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Am J Physiol Regul Integr Comp Physiol 291: R391-R399, 2006. First published March 30, 2006; doi:10.1152/ajpregu.00029.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Neonatal sympathectomy reduces NADPH oxidase activity and vascular resistance in spontaneously hypertensive rat kidneys

Torsten Schlüter,1 Rita Grimm,1 Antje Steinbach,1 Gerd Lorenz,2 Rainer Rettig,1 and Olaf Grisk1

Institutes of 1Physiology and 2Pathology, University of Greifswald, Greifswald, Germany

Submitted 12 January 2006 ; accepted in final form 9 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neonatal sympathectomy reduces arterial pressure in spontaneously hypertensive rats (SHR). In SHR transplanted with a kidney from sympathectomized SHR, arterial pressure was lower and less Na+ sensitive than in SHR transplanted with a kidney from hydralazine-treated SHR. This study was performed to identify underlying renal mechanisms. Tests for differential renal mRNA expression of nine a priori selected genes revealed robust differences for renal medullary expression of the NADPH oxidase subunit p47phox. Therefore, we investigated the effects of neonatal sympathectomy on renal mRNA expression of NADPH oxidase subunits, NADPH oxidase activity, and renal function. In 10-wk-old sympathectomized SHR fed a 0.6% NaCl diet, medullary p47phox and gp91phox expression was 40% less than in hydralazine-treated SHR. Also, after a 1.8% NaCl diet, medullary p47phox mRNA expression was lower in sympathectomized than in hydralazine-treated SHR. We found lower cortical (–30%, P < 0.01) and medullary (–30%, P < 0.05) NADPH oxidase activities in sympathectomized than in hydralazine-treated or untreated SHR. Glomerular filtration rate, renal blood flow, medullary blood flow, and fractional Na+ excretion in kidney grafts from sympathectomized and hydralazine-treated donors (n = 8 per group) were similar at baseline and in response to a 20-mmHg rise in renal perfusion pressure. Renal vascular resistance was lower in kidneys from sympathectomized than hydralazine-treated donors (25 ± 2 vs. 32 ± 4 mmHg·min·ml–1, P < 0.05). The results indicate that the sympathetic nervous system contributes to the level of renal NADPH oxidase activity and to perinatal programming of alterations in renal vascular function that lead to elevated renal vascular resistance in SHR.

inbred strains; kidney; sympathetic activity; gene expression

THE KIDNEY AND THE SYMPATHETIC nervous system contribute to the pathogenesis of arterial hypertension in spontaneously hypertensive rats (SHR) (12). Arterial pressure can be chronically lowered in SHR by neonatal sympathectomy (19, 21), and renal mechanisms are involved in this chronic arterial pressure reduction (14). We previously showed lower arterial pressure in SHR transplanted with a kidney from a sympathectomized SHR donor than in SHR recipients of a kidney from a hydralazine-treated SHR donor (14). In addition, Na+ sensitivity of arterial pressure was lower in recipients of a kidney graft from sympathectomized donors than in recipients of a graft from hydralazine-treated donors (14). The present study was performed to identify mechanisms that may explain these findings; therefore, we focused on kidneys from sympathectomized and hydralazine-treated SHR. Limited data are available on chronic effects of neonatal sympathectomy on SHR kidneys. Thus we initially tested for differential mRNA expression of nine a priori selected genes in kidneys from sympathectomized and hydralazine-treated SHR exposed to three different experimental conditions. Our aim was to identify alterations in gene expression that persist under different conditions and, therefore, may be involved in chronic effects of neonatal sympathectomy. The choice of genes was based on the results from comparative studies in SHR and normotensive rats relevant to the present experimental paradigm.

From the renin-angiotensin system, we chose the angiotensin type 1 (AT1) receptor and renin. Glomerular and tubular AT1 receptor expression was higher in newborn and juvenile SHR than in normotensive rats (5, 15). Renal renin mRNA expression was more sensitive to renal denervation in young SHR than in normotensive rats (25). The renal endothelin (ET) system contributes to the pathogenesis of Na+-sensitive hypertension (27), including SHR (31). Therefore, we tested for differential ET-1 and ET type A and type B receptor mRNA expression. From oxygen radical-generating systems, we included the p47phox subunit of the NADPH oxidase, because its renal expression and the NADPH oxidase activity were higher in SHR than in normotensive rats (1, 4). Furthermore, we tested for differential expression of cyclooxygenase 2 (COX-2), endothelial nitric oxide synthase (NOS), and neuronal NOS. Altered renal expression and function of these enzymes or decreased renal availability of their products is involved in the pathogenesis of experimental hypertension (9, 23, 39, 41, 44). COX-2 expression is developmentally regulated in rat kidneys (45), and renal neuronal NOS expression and function depend on renal innervation (16, 41).

Screening for differential mRNA expression revealed the most prominent changes for the NADPH oxidase subunit p47phox. NADPH oxidase is a major source of renal oxygen radical production (39), and arterial hypertension can be efficiently treated with antioxidants in SHR (32, 43). Increased renal oxygen radical formation can contribute to the development and maintenance of hypertension by elevating renal vascular resistance (RVR), tubuloglomerular feedback sensitivity, and tubular Na+ reabsorption (26, 32, 39). Little information is available on the influence of the sympathetic nervous system on renal NADPH oxidase expression and activity. We therefore tested the hypothesis that neonatal sympathectomy reduces renal mRNA expression of NADPH oxidase subunits other than p47phox that are necessary for NADPH oxidase enzyme activity. Furthermore, we tested the hypothesis that neonatal sympathectomy causes a reduction in renal NADPH oxidase activity in young SHR. We found that NADPH oxidase activity was reduced in the renal cortex and medulla of young sympathectomized SHR. Therefore, we tested whether this reduction of renal NADPH oxidase activity is associated with chronic changes in renal function that would persist in transplanted kidneys and may help explain the long-term renal effects of neonatal sympathectomy on arterial pressure in SHR (14).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male and female SHR were obtained from M & B (Ry, Denmark). First-generation offspring were bred in our temperature- and humidity-controlled animal facility with lights on from 0600 to 1800. If not stated otherwise (protocol 2), animals were fed a standard rat chow containing 0.6% NaCl. All diets were obtained from Ssniff (Soest, Germany). Rat pups were subjected to neonatal sympathectomy consisting of injections of guanethidine (50 µg/g body wt ip) and surgical removal of adrenal medullary tissue on postnatal day 28 or sham treatment as described and validated previously (14). Hydralazine (50 mg/kg body wt) was administered via the drinking water starting on postnatal day 28 and continuing until the end of postnatal week 10. Male animals were used for experimentation. All procedures were approved by a governmental committee on animal welfare and conformed to the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." The animals were subjected to three different experimental protocols.

Protocol 1. Ten-week-old sympathectomized and hydralazine-treated SHR were anesthetized with ether, and kidneys were rapidly removed, blotted, and cut in slices on a petri dish cooled with ice. Tissue slices were dissected into cortex and medulla. Medullary specimens contained the inner stripe of the outer medulla and the inner medulla. After dissection, the tissue samples were snap frozen in liquid nitrogen and stored at –70°C. Identically treated 10-wk-old SHR had been used as kidney donors in previous renal transplantation studies (14). At 7 wk after transplantation, the kidney grafts of that study (14) had been dissected into cortex and medulla and stored at –70°C. These tissue samples were included in the gene expression analyses of the present study. NADPH oxidase activity was measured in tissues of a separate set of animals consisting of sympathectomized, hydralazine-treated, and untreated SHR. The kidneys of untreated SHR consisted of the left native kidneys, which were removed from the recipients before renal transplantation (protocol 3) (13, 14).

Protocol 2. To investigate the effect of an Na+ load on arterial pressure and renal gene expression, sympathectomized, hydralazine-treated, and sham-treated SHR were implanted with radio-telemetric arterial pressure-recording devices (model TA11PA-C40, Data Sciences International, St. Paul, MN) at 10 wk of age. Hydralazine treatment was stopped on the day of transmitter implantation. The sham-treated group was included to verify that SHR were, indeed, hypertensive and to quantify the antihypertensive effects of hydralazine treatment and neonatal sympathectomy by telemetry. After 1 wk of recovery, only sympathectomized and hydralazine-pretreated animals were transferred to metabolism cages and placed on a diet containing 0.12% NaCl. After a 3-day adaptation period, urine was collected and telemetric recordings were performed. After a 3-day data collection period during which the animals were fed the 0.12% NaCl diet, the animals were fed a 1.8% NaCl diet for 4 days, similar to the preceding study in renal transplanted SHR (14). Arterial pressure was recorded and analyzed as described previously (13, 14). Telemetric data are given as 12-h mean values. At the end of these experiments, the kidneys were harvested as described for protocol 1 for gene expression analysis.

Protocol 3. To investigate renal function in greater detail than possible in conscious, chronically instrumented rats (14), kidneys from sympathectomized and hydralazine-treated SHR were transplanted into untreated SHR recipients. Both native kidneys of the recipients were removed (13, 14). At 7 wk after transplantation, the animals were instrumented for acute renal function studies after an overnight fast. At this time, the animals were 17–18 wk old and body weight was 300–350 g; there was no difference between the groups. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and placed on a servo-controlled heating pad. Catheters for infusion and supplemental administration of anesthetics were inserted into the right jugular and right femoral vein. Arterial pressure was recorded via a catheter placed in the right carotid artery. A cannula was placed in the trachea, and the animals were artificially ventilated after administration of pancuronium bromide (1 mg/kg). The renal graft was approached via an abdominal midline incision, carefully cleaned from surrounding tissue with care taken to prevent damage to the renal capsule, and placed in a small plastic holder filled with gauze to prevent movements. The kidney was covered and kept moist with prewarmed isotonic saline. A transit time ultrasound probe (type 1RB, Transonic Systems, Ithaca, NY) was placed around the renal artery of the graft. Proper acoustic coupling was achieved with ultrasonic gel. The superior mesenteric artery was cleared from surrounding tissue, and a suture was placed loosely around the vessel to facilitate rapid occlusion of the vessel with a small vessel clamp to elevate arterial pressure. With the aid of a stereotaxic apparatus, a laser-Doppler probe (type 418-2, Perimed, Järfälla, Sweden) was advanced rectangular to the frontal plane 5 mm deep into the renal tissue to record renal medullary flow. Before insertion of the laser-Doppler probe, the renal capsule was perforated with a 26-gauge needle at the insertion site. A PE-10 catheter was placed in the ureter to collect urine for measurements of inulin clearance and renal Na+ excretion.

Animals were infused with isotonic saline containing inulin (3.3 mg/ml) and 2% BSA at a rate of 5.66 ml/h starting during abdominal surgery. After 45 min of postsurgical equilibration, arterial pressure and renal function data were collected over a 20-min period. Thereafter, occlusion of the superior mesenteric artery caused a stable 20- to 25-mmHg arterial pressure elevation, and data were collected for another 20 min. At the end of the experiments, a final blood sample was taken from the arterial catheter, and the animals were killed with an overdose of pentobarbital. The background signal from the laser-Doppler flowmeter was recorded when renal blood flow was zero (~2 min after the overdose of pentobarbital). Proper location of the probe tip in the inner medulla was verified postmortem, and the grafts were rapidly dissected into cortex and medulla for NADPH oxidase activity measurements.

Arterial pressure, renal blood flow, and renal medullary blood flow (MBF) signals were fed into a personal computer, digitized, sampled at 500 Hz, and averaged over 1-s intervals. Hardware and software (LabView 5.1) for data acquisition were obtained from National Instruments (Austin, TX).

Renal mRNA expression. Total RNA was isolated with the acid guanidinium thiocyanate-phenol-chloroform procedure using the TRIzol reagent (Invitrogen, Paisley, UK). RNA was dissolved in RNA storage solution (Ambion, Huntingdon, UK). For removal of residual genomic DNA, total RNA was treated with DNase (DNA-free, Ambion) and stored at –70°C. Concentration and purity of DNase-treated RNA were determined spectrophotometrically by measurement of absorbance at 260 nm and the ratio of absorbance at 260 nm to absorbance at 280 nm. Integrity of the prepared RNA was assessed by agarose gel electrophoresis followed by ethidium bromide staining.

Reverse transcription was performed with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). RNA samples (1 or 2 µg) were reverse transcribed into cDNA with random hexamer primers in a total volume of 100 µl according to the manufacturer's instructions. Expression levels were determined by TaqMan real-time PCR with porphobilinogen deaminase as endogenous reference. Real-time PCR was performed with ready-to-use TaqMan Universal PCR Master Mix (Applied Biosystems) with 1 µl of cDNA as template and the GenAmp 5700 sequence detection system (Applied Biosystems). Design of synthetic oligonucleotide primers and fluorescent probes for each gene was determined on the basis of published sequences (Table 1). The PCR included 10 min of activation of the DNA polymerase at 95°C, 40 cycles at 95°C for 15 s for denaturation, and 1 min at 60°C for annealing and extension.


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  		Table 1. Renal cortical gene expression in native kidneys from 10-wk-old SHR and 13-wk-old SHR after exposure to high-salt diet and in renal grafts obtained 7 wk after transplantation

 
PCRs were performed in triplicate. Gene expression was quantified with the standard curve method. Standard curves (cycle threshold vs. logarithm of concentration) for target genes and for the endogenous reference were generated from a serially diluted cDNA pool of all tested samples. Expression levels are given relative to porphobilinogen deaminase mRNA. In each experimental plate, a no-template control was included to check for any contamination of the PCR. It was also confirmed that no amplification occurred when samples were not subjected to reverse transcription, indicating the absence of genomic DNA after DNase treatment. [The online version of this article contains supplemental information about sequences of primers and TaqMan probes.]

Identity of the gene-specific PCR products was confirmed by size estimation using standard agarose gel electrophoresis and ethidium bromide staining, as well as by sequencing of PCR products (Invitek & Biodesign, Berlin, Germany; SEQLAB, Göttingen, Germany). For purification of PCR products, commercial gel band extraction kits (MinElute Gel extraction kit, Qiagen, Hilden, Germany) were used. Sequencing was performed with the oligonucleotide primers that were used for real-time PCR.

NADPH oxidase activity. Renal NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence in renal tissue from 10-wk-old SHR. The membrane fraction was obtained by tissue preparation according to an established procedure (24). Tissues where homogenized in PBS containing a proteinase inhibitor cocktail (Sigma), 0.1% (vol/vol) 2-mercaptoethanol, 0.01 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, and 1% (wt/vol) BSA. After stepwise centrifugation of the homogenate at 500, 25,000, and 100,000 g, the resulting pellet was resuspended in PBS containing 1% BSA. Enzyme activity was measured in triplicate in aliquots of the pellet corresponding to 18 mg (renal cortex) or 7.2 mg (renal medulla) wet tissue weight with a luminometer (Microlumat Plus, Berthold, Bad Wildbad, Germany) in the presence of 5 µmol/l lucigenin, 100 µmol/l nitro-L-arginine methyl ester, 100 µmol/l allopurinol, and 50 µmol/l rotenone. After 60 s of baseline recording, NADPH (300 µmol/l final concentration) was added to the reaction mixture. Thereafter, a luminescence reading was obtained at 6-s intervals for an overall measuring time of 9 min. To control for specificity, simultaneous runs containing 50 µmol/l diphenyliodonium or 20 mmol/l tiron were performed for each measurement. Enzyme activity is expressed in relative light units per minute and milligrams wet tissue weight.

Histology. Paraffin-embedded tissue samples were cut into 2-µm slices and stained with hematoxylin-eosin. For specific visualization of leukocytes, tissue sections were also stained for leukocyte-specific naphtole AS-D chloracetate esterase and counterstained with hematoxylin. Histological examination was performed by a pathologist who was blinded to the experimental design.

Statistics. Data were analyzed by ANOVA (2-way ANOVA, 2-way ANOVA for repeated measurements, and 1-way ANOVA) as appropriate followed by post hoc t-tests with Bonferroni's correction. Because the three sets of tissue samples for gene expression analyses were obtained on separate occasions, statistical analyses of renal gene expression data were restricted to group comparisons within each experimental protocol. Differences were considered statistically significant when P < 0.05. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Renal gene expression. Analysis of renal cortical gene expression revealed no statistically significant differences between 10-wk-old sympathectomized and hydralazine-treated animals and no differences between kidney grafts from sympathectomized and hydralazine-treated donors (protocol 1) for the nine selected genes (Table 1). After high-NaCl diet and hydralazine withdrawal (protocol 2), cortical COX-2 expression was 138% (P < 0.01) and renin expression was 74% (P < 0.05) in cortices of sympathectomized animals relative to hydralazine-treated animals (Table 1). At 1 wk after implantation of radio-telemetric devices (protocol 2), average daytime arterial pressure was 149 ± 7/107 ± 6 mmHg in sympathectomized SHR and 142 ± 7/104 ± 8 mmHg in SHR that had been taken off hydralazine at the time of transmitter implantation. Arterial pressure was significantly higher in sham-treated animals (165 ± 5/127 ± 5 mmHg) than in the other groups (P < 0.01). Changes in NaCl intake did not differentially affect arterial pressure in sympathectomized and hydralazine-pretreated animals. Sympathectomized animals transiently retained more Na+ after initiation of the high-salt diet (P < 0.05; Fig. 1), which is consistent with the established fact that intact regulation of renal innervation facilitates renal excretion of an acute Na+ load (7). Heart rate was higher in sympathectomized than in hydralazine-treated animals to compensate for compromised sympathetic regulation of peripheral vascular resistance (P < 0.001).


Figure 1
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  		Fig. 1. Locomotor activity, heart rate (HR, beats/min), mean arterial pressure (MAP), and 24-h renal Na+ balance (mmol·100 g body wt–1·day–1) in sympathectomized (n = 7, bullet) and hydralazine-treated (n = 7, {circ}) spontaneously hypertensive rats (SHR). *Significant difference between groups: P < 0.001 for heart rate and P < 0.05 for renal Na+ balance.

 
More differences in gene expression were observed in renal medullary than cortical samples obtained from these experimental groups (Table 2). Medullary renin mRNA expression in sympathectomized SHR was 56% of the expression level in hydralazine-treated SHR at 10 wk of age (P < 0.005). Furthermore, medullary prepro-ET-1 and ET type A receptor mRNA expression was lower in kidneys from sympathectomized than hydralazine-treated animals, whereas ET type B receptor mRNA expression was less in renal grafts from sympathectomized animals. The between-group differences in mRNA expression of the ET system components were in the range of 20–25%. Depending on the experimental conditions, we observed differences also in medullary neuronal and endothelial NOS expression. Under all experimental conditions, renal medullary p47phox mRNA expression was lower in sympathectomized than hydralazine-treated SHR. In kidney grafts, this difference failed to reach statistical significance.


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  		Table 2. Renal medullary gene expression in native kidneys from 10-wk-old SHR and 13-wk-old SHR after exposure to high-salt diet and in renal grafts obtained 7 wk after transplantation

 
Among the nine genes investigated, renal medullary p47phox showed the largest differences in mRNA expression levels between sympathectomized and hydralazine-treated groups. These differences were robust and were observed under three different experimental conditions: 10-wk-old SHR, 13-wk-old SHR after 1.8% NaCl, and transplanted SHR. These observations prompted us to investigate the effects of neonatal sympathectomy on NADPH oxidase subunits in greater detail (Table 3). In addition to medullary p47phox mRNA expression, medullary gp91phox mRNA expression was also reduced in native kidneys from 10-wk-old sympathectomized rats compared with hydralazine-treated animals. Among the NOX isoforms, NOX4 expression was highest in the cortex and lowest in the medulla in all experimental groups (Table 3). Furthermore, p22phox mRNA expression was less in the medulla than in the cortex.


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  		Table 3. Renal cortical and medullary mRNA expression of NADPH oxidase subunits normalized to PBGD mRNA expression

 
Histological examination was performed to exclude the possibility that the differences in medullary p47phox and gp91phox mRNA expression were due to differential leukocyte infiltration in the kidneys of sympathectomized and hydralazine-treated animals. There were no apparent morphologicaldifferences in kidneys from sympathectomized and hydralazine-treated SHR (Fig. 2). Morphology of the grafts used for gene expression analysis has been shown previously (14). Measurements of renal copper-zinc (SOD1) and extracellular (SOD3) superoxide dismutase (SOD) mRNA expression revealed no significant group differences in 10-wk-old animals (Tables 1 and 2).


Figure 2
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  		Fig. 2. Kidney sections stained for leukocyte-specific naphtole AS-D chloracetate esterase and counterstained with hematoxylin. Scale bars, 100 µm. A and B: cortex and medulla from sympathectomized SHR. C and D: cortex and medulla from hydralazine-treated SHR.

 
NADPH oxidase enzyme activity. To examine the impact of neonatal sympathectomy on NADPH oxidase function, enzyme activity was measured in kidneys from 10-wk-old SHR. NADPH oxidase activity was lower in renal cortex and medulla of sympathectomized than hydralazine-treated animals and untreated controls (Fig. 3). NADPH oxidase activities in kidney grafts from sympathectomized and hydralazine-treated donors obtained 7 wk after transplantation and after completion of acute experiments (see below) were not significantly different (data not shown).


Figure 3
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  		Fig. 3. Renal NADPH oxidase activity as measured by lucigenin-enhanced chemiluminescence in the presence of inhibitors for other oxygen radical-generating systems [relative light units (RLU)·min–1·mg wet tissue wt–1]. A: cortical NADPH oxidase activity in untreated (n = 12, shaded bar), sympathectomized (n = 11, solid bar), and hydralazine-treated (n = 11, open bar) SHR. *Significantly different from untreated and hydralazine-treated SHR (P < 0.01). B: medullary NADPH oxidase activity in untreated (n = 10, shaded bar), sympathectomized (n = 8, solid bar), and hydralazine-treated (n = 9, open bar) SHR. *Significantly different from untreated and hydralazine-treated SHR (P < 0.05).

 
Renal function. Studies of renal function in transplanted kidneys showed that renal blood flow, glomerular filtration rate, and fractional Na+ excretion were similar in both groups under baseline conditions and after nonpharmacological arterial pressure elevation (Fig. 4). In both groups, fractional Na+ excretion was significantly increased in response to increased renal perfusion pressure (P < 0.05), whereas MBF did not significantly change (Fig. 4). RVR was significantly lower (P < 0.05) in kidney grafts from sympathectomized animals than in grafts from hydralazine-treated animals (Fig. 5).


Figure 4
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  		Fig. 4. Renal blood flow (RBF, ml·min–1·g kidney wt–1), medullary blood flow (MBF) perfusion units (PU), glomerular filtration rate (GFR, ml·min–1·g kidney wt–1), and fractional Na+ excretion (FENa) in transplanted kidneys from sympathectomized (n = 8, bullet) and hydralazine-treated SHR donors (n = 8, {circ}). Data were obtained over two 20-min recording periods under baseline conditions and after nonpharmacological elevation of arterial pressure. *Significantly different from baseline (P < 0.05).

 

Figure 5
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  		Fig. 5. Renal vascular resistance (RVR) in kidney grafts from sympathectomized (n = 8, solid bar) and hydralazine-treated (n = 8, open bar) donors. *Significant difference (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study was performed to identify renal mechanisms that are involved in the chronic arterial pressure reduction in SHR induced by neonatal sympathectomy (14). Because limited data are available on the effects of neonatal sympathectomy on SHR kidneys, the study was initiated with a screening for differentially expressed genes to allow simultaneous investigation of multiple physiological systems in the tissue samples. The screening for differential gene expression revealed that medullary p47phox mRNA expression was lower in kidneys of sympathectomized SHR than in kidneys of hydralazine-treated SHR under three different experimental conditions. This was not observed for the other eight mRNA species included in the screening. Because of 1) the consistent responses of p47phox mRNA expression to neonatal sympathectomy, 2) the role of the NADPH oxidase in the pathogenesis of hypertension, and 3) the paucity of in vivo data on the influence of the sympathetic nervous system compared with other regulators such as the renin-angiotensin system (3, 20, 29, 39) on renal and vascular NADPH oxidase, we investigated the effect of neonatal sympathectomy on NADPH oxidase subunit mRNA expression in greater detail.

The subunits gp91phox (NOX2), p22phox, and p67phox, which are required for NADPH oxidase activity (20, 39), as well as the electron-transporting NOX isoforms NOX1 and NOX4, were included in the expression analyses. With the exception of gp91phox, which showed a decreased medullary mRNA expression in sympathectomized vs. hydralazine-treated animals, neonatal sympathectomy did not alter the renal mRNA expression levels of any of these subunits. These findings indicate that, among the renal NADPH oxidase subunits in SHR, only gp47phox and gp91phox respond to a chronic reduction in sympathetic tone with altered mRNA expression levels. In contrast, cortical p22phox, NOX1, and NOX4 mRNA expression is sensitive to systemic angiotensin II administration (3). Our data show that the renal cortical mRNA expression pattern of NOX isoforms in SHR is similar to that reported for Sprague-Dawley rats (3).

NADPH oxidase activity was significantly lower in kidneys of sympathectomized SHR than in kidneys of untreated or hydralazine-treated controls. It has been shown that 5 days of angiotensin II, but not norepinephrine, infusion activates vascular NADPH oxidase activity in conduit arteries of Sprague-Dawley rats (29), indicating that vascular NADPH oxidase does not respond to short-term elevations in adrenergic tone. In the present study, we show a clear dependence of renal NADPH oxidase activity on sympathetic tone. The data indicate that the sympathetic nervous system exerts its regulatory effect mainly on the level of NADPH oxidase function and/or protein expression in the cortex but also elicits alterations in p47phox and gp91phox mRNA expression in the medulla. Whether the reduction of medullary p47phox and gp91phox expression is a direct effect of reduced sympathetic activity or is caused indirectly by a concomitant reduction in medullary renin-angiotensin system activity remains to be established. The reduction in medullary renin mRNA expression in sympathectomized animals is consistent with the latter possibility. Recently, it has been shown that angiotensin II stimulates medullary renin expression via an AT1 receptor-dependent mechanism in rat kidneys (28). Our findings indicate that, in SHR, renal medullary renin mRNA expression depends on sympathetic activity. A direct pathway of renal NADPH oxidase activation by the sympathetic nervous system may involve {alpha}1-adrenoceptor-mediated activation of phospholipase C and PKC. This pathway is functioning in renal vasculature and epithelia (7), and PKC-dependent p47phox phosphorylation is a signaling step of NADPH oxidase activation by several agonists (20). In vitro experiments have shown that {alpha}1-adrenoceptor activation increases NADPH oxidase activity and p47phox mRNA expression in vascular smooth muscle cells (2).

Neonatal sympathectomy reduced arterial pressure by ~20 mmHg, but not to normotensive levels, indicating that, in addition to elevated sympathetic activity, other mechanisms contribute to hypertension in SHR. Chronically instrumented rats studied 10 days after drug washout showed no difference in Na+ sensitivity of arterial pressure. Interestingly, hydralazine-treated animals showed a persistent arterial pressure reduction after treatment was stopped. This finding indicates that, in SHR, hydralazine given early after birth has a prolonged antihypertensive action similar to that of sympatholytic drugs (14, 19) or angiotensin-converting enzyme inhibitor treatment (34). Consistent with this observation, radio-telemetric studies have shown that hydralazine with a diuretic and a Ca2+ antagonist lowers arterial pressure for several weeks beyond cessation of drug administration in adult SHR (40). The different potency of these treatments to induce chronic changes in renal function that contribute to the maintenance of the arterial pressure reduction becomes apparent in transplantation experiments (14, 34). Although transplantation of a kidney from sympathectomized or angiotensin-converting enzyme inhibitor-treated SHR donors persistently lowers arterial pressure in SHR recipients (14, 34), transplantation of a kidney from hydralazine-treated SHR donors is associated with a slow but distinct recovery of arterial pressure in SHR recipients (14).

The mechanisms underlying the chronic arterial pressure-lowering effect of renal grafts from sympathectomized SHR (14) are largely unknown. The present transplantation experiments were performed to investigate potentially blood pressure-relevant effects of neonatal sympathectomy on renal function in SHR. The transplantation approach has the distinct advantage that the function of kidneys from sympathectomized and hydralazine-treated SHR can be studied in a neurohormonal environment that is as similar as possible in both experimental groups (13). Confounding effects of differences in sympathetic tone that are difficult to eliminate by hormone clamping when native kidneys are studied can be avoided. Under acute experimental conditions with anesthesia and extensive instrumentation, the difference in mean arterial pressure between recipients of a kidney from sympathectomized and hydralazine-treated donors was only 10 mmHg and did not reach statistical significance, whereas the difference was 20 mmHg in conscious freely moving rats (14). RVR was reduced in kidney grafts from sympathectomized donors, whereas glomerular filtration rate, renal blood flow, and fractional Na+ excretion did not differ significantly between the groups. These results indicate that neonatal sympathectomy induces a persistent reduction in RVR that is maintained for at least several weeks after restoration of the extrarenal neurohormonal environment by means of kidney transplantation. Detailed investigation of isolated distal interlobar artery function from SHR did not reveal major effects of neonatal sympathectomy (11). Therefore, it is likely that major effects of sympathectomy on renal preglomerular vessel function are exerted at the level of interlobular arteries or afferent arterioles. Renal MBF scarcely responded to a 20-mmHg elevation of renal perfusion pressure in both groups, which corresponds with findings obtained under similar experimental conditions in untreated SHR (8), indicating that neonatal sympathectomy does not induce long-lasting effects on the pressure dependency of MBF in SHR kidneys.

Perspectives

In rats, nephrogenesis and the development of renal sympathetic innervation continue through postnatal weeks 2–3, and both processes may influence each other (10, 18, 22, 37). Activation of beta-adrenoceptors reduced renal RNA and protein synthesis in 5- and 9-day-old rats (35), and an elevated sympathetic tone has been suggested to inhibit cell replication in the developing rat kidney (36). On the other hand, the trophic effects of sympathetic innervation on the cardiovascular system are well established (19, 21).

We provide evidence that neonatal sympathectomy lowers renal NADPH oxidase activity and renal medullary mRNA expression of two major NADPH oxidase subunits. Thus oxygen radicals activate sympathetic nerve activity in SHR (33), and the elevated sympathetic nerve activity may contribute to enhanced renal oxygen radical formation by increasing NADPH oxidase activity under pathological conditions. RVR was reduced in kidneys from sympathectomized SHR (14). This effect persisted for several weeks after restoration of sympathetic tone by transplantation of these kidneys into untreated SHR. A role of NADPH oxidase activation in {alpha}1-adrenoceptor-mediated vascular smooth muscle cell growth and proliferation has been shown (2). Conversely, neonatal sympathectomy reduced the media thickness of mesenteric arteries in SHR (21) and lowered rat vascular smooth muscle actin synthesis (6). A long-term reduction of arterial pressure and RVR was also induced with the SOD mimetic tempol (32); however, the effects of tempol may not be due exclusively to oxygen radical dismutation (42). Activation of the NADPH oxidase by enhanced sympathetic innervation density and activity in SHR (7, 10, 12) may be involved in the programming of elevated RVR and contribute to subtle alterations during perinatal programming of renal function that lead to chronic arterial hypertension (17).


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This study was supported by Deutsche Forschungsgemeinschaft Grant GR 1430/2-4.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Grisk, Institute of Physiology, Dept. of Cardiovascular Medicine, Univ. of Greifswald, Greifswalder Strasse 11c, D17495 Karlsburg, Germany (e-mail: grisko{at}uni-greifswald.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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