanted subcutaneously between the scapulae by 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 metabolic cages on days 2, 5, and 7 for 24 h; urine collections and corresponding plasma samples were taken at the end of the 24-h 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) was determined by measuring the volume of the 24-h urine collection, and glomerular filtration rate (ml/min) 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 MSA (Sigma Chemical) were labeled with [14C]formaldehyde [56 mCi/mmol; New England Nuclear (NEN) Life Science Products, Boston, MA] according to a reductive methylation procedure described by Eng (7). The specific activity of RSA was 6.9 x 106 dpm/mg and that of MSA was 7.9 x 106 dpm/mg.

Polydisperse Ficoll 70 (70,000 mol wt; Sigma Chemical) and negatively charged CM-Ficoll 40 (40,000 mol wt; TdB Consultancy, 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 x 107 dpm/mg and that of [3H]CM-Ficoll was 6.33 x 107 dpm/mg.

Characterization of CM-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 0.34–0.54.

Column chromatography. Plasma samples containing polydisperse Ficoll or CM-Ficoll were fractionated on a Sephacryl S-300 column (2 x 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. Ninety-five fractions of ~1.7 ml were collected with recoveries >90%. The column was calibrated using blue dextran (2 mg/ml) and tritiated water (4 x 104 dpm/ml) to determine the void volume (V0) and the total volume (Vt), respectively. The available volume of material fractionated on the column (Kav) was determined by the following formula: Kav = (Ve – V0)/(Vt – V0), where Ve is elution volume of material.

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)] were used to construct a calibration curve for the column. A linear relationship was obtained for the plot of radii vs. Kav (r = 0.993). Other radii estimates were obtained by interpolation and extrapolation of this plot.

Radioimmunoassay for albumin. The concentration of albumin in urine and plasma samples was determined using 125I-labeled RSA or MSA, prepared using the chloramine T method (10), along with rabbit antiserum (polyclonal) to rat or mouse albumin (ICN Biomedicals, Aurora, OH) and sheep anti-rabbit antibodies (generously supplied by David Casley, Dept. of Medicine, Austin and Repatriation Medical Center, Victoria, Australia). The urinary albumin concentration measured by this double-antibody radioimmunoassay had an interassay coefficient of variation of 7% at a concentration of 180 ng/ml. The detection limit of the assay was 31.2 ng/ml. The standard curve was prepared using albumin standard (1 mg/ml), which was diluted to give a range of 4,000 to 31.2 ng/ml.

Total urinary protein. All collected blood and urine samples were centrifuged at 1,600 g for 10 min in a bench-top centrifuge (model KS-5200C, Kubota, Tokyo, Japan) to obtain plasma and sediment-free urine, respectively. Total urinary protein was determined by the biuret assay (9), with BSA as a standard.

Tissue uptake of [3H]Ficoll. To determine organ uptake of polydisperse Ficoll, organ tissues (kidneys, spleen, liver, and muscle) were excised from the rats after cardiac puncture 24 h after injection 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 were added to decolorize the samples, and the volume was made up to 1 ml with 850 µl of water. Scintillation fluid (4 ml) was added to the samples, which 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 was determined as percentage of injected dose.

Counting of radioactivity. Radioactivity from 14C- and tritium-labeled material was determined by beta scintillation counting in an LKB Wallac 1409 liquid scintillation analyzer with a 1:3 aqueous sample-to-Optiphase scintillation cocktail ratio.

Statistical analysis. All experimental data are means (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 P < 0.05. Linear regression analysis was performed using the computer program SigmaPlot (version 4 for Windows 98, Jandel, San Rafael, CA) or Microsoft Excel.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Integrity of radiolabeled probes in the plasma. [14C]albumin and [3H]Ficoll used in the study were not biochemically altered over the course of 24 h in the circulation, as determined by size-exclusion chromatography (Fig. 1), which could otherwise affect the determination of their elimination rate from the plasma. Chromatographic analysis of plasma samples also demonstrated no binding of any of the tracer molecules to other plasma components to generate higher-molecular-weight material.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Size exclusion chromatography profile of [3H]Ficoll integrity in plasma [in disintegrations per minute (dpm)] 24 h after administration in control (A) and puromycin aminonucleoside (PA)-treated (B) rats. Kav, available volume of material fractionated on the column.

 
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 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 UFR, as observed previously (12, 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, inasmuch as albumin catabolism has been significantly reduced. Other studies had established reduced albumin catabolism in PA-treated rats (12, 22).


View this table:
[in this window]
[in a new window]
  		Table 1. Physiological parameters in the control and PA-treated groups

 
Plasma elimination rate of Ficoll. Plasma elimination rate of polydisperse [3H]Ficoll mixture was 0.020 (SD 0.002) h–1 (n = 5) in controls and did not change significantly in PA-treated rats [0.021 (SD 0.003) h–1, n = 7]. Elimination rates of Ficoll of individual radii were compared with the plasma elimination rate of albumin in control and PA-treated groups (Fig. 2). Figure 2 clearly demonstrates a lack of significant difference in the plasma elimination rate of [3H]Ficoll in PA-treated rats compared with controls at any of the radii examined (36–85 Å; n = 5).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2. Plasma elimination rates of [14C]albumin in control ({blacktriangledown}) and PA-treated ({triangledown}) rats (n = 18–19) and plasma elimination rate of [3H]Ficoll as a function of hydrodynamic radius in control ({bullet}) and PA-treated ({circ}) rats (n = 5 at each radius). *P < 0.05 vs. control.

 
In control rats, the elimination rate of albumin was comparable to that of ≥65-Å Ficoll. In PA-treated rats, the plasma elimination rate of albumin was comparable to that of 36-Å Ficoll.

Volume of distribution and total clearance of Ficoll. The volume of distribution of Ficoll was compared with that of albumin in control and PA experimental groups (Fig. 3). Despite a significant increase in albumin volume of distribution in PA-treated rats, the volume of distribution did not change significantly for 36- to 85-Å Ficoll (n = 4–5).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Volume of distribution [injected dose of tracer (dpm) ÷ plasma concentration of tracer (dpm/ml)] of albumin in control ({blacktriangledown}) and PA-treated ({triangledown}) rats (n = 18–19) and [3H]Ficoll as a function of hydrodynamic radius in control ({bullet}) and PA-treated ({circ}) rats (n = 4–5 at each radius). *P < 0.05 vs. control.

 
Total clearances of Ficoll and albumin in control and PA-treated animals are shown in Fig. 4. The total clearance of 36- to 85-Å Ficoll did not change significantly (n = 4–5) in the nephrotic group, except for 55-Å Ficoll (P < 0.05). This was contrary to the significant increase in the total clearance of albumin in the PA-treated group.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 4. Plasma clearance of [14C]albumin in control ({blacktriangledown}) and PA-treated ({triangledown}) rats (n = 18–19) and [3H]Ficoll as a function of hydrodynamic radius in control ({bullet}) and PA-treated ({circ}) rats (n = 4–5 at each radius). Total clearance = plasma elimination rate (h–1) x volume of distribution (ml). *P < 0.05 vs. control.

 
Tissue accumulation of Ficoll. Table 2 shows the accumulation of radiolabeled material in the tissues compared with plasma and urine 24 h after bolus injection of [3H]Ficoll. The relative changes in uptake for each tissue were generally moderate, i.e., 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 a small percentage of the initial dose. These results are supported by the lack of change in the volume of distribution of Ficoll across the examined radii.


View this table:
[in this window]
[in a new window]
  		Table 2. Accumulation of [3H]Ficoll in tissues, plasma, and urine

 
Plasma elimination rate of CM-Ficoll. Control plasma elimination rate of albumin (hydrodynamic radius = 36 Å) was comparable to that of ≥65-Å Ficoll. This suggests that albumin elimination from the circulation is minimized by some mechanism. One mechanism proposed to account for this is charge repulsion of the negatively charged albumin by the fixed charges of the capillary wall. To test this possibility, we examined the elimination of CM-Ficoll (valence = –60). Results in Fig. 5 show that plasma elimination rate of 50- to 80-Å [3H]CM-Ficoll (n = 5) decreased in PA-treated rats compared with controls, with statistical significance (P < 0.05) for 75- and 80-Å CM-Ficoll. The elimination rate of this negatively charged probe was significantly higher (P < 0.001) than that of uncharged Ficoll (in control and PA-treated rats) and also that of albumin (in controls). These results demonstrated that charge interactions associated with electrostatic repulsion were not responsible for the minimization of albumin plasma elimination.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Plasma elimination rate of [14C]albumin in control ({blacktriangledown}) and PA-treated ({triangledown}) rats (n = 18–19) and [3H]carboxymethyl (CM)-Ficoll as a function of hydrodynamic radius in control ({bullet}) and PA-treated ({circ}) rats (n = 4–5 at each radius). *P < 0.05 vs. control.

 
{beta}2M and FcRn KO mice. Hypoalbuminemic states were apparent in {beta}2M and FcRn KO mice (Table 3) and comparable to the levels in PA-treated rats (Table 1). Albumin excretion did not increase in these KO mice, in agreement with results 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 a decrease in the amount of albumin-derived material excreted in the FcRn-deficient mice. We did observe, particularly with the {beta}2M mice, a gradual increase in plasma albumin with age (Fig. 6), but it was always lower than that of controls.


View this table:
[in this window]
[in a new window]
  		Table 3. Physiological parameters and albumin excretion in control and FcRn-deficient mice

 


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 6. Plasma albumin concentration in {beta}2-microglobulin ({bullet}) and Fc receptor ({circ}) knockout mice (n = 2–9 at each time point) as a function of age. §P < 0.05 vs. 6-wk-old Fc receptor knockout mice; *P < 0.05 vs. 7-wk-old {beta}2-microglobulin knockout mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The extent to which albumin is retained in the circulation compared with other molecules of equivalent size has not been measured. 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 = 36 Å) was comparable to that of ≥65-Å Ficoll. In PA-treated rats, the plasma elimination rate of albumin was comparable to that of Ficoll with a radius of 36 Å, which is 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 of 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 percentage 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 a heterogeneous effect associated with the clearance of Ficoll, i.e., an increase in uptake in some tissues and a decrease in others, inasmuch as 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 also with other recent studies that have not been able to identify electrostatic charge repulsion of albumin (12, 14, 25, 26).

Measurements of the plasma elimination rate and clearance of albumin are a function of albumin distribution from plasma into various nonrenal tissues as well as urinary excretion. We previously showed that, after 24 h, 10–20% of albumin from the initial injected dose is found in the liver, muscle, and urine (12). 125I-labeled 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 the 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 seven-eights nephrectomized rats, total albumin plasma clearance in the control was 4.5 mg·100 g body wt–1·h–1, which increased to 5.99 mg·100 g 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·100 g body wt–1·h–1, which would account for 78% of the increase in plasma clearance (17). In another study (16), it was demonstrated that, in Heymann nephritis, where plasma albumin concentrations were reduced by up to 70% and plasma elimination rates increased up to twofold, 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 the primary organ responsible for albumin loss in PA-treated rats. Importantly, they found no major change in the albumin permeability/uptake by nonrenal tissues. Although hypoalbuminemia in PA-treated rats is due to increased albumin loss into the urine, it is not due to altered capillary wall permeability, as suggested by the lack of change in the plasma elimination rate of different-sized Ficolls.

It has been recently suggested that albumin elimination from the circulation may be governed by the FcRn-mediated mechanism (5). Major histocompatibility complex molecules are involved in the development of several autoimmune diseases of the kidney, and it was found that the mice that lacked FcRn receptor ({beta}2M 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. Table 3 demonstrates 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 PA-treated rats are quite different from 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, inasmuch as {beta}2M and FcRn KO mice exhibit similar albumin excretion, despite having considerably different plasma concentrations (Table 3). The plasma concentrations are also age dependent (Fig. 6). Chaudhury et al. (5) reported that albumin biosynthetic rate was lower in FcRn-deficient mice, implicating FcRn in the albumin biosynthetic pathway.

Although the results of nephrotic studies focus on a renal centric mechanism that confers major control of plasma albumin levels, this mechanism appears cellularly mediated, inasmuch as capillary wall permeability seems unaltered. It had been suggested earlier that a high-capacity postfiltration retrieval pathway exists for albumin (8); when inhibited by PA treatment, this pathway could account for the changes observed in this study.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent technical assistance of Lynette Pratt (Dept. of Biochemistry and Molecular Biology, Monash University) and technical assistance with the radioimmunoassays of Dr. Tanya Osicka and Steve Sastra (AusAm Biotechnologies).


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. D. Comper, Dept. of Biochemistry and Molecular Biology, Monash Univ., Wellington Rd., Clayton, Victoria 3800, Australia (E-mail: wayne.comper{at}med.monash.edu.au)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ballmer PE. Causes and mechanisms of hypoalbuminaemia. Clin Nutr 20: 271–273, 2001.[CrossRef][Web of Science][Medline]
  2. Bohrer MP, Deen WM, Robertson CR, Troy JL, and Brenner BM. Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall. J Gen Physiol 74: 583–593, 1979.[Abstract/Free Full Text]
  3. Burne MJ, Osicka TM, and Comper WD. Fractional clearance of high molecular weight proteins in conscious rats using a continuous infusion method. Kidney Int 55: 261–270, 1999.[CrossRef][Web of Science][Medline]
  4. Caulfield JP, Reid JJ, and Farquhar MG. Alterations of the glomerular epithelium in acute aminonucleoside nephrosis. Evidence for formation of occluding junctions and epithelial cell detachment. Lab Invest 34: 43–59, 1976.[Web of Science][Medline]
  5. Chaudhury C, Mehnaz S, Robinson JM, Hayton WL, Pearl DK, Roopenian DC, and Anderson CL. The major histocompatibity complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 197: 315–322, 2003.[Abstract/Free Full Text]
  6. Di Giorgio J. Non protein nitrogenous constituent. In: Clinical Chemistry Principles and Techniques, edited by Henry R, Canon D, and Winkelman J. Hagerstown, MD: Harper and Row, 1974, p. 503–563.
  7. Eng LA. Production and characterization of [14C]protein A, a long-lived immunological reagent. J Immunol Methods 81: 239–243, 1985.[CrossRef][Web of Science][Medline]
  8. Eppel GA, Osicka TM, Pratt LM, Jablonski P, Howden BO, Glasgow EF, and Comper WD. The return of glomerular-filtered albumin to the rat renal vein. Kidney Int 55: 1861–1870, 1999.[CrossRef][Web of Science][Medline]
  9. Gornall AG, Bardawill CJ, and David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 177: 751–766, 1949.[Free Full Text]
  10. Greenwood FC, Hunter WM, and Glover JS. The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem J 89: 114–123, 1963.[Web of Science][Medline]
  11. Greive KA. The Renal Processing of Proteins in Health and Disease (Thesis). Melbourne, Australia: Department of Biochemistry and Molecular Biology, Monash University, 2001.
  12. Greive KA, Nikolic-Paterson DJ, Guimarães MAM, Nikolovski J, Pratt LM, Mu W, Atkins RC, and Comper WD. Glomerular permselectivity factors are not responsible for the increase in fractional clearance of albumin in rat glomerulonephritis. Am J Pathol 159: 1159–1170, 2001.[Abstract/Free Full Text]
  13. Greive KA, Russo LM, Osicka TM, and Comper WD. Nephrotic-like proteinuria in experimental diabetes. Am J Nephrol 23: 38–46, 2003.[CrossRef][Web of Science][Medline]
  14. Guimarães MAM, Nikolovski J, Pratt LM, Greive K, and Comper WD. Anomalous fractional clearance of negatively charged Ficoll relative to uncharged Ficoll. Am J Physiol Renal Physiol 285: F1118–F1124, 2003.[Abstract/Free Full Text]
  15. Kaysen GA. Non renal complications of the nephritic syndrome. Annu Rev Med 45: 201–210, 1994.[CrossRef][Web of Science][Medline]
  16. Kaysen GA, Kirkpatrick WG, and Couser WG. Albumin homeostasis in the nephrotic rat: nutritional considerations. Am J Physiol Renal Fluid Electrolyte Physiol 247: F192–F202, 1984.[Abstract/Free Full Text]
  17. Kaysen GA and Watson JB. Mechanism of hypoalbuminemia in the 7/8-nephrectomized rat with chronic renal failure. Am J Physiol Renal Fluid Electrolyte Physiol 243: F372–F378, 1982.[Abstract/Free Full Text]
  18. Mixter PF, Russel JQ, Durie FH, and Budd RC. Decreased CD4–CD8– TCR-{alpha}{beta}+ cells in Ipr/Ipr mice lacking {beta}2-microglobulin. J Immunol 154: 2063–2074, 1995.[Abstract]
  19. Mozes E, Kohn LD, Hakim F, and Singer DS. Resistance of MHC class I-deficient mice to experimental systemic lupus erythematosus. Science 261: 91–93, 1993.[Abstract/Free Full Text]
  20. Nakayama K, Natori Y, Sato T, Kimura T, Sugiura A, Sato H, Saito T, Ito S, and Natori Y. Altered expression of NDST-1 messenger RNA in puromycin aminonucleoside nephrosis. J Lab Clin Med 143: 106–114, 2004.[CrossRef][Web of Science][Medline]
  21. Öqvist BW, Rippe A, Tencer J, and Rippe B. In vivo clearance of albumin in renal and extrarenal tissues in puromycin aminonucleoside (PAN) induced nephrotic syndrome. Nephrol Dial Transplant 14: 1898–1903, 1999.[Abstract/Free Full Text]
  22. Osicka TM, Hankin AR, and Comper WD. Puromycin aminonucleoside nephrosis results in a marked increase in fractional clearance of albumin. Am J Physiol Renal Physiol 277: F139–F145, 1999.[Abstract/Free Full Text]
  23. Peters T. Serum albumin. Adv Protein Chem 37: 161–245, 1985.[Web of Science][Medline]
  24. Rowland M and Tozer TN. Clinical Pharmacokinetics: Concepts and Applications. Philadelphia, PA: Lippincott, Williams & Wilkins, Wolters Kluwer, 1995, p. 18–33.
  25. Russo LM, Bakris GL, and Comper WD. Renal handling of albumin: a critical review of basic concepts and perspective. Am J Kidney Dis 39: 899–919, 2002.[CrossRef][Web of Science][Medline]
  26. Schaeffer RC, Gratrix ML, Mucha DR, and Carbajal JM. The rat glomerular filtration barrier does not show negative charge selectivity. Microcirculation 9: 329–342, 2002.[CrossRef][Web of Science][Medline]
  27. Sterling K. The turnover rate of serum albumin in man as measured by I131-tagged albumin. J Clin Invest 43: 1228–1237, 1951.
  28. Strobel JL, Cady SG, Borg TK, Terracio L, Baynes JW, and Thorpe SR. Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues. J Biol Chem 261: 7989–7994, 1986.[Abstract/Free Full Text]
  29. Strober W and Waldmann TA. The role of the kidney in the metabolism of plasma proteins. Nephron 13: 35–66, 1974.[Web of Science][Medline]
  30. Van Damme MP, Comper WD, and Preston BN. Experimental measurements of polymer unidirectional fluxes in polymer + solvent systems with non-zero chemical potential gradients. J Chem Soc Faraday Trans I 225: 8842–8847, 1982.
  31. Waldman TA. Albumin catabolism. In: Albumin Structure, Function and Uses, edited by Rosenoer VM, Oratz M, and Rothschild MA. New York: Pergamon, 1977, p. 255–273.
  32. Watson PD, Sloop CH, and Renkin EM. Disappearance of fluorescein-labelled dextran from dog plasma. Bibl Anat 16: 453–456, 1977.[Medline]
  33. Yedgar S, Carew TE, Pittman RC, Beltz WF, and Steinberg D. Tissue sites of catabolism of albumin in rabbits. Am J Physiol Endocrinol Metab 244: E101–E107, 1983.[Abstract/Free Full Text]
  34. Zurovsky Y, Ovadia M, and Shkolnik A. Disappearance of dextran 70,000 from plasma of Hamadryas baboons. Comp Biochem Physiol A 80: 373–375, 1985.



This article has been cited by other articles:


Home page
 J. Am. Soc. Nephrol.Home page
M. Sarav, Y. Wang, B. K. Hack, A. Chang, M. Jensen, L. Bao, and R. J. Quigg
Renal FcRn Reclaims Albumin but Facilitates Elimination of IgG
J. Am. Soc. Nephrol., September 1, 2009; 20(9): 1941 - 1952.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
W. D. Comper, L. M. Hilliard, D. J. Nikolic-Paterson, and L. M. Russo
Disease-dependent mechanisms of albuminuria
Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1589 - F1600.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
T. Fujita, S. Yasuda, Y. Kamata, K. Fujita, Y. Ohtani, Y. Kumagai, and M. Majima
Contribution of Down-Regulation of Intestinal and Hepatic Cytochrome P450 3A to Increased Absorption of Cyclosporine A in a Rat Nephrosis Model
J. Pharmacol. Exp. Ther., November 1, 2008; 327(2): 592 - 599.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
D. B. Tesar, E. J. Cheung, and P. J. Bjorkman
The Chicken Yolk Sac IgY Receptor, a Mammalian Mannose Receptor Family Member, Transcytoses IgY across Polarized Epithelial Cells
Mol. Biol. Cell, April 1, 2008; 19(4): 1587 - 1593.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/H1604    most recent
00808.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koltun, M.
Right arrow Articles by Comper, W. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koltun, M.
Right arrow Articles by Comper, W. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.