Departments of 1 Nephrology and 3 Clinical Chemistry, University Hospital, S-221 85 Lund, Sweden; and 2 Department of Physiology, University of Bergen, N-5509 Bergen, Norway
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ABSTRACT |
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The size and charge-selective properties
of the glomerular barrier are partly controversial. Glomerular sieving
coefficients () for proteins have rarely been determined
noninvasively before in vivo. Therefore,
was assessed vs.
glomerular filtration rate (GFR; 51Cr-EDTA clearance) in
intact rats for radiolabeled myoglobin,
-dimer, neutral horseradish
peroxidase (nHRP), neutral human serum albumin (nHSA), and native
albumin (HSA). To obtain
, glomerular tracer clearance, assessed
from the 7- to 8-min kidney uptake of protein, was divided by the GFR.
The data were fitted with a two-pore model of glomerular permeability,
where the small-pore radius was 37.35 ± 1.11 (SE) Å, and the
"unrestricted pore area over diffusion path length"
(A0/
X) 1.84 ± 0.43 · 106 cm. Although seemingly
horizontal for nHRP and nHSA, the log
vs. GFR curves showed
slightly negative slopes for the proteins investigated in the GFR
interval of 2-4.5 ml/min. Strong negative (linear) correlations
between (log)
and GFR were obtained for myoglobin
(P = 0.002) and HSA (P = 0.006),
whereas they were relatively weak for nHRP and nHSA and nonsignificant
for
-dimer.
for nHSA was markedly higher than that for HSA. In
conclusion, there were no indications of increases in
vs. GFR, as
indicative of concentration polarization, for the proteins investigated
at high GFRs. Furthermore, the glomerular small-pore radius assessed
from endogenous (neutral) protein sieving data was found to be smaller
than previously determined using dextran or Ficoll as test molecules.
glomerular permeability; macromolecules; reflection coefficient; transport
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INTRODUCTION |
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THE GLOMERULAR BARRIER SELECTS molecules based on their size, shape, and charge, and it almost completely prevents large macromolecules from reaching Bowman's space (8). The fenestrated endothelium with its glycocalyx, the glomerular basement membrane (GBM), and the epithelial filtration slits are arranged in series to produce this highly selective sieving filter. There is little agreement as to where the major barrier function is located (8). It has been suggested that the most size-selective portion of the glomerular barrier be represented by the podocyte slit membrane (PSM), especially by a zipper-like arrangement of structures in this membrane (24), conceivably made up (partly) of nephrin molecules (38). Some authors have brought attention to the fact that the most charge-selective barrier may be located close to the plasma compartment, possibly in the endothelial glycocalyx (25), whereas the most size-selective barrier may be more distally located (18). That the charge selectivity may be located in the endothelial glycocalyx has become even more evident after measurements of the charge-barrier properties of isolated GBMs, which were similar for neutral and negatively charged Ficoll molecules (2) or for native (anionic) and cationized albumin (1).
If the PSM were the major sieving barrier of the glomerular filter,
this arrangement would result in concentration polarization of proteins
in the GBM at high glomerular filtration rates (GFRs) (9).
According to the fact that the relative contribution of diffusional
transport decreases with increasing GFRs, high filtration rates will
normally lead to reductions in the glomerular sieving coefficients
() for (small) macromolecules (4, 20, 21, 23, 33). By
contrast, if concentration polarization occurs, then increases in
may instead be expected for the highest filtration rates
(9). However, only very few studies have been performed, particularly in vivo, in which the GFR dependence of
for
macromolecules has been systematically investigated.
In view of the paucity of data on fractional clearances of
macromolecules, especially of proteins, as a function of GFR, we assessed the glomerular for a number of neutral proteins and albumin at normal and high GFRs using a noninvasive technique in intact
rats (35, 36). Measured
values were consistent with a
two-pore model of glomerular permselectivity (23, 33), in
which the small-pore radius was ~37.4 Å, when the
(negatively charged) large-pore radius was set at 110 Å. Whereas the
small-pore radius was smaller than that usually obtained using Ficoll
or dextran as test molecules, measured diffusional small-solute
capacities, i.e., the effective area for diffusion over unit path
length (A0/
X), were largely
consistent with the calculated glomerular filtration coefficient
(LpS). Furthermore,
A0/
X (and
LpS) remained stable as a function of GFR. In
addition, there were no indications of concentration polarization
(increases in
) occurring at the highest filtration rates.
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METHODS |
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Experiments were performed in 85 male Wistar rats (Møllegaard, Stensved, Denmark) weighing 270 ± 8 (SE) g. The rats were kept on standard chow and had free access to water before the start of the experiments. Experiments were approved by the Animal Ethics Committee at Lund University.
Anesthesia was induced using pentobarbital sodium (50 mg/kg ip), and a thermostatically controlled heating pad maintained the body temperature at 37°C. A tracheotomy was performed to ensure free airways. The tail artery was cannulated for recording the arterial pressure (PA) and for subsequent administration of drugs. The right jugular artery and the left jugular vein were cannulated for infusion and sampling purposes. Via an abdominal incision, a catheter was placed in the urinary bladder for continuous urine sampling, the abdominal insertion being sealed with Histoacryl (Melsungen, Germany).
Tissue uptake technique.
The technique has been described in detail and validated by Tenstad et
al. (35, 36). When a tracer protein is added to the plasma
compartment, it will mix with the plasma, dissipate within the
extracellular space, and filter across the glomerular barrier. After
appearing in Bowman's space, it will be reabsorbed, more or less
completely, by the renal proximal tubules to be processed by the
tubular cells. During the first 7-9 min of protein reabsorption, the breakdown of the protein and the subsequent reabsorption to the
plasma of split products will be negligible, whereas a tiny fraction of
the tracer will appear in the urine. This is the principle utilized in
the present experiments. Glomerular protein clearance was assessed as
the timed total (cortical) kidney uptake plus the (precipitable) urine
excretion of protein tracer divided by the average plasma tracer
protein concentration. Was calculated from the protein clearance
divided by GFR, determined by the simultaneous assessment of the
plasma-to-urine clearance of 51Cr-EDTA.
Tracers and labeling procedures.
The protein probes were labeled with 125I by using
1,3,4,6-tetrachloro-3,6
-diphenylglycouril (Iodo-Gen)
(10). Briefly, 0.1 mg Iodo-Gen (T0656, Sigma) dissolved in
0.1 ml chloroform was dispersed in a 1.8-ml Nunc vial (Nunc-Kamstrup,
Roskilde, Denmark). A film of the virtually water-insoluble Iodo-Gen
was formed in the Nunc vial by allowing the chloroform to evaporate to
dryness under nitrogen. Then, 1 ml 0.05 M PBS solution, pH 7.5, containing 1-2 mg protein to be labeled, 5 MBq 125I
(Institute for Energy Technique, Kjeller, Norway), and 15 µl 0.01 M
NaI were added, and the iodinating tube was gently agitated for 10 min
before the reaction was terminated by removing the solution from the
Iodo-Gen tube. Unincorporated iodine isotope accounting for <10% of
the total radioactivity, as estimated by TCA precipitation, was removed
by dialyzing the tracer against 1,000 ml 0.9% saline containing 0.02%
azide. The stock solution was stored in the dark at 4°C and dialyzed
for at least 24 h before use.
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Neutralization of HSA.
nHSA was obtained by a graded modification of the COOH groups using a
procedure modified from that described by Hoare and Koshland
(11) as follows. HSA (1.5 g) was dissolved in 15 ml 0.133 M glycine methyl ester at pH 4.75 (at room temperature). A solution of
5 ml of 0.04 M N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was then added to the mixture to
initiate the reaction. The pH was continuously recorded and kept at
4.75 by addition of 0.1 M NaOH. Aliquots (1 ml) were removed every 5 min for 60 min and immediately added to 1 ml of 4.0 M acetate buffer at
pH 4.75 to quench the reaction. After being kept for a few minutes at
room temperature, these solutions were dialyzed overnight against two
changes of 10 liters of distilled water, and the dialysate was
freeze-dried and stored at 20°C. The effect of the reaction was
evaluated by isoelectric focusing using a vertical minigel system (CBS
Scientific) and Novex (Novel Experimental Technology, San Diego, CA)
precast gels. It turned out that a 45-min reaction time produced
albumin with an average isoelectric point close to 7.4 without any
significant change in hydrodynamic radius, as measured by HPLC
(Superdex 75 HR and Superose 12 HR).
Calculations.
Renal tracer protein clearance was calculated from the amount of tracer
radioactivity accumulated in both kidneys plus the TCA-precipitable
urine tracer activity (collected during the tracer infusion period)
divided by the average venous plasma tracer concentration and by the
tracer infusion time until death. In washout experiments, clearance was
assessed by the amount of tracer in kidneys plus urine divided by the
area under the curve of the plasma tracer concentration vs. time
function. Protein values were calculated by dividing the measured
protein clearance by the simultaneously assessed GFR.
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
Statistics. Values are given as means ± SE. Differences among groups were detected using ANOVA. Calculating the variance-covariance matrix (see APPENDIX A) assessed the SE of the fitted parameters.
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RESULTS |
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In Fig. 1, fractional clearances
(uncorrected values or "
raw") for the neutral
proteins investigated and for native albumin are plotted vs. GFR in a
semilogarithmic diagram. In the GFR interval of 2.0-4.5 ml/min,
correlated negatively with GFR for all proteins investigated,
except for the
-dimer (see below). Values for
corrected
according to Eq. 1 are plotted vs. GFR in Fig.
2, and, furthermore, the best fitting
two-pore parameters were here fitted to the data. According to the
two-pore model, rs was calculated to be
37.35 ± 1.11 (SE) Å (for uncorrected data we obtained 37.55 ± 0.75 Å) when rL was fixed at 110 Å.
Furthermore, A0/
X was 1.84 · 106 ± 4.29 · 105 cm (2.40 · 106 ± 5.19 · 105 cm for uncorrected data), and
l was 4.86 · 10
4 ± 2.34 · 10
4 (4.61 · 10
4 ± 6.25 · 10
5 for uncorrected data). Average data, corrected and
uncorrected (or raw), for
(at an average GFR of ~3 ml/min) and
for myoglobin,
-dimer, neutral HRP, and neutral and negative
albumin are shown in Table 2.
LpS calculated from
A0/
X (corrected data) was 0.36 ml · min
1 · mmHg
1
(both kidneys). For a GFR of 3 ml/min, with the assumption of a net
transglomerular pressure gradient on the order of 10 mmHg in our
experimental animals, LpS can be estimated to be
0.3 ml · min
1 · mmHg
1.
Thus the A0/
X calculated from
sieving data for small and intermediate size solutes was largely
consistent with the filtration coefficient of the glomerular filtration
barrier. Furthermore, these two entities could be set constant (and
independent of GFR) in all simulations.
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Figures 1 and 2 indicate the presence of a negative dependence of on GFR. By applying a simple linear regression analysis of log
vs.
GFR, we obtained a highly significant negative correlation for
myoglobin (P = 0.002 for corrected
), HSA
(P = 0.006), and nHSA (P = 0.01), but a
barely significant one for nHRP (P = 0.04), whereas it
was nonsignificant for the
-dimer. The regression coefficients with
their 95% confidence intervals (for corrected and raw data) are listed
in Table 3. The
-GFR relationships are
largely consistent with models in which diffusion and convection occur
simultaneously across a size (and/or charge)-selective barrier. Thus
for small proteins, the reduction in the diffusional component of
transport with increasing filtration rates will cause reductions in
. However, for large proteins, and at high filtration rates, the
impact of diffusion is small. This results in an essentially flat
vs. GFR curve, where
approximates (1
) at high GFRs. Note that for solutes with radii larger than the small-pore radius (37.35 Å), one would, according to the two-pore model, also expect a
dependency of
on GFR. In a heteroporous model, this phenomenon results from the fact that the fractional large-pore volume flow (Jv, L/GFR) will asymptotically fall
with increases in GFR to approach
L at high GFRs. This
behavior is expected for HSA, because the presence of charge
interactions may completely prevent HSA from entering the small pores,
whereas nHSA may filter through both small and large pores. Note also
that nHSA transport was one order of magnitude higher than that of
native (negatively charged) albumin. Finally, for all proteins,
including the two largest investigated (nHSA and HSA), there were no
indications of concentration polarization occurring at any filtration
rates.
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Figure 3 illustrates (log) vs. both
GFR and solute radius in a three-dimensional diagram simulated using
the present two-pore parameters (for corrected data). Here, it is again
evident that, for solutes with radii <15 Å ,
is unity and
completely independent of GFR, whereas
for solutes with radii
ranging between 15 and 30 Å
is dependent on GFR. In the GFR
interval of 2-5 ml/min, however, solutes with radii of 30-37
Å exhibit rather stable
values, which are close to their (1
) values. For solutes with radii larger than the small-pore
radius, there is again a dependency of
on GFR, determined by the
JV, L/GFR ratio, as mentioned above.
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DISCUSSION |
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The essential result of this study is that the fractional
clearances () of small endogenous neutral proteins and of albumin in
rats, assessed as a function of GFR in vivo, slightly declined with
increases in GFR in the GFR interval of 2.0-4.5 ml/min. This was
particularly evident for myoglobin. The results essentially agree with
dextran sieving data obtained during isoncotic volume expansion,
performed to increase GFR, in Munich-Wistar rats (4). With
increases in GFR, dextrans with radii of 20-38 Å clearly decreased their
values. In the present study, there was no evidence for concentration polarization (increasing
values) occurring at
high GFRs, either for the small proteins investigated or for neutral or
native albumin. Furthermore, the
values for small proteins and
albumin were lower than previously obtained using Ficoll or dextran of
equal hydrodynamic radii as probes for testing glomerular
permselectivity. Also, there was a marked charge dependency of
glomerular transport, as evidenced by the large difference in
for
neutralized vs. negatively charged albumin.
There have been very few previous analyses of the dependence of protein
values on GFR, at least in vivo. In the isolated perfused rat
kidney (IPK) at 8°C, however, there is one recent set of
measurements of this kind for "asymmetrical" proteins (19). Although in the IPK the GFR (per kidney) was only
20% of those obtained at 37°C in the intact rat in vivo, the
observed
for the most permeable proteins investigated (hyaluronan
and bikunin) showed a similar asymptotic reduction as a function of GFR, as found in the present study. This is indeed the expected behavior of
when the diffusional component of transport is high, because this component will theoretically decrease asymptotically with
increases in GFR according to nonlinear transport formalism (20). Only at high GFRs, the impact of the diffusional
component will become negligible, so that the protein
will equal
(1
) (7, 8, 20, 21, 23, 33). Contrary to
results from the IPK (19), there were no indications of
increases in microvascular permeability occurring for albumin at high
intraglomerular hydrostatic pressures in the present study. A tentative
explanation could be that the IPK, although partly protected from
inflammatory mediators or ischemia-reperfusion injury by the
temperature reduction, might be more vulnerable to high intraluminal
pressures than is the intact kidney under in vivo conditions.
One important consequence of the presence of a large diffusional
component of small protein transport across the glomerular filter,
i.e., a large A0/X for the renal
microcirculation, is that assessments of
for small proteins must be
standardized to rather narrow GFR ranges to be compared between
different experimental conditions. For example, if an
ischemia-reperfusion insult per se results in a fall in GFR,
then by necessity
for a small protein, such as myoglobin or
2-microglobulin, must increase, even if the glomerular
permeability is unaffected. On the other hand, if GFR is increased but
the permeability is unchanged, then
for a protein will fall. For
the largest neutral molecules investigated in the present study
(
-dimer, nHRP, and nHSA), however, the diffusional contribution to
was expected to be low in the whole GFR range investigated. Indeed,
was independent of GFR changes for the
-dimer and nearly so for
nHRP. Unexpectedly, there was, however, a slight decline of the log
vs. GFR relationship for nHSA.
Theoretically, of a magnitude measured for HRP and nHSA would
remain stable as a function of GFR even at high filtration rates, if
the major barrier to solute sieving were close to the blood side of the
membrane. However, if the major barrier function were instead located
close to Bowman's space, e.g., at the PSM, then one would expect at
least some degree of concentration polarization to occur at high
filtration rates. According to a recent modeling study of the sieving
behavior of the glomerular capillary wall (9), it was
assumed that the glomerular barrier exhibited three transport
resistances arranged in series, with a major portion of the overall
transport resistance to macromolecules present at the level of the PSM.
Furthermore, it was assumed that the resistance of the transport of
large solutes was low (negligible) at the fenestrae. Under such
assumptions, a rise in single-nephron GFR from 40-45 to ~80
nl/min, corresponding to a rise in whole rat (300 g) GFR from ~2.5 to
5 ml/min, caused a significant rise in
for (neutral) solutes having
a
similar to that of albumin (nHSA and HSA) in the present study.
However, because we were not able to detect any signs of concentration
polarization occurring in the GFR interval of 2-4.5 ml/min, we are
inclined to conclude that the case for a major sieving barrier located
at the PSM is rather weak. In case the slit membrane would still be the
major filtration barrier, the present data indicate that serial
barriers proximal to the PSM must be very highly permeable to
macromolecules to prevent the buildup of concentration polarization
layers at the PSM.
The present study essentially confirms and extends previous
measurements of using micropuncture techniques. Micropuncture techniques have been criticized, because they imply exposure of and
mechanical interactions with an intact kidney. Furthermore, proteins
sampled from the tubules may bind to the glass pipette, and
interstitial proteins may leak into the tubules during the micropuncture. Moreover, because the tubular micropuncture procedure has to be performed at sites distally to Bowman's capsule, primary urine cannot be directly assessed (15). Indeed, tubular
protein concentration falls along the distance of the proximal tubule, because protein reabsorption is usually more avid than that of water.
In an attempt to avoid all these sources of error, Tojo and Endou
(37) used a double-barrel pipette technique, which made it
possible to seal the punctured (rat) proximal tubule from the
interstitium. Furthermore, they assessed the tubular concentration of
protein together with that of a filtration marker (inulin) at various
distances from Bowman's capsule (37). With this
technique, they were able to quite precisely estimate the urinary
albumin protein concentration of Bowman's capsule by an extrapolation procedure. Using this careful technique, they estimated the
value
for native albumin to be 6.2 · 10
4, which is almost identical to that assessed by the
present technique in vivo (6.6 · 10
4). Also, our assessments of
values for myoglobin,
dimeric
-chain (Bence Jones proteins), and nHRP are remarkably close
to estimates previously obtained using micropuncture techniques
(14).
All measured values of for neutral proteins in the present study
are much lower than the corresponding
values previously obtained
for neutral Ficoll, which, in turn, are much lower than
values for
dextran (15). The marked discrepancy between glomerular protein sieving data and glomerular dextran sieving data was discussed at some length in the classic review by Renkin and Gilmore
(21). It may be due to the fact that dextrans are flexible
molecules, and thereby hyperpermeable in vivo, so that they may
actually transmigrate through pores, which are even smaller than their Stokes-Einstein radii, sometimes denoted "reptation"
(17). Moreover, recent data indicate that the more ideal
Ficoll molecule, a copolymer of epichlorhydrine and sucrose, may not
behave in all aspects as an ideal ridgid sphere (12, 27),
but we will return to this issue in a forthcoming publication. At any
rate, the permselectivity of the glomerular barrier, in terms of the
small-pore radius, for example, seems to be dependent on the physical
properties of the probe used for testing permeability. Using neutral
dextran as a probe, the average glomerular rs
has been determined to be on the order of 50-55 Å (15). Using Ficoll at normal ionic strength, rs has been determined to be on the order of 45 Å (18, 19), whereas at low ionic strengths
rs was only 41 Å (30). This
value is similar to the rather low rs estimate
of the present study and to earlier estimates using proteins for
probing glomerular permeability (21).
It has been well established since the 1970s and 1980s that the
glomerular filter discriminates among macromolecules based on both
their net charge as well as their size (15). Much of the
evidence in favor of charge selectivity of the glomerular filter has
been based on comparisons between sieving data for uncharged and
anionic dextran (dextran sulfate) (3). Although vivid
arguments against glomerular charge selectivity have been raised during
the last decade (5, 26, 32, 42), strong evidence
supporting the classic view was recently given by comparing neutral and
anionic lactate dehydrogenase or neutral and anionic HRP in the IPK
(13, 31). These studies largely confirm the classic
studies by Rennke et al. (22) for differently charged HRP
(22). The present data are entirely consistent with the glomerular filter as a charge-selective barrier, producing a near 10-fold difference in for neutralized vs. negatively charged (native) albumin.
The present tissue uptake technique has been validated for small
proteins in previous publications (35, 36). For proteins with very low renal clearances, such as albumin, it is crucial that the
kidneys are completely washed free of intravascular tracer and that the
bulk of interstitially accumulated tracer (and free iodine) is to a
large extent cleared by back-diffusion to the rinse fluid. The washout
procedure is thus crucial to the success of the technique. Even though
we consider the washout to have been more or less complete, we cannot
completely rule out that some tracer remained either intravascularly or
extracellularly after tracer washout. From that point of view, the
present for native albumin of 6.6 · 10
4 may represent an overestimate. Still, the value
obtained is in agreement with the recent careful micropuncture study by
Tojo and Endou (37) referred to above. Therefore, we feel
confident that the degree of overestimation of
for native albumin
was, after all, rather moderate.
In conclusion, there was a dependence of glomerular small-protein on GFR for neutral molecules with molecular radii ranging between 15 and 30 Å and also for native albumin. The data were readily fitted to
a two-pore model of glomerular permeability where
rs was found to be ~37-38 Å. Neither for
small proteins nor for albumin was there any evidence for concentration
polarization present at high GFRs. Furthermore, the glomerular filter
showed properties of a negative charge barrier. Taken together, the
present in vivo data may be interpreted to indicate that the
endothelial glycocalyx-filled fenestrae are playing a greater role than
previously thought, and the epithelial slit diaphragms a lesser role,
in determining the sieving properties of the glomerular filtration barrier.
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APPENDIX A |
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The fractional clearance data () were fitted to a two-pore
model of membrane permeability (23, 34) using a weighted
nonlinear least squares regression analysis. In detail, the function to be minimized was
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(A1) |
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(A2) |
The estimated parameters were rs,
A0/X, and the fractional
LpS accounted for by the large pores
(
L). However, because the numerical values of these
parameters differ by several orders of magnitude (9 from
A0/
X to
L), a set
of scaling multipliers was introduced, so that the minimization
algorithm had to deal with parameters near to unity.
SE of the fitted parameters was assessed by calculating the variance-covariance matrix according to the method described by Smith et al. (29).
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ACKNOWLEDGEMENTS |
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We are grateful to Kerstin Wihlborg for skillful typing and editing of the manuscript. The expert technical assistance by Veronica Lindström (Dept. of Clinical Chemistry, University Hospital, Lund, Sweden) is acknowledged.
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FOOTNOTES |
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This study was supported by Swedish Medical Research Council Grant 08285 and by European Union Contract FMRX-CT98-0219.
This study has been published in abstract form (J Am Soc Nephrol 12: 503A, 2001).
Address for reprint requests and other correspondence: B. Rippe, Dept. of Nephrology, Univ. Hospital of Lund, S-211 85 Lund, Sweden (E-mail: Bengt.Rippe{at}njur.lu.se).
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.
First published March 4, 2003;10.1152/ajprenal.00316.2002
Received 3 September 2002; accepted in final form 22 February 2003.
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