Abstract A low level of sympathetic nerve activity (SNA) to brown adipose tissue has been found in genetically obese Zucker rats and may promote obesity through decreased thermogenesis. In contrast, acquired obesity is reportedly associated with increased SNA. To determine whether low SNA levels in obese Zucker rats extend to the kidney, we compared baseline levels of renal SNA in obese and lean conscious unrestrained Zucker rats fed for 2 weeks on low salt (0.4% NaCl) and high salt (8.0% NaCl) diets. Baseline renal SNA was calculated from multifiber recordings obtained before death under conscious, resting conditions and after death. Body weight averaged 490±12 g (mean±SEM) in obese rats (n=17) and 339±7 g in lean rats (n=19). Mean arterial pressure did not differ in obese and lean Zucker rats fed the low salt diet. However, on the high salt diet, mean arterial pressure was significantly higher in obese rats (n=8) than in lean rats (n=9) (113±3 and 101±3 mm Hg, respectively; P<.05). Baseline renal SNA was approximately 2 to 2.5 times higher (P<.05) in obese rats than in lean rats in all groups. These studies suggest that obese Zucker rats have heightened levels of SNA to the kidney in contrast to reduced SNA to brown adipose tissue.
Genetically obese Zucker rats have a low level of sympathetic nerve activity (SNA) to brown adipose tissue measured either as norepinephrine turnover and synthesis1 2 or as direct recordings of SNA.3 This alteration in sympathetic function reportedly occurs in young preobese Zucker rats.4 The reduced level of SNA is postulated to reduce thermogenesis in brown adipose tissue and thereby to promote obesity.5 In contrast to the low norepinephrine synthesis in brown adipose tissue, norepinephrine turnover in the heart is normal.1 2 This indicates that alterations in sympathetic function in obese Zucker rats are tissue specific.
In contrast to the finding of low levels of SNA to brown adipose tissue in obese Zucker rats, there is evidence that SNA to muscle in humans is directly related to percent body fat.6 7 In addition, Kassab et al8 have recently reported that renal SNA plays an important role in obesity-induced hypertension.
The goal of the current study was to compare renal SNA in obese and lean Zucker rats consuming low and high salt diets. We wanted to determine whether obese Zucker rats have low renal SNA, as might be predicted from studies of SNA to brown adipose tissue in these rats, or have high renal SNA, as might be predicted from models of acquired obesity.
Male Zucker obese (fa/fa) and Zucker lean (+/+ or +/?) rats 12 to 13 weeks of age were obtained from Charles River Laboratories. They were fed either a low salt diet (0.4% NaCl, 1.06% KCl) or a high salt diet (8.0% NaCl, 1.06% KCl) for 14 to 17 days. A separate group of Zucker obese rats was fed either the low salt or the high salt diet for 28 days. Animal care and all procedures were approved by the University of Iowa and the Veterans Affairs Animal Research Committee.
Conscious Renal SNA Recording
At the end of the diet period, rats were anesthetized with methohexital sodium (40 mg/kg IP; Brevital, Eli Lilly). A catheter was inserted into a femoral artery for measurement of arterial pressure and another catheter was introduced into a femoral vein for administration of anesthetic and drugs. Both catheters were tunneled subcutaneously and exited out of the nape of the neck. Anesthesia was sustained by continuous infusion of methohexital sodium (5 mg · kg−1 · min−1 IV) while a left retroperitoneal incision was made. From this dissection, a nerve fascicle to the left kidney was isolated and placed on a bipolar platinum-iridium electrode (Cooner Wire). After an optimal nerve recording was obtained, as described previously in a report from our laboratory,9 silicone gel (Sil-Gel 604, Wacker-Chemie) was applied and allowed to harden before securing of the electrode to surrounding muscle. The other end of the electrode was carefully tunneled underneath the skin and exited through the same incision as the femoral catheters. All incisions were closed and the rat was allowed to recover until the next day.
After 16 to 24 hours of recovery, the femoral arterial catheter was connected to a pressure transducer (model p231D, Gould Electronics) for measurement of systolic, diastolic, and mean arterial pressures and heart rate on a thermal array recorder (TA-4000, Gould Electronics). The femoral venous catheter was connected to a PE-10 extension for later administration of drugs. The nerve electrode was attached to a high-impedance probe (HIP-511, Grass Instruments), amplified by 105, and filtered at low- and high-frequency cutoffs of 700 and 2000 Hz, respectively, with a nerve traffic analysis system (model 662-C, University of Iowa Bioengineering). The filtered, amplified nerve signal was routed (1) to an oscilloscope (model 54501A, Hewlett-Packard) for monitoring and to a DC amplifier for permanent display of the neurogram on the Gould TA-4000 recorder, (2) to a resetting spike counter that counts 100 action potentials before resetting to zero, and (3) to a resetting voltage integrator (model B600C, University of Iowa Bioengineering) that sums the total voltage output to a unit of 1 V · s before resetting to zero.
To measure renal SNA, we made recordings of the renal nerve signal while the animals were in the conscious resting state and again after ganglionic blockade with chlorisondamine (30 mg/kg IV) and after the rat was killed with methohexital sodium (150 mg/kg IV). Using tapes of the recordings, we set the upper margin of the activity after death as a cursor in analyzing the recordings in the conscious state for spike counting. Action potentials exceeding this cursor were counted. In the measurement of renal SNA as integrated voltage, all the activity in the filtered neurogram was analyzed. The integrated voltage after death was subtracted from the integrated voltage in the conscious state to calculate renal SNA (Fig 1⇓). Although not presented in this article, similar results for renal SNA were obtained when the signal after ganglionic blockade was used as noise rather than the signal after death.
Simultaneous measurements of heart rate and systolic, diastolic, and mean arterial pressures, as well as basal renal SNA as measured by the spike counter (spikes per second) and by the resetting integrator (V · s · min−1), were obtained from conscious unrestrained Zucker obese and lean rats. Recordings were continuously displayed on the Gould TA-4000 recorder and stored for later analysis on videotape (model 4000a, AR Vetter). These parameters were selectively recorded during the time the rat was quiescent for up to 20 minutes.
Renal Nerve Morphology
To determine whether there were differences in renal nerve morphology that might explain differences in the nerve recordings, we performed additional experiments in which a renal nerve fascicle to the left kidney was isolated as if to perform a nerve recording. This was done in four obese and four lean Zucker rats. The fascicle was immersion fixed for 20 minutes with 1.0% paraformaldehyde and 1.0% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.6). After sectioning, each fascicle was immersed in the same fixative for an additional 60 minutes, then postfixed in 1.0% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 mol/L sodium cacodylate buffer for 2 hours. The fascicle was then en bloc stained for 20 minutes in 2.5% uranyl acetate, dehydrated by graded ethanol solutions, and embedded in Polybed 812. One-millimeter light level sections were cut from the Polybed 812 block on an ultramicrotone (Reichert-Jung Ultracut E), stained with Richardson’s biological stain, and digitized under the light microscope (×100) for cross-sectional dimensions.
Statistical comparisons of baseline values of systolic, diastolic, and mean arterial pressures and renal SNA were performed to compare data from the Zucker obese rats with those from the lean rats fed one of the two diets using an unpaired t test and by ANOVA with the Bonferroni method for multiple comparison. P<.05 was considered significant. Results are expressed as mean±SEM.
Body Weight and Arterial Pressure
Zucker obese rats weighed significantly more (P<.05) than the age-matched lean rats (Table⇓). Arterial pressure did not differ significantly in obese and lean rats fed the low salt diet (Table⇓). Arterial pressure was significantly (P<.05) although not strikingly higher in obese rats fed the high salt diet compared with lean rats on the same diet (Table⇓).
To determine whether the differences in arterial pressure would be greater with longer duration of the high salt diet, we measured arterial pressure in separate groups of obese rats fed the low salt or the high salt diet for 4 weeks. Mean arterial pressure in eight obese rats fed the high salt diet was significantly, albeit modestly, higher (120±4 mm Hg) than in eight obese rats fed the low salt diet (110±2 mm Hg, P<.05).
Heart rates did not differ in the four groups except for a slightly but significantly (P<.05) lower heart rate in obese rats fed the high salt diet (Table⇑).
Fig 2⇓ shows the filtered renal neurograms and the output from the spike counter and voltage integrator during the conscious state and at 30 minutes after death from a lean and an obese rat fed the low salt diet. Fig 3⇓ shows the filtered neurograms from obese and lean rats fed either the low salt or the high salt diet. Summary data are presented in Fig 4⇓. Renal SNA calculated as either spikes per second or integrated voltage was higher in obese rats than in lean rats on either diet. In lean rats, renal SNA was similar during the low salt and the high salt diet. In obese rats, renal SNA calculated as spikes per second did not differ during the low salt and the high salt diet, but renal SNA calculated as integrated voltage was greater (P<.05) during the high salt diet.
There were no qualitative differences in the morphology of renal nerve fascicles from obese and lean rats. Cross-sectional area tended to be lower in obese rats, but there was substantial overlap in the two groups (Fig 5⇓).
This study indicates that, in contrast to previous reports of low SNA to brown adipose tissue, obese Zucker rats have high levels of renal SNA. In addition, in obese Zucker rats, renal SNA expressed as integrated voltage is greater during a high salt diet than during a low salt diet.
The discussion will focus on several issues: (1) the validity of the measurement of renal SNA, (2) potential mechanisms for higher renal SNA, and (3) regulation of arterial pressure.
The results of this study depend on the validity of a comparison of multifiber recordings of SNA between groups of animals. Some investigators have suggested that variables involved in the technique limit the validity of measurements of basal SNA from multifiber recordings. In contrast, there have been a number of reports by various investigators comparing basal SNA between animals.10 11 12
Our view was that there is no a priori reason why multifiber recordings of renal SNA could not be used for comparison of basal SNA between animals if the techniques were standardized and if there were no major differences in renal nerve morphology that would introduce systematic bias in the measurements in the two groups. The techniques used in our study were highly standardized from animal to animal. Filter settings and amplification were standardized, as were the methods of analyzing the recordings. There were also no qualitative or systematic differences in renal nerve fascicle morphology that would explain differences in the nerve recordings. It might be argued that the apparent differences in renal SNA reflect differences in afferent rather than efferent activity, but this seems unlikely because eliminating renal SNA with ganglionic blockade (not death) also revealed higher SNA in obese animals than in lean animals. We conclude that the higher values for renal SNA in the obese rats reflect a higher level of SNA and not an artifact of the method.
Potential Mechanisms for Higher Renal SNA
In a broad sense, there are three potential mechanisms for higher renal SNA in the obese rats. First, the elevated renal SNA could be a direct result of the genetic mutation responsible for obesity in the Zucker rats. As mentioned above, it is thought that the fatty mutation in the Zucker rats results in a lower level of SNA to brown adipose tissue that in turn promotes decreased thermogenesis and thereby obesity. It has been known that the altered SNA in obese Zucker rats is tissue specific.2 It is possible that the higher renal SNA is also a result of the fatty mutation (independent of the obesity). Second, the elevated SNA could be secondary to increased body weight or fat and not a direct result of the genetic mutation that causes obesity in the Zucker rats. There have been reports that muscle SNA is directly related to percent body fat in humans.6 The precise mechanisms linking increased body fat and SNA in either humans or animals are not known. Insulin resistance and hyperinsulinemia promote sympathetic activation,13 and obese Zucker rats are insulin resistant and hyperinsulinemic.14 15 However, it is not known whether the higher renal SNA reflects hyperinsulinemia or other possible effects of obesity. Studies in young genetically obese Zucker rats before the development of obesity would help to determine whether the elevated renal SNA is a direct result of the genetic differences between the obese and lean Zucker rats or the result of increased body fat. Third, the higher renal SNA in the obese rats might reflect genetic differences between the obese and lean rats that are unrelated to the fatty mutation or obesity, ie, that are due to genetic drift.
Regulation of Arterial Pressure
Obese Zucker rats have been reported by some investigators to have higher arterial pressure than the lean rats16 17 and to develop higher arterial pressure during a high salt diet.18 In contrast, other investigators have failed to find a difference in arterial pressure between obese and lean Zucker rats.19 20 In addition, even in those studies in which arterial pressure was higher in obese than in lean Zucker rats, the elevation in arterial pressure was not impressive. We did not observe a difference in arterial pressure between the obese and lean rats during the low salt diet. During the high salt diet, mean arterial pressure was significantly higher in the obese rats than in the lean rats, but the difference was not striking.
The reasons for the lack of significant or striking hypertension in the obese rats are not clear and are beyond the scope of this study. We would note, however, that the rats in our study were obese and thus had the phenotypic expression of the fatty mutation. We therefore speculate in a broad way that either the fatty mutation does not contribute importantly to hypertension even during the high salt diet or, alternatively, that the genetic background of the obese Zucker rat modulates a hypertensive effect of the fatty mutation and obesity.
Relationship of Renal SNA and Arterial Pressure
The renal nerves have been reported to contribute to sodium retention and hypertension in obesity.8 21 In addition, exaggerated stress-induced increases in renal SNA have been shown to contribute to exaggerated sodium and water reabsorption in Okamoto spontaneously hypertensive rats, particularly during a high salt diet.22 23 There are several possible interpretations for our finding of higher basal renal SNA in the absence of significant elevation of arterial pressure. First, the elevated levels of SNA may not have reached the threshold for a significant pathophysiological effect. Second, basal levels may be less important than stress-induced levels of SNA. Third, high levels of renal SNA may contribute to elevated arterial pressure but may not be a sufficient factor in the absence of other prohypertensive influences.
This study indicates that obese Zucker rats have higher levels of basal renal SNA than do lean Zucker rats.
Research from this laboratory that was cited in this article was supported by grants HL-44546 and HL-43514 from the National Heart, Lung, and Blood Institute and by funds from the Department of Veterans Affairs. The authors would also like to acknowledge Dr Steven Moore and Julie Nessler for assistance with nerve morphology and Nancy Davin and Kathleen Romero for secretarial assistance.
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