INTRODUCTION

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NEUROPEPTIDE Y (NPY) is a cotransmitter in the sympathetic nervous system and is involved in the regulation of cardiovascular functions at the central, cardiac, vascular, and renal levels (18). Systemic infusion of NPY elevates blood pressure, reduces renal blood flow (RBF), and concomitantly causes diuresis, natriuresis, and calciuresis in anesthetized and conscious rats (5). The tubular NPY actions occur at least partly independently from the pressure-natriuresis mechanism and from alterations of RBF and glomerular filtration rate (3, 4, 6, 27). Studies with several subtype-selective agonists and with the Y₁-selective antagonist BIBP-3226 have demonstrated that RBF reductions occur via a Y₁ receptor. On the other hand, tubular effects occur via a Y₅ receptor (2), although no mRNA for this subtype is detectable in rat kidney (5). Moreover, the RBF reductions were markedly enhanced upon intrarenal administration relative to systemic administration. In contrast, direct intrarenal NPY infusion caused either no enhancement of diuresis and natriuresis or much less enhancement than intravenous administration produced (3). This indicates that an extrarenal NPY receptor and a formed or released mediator of unknown nature may interact to regulate tubular function. This potential mediator may reach the kidney either by release from renal nerves or via the blood stream. A mediator could be released from the sympathetic nerve fibers that densely innervate the kidney and reach intrarenal blood vessels and basement membranes of proximal and distal tubules (10). Possible humoral mediators include components of the renin-angiotensin, kallikrein-kinin, and vasopressin systems. The renin-angiotensin system is a candidate because NPY can inhibit renin release in vitro and reduce the plasma renin activity in vivo (5). Renin-release inhibition and enhancements of diuresis and natriuresis are mediated by an NPY receptor with similar pharmacological characteristics (2), and both are sensitive to pertussis toxin (15, 27). Thus lowering of angiotensin tone to the kidney may contribute to the tubular NPY effects. The kallikrein-kinin system is a candidate because NPY (5) and bradykinin (7) both cause diuresis and natriuresis mainly by altering the function of distal nephron segments. Moreover, the diuretic effects of both agents can be blocked by cyclooxygenase inhibitors (7, 8, 29). Finally, vasopressin can be considered as a candidate because it also acts on distal nephron segments; however, vasopressin is not a very likely mediator of NPY effects because it enhances free water clearance (12) whereas NPY does not (3, 27).

To examine the role of neuronally released mediators or one of the above humoral mediators, we have used two experimental designs. First, we have compared the effects of NPY on both kidneys after acute unilateral renal denervation. Second, we have compared the effects of NPY in rats treated with the angiotensin II-receptor antagonist losartan, the bradykinin B₂-receptor antagonist icatibant, the converting-enzyme inhibitor ramiprilat, and vehicle. In animals from the second study, we have also investigated whether NPY infusion alters plasma vasopressin concentrations.

	MATERIALS AND METHODS

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Two studies were performed following approval by the state animal welfare board at the Regierungspräsident Düsseldorf. In study 1 the animals underwent acute renal denervation, and in study 2 they were treated with pharmacological inhibitors. For both studies male Wistar rats (strain, Hsd/Cpb:WU; weight, 250-350 g in study 1 and 300-450 g in study 2) were obtained from Harlan CPB (Zeist, Netherlands). They were kept under standardized conditions with free access to standard lab chow and tap water. On the day of the experiment, the animals were anesthetized with an initial intraperitoneal injection of pentobarbital sodium (60 mg/kg). Additional anesthetic was administered in doses of 3 mg iv every 30 min. The animals were placed on a heating pad to maintain the body temperature at 37°C. Catheters were placed into the femoral vein (for saline and drug infusion), the femoral artery (for blood pressure measurements), and the ureter (for urine collection). In addition, an electromagnetic flow probe was placed on the renal artery for measurement of RBF and heart rate. The construction of the catheters and the preparation of the rats have previously been described in detail (3).

Study 1: Acute renal denervation. During these experiments, both kidneys remained in situ and both ureters were cannulated. After careful dissection of both renal arteries, one of the vessels was denervated in random order as described (1, 19, 20). Briefly, the adventitia and all visible nerve fibers were removed with a small forceps. Thereafter, the artery was surrounded by a piece of silk soaked with ethanol-phenol solution (10:1) for 4 min. After removal of the silk cloth, electromagnetic blood flow sensors (Skalar MDL 1401; Föhr Medical Instruments, Seeheim/Ober Beerbach, Germany) were placed on the treated and the contralateral renal arteries. Before the start of the experiment, animals were allowed 3 h of recovery, during which 80 µl/min of 0.9% saline were infused via the femoral vein. Thereafter, saline (n = 8) or NPY (1 µg · kg¹ · min¹; n = 10) was infused via the femoral vein for 90 min.

Study 2: Pharmacological inhibitors. In study 2, the left kidneys were removed under ketamine and xylazine anesthesia (100 and 6 mg/kg, respectively) 7-10 days before the experiment. On the day of the experiment, two catheters were inserted into the femoral vein, one for saline and NPY and one for inhibitor infusion. The right ureter was catheterized for urine collection. Thereafter, the electromagnetic flow sensor was placed on the right renal artery. Before the start of the experiment, animals were allowed 3 h of recovery, during which 60 µl/min of 0.9% saline was infused.

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Biochemical analysis. Urine formation was quantitated gravimetrically by assuming a specific gravity of 1.0 kg/l, and samples were stored at 4°C until analysis. Serum and plasma were prepared from the aortic blood sample by centrifugation (10 min at 5,000 g) and stored at 20°C until analysis for creatinine and vasopressin levels, respectively. Urinary sodium and calcium concentrations were determined with an Eppendorf flame photometer. Urinary and serum creatinine levels were determined with an automated analysis system for clinical chemistry (Hitachi 707). Vasopressin immunoreactivity was determined by a radioimmunoassay as previously described (22).

Chemicals. Rat and human NPY, angiotensin I, angiotensin II, and bradykinin were obtained from Saxon Biochemicals (Hannover, Germany); pentobarbital sodium (Nembutal) from Sanofi (Hannover, Germany); ketamine from Pittman-Moore (Burgwedel, Germany); and xylazine (Rompun) from Bayer (Leverkusen, Germany). Losartan was a kind gift from DuPont Merck (Wilmington, DE), and ramiprilat and icatibant (also known as HOE-140) were provided by Hoechst (Frankfurt-am-Main, Germany).

Data analysis. The averages of mean arterial pressure, heart rate, and RBF during the last 30 min and the average of urine formation during the last 45 min before the start of the experiment in each animal were taken as baseline (Tables 1 and 2). All other experimental data are expressed as alterations relative to the baseline values. Data are means ± SE of n experiments. Statistical significance of the differences in basal values was determined by paired two-tailed t-tests relative to the values from the contralateral kidney (study 1) and one-way analysis of variance (study 2). Statistical significance of the differences in vascular or tubular NPY effects between rats treated with losartan, ramiprilat, or icatibant relative to control rats was determined by a two-way analysis of variance. Additionally, a one-way analysis was performed for the total amount of excreted urine and electrolytes during the observational period. P < 0.05 was considered significant. Statistical calculations were performed with the Prism program (GraphPAD Software, San Diego, CA).

	RESULTS

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Study 1: Acute renal denervation. At the end of the equilibration period, basal RBF in denervated kidneys was 50% higher and renovascular resistance (RVR) was 30% lower than in innervated kidneys (Table 1). Water and electrolyte excretion also tended to be higher in denervated kidneys, but these differences failed to reach statistical significance with the given number of animals. Creatinine clearance of both kidneys was very similar (Table 1).

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View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effects of systemic neuropeptide Y (NPY) infusion (1 µg · kg1 · min1) on renal blood flow (RBF; left) and renovascular resistance (RVR; right) in innervated (top) and denervated (bottom) kidneys in anesthetized rats (study 1). Data are means ± SE of alterations relative to baseline values shown in Table 1. NPY was infused from 0 to 90 min. Despite the differences of basal values in innervated and denervated kidney, systemic infusion of NPY rapidly reduced RBF and increased RVR in both kidneys.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of systemic NPY infusion (1 µg · kg1 · min1) on urine (top), Na+ (middle), and Ca2+ excretion (bottom) in innervated and denervated kidneys in anesthetized rats (study 1). Data are means ± SE of alterations relative to baseline values shown in Table 1. NPY was infused from 0 to 90 min. Urine formation and electrolyte excretion were significantly increased by NPY infusion compared with vehicle in a 2-way analysis of variance (P < 0.05). NPY-induced elevations of diuresis, natriuresis, and calciuresis were similar in innervated and denervated kidneys.

Study 2: Pharmacological inhibitors. Icatibant treatment did not significantly affect any of the basal parameters relative to saline-treated rats (Table 2). In contrast, losartan treatment increased basal RBF by 20% and reduced RVR by 25%. Mean arterial pressure and water and electrolyte excretion also tended to be lower, but this failed to reach statistical significance with the given number of animals (Table 2). Ramiprilat treatment enhanced basal RBF by 14% and lowered RVR by 22% but did not significantly alter urine or electrolyte excretion (Table 2).

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View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effects of systemic infusion of 2 µg · kg1 · min1 NPY with concomitant infusion of inhibitory substances on mean arterial pressure in anesthetized rats (study 2). In study 2, 4 experimental groups (control, top left; losartan, bottom left; icatibant, top right; ramiprilat, bottom right) were investigated. Data are means ± SE of alterations relative to baseline values shown in Table 2. NPY was infused from 0 to 120 min. Systemic NPY infusion significantly elevated mean arterial pressure in all experimental groups compared with vehicle (P < 0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effects of systemic infusion of 2 µg · kg1 · min1 NPY with concomitant infusion of inhibitory substances on RVR in anesthetized rats (study 2). In study 2, 4 experimental groups (control, top left; losartan, bottom left; icatibant, top right; ramiprilat, bottom right) were investigated. Data are means ± SE of alterations relative to baseline values shown in Table 2. NPY was infused from 0 to 120 min. Systemic NPY infusion significantly elevated RVR in all experimental groups compared with vehicle (P < 0.05). Maximal peak elevations were not affected by any treatment, whereas the decline in the renovascular response with time was lowered by ramiprilat and losartan treatment and accelerated by icatibant treatment compared with control (P < 0.05 in a 2-way analysis of variance).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of systemic infusion of 2 µg · kg1 · min1 NPY with concomitant infusion of inhibitory substances on urine formation in anesthetized rats (study 2). In study 2, 4 experimental groups (control, top left; losartan, bottom left; icatibant, top right; ramiprilat, bottom right) were investigated. Data are means ± SE of alterations relative to baseline values shown in Table 2. NPY was infused from 0 to 120 min. Losartan treatment did not alter NPY-induced diuresis. Ramiprilat treatment enhanced the NPY effect, whereas icatibant treatment attenuated it. Both alterations were statistically significant compared with the control in a 2-way analysis of variance (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effects of systemic infusion of 2 µg · kg1 · min1 NPY with concomitant infusion of inhibitory substances on Na+ excretion in anesthetized rats (study 2). In study 2, 4 experimental groups (control, top left; losartan, bottom left; icatibant, top right; ramiprilat, bottom right) were investigated. Data are means ± SE of alterations relative to baseline values shown in Table 2. NPY was infused from 0 to 120 min. Losartan treatment did not alter NPY-induced natriuresis. Ramiprilat treatment enhanced the NPY effect, whereas icatibant treatment attenuated it. Both alterations were statistically significant compared with the control in a 2-way analysis of variance (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of systemic infusion of 2 µg · kg1 · min1 NPY with concomitant infusion of inhibitory substances on Ca2+ excretion in anesthetized rats (study 2). In study 2, 4 experimental groups (control, top left; losartan, bottom left; icatibant, top right; ramiprilat, bottom right) were investigated. Data are means ± SE of alterations relative to baseline values shown in Table 2. NPY was infused from 0 to 120 min. Losartan treatment did not alter NPY-induced calciuresis. Ramiprilat treatment enhanced the NPY effect, whereas icatibant treatment abolished it. Both alterations were statistically significant compared with the control in a 2-way analysis of variance (P < 0.05).

	DISCUSSION

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Previous work has demonstrated that systemic infusion of NPY concomitantly enhances mean arterial pressure and causes diuresis, natriuresis, and calciuresis (5). The simplest explanation of these data would be an activation of the pressure-natriuresis mechanism (11, 24). However, several lines of evidence demonstrate that this mechanism only plays a small role in NPY-induced diuresis and natriuresis (2-4, 6). First, NPY-induced diuresis and natriuresis are largely maintained when elevations of renal perfusion pressure are prevented mechanically (using an adjustable clamp on the abdominal aorta or renal decapsulation) or pharmacologically (stabilizing blood pressure by concomitant sodium nitroprusside infusion). Second, NPY-receptor antagonists, which block blood pressure elevations, do not affect diuresis and natriuresis. Third, analogs of NPY that do not increase blood pressure [e.g., PYY-(3-36)] can cause diuresis and natriuresis. These data exclude a major role of the pressure-natriuresis mechanism in NPY-induced diuresis and natriuresis.

The tubular effects of systemic NPY infusion are not mimicked by direct intrarenal NPY administration, and the receptor subtype that mediates them is not detectable in the kidney (5). Therefore, NPY appears to modify tubular function by stimulating a receptor located in an extrarenal site and using a neuronal and/or humoral mediator. Therefore, the present study was designed to identify any such mediators.

Acute denervation experiments were performed to test whether this mediator may be released from the renal nerves. The renal denervation was produced by ethanol-phenol treatment of the dissected renal arteries (1, 19, 20). Its effectiveness in our study was demonstrated by a significantly enhanced RBF and lowered RVR, possibly a consequence of the withdrawal of -adrenoceptor-mediated renal vasoconstriction (10). Acute denervation had no major effects on NPY-induced reductions of RBF or enhancements of RVR, diuresis, natriuresis, or calciuresis. Thus the mediator of tubular NPY effects does not appear to be released from the renal nerves. Therefore, our further experiments were designed to identify a possible humoral mediator of tubular NPY effects.

Three possible humoral mediator systems were investigated: the renin-angiotensin, kallikrein-kinin, and vasopressin systems. Because we have not detected significant effects of NPY on plasma vasopressin levels, our data do not support a role for vasopressin in tubular NPY effects. This is consistent with the observation that a lowering of vasopressin tone to the kidney primarily causes diuresis without major alterations to sodium excretion, whereas NPY concomitantly causes diuresis, natriuresis, and calciuresis [i.e., it does not alter free water clearance (5)].

A role for the renin-angiotensin system in tubular NPY effects was investigated by use of the angiotensin II-receptor antagonist losartan and the converting-enzyme inhibitor ramiprilat. Whereas losartan is selective for the AT₁ subtype of angiotensin II receptors (28), ramiprilat suppresses the formation of angiotensin II and should therefore prevent the effects of angiotensin II regardless of the receptor subtype by which they occur. The effectiveness of our losartan and ramiprilat treatment regimens throughout the experimental period was demonstrated by marked attenuation of the effects of bolus injections of angiotensin II and angiotensin I, respectively. Because both losartan and ramiprilat abolished the decline of renovascular NPY effects with time, angiotensin II, acting on an AT₁ receptor, might contribute to this decline. These data indicate that compensatory mechanisms, rather than NPY receptor desensitization, may be prominent in the reduction of renovascular NPY effects over time. Because losartan or ramiprilat did not inhibit the NPY-induced enhancements of water and electrolyte excretion, a withdrawal of angiotensin II tone to the kidney does not appear to mediate the tubular NPY effects.

Ramiprilat actually magnified the diuresis, natriuresis, and calciuresis caused by NPY. Because this cannot be attributed to the withdrawal of angiotensin, it is likely to occur by the other known effect of converting-enzyme inhibition, reduced bradykinin degradation. This is supported by our data with the bradykinin B₂-receptor antagonist icatibant, which inhibited NPY-induced diuresis and natriuresis and abolished enhancements of calcium excretion. Therefore, the combined ramiprilat and icatibant data suggest that bradykinin may be involved in the tubular NPY effects. Its role in the control of calciuresis may be even more important than its role in the control of diuresis and natriuresis.

Several other findings also support the idea that bradykinin may mediate tubular NPY effects. Bradykinin causes diuresis and natriuresis on intrarenal infusion in conscious and anesthetized dogs (7, 13, 16, 26). This is also seen on systemic infusion in conscious rats, which had been rendered hypertensive by deoxycorticosterone treatment (21). The diuretic and natriuretic effects of bradykinin mainly occur in distal nephron segments (9, 25). NPY also appears to cause diuresis mainly by altering the function of distal nephron segments (5). Finally, bradykinin stimulates renal prostaglandin formation (14, 17), and its diuretic and natriuretic effects can be inhibited by cyclooxygenase inhibitors (7, 8). Similarly, the cyclooxygenase inhibitor indomethacin also inhibits the diuretic and natriuretic NPY effects (4). Taken together these data demonstrate that bradykinin may be an important mediator of tubular NPY effects. In this model it is theoretically possible that stimulation of the extrarenal Y₅ NPY receptors causes bradykinin release, which then reaches the kidney via the bloodstream to act intrarenally on prostaglandin formation. The very short plasma half-life of bradykinin (23), however, casts doubt on this mode of action. Alternatively, it is possible that the extrarenal Y₅ receptor activates yet another mediator that causes intrarenal bradykinin formation and/or release. Discrimination of these possibilities will require additional studies.

In summary, our data demonstrate that renal nerves, vasopressin, and the renin-angiotensin system do not play an important role in tubular NPY effects. In contrast, enhancements and inhibition of NPY-induced diuresis, natriuresis, and calciuresis by ramiprilat and the bradykinin-receptor antagonist icatibant, respectively, demonstrate that bradykinin may be involved in the tubular NPY effects.