Departments of 1 Physiology and 2 Nephrology, Göteborg University, SE-405 30 Göteborg, Sweden
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ABSTRACT |
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The fractional clearances () for
FITC-Ficoll and albumin were estimated in isolated perfused rat kidneys
in which the tubular activity was inhibited by low temperature (8°C)
and/or 10 mM NH4Cl. The Ficoll data were analyzed according
to a two-pore model giving small and large pore radii of 46 Å and
80-87 Å, respectively. The estimated negative charge density was
35-45 meq/l at 8°C. Perfusion with erythrocyte-free solutions of
kidneys at 37°C reduced glomerular size and charge
permselectivity. Thus the large pore fraction of the glomerular
filtrate (fL) was 1.64% at 37°C compared with 0.94% at
8°C. The
for albumin was four times higher at 37°C than at
8°C (0.86% vs. 0.19%, respectively). NH4Cl caused further irreversible damage to the glomerular barrier. We conclude that
there are no deleterious effects on the glomerular barrier of a
reduction in temperature from 37°C to 8°C. Therefore our data seem
to disprove the hypothesis of low glomerular permselectivity and
transtubular uptake of intact albumin and support the classic concept of a highly selective glomerular barrier.
capillary permeability; macromolecular transport; two-pore model
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INTRODUCTION |
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THE MAINTENANCE OF AN INTACT glomerular barrier to macromolecules and in particular albumin plays a pivotal role for the electrolyte and fluid balance in the body. However, the properties of this intricate membrane are not yet completely understood, and even less is known about the disturbances leading to proteinuria. There are, for example, controversies concerning which glomerular layer constitutes the principal barrier: the endothelium, the basement membrane, or the podocytes.
The classic dextran studies by Chang and associates
(5-7) revealed that small molecules are freely
filtered across the membrane, whereas the fractional clearance ()
for molecules with Stokes-Einstein radius (aSE)
>42 Å approaches zero (6). In this context, the term
"Stokes-Einstein radius" is always used to describe molecular size,
since it is derived from the free diffusion constant. Also, the passage
of anionic molecules is restricted, whereas that of cationic ones seems
to be enhanced (5).
Recently, an alternative hypothesis of glomerular permselectivity was
presented (29). Isolated kidneys, removed by en bloc dissection, were perfused with recirculated filtered 5% BSA in a
Krebs-Henseleit buffer at 37°C. The tubular reabsorption was inhibited by the use of various drugs, i.e., NH4Cl.
Dextrans with aSE of 26-50 Å were used to
determine whether the glomerular permselectivity was unaffected by the
drugs. The for albumin in the control situation was
0.75-0.9%, increasing to ~7% when the drugs were used. To
explain these high
values, the authors suggested that a new
transtubular cell pathway must be responsible for the return of intact
albumin from the tubular lumen to the blood (29). Also,
the glomerular charge selectivity was suggested as being insignificant.
However, the use of dextran as a transport probe for the ideal neutral
sphere has been questioned. Rennke et al. (30) used the
protein horseradish peroxidase (HRP) and found that the values for
its cationic, neutral, and anionic forms were less than those for
dextran with similar size and charge. Dextrans form random-coiled
spheres in free solution and are vulnerable to deforming forces. It was
suggested that dextran is subjected to unfolding during convectional
transport across the glomerulus. Dextran would thus behave as if it was
of smaller dimensions than assumed. Oliver et al. (27)
found that Ficolls of various radii had a lower
than dextrans of
equal aSE. This implies that Ficoll might be a
better probe for the measurement of the equivalent small and large pore
radii. Also, Blouch et al. (3) found that over a molecular
radius interval of 20-70 Å,
for a given Ficoll was uniformly
lower than the corresponding
for a dextran of equal molecular
radius, both in healthy and nephrotic humans. Solute shape is in fact
highly important for its glomerular passage and may actually outweigh
size and charge (22).
One way of inhibiting the tubular activity is low temperature. Reduced temperature inhibits tubular function as well as energy consumption and myogenic tone (8, 12), and it reduces protease activity without detectable changes on capillary permeability (33).
Thus at present there are two dramatically different views of glomerular permeability: the classic highly selective barrier, and a new hypothesis of "leaky" glomerular capillaries with massive tubular uptake. To understand the reason for the disparate results, we decided to test some of the experimental conditions used by Osicka et al. (29). We used the isolated perfused kidneys (IPK) to estimate the clearances for albumin and Ficoll with or without inhibited tubular activity, obtained by low temperature (8°C) and/or NH4Cl.
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METHODS |
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Kidney Perfusion Technique
Fifteen male rats weighing between 250 and 310 g (Wistar strain; Møllegaard, Stensved, Denmark) were used. The rats were kept on standard chow and had free access to water prior to the experiments. The local ethics committee approved the experiments.We used a modification of the isolated perfused rat kidney preparation described by Johnsson and Haraldsson in 1992 (18) that has been described in detail previously (37). Anesthesia was induced by an intraperitoneal injection of pentobarbital (50 mg/ml; Apoteksbolaget, Umeå, Sweden), and a thermostatically controlled heating pad maintained the body temperature of the rat at 37°C. The tail artery was cannulated to establish a route for subsequent administration of drugs and for recording of the arterial pressure (PA). The experiments were performed at either 8°C or 37°C.
Both kidneys from six rats were used in the cold experimental setup. The rat was eviscerated, and the intestines were removed. Cannulation of both ureters (PE-25 cannulas) was facilitated by enhanced diuresis after injection of furosemide (2 mg/kg; Benzon Pharma, Copenhagen, Denmark) and saline (0.4 ml). The rat was heparinized (1,000 IU; Lövens Läkemedel, Malmö, Sweden), and the aorta was thereafter cannulated in a retrograde direction, thus allowing for artificial perfusion of the kidneys by use of a peristaltic pump (model IPC-04 V1.32; Ismatech, Zurich, Switzerland). A second cannula was inserted into the thoracic aorta with the tip close to the right renal artery. The two kidneys were perfused with completely separate perfusion lines by ligating the aorta between the renal arteries.
Nine kidneys from nine rats were used in the warm experiments. The first part of the preparation did not differ from the cold setup, except that only the left ureter was cannulated. The aorta was ligated distal to the renal arteries and cannulated in a retrograde direction, providing the kidneys with both perfusate and their own circulation. Following a ligature on the aorta proximal to the left renal artery, the caval vein was cut open.
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.
Near the aortic inlets, T-tubes connected to pressure transducers (PVB Medizintechnik, Kirchenseeon, Germany) recorded mean PA values. The urine was collected in small vials and continuously weighed for calculation of urine flow. A computer (PC 586), using Labview computer software, monitored PA and urine weight changes as well as urine flow and pump speed.
Perfusates
Three different perfusates were used, all based on a modified Tyrode solution containing human albumin (18 g/l; Pharmacia & Upjohn, Uppsala, Sweden). The standard perfusate contained the following: 113 mM NaCl, 4.3 mM KCl, 2.5 mM CaCl2, 0.8 mM MgCl2, 25.5 mM NaHCO3, 0.5 mM NaH2PO4, 5.6 mM glucose, nitroprusside (0.9 mM; Merck, Darmstadt, Germany), and furosemide (10 mg/l, Benzon Pharma). For the second perfusate, 2 g/l of FITC-labeled Ficoll (Ficoll-70; Bioflor, Uppsala, Sweden) was added to the standard solution. The third perfusate was obtained by adding 2 g/l of FITC-Ficoll and 10 mmol/l NH4Cl. The perfusates were protected from light and bubbled with 5% CO2 in O2. All solutions were made with fresh distilled water (Millipore) with a resistivity of 18.2 MExperimental Protocol
The perfusion started with the standard solution for 20 min. Then the perfusate with FITC-Ficoll was used for about 15 min, after which the perfusate containing both FITC-Ficoll and NH4Cl was used for another 15 min. Finally, the kidneys were perfused with the standard solution with FITC-Ficoll. Total perfusion time was approximately 1 h. During each perfusion period, three to five urine samples were collected for determination of glomerular filtration rate (GFR) andAnalysis of Ficoll Concentrations
The perfusates and the urine samples were subjected to gel filtration on a Superose 12 PC 3.2/30 column (SMART HPLC; Amersham Pharmacia Biotech, Uppsala, Sweden) for calculation of the sieving coefficients for FITC-Ficoll. The total bed volume of the column was 2.4 ml, and the void volume was 0.97 ml. A 0.05 M phosphate buffer with 0.15 M NaCl with pH 7.0 was used as an eluent. A 20-µl sample was analyzed at 490 nm with a flow of 40 µl/min. The pressure was ~1.2 MPa and the temperature was kept at 8°C during the analysis. The distribution of FITC-Ficoll in the perfusate and in the urine, from a representative experiment at 37°C (control situation), is shown in Fig. 1. The estimation of
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Calibration Curve
Six monodisperse samples of Ficoll with known molecular radii, generously provided by Dr. Torvald Andersson (Amersham Pharmacia Biotech, Uppsala, Sweden), were marked with FITC. The labeling was carried out by dissolving 1 g of Ficoll in 20 ml dimethyl sulfoxide (DMSO), to which 20 mg of sodium bicarbonate and 100 mg of FITC were added. The solution was heated for 15 min in a boiling water bath and was then poured slowly into 200 ml ethanol for precipitation overnight. The FITC-Ficoll was resolved in 20 ml distilled water, and pH was adjusted to 6.5-7.0. The labeled Ficoll was then eluted on an equilibrated desalting column (Sephadex G-25 PD-10) to reduce the free FITC content. The FITC-marked monodispere Ficolls were run on a Superose 6 PC 3.2/30 column (SMART HPLC) and their molecular radii were unaltered after the FITC labeling.The labeled FITC-Ficoll molecules and a selection of protein
standards (Calibration Kit; Amersham Pharmacia Biotech, Uppsala, Sweden); blue dextran 2000, vitamin B12, thyroglobulin,
ferritin, albumin, ovalbumin, aldolase, chymotrypsinogen A, and RNase
A, were used to obtain a calibration curve on the Superose 12 PC 3.2/30
column (SMART HPLC). The calibration curve is shown in Fig.
2, where the aSE
of the molecule on the x-axis corresponds to the eluted
volume on the y-axis.
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Calculations
Glomerular filtration rate. GFR was calculated according to the formula urine over plasma concentration ratio (CU/CP) of 51Cr-EDTA times urine flow (QU), i.e., GFR = (CU/ CP)Cr-EDTA · QU.
Fractional clearance for albumin and Ficoll.
The renal clearance (Cl) of a solute, x, can be estimated
from the amount excreted in the urine (CU) during a certain
time period over the plasma concentration (CP), i.e.,
Cl = (CU/CP)x · QU. The of a solute is given by its Cl over GFR.
The two-pore model.
The exchange can be described using the following parameters: small
pore radius, rs, large pore radius,
rL, the large pore fraction of the glomerular
filtrate, fL, and finally the unrestricted pore area over
diffusion distance, A0/x. The net
fluxes of fluid and solutes are calculated for each pore pathway
separately using nonlinear flux equations (32). In the
analysis, A0/
x was assumed to be
equal to or larger than 10,000 cm. The viscosity of water, unique for
every molecular radius, was included in the model. The temperature will
influence charge interactions as evident from the equations for Debye
length (see Ref. 37). The effect is, however, small (5%) and
was therefore not included in the analysis.
Charge selectivity.
The effects of molecular charge were estimated using the Donnan concept
of charge-charge interactions as described in a previous report
(37). The crucial parameter to determine is the
concentration of fixed negative charges within the gel, .
Statistics
Results are presented as means ± SE, and differences were tested using the Wilcoxon signed rank sum test or Student's t-test where appropriate. ![]() |
RESULTS |
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General
Mean values ± SE for PA, pump flow (Q), vascular resistance (PRU100), GFR, and
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Sieving Coefficients for Ficoll
The sieving coefficients for FITC-Ficoll were not significantly different between 8°C and 37°C for the various perfusion periods used. Figure 4 shows the sieving coefficients for FITC-Ficoll after perfusion with 10 mM NH4Cl. The
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Two-Pore Model Analysis
The Ficoll data were analyzed according to a two-pore model (32). The results from that analysis are presented in Table 3. The small pore radius was rather precise at both 8°C and 37°C, being 45.5 ± 0.36 and 46.3 ± 0.25 Å, respectively. Moreover, the small pore radius did not differ between the different perfusion periods (Fig. 5). The large pore radius varied between 80 and 87 Å, at the different temperatures and for the different perfusion periods that were used. The large pore fraction of the glomerular filtrate (fL) was 1.64 ± 0.12% at 37°C compared with 0.94 ± 0.13% at 8°C (P < 0.05, n = 8) (Fig. 6). Perfusion with NH4Cl increased fL even further to 2.42 ± 0.15% at 37°C (P < 0.05, n = 8) and 1.72 ± 0.10% at 8°C (P < 0.01, n = 8) (Fig. 6). The increase in fL was not completely reversible when returning to the perfusate without NH4Cl. During control, both at 8°C and 37°C, the pore area over diffusion distance (A0/
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Sieving Coefficients for Albumin
During control, the
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Glomerular Charge Density
Fractional clearance ratios were calculated from the
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Stability of the Two-Pore Parameters
The standard errors of the four parameters from the pore analysis are shown in Table 3. They are based on the mean ![]() |
DISCUSSION |
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This study supports and extends the classic view of the highly selective glomerular barrier.
First, our data seem to disprove the recently launched "albumin
retrieval" hypothesis (29), which suggests a low
glomerular permeability together with massive tubular uptake of intact
albumin. The low albumin at 8°C, a temperature
expected to strongly inhibit endocytotic uptake of filtered proteins
(25, 26), provides strong evidence in favor
of the classic view of high glomerular permselectivity.
The present albumin of 0.19% is far less than that
reported from Osicka et al. (29), where the tubular uptake
of proteins was inhibited by various toxins, i.e., 150 mM lysine, 10 mM
NH4Cl, 0.1 mM chloroquine, and 0.02 mM cytochalasin B. Under such conditions,
albumin reached 7%, which led
the authors to postulate the presence of a transtubular cell pathway
transporting intact albumin from the tubular lumen to the blood. The
present study offers limited scope for such mechanisms.
Indeed, previous morphological studies in rat (2) and in humans (34) indicate that almost no endogenous albumin enters the urine. Also, Ryan and Karnovsky (35) showed that as long as the kidneys were perfused, no detectable amounts of albumin entered the urine (35). Moreover, albumin entering the proximal tubule seems to be degraded (24). Against this background, we suggest that the glomerular barrier may have been damaged in the studies by Osicka et al. (29), resulting from the ischemia-reperfusion, accumulation of substances in the perfusate due to recirculation, and ultimately resulting from toxic effects of the drugs used to inhibit tubular uptake.
Second, the Ficoll data show that glomerular size selectivity is well described by a two-pore model. The functional small pore radius is close to 46 Å, whereas the less frequent large pores have a functional radius of 80-90 Å. Our results are well in accordance with the previously noted clearance values for Ficoll in humans (3) and in rat (28). The large pore radius found in the present study is similar to that found when using two lactate dehydrogenase (LDH) isoforms as tracer molecules (23), being 75-85 Å. Note, however, that the functional pore radii in the classic dextran studies were considerably higher (10). Indeed, there are few dextran studies that suggest lower pore radii (21).
Third, the glomerular barrier does not seem to be significantly
affected by a reduced temperature. Thus the small and large pore radii
were similar in kidneys perfused either at 8°C or at 37°C (Fig. 5).
Low temperature does not affect the permselective properties of the
glomerular barrier, which are in accordance with data from skeletal
muscle (31). Moreover, the reported results cannot be due
to toxic effects of FITC on the glomerular cells, since
albumin was measured before and after FITC-Ficoll administration both at 8°C and at 37°C. There was, however, a slight time-dependent increase in
for albumin of 0.30% per hour at
8°C and 1.4% per hour at 37°C. Indeed, the present
albumin is only slightly higher than that reported by
Tojo and Endou (38) from a micropuncture study. That study
is particularly interesting since it was designed to correct for
tubular modification of the urine composition. Thus albumin and inulin
U/P ratios were determined, and the U/P for albumin was then plotted
vs. U/P for inulin. Extrapolation to U/P = 1 for inulin would
represent the true glomerular filtrate
albumin.
Fourth, the classic notion of a charge barrier is supported in the
present study by the much lower sieving coefficient of albumin compared
with Ficoll of equivalent size (36 Å). The sieving data for Ficoll and
albumin were analyzed according to a simplified model of charge-charge
interactions (37). The charge density, , in the gel (or
membrane) was 43 meq/l in the experiments performed at 8°C. Similar
charge density values for the glomerular wall have been found using
native and charge-modified myoglobin (42), LDH isoenzymes
(23), and native and charge-modified HRP
(37). Interestingly, Huxley et al. (17)
estimated a charge density of 34 meq/l in single mesenteric capillaries
perfused with plasma. Thus peripheral (13) and glomerular
capillaries may actually have similar charge-selective properties but
differ in the radii of the small and large pore pathways. Thus the
charge density was probably overestimated using dextran sulfate, with
values of 120-170 meq/l (11).
Fifth, hypoxia and/or ammonium chloride may damage the glomerular barrier. Perfusion with erythrocyte-free solution at 37°C increased the number of large pores. Thus the large pore fraction of the glomerular filtrate (fL) was 1.64% at 37°C and 0.94% at 8°C. Perfusion with ammonium chloride increased the number of large pores even further, with fL values of 2.42% and 1.72% at 37°C and 8°C, respectively. These changes were not completely reversible when returning to the perfusate without NH4Cl. It is well known from other organs that inflammatory reactions increase the number of large pores rather than the average pore radii (14, 31). These effects are caused by endothelial cell contraction, which has been extensively described (39). For the glomerular barrier, the exact position of the size selectivity is debated (basement membrane or podocyte slit-membrane?), making interpretations highly speculative.
Both high temperature and NH4Cl reduced the charge
selectivity. Thus the for albumin was four times higher in the warm
experiments than in the cold ones (0.86% and 0.19%, respectively).
Ammonium chloride increased the
for albumin to 1.9% at 37°C and
to 0.28% at 8°C. Again, these changes were not completely reversible
when returning to the standard perfusate. Figure 8 shows that the
isolated kidneys perfused at 37°C had significantly less fixed
negative charges (29 meq/l) compared with the charge density (
) at
8°C (43 meq/l). Ammonium chloride seemed to reduce
even further.
The effects of temperature on the number of large pores and on the charge density are probably due to ischemic damage in both the size and charge restrictivity of the glomerular barrier induced by artificial perfusion of the kidneys at 37°C. Indeed, erythrocyte-free perfusion at 37°C is known to cause hypoxic cell damage in the proximal tubule (1, 16, 36) as well as in the thick ascending limb cells (4). Moreover, the endothelial glycocalyx (9, 41) as well as the glomerular basement membranes (40) have been shown to be sensitive to hypoxia/reperfusion, which leads to loss of proteoglycans (40).
In summary, the present study supports the classic view of glomerular permselectivity. We found no support for the so-called "albumin retrieval hypothesis." On the contrary, the glomerular membrane is highly permselective to macromolecules. Artificial perfusion of the kidney at 8°C does not affect glomerular permselectivity. Ammonium chloride and erythrocyte-free perfusion at 37°C increased the number of large pores and reduced the charge density. Taken together with our recent findings on the effects of ionic strength on the charge barrier (37), the present data are compatible with the following notions of the glomerular barrier: 1) The glomerular membrane is composed of two separate barriers in series, one charge selective and one size selective. 2) The size barrier is heteroporous with numerous small pores (radius 46 Å) and far less frequent large pores (80-87 Å). 3) The charge barrier behaves as a gel containing 35-45 meq/l of fixed negative charges. 4) Glomerular charge selectivity is exceeded by a structure that is more dynamic and vulnerable than the size barrier. 5) Finally, we propose that proteoglycans produced by the endothelial cells play a much more important role for glomerular charge selectivity than hitherto suggested.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Torvald Andersson (Amersham Pharmacia Biotech, Uppsala, Sweden) for the generous gift of the well-characterized monodispere Ficoll molecules and for the determination of their size after the FITC labeling.
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FOOTNOTES |
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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 the Gothenburg Medical Society.
Address for reprint requests and other correspondence: M. Ohlson, Dept. of Physiology, Göteborg Univ., Box 432, SE-405 30 Göteborg, Sweden (E-mail: maria.ohlson{at}fysiologi.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. §1734 solely to indicate this fact.
Received 24 August 1999; accepted in final form 27 January 2000.
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