Vol. 274, Issue 4, F642-F649, April 1998
Downregulation of nitric oxide synthase in chronic renal
insufficiency: role of excess PTH
N. D.
Vaziri,
Z.
Ni,
X. Q.
Wang,
F.
Oveisi, and
X. J.
Zhou
Division of Nephrology, Department of Medicine, University of
California, Irvine, California 92868
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ABSTRACT |
The available data
on the effect of chronic renal failure (CRF) on nitric oxide (NO)
metabolism are limited and contradictory. We studied rats with CRF 6 wk
after a five-sixths nephrectomy and compared the results with those in
the sham-operated controls, felodipine-treated CRF, and
parathyroidectomized (CRF-PTX) animals. CRF was produced by surgical
resection of the upper and lower thirds of the left kidney, followed by
contralateral nephrectomy. We chose this model, as opposed to that
produced by renal artery branch ligation, because the latter causes
exuberant hypertension (HTN), which independently affects NO
metabolism. The CRF group exhibited a mild HTN coupled with elevated
basal platelet cytosolic Ca2+
concentration
([Ca2+]i),
blunted hypotensive response to
L-arginine, decreased
hypertensive response to NO synthase (NOS) inhibitor,
NG-monomethyl-L-arginine, and normal
hypotensive response to NO donor, sodium nitroprusside. This was
associated with a significant reduction in urinary excretion of stable
NO metabolites (NOX) and depressed NOS activity, as well as endothelial
and inducible NO synthase (eNOS and iNOS, respectively) protein
contents of thoracic aorta and the remnant kidney in the CRF animals.
Calcium channel blockade and PTX lowered blood pressure, increased
urinary NOX, and enhanced vascular NOS activity, as well as eNOS and
iNOS protein expressions in the tested tissues. Thus CRF animals
exhibited significant reductions in vascular NOS activity and eNOS and
iNOS expressions. These abnormalities were reversed by calcium channel blockade and PTX, suggesting the possible causal role of CRF-induced dysregulation of
[Ca2+]i.
uremia; hypertension; blood pressure; endothelial-derived relaxing
factor; parathyroid hormone; calcium channel blockers; anemia
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INTRODUCTION |
NITRIC OXIDE (NO), once known as a toxic environmental
pollutant, is now recognized as an important endogenous biological modulator with diverse physiological actions. NO modulates vascular resistance, tissue perfusion, blood pressure, and platelet function and
serves as a neurotransmitter, a host defense agent, an
antiproliferative factor, and an oxygen free radical scavenger, while
being a free radical itself (17, 20).
In vascular smooth muscle, NO stimulates production of cGMP, which
promotes vasodilation by lowering intracellular
Ca2+ concentration
([Ca2+]i)
and reducing sensitivity of the contractile proteins to
Ca2+ (11, 13). NO is synthesized
from conversion of L-arginine to
L-citrulline by a family of NO
synthases (NOS), which include constitutive and inducible isoforms
(21). Basal release of NO by endothelial cells contributes to the
maintenance of normal blood pressure in the resting state. Conversely,
relative NO deficiency leads to a rise in arterial blood pressure (15).
It has been demonstrated that
L-arginine:NO pathway is
impaired in animal models and patients with advanced hypertension (HTN)
(6, 14, 15, 27).
The effect of chronic renal failure (CRF) on NO metabolism is
incompletely understood. Both increased and decreased activities of
L-arginine:NO pathway have been
suggested in rats and humans with CRF (1, 2, 7, 16, 24, 29, 30). In
this regard, several studies have pointed to the possible reduction of
NO production in CRF. For instance, renal insufficiency has been shown
to cause accumulation of endogenous NOS inhibitors (methylated
L-arginine derivatives and
guanidino compounds), which can potentially depress NO production in
CRF (16, 30). In addition, rats with renal mass reduction show reduced
urinary excretion of NO metabolites (total nitrates + nitrites) and
diminished histochemically detectable NOS in the remnant kidney,
suggesting reduced renal NO production (2). In fact, chronic
administration of an NO donor has been shown to retard progressive
deterioration of renal function and structure in experimental CRF (7).
Although the above observations point to impaired local or systemic NO
production in CRF, a number of other studies have offered direct or
indirect evidence for increased vascular NO production in CRF (2, 24,
29). First, pharmacological inhibition of NOS has been shown to improve
platelet dysfunction in patients and animals with CRF (24, 29). These observations suggest that uremic platelet dysfunction may be due to
increased systemic vascular NO production (24, 29). Finally, Aiello et
al. (2) recently showed that reduced urinary nitrate/nitrite excretion
and decreased NOS appearance in the remnant kidney of rats with renal
mass reduction is coupled with increased plasma nitrate/nitrite
concentration and histochemically detectable NOS in the aorta. The
authors concluded that, in this model of CRF, renal NO production is
reduced, but systemic vascular NO production is increased (2). They
attributed the latter finding to the known stimulatory action of high
blood pressure on vascular NOS activity, citing several in vivo and in
vitro studies (4, 22, 25, 28). It should be noted that the CRF model
employed by this group was produced by ligation of the upper and lower
branches of the renal artery, leading to partial infarction of one
kidney, followed by contralateral nephrectomy (2). This model of renal mass reduction is associated with an exuberant HTN, which can independently stimulate
L-arginine:NO pathway. In the
present study, we accomplished renal mass reduction by surgical
resection of the upper and lower thirds of one kidney, followed by
contralateral nephrectomy, which produces CRF with only a mild HTN.
This was intended to demonstrate the effect of CRF, per se, on
L-arginine:NO pathway by
minimizing that of severe HTN. The results showed that, in contrast to
the severely hypertensive renal infarction model, the mildly
hypertensive surgical-resection model of CRF is associated with
significant downregulation of vascular NOS activity and tissue NOS
isoform protein expressions. The study further suggests that the
observed downregulation of vascular NOS in this model of CRF is related
to secondary hyperparathyroidism.
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MATERIALS AND METHODS |
Animal Models
Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) with
an average body weight of 300 g were used. The animals were fed a
standard laboratory diet (Purina Rat Chow; Purina Mills, Brentwood, MO)
and water ad libitum. Animals were randomly assigned to the CRF and
sham-operated normal control groups. Animals assigned to the CRF group
underwent five-sixths nephrectomy as follows. Under general anesthesia
(pentobarbital sodium, 50 mg/kg ip), the animals underwent surgical
resection of the upper and lower thirds of the left kidney, followed by
a right nephrectomy 4 days later to produce CRF. The nephrectomies were
carried out extraperitoneally through a dorsal incision. Strict
hemostasis and aseptic measures were observed during the surgical
procedures.
To examine the possible effect of previously demonstrated elevation of
basal cytosolic Ca2+ in CRF (32),
a group of CRF animals was treated with the calcium channel blocker,
felodipine (CRF-FEL group). These animals received felodipine (7 mg · kg
1 · day1;
Astra-Merck, Wayne, PA), using osmotic pumps implanted subcutaneously (Alza, Palo Alto, CA).
Excess parathyroid hormone (PTH) has been shown to depress expression
of a number of enzymes, i.e., lipoprotein lipase and hepatic lipase, as
well as several hormone receptors in CRF (3, 12, 19, 31). These
abnormalities have been attributed to a PTH-induced rise in
[Ca2+]i.
Parathyroidectomy has been demonstrated to lower
[Ca2+]i
and reverse the abnormalities of hepatic lipase, lipoprotein lipase,
and hormone receptor expression in CRF (3, 12, 19, 31). In an attempt
to explore the possible role of excess PTH, a group of animals was
subjected to surgical parathyroidectomy prior to nephrectomy (CRF-PTX
group). Animals exhibiting serum calcium concentrations >6.0 mg/dl
(while receiving no calcium supplementation) were excluded. In an
attempt to dissect the possible effect of hypocalcemia, per se, the
drinking water consumed by these animals was supplemented with calcium
gluconate (Mallinckrodt Chemical, Paris, KY) at 5% concentration,
which has been shown to correct hypocalcemia in this model (12). After
seven days of stabilization, the animals were subjected to five-sixths
nephrectomy and studied as described above.
Systolic arterial blood pressure was monitored once a week using a tail
sphygmomanometer (Harvard Apparatus, South Natick, MA). At the end of
the 6-wk observation period, the animals were placed in metabolic cages
for a 24-h urine collection. Subgroups of animals were then employed
for various experiments described below.
Response to L-Arginine,
NG-monomethyl-L-arginine, and Sodium
Nitroprusside
Under general anesthesia with pentobarbital sodium (50 mg/kg ip), the
left jugular vein and carotid artery were cannulated with polyethylene
tubing (PE-50). A tracheal cannula was also inserted. Rats were then
placed on heating pads. Arterial blood pressure was monitored directly
via the arterial catheter, which was connected to a Gould P-50 pressure
transducer and recorded on a Dynograph R511A recorder (Sensor Medics,
Anaheim, CA). Once animals were stable, blood pressure was recorded for
5 min to determine the baseline value. Subsequently, NO precursor,
L-arginine (100 mg/kg; Sigma
Chemical, St. Louis, MO), or
NG-monomethyl-L-arginine
(L-NMMA, 10 mg/kg; Sigma
Chemical), a competitive inhibitor of NOS, were given as bolus
intravenous injections. In addition, the hypotensive response to
intravenous bolus injection of NO donor, sodium nitroprusside (SNP, 0.5 µg/kg; Sigma Chemical), was determined. Response to each drug was
calculated as peak change in blood pressure from baseline. Mean
arterial pressure was calculated as the sum of diastolic
blood pressure and one-third of the pulse pressure. Each drug was
injected at least twice, and the average of the values obtained was
used.
Measurements of
/
The concentration of total nitrate and nitrite (NOX) in the test
samples was determined by a modification of the procedure described
previously (5), using the purge system of a Sievers Instruments model
270B Nitric Oxide Analyzer (Sievers Instruments, Boulder, CO). Briefly,
urine samples were diluted ten times in distilled water prior to
analysis. A saturated solution of
VCl3 in 1 M HCl was prepared and
filtered prior to use. Five milliliters of this reagent were added to
the purge vessel and purged with nitrogen gas for 5-10 min prior
to use. The purge vessel was equipped with a cold water condenser and a
water jacket to permit heating of the reagent to 95°C, using a
circulating water bath. The hydrochloric acid vapors were removed by a
gas bubbler containing ~15 ml of 1 M NaOH. The gas flow rate into the
chemiluminescence detector was controlled, using a needle valve
adjusted to yield a cell pressure of ~7 Torr. The flow rate of
nitrogen into the purge vessel was adjusted to prevent vacuum
distillation of the reagent.
Samples were injected into the purge vessel to react with the
VCl3/HCl reagent, which converted
nitrate, nitrite, and S-nitroso compounds to NO. The NO produced was stripped from the reaction chamber
(by purging with nitrogen and vacuum) and detected by ozone-induced
chemiluminescence in the chemiluminescence detector. The signal
generated (NO peak and peak area) was recorded and processed by a
Hewlett- Packard model 3390 integrator. In a typical assay, 5 µl of
the test sample were injected to the purge vessel, and all samples were
run in triplicate.
Standard curves were constructed using various concentrations of
(5-100 µM) relating the
luminescence produced to the given
concentrations of the standard solutions. The amount of
/ in the test sample was determined by interpolation of the result into
the standard curve.
Tissue Preparation
Thoracic aorta and remnant kidneys were used for determination of NOS.
Rats were killed by decapitation, and thoracic aorta and remnant
kidneys were immediately excised, cleaned with PBS, frozen in liquid
nitrogen, and stored at 
75°C. Homogenates (25% wt/vol) were
prepared in 10 mM HEPES buffer, pH 7.4, containing 320 mM sucrose, 1 mM
EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, and 2 µg/ml
aprotinin at 0-4°C, with the aid of a tissue grinder fitted
with a motor-driven ground-glass pestle. Homogenates were centrifuged
at 12,000 g for 5 min at 4°C to
remove tissue debris without precipitating plasma membrane fragments.
The supernatant was used for determination of NOS activity.
For Western blot analysis, tissue was homogenized in five volumes of
lysis buffer (1% SDS, 1.0 mM sodium vanadate, 10 mM
Tris · HCl, pH 7.4) containing 10 µM pepstatin, 13 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The homogenate
was centrifuged at 12,000 g for 5 min
at 15°C to remove tissue debris without precipitating plasma
membrane fragments (8, 18). Protein concentration was determined by
using a Bio-Rad kit (Bio-Rad, Hercules, CA).
Western Blot Analysis
These measurements were carried out to determine the endothelial and
inducible NOS (eNOS and iNOS, respectively) protein mass, as previously
described (8, 18). Anti-eNOS monoclonal antibody, anti-Mac NOS-I, human
endothelial positive control, and mouse macrophage positive control
were supplied by Transduction Laboratories (Lexington, KY).
Peroxidase-conjugated goat anti-mouse IgG antibody was purchased from
Pierce (Rockford, IL). Briefly, tissue preparations (50 µg of
protein) were size fractionated on 4-12% Tris-glycine gel (Novex,
San Diego, CA) at 120 V for 3 h. After electrophoresis, proteins were
transferred onto Hybond-ECL enhanced chemiluminescence membrane
(Amersham Life Sciences, Arlington Heights, IL) at 500 mA for 120 min,
using the Novex transfer system. The membrane was prehybridized in 25 ml of Super Block blocking buffer (Pierce) for 1 h and then hybridized
for an additional 1-h period in the same buffer containing 10 µl of
the given anti-NOS monoclonal antibody (1:2,500). The membrane was then
washed for 60 min in a shaking bath, with the wash buffer
(Tris-buffered saline containing 0.1% Tween 20) changed every 10 min
prior to 1 h of incubation in Super Block blocking buffer plus goat
anti-mouse IgG-horseradish peroxidase at the final titer of 1:100,000.
Experiments were carried out at room temperature. The washes were
repeated before the membrane was developed with a light emitting
nonradioactive method using Ultraluminal Enhanced Solution (Pierce).
The membrane was then subjected to autoluminography for 10 s. The
autoluminographs were scanned with a laser densitometer (model PD1211;
Molecular Dynamics, Sunnyvale, CA) to determine the relative optical
densities of the bands. In all instances, the membranes were stained
with Ponceau stain, which verified the uniformity of protein load and
transfer efficiency across the test samples.
NOS Activity Assay
NOS activity was measured as previously described (9). In brief, enzyme
reactions were conducted at 37°C for 30 min in 40 µl of the
supernatant and 100 µl of 40 mM potassium phosphate buffer, pH 7, containing 4.8 mM DL-valine, 1 mM NADPH, 1 mM MgCl2, 2 mM
CaCl2, 20 µM
L-arginine, 1 µg/ml
calmodulin, and 1.25 µl/ml L-[3H]arginine
(59 Ci/mM, Amersham). On each occasion, parallel measurements were
obtained in the presence and absence of 1 mM
NG-methyl-L-arginine.
The reactions were terminated by 0.86 ml ice-cold stop buffer
containing 0.2 mM EDTA. Two hundred and fifty (250) milligrams of the
Na+ form of Dowex 50W-X8 resin was
added to a 0.25-ml aliquot of the reaction mixture and shaken for at
least 5 min prior to microcentrifugation to remove the remaining
L-arginine. The
Na+ form of Dowex 50W was prepared
by washing the H+ form of the
resin (100-200 mesh, Bio-Rad) with 1 M NaOH four times and then
washing with H2O until pH < 7.5. A 100-µl aliquot of the latter supernatant (containing
L-citrulline) was mixed with 10 ml of scintillation cocktail in a 20-ml scintillation vial and counted
by a Beckman LS-9000 counter (Beckman Instruments, Fullerton, CA). Net
radioactivity was determined by substrating the cpm observed in the
presence of
NG-methyl-L-arginine
from that observed in the absence of
NG-methyl-L-arginine.
NOS activity was determined from the production of
[3H]citrulline per
minute per milligram of protein.
Platelet Cytosolic Calcium Measurements
At the end of the 6-wk observation period, a subgroup of rats was
killed, and platelets were isolated using a procedure described previously (10). The isolated platelets were suspended in
HEPES-buffered saline (HBS) with the following composition (in mmol/l):
145 NaCl, 5 KCl, 0.8 Na2HPO4,
0.2 KH2PO4,
1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. This resulted in a platelet suspension containing ~2 × 108 cells/ml.
[Ca2+]i
was determined with the fluorescent calcium indicator, fura 2-AM. The
suspended platelets were loaded with 4 mol fura 2-AM in the presence of
0.02% Pluronic F-127 to facilitate entry of the indicator into the
cells. In addition, 2 mM probenecid was added to minimize leakage of
fura 2 out of the platelets (10). Cells were incubated for 60 min at
37°C, then centrifuged at 400 g
for 20 min. The supernatant was decanted, and an equal volume of HBS
added. The cells were incubated at 37°C for 1 h to allow complete
hydrolysis of the AM group. The dye-loaded cells were suspended in HBS
containing 2 mmol CaCl2 and kept
under constant magnetic stirring in a thermostatically controlled
cuvette of a spectrofluorometer (DMX 1000; SLM Instruments, Urbana,
IL). Alternating excitation wavelengths of 340 and 380 nm were used, with an emission wavelength of 510 nm. Ratios of fluorescence (R = 340/380 nm) were measured every second, automatically corrected for
autofluorescence, and plotted graphically for each sample analyzed.
Values of autofluorescence were <5% of the fluorescence of the
dye-loaded cells and were measured for every experiment.
[Ca2+]i
was calculated as described earlier (33), using the following formula:
[Ca2+]i = (Kd)(B)(R Rmin)/(Rmax R), where B is the ratio of 380 nm fluorescence in the absence
and presence of saturating concentration of calcium,
Kd is the
dissociation constant for fura 2 (assumed to be 225 nM), and R is the
ratio of fluorescence, as defined above. The cells were lysed with
Triton (0.05%) to obtain Rmax in
the presence of 2 mmol calcium, and
Rmin was obtained by the addition
of 10 mmol EGTA and sufficient NaOH to raise the pH to 8.5.
Data Presentation and Analysis
Data are presented as means ± SE. ANOVA and Duncan multiple range
test were used as appropriate. P 
0.05 was considered statistically significant.
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RESULTS |
General
As shown in Table 1 and Fig.
1, the body weight and hematocrit obtained
at the conclusion of the study were significantly lower in the CRF,
CRF-FEL, and CRF-PTX groups, compared with the normal control group,
despite comparable values at the time of randomization. As expected,
the CRF groups showed a significant rise in serum creatinine and a
significant decline in creatinine clearance. In addition, the CRF group
exhibited a significant increase in arterial blood pressure, compared
with both the baseline value and those found in the normal control,
CRF-FEL, and CRF-PTX groups. The CRF-PTX group showed marked
hypocalcemia (5.46 ± 0.5 mg/dl) prior to initiation of calcium
supplementation. The associated hypocalcemia was corrected by
addition of calcium gluconate to the drinking water, yielding a serum
calcium concentration comparable to values found in the other groups
(CRF-PTX, 9.6 ± 0.31; CRF, 9.0 ± 0.35; and normal control
group, 9.7 ± 0.14 mg/dl).
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Table 1.
Body weight, plasma creatinine concentration, plasma creatinine
clearance, and hematocrit in CRF and normal control groups
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Fig. 1.
Systolic blood pressure measurements obtained in chronic renal failure
rats treated with placebo (CRF), felodipine (CRF-Fel), or
parathyroidectomy (CRF-PTX) and the normal (NL) control group during
the 6-wk observation period. N = 6 in
each group. * P < 0.01 (multiple measure ANOVA).
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Effect of CRF
Blood pressure response to
L-arginine,
L-NMMA, and
SNP. In both the normal and CRF animals, blood pressure
declined within a few seconds after bolus injection of
L-arginine and returned to
baseline promptly thereafter (Fig. 2). The
magnitude of the L-arginine-induced fall in
arterial blood pressure was ~1.5-fold greater in the control group
than that observed in the CRF group (P < 0.05). L-NMMA administration
led to a rise in blood pressure lasting 10-20 min in
both groups. Maximal pressor response to L-NMMA was significantly lower
(P < 0.01) in the CRF group compared with the normal controls. However, the magnitude of hypotensive response to NO donor, SNP, in the CRF group was similar to that found
in the control group.
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Fig. 2.
Change in mean arterial pressure ( MAP) from baseline in response to
intravenous bolus injections of
L-arginine
(L-Arg, 100 µg/kg),
NG-monomethyl-L-arginine
(L-NMMA, 10 µg/kg), and sodium
nitroprusside (SNP, 0.5 µg/kg) in rats with CRF and sham-operated
controls. Each column represents mean values obtained in 6 animals.
Bars denote SEM. * P < 0.05 vs. control group.
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Urinary excretion and plasma concentration of
NOX. Data are given in Table
2 demonstrate that the CRF group showed a
significant reduction in urinary excretion of total nitrates and
nitrites. This was accompanied by normal plasma NOX concentration.
These findings point to a possible reduction in total body NO
production.
NOS activity and protein mass. The
results in Figs.
3-7
indicate that the CRF group showed a significant reduction in aorta NOS
activity. The reduction in the thoracic aorta NOS activity in the CRF
group was accompanied by parallel reductions in the aorta eNOS and iNOS
protein contents. The concomitant reductions in vascular tissue NOS
activity with those of eNOS and iNOS proteins point to the quantitative
deficiency, as opposed to the mere functional impairment of NOS in this
model of CRF. As with the aorta, eNOS and iNOS protein abundance was
significantly reduced in the remnant kidneys of the CRF animals.
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Fig. 3.
Thoracic aorta tissue nitric oxide synthase (NOS) activity in NL,
placebo-treated CRF, felodipine-treated CRF (CRF/FEL), and
parathyroidectomized CRF (CRF/PTX) animals.
N = 6 in each group.
* P < 0.01 vs. other groups.
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Fig. 4.
A: representative Western blot of the
thoracic aorta endothelial NOS (eNOS) in 2 NL, 2 placebo-treated CRF
(CRF), two CRF/FEL, and 2 CRF/PTX animals.
B: group data illustrating relative
optical densities of eNOS protein bands in the study animals.
N = 6 in each group.
* P < 0.01 relative to other
groups.
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Fig. 5.
A: representative Western blot of the
thoracic aorta inducible NOS (iNOS) in 2 NL, 2 placebo-treated CRF, 2 CRF/FEL, and 2 CRF/PTX animals.
B: group data illustrating relative
optical densities of the iNOS protein bands in the study animals.
N = 6 in each group.
* P < 0.05 relative to other
groups.
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Platelet cytosolic
[Ca2+].
Compared with the normal control group, resting platelet
[Ca2+]i
was significantly elevated (P < 0.05) in the CRF animals. In contrast, thrombin-stimulated rise in
[Ca2+]i
was significantly attenuated in the CRF group (Fig. 8).
Thus the magnitude of change from basal to peak value was greatly
diminished in CRF animals.
Effect of Calcium Channel Blockade
Data in Table 2 and Figs. 1 and 3-6 show that calcium channel
blockade with felodipine mitigated the CRF-induced HTN and abrogated the CRF-associated elevation of basal
[Ca2+]i.
However, calcium channel blockade did not interfere with
thrombin-stimulated rise in platelet
[Ca2+]i.
Thus the difference between the peak-stimulated and basal values
([Ca2+]i)
was improved with this therapy. Calcium channel blockade resulted in a
significant rise in urinary excretion and plasma concentration of NOX
to supranormal values. This was accompanied by a complete restoration
of renal and vascular tissue eNOS and iNOS proteins, as well as
vascular NOS activity in the CRF-FEL group. This phenomenon suggests
that the CRF-associated downregulation of vascular eNOS and iNOS
expressions may be due to CRF-induced dysregulation of [Ca2+]i.
In addition, the data suggest that the antihypertensive effect of
calcium channel blockade in this model may be, in part, mediated by
upregulation of vascular NOS expression and NO production.
Effects of Parathyroidectomy
Data in Table 2 and Figs. 3-7 show that, as with calcium channel
blockade, PTX increased urinary excretion of NOX and enhanced vascular
tissue NOS activity, as well as renal and vascular tissue eNOS and iNOS
protein expressions. This was accompanied by a significant amelioration
of CRF-associated HTN. Thus the effects of PTX were directionally
similar to those of calcium channel blockade.
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Fig. 6.
A: representative Western blot of the
remnant kidney eNOS in 2 NL, 2 placebo-treated CRF, 2 CRF/FEL,
and 2 CRF/PTX animals. B: group
data illustrating relative optical densities of the eNOS protein bands
in the study animals. N = 6 in each
group. * P < 0.01 relative to
other groups. ** P < 0.05 relative to the NL group.
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DISCUSSION |
As expected, the CRF animals exhibited a mild but significant rise in
arterial blood pressure, coupled with a significant increase in basal
[Ca2+]i.
Investigation of the
L-arginine:NO pathway in the CRF
animals revealed a blunted hypotensive response to the NO precursor,
L-arginine, in the face of
normal response to NO donor, SNP. These observations suggest the
presence of a possible defect in NO production from L-arginine, as opposed
to a resistance to the action of NO. This supposition is further
supported by the blunted hypertensive response to NOS inhibitor,
L-NMMA, which points to reduced
NO dependence/availability in the CRF animals. In addition, the
combination of normal response to NO donor and diminished response to
NOS inhibitor in this model argues against excess vascular NO
production, which would have been marked by reduced sensitivity to
exogenous NO and exaggerated response to NOS inhibition, not seen
here.
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Fig. 7.
A: representative Western blot of the
remnant kidney iNOS in 2 NL, 2 placebo-treated CRF, 2 CRF/FEL,
and 2 CRF/PTX animals. B: group
data illustrating relative optical densities of the iNOS protein bands
in the study animals. N = 6 in each
group. * P < 0.05 relative to
other groups. ** P < 0.05 relative to the NL group.
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Fig. 8.
Basal cytosolic calcium (top) and
change from basal to peak value
(bottom) observed following thrombin
stimulation in the placebo-treated CRF, CRF/FEL, and normal control
animals. Each column represents mean values obtained from 20-25
experiments. Bars denote SEM.
* P < 0.05 vs. control
group.
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Depressed L-arginine:NO pathway
in this model could be due to decreased
L-arginine availability.
However, the blunted response to
L-arginine administration shown
here argues against this possibility. Alternatively, accumulation of
endogenous NOS inhibitors in uremia can account for the observed
depression of L-arginine:NO
pathway activity. In fact, increased concentration of methylated
arginine and guanidino compounds with NOS inhibitory properties has
been previously shown in patients with end-stage renal disease (16, 30). Although this is in part true, competitive inhibition of the
enzyme should not lead to the reduction of immunodetectable quantity of
the NOS proteins as shown here. In fact, eNOS and iNOS proteins were
significantly reduced in the CRF animals, suggesting decreased enzyme
availability as opposed to a mere competitive inhibition of the enzyme.
Thus the present study provides evidence for acquired NOS deficiency in
the vascular and renal tissue of mildly hypertensive rats with CRF
produced by five-sixths nephrectomy using surgical resection. Further
support for the downregulation of
L-arginine:NO pathway in this
model comes from the marked reduction in urinary NOX excretion in the
CRF animals. The reduction in urinary NOX excretion in the CRF animals
was accompanied by normal plasma NOX concentration, suggesting
depressed total body NO generation. This is because, if systemic NO
generation were normal, CRF would have raised plasma NOX concentration,
as exemplified by various other nitrogenous end products whose plasma
concentrations rise in CRF (azotemia). Thus plasma NOX level more
likely reflects renal function and volume distribution rather than
systemic NO generation.
These findings, in part, differ from those reported by Aiello et al.
(2), who used a different CRF model produced by ligation of the upper
and lower renal artery branches of one kidney followed by contralateral
nephrectomy. Unlike the model employed in the present study, in which
CRF is associated with mild HTN, the CRF model of renal infarction is
associated with severe exuberant HTN. The authors rightfully attributed
the upregulation of vascular NOS in their CRF animals to the well-known
stimulatory action of HTN, citing several in vivo and in vitro studies
of the direct effects of pressure and flow (4, 22, 25, 28). In contrast to the renal infarction model employed by Aiello et al., the surgical resection model of five-sixths nephrectomy is associated with a
relatively mild HTN. Thus the intense stimulation of vascular NOS by
severe HTN was far greater in the CRF animals employed in the latter
study than in those employed in the present study. Based on the results
of the present study, we believe that CRF, per se, leads to a
reversible downregulation of vascular NOS. Consequently, removal of the
suppressive factor (e.g., excess PTH) can correct this abnormality.
Similarly, the presence of intense stimulatory influences such as
severe HTN, volume expansion, and increased shear stress can override
the downregulatory action of CRF, leading to a net increase in vascular
NOS activity and protein mass. This was clearly exemplified by CRF
animals described by Aiello et al. (2).
The CRF animals exhibited a significant elevation of basal
[Ca2+]i
and a defective rise in stimulated
[Ca2+]i,
consistent with our earlier observations (32). Excess production of PTH
has been linked to elevation of
[Ca2+]i
in a variety of cell systems in CRF. In addition, increased PTH
production and the associated elevation of
[Ca2+]i
in CRF has been found to depress expression of several enzymes, including hepatic lipase and lipoprotein lipase, as well as several hormone receptors. Furthermore, parathyroidectomy has been shown to
reverse these abnormalities in patients and animals with CRF (3, 12,
19, 31). We therefore sought to examine the role of secondary
hyperparathyroidism in downregulation of renal and vascular NOS
expression by including a group of CRF-PTX animals. The CRF-PTX animals
showed a significant increase in remnant kidney and vascular tissue
eNOS and iNOS protein expressions together with a significant rise in
urinary NOX excretion. These results point to the important role of
excess PTH in the pathogenesis of CRF-associated depression of vascular
NOS.
In an attempt to determine whether the effect of excess PTH on
L-arginine:NO pathway in CRF
animals is mediated by the change in
[Ca2+]i,
we tested the effect of pharmacological modification of
[Ca2+]i.
To this end, a subgroup of rats was treated with calcium channel blockade, using implanted osmotic pumps loaded with felodipine. The
results showed an expected amelioration of CRF-associated hypertension,
coupled with the restoration of normal basal
[Ca2+]i.
Interestingly, remnant kidney and vascular eNOS and iNOS protein contents and vascular NOS activity were increased significantly and
urinary NOX excretion reached supranormal levels by this therapy in the
CRF animals. The directional similarities of the effects of felodipine
and parathyroid ablation (which both lower resting [Ca2+]i)
on vascular NOS expression and NO production in rats with CRF suggest
that these abnormalities may be linked to the CRF-induced dysregulation
of
[Ca2+]i.
It should be noted that hemodynamic alterations may have also contributed to the observed improvement of NO production and NOS expression with PTX and calcium channel blockade in the CRF animals. Our data further indicate that the antihypertensive action of calcium
channel blockade in this model may be, in part, mediated by NO.
Interestingly, calcium channel blockade and to a lesser extent PTX
resulted in the rise of urinary excretion and plasma concentration of
NOX to supranormal levels. This was accompanied by similar rises in NOS
proteins of the tested tissues. The available data do not allow
definitive explanation for this exaggerated response. However, lifting
of the inhibitory action of abnormal
[Ca2+]i
may have unmasked the upregulatory action of the associated anemia (23,
26) in CRF animals. Alternatively, the observed phenomenon may have
been mediated by hemodynamic alterations caused by these interventions.
In conclusion, the present study demonstrated marked reductions of eNOS
and iNOS proteins in mildly hypertensive CRF rats employed here. These
abnormalities were reversible with parathyroidectomy and calcium
channel blockade, suggesting the possible causal role of CRF-induced
dysregulation of cytosolic Ca2+.
We wish to note that although CRF, per se, downregulates remnant kidney
and vascular NOS, several conditions that frequently accompany CRF,
such as severe HTN, volume expansion, inflammatory disorders, immune
activation, anemia, and pharmacological agents, can independently affect NOS in the opposite direction. Thus NO production and NOS activity/expression in a given CRF patient or animal model is determined by the collective actions of these influences.
| |
FOOTNOTES |
Address for reprint requests: N. D. Vaziri, Division of Nephrology,
UCI Medical Center, 101 The City Drive, Orange, CA 92668.
Received 10 September 1997; accepted in final form 12
December 1997.
| |
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