INTRODUCTION

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URETERAL OBSTRUCTION INCREASES renal pelvic pressure and activates mechanosensitive neurons in the renal pelvic wall, resulting in an increase in ipsilateral afferent renal nerve activity (ARNA). The increase in ARNA causes a fall in contralateral efferent renal nerve activity and a contralateral natriuresis, known as the renorenal reflex (27). A similar renorenal reflex response is elicited by administration of bradykinin into the renal pelvis (24).

PGE₂ and substance P are important mediators of the neural signal elicited by increased renal pelvic pressure and bradykinin. Increasing renal pelvic pressure or renal pelvic administration of bradykinin increases renal pelvic release of PGE₂ and substance P (24, 25). Blocking renal PG synthesis abolishes the increases in ARNA and renal pelvic release of substance P. In vitro studies using an isolated renal pelvic wall preparation showed that PGE₂ increases the release of substance P by a calcium-dependent mechanism that requires activation of N-type calcium channels (22). Further studies in vivo showed that renal pelvic administration of a substance P-receptor antagonist blocked the increases in ARNA produced by either increased renal pelvic pressure or bradykinin (24, 29). Taken together, our findings suggest that activation of renal sensory neurons by either increased renal pelvic pressure or bradykinin increases the release of PGE₂, which in turn increases the release of substance P. The released substance P activates substance P receptors with a resultant increase in ARNA.

The similar mechanisms involved in the activation of renal sensory neurons by increased renal pelvic pressure and bradykinin suggest that bradykinin contributes to the activation of renal sensory neurons produced by increased renal pelvic pressure. Therefore, we examined whether the increase in ARNA produced by increased renal pelvic pressure could be reduced by a blocker of the bradykinin B₂ receptors.

The present study showed that the ARNA response to increased renal pelvic pressure was abolished by a B₂-receptor antagonist, suggesting a common pathway in the activation of renal sensory neurons by increased renal pelvic pressure and bradykinin. Bradykinin is a known activator of the phosphoinositide system (5). Activation of phosphoinositidase C leads to increased intracellular calcium and activation of protein kinase C (PKC) (10, 38). PKC activates phospholipase A₂, which leads to a release of arachidonic acid with subsequent formation of eicosanoids. PKC activation has been implicated in depolarization of afferent neurons in various tissues, including vagal afferent neurons (39), ischemically sensitive abdominal afferent neurons (18), and dorsal root ganglionic neurons (6). A role for PKC in depolarization of renal sensory neurons was demonstrated by our previous studies. Renal pelvic perfusion with the phorbol ester 4-phorbol 12,13-dibutyrate (PDBu), known to activate PKC (10, 21), increased ARNA (28). Furthermore, the ARNA response to bradykinin was blocked by a selective PKC inhibitor (28). Therefore, we tested the hypothesis that stimulation of renal sensory neurons by increased renal pelvic pressure involved activation of PKC.

Although the signal transduction system involved in the renal pelvic release of PGE₂ and substance P produced by increased renal pelvic pressure is unknown, several lines of evidence would implicate the phosphoinositide system. The results of our current studies would suggest that increasing renal pelvic pressure activates PKC. Our previous studies showing a lack of an increase in ARNA in response to PDBu in rats fed an essential fatty acid-deficient diet (26) would suggest an important role for arachidonic acid metabolites in the PKC-mediated activation of renal sensory neurons. The effects of PKC on neuronal cell membrane depolarization are suggested to be due to its modulatory effects on ion channel conductivity and transmitter release (11). In vitro studies in dorsal root ganglionic neurons and spinal cord have shown that PKC activation leads to increases in the release of substance P (4, 15). To test our hypothesis that activation of renal mechanosensory neurons involves PKC-mediated release of PGE₂ and substance P, we examined whether the increase in ARNA produced by PDBu was associated with renal pelvic release of PGE₂ and substance P and could be blocked by a cyclooxygenase inhibitor and a substance P-receptor antagonist.

	METHODS

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The study was performed on male Sprague-Dawley rats weighing 242-443 g (mean 327 ± 4 g). Anesthesia was induced with pentobarbital sodium, 0.2 mmol/kg ip, and maintained with an intravenous infusion of pentobarbital sodium, 0.04 mmol · kg¹ · h¹, in isotonic saline at 50 µl/min. Catheters were placed in the femoral artery for continuous arterial pressure recordings and in the femoral vein for pentobarbital sodium infusion. Heart rate was recorded with a linear cardiotachometer triggered by the arterial pressure wave form.

All recordings were made on a Grass 7D polygraph that was connected to an IBM PS/2 via a Data Translation analog-digital board (model DT2801) for online data acquisition.

A left flank incision was performed, and a PE-10 catheter was inserted into the right ureter for collection of urine.

Renal pelvic administration of vehicle and experimental agents. A PE-60 catheter was placed in the left renal pelvis. To administer various agents into the left renal pelvis, a PE-10 catheter was inserted into the PE-60 catheter and advanced into the renal pelvis so that its tip extended 1-2 mm beyond the tip of the PE-60 catheter (23-30). The renal pelvis was perfused at 20 µl/min. The renal pelvic effluent was drained via the PE-60 catheter, except in the groups of rats in which PGE₂ and substance P were measured in the renal pelvic effluent.

Increased renal pelvic pressure. Renal pelvic pressure was increased by raising the PE-60 catheter, inserted into the left ureter, above the level of the kidney (23, 25-30). The PE-60 catheter was filled with vehicle or various experimental agents as outlined below.

Collection of renal pelvic effluent for PGE₂ and substance P determination. A PE-50 catheter was inserted through the renal parenchyma into the renal pelvis to collect renal pelvic effluent. The open end of the PE-60 ureteral catheter was clamped to allow drainage of all effluent via the PE-50 catheter inserted through the renal parenchyma (24, 25).

Recording of ARNA. One renal nerve branch was isolated at the angle between the aorta and the left renal artery and placed on a bipolar silver wire electrode for recordings of multifiber renal nerve activity. The signals were led by a high-impedance probe (Grass HIP511) to a band-pass amplifier (Grass P511) with a high-frequency cutoff at 3,000 Hz and a low-frequency cutoff at 30 Hz; they were amplified 20,000 times. The output of the band-pass amplifier was fed to an oscilloscope (Tektronix 5113) and to a resetting voltage integrator (Grass 7P10). Renal nerve activity was integrated over 1-s intervals, the unit of measure being microvolts times seconds per second. Assessment of renal nerve activity was done by its pulse-synchronous rhythmicity. After renal nerve activity was identified and verified, the renal nerve was sectioned and the distal part placed on the electrode for recording ARNA. The electrode was fixed to the renal nerve with Wacker Sil-Gel 604. Postmortem renal nerve activity, which was assessed by crushing the decentralized renal nerve bundle peripheral to the recording electrode, was subtracted from all values of renal nerve activity. ARNA was expressed as a percentage of its baseline value during the control period (23-30).

Measurement of PKC activity. Pilot studies were performed to examine whether 1 µM PDBu activates PKC in the renal pelvic wall of Sprague-Dawley rats. Renal pelvises were dissected from both kidneys of seven rats anesthetized with pentobarbital sodium (Abbott Laboratories), 0.2 mmol/kg ip, and placed in HEPES buffer (in mM: 25 HEPES, 135 NaCl, 3.5 KCl, 2.5 CaCl₂, 1 MgCl₂, 3.3 D-glucose, and 0.1 ascorbic acid, pH 7.45), containing either the phorbol ester PDBu at 1 µM or vehicle (0.1% DMSO) for 10 min (23). Activation of PKC was assessed by measuring its translocation from the cytosol to the plasma membrane, as previously described (13, 23). Briefly, after incubation with PDBu and DMSO, respectively, the renal pelvises were rinsed in ice-cold PBS, then placed into ice-cold homogenizing buffer, and homogenized for 2 × 10 s. The resulting homogenate was centrifuged at 1,000 g to pellet cell debris and nuclei. The supernatant was removed and again centrifuged at 100,000 g to obtain the cytosolic fraction (supernatant). The particulate (membrane) fraction was resuspended in the homogenizing buffer containing 0.1% Triton X-100, and PKC was extracted with repeated vortexing. PKC activity in the membrane and particulate fraction was assayed by measuring ³²P incorporation into the substrate histone IIIs.

Experimental protocols. Approximately 1.5 h elapsed after the end of surgery and the start of the experiment to allow the rat to stabilize as evidenced by 30 min of steady-state urine collections and ARNA recordings.

Effects of a bradykinin B₂-receptor antagonist on the ARNA responses to increased renal pelvic pressure and substance P. Pilot experiments were performed to determine the concentration of the B₂-receptor antagonist HOE 140 (19) that blocked the ARNA responses to renal pelvic perfusion with bradykinin. The experiment consisted of four parts separated by 10-min intervals. Each part consisted of a 10-min control, 5-min experimental, and 10-min recovery period. Bradykinin (20 µM) was administered during each experimental period. Renal pelvis was perfused with vehicle during the first part and HOE 140 at 1.5, 15, and 150 µM during the second, third, and fourth parts, respectively (n = 4). The pelvic perfusates were switched immediately after each recovery period. Because these experiments showed that HOE 140 at 15 and 150 µM abolished the ARNA response to bradykinin, additional experiments were performed (n = 6) in which HOE 140 was administered at 1.5, 4.6, and 15 µM with the same experimental protocol. Previous time-control studies have shown that in the presence of vehicle, four administrations of bradykinin into the renal pelvis at similar intervals as in the present studies result in reproducible increases in ARNA (28).

Because the results of these experiments showed that HOE 140 at 15 µM produced a maximum blockade of the ARNA response to bradykinin, this concentration was used in the subsequent experiments. Two groups of rats were studied. In the first group (n = 7), the experiment consisted of three parts separated by 15-min intervals. Each part consisted of a 10-min control, 5-min experimental, and 10-min recovery period. Renal pelvic pressure was increased during each experimental period. Renal pelvis was perfused with vehicle (0.15 M NaCl) during the first part, HOE 140 (15 µM) during the second part, and vehicle during the third part. The renal pelvic perfusates were switched immediately after each recovery period. Thus renal pelvis was perfused with HOE 140 for 25 min before renal pelvic pressure was increased during the second experimental period. In the second group of rats (n = 7), the experiment consisted of two parts separated by a 20-min interval. Each part consisted of a 10-min control, 5-min experimental, and 10-min control period. Substance P (0.15 nmol) was added to the renal pelvic perfusate during the two experimental periods. Renal pelvis was perfused with vehicle during the first part and HOE 140 (15 µM) during the second part.

Effects of PKC inhibition on the ARNA response to increased renal pelvic pressure. The experiment consisted of two parts separated by a 15-min interval (n = 7). Each part consisted of a 10-min control, 3-min experimental, and 10-min recovery period. Renal pelvic pressure was increased during each experimental period. Renal pelvis was perfused with vehicle (0.1% DMSO) during the first part and the PKC selective inhibitor calphostin C (1 µM) (42), during the second part. The renal pelvic perfusate was switched immediately after the recovery period. Thus the renal pelvis was perfused with calphostin C for 25 min before renal pelvic pressure was increased during the second part of the experiment. In an additional seven rats that served as time controls, a similar protocol was performed with the exception of the renal pelvis being perfused with vehicle during both parts of the experiment.

Effects of PKC activation on ARNA and renal pelvic release of PGE₂ and substance P. The experiment consisted of a 10-min control, 10-min experimental, and 40-min recovery period. Three groups of rats were studied. In the first group (n = 15), the renal pelvis was perfused with 0.2 mM Na₂CO₃ in 0.1% DMSO (vehicle) during the control and recovery periods and PDBu (1 µM) during the experimental period. In the second group (n = 11), the renal pelvis was perfused with indomethacin (140 µM) during the control and recovery periods and PDBu plus indomethacin during the experimental period. In the third group (n = 11), the experimental protocol was similar to that in the first group, except the inactive phorbol ester 4-phorbol 12,13-didecanoate (PDD, 1 µM) was administered into the renal pelvis instead of PDBu. In all groups, the endopetidase inhibitor thiorphan (10 µM) (34) was added to the renal pelvic perfusate to minimize substance P catabolism. Renal pelvic effluent from the left perfused kidney was collected on ice in 5-min periods throughout the experiment and stored at 80°C for later analysis of PGE₂ and substance P.

Effects of a substance P-receptor antagonist on the ARNA response to PKC activation. The experiment consisted of a 10-min control, 10-min experimental, and 40-min recovery period. Two groups of rats were studied. In the first group (n = 7), the renal pelvis was perfused with the substance P-receptor antagonist RP 67580 (0.1 mM) (40) during the control and recovery periods and PDBu (1 µM) plus RP 67580 during the experimental period. In the second group (n = 5), the experimental protocol was similar, except renal pelvis was perfused with RP 68651 (0.1 mM) instead of RP 67580. RP 68651 is the inactive enantiomer of RP 67580.

Drugs. RP 67580 and RP 68651 were supplied by Rhône-Poulenc Rorer Recherche et Developpement (Vitry Sur Seine, France), and HOE 140 was supplied by Hoechst Marion Roussel (Cincinnati, OH). All other agents were from Sigma Chemical (St. Louis, MO). Indomethacin was dissolved together with Na₂CO₃ (2:1 weight ratio) in 0.15 M NaCl. Thiorphan was dissolved in 100% ethanol and further diluted with either 0.2 mM Na₂CO₃ or 140 µM indomethacin, the final ethanol concentration being 0.1%. RP 67580 and RP 68651 were dissolved in 0.0005 N HCl. PDBu and PDD were dissolved in DMSO and further diluted in the various renal perfusates in the different experimental protocols, the final DMSO concentration being 0.1%. Calphostin C was dissolved in DMSO and further diluted in 0.15 M NaCl to a final DMSO concentration of 0.1%. HOE 140, bradykinin, and substance P were dissolved in 0.15 M NaCl.

Analytic procedure. Contralateral right urinary sodium concentrations were determined with a flame photometer. Right urinary sodium excretion was expressed per gram of kidney weight.

Activation of PKC was assessed by measuring its translocation from the cytosol to the plasma membrane as we have described previously in detail (13, 23).

PGE₂ and substance P in the renal pelvic effluent were measured by either RIA or ELISA. Both methods gave similar results, so the data have been pooled. In short, RIA for PGE and substance P was determined as previously established and validated (24, 25, 47). The PGE antibody used (Iowa RAB 66) cross-reacted 100% with PGE₁ and PGE₂ but showed <2% cross-reactivity with other arachidonic acid metabolites. The substance P antibody (RIN 7451, Peninsula, San Carlos, CA) demonstrated 100% cross-reactivity with fragments 2-11, 3-11, 4-11, 5-11, <5% with 6-11, and <1% with fragment 7-11, neuropeptide K, neurokinins B and A, endothelin-1, somatostatin, and vasoactive intestinal peptide. ELISA for PGE₂ was determined using a kit from Cayman Chemical (Ann Arbor, MI). ELISA for substance P was performed as previously described in detail (22). The rabbit substance P antibody (IHC 7451 Peninsula) demonstrated similar cross-reactivity as that used in the RIA.

Statistical analysis. Systemic hemodynamics and renal excretion were measured and averaged over each period. The effects of renal sensory-receptor stimulation were calculated by comparing the experimental value with the average value of the bracketing control and recovery periods. The ARNA responses to increased renal pelvic pressure, bradykinin, and substance P were calculated as the area under the curve of time vs. ARNA (AUC), where ARNA was expressed as a percentage of its baseline value during the 10-min control period preceding each experimental period. Release of PGE₂ and substance P into the renal pelvic effluent was calculated as concentration times volume divided by duration of the collection period. Friedman two-way analysis of variance, shortcut analysis of variance, Mann-Whitney U-test, and Wilcoxon matched-pairs signed-rank test were used (43, 45). A significance level of 5% was chosen. Data in text and figures are expressed as means ± SE.

	RESULTS

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Effects of a bradykinin B₂-receptor antagonist on the ARNA responses to increased renal pelvic pressure and substance P. The similar mechanisms involved in the activation of renal sensory neurons by increased renal pelvic pressure and bradykinin (24, 25) suggested that bradykinin contributes to the activation of renal mechanosensitive neurons. We tested this idea by increasing renal pelvic pressure in the absence and presence of the B₂-receptor antagonist HOE 140. Pilot experiments showed that HOE 140 at 0.15, 15, and 150 µM blocked the ARNA response to bradykinin by 30 ± 13, 93 ± 5, and 100 ± 0%, respectively. Further experiments showed that 15 µM HOE 140 was the concentration required to produce maximal blockade of the ARNA response to bradykinin (Table 1). Before renal pelvic perfusion with 15 µM HOE 140, increasing renal pelvic pressure 15 ± 1 mmHg increased ipsilateral ARNA (Fig. 1) and contralateral urinary sodium excretion from 1.1 ± 0.2 to 1.4 ± 0.3 µmol · min¹ · g¹ (P < 0.02). HOE 140 did not affect basal ARNA but produced a reversible blockade of the ipsilateral ARNA (Fig. 1) and the contralateral natriuretic responses to increased renal pelvic pressure. Mean arterial pressure (114 ± 3 mmHg) and heart rate (289 ± 19 beats/min) were unaffected by renal pelvic perfusion with HOE 140. 

View this table: [in this window] [in a new window]   Table 1.   Effects of renal pelvic perfusion with HOE 140 on the ARNA responses () to renal pelvic administration of bradykinin

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Afferent renal nerve activity (ARNA) responses () to increased renal pelvic pressure in presence of renal pelvic perfusion with vehicle and bradykinin B2-receptor antagonist HOE 140 (15 µM). AUC, area under the curve of ARNA vs. time. ** P < 0.01,  P < 0.02, * P < 0.05.

To exclude the possibility that the blockade of the ARNA response to increased renal pelvic pressure produced by HOE 140 was related to mechanisms involved in the activation of substance P receptors, we tested the effects of HOE 140 on the ARNA response to renal pelvic administration of substance P. As shown in Fig. 2, HOE 140 failed to affect the increase in ARNA produced by substance P. 

View larger version (19K): [in this window] [in a new window]   Fig. 2.   ARNA responses to renal pelvic administration of substance P (0.15 nM) in presence of renal pelvic perfusion with vehicle and bradykinin B2-receptor antagonist HOE 140 (15 µM).  P < 0.02.

Effects of PKC inhibition on the ARNA response to increased renal pelvic pressure. To examine whether the intracellular mechanisms involved in the stimulation of renal mechanosensory neurons entailed activation of PKC, renal pelvic pressure was increased in the absence and presence of the PKC selective inhibitor calphostin C at 1 µM, a concentration that blocks the ARNA response to bradykinin (28). Before administration of calphostin C, increasing renal pelvic pressure 16 ± 0 mmHg increased ipsilateral ARNA (Fig. 3) and contralateral urinary sodium excretion from 0.8 ± 0.3 to 1.1 ± 0.4 µmol · min¹ · g¹ (P < 0.02). Calphostin C did not affect basal ARNA but blocked the increases in ipsilateral ARNA (Fig. 3) and contralateral urinary sodium excretion from 1.1 ± 0.2 to 1.2 ± 0.3 µmol · min¹ · g¹, produced by increased renal pelvic pressure. Mean arterial pressure (122 ± 4 mmHg) and heart rate (335 ± 10 beats/min) remained unaltered throughout the experiment. In the time control experiments, repeated increases in renal pelvic pressure of 15 ± 0 mmHg resulted in reproducible increases in ARNA, 5,300 ± 271 and 4,672 ± 836% · s (AUC, both P < 0.02).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   ARNA responses to increased renal pelvic pressure in presence of renal pelvic perfusion with vehicle and protein kinase C (PKC) inhibitor calphostin C (1 µM). ** P < 0.01.

Effects of PKC activation on ARNA and renal pelvic release of PGE₂ and substance P. The results from the in vitro studies examining the effects of 1 µM PDBu on PKC activity in isolated renal pelvises are shown in Table 2. Compared with vehicle-treated pelvises, treatment with PDBu produced a reciprocal decrease in cytosolic and increase in membrane-bound PKC activity, consistent with translocation and activation of PKC. In PDBu-treated pelvises, the percent of total PKC that was membrane bound was significantly greater than that in vehicle-treated pelvises (P < 0.02).

To examine whether the increased renal pelvic release of PGE₂ and substance P produced by increased renal pelvic pressure or bradykinin was related to activation of PKC, we measured whether activation of renal pelvic PKC increased renal pelvic release of PGE₂ and substance P. Renal pelvic perfusion with PDBu resulted in a gradual increase in ARNA that peaked at the end of the PDBu perfusion (Fig. 4). ARNA returned toward control values 20 min after the end of the perfusion. The increase in ARNA was associated with a renal pelvic release of PGE₂ and substance P, which was maximum during the last 5-min period of the PDBu perfusion or the first 5-min period after the PDBu perfusion. Renal pelvic release of PGE₂ and substance P returned to baseline values 30-40 min after the PDBu perfusion (Fig. 5).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of PKC activation by phorbol ester 4-phorbol 12,13-dibutyrate (PDBu) on ARNA in kidneys pretreated with renal pelvic perfusion with vehicle or indomethacin (140 µM). ** P < 0.01.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Renal pelvic release of PGE2 and substance P (SP) from kidneys pretreated with renal pelvic perfusion with vehicle (A) and indomethacin 140 µM (B). CNT, last 5-min control period before PDBu perfusion; PDBu, maximum value of released PGE2 and SP during last 5-min period of PDBu perfusion and first 5-min after PDBu perfusion; REC, last 5-min recovery period 40 min after PDBu perfusion. ** P < 0.01 vs. average of control and recovery periods.

Renal pelvic perfusion with the inactive phorbol ester PDD failed to increase ARNA and renal pelvic release of substance P (Table 3). There was a small increase in renal pelvic release of PGE₂, the magnitude being significantly less than that produced by PDBu in vehicle-treated rats (P < 0.01).

The importance of PGE₂ in the increases in ARNA and renal pelvic release of substance P was examined by perfusing the renal pelvis with PDBu in the presence of the cyclooxygenase inhibitor indomethacin. Renal pelvic perfusion with indomethacin reduced basal renal pelvic release of PGE₂ and the increase in the release of PGE₂ produced by PDBu (Fig. 5, both P < 0.01). In the presence of indomethacin, PDBu failed to increase ARNA (Fig. 4) and renal pelvic release of substance P (Fig. 5). Mean arterial pressure (109 ± 3 and 116 ± 4 mmHg) and heart rate (329 ± 34 and 338 ± 10 beats/min) were similar in the two groups and were not altered by PDBu.

Effects of a substance P-receptor antagonist on the ARNA response to PKC activation. We tested the idea that the PDBu-mediated release of substance P contributed to the increased ARNA produced by PDBu by examining the effect of the substance P-receptor antagonist RP 67580 (Fig. 6). In the presence of a renal pelvic perfusion with RP 67580, PDBu failed to increase ARNA. In contrast, in the presence of RP 68651, the inactive enantiomer of RP 67580, PDBu produced a similar increase in ARNA, as seen in vehicle-treated pelvises (Fig. 4). Mean arterial pressure (112 ± 3 and 112 ± 4 mmHg) and heart rate (302 ± 14 and 300 ± 14 beats/min) were similar in the two groups.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of PKC activation by PDBu on ARNA in kidneys pretreated with renal pelvic perfusion with substance receptor antagonist RP 67580 (0.1 mM) and its inactive enantiomer RP 68651 (0.1 mM). ** P < 0.01, * P < 0.05.

	DISCUSSION

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The results of these experiments show that the increase in ARNA produced by increased renal pelvic pressure was blocked by a B₂-receptor antagonist and a PKC inhibitor. The phorbol ester PDBu increased PKC activity in the membrane fraction of an isolated renal pelvic wall preparation. The increase in ARNA produced by renal pelvic perfusion with PDBu was associated with increases in renal pelvic release of PGE₂ and substance P. Indomethacin blocked the increases in ARNA and renal pelvic release of PGE₂ and substance P. The PDBu-mediated increase in ARNA was also blocked by a substance P-receptor antagonist. Taken together, these studies suggest that bradykinin facilitates the activation of renal mechanosensitive neurons by activating PKC. Activation of PKC leads to a PGE₂-mediated release of substance P with a resultant increase in ARNA.

Activation of renal mechanosensitive neurons: role of bradykinin. Bradykinin binding sites have been localized to the outer and inner medulla and the renal pelvis (32). There is considerable evidence for bradykinin receptors being located on sensory neurons in the central nervous system (17). Morphological evidence is currently lacking for bradykinin receptors on peripheral sensory nerve endings. However, numerous functional studies have shown that bradykinin activates peripheral sensory neurons (for example, see Refs. 17 and 46) and increases neuropeptide release (17). Increasing renal pelvic pressure and bradykinin elicit similar renorenal reflex responses by activating mechanisms involving renal pelvic release of PGE₂ and substance P. Therefore, we hypothesized that bradykinin contributes to the activation of renal mechanosensitive neurons. The present results confirmed our hypothesis. Perfusing the renal pelvis with HOE 140 abolished the ipsilateral ARNA and contralateral natriuretic responses to increased renal pelvic pressure. Importantly, HOE 140 failed to affect the ARNA response to substance P (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Increasing renal pelvic pressure increases release of bradykinin, which activates PKC via stimulation of renal pelvic bradykinin B2 receptors. PKC activation leads to renal pelvic release of PGE2, which elicits a release of SP via activation of N-type calcium channels. SP activates substance P receptors in renal pelvic area with a resultant increase in ARNA (current study and Refs. 21, 24, 25).

There is extensive evidence for bradykinin activating and/or sensitizing mechanosensitive nerve endings in various tissues (5, 17). Analogous to renal mechanosensitive neurons, serosal mechanosensitive afferent neurons supplying the jejunum are activated by bradykinin-mediated PGE₂ release (33). Whether increased renal pelvic pressure increases the release of bradykinin was not determined in the present study. However, studies in urinary bladder strips show that spontaneous contractions are associated with a release of both bradykinin and substance P (41). Bladder contractions elicited by bradykinin are abolished by inhibiting PKC activity (8) or PG synthesis (36). Kinins are excreted in significant amount in the urine. In the kidney, the highest concentration of kinins is found in the terminal segments of the nephron and in the pelvis (5). A physiological role for kinins in the renal regulation of body water and sodium has been suggested by numerous studies. Urinary kinin and kallikrein excretion have been shown to be positively correlated with urine flow rate (3, 35). This is of particular interest, because increases in urine flow rate increase ARNA, the increase in ARNA being correlated with the increased pelvic pressure produced by the increased flow rate (16).

Activation of renal mechanosensitive neurons: role of PKC. PKC is widely distributed in various tissues and organs. The high concentration of PKC in the nervous system, compared with many other tissues, suggests that PKC plays an important role in the control of neuronal activity (10, 20, 21). In the rat vagus nerve and dorsal root ganglionic neurons, PKC activation results in increases in sodium conductance with subsequent cell membrane depolarization and increased calcium uptake (6, 39). Our previous studies have shown that the bradykinin-mediated increase in ARNA is blocked by various PKC inhibitors or downregulation of PKC by pretreatment with PDBu (28). These findings together with our current findings suggested that bradykinin contributes to the stimulation of renal mechanosensitive neurons by activating the phosphoinositide system. We tested this hypothesis by examining the effect of calphostin C on the ARNA response to increased renal pelvic pressure. Calphostin C abolished the ARNA response as well as the contralateral natriuretic response to increased renal pelvic pressure, suggesting an important role for PKC activation in the stimulation of renal pelvic mechanosensitive neurons (Fig. 7). Of interest in this context are studies in urinary tract smooth muscle cells. Exposing these cells to cyclic stretch to study the increased production of nerve growth factor occurring during urethral obstruction (44) showed an important role for PKC in the stretch-induced increase in nerve growth factor (37). Elevated nerve growth factors have been shown to lower the activation threshold for visceral afferent nerves deriving from the bladder (14). Furthermore, the findings that PDBu increases bladder contractility (51) are of interest because our previous studies have shown that the renal pelvic mechanosensitive neurons are activated by increases in pelvic pressure seen during spontaneous pelvic contractions (30).

PKC-mediated activation of renal pelvic sensory receptors: release of PGE₂. In agreement with our previous study in spontaneously hypertensive and normotensive Wistar-Kyoto rats (23), the phorbol ester PDBu produced a translocation of PKC from the cytosol to the cell membranes of the renal pelvis in Sprague-Dawley rats, indicating an activation of PKC. The increase in PKC activation was of a similar magnitude as in the other two rat strains. Several lines of evidence would implicate the phosphoinositide system as being the link between bradykinin and renal pelvic release of PGE₂ in the activation of renal mechanosensory neurons. Phorbol esters induce a release of various cyclooxygenase products, including PGE₂ (for example, see Refs. 1, 7, and 50). The importance of arachidonic acid metabolites in the PKC-mediated activation of renal sensory neurons was shown by the lack of an ARNA response to PDBu in rats fed an essential fatty acid-deficient diet (26). The results of our current studies extend our previous findings by showing that PDBu resulted in an increase in ARNA that was associated with a renal pelvic release of PGE₂ and blocked by cyclooxygenase inhibition (Fig. 7). Baseline PGE₂ release was lower in the indomethacin-treated rats. However, it is unlikely that the lower baseline release of PGE₂ influenced the effect of PDBu on PGE₂ release, because the magnitude of the PDBu-mediated PGE₂ release was not related to baseline PGE₂ in vehicle-treated rats (r = 0.06). Our data do not exclude the possibility that other cyclooxygenase products contribute to the PKC-mediated activation of renal sensory neurons. However, the concurrent indomethacin-mediated blockade of the increases in PGE₂ release and ARNA produced by increased renal pelvic pressure, bradykinin, and PDBu lends support to the notion that PGE₂ contributes importantly to the activation of renal sensory neurons. Furthermore, PGE₂, being one of the major cyclooxygenase products in the renal medulla, is present and synthesized in the renal pelvic wall (25), which contains the majority of the renal sensory neurons (31).

PKC-mediated activation of renal pelvic sensory receptors: release of substance P. Activation of PKC facilitates the depolarization-induced release of various neurotransmitters and neuropeptides (4, 11, 15). Likewise, renal pelvic perfusion with PDBu elicited a release of substance P from renal pelvic sensory nerves. The mechanisms of PKC-mediated transmitter release are not known. Because PKC is present in presynaptic nerve terminals of various neuronal types and found in synaptic vesicles, it is plausible that PKC phosphorylates synaptic proteins involved in exocytosis (11). Moreover, PKC-mediated transmitter release may also be related to the modulating effect of PKC on various ion channels (6, 11, 39).

Our previous in vitro studies showing that PGE₂ produces a calcium-dependent release of substance P from the renal pelvic sensory nerves (22) suggest that the PKC-mediated release of PGE₂ into the renal pelvic effluent may contribute to the renal pelvic release of substance P. The results of the present study would support this hypothesis. Blocking renal pelvic PGE₂ synthesis with indomethacin abolished the renal pelvic release of substance P elicited by PDBu (Fig. 7).

PKC-mediated activation of renal pelvic sensory receptors: role of substance P. The majority of renal substance P-containing neurons are located in the subepithelial layer of the renal pelvic wall (31). Autoradiographic studies have provided evidence for substance P receptors in the renal pelvic area (9). To examine whether the PDBu-mediated increase in ARNA was related to the renal pelvic release of substance P, PDBu was administered into renal pelvises treated with the substance P-receptor antagonist RP 67580. RP 67580, which has a high affinity for rat substance P receptors (40), was administered at a concentration (0.1 mM) that abolishes the ARNA response to renal pelvic administration of substance P (24). Our results showed that PDBu failed to increase ARNA in the presence of RP 67580. To rule out the possibility that the effect of RP 67580 was related to blockade of calcium channels, we examined the effect of the racemic enantiomer RP 68651 on the PDBu-mediated increase in ARNA. RP 68651 has the same affinity for the calcium channels as RP 67580 but is totally devoid of affinity for the substance P receptors (40). The inactive enantiomer had no effect on the ARNA response to PDBu. Taken together, these data suggest an important role for substance P in the PKC-mediated activation of renal sensory neurons (Fig. 7).

The advantage in using an in situ perfused renal pelvic preparation in dissecting the various mechanisms involved in the activation of renal sensory neurons lies in the possibility of measuring physiological parameters, e.g., renal pelvic release of PGE₂ and substance P, ARNA, and sodium excretion, during various experimental conditions. However, the nature of this preparation does not allow us to conclude that the chain of events elicited by the various stimuli is limited to events that occur only within the renal sensory nerves.

In summary, the present data show that increasing renal pelvic pressure results in an increase in ARNA, which is blocked by a B₂-receptor antagonist and a PKC inhibitor. These studies taken together with our previous studies suggest that bradykinin contributes to the stimulation of renal mechanosensitive neurons by activating PKC. Renal pelvic administration of PDBu results in increases in ARNA and renal pelvic release of PGE₂ and substance P, which are blocked by indomethacin. These data together with those showing that the PDBu-mediated increase in ARNA is blocked by a substance P-receptor antagonist suggest that activation of renal pelvic PKC stimulates renal sensory neurons by a PGE₂-induced release of substance P. PKC activation leads to a release of PGE₂, which elicits a calcium-dependent release of substance P, which, in turn, activates renal pelvic substance P receptors with a resultant increase in ARNA (Fig. 7).

Perspectives