1 Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, District of Columbia 20007; 2 Second Department of Internal Medicine, University of Tokyo, Japan; and 3 Department of Physiology, Chonnam University Medical School, Kwangju, Korea
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ABSTRACT |
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The spontaneously
hypertensive rat (SHR) has an enhanced tubuloglomerular
feedback (TGF) and a diminished buffering by juxtaglomerular apparatus
(JGA)-derived NO. We examined the hypothesis that these effects are due
to decreases in nitric oxide synthase (NOS) expression or limited
availability of L-arginine or
tetrahydrobiopterin (BH4). SHR
had significantly (P < 0.05) greater
mRNA abundance (by RT-PCR) or protein (by Western analysis) for
neuronal NOS (nNOS, or type I) and endothelial cell NOS (ecNOS, or type
III) in renal cortex or isolated glomeruli, respectively. There was
prominent expression of ecNOS in glomerular endothelium and nNOS in
macula densa. Maximal TGF responses, assessed from changes in proximal
stop-flow pressure during orthograde loop of Henle (LH) perfusion, were
greater in SHR [Wistar-Kyoto (WKY), 8.1 ± 0.3 (n = 46) vs. SHR, 10.3 ± 0.3 mmHg
(n = 57);
P < 0.001]. Unlike WKY, TGF
responses of SHR were unresponsive to microperfusion of the nNOS
inhibitor, 7-nitroindazole (7-NI,
104 M) [WKY, 9.5 ± 0.5 to 13.2 ± 0.7 (n = 13, P < 0.001) vs. SHR, 11.8 ± 0.7 to 12.5 ± 0.6 mmHg (n = 19, not
significant)], or to L-arginine
(10
3 M) [WKY, 7.7 ± 0.8 to 6.3 ± 0.4 (n = 10, P < 0.05) vs. SHR, 10.4 ± 0.7 to
10.6 ± 0.7 mmHg (n = 10, not
significant)]. Neither BH4 (10
4 M) nor sepiapterin
(10
4 M), its stable
precursor, modified TGF responses in WKY or in SHR, nor did they
restore a response to microperfusion of 7-NI in SHR. In conclusion,
there is a diminished role for NO from nNOS in blunting of TGF in SHR
which cannot be ascribed to limited NOS expression or availability of
substrate or BH4.
L-arginine; 7-nitroindazole; sepiapterin; tetrahydrobiopterin; nitric oxide; spontaneously hypertensive rat; juxtaglomerular apparatus
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INTRODUCTION |
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MOST STUDIES (25), although not all (11), have demonstrated blunted agonist-stimulated, endothelium-dependent vasorelaxation in the peripheral circulation of patients with essential hypertension. The spontaneously hypertensive rat (SHR) also shows impaired endothelium-dependent relaxation responses in the aorta that may represent a decreased release of endothelium-dependent nitric oxide (NO) or an enhanced generation of an endothelium-dependent contraction factor. In contrast to the aorta, the coronary artery of the SHR has a normal endothelium-dependent vasodilation and an enhanced expression of endothelial cell NOS (ecNOS, or type III) (23). Thus the expression of NOS and the action of NO appear to be organ specific in the SHR. A comprehensive analysis of NOS isoform expression and function in the juxtaglomerular apparatus (JGA) of the SHR has not been undertaken previously.
The neuronal or type I isoform of NOS (nNOS) is expressed densely in macula densa cells where it functions to blunt the tubuloglomerular feedback (TGF) response (41). Studies in intact SHR kidneys suggest a diminished role for NO in tubular and vascular regulation. Thus SHR have impaired pressure natriuresis that is corrected by infusion of L-arginine (18) and enhanced TGF responses that have been ascribed to diminished blunting by macula densa-derived NO generated from nNOS (29). Any defect in NO generation in the JGA could contribute to heightened TGF responses, enhanced renal vascular resistance, and hypertension (24).
Therefore, the present studies were designed to investigate the role of NO in blunting TGF responses in the JGA of the intact SHR kidney. The hypothesis that there is a defect in constitutive NOS isoform expression in the JGA of the SHR kidney was assessed from studies of NOS isoform gene and protein expression. NO function was assessed from the enhancement of TGF during microperfusion into the JGA of the relatively nNOS-selective inhibitor, 7-nitroindazole (7-NI) (20, 22, 43). The hypothesis that a blunted TGF response to NOS blockade is due to defective L-arginine delivery to the JGA was assessed in studies of the TGF response to local microperfusion of L-arginine into the macula densa. This was tested because of our recent finding that an absent TGF response to NOS blockade in the salt-restricted Sprague-Dawley rat could be restored by local microperfusion of L-arginine into the JGA (41). The potential role of diminished tetrahydrobiopterin (BH4), which is an essential NOS cofactor, was assessed by perfusion of BH4 or its stable parent compound, sepiapterin, which can be taken up into renal tubular epithelium to stimulate NO generation during NOS induction (1). A recent study in aorta has shown that BH4 can be limiting for NO generation in the SHR (12).
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METHODS |
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Studies were undertaken on male SHR and Wistar-Kyoto (WKY), weighing 235-275 g (obtained from Harlan-Sprague Dawley, Madison, WI; at 175-200 g), and maintained on a standard rat chow (Purina, St. Louis, MO) with a sodium content of 0.3 g/100 g. Rats were allowed free access to food and water until the day of study.
Series 1: Comparison of mRNA abundance of ecNOS and nNOS transcripts in glomeruli or renal cortex, respectively, of SHR and WKY. These studies were designed to test the hypothesis that transcripts for constitutive NOS are diminished in the cortex of the SHR. nNOS transcripts and protein are expressed abundantly in the macula densa of the renal cortex. There is a close correlation between nNOS mRNA transcript abundance in renal cortex and nNOS transcript abundance in isolated vascular poles containing macula densas (28). Accordingly, studies of nNOS mRNA expression were undertaken in renal cortical tissue with the assumption that differences likely reflect predominantly changes in the macula densa. ecNOS mRNA is more widely expressed in the vasculature, and therefore its abundance was assessed in individual glomeruli that were microdissected from outer cortical nephrons.
Under thiobarbital anesthesia (pentobarbital, 100 mg/kg ip), the
abdomen was opened and the aorta was cannulated to allow flushing of
the kidneys with ice-cold dissection solution. This contained 135 mM
NaCl, 1 mM
Na2HPO4,
1.5 mM MgSO4, 2 mM
CaCl2, 5 mM glucose, and 5 mM
HEPES at pH 7.4. For isolation of renal cortical RNA, one kidney from
six SHR and one from six WKY were cut longitudinally, and a segment of
renal cortex was removed and digested with collagenase (1%) for 30 min
at 37°C. Glomeruli were dissected under a stereomicroscope at
4°C in dissection solution containing 5 mM dithiothreitol (DTT) and
10 mM vanadyl ribonucleoside complex. Dissected glomeruli were further
cleaned in dissection solution containing 5 mM DTT and 2 U/µl RNase
inhibitor under a stereomicroscope at 4°C. Glomeruli were
transferred to centrifuge tubes containing deionized water, 2% Triton
X-100, 5 mM DTT, and 2 U/µl RNase inhibitor. Total RNA was extracted
using RNA ATAT-60 (Tel-Test B, Friendswood, TX). It was reverse
transcribed (RT) with oligo(dT)16
as primer and Moloney murine leukemia virus reverse transcriptase
using an RNA PCR Kit (Perkin-Elmer, Branchburg, NJ). The
primers used for PCR of the nNOS gene product were those described
previously (21) based on the published sequence for rat nNOS (6). For
nNOS, the sense primer was
5'-GTCGAATTCCGAATACCAGCCTGATCCATGGAA-3', and the
antisense primer was
5'-CGCGGATCCCATGCGGTGGACTCCCTCCTGGA-3'. The predicted
product has a length of 599 bp. -Actin was selected as a
"housekeeper gene" for comparison. The primers used for
-actin mRNA were as follows: sense primer,
5'-GATCAAGATCATTGCTCCTC-3'; and antisense primer,
5'-TGTACAATCAAAGTCCTCAG-3'. The PCR product has a predicted
length of 426 bp. The amounts of NOS cDNAs were normalized by the
amounts of
-actin cDNA. The reaction mixture contained 50 pmol of
each primer, 1.25 mM deoxynucleotide mixture, 2.5 µl
Taq DNA polymerase, 10 mM
Tris · HCl (pH 10), 50 mM KCl, 1.5 mM
MgCl2, and 0.001% (wt/vol)
gelatin in a final volume of 50 µl. The PCR was carried out by the
following protocol: after an initial melting temperature of 94°C
for 4 min, there was 30 s of denaturation at 94°C, 45 s of
annealing at 60°C, and 45 s of extension at 72°C for repeated
cycles of amplification, followed by a final extension at 72°C for
7 min. The PCR product was analyzed on a 1.5% agarose gel stained with
ethidium bromide and visualized under ultraviolet light. The size of
the product was compared with a rat kidney cDNA probe for nNOS
constructed in our laboratory (21). To verify the authenticity of the
PCR products, the amplified nNOS cDNAs from the rat kidney cortex of an
SHR and WKY rat were purified by Microcon (Amicon, Beverly, MA) and
sequenced with an AmpliTaq cycle
sequencing kit (Perkin-Elmer, Branchburg, NJ).
The transcript abundance for ecNOS was assessed in a single outer cortical glomerulus, isolated using the method of Pelayo et al. (26). Separate groups of SHR (n = 6) and WKY (n = 6) were prepared as described above. For these studies, abundance was examined from 25% of the mRNA extracted from a single glomerulus. After anesthesia and preparation of the animal, blue 1-5 µm latex microspheres (Polysciences, Warrington, PA) were infused in HEPES buffer (pH 7.4) into the left kidney. After perfusion, the kidney was excised, cut into coronal slices, and placed on ice, and a glomerulus from the outer cortex was microdissected under stereomicroscopy. Thereafter, the mRNA was extracted, subjected to RT, and amplified as described above. The primers used for ecNOS were sense primer 5'-GTCGAATTCCTGGCGGCGGAAGAGAAGGAGTC-3' and antisense primer 5'-CGCGGATCCGGGGCTGGGTGGGGAGGTGATGTC-3'. The predicted product has a length of 691 bp. It was compared with a rat kidney cDNA probe for ecNOS constructed in our laboratory (21).
Care was taken to optimize conditions for the RT-PCR. For each study, initial analyses were undertaken of serially diluted amounts of cDNA to ensure that product (as assessed by densitometry) increased log-linearly with cDNA amount in the ranges used. Negative controls were undertaken by PCR without prior RT and by RT-PCR of the buffer used. Positive controls were obtained by amplifying the respective cDNAs.
Series 2: Comparison of ecNOS and nNOS protein
expression in renal cortex of SHR and WKY. These
studies were undertaken to assess the hypothesis that changes in renal
cortical gene transcript abundance for nNOS and ecNOS were accompanied
by changes in gene translation products. For these studies, six SHR and
six WKY rats were anesthetized, and their kidneys were prepared as
described above and stored at 80°C until analyzed. Slices of
kidney cortex were dissected on ice and homogenized using a Polytron
tissue homogenizer at 3,000 rpm in buffer containing 250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 50 mM potassium phosphate at pH 7.6. Large tissue debris and nuclear fragments were
removed by two consecutive low-speed centrifuge spins (3,000 g, 5 min; 10,000 g, 10 min). The supernatant was
centrifuged at 100,000 g for 60 min.
This was used for blotting nNOS, and the remaining pellet was
resuspended in 20 mM Tris · HCl buffer (pH 7.5) and
used for blotting ecNOS. The protein concentration was determined
colorimetrically (4) with bovine serum albumin as a standard.
Protein samples were electrophoretically size-separated with a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. High-range molecular weight markers (Bio-Rad, Hercules, CA) were loaded as size standards. A precise amount of total tissue protein (100 µg) was loaded on each lane. After separation, the proteins were electrophoretically transferred to a nitrocellulose membrane at 20 V overnight. The membranes were washed in Tris-based saline buffer (pH 7.4) containing 0.1% Tween 20 (TBST), blocked with 5% nonfat milk in TBST for 1 h, and incubated with a 1:2,000 dilution of monoclonal mouse anti-nNOS and anti-ecNOS antibodies (Transduction Laboratories, Lexington, KY) in 2% nonfat milk/TBST for 1 h at room temperature. The membranes were incubated with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:1,000) in 2% nonfat milk in TBST for 2 h. The bound antibody was detected by enhanced chemiluminescence on Hyperfilm (Amersham, Little Chalfont, Buckinghamshire, UK). Positive controls were obtained for ecNOS from protein extracts from endothelial cells in culture and for nNOS from rat pituitary gland.
Series 3: Immunohistochemical study of ecNOS and nNOS distribution in the kidney cortex of SHR and WKY. These studies were undertaken to assess the distribution of ecNOS immunoreactivity in vascular and glomerular capillary endothelium and nNOS in macula densa cell cytoplasm in SHR and WKY rats. After anesthesia, the abdominal aortas of five SHR and five WKY were cannulated and the kidneys perfused retrogradely with 0.154 M NaCl followed by paraformaldehyde-lysine-periodate (PLP) solution for 5 min, cut into slices, and immersed into PLP overnight at 4°C before embedding in wax (polyethylene glycol 400 disterate; Polysciences, Warrington, PA).
Two-micrometer wax sections were processed for light microscopic immunohistochemistry using the streptavidin-biotin-HRP complex technique (LSAB kit, Dako) as previously reported (31). Briefly, sections were dewaxed, rehydrated, and incubated with 3% H2O2 for 10 min to eliminate endogenous peroxidase activity. After rinsing in Tris-buffered saline with 0.1% TBST, sections were treated with blocking serum for 10 min and incubated with the primary mouse monoclonal antibody for nNOS and ecNOS (both from Transduction Laboratories) in a dilution of 1:100 for 1 h. After rinsing with TBST, the sections were incubated with the secondary antibody, rabbit biotinylated polyclonal antibody against mouse immunoglobulin (Dako, Glostrup, Denmark), in a dilution of 1:600 for 30 min, rinsed, and incubated for 20 min with HRP-labeled streptavidin. After rinsing with TBST, HRP was detected by diaminobenzidine with H2O2. The sections were counterstained with hematoxylin and examined under light microscopy.
For electron microscopic (EM) immunocytochemistry using the postembedding immunogold procedure, 1-mm3 blocks of kidney cortex were dehydrated and embedded in Lowicryl. Ultrathin sections were cut on an ultramicrotome, mounted on colloidin-coated nickel grids, and processed for immunogold labeling (32). The sections were incubated with 0.1 M NH4Cl for 1 h, rinsed with buffer solution (0.02 M Tris · HCl, 0.15 M NaCl, and 0.05% TBST adjusted to pH 7.2) for 15 min, and incubated with primary mouse monoclonal antibody against ecNOS (Transduction Laboratories) at a concentration of 1:100 overnight at 4°C. After three 10-min buffer washes, 30-nm gold-labeled goat anti-mouse IgG secondary antibody (Amersham Life Science, Buckinghamshire, UK) was applied for 2 h at a dilution of 1:50. Thereafter, the sections were washed with 0.01 M PBS, incubated with 2% glutaraldehyde/PBS solution for 30 min, rinsed with distilled water, counterstained with uranyl acetate and lead citrate, and examined with an electron microscope (Hitachi 7000 transmission electron microscope). To evaluate semi-quantitatively the degree of ecNOS immunogold labeling, a blinded observer assessed EM pictures of sections from three SHR and three WKY rats. The number of immunogold particles overlying endothelial cells were counted and expressed as the number of particles per micrometer of glomerular basement membrane.
Series 4: Maximal TGF responses and effects of microperfusion of the nNOS inhibitor, 7-NI, in SHR and WKY. These experiments were designed to test the hypothesis that the enhanced TGF responses of the SHR kidney could be ascribed to a blunted generation of NO by nNOS in the macula densa. Groups of SHR and age-matched WKY rats were prepared for in vivo micropuncture, microperfusion, and TGF studies as described in detail previously (35). In brief, animals weighing 235-300 g were anesthetized with thiobarbital (Inactin, 100 mg/kg ip; Research Biochemicals, Natick, MA). A catheter was placed in a jugular vein for fluid infusion and in a femoral artery for recording of mean arterial pressure from the electrically damped output of a pressure transducer (Statham). A tracheotomy tube was inserted, and the animals were allowed to breathe spontaneously. The left kidney was exposed by a flank incision, cleaned of connective tissue, and stabilized in a Lucite cup. This kidney was bathed in 0.154 M NaCl maintained at 37°C. After completion of surgery, rats were infused with a solution of 0.154 M NaCl and 1% albumin at 1.5 ml/h to maintain a euvolemic state (35). Micropuncture studies were begun after 60 min for stabilization.
For orthograde microperfusion of the loop of Henle (LH), a micropipette (8 µm OD) containing artificial tubular fluid (ATF) stained with FD&C dye was inserted into a late proximal tubule (35). Injections of the colored ATF identified the nephron and the direction of flow. An immobile bone wax block was inserted into this micropuncture site via a micropipette (10-15 µm) connected to a hydraulic drive (Trent Wells, La Jolla, CA) to halt tubular fluid flow. A perfusion micropipette (6-8 µm) containing ATF with test compounds or vehicle was inserted into the proximal tubule downstream from the wax block and connected to a nanoliter perfusion pump (WPI, Sarasota, FL). A pressure micropipette (1-2 µm) was inserted into the proximal tubule upstream from the wax block to measure proximal stop-flow pressure (PSF). The pressure was recorded by a servo-null instrument (Instruments for Physiology and Medicine, La Jolla, CA). Changes in PSF are an index of changes in glomerular capillary hydraulic pressure. Measurements of PSF were made in each nephron during zero loop perfusion and during perfusion with ATF at 40 nl/min, which produces a maximal TGF response, defined as the difference between PSF values recorded during perfusion of the loop with ATF at 0 and 40 nl/min.
The maximal TGF responses were determined in one to three nephrons from
SHR (n = 7) and WKY rats
(n = 6) during perfusion of the LH
with ATF + vehicle and contrasted with ATF + 7-NI
(104 M). In preliminary
studies, this was found to be a maximally effective dose. For
retrograde microperfusion studies of TGF, an early distal tubule was
identified and a wax block placed to prevent downstream flow. A
perfusion pipette was inserted immediately upstream from the block. To
allow retrograde flow, the late proximal tubule of the test nephron was
vented. The PSF response was
recorded before and during retrograde microperfusion of ATF + vehicle
or ATF + 7-NI (10
4 M ) at a
maximal rate of 40 nl/min.
Series 5: Maximal TGF responses during
microperfusion of L-arginine,
BH4, or sepiapterin in SHR and
WKY.
This series was designed to test the hypothesis that a reduced delivery
to the macula densa of the NOS substrate,
L-arginine, or NOS cofactor
BH4 (1) could underlie diminished
NO generation, as assessed in series
4. Groups of SHR (n = 5) and WKY rats (n = 4) were prepared
for microperfusion of
L-arginine.
PSF was recorded during orthograde
LH perfusion at 0 and 40 nl/min with ATF + vehicle and ATF + L-arginine
(103 M). Previous studies
have shown that this dose of
L-arginine is maximally
effective (36). Other groups of SHR were studied during loop perfusion
of vehicle, BH4
(10
4 M), or its stable
precursor sepiapterin (10
4
M, n = 5) in ATF microperfused
orthogradely at 40 nl/min. Measurements of
PSF were made at 5 min of
perfusion and compared with ATF + vehicle. Some further nephrons in SHR
(n = 4) that had received a 5-min
perfusion of sepiapterin, which was considered to have corrected any
potential defect in BH4, were
perfused with ATF + 7-NI
(10
4 M) to determine
whether the local sepiapterin administration had effected the response
to 7-NI.
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RESULTS |
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For series 1, as shown in Fig.
1, nNOS mRNA abundance as measured by
RT-PCR was consistently greater in outer cortex from SHR than WKY,
whereas the abundance for -actin mRNA was similar in the two
strains. Analysis of the PCR product from one kidney confirmed that it
corresponded to the published sequence for rat nNOS (6).
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RT-PCR products corresponding to cDNAs for ecNOS were obtained from
individual outer cortical glomeruli of six SHR and six WKY rat kidneys.
The density of the bands obtained from SHR was consistently greater
than that for WKY, although similar densities were apparent for
-actin. The cDNA obtained from one glomerulus was analyzed and found
to correspond to the published sequence for rat ecNOS (33). The
enhanced expression in SHR kidneys of RT-PCR products corresponding to
mRNA for ecNOS and nNOS was confirmed by densitometric analysis (Fig.
2, A and
B).
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For series 2, Western analysis of
proteins extracted from the renal cortex of kidneys of SHR and WKY
demonstrated that the monoclonal anti-nNOS and anti-ecNOS antibodies
hybridized with proteins of 155 and 140 kDa, respectively. Data from
six SHR and six WKY kidneys are shown in Fig.
3. Compared with WKY, SHR showed significantly higher content for nNOS of 67% (Fig.
3A) and for ecNOS of 120% (Fig.
3B).
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The distribution of ecNOS and nNOS immunoreactivity in the kidney
cortex of SHR and WKY corresponded to previous published series in
Sprague-Dawley rats (series 3) (31,
42). Examination of nNOS immunoreactivity showed heavy staining of the
macula densa cell plaque, as described previously in Sprague-Dawley
rats (32). This appeared to be less prominent in WKY (Fig.
4A)
compared with SHR (Fig. 4B). Kidneys
from five SHR and five WKY rats were tested systematically for
immunocytochemical staining. The results showed stronger macula densa
staining for nNOS in SHR compared with WKY in each pair examined by a
blinded observer.
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As described previously in Sprague-Dawley rats (32), ecNOS
immunoreactivity was readily demonstrable in the endothelium of arcuate
arteries and arterioles in the renal cortex of WKY and SHR (data not
shown). EM immunocytochemistry was used to assess ecNOS immunoreactive
expression in the glomerular capillaries (Fig.
5). The number of immunogold particles
along the capillary walls of outer cortical glomeruli was significantly
greater in SHR than WKY [SHR, 0.51 ± 0.05 (n = 41) vs. WKY, 0.32 ± 0.05 (n = 40), gold particles/µm;
P < 0.01].
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The baseline data for the micropuncture/microperfusion studies of rats
of series 4-8 are shown in Table
1. It is apparent that, compared with WKY,
SHR rats were of similar body and kidney weight but had consistently
higher levels of blood pressure (BP) and slightly greater
heart rates. TGF parameters showed higher values for proximal
PSF during perfusion of the LH at
0 and 40 nl/min and a greater maximal TGF response, as assessed from
differences between PSF during
perfusion at 0 and 40 nl/min in SHR, which averaged 127%
(P < 0.001) of the WKY control.
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For series 4, maximum TGF responses
were contrasted in SHR and WKY rats during addition of vehicle or 7-NI
to orthograde LH perfusates. As shown in Table
2, the maximum TGF responses again were
greater in SHR than WKY during perfusion of ATF + vehicle. The addition
of 7-NI increased maximal TGF responses consistently in WKY by an
average of 39% but had no significant effects on TGF responses of SHR.
Retrograde perfusion showed similar results; 7-NI increased TGF in WKY
(8.0 ± 0.7 to 9.8 ± 0.7 mmHg;
n = 6, P < 0.01) but had no effect on SHR
(10.3 ± 1.0 to 10.3 ± 0.7 mmHg; n = 8).
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For series 5, TGF responses were
contrasted in SHR and WKY during addition of
L-arginine to orthograde LH
perfusates. As shown in Table 3, the
maximum TGF responses again were greater in SHR compared with WKY
during perfusion of ATF + vehicle. Addition of
L-arginine blunted ATF responses
of WKY significantly by an average of 18% but had no significant
effects on TGF responses of SHR. The effects of orthograde
microperfusion of BH4
(104 M) or sepiapterin
(10
4 M) are shown in Table
4. Neither agent had a significant effect on maximal TGF responses of WKY or SHR. During microperfusion with
BH4 or sepiapterin, coperfusion of
7-NI increased TGF responses in WKY by 25-50%, which was quite
similar to the mean increase of 39% seen in WKY rats of
series 4 to
microperfusion of 7-NI alone (Table 2). Microperfusion of
BH4 and sepiapterin failed to
elicit a TGF response in SHR to coperfusion with 7-NI. Moreover, when
tested in nephrons that had received a prior microperfusion of
sepiapterin for 5 min, there was still no significant effect in SHR of
microperfusion with 7-NI. These data indicate that the failure of SHR
nephrons to show evidence of a functional response to nNOS inhibition
cannot be readily ascribed to a failure to deliver NOS substrate or
cofactor.
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Since there were differences in basal TGF responses, the percentage
changes in maximal TGF responses obtained during addition of 7-NI or
L-arginine to LH microperfusion
with ATF are compared in nephrons of SHR and WKY in Fig.
6 (16). It is apparent that the SHR had no
response to 7-NI or L-arginine,
whether expressed in percentage terms (Fig. 6) or absolute terms
(Tables 2 and 3).
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DISCUSSION |
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The main new findings of this study are that the expressions of nNOS and ecNOS isoforms are increased in the kidney cortex and JGA of the SHR. This increase in constitutive NOS isoforms appears to be transcriptionally regulated, since it is accompanied by an increase in mRNA abundance. Despite enhanced NOS expression, the exaggerated TGF responses of SHR are unresponsive to local blockade of nNOS by microperfusion of 7-NI into the macula densa segment. The failure to respond to nNOS inhibition cannot be ascribed to a failure to supply NOS substrate to the macula densa, since local microperfusion of L-arginine did not blunt TGF responses of SHR. Nor can it be ascribed to a failure to supply NOS cofactor, BH4, since local microperfusion of BH4 or its precursor, sepiapterin, did not blunt TGF responses of SHR nor restore a response to blockade with 7-NI.
Our results contrast with other studies in the SHR that show a normal (19) or enhanced (8) endothelium-dependent renal vasodilator response to bradykinin or acetylcholine and an enhanced vasoconstrictor response of isolated afferent arterioles to NOS inhibition (15). Young SHR have enhanced plasma NO2 and NO3 (NOx) concentrations, enhanced renal NOx excretion, and enhanced expression of ecNOS and inducible NOS proteins in the aorta and kidney (34). These changes in young SHR are unaffected by the development of hypertension as they age. Our results confirm these findings of enhanced ecNOS expression in the kidney. They show further that the enhanced ecNOS expression is transcriptionally regulated and involves the glomerular capillary endothelium. However, these findings of enhanced NO generation in the whole animal or blood vessels contrast sharply with our findings of absent nNOS function in regulation of TGF. These differences suggest that the defect in NOS function in the SHR is likely specific for nNOS, although we did not specifically study the function of ecNOS in our protocols. Moreover, ecNOS is enhanced in females, and therefore our results on male rats could be influenced by this effect of gender.
Our results confirm previous studies that have shown enhanced TGF responsiveness and sensitivity in the SHR (13, 29). These exaggerated responses persist after normalization of the renal perfusion pressure (13). Recently, Thorup and Persson (29) have shown that microperfusion of nitro-L-arginine (L-NA) into the macula densa enhances TGF responses more in WKY than SHR. Our results with the relatively nNOS-selective antagonist, 7-NI (2), show further that this defect in the response to NOS blockade presumably involves nNOS that is located in the macula densa. 7-NI given systemically to rats provides substantial inhibition of nNOS in the brain within 30 min (2, 20, 22, 43) without alteration in BP (22, 38) or the vasodilator action of acetylcholine that is mediated via ecNOS (22). We found that 7-NI given to rats on a high salt intake specifically increased renal vascular resistance without changing BP (38). This suggests a selective renal action during high salt intake consistent with our finding that the dependence of TGF on macula densa NOS is enhanced in rats adapted to high salt intake (37, 40). Ollerstam et al. (24) showed that 7-NI given systemically did not raise BP acutely, unlike L-NA, but increased TGF similarly to nonselective NOS inhibitors. They concluded that the pool of NO that acts on TGF is sensitive to 7-NI and that inhibition of ecNOS by 7-NI, if indeed that occurs at all, has little or no effect on TGF. Our results support their conclusion.
A similar dissociation between NOS expression and response to NOS inhibition in the JGA is seen in Sprague-Dawley rats during changes in salt intake. Dietary salt restriction enhances nNOS mRNA (28) and protein (31) expression in macula densa yet abolishes the enhancement of TGF by local microperfusion of NG-methyl-L-arginine (L-NMA) into the macula densa (36, 39). Microperfusion of L-arginine into the efferent arteriole of the salt-restricted Sprague-Dawley rat blunts TGF and restores a TGF response to L-NMA (36). Arginine appears to be acting via NOS, since the response to L-arginine is stereospecific and is prevented by inhibition of NOS with L-NMA (36). In the present series, microperfusion of L-arginine into the JGA blunted maximal TGF responses in WKY similarly to that shown in Sprague-Dawley rats (36) yet did not significantly modify responses in SHR. This implies that L-arginine delivery is not limiting for NO generation in the JGA of the SHR. This is consistent with the finding of Chen and Sanders (9) that L-arginine does not improve the glomerular filtration rate of the SHR.
BH4 dimerizes and allosterically activates nNOS (17). Sepiapterin is more stable than BH4. It is taken up into renal tubules, where it is metabolized by dihydrofolate reductase and can enhance NO generation during induction of NOS by cytokines (1). A diminished release of NO by the aorta from SHR can be improved by exogenous BH4 (12). However, microperfusion of BH4 or sepiapterin into the macula densa segment did not blunt TGF responses of the WKY or SHR nephron, nor did it restore a response to nNOS inhibition with 7-NI in the SHR. Therefore, it seems unlikely that BH4 or sepiapterin availability limits NO generation in the macula densa of the SHR.
It is unlikely that the failure to respond to 7-NI is due to a failure of SHR arterioles to dilate in response to NO. Previous studies have reported an intact vasodilator response to sodium nitroprusside in the isolated kidneys of SHR (8, 14).
The cause for the dissociation between NOS expression and response to
NOS inhibition in the JGA of the SHR was not established in these
studies. nNOS itself may function abnormally, since pressor responses
to central NOS inhibitors that are mediated via nNOS are deficient in
stroke-prone SHR (7). nNOS activity is inhibited in vitro
by protein kinase A- or C-dependent phosphorylation (5), by binding to
PIN-peptide (10) or by endogenously formed asymmetric dimethylarginine (ASDA) (32). It is activated by calcineurin-dependent dephosphorylation and by dystrophin-associated, postsynaptic density (PSD), membrane-bound proteins (10). We have recently detected the
enzyme,
NG, NG-dimethylarginine
dimethylaminohydrolase (DDAH), which degrades ASDA (32),
in the macula densa. We have also found that a portion of nNOS is
colocalized ultrastructurally with PSD-95 in macula densa cell
cytoplasm (30). Therefore, changes in macula densa DDAH or PSD-95
expression, localization, or function could contribute to altered nNOS
activity in the macula densa. NO action is curtailed in vivo by
interaction with oxygen radicals
(O2) that are enhanced in the
aorta of SHR (3). Renal vasoconstriction and hypertension in the SHR
can be reversed by a membrane-permeable scavenger of
O
2 (27). This provides another potential explanation for a dissociation between nNOS expression and
functional activity in the macula densa cells of the SHR nephron that
remains to be explored.
In conclusion, our data demonstrate a defect in nNOS function in the JGA of SHR, despite abundant transcriptional and translational expression. This cannot readily be ascribed to a deficient availability of arginine or BH4.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Bo Peng for technical assistance and to Marly Davidson for typing the manuscript.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36079 and DK-49870 (to C. S. Wilcox) and by funds from the George E. Schreiner Chair of Nephrology. C. G. Schnackenberg was supported by a National Research Service Award grant from the National Institutes of Health.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. S. Wilcox, Division of Nephrology and Hypertension, Georgetown Univ. Medical Center, 3800 Reservoir Road, N.W., PHC F6003, Washington, D.C. 20007 (E-mail: donohume{at}gunet.georgetown.edu).
Received 18 August 1998; accepted in final form 1 April 1999.
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