INTRODUCTION

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UNDER RESTING CONDITIONS, blood flow to the kidney is among the highest to any organ, whether compared as a percentage of cardiac output or relative to organ weight (27, 30). The majority of this blood flow is fundamental to the filtration processes of the kidney rather than to satisfy tissue oxygen demand (27, 30). In humans, exercise does not produce increases in renal tissue oxygen consumption (27); therefore, it is not surprising that during dynamic exercise there is a redistribution of blood flow away from the kidney to metabolically active skeletal muscle (26, 28). The resulting vasoconstriction in the kidney produces a decrease in renal blood flow in most species studied, including humans (11, 32, 35). One exception is the dog, in which renal blood flow is maintained during dynamic exercise unless the studies are performed under conditions of circulatory compromise such as heart block, heart failure, or splenectomy (19, 33, 34). Recently, it has been shown that renal vasoconstriction occurs in rabbits during dynamic exercise (4, 7, 14). Furthermore, renal sympathetic nerve activity increases at the onset of dynamic exercise in rabbits (5, 22) and is related to intensity (22). It has been presumed that the decrease in renal blood flow is due to an increase in renal sympathetic nerve activity, although only a single study has addressed this issue in intact animals. Hohimer and Smith (9) demonstrated that renal denervation abolished decreases in renal blood flow observed in baboons during mild-intensity leg exercise. Nevertheless, there is a paucity of data regarding the time course of renal hemodynamic responses to exercise and its relationship to exercise intensity and the role of renal nerves.

Therefore, the primary purpose of the present study was to determine the role of renal nerves in the control of blood flow to the kidney during dynamic exercise. Specifically, we compared renal hemodynamic responses to graded levels of exercise in rabbits with one surgically denervated and one intact kidney. We also compared responses at a heavy workload before and after systemic -adrenergic-receptor blockade. We hypothesized that exercise would produce intensity-dependent renal vasoconstriction in the innervated kidney but not in the denervated kidney. Furthermore, we postulated that the exercise-induced vasoconstriction would be mediated by -adrenergic receptors.

	METHODS

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All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the American Physiological Society's Guiding Principles in the Care and Use of Animals. Female New Zealand White rabbits (weight 3-4.5 kg, n = 9) were selected for their willingness to run on a motor-driven treadmill. After selection, rabbits were acclimated to the treadmill by running one to two times per week. These sessions consisted of one to two short (2- to 5-min) bouts of exercise at 15 m/min and 17% grade (10°). These nine rabbits were included in another study that examined renal hemodynamic responses to activation of the nasopharyngeal reflex with formaldehyde vapor (21).

Surgical Preparation

In each animal, ultrasonic transit-time flow probes (Transonic Systems, Ithaca, NY) were implanted around each renal artery according to previously published methods (2). Briefly, each kidney was exposed via a retroperitoneal approach, and a 1 × 2-cm piece of silicone rubber medical-grade sheeting (0.007-in. thickness, Technical Products, Decatur, GA) was placed under the renal artery and in some cases the renal vein. The flow probe was then placed delicately around the renal artery to avoid damaging the renal nerves. The ends of the silicone sheeting were brought together around the probe and sutured together for probe stabilization. The connector ends of the probes were wrapped in sterile parafilm and tunneled to subcutaneous pockets for later retrieval. In each animal, one kidney was surgically denervated before probe implantation by stripping the renal artery and vein near the hilus of the kidney and painting the vessels with 5% phenol. Probe implantation on the contralateral kidney served as a sham surgery for the renal denervation. Rabbits were allowed 2-5 wk to recover before being studied.

Experimental Procedures

Instrumentation. On the day of experimentation, arterial blood pressure was obtained from a small Teflon catheter (24 gauge, Angiocath, Deseret, Sandy, UT) placed into the central ear artery via a percutaneous insertion. In some cases, a cutdown under a local 4% lidocaine block was used. A short pressure line was connected to a solid-state pressure transducer (Viggo-Spectramed, Oxnard, CA) that was strapped to the animal's back. Venous access was obtained by placing a 24-gauge Teflon catheter percutaneously into the contralateral ear vein. The connector ends of the flow probes were retrieved from their subcutaneous pockets and connected to the flowmeter (model T206, Transonics, Ithaca, NY).

Nasopharyngeal reflex. The nasopharyngeal reflex (18, 36) was activated by placing a cotton swab, soaked in 37% formaldehyde (JT Baker Chemical, Philipsburg, NJ), in front of the animal's nose for 5-10 s. The cotton swab was presented until a substantial bradycardia and a decrease in renal blood flow in the innervated kidney were observed. Because the nasopharyngeal reflex has been shown to produce a large decrease in renal blood flow (6, 36) mediated by renal nerves (6), only those animals with decreases in renal blood flow in the sham-operated kidney and little or no change in renal blood flow in the surgically denervated kidney were selected for this study. Tissue catecholamines were also used to verify renal innervation status (see Tissue catecholamine analysis).

Exercise protocol. Three exercise intensities were used in this study, and the animals were given a minimum of 45 min of rest between exercise bouts. At one intensity, the treadmill was set at 7 m/min (0% grade), and the rabbits ran for 5 min. Rabbits exercising at this intensity and duration utilize ~84% of their aerobic capacity and plasma lactate levels remain <4 mM (10). At the second exercise intensity, the treadmill was set at 12 m/min (0% grade), and the rabbits ran for 2 min. At this intensity rabbits are exercising at ~92% of estimated aerobic capacity (10). These two exercise intensities have been used in a previous study examining renal sympathetic nerve activity during treadmill exercise in rabbits (22).

Tissue catecholamine analysis. On completion of the experiments, rabbits were overdosed with a commercial euthanasia solution (Succomb, Butler, Columbus, OH), and each kidney was removed, blotted dry on gauze, weighed, and flash frozen in liquid nitrogen. Samples were kept at 70°C until assayed for norepinephrine content.

Data analysis.. Phasic arterial pressure and blood flows were stored simultaneously on magnetic tape with a videocassette data recorder (Vetter, Rebersburg, PA) and written to paper on a polygraph (Grass, West Warwick, RI). Data were analyzed off line by using a computer (Apple 8500 Power Personal Computer) and MacLab System at 100 Hz (ADInstruments, Castle Hill, Australia) to calculate mean arterial pressure, heart rate (HR), mean renal blood flow, and mean renal vascular conductance (mean renal blood flow/mean arterial pressure). Renal vascular conductance was used instead of renal vascular resistance because it has been argued that conductance better reflects changes in vascular tone in vivo, especially when the experimental manipulation causes larger changes in organ blood flow than in systemic arterial blood pressure (15, 23). Baseline variables were averaged over a 30-s period before the experimental manipulation. Responses to formaldehyde were averaged over 10-s intervals, and responses to phenylephrine were averaged over 5-s intervals. Averages were chosen at the peak change in renal vascular conductance. After phentolamine administration, the peak responses for the subsequent phenylephrine infusion were averaged over the time interval corresponding to the peak responses for the control phenylephrine infusion. For the exercise trials, the data were averaged in 10-s intervals for the first 30 s of exercise (3 data points). Data were also obtained in 10-s averages after 1 and 2 min of exercise at all intensities, after 5 min of exercise at 7 m/min, and 10 s before the point of exhaustion at 15 m/min (17% grade).

Statistical analysis. Paired t-tests were used to evaluate differences in kidney weights and renal norepinephrine concentration between the innervated and denervated kidneys and to compare the duration of exercise at 15 m/min (17% grade) intensity under control and -adrenergic blocked conditions. Repeated-measures ANOVA procedures were used to assess differences in cardiovascular variables in response to exercise and to evaluate the hemodynamic responses to phenylephrine infusion. For the exercise analyses, only the data from the first 2 min of exercise were utilized. Where significant F-ratios were found with the ANOVA, post hoc analysis was completed with Tukey's procedure. Differences were considered significant at P < 0.05. All data are expressed as means ± SE.

	RESULTS

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Responses to Formaldehyde

Renal Norepinephrine Content

Responses to Phenylephrine Before -Adrenergic Blockade (Table 1)

Effect of Exercise

The largest exercise-induced changes in cardiovascular variables were observed at 15 m/min (17% grade). Figure 1 is a raw-data tracing taken from one rabbit exercising on the treadmill at 15 m/min (17% grade). As expected, HR increased rapidly at the start of exercise and appeared to reach a steady state by 2 min. Because arterial pressure increased and renal blood flow in the denervated kidney remained essentially unchanged, there was a slight reduction in renal vascular conductance in the denervated kidney. In sharp contrast, renal blood flow and renal vascular conductance in the innervated kidney decreased dramatically at the onset of exercise, reached a steady state after 2 min, and remained depressed until the animal refused to run any longer.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Hemodynamic responses during treadmill exercise at 15 m/min (17% grade) in 1 rabbit with 1 surgically denervated and 1 intact kidney. Note dramatic difference in renal blood flow and calculated renal vascular conductance changes between innervated and denervated kidney. Marker depicts beginning and end of exercise trial.

Figure 2 illustrates the blood flow responses at each exercise intensity in denervated and innervated kidneys. Resting blood flow was higher in the innervated vs. denervated kidney before each exercise bout. During exercise at all three workloads, renal blood flow decreased in the innervated kidney within the first 10 s of exercise (range 10 to 17%) and remained depressed throughout each exercise bout, and the response was intensity dependent (P < 0.01). In contrast, renal blood flow in the denervated kidney remained essentially unchanged at the onset of exercise at each workload (range 1 to +3%) and remained stable throughout each exercise bout, and this response was not related to intensity (P > 0.05). At 7 m/min, blood flow in the innervated kidney decreased but was still significantly higher than blood flow in the denervated kidney. At the two higher exercise workloads, blood flow in the innervated kidney decreased to levels similar to those observed in the denervated kidney.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Renal blood flow responses in innervated () and denervated kidney () at rest and during 3 different exercise intensities [7 m/min, 0% grade (A); 12 m/min 0% grade (B); and 15 m/min, 17% grade (C)]. Values are means ± SE for 9 rabbits. * Significant change from rest in innervated kidney, P < 0.01. # Significant difference between innervated and denervated, P < 0.01. Note: a decrease in renal blood flow occurs only in innervated kidney.

Figure 3 shows the changes in vascular conductance in the denervated and innervated kidneys at each exercise intensity. Resting vascular conductance was significantly lower in the denervated kidney. Renal vascular conductance decreased significantly in the innervated kidney within 10 s of the onset of exercise (17.7 to 22.7%) and remained depressed over the entire time course of exercise, and the response was intensity dependent (P < 0.01). In the denervated kidney, renal vascular conductance was not altered during the first 30 s of exercise at any workload. However, renal vascular conductance significantly decreased by 2 min of exercise at 7 m/min (17 ± 3%) and 12 m/min (27 ± 3%). At 15 m/min (17% grade), the decrease in renal vascular conductance in the denervated kidney occurred earlier, at 1 min after the onset of exercise (31 ± 4%). During exercise at all three workloads, the values for renal vascular conductance were similar between the innervated and denervated kidneys (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Renal vascular conductance responses in innervated () and denervated kidney () at rest and during 3 different exercise intensities [7 m/min, 0% grade (A); 12 m/min, 0% grade (B); and 15 m/min, 17% grade (C)]. Values are means ± SE for 9 rabbits. * Significant change from rest in innervated kidney (above lines) or denervated kidney (below lines), P < 0.01. # Significant difference between innervated and denervated kidneys, P < 0.01. Note decrease in renal vascular conductance at onset of exercise (0-30 s) in innervated kidney and a slowly developing vasoconstriction in denervated kidney that is statistically significant (P < 0.01) at 1 min (15 m/min, 17% grade) and 2 min (all workloads) of exercise.

Effect of -Adrenergic Blockade on the Responses to Exercise

There were no significant differences in baseline mean arterial pressure or HR after phentolamine (Table 2). Unlike exercise in the absence of -adrenergic blockade, arterial pressure decreased significantly at the onset of exercise with phentolamine pretreatment. The depressor response was sustained through 2 min of exercise but was not different from its preexercise value at the time of exhaustion. HR responses to exercise appeared larger after -adrenergic blockade but did not reach statistical significance.

Figure 4 depicts the changes in renal blood flow during exercise at 15 m/min in the presence and absence of -adrenergic blockade. Phentolamine did not alter resting blood flows in the innervated (40 ± 6 vs. 42 ± 6 ml/min) or denervated kidney (29 ± 5 vs. 26 ± 5 ml/min). Therefore, similar to the other exercise conditions, resting renal blood flow was higher in the innervated kidney compared with the denervated kidney after -adrenergic blockade. During exercise at 15 m/min, the reduction in renal blood flow in the innervated kidney was not affected by -adrenergic blockade. However, in the denervated kidney there was an abrupt decrease in renal blood flow at the onset of exercise with -adrenergic blockade, which was not present in the absence of -adrenergic blockade.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Renal blood flow responses in innervated (A) and denervated (B) kidney during treadmill exercise at 15 m/min (17% grade) in the presence () and absence () of -adrenergic-receptor blockade with phentolamine mesylate (5 mg/kg iv). Values are means ± SE for 9 rabbits. * Significant change from rest in presence (below lines) and absence (above lines) of phentolamine, P < 0.01. # Significant differences between exercise with and without -adrenergic blockade, P < 0.01.

The effects of -adrenergic blockade on renal vascular conductance responses to exercise at 15 m/min are shown in Fig. 5. Resting vascular conductance was not altered by phentolamine in either the innervated (0.49 ± 0.07 vs. 0.54 ± 0.07 ml · min¹ · mmHg¹) or denervated kidney (0.36 ± 0.06 vs. 0.33 ± 0.05 ml · min¹ · mmHg¹). Phentolamine abolished the abrupt decrease in renal vascular conductance in the innervated kidney at the onset of exercise (0-30 s). At the onset of exercise, renal vascular conductance increased slightly in the denervated kidney. Thus the fall in renal blood flow observed in both the innervated and denervated kidney at the onset of exercise with phentolamine treatment was likely a passive response to the abrupt fall in arterial pressure.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Renal vascular conductance responses in innervated (A) and denervated (B) kidney during treadmill exercise at 15 m/min (17% grade) in presence () and absence () of -adrenergic-receptor blockade with phentolamine mesylate (5 mg/kg iv). Values are means ± SE for 9 rabbits. * Significant change (P < 0.01, except 30 s in denervated without phentolamine, P < 0.05) from rest in presence (above lines) and absence (below lines) of phentolamine. # Significant differences between exercise with and without -adrenergic blockade, P < 0.01.

As exercise continued past 30 s, a significant, sustained reduction in renal vascular conductance developed in the innervated kidney despite -adrenergic blockade. In contrast, phentolamine abolished the slowly developed reduction in renal vascular conductance observed in the denervated kidney during exercise under the unblocked condition. Thus a combination of renal denervation and -adrenergic blockade eliminated the renal vasoconstriction associated with dynamic exercise in rabbits.

Time to Exhaustion

	DISCUSSION

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The results of this study demonstrate the importance of renal nerves in control of blood flow to the kidney at the onset of exercise. At all three exercise intensities, a decrease in renal blood flow due to renal vasoconstriction was observed in the innervated kidney within the first 10 s of treadmill exercise and was sustained as long as exercise continued. In contrast, neither a decrease in renal blood flow nor vasoconstriction occurred in the denervated kidney at the onset of exercise. A slowly developing vasoconstriction did occur in the denervated kidney as exercise continued, which was blocked by -adrenergic blockade. These results suggest that circulating catecholamines can contribute to the renal vasoconstrictor response during exercise.

At the highest workload, -adrenergic blockade abolished the initial renal vasoconstriction in the innervated kidney, supporting a role for neurally released norepinephrine in the vasoconstrictor response. As exercise continued past 30 s, a residual vasoconstrictor response developed in the innervated kidney but not in the denervated kidney. That residual vasoconstriction occurred after -adrenergic block in the innervated kidney but not in the denervated kidney suggests that a neurally mediated renal vasoconstriction can occur independently of -adrenergic receptors.

Although resting blood flow and vascular conductance were lower in the denervated kidney, the denervated vasculature was capable of responding adequately to -adrenergic stimulation. Indeed, the vasoconstrictor response to phenylephrine was potentiated in the denervated kidney, as indicated by the greater relative change in vascular conductance. When significant differences in baseline flow occur, the relative change in conductance is the most appropriate index of blood vessel radius. This is due to the fact that absolute changes in conductance can vary greatly when identical changes in vessel radius are imposed on differing baseline blood flows, whereas a given percent reduction in conductance always reflects a predictable percent reduction in the radius of the vessel despite differing baseline blood flows. In the present study, the percent change in renal vascular conductance to phenylephrine was significantly greater in the denervated kidney, which suggests the possibility of denervation supersensitivity (8, 12).

The minor reductions in renal blood flow and renal vascular conductance in the denervated kidney coupled with the striking vasoconstriction in the innervated kidney resulted in similar levels of absolute flow and conductance during exercise. If renal nerves were not important in mediating the initial vasoconstriction, we would have expected vascular conductance in the denervated kidney to decrease at the onset of exercise; in other words, the denervated kidney would have demonstrated a response similar to the innervated kidney. In contrast, the significant interaction between innervation status and renal hemodynamic responses to exercise demonstrates that the kidneys responded differently to the stimulus of exercise and that the state of innervation is important in renal vascular responses to exercise.

In humans, dynamic exercise produces an intensity-dependent decrease in renal blood flow (32). Previous studies examining renal blood flow responses during dynamic exercise in rabbits have reported that decreases in renal blood flow occur within the first 20 s of exercise (7) and that renal vasoconstriction continues through at least 2 min of exercise (4). The results of the present study complement and extend these previous findings, in that intensity-dependent renal vasoconstriction was observed within the first 10 s of exercise in the innervated kidney and was sustained throughout each exercise bout. More importantly, our data suggest that the immediate decrease in renal blood flow is due to neurally mediated renal vasoconstriction, because no changes in renal blood flow or vascular conductance were observed in the denervated kidney at the onset of exercise. This study supports a physiological role for the increase in renal sympathetic nerve activity observed at the onset of dynamic exercise in rabbits (5, 22).

To our knowledge, there is only one other report (9) that has examined the effects of renal denervation in an intact-animal model that exhibits sustained decreases in renal blood flow during dynamic exercise. Hohimer and Smith (9) reported that unilateral denervation abolished the initial renal vasoconstriction observed in baboons performing light exercise. Therefore, the evidence to date demonstrates that renal nerves are responsible for the initial renal vasoconstriction during exercise across a wide range of intensities.

There have been a number of studies performed in dogs that have examined the renal blood flow response to dynamic exercise (16, 19, 29, 33, 34). Renal denervation has been shown to block the decrease in renal blood flow in dogs with circulatory compromise such as splenectomy or pacing-induced heart failure (19, 33). Because healthy dogs do not exhibit a sustained decrease in renal blood flow even at exhaustive workloads (16, 19, 34), other investigators have questioned the validity of the dog as an appropriate model for studying renal hemodynamic responses to exercise (9, 29). There is an increase in renal vascular resistance during exercise in dogs, indicating renal vasoconstriction (16, 19, 34). However, the increase in renal vascular resistance is unaffected by renal denervation or -adrenergic blockade, suggesting an autoregulatory phenomenon (16, 19, 34). In contrast, in the present study the combination of renal denervation and -adrenergic block abolished renal vasoconstriction during exercise. These results argue against a strong role for autoregulation in the renal vasoconstrictor response to short-term exercise in the rabbit.

In the present study, there was significant renal vasoconstriction in the denervated kidney by 2 min of exercise at each workload and by 1 min at the highest workload. These data suggest that compensatory mechanisms exist in the renal vasculature that act independently of direct renal innervation to permit redistribution of blood flow away from the kidneys as exercise duration or intensity increases. Because phentolamine pretreatment abolished the slowly developing vasoconstrictor response, -adrenergic receptors appear to produce renal vasoconstriction in the denervated kidney via circulating catecholamines. Interestingly, the intact kidney is thought to contribute a significant portion of the elevated plasma norepinephrine during dynamic exercise in humans (32). Norepinephrine is the dominant catecholamine released from the kidney in the rabbit (3). Therefore, it is possible that, in the rabbit, norepinephrine spillover from the innervated kidney contributed to circulating catecholamines that were responsible for the vasoconstriction in denervated kidney. It is important to note that the magnitude of the vasoconstriction observed in the denervated kidney could be influenced by an enhanced sensitivity to circulating catecholamines (8, 12). Denervation supersensitivity has been shown to occur in denervated rat kidneys (8, 12, 13) and may be due to an increase in the number of -adrenergic receptors in the renal vasculature (37).

The residual vasoconstriction apparent in the innervated kidney during exercise with -adrenergic blockade suggests that a nerve-dependent mechanism produced vasoconstriction independent of -adrenergic receptors. The classic neurotransmitter at postganglionic sympathetic nerves is norepinephrine, but more recently it has been shown that sympathetic nerve stimulation produces corelease of other vasoconstrictor substances, such as neuropeptide Y (24). There are now several studies that provide evidence for an increase in neuropeptide Y during physical activity in humans (1, 17, 25) that may be related to decreases in renal blood flow (32). Exogenous neuropeptide Y causes renal vasoconstriction in rabbits that is independent of adrenergic receptors (20). Because renal sympathetic nerve activity increases in an intensity-dependent manner in rabbits (22), we speculate that higher intensity exercise will produce greater activation of renal sympathetic nerves, resulting in corelease of norepinephrine and neuropeptide Y.

Alternatively, the -adrenergic-independent vasoconstriction in the innervated kidney may be mediated by the renin-angiotensin system. Stimulation of renal nerves results in renal release of renin, which is mediated by ₁-adrenergic receptors (3). The hypothesis that angiotensin II may be involved in renal vasoconstriction during exercise is supported by studies in which dynamic exercise was associated with increases in renal overflow of renin and angiotensin II (32). In addition, Stebbins and Symons (31) reported that losartan, an angiotensin AT₁-receptor antagonist, attenuated decreases in renal blood flow and increases in renal vascular resistance observed in exercising miniature swine.

In the present study, -adrenergic blockade abolished the slowly developing vasoconstriction observed in the denervated kidney, which argues against a significant role for circulating angiotensin II in the renal vascular response to exercise in the rabbit. However, we did not measure plasma levels of renin or angiotensin II and thus cannot evaluate whether chronic unilateral renal denervation or phentolamine pretreatment altered circulating levels of angiotensin II during exercise in the rabbit. Although unilateral denervation should decrease ₁-adrenergic-mediated renin release from the ipsilateral kidney, the abrupt fall in renal perfusion pressure at the onset of exercise with -adrenergic block should have been a potent stimulus for renin release from both kidneys (3).

In summary, our study demonstrates that renal nerves, via activation of -adrenergic receptors, are primarily responsible for the abrupt decreases in renal blood flow and renal vascular conductance at the onset of dynamic exercise in rabbits. In the absence of renal nerves, renal blood flow does not decrease during moderate to heavy dynamic exercise. The renal vasoconstriction observed during steady-state exercise in the innervated kidney appears to have a neurally mediated, -adrenergic-independent component.