1 Renal Division, Department of Clinical Medicine, and 2 Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo 01246-903; and 3 Department of Pharmacology, State University of Campinas School of Medicine, Campinas, 13083-970 Brazil
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ABSTRACT |
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Chronic nitric oxide (NO) inhibition causes hypertension and
renal injury. Concomitant salt overload promotes massive albuminuria. We investigated the mechanisms whereby these treatments impair glomerular permselectivity. Adult male Munich-Wistar rats received either a standard-salt (SS; 0.5% Na) or high-salt (HS; 3.1% Na) diet
and either no treatment or the NO inhibitor
N-nitro-L-arginine methyl ester
(L-NAME). At 30 days, albuminuria was moderate, the density
of fixed anionic sites at the glomerular basement membrane (GBM),
estimated by cationic ferritin binding, declined by ~35%, and the
fractional clearance of 70-kDa neutral dextran (
) rose moderately in
rats receiving L-NAME and SS. Rats given L-NAME and HS exhibited massive albuminuria,
whereas
was nearly tripled. Depletion of GBM anionic sites was also
seen in these rats. The GBM was thickened in both
L-NAME-treated groups. These abnormalities were largely
reversed after cessation of treatments. These results indicate that
chronic L-NAME treatment promotes reversible albuminuria by
impairing both glomerular size and charge selectivity. These effects
likely reflect functional rather than structural disruption of the
glomerular wall.
kidney glomerulus; kidney physiopathology; capillary permeability; sodium; dietary
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INTRODUCTION |
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CHRONIC NITRIC OXIDE (NO) inhibition promotes severe and progressive arterial hypertension and renal structural injury, consisting of glomerular ischemia, glomerulosclerosis, and interstitial expansion (4, 34). In addition, urinary albumin losses are markedly increased in these animals (4, 39), reaching nephrotic levels when NO inhibition is associated with dietary salt overload (14, 39). The mechanisms underlying the severe impairment of the glomerular barrier observed in this model have not been investigated.
Under normal circumstances, restriction to the filtration of macromolecules occurs mostly at the glomerular basement membrane (GBM) and the slit membrane (10, 12, 32). Macromolecules can be hindered by size, due to the existence of restrictive "pores" in the GBM and, particularly, in the slit membrane (8, 31, 36). In addition, because most of these molecules are negatively charged, they are repelled by the fixed anionic sites distributed throughout the glomerular wall, especially in the GBM (12, 32).
The pathogenesis of proteinuria in clinical and experimental renal disease may involve a glomerular size defect (6, 38), the depletion of fixed negative charges at the glomerular wall (7), or an association between these two abnormalities (28). In the present study, we examined rats subjected to chronic inhibition of NO synthesis, alone or in combination with dietary salt excess, to determine 1) whether the massive albuminuria associated with this model is mediated by a size defect, by a charge defect, or by a combination of both mechanisms; and 2) whether the increased glomerular permeability observed in this model can be reversed on discontinuation of NO inhibition or, instead, reflects permanent damage to the capillary wall.
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METHODS |
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Forty adult male Munich-Wistar rats, obtained from a local colony at the University of São Paulo and weighing 240-260 g, were used in this study. The animals were maintained in groups of three or four at 23 ± 1°C on a 12:12- light-dark cycle, with free access to food and water. All rat food was purchased from Harlan Teklad (Madison, WI). All experimental procedures were conducted in accordance with our institutional guidelines.
Experimental groups.
Rats were distributed among four experimental groups as described
below. Standard-salt (SS; n = 8) untreated control rats received ad libitum tap water and rat chow containing standard amounts
of sodium (0.5%) and protein (25%) during 4 wk. SS+NAME (n = 8) rats were treated like SS rats but received the
NO inhibitor N-nitro-L-arginine
methyl ester (L-NAME; 700 mg/µl) in the drinking water,
corresponding to an expected ingestion of ~70
mg · kg
1 · day
1. This
treatment was maintained for 4 wk. High-salt (HS; n = 8) untreated rats received ad libitum tap water and rat chow containing 25% protein and high (3.1%) sodium content during 4 wk. HS+NAME (n = 8) rats were treated like HS rats but received
L-NAME in the drinking water for 4 wk. Because HS rats
drink twice as much water as SS rats, L-NAME was dissolved
in the drinking water at 350 ml/l to ensure that the L-NAME
dose was similar between the HS+NAME and SS+NAME groups. To ascertain
the reversibility of the glomerular functional abnormalities associated
with treatments, an additional group was studied (HS+NAME-REC),
consisting of eight rats receiving a HS diet and L-NAME as
described above during 4 wk. At the end of this period, all treatments
were discontinued. Rats were then allowed to recover during the ensuing
4 wk.
Renal functional studies.
For assessment of renal hemodynamics and glomerular permeability to
dextran, eight rats from each group were anesthetized with inactin (100 mg/kg body wt ip) and placed on a temperature-controlled surgical
table. Rectal temperature was maintained at 37 ± 0.5°C. The
left femoral artery was catheterized with PE-50 polyethylene tubing for
determination of baseline hematocrit, for continuous monitoring of mean
arterial pressure (MAP) by a Statham P23Db pressure transducer
connected to a chart recorder (model A8200, Anamed Instruments,
São Paulo, Brazil) and for collection of blood samples during the
experiment. After tracheotomy, both jugular veins were catheterized
with PE-50 tubing. Homologous plasma (to replace surgical losses) was
infused through the left jugular, while a saline solution, containing
inulin at 25 mg/ml and 14C-labeled neutral dextran, 70 kDa
molecular mass, at 0.1 µCi/ml, was infused through the right jugular
at 1.5 ml/h throughout the experiment. The left ureter was catheterized
with PE-10 tubing. About 2.5 h after anesthesia, two timed (20-in)
urine collections were performed for determination of flow rate, inulin
concentration, and radioactive dextran activity. A 50-µl blood sample
was collected at the middle of each period to assess the glomerular
filtration rate (GFR) by inulin clearance and to determine 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 glomerular sieving
coefficient for this compound (7), was calculated as
= CD/GFR · 100.
Ultrastructural studies. At the end of each experiment, the abdominal aorta was ligated above, while an 18-gauge needle connected to a PE-50 polyethylene tubing was inserted below, the emergence of the renal arteries. A vent was made on the renal vein. The kidneys were then perfused at the measured blood pressure with a saline solution containing 2 mg of cationic ferritin (CF; Sigma, St. Louis, MO) after a brief washout with pure saline. After excision of the kidneys, 1-mm3 fragments of renal cortex were fixed in 2% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4) for 2 h at room temperature. The fragments were rinsed in 0.9% sodium chloride solution containing 17.8 mg of sucrose/ml and buffered to pH 7.3 with sodium bicarbonate. Postfixation was carried out in 1% OsO4 in 0.08 M cacodylate buffer at pH 7.3 for 1 h. After being rinsed in 0.9% sodium chloride solution, the fragments were immersed in 0.5% aqueous uranyl acetate for 12-18 h. After dehydration in an ethanol series starting at 70%, the specimens were embedded in Araldite. Sections (0.5 µm) were stained with 1% toluidine blue and 1% azur II. Silver ultrathin 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 that included properly oriented GBM sections were taken for each rat. The density of fixed anionic sites at the GBM, indirectly assessed by the binding of CF, was quantified by superimposing a 300-point grid on enlarged glomerular micrographs. The fraction of the GBM area covered by CF (%CF) was then estimated as %CF = F/T, where F is the number of points hitting areas of CF deposition and T represents the total of points hitting the GBM. The average thickness of the GBM was estimated by randomly superimposing a test line on enlarged photographs of the glomerular wall and measuring the segment of this line comprised within the GBM area (1).Urinary protein analysis. Four weeks after treatments were started, rats were placed in metabolic cages and urine was collected during 24 h. Those rats whose treatments were discontinued after 4 wk (HS+NAME-REC) were subjected to the same procedure at the end of an additional 4 wk. Total urinary protein concentration was measured by the sulfosalicylic method, whereas urinary albumin concentration was determined by radial immunodiffusion (24).
The composition of urinary proteins was estimated by SDS-PAGE, utilizing an adaptation of the discontinuous system described by Laemmli (23). The volume of either urine samples or standard solutions placed in each well was individually adjusted so that its calculated protein content was always 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, 10% acetic acid in water.Statistics.
Differences among groups were assessed by one-way ANOVA, with posttest
pairwise comparisons between groups according to the Bonferroni
formulation (37). Because urinary albumin excretion rate
(UalbV) and were not normally distributed, log
transformation of these parameters was performed before statistical
analysis. Interaction between dietary salt and L-NAME
treatment was assessed by performing two-way ANOVA of the SS, SS+NAME,
HS, and HS+NAME groups. The Pearson correlation coefficient was
calculated to assess linear correlation between log (UalbV)
and either %CF, GBM thickness (
), or log
. For all statistical
tests, P
0.05 was considered significant.
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RESULTS |
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As expected, individual water consumption was about twice as high
in HS (76 ± 6 ml/day) and HS+NAME (74 ± 2 ml/day) as in SS
(35 ± 3 ml/day) and SS+NAME (38 ± 3 ml/day) rats. Thus
L-NAME intake was 78 ± 6 and 76 ± 2 mg · kg1 · day
1 in SS+NAME
and HS+NAME rats, respectively. These values were not significantly
different when compared by using the unpaired Student's
t-test. Individual food consumption was 20 ± 2 in the SS group, 18 ± 1 in the SS+NAME group, 18 ± 2 in the HS
group, and 11 ± 2 g/day in the HS+NAME group. The respective
daily sodium intakes, estimated from the known composition of the diets
(0.5% Na for SS and 3.1% for HS) were therefore 4.3 ± 0.4 mmol/day in SS, 3.9 ± 0.2 mmol/day in SS+NAME, 24 ± 3 mmol/day in HS, and 15 ± 4 mmol/day in HS+NAME rats.
Renal functional studies.
Renal and systemic hemodynamic parameters measured at 4 wk of treatment
are displayed in Table 1. As described
previously (14, 34, 39), MAP was markedly elevated in rats
receiving L-NAME at either dietary salt regimen. Blood
pressure returned to control levels 30 days after treatments were
withdrawn (HS+NAME-REC). GFR was markedly decreased in the SS+NAME and
HS+NAME group. Interruption of treatments was associated with
normalization of GFR after 4 wk. The fractional dextran clearance,
i.e., , was increased by 81% in the SS+NAME compared with the SS
group. However,
more than tripled in the HS+NAME compared with the
HS group. This abnormality was almost entirely reversed after
treatments were interrupted.
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Ultrastructural studies. Virtually no abnormalities were noted in electron microscopic sections from the SS+NAME group. In the HS+NAME group, simplification of pedicles was observed in most electron microscopic sections. However, severe podocyte injury and denudation of the external aspect of the GBM, such as described in other models (25, 28), were not observed.
L-NAME treatment significantly decreased the density of anionic sites at the GBM (%CF), as estimated by the extent of CF deposition (Table 2 and Fig. 1). %CF was numerically lower in the HS+NAME compared with the SS+NAME group, although this difference did not reach statistical significance. CF deposition at the endothelial layer also appeared consistently reduced in this group compared with control (Fig. 1). After withdrawal of treatments, the CF deposition density at the glomerular wall was nearly completely restored.
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Correlation analysis and two-way ANOVA.
There was a strong positive linear correlation between log
(UalbV) and log (r = 0.73, P < 0.0001). A similar correlation was found between
log (UalbV) and
(r = 0.62, P < 0.0001). A negative, equally tight linear
correlation was observed between log (UalbV) and the
density of GBM anionic sites (%CF; r = 0.78, P < 0.0001) (Fig.
2).
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Urinary protein analysis.
The results obtained after urine analysis by SDS-PAGE electrophoresis
are illustrated in Fig. 3. As expected,
rats receiving no drug treatment excreted mostly low-molecular-weight
proteins. In L-NAME-treated groups, proteinuria consisted
essentially of albumin, with only trace amounts of IgG appearing in the
urine, suggesting that no major structural disruption of the capillary wall occurred in these animals.
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DISCUSSION |
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Chronic NO inhibition resulted in a 20-fold increase in
UalbV. The relatively moderate albuminuria observed in this
group may have resulted from two distinct, independent abnormalities: 1) a charge defect, as indicated by the 35% reduction in
the density of CF binding sites at the GBM observed in these animals;
and 2) a size defect, as suggested by the 81% increase in
compared with control values.
Concomitant L-NAME treatment and dietary salt excess
aggravated the glomerular barrier dysfunction already observed in
SS+NAME rats, raising albuminuria to nephrotic levels at 30 days of
treatment. These two maneuvers interacted strongly to augment
albuminuria, as revealed by two-way ANOVA. One possible mechanism for
this cooperation might be a disruption of the glomerular size-selective properties, because more than tripled in HS+NAME rats compared with
HS. Accordingly, two-way ANOVA disclosed a specific interaction between
L-NAME and salt overload to increase
. Theoretically, this effect could have resulted from the concomitant decrease in GFR,
which would tend to increase
by enhancing the relative importance
of transcapillary diffusion (7, 30). However, even a 50%
decrease in GFR would be predicted to augment
by no more than 50%
(7), as opposed to the 206% increase observed in the
HS+NAME relative to the HS group. In addition, GFR was reduced by a
similar extent in rats receiving SS and L-NAME, which exhibited a much smaller increase in
, suggesting that a true size
defect occurred in the HS+NAME group. Impairment of glomerular size
selectivity might result from the appearance of large nonselective openings at the glomerular wall, creating a "shunt pathway" for the
passage of albumin and other macromolecules. Because the urinary excretion of IgG was negligible compared with that of albumin, the
radius of these hypothetical "large pores" is unlikely to have
substantially exceeded 60 Å (the approximate radius of the IgG
molecule). Accordingly, we were unable to detect podocyte detachment
from the GBM, or any epithelial abnormality other than foot process
"fusion" that might constitute an anatomic basis for a large,
unselective shunt pathway, as shown in other proteinuric conditions
(6, 28). The finding of a size defect in
L-NAME-treated rats in the absence of an obvious
ultrastructural abnormality of the glomerular wall suggests an
alteration of the molecular composition and/or conformation at the GBM
and/or the slit membrane. The increase in
found in
L-NAME-treated rats and the tight correlation observed
between
and UalbV suggest that disruption of the GBM architecture may indeed have occurred in these animals.
In HS+NAME rats, the density of anionic charges at the GBM (%CF) was
numerically decreased compared with in SS+NAME rats, falling to 50% of
baseline. This difference between results in HS+NAME and SS+NAME rats
did not attain statistical significance. It must be noted, however,
that even a small (hence difficult to detect) further loss of GBM
negative charge may have exerted a disproportionate effect on
albuminuria, because the passage of polyanions is expected to grow
exponentially, with linear decreases in the fixed anion density at the
glomerular wall (9). Accordingly, in this study log
(UalbV) exhibited a strong negative linear correlation with
%CF. Moreover, it should be stressed that, due to its large molecular
dimensions, the penetration of CF into the GBM was always mostly
confined to the lamina rara interna. Because the density of anionic
sites may be higher at the outermost layers of the GBM
(12), it is conceivable that more severe charge depletion occurred in this group than revealed by examination of CF deposition. In addition, albuminuria may have been aggravated in
L-NAME-treated rats by the loss of anionic sites at the
endothelial layer. Finally, it must be noted that anionic depletion of
the GBM was most likely associated with loss of charged molecules such
as glycosaminoglycans, which may have contributed to loosen the GBM
meshwork, thus increasing and impairing both the charge- and the
size-selective properties of the glomerular wall (18).
The functional and structural abnormalities of the glomerular wall in rats undergoing chronic NO inhibition, particularly when associated with salt overload, may have resulted from the marked glomerular hypertension observed in these animals (4), which is aggravated by dietary salt overload (14, 14). Intracapillary hypertension may directly enhance the filtration of proteins by increasing the force that governs the passage of macromolecules across the glomerular wall (8, 29). Besides this direct physical effect, glomerular hypertension may damage the endothelial (13, 27) and epithelial (25, 33) layers of the glomerular wall, thus facilitating the traffic of macromolecules. Mechanical stress may also affect the GBM and the slit membrane, increasing the size of the pores, or equivalent structures, that function as pathways for macromolecule filtration (35). Moreover, the density of anionic charges in the GBM was shown to decrease in response to elevations in perfusion pressure (16, 40). In rats receiving chronic NO inhibitors, angiotensin II receptor blockers lower glomerular pressure and strongly attenuate albuminuria, regardless of whether the rats are concomitantly given salt overload (14, 34). These observations suggest that intracapillary hypertension may indeed mediate the increase in glomerular permeability observed in these rats. However, it must be noted that, in other experimental models, severe glomerular hypertension is not necessarily associated with massive albuminuria. In rats with diabetes mellitus, despite persistent glomerular hypertension, albuminuria remains low for nearly 1 yr before exceeding 100 mg/day (15). In rats with puromycin aminonucleoside nephrosis, albuminuria reaches nephrotic levels at a time when glomerular pressure is normal, then nearly disappears when glomerular pressure rises (2). Therefore, factors in addition to glomerular hypertension may have also participated in the pathogenesis of the massive albuminuria associated with this model.
A host of experimental findings obtained in several microcirculatory territories suggests that local, continuous NO production may play an important physiological role by directly restricting the capillary permeability to macromolecules. In murine (22) and feline (20) mesenteric circulations, acute inhibition of NO synthesis led to marked extravasation of albumin, indicating a corresponding increase in capillary leakiness. Conversely, the administration of a NO donor or a cGMP analog prevented albumin leakage induced by histamine or other inflammatory mediators (17). In rat coronary circulation (11), intravenous infusion of L-NAME markedly increased vascular permeability, an effect unrelated to the concomitant elevation of blood pressure. Evidence that NO limits microvascular permeability to macromolecules has also been obtained in the human lung (5) and in the rat hind paw (19). The results obtained in the present study suggest that this effect of NO may occur at the glomerular microcirculation as well. The mechanisms by which NO inhibition might enhance capillary permeability are poorly understood. In intestinal capillaries, NO inhibition may disorganize the endothelial cytoskeleton and, consequently, disrupt cell-cell junctions, leading to the formation of intercellular leaks and allowing the passage of large molecules (3). NO inhibition may also trigger inflammatory events such as leukocyte adherence (21) and mast cell degranulation (22). We have recently shown preliminary evidence (26) of lymphocyte and macrophage infiltration in the renal parenchyma of rats receiving chronic L-NAME treatment and that this abnormality is markedly aggravated by dietary salt overload. These events may contribute to increased glomerular permeability in this model, perhaps owing to the release of inflammatory mediators by these infiltrating leukocytes. Whether this effect involves a glomerular cytotoxic action, resulting in deficient synthesis of GBM/slit-membrane constituents, is presently unknown.
The massive albuminuria observed at 30 days of combined HS and L-NAME treatment was almost completely reversed 30 days after this regimen was stopped. Recovery of the glomerular wall properties was accompanied by near normalization of the GBM negative charge density and of the sieving coefficient for 70-kDa dextran, indicating that both the size and charge defects observed at 30 days of HS+NAME treatment were reversed. This finding is consistent with the view that a functional defect (for instance, impaired biosynthesis of glomerular wall constituents), rather than permanent structural damage, was responsible for the severe glomerular barrier dysfunction observed in this study. Restoring NO synthesis and/or normalization of glomerular hemodynamics may have governed this recovery process. It remains to be established whether rats surviving L-NAME and HS treatment for substantially longer than 30 days would also recover or whether the process leading to albuminuria in these animals would perpetuate itself if treatments were maintained for a sufficiently long period. Equally undetermined is whether recovery would still occur if the L-NAME dose employed in this study were substantially higher.
In summary, the present observations suggest that the albuminuria observed in the chronic NO inhibition model is associated with a size defect as well as with depletion of electronegative charges at the GBM. The massive albuminuria that develops when salt overload is superimposed on NO inhibition involves exacerbation of the size and, in all likelihood, the charge defect. Albuminuria almost completely disappears on interruption of treatments, suggesting that it results from a glomerular functional, rather than structural, defect.
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ACKNOWLEDGEMENTS |
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We are indebted to Wagner Vasques Dominguez and Laura Araújo Tomé for expert technical assistance.
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FOOTNOTES |
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This work was supported by Grant 95/4710-2 from the São Paulo Foundation for Research Support. Preliminary observations obtained in this study were presented at the 29th Congress of the American Society of Nephrology, New Orleans, LA, November 3-6, 1996, and published in abstract form (J Am Soc Nephrol 7: 1558, 1996). During these studies, R. Zatz was the recipient of Research Award 326.429/81 from the Brazilian Council of Scientific and Technologic Development.
Address for reprint requests and other correspondence: R. Zatz, Laboratório de Fisiopatologia Renal, 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 23 March 2000; accepted in final form 3 August 2000.
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