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J Am Soc Nephrol 13:1872-1883, 2002
© 2002 American Society of Nephrology

Impaired Renal Sensory Responses after Renal Ischemia in the Rat

Ming-Chieh Ma*, Ho-Shiang Huang{dagger}, Ming-Shiou Wu{ddagger}, Chiang-Ting Chien§ and Chau-Fong Chen*

*Department of Physiology, College of Medicine, National Taiwan University; {dagger}Department of Urology, {ddagger}Department of Internal Medicine, and §Office for Clinical Research, National Taiwan University Hospital, Taipei, Taiwan, ROC.

Correspondence to Dr. Chau-Fong Chen, Department of Physiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan, ROC. Phone: 886-2-23222954; Fax: 886-2-23222954; E-mail: chfochen{at}ha.mc.ntu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Renal sensory responses and reflex function were examined in rats 24 h after 45 min of ischemic injury caused by unilateral renal arterial occlusion (RAO). The integrity of renal pelvic mechanoreceptor (MRu)-mediated renorenal reflex was examined. An increase in ipsilateral afferent renal nerve activity (ARNA) and a reflex decrease in efferent renal nerve activity (ERNA) and contralateral diuresis and natriuresis produced by increasing the intrapelvic pressure were seen in sham-operated (Sham) rats, but it was largely attenuated in RAO rats. Using single-fiber recordings of the renal MRu discharge, graded increases in intrapelvic pressure or renal pelvic administration of substance P (SP) resulted in pressure- or concentration-dependent increases in ARNA in the control kidney of Sham rats, whereas attenuated responses were seen in the postischemic kidney of RAO rats. The unresponsiveness of renal MRus in RAO rats was accompanied by an insufficient release of SP. However, the baseline SP release is higher in RAO kidneys due to a reduced neutral endopeptidase (NEP) activity in the renal pelvis of the postischemic kidney. No changes in NK-1 receptor mRNA levels were demonstrated; however, the expression of NK-1 receptors in the plasma membrane of RAO pelvis were decreased, possibly resulting from the internalization of the receptors associated with {beta}-arrestin trafficking. Renal excretory responses after saline loading were significantly lower in the postischemic kidney of RAO rats than in Sham rats. Responses of ARNA and ERNA were also lower. It is concluded that the defective activation of renal sensory mechanoreceptors in the postischemic kidney results from an inadequate release of SP after mechanostimulation and the reduced functional NK-1 receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ischemia-induced acute renal failure is characterized by renal hypoperfusion, decreased renal function, and nitrogen waste retention (1). Complete renal artery occlusion has been used in animal models to determine the mechanisms involved in the pathogenesis of acute renal failure (2). Previous studies of rats after 45 min of unilateral renal arterial occlusion (RAO) and 24 h of reperfusion have demonstrated renal hypofunction in the postischemic kidney (3,4) and abnormal renal nerve activity (5). Certain experimental approaches, such as infusion of atrial natriuretic peptide (ANP) (6) or saline (5,7), can restore renal function after renal ischemia, but recovery of urinary excretion is never complete. Surgical and pharmacologic renal denervation improves renal excretion in response to natriuretic stimuli and prevents the development of acute renal failure (8). These findings suggest that the defective natriuretic response seen in ischemic acute renal failure is frequently associated with increased renal sympathetic nerve activity; however, the underlying mechanism affecting renal nerve activity in this condition, especially the role of afferent renal nerve activity, is not known.

We have demonstrated an impaired renal sensory response in rat models associated with various kidney disorders (9–12) and have suggested that sensory receptors on renal afferent nerves play an important role in the reflex control of renal function. The majority of renal sensory neurons are located in the renal pelvis (13). Substance P (SP)-immunoreactive nerves (14) and SP receptors, neurokinin 1 (NK-1) receptors (15), are found in the renal pelvis, suggesting that all components required for renal sensory transmission are present. In rats, activation of renal sensory neurons via the action of SP-containing neurons (16) elicits an inhibitory renorenal reflex.

Biologic responses to SP are rapidly attenuated and degraded by a cell-surface protease, neutral endopeptidase (NEP), and receptor endocytosis (17). Numerous in vivo studies have shown that exogenous or endogenous SP can induce NK-1 receptor internalization (18–20). A detailed mechanism has been suggested in which the trafficking of NK-1 receptors into the endosome is dependent on the translocation of the cytosolic protein, {beta}-arrestin, subsequent to the binding of SP and receptor phosphorylation (17). It has been suggested that change in NK-1 receptor expression participates in certain pathophysiologic states (20) and that its desensitization prevents noxious stimuli from acting on cells.

The present study was therefore undertaken to explore the underlying mechanisms in the decreased responsiveness of renal excretion in the postischemic kidney. We first compared the responsiveness of renal pelvic mechanoreceptor (MRu)-induced renorenal reflex in the normal and postischemic kidney. SP plays a crucial role in renal sensory transmission (21); we therefore studied the effects of intraplevic pressure and SP on MRu and the individual components involved in renorenal reflex, including the amount of renal pelvic SP release, the activity of NEP, its catabolizing enzyme, and the regulation of NK-1 receptor expression. Finally, we examined whether renal nerve activity changed in parallel with renal excretion in response to a diuretic stimulus in the normal and postischemic kidney.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Care and Experimentation
Female Wistar rats weighing 200 to 220 g were used. All animal experiments and care were performed in accordance with the Guide for the Care and Use of Laboratory Animals (published by National Academy Press, Washington DC, 1996). All protocols used in this study were approved by the Laboratory Animal Care Committee of the National Taiwan University College of Medicine.

Induction of Unilateral Renal IschemicReperfusion Injury
As in our previous study (5), injury was produced by 45 min of renal ischemia followed by a 24-h reperfusion recovery period. Briefly, rats were anesthetized with a combination of ketamine (50 mg·kg-1, intraperitoneally) and sodium pentobarbital (15 mg·kg-1, intraperitoneally), the abdomen was opened, and the left renal artery was carefully isolated and occluded for 45 min (RAO rats). The abdominal wound was then closed under aseptic conditions. Sham-operated (Sham) rats were treated similarly, but they did not undergo RAO of the left kidney. The rats were allowed to recover in individual cages for 24 h after surgery. In the RAO rats, the ischemic injured kidney is known as the postischemic kidney. The choice of a 24-h reperfusion period is based on the altered excretory responses that are suggested to occur after this period (3,22,43).

General Surgical Preparation
After 24-h recovery from the above treatment, the rats were anesthetized with sodium pentobarbital (35 mg/kg of body weight, intraperitoneally) and the trachea was exposed and intubated for spontaneous ventilation. Catheters (PE-50) were placed in the external jugular vein for continuous saline infusion and in the carotid artery to measure mean arterial BP (MABP). The left kidney was exposed via a left flank incision, and both ureters were cannulated with PE-50 catheters for urine collection. The MABP was recorded on a Gould polygraph (RS 4300; Gould, Valley View, OH). The kidney was then bathed with warmed paraffin oil (38°C) to prevent drying and illuminated with a fiberoptic light source. To record renal nerve activity, the left renal nerve fiber at the angle between the abdominal aorta and the renal artery was carefully isolated from the surrounding tissue by using a stereoscopic dissecting microscope (SZ-STU2; Olympus, Tokyo, Japan).

Recording of Multi-Unit and Single-Unit RenalNerve Activity
The recording techniques for multi-unit and single-unit renal nerve activities have been described previously (5,9–11). Briefly, multi-unit nerve recordings of afferent renal nerve activity (ARNA) and efferent renal nerve activity (ERNA) were recorded simultaneously by placing two intact nerve fibers on two pairs of thin bipolar electrodes. The electrical signals were amplified and filtered by using a P511 AC amplifier (Grass, Quincy, MA) and continuously displayed on an oscilloscope (Model 1604, Gould). The amplified signals were fed into a window discriminator (Model 121; World Precision Instrument, Sarasota, FL) and counted on an integrator amplifier (Model 13–4615–70, Gould). The neural activity was transformed into spike counts on a Gould polygraph. After assessing renal nerve activity by its pulse synchronous rhythmicity with the heart beat, the distal and proximal parts of the nerve fibers were transected for the individual recording of ipsilateral ERNA and ARNA. After recording multi-unit ARNA, the nerve bundle was repeatedly split with fine forceps until a single-unit impulse of ARNA was apparent on the oscilloscope trace (Figure 2, A and B). Single-unit recordings of MRu activity were identified by using an increase in intrapelvic pressure, which does not act as a specific stimulus for other subtypes of renal sensory receptor.



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  		Figure 2. Original tracings showing the effects of stimulation of a single-unit renal pelvic mechanoreceptor (Mru) neuronal response in the left kidney after an increase in IPP or intrapelvic infusion of SP on the arterial pressure (AP) and single-unit ARNA of a Sham (A) and an RAO (B) rat. The units shown in the figure are similar amplitudes of ARNA discharges continuously selected by a window discriminator. (C) Original tracings of the effects of single-unit MRu discharges in the left kidney as a result of graded increases in IPP on the AP and single-unit ARNA in a Sham rat. (D) Original tracings show the effects of increasing concentrations of SP (1, 5, and 10 µg·ml-1, administered at the time marked by the horizontal bars) on the single-unit MRu discharge in a Sham rat, identified by the response to increased IPP before and after SP administration.

 
Renorenal Reflex
The renorenal reflex was studied in 12 rats from each of the Sham and RAO groups. Increasing the intrapelvic pressure (IPP) resulted in stimulation of the renal MRu of the left kidney (9,10,23) and changes in the ERNA and ARNA, which are described as follows. Contralateral (right kidney) urine samples were collected. The renal pelvis was perfused throughout the experiment with normal saline at a rate of 20 µl·min-1 to control the chemical composition of the urine produced by both groups, a procedure that did not affect the ureteral pressure. Changes in renal IPP were recorded on a Gould polygraph with a transducer connected to the PE-50 ureteral catheter by a T-tube connector. The T-tube connector was also connected to a 50-cm PE-50 catheter to increase the IPP by about 20 mmHg, the pressure being maintained for 3 min with a 10-min interval between changes.

Renal Mechanoreceptor Stimulation and Intrapelvic Administration of SP
Renal pelvic mechanostimulation was studied in ten rats from each group, as described above. Raising the 50-cm PE-50 catheter increased the IPP by 4, 8, 12, 16, and 20 mmHg, each pressure being maintained for 3 min with a 10-min interval between changes. After verification of the single-unit ARNA response to renal pelvic mechanostimulation, SP (Peninsula Laboratories, Belmont, CA) dissolved in normal saline, was perfused as described above at concentrations of 1, 5, and 10 µg·ml-1, each concentration being perfused for 3 min with a 10-min interval between concentration changes. The SP doses that were used have been shown to elicit a fully activated ARNA response in normal rats (23).

Renal Pelvic SP Release
To assay SP in the renal pelvic effluent in response to increasing IPP in the part experiment of renorenal reflex, the perfused saline containing 10 µmol·L-1 of the endopeptidase inhibitor thiorphan (Sigma, Saint Louis, MO) was used to minimize SP degradation (9,10,23). During the periods of increased IPP (approximately 20 mmHg), the renal pelvic effluent was collected after lowering the 50-cm PE-50 catheter to the same level as the left kidney for 1 min. The amount of SP in the renal pelvic effluent was determined by enzyme-linked immunoassay, as described previously (9,10).

Determination of Renal Pelvic NEP Activity
After the acute animal studies, both groups of rats (n = 10 for each) were sacrificed by anesthetic overdose. The tissues containing the renal pelvis and the proximal end of the ureter in the left kidney were sampled in both groups to determine NEP activity using a previously described method (9,24). Briefly, the sampled tissues were immediately homogenized then centrifuged, and the supernatants were aspirated for analysis. Incubation mixtures contained glutaryl-Ala-Ala-Phe-4-MeOH-naphthylamine (Sigma), tissue extract, and leucine aminopeptidase M (Sigma). After incubation, a solution of Fast garnet GBC in Brij 35 solution (both from Sigma) was added to stop the enzymatic reaction and to develop the color at room temperature. The 546-nm absorbance of the colored product, 2-naphthylamine (2-NA)-fast garnet GBC, was measured on a spectrophotometer (Eclipse; Vitalab, Dieren, Netherlands). Another 50-µl sample of each tissue extract was also analyzed for NEP activity in the presence of phosphoramidon (RBI, Natick, MA). The total protein concentration in each sample was determined using the Bradford dye-binding assay method (Bio-Rad, Hercules, CA).

Immunoblotting of NK-1 Receptors and {beta}-Arrestin
After sacrificing the rats, the left kidney was exposed and the region of the renal pelvis and the proximal end of the ureter was sampled for the preparation of plasma membrane (9,10,25) and endosomal (26) fractions. Protein samples were separated and electrophoretically transferred to nitrocellulose membranes (Amershan-Pharmacia Buckingham, UK). After blocking, the membranes were then incubated overnight at 4°C with rabbit anti-NK-1 receptor antiserum (Novus Biologicals, Littleton, CO; 1:1000), mouse monoclonal anti-{beta}-arrestin antibody (Transduction, Lexington, KY; 1:250), or rabbit polyclonal anti-transferrin receptor (TfR) antibody (Santa Cruz Biochemicals, Santa Cruz, CA; 1:100). After wash, the membranes were incubated for 1 h at room temperature with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Vector, Burlingame, CA) or HRP-conjugated goat anti-mouse IgG (Lenico, St. Louis, MO) as appropriate. They were then washed, and the bound antibody was visualized by using a commercial DAB (3,3'-diaminobenzidine) peroxidase substrate kit (Vector). The densities of the bands with molecular masses of about 79 kD (NK-1 receptor), 55 kD ({beta}-arrestin), or 95 kD (TfR) were determined semiquantitatively by densitometer using an image analytic system (Alpha Innotech, San Leandro, CA).

Quantitative Real-Time Reverse Transcriptase-PCR (RT-PCR) to Measure NK-1 Receptor mRNA Levels
The theoretical basis and methodology of the ABI PRISM 7700 Sequence Detection System (TaqMan) real-time quantitative PCR (Perkin-Elmer Applied Biosystem, Foster City, CA) have been described by Johnson et al. (27). Briefly, samples with a high starting copy number of the genes of interest show increased fluorescence early in the PCR process, resulting in a low threshold cycle (CT) number.

The primers and fluorogenic probes of NK-1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total RNA extraction, and the PCR procedure were designed as described previously (9,25). The NK-1 receptor primers and probe were as follows: forward primer 5'-GGC CAG AGG ACC AGA ACT TTT-3', reverse primer 5'-GCT AGC AAC TCC CAC TAA CAT ACG T-3', and probe 5'-6-carboxyflurescein (FAM)-CAA GCA ACA CTG CAC TGC GAG CA-6-carboxy-tetramethylrhodamine (TAMRA)-3'. The GAPDH primers and probe were as follows: forward primer 5'-TTT CTC GTG GTT CAC ACC CA-3', reverse primer 5'-GTC ATC ATC TCC GCC CCT T-3', and probe 5'-FAM-CGC TGA TGC CCC CAT GTT TGT G-TAMRA-3'.

Acute Saline Loading
After 1 h of equilibrium, acute saline loading was applied to 14 rats from each of the Sham and RAO groups by intravenous infusion of an amount of isotonic saline equal to 5% of the body weight over a period of 10 min (time 0 to 10 min) (9,10). MABP, ERNA, and ARNA were continuously monitored, and urine samples were collected from the left kidney at time-points 5, 10, 20, 30, 45, 60, and 90 min after the start of infusion.

Chemical Analyses and Data Treatment
Urine volume was determined gravimetrically. The urinary sodium concentration was measured by flame photometry (FCM 6341; Eppendorf, Hamburg, Germany). Urine flow rate (UV) and urinary sodium excretory rate (UNaV) were expressed per gram of kidney weight.

Systemic hemodynamic and renal excretory functions were averaged over each period. Renal nerve activities were also averaged over each period, and the effects of acute saline loading, increased IPP, and renal pelvic perfusion of SP on renal nerve activities were calculated by comparing the experimental value with the average value for the control period. In the multifiber nerve recordings, there are possible differences in the numbers of nerve fibers and the degree of nerve-electrode contact; absolute values of integrated voltages cannot therefore be reliably compared between rats or groups of rats. As a result, the data were analyzed as the percentage change from the basal value during the control period.

The amount of 2-NA released was calculated from the absorbance and the extinction coefficient of 27,000 L·cm-1·mol-1 (9,28). Specific NEP activity was calculated by subtraction of the phosphoramidon-inhibitable NEP activity and was expressed in terms of nanomoles of 2-NA released per milligram of tissue protein per hour (nmol·mg-1 of protein·h-1).

The comparative CT ({Delta}{Delta}CT) method was used to quantify NK-1 receptor mRNA levels as described previously (9,23). The calculation used was:

{Delta}{Delta}CT = [CT NK-1 receptor (unknown sample) - CT GAPDH (unknown sample)] - [CT NK-1 receptor (calibrator sample) - CT GAPDH (calibrator sample)]

with the Fold induction being equal to 2-[{Delta}{Delta}CT] (29).

The data in the text, tables, and figures are expressed as the mean ± SEM. Statistical analyses was performed using the Newman-Keuls test of ANOVA for multiple comparison. A significance level of 5% was chosen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After the 24-h reperfusion period, the kidney-to-body weight ratio (%) of the postischemic kidney in RAO rats (0.47 ± 0.01) was significantly higher than that of the control left kidney in Sham rats (0.40 ± 0.02). Before acute saline loading, both the baseline urine flow rate and urinary sodium excretion was significantly lower in the postischemic kidney of RAO rats (UV, 0.8 ± 0.1 µl·min-1·g-1; UNaV, 0.07 ± 0.02 µl·min-1·g-1) than that in the control left kidney of Sham rats (UV, 4.3 ± 0.6 µl·min-1·g-1; UNaV, 0.34 ± 0.06 µmol·min-1·g-1).

Renorenal Reflex Function
Figure 1 shows that stimulation of renal MRu elicited an inhibitory renorenal reflex. In both the Sham (left panels) and RAO (right panels) groups, the MABP was unchanged during the increased IPP and recovery periods. In Sham rats, an IPP increase from 0.6 ± 0.8 mmHg to 21.4 ± 2.6 mmHg in the ipsilateral left kidney resulted in an increase in the UV and UNaV accompanied by a decrease and an increase in the ipsilateral ERNA and ARNA, respectively, in the contralateral right kidney. In contrast, contralateral diuretic and natriuretic responses were not seen in RAO rats after a similar IPP increase (from 0.5 ± 0.7 mmHg to 20.7 ± 1.9 mmHg) in the postischemic kidney, despite mild changes in ipsilateral renal nerve activity.



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  		Figure 1. Renorenal reflex and renal pelvic substance P (SP) release from the left kidney in sham-operated (Sham) and renal arterial occlusion (RAO) rats. IPP, intrapelvic pressure; MABP, mean arterial BP; UV and UNaV, urine flow rate and urinary sodium excretion recorded in the contralateral right kidney; ARNA and ERNA, afferent and efferent renal nerve activity recorded in the left kidney; Rec, recovery period. *Significant difference (P < 0.05) compared with the basal state; {dagger}Significant difference (P < 0.05) compared between groups.

 
Single-Unit Renal Mechanoreceptor Responses: Mechanostimulation and Effect of SP
Typical simultaneous recordings of the single-unit MRu activity, arterial pressure, transformed spike unit, and IPP in Sham and RAO rats are shown in Figures 2A (Sham rat) and 2B (RAO rat). A window discriminator was used to select the transformed spike unit to represent the MRu discharge on ARNA. The single-unit MRu responses in both groups were a spontaneously firing type (left panels of Figures 2A and 2B) with a high signal-to-noise ratio. The data in Figures 2A and 2B (left panel) and Table 1 show that the postischemic kidney in RAO rats had a lower basal activity of MRu discharge than the control kidney in Sham rats. Original tracings for a Sham rat (Figure 2C) showed that raising the ureteral catheter to various levels above the left kidney increased the IPP and activated single-unit MRu discharge. The IPP changes did not affect the AP. Increasing the IPP to about 15 mmHg resulted in regular firing of afferent nerve activity by the MRu in Sham rats (Figure 2A, center panel), whereas a similar IPP increase applied to the postischemic kidney of RAO rats did not enhance the firing activity of the MRu (Figure 2B, center panel). IPP increases resulted in a dose-dependent increase in MRu spike activity in Sham rats, but only at the highest IPP in the RAO rats (Table 1). The increased single-unit MRu activity was reversible when the IPP returned to the basal level. The IPP threshold for a significant increase in MRu activity in the test kidney was 7.8 ± 0.3 mmHg in Sham rats and 20.7 ± 1.0 mmHg in RAO rats. At an IPP level of approximately 20 mmHg, MRu activity was increased by 409 ± 138% in Sham rats and by 42 ± 19% in RAO rats (P < 0.05).


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  		Table 1. Responses of renal pelvic mechanoreceptors to graded IPP increases in Sham and RAO ratsa
 
Figure 2D shows typical tracings showing the effect of intrapelvic SP perfusion on single-unit MRu activity in a Sham rat. SP induced the MRu to generate firing activity similar to that seen with IPP-induced activation. Perfusion with SP did not significantly alter the AP. As with mechanostimulation, the increased MRu activity due to SP was reversible when perfusion with SP was stopped. In animals receiving 10 µg·ml-1 of SP, the MRu produced trained firing of single-unit ARNA in Sham rats (Figure 2A, right panel), but not in RAO rats (Figure 2B, right panel). The SP-evoked MRu activation was dose-dependent in Sham rats (Table 2), and the MRu activity increased significantly by 72 ± 8%, 233 ± 29%, and 538 ± 65%, respectively, at SP concentrations of 1, 5, and 10 µg·ml-1. In contrast, a blunted response to graded SP concentrations was seen in RAO rats, a significant increase in spike activity only being seen at the dose of 10 µg·ml-1 of SP (Table 2), at which concentration MRu activity was increased by 78 ± 37%.


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  		Table 2. Responses of renal pelvic mechanoreceptors to substance P (SP) in Sham and RAO rats
 
Renal Pelvic SP Release and NEP Activity
In the part experiment of renorenal reflex, the basal values for ipsilateral (left side) renal pelvic SP release (n = 12 for each group) were significantly different in Sham (4.9 ± 0.9 pg·min-1) and RAO (23.4 ± 2.7 pg·min-1) rats. An IPP increase resulted in a significant increase in SP release in Sham rats to 29.8 ± 6.0 pg·min-1, but not in RAO rats (26.9 ± 5.4 pg·min-1). When the IPP was reduced, SP release returned to 4.2 ± 0.8 pg·min-1 in Sham rats, but not in RAO rats, in which it increased to 35.9 ± 5.9 pg·min-1.

NEP activity in renal pelvic samples from the control kidney of Sham rats was 206.9 ± 17.3 nmol·mg-1 of protein·h-1, significantly higher than that in the postischemic kidney of RAO rats (97.2 ± 8.0 nmol·mg-1 of protein·h-1).

Different Subcellular Distributions of NK-1 Receptors and {beta}-Arrestin in the Renal Pelvis as Demonstratedby Immunoblotting
To explore the role of renal pelvic NK-1 receptors in the impaired renal sensory response and reflex function, we quantified NK-1 receptor expression in the Sham and RAO groups. As shown in Figure 3A, the NK-1 receptor antiserum recognized a protein band with a molecular mass of 79 kD (lanes 1 and 2) in the plasma membrane fraction prepared from rat ileum and brain cortex. The same band was recognized in plasma membrane fractions prepared from the renal pelvic tissues of the left kidney, but the band intensity was lower in the samples from the RAO rats (lanes 6 to 8). Figure 3D shows the semiquantitative density of the 79-kD band for membrane NK-1 receptors; the integrated digital value (IDV) in Sham rats was 2150 ± 165, significantly higher than that of 164 ± 23 in RAO rats.



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  		Figure 3. Neurokinin 1 (NK-1) receptor expression in the plasma membrane and endosomal fractions of renal pelvic tissues. (A) Western blot of the plasma membrane fraction prepared from three Sham (lanes 3 to 5) and three RAO (lanes 6 to 8) rats using anti-NK-1 antiserum. Positive controls of membranes prepared from rat brain cortex and ileum are shown in lanes 1 and 2. Each lane contains 80 µg of membrane protein, except for lanes 1 and 2 (10 µg). Protein staining showed that equal amounts of protein were loaded in lanes 3 to 8. Lane M, prestained low-molecular weight standards (Bio-Rad, Burlingame, CA); phosphorylase B (106 kD), bovine serum albumin (81 kD), ovalbumin (47.5 kD), carbonic anhydrase (35.3 kD), soybean trypsin inhibitor (28.2 kD), and lysozyme (20.8 kDa). (B) Western blot of the endosomal protein fraction prepared from same three Sham (lanes 3 to 5) and three RAO (lanes 6 to 8) rats. Positive controls of membrane protein prepared from rat brain cortex and ileum are shown in lanes 1 and 2. Each lane contains 200 µg of endosomal protein, except for lanes 1 and 2 (10 µg). (C) The same endosomal protein fractions stained for transferrin receptor (TfR). Lane M, prestained standards as in Part A. (D) Semiquantitative densitometry showing the amount of NK-1 expression in both groups in different protein fractions. {dagger}Significant difference (P < 0.05) compared between groups.

 
The reduced levels of NK-1 receptors in plasma membrane preparations from the renal pelvis and high SP release of RAO rats suggested possible downregulation of NK-1 receptors. Figure 3B shows Western blots of NK-1 receptor in the endosomal fraction of the renal pelvis in both groups; lanes 1 to 2 again plasma membrane of brain cortex and ileum samples to serve as positive controls. Figure 3C shows that the same endosomal preparations contained TfR, an endosomal marker. Figure 3D shows the IDV value for endosomal NK-1 receptors was 511 ± 36 in Sham rats, significantly lower than that of 2277 ± 139 in RAO rats.

The NK-1 receptor interacts with {beta}-arrestin and then uncouples from G-proteins to terminate receptor signaling (17). We therefore investigated the role of {beta}-arrestin in the trafficking and downregulation of the renal pelvic NK-1 receptor in the postischemic kidney. Figure 4A shows {beta}-arrestin expression in the renal pelvis in both groups. Lane 1 and 2 show, respectively, positive controls of cytosolic proteins from the rat ileum and commercially available {beta}-arrestin. {beta}-Arrestin immunoreactivity was found in the plasma membrane and cytosolic fractions of the renal pelvis in rats from both groups. As shown in Figure 4B, the {beta}-arrestin density for the plasma membrane fraction in Sham rats was significantly lower than that of in the cytosolic fraction; the converse was seen in RAO rats.



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  		Figure 4. Translocation of {beta}-arrestin in renal pelvic samples. (A) Western blot showing {beta}-arrestin in the renal pelvic plasma membrane fraction (lanes 3 to 5) and the cytosolic protein fraction (lanes 6 to 8) prepared from three Sham rats. Lanes 9 to 11 and 12 to 14 show results for the same cellular fractions from three RAO rats. Positive controls of a cytosolic protein fraction prepared from the rat ileum and commercial available {beta}-arrestin (Transduction, Lexington, KY) are shown in lanes 1 and 2, respectively. Each lane contains 120 µg of protein, except for the positive controls. Lane M, the same prestained standards as in Figure 3. (B) Semiquantitative densitometry showing the amount of {beta}-arrestin in the different protein fractions in both groups.

 
Renal Pelvic Expression of NK-1 Receptor mRNA
Figure 5 shows original amplification plots for GAPDH and NK-1 receptor mRNA in one Sham and one RAO rat. Table 3 shows the raw data for the CT values for the NK-1 receptor and GAPDH mRNAs, the calculated {Delta}{Delta}CT values, and the fold induction of NK-1 receptor mRNA in the Sham and RAO groups. The CT results for the housekeeping GAPDH analyses of a variety of samples were similar; the standard error values were small, indicating the high precision of the quantitative real-time RT-PCR method. The NK-1 receptor CT values showed that NK-1 receptor mRNA was expressed in the renal pelvis in both groups. The values for the fold induction of renal pelvic NK-1 receptor mRNA, relative to the ileum, showed no significant difference between the two groups.



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  		Figure 5. A typical amplification plot for NK-1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. The four amplification plots show the RNA samples from one Sham and one RAO rat amplified with specific GAPDH and NK-1 receptor primers. The graph shows the fluorescence intensity above the baseline ({Delta}Rn) for each PCR plotted against cycle number. The y axis ({Delta}Rn) is a logarithmic scale. The no-template control (background) has been subtracted. The baseline (0.082) is the threshold setting. The CT value was determined as the cycle number at which the amplification plot exceeded the threshold setting. The baseline was set at cycles 3 to 15. The average value from cycles 3 to 15 was taken as the background fluorescence and was subtracted from the plotted values.

 

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  		Table 3. Analysis of NK-1 receptor mRNA expression in Sham and RAO rats using the comparative CT methoda
 
Acute Saline Loading
Original tracings of the renal nerve responses to acute saline loading in the control kidney of one Sham rat and in the postischemic kidney of one RAO rat are shown, respectively, in Figures 6A and 6B. In both groups, there was a similar AP response during saline loading, with a transient increase followed by a decrease. In response to saline loading, ARNA and ERNA were activated and suppressed, respectively, in the Sham rat, but not in the RAO rat. Figure 6C shows original tracings of ARNA and ERNA discharge patterns in one Sham rat. The firing pattern of the ERNA discharge before acute saline loading (VE) was synchronized with the heartbeat, but the ARNA discharge was not. During saline loading, the ARNA discharges increased and the amplitude of the ERNA discharges became smaller in the Sham rats. In contrast, the difference was seen in the RAO rat, with no change in either ARNA or ERNA discharge on acute saline loading (Figure 6D). Figure 6E shows the summarized data for renal excretory and nerve responses to acute saline loading in both groups. As shown in panel 1, the systemic MABP was not significantly altered by acute saline loading in either group, although a downward, but insignificant, trend was seen after 5 and 10 min of saline loading. The UV (panel 2) and UNaV (panel 3) increased in both groups in response to saline loading, but both the diuretic and natriuretic responses were markedly attenuated in the postischemic kidney of RAO rats. Cumulative urine output and sodium excretion in the postischemic kidney of RAO rats were 17.4 ± 2.5% and 10.4 ± 3.0%, respectively, of those in the control left kidney of Sham rats (P < 0.05). Changes in ERNA and ARNA responses in both groups are shown in panels 4 and 5. In Sham rats, the ERNA or ARNA showed, respectively, a significant decrease or increase at the 5th, 10th, 20th, and 30th min during the course of acute saline loading; however, both were markedly attenuated in RAO rats.



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  		Figure 6. Responses to saline load in the control left kidney of Sham rats and the postischemic kidney of RAO rats. (A and B) Original tracings showing the effect of acute volume expansion (VE) by intravenous infusion of saline on the AP and integrated multifiber ERNA and ARNA of one Sham (A) and one RAO (B) rat. (C and D) Simultaneous recordings of AP and ERNA and ARNA discharges in one Sham (C) and one RAO (D) rat at the basal state (before VE) and after saline infusion (during VE 5 min). (E) Statistical results for changes in MABP, UV, UNaV, and the percentage change in ERNA and ARNA in both groups. {dagger}Significant difference (P < 0.05) compared between groups at the same time point. The horizontal bar indicates the time of saline infusion.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Renal Mechanoreceptors Dysfunction
Several studies have demonstrated the existence of renal mechanoreceptors (MR) in rats, cats, rabbits, dogs, and monkeys (30). One type of renal mechanoreceptor, termed the MRu, which is sensitive to changes in ureteral pressure, exists in all of the above-named species. We evaluated the function of single MRus by graded increases in IPP and found that applying pressure to the MRu resulted in dose-dependent ARNA activation in control kidneys, as previously reported in multi-fiber ARNA recordings (16,23), whereas a blunted response was seen in the postischemic kidney. The IPP threshold for mechanosensitive neurons in the renal pelvic area was found to be within the physiologic range (31). In the present study, the activation threshold was <8 mmHg in the control kidney, but approximately 21 mmHg in the postischemic kidney (Table 1). These results also explain the observation of defective ARNA responses on saline loading in the postischemic kidney, because the unresponsive MRu cannot sense the increased renal pelvic or ureteral pressure well, especially after massive urine formation after diuretic stimulation, leading to fluid retention in acute renal failure.

Our results using SP administered by the intrapelvic route showed dose-dependent discharge of the single-unit ARNA similar to that seen by Kopp et al. (23) in multi-unit recording preparations. It was of interest in the present study to find that intrapelvic perfusion of SP generated the same single-unit discharge of MRu neurons on ARNA as an increase in IPP. SP is an important mediator of the ARNA response to increase renal pelvic pressure (16) and that the ARNA response can be blocked by a SP receptor antagonist (21). Furthermore, the present study provided direct evidence that SP elicits activation of single renal pelvic MRs. Considering these results together, we can say that SP is the neuropeptide that activates the MR in the renal pelvis. Whether SP can activate other types of renal MR or chemoreceptor is not yet known.

Abnormal SP Release, Renal Pelvic NEP Activity, and NK-1 Receptors Expression
In our control rats, an increase in renal intrapelvic pressure resulted in a significant increase in ipsilateral ARNA as well as SP release and a reflex decrease in ERNA accompanied by contralateral diuresis and natriuresis. In rats, neural activity generated by stimulation of renal afferent nerves elicits a diuretic signal to the integrative site to reflexively inhibit ERNA in both kidneys via the spinal to supraspinal sites (32) and plays a role in the fluid balance (33). These results were consistent with those of a previous study by Kopp et al. (34), and are termed an inhibitory renorenal reflex. Depletion of SP in neurons by capsaicin pretreatment abolishes the SP-mediated and MR-induced renorenal reflex (16). It is apparent that the release of SP into the renal pelvis plays an important role in driving the inhibitory renorenal reflex. However, the renorenal reflex in our RAO rats was not working properly. Despite basal SP release from renal pelvis was higher in the postischemic kidney, however, it is likely that the lack of an increase in SP release in response to increased IPP contributed to the impaired responsiveness of the afferent renal nerves in RAO rats.

The higher concentration of SP found in the renal pelvis of RAO rats might be due to inadequate catabolism. NEP, a cell-surface enzyme originally discovered in the kidney, is a major inactivator of SP in the extracellular fluid (35). Sakakibara et al. (36) found that renal NEP was localized mainly in the proximal tubule, with a small amount in the distal tubule. Edwards et al. (37) found that, of various nephron segments dissected from rat kidney, only the proximal tubule and glomerulus contained measurable NEP activity. The present study demonstrates measurable NEP activity in the rat renal pelvis and that the postischemic kidney showed a decrease in renal pelvic NEP activity of about 47% compared with the control kidney. In a previous study of the rat kidney after 30 min of RAO treatment and 24 h of reperfusion, Nambi et al. (38) found a decreases of approximately 58% in NEP activity and approximately 90% in NEP mRNA levels in the renal cortex; they suggested that NEP downregulation is one of the mechanisms leading to the increased tissue levels of endothelin that exacerbate kidney damage after ischemic injury. In the present study, it is clearly shown that the excess SP in the renal pelvis is associated with decreased NEP activity. Decreased SP degradation in NEP knockout mice or animals treated with NEP inhibitor has been found to exacerbate the inflammatory response in many organs, and downregulation of NEP activity is seen in the inflamed intestine and airway (17). There is a lack of direct evidence linking the SP and NEP systems; the possibility that other events resulting from ischemic insult may influence the metabolism of SP and NEP activity cannot therefore be excluded.

Another explanation for the impairment of the renorenal reflex could be the reduced number of SP receptors in the renal pelvis. NK-1 receptor expression in the plasma membrane fraction of the renal pelvis of the postischemic kidney was significantly decreased compared with that in the control kidney of Sham rats. This mismatch of higher basal SP release and reduced numbers of NK-1 receptors in the postischemic kidney clearly indicates that receptor desensitization (i.e., the number of receptors is decreased when the concentration of the ligand is increased) occurred in the renal pelvis. We further explored the underlying mechanism at the cellular level. Our results showed that the endosomal fraction from the postischemic kidney had a higher content of NK-1 receptors than the equivalent fraction from the control kidney, suggesting that NK-1 receptors be internalized from the plasma membrane into the endosome. Assuming that the total amount of NK-1 receptor in the renal pelvis was the sum of that found in the plasma membrane and endosomal fractions, 7% and 93% of the NK-1 receptor in the postischemic kidney was found in the membrane and endosomal fractions, respectively, contrasting with the distribution of 81% and 19% in the corresponding fractions in the normal kidney. The NK-1 receptor distribution in the postischemic kidney is consistent with the results of a previous study by Mann et al. (19), who showed that, after exogenous SP treatment of myenteric neuronal cells, 72% of NK-1 receptors were found in intracellular compartments. The same sample of renal pelvis from the postischemic kidney showed that {beta}-arrestin trafficking from the cytosol to the plasma membrane was about 47% of that seen in the control kidney (Figure 4B). The present results showing NK-1 receptor desensitization in the renal pelvis are very similar to those of previous studies using cultured cells (17–19), but ours is the first study to show that this was not due to changes in NK-1 receptor mRNA levels (Table 3). The dynamic change in NK-1 receptor mRNA levels in response to ischemic insult was not evaluated in this study. In general, mRNA levels in a tissue are dependent on two processes: the rate of transcription of the gene and the rate of degradation within the cytosol. It is therefore possible that an increase in both transcription and degradation might result in unchanged NK-1 receptor mRNA levels. In cells expressing NK-1 receptors, SP was found to induce an intracellular Ca2+ increase and phosphorylation of the NK-1 receptor followed by promotion of {beta}-arrestin trafficking and NK-1 receptor internalization into the early endosome, co-localizing with the translocated {beta}-arrestin (17). SP-induced internalization of NK-1 receptors in the renal pelvis after ischemic insult is similar to that in previous in vivo studies on the inflammatory response (17,39,40) and in spinal injury (41). Internalization of renal pelvic NK-1 receptors as a result of endogenous SP release suggested playing a regulatory role on sensory neurotransmission, preventing noxious stimuli from acting on the kidney after ischemic reperfusion damage. There is no denying that ischemia can alter the metabolism and processing of the NK-1 receptor or that other contributory systems may also be changed in different ways. The {beta}-arrestin-mediated trafficking of the NK-1 receptor postulated here is one potential mechanism that could result in impaired activation of renal sensory nerves after ischemic insult.

Impaired Renal Nerve Responses during Saline Load
It is widely accepted that a number of intrarenal vasoactive factors are responsible for the defective renal excretion seen in the kidney after ischemic injury. Brief clamping of the renal artery for 20 to 60 min does not cause irreversible kidney injury in rats (42), and oliguria usually occurs in this postischemic situation (3,4,43,44). Mediators of reduced renal function suggested include increased adrenergic activity, altered vascular reactivity, renin-angiotensin stimulation, elevated plasma vasopressin, enhanced synthesis of thromboxane, adenosine and endothelin, and intravascular coagulation in kidney (22). Applied natriuretic maneuvers, such as administration of ANP (6) or volume expansion with saline (5,7), reduce the severity of renal injury in the postischemic kidney, but the excretory response never fully recovered. Our results confirmed the observation of a defective excretory response in the postischemic kidney.

Overactivity of the renal sympathetic nervous system, evidenced by accumulation of intrarenal vasoconstrictor catecholamines (44,45), has been suggested to contribute to the development of or interfere with recovery from ischemic injury (8,46). In a different experimental setting, prior administration of a ganglion blocker or the application of a local anesthetic to the renal pedicle (8) or the interruption of renal nerve traffic (47) prevents the development of acute renal failure. Our findings of increased ERNA and less reflexive suppression in the postischemic kidney tested by saline loading (Figure 6, B and E) suggest that the increased ERNA resulted in worsened renal function. An increase in renal nerve activity leads to increased renal tubular reabsorption of sodium and activation of the renin-angiotensin system (32); both may serve to modulate the antinatriuretic response in the postischemic kidney. However, the factors responsible for the increase in renal tissue norepinephrine and nerve activity in the postischemic kidney are not known. Our study provides evidence that another intrarenal factor, which attenuates activation of renal sensory nerves on the basis of its role in neural reflex control of ERNA, might participate in the neurogenic control of renal nerves after one-side renal injury.

We have shown that ARNA unresponsiveness in the ischemia-injured kidney occurred in parallel with impaired urinary excretion and efferent renal sympathetic nerve activity in response to acute saline loading. Transient inhibition of sensory nerve functions of the lower limb has been found in patients with uremia (48). A previous study has suggested that ARNA activation via an increase in renal pelvic pressure results from increased urine flow rates that stretch the renal pelvic wall (49). Another possibility that cannot be excluded is that persistent vasoconstriction in the postischemic kidney, which has a profound effect on urine flow formation and activation of renal MRus, attenuates afferent firing activity and consequently reflex efferent activity, thus exacerbating renal damage. However, the results of direct renal MRu stimulation showed lower ARNA discharges, supporting functional defects of mechanosensitive neurons located in the renal pelvis of RAO kidneys.

In summary, the present study shows that a blunted renal excretory function in the postischemic kidney is associated with abnormal activity of the renal nerves. Renal mechanoreceptor activation in the postischemic kidney fails to elicit a full inhibitory renorenal reflex. The attenuation of the renorenal reflex response is not due to changes in NK-1 receptor mRNA expression; it is, however, associated with a decrease in renal pelvic NEP activity, leading to an inability to catabolize the released SP, which results in the downregulation of NK-1 receptors in sensory neurons.


   Acknowledgments
 
This study was supported by a grant from the National Science Council of the Republic of China (NSC90-2320-B002–161).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 12, 2002. Accepted for publication April 24, 2002.




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