Clinical Science (1997) 93,557-564 (Printed in Great Britain)
557
Fractional clearance of albumin is influenced by its degradation
during renal passage
Tanya M. OSICKA, Sianna PANAGIOTOPOULOS*, George ]ERUMS* and Wayne D. COMPER
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3 168, Australia,
and *Endocrine Unit, Austin and Repatriation Medical Centre, Department of Medicine, University of
Melbourne, Heidelberg, Victoria 3084, Australia
(Received I May/26 June 1997; accepted I3 August 1997)
1. The fractional clearance of intact albumin as
determined by fractionation of urine by gel chromatography gave a value of 3.9k1.6 x
for the isoin vivo
lated perfused kidney and 2.1 k0.6 x
using ALZET osmotic pumps.
2. Albumin fractional clearance as measured by
detection of the tritium label on the albumin molecule by radioactivity analysis gave a value of 7.5k
3.9 x
for the isolated perfused kidney and 2.3
ko.9 x 10-3 in viva.
3. The major differences between assays that detect
intact albumin compared with non-specific assays in
the estimates of the fractional clearance of albumin
can be explained by the degradation of approx. 90%
of albumin to small peptides during its renal passage. This has been demonstrated by size exclusion
chromatography of urine samples from experiments
where (i) exogenous tritium-labelled albumin was
used in isolated perfused kidneys, (ii) exogenous
tritium-labelled albumin was administered intravenously and (iii) analysis was made with metabolically labelled endogenous albumin in vivo.
INTRODUCTION
The apparent marked restriction of albumin transport across the glomerular capillary wall compared
with other proteins or dextrans of similar size is
unique. Transport studies in both continuous and
fenestrated capillaries in the body demonstrate that
albumin transport is not excessively restricted in
relation to other proteins or dextrans [l]. For
example, the peritubular capillary sieving coefficient
for albumin is 0.33 as calculated from the lymph/
plasma ratio [2], while the sieving coefficient of
albumin has been found to be 0.51 to 0.74 in the
gastrointestinal tract [2] and 0.23 to 0.38 in subcutaneous tissue [2]. In all these capillary beds the
restriction to dextran of equivalent hydrodynamic
radius to albumin is similar to that observed across
the glomerular capillary wall [l].
The relatively high sieving coefficient values for
albumin in non-glomerular capillary beds are supported by other studies where the glomerular fractional clearance of albumin was measured as 0.02 to
0.06 by glomerular volumetric analysis in isolated rat
glomeruli [3, 41. Furthermore, the relative difference
in permeability between albumin and dextran across
isolated glomerular basement membrane systems
does not reflect the differences seen in vivo [5 ,6], as
sieving coefficients across isolated basement membranes have been found to be between 0.05 and 0.1
[6-81.
It appears that one of the major difficulties in elucidating the mechanism of the renal processing of
albumin has been the marked variation in the published values of its glomerular sieving coefficient
(GSC) and urinary fractional clearance (combination of GSC and tubular reabsorption). The GSC
for albumin has been obtained mainly from micropuncture experiments which sample the post-glomerular early proximal tubular space. It has been
assumed that the ratio of plasma concentration to
early proximal tubular lumen concentration equates
with the GSC. Micropuncture studies have estimated the concentration of filtered albumin in proximal tubules to be in the range of 0.1 to
50mg/100ml for normal kidneys [9-221 and 3.0 to
650 mg/100 ml for diseased kidneys [13-18, 211.
Initial micropuncture studies determined the GSC
and 10.0 x
of albumin to be between 1.0 x
when protein in the early proximal fluid was measured by non-specific total protein assays. More
recent measurements of GSC, using specific radioimmunoassay or disc electrophoresis for albumin
analysis, have yielded lower values of 2 3.0 x
[19-22]. These values represent a partitioning of
albumin (i.e. the concentration ratio of albumin in
the Bowman’s space as compared with plasma) at
the glomerular capillary wall of > 1:3000.
The micropuncture studies give GSCs of albumin
which in some cases are quantitatively lower than
those obtained through urinary fractional clearance
studies on kidneys. Measurements in vivo of the
Key words: albumin, degraded albumin, fractional clearance, isolated perfused kidney, osmotic pumps.
Abbreviations: GSC, glomerular sieving coefficient; GFR, glomerular filtration rate; IPK, isolated perfused kidney. TCA, trichloroaceticacid; UFR, urine flow rate.
Correspondence: Dr Wayne D. Cornper.
558
T.M. Osicka et al.
GSC of albumin in rats from fractional clearance
by Bertolatus
studies have varied from 6.0 x
and Hunsicker [23] to 3.0 x
by Bertolatus et al.
[24], where allowance was made for tubular reabsorption by assuming that albumin accumulated in
the tubules of the whole kidney as measured by trichloroacetic acid (TCA)-insoluble radiolabelled
albumin. Purtell et al. [25], who did not correct for
tubular reabsorption, found a fractional clearance
(equivalent to the GSC) of 5.0-7.0 x lop3. The fractional clearance of albumin as measured by Lowry
assay was 1 . 0 ~ 1 0 -for
~ studies in vivo in rat [19]
and 7.6-72.0 x
in the isolated perfused kidney
(IPK) [19, 261. This compares with micropuncture
in vivo and
studies which gave a GSC of 2.7 x
in the IPK [19]; in both cases the albumin
2.3 x
concentration was estimated by ultra-microdisc
electrophoresis with Coomassie Blue stain.
Rationalization of some of this variation, particularly for the fractional clearance studies, can be
made with the recognition that albumin which
appears in the urine is significantly degraded [27, 281
and that the degraded protein may not be detected
by specific albumin assays or included in TCA precipitates. The degradation which has now been shown
to occur for a number of proteins [28] may considerably influence clearances, particularly for assays
designed for the specific analysis of intact albumin
or other intact protein. Under such circumstances
the assay may vastly underestimate the total renal
clearance of the protein.
The above range of values for the clearance of
albumin is still too large to permit conclusions to be
drawn about the degree of glomerular sieving and
the renal turnover rate of albumin. Thus, the aim of
this study was to compare various techniques to
measure urinary albumin concentration. We
examine the fractional clearance of albumin by the
fractionation of urine samples, obtained from both
studies in the IPK and in vivo using ALZET osmotic
pumps, by size exclusion chromatography using
tritium-labelled albumin. The fractional clearance of
albumin was also determined using various nonspecific protein assays. The processing of exogenous
labelled albumin was then compared with that of
endogenous labelled albumin.
METHODS
Materials
Male Sprague-Dawley rats (300-350 g) were
obtained from the Monash University Central
Animal House. Catalase (EC 1.11.1.6) (1600 i.u./mg
solid from bovine liver), sulphosalicylic acid, thimerosal, sodium azide, benzoylated dialysis tubing
(molecular mass cut-off of 2000 Da) and the amino
acids lysine, tyrosine, serine, cysteine, aspartate,
glutamate, asparagine and glutamine were obtained
from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
BSA (Fraction V, fatty acid free) and superoxide
dismutase (EC 1.15.1.1) (5000 i.u./mg solid from
bovine erythrocytes) were purchased from
Boehringer Mannheim GmbH Biochemica (Mannheim, Germany). Sephadex G-100, Sephadex G-25
in PD-10 columns and blue dextran T2000 were purchased from Pharmacia Fine Chemicals (Uppsala,
Sweden). Nembutal (60 mg/ml) was from Cera
Chemicals Australia Pty Ltd (Hornsby, NSW,
Australia). Synthamin (a source of amino acids) was
from Travenol Laboratories (NSW, Australia).
Sodium heparin was from Commonwealth Serum
Laboratories (Melbourne, Australia). Mannitol was
from CSR Chemicals Ltd (Rhodes, NSW,
Australia). Tritiated water (0.25 mCi/g), [carboxyl14C]inulin (2.7 mCi/g) and [3H]lysine (108.4 Ci/
mmol) were obtained from Du Pont (Wilmington,
Detroit, U.S.A.) and sodium b~ro[~H]hydride
(132 mCi/mg) was from Amersham International
(Bucks, U.K.). Sodium [1251]iodidewas purchased
from Australian Radioisotopes (Lucas Heights,
NSW, Australia). ALZET osmotic pumps (model
2001) were purchased from ALZA Corporation
(California, U.S.A.). Rabbit anti-cow albumin antibody was from DAKO Australia Pty Ltd (Botany,
NSW, Australia) and sheep anti-rabbit antibody was
a gift from David Casley, Austin and Repatriation
Medical Centre, Heidelberg, Victoria, Australia.
Solutions and buffers
PBS, pH 7.4, contained 136.9 mmol/l NaCl, 2.68
mmol/l KCl, 8.1 mmol/l NazHP04 and 1.5 mmol/l
KHzP04. Krebs-Henseleit buffer, pH 7.4, contained
122 mmol/l NaCl, 4.6 mmol/l KCl, 0.115 mmol/l
KHzPO4, 0.115 mmol/l MgS04, 24.9 mmolfl NaHC03
and 0.1 mmol/l CaClz.Hz0. Sodium phosphate
buffer (0.067 mol/l, pH 6.2) contained 18.5 ml of
0.067 mol/l NazHP04 and 81.5 ml of 0.067 mol/l
NaHzP04.2H20. Kidney perfusate solution was 5%
BSA in Krebs-Henseleit buffer that contained
5 mmol/l glucose and 36 mg of mannitol, 10 600 i.u.
of superoxide dismutase and 110 000 i.u. of catalase
(all per 200ml of perfusate), and amino acids
(mmol/l) tyrosine (0.2), serine (l.O), cysteine (0.5),
aspartate (0.2), glutamate ( O S ) , asparagine (0.2),
glutamine (2.0), leucine (0.4), phenylalanine (0.32),
methionine (0.33), lysine (l.O), isoleucine (0.3), valine (0.33), histidine (0.24), threonine (0.24), tryptophan (0.07), alanine (2.0), glycine (2.3), arginine
(0.5) and proline (0.31).
Tritium labelling
BSA was labelled with tritium by the reductive
methylation procedure of Tack et al. [29]. This reaction involves a brief exposure to formaldehyde and
sodium b~ro[~H]-hydride.
The tritium labelling is
specific for the cc-amino groups of the amino terminal residues and the &-amino groups of lysyl residues. The labelled preparation was applied to a
Fractional clearance of albumin
Sephadex G-25 PD-10 column to separate it from
free label. The purity of the [3H]BSA preparation
was ensured by extensive dialysis of the sample
(approx. lo9 d.p.m./ml; 3.5 ml) using dialysis tubing
with a molecular mass cut-off of 2000Da until no
tritium was found in the dialysate (volume = 4 1).
Assuming that there is less than 100 d.p.m./ml in the
dialysate, this indicates that less than 0.0025% of the
original sample could be contaminated with free
tritium or low-molecular-mass-labelled material. The
dialysed preparation was again applied to a PD-10
column immediately before use. The activity of
[3H]BSAwas 2.31 x lo8 d.p.m./mg.
Kidney petfusion
Male Sprague-Dawley rats were anaesthetized by
a 1ml intraperitoneal injection of 18 mg/ml pentobarbitone (Nembutal). (Male rats were used as the
cannulation of the renal artery is simpler due to the
proximity of the mesenteric artery.) PBS (1 ml) containing 10% mannitol and 200 i.u. of sodium heparin
was injected into the femoral vein. A laparotomy
was performed and the right ureter cannulated with
polyethylene tubing (PE-10; Dural Plastics and
Engineering, Auburn, NSW, Australia). The right
renal artery was cannulated via the superior mesenteric artery and the kidney was removed by en bloc
dissection. This whole procedure took no longer
than 10 min. The perfusion pressure was maintained
at 90-100 mmHg with a peristaltic pump monitored
by a calibrated aneroid manometer, while kidneys
had an average flow rate of approximately 25 ml/min
as monitored by a ball flowmeter. Kidneys were perfused with 160 ml of recirculated filtered 5% BSA in
Krebs-Henseleit buffer containing glucose, essential
amino acids [30-321 and oxygen radical scavengers
to prevent partial ischaemia [32]. The system was
maintained at 37°C and the perfusate was continually gassed with 95% 02-5% C02. The kidney was
allowed to equilibrate for 10 min and urine and perfusate samples collected after 40 and 60 min of perfusion. The perfusate contained approx. 1x lo6
d.p.m./ml [3H]BSA. Measurement of glomerular
filtration rate (GFR) was made with [carboxyl14C]inulin. Determination of fractional clearance
was made by Lowry assay [33] (controls for the
Lowry assay were performed by subtracting the
chromogen in the supernatant after TCA precipitation of the urine sample), Biuret assay [34], sulphosalicylic acid turbidity method [35], radioimmunoassay (all using BSA as standard) or radioactive counting for tritium-labelled protein.
To determine the fractional clearance by the
specific analysis of intact albumin in urine and perfusate, samples were analysed on a Sephadex G-100
column (1.6 x 60 cm2) eluted with PBS at 20 ml/h at
4°C. One-millilitre samples were loaded and 95 fractions of 1.65 ml each were collected. These samples
were then analysed for radioactivity. The void
559
volume (V,) was determined with blue dextran
T2000 and the total volume (K) with tritiated water.
Processing of [)H]BSA in vivo
An implantable osmotic pump (model 2001) was
used for the measurement of the fractional clearance of [3H]BSA in vivo [36, 371. A steady-state
tracer concentration was reached in the plasma,
achieved by the slow, continuous infusion of tracer
by the pump at 1pl/h for 7 days. Pumps were filled
with 200p1 of [3H]BSA at a concentration of
4 x lo7 d.p.m./ml containing thimerosal at 1/5000
(wt/vol.) to inhibit bacterial growth. The pump was
incubated in PBS containing 0.02% sodium azide at
37°C for 4 h before implantation. The pump was
wiped with 70% isopropanol and implanted subcutaneously on the back of an anaesthetized male
Sprague-Dawley rat, slightly posterior to the scapulae, using sterile technique. Rats were maintained
in a metabolic cage with free access to food and
water. Urine was collected for 24 h periods with a
corresponding blood sample from the tail vein.
Urine and blood samples were centrifuged at
3500rpm in a KUBOTA bench top centrifuge for
10 min and then analysed for radioactivity. GFR was
estimated by creatinine assay.
A separate group of male Sprague-Dawley rats
were also injected with 1x los d.p.m./ml [3H]BSA in
O.8ml PBS in the tail vein and maintained in a
metabolic cage with free access to food and water.
Urine samples were collected after 2 h and were
analysed on a Sephadex G-100 column as described
above.
Synthesis of endogenous [3H]albumin in vivo and its
appearance in urine
Male Sprague-Dawley rats were injected with
0.3 ml (300 pCi) of [3H]lysine via the tail vein and
then maintained in a metabolic cage with free access
to food and water. Urine was collected after 1h; the
rats were then killed and blood was collected by
cardiac puncture. Ten millilitres of blood were collected into a syringe containing 1ml of 3.8% sodium
citrate. This suspension was centrifuged at 3000 rpm
for 30 min and the serum was removed. Samples of
urine and serum were analysed on a Sephadex
G-100 column as described above.
Radioimmunoassay
The 12sI-BSAwas prepared by the Chloramine T
method [38]. One-hundred microlitres of sample,
100 pl of the first antibody, rabbit anti-cow albumin,
100 p1 of [12sI]BSA and 30Opl of PBS containing
0.4% gelatine were added to each tube. After vortexing, the tubes were incubated at room temperature for 4 h. One-hundred microlitres of the second
560
T. M. Osicka et al.
antiserum, sheep anti-rabbit, was then added to each
tube which were again vortexed and incubated overnight at room temperature. Polyethylene glycol
(750~1,8%) was added to tubes which were then
vortexed and centrifuged at 3500rpm for 30min at
4°C. Supernatants were decanted and precipitates
estimated for radioactivity. The standard curve was
prepared by diluting a solution (1 mg/ml) of BSA in
PBS to give a range of 4000 to 31.2 ng/ml.
Counting of radioactivity
3H and 14C radioactivity were determined in 1 ml
aqueous samples with 3 ml of scintillant [39] and
recorded on a Wallac 1410 liquid scintillation counter (Wallac Oy, Turku, Finland). 1251radioactivity
was recorded by a Packard crystal 5412 gamma
counter.
only from the original material that has a profile
similar to that shown in Fig. la) has been used in
vivo and in the IPK [28]. When the fractional clearance of [3H]BSA was determined by the specific
analysis of intact albumin from the area under the
bell-shaped curve of the chromatographic profiles
(centred at fraction 41 in Figs l a to lc), a value of
(n = 6) was obtained.
3.9 rfi 1.6 x
When the fractions eluted from the Sephadex
G-100 column were assayed for BSA by radioimmunoassay (Fig. 2) and absorption at 280nm,
only the intact BSA peak was detected by radioimmunoassay with no evidence of any low-molecu-
I
I
a
Calculations
All quantitative data are expressed as means k SD
where n represents the number of determinations.
RESULTS
Fractional albumin clearance in the isolated perfused rat
kidney
The IPK preparation, when studied over a 1 h
period, has been shown by this laboratory to be a
steady-state, stable preparation over this time period
[27] with an average GFR and urine flow rate
(UFR) of 0.828 k0.166 ml/min and 0.148 k0.065
ml/min (n = 6), respectively. BSA is the only filterable protein in the perfusate used in IPK experiments. The other proteins added to the perfusate,
namely catalase and superoxide dismutase at conmg/ml
centrations of 0.196 mg/ml and 5.5 x
respectively, are too low in concentration and too
high in molecular mass to be filtered in detectable
quantities.
The gel chromatographic profile on Sephadex
G-100 of [3H]BSA from a 60min perfusate sample
(Fig. l a ) appears to be essentially the same as the
original material [27], both preparations eluting as a
single major peak with a K,, [ = (Vc-Vo)/(Vt-Vo)
where Vc is the elution volume] of 0.190. The small
leading peak in the profile represents the albumin
dimer. There is no low-molecular-mass material
present in the perfusate. The elution profiles of the
urine obtained from isolated rat kidneys with
[3H]BSA, shown in Fig. lb, reveal that most of the
radioactivity is associated with low-molecular-mass
material. The degradation of the [3H]BSA molecule
is not due to the IPK system as degradation was also
observed in vivo (Fig. lc). Similar profiles have been
demonstrated when ultrapure [3H]BSA (fraction 41
0 10 20 30 40 50 60 70 80 90
FRACTION NO.
Fig. I. Representative profiles of the size exclusion chromatography
on Sephadex G-100 of ['HIBSA in perfusate and urine samples from
isolated perfused kidneys and in vivo. rH]BSA in (a) perfusate (135000
d.p.m., n = 6), (b) 40 to 60 min urine (75000 d.p.m., n = 6) and (c) urine
(69 000 d.p.m.) collected 2 h after a bolus intravenous injection of [)H]BSA
in rats (n = 4). Perfusatewas sampled after 60 min of perfusion. The vertical
line shows the position of the albumin monomer peak. VO represents the
void volume as determined with Blue Dextran T2000 and V, the total
volume as determined with tritiated water. Fractionvolume = I .65 ml.
Fractional clearance of albumin
56 I
1200
1000
9 30000
3
\
kz
800
a
p
I-
p
600
'3
Y
400
C
I
200
25000
20000
15000
10000
n
0
0
2
10 20 30 40 50 60 70 80 90
FRACTION NO.
Fig. 2. Representative profile of the size exclusion chromatography
on Sephadex G-100 of BSA in urine samples from isolated petfused
kidneys as detected by radioimmunoassay (0)(n = 5) and by absorbance at 280 nm (0)
lar-mass material in spite of its presence as detected
by absorption at 280nm. This suggests that the
radioimmunoassay is specific for intact albumin and
does not detect small BSA fragments.
When urine is precipitated, as in the sulphosalicylic acid turbidity assay, only intact albumin and
its large fragments would be detected. This resulted
in a measured fractional clearance from IPK experi(n = 6) which is higher
ments of 9.2f2.4 x
than that obtained for intact albumin as determined
by gel chromatography in the IPK. It is of interest to
note that the TCA-precipitable fraction in urine
from IPKS can only recover 56.0+ 16.6% (n = 5) of
the radioactivity due to the [3H]BSAmolecule [27].
The fractional clearance of [3H]BSA as measured
by total radioactivity in the urine was 7.5 k3.9 x
whereas the fractional clearance as measured
by Biuret assay (detects peptide bonds) of urine
The Lowry assay gave
samples was 6.0k3.3 x
(n = 6 for all assays). The
a value of 1.8 0.7 x
good agreement between the fractional clearance
determined by radioactivity and that determined by
the Biuret assay provides strong support that the
two types of measurements are essentially measuring
the clearance of material derived from albumin
alone. It also demonstrates that the Lowry assay
underestimates fractional clearance, most probably
due to the influence on this assay of contaminants/
conditions of urine samples as noted also by Tojo
and Endou [22].
Fractional clearance in vivo
The fractional clearance of rH]BSA in vivo was
determined by using implanted ALZET osmotic
pumps which release a slow, continuous infusion of
tracer. Figure 3 shows a time course of plasma levels
*
a
ht
5000
o
1
L L I 4 1
2
3
4
u
5
6
7
DAY
Fig. 3. Plasma concentrations of ['HIBSA with continuous infusion
using A U E T osmotic pumps over a period of 7days. Tracer was
released at a rate of I plh.
of rH]BSA which tended to increase slowly until
they plateaued at day 5. Hence fractional clearances
were calculated after this time at day 6. The GFR
was determined by creatinine assay and was found
to be 1.4420.39 ml/min (n = 4) and the UFR was
5.9 k 1.7 x
ml/min (n = 4). The fractional clearance of total [3H]BSA was found to be
(n = 4) and 2.1 k0.6 x
2.3 k0.9 x
(n = 4)
for intact [3H]BSA as shown in Table 1. Since the
d.p.m. present in urine fractions collected from the
osmotic pump experiments was too low to be
analysed by size exclusion chromatography, the fractional clearance for intact [3H]BSA was calculated
by assuming that the degradation profile of urinary
[3H]BSA in osmotic pump experiments was similar
to that obtained from injecting [3H]BSA intravenously.
It is of interest to note that the differences in the
chemical assay results for IPK urine described above
are also reflected in the total protein fractional
clearances measured in viva Measurements of the
fractional clearance of albumin in vivo with the
Biuret assay gave a value of 1.25 k0.39 x
the
Lowry assay gave 0.70k0.16 x
whereas the sulphosalicylic acid turbidity assay gave 0.13 k0.03 x
(this assumes that the plasma protein concentration in the rat is 70 mg/ml) (n = 10 for all assays).
Table 1. Fractional clearance of albumin in vivo (n = 4).
Fractional clearance ( x lo-')
Radioactivity (METpump)
Intact albumin
2.30k0.90
0.21 k0.06
T. M. Osicka et al.
562
Synthesis and excretion of endogenous [3H]lysinelabelled albumin in vivo
The evidence that is presented in this paper and
elsewhere [27, 281 supports the concept that the
normal turnover of rat albumin is, in part, associated with its excretion in the urine as a mixture of
intact and degraded peptides. In an effort to demonstrate this in vivo, we injected [3H]lysine intravenously in order to synthesize labelled albumin and
examine its possible urinary excretion. Figure 4
shows the gel chromatographic profiles of labelled
material in both plasma and urine collected 1 h
post-injection. The plasma profile demonstrates that
within 1 h albumin is synthesized and is detectable
by column chromatography. The peak eluting at
fraction 35 was identified as albumin by radioimmunoassay adapted for the detection of rat serum
albumin (note that this column has slightly different
dimensions to that used in Figs. 1 and 2). The peak
eluting at the void volume of the column may repre-
50000
1-
40000
30000
W
I3
g
20000
10000
5n.
0
v)
z
P
8000
E
I-
z
v,
6000
n
4000
2000
0
0
10 20 30 40
50 60 70 80 90
FRACTION NO.
Fig. 4. Representative profiles of the size exclusion chromatography
on Sephadex G-100 of [3H]lysine-labelled material obtained I h in
vivo after injection in (a) plasma (600000d.p.m.) and (b) urine
(2 X
d.p.m.). The peak eluting at fraction 35 has been identifiedas albumin by radioimmunoassay. VO was fraction 29 and V, was fraction 74. Fraction volume = 1.75 ml.
lo6
sent synthesized immunoglobulins whereas the
material eluting at the total volume is free lysine.
The major feature of the gel chromatographic profile was its marked similarity to the profiles obtained
from filtered [3H]BSA in vivo (Fig. lc) and in the
IPK (Fig. lb). In both experiments, a large amount
of low-molecular-mass macromolecular material was
excreted. In vivo, it is most probably derived from
albumin but we cannot eliminate the possibility of a
contribution from synthesized low-molecular-mass
proteins that exist in plasma.
DISCUSSION
It is commonly stated in the literature that filtered
albumin is taken up by the tubules, degraded in the
lysosomes and the resulting amino acids are
returned to the circulation. Surprisingly, this pathway has never been demonstrated in vivo, particularly in terms of the presence of tubular degradation
products in the circulation [40]. Studies in both the
IPK and in vivo have demonstrated that albumin is
degraded to peptides (not amino acids) that are
excreted in the urine. There is no return of albumin
degradation products to the circulation [27, 281.
These conclusions have been primarily derived from
the use of [3H]albumin which has been shown to be
processed in the same manner as unlabelled albumin [27, 281. Further evidence for this is shown in
Fig. 2 where unlabelled peptides are seen to be
excreted in the IPK. Note also that we have failed to
identify any extrarenal degradation of albumin leading to degradation products present in the plasma
[28] that may be ultimately excreted.
This study demonstrates that the degradation of
albumin has a marked effect on the estimation of
the fractional renal clearance of albumin. The fractional clearance of total [3H]BSA in the IPK as
determined by total radioactivity was found to be
7.5 k3.9 x lop3. This is approximately 20-fold higher
than the fractional clearance of intact [3H]BSA of
3.90 & 1.6 x
as determined by size exclusion
chromatography. This trend is also observed in vivo
using osmotic pumps where the fractional clearance
of total [3H]BSA was 2.3 k0.9 x lop3, and 2.1 f
0.6 x
for intact [3H]BSA. The similarity
between the fractional clearance as determined by
total radioactivity and the Biuret assay in the IPK
demonstrates that the radioactivity is associated with
degraded albumin peptides. Albumin appears to be
degraded in the tubules and this may be associated
with internalization by tubular cells [27, 281. This
may ultimately affect the size of the material
released and excreted as compared with that
retained within the tubular cell. In any case, our
results suggest that any unlabelled peptide material
derived from the labelled material during perfusion
is negligible.
The fractional clearance of [3H]BSA as measured
by the specific analysis of intact albumin in the urine
Fractional clearance of albumin
of the IPK by gel chromatography (3.90+
1.6 x
is comparable to the fractional clearance
of intact albumin in vivo (2.1 10.6 x loM4).Furthermore, it is similar to a previous study by Stolte et al.
[19] who used the Lowry assay (which may underestimate clearance [22, 271) to give a value of
1.50 10.14 x
This demonstrates that the IPK
technique closely mimics the processing of intact
albumin in vivo.
While low-molecular-mass proteins are seen in
fluid from the early proximal tubule obtained
through micropuncture [22], the amount observed
would not account for the ratio of intact to
degraded albumin that has been observed in urine
collected from both the IPK and in vivo [27, 281.
This may be due to the underestimation of albumin
in these samples through mixing/convection in the
tubule where albumin may be reabsorbed before
collection. Degradation may also occur later in the
tubule region. Surface glomeruli used for micropuncture may also not represent a true average of
all the glomeruli associated with filtration.
In an effort to compare the processing of exogenous with endogenous albumin we examined [3H]albumin synthesized in vivo. The chromatographic
profiles of the urine were remarkably similar to
those obtained with exogenous [3H]albumin. It is of
interest too that previous studies on the turnover of
albumin in rats have been unable to account for the
differences in albumin synthesis (as measured by a
specific electrophoresis assay) of 3.6 mg h-l 100 g-'
body wt and catabolism measured as 4.36mg
h-l100g-' body wt [41]. Albuminuria was measured for intact albumin and gave a value of
0.022 mg h-' 100 g-l body wt [41] which would
translate to 2.32 mg h-' 100 g-' body wt if we used
the fractional clearance from radioactivity as measured in vivo (Table l). We would suggest that part
of the difference between albumin synthesis and catabolism could be accounted for by the rapid synthesis and excretion of degraded albumin where the
latter would not be detected by specific (intact)
albumin assays. This would result in an underestimation of the amount of albumin synthesized.
Finally, it is apparent that measuring intact albumin alone in urine will severely underestimate the
albumin associated with glomerular filtration. When
the excretion of degraded albumin is taken into
account the fractional clearance may increase by
20-fold. This is not enough, however, to explain why
albumin has such a highly restricted transport across
the glomerular capillary wall when compared with
its transport in other capillary beds.
ent of the Monash University Postgraduate Writing
up award. We gratefully acknowledge Mrs Lynette
Pratt and Mr Steve Sastra for their expert technical
assistance.
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