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1 Department of Physiology, University College Cork, Cork, Republic of Ireland
2 Laboratory of Renal and Body Fluid Physiology, M. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
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(Received 29 August 2005;
accepted after revision 18 October 2005;
first published online 20 October 2005)
Corresponding author E. J. Johns: Department of Physiology, Aras Windle, University College Cork, Cork, Republic of Ireland. Email: e.j.johns{at}ucc.ie
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Introduction |
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At the neuro-effector junction, NO has the potential of exerting both pre- and postsynaptic actions, which complicate studies of its role in neuro-transmission. There is evidence in the anaesthetized dog that in response to low-level renal nerve stimulation, noradrenaline output is enhanced following NOS blockade, and suppressed by exogenous NO, suggesting that NO exerts a tonic inhibitory action at the presynaptic level (Egi et al. 1994; Maekawa et al. 1996). In contrast, in the rat, it has been reported that sympathetic nerve-induced noradrenaline release from the heart is enhanced (Schwartz et al. 1995) but is suppressed from the mesenteric vasculature (Yamamoto et al. 1994, 1997) as a consequence of NOS inhibition. Tanioka et al. (2002), using the isolated pump perfused rat kidney, reported that NOS blockade suppressed sympathetic nerve-mediated noradrenaline release and the vasoconstrictor responses to high levels of renal nerve stimulation consistent with a presynaptic facilitatory action of NO. Taken together, these divergent findings mean that there is a lack of clarity as to the exact action of NO at neuroeffector junctions and would suggest that the role played by NO is dependent upon the tissue, organ and species studied.
Most reports have focused on the role of NO in modulating neurotransmission at the renal vasculature, but another major site of neural action is at the tubular epithelia to stimulate sodium reabsorption. Recent reports from this laboratory have shown that administration of L-NAME into the proximal tubules increased, and an NO donor decreased, fluid reabsorption, indicative of NO having a direct action to inhibit fluid reabsorption (Wu et al. 1999). Moreover, low-level RNS, which increased fluid reabsorption in a frequency-related way, was prevented following intraluminal administration of L-NAME and 7-nitroindazole (7-NI), which is a relatively selective nNOS blocker (Wu & Johns, 2002, 2004). However, whether this local application of the NOS blockers was sufficient to penetrate into the neuro-effector junction was unclear. These observations were, in part, compatible with NO having a facilitatory action at the neuro-effector junction.
The present study aimed to investigate the importance of NO in mediating neurally induced sodium reabsorption in a whole-kidney setting and to evaluate the isoform of NOS involved. This was done by directly stimulating the renal nerves at low frequencies that had no effect on renal haemodynamics, and determining how the antinatriuretic and antidiuretic responses were affected following relatively selective blockade of different NOS isoforms.
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Methods |
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The experimental protocol comprised five 15 min clearance periods, two before and two following a period in which the renal sympathetic nerves were stimulated at 15 V, 2 ms duration and at a frequency which was just subthreshold for causing a reduction in renal blood flow (0.5–1.0 Hz). The nerves were stimulated for a total of 20 min, but urine collections were not begun until 5 min after the start of stimulation in order to clear preformed urine from the ureteral cannula. The rats were killed humanely with a 1 ml overdose of anaesthetic at the end of the experiment.
Four groups of rats were studied. Group I received an infusion of saline throughout, and acted as a control group. Group II received a continuous I.V. infusion of N
-nitro-L-arginine methyl ester (L-NAME) at 10 µg kg–1 min–1 for 30 min prior to the start of the clearance protocol. This infusion rate of L-NAME has been reported to block all NOS isoforms, but without increasing blood pressure or reducing renal haemodynamics (Lahera et al. 1991). Group III was given 1400W at 20 µg kg–1 min–1I.V. for 30 min before beginning the sequence of clearance measurements. This compound has a 500-fold selectivity for blocking iNOS compared with eNOS, and at this infusion rate it has been demonstrated to prevent iNOS-mediated events (Garvey et al. 1997). Group IV was infused with S-methyl thiocitrulline (SMTC) at 20 µg kg–1 min–1I.V. for 30 min before the start of the clearance collections. SMTC is a relatively selective nNOS blocker having a 17-fold greater inhibitory action for nNOS than eNOS (Furfine et al. 1994), and at this dose level it has been reported to depress renal levels of NO (Walkowska et al. 2005). All drugs were obtained from ‘Sigma, (Dorset, UK)’.
All data are the average values calculated from individual animals and are given as means ±S.E.M. The renal responses to stimulation were calculated by averaging the two clearances before and the two following renal nerve stimulation and comparing the values to those obtained whilst the nerves were stimulated. Comparisons were undertaken using a two-way ANOVA (SuperANOVA Software, Abacus, CA, USA). Significance was taken when P < 0.05.
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Discussion |
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The 1400W compound is one that has a 500-fold greater selectivity of inhibition for iNOS as against eNOS (Garvey et al. 1997), and the dose chosen for the present study has been used previously to prevent iNOS-mediated activities (Rocha et al. 2002). There was no evidence that 1400W given in this way had any effect on blood pressure or kidney function. The SMTC was used to cause a relatively selective blockade of nNOS and the dose used in the present study was aimed at causing an inhibition of nNOS with minimal action on eNOS. Furfine et al. (1994) have demonstrated that SMTC has a 17-fold inhibitory selectivity for nNOS as against eNOS, and at these doses. This compound has been reported to have differential actions on the renal vasculature (Ichihara et al. 1998), to selectively increase sodium reabsorption-dependent oxygen consumption (Deng et al. 2005) and to cause a decrease in the renal content of NO (Walkowska et al. 2005) at concentrations where it is probably acting preferentially on nNOS. This compound was utilized in preference to 7-NI used previously (Wu et al. 1999; Wu & Johns, 2002), which has only a 5-fold greater inhibitory action on nNOS compared with eNOS, and has to be dissolved in oil and given I.P. to obtain sufficient systemic concentrations (Wongmekiat & Johns, 2001a,b,c). Again, it was apparent from the basal values that the compound did not result in any major differences in renal haemodynamic or excretory function compared with the control group of rats receiving saline, although blood pressure was higher. There have been several reports of SMTC being used in a range of studies, and all appear to report small but variable effects on blood pressure which may be dependent on the dose of drug used, type of anaesthesia and the experimental conditions (Walkowska et al. 2005).
Electrical stimulation of the left renal nerves had no consistent effect on renal blood flow or glomerular filtration rate, but resulted in a 40–50% reduction in urine flow and sodium excretion. These observations are very comparable with those we previously reported in the anaesthetized rat and rabbit (Hesse & Johns, 1984; Manitius & Johns, 1987), and those reported by others in the dog (Slick et al. 1975). Micropuncture studies have demonstrated that such low-level activation of the renal sympathetic nerves, which has no haemodynamic action, directly stimulates fluid reabsorption at the proximal tubular epithelial cells (Bello-Reuss et al. 1976; Wu & Johns, 2002, 2004) and at the thick ascending loop of Henle (DiBona & Sawin, 1982). It is very likely that the whole-kidney antinatriuresis and antidiuresis in response to renal sympathetic nerve stimulation, reported herein, reflects this tubular action of the renal nerves. It is also important to point out that the action of the renal nerve stimulation on sodium and water excretion was limited to the left kidney, since over this time neither water nor sodium excretion from the right kidney was changed.
Infusion of L-NAME resulted in blood pressures which were comparable with those of control rats receiving saline, and this was important as it meant that there were no confounding effects of pressure on the level of sodium and water excretion (Granger, 1992). It was clear that following the infusion of L-NAME, the antinatriuresis and antidiuresis associated with the low-level renal nerve stimulation was prevented. This observation would suggest that the presence of NO was necessary for the nerves to exert their effect at the epithelial cells. The question arises as to whether this action represents a pre- or postsynaptic action of the NO. Interestingly, in previous reports from this laboratory, a role for NO in adrenergically mediated proximal tubular fluid reabsorption became apparent. The first indication of an interaction was that local infusion of L-NAME into the proximal tubular lumen for a few seconds resulted in a significant increase in proximal tubular fluid reabsorption (Wu et al. 1999). This suggested that the basal level of proximal tubular fluid reabsorption was under the influence of NO which exerted a tonic inhibitory action (Ortiz & Garvin, 2002). Interestingly, this action of L-NAME to increase tubular fluid reabsorption occurred only if the renal sympathetic nerves were intact. It was unclear how NO might interact with the renal nerves, but in a subsequent study it was reported that the effect of low-level renal nerve stimulation, which caused a frequency-dependent increase in proximal tubule fluid reabsorption, was also blocked by L-NAME (Wu & Johns, 2002). This finding was interpreted as indicating a potential second site of action, i.e. at the neuro-effector junction, where NO might be important in facilitating the release of noradrenaline. This latter concept was supported by the observations in the rat mesenteric bed where L-NAME blunted the noradrenaline release in response to stimulation of the sympathetic nerves (Yamamoto et al. 1994, 1997). Moreover, Tanioka et al. (2002) demonstrated not only that noradrenaline release from the isolated pump-perfused kidney was suppressed following NOS inhibition with L-NAME, but also that the nerve-mediated vasoconstrictions were attenuated. However, there is a body of evidence indicating that NO has a negative action to inhibit noradrenaline release at the neuro-effector junction and to depress adrenergically mediated actions (Egi et al. 1994; Schwartz et al. 1995; Maekawa et al. 1996; Chowdhary & Townend, 1999). Exactly why there should be such divergent reports in the literature is unclear at the present time, but it does indicate that the situation at each neuro-effector junction may be very different.
Together, the micropuncture studies (Wu et al. 1999; Wu & Johns, 2001) suggested two sites at which NO could have been exerting an action. However, there were concerns that in these micropuncture studies, because the NOS blockers were given into the tubular lumen and were present for a relatively short period, uncertainty arose as to whether there was sufficient time for the NOS enzymes to be blocked in sites beyond the epithelial cell and in the neuro-effector junction. Based on these considerations, it was hypothesized that the NO could have been acting not only on basal rates of fluid reabsorption where it had a tonic inhibitory action, but also on the noradrenaline-stimulated fluid reabsorption, where it had a facilitatory action. The question remained as to whether there might have been a presynaptic site where the NO could have been acting.
The present study was an attempt to provide further evidence for this interaction. In a whole-kidney setting, the NOS blocker L-NAME was given I.V., with the aim of providing blockade of NOS, including that involved in noradrenaline release and transmission at the neuro-effector junction. Clearly in this whole-kidney setting the presence of NO was necessary if the low-level renal nerve stimulation was to cause an antinatriuresis and an antidiuresis. These observations reinforce the earlier findings and provide supportive evidence for NO being importantly involved in allowing adrenergically mediated sodium reabsorption to occur within the kidney.
A further objective concerned the identification of the NOS isoform involved in generating the NO necessary to ensure effective adrenergic regulation of fluid reabsorption. It was evident from the results that it was unlikely to be a consequence of iNOS activity, since in the presence of 1400W, at dose levels which have been shown to inhibit iNOS activity in vivo (Garvey et al. 1997; Rocha et al. 2002), the magnitudes of the antinatriuresis and antidiuresis were the same as in the control rats infused with saline. Thus, in spite of reports that iNOS was constitutively active in the medullary regions of the kidney (Kone & Baylis, 1997), any NO generated by iNOS was unlikely to be involved in the neural control of fluid handling. The study with SMTC aimed to investigate whether nNOS provided a source for the NO. It was clear that following administration of the SMTC the magnitude of the reductions in sodium and water excretion were markedly blunted, although not totally prevented. This would suggest that the nNOS isoform was, to a degree, responsible for the generation of NO required to allow an effective action of the renal nerves on fluid reabsorption. However, the renal nerve-mediated antinatriuretic and antidiuretic responses were not completely blocked and it remains uncertain as to whether this could have been due to an insufficient dose of SMTC used or whether a component of the NO did indeed arise from activity of eNOS. This is a limitation consequent on the relative lack of selectivity of the available compounds, as even if a higher dose of SMTC were used, the possibility would still arise that the compound was at a concentration at which it would begin to block eNOS
This study investigated the interaction between NO and the renal nerves to cause a retention of sodium and water. Using the anaesthetized rat, low-level stimulation of the renal nerves decreased urine flow and sodium excretion by almost one-half. In the presence of L-NAME, the renal nerve-induced antinatriuresis and antidiuresis was blocked but this was not the case in the presence of the relatively selective iNOS blocker 1400W. In the presence of SMTC, the magnitudes of the renal nerve-mediated excretory responses were markedly reduced. Together these findings show that NO is importantly involved in allowing the renal nerves to stimulate tubular fluid reabsorption. This action of NO may be at a presynaptic as well as a postsynaptic site at the neuro-effector junction, and may involve both nNOS and eNOS isoforms.
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References |
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