Departments of 1 Physiology and 2 Nephrology, Göteborg University, SE-405 30 Gothenburg, Sweden
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ABSTRACT |
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Modifying the ionic strength (I) is a gentle way to alter charge
interactions, but it cannot be done for studies of the glomerular sieving of proteins in vivo. We therefore perfused 18 isolated rat
kidneys with albumin solutions of different ionic strengths at a low
temperature (cIPK) to inhibit tubular uptake and protease activity.
Four anionic proteins were studied, namely albumin (Alb), orosomucoid
(Oro), ovalbumin (Ova), and anionic horseradish peroxidase (aHRP),
together with the neutral polymer Ficoll. With normal ionic strength of
the perfusate (152 mM), the fractional clearance () was 0.0018 ± 0.0003 for Alb, 0.0033 ± 0.0003 for Oro, 0.090 ± 0.008 for Ova, and 0.062 ± 0.002 for aHRP. These
values were all
lower than for Ficoll of similar hydrodynamic size; e.g.,
Ficoll 36 Å was >20 times higher than
for albumin. Low ionic strength (34 mM) increased size selectivity as
for anionic proteins and Ficoll fell, suggesting a reduction in
small-pore radius from 44 ± 0.4 to 41 ± 0.5 Å,
P < 0.01. In contrast, low I reduced the charge
density of the membrane,
, to one-quarter of the 20-50 meq/l
estimated at normal I. These dynamic changes in
seem to be due to
volume alterations of the charged gel, fluid shifts that easily are
accounted for by the changes in electroosmotic pressures. The finding
that low ionic strength induces inverse effects on size selectivity and
charge density strongly suggests that separate structures of the
glomerular wall are responsible for the two properties.
capillary permeability; kidney glomerulus physiology; charge; endothelium
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INTRODUCTION |
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THERE IS AN ONGOING
DEBATE as to how the restriction of molecules is carried out in
the kidney. In several studies using neutral and charge-modified
dextran polymers, the glomerular barrier has been shown to be both size
and charge selective (2, 5). A significant charge barrier
was also found for horseradish peroxidase (HRP) of different net
charges (42). These classic studies suggest a charge
density of 120-170 meq/l (10). However, the validity of the dextran data has been questioned, because some fractions of
sulfated dextran seem to bind to glomerular cells (8) or to plasma proteins (13). Also, the data based on HRP
protein clearance (42) have been criticized because of the
effects of tubular degradation (39). The term "charge
selectivity" refers to the phenomenon in which the fractional
clearance () of anionic macromolecules is less than that of
otherwise similar uncharged solutes under similar hemodynamic conditions.
Several recent studies support the classic view of glomerular charge
selectivity, albeit with a smaller charge density. Thus the of
neutral and anionic myoglobin in rats in vivo suggests a charge density
of 32 meq/l (54). We found similar results in isolated rat
kidneys (IPK) perfused at low temperature (cIPK) for HRP
(50), lactate dehydrogenase (28), and albumin
(33).
Not all investigators share the notion of a significant glomerular
charge barrier (8). Indeed, there are reports suggesting the glomerular barrier to be just as permeable as capillaries in
skeletal muscle (40). However, the latter observation was performed in IPK at 37°C using lysine or NH4Cl to inhibit
tubular activity, a procedure that recently has been shown to affect
permeability per se (33). Another important factor for
transglomerular passage is the shape of a molecule (38).
In fact, elongated solutes have much higher than spherical
molecules of similar size and charge (27, 35).
In a previous paper we studied the of anionic (aHRP) and neutral
HRP. Neutral HRP had a
twice as high as that of the aHRP of similar
hydrodynamic size, supporting significant charge selectivity (50). In that study, we reduced the ionic strength of the
perfusate without affecting osmolarity. As expected, the
for aHRP
fell during perfusion with low I, but the effects were rather small and
fully reversible. The
for neutral HRP was unaffected by a reduction
in ionic strength.
In the present study, we wanted to quantify the glomerular charge density using several different anionic proteins and neutral spherical Ficolls of similar hydrodynamic sizes. The use of a broad fraction of Ficoll also allowed pore analysis, thus giving a detailed description of glomerular size selectivity. The experiments were conducted in isolated rat kidneys perfused at low temperature to reduce tubular cell and protease activity. The experimental model also allows more dramatic alterations of perfusate composition than would be possible in vivo. Thus increasing or reducing perfusate ionic strength, without affecting osmolarity, would be expected to markedly affect charge interactions, in line with the everyday experience of ion exchange chromatography. The two main questions at issue were the following. First, is there any evidence for charge selectivity when studied by using a variety of anionic proteins? Second, could the charge and/or size selectivity be affected by alterations of ionic strength?
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METHODS |
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A total of 18 female rats of Wistar strain (Møllegaard, Stensved, Denmark) were used in the study. The rats were kept on standard food and had free access to water before the experiments. Anesthesia was induced by intraperitoneal injection of pentobarbitone (50 mg/ml, Apoteksbolaget, Umeå, Sweden). The tail artery was cannulated for recordings of arterial pressure, also serving as a route for subsequent administration of drugs. The body temperature of the rat was kept at 37°C during the preparation by means of a thermostatically controlled heating pad. The total preparation time was ~1 h. The local Ethical Committee approved the experiments.
Kidney Perfusion Technique
We used a modification of the isolated perfused rat kidney preparation described by Johnsson and Haraldsson in 1992 (19). Care was taken not to touch the kidneys during the preparation, and the kidneys were fully perfused with either blood or perfusate during the entire preparation. The temperature of the perfusates was kept at 8°C to inhibit tubular function as well as energy consumption and myogenic tone (6, 11) without altering capillary permeability (33, 45).Perfusates
Modified Tyrode solutions containing human albumin (18 g/l, Immuno, Vienna, Austria), containing orosomucoid (15) were used to perfuse the kidneys. The perfusates differed in ionic strength, the standard being 152 mM, the low ionic strength 34 mM, and the high ionic strength 292 mM. All the perfusates were kept at the same high osmolarity (590 mosmol/kgH2O) to enable variations in ionic strength without altering osmolarity. This was obtained by adding mannitol, which has also been shown to protect against acute renal failure (14) and act as a free radical scavenger (16). The composition of the standard perfusate was as follows: (in mM) 148 Na, 4.3 K, 2.5 Ca, 131 Cl, 0.8 Mg, 25 HCO3, 0.5 H2PO4, 5.6 glucose, and 285 mannitol. The low-ionic-strength perfusate contained (in mM) 26 Na, 4.3 K, 2.5 Ca, 8.4 Cl, 0.8 Mg, 25 HCO3, 0.5 H2PO4, 5.6 glucose, and 559 mannitol. High-ionic-strength perfusate contained (in mM) 284 Na, 4.3 K, 267 Cl, 2.5 Ca, 0.8 Mg, 25 HCO3, and 0.5 H2PO4. In addition, nitroprusside (0.27 g/l, Merck, Darmstadt, Germany) and furosemide (10 mg/l, Benzon Pharma, Copenhagen, Denmark) were added to the perfusates to ensure maximum vasodilatation and further reduce tubular reabsorption of sodium and water. Two grams per liter of FITC-labeled Ficoll70 (14-70 Å, Bioflor, Uppsala, Sweden) was added to all perfusates in the second set of experiments. The perfusates were protected from light and bubbled with 5% CO2 in O2. Radiolabeled 125I-aHRP tracer in the first series and 125I-orosomucoid and 131I-ovalbumin in the second series, was eluted on an equilibrated desalting column (PD-10, Amersham Pharmacia Biotech, Uppsala, Sweden) before the experiments to reduce the free iodide content and then added to both perfusates. The solutions were made with fresh distilled water (Millipore) with a resistivity of 18.2 MTracers
The physicochemical properties of the molecules used as tracers are reviewed in Table 1.
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aHRP.
HRP is a well-characterized protein with a molecular mass of 40 kDa. Neutral HRP (303 U/mg, Genzyme Biochemicals, Maidstone, UK) was charge modified by succinylation (25, 37, 43) as previously described (50) to obtain an anionic form. The
modified molecules were tested and compared with neutral HRP by agarose gel electrophoresis. There was a marked alteration of the charge, and
the resulting aHRP seemed to be homogenous. The material was dialyzed
and freeze-dried. The two HRP proteins had similar molecular radii as
determined by gel filtration on a Superose 12 PC 3.2/30 column (SMART
HPLC, Amersham Pharmacia Biotech), using 50 mM phosphate buffer with
115 mM sodium chloride and pH 7.0 as the mobile phase. The pI was 7.3 for neutral HRP and <3.5 for aHRP as determined by isoelectric
focusing. The labeling of aHRP was done by using the chloramine-T
method. The activity concentration was 7.8 MBq/ml for anionic
125I-labeled HRP. The net charge was 6 as judged from the
reduced electrophoretic mobility after this labeling method was used. Analyses were performed on a Superdex 200 HPLC column (Amersham Pharmacia Biotech), whereby a 0.1 M phosphate buffer (pH 7.4) was used
as the mobile phase. There was no measurable change in molecular size
after the iodination.
Ovalbumin. Ovalbumin (Sigma) has a molecular mass of 43 kDa. The ovalbumin was labeled with 131I by using N-succinimidyl · 3-trimethylstannyl) benzoate, and the activity concentration was 9.4 MBq/ml. The tracer analysis was done as described for aHRP above.
Orosomucoid. The serum protein orosomucoid (kindly provided by Immuno) has a molecular mass of 40 kDa. The labeling with 125I was made by using Iodogen, and the activity concentration was 4.6 MBq/ml. The tracer analysis was done as for aHRP.
Ficoll. The neutral polymer Ficoll labeled with FITC was used in molecular radii of 14-70 Å. Ficoll is a spherical molecule (frictional ratio close to 1.0) and had no electrophoretic mobility after labeling with FITC (33).
Experimental Protocol
Group A.
Nine rats, weighing 240 ± 4 g, were used in the first set of
experiments by using 125I-aHRP and albumin as tracers.
After preparation, perfusion started with the standard solution, and
then low ionic strength was introduced followed by high ionic strength
and finally back to standard again. The low- and high-ionic-strength
values were compared with the mean value of the two standard perfusion
periods. The total perfusion time was ~1 h. During each perfusion
period in the first set of experiments, three urine samples were
collected for determination of glomerular filtration rate (GFR) and for aHRP and albumin. The perfusion pressure was kept constant. The
urine and perfusate samples were subjected to gel filtration on a
Sephadex PC 3.2/10 column (SMART HPLC, Amersham Pharmacia Biotech) for
assessment of tracer-bound radioactivity shortly after each experiment.
Radioactivity was determined by analysis of the amounts of
125I-aHRP in a gamma counter (Cobra, Auto-Gamma Counting
systems, Packard Instrument, Meriden, CT). Corrections were made for
background activity. Albumin concentrations were measured by
radioimmunoassay (PharmaciaUpJohn Diagnostics Sverige, Uppsala, Sweden).
Group B. In the second set of experiments with orosomucoid, ovalbumin, albumin, and Ficoll (14-70 Å) as tracers, we used nine rats weighing 263 ± 4 g. The kidneys were perfused with standard perfusate, low ionic strength, standard, high ionic strength, and finally the standard perfusate again. Low-ionic-strength values were compared with a mean value of the first and second standard period and high ionic strength to a mean value of the second and third standard perfusion. Pump flow was changed to maintain a constant perfusion pressure. Otherwise, the urine and perfusate samples were handled the same way as in the first set of experiments. The amount of tracer-bound radioactivity was determined in a gamma counter (Cobra, Auto-Gamma Counting systems, Packard Instrument). Corrections were made for background activity, decay, and spillover from 131I to 125I.
Analysis of Ficoll Concentrations
For calculation of the sieving coefficients for FITC-Ficoll, perfusate and urine samples were subjected to gel filtration (BioSep-SEC-S3000, Phenomenex, Torrance, CA) and fluorescence detection (Dionex fluorimeter RF-2000, Dionex Softron, Germering, Germany) by using Chromeleon (Gynkotek, Germering, Germany) software. A 0.05 M phosphate buffer with 0.15 M NaCl with pH 7.0 was used as eluent. A 5- to 10-µl sample was analyzed at an excitation wavelength of 492 nm, and an emission wavelength of 520 nm with a flow of 1 ml/min by using a fixed sampling frequency of 1 sample/s. The pressure was ~4 MPa, and the temperature was kept at 8°C during the analysis. Estimated errors in urine-to-plasma concentration ratio (CU/CP) values for Ficoll were <1%, except for the larger molecules where the wavelength noise increased and thus probably resulted in slight overestimation of the large pore radius.The Ficoll Calibration Curve
Six monodisperse samples of Ficoll with known molecular radii were marked with FITC (for detailed information about the marking procedure, see Ref. 33). The FITC-labeled Ficoll molecules were used to obtain a calibration curve on the BioSep-SEC-S3000 column (Phenomenex).Calculations
GFR.
GFR was calculated as CU/CP of
51Cr-EDTA times urine flow (QU), i.e., GFR = (CU/CP)CrEDTAQU. In
this study, the (CU/CP)Cr
EDTA was
assumed to be 1.15. We have performed numerous cIPK experiments at
8°C with Cr
EDTA, and the inhibition of tubular reabsorption is so
reproducible that a constant value of 1.15 is justified (27, 15,
19).
Fractional clearance for albumin, orosomucoid, and ovalbumin.
The renal clearance, Cl, for a molecule X is given by
an equation analogous to that for calculation of GFR, that is, Cl = (CU/CP)XQu.
The fractional clearance, , of a solute X is given by
dividing its clearance with GFR giving the following simple equation
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(1) |
Charge Density
We determined the charge density of the membrane during perfusion with solutions of different ionic strengths. As previously reported, this was done by using equations to describe the glomerular membrane as a gel with homogenous distribution of fixed charges (50). Such a model represents an oversimplification for several reasons, as outlined by Deen et al. (10). However, an extended and more precise analysis is presented in the APPENDIX. Briefly, the analysis is reduced to three fundamental equations with three unknown parameters, namely, the distribution of sodium (Nagel) and chloride (Clgel) in the gel, and the charge density of the gel,
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(2) |
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(3) |
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(4) |
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(5) |
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(6) |
The imbalance between the concentrations of free ions in the gel
and those in plasma will give rise to an electroosmotic pressure (Eo) (53) that amounts to
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(7) |
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(8) |
Statistics
Results are presented as means ± SE, and differences were tested by using Student's t-test, paired design. In the case of uneven distribution, i.e., for the pore analysis parameters, the data were transformed and the geometric mean and SE values were calculated. The 95% confidence intervals for the charge densities were calculated for the fractional clearance of the anionic proteins and their size-matched neutral Ficoll control. ![]() |
RESULTS |
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General
Nine rats, weighing 240 ± 6 g, were used in group A, and nine rats, weighing 263 ± 4 g, were used in group B. Values for GFR, perfusion pressure, and pump flow are shown in Table 2. The mean wet weight of the kidneys (immediately after perfusion) was 1.07 g (n = 9). There was no evident difference between the two groups, and the results were therefore pooled for analysis.
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Albumin
During control, with normal ionic strength and high osmolarity, the
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aHRP
During control, with normal ionic strength and high osmolarity, the fractional clearance for aHRP was 0.062 ± 0.002 (n = 9). TheOvalbumin
During the control period, theOrosomucoid
TheFicoll
TheProtein-To-Ficoll Clearance Ratios
For orosomucoid, albumin, and ovalbumin, the
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Results of Two-Pore Analysis
A neutral two-pore model based on the sieving data of Ficolls (14-70 Å) was carried out. The results are summarized in Table 4. At normal ionic strength (152 mM), the small-pore radius was 44 ± 0.4 Å, the large-pore radius was 98 ± 3 Å, the large-pore fraction of the hydraulic conductance was 0.33 ± 0.04%, and the total pore area-to-diffusion distance ratio (A0/
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Estimated Wall Charge Density,
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During perfusion by the two solutions with different ionic strengths,
the gel reversibly changed. Thus for perfusates with low ionic
strength,
fell to 22% of control, reaching values of ~7 meq/l
(see Fig. 4). For high ionic strengths, the apparent
increased
almost twofold to reach values of ~52 meq/l.
Estimated Electroosmotic Pressure, Eo
Comparison of Experimental Data to Theoretical Models
We have analyzed the
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DISCUSSION |
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This report is the hitherto most extensive analysis of glomerular
charge selectivity in that four anionic proteins, and a range of
neutral Ficolls, are studied. In addition, charge interactions are
rapidly and reversibly manipulated by altering the ionic strength in
the surrounding environment. This approach, used in everyday ion-exchange chromatography, is readily performed in the IPK but is of
course inaccessible for studies in vivo. Our main findings are the
following. First, the glomerular barrier is highly selective and
discriminates molecules on the basis of their size, charge, and shape,
in qualitative agreement with the classic view. Thus all four anionic
proteins studied had significantly lower values than their
size-matched neutral Ficolls. The estimated glomerular
was
30-50 meq/l, which is considerably less than the 120-170 meq/l predicted from the dextran data (10). In terms of
Eo, the difference represents several hundred
millimeters mercury (see Eq. 8). Second, altering perfusate
ionic strength had far fewer effects on the
values of negatively
charged proteins than predicted for a charged gel (or membrane) with
constant
. In fact, the glomerular charge selectivity was almost
independent of ionic strength, because the protein-to-Ficoll clearance
ratios did not change, a phenomenon that contrasts to the predictions of two different theories of charge interactions (see the
APPENDIX). Third, low ionic strength caused opposite
effects on glomerular size selectivity and
. The
values for
proteins and size-matched Ficolls were all reduced during perfusion
with low-ionic-strength solutions. Pore analysis revealed a significant
reduction of the small-pore radius without any other alterations of the
size barrier. Hence, low ionic strength significantly increased
glomerular size selectivity. In contrast, the
was markedly reduced
during perfusion with low ionic strength. High ionic strength increased
without detectable alterations of the size barrier. Fourth, the
inverse alteration of glomerular size selectivity and
clearly
indicates that different components of the glomerular barrier are
responsible for the two selective properties. There is no theory that
would allow opposite changes in size and charge discrimination in one single structure, at least not at present. Fifth, a gel membrane model
of size- and charge-selective barriers in series adequately describes
the transglomerular passage of proteins and Ficoll at various ionic
strengths. Sixth, the effects of low ionic strength on glomerular
can be explained by the concomitant increase of
Eo in
the gel. As a consequence of the increased
Eo, fluid
will move into the gel and dilute the concentration of fixed charges.
The presence of significant charge selectivity is evident from the fact
that the values for all four anionic proteins (albumin, ovalbumin, orosomucoid, and aHRP) were significantly lower than for
their neutral counterparts (size-matched Ficolls). Thus the
for
neutral Ficoll was >20 times higher than albumin despite similar
hydrodynamic size. Moreover, ovalbumin had a
that was less than
one-third of that for the neutral Ficoll of similar size. Neutral
Ficoll the size of HRP had a
almost identical to the value we
reported for neutral HRP in our previous study (50).
However, a higher value was found for the
for aHRP in the present
work, probably reflecting the use of different labeling techniques.
Thus the present chloramine-T approach was found to reduce the net
charge (51) by half from
11 to approximately
6.
Finally, orosomucoid had a
that was one-third of that for the
neutral Ficoll of similar size. Orosomucoid had a rather high
considering its size (40 Å compared with 36 Å for albumin) and its
negative charge (
24 compared with the albumin value of
23). However, the protein is elongated, with a frictional ratio of 1.5 (see
Table 1), a factor that has been found to drastically increase the
transglomerular passage of a molecule (27, 35). These
biological data clearly demonstrate significant charge selectivity regardless of what kind of theoretical model is used for the analysis.
From our results, we conclude that negative charge is an important
determinant of glomerular capillary permselectivity. The estimated
glomerular was found to be 30-40 meq/l by using perfusate with
normal ionic composition. Studies that confirm these magnitudes of the
have been performed by using myoglobin in vivo (32 meq/l) (54), aHRP (34 meq/l) (50), and lactate
dehydrogenase (LDH; 35 meq/l) (28) in isolated cooled
perfused rat kidneys, and with albumin in fixed perfused kidneys (43 meq/l) (7).
Regarding glomerular size selectivity, the two-pore analysis showed slightly lower values for the small-pore radius compared with our previous studies (33, 36). Also, the large-pore radius was somewhat larger. These discrepancies could occur by pure chance but could also be due to the use of hyperosmolar solutions in the present study. Thus to maintain osmolarity over a wide range of ionic strengths, we used a high osmolality of 590 mosmol/kgH2O. Indeed, the changes in pore radii resemble those found with increasing glomerular hydrostatic pressures and/or GFRs (35).
We appear to have been the first group to use the concept of altering ionic composition as a means of reversibly modifying the functional properties of the glomerular charge barrier (49, 50). The same approach was used on isolated basement membranes (3). Also, Kanwar and Rosenzweig (21) studied the effects of high ionic strength on GFR, inulin clearance, and morphological parameters. GFR was constant as the salt concentration was 500 mM or less but fell to 50% at 1.0 M and was only 5% of normal with a 2.0 M solution. The morphological changes suggested that high-salt solutions induced "clogging" of the glomerular wall, but the extremely high osmolarity might have had effects per se.
Lowering the ionic strength of the perfusate would induce powerful
changes in terms of increasing the charge-charge interactions according
to the Debye-Hückel theory. Thus the Debye length (at 8°C)
would be expected to increase from 8 to 17 Å when normal perfusate is
switched for one with low ionic strength and to fall to 6 Å when a
high-ionic-strength perfusate is introduced. These effects would be
expected to drastically affect the values of anionic solutes, but
that was not seen in this study. In contrast, lowering or increasing
the ionic strength gave rise to only minor alterations in the
for
the anionic molecules. The low ionic perfusate induced an average
decrease in the
for various proteins of 35%. A similar fall in
was noted for the neutral Ficolls. This is in contrast with the
findings in our previous study (50), where we found no
effects on neutral HRP of a low ionic strength of the perfusate. Note,
however, that the conditions were somewhat different because isotonic
solutions were used in the previous paper. All changes occurred
immediately, were reversible, and, except for a small time-dependent
increase in
for albumin and large Ficolls (>45 Å), there was no
apparent damage to the barrier.
The glomerular charge selectivity was remarkably constant as ionic
strength was altered, as reflected by the unchanged protein-to-Ficoll clearance ratios. This is in contrast to the marked effects predicted from the altered Debye lengths. Therefore, it seems as if the glomerular changed with the ionic strength. Could this reflect true
changes in
? If so, what are the underlying mechanisms? To answer
these pertinent questions, it must be remembered that the concentration
of fixed charges is reversibly decreased by low ionic strength, whereas
the opposite occurs with high ionic strength. The reversibility can
hardly be explained by loss of charges followed by renewed synthesis,
because the alterations are rapid even at 8°C. We therefore conclude
that the volume of the gel must change. Fluid is driven into the gel
during perfusion with low ionic strength and the opposite occurs with
high ionic strength. This is in total agreement with the predicted
effects of ionic strength on the
Eo. Thus at a normal
ionic strength of 152 mM, the
Eo is 36 mmHg. Reducing
ionic strength to 34 mM will cause an increase in
Eo to
162 mmHg, which in turn will reduce the electrochemical potential for
water and cause a fluid shift into the gel. Hereby, the charge density
will fall from 34 to 7 meq/l (SEE RESULTS). Elevated ionic
strength will give the opposite result. In addition, low ionic strength
will double the Debye length, which would be expected to increase the
repulsion of negatively charged groups, and hence expand the gel.
Many researchers argue that the permselective part of the glomerular
barrier must lie within the basement membrane or the podocyte layer,
because the endothelial cells are so highly fenestrated. The podocyte
slit membrane has attracted special interest, and several new proteins
have been identified such as podocalyxin (23) and nephrin
(47). The latter seems to be pivotal for podocyte
integrity and has been shown to be mutated in congenital nephrotic
syndrome of the Finnish type (24), a condition with massive proteinuria. Regarding the glomerular basement membrane, GBM,
the seems to be rather low, at least in isolated fractions in vitro
(3).
As stated, the role of the endothelium has been neglected. However, there are a number of reports that recognize the presence of a cell coat or glycocalyx covering the fenestrated endothelial cells (29, 48, 52). This cell coat consists of a mesh of sialylated glycoproteins anchored in the endothelial cell surface and reinforced by proteoglycans and plasma proteins. It can be made visible by staining with a cationic dye, i.e., ruthenium red (29). Indeed, the cell coat may be a rather thick structure, in the range of 50-100 nm, covering the fenestrae and surrounding domains of the capillary wall, as demonstrated by using nonaqueous fixative (46). Such a periendothelial layer of proteoglycans may behave like the ion-exchange gel described in our model (36). The increased ion-ion interactions induced by low ionic strength may reduce charge density by simply expanding the gel volume. Figure 4 would support such an interpretation and suggests that some nephrotic syndromes may be due to endothelial dysfunction rather than kidney-specific disorders.
It may be argued that there are alternative explanations for the present findings. Could there, for example, be an uptake of negatively charged molecules by the tubular cells? This is highly unlikely. Because we perfused the kidneys at 8°C, there should be no significant cellular metabolism or uptake (31). There are, however, changes in viscosity at 8°C (~2 times higher than at 37°C); the resistance to flow and filtration is twice as high, and diffusion is reduced by 50%. Also, the Debye length is decreased by ~0.6% at 8°C. In addition, there is an increase in viscosity due to the increased concentration of mannitol in the low-ionic perfusate, to maintain the same osmolarity compared with neutral perfusate. This accounts for the slight decrease in GFR during low-ionic-strength perfusion. There is, however, no evidence that the reduced temperature affects permeability per se (33).
In summary, this study confirms that the glomerular barrier is highly
charge selective. However, the magnitude of the is less than
predicted from dextran data and amounts to 30-50 meq/l. Our
results strongly suggest that there are two separate barriers in series
in the glomerular wall. This is evident from the inverse changes of
glomerular size selectivity and
that occurred during perfusion with
low-ionic-strength solutions. Thus there is a charge-selective gel with
highly dynamic properties, the morphological counterpart of which may
well be the endothelial cell coat and/or the glomerular basement
membrane. This layer may act as an ion exchanger, reducing the
concentration of albumin in the gel to 5% of that in plasma. A second,
mainly size-discriminating component acts to further reduce the
concentration of albumin in the primary urine to ~4% of that in the
gel, or 0.2% of that in plasma. At present, we consider this second
barrier to be situated in the podocyte slit membrane, where nephrin is
a key component.
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APPENDIX |
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There have been few attempts in the literature to combine size and charge selectivity because the equations derived are highly complex. In particular, the concept of a porous membrane is difficult to reconcile with the requirements of homogenous distribution of charges (10). Recently, Johnson and Deen (17) developed such a theory, which predicts the effects of electrostatic interactions on the partition coefficients of spherical macromolecules in gels with random arrays of fibers. At present, their model seems to provide the most accurate description of how macromolecules interact with a fiber matrix or gel structure.
We have analyzed our glomerular sieving data according to the theoretical model of Johnson and Deen (17). Here we will first present some of the most important equations used in the analysis and then the results of applying the theory on our data. (For a more detailed description of the model, consult Ref. 17).
The partition coefficient () of a spherical macromolecule in a
random array of straight fibers was first modeled by Ogston (32)
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(A1) |
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(A2) |
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(A3) |
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(A4) |
Our first step was to reproduce Figs. 5-8 in the original
publication (17). The second step was to adjust the model
parameters to describe the partition coefficients, , for albumin and
the size-matched Ficoll in a normal-ionic-strength solution. Finally, we observed the theoretical effects on
albumin
and
Ficoll 36 Å of altering the ionic strength.
We assumed the fiber radius, rf, to be 4 Å,
as to be 35.5 Å, = 0.07, and the surface
charge densities (q) of fiber and albumin to be
0.08 and
0.022 C/m2, respectively. Under these conditions, the
partition coefficients for albumin were estimated to be 3.4 10
5 at normal ionic strengths compared with 7.6 10
4 for neutral Ficoll of similar size, giving a
ratio of 22. At low ionic strength,
albumin was 6.3 10
8 and
Ficoll 36Å was 3.6 10
4, giving a ratio of 5,700. High ionic strength gave a
albumin of 1.3 10
4 and
Ficoll
36Å of 8.8 10
4, giving a
ratio of 7. Altering
the various parameters in the model changes the absolute values for the
partition coefficients, but the effects of ionic strength are
qualitatively the same.
To compare these partition coefficients to our experimental data, one
must calculate fractional clearances. Curry and Michel (9)
introduced the concept of a "fiber matrix" (32) into the field of microvascular research. In so doing they used the expression of Anderson and Malone (1) to calculate the
reflection coefficient, , from the partition coefficient,
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(A5) |
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(A6) |
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(A7) |
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ACKNOWLEDGEMENTS |
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This study was supported by Swedish Medical Research Council Grants 9898 and 2855, the Knut and Alice Wallenberg Research Foundation, the Ingabritt and Arne Lundberg Research Foundation, the National Association for Kidney Diseases, and by Sahlgrenska University Hospital LUA-B31303.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Haraldsson, Dept. of Physiology, Göteborg Univ., PO Box 432, SE-405 30 Gothenburg, Sweden (E-mail: bh{at}kidney.med.gu.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.
Received 8 August 2000; accepted in final form 5 December 2000.
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