Department of Physiology, The Medical School, Birmingham, UK, 1 Pfizer Global Research and Development, Sandwich, Kent, UK and 2 Department of Physiology, University College Cork, Cork, Republic of Ireland
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Abstract |
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Methods. The animals were anaesthetized, drug infusion commenced, and the renal artery occluded for 30 min. The endothelin antagonists were given for 30 min before, during, and 60 min after the ischaemic period, at 10, 30 and 100 µg/kg/min or for 60 min after the start of reperfusion.
Results. On day 1, following 30 min renal artery occlusion, there was a 95% reduction in glomerular filtration rate, an 810-fold increase in plasma creatinine, and 1015-fold increases in fractional excretions of sodium and potassium, which were partially resolved on day 3 and normalized on day 8. The lowest dose of SB209670 was without effect on the renal functional responses but they were blunted (all P<0.05) by the highest dose. At 30 and 100 µg/kg/min UK-350,926, the decreases in renal function subsequent to the ischaemic challenge were attenuated. Administration of UK-350,926 at 100 µg/kg/min for 1 h starting 60 min after the start of reperfusion, had no effect on the magnitude of the renal disturbances over the first 3 days.
Conclusions. The data show that both the ETA/ETB and selective ETA-receptor antagonist ameliorated the ischaemia-reperfusion injury when given in the peri-ischaemic period but not when the ETA-receptor antagonist was given for 60 min at 100 µg/kg/min after the ischaemic period.
Keywords: acute renal failure; endothelin antagonists; renal haemodynamics; urinary sodium excretion
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Introduction |
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A number of models of acute renal failure have been developed, most of which involve a period of kidney renal artery occlusion followed by reperfusion [12]. This manoeuvre induces a period of anoxia during which there is build up of ischaemic metabolites, which on reperfusion initiate tissue damage. It is under these conditions that the kidney fails, as reflected by oliguria plus elevated plasma creatinine levels, for 23 days. A number of studies have indicated that if endothelin antagonists, both the mixed ETA/ETB non-selective and ETA-selective compounds, were given during the ischaemic period the rate of recovery in renal function was enhanced [1315]. These findings suggested that these compounds might be potentially useful in ameliorating the level of acute renal failure.
The aim of the present study was 2-fold. First, to compare the effectiveness of a mixed ETA/ETB-receptor antagonist with that of a novel ETA-selective receptor antagonist in blunting the deterioration in kidney function in response to an ischaemic challenge. Secondly, to examine whether potential reno-protective effects exhibited by these compounds only occurred if they were present before and during the ischaemic challenge, or whether they could be useful if given at some time after the period of ischaemia. This was done utilizing the non-selective endothelin antagonists SB209670 and the novel non-peptide ETA-selective compound UK-350,926.
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Subjects and methods |
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Model generation
A rat model was used, in which following a prior unilateral nephrectomy the remaining kidney was subjected to a period of anoxia. Briefly, male Wistar rats, 200225 g, were anaesthetized with a mixture of halothane/O2/N2O (4/2 l/min), the right kidney was removed retroperitoneally and on recovery the animals were given antibiotics and analgesics in the immediate post-operative period. The animals were used 2 weeks later when the renal hypertrophy had been completed and the animals re-established their growth rate.
Renal ischaemia-reperfusion injury
On the experimental day, the animals were again anaesthetized (Domitor/ketamine i.p., an initial dose of 0.24 mg/kg medetomidine and 58 mg/kg ketamine followed by supplementary doses every 2040 min of half the initial dose as indicated by the pedal withdrawal reflex), a butterfly needle inserted in the tail vein and saline (150 mmol NaCl) infused at approximately 3 ml/h. The left renal artery was exposed retroperitoneally and occluded with a non-traumatic clamp for 30 min. On completion of the drug infusion protocol, the animals were allowed to recover (antisedan i.p. at a dose of 0.1 mg/kg) again with appropriate analgesics being administered.
Renal function
Blood samples (0.5 ml) were removed from the animals at various stages from the tail vein while they were briefly anaesthetized with a halothane/O2/N2O mixture. Plasma and urine creatinine levels were measured with a Sigma creatinine kit (Poole, Dorset, UK). GFR was estimated as endogenous creatinine clearance. Electrolytes in plasma and urine were measured by flame photometry (Corning 410C).
Protocol
From 2 to 3 days before the ischaemic challenge the animals were placed in Technoplast metabolic cages for 24 h to measure basal levels of water intake and fluid output. Water intake and urine flow were measured and on removal from the cage, a tail vein blood sample was collected for estimation of plasma electrolytes and creatinine. On the experimental day, 46 h after the renal artery clamping, the rats were placed back into the metabolic cages for a further 3 days. Urine flow and water intake were continuously monitored and blood samples were taken at 18 and 72 h after entry into the cages. At the end of 3 days, the animals were returned to their home cages, but in some groups on day 8, they went back into the metabolic cages for a further 24 h, at the end of which a final blood sample was taken. The animals were killed with an overdose of sodium pentobarbitone, the kidney removed and weighed.
The following groups of animals were studied.
Determination of ETA-receptor antagonism in anaesthetized rats
Male rats (200250 g) were anaesthetized with inactin (120 mg/kg, i.p.) and placed on a homeothermic blanket to maintain body temperature. The trachea was cannulated and the animals were ventilated with room air. The right and left jugular veins were cannulated for administration of anaesthetic, endothelin-1, and infusion of endothelin antagonists. The external carotid artery was cannulated and connected to a pressure transducer for measurement of arterial pressure. Infusion at 1 ml/h of an endothelin antagonist or vehicle was started. After 70 min, 4.3 mg/kg of pentolinium was administered intravenously, followed 10 min later by 3 mg/kg of the ET antagonists, BQ-788, SB209670 or UK-350,926. After a further 10 min, cumulative i.v. doses of ET-1 in saline containing 1% BSA were administered from 1 pmol/kg to 3 nmol/kg. Each dose was administered after the response to the previous dose had reached a plateau. Pressor doseresponse curves to ET-1 in vehicle- and drug-treated animals were compared and the ratio of agonist dose required to increase blood pressure by 20 mmHg in antagonist treated animals compared with that in vehicle treated animals was calculated.
Determination of selectivity of UK-350,926 for ETA receptors in isolated tissues
Rat isolated aortic rings were used for the assessment of ETA-receptor antagonism. The rings were denuded of endothelium by gentle rubbing and set up under 1 g tension in organ chambers (37°C) containing Krebs solution (composition in millimolar: NaCl 115, KCl 4.7, NaHCO3 25, glucose 5.6, NaH2PO4 1.2, CaCl2 2.5, MgCl2 1.2) for isometric tension recording. After 1 h equilibration period, the tissues were contracted twice with KCl (60 mM) and following washout were exposed to UK-350,926 (30, 100 and 300 nM) or vehicle for 90 min. The tissues were then exposed to cumulative doses of ET-1 and the contractions expressed as a percentage of the KCl evoked response in that tissue.
Rabbit isolated pulmonary artery rings were used for the assessment of ETB-receptor antagonism and were set up under similar conditions to the aortic rings. After a 1 h equilibration period the tissues were contracted twice with KCl (60 mM) and following washout were exposed to UK-350,926 (1, 3 and 10 mM) or vehicle for 90 min. The tissues were then exposed to cumulative concentrations of the ETB agonist, sarafotoxin S6c (Cambridge Bioscience).
Materials
BQ788 (RBI), ET-1, sarafotoxin S6c, SB209670 was a gift from Smith Kline Beecham, kindly provided by Dr David Brooks. UK-350,926 was generated by Pfizer.
Statistics
Values are expressed as mean±SEM. Data were compared using ANOVA followed by a Bonferroni Dunn post-hoc test where necessary. Significance was taken at P<0.05.
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Results |
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The haemodynamic and excretory responses to the 30 min period of renal ischaemia for the 8 days subsequent to the insult are shown in Figures 1 and 2
, respectively. In the group of rats, which received no drug, there was an approximate 78-fold increase in plasma creatinine at day 1, which had decreased somewhat at day 3 and had returned almost to baseline levels at day 8. This translated into a decrease in GFR of some 96% at day 1, of 80% at day 3 but was virtually normal by day 8 (Figure 1
). There was only a modest reduction in urine flow over the first day, followed by a slight increase in this variable at days 3 and 8. However, absolute sodium excretion decreased by 75% at days 1 and 3 but was restored to just over baseline levels at day 8 (Figure 2
).
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It can be seen that in the group of rats given SB209670 at 10 µg/kg/min in the peri-ischaemic period, the responses in plasma creatinine and GFR were very similar in pattern and magnitude to those observed in the group receiving saline (Figure 1). Similarly, the decreases in both absolute sodium and potassium excretions were comparable with those obtained in the saline infused group (Figure 2
). However, the increases in fractional sodium and potassium excretions were slightly smaller than those observed in the saline infused animals, with a marginally faster recovery (Figure 1
). The results from the group of rats receiving 30 µg/kg/min SB209670 showed that the magnitude of the haemodynamic and excretory changes were somewhat smaller than those evident in the saline treated rats (Figures 1
and 2
). Thus, the day 1 increases in plasma creatinine and decreases in GFR were smaller while the decreases in absolute sodium excretion and rises in fractional sodium excretion were less. However, the levels of both absolute and fractional potassium excretion in the 8 days following the ischaemic insult were very similar to those observed in the untreated saline infused animals.
It was evident that in the group of rats receiving 100 µg/kg/min SB209670, the increase in plasma creatinine was markedly less at days 1 and 3 (both P<0.05) and was normalized by day 8 and this reduced response to ischaemia was reflected in GFR which was only decreased by about 80% on day 1, significantly less (P<0.05) than the saline/vehicle group, and had recovered by day 8. Interestingly, there was no consistent change in urine flow or absolute sodium excretion (Figure 2) although there was a 4-fold increase in fractional sodium excretion on day 1, a somewhat smaller (P<0.05) response than that of the saline vehicle group and it was virtually at basal levels on day 3 (Figure 1
). This pattern of excretory change was roughly paralleled when considering potassium excretion, that is a decrease in absolute and an increase in fractional excretion on day 1, the former being at basal levels by day 3 and both variables being normalized by day 8.
The data from the animals given UK-350,926 are presented in Figures 3 and 4
but includes the same data for the control study as shown in Figures 1
and 2
, for comparative purposes. When UK-350,926 was infused at 10 µg/kg/min, the haemodynamic and excretory variables responded to the ischaemic challenge, in both magnitude and pattern, to the same degree as those obtained in the group infused with saline and this comparison is shown in Figures 3
and 4
.
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Moreover, the increase in fractional sodium and potassium excretions and days 1 and 3 (P<0.05) were smaller than those in the untreated group and they had returned to basal levels by day 8. Although the ischaemic insult in the animals receiving UK-350,926 at 30 µg/kg/min (Figure 3) had no effect on urine flow, absolute excretions of sodium and potassium were decreased at days 1 and 3 these were significantly (P<0.05) less when compared with the saline infused group.
In the animals which received UK-350,926 at 100 µg/kg/min the rise in plasma creatinine and decrease in GFR induced by the ischaemic insult over the 8-day period was much less at each time point than those which occurred in the untreated animals given saline (Figure 3). This was also reflected in smaller responses (all P<0.05) in fractional sodium and potassium excretions over the 8-day period compared with the animals, receiving only saline (Figure 3
). There were reductions in both absolute sodium and potassium excretions 1 and 3 days following the ischaemic insult in this group of rats receiving the 100 µg/kg/min UK-350,926 (Figure 4
).
Figure 5 presents the renal haemodynamic and excretory values for a further group of rats over the first 3 days following renal artery clamping for 30 min in which the animals received only saline.
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Discussion |
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Administration of the non-selective ETA/ETB-endothelin-receptor antagonist SB209670 before, during, and following, the ischaemic challenge at 10 and 30 µg/kg/min had little effect on either the increase in plasma creatinine or decreases in GFR on days 1 and 3. However, the 100 µg/kg/min dose of SB209670 blunted these changes in plasma creatinine and GFR over the first 3 post-ischaemic days such that recovery in renal haemodynamics was virtually complete by day 8. An important point to acknowledge is that the ability of SB209670 at the highest dose to blunt the renal haemodynamic deterioration following the period of ischaemia was very comparable with that reported earlier [14,15] for SB209670 and tezosartan [16]. This would be compatible with the endothelins being released in response to the ischaemia and causes the reduced renal haemodynamics. In terms of the excretory responses to ischaemia, it was only the highest dose of SB209670, which reduced the magnitude of the responses in urine flow, absolute and fractional sodium, and potassium excretions. Moreover, this action of the endothelin antagonist would suggest that part of damage to the renal sodium and water reabsorptive processes is caused by an action of the endothelins, probably at the level of the tubules. Whilst endothelin antagonism can alleviate the severity of the acute renal failure in the short term, of 18 days (present study, 1416), Forbes et al. [17] have reported that the endothelin antagonists have long-term proliferative actions approximately 6 months post-ischaemic challenge.
A significant issue is whether these haemodynamic and excretory actions of endothelins resulting from the ischaemia are mediated via ETA, ETB or both receptor subtypes. UK-350,926 has a similar potency in vivo to that of SB209670 as an antagonist at endothelin receptors mediating pressor responses in the rat but has a marked selectivity for ETA receptors. Thus, a further series of studies were undertaken using the same dose levels of UK-350,926 as had been used for SB209670. It was apparent that at the highest dose, UK-350,926 attenuated the increase in plasma creatinine and the fall in GFR following the ischaemic challenge to the kidney on both days 1 and 3. The 30 µg/kg/min dose of UK-350,926 also prevented the rise in plasma creatinine (Figure 3). At the highest dose of UK-350,926, the magnitude and pattern of the blunted haemodynamic responses to the period of ischaemia subsequent to the use of the selective ETA-receptor antagonist was very comparable with that observed with the same dose of the non-selective receptor antagonist. This would suggest that in the rat the deterioration in renal haemodynamics following the ischaemia-reperfusion injury was due mainly to endothelin acting on ETA receptors. Interestingly, at both the 30 and 100 µg/kg/min doses, UK-350,926 prevented the marked increases in fractional sodium excretion on day 1 following the ischaemia. This was due not only to the smaller reductions in filtration rate, but also to the fact that absolute sodium excretion remained reduced on day 1. This observation contrasts with that observed following the mixed antagonist at the high dose on day 1 in which absolute sodium excretion was significantly higher and suggests that blockade of ETB receptors might have delayed the normalization of the sodium reabsorptive processes following the ischaemic damage. The amelioration of the severity of acute renal failure by UK-350,926 was comparable with that reported earlier with another ETA antagonist, LU 135252 [18]. At a clinical level, it has been reported [19] that blockade of ET receptors with a mixed antagonist exacerbated the increase in serum creatinine in the first 48 h post-radiocontrast dye administration, but it is important to emphasize that these observations were made in patients with chronic renal failure. The situation in normal subjects exposed to ischaemic challenges and the effectiveness of endothelin-receptor blockade remains to be resolved.
The studies in which the endothelin antagonists were given over the peri-ischaemia period, that is before, during, and for 1 h following the renal artery clamping, clearly demonstrated that they decreased the severity of the reductions in renal haemodynamics and fluid handling. Another important issue to be addressed was whether administration of an endothelin antagonist after the ischaemic challenge had occurred could also ameliorate the marked renal haemodynamic and excretory responses over the first few days. The experimental design chosen was to infuse the highest dose of the ETA-selective antagonist for 1 h, beginning 1 h after removal of the renal artery clamp. It was very clear that the magnitudes of the increase in plasma creatinine and reductions in GFR were not different whether the animals were given saline or the ETA-receptor antagonist. Moreover, the pattern of the sodium and potassium excretory responses was unchanged by the endothelin antagonist. Thus, the findings clearly demonstrated that if an antagonist was given following the ischaemic challenge the pattern and progression of the acute renal failure over the first 3 days was unchanged. This suggested that the endothelin receptors had to be blocked before the ischaemia took place in order to offer maximum protection from an action of endothelins produced as a consequence of the ischaemia-reperfusion injury. These findings, to some extent, are in contrast with earlier observations [14], which demonstrated that if a comparable dose of the mixed ETA/ETB antagonist was given over 3 h, 24 h after the ischaemic period, it caused a marked increase in survival rate, albeit without significantly enhancing renal haemodynamics. Moreover, Wilhelm et al. [16], using a similar rat model of acute renal failure, found that administration of a very high dose of endothelin antagonist in the immediate post-ischaemic period blunted the elevation in serum creatinine levels. Taken together, the data of the present study indicate that administration of the endothelin antagonists at the dose level of 100 µg/kg/min for 60 min shortly after the ischaemic challenge was unlikely to alter the progress of the acute renal failure. This may be due to the markedly elevated levels at this time when the chosen dose of antagonist might not be sufficient to overcome the actions of the peptide. It may be that 24 h after the challenge, the dose of antagonist, 15 mg/kg i.v. [16] or 30 µg/kg/min for 3 h [14], and the length of time over which it was given was sufficient to overcome the action of the decreasing level of endothelin compatible with enhancing survival. These interactions deserve further investigation and could provide some indication of how these potential therapeutic compounds may be applied once the ischaemic insult to the kidney has occurred.
These studies were designed to examine the effectiveness of two endothelin antagonists in blunting the renal haemodynamic and excretory deficits occurring following a 30-min period of renal ischaemia. The findings showed that at the highest doses used, 100 µg/kg/min, given before, during and after the challenge, both compounds were equally effective in blunting the haemodynamic and excretory responses to the ischaemic challenge. In contrast, if the ETA-selective antagonist was given 1 h after the ischaemic period, it was unable to alter the magnitude or the pattern of the haemodynamic and excretory responses characteristic of the acute renal failure. This suggests the compounds might be effective prophylactically but their therapeutic potential remains to be explored.
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Acknowledgments |
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Notes |
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References |
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