Temporal decrease in renal sensory responses in rats after chronic ligation of the bile duct

Ming-Chieh Ma1, Ho-Shiang Huang2, Chiang-Ting Chien3, Ming-Shiou Wu4, and Chau-Fong Chen1

1 Department of Physiology, College of Medicine, National Taiwan University, 3 Office for Clinical Research, 2 Department of Urology, and 4 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renal responses to renal sensory receptor activation were examined in rats after 1 and 4 wk of common bile duct ligation (CBDL). Compared with sham-operated rats (Sham), urine and sodium excretion after acute saline loading was significantly reduced at both times after CBDL. The blunted excretory responses in CBDL rats, accompanied by less activation of afferent renal nerve activity (ARNA), were already apparent at 1 wk and became severe at 4 wk. The defect in ARNA activation in CBDL rats was further studied using specific stimuli to activate renal sensory receptors. Graded increases in intrapelvic pressure or renal pelvic perfusion of substance P (SP) elicited an increase in ARNA in Sham rats, these responses being temporally attenuated in CBDL rats. Despite no significant change in renal pelvic SP release, no renorenal reflex was demonstrable in 4-wk CBDL rats. Immunoblotting showed that expression of renal pelvic neurokinin 1 (NK-1) receptors was 32 and 47% lower in 1- and 4-wk CBDL rats, respectively, than in Sham rats, this decrease correlating well with plasma SP levels. The quantitative real-time RT-PCR showed similar levels of NK-1 receptor mRNA in the renal pelvis in the Sham and 4-wk CBDL groups. We conclude that impairment of renal excretory and sensory responses increases with the duration of cirrhosis. An impaired renorenal reflex in cirrhotic rats is involved in the defective activation of the renal sensory receptors could be due, in part, to the low expression of NK-1 receptors, which is dependent on the duration of CBDL. The decrease in NK-1 receptor protein levels is not due to a decrease in mRNA levels.

afferent renal nerve activity; renorenal reflex; substance P; neurokinin 1 receptor; cirrhosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH SUBSTANCE P (SP)-IMMUNOREACTIVE nerves (14) and the SP receptor, the neurokinin 1 (NK-1) receptor (6), are found in the subepithelial area of the renal pelvis, suggesting that all components required for renal sensory transmission are present. Activation of renal sensory neurons by SP-containing or other capsaicin-sensitive neurons (24) elicits an inhibitory renorenal reflex (20), which plays an important physiological role in enhancing urine flow by tonically inhibiting renal sympathetic nerve activity in the urinary tract (11). Furthermore, renal sensory impairment has been extensively studied in two-kidney, one-clip hypertensive rats (19), spontaneously hypertensive rats (13, 23, 25, 26), chronically hypoxic rats (8), and streptozotocin-induced diabetic rats (7), and the results suggest that the inability of renal afferents to transmit diuretic signals from the kidney might contribute to the abnormal fluid retention.

Cirrhosis is characterized by resistance to natriuretic stimuli (35). The basis for this resistance is not well established, although some neurohormonal mechanisms have been discussed. In the experimental rat model of biliary cirrhosis induced by common bile duct ligation (CBDL), previous studies have shown blunted excretory responses to natriuretic stimuli, and renal denervation performed in CBDL rats improves their excretory ability in response to these different natriuretic maneuvers, suggesting a role of the renal nerve in the pathogenesis of cirrhosis (10, 12, 18, 33).

Because little is known about renal sensory function in experimental cirrhosis, this study was carried out to investigate temporal changes in renal function and renal sensory neuron activation caused by acute saline loading. We also evaluated the responsiveness of renal receptors to mechanostimuli and to SP, as well as the renal pelvic expression of NK-1 receptors, and tested the function of the SP-mediated inhibitory renorenal reflex.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care and experimentation. Male Wistar rats weighing 200-250 g were used. All animal experiments and animal care were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

Induction of cirrhosis. Hepatic cirrhosis was induced by CBDL (27). Briefly, the abdomen was opened under anesthesia, and the common bile duct was identified and cut between double ligatures (CBDL rats). Sham-operated rats (Sham) were treated similarly, but without bile duct ligation and resection. The rats were allowed to recover in individual cages and were then studied at 7-10 days (1-wk) and 28-35 days (4-wk) after surgery, at which times ongoing cholestasis and edema formation were evident in the CBDL rats. Biochemical indexes of hepatic function, such as the levels of plasma bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP), were measured using routine clinical laboratory procedures. In addition, six rats in each group were housed in individual metabolic cages for 24-h urine collection, as previously described (5). Because no differences between 1- and 4-wk Sham rats were seen in any of the studied parameters, and the data for all Sham rats were pooled.

General surgical procedures. On the day of the experiment, the rats were anesthetized with urethane (1 g/kg body wt ip), and the trachea was exposed and intubated for spontaneous ventilation. Catheters (PE-50) were placed in the external jugular vein for saline infusion and in the femoral artery to measure the mean arterial blood pressure (MABP). The rat was placed on its right side, the left kidney was exposed via a left flank incision, and both ureters were cannulated for urine collection. Renal blood flow (RBF) was continuously monitored using an electromagnetic flowmeter (model FM701, Carolina Medical Electronics, King, NC) or determined using the p-aminohippurate-clearance method. All hemodynamic responses were recorded on a Grass 7P4 polygraph (Quincy, MA). To record afferent renal nerve activity (ARNA), another group of rats underwent the same surgical procedure, but, in this case, after the left-flank incision was performed, the kidney was fixed and bathed with warmed paraffin oil (38°C) to prevent drying. With the use of a stereoscopic dissecting microscope (Olympus, SZ-STU2, Tokyo, Japan), the left renal nerve at the angle between the abdominal aorta and the renal artery was carefully isolated from the renal artery to record renal nerve activity.

Recording of ARNA. The recording technique has been previously described (8). Briefly, multifiber nerve activity was recorded by placing intact nerve fibers on a pair of thin, bipolar stainless steel electrodes. The electrical signals were amplified and filtered by a Grass model P511 AC amplifier, and the amplified signals were selected using a window discriminator (World Precision Instrument 121, Sarasota, FL) and counted on a Gould integrator amplifier (13-4615-70, Valley View, OH). Neural activity was transformed into spike counts and displayed continuously on a Gould oscilloscope (model 1604). Renal nerve activity was assessed using its pulse synchronous rhythmicity with the heartbeat. After the identification and verification of renal nerve activity, the distal parts of the nerve fibers were transected to record ipsilateral ARNA (29).

Acute saline loading. After 1 h of equilibration, acute saline loading was performed by intravenous infusion over 10 min of an amount of isotonic saline equal to 5% of body weight. MABP, RBF, and ARNA (in a separate group) were continuously monitored. Urine samples were collected from the left kidney at 5, 10, 20, 30, 45, 60, and 90 min after the start of infusion.

Renal pelvic mechanoreceptor stimulation and SP perfusion. Renal pelvic mechanostimulation was studied in the Sham (n = 12), 1-wk CBDL (n = 8), and 4-wk CBDL (n = 12) groups as described previously (26, 29). Because little is known about the chemical composition of the urine produced by cirrhotic rats, the renal pelvis in all groups was perfused throughout the experiment as described below, using saline at a rate of 20 µl/min, a perfusion rate that did not affect ureteral pressure. A PE-10 catheter with a heat-pulled tip was placed inside a PE-50 catheter extending beyond the tip of the PE-50 catheter. The tips of the two catheters were placed together in the left ureter near the renal pelvis, allowing the renal pelvis to be perfused via the PE-10 catheter, and the effluent was drained away by the PE-50 catheter. Changes in intrapelvic pressure (IPP) were recorded on a Gould polygraph with a transducer connected to the PE-50 ureteral catheter by a T tube connector. The third end of the T tube connector was connected to a 50-cm length of PE-50 tubing to increase the IPP by 2, 4, 8, 12, 16, or 20 mmHg, each value being maintained for 3 min, with 10-min intervals between changes.

The ARNA response was also tested by renal pelvic perfusion with graded concentrations of SP (1, 5, and 10 µg/ml) via the inserted PE-10 catheter, as described above. The experiment consisted of one 10-min basal period, three consecutive 10-min periods at the different SP concentrations, and one or two 10-min recovery periods.

Renorenal reflex test. The renorenal reflex was studied in Sham rats (n = 14), 1-wk CBDL rats (n = 10), and 4-wk CBDL rats (n = 14). The renal pelvic mechanoreceptors of the left kidney were stimulated by increasing the IPP by ~20 mmHg, and changes in ARNA were produced, as described above. Contralateral (right kidney) urine samples were collected. To assay SP in the renal pelvic effluent, the renal pelvis was perfused at 20 µl/min with saline containing 10 µmol/l of the endopeptidase inhibitor thiorphan (Sigma, St. Louis, MO), to minimize SP degradation (20, 29). The amount of SP in the renal pelvic effluent and in the plasma was determined by enzyme-linked immunoassay (29).

Immunoblotting of NK-1 receptors in the renal pelvis. After the physiological studies, the rats were killed, both kidneys were exposed, and the regions of the renal pelvis and the proximal end of the ureter were sampled to prepare membrane fractions, as described previously (28, 29). Membrane proteins (80 µg) were separated on 12% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Amersham-Pharmacia, Buckinghamshire, UK). Positive controls of rat ileal and brain cortical membranes (10 µg of protein) were run in parallel. After being blocked, the membranes were incubated overnight at 4°C with rabbit anti-NK-1 receptor antiserum (Novus Biologicals, Littleton, CO) diluted 1,000-fold. After washes, the membrane was incubated for 1 h at room temperature with goat anti-rabbit IgG conjugated to horseradish peroxidase (Vector Labs, Burlingame, CA) and then washed, and the bound antibody was visualized using a commercial peroxidase substrate kit (Vector Labs). The density of the band with a molecular mass of ~79 kDa was determined semiquantitatively by densitometry using an image-analysis system (Alpha Innotech, San Leandro, CA).

Quantitative real-time RT-PCR to measure NK-1 receptor mRNA levels. The theoretical basis of the real-time quantitative PCR and the methodology of the ABI PRISM 7700 Sequence Detection System (PerkinElmer Applied Biosystems, Foster City, CA) have been described by Johnson et al. (17). 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 for the NK-1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed, and total RNA extraction and the PCR procedure were performed, as previously described (28, 29). The NK-1 receptor primers and probe were 5'-GGC CAG AGG ACC AGA ACT TTT-3' (forward primer) 5'-GCT AGC AAC TCC CAC TAA CAT ACG T-3' (reverse primer) and 5'-6-carboxyfluorescein (FAM)-CAA GCA ACA CTG CAC TGC GAG CA-6-carboxy-tetramethylrhodamine (TAMRA)-3' (probe). The GAPDH primers and probe were 5'-TTT CTC GTG GTT CAC ACC CA-3' (forward primer), 5'-GTC ATC ATC TCC GCC CCT T-3' (reverse primer), and 5'-FAM-CGC TGA TGC CCC CAT GTT TGT G-TAMRA-3' (probe).

Chemical analysis and data treatment. In the acute or 24-h collection, urine volume was determined gravimetrically. The urinary sodium concentration was measured by flame photometry (FCM 6341, Eppendorf, Hamburg, Germany). Urinary and plasma p-aminohippurate levels were measured by the colorimetric method, and the hematocrit was determined to calculate the RBF, as previously described (5). The RBF, urinary flow rate (UV), and urinary sodium excretory rate (UNaV) were expressed per gram of kidney weight.

Systemic or renal hemodynamics and excretory functions were averaged over each period. ARNA was also averaged over each period, and the effects of acute saline loading and increased IPP on ARNA were calculated by comparing the experimental value with the average value for the control period (29).

The comparative CT (Delta Delta CT) method was used to quantify NK-1 receptor mRNA levels, as previously described (38). The calculation used was Delta Delta CT = [NK-1 receptor CT (unknown sample) - GAPDH CT (unknown sample)] - [NK-1 receptor CT (calibrator sample) - GAPDH CT (calibrator sample)], the degree of induction being equal to 2<SUP>−&Dgr;&Dgr;C<SUB>T</SUB></SUP>.

Data are expressed as means ± SE. Statistical analysis was performed using analysis of variance (Newman-Keuls test) for multiple comparisons and linear regression between groups. A significance level of 5% was chosen.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CBDL model. The 24-h urine output (ml/day) was similar in Sham and both CBDL groups (Sham: 12.9 ± 2.1; 1-wk CBDL: 14.7 ± 3.0; 4-wk CBDL: 9.1 ± 1.8 ml/day), whereas sodium excretion (ml/day) was significantly decreased in 4-wk CBDL group (Sham: 3.8 ± 0.2; 1-wk CBDL: 4.1 ± 0.4; 4-wk CBDL: 2.5 ± 0.1 ml/day). These results are consistent with those in a previous study (12). Table 1 shows the basal data for hepatic and renal functions in Sham rats and both groups of CBDL rats. Systemic hypotension was seen in the 4-wk, but not the 1-wk, CBDL rats. Significant increases in plasma SP levels were seen in both groups of CBDL rats. These results are consistent with those from a previous study by Chu et al. (9), who suggested that the major factor resulting in the plasma SP increase seen in cirrhosis appears to be the reduced ability of the liver to degrade SP rather than increased SP release from the splanchnic vascular bed. A higher liver weight-to-body weight ratio, accompanied by high biochemical indexes of hepatic function (bilirubin, AP, AST, and ALT), was also noted in both CBDL groups. The kidney weight-to-body weight ratio and baseline UV values for both CBDL groups were similar to those in Sham rats. The baseline RBF and UNaV values in 1-wk CBDL rats did not differ from those in Sham rats but were significantly lower in 4-wk CBDL rats.

                              
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Table 1.   Summary data for hepatic and renal parameters in Sham and CBDL rats

Acute saline loading. Figure 1 shows the response to acute saline loading. As shown in Fig. 1A (panel 1 and 2, respectively), the systemic MABP was significantly increased by saline loading in all groups, whereas the RBF was not significantly increased. The MABP and RBF values in the Sham and 1-wk CBDL groups were not significantly different but were lower in the 4-wk CBDL rats at all time points. The increase in blood pressure after saline loading in the 4-wk CBDL rats (36 ± 5%) was significantly greater than that in Sham rats (15 ± 4%). The UV (panel 3) and UNaV (panel 4) increased in all groups in response to saline loading. However, diuresis and natriuresis were both attenuated in the CBDL rats, and severe impairment was seen in 4-wk CBDL rats. In the 1-wk CBDL rats, the cumulative urine output and sodium excretion values were, respectively, 63 ± 5 and 78 ± 7% of those in Sham rats (P < 0.05) and fell to 46 ± 2 and 67 ± 8% of the Sham values in the 4-wk CBDL rats (P < 0.05). Cumulative urine and sodium excretion was also significantly different between the CBDL groups (P < 0.05). This difference in response was paralleled by a similar difference in ARNA activation. As shown in Fig. 1B, ARNA activation of the left kidney in all groups was significantly increased at 5 and 10 min after the start of acute saline loading and at 10 and 20 min after the end of loading; however, both groups of CBDL rats showed blunted ARNA responses.


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Fig. 1.   Responses to saline loading in the left kidney. A: changes in mean arterial blood pressure (MABP), renal blood flow (RBF), urinary flow rate (UV), and urninary sodium excretion rate (UNaV) in sham-operated (Sham) and rats after 1 and 4 wk of common bile duct ligation (CBDL). B: a separate experiment in Sham (n = 12), 1-wk CBDL (n = 8), and 4-wk CBDL rats (n = 12) showing the percent change (%Delta ) in afferent renal nerve activity (ARNA). VE, time of saline infusion. *Significantly different (P < 0.05) from time 0.

Stimulation of afferent renal nerve activity by mechanostimulation and SP. Raising the ureteral catheter to various levels above the left kidney increased the IPP and stimulated mechanosensitive neurons in the renal pelvis. Original tracings are shown in Fig. 2. Increases in the IPP resulted in a dose-dependent increase in ARNA in Sham rats (Fig. 2A), but this was reduced in 1-wk CBDL rats (Fig. 2B) and greatly attenuated in 4-wk CBDL rats (Fig. 2C). The RNA increase was reversed when the IPP returned to the basal level. The grouped data are shown in Fig. 2D. The threshold IPP for a significant ANRA increase in the test kidney was 4.7 ± 0.1 mmHg in Sham rats, 12.4 ± 0.3 mmHg in 1-wk CBDL rats, and 19.2 ± 0.3 mmHg in 4-wk CBDL rats. At pressures below 16 mmHg, the IPP-induced ARNA increase in 1-wk CBDL rats was significantly lower than that in Sham rats. At the highest pressure, the increase in the ARNA in 4-wk CBDL rats was significantly reduced compared with that in Sham rats and 1-wk CBDL rats.


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Fig. 2.   Effects of graded increases in intrapelvic pressure (IPP) on ipsilateral ARNA in Sham (A) and 1-wk (B) and 4-wk (C) CBDL rats. A: original traces showing the effect of renal mechanoreceptor stimulation by graded IPP increases on integrated multifiber ARNA. D: grouped data for the effect of graded IPP increases on ipsilateral ARNA. *Significantly different (P < 0.05) from initial value.

Administration of exogenous SP into the renal pelvis was used to directly test whether the impaired ARNA activation in CBDL rats might be related to SP receptor function. Figure 3A shows typical traces of the effect of intrapelvic SP perfusion on ARNA. SP caused an increase in ARNA in Sham rats (top trace); this response was attenuated in both 1-wk (middle trace) and 4-wk (bottom trace) CBDL rats. As with the mechanoreceptors, the increase in ARNA was reversible when SP perfusion was stopped. The grouped data are shown in Fig. 3B. In both Sham and 1-wk CDBL rats, graded increases in SP resulted in a concentration-dependent increase in the ARNA, whereas a severely blunted response was seen in 4-wk CBDL rats. At the SP dose of 1 µg/ml, the 1-wk and 4-wk CBDL rats showed a significantly attenuated ARNA increase compared with Sham rats. At 5 and 10 µg/ml of SP, although the 1-wk CBDL rats displayed a lower ARNA increase than Sham rats, the difference was not statistically significant, whereas 4-wk CBDL rats showed a very low ARNA increase.


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Fig. 3.   Effects of graded SP concentrations on the ipsilateral ARNA in Sham and 1-wk and 4-wk CBDL rats. A: original traces showing the effect of renal chemoreceptor stimulation by intrapelvic perfusion with graded SP concentrations (horizontal bars) on integrated multifiber ARNA. B: grouped data for the effect of SP on the ipsilateral ARNA. *Significantly different (P < 0.05) from the initial value.

Renorenal reflex and SP release. Figure 4 shows that stimulation of renal mechanoreceptors elicited an inhibitory renorenal reflex. The MABP in all groups was unchanged during the increased IPP and recovery periods (data not shown). In Sham rats (left), an increase in IPP in the ipsilateral kidney to ~20 mmHg resulted in significant increases in the contralateral kidney of the UV and the UNaV, which were accompanied by an increase in ipsilateral ARNA. The 1-wk CBDL rats also displayed a reflex response (middle) with a similar IPP increase, the contralateral UV and UNaV and the ipsilateral ARNA being significantly increased. In contrast, contralateral diuretic and natriuretic responses were not seen in 4-wk CBDL rats (right), despite a moderate increase of 40 ± 8% in ARNA.


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Fig. 4.   Renorenal reflex and renal pelvic substance P (SP) release from the left kidney in Sham and 1-wk and 4-wk CBDL rats. UV and UNaV values are from the contralateral right kidney (A); Delta ARNA, change in the afferent renal nerve activity recorded in the left kidney (B); IPP, increased intrapelvic pressure in the left kidney; Rec, recovery period. *Significantly different (P < 0.05) from the basal state. dagger Significantly different (P < 0.05) from the Sham group.

As shown in Fig. 4 (bottom), the basal values for renal pelvic ipsilateral (left side) SP release did not differ significantly between the groups. An increase in IPP resulted in significant increases in SP release in all three groups, with no significant difference between the groups. SP release returned to the basal level after the IPP was reduced.

Immunoblotting of NK-1 receptors in the renal pelvis. As shown in Fig. 5A, in rat ileal and brain cortical membranes, the anti-NK-1 receptor antiserum recognized a protein band with a molecular mass of 79 kDa (lanes 1 and 2). Renal pelvic tissue membranes from six animals from each group showed a band with the same molecular mass as that in the brain cortical and ileal membranes, this band being weaker in the CBDL rat membranes than in those from Sham rats. Figure 5B shows the semiquantitative data; the band density in Sham rats was significantly higher than the values in 1-wk CBDL rats and in 4-wk CBDL rats. Figure 5C shows that there was a strong negative correlation between plasma SP levels and renal pelvic NK-1 receptor expression in the three groups (P < 0.05).


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Fig. 5.   A: Western blots of neurokinin 1 (NK-1) receptor expression in renal pelvic membranes. Membranes were prepared from 6 animals each from the Sham (lanes 3-8), 1-wk CBDL (lanes 9-14), and 4-wk CBDL (lanes 15-20) groups; lanes 1 and 2, 2 internal positive controls of rat ileum and brain cortex, respectively. Protein staining showed that equal amounts of protein were loaded in lanes 3-20 (data not shown). Lane M: prestained low-molecular-mass standards (Bio-Rad, Burlingame, CA) consisting of phosphorylase B (106 kDa), BSA (81 kDa), ovalbumin (47.5 kDa), carbonic anhydrase (35.3 kDa), soybean trypsin inhibitor (28.2 kDa), and lysozyme (20.8 kDa). B: semiquantitative densitometry showing decreased NK-1 receptor expression as a function of duration of CBDL. *Significantly different (P < 0.05) from the Sham group. C: relationship between plasma SP levels and renal pelvic NK-1 receptor expression in 6 rats/group.

Quantification of NK-1 receptor mRNA expression. The raw data for the CT values for NK-1 receptor and GAPDH mRNAs, the calculated Delta Delta CT values, and the degree of induction of NK-1 receptor mRNA in the Sham and 4-wk CBDL groups are shown in Table 2. The CT results for the housekeeper GAPDH mRNA from a variety of samples were similar, indicating the high precision of the quantitative real-time RT-PCR. The CT values showed that NK-1 receptor mRNA was expressed in the renal pelvis in both groups, and the values for the degree of induction of renal pelvic NK-1 receptor mRNA, relative to the ileum, showed no significant difference between the groups.

                              
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Table 2.   Analysis of NK-1 receptor mRNA expression in Sham and 4-wk CBDL rats using the comparative CT method


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Temporal decrease in renal excretion and sensory nerve activity in CBDL rats. In response to saline loading, the CBDL rats had an impaired renal sodium and urine excretory capacity. These alterations were very similar to those seen in various rat models of cirrhosis (2, 12, 33). Plausible mechanisms include enhanced renal sympathetic nerve activity (10, 12, 39), atrial natriuretic peptide (ANP) resistance (33), and a reduced sensitivity of the pressure-diuretic response (1). Our present results agree with these mechanisms. First, when we simultaneously recorded efferent renal nerve activity and ARNA in 4-wk CBDL rats (not shown), we found that the ARNA activation defect seen during acute saline loading was accompanied by increased efferent renal nerve activity as a result of less reflex inhibition. Second, acute saline loading is known to stimulate endogenous ANP release, and ANP resistance in CBDL rats is suggested to result from increased cGMP-dependent phosphodiesterase activity in the cirrhotic kidney (33). Interestingly, we also noted that, during acute saline loading, the increase in the blood pressure in 4-wk CBDL rats was greater than that in Sham rats (Fig. 1A). However, because of insufficient pressure-diuresis, the cirrhotic kidney could not excrete sodium and water efficiently in response to the increased arterial pressure; this may be due to intrarenal factors, rather than to the effect of reduced systemic blood pressure (1).

Atucha et al. (2) also showed that the blunted volume expansion response in cirrhotic rats might result from an attenuated increase in renal interstitial hydrostatic pressure. This raises the possibility that the lower excretory response and the attenuated increase in ARNA after saline loading in our cirrhotic rats may be related, at least in part, to impaired transmission of intrarenal pressure, caused by hypoperfusion of the kidney. Despite the lower MABP in 4-wk CBDL rats than in the Sham group throughout the experiment, we can rule out an effect of renal hypoperfusion on the ARNA response, because, although the 1 wk-CBDL rats (i.e., early stage of cirrhosis) showed a similar change in MABP to that in Sham rats, their renal excretion and ARNA activation by saline loading were already affected (Fig. 1A). This suggested that some intrarenal factors, rather than systemic hypotension, might contribute to impaired renal excretory response in cirrhotic kidneys.

Normally, body fluid expansion activates the renal afferent nerves, which trigger a diuretic signal for reflex inhibition of the renal sympathetic nerve, resulting in diuresis and natriuresis, thereby overcoming the fluid imbalance. Because we found that acute saline loading resulted in reduced excretory ability and lower activation of renal sensory nerves and these effects increased with the duration of CBDL (Fig. 1B), these changes seem to be temporal events during the pathogenesis of cirrhosis. Taking these results together, we conclude that attenuated renal sensory activation contributes to impaired renal excretory function and worsens fluid retention in cirrhosis, especially under fluid-overloading conditions.

In the rat kidney, the majority of renal sensory neurons are found in the renal pelvis (14), and their responses can be evaluated using specific stimuli, namely, graded increases in IPP (26). As previously reported (20, 26), mechanostimuli resulted in dose-dependent ARNA responses in the Sham rats, whereas CBDL rats displayed blunted responses, the extent of which was dependent on the duration of CBDL, with a shift of the threshold for ARNA activation to a higher pressure (Fig. 2B). These results directly show a temporal deficiency in mechanosensitive response and also support the observation of defective ARNA responses on saline loading.

SP-mediated renal mechanoreceptor activation can be blocked by a SP receptor antagonist (22), suggesting a role for the SP receptor in the renal sensory response. Our results showing that SP increased ARNA in control rats are similar to those of a previous study (20), and the fact that these responses were attenuated in both CBDL groups (Fig. 4B) suggests a deficiency in the action of SP on NK-1 receptors involved in renal pelvic sensory function. Although the ARNA increase seen in 1 wk-CBDL rats at the SP dose of 10 µg/ml was not significantly different from that in Sham rats (Fig. 4B), which can be explained by an excess of SP, at SP doses of 1 and 5 µg/ml the temporal defect in ARNA responses in both CBDL groups was apparent.

Renorenal reflex: role of SP. The existence of a contralateral inhibitory renorenal reflex in rats was initially demonstrated by activating renal mechanoreceptors (21). This mechanoreceptor-induced renorenal reflex is mediated by SP and abolished by capsaicin pretreatment (24). SP is found at high concentrations in the spinal cord, where it plays a role as a transmitter (34), and the presence of SP in the area of the renal pelvis also suggests a role in renal sensory transmission (14). Our results showed that 1-wk CBDL rats displayed a reflex function after an ipsilateral ARNA increase, but the percent changes in the increase in contralateral urinary (1-wk CBDL vs. Sham: 49 ± 7 vs. 20 ± 8%) and sodium (1-wk CBDL vs. Sham: 98 ± 11 vs. 60 ± 6%) excretion were still significantly diminished. The temporal changes in renorenal reflex function in CBDL rats led us to suspect that SP secretion in the renal pelvis would be decreased, but, in fact, a higher, although statistically insignificant, amount of SP was released in CBDL rats on mechanoreceptor stimulation compared with in Sham rats. Therefore, it seems that the extent of renal pelvic SP release is not related to the impaired renorenal reflex response in cirrhotic rats.

NK-1 receptors in the renal pelvis. Vigna et al. (37) have demonstrated the specificity of the antiserum used in the present study and shown that, in Western blot analysis, the anitserum recognizes rat NK-1 receptors with molecular masses ranging from 80 to 90 kDa. Our results show that the NK-1 receptor is expressed in the renal pelvis, the pattern of expression being similar to that seen for NK-1 receptors in the ileum and brain cortex. NK-1 receptor expression was lower in the 1-wk CBDL group (~68% of that in Sham rats) and was again reduced in the 4-wk CBDL group (~53% of that in the Sham group).

Although we did not study in detail the mechanism responsible for the reduced expression of NK-1 receptors in cirrhosis, the higher plasma SP concentration seems to indicate downregulation of NK-1 receptors in CBDL rats (Fig. 5C). The quantitative RT-PCR analysis showed the expression of renal pelvic NK-1 receptor mRNA to be similar in the Sham and 4-wk CBDL groups, ruling out the possibility of an effect of NK-1 receptor mRNA levels on receptor protein expression in the cirrhotic renal pelvis. The reduction in NK-1 receptors could be due to an increase in plasma SP levels in CBDL rats. Previous studies have examined the effect of SP-evoked desensitization or internalization of NK-1 receptors in vitro in rat kidney epithelial cells (36) or other cell lines (15, 16) and in vivo in tracheal endothelial cells (3), ileal myenteric neurons (30), spinal neurons (32), and the striatum (31). Further studies are needed to evaluate the roles of SP-mediated regulation in renal pelvic NK-1 receptor expression and in the renal sensory response in the cirrhotic kidney.

In summary, the present study shows that renal sensory impairment in CBDL rats is a time-dependent event, the defect being most severe at the cirrhotic stage. The renal mechanoreceptor-mediated inhibitory renorenal reflex is attenuated in cirrhotic rats, in which a temporal reduction in renal pelvic NK-1 receptors, but no change in renal pelvic SP release, is seen. This reduction is strongly correlated with increased plasma SP levels, but it is not due to altered mRNA expression. Taking these results together, we conclude that the physiological role of decreased renorenal reflex in CBDL rats may contribute to the overall dysfunction of sodium and water handling present in cirrhosis.


    ACKNOWLEDGEMENTS

We thank Dr. Y. C. Lin, Hawaii University, for carefully reading the manuscript.


    FOOTNOTES

The present study was supported by grants from the National Science Council of the Republic of China (NSC89-2320-B002-123) and the Liver Disease Prevention and Treatment Research Foundation.

Address for reprint requests and other correspondence: C. F. Chen, Dept. of Physiology, College of Medicine, National Taiwan University, No. 1, Sect. 1, Jen-Ai Rd., Taipei, Taiwan (E-mail: chfochen{at}ha.mc.ntu.edu.tw).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 19, 2002;10.1152/ajprenal.00231.2001

Received 30 July 2001; accepted in final form 8 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Atucha, NM, Cegarra M, Ramirez A, Quesada T, and Garcia-Estan J. Pressure diuresis and natriuresis in cirrhotic rats. Am J Physiol Gastrointest Liver Physiol 265: G1045-G1049, 1993[Abstract/Free Full Text].

2.   Atucha, NM, Quesada T, and Garcia-Estan J. Reduced renal papillary plasma flow in non-ascitic cirrhotic rats. Clin Sci (Colch) 85: 139-145, 1993[ISI][Medline].

3.   Bowden, JJ, Garland AM, Baluk P, Lefevre P, Grady EF, Vigna SR, Bunnett NW, and McDonald DM. Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation. Proc Natl Acad Sci USA 91: 8964-8968, 1994[Abstract].

4.   Chen, CF. Renal functional response to ischaemic renal failure in chronic hypoxic rats. Clin Sci (Colch) 85: 123-127, 1993[ISI][Medline].

5.   Chen, CF, Chen LW, Chien CT, Wu MS, and Tsai TJ. Renal kallikrein in chronic hypoxic rats. Clin Exp Pharmcol Physiol 23: 819-824, 1996[ISI][Medline].

6.   Chen, Y, and Hoover DB. Autoradiographic localization of NK1 and NK3 tachykinin receptors in rat kidney. Peptides 16: 673-681, 1995[ISI][Medline].

7.   Chien, CT, Chien HF, Cheng YJ, Chen CF, and Hsu SM. Renal afferents signaling diuretic response is impaired in streptozotocin-induced diabetic rats. Kidney Int 57: 203-214, 2000[ISI][Medline].

8.   Chien, CT, Fu TC, Wu MS, and Chen CF. Attenuated response of renal mechanoreceptors to volume expansion in chronically hypoxic rats. Am J Physiol Renal Physiol 273: F712-F717, 1997[Abstract/Free Full Text].

9.   Chu, CJ, Lee FY, Wang SS, Chang FY, Tsai YT, Lin HC, Hou MC, Wu SL, Tai CC, and Lee SD. Hyperdynamic circulation of cirrhotic rats: role of substance P and its relationship to nitric oxide. Scand J Gastroenterol 32: 841-846, 1997[ISI][Medline].

10.   DiBona, GF, Herman PJ, and Sawin LL. Neural control of renal function in edema-forming states. Am J Physiol Regulatory Integrative Comp Physiol 254: R1017-R1024, 1988[Abstract/Free Full Text].

11.   DiBona, GF, and Kopp UC. Neural control of renal function. Physiol Rev 77: 75-165, 1997[Abstract/Free Full Text].

12.   DiBona, GF, and Sawin LL. Role of renal nerves in sodium retention of cirrhosis and congestive heart failure. Am J Physiol Regulatory Integrative Comp Physiol 260: R298-R305, 1991[Abstract/Free Full Text].

13.   DiBona, GF, Susan Y, and Kopp UC. Renal mechanoreceptor dysfunction. An intermediate phenotype in spontaneously hypertensive rats. Hypertension 33: 472-475, 1999[Abstract/Free Full Text].

14.   Ferguson, M, and Bell C. Ultrastructural localization and characterization of sensory nerves in rat kidney. J Comp Neurol 247: 9-16, 1988.

15.   Garland, AM, Grady EF, Lovett M, Vigna SR, Frucht MM, Krause JE, and Bunnett NW. Mechanisms of desensitization and resensitization of G protein-coupled neurokinin 1 and neurokinin 2 receptors. Mol Pharmacol 49: 438-446, 1996[Abstract].

16.   Garland, AM, Grady EF, Payan DG, Vigna SR, and Bunnett NW. Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells. Biochem J 303: 177-186, 1994[ISI][Medline].

17.   Johnson, MR, Wang K, Smith JB, Heslin MJ, and Diasio RB. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem 278: 175-184, 2000[ISI][Medline].

18.   Koepke, JP, Jones S, and DiBona GF. Renal nerves mediate blunted natriuresis to atrial natriuretic peptide in cirrhotic rats. Am J Physiol Regulatory Integrative Comp Physiol 252: R1019-R1023, 1987[Abstract/Free Full Text].

19.   Kopp, UC, and Buckley-Bleiler RL. Impaired renorenal reflexes in two-kidney, one-clip hypertensive rats. Hypertension 14: 445-452, 1989[Abstract].

20.   Kopp, UC, Cicha MZ, Farley DM, Smith LA, and Dixon BS. Renal substance P-containing neurons and substance P receptors impaired in hypertension. Hypertension 33: 815-822, 1998.

21.   Kopp, UC, Olson LA, and DiBona GF. Renorenal reflex responses to mechano- and chemoreceptor stimulation in the dog and rat. Am J Physiol Renal Fluid Electrolyte Physiol 246: F67-F77, 1984[Abstract/Free Full Text].

22.   Kopp, UC, and Smith LA. Effects of the substance P receptor antagonist CP-96,345 on renal sensory receptor activation. Am J Physiol Regulatory Integrative Comp Physiol 264: R647-R653, 1993[Abstract/Free Full Text].

23.   Kopp, UC, and Smith LA. Bradykinin and protein kinase C activation fail to stimulate renal sensory neurons in hypertensive rats. Hypertension 27: 607-612, 1996[Abstract/Free Full Text].

24.   Kopp, UC, and Smith LA. Inhibitory renorenal reflexes: a role for substance P or other capsaicin-sensitive neurons. Am J Physiol Regulatory Integrative Comp Physiol 260: R232-R239, 1991[Abstract/Free Full Text].

25.   Kopp, UC, and Smith LA. Renorenal reflexes present in young and captopril-treated adult spontaneously hypertensive rats. Hypertension 13: 430-439, 1989[Abstract].

26.   Kopp, UC, Smith LA, and Pence AL. Na+-K+-ATPase inhibition sensitizes renal mechanoreceptors activated by increases in renal pelvic pressure. Am J Physiol Regulatory Integrative Comp Physiol 267: R1109-R1117, 1994[Abstract/Free Full Text].

27.   Kountouras, J, Billing BH, and Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65: 305-311, 1984[ISI][Medline].

28.   Lai, YL, Chu SJ, Ma MC, and Chen CF. Temporal increase in the reactivity of pulmonary vasculature to substance P in chronically hypoxic rats. Am J Physiol Regulatory Integrative Comp Physiol 282: R858-R864, 2002[Abstract/Free Full Text].

29.   Ma, MC, Huang HS, and Chen CF. Impaired renal sensory responses after unilateral ureteral obstruction in the rat. J Am Soc Nephrol 13: 1008-1016, 2002[Abstract/Free Full Text].

30.   Mann, PT, Southwell BR, and Furness JB. Internalization of the neurokinin 1 receptor in rat myenteric neurons. Neuroscience 91: 353-362, 1999[ISI][Medline].

31.   Mantyh, PW, Allen CJ, Ghilardi JR, Rogers AD, Mantyh CR, Liu H, Basbaum AI, Vigna SR, and Maggio JE. Rapid endocytosis of a G protein-coupled receptor: substance P-evoked internalization of its receptor in the rat striatum in vivo. Proc Natl Acad Sci USA 92: 2622-2626, 1995[Abstract].

32.   Mantyh, PW, DeMaster E, Malhotra A, Ghilardi JR, Rogers SD, Mantyh CR, Liu H, Basbaum AI, Vigna SR, Maggio JE, and Simone DA. Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation. Science 268: 1629-1632, 1995[ISI][Medline].

33.   Ni, X, Cheng Y, Cao L, Gardner DG, and Humphreys MH. Mechanisms contributing to renal resistance to atrial natriuretic peptide in rats with common bile-duct ligation. J Am Soc Nephrol 7: 2110-2118, 1996[Abstract].

34.   Quartara, L, and Maggi CA. The tachykinin NK1 receptors. Part II: Distribution and pathophysiological roles. Neuropeptides 32: 1-49, 1998[ISI][Medline].

35.   Vallance, P, and Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide? Lancet 337: 776-778, 1991[ISI][Medline].

36.   Vigna, SR. Phosphorylation and desensitization of neurokinin-1 receptor expressed in epithelial cells. J Neurochem 73: 1925-1932, 1999[ISI][Medline].

37.   Vigna, SR, Bowden JJ, McDonald DM, Fisher J, Okamoto A, McVey DC, Payan DG, and Bunnett NW. Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells. J Neurosci 14: 834-845, 1994[Abstract].

38.   Winer, J, Jung CKS, Shackel I, and Williams PM. Development and validation of real-time reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270: 41-49, 1999[ISI][Medline].

39.   Zambraski, EJ. Renal nerves in renal sodium-retaining states: cirrhotic ascites, congestive heart, nephrotic syndrome. Miner Electrolyte Metab 15: 88-96, 1989[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 283(1):F164-F172
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society




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