Dynamic alterations of glomerular charge density in fixed rat kidneys suggest involvement of endothelial cell coat

Giuliano Ciarimboli,1,2 Clara Hjalmarsson,3 Arend Bökenkamp,4 Hans-Joachim Schurek,1 and Börje Haraldsson5

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a previous paper, we found that low ionic strength (I) reversibly reduced the glomerular charge density, suggesting increased volume of the charge-selective barrier. Because glutaraldehyde makes most structures rigid, we considered the isolated, perfusion-fixed rat kidney to be an ideal model for further analysis. The fixed kidneys were perfused with albumin solutions containing FITC-Ficoll at two different Is (I = 151 and 34 mM). At normal I, the fractional clearance ({theta}) for albumin was 0.0049 (SE –0.0017, +0.0027, n = 6), whereas {theta} for neutral Ficoll35.5Å of similar size was significantly higher 0.104 (SE 0.010, n = 5, P < 0.001). At low I, {theta} for albumin was 0.0030 (SE –0.0011, +0.0018, n = 6, not significant from {theta}albumin at normal I) and {theta} for Ficoll35.5Å was identical to that at normal I, 0.104 (SE 0.015, n = 6, P < 0.01 compared with {theta}albumin at low I). According to a heterogeneous charged fiber model, low I reduced the fiber density from 0.056 to 0.0315, suggesting a 78% gel volume expansion. We conclude that 1) there is a significant glomerular charge barrier. 2) Solutions with low I increase the volume of the charge barrier even in kidneys fixed with glutaraldehyde. Our findings suggest that polysaccharide-rich structures, such as the endothelial cell coat, are key components in the glomerular barrier.

glomerular permeability; charge selectivity


TO ACHIEVE THE rapid rate of filtration required to regulate the composition and volume of body fluids, glomerular capillaries possess unique functional and structural characteristics. A striking example is an extraordinarily high hydraulic permeability. Glomerular capillaries are one to two orders of magnitude more permeable to water than are capillaries from various other microvascular beds (9). Nevertheless, the same structure normally imposes an extremely efficient barrier to the passage of plasma proteins so that the concentrations of albumin and larger proteins are minute.

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 ({theta}) 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 120–170 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).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals

Experiments were performed on male Sprague-Dawley rats weighing 200–300 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 5–10 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, {theta}. The fractional clearance {theta} 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 {theta} 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 {theta} for albumin and its neutral counterpart of similar size, Ficoll35.5Å, giving a density of fixed charges, {omega} (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/{Delta}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 ({Phi})

(3)
where g(h) is the probability of finding the closest fiber at a distance, h, from a spherical solute in a dilute solution

(4)
where {phi} is the volume fraction of fibers, rs is the solute radius, and rf is the fiber radius. By integrating Eq. 4, Ogston (23) reached the following expression for {Phi}

(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

(6)
where E(h) is the electrostatic free energy of the interactions between the solute and nearest fiber divided by kT (k is Boltzmann's constant and T is the absolute temperature). E is dependent of one position variable, h, only. In a true system, the solute would interact with multiple fibers (and other solutes). Johnson and Deen (16) solved Eq. 6 using a linearized Poisson-Boltzmann equation with dimensionless parameters scaled by the electrical potential RT/F (R is the gas constant and F is Faraday's constant).

Interactions between solute and fiber cause changes in the free electrochemical energy

(7)
where the subscripts s, f, and sf refer to the isolated solute, isolated fiber, and combined solute-fiber system. Note that to obtain {Delta}G, nested polynomial equations are required making the calculations much more complex (16).

The energy (E) needed in Eq. 6 is given by

(8)
where {epsilon} is the dielectric permittivity for the solvent. R, T, and F were previously explained. The dielectric permittivity is the relative dielectric constant multiplied by {epsilon}0, the constant for vacuum ({epsilon}0 = 8.8542 · 10–12 C · V1 · m1). In the case of uncharged solutes or fibers, the low dielectric constant will change the potential field of the charged solute or fiber surrounding, thus increasing the electrostatic free energy. For further details of the equations, please consult Johnson and Deen (16).

To apply the partition coefficients to experimental data, one must calculate fractional clearances ({theta}). In the concept of "fiber matrix," Curry and Michel (7) used the expression of Anderson and Malone (2) to calculate the reflection coefficient ({sigma}) from the partition coefficient ({Phi})

(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)
where A0/{Delta}x is the unrestricted exchange area over diffusion distance and D is the free diffusion constant. D/D0 is the relative diffusivity in a gel as presented by Phillips (29)

(11)

The fractional clearance ({theta}) is obtained using a nonlinear flux equation (30)

(12)
where GFR was 0.1 ml · min1 · g kidney1 (wet wt) in this study.

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 {theta} through the main gel ({theta}main gel) and that occurring through the large pore discontinuities ({theta}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/{Delta}x). Hence, {theta}L is calculated as for {theta}main gel except for the fact that

which will affect {sigma} and PS and the resulting fractional clearance can be written as

(13)

The important parameters in the model are: the fiber radius (rf), the relative concentration of fibers in the gel ({phi}), the surface charge densities of solute (qs) and fiber (qf), the unrestricted exchange area over diffusion distance (A0/{Delta}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/{Delta}x, {phi}, 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/{Delta}x, {phi}, 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 {theta}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.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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Reperfusion Experiments of the Fixed Kidney

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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Table 1. GFR and RPF

 

Fractional Clearance of BSA and Ficoll35.5Å

At normal I, the sieving coefficient, {theta}, 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, {theta} for albumin was 0.0030 (SE –0.0011, +0.0018, n = 6), not significant compared with that at normal I. {theta} For Ficoll35.5Å was 0.104 (SE 0.015, n = 6, not significant compared with normal I). Thus {theta} for Ficoll35.5Å was significantly higher than {theta} for albumin at low I as well (P < 0.01). For details, see Fig. 1.



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Fig. 1. Fractional clearance ({theta}) ± SE for Ficoll35.5Å (right) and albumin (left) with normal (151 mM, open bars) and low (34 mM, closed bars) ionic strength perfusates. The {theta} for Ficoll was higher than {theta} for albumin at both low (**P < 0.01) and normal (***P < 0.001) ionic strengths.

 

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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Fig. 2. Fractional clearance of Ficoll35.5Å plotted against the fractional clearance for albumin in the perfusion-fixed rat kidney. Data are shown for kidney perfusion with low ({bullet}) and normal ({circ}) ionic strength solutions.

 


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Fig. 3. Urine over plasma concentration ratios for Ficoll plotted against the Stokes-Einstein radius. Data are shown for kidney perfusion with low ({bullet}) and normal ({circ}) ionic strength solutions.

 

Gel-Membrane Model Analysis

The functional small pore radius was 36 Å (27–44 meq/l, 95% CI) at normal I and 33 Å (23–43 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, {omega}, was estimated to be 38 meq/l (28–71 meq/l, 95% CI) for normal I and 13 meq/l (11–16 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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Fig. 4. Estimated charge density using the gel-membrane model at normal and low ionic strength perfusion (**P < 0.01).

 

Heterogeneous Charged Fiber Model

In this model, the unrestricted exchange area over diffusion distance, A0/{Delta}x, was 100,000 cm. The fiber radius was 4.5 , the relative fiber volume, {phi}, 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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Table 2. Parameters of the hetergenous charged fiber model

 


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Fig. 5. Blandt-Altman plot describing the agreement between measured and modeled U/P concentration ratios for Ficolls of different sizes. At normal ionic strength (I), there is an acceptable agreement. For low ionic strength, however, the fitting is poor unless the fiber density is markedly reduced as for the diluted model.

 

In contrast to the Ficoll data, the U/P ratios for albumin were not adequately described by the heterogeneous charged fiber model, which overestimated {theta} for albumin six to eight times. Possible explanations are presented in DISCUSSION.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, the functional properties of the glutaraldehyde-fixed glomerular barrier were evaluated using a broad fraction of neutral spherical Ficoll molecules together with albumin using perfusate solutions of variable I. This experimental model may be considered highly artificial, but it is actually ideal for the purpose of this study, as previous studies suggested marked volume changes to occur in the charge-selective compartment in response to alterations of perfusate I (38).

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, {theta}, 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 {theta}albumin and {theta}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., {theta}tot = {theta}1 · {theta}2 · {theta}3 · {theta}4...{theta}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 {theta} 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 {theta} 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 {theta}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.


    DISCLOSURES
 
This study was supported by Swedish Medical Research Council Grants 9898, the Knut and Alice Wallenberg Research Foundation, the IngaBritt and Arne Lundbergs Research Foundation, and Sahlgrenska University Hospital Grant LUA-S71133.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. Hjalmarsson, Dept. of Physiology, Göteborg Univ., Box 432, SE-405 30 Göteborg, Sweden (E-mail: Clara.Hjalmarsson{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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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