First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan
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Abstract |
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Methods. Ischaemic renal injury was provoked by 60-min left renal artery occlusion (RAO). Removal of the right kidney was performed just before RAO in the Nx group. Forty-eight hours after release of the clamp, renal ET-1 content was measured in both non-nephrectomized and unilaterally nephrectomized rats. We also examined the effects of a selective ETA receptor (FR139317) and monoclonal ET antibody (AwETN40) on the RAO-induced changes in renal haemodynamics at 2 and 48 h after RAO respectively.
Results. The plasma concentration of ET-1 did not change in the two groups of ARF rats, but the cortical content of ET-1 increased to a lesser extent in Nx animals after ischaemia. Prior removal of the right kidney significantly facilitated the percentage recovery of left renal blood flow (RBF) during the first 2 h after release of the clamp. The percentage recovery of inulin clearance (Cin) by the kidney was also significantly better in Nx than sham-Nx rats at 48 h after RAO. Continuous administration of FR139317 (50 mg/kg/day) using osmotic minipumps for 3 days significantly attenuated exogenous ET-1-induced decrease in Cin and RBF. Infusion of FR139317 restored the decrease in RBF to control values during first 2 h in sham-Nx rats. However, FR139317 and AwETN40 did not ameliorate the RAO-induced decline of Cin in sham-Nx or Nx rats at 2 and 48 h after ischaemia respectively.
Conclusions. Contralateral uninephrectomy prior to ischaemia-induced ARF attenuated the increase in cortical ET-1 content and subsequent renal response to ischaemic injury. This beneficial effect of unilateral nephrectomy, however, was not mediated through well-preserved RBF due to reduced intrarenal ET-1 action.
Keywords: endothelin-1; ETA receptor antagonist; monoclonal antibody; uninephrectomy
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Introduction |
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Endothelin-1 (ET-1), a 21-amino-acid peptide, is one of the most potent vasoconstrictors. Exogenous administration of ET-1 acutely induces a decline in renal blood flow (RBF) and glomerular filtration rate (GFR) [7]. Recent studies suggest the importance of enhanced renal production of ET-1 in the pathogenesis of ischaemic acute renal failure (ARF). For example, plasma ET-1 levels are elevated in ARF patients, and renal ischaemia increases renal ET-1 content [8,9] and ET receptor affinity [10,11]. Wilhelm et al. [12] noted that renal artery occlusion without reperfusion increased prepro-ET-1 mRNA synthesis by about fivefold in the kidney. ET-1 peptide expression is found in the cortical peritubular capillaries of the post-ischaemic kidney [12]. Moreover, several investigators have noted that exogenous monoclonal or polyclonal antibody to ET [7,8,13,14], ETA receptor antagonists [9,1518], or ETA/ETB dual receptor antagonists [1921] ameliorated declines in GFR and tubular damage in the setting of ischaemiareperfusion injury. Therefore it is possible that reduced severity of post-ischaemic renal injury in uninephrectomized animals is related to an attenuation of renal ET-1 production. It remains to be clarified, however, whether ET-1 production in the post-ischaemic kidney is modified by previous contralateral nephrectomy.
The present work was designed to determine whether contralateral nephrectomy lessens an increase in ET-1 production in the post-ischaemic kidney of the rat, and if so, whether reduced severity of post-ischaemic renal injury in uninephrectomized rats is due to attenuated ET-1 production. We measured renal ET-1 content in both non-nephrectomized and right-nephrectomized rats. The results indicate that increases in the cortical ET-1 content in the post-ischaemic kidney were less pronounced in uninephrectomized rats compared with sham-nephrectomized animals. Exogenous administration of FR139317, an ETA receptor antagonist, restored a decline in RBF during the first 2 h after release of the clamp. However, neither FR139317 nor monoclonal ET antibody improved the recovery of GFR after renal ischaemia in uninephrectomized or in sham-nephrectomized rats.
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Subjects and methods |
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Renal blood flow measurements and clearance studies
Clearance studies were performed in 115 rats taken from all groups. Renal blood flow (RBF) and inulin clearances (Cin) were estimated in 29 sham-Nx and 29 Nx rats during the first 2 h (group 2, n=14; group 4, n=14) or at 48 h (group 2, n=15; group 4, n=15) following renal-artery clamping. Thirty-one rats from group 1 and 26 animals from group 3 were used as controls both at 2 h (group 1, n=14; group 3, n=14) and 48 h (group 1, n=17; group 3, n=12) after sham-RAO. Animals were anaesthetized with intraperitoneal sodium pentobarbital (50 mg/kg) and tracheotomy was performed. A carotid arterial catheter was used for blood sampling and for blood-pressure monitoring using a pressure transducer (MPU 05A, Nihon Kohden, Tokyo, Japan). RBF was measured with an electromagnetic flowmeter (MFV-1200, Nihon Kohden) placed on the left renal artery. All transducers were connected to appropriate amplifiers in a pen-writing recorder (WI-180M, Nihon Kohden). To estimate GFR in the left kidney, [14C]inulin saline solution (1 µCi/ml) was infused at a constant rate (20 µl/min) through a jugular vein catheter. A catheter was also inserted into the left ureter for urine sampling. After a 40-min equilibration period, two 10-min clearance studies were performed. Blood samples were collected at the mid-point of the clearance period. For measurements of [14C]inulin in blood and urine, 50 µl of the blood sample and 10 µl of the urine sample were added to 10 ml of ACS® (Amersham Corporation, Canada), and radioactivity was determined in a liquid scintillation counter (LS5000TS, Beckman, Chicago, USA). Average values of RBF and Cin during the two clearance periods were expressed as ml/min/kidney and as a percentage of their respective controls.
ETA receptor antagonist studies
To elucidate the contribution of ET-1 to post-ischaemic renal injury, the effects of FR139317 (Fujisawa Pharmaceutical Co., Osaka, Japan), an ETA receptor antagonist [22], and AwETN40 (Tsukuba Research Laboratories, Takeda Chemical Industries, Osaka, Japan), a monoclonal ET antibody were examined in sham-Nx plus RAO and Nx plus RAO rats. FR139317 (1 mg/kg, i.v.) has been shown to inhibit the exogenous ET-1 (3.2 µg/kg, i.v.)-induced elevation in blood pressure [22]. To determine an adequate dose of FR139317 to reduce MAP and ameliorate renal function, we first examined the effect of FR139317 (5 mg/kg in a bolus and then 0.1 mg/kg/min) on ET-1 (1.2 nmol/kg, i.v.)-induced changes in GFR and RBF in five normal rats. Intravenous infusions of FR139317 completely prevented the ET-1-induced increase in MAP and decrease in RBF and Cin at 15 min after injection (data not shown). Therefore, seven rats per group were treated with the same protocol of an i.v. bolus of FR139317 (5 mg/kg) 5 min before clamping and then infused thereafter at a constant rate (0.1 mg/kg/min). The other seven rats were treated with vehicle (isotonic saline). In these animals, renal haemodynamic and clearance studies were performed before and during the first 2 h after renal-artery clamping.
We also examined the long-term effect of continuous administration of FR139317 for 3 days during ET-1-induced alterations in renal haemodynamics. An s.c. infusion of the agent (50 mg/kg/day) using an osmotic minipump (Alzet 1003D, Alza Co., Palo Alto, USA) was started 24 h before the clamping and was continued until the end of the clearance studies. This administration of FR139317 significantly attenuated the increase in MAP and decrease in RBF and Cin after i.v. injection of ET-1 of 0.080.4 nmol/kg (Figure 1). Thus, five rats in group 2 and seven rats in group 4, in which clearance studies were carried out 48 h after the clamping, were treated with s.c. continuous FR139317 for 3 days. As ARF controls, another six rats in group 2 and five rats in group 4 were treated with vehicle (dimethyl sulphoxide, DMSO) alone.
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Anti-ET antibody studies
AwETN40 is a monoclonal antibody against the N-terminal loop domain of ET-1 and ET-2. Since a dose of 33 nmol/kg of AwETN40 was sufficient to protect against azotaemia caused by intravenous ET-1 (1.0 nmol/kg) in normal rats [8], we administered this dose i.v. four times according to the regimen of Shibouta et al. [8]: 5 min before renal-artery clamping, and 5 min, 1 h, and 5 h after release of the clamp in three rats from groups 2 and 4. Control sham-RAO rats received four identical i.v. injections of saline (group 1, n=9; group 3, n=7). RBF and Cin measurements were carried out 48 h after the clamping.
Measurements of ET-1
An additional 31 rats from groups 1, 2 and 4 were used for the measurements of blood, urinary, and renal ET-1 concentrations (group 1, n=8; group 2, n=7; group 4, n=8). Forty-eight hours after sham operation or RAO, blood was drawn into plastic tubes containing EDTANa (1.5 mg/ml) for the measurement of plasma ET-1. The left kidney was then excised and immediately frozen in liquid nitrogen. For the measurement of renal ET-1 content, the kidney was divided into cortical and medullary portions and slices from each portion were homogenized in four volumes of chloroform:methanol (2:1) at 4°C for 4 min. Then 0.2 volume of water was added to the homogenate and the mixture was centrifuged at 2500 g for 25 min at 4°C. The aqueous phase was used for the measurement of ET-1. To estimate urinary excretion of ET-1, urine sample from 48 h after RAO were collected for 5 to 12 h from indwelling left ureter catheters in 23 rats (group 1, n=8, group 2, n=7, group 3, n=8). Plasma, kidney, and urine samples were frozen at -80°C until the assay. The ET-1 concentration in the sample was measured by specific radioimmunoassay.
Statistical analysis
All data are expressed as means±SE. Comparisons between two groups were performed using unpaired Student t-test. For multiple comparisons, analysis of variance (ANOVA) followed by protected Student's t-test were used.
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Results |
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Renal haemodynamic and clearance studies
After release of renal artery occlusion, RBF returned to near basal levels (3.6±0.4 ml/min/kidney, n=7) within the first hour in rats from group 4 (% recovery of RBF, 87.3±6.1% of basal levels, Figure 3A). Renal vascular resistance (RVR) was also returned to 120.7±10.3% of basal values within the first 2 h. Urine output was gradually increased from 3.3±0.8 to 8.8±3.1 µl/min at 2 h after reperfusion. In contrast, in group 2, the percentage recovery of RBF was significantly less (63.9±6.1% of basal values, P<0.05, vs group 4, n=7, Figure 3A
). RVR also remained increased at 2 h after clamp release (149.5±22.1% of basal values, P<0.05, vs group 4). At this early stage, Cin was reduced from 1.08±0.15 to 0.21±0.06 ml/min/kidney in group 2 and from 1.01±0.09 to 0.31±0.08 ml/min/kidney in group 4 at 2 h after ischaemia. The percentage recovery of Cin was higher in group 4 than in group 2, but the difference did not reach statistical significance (group 2, 19.9±6.4, vs group 4, 36.0±10.6% of basal values, P=NS, Figure 3B
).
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Effects of FR139317 on ischaemic ARF
The intravenous infusion of FR139317 facilitated the percentage recovery of RBF to control levels within 15 min and maintained RBF for 2 h after release of the clamp in group 2 (n=7) but not group 4 (n=7) (Figure 3A). In contrast, FR139317 did not accelerate the recovery of Cin in either ARF group at this initial stage (Figure 3B
). Continuous administration of FR139317 for 3 days did not change RBF or Cin at 48 h after RAO (Table 1
). The percentage recovery of RBF of respective sham-RAO controls by FR139317 was more evident in group 2 (69.6±5.3 vs 87.2±7.5%, n=6, P=0.08) compared with group 4 (85.9±10.9 vs 91.2±4.6%, n=5, P=NS) at 48 h after ischaemia. In contrast, FR139317 did not accelerate the percentage recovery of Cin at 48 h after RAO in groups 2 (0.3±0.2 vs 1.3±1.2%, n=5, P=NS) or 4 (6.9±2.6 vs 14.0±7.0%, n=7, P=NS) respectively.
Effect of AwETN40 on ischaemic ARF
The administration of AwETN40 did not change RBF in sham-Nx plus RAO rats (group 1, 3.8±0.2, n=9, vs group 2, 2.6±1.0 ml/min/kidney, n=3, P=NS) or in Nx plus RAO animals (group 3, 4.8±0.4, n=7, vs group 4, 5.5±0.3 ml/min/kidney, n=3, P=NS) at 48 h after clamping. Moreover, AwETN40 did not ameliorate the decline in Cin either in sham-Nx groups (group 1, 0.076±0.052 vs group 2, 0.019±0.015 ml/min/kidney, P=NS) or in Nx groups (group 3, 0.412±0.074 vs group 4, 0.167±0.076 ml/min/kidney, P=NS).
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Discussion |
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To assess this possibility, we first treated rats with intravenous FR139317, a highly selective ETA receptor antagonist [22], before, during, and after renal artery clamping. At the initial stage of ARF, intravenous FR139317 accelerated the recovery of RBF but not Cin in post-ischaemic kidneys from sham-Nx plus RAO rats. In addition, continuous administration of the agent did not ameliorate the decline in Cin at 48 h after the clamping either in sham-Nx plus RAO or in Nx plus RAO animals, despite the well-maintained RBF. These data suggest that the action of local ET-1 on ETA receptors seems not to play a fundamental role in producing and maintaining ischaemic ARF.
Renal ischaemia also up-regulates the expression of ETB receptors in the kidney [10,11]. Since the ETB receptor predominantly mediates vasoconstrictive actions of ET-1 in rat kidney [23], there is a possibility, that the action of ET-1 on ETB receptors may play an important role in producing and maintaining ischaemic ARF. Therefore, we next employed a monoclonal ET antibody (AwETN40) to block the action of ET-1 using the regimen used by Shibouta et al. [8], because the effect of selective ETB receptor antagonists [24] or ETA/ETB receptor antagonists [19] on ischaemic ARF is absent or limited. However, our exogenous anti-ET monoclonal antibody did not improve, but rather aggravated the decline in Cin at 48 h after renal artery clamping in the present study.
The exact reasons for the discrepancies between the present study and previous studies remain unknown. It is unlikely, however, that the doses of ETA receptor antagonist that we employed were insufficient to block the effect of ET-1, since we confirmed that continuous FR139317 administration via osmotic minipump for 3 days prevented the exogenous ET-1-induced decreases in RBF and Cin (Figure 1). In addition, we had previously found that thromboxane A2 synthetase inhibitor attenuated ischaemic renal injury in this model, indicating that our ARF model can be treatable. Thus, the present data may indicate a minor role for ET-1 in uninephrectomy-induced attenuation of ischaemic injury rather than the problem of experimental protocols.
There are notable differences between previous ischaemic ARF models and experimental protocols and the present study. Previous studies demonstrating the effectiveness of ETA receptor antagonist or anti-ET antibody carried out renal artery occlusion within 45 min in rats [810,1320]. In this study we occluded the renal artery for 60 min, because the effect of uninephrectomy was not evident when the renal artery was clamped for 45 min (data not shown). Since a longer duration of renal artery occlusion induces more severe renal damage, the discrepancy between the present work and previous studies may be partly due to the difference in ischaemic ARF models. In addition, we gave anti-ET antibody intravenously, contrasting with intra-arterial administration in other studies [7,14]. We also used a monoclonal ET antibody, as opposed to the polyclonal ET antibody in other studies [13,14]. Furthermore, BQ-123, an ETA receptor antagonist often used for the treatment of ischaemic ARF in rats [15,17,23], is known to inhibit angiotensin II-mediated vascular effects, which could be a source of misinterpretation [25].
To date, the effect of ET receptor antagonism on the development of ischaemic ARF is controversial. Brooks et al. [21] found that BQ-123 did not improve the decline in creatinine clearance at the initial stage of ischaemic ARF in rats. In addition, Chan et al. [15] did not find any beneficial effect of BQ-123 when the agent was administered during the reflow phase in isolated rat kidneys. Interestingly, Forbes et al. [26] recently found that treatment with ETA (PD 156707) or a non-selective ET receptor antagonist (SB 209760) did not protect against the increment of serum creatinine levels at 48 h after renal ischaemia. These antagonists, however, significantly increased the number of proliferating cell nuclear antigen (PCNA)-positive cells and decreased apoptotic cell death at 7 days. Both ET receptor antagonists also reduced post-ischaemic macrophage infiltration in the kidney, which may shorten the duration of the inflammation phase. Gellai et al. [27] also noted that BQ-123 markedly reduced the mortality rate during 3 days without changing maximal serum creatinine values. In addition, they found that SB 209760 did not prevent the 45-min RAO-induced ARF, but reduced mortality and tubular dysfunction up to 14 days after ischaemia in rats [20].
These findings convincingly suggest that blockade of ET-1 may be more beneficial for recovery rather than prevention of ischaemic renal damage. In this study, however, we could not assess a long-term effect of AwETN40 for ischaemic renal injury in ARF rats because of a limited quantity of drug. Since we previously found that Nx increased the number of PCNA-positive cells in the outer stripe of the outer medulla after renal ischaemia [4,5], future studies are needed to determine whether lesser increases in cortical ET-1 content may be associated with the Nx-induced facilitation of recovery from ischaemic renal injury.
Our previous studies [68] showed that at 48 h after 60-min of renal artery clamping, the degree of tubular necrosis, apoptosis, and cast formation in the post-ischaemic kidney was less remarkable in Nx than in sham-Nx rats, but no significant difference in morphological alterations of glomerular capillary ultrastructure was found between the groups. In this study, a restoration of RBF to control levels by the ETA receptor antagonist did not improve Cin at the initiation and maintenance stages in our ischaemic ARF models. Taken together, our data support the possibility that an attenuated decline of Cin in ischaemic kidneys of Nx rats can be exclusively attributed to reduced tubular damage rather than to a lesser decrease in RBF through ET-1 suppression.
The smaller increase in cortical ET-1 content in Nx rats might be due to a less enhanced local production and/or significantly increased urinary ET-1 excretion, since plasma ET-1 levels did not change but the urinary excretion rate of this peptide was significantly enhanced following renal artery clamping. We found previously that a reduced production of thromboxane A2 (TxA2) plays an important role in the Nx-induced attenuation of ischaemic renal injury [4]. Since a TxA2 analogue stimulates ET-1 mRNA expression in cultured renal cells [28], it is possible that the reduced TxA2 synthesis is the cause of lower levels of ET-1. Blockade of TxA2 is also reported to lessen the ET-1-induced ischaemic vascular permeability in rats [29].
In this study, as described previously [24,6], renal ischaemia induced polyuria in Nx rats but oliguria in sham-Nx rats. Although RAO induced a greater loss of body weight in Nx rats compared with sham-Nx rats, we found that plasma renin activity and cortical renin content were rather increased in sham-Nx animals compared to Nx animals at 48 h after 60 min of renal ischaemia [6]. Thus, polyuria-induced fluid deprivation seems not to play a role in the severity of ischaemic ARF in this model. A reduced activation of the reninangiotensin system, independent of fluid status, may contribute to the Nx-induced attenuation of ischaemic renal injury.
In summary, contralateral nephrectomy attenuated the decline in Cin and the increase in the cortical ET-1 content in post-ischaemic kidneys of rats. Although the ETA receptor antagonist FR139317 facilitated percentage recovery of RBF in sham-Nx rats, the administration of either FR139317 or monoclonal ET antibody AwETN40 failed to attenuate the decline in Cin following a 60-min renal artery clamping in sham-Nx and Nx animals. It is unlikely, therefore, that the protective effect of unilateral nephrectomy on the development of ischaemic ARF is mediated exclusively through a smaller increase of ET-1 production in the renal cortex.
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Acknowledgments |
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Notes |
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References |
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