2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3800; and 1Laboratory of Immunopathology, School of Medical Sciences, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Submitted 15 October 2002 ; accepted in final form 17 July 2003
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ABSTRACT |
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negatively charged macromolecules; albumin transport; glomerular capillary wall; facilitated clearance; charge selectivity
Recent published studies reported that charge selectivity can be measured in low-temperature perfusion of rat kidneys. The approach used in these studies was to measure the differences in the clearance of Ficoll and albumin, which were then used to calculate an apparent fixed charge density of the glomerular barrier (17). Other negatively charged proteins have also been examined, and the corresponding fixed charge on the glomerular barrier has been calculated (27). The major concern with the conclusions of these studies is that the calculated charge concentration of the glomerular barrier has not been confirmed by direct experimental measurement together with the fact that potential temperature-dependent interactions of the charged proteins with components of the perfusate and/or the kidney that may influence urinary excretion have not been eliminated.
The issue of whether charge selectivity exists is an important one in renal physiology. It is to be noted that no previously published biophysical study has ever demonstrated any significant electrostatic repulsion of albumin by any polyanion under physiological conditions (8, 12, 16, 25). The hypothesis to be tested in this study is whether the fractional clearance of a stable, negatively charged molecule, carboxymethyl Ficoll, which has similar globular conformation and charge to albumin, is lower than that of uncharged Ficoll of the same hydrodynamic radius. These studies will address the issue of whether glomerular charge selectivity is significant over and above that of kidney uptake of the transport probes and their potential binding by plasma components.
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MATERIALS AND METHODS |
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Preparation of Tritiated Polysaccharides
The radiolabeled Ficolls were prepared using a reductive technique with sodium boro-[3H]hydride, as described by Van Damme et al. (28). The labeled preparations were separated from free label by extensive dialysis against 0.15 M NaCl and chromatography on Sephadex G-25.
In Vivo Fractional Clearance Studies of Radiolabeled Polydisperse Ficoll 70 and Carboxymethyl Ficoll Using Short-Term Steady-State Method
Method 1: using [99mTc]DTPA to measure glomerular filtration rate. Male Sprague-Dawley rats (400450 g) were injected in the tail vein with either 1 x 108 dpm [3H]Ficoll (1.8 mg) plus 0.26 ml [99mTc]DTPA [for glomerular filtration rate (GFR) measurement] or 2 x 107 dpm [3H]carboxymethyl Ficoll (6.9 mg) plus 0.26 ml [99mTc]DTPA. The rats were then placed in a metabolic cage for urine collection. Exactly 43 min after the injections, the rats were bled from the tail vein for the GFR measurement (15). Two hours after the injection, the rats were placed under an infrared lamp, wrapped in a towel, and 0.5 ml blood were collected from the tail vein of rats into an Eppendorf with 5 µl heparin (1,000 IU/ml) for initial [3H]Ficoll or [3H]carboxymethyl Ficoll measurement in plasma. The rats emptied their bladder with this procedure. At 6 h after injection, rats were anesthetized with 0.40 ml Nembutal and bled by cardiac puncture into a heparinized 10-ml syringe for the 6-h measurement. Urine was collected over the 4-h period from 2 to 6 h. Blood and urinary samples were centrifuged for 10 min at 3,000 rpm, and plasma and urinary samples were counted for tritium using a -scintillation counter. As the 99mTc interferes with the tritium radioactivity analysis, it was found that samples for tritium should be stored for 3 days before counting began. Plasma and urinary samples were applied to a Sephacryl S-300 column for fractional clearance calculations. Urine flow rate (UFR) was calculated from the volume of urine collected over the 2- to 6-h period including any urine present in the bladder. GFR was measured by a single-injection isotopic technique using [99mTc]DTPA as previously described (15). There was no significant difference in the average UFR and GFR for both types of experiments, where for [3H]Ficoll 70 UFR was 0.0063 ± 0.0032 ml/min (n = 5) and GFR 3.31 ± 0.56 ml/min (n = 5) and for experiments with [3H]carboxymethyl Ficoll UFR was 0.0080 ± 0.0029 ml/min (n = 5) and GFR 4.02 ± 0.35 ml/min. There was a small reduction in the plasma radioactivity over the 2- to 6-h period (Fig. 1); the plasma concentration for molecules was taken as the mean of 2- and 6-h plasma radioactivity.
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Method 2: using creatinine to measure GFR. Sprague-Dawley rats were injected in the tail vein with 4.0 x 107 dpm of [3H]Ficoll 70 (0.4 mg) or 2.0 x 107 dpm of [3H]carboxymethyl Ficoll (7 mg) and placed in individual metabolic cages. For these experiments, the specific activity of [3H]Ficoll was 0.95 x 108 dpm/mg and for [3H]carboxymethyl Ficoll was 2.9 x 106 dpm/mg. The 1-ml dose of radiolabeled Ficoll or carboxymethyl Ficoll was determined to maximize radioactivity concentration in the plasma but with relatively low concentrations of circulating Ficoll and carboxymethyl Ficoll. Urine was collected between 0 and 4 h and between 4 and 6 h (around midday to 2 PM) after the injection, by the urine container in the metabolic cage and by collection from the bladder at 6 h. There was no significant difference in the average UFR and GFR for both types of experiments, where for [3H]Ficoll 70 UFR was 0.0123 ± 0.0035 ml/min (n = 5) and GFR 3.22 ± 1.16 ml/min (n = 5) and for experiments with [3H]carboxymethyl Ficoll UFR was 0.0135 ± 0.0054 ml/min (n = 6) and GFR 3.11 ± 2.38 ml/min (n = 6). There was a small reduction in the plasma radioactivity over the 4- to 6-h period (Fig. 1); the plasma concentration for molecules was taken as the mean of 4- and 6-h plasma radioactivity. Blood was collected via the tail vein into a heparinized syringe at 4 h, and a cardiac puncture was performed at 6 h with a heparinized syringe. Plasma and urinary samples at 6 h were analyzed for creatinine (7). The plasma and urinary samples were fractionated on a Sephacryl S-300 column, and the fractional clearance as a function of molecular radii was determined.
In Vivo Clearance Studies of Radiolabeled Carboxymethyl Ficoll and Uncharged Ficoll Using the Osmotic Pump Method
The Alzet osmotic pumps were filled with [3H]carboxymethyl Ficoll and implanted to individual rats. The procedure for using the osmotic pumps in rats has been described previously (5). GFR was determined by creatinine assay (7). UFR was determined by measuring the volume of the 24-h urine collection.
Chromatographic Analysis
Plasma and urinary samples were analyzed using a Sephacryl S-300 column (column dimensions 2 x 66 cm2). The Kav was determined by the formula (Ve Vo)/(Vt Vo), where Vo is the void volume, Ve the elution volume, and Vt is the total volume of the column. The column was run at 4°C with phosphate-buffered saline solution containing 2 mg/ml bovine serum albumin (used to prevent adsorption) and 0.02% azide. The column was calibrated with radiolabeled globular proteins albumin, transferrin, and immunoglobulin G of known radius. For Sephacryl S-300, a linear relationship was apparent between the semilog plot of radii vs. Kav. Other radii estimates were obtained by both interpolation and extrapolation of this graph.
Samples were also analyzed by ion-exchange chromatography using a Sepharose Q column (1.0 x 21 cm2). The samples were applied in 6 M urea, 0.05 M Tris, 0.05% (wt/vol) CHAPS, pH 7.0, and eluted with a linear gradient of 0.152.5 M NaCl in the same buffer at a flow rate of 0.5 ml/min.
Fractional Clearance Measurements
The fractional clearance of molecules eluted from the size exclusion chromatography column with the same Kav in plasma and urine was determined by radioactive counting, using samples collected at day 7 of osmotic pump implantation or 6 h after the bolus injection. Fractional clearance is defined as the product of the ratio of disintegrations per minute of a labeled urinary test molecule of a particular hydrodynamic radius to disintegrations per minute of a labeled plasma test molecule with the same hydrodynamic radius, times the ratio of UFR to GFR.
Kidney Digestion
The radiolabeled material accumulated in the kidney at the end of day 7 of the osmotic pump period was analyzed by removing the kidneys. They were then weighed, minced, and 1.4 M NaOH was added to make a final volume of 6 ml. The samples were then heated in boiling water for 20 min. Before counting of radioactivity began, 50 µl of hydrogen peroxide were added to each 100-µl sample to decolorize the solution.
Counting of Radioactivity
Radioactivity from tritium-labeled material was determined by scintillation counting in a LKB Wallac 1410 liquid scintillation analyzer, using a 1:4 aqueous sample-to-Optiphase scintillation ratio. 22Na and [99mTc]DTPA were determined using a United Technologies Packard Model Minaxi 5530.
Calculations
All quantitative data are expressed as means ± SD, where n represents the number of determinations. Significance of the results was determined using Student's t-test.
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RESULTS |
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Five milliliters of Ficoll and carboxymethyl Ficoll at 16.7 mg/ml in 0.001 M NaCl were dialyzed for 48 h at 4°C against 250 ml of 0.001 M NaCl containing 1 x 106 dpm 22Na. The average (n = 3) disintegrations per minute per milliliter in the dialysate was 2,635 ± 26 dpm/ml, whereas for the Ficoll solution it was 2,569 ± 173 dpm/ml, and for carboxymethyl Ficoll it was 55,314 ± 948 dpm/ml. Assuming that the 22NaCl concentration in the dialysis tube is negligible (it will be significantly less than 2,635 dpm/ml), then the degree of carboxyl substitution per sucrose on the carboxymethyl Ficoll from 22Na partitioning is calculated to be 0.54 (compared with 0.34 from manufacturer's titration). This demonstrates the high negative charge valence of the carboxymethyl Ficoll preparation by binding relatively large quantities of the sodium counter ion. The carboxymethyl Ficoll elutes on the Sepharose Q ion-exchange column with 0.45 M NaCl (see also Fig. 5). A Ficoll with MW of 48,000, which would be equivalent to albumin in size based on partial specific volumes, substituted with 0.34 carboxyl groups per sucrose residue, would have a valence of 50.
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Short-Term Fractional Clearance Studies
[3H]carboxymethyl Ficoll used in the short-term steady-state experiments was not biochemically altered in plasma or in urine as determined by ion-exchange chromatography (not shown, although ion-exchange analysis for long-term osmotic pump experiments is demonstrated in Fig. 5). Both ion-exchange and size exclusion chromatographic analysis of plasma samples demonstrated that there was no binding of the carboxymethyl Ficoll to plasma components to generate higher MW components (not shown). Kidney uptake of Ficoll was 3.52 x 105 ± 1.68 x 105 dpm/kidney (n = 4) compared with plasma of 1.61 x 106 ± 0.55 x 106 dpm/ml (n = 4) and that of carboxymethyl Ficoll was 0.85 x 105 ± 0.35 x 105 dpm/kidney (n = 6) compared with plasma of 3.16 x 105 ± 1.22 x 105 dpm (n = 6). The circulating plasma concentration of carboxymethyl Ficoll was <1 mg/ml and so would have a negligible effect on the osmotic properties and net charge concentration of plasma.
Fractional clearances of both Ficolls were examined by two short-term methods differing essentially in the manner that GFR was measured, which was either through the use of creatinine (Fig. 2A) or [99mTc]DTPA (Fig. 2B) or long-term osmotic pump studies (Fig. 2C). In general, the fractional clearance measurements were higher for GFR determined by creatinine clearance, but otherwise the relative differences in the fractional clearances of carboxymethyl Ficoll and Ficoll were the same. The fractional clearances corresponding to a radius of 36 Å gave similar values for both Ficoll 70 and carboxymethyl Ficoll (Fig. 2). On the other hand, the fractional clearances as a function of molecular radius as shown in Fig. 2 demonstrate that irrespective of the method of GFR measurement, it is evident that [3H]carboxymethyl Ficoll facilitated fractional clearance for radii >45 Å compared with [3H]Ficoll. This is even more apparent from the size exclusion chromatographic analysis on Sephacryl S-300 for both plasma [3H]carboxymethyl Ficoll and [3H]Ficoll as shown in Fig. 3. Both preparations have a similar distribution of radiolabeled plasma material as a function of hydrodynamic radius (determined by calibrating the column with proteins of known radii; Fig. 3A). There was no depolymerization of the material in the circulation. However, size exclusion analysis profiles of urinary material (Fig. 3B) demonstrated that only for [3H]Ficoll was there a marked shift to material being excreted with lower molecular radii compared with [3H]carboxymethyl Ficoll (Fig. 3B). The size exclusion profile of urinary [3H]Ficoll was not altered when [3H]Ficoll was studied in the presence of excess quantities of unlabeled carboxymethyl Ficoll (Fig. 4). This demonstrates that renal function was not compromised by the presence of carboxymethyl Ficoll.
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A similar distribution for plasma and urinary carboxymethyl Ficoll was obtained from the osmotic pump studies after 7 days (not shown). The fractional clearances estimated from using this technique are shown in Fig. 2. These fractional clearances, obtained after steady-state clearances at day 7, also exhibit significant facilitated clearance as noted with the short-term studies. Ion-exchange analysis (Fig. 5) demonstrated that there was no significant decarboxylation of the [3H]carboxmethyl Ficoll sample collected from either plasma or urinary samples at day 7. Both samples eluted from the ion-exchange column at a NaCl concentration of 0.45 M.
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DISCUSSION |
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It is surprising that experimental evidence to demonstrate the direct effect of charge repulsion of the GCW on negatively charged transport probes has yet to be published. There have been numerous investigations reporting apparent restricted transport of polyanions compared with their neutral counterparts. Initially, there were studies of the apparent restriction of negatively charged electron-dense probes (23, 24). However, a good deal of caution should accompany the interpretation of these types of results particularly when conclusions concerning transglomerular passage are made. There is no a priori relationship between localization of the probe and fractional clearance. The ultrastructural localization is performed under non-equilibrium conditions, whereas fractional clearance is a steady-state measurement. Localization may mean genuine transport restriction but then it may also represent a binding interaction. Furthermore, the presence of an exogenous probe may exert cooperative effects to influence further localization through changes in filter structure. Overall, it is very difficult to interpret the ultrastructural data alone in terms of transglomerular transport.
More quantitative studies of labeled probes appearing in the urine have been put in doubt as the probes have subsequently been shown to be significantly modified during filtration and renal passage (21). More direct efforts to measure charge repulsion by the GCW or its critical components demonstrated that its effect is negligible. Bolton et al. (4) showed that transport of Ficoll sulfate is not charge affected by the glomerular basement membrane. Studies using the isolated, perfused kidney demonstrated that dextran sulfate with degrees of substitution (1.7) shows apparent charge selectivity or transglomerular-restricted clearance when used at low concentrations in the perfusate but apparent charge selectivity or restricted passage disappears when the concentration of dextran sulfate in the perfusate is increased (29). This is consistent with other results for dextran sulfate with degrees of substitution <1.0, which indicate that charge repulsion by the GCW to polyanions is negligible (29).
Accurate thermodynamic interaction studies of albumin interaction with charged polysaccharides and the partitioning of albumin in nonrenal extracellular matrix tissues containing high concentrations of charged polysaccharides have all been demonstrated to be independent of charge effects under physiological conditions (8, 12, 13, 16, 25, 26). Furthermore, the thermodynamic interaction of negatively charged albumin with itself, at relatively high concentrations, is purely governed by nonelectrostatic excluded volume effects (8, 25). Specifically, experimental studies of albumin partitioning from compartments containing the highly charged glycosaminoglycan hyaluronan at a charge concentration of 37 meq/l yielded partition coefficients of 0.2 to 0.4 (12, 25). These partition coefficients included the effects of both steric exclusion and potential charge interactions. The investigators showed the latter was negligible because the partitioning does not change by increasing ionic strength. This compares with the estimate of the partition coefficient at the interface between the GCW and perfusate of 0.05 that comes from isolated, perfused kidney studies when albumin clearance is compared with uncharged Ficoll clearance (17). Other studies indicated that if there is an electrostatic interaction of anionic polysaccharides with albumin, it is a close proximity binding one (2, 9) rather than one of electrostatic repulsion.
In the cold perfusion studies of Haraldsson et al. (11, 17, 18, 27), there are a number of unexplained findings with the technique as well as some inconsistencies with the classic charge selectivity concept. No experimental studies have been forthcoming to explain why albumin clearance, as studied using their technique, is markedly temperature dependent because perfusion studies yield fractional clearance of albumin of 0.022 at 37°C, whereas at 8°C it was 0.002; for Ficoll clearance, there was no effect of temperature and that apparent charge selectivity only occurs at 8°C but not at 37°C. Apart from the potential involvement of albumin in temperature-dependent interactions with components of the perfusate or the kidney, proof that 8°C perfusate perfusing an in situ kidney in a rat maintained at 37°C inhibits renal cell uptake of proteins has not been provided. Recent studies demonstrated cellular protein uptake may occur at 4°C (14), so assumptions of zero uptake in the low-temperature perfusion studies have to be tested. Perhaps the most surprising result of the low-temperature perfusion is that albumin clearance is only minimally affected by increasing the ionic strength of the perfusion medium to that containing 0.3 M NaCl (27). A further issue is that human albumin used in their studies is not characterized for charge density. The charge valence on the molecule may vary greatly depending on whether the albumin is carrying fatty acids (22).
Apart from their own cold perfusion work, Haraldsson et al. cite studies where charge selectivity
has been measured using the relative clearance differences of neutral and negatively charged myoglobin (30). Although the relative difference in transport was only 3%, these authors eventually calculated a glomerular charge barrier concentration of
30 meq/l. Another study cited to support charge selectivity is that of dextran sulfate clearance for a dextran sulfate radius range of only 1025Å (10). These studies did not analyze the desulfation of dextran sulfate during renal passage.
The conclusion from the studies reported here is that exclusion of albumin, as modeled by negatively charged Ficoll, from the GCW is not based on charge repulsion, the basis of conventional theories associated with charge selectivity. The charge repulsion interaction of albumin with another polyanion has yet to be demonstrated under physiological conditions. The facilitated clearance of negatively charged Ficoll across the GCW adds to the growing observations reported in the literature that many stable nonproteinaceous polyanions have facilitated clearance. At this stage, there is no evidence to suggest that albumin transport across the GCW is governed by repulsive charge interactions with the negatively charged GCW.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Since the submission of this article, another publication has demonstrated that stable negatively charged dextran and hydroxyethyl starch also do not exhibit negative charge selectivity associated with renal filtration in rats (Schaeffer RC, Gratrix ML, Mucha DR, and Carbajal JM. The rat glomerular filtration barrier does not show negative charge selectivity. Microcirculation 9: 329342, 2002).
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REFERENCES |
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