1 Renal Division, Department of Clinical Medicine, and 2 Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo 01246-903, Brazil
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ABSTRACT |
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Plasma albumin restricts capillary water filtration. Accordingly, the glomerular ultrafiltration coefficient is higher in Nagase analbuminemic rats (NAR) than in Sprague-Dawley controls. We investigated whether the glomerular permeability to macromolecules is also enhanced in NAR. SDS-PAGE fractionation of urine proteins showed several bands with molecular masses between 60 and 90 kDa in NAR only. Acute administration of BSA to NAR led to nearly complete disappearance of these proteins from urine, an effect partially reversed when most of the exogenous albumin was cleared from circulation. The fractional clearance of 70-kDa dextran was increased in NAR, indicating a size defect. Binding of cationized ferritin to the glomerular basement membrane was decreased in NAR, suggesting associated depletion of fixed anions. The magnitude of cationic ferritin binding correlated negatively with the fractional clearance of 70-kDa dextran, suggesting that the two abnormalities may share a common pathogenic mechanism. Collectively, these results suggest enhanced glomerular permeability to macromolecules in NAR. Albumin may be necessary to maintain the normal glomerular permselectivity properties.
glomerulus; analbuminemia; albumin; capillary permeability; permselectivity
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INTRODUCTION |
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IN ADDITION TO ITS WELL-KNOWN contribution to plasma oncotic pressure, circulating albumin may directly influence the physical properties of the capillary walls. Studies of artificially perfused capillaries (6, 14, 15) showed that removal of albumin from the perfusing solution led to a massive increase in the capillary ultrafiltration coefficient (Kf). These findings prompted the hypothesis that small amounts of albumin are adsorbed to the capillary walls and directly influence their physiological properties by some interaction with its constituents, such as collagen and proteoglycan fibrils (3, 18).
Albumin appears to influence the physical properties of glomerular capillaries as well. In Nagase analbuminemic rats (NAR), a mutant of the Sprague-Dawley (SD) strain devoid of circulating albumin (19), we showed increased glomerular Kf (8), consistent with the findings obtained in nonrenal capillaries. However, the glomerular permeability of these rats to macromolecules has not been examined.
The glomerulus differs from other fenestrated and nonfenestrated capillary beds in several aspects. First, the magnitude of the glomerular hydraulic conductance and the pressures and flows prevailing at the glomerular microcirculation are considerably higher than in nonrenal capillaries. Second, the glomerular capillary wall is unique from an anatomic point of view, because barriers nonexistent in other territories, such as the slit membrane (4, 21), are crucial to limit the passage of fluid and solutes. Given these differences, it is uncertain whether plasma albumin influences the permselectivity properties of the glomerular capillary in the same manner as it does elsewhere.
In the present study, we verified directly and in the whole animal the hypothesis that circulating albumin, in addition to its effect on hydraulic conductance, also influences the glomerular barrier function. For this purpose, we analyzed the composition of urinary proteins in NAR and SD rats as well as the effect of exogenous albumin administration. We also evaluated the glomerular size-selective properties of NAR and SD rats by assessing the fractional clearance of 70-kDa neutral dextran. In addition, we estimated the glomerular fixed anionic charge density of these animals by quantifying the deposition of cationized ferritin (CF) at the glomerular basement membrane (GBM).
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METHODS |
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Adult male rats (32 SD rats and 34 NAR) obtained from a local colony were given free access to regular (25% protein) rat chow and tap water before the study. NAR were originally purchased from CLEA Japan (Tokyo, Japan).
Protocol 1: Fractionation of Proteins in Plasma and Urine
Eight SD rats and 8 NAR were placed in metabolic cages during 24 h for urinary protein analysis. At the end of this period, blood samples were collected from a tail vein. Total protein concentrations in plasma and urine were determined by the biuret reaction. The composition of plasma and urinary proteins was assessed by SDS-PAGE, utilizing an adaptation of the discontinuous system described by Laemmli (13), with a 5-15% polyacrylamide gradient. The volume of urine samples and standard solutions placed in each well was individually adjusted to achieve a total protein content equal to 15 µg. Protein standards (Pharmacia, Uppsala, Sweden) ranged from 10 to 100 kDa. Purified rat IgG was used as an additional, high-molecular-weight standard. The plates were stained with 0.1% Coomassie blue R-250 (Pharmacia), dissolved in 25% methanol and 10% acetic acid in water.Effect of acute albumin administration on proteinuria. In a separate cohort of 13 SD rats and 16 NAR, a blood sample was collected from a tail vein for baseline measurements. Shortly thereafter, rats were placed in metabolic cages for urine collection during a 12-h overnight period. On the following evening, 8 NAR and 8 SD rats from this cohort received a single injection of BSA (250 mg/kg in 0.5 ml saline, Sigma, St. Louis, MO) through a tail vein. The remaining 5 SD rats and 8 NAR received saline only. Rats were then returned to the metabolic cages, and urine was collected during a second overnight period, at the end of which a second blood sample was taken. Rats were again returned to the metabolic cages on the following evening, and a third overnight urine collection was performed, at the end of which a third tail blood sample was obtained. Assessment of total protein in plasma and urine, as well as protein fractionation, was then performed as described above.
Protocol 2: Measurement of Fractional Dextran Clearance
To assess the GFR and the glomerular permeability to 70-kDa neutral dextran, 11 SD rats and 10 NAR were anesthetized with Inactin (100 mg/kg body wt ip) and prepared on a surgical table for clearance studies. Rectal temperature was maintained at 37 ± 0.5°C. The left femoral artery was cannulated with PE-50 polyethylene tubing for determination of baseline hematocrit, for continuous blood pressure monitoring, and for blood collection throughout the experiment. A tracheotomy was performed, and both jugular veins were catheterized with PE-50 tubing. Respective SD rat or NAR plasma was infused through the left jugular to replace surgical losses. The right jugular received saline containing inulin at 25 mg/ml and 14C-labeled neutral dextran, 70-kDa molecular mass, at 0.1 µCi/ml. This solution was infused at 1.5 ml/h throughout the experiment. The left ureter was cannulated with PE-10 tubing. About 2.5 h after anesthesia, two 20-min urine collections were performed to measure flow rate, inulin concentration, and radioactive dextran activity. A 50-µl blood sample was obtained at the middle of each period to determine glomerular filtration rate (GFR) by inulin clearance, as well as the 70-kDa neutral dextran clearance (CD). Plasma and urinary inulin concentrations were measured by the anthrone technique, whereas the activities of 14C were determined in a beta counter (Beckman Instruments, Shiller Park, IL). The fractional dextran clearance, assumed to be identical to the respective glomerular sieving coefficient (Protocol 3: Ultrastructural Studies with CF
At the end of the clearance experiments, the abdominal aorta was ligated above the emergence of the renal arteries while an 18-gauge needle connected to PE-50 tubing was inserted ~0.5 cm below this level. A vent was made on the left renal vein. The kidneys were then perfused at the measured blood pressure with saline containing 2 mg of CF (Sigma) after a brief washout with pure saline. The kidneys were then removed, and 1-mm3 renal cortical fragments were fixed by immersion in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature. The fragments were rinsed in 0.9% sodium chloride containing 17.8 mg/ml sucrose, buffered to pH 7.3 with sodium bicarbonate, and postfixed for 1 h in 1% OsO4 in 0.08 M cacodylate buffer at pH 7.3. After being rinsed again in 0.9% sodium chloride, the fragments were immersed in 0.5% aqueous uranyl acetate for 12-18 h, dehydrated in an ethanol series starting at 70%, and embedded in Araldite. Sections (0.5 µm thick) were stained with 1% toluidine blue and 1% azur II. Ultrathin silver sections were contrasted with uranyl acetate and lead citrate and examined under a Jeol 1010 transmission electron microscope at 80 kV.The glomerular ultrastructure was examined at a final magnification of ×80,000, and at least three micrographs including adequately oriented GBM sections were obtained for each rat. The density of fixed anionic sites at the GBM, indicated by the binding of CF, was estimated by superimposing a 300-point grid on enlarged micrographs. The fraction of the GBM area covered by CF (%CF) was expressed as %CF = F/T, where F is the number of points hitting areas of CF deposition and T represents the total number of points hitting the GBM.
Statistics
An unpaired Student's t-test was employed to compare values obtained in SD rats and NAR (24). Values obtained on consecutive days from rats receiving exogenous BSA were compared by using one-way ANOVA (24). The Spearman correlation coefficient, r, was calculated by standard methods to assess linear correlation between %CF and ![]() |
RESULTS |
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Fractionation of Proteins in Plasma and Urine
Total plasma protein concentration was lower in NAR than in SD rats (6.5 ± 0.1 vs. 6.8 ± 0.1, respectively, P < 0.05). Total urinary protein excretion rate was similar in SD rats and NAR (0.8 ± 0.1 vs. 0.9 ± 0.1 mg/h, respectively, P > 0.4). Typical SDS-PAGE fractionation patterns of serum and urine proteins from NAR and SD rats are shown in Fig. 1. As reported previously, albumin was clearly absent in NAR serum. Striking differences between the urine of NAR and SD rats were found as well. As expected, low-molecular-weight fractions, presumably of tubular origin, constituted most urinary protein in SD rats. Albumin appeared in small amounts, whereas only traces of other plasma proteins were detected in the urine of SD rats. In the urine of NAR, several protein bands, with molecular masses ranging from ~60 to ~90 kDa, were seen in addition to tubule-derived material. No urinary protein with a molecular mass higher than 90 kDa was detected in NAR.
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Effect of acute albumin administration on proteinuria.
In NAR, injection of BSA resulted in a mean plasma albumin
concentration of 0.32 ± 0.0 g/dl after 12 h and 0.12 ± 0.04 g/dl after 24 h. Urinary protein excretion rate was markedly
depressed in NAR during the 12 h after BSA injection (0.17 ± 0.01 vs. 1.2 ± 0.22 mg/h before injection, P < 0.02 vs. baseline), whereas saline injection had little effect on
proteinuria (Fig. 3). This effect was partially reversed 24-36 h
after BSA injection, with protein excretion reaching 0.43 ± 0.14 mg/h during this period (P > 0.05 vs. proteinuria
measured 12 h after BSA injection). SDS-PAGE fractionation (Fig.
2) revealed that the depression of proteinuria observed in NAR after BSA injection occurred almost exclusively at the expense of plasma-derived proteins, which were now
largely excluded from urine. Plasma-derived proteins returned partially
to urine 24-36 h after BSA injection (Fig. 2). Neither BSA nor
saline administration had any significant effect in SD rats (Fig.
3).
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Measurement of
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Ultrastructural Studies with CF
Electron microscopic examination of the glomerulus showed no difference between SD rats and NAR. The ultrastructural aspect of proximal tubular cells also exhibited similar aspect in both groups, with no perceptible difference regarding endocytic vacuoles, lysosomes, or mitochondria.The density of anionic sites at the GBM (%CF), estimated by the
intensity of CF deposition, was significantly decreased in NAR compared
with SD rats (8.1 ± 1.0 vs. 14.4 ± 1.7, respectively, P < 0.02) (Table 1 and Fig.
4). The endothelial glycocalyx also appeared depleted of CF binding sites. A significant statistical correlation (r = 0.54, P < 0.05) was
observed between individual values of %CF and the respective values (Fig. 5).
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DISCUSSION |
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Previous studies from this laboratory (8) showed that Kf is increased in NAR compared with SD controls. In other studies, the capillary hydraulic conductivity in the dog glomerulus (6), frog mesentery (15), and rabbit myocardium (14) was shown to be sharply and reversibly increased on perfusion of these vessels with protein-free solutions. The mechanisms whereby protein depletion increases capillary hydraulic conductance have not been determined. Curry and co-workers (2, 16) have postulated that the hydraulic conductivity of both fenestrated and nonfenestrated capillaries is largely influenced by the physical characteristics of the fiber matrix that exists in the glycocalyx between endothelial cells, in the endothelial fenestrae, and in the GBM. Albumin and other plasma-derived proteins are believed to bind to constituents of these structures, such as collagen and glycosaminoglycans, hindering the passage of water and small solutes by physically restricting the space between fibers and/or by helping to organize these fibers into a highly ordered matrix (3, 18).
Previous studies suggested that, in addition to its effect on hydraulic conductance, adsorption of albumin and other serum proteins to the capillary walls also restricts the transcapillary passage of macromolecules. Evidence of this effect was obtained in frog mesentery (17) and in rat lung (22). In NAR, Renkin and co-workers (20) showed that the clearance of bovine albumin from the circulation was moderately increased in skin, myocardium, and skeletal muscle and decreased to a similar extent in intestinal capillaries. However, the glomerular permeability to macromolecules was not evaluated in that study.
The present study strongly suggests, on the basis of three independent sets of data, that the glomerular permeability to macromolecules is increased in NAR. First, the spectrum of urinary proteins observed in NAR differed markedly from that seen in SD rats. In the latter, urinary proteins comprised, as expected, a low-molecular-weight fraction, most likely originating in the tubules and dependent on sexual hormones, because it is absent in females (12), a small amount of plasma albumin, and only traces of other plasma-derived proteins. In NAR, tubular proteins coexisted with several proteins of plasma origin, with molecular masses ranging from 60 to 90 kDa, suggesting that these molecules were more promptly filtered than in SD rats. It should be noted that, as shown previously (11), the SDS-PAGE fractionation of NAR plasma revealed the presence of several 60- to 90-kDa proteins, not readily apparent in SD rats, which might contribute to the enhanced filtration of macromolecules in this range. However, the administration of bovine albumin to NAR so as to achieve plasma concentrations of 0.25 g/dl resulted in nearly complete removal of plasma proteins from urine, generating a urinary profile strongly reminiscent of that found in SD rats. This finding suggests that, as in other capillary beds, albumin specifically restricts the filtration of macromolecules across the glomerular wall. This view is strengthened by the observation that this effect was largely reversed at 36 h, when most of the exogenous protein had been cleared from the circulation.
Enhanced glomerular permeability to macromolecules in NAR is also
indicated by the finding of an increased , suggesting the presence
of a glomerular-size defect in these animals. The intimate nature of
this abnormality cannot be ascertained on the basis of the present
data. The pores, slits, or equivalent passages across the glomerular
wall could be individually enlarged in NAR or, alternatively, the total
number of pores/slits could be augmented, leading to an increase in the
total area available for the filtration of water and, especially,
macromolecules. It must be noted that no IgG appeared in NAR urine and
that the molecular mass of urinary proteins never exceeded 90 kDa in
these animals. This finding suggests that large openings in the
glomerular wall, which might extend the nonselective "shunt
pathway" (5), were absent in NAR and that the size
defect observed in these animals is caused by a diffuse abnormality,
rather than by focal disruption, of the glomerular wall.
Enhanced glomerular permeability to plasma proteins in NAR may also have resulted from decreased density of glomerular anionic sites. Huxley and co-workers (10) and Haraldsson and co-workers (9) reported evidence that plasma proteins, such as albumin and orosomucoid, contribute to maintain normal permeability in frog mesenteric capillaries and glomerular microcirculation, respectively, by conferring to these vessels a substantial fraction of the negative charge they contain. In the present study, the labeling of the glomerular wall by CF was ~40% lower in NAR than in SD rats, suggesting that the lack of albumin in the former led to a relative depletion of glomerular anionic sites. In both SD rats and NAR, most of the CF tended to locate at the endothelial aspect of the GBM, consistent with recent observations (23). However, it should be noted that this confinement might be explained in part by size restriction, owing to the high molecular mass of the CF employed in this study. In addition, significant CF binding has been described at the epithelial aspect of the GBM as well (7). Therefore, the magnitude of the glomerular anionic rarefaction in NAR may be even larger than estimated in the present study.
The mechanisms by which the lack of circulating albumin might decrease
the glomerular anionic density are unclear. However, it is interesting
to note that the density of CF binding sites at the GBM correlated
negatively with . This finding is consistent with the view that the
size defect and the depletion of fixed anionic sites observed in NAR
share a common pathogenic mechanism. It is conceivable that the binding
of albumin to the extracellular matrix and the endothelial
glycocalyx serves not only to limit Kf but also
to impart a negative charge to these structures. As polyanions, albumin and other plasma proteins may
contribute to enhance the anionic density of the glomerular
wall, thus restricting the further passage of negatively charged
macromolecules. Alternatively, plasma proteins could simply help
to retain glycosaminoglycans and other polyanionic chains in the
matrix, thus conserving the negative charge in the glomerular wall.
Further investigation is necessary to verify these hypotheses.
Despite the clear evidence of enhanced glomerular permeability to macromolecules in NAR, the total urinary protein excretion rate was not increased in these animals, indicating that the abnormally high filtration of macromolecules was nearly completely compensated for by tubular absorption. It should be considered that the increase in glomerular permeability in NAR was much less pronounced than observed in capillaries perfused with protein-free solutions (17, 22) and comparable to the modest changes reported by Renkin and co-workers (20) in NAR skin, myocardium, and skeletal muscle capillaries. This observation is also consistent with that reported by Joles and co-workers (12) in ovariectomized female NAR (in which the confounding effect of tubular-derived proteinuria is absent), which exhibit only minimal protein excretion. Collectively, these findings support the notion that, as in nonrenal capillaries, plasma-derived proteins other than albumin may also contribute to restrict the glomerular permeability to macromolecules. In NAR, this limiting effect may receive the additional contribution of proteins synthesized in lieu of albumin, such as those in the 60- to 90-kDa range, described previously (11) and shown in Fig. 1. Of note, previous observations from this laboratory indicated that much more intense proteinuria developed in NAR than in SD rats after 5/6 nephrectomy (8). The modest permeability defect noted in this study may constitute one of the factors predisposing these animals to more severe glomerular injury under stress conditions.
In summary, the present study provides evidence that, in NAR, not only
Kf but also the glomerular permeability to
macromolecules is increased compared with in SD controls. This
abnormality is mediated by a size defect, evidenced by an increase in
and by the depletion of glomerular anionic sites, as indicated by
decreased binding of CF at the GBM. Small amounts of circulating
albumin and other plasma-derived proteins may be necessary to fine-tune the normal permeability characteristics of the glomerular capillary wall.
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ACKNOWLEDGEMENTS |
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This study was supported by Research Grant 85/0244-5 from the State of São Paulo Foundation for Research Support. During these studies, R. Zatz was the recipient of Grant 300.426-81 from the National Scientific and Technologic Development Council.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Zatz, Laboratório de Fisiopatologia Renal, Faculdade de Medicina, Av. Dr. Arnaldo, 455, 3-s/3342, 01246-903 São Paulo SP, Brazil (E-mail: rzatz{at}usp.br).
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 20 March 2001; accepted in final form 17 July 2001.
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