Introduction

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Complete or partial ureteral obstruction can be caused by urinary calculi and can lead to edema, inflammation, and infection of the upper urinary tract, as well as to changes in ureteral functions (Biancani et al., 1976; Crowley et al., 1990). The resulting increase in intraluminal pressure in the upper urinary tract is the major cause of ureteral colic (Michaelson, 1974; Moriel et al., 1990). Previous clinical studies have shown that antispasmodics (e.g., anticholinergic agents, calcium antagonists, or papaverine) are effective for the relief of ureteral colic, and possibly for the promotion of stone passage (Ross et al., 1967; Jönsson et al., 1987; Borghi et al., 1994). However, a relaxant with more relative ureteral smooth muscle specificity compared with the cardiovascular system would result in potential clinical benefits.

The autonomic nervous system is known to play an important role in modulation of ureteral motility (Schulman, 1974; Morita et al., 1987), but the functions of the parasympathetic supply to the ureter have not been well defined. Muscarinic receptors and cholinergic nerves can be demonstrated in mammalian ureters, with the innervation being rich in the intravesical ureter but scarce in the proximal ureter (del Tacca, 1978; Hernández et al., 1993), and the excitatory effects of cholinergic agonists are believed to result from both a direct muscarinic stimulation (Hernández et al., 1993) and an indirect release of catecholamines (Rose and Gillenwater, 1974). In the case of the sympathetic nervous system, adrenoceptors (ARs) and adrenergic nerves have been demonstrated in mammalian ureters (Latifpour et al., 1990; Edyvane et al., 1994), and in vitro and in vivo studies have both shown that -AR agonists stimulate, whereas -AR agonists inhibit, ureteral motility (Deane, 1967; Malin et al., 1970; Weiss et al., 1978; Morita et al., 1987). In the rabbit ureter, isoproterenol increases adenylate cyclase activity and the cAMP level, and these are reduced by propranolol (Weiss et al., 1977).

Recently, Tomiyama et al. (1998) indicated that there are significant species differences in the -AR subtypes mediating the relaxation of ureteral smooth muscle: ₁-ARs in the rat, ₂-ARs in the rabbit, and ₃-ARs in the dog. In the case of the isolated dog ureter, (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243, a ₃-AR agonist) and isoproterenol were both effective at relaxing the KCl-induced contraction than was either dobutamine (a ₁-AR agonist) or procaterol (a ₂-AR agonist). Park et al. (1997) showed the coexistence of ₃-ARs with ₂-ARs in the human ureter; CGP-12177 (a ₃-AR agonist), procaterol, and isoproterenol were more effective at relaxing either the spontaneous rhythmic contraction or the KCl-induced contraction than was dobutamine in the isolated human ureter. In addition, like in the human ureter, ₃-ARs are present in the human detrusor, and the relaxation induced by adrenergic stimulation is mediated mainly by ₃-ARs (Igawa et al., 1999).

In the present study, we attempted to evaluate the effects of a selective ₃-AR agonist on obstructive dysfunctions in the upper urinary tract using an acute ureteral obstruction model in the anesthetized dog. To this end, we compared the effects of isoproterenol (a nonselective -AR agonist), CL 316243 (a ₃-AR agonist; Largis et al., 1994), and butylscopolamine (an anticholinergic agent) on the elevated intraluminal ureteral pressure (UP) and impeded urine flow in the obstructed ureter; we also compared the hemodynamic effects of these drugs. In addition, we observed obstructive changes in the kidney after the administration of isoproterenol, CL 316243, or butylscopolamine.

	Materials and Methods

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Animals. The animal experiments in this study were conducted in accordance with guidelines issued by the Kissei Pharmaceutical Co. Ltd. Animal Care and Use Committee. Adult beagle dogs (Nihon Nosan Kogyo, Yokohama, Japan) were housed individually with free access to tap water and commercial food pellets (CD-5; Nihon Clea, Osaka, Japan). The room temperature and humidity were ~ 23°C and ~55%, respectively, and a 12-h light/dark cycle was maintained.

Acute Ureteral Obstruction in Anesthetized Dogs. Dogs of either sex, weighing 8.0 to 14.0 kg, were anesthetized with sodium pentobarbital (30 mg/kg i.v.) and immediately intubated. Artificial ventilation with room air was maintained using a volume-limited ventilator (SN-480-3; Shinano Seisakusyo, Tokyo, Japan: 20 ml/kg, 15 strokes/min). The dog was placed on a heating pad to minimize heat loss. Figure 1 shows a schematic representation of the experimental model. After an abdominal midline incision, the left ureter was identified. The left kidney was minimally dissected free from adjacent tissues, and a small nephrotomy was made on the convex border. A catheter was inserted using a indwelling needle (23 gauge × 200 mm; Hakko, Tokyo, Japan) into the upper ureter via the renal pelvis, with the tip being placed ~5 cm above the ureterovesical junction. The other end of the catheter was connected to a pressure transducer (P23XL; Gould, Valley View, OH) for the measurement of UP. The distal portion of the left ureter was isolated at a site within 1 cm of the ureterovesicular junction, and a small ureterotomy was made. A balloon catheter (Fogarty 2F balloon embolectomy catheter; Baxter, Irvine, CA) filled with water was advanced up into the lower ureter, so the distance between the balloon and the tip of the catheter used for UP recording was ~2 cm. Another catheter (PE 50; Becton Dickinson, Parsippany, NJ) was inserted in parallel with the balloon catheter, with the tip being placed just proximal to the bladder side of the balloon for the measurement of the urine flow before ureteral obstruction (UF). The ureter was ligated with a thread distally to prevent passage of urine from the obstructed ureter into the bladder, and another thread was tightened around the ureter with two catheters. We ensured there was no leakage from the ureterotomized site by infusing a small amount of physiological saline into the ureter via the catheter used for UP recording. After ureteral obstruction, we noted the magnitude of the urine flow leaking around the balloon and draining to below the obstructed site [urine outflow (UFo)]. The right femoral artery was cannulated, and arterial blood pressure (BP) was recorded via a pressure transducer (P23XL; Gould). Heart rate (HR) was obtained from the arterial pulse wave by means of a cardiotachometer (AT-601G; Nihon Kohden, Tokyo, Japan). UP, BP, and HR were all recorded on a thermowriting rectigraph (WS-681G; Nihon Kohden). Throughout the experiment, physiological saline (10 ml/kg/h) was infused intravenously to ensure a stable urine flow. Anesthesia was maintained with an infusion of sodium pentobarbital (3-5 mg/kg/h i.v.).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the acute ureteral obstruction model in the anesthetized dog.

Experimental Protocol. After a rest period (~120 min) to allow stabilization of UP, UF, and hemodynamic parameters, the balloon was gradually inflated with water using an infusion pump (KN-201; Natsume Seisakusyo, Tokyo, Japan). Complete ureteral obstruction was considered to have been achieved when UF stopped. The volume of water infused into the balloon was stable in each dog (~20 µl). When UP had risen and barely changed for more than 10 min, it was considered to be the plateau UP, and vehicle or one dose of one drug was injected i.v. The dogs were divided into seven groups depending on whether they received physiological saline (vehicle, n = 6); isoproterenol at 1 µg/kg (n = 4) or 10 µg/kg (n = 5); CL 316243 at 0.1 µg/kg (n = 4), 0.3 µg/kg (n = 4), or 1 µg/kg (n = 4); or butylscopolamine at 1000 µg/kg (n = 4). UP, BP, and HR were recorded for 120 min after drug administration because elevated UP sometimes decreased gradually after that time without development of UFo in vehicle-treated dogs (in a preliminary experiment). UF was measured for the 20 min immediately before ureteral obstruction, and UFo was measured for one 20-min period just before and six consecutive 20-min periods after drug administration.

Histological Determination. Dogs were sacrificed with the administration of an excess dose of sodium pentobarbital 120 min after drug administration. The left and right kidneys were immediately excised and weighed and then immersed in buffered 10% formalin. After fixation, the kidneys were embedded in paraffin wax and cut into sections 3 to 4 µm thick. The sections were stained with H&E. Histopathological findings were classified into four grades: no remarkable change (0), slight change (1), moderate change (2), and severe change (3). The grading of histopathological findings was performed by an observer who was blind to the treatment group from which the tissue was obtained. Both the rate and the degree of tubules with dilated lumen and flatted epithelium existing in a section were completely judged.

Drugs. Isoproterenol [()-isoproterenol bitartrate] and butylscopolamine [()-scopolamine N-butyl bromide] were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium pentobarbital was obtained from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). Sodium heparin was obtained from Marion Merrell Dow Co. Ltd. (Tokyo, Japan). CL 316243 was synthesized by Kissei Pharmaceutical Co. Ltd. (Hotaka, Japan). All drugs were dissolved in physiological saline.

Statistical Analysis. The data are expressed as mean ± S.E. A one-way ANOVA was used for the statistical analysis of multiple comparisons within each group. When a significant difference was detected by the one-way ANOVA, the data were further analyzed with Dunnett's test. A Student's t test for unpaired data was used when comparisons were made between two groups. The difference between UFo values was analyzed with Wilcoxon's sign rank test because the data were not normally distributed. A value of P < .05 was considered to indicate statistical significance.

	Results

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Effects of Isoproterenol, CL 316243, and Butylscopolamine on UP, BP, and HR. Before ureteral obstruction, rhythmic UP waves associated with each ureteral peristalsis were observed in anesthetized dogs. In terms of the baseline UP, mean blood pressure (MBP), and HR, there were no significant differences among the vehicle-, isoproterenol (1 and 10 µg/kg i.v.)-, CL 316243 (0.1, 0.3, and 1 µg/kg i.v.)-, and butylscopolamine (1000 µg/kg i.v.)-treated groups (Table 1). Within 5 min of complete ureteral obstruction, both the peristaltic rate and the peak UP increased transiently (Fig. 2). The baseline UP then reached a plateau, and the ureteral peristalsis disappeared. The plateau UP was 52.5 ± 6.9 mm Hg (n = 6) and the time required to reach the plateau after ureteral obstruction was 86.2 ± 4.8 min (n = 6) in vehicle-treated dogs. Neither the UP, MBP, and HR values recorded before drug administration nor the time between ureteral obstruction and drug administration was significantly different among the seven treatment groups (Table 1). In the vehicle-treated group, UP, like MBP and HR, barely changed for 120 min after its administration (see Figs. 4-6).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A typical tracing of UP after complete ureteral obstruction produced by inflation of the balloon catheter in an anesthetized dog.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Typical tracings of the effects of isoproterenol (10 µg/kg i.v.; A), CL 316243 (1 µg/kg i.v.; B), and butylscopolamine (1000 µg/kg i.v.; C) on UP, BP, and HR in anesthetized dogs. Arrow, point of the injection of each drug.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of isoproterenol on UP, BP, and HR in anesthetized dogs. , vehicle i.v. (n = 6). , isoproterenol, 1 µg/kg i.v. (n = 4). , isoproterenol, 10 µg/kg i.v. (n = 5). Vehicle or isoproterenol was injected at time 0. *P < .05, significant difference from vehicle-treated controls.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of CL 316243 on UP, BP, and HR in anesthetized dogs. , vehicle i.v. (n = 6). , CL 316243, 0.1 µg/kg i.v. (n = 4). , CL 316243, 0.3 µg/kg i.v. (n = 4). , CL 316243, 1 µg/kg i.v. (n = 4). Vehicle or CL 316243 was injected at time 0. *P < .05, significant difference from vehicle-treated controls.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of butylscopolamine on UP, BP, and HR in anesthetized dogs. , vehicle i.v. (n = 6). , butylscopolamine, 1000 µg/kg i.v. (n = 4). Vehicle or butylscopolamine was injected at time 0. *P < .05, significant difference from vehicle-treated controls.

Effects of Isoproterenol, CL 316243, and Butylscopolamine on UFo. Figure 7 shows the effects of isoproterenol, CL 316243, and butylscopolamine on UFo from the obstructed ureter in anesthetized dogs. Before ureteral obstruction, UF values were not significantly different among the vehicle-, isoproterenol (1 and 10 µg/kg i.v.)-, CL 316243 (0.1, 0.3, and 1 µg/kg i.v.)-, and butylscopolamine (1000 µg/kg i.v.)-treated groups. After ureteral obstruction, UFo was not observed for 120 min in vehicle-treated dogs (with the exception of a small UFo from 80 to 120 min in one of the six animals). We also measured the urine flow from the right (nonobstructed) ureter in the same dogs; it did not change significantly for 120 min (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Effects of isoproterenol, CL 316243, and butylscopolamine on UFo in anesthetized dogs. UFo was measured every 20 min after drug administration. A, vehicle, i.v. (n = 6). B, isoproterenol, 1 µg/kg i.v. (n = 4). C, isoproterenol, 10 µg/kg i.v. (n = 5). D, CL 316243, 0.1 µg/kg i.v. (n = 4). E, CL 316243, 0.3 µg/kg i.v. (n = 4). F, CL 316243, 1 µg/kg i.v. (n = 4). G, butylscopolamine, 1000 µg/kg i.v. (n = 4). Vehicle, isoproterenol, CL 316243, or butylscopolamine was injected at time 0. *P < .05, significant difference from vehicle-treated controls. First column in each panel shows UF for the 20 min before ureteral obstruction (a); UFo is expressed as a percentage of UF in each dog.

Effects of Isoproterenol, CL 316243, and Butylscopolamine on Kidney. Table 2 shows the left (obstructed) kidney weight (LKW), right (nonobstructed) kidney weight (RKW), and the LKW/RKW ratio 120 min after the administration of vehicle, isoproterenol (1 and 10 µg/kg i.v.), CL 316243 (0.1, 0.3, and 1 µg/kg i.v.), and butylscopolamine (1000 µg/kg i.v.). In addition, we weighed both kidneys in normal (without ureteral obstruction or drug administration) dogs. LKW/RKW was significantly greater in vehicle-treated dogs than in normal dogs. CL 316243 at doses of 0.3 and 1 µg/kg significantly and dose dependently suppressed the increase in LKW/RKW to an extent close to that in normal dogs and also tended to decrease LKW, indicating that CL 316243 attenuated the increase in LKW seen after ureteral obstruction.

View larger version (140K): [in this window] [in a new window]   Fig. 8.   Typical light microscope images of renal tubules. A, right (nonobstructed) kidney from a vehicle-treated dog; histopathological grade 0. B, left (obstructed) kidney from a vehicle-treated dog; histopathological grade 3. C, left kidney from a 10 µg/kg isoproterenol-treated dog; histopathological grade 1. D, left kidney from a 1 µg/kg CL 316243-treated dog; histopathological grade 1. H&E stain: original magnification 50×.

	Discussion

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Acute Ureteral Obstruction in Dogs. Acute ureteral obstruction increases the intraluminal pressure in the upper urinary tract, and the elevated UP is responsible for inducing changes in both ureteral functions (Biancani et al., 1976; Crowley et al., 1990) and renal hemodynamics (Moody et al., 1975). In the present study, we achieved acute ureteral obstruction in dogs through the inflation of an intraluminal balloon catheter to simulate the effects of an incarcerated calculus on ureteral functions; the technique we used involving minor modifications of previously reported methods (Stower et al., 1986; Crowley et al., 1990). UP rose gradually and reached a plateau at 52.5 mm Hg within 86.2 min of ureteral obstruction and then showed no decline for an additional 120 min in vehicle-treated dogs (Table 1, Figs. 4-6). This time course is consistent with previous observations in dogs with a complete ureteral obstruction (Darracott Vaughan et al., 1971; Moody et al., 1975). Thus, our model mimicked quite closely the clinical situation in which obstructive dysfunctions result from the presence of incarcerated calculi, including elevated intraluminal pressure and stagnated urine in the upper urinary tract. In addition to our measurements of UP, we observed a resumption in urine flow after its interruption by the balloon (UFo), obstructive changes in the kidney, and hemodynamic parameters. By using this model of acute ureteral obstruction in dogs, we could study the effects of -adrenergic stimulation on ureteral, renal, and cardiovascular functions.

Effects on UP. Previous in vitro and in vivo studies have demonstrated that -adrenergic stimulation inhibits ureteral motility. Isoproterenol produces relaxation of isolated ureters in a number of species (Malin et al., 1970; Weiss et al., 1978; Morita et al., 1987; Tomiyama et al., 1998), whereas it decreases both ureteral peristalsis and UP in anesthetized dogs (Rose and Gillenwater, 1974; Mayo and Halbert, 1981; Morita et al., 1987). In the present study, as in previous in vivo studies, isoproterenol (1 and 10 µg/kg i.v.) decreased the elevated UP seen after acute ureteral obstruction (Fig. 4), indicating that relaxation of ureteral smooth muscle by -adrenergic stimulation actually does reduce the intraluminal pressure in the upper urinary tract. It has recently become clear that there are significant species differences in the functional -AR subtypes mediating relaxation of the ureter: ₁-ARs in the rat, ₂-ARs in the rabbit, ₃-ARs in the dog, and ₂-/₃-ARs in the human (Park et al., 1997; Tomiyama et al., 1998). In the present in vivo study, the specific contribution of ₃-ARs to the relaxation of the dog ureter was confirmed; CL 316243 (0.3 and 1 µg/kg i.v.) produced an evident decrease in UP with relative smaller hemodynamic effects (mediated mainly by ₁- and ₂-ARs) than with isoproterenol (Fig. 5). It is therefore suggested that the selective adrenergic stimulation of ureteral -ARs may prove to be useful for relieving the ureteral colic that follows ureteral obstruction. Furthermore, it is also possible that the relaxation of ureteral smooth muscle by -adrenergic stimulation would affect the mechanical factors relating to the movement of intraluminal calculi down the ureter. Indeed, a decrease in friction between the calculus and the ureteral mucosa as a result of relaxation at the obstructed site (decrease in ureteral wall tension) is considered to be one of the factors promoting stone passage (Holmlund, 1968; Weiss, 1997).

Effects on UFo. Based on the observation that both isoproterenol (10 µg/kg i.v.) and CL 316243 (0.3 and 1 µg/kg i.v.) significantly increased UFo from the obstructed ureter (Fig. 7), it seems that stagnated urine above the obstructed site could leak around the balloon and drain to below the obstructed site due to a drug-induced relaxation of ureteral smooth muscle. In this model of acute ureteral obstruction, a significant increase in UFo was seen 40 min after drug administration (i.e., some time after the maximum reduction in UP induced by isoproterenol or CL 316243). Similarly, in 1 µg/kg CL 316243-treated dogs, in which UP was decreased by 77.2% at 20 min with no recovery for an additional 100 min, UFo was rather less than that in 0.3 µg/kg CL 316243-treated dogs, in which UP was decreased by 54.3% at 20 min followed by a gradual recovery. Although it is difficult to explain the lack of a strict correlation between the reduction in UP and the level of UFo with the data obtained in the present study, it is likely that the leakage of urine around the balloon depends both on the hydrostatic pressure above the obstructed site and the relaxation at the obstructed site itself. Furthermore, other effects of -adrenergic stimulation could possibly modify UFo, such as a decrease in ureteral peristalsis or an inhibition of urine bolus formation (Mayo and Halbert, 1981; Morita et al., 1987). On the basis of these results, it is speculated that relaxation of ureteral muscle by -adrenergic stimulation may promote urine flow around incarcerated calculi; by decreasing ureteral wall tension, there may be decreased force of coaptation between the point of obstruction and the ureteral wall, which may actually decrease the pressure gradient across the obstructed site. Such a resumption in urine flow would contribute to a sustained reduction in UP and could possibly promote stone passage, although further experimental studies, including the development of new ureteral obstruction models using an artificial calculus, are needed to clarify the effects of -adrenergic stimulation on urine flow and stone passage. In addition, when taken together with the fact that UFo developed variably in each preparation after drug administration, we must consider the existence of uncontrolled/uncontrollable renal and prerenal factors affecting UFo and UP (e.g., an extravasation of urine at calices, a hydration status, or an osmotic diuresis) in this model of acute ureteral obstruction in dogs.

Effects on Kidney. The anatomic changes occurring in the upper urinary tract with obstruction have been investigated experimentally in rabbits; hydronephrosis is known to develop as early as 1 day after ureteral obstruction in association with an increase in kidney weight, with the tubules initially undergoing dilatation of the lumen with flattening of the epithelium (Sheehan and Davis, 1959). In the present study, we were surprised to find that we could observe weight gain and dilatation of tubules in the obstructed kidney only ~210 min after ureteral obstruction in our acute preparation in dogs. CL 316243 (0.3 and 1 µg/kg i.v.) significantly suppressed the increase in LKW/RKW (Table 2), and the histopathological findings in the obstructed kidney of either isoproterenol (10 µg/kg i.v.)- or CL 316243 (1 µg/kg i.v.)-treated dogs were, if anything, less severe than those of vehicle-treated dogs (Table 3, Fig. 8). It is likely that these effects were due both to a sustained decrease in intraluminal pressure and the resumption in urine flow in the upper urinary tract after the administration of isoproterenol or CL 316243. On the basis of these results, it is possible that a secondary effect of -adrenergic stimulation would be an attenuation of the early development of renal edema that follows ureteral obstruction, although further chronic studies are required to substantiate this idea.

Hemodynamic Effects. Previous observations in conscious dogs have shown that the positive chronotropic effect of ₃-AR agonists is attributable to a baroreceptor-mediated reflex triggered by the fall in BP resulting from their direct vasodilator action (Tavernier et al., 1992; Shen et al., 1994). In the present study, isoproterenol at a dose of 10 µg/kg, which decreased UP by 74.1%, maximally reduced MBP by 51.7% and increased HR by 57.9% (2 min after its administration; Fig. 4), whereas CL 316243 at a dose of 1 µg/kg, which decreased UP by a similar extent (75.8%), maximally reduced MBP by only 12.7% and increased HR by 28.2% (30 min after its administration; Fig. 5). These results demonstrate that CL 316243 gradually decreases UP with a relatively smaller hypotensive effect than isoproterenol. CL 316243 shows a >10,000-fold selectivity for ₃-ARs compared with both ₁- and ₂-ARs (Largis et al., 1994) and a slow relaxing kinetic in the isolated rat colon (Kaumann and Molenaar, 1996). It is therefore suggested that in dogs, the CL 316243-induced reduction in UP may be mainly mediated via adrenergic stimulation of ureteral ₃-ARs and bears little dependence on its effect on BP.

Conclusion. We have demonstrated that the relaxation of ureteral smooth muscle by CL 316243, a selective ₃-AR agonist, decreases the elevated UP and resumes urine flow in the acutely obstructed ureter in dogs. These findings are consistent with the idea that selective adrenergic stimulation of ureteral -ARs may prove to be useful for relieving ureteral colic and promoting stone passage in patients with urolithiasis. In the human ureter, the relaxation induced by adrenergic stimulation is mediated by both ₂- and ₃-ARs (Park et al., 1997). Because -ARs are widely distributed in the cardiovascular system, we believe that a specific agonist for human ureteral -ARs with comparatively weak hemodynamic effects would be extremely useful for the drug treatment of urolithiasis; additional acute and chronic studies are needed to confirm this concept.