Division of 1Nephrology, 2Children's Hospital, Hannover Medical School, D-30623 Hannover; 4Children's Hospital, Bonn University, D-53113 Bonn, Germany; and Departments of 3Physiology and 5Nephrology, Göteborg University, SE-405 30 Göteborg, Sweden
Submitted 20 July 2001 ; accepted in final form 14 June 2003
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ABSTRACT |
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glomerular permeability; charge selectivity
The transport of solutes across microvascular walls can be described by a
two-pore theory of capillary permeability
(30). Measurements of
steady-state sieving coefficients () of proteins from plasma to
interstitium or lymph are used to predict the pore radius and distribution. In
glomerular capillaries, the presence of tubular reabsorption and secretion
processes that modify final urinary composition is a formidable obstacle to
the determination of sieving coefficients for proteins and, consequently, to
the study of glomerular permselectivity. Experimental in vitro systems such as
the isolated nephron or the isolated perfused glomerulus are free from the
influence of tubular transport processes
(26,
37). These systems, however,
are less suitable for studies of macromolecular transport due to the small
amounts of solute filtrated in minute volumes. With a normal albumin fraction
of only a few tenths of a percent, the measurements are less accurate than for
smaller solutes.
Oliver et al. (27) found that Ficolls (globular uncharged cross-linked copolymer of sucrose and epichlorohydrin that is neither secreted nor reabsorbed by the renal tubules) of various radii had a lower fractional clearance than dextrans of equal Stokes-Einstein radius (aSE). This implies that Ficoll may be a reliable transport probe for the measurement of small and large pore radii.
Fractional clearance experiments of charged dextrans substantiated the hypothesis of a charge-dependent glomerular filtration of macromolecules (4). On the basis of fractional clearance data of dextran in the rat, in a now classic work Deen et al. (10) calculated an apparent fixed charge concentration on the glomerular capillary wall (GCW) of 120170 meq/l. However, when using charged dextran in permselectivity studies, uptake and desulphation of dextran sulfate by the glomerular and tubular cells (6, 42, 43) and the ability of certain dextran sulfates to bind to plasma proteins (13) complicate the interpretation of the results.
We developed a modified isolated rat kidney model in which the tubular reabsorption processes were eliminated by glutaraldehyde fixation (5). Gluteraldehyde is a fixative that acts rapidly and offers accurate tissue preservation. There is evidence that in the isolated kidneys fixed by perfusion with glutaraldehyde, no ultrastructural alteration of the GCW is detectable; an intact organization of the glomerular cells and an unaltered distribution of glomerular polyanions were reported (36, 37). In one of the most detailed descriptions of the fine structure of isolated kidneys after perfusion fixation with glutaraldehyde, Kriz et al. (19) showed that the integrity of the barrier is remarkably preserved. No cell lysis is noticed, and the structure of the glomerular capillaries is undistorted (11, 12), whereas the metabolic processes are eliminated. With the use of this model, the glomerular permeability properties can be directly studied, without interferences of the tubular apparatus and without influence of hemodynamic factors and blood constituents such as hormones. The charge of the GCW was determined (5) using albumin solutions buffered at different pHs spanning the isoelectric points of albumin and of the glomerular basal membrane.
In the present work, the fractional clearance of FITC-Ficoll was measured to determine the glomerular size and charge selectivity in the perfusion-fixed, isolated rat kidneys. Previous studies suggest low ionic strength (I) to reversibly reduce the glomerular charge density, most likely due to volume expansion of the compartment responsible for charge selectivity.
Therefore, it was of particular interest to estimate the charge density at different Is of the perfusate. Our hypothesis was that lowering I would not induce dynamic alterations of the estimated charge density in the fixed kidneys if the glomerular charge selectivity were to reside in the basement membrane and/or in the podocyte slit membrane. On the other hand, marked changes in charge density could be expected in the fixed kidneys if glomerular charge selectivity were dependent on the endothelial cell coat barrier, which is more resistant to glutaraldehyde (31, 35).
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MATERIALS AND METHODS |
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Experiments were performed on male Sprague-Dawley rats weighing 200300 g. The animals had free access to food (standardized pellets, Altromin, Altromin Gesellschaft für Tierernährung mbH Lage, Germany) and tap water until the experiment. The rats were anesthetized intraperitoneally with 100 mg/kg body wt thiopental-sodium (Trapanal, Byk-Gulden, Konstanz, Germany). The local ethics committee approved the experiments.
Kidney Isolation
Surgery. The rats were placed on a temperature-regulated table. The surgical procedure was a modification of that reported by Weiss et al. (40) and by Nishiitsutsuji-Uwo et al. (22). The right kidney was always used for perfusion. As ureter catheters, we used short (10 mm) polypropylene catheters (PP-10, Portex, Hythe, UK) connected to larger polyethylene catheters (PE-50, Portex), thereby preventing a buildup of ureteral backpressure. After heparin injection (Liquemin, Hoffmann-LaRoche Grenzach-Wyhlen, Germany), the kidney was placed in a temperature-controlled metal chamber. Before the perfusion was started, the aorta was clamped distal to the right renal artery, and a double-barreled cannula was inserted into the abdominal aorta distal to the clamp. Perfusion was started in situ by opening the clamp and tying the proximal aortic ligature. Thus zero perfusion of the experimental kidney never occurred.
Perfusion apparatus and technique. The apparatus was designed as a recirculation system with dialysis because of a higher stability. The perfusion technique and apparatus have been previously described in detail (33, 34). Experiments were performed using a substrate-enriched Krebs-Henseleit bicarbonate solution containing 50 g/l BSA (Fraction V, Sigma, Deisenhofen, Germany) (34). Verapamil (Isoptin, Knoll, Minden, Germany) at a dose of 4.4 µmol/l was added to the perfusion medium. The effective perfusion pressure was 100 mmHg.
Kidney Fixation
After isolation, the kidney was perfusion-fixed with a 1.25% monomeric glutaraldehyde (Polyscience, Warrington, PA) solution in 0.1 M phosphate buffers (final pH 7.2). The fixation solution was made isooncotic to plasma by addition of hydroxyethyl starch (Plasmasteril, Fresenius, Bad Homburg, Germany) to a final concentration of 60 g/l. In previous experiments, it had been observed that the perfusion resistance was increasing dramatically when the perfusate was colloid free. For fixation, the kidney was perfused for 6 to 8 min at a pressure of 150 mmHg.
Reperfusion of the Fixed Kidney
Before reperfusion experiments were started, the fixed kidney was washed free from glutaraldehyde by a 60-min single-pass perfusion with 0.9% saline at a pressure of 100 mmHg. This step was necessary to avoid the formation of protein-glutaraldehyde aggregates, which can significantly reduce the perfusion flow rate in protein perfusion experiments (not shown).
Every solution used in perfusion experiments of the fixed kidney contained 100 mg/l polyfructosan. Experiments were performed at a perfusion pressure of 100 mmHg.
Study Design
The fixed kidneys were perfused successively with phosphate-buffered (136.9 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.9 mM CaCl2, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) solutions containing 10 g/l BSA and 70 mg/l FITC-Ficoll (Ficoll-70, Bioflor, Uppsala, Sweden) containing Ficoll molecules of different size. The I of the "normal" perfusate was 151 mM. The low I perfusates (I = 34 mM) contained the same concentrations of BSA and Ficoll, respectively, but otherwise had the following composition: 26 mM Na, 4.3 mM K, 2.5 mM Ca, 8.4 mM Cl, 0.8 mM Mg, 25 mM HCO3, 0.5 mM H2PO4, 5.6 mM glucose, and 241 mM mannitol.
Electrolytes
Na+ and K+ concentrations in perfusate and urinary samples were determined with an ion-selective electrode analyzer (System E2A electrolyte analyzer, Beckman, Brea, CA).
Analysis of Ficoll Sieving
For calculating the sieving coefficients for FITC-Ficoll, all perfusate and urine samples were subjected to gel filtration (BioSep-SEC-S3000, Phenomenex, Torrance, CA) and detection of fluorescence (RF 1002 Fluorescence HPLC Monitor, Gynkotek, Germering, Germany) using Chromeleon (Gynkotek) software. As eluent, we used a 0.05 M phosphate buffer with 0.15 M NaCl with pH 7.0. From each sample, a volume of 510 µl was analyzed at an emission wavelength of 520 nm and an excitation wavelength of 492 nm; during analysis flow rate (1 ml/min), sampling frequency (1 per second), pressure (4 MPa), and temperature (8°C) were maintained constant. We estimated the error in the CU/CP ratios for Ficoll to be <1% for most molecular sizes.
Other Analytic Methods
Total protein was determined using the Bradford method (3). Inulin was measured after acid hydrolysis by the hexokinase/glucose-6-phosphate dehydrogenase method (32) by including a phosphohexose isomerase reaction into the assay.
Calculations
Glomerular filtration rate. The glomerular filtration rate (GFR)
of the isolated and of the fixed kidney was determined by measuring inulin
(polyfructosan) clearance. For calculating the GFR, we used the following
formula
![]() | (1) |
Cp is the concentration of inulin in plasma, and Cu represents its concentration in urine; Qu is urine flow rate.
Fractional clearances of albumin and Ficoll, . The
fractional clearance
for solute X was calculated as
![]() | (2) |
Models of Glomerular Size and Charge Selectivity
We used two different theoretical models for analysis of glomerular size and charge selectivity, namely the gel-membrane model (24) and a charged fiber model (16) with small discontinuities with extremely low concentrations of fibers (large pores).
Gel-Membrane Model
The gel-membrane model (24)
assumes the glomerular barrier to be composed of two separate compartments in
series: one charge selective (gel) and one size selective (membrane). The gel
contains fixed negative charges, and the concentration of an anionic molecule
such as albumin will be lower in the gel than in the plasma. The second
compartment of the barrier behaves as a membrane exerting size discrimination
but no charge selectivity. Thus, in this model, the effects of size and charge
are treated in two different compartments, which greatly facilitate the
calculations but naturally represent a gross oversimplification, because the
sieving coefficient for a certain solute is given by the product of
for each individual component of a serial barrier
(8). Furthermore, it can be
argued that the limitations of the model affect the results leading to
erroneous conclusions. However, if we consider the "gel" to be a
part of the plasma compartment rather than the barrier, the model may still be
valid.
Charge selectivity is estimated from the for albumin and its
neutral counterpart of similar size, Ficoll35.5Å, giving a
density of fixed charges,
(see Ref.
24). The fractional clearance
for Ficolls of Stokes-Einstein radii between 30 and 70 Å (180 data
pairs) allows estimates of size selectivity using a two-pore model, which has
the following four parameters: the functional small pore radius
(rS), the large pore radius (rL), the large pore
fraction of the hydraulic conductance (fL), and the unrestricted
exchange area over diffusion distance (A0/
x). For more
details, please consult Ref.
24.
Charged Fiber Model with Discontinuities of Low Fiber Density
To combine size and charge selectivity in one model is highly complicated, but Johnson and Deen (16) extended the partitioning theory of Ogston (23) to develop a charged fiber model to predict the concentration ratio of a solute at equilibrium in and outside a gel. The endothelial surface layer (glycocalyx) and the glomerular basement membrane are examples of such more or less charged gels. We previously used the model in a quantitative analysis of charge selectivity (38), but the present analysis differs in two important aspects. First, the present analysis takes into account that there may be heterogeneous fiber densities with regions with low fiber concentrations, i.e., large pores. Second, due corrections are made for the diffusivity in a gel (29).
The gel/plasma concentration ratio at equilibrium in a fiber matrix is
described by the partition coefficient ()
![]() | (3) |
![]() | (4) |
![]() | (5) |
Johnson and Deen (16)
introduced a Boltzmann factor to describe the relative probability at
different energy states in charged gels. Multiplying g(h) by this factor gives
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Interactions between solute and fiber cause changes in the free
electrochemical energy
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The energy (E) needed in Eq. 6 is given by
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To apply the partition coefficients to experimental data, one must
calculate fractional clearances (). In the concept of "fiber
matrix," Curry and Michel
(7) used the expression of
Anderson and Malone (2) to
calculate the reflection coefficient (
) from the partition coefficient
(
)
![]() | (9) |
Note that experimental observations in agarose gels give reflection coefficients that differ somewhat from those of Eq. 9 (17). There are, however, limited experimental studies of reflection coefficients in biological gels, and, to our knowledge, this is the best equation available.
Finally, the diffusion capacity (PS) is given by
![]() | (10) |
![]() | (11) |
The fractional clearance () is obtained using a nonlinear flux
equation (30)
![]() | (12) |
In a previous study (38),
we noted that the charged fiber model did not adequately describe the effects
of changing I. However, introducing large pores improves the precision
significantly. Moreover, introducing a heterogeneous fiber network with small
regions with low fiber concentrations (1/20th of the average) further improves
the agreement between theory and experimental data. Thus the total fractional
clearance for a solute is the sum of the through the main gel
(
main gel) and that occurring through the large pore
discontinuities (
L). The large pores represent a small
fraction (fL) of the total hydraulic conductance and an even
smaller fraction (f 2L) of the exchange area
(A0/
x). Hence,
L is calculated as for
main gel except for the fact that
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![]() | (13) |
The important parameters in the model are: the fiber radius
(rf), the relative concentration of fibers in the gel (), the
surface charge densities of solute (qs) and fiber (qf),
the unrestricted exchange area over diffusion distance
(A0/
x), the large pore fraction of the hydraulic conductance
(fL), and the dilution factor for the fiber density in the large
pores. Some of these parameters were constant (rf, qs
for albumin and Ficoll, the large pore dilution factor), whereas others were
modified (A0/
x,
, qf, fL) to
achieve a good fit between experimental and theoretical data.
Curve-Fitting Procedures
In the present study, the fractional clearances for Ficolls of different
molecular sizes were modeled using Mathcad 2001i (MathSoft Engineering &
Education, Cambridge, MA). First, different values for A0/x,
, qf, fL were tested to achieve acceptable fitting
between modeled and experimental data at normal I (151 mM). Second, the same
parameter values were used to calculate the fractional clearance for Ficoll at
low I, 34 mM. This resulted, however, in poor fitting between experimental and
modeled data particularly for the smaller solutes. Finally, the concentration
of fibers was gradually reduced by low I until acceptable agreement was
obtained between the experimentally determined fractional clearance for Ficoll
and the values obtained by the heterogeneous charged fiber model. Details of
the calculations are given in a separate PDF file available at the Journal
website
(http://ajprenal.physiology.org/cgi/content/full/00227.2001/DC1).
Statistics
Data are presented as means ± SE or with 95% confidence intervals
(CIs). For the two-pore model parameters and for albumin,
the statistical analysis was based on the logarithmic values, due to the
skewed distribution of data. Differences were tested using Student's
t-test paired design.
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RESULTS |
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The urine concentrations of sodium and potassium were equal to those in perfusate (data not shown). The inulin concentration ratio between perfusate and urine was 1.01 ± 0.02 (n = 10), i.e., not significantly different from unity.
GFR and Renal Perfusate Flow
The values for the GFRs and the renal perfusate flow (average ± SE) are reported in Table 1.
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Fractional Clearance of BSA and Ficoll35.5Å
At normal I, the sieving coefficient, , for albumin was 0.0049 (SE
0.0017, +0.0027, n = 6), i.e., about 1/20th of that for
neutral Ficoll of similar size (aSE = 35.5 Å) 0. 104
(SE = 0.010, n = 5, P < 0.001). At low I perfusion,
for albumin was 0.0030 (SE 0.0011, +0.0018, n = 6),
not significant compared with that at normal I.
For
Ficoll35.5Å was 0.104 (SE 0.015, n = 6, not
significant compared with normal I). Thus
for
Ficoll35.5Å was significantly higher than
for albumin
at low I as well (P < 0.01). For details, see
Fig. 1.
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Figure 2 shows the sieving coefficients obtained in individual reperfusion experiments of the fixed kidney for BSA compared with that of Ficoll of aSE 35.5 Å. All data fall to the right of the line of identity indicating restriction of the anionic albumin compared with the neutral Ficoll of similar hydrodynamic size, i.e., a significant glomerular charge barrier is evident. Figure 3 illustrates the U/P concentration ratios for Ficoll of various molecular radii.
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Gel-Membrane Model Analysis
The functional small pore radius was 36 Å (2744 meq/l, 95% CI)
at normal I and 33 Å (2343 meq/l, 95% CI) at low I. The large
pore radius was 137 ± 8 and 197 ± 30 Å for normal and low
I, respectively. The large pore fraction of the total hydraulic conductance
was 2% for normal and 3% for low I. The glomerular charge density, ,
was estimated to be 38 meq/l (2871 meq/l, 95% CI) for normal I and 13
meq/l (1116 meq/l, 95% CI) during perfusion with low I perfusate,
suggesting a threefold increase in volume of the "charged gel"
(Fig. 4).
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Heterogeneous Charged Fiber Model
In this model, the unrestricted exchange area over diffusion distance,
A0/x, was 100,000 cm. The fiber radius was 4.5
, the relative fiber volume,
, was 5.6%
and the fiber surface charge density was 0.3 C/m compared with
0.022 C/m for albumin (Table
2). The gel was heterogeneous with discontinuities with 1/20th of
the fiber density accounting for 8.5% of the hydraulic conductivity or 0.72%
of the total area. With these parameters, there was an acceptable fit between
the 180 Ficoll data pairs (U/P ratio vs. Stokes-Einstein radius) obtained at
normal I and the modeled values. As the I was reduced, however, the modeled
values deviated from the measured data and more so for smaller solutes, i.e.,
higher U/P ratios. Figure 5 shows a Blandt-Altman plot demonstrating the deviations between measured and
modeled U/P ratios. To achieve a better fit at low I, a twofold (+78%)
expansion of the gel was assumed, reducing the fiber density to 3.15%
(Fig. 5).
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In contrast to the Ficoll data, the U/P ratios for albumin were not
adequately described by the heterogeneous charged fiber model, which
overestimated for albumin six to eight times. Possible explanations
are presented in DISCUSSION.
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DISCUSSION |
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Our hypothesis was that if cellular structures and/or the collagen IV-rich glomerular basement membrane were responsible for charge selectivity, then fixation would abolish dynamic changes of the charge density in response to alterations of I. On the other hand, if fixation does not affect the dynamics of charge density, then mucous structures such as the endothelial cell coat are likely to be involved because they are more resistant to glutaraldehyde fixation (1, 35).
Our main findings were that the fractional clearance, , for albumin
was one order of magnitude less than that for a neutral Ficoll of similar
hydrodynamic size (35.5
). From this charge
selectivity, a charge density of 38 meq/l could be calculated using the
gelmembrane model. The value is surprisingly similar to those estimated in
vivo (41) and in vitro
(15,
21,
25,
38), as noted in a previous
study on fixed kidneys (5).
Data could also be interpreted in terms of charged fiber densities assuming a
certain degree of heterogeneity. Reducing the I of the perfusate did not
affect the relationship between
albumin and
Ficoll35.5Å as much as expected based on the increased
charge-charge interactions. Consequently, both theoretical models predict that
the volume of the gel did increase during low I perfusion. The more accurate
heterogeneous charge fiber model suggests a volume expansion of 78%, whereas
the gel-membrane model suggests a threefold volume expansion. These dynamic
alterations of the charge density in a fixed kidney suggest that the structure
responsible for glomerular charge selectivity is a polysaccharide-rich layer
resistant to fixation such as the endothelial cell coat (or possibly the
glomerular basement membrane).
Permeability Characteristics of the Fixed Kidney
The sieving coefficient for albumin obtained at neutral pH in the fixed kidney is higher than in vivo but similar to that reported for the unfixed isolated, perfused rat kidney (37). The glomerular permeability is, however, heterogeneous (5).
The sieving coefficient for BSA at a concentration of 50 g/l does not significantly differ from that obtained at a concentration of 10 g/l (44). Similar results have been obtained in micropuncture experiments of the isolated rat kidney (37). Therefore, perfusion experiments of the fixed kidney were performed at an albumin concentration of 10 g/l to maintain the costs of the experiments low. Recent experiments showed, however, that the albumin concentration indeed may affect the sieving of tracer macromolecules (20). This deviation from the normal physiological protein concentration, albeit disturbing, will, however, not affect the conclusions of the study.
Glomerular Barrier
The finding that the sieving coefficient of albumin is much lower than that
of Ficoll of equivalent size (35.5 Å) supports the classic notion of a
charge barrier (4). Recently,
this notion has been challenged due to some limitations of the dextran used as
a tracer (28). The calculation
of the GCW charge distribution according to a simplified model of
charge-charge interactions
(24) gave a charge density of
38 meq/l. Thus glomerular charge selectivity was overestimated in the
classical studies (9) due to
the use of sulfated dextrans. The gel-membrane model has the virtues of being
able to describe glomerular permeability in a variety of situations, and the
calculations are rather straightforward. It is, however, an oversimplified
view of the reality because charge and size interactions cannot really be
separated. Thus barriers in series will contribute to the overall sieving
coefficient of a tracer as products (i.e., tot =
1 ·
2 ·
3
·
4...
n), which suggests that there
must be some degree of size restriction in the gel compartment as well
(20). The charged fiber model
is theoretically more correct, but it suffers from being highly complex.
Indeed, some of the fiber matrix equations have not yet been fully developed.
The equations required to estimate the partition coefficients are
sophisticated but do nevertheless have certain limitations. They do, for
example, assume random interactions between a solid sphere and one fiber. In
reality, the glomerular barrier is composed of multiple fibers and plasma
proteins in an orderly fashion. Moreover, the equations to estimate the
reflection coefficient and the diffusion capacity in the gel are crude at
present. Still, we consider the charged fiber model to be the most accurate
theory for analysis of glomerular permeability.
The gel-membrane model adequately describes both albumin and Ficoll data.
The heterogeneous charged fiber model grossly overestimated the fractional
clearance for albumin. This probably indicates that the latter model, despite
its complexity, has limitations. Alternatively, it may suggest that albumin
binds to tubular structures in the fixed kidney causing underestimations of
for albumin. There are, however, no indications of such binding
problems in these kidneys that have been extensively prewashed.
In the present study, we introduce heterogeneity into the charged fiber model. Hereby, the adaptation to the experimentally determined Ficoll data improved dramatically. It is important to note that similar conclusions were drawn using the two different models of glomerular charge and size selectivity namely that perfusion of rat kidneys with low I seems to induce a volume expansion of the gel (by 78% or more).
Fixation with Glutaraldehyde
The urine-to-perfusate ratio of one for both inulin and the electrolytes demonstrates that the urine collected represents glomerular ultrafiltrate. The tubules are therefore part of an inert system in which the metabolic processes have been eliminated. The fixed kidney can thus be regarded as a pure "membrane." Histological studies (36) have shown that glomerular structures of isolated, perfusion-fixed kidneys were similar to those in vivo, including the distribution of anionic sites in the glomerular basement membrane as characterized with Ruthenium red. Moreover, in rat hindquarter preparations, fixation with glutaraldehyde reduced surface area for capillary exchange but had no effect on capillary permeability (14).
Mucous structures rich in polysaccharides, however, are more resistant to regular fixation techniques and studies on the microanatomy of such structures must employ special, nonconventional fixation regimes (1, 18, 31, 35). For this reason, it would be expected that the endothelial cell coat, a polysaccharide-rich layer creating an interface between plasma and endothelial cells, should allow volume changes even in a fixed kidney. In our study, the low I perfusion did indeed reduce the estimated charge fiber density in the isolated, perfusion-fixed kidneys. As all fixed structures, except the endothelial cell coat, are rigid and incapable of undergoing the large volume changes required to alter the charge density observed in our experiment, this supports the hypothesis that glomerular charge selectivity is related to the cell coat covering the endothelial cells.
Finally, we have to consider some alternative interpretations of our
results. Could, for example, the observed alteration in estimated charge
density be due to something else than volume changes of the glomerular charge
barrier? Indeed, the biophysical models for transport of charged solutes
across charged membranes or gels are far less precise than the theories
dealing with transport of neutral solutes. However, in a recent study, we
compared different models including the most advanced charged fiber-matrix
analysis (38) and the results
are more or less the same. All current theories predict that the for
albumin should be reduced by more than one order of magnitude when the I is
reduced from 151 to 34 mM. The experimental observations suggest a modest, but
statistically significant, reduction of
albumin at low I. At
present, the only plausible explanation is that the glomerular charged fiber
density is reduced. The reversibility of this process demonstrated by
Sörensson et al. (39)
seems to rule out other possibilities than volume changes of the charge
barrier with a constant number of fixed charges. Indeed, dramatic fluid shifts
are to be expected because the electroosmotic pressure of the gel increases
drastically (reaching 160 mmHg) as the I is reduced (for more details, see
Ref. 38).
In conclusion, the glomerular barrier is size and charge selective. Perfusion with solutions of low I reduced the estimated charged fiber density by at least 78%, probably due to volume expansion of gel. Because almost all constituents of the glomerular barrier, except the polysaccharide-rich endothelial cell coat, are rigid in the fixed kidney, our findings support the view that the endothelial cell coat can be an important component of the glomerular barrier.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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