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1 The Medical School, Birmingham B15 2TT, UK2 Department of Physiology, University College Cork, Ireland
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Abstract |
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-nitro-L-arginine methyl ester (L-NAME) had no effect on baseline Jva or the pattern of response to renal nerve stimulation which contrasted with earlier reports in the Wistar rat. Intraluminal superoxide dismutase (SOD) had no effect on basal Jva in the Wistar rats but increased it in the SHRSP (P < 0.05) while the pattern of change in Jva during nerve stimulation was unaltered in both rat strains. By contrast, in the SHRSP, intraluminal sodium nitroprusside (SNP) resulted in a frequency related increase in Jva comparable to that obtained in the vehicle treated Wistar rats. These data suggest that in the hypertensive rats, superoxide anion production is raised which depresses Jva and interacts with NO preventing a normal Jva response to renal nerve stimulation.
(Received 6 August 2003;
accepted after revision 3 February 2004)
Corresponding author E. J. Johns: Sir Bertram Windle Building, University College Cork, College Road, Cork, Republic of Ireland. Email: e.j.johns{at}ucc.ie
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Introduction |
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-adrenoceptors of the proximal epithelial cells to stimulate both the basolateral sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and the sodium/hydrogen (Na+/H+) exchanger at the apical membrane, causing an increase in sodium and water reabsorption (Aperia et al. 1992; Nord et al. 1987). Low level stimulation of the renal sympathetic nerves, either directly or reflexly via activation of low or high pressure cardiovascular baroreceptors (DiBona & Kopp, 1997) or the somatosensory system, has been shown to primarily stimulate renin secretion and sodium reabsorption rather than cause major changes in renal haemodynamics.
A number of studies have indicated that in the rat genetic model of hypertension, the spontaneously hypertensive rat, renal sympathetic nerve activity is elevated compared to normotensive controls (Lundin et al. 1984; Lundin & Thoren, 1982) and this might be expected to exert an enhanced neural regulation of kidney function. However, in a number of studies using reflex activation of the renal sympathetic nerves, the renal nerve-dependent antinatriuresis and antidiuresis was very much attenuated (Davis & Johns, 1994; Zhang et al. 1997). The reason for this blunted neural regulation of sodium handling is not yet clear but may reside either within the central nervous system or at the neuroeffector junction, or both. We reported recently (Wu et al. 1999) that in normotensive Wistar rats, intraluminal administration of N-nitro-L-arginine methyl ester (L-NAME), to block nitric oxide (NO) formation, increased proximal tubular fluid reabsorption while coadministration of a nitric oxide donor normalized proximal fluid reabsorption. Importantly, these responses were prevented if the kidney had been acutely denervated, indicating some interaction between the nerves and NO production in modulating proximal epithelial cell transport processes. By contrast, in the stroke prone spontaneously hypertensive rat (SHRSP) we found (Wu et al. 1999) that neither L-NAME nor the NO donor sodium nitroprusside (SNP) had any influence on proximal tubular fluid reabsorption, even if the kidneys were innervated. Together, these findings suggested that there might be some defect in the role played by NO in the hypertensive rat model.
It is recognized that NO is one of a number of active oxidant molecules which includes superoxide anions (O2–), hydrogen peroxide (H2O2), hydroxyl radicals (OH–), peroxynitrite (ONOO–) and lipid-derived radicals. Investigations have shown that O2– can react with NO to generate ONOO– (Beckman & Koppenol, 1996) and thereby is able to modulate the potency of NO at its site of action. Immunohistochemical studies have revealed staining for the superoxide dismutase (SOD), which is a scavenger for O2–, in neuronal nitric oxide synthase (nNOS)-containing nerves, and would have the potential of protecting NO from destruction by O2– (Liu et al. 1997). The observation by Liu et al. (1996), that there are nitrergic nerve fibres in the kidney which colocalize with sympathetic fibres, has been taken to suggest that a relationship exists between O2– and NO at the neuroeffector junction. The oxidative and antioxidant systems are normally in balance but under pathophysiological conditions, for example in hypertensive states, there are often altered levels of NO or O2– generation giving rise to accelerated NO removal and a blunting of its physiological role. Indeed, in the spontaneously hypertensive rat there is evidence of an up-regulation of superoxide dismutase suggesting enhanced production and removal of O2– (Grunfeld et al. 1995).
The aim of this investigation was to evaluate the contribution of NO in mediating the renal sympathetic nerve-induced increases in proximal tubular fluid reabsorption in a genetic rat model of hypertension, the stroke prone spontaneously hypertensive rat. A second aim was to examine whether O2– was involved in modulating the influence of NO in the neurally stimulated increases in proximal tubular fluid reabsorption. In this way it was hoped to reveal possible interactions which might exist between NO and O2– in the hypertensive rats at this level of kidney function.
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Methods |
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The measurement of proximal tubular fluid reabsorption was performed as previously described (Wu et al. 1999). Briefly, tubules were punctured with a double barrelled micropipette and a column of Sudan black castor oil, of some 20 tubule diameters in length, was injected and then a small volume of artificial proximal tubular fluid (APTF) injected to split the column. Images were captured of the shrinking split droplet at 2 s intervals using a video camera (Leica, UK). A digital image capture programme stored and analysed the images which then calculated the rate of proximal tubular fluid reabsorption expressed per unit area of epithelium (Jva x 10–4 min3 mm–2 s–1; Harris et al. 1987). Each tubule was subjected to the shrinking split droplet procedure two to three times to ensure that measurements were consistent and average values were taken.
Whole kidney glomerular filtration rate was evaluated using 15 min urine collections and the calculation of inulin clearance (Zhang et al. 1997; Wu et al. 1999). Urine flow rate was estimated gravimetrically and sodium content assessed using flame photometry (Corning model 410C, Halstead, Essex, UK). Blood pressure was measured via a simulated polygraph using LabVIEW software (National Instruments, Austin, Texas, USA).
Groups of rats (n= 6–8) were studied to determine control responses to renal nerve stimulation and then when either NO production was blocked, a NO donor given or superoxide anion production was minimized:
Wistar rats
SHRSP rats
A minimum of one pair of surface nephrons was used per rat. Basal measurements of Jva were performed and then the second and third measurements were taken from the same nephron, during which the renal sympathetic nerves were stimulated at either 0.75 or 1 Hz (2 ms, 15 V) in random order. After a recovery period of 15 min, a second set of estimations were undertaken using a different nephron. The control whole kidney and Jva measurements for the Wistar rats have been previously published (Wu & Johns, 2002) but are included to allow more informative comparisons to be undertaken. The drugs under investigation were presented to the tubules in random order on either the first or second set of measurements. Once the Jva estimations were completed, 15 min clearance periods were undertaken for whole kidney function measurements. Arterial blood samples were taken for inulin and electrolyte evaluation. At the end of each study, the animals were killed humanely using anaesthetic overdose.
L-NAME, sodium nitroprusside (SNP), superoxide dismutase (SOD) and castor oil were purchased from Sigma (Poole, Dorset, UK) and other compounds were obtained from BDH (Poole, Dorset, UK).
Statistics
Data are presented as means ±S.E.M. Comparisons were undertaken using ANOVA for differences between groups followed by a Bonferroni-Dunn post hoc test for within group variations. The percentage changes reported were calculated from the absolute values obtained. Significance was taken when P < 0.05.
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Results |
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In the SHRSP in which APTF was given intraluminally, renal nerve stimulation at 0.75 Hz increased Jva by 27% (P < 0.05) but when the stimulation frequency was increased to 1.0 Hz, Jva was significantly higher than basal values (P < 0.01) but similar to that achieved at 0.75 Hz (Fig. 2). This pattern of response to graded renal nerve stimulation was different from that obtained in the Wistar rats when APTF was present in the tubules. The intratubular administration of L-NAME had no effect on the baseline values of Jva but during renal nerve stimulation at 0.75 Hz it was increased (P < 0.01) by 34%, but at 1.0 Hz was slightly lower and not different from the basal value. The pattern of Jva responses to the graded rise in renal nerve stimulation was no different from that obtained when APTF was given alone. In the presence of SOD, basal Jva rose by 33% (2.05 ± 0.17 versus 2.72 ± 0.13 x 10–4 mm3 mm2 s–1, P < 0.01), which was in contrast to the Wistar rats where SOD was without effect on Jva. During renal nerve stimulation with SOD present intraluminally, Jva increased by 21% at 0.75 Hz (P < 0.05), but at 1.0 Hz was lower than at the 0.75 Hz stimulation and not significantly different from basal Jva. Again, these responses parallel those obtained during renal nerve stimulation when APTF or APTF plus L-NAME was present in the tubule. When SNP was present intraluminally, basal Jva was similar to that when APTF was used, but under these conditions renal nerve stimulation increased Jva by 20% at 0.75 Hz and 45% at 1.0 Hz (P < 0.05; Fig. 2) which was a pattern very different from that observed when only APTF was present in the tubules.
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Discussion |
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It was evident in the normotensive rats that the renal nerve stimulation parameters were such that there were no measurable changes in whole kidney glomerular filtration rate but they were able to cause frequency related decreases in urine flow and absolute sodium excretion. These observations are similar to those reported earlier by ourselves and others (Johns & Manitius, 1987; DiBona & Sawin, 1982; Bello-Reuss et al. 1976) indicative of a direct action of the neurotransmitter noradrenaline on the tubular reabsorptive processes. Indeed, this was shown to be the case more directly in that Jva increased in proportion to the degree of renal nerve stimulation which was consistent with our earlier report (Wu & Johns, 2002). The situation in the SHRSP was somewhat different in that although basal blood pressure was higher, glomerular filtration rate, urine flow and sodium excretion were lower than the corresponding basal values in the normotensive Wistar rats. These altered levels of renal function are similar to those reported earlier in this strain (Davis & Johns, 1994; Zhang et al. 1997) and may be due to both the chronically elevated blood pressure as well as genetic differences within the kidney. Electrical stimulation of the renal sympathetic nerves in the SHRSP caused no change in glomerular filtration rate but an antinatriuresis and antidiuresis was observed as in the Wistar rats. However, it was clear that the excretory responses to renal nerve stimulation in both absolute and percentage terms were substantially blunted in the SHRSP. Indeed, this corresponded to earlier observations in this strain when the renal sympathetic nerves were reflexly activated (Davis & Johns, 1994; Zhang et al. 1997). This lack of responsiveness to electrical stimulation of the renal nerves was also reflected at the proximal tubular level in that although stimulation at 0.75 Hz caused a small increase in Jva, there was no further rise at the higher stimulation frequency of 1.0 Hz as occurred in the Wistar rats. The reason for this blunted frequency-response relationship in the SHRSP was not clear but suggested some feedback interaction either at the neuroeffector junction, limiting the action of the renal nerves, or within the epithelial cells themselves.
More recently, we demonstrated that low level electrical renal nerve stimulation causing an increase in Jva also required the presence of NO (Wu & Johns, 2002) suggesting another site of action for NO in the neural regulation of tubular fluid handling. Further evidence for a deficit in the role of NO in the proximal tubule of the SHRSP was provided by the present study using intraluminal L-NAME. This study gave rise to two important findings: firstly, that the intraluminal L-NAME had no effect on basal levels of Jva, consistent with our earlier report in the SHRSP (Wu et al. 1999); and secondly that it had no influence on the pattern of the nerve-mediated increases in Jva, that is a small increase at 0.75 Hz, and no further change at 1.0 Hz. Indeed, this lack of effect of L-NAME contrasts with the observation that it blocked the renal nerve-induced increases in Jva in the Wistar rats (Wu & Johns, 2002). This would suggest that NO was not able to contribute to the noradrenergic control of tubular fluid reabsorption in the SHRSP. Interestingly, the expression of nitric oxide synthase (NOS) isoforms in the kidneys of hypertensive rats has been reported to be up-regulated (Welch et al. 1999; Varizi et al. 1998) which would suggest increased NO production in this pathophysiological state. In spite of this, the observations of the present study in the SHRSP indicate that at a functional level NO was unable to exert its normal physiological role. Alternatively, at the proximal epithelial cells, there may still be a lack of NO as L-NAME was without effect whereas in the presence of a NO donor (SNP) there was a restoration of the Jva response to renal nerve stimulation.
One of the other ways in which NO activity and production may be altered is as a result of a deficit in cofactors which modify the end product of NOS activity. The cellular function of NOS enzymes is complex and the end product of their action, NO, only occurs when there are adequate supplies of cofactors and substrate, particularly pteridine tetrahydrobiopterin and L-arginine (Andrew & Mayer, 1999; Govers & Rabelink, 2001). Should the availability of either or both these factors be limited in the environment, either O2– or ONOO– may be generated with the outcome that less NO is available and dependent mechanisms will be defective. Thus, the possibility exists that there may be an overproduction of O2– or ONOO– rather than NO and that these radicals could degrade NO within the epithelial cells before it was able to exert its normal physiological action. This possibility was tested in the present study by administering SOD intraluminally in an attempt to scavenge all O2– that might be produced and which might be responsible for degrading NO. It was apparent that SOD given into the Wistar rats was without effect on either basal levels of Jva or on the ability of the renal nerves to increase proximal tubular fluid reabsorption. This observation supports a recent report of Varizi et al. (1998) who found that antioxidant administration to normotensive rats had no effect on nitrite/nitrate excretion in the urine.
The situation in the SHRSP was somewhat different in that intraluminal SOD increased basal levels of Jva to values comparable to those observed in Wistar rats which would imply that the production of the reactive oxygen species was enhanced in the hypertensive state and that these radicals could in some way be suppressing basal levels of fluid transport. Again, support for this view can be drawn from the report of Varizi et al. (2000) who demonstrated increased production of reactive oxygen species in the SHR. Moreover, these authors also found that there was increased expression of eNOS, iNOS and nNOS in a number of tissues of the SHR, including the kidney. However, an increase in expression of the enzymes does not address the question as to whether post transcriptional or translational activity is parallel under these conditions. In spite of this observation, stimulation of the renal nerves increased Jva at the lower, but not higher, frequency of stimulation, in a pattern comparable when only APTF was present in the lumen. Thus, one conclusion could be that the deficit in the renal nerve-mediated increases in tubular fluid reabsorption in the hypertensive rat was not entirely dependent upon a raised production of reactive oxygen species.
In an attempt to explore these relationships further, another set of studies was performed in which the NO donor SNP was given intraluminally in the SHRSP, and under these conditions it had no effect on basal levels of Jva. However, when the renal nerves were stimulated there was a frequency related increase in Jva, the pattern of which was comparable to that found in the Wistar rats. This would suggest that NO availability was also an important rate limiting step but not the only factor(s) that determined the epithelial cell response to the nerve stimulation.
This series of studies has shown that the neurally induced increases in proximal tubular fluid reabsorption were blunted in the SHRSP compared to the normotensive Wistar rats. They showed that in the SHRSP neither the pattern nor magnitude of response was altered when NO production was prevented with L-NAME and that intraluminal administration of a NO donor altered the pattern of renal nerve mediated increases in Jva to more like that of the Wistar rats. The apparent deficient availability of NO did not appear to be due to raised production of reactive oxygen species as intraluminal SOD had no effect on the way in which renal nerve stimulation increased proximal fluid reabsorption. Together, these data suggest that in the hypertensive rat a complex interaction exists leading to a NO-related deficit which prevents the renal nerves from exerting their normal actions on fluid handling at the proximal tubule.
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