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REGULAR ARTICLES |
*
Cardiology, University Hospital Zürich and
Cardiovascular Research, Institute of Physiology, University of
Zürich, Switzerland
Department of Clinical Research, University Hospital Bern,
Switzerland
Nephrology and Hypertension, University Hospital Utrecht, The
Netherlands
§
Department of Pathology, Philipps-University Marburg, Germany.
Correspondence to Dr. Thomas F. Lüscher, University Hospital, Rämistrasse 100, CH-8091 Zürich, Switzerland. Phone: +41 1 255 21 21 or 21 77; Fax: +41 1 255 42 51; E-mail: 101527.3476{at}compuserve.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal studies using infusions or oral treatment with NO synthase (NOS) inhibitors have suggested that NO plays an important role in maintaining renal function and structure. Indeed, short-term treatment with NOS inhibitors increases BP and inhibits sodium excretion (10), whereas a more prolonged inhibition also causes fibrinoid deposition in both the vasculature and renal parenchyma (11). Genetic studies (12,13) as well as indirect functional evidence suggested a role of the L-arginine/NO pathway for hypertension also in salt-sensitive Dahl (DS) rats, because both oral L-arginine treatment (14) and intramedullary L-arginine infusion (15) normalize BP and improve renal hemodynamics (16). High salt diet increases protein expression of Nos2 in the kidney of Sprague Dawley rats without affecting BP (17), suggesting a potential compensatory mechanism to counteract the increase in volume load and pressure. Consistent with this notion, Sprague Dawley rats treated with the Nos2 inhibitor aminoguanidine develop hypertension, which can be reversed by L-arginine treatment (18). These data, together with the observation that dietary sodium aggravates hypertension and renal injury during chronic NOS inhibition (19), suggest that the effects of sodium on BP may be mediated through interaction with NO synthesis. Of note, ET-1, a potent vasoconstrictor and mitogen synthesized by the vasculature and the kidney which interacts with the L-arginine/NO pathway (11) (20,21), has recently been implicated in the pathogenesis of salt-induced Dahl hypertension (9,22). Although the ET-3 gene locus has been linked to hypertension in Dahl rats (23), it is unknown whether changes in ET-3 expression occur in hypertensive animals.
The aims of this study were: (1) to characterize potential mechanisms of endothelial function in the renal artery in normotensive and hypertensive Dahl rats; and (2) to determine whether and to what extent the L-arginine/NO pathway and/or the endothelin system play a role for renovascular functional and structural changes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arterial and Renal Tissue Preparations
Rats were anesthetized (thiopental, 50 mg/kg body wt, intraperitoneally)
and sacrificed. The left and right main renal artery and the thoracic aorta
were isolated, removed, and placed into cold (4°C) Krebs Ringer
bicarbonate solution (in mmol/L): 118.6 NaCl, 4.7 KCl, 2.5 CaCl2,
1.2 KH2PO4, 1.2 MgSO4, 25.1
NaHCO3, 0.026 ethylenediaminetetra-acetic acid (EDTA) calcium
disodium, and 11.1 glucose. Arteries were dissected in cold Krebs solution
under a microscope (Wild-Heerbrugg, Switzerland), cleaned from perivascular
tissue, and rinsed with a cannula to remove residual blood cells. Two rings of
each renal artery (length: DS 2.37 ± 0.03 mm, n = 67 rings; DR
2.43 ± 0.03 mm, n = 51 rings) were used for organ chamber
experiments. For endothelium-dependent contractions, aortic rings were used
for comparison
(9,25).
Remaining renal artery tissue was snap-frozen in liquid nitrogen and stored at
-80°C. The right kidney was cut in half, and one half was fixed in
phosphate-buffered saline-buffered paraformaldehyde (4%) at 4°C, processed
for histologic analysis, and embedded in paraffin. The other half and the left
kidney were decapsulated, sliced horizontally, separated between outer medulla
and inner cortex, immediately snap-frozen in liquid nitrogen, and kept at
-80°C until assayed.
Renal Artery Endothelial Function and Structure
Renal artery rings were suspended to fine tungsten holders (diameter, 100
µm) in organ chambers containing 25 ml of Krebs-bicarbonate solution
(37°C, pH 7.4, 95% O2 and 5% CO2) and equilibrated
for 1 h. Resting tension was gradually increased, and rings were repeatedly
exposed to 100 mmol/L KCl until the optimal tension for generation of force
during isometric contraction was reached (DS group: 1.48 ± 0.01 g,
n = 90 rings; DR group: 1.46 ± 0.01 g, n = 72 rings,
NS). After equilibration for 30 min, rings were exposed to cumulative
concentrations of ET-1 (10-11 - 3 x 10-7 mol/L) or
norepinephrine (10-10 3 x 10-5 mol/L). In
quiescent renal artery and aortic rings, basal NO release was assessed by
contractions to NOS inhibitor NG-nitro-L-arginine methyl
ester (L-NAME; 10-5 - 3 x 10-4 mol/L). In rings
pretreated with L-NAME (3 x 10-4 mol/L, 30 min of
preincubation), acetylcholine (10-8 - 3 x 10-4
mol/L) in the presence or absence of the thromboxane
A2/prostaglandin H2 receptor antagonist SQ 30741
(10-7 mol/L, 30 min of preincubation) or cyclooxygenase inhibitor
indomethacin (10-5 mol/L, 30 min of preincubation) was used to
study endothelium-dependent contractions. Other rings were precontracted with
norepinephrine (1 x 10-8 - 2 x 10-7 mol/L)
until a stable plateau was reached (approximately 70% of contraction induced
by 100 mmol/L KCl); precontraction was not different between groups (data not
shown). Relaxations to acetylcholine (10-10 - 3 x
10-5 mol/L)—with or without indomethacin (10-5
mol/L, 30 min of preincubation)— and to sodium nitroprusside
(10-10 - 3 x 10-5 mol/L) were then performed. In
some preparations, the endothelium was removed and its absence was confirmed
by the lack of relaxation in response to acetylcholine (3 x
10-6 mol/L). For the assessment of vascular hypertrophy, rings were
blotted dry after the experiments and weighed, and arterial surface area
(mm2) was calculated by microscopic planimetry as described
previously (9). Arterial
diameter of renal artery rings was calculated using the following equations:
Given that the circumference (c) of the artery equals the length of
the transversely cut, opened arterial strip, the formula (c =
2
r) was used ( = 3.1415, r = vessel radius). Renal
artery diameter (2r = c/) was calculated by use of
formulas for diameter (d = 2r) and radius (r =
c/2) of a cylinder for each individual ring, and values were
averaged. Mean diameter of renal arteries was 444 ± 14 µm (DS rats,
n = 67 rings) and 428 ± 11 µm (DR rats, n = 51
rings), with no statistical difference between treatment groups
(Table 1).
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Determination of NOS Activity in Renal Tissue
NOS activity was determined as the formation of
L-[2,3,4,5-3H]-citrulline from L-[2,3,4,5-3H]-arginine
of tissue homogenates from renal cortex and medulla. After excision, kidneys
were dissected into cortex and medulla, frozen in liquid nitrogen, and stored
at -80°C until assay. For determination, tissue samples were homogenized
using an Ultra-Turrax in 1.5 ml of buffer (pH 7.4) containing Tris (50
mmol/L), sucrose (320 mmol/L), EDTA (1 mmol/L), dithiothreitol (1 mmol/L),
aprotinin (2 mg/L), and phenylmethylsulfonyl fluoride (100 mg/L). The
homogenates were used for measuring protein concentration using the Bradford
method and NOS activity. All measurements were performed in duplicate. Total
(Ca2+-dependent plus Ca2+-independent) activity was
measured by incubating 50 µl of homogenate during 30 min at 37°C with
50 µl of KH2PO4 buffer (50 mmol/L, pH 7.2) containing
(final concentrations) dithiothreitol (1 mmol/L), NADPH (0.5 mmol/L),
tetrahydrobiopterin (0.3 mmol/L), calmodulin (300 U/ml), CaCl2 (1
mmol/L), L-citrulline (1 mmol/L), L-arginine (0.01 mmol/L), and
L-[2,3,4,5-3H]-arginine (3.7 KBq). Ca2+-independent
activity was measured by incubation in the presence of EDTA (10 mmol/L)
instead of CaCl2, and nonspecific activity was determined by
incubation in the presence of L-NAME (100 mmol/L) instead of NADPH. Lower
concentrations of EDTA and L-NAME were found to give incomplete inhibition of
Ca2+-dependent and total NOS activity, respectively. Incubations
were stopped by placing the incubation mixtures in ice, followed by addition
of 1 ml of ice-cold Hepes buffer (20 mmol/L, pH 5.5). The mixtures were loaded
onto 1-ml Dowex 50x8-200 (Na+ form) columns, followed by
washing with 1 ml of stop buffer. Citrulline was eluted with 2 ml of stop
buffer, collected into scintillation vials, and counted in a beta counter in
the presence of 15 ml of scintillation fluid. By addition of
L-[14C]-citrulline before or after the incubation step, the
recovery of this procedure was found to be >95%. After correction for
nonspecific counts, total and Ca2+-independent NOS activity was
calculated from the percent conversion of [3H]-arginine into
[3H]-citrulline in the presence of CaCl2 or EDTA,
respectively, and expressed as picomoles per milligram of protein per minute.
Ca2+-dependent activity was calculated by subtraction of
Ca2+-independent activity from total activity. In additional
experiments, we compared NOS enzyme activity in membrane fractions from 20,000
x g supernatants with the activity measured in crude tissue
homogenates.
Morphologic Analysis of Glomerular Injury
Renal injury was assessed as described previously
(26). Briefly,
paraffin-embedded sections of whole kidneys (5 to 7 µm) stained with
periodic acid-Schiff reagent were viewed by light microscopy at a
magnification of x40 using a Zeiss microscope. One hundred glomeruli per
slide were evaluated. Morphologic evaluation of glomerular injury was
performed by two of the authors (I.V., H.J.G.) blinded to the groups, using
semiquantitative scoring methods. Lesions were graded by glomerulosclerosis
(grade 0 to 4, i.e., 1 to 25, 26 to 50, 51 to 76, and 76 to 100%
sclerosis), mesangiosclerosis, mesangiolysis, and mesangioproliferation (grade
1 to 3), ischemia, and thrombosis. The glomerular injury score was calculated
by summarizing the products of severity score (index) and the percentage of
glomeruli or arteries displaying the same degree of severity. The total injury
index was defined as total amount of morphologic changes.
Quantification of Tissue Endothelin Protein Content
Determination of tissue ET-1 and ET-3 protein content was performed in a
blinded manner. Frozen renal artery tissue was pulverized and homogenized
using a polytron for 60 s in ice-cold chloroform: methanol (2:1 dilution)
containing 1 mmol/L N-ethylmaleimide and 0.1% trifluoroacetic acid.
Frozen tissue from renal cortex and medulla (300 mg) was homogenized without
pulverization. ET-1 and ET-3 protein was extracted from tissue homogenates as
described (24). RIA
measurements and reversed-phase HPLC was used for ET-1 and ET-3 protein
content determination according to previously published protocols
(24,27),
and vascular and renal ET-1 and ET-3 tissue content was related to tissue
weight (pg/g) (24).
Materials
Acetylcholine chloride, calmodulin, CaCl2,
dichlorodiphenyltri-chloroethane, Dowex resin AG 50W-X4, EDTA, ET-3,
indomethacin (dissolved in 5 mmol/L sodium carbonate), L-arginine, L-NAME,
NADPH, norepinephrine bitartrate salt, phenylmethylsulfonyl fluoride,
potassium chloride, sucrose, sodium nitroprusside dihydrate, and Tris salt
were purchased from Sigma Chemical Co. (St. Louis, MO). ET-1 was from
Calbiochem/Novabiochem AG (Läufingen,
Switzerland). Rabbit antibodies against synthetic ET-1 and ET-3 were from
Peninsula Laboratories (San Carlos, CA). Radiolabeled 125I-ET-1,
125I-ET-3, and L-[2,3,4,5-3H]-arginine was purchased
from Amersham (Amersham, Buckinghamshire, United Kingdom). SQ 30741 was a gift
of Bristol-Myers Squibb (Princeton, NJ), and pentobarbital was from Abbott
Laboratories (Chicago, IL).
Statistical Analyses
Data are given as mean ± SEM, and n equals the number of
animals used. Relaxations are expressed as percent contraction to
norepinephrine, and contractions are given as percent contraction to potassium
chloride. For multiple comparisons, results were analyzed using ANOVA followed
by Bonferroni correction. For comparison between two values, the unpaired
t test or the nonparametric Mann-Whitney test were used when
appropriate. The Pearson correlation coefficient was calculated by linear
regression analysis. A P value <0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelium-Dependent Relaxations to Acetylcholine
Despite hypertension, relaxations to acetylcholine were unaffected in DS
rats (NS) (Figure 2); however,
concomitant treatment with LU135252 slightly enhanced relaxations in DS rats
(P < 0.05 versus salt). Surprisingly, relaxations were
reduced in salt-treated DR rats after chronic treatment with LU135252
(P < 0.05) (Figure
2A, right panel), which could be prevented by cyclooxygenase
inhibition with indomethacin in vitro (P < 0.05)
(Table 2).
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Endothelium-Dependent Contractions to NOS Inhibitor L-NAME
NOS inhibitor L-NAME caused concentration-dependent contractions in
quiescent renal artery and aortic rings. Treatment with high salt diet
attenuated contractile responses to L-NAME in DS rats, while contractions
increased in DR rats (Figure
2B) (P < 0.05 versus low salt). In DS rats,
LU135252 treatment normalized the attenuated response (P < 0.05
versus salt). In comparison, maximal contractions to L-NAME (300
µmol/L) in the aorta of DS control rats were about one-third in magnitude
compared with the renal artery (10 ± 2% of KCl) (P < 0.01
versus renal artery) and unaffected by high-salt diet or LU135252
(Table 2).
Endothelium-Dependent Contractions to Acetylcholine
The magnitude of responses was similar in renal arteries from DS and DR
control rats (NS) (Figure 3).
High salt diet markedly potentiated contractions in DS rats (approximately
threefold, from 25 ± 3 to 78 ± 4%, P < 0.05
versus control) and, to a lesser extent, in DR rats (approximately
1.6-fold) (Figure 3).
Contractions were correlated with vascular ET-1 protein content in DS rats
only (r = 0.7, P < 0.01). LU135252 treatment completely
normalized the increased contractions
(Figure 3) (P <
0.05 versus high salt). Responses to acetylcholine were markedly
inhibited by pretreatment with the thromboxane receptor antagonist SQ 30741
(10-7 mol/L, P < 0.05)
(Table 2) or indomethacin
(10-5 mol/L) in all groups (data not shown). In the aorta of DS
rats, contractions to acetylcholine were only about 1/20 in magnitude compared
with the renal artery (DS control rats 3 ± 2% and DS rats 4 ± 2%
on a high salt diet, NS) and unaffected by concomitant LU135252 treatment
(Table 2).
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Endothelium-Independent Responses
Potassium chloride-induced contractions were comparable between groups (NS)
(Table 2). Contractions to ET-1
but not those to norepinephrine were attenuated in salt-loaded DS rats;
however, this was prevented by concomitant LU135252 treatment
(Figure 4, A and B). LU135252
normalized the response to ET-1 (P < 0.05 versus salt)
(Figure 5, right panel).
Endothelium-independent relaxation to sodium nitroprusside was unaffected by
the treatments (NS) (Table
2).
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Renal Artery Structure
Vascular hypertrophy of the renal artery (expressed as ratio of tissue
weight in mg per mm2 vascular surface area) was observed after salt
loading in DS rats only. The ratio increased from 0.17 ± 0.01 to 0.23
± 0.01 mg/mm2, and was completely normalized by concomitant
LU135252 treatment (0.18 ± 0.01 mg/mm2, P <
0.001 versus salt). High salt diet with or without LU135252 had no
effect on vascular structure in DR rats (NS)
(Table 1).
Renal NOS Activity
Total NOS activity is given as the L-NAME-inhibitable portion of
L-citrulline formation. L-NAME concentrations <100 mmol/L showed incomplete
inhibition of total NOS activity. Total activity was comparable between
untreated DS and DR rats and higher in the medulla than in the cortex
(Figure 5). Total NOS activity
was of the same order of magnitude as reported previously in Dahl rats, Wistar
Kyoto rats, and spontaneously hypertensive rats
(8,28).
The majority of renal NOS activity was calcium-independent
(Table 3). Salt loading
markedly increased total NOS activity (about three-fold) in DR rats
(P < 0.05). In contrast, DS rats failed to increase total NOS
activity after salt loading (Figure
5). Interestingly, LU135252 treatment was associated with an
increase of total NOS activity in both cortex (74 ± 10%) and medulla
(58 ± 8%) in DS rats only (P < 0.05)
(Figure 5). Using membrane
fractions instead of crude homogenates resulted in no net difference with
regard to enzyme activity and had no effect on the ratio of calcium-dependent
and -independent conversion (data not shown).
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Glomerular Injury
Compared with DS control animals, the number of structurally normal
glomeruli (grade 0 renal injury) in hypertensive DS rats was reduced to 49
± 2% (P < 0.05). Hypertension in DS rats was associated
with pronounced glomerular damage with thickening of Bowman's capsule,
adhesion formation, mesangial proliferation, and hypertrophy of the
preglomerular artergial and arterioles
(Figure 6, middle panels).
Concomitant LU135252 treatment increased the number of healthy glomeruli from
49 ± 2 to 79 ± 4% (P < 0.05 versus high
salt) (Figure 7) and markedly
reduced structural glomerular changes (P < 0.05)
(Figure 6, right panels and
Figure 7). In salt-treated DR
rats, renal morphology was essentially identical to untreated DR rats, which
showed no signs of renal damage (authors' unpublished observation and
reference 26).
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Endothelin Protein Content in Renal Artery and Kidney
In hypertensive DS rats, renal artery ET-1 protein content was increased
from 165 ± 33 to 484 ± 57 pg/g tissue (P < 0.05).
This increase was prevented by chronic LU135252 treatment (124 ± 11
pg/g tissue, P < 0.05 versus salt). No changes occurred
in DR rats (Table 1). LU135252
also prevented the increase in ET-1 protein content in renal cortex and
medulla of DS rats only (Figure
8) (P < 0.05). Renal ET-3 protein content, which was
predominantly expressed in the medulla of both DS and DR rats, was unaffected
by high salt diet (NS) (Figure
8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Salt-Induced Hypertension: A Failure to Increase Renal NOS
Activity
The key finding of our study was the observation that DS rats failed to
increase renal NOS activity in response to salt loading. In contrast, and
consistent with increased renal NOS2 protein expression in salt-loaded Sprague
Dawley rats (17), renal NOS
activity in DR rats increased markedly after high salt diet treatment. Neither
in Sprague Dawley rats (17)
nor in DR rats in the present study did salt loading affect BP. The observed
failure in DS rats to adequately increase NOS activity could be linked to a
genetically determined defect of NOS or its regulation. In this context, it is
of interest that the gene of calcium-independent Nos2 cosegregates with BP in
DS rats
(12,13).
A functional defect of NOS is also suggested by studies demonstrating that
treatment with the NOS substrate L-arginine abolishes hypertension and
inhibits renal injury in this model
(14,15,29).
An unexpected and novel finding in contrast to previous studies (8,28) was that in all groups more than half of renal NOS activity was calcium-independent. It is important to note that in our experiments L-NAME concentrations <100 mmol/L did not completely block NOS activity in renal tissue. The concentration used was 100-fold higher compared to previously published protocols (8,28), and may in part explain the difference between calcium-dependent and -independent NOS activity compared with these previous studies. Unlike other calcium-independent NOS enzymes, expression of Nos2 is constitutive in Dahl and Wistar-Kyoto rat kidneys (30,31), and can be stimulated by salt loading (17). Also, sodium-mediated effects in humans may involve the L-arginine/NO pathway, as L-arginine-induced increases in sodium excretion and inhibition of fractional sodium reabsorption are dependent on salt loading (32). NOS2 regulates BP under normal conditions (18), and chronic inhibition of NOS2 is associated with reduced urinary nitrate excretion and proteinuria (33), indeed suggesting a role for NOS2-derived NO in renal function. Thus, compensatory upregulation of Nos2 expression and/or NOS activity in the kidney (as observed in DR rats in the present study) may represent a novel mechanism regulating the increased volume load and BP. Two distinct forms of Nos2 mRNA—macrophage Nos2 and vascular smooth muscle cell Nos2—have been identified in the rat kidney (30). In addition, evidence from a very recent study indicates that Nos2 mRNA is present in both rat cortex and medulla and that the enzyme is functionally active (34). Also, Nos2 is present in vascular smooth muscle of intrarenal vessels (30), and reduced nitrate production by Nos2 has been demonstrated in vascular smooth muscle cell of DS rats (13).
Future studies will determine whether the increase in calcium-independent NOS activity observed in DR rats is derived from NOS2. In this context, it is interesting to note that the paradigm of calcium dependency of NOS isoenzymes has recently been modified by studies showing that Nos2 activity—although completely calmodulin-independent—can be partly inhibited by removal of calcium (35). On the other hand, calcium-independent actions of Nos3 have recently been reported (36,37). Although we did not investigate gene expression of NOS isoenzymes, our study unequivocally demonstrates that salt sensitivity appears to involve a failure to increase renal NOS activity in response to salt loading, and that a substantial portion of the activity is calcium-independent. These findings are in agreement with the previously mentioned molecular data and a recent preliminary report (38).
Renal Artery Endothelial Dysfunction: Role of NO and Vasoconstrictor
Prostanoids
Impaired renal vasodilatory capacity after salt loading has been described
in Dahl hypertension (6). Here,
we provide evidence that "selective" endothelial dysfunction may
be one of the mechanisms interfering with renal artery vasodilation.
Unexpectedly, and in contrast to aorta
(9,25)
and mesenteric artery (39),
endothelium-dependent relaxations in the renal artery were unaffected by
hypertension. However, endothelial dysfunction due to abnormal increased basal
NO release and prostanoid-mediated contractions was present, which could
result in inappropriate vascoconstriction. To our knowledge, this is the first
study reporting the release of endothelium-derived vasoconstrictor prostanoids
as part of endothelial dysfunction in Dahl hypertension. These mechanisms are
likely to contribute to impaired renal vasodilatory capacity
(6). Furthermore, our data
suggest that endothelium-dependent relaxation per se should not be
used as the sole indicator to indicate "preserved" endothelial
function.
Interestingly, our study also shows that endothelin plays an important role in these functional changes. Although chronic ETA receptor blockade only partly reduced hypertension in DS rats, the treatment completely prevented the attenuation of basal NO release and the enhanced vasoconstrictor prostanoid release. Renal NOS activity was also slightly increased, suggesting that ETA receptor blockade can interfere with the L-arginine/NO pathway in vivo, in line with recent in vitro observations in mesangial (40) and vascular (41) cells in culture and normotensive animals in vivo (20). Both ET-1 (through ETA receptor-mediated mechanisms) (42,43) and NO deficiency (44) have been implicated in vasoconstrictor prostanoid formation in vitro. As chronic treatment with LU135252 completely normalized prostanoid-mediated vaso-constriction and increased tissue ET-1 levels, increased vascular ET-1 concentrations may well contribute to endothelial vasoconstrictor prostanoid release in vivo. As observed previously in other vascular beds (9,39), contractions to exogenous ET-1 but not those to norepinephrine were reduced in the renal artery, and this was likely caused by agonist-induced receptor downregulation due to increased vascular ET-1 content. Again, treatment with LU135252 prevented both the increase of ET-1 protein and the attenuation of contractile responses.
Endothelin: Mediator of Renal Injury and Endothelial Dysfunction
In this study, we have demonstrated that both renal and vascular ET-1
protein levels, but not renal ET-3 protein, markedly increase in DS rats after
salt loading. This was associated with renal artery hypertrophy and
glomerulosclerosis in hypertensive DS rats, confirming previous reports
(26,45).
Although genetic analyses have linked the ET-3 gene locus
(23) but not the ET-1 gene
locus (46) to hypertension in
DS rats, and despite altered renal ET-3 expression in other forms of renal
injury (47), we found no
evidence that ET-3 expression is modulated in hypertensive DS rats. Thus, ET-1
but not ET-3 appeared to be the likely mediator of salt-mediated injury.
Consistent with this hypothesis, the structural and particularly the
functional changes associated with hypertension were largely normalized by
concomitant treatment with an orally active ETA antagonist, which
had only moderate effects on the salt-induced BP increase. It is noteworthy
that no activation of the ET system was observed in DR rats in which NOS
activity (in contrast to DS rats) almost tripled. We speculate that the
failure to adequately increase NOS activity may facilitate activation of the
ET system in hypertensive DS rats, which is regulated by NO
(16,48).
This "intrinsic" NO deficiency in DS rats, in turn, may promote
the detrimental effect of ET-1 on renal structure and function. Indeed,
chronic NO deficiency induced pharmacologically by L-NAME treatment is
associated with hypertension
(2) and with pronounced changes
in renal morphology and function, which can be attenuated by ET receptor
antagonism
(11,49.
One of the limitations of our study is that we did not use another
antihypertensive agent to examine the effect of BP lowering. However, it is
unlikely that normalization of the observed changes was due to the
antihypertensive effects of LU135252, which lowered BP only in part. In line
with this notion, a previous study demonstrated that antihypertensive therapy
with the diuretic indapamide actually normalized BP, but did not provide
effective renal protection (8).
Unfortunately, these investigators did not examine vascular hypertrophy.
Schiffrin and colleagues have suggested that the effects of ET-1 on vascular
structure may be, at least in part, pressure-independent
(50), which is in keeping with
the present study and our observations in atherosclerotic mice
(20).
We have reported previously that chronic ETA receptor blockade normalizes elevated tissue levels of ET-1 protein in hypertension and atherosclerosis (9,20,24). We have confirmed these findings in the present study, in which ET-1 tissue levels in LU135252-treated animals were comparable to those seen in untreated DS control rats. The underlying mechanism is presently unknown. It is possible, however, that increased endothelial NO activity, which regulates ET-1 production (51) and was observed after LU135252 treatment, was involved. The effects of LU135252 may also involve ETB receptor-mediated mechanisms such as NO formation, because ETB blockade worsens renovascular function and injury in deoxycorticosterone acetate-salt hypertension (52). Other possible mechanisms include ETA receptor-coupled autocrine regulation of ET-1 synthesis at the transcriptional (53) or protein (54,55) level.
Clinical Implications
Salt sensitivity in patients with essential hypertension is associated with
increased cardiovascular risk
(1) and is particularly common
among African-Americans. These patients are often resistant to conventional
antihypertensive therapy (56)
and commonly develop hypertensive renal injury. Two recent studies in this
population demonstrated both endothelial dysfunction
(57) and increased renal
vasoconstriction in response to salt loading
(58), consistent with our
observations in experimental salt-sensitive hypertension. The ET system
appears to be activated in these patients because plasma levels of ET-1 are
extraordinarily high (59).
Therefore, it is reasonable to speculate that selective ETA
receptor antagonism will provide additional therapeutic benefit for the
treatment of salt-sensitive forms of essential hypertension.
In conclusion, we have demonstrated: (1) a failure to increase NOS activity; (2) enhanced renal artery vasoconstriction due to selective endothelial dysfunction; and (3) a role for ET-1 as mediator of functional and structural changes that act as novel mechanisms in the pathogenesis of experimental salt-sensitive hypertension. Clinical studies will show whether ETA receptor blockade has the therapeutic potential to inhibit endorgan damage in patients with salt-sensitive hypertension.
Note Added in Proof: While this manuscript was under revision, a study was published reporting that chronic inhibition of Nos2 by use of several isoform-specific enzyme inhibitors resulted in salt-induced hypertension without affecting endothelial function in resistance arteries in formerly salt-resistant DR rats (60).
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Acknowledgments |
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Footnotes |
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American Society of Nephrology
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Fang, J.-J. Mu, L.-C. He, S.-C. Wang, and Z.-Q. Liu Salt Loading on Plasma Asymmetrical Dimethylarginine and the Protective Role of Potassium Supplement in Normotensive Salt-Sensitive Asians Hypertension, October 1, 2006; 48(4): 724 - 729. [Abstract] [Full Text] [PDF]
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 M. Feletou and P. M. Vanhoutte Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture) Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H985 - H1002. [Abstract] [Full Text] [PDF]
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 M. J. A. Kocks, S. Gschwend, D. de Zeeuw, G. Navis, and H. Buikema Low Sodium Modifies the Vascular Effects of Angiotensin-Converting Enzyme Inhibitor Therapy in Healthy Rats J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1183 - 1189. [Abstract] [Full Text] [PDF]
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 C. Kitiyakara, T. Chabrashvili, Y. Chen, J. Blau, A. Karber, S. Aslam, W. J. Welch, and C. S. Wilcox Salt Intake, Oxidative Stress, and Renal Expression of NADPH Oxidase and Superoxide Dismutase J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2775 - 2782. [Abstract] [Full Text] [PDF]
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Y. Nishimoto, T. Tomida, H. Matsui, T. Ito, and K. Okumura Decrease in Renal Medullary Endothelial Nitric Oxide Synthase of Fructose-Fed, Salt-Sensitive Hypertensive Rats Hypertension, August 1, 2002; 40(2): 190 - 194. [Abstract] [Full Text] [PDF]
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T. QUASCHNING, L. V. D'USCIO, S. SHAW, H.-J. GRONE, F. RUSCHITZKA, and T. F. LUSCHER Vasopeptidase Inhibition Restores Renovascular Endothelial Dysfunction in Salt-Induced Hypertension J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2280 - 2287. [Abstract] [Full Text] [PDF]
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