INTRODUCTION

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A POPULAR METHOD for discerning the role of the renal sympathetic nerves in the regulation of renal function has been to chronically denervate one kidney (1, 7). One concern with this approach is that increased renal responsiveness to plasma levels of norepinephrine may develop over time, often termed denervation supersensitivity. This may reduce the apparent magnitude of the effect of the renal nerves or indeed completely mask their effect.

While the issue of denervation supersensitivity is clearly of some importance, unfortunately it is also one of debate. Krayacich and co-workers (3) examined the renal responses to norepinephrine infusions of the chronically denervated (7-10 days) and the contralateral ganglionically blocked kidney in rats. They found that with norepinephrine infusions, the denervated kidney showed significantly greater decreases in renal blood flow (RBF) and glomerular filtration rate than the ganglionically blocked kidney. When the mean arterial pressure (MAP) was maintained at preganglionic blockade levels with an infusion of arginine vasopressin, the denervated kidney still showed greater responses in RBF, glomerular filtration rate, and sodium excretion than the ganglionically blocked kidney (4). However, the methodology in these studies involved comparing the responses in the chronically denervated kidney with the acutely sympathetically blocked kidney using ganglionic blockade, which may have produced nonspecific compensation in other cardiovascular control mechanisms. Conversely, other reports indicate the denervated kidney does not become supersensitive to physiological levels of circulating norepinephrine. Lohmeier et al. (9) observed no differences in sodium excretion between the innervated and chronically denervated kidneys (2 wk postdenervation) in response to infusions of norepinephrine at physiologically relevant levels. However, at supraphysiological levels of norepinephrine, they observed sustained differences in sodium excretion from the innervated and the denervated kidneys.

Chronic renal denervation has also been used to look at the role of the renal nerves in pathological conditions such as hypertension (6-8). In this case, other mediators of vascular tone may interact with the ability of the sympathetic nervous system to regulate renal function. The vasoconstrictor responses to adrenergic nerve stimulation have been shown to be augmented by ANG II (11), and ANG II affects transmission at the nerve terminals (15). The response of the blood vessels to norepinephrine may also be potentiated by ANG II (16). It is conceivable that while the chronically denervated kidney may not be supersensitive to norepinephrine in the normotensive situation, with high ANG II levels occurring during some hypertensive models, the denervated kidney may become supersensitive to norepinephrine. If indeed this is the case, then the use of the unilateral denervated model to examine sympathetic responses in high-ANG II models of hypertension is questionable.

In the present experiment, we examined the acute RBF responses to phenylephrine to determine if there were differences between the responses in chronically denervated and either intact or acutely denervated kidneys. In addition, we examined the responses in rabbits that had been made hypertensive using a continuous infusion of ANG II for 7 wk.

	METHODS

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Animal preparation. Experiments were conducted on New Zealand White rabbits (mean wt = 2.7 ± 0.2 kg) and were approved by the University of Auckland Animal Ethics Committee. Throughout the study, the rabbits were fed daily (100 g standard rabbit pellets supplemented with hay, carrots, and apples) at 0900, and water was available ad libitum. The room was kept at a constant temperature (18°C) and dark-light cycle (lights on from 0600 to 1800).

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Experimental preparation. After 7 wk of either continuous ANG II or control, all rabbits were subjected to an acute experiment. Induction of anesthesia was by intravenous administration of pentobarbital sodium (90-150 mg Nembutal; Virbac Laboratories) and immediately followed by endotracheal intubation and artificial respiration. Anesthesia was maintained throughout the surgery and experiment by pentobarbital sodium infusion (30-50 mg/h). A catheter was inserted into the central ear artery to monitor arterial pressure. In addition, the marginal veins in both ears were also cannulated. During surgery, Hartman's solution was continuously infused at a rate of 10.8 ml · kg¹ · h¹ to replace fluid losses. A heated blanket was used throughout the surgery and experiment to maintain body temperature at 36°C. The left kidney was approached via a retroperitoneal incision, and the renal artery and nerves were carefully exposed. A transit time flow probe (type 2SB, connected to a T206 flowmeter; Transonic Systems) was placed around the renal artery. The right kidney was also approached via a retroperitoneal incision, and a transit time flow probe was placed around the right renal artery. Animals were left for 30 min to enable all variables to stabilize before commencing the experimental protocol.

Experimental protocol. RBF responses of both the left (renal nerve intact) and right (chronically denervated) kidneys were recorded to bolus intravenous injections of phenylephrine (2.5, 5, 10, 25, 50, and 75 µg) (Sigma) in random order. The different doses were made up in 200 µl of saline, and each dose was flushed with 0.5 ml of saline. After the doses were complete, the left renal nerve was carefully sectioned distal to the flow probe. Subsequently, the RBF responses to the phenylephrine injection were repeated. Thus comparison was made in the RBF response between acute and chronically denervated kidneys to phenylephrine.

Data acquisition. BP and RBF to both left and right kidney signals were digitized at 500 Hz and continuously displayed on a computer, allowing continuous sampling of MAP (mmHg) and RBF (ml/min). Heart rate (HR; beats/min) was derived from the MAP waveform. During each experiment, data from each variable were saved continuously at 500 Hz.

Statistical analysis. Mean RBF, BP, and HR were calculated for the 1-min period before phenylephrine and the maximum response during each injection of phenylephrine. Comparisons between the intact and chronically denervated kidneys were done using an ANOVA. For comparisons with the acutely denervated kidney, results were compared using an ANOVA with a double repeated-measures design. The independent variable was the RBF after acute left renal denervation. The model used the RBF before acute left renal denervation as a covariant for the RBF after denervation. The left and right RBFs were assigned as repeated measures to account for both of them being measures in the same animal. The other factors were the group and the dose of phenylephrine. Data are presented as means ± SE. P values <0.05 were considered significant.

	RESULTS

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Normotensive animals (n = 9). Baseline RBF between the chronically denervated right kidney and the left kidney (initially renal nerve intact) was not significantly different (26 ± 5 and 25 ± 3 ml/min, respectively) for the normotensive group. The mean dose responses to phenylephrine infusion in the innervated and denervated kidneys are shown in Fig. 1. No significant difference in the RBF response to phenylephrine infusion was observed between the intact and chronically denervated kidneys.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Blood pressure and renal blood flow (RBF) responses to increasing doses of phenylephrine in the normotensive animals (n = 6). , Left innervated kidney; , right chronically denervated kidney. Values are means ± SE. When the RBF response between the innervated and the chronically denervated kidneys was compared, either as the change in absolute RBF or as the percentage change in RBF from control, no significant differences were observed between the kidneys.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Blood pressure, heart rate, and left and right kidney RBF responses to 25 µg of phenylephrine in a normotensive animal. Dashed line indicates time at which phenylephrine was infused. The range of resting blood pressures in these animals was 65-92 mmHg. bpm, Beats/min.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Blood pressure and RBF responses to increasing doses of phenylephrine in normotensive animals (n = 9). , Left acutely denervated kidney; , right chronically denervated kidney. Values are means ± SE. When the RBF response between the acutely denervated and the chronically denervated kidneys was compared, either as the change in absolute RBF or as the percentage change in RBF from control, no significant differences were observed between kidneys.

Hypertensive animals (n = 6). In animals that received continuous ANG II, BP increased from 77 ± 1 mmHg before ANG II to 104 ± 6 mmHg (conscious BP) after 6 wk of ANG II. There was no significant change in HR (214 ± 11 beats/min before ANG II and 198 ± 7 beats/min after 6 wk of ANG II). Baseline RBF between the chronically denervated right kidney and the intact left kidney was not significantly different (26 ± 6 and 28 ± 6 ml/min, respectively) in the hypertensive group. Following the same protocol as the normotensive animals, the blood flow responses to phenylephrine were measured in the two kidneys. Under anesthesia, the BP of the hypertensive animals fell to a mean of 82 ± 6 mmHg. The RBF responses of the innervated and the denervated kidneys to phenylephrine are shown in Fig. 4. Again, no significant difference was observed in the RBF responses to phenylephrine in the innervated and chronically denervated kidneys.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Blood pressure and RBF responses to increasing doses of phenylephrine in hypertensive animals (n = 6). , Left innervated kidney; , right chronically denervated kidney. Values are means ± SE. When the RBF response between the innervated and the chronically denervated kidneys was compared, either as the change in absolute RBF or as the percentage change in RBF from control, no significant differences were observed between the kidneys.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Blood pressure and RBF responses to increasing doses of phenylephrine in hypertensive animals (n = 6). , Left acutely denervated kidney; , right chronically denervated kidney. Values are means ± SE. When the RBF response between the acutely denervated and the chronically denervated kidneys was compared, either as the change in absolute RBF or as the percentage change in RBF from control, no significant differences were observed between kidneys.

	DISCUSSION

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In the present study, we found that chronic denervation did not result in increased renal responsiveness to phenylephrine compared with either the renal nerve-intact or acutely denervated kidneys, suggesting that differences in renal function between intact and denervated kidneys observed in previous studies are unlikely to be confounded by supersensitivity (1, 6). Previously, it has been shown that denervation results in increased expression of -receptors for norepinephrine in the rat kidney (14). Blockade of -receptors has also been shown to blunt the increased response to norepinephrine in sodium excretion of the denervated kidney compared with the innervated kidney in the dog (12). Thus it was logical to propose that renal denervation resulted in increased sensitivity to circulating norepinephrine. If indeed chronic denervation does lead to supersensitivity, differences between the innervated and denervated kidneys will be confounded by this supersensitivity. Given the number of research groups that have used chronic denervation (1) as a means to assess the role of the renal nerves, this issue is potentially a major confounding variable.

Our results agree with the previous studies by Lohmeier et al. (9), who used the unilaterally denervated kidney preparation in the dog. Sodium excretion was measured in the intact and denervated kidneys in response to continuous norepinephrine infusions. While there were transient changes in sodium excretion between the two kidneys, there were no sustained differences in the sodium excretions between the two kidneys in response to high physiological levels of norepinephrine. The transient increases in sodium excretion by the innervated kidney were attributed to sympathoinhibition. These authors compared the denervated kidney with the innervated kidney. In addition to supersensitivity of the chronically denervated kidney, another difference between the two kidneys under this condition is the presence of changes in renal sympathetic nerve activity during changes in BP. Baroreflex-induced changes in RBF resulting from changes in BP are a potential confounding factor in examining the supersensitivity of the chronically denervated kidney. For this reason, in the present study we compared the sensitivity of the chronically denervated kidney to the renal nerve-intact and acutely denervated condition. We found no difference in the response to phenylephrine infusion in the chronically denervated kidney compared with either the intact or acutely denervated contralateral kidney. Our results suggest that the presence of the renal nerves does not confound the results when comparing the sensitivity of intact and chronically denervated kidneys.

Our finding of a lack of denervation supersensitivity is in contrast to the conclusions reached by Kline and Mercer (2) and Krayacich and co-workers (3, 4), who found that the chronically denervated kidney had a greater decrease in blood flow with norepinephrine infusions. It must be mentioned that the above authors used a continuous infusion of norepinephrine, whereas we used boluses of phenylephrine. The above authors also used ganglionic blockade to eliminate reflex control of renal function in the innervated kidney. We chose to physically denervate the left kidney to eliminate the decrease in pressure that would occur with ganglionic blockade. Unlike ganglionic blockade, physical denervation of the nerves to the left kidney did not result in a fall in BP.

In previous studies, the issue of supersensitivity has been examined generally up to 15 days after denervation of the kidney (2-4, 9). One possibility is that reinnervation of the kidney can occur after extended time periods, e.g., 2-3 mo. While evidence suggests regeneration of the adrenergic nerves does occur, there is no evidence of regeneration even after 3 mo in the dog (10). Previously, we have used chronic renal denervation for 4 wk and measured the RBF differences between intact and denervated kidneys (1). Besides denervation supersensitivity, one potential criticism is that the renal nerves may reinnervate during this time. However, in the present study we showed significant reductions in the norepinephrine content of the chronically denervated kidney, suggesting reinnervation was not a confounding issue.

Renal denervation has been shown to increase ANG II receptors in the glomeruli of the denervated kidney (13). This is a potentially confounding factor if chronic denervation is a method used in studies involving ANG II. However, in our study, chronic ANG II infusion did not result in increased sensitivity of the chronically denervated kidney to phenylephrine. The finding of comparable RBFs in both the innervated and denervated kidneys during ANG II infusion in both the present study in rabbits, and as has previously been described in dogs (6), also argues against an exaggerated response of the denervated kidney to ANG II.

Limitations. In the present study, we used infusions of phenylephrine and recorded the RBF responses. The RBF responses we recorded are comparable to that observed using electrical stimulation of the renal nerves in the rabbit (5). Furthermore, we have observed similar decreases in RBF under reflex activation of sympathetic activity using hypoxia, suggesting the responses to phenylephrine were well within the physiological range.

Perspectives