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Intrathecal injection of an oxytocin-receptor antagonist attenuated postnephrectomy natriuresis in the male rat
ELSEVIER
Neuroscience Letters 195 (1995) 33-36
N[URgSCI[NC[ LETTERS
Intrathecal injection of an oxytocin-receptor antagonist attenuated postnephrectomy natriuresis in the male rat W. Huang a, Q.Z. Zhai b, M.
S j 6 q u i s t a,*
aDepartment of Physiology and Medical Biophysics, Biomedicum, Uppsala University, Box 572, S-751 23 Uppsala, Sweden bDepartment of Experimental Alcohol and Drug Addiction Research, Karolinska Institute, Stockholm, Sweden Received 1 March 1995; revised version received 12 June 1995; accepted 15 June 1995
Abstract
We have previously shown that oxytocin (OT) is a major humoral mediator in postnephrectomy natriuresis. As immunoassayable OT has been demonstrated in the spinal cord, the aim of this investigation was to determine whether OT receptors in the spinal cord are also involved in this natriuresis. The experiments were performed on anesthetized male rats. Before acute unilateral nephrectomy, an oxytocinreceptor antagonist was injected intrathecally in the thoracolumbar region in rats. Postnephrectomy natriuresis was attenuated by this injection but not by intrathecal injection of artificial cerebrospinal fluid. Our results suggest that OT receptors within the spinal cord may influence the autonomic nervous regulation of renal function. In an additional experiment, intravenously infused hexamethonium did not prevent the adaptive natriuresis in the remaining kidney. We conclude that OT receptors in the spinal cord are involved in the postnephrectomy natriuresis, possibly as a component in the afferent signal pathway.
Keywords: Renal sodium excretion; Acute unilateral nephrectomy; Intrathecal injection; Oxytocin-receptor antagonist; Hexamethonium
It has been suggested that postnephrectomy natriuresis may be a consequence of a neurohormonal reflex involving renal nerves, renorenal and baroreceptor reflex arcs and humoral factors [5,14]. Oxytocin (OT) is a neurohypophyseal hormone produced mainly in the paraventricular nucleus of the hypothalamus. In a previous study it was found that an intravenous infusion of an oxytocinreceptor antagonist (OT-ant) could depress the natriuresis occurring after acute unilateral nephrectomy (AUN). The plasma OT concentration increased after A U N and a similar elevated plasma level of OT, induced by OT infusion, was accompanied by natriuresis mimicking that observed postnephrectomy. Thus, it seems as if circulating OT is an essential component of the postnephrectomy natriuresis [3]. This is also true for the natriuresis elicited by a hypertonic NaC1 load [4]. On the other hand, OT may also act as a neurotransmitter and a neuromodulator in the nervous system (reviewed in Refs. [7,15]). Radioimmunoassay data have shown that in the human spinal cord oxytocin predominates over vasopressin [6], and that * Corresponding author, Tel.: +46 18 174181; Fax: +46 18 553541; E-mail: matss @bmc.uu.se.
the concentration of OT in the subarachnoid space is much higher than that of vasopressin [13]. The present investigation was undertaken to determine whether OT receptors in the spinal Cord, accessible via intrathecal injections, are involved in the postnephrectomy natriuresis. Adult male F1 hybrids of Lewis x DA rats weighing 250-380 g were used (n = 19). Female Lewis and male DA rats were purchased from ZFV, Hanover, Germany. They were mated and bred at our animal department. The experiments were approved by the Local Ethics Committee in Uppsala. Prior to the experiments, all animals had free access to tap water and a standardized chow (R3, Ewos, Sfdert~ilje, Sweden) containing 3 g Na/kg, 8 g K/kg and 13 MJ/kg. A selective OT antagonist [1-(3-mercaptopropionic acid),2-O-ethyl-D-Tyr,4-Thr, 8-Orn]-oxytocin (Atosiban, Ferring, Malm6, Sweden) was used in this study. This OT antagonist was designed and tested for its lack of agonistic properties (P. Melin, Ferring, pers. commun.). In a previous study [3], the specificity of the OT-ant was investigated in an in vivo bioassay system and is summarized in another report [4]. We found high antioxytocic
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 7 7 3 - M
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W. Huang et al. / Neuroscience Letters 195 (1995) 33-36
activity, without antivasopressor (anti-V 1) and antiantidiuretic (anti-V2) activities, when using a dose 100 times that used in the present study [3]. These findings are in good agreement with previously published pharmacological data [10]. In an additional experiment, we infused the ganglionic blocker hexamethonium (Hex) (Sigma, USA) intravenously. The animals were anesthetized intraperitoneally with Inactin R (Byk-Gulden, Konstanz, Germany), 120 mg/kg body weight (BW). A polyethylene catheter (PE-10) was inserted into the subarachnoid space via an incision in the cisterna magna guided by stereotaxic apparatus [ 18]. The catheter was introduced ~7.5 cm, with its tip placed in the lower thoracic region (TlrT13). At the end of each experiment, the position of the catheter was verified by laminectomy. The rat was placed on a servo-controlled heating pad which maintained its rectal temperature at 37.5°C. After tracheostomy, the left femoral vein was cannulated for continuous infusion (5 ml h -l kg -1 BW) of a Ringer solution containing (in mM) 129 NaCI, 2.5 KCI, 25 NaHCO3 and 0.75 CaCI2, to compensate for fluid losses during the experiment. The right femoral artery was cannulated for continuous recording of blood pressure. The bladder was catheterized through a suprapubic incision. Through a subcostal flank incision the left kidney was exposed and a ligature was placed loosely around its pedicle. The incision was then closed with clips. A 60min recovery period was allowed, followed by four collection periods lasting 15 min each. Urine was collected into preweighed plastic vials. After the completion of these control collections, the subcostal incision was opened again. The renal pedicle was tied with the previously placed ligature, without removing the kidney. Two groups were studied: (1) AUN and intrathecal pre-treatment with OT-ant (OT-ant AUN group) (n = 6); (2) AUN and intrathecal pre-treatment with vehicle (CSF AUN group) (n = 6). In the OT-ant AUN group, OT-ant in a dose of 0.4/~g kg -1 BW was injected intrathecally in a volume of 10/A followed by a 10/~1 injection of CSF to wash out the catheter. This was done 10 min before the AUN. In the CSF AUN group, 10 + 10/A of artificial CSF was injected intrathecally. Immediately after the completion of AUN, four urine samples were collected at 15-min intervals. CSF contains (in mM) 126 NaC1, 3 KC1, 25 NaHCO3, 0.8 MgC1, 0.5 NaH2PO 4 and 1.14 CaC12. In an additional experiment, the Ringer solution was switched at the beginning of the control periods to one supplemented with hexamethonium yielding a dose of 20 mg h -1 kg -1 BW. The hexamethonium solution was infused intravenously throughout the experiment in the HEX AUN group (n = 7). The urine volume was measured gravimetrically and was calculated uniformly in terms of excretion of a single kidney. Urinary sodium and potassium concentrations
were determined by flame photometry (FLM3, Radiometer, Denmark). The osmolality was measured as freezing point depression (Model 3MO, Advanced Instr. Inc, MA, USA). All data are presented as means + SEM. The effects of AUN on renal function were analyzed by one-way analysis of variance (ANOVA) with a repeated measures design within all groups. Comparisons between groups were made with one-way ANOVA using Bonferroni adjusted t values. A P value of less than 0.05 was considered to be statistically significant. AUN in rats injected intrathecally with artificial CSF caused an increase in renal electrolyte and water excretion without a change in osmolality (Fig. 1). Although the basal level of renal excretion was reduced by the intrathecal catheterization per se, the excretory data and the transient increase in MAP shortly after AUN (Figs. 1 and 2) are in a good agreement with previous observations [3]. Intrathecal administration of OT-ant attenuated the renal cation excretion in response to AUN, but not the renal 0.5
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Fig. 1. Sodium excretion (UNaV), potassium excretion (UK~/), urine flow rate ( V ) and urine osmolality (Uosrn) in the animals pretreated with intrathecal injection of CSF (CSF AUN, n = 6) or of OT-ant (OTant AUN, n = 6) prior to acute unilateral nephrectomy (AUN). The arrows indicate the time of nephrectomy. Values are means _+ SEM. *P < 0.05, **P < 0.01 compared with the corresponding value in the control period within each group. * P < 0.05, * * P < 0.01 for differences between CSF AUN and OT-ant AUN groups.
W. Huang et al. / Neuroscience Letters 195 (1995) 33-36
130
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Fig. 2. The mean (±SEM) arterial blood pressure (MAP) in the animals pretreated with intratheeal injection of CSF (CSF AUN, n = 6) or of OT-ant (OT-ant AUN, n = 6) prior to acute unilateral nephrectomy (AUN). The arrows indicate the time of nephrectomy. Values are means ± SEM.
water excretion and the transient increase in MAP (P < 0.01) (Figs. 1 and 2). The effects of intravenous infusion of hexamethonium are shown in Figs. 3 and 4. Despite a fall in MAP (by approximately 50-60 mmHg), the excretory responses of the remaining kidney to AUN still remained. An interesting finding was a uniform transient rise in MAP (from 72 _+2.2 to 94 _+ 3.2 mmHg, P < 0.01) 1 or 2 min after ligation of the left renal pedicle. The results of our current study indicate that OT receptors exist within the thoracolumbar region of the spinal cord and that they may contribute to the initiation of postnephrectomy natriuresis (Fig. 1). We used the method of Yaksh et al. [ 18] to catheterize the lower thoracic subarachnoid space, which is the level on which both afferent and efferent renal nerves are connected with the spinal cord [9]. Yaksh et al. tested this method with an intrathecal injection of the dye bromophenol blue and showed that 10/~1 of dye had diffused about 2.5 cm within the subarachnoid space from the tip of the catheter in 10 min. The dosage of OT-ant employed in the present study was 1% of the intravenous infusion dosage as used in our previous study. Given that the lipid solubility of OT-ant is very low, it appears unlikely that intrathecally administered OT-ant might have gained access to either supraspinal or peripheral systems. It has also been shown that the turnover of the spinal fluid is so slow that antagonist injected into the thoracolumbar region of the spinal cord can be assumed to exert its effect locally [18]. OT has been claimed to be capable of influencing sympathetic preganglionic neurons through the paraventriculo-spinal pathway [2]. To investigate whether OT receptors are involved in the afferent or the efferent renal neural pathways, we used the ganglionic blocker hexamethonium in our additional experiment. Hexametho-
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Fig. 3. Sodium excretion (UNaV), potassium excretion (UK~/), urine flow rate ( V ) and urine osmolality (Uosm) in the animals pretreated with intravenous infusion of hexamethonium (HEX) prior to acute unilateral nephrectomy (AUN) (HEX AUN, n = 7). The arrows indicate the time of nephrectomy. Values are means ± SEM. * P < 0 . 0 5 , **P < 0.01 compared with the corresponding value in the control period.
nium blocks the efferent nerve traffic to the kidneys and can be used to differentiate renal afferent nerve activity from efferent activity [12]. The dose we used has been
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36
w. Huang et al. / Neuroscience Letters 195 (1995) 33-36
shown to cause a total ganglionic blockade in rats [8]. Despite a fall in M A P after intravenous administration of hexamethonium, the renal functional adaptation of the remaining kidney to the loss of its partner still remained and the natriuresis was accompanied by a uniform transient rise in M A P immediately after A U N (Figs. 3 and 4). It has been assumed that the renorenal reflex and baroreceptor reflexes participate in the rapid adaptation of the remaining kidney after loss of its counterpart [5,14]. If this is true, the efferent limb of the reflex arc might involve not only the efferent renal nerves, but also a humoral effector, since i.v. hexamethonium did not sufficiently block the adaptation. This is supported by experiments performed on transplanted kidneys, in which postnephrectomy natriuresis persisted in newly transplanted kidneys [11]. In a previous study concerning the effects of neurohypophyseal antagonists in postnephrectomy natriuresis, evidence was obtained that circulating OT was such a humoral effector [3]. In an investigation involving the destruction of the paraventricular nucleus, Webb and Brody [17] pointed out the close relationship between the afferent renal projections and the paraventricular nucleus. The latter could be a site of integration of neuroendocrine and autonomic mechanisms [16]. Our results suggest that OT receptors may exist in the afferent renal signal route receiving the renal sensory signals. The contribution of the renal sympathetic nerves to hypertension has been summarized by DiBona [1]. The afferent renal nerves, conveying information from renal sensory receptors to the hypothalamus, are important modulators of integrative centres involved in the regulation of peripheral sympathetic nervous system activity. Thus, it seems that the kidneys may not only act under hypothalamic neurohumoral control, but also initiate stimuli to the hypothalamus to integrate the circulatory and endocrine responses to perturbation of fluid balance. Together with our previous observations [3,4], the present results indicate that oxytocin can function at sites both in the central nervous system and in the peripheral circulation, thus contributing to sodium homeostasis. The fact that the acute renal response to unilateral nephrectomy can be attenuated by intrathecal injection of the selective OT-receptor antagonist, but not by intravenous infusion of hexamethonium, implies that oxytocin receptors in the spinal cord may influence the acute renal adjustment of sodium excretion mainly through afferent mechanisms. We are grateful to Britta Isaksson for technical assistance, to Eva Jacobsson for help with the statistics, and to
Dr. Per Melin (Ferring) for supplying the OT-receptor antagonist. Financial support for this study was provided by the Swedish Medical Research Council project 00140, the Wiberg and the T. & R. S6derberg Foundation. [1] DiBona, G.F., Sympathetic neural control of the kidney in hypertension, Hypertension,19 (suppl. I) (1992) 128-I35. [2] Gilbey, M.P., Coote, J.H., Fleetwood-Walker, S. and Peterson, D.F., The influence of the paraventriculo-spinalpathway, and oxytocin and vasopressinon sympatheticpreganglionicneurones, Brain Res., 251 (1982) 283-290. [3] Huang, W., Lee, S.L. and Sj6quist, M., Effects of neurohypophyseal antagonists in postnephrectomynatriuresis in male rats, Kidney Int., 45 (1994) 692-699. [4] Huang, W., Lee, S.L. and Sj6quist, M., Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaCI, Am. J. Physiol.,268 (1995) R634-R640. [5] Humphreys, M.H., Lin, S.Y. and Wiedemann, E., Renal nerves and the natriuresisfollowing unilateral renal exclusion in the rat, Kidney Int., 39 (1991) 63-70. [6] Jenkins, J.S., Ang, V.T.Y., Hawthorn, J., Rossor, M.N. and Iversen, L.L., Vasopressin, oxytocin and neurophysins in the human brain and spinal cord, Brain Res., 291 (1984) 111-117. [7] Jones, P.M. and Robinson, I.C.A.F., Differential clearance of neurophysin and neurohypophysialpeptides from the cerebrospinal fluid in conscious guinea pigs, Neuroendocrinology, 34 (1982) 297-302. [8] Krueger, A.D., Lee, J.Y., Yang, P-C., Papaioannou, S.E. and Walsh, G.M., Selective vasodilation produced by renal denervation in adult spontaneously hypertensive rats., Hypertension, 8 (1986) 372-378. [9] Kuo, D.C., Nadelhaft, I., Hisamitsu, T. and de Groat, W.C., Segmental distributionand central projections of renal afferent fibers in the cat studied by transganglionictransport of horseradish peroxidase, J. Comp. Neurol., 216 (1983) 162-174. [10] Melin, P., Oxytocin antagonists in preterm labour and delivery, Bailli~res Clin. Obstet. Gynaecol.,7 (1993) 577-600. [11] Miiller-Suur,R., Norlen, B.J. and Persson, A.E.G., Resetting of tubuloglomerular feedback in rat kidneys after unilateral nephrectomy, Kidney Int., 18 (1980)48-57. [12] Osbom, Jr., J.W., Livingstone,R.H. and Schramm, L.P., Elevated renal nerve activity after spinal transection:effects on renal function, Am. J. Physiol.,253 (1987) R619-R625. [13] Pittman, Q.L, Riphagen, C.L. and Lederis, K., Release of immunoassayable neurohypophysealpeptides from rat spinal cord, in vivo, Brain Res., 300 (1984) 321-326. [14] Ribstein, J. and Humphreys, M.H., Renal nerves and cation excretion after acute reduction in functioning renal mass in the rat, Am. J. Physiol., 246 (1984) F260-F265. [15] Richard, P., Moos, F. and Freund-Mercier,M.-J., Central effects of oxytocin,Physiol. Rev., 71 ( 1991) 331-370. [16] Swanson, L.W. and Sawchenko, P.E., Paraventricularnucleus: a site for the integration of neuroendocrineand autonomic mechanisms, Neuroendocrinology,31 (1980) 410-417. [17] Webb, R.L. and Brody, M.J., Functional identification of the central projectionsof afferent renal nerves, Clin. Exp., A9 (suppl. 1.) (1987) 47-57. [18] Yaksh, T.L. and Rudy, T.A., Chronic catheterizationof the spinal subarachnoid space, Physiol. Behav. 17 (1976) 1031-1036.
Neuroscience Letters 195 (1995) 33-36
N[URgSCI[NC[ LETTERS
Intrathecal injection of an oxytocin-receptor antagonist attenuated postnephrectomy natriuresis in the male rat W. Huang a, Q.Z. Zhai b, M.
S j 6 q u i s t a,*
aDepartment of Physiology and Medical Biophysics, Biomedicum, Uppsala University, Box 572, S-751 23 Uppsala, Sweden bDepartment of Experimental Alcohol and Drug Addiction Research, Karolinska Institute, Stockholm, Sweden Received 1 March 1995; revised version received 12 June 1995; accepted 15 June 1995
Abstract
We have previously shown that oxytocin (OT) is a major humoral mediator in postnephrectomy natriuresis. As immunoassayable OT has been demonstrated in the spinal cord, the aim of this investigation was to determine whether OT receptors in the spinal cord are also involved in this natriuresis. The experiments were performed on anesthetized male rats. Before acute unilateral nephrectomy, an oxytocinreceptor antagonist was injected intrathecally in the thoracolumbar region in rats. Postnephrectomy natriuresis was attenuated by this injection but not by intrathecal injection of artificial cerebrospinal fluid. Our results suggest that OT receptors within the spinal cord may influence the autonomic nervous regulation of renal function. In an additional experiment, intravenously infused hexamethonium did not prevent the adaptive natriuresis in the remaining kidney. We conclude that OT receptors in the spinal cord are involved in the postnephrectomy natriuresis, possibly as a component in the afferent signal pathway.
Keywords: Renal sodium excretion; Acute unilateral nephrectomy; Intrathecal injection; Oxytocin-receptor antagonist; Hexamethonium
It has been suggested that postnephrectomy natriuresis may be a consequence of a neurohormonal reflex involving renal nerves, renorenal and baroreceptor reflex arcs and humoral factors [5,14]. Oxytocin (OT) is a neurohypophyseal hormone produced mainly in the paraventricular nucleus of the hypothalamus. In a previous study it was found that an intravenous infusion of an oxytocinreceptor antagonist (OT-ant) could depress the natriuresis occurring after acute unilateral nephrectomy (AUN). The plasma OT concentration increased after A U N and a similar elevated plasma level of OT, induced by OT infusion, was accompanied by natriuresis mimicking that observed postnephrectomy. Thus, it seems as if circulating OT is an essential component of the postnephrectomy natriuresis [3]. This is also true for the natriuresis elicited by a hypertonic NaC1 load [4]. On the other hand, OT may also act as a neurotransmitter and a neuromodulator in the nervous system (reviewed in Refs. [7,15]). Radioimmunoassay data have shown that in the human spinal cord oxytocin predominates over vasopressin [6], and that * Corresponding author, Tel.: +46 18 174181; Fax: +46 18 553541; E-mail: matss @bmc.uu.se.
the concentration of OT in the subarachnoid space is much higher than that of vasopressin [13]. The present investigation was undertaken to determine whether OT receptors in the spinal Cord, accessible via intrathecal injections, are involved in the postnephrectomy natriuresis. Adult male F1 hybrids of Lewis x DA rats weighing 250-380 g were used (n = 19). Female Lewis and male DA rats were purchased from ZFV, Hanover, Germany. They were mated and bred at our animal department. The experiments were approved by the Local Ethics Committee in Uppsala. Prior to the experiments, all animals had free access to tap water and a standardized chow (R3, Ewos, Sfdert~ilje, Sweden) containing 3 g Na/kg, 8 g K/kg and 13 MJ/kg. A selective OT antagonist [1-(3-mercaptopropionic acid),2-O-ethyl-D-Tyr,4-Thr, 8-Orn]-oxytocin (Atosiban, Ferring, Malm6, Sweden) was used in this study. This OT antagonist was designed and tested for its lack of agonistic properties (P. Melin, Ferring, pers. commun.). In a previous study [3], the specificity of the OT-ant was investigated in an in vivo bioassay system and is summarized in another report [4]. We found high antioxytocic
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 7 7 3 - M
34
W. Huang et al. / Neuroscience Letters 195 (1995) 33-36
activity, without antivasopressor (anti-V 1) and antiantidiuretic (anti-V2) activities, when using a dose 100 times that used in the present study [3]. These findings are in good agreement with previously published pharmacological data [10]. In an additional experiment, we infused the ganglionic blocker hexamethonium (Hex) (Sigma, USA) intravenously. The animals were anesthetized intraperitoneally with Inactin R (Byk-Gulden, Konstanz, Germany), 120 mg/kg body weight (BW). A polyethylene catheter (PE-10) was inserted into the subarachnoid space via an incision in the cisterna magna guided by stereotaxic apparatus [ 18]. The catheter was introduced ~7.5 cm, with its tip placed in the lower thoracic region (TlrT13). At the end of each experiment, the position of the catheter was verified by laminectomy. The rat was placed on a servo-controlled heating pad which maintained its rectal temperature at 37.5°C. After tracheostomy, the left femoral vein was cannulated for continuous infusion (5 ml h -l kg -1 BW) of a Ringer solution containing (in mM) 129 NaCI, 2.5 KCI, 25 NaHCO3 and 0.75 CaCI2, to compensate for fluid losses during the experiment. The right femoral artery was cannulated for continuous recording of blood pressure. The bladder was catheterized through a suprapubic incision. Through a subcostal flank incision the left kidney was exposed and a ligature was placed loosely around its pedicle. The incision was then closed with clips. A 60min recovery period was allowed, followed by four collection periods lasting 15 min each. Urine was collected into preweighed plastic vials. After the completion of these control collections, the subcostal incision was opened again. The renal pedicle was tied with the previously placed ligature, without removing the kidney. Two groups were studied: (1) AUN and intrathecal pre-treatment with OT-ant (OT-ant AUN group) (n = 6); (2) AUN and intrathecal pre-treatment with vehicle (CSF AUN group) (n = 6). In the OT-ant AUN group, OT-ant in a dose of 0.4/~g kg -1 BW was injected intrathecally in a volume of 10/A followed by a 10/~1 injection of CSF to wash out the catheter. This was done 10 min before the AUN. In the CSF AUN group, 10 + 10/A of artificial CSF was injected intrathecally. Immediately after the completion of AUN, four urine samples were collected at 15-min intervals. CSF contains (in mM) 126 NaC1, 3 KC1, 25 NaHCO3, 0.8 MgC1, 0.5 NaH2PO 4 and 1.14 CaC12. In an additional experiment, the Ringer solution was switched at the beginning of the control periods to one supplemented with hexamethonium yielding a dose of 20 mg h -1 kg -1 BW. The hexamethonium solution was infused intravenously throughout the experiment in the HEX AUN group (n = 7). The urine volume was measured gravimetrically and was calculated uniformly in terms of excretion of a single kidney. Urinary sodium and potassium concentrations
were determined by flame photometry (FLM3, Radiometer, Denmark). The osmolality was measured as freezing point depression (Model 3MO, Advanced Instr. Inc, MA, USA). All data are presented as means + SEM. The effects of AUN on renal function were analyzed by one-way analysis of variance (ANOVA) with a repeated measures design within all groups. Comparisons between groups were made with one-way ANOVA using Bonferroni adjusted t values. A P value of less than 0.05 was considered to be statistically significant. AUN in rats injected intrathecally with artificial CSF caused an increase in renal electrolyte and water excretion without a change in osmolality (Fig. 1). Although the basal level of renal excretion was reduced by the intrathecal catheterization per se, the excretory data and the transient increase in MAP shortly after AUN (Figs. 1 and 2) are in a good agreement with previous observations [3]. Intrathecal administration of OT-ant attenuated the renal cation excretion in response to AUN, but not the renal 0.5
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Fig. 1. Sodium excretion (UNaV), potassium excretion (UK~/), urine flow rate ( V ) and urine osmolality (Uosrn) in the animals pretreated with intrathecal injection of CSF (CSF AUN, n = 6) or of OT-ant (OTant AUN, n = 6) prior to acute unilateral nephrectomy (AUN). The arrows indicate the time of nephrectomy. Values are means _+ SEM. *P < 0.05, **P < 0.01 compared with the corresponding value in the control period within each group. * P < 0.05, * * P < 0.01 for differences between CSF AUN and OT-ant AUN groups.
W. Huang et al. / Neuroscience Letters 195 (1995) 33-36
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Fig. 2. The mean (±SEM) arterial blood pressure (MAP) in the animals pretreated with intratheeal injection of CSF (CSF AUN, n = 6) or of OT-ant (OT-ant AUN, n = 6) prior to acute unilateral nephrectomy (AUN). The arrows indicate the time of nephrectomy. Values are means ± SEM.
water excretion and the transient increase in MAP (P < 0.01) (Figs. 1 and 2). The effects of intravenous infusion of hexamethonium are shown in Figs. 3 and 4. Despite a fall in MAP (by approximately 50-60 mmHg), the excretory responses of the remaining kidney to AUN still remained. An interesting finding was a uniform transient rise in MAP (from 72 _+2.2 to 94 _+ 3.2 mmHg, P < 0.01) 1 or 2 min after ligation of the left renal pedicle. The results of our current study indicate that OT receptors exist within the thoracolumbar region of the spinal cord and that they may contribute to the initiation of postnephrectomy natriuresis (Fig. 1). We used the method of Yaksh et al. [ 18] to catheterize the lower thoracic subarachnoid space, which is the level on which both afferent and efferent renal nerves are connected with the spinal cord [9]. Yaksh et al. tested this method with an intrathecal injection of the dye bromophenol blue and showed that 10/~1 of dye had diffused about 2.5 cm within the subarachnoid space from the tip of the catheter in 10 min. The dosage of OT-ant employed in the present study was 1% of the intravenous infusion dosage as used in our previous study. Given that the lipid solubility of OT-ant is very low, it appears unlikely that intrathecally administered OT-ant might have gained access to either supraspinal or peripheral systems. It has also been shown that the turnover of the spinal fluid is so slow that antagonist injected into the thoracolumbar region of the spinal cord can be assumed to exert its effect locally [18]. OT has been claimed to be capable of influencing sympathetic preganglionic neurons through the paraventriculo-spinal pathway [2]. To investigate whether OT receptors are involved in the afferent or the efferent renal neural pathways, we used the ganglionic blocker hexamethonium in our additional experiment. Hexametho-
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I
30
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Fig. 3. Sodium excretion (UNaV), potassium excretion (UK~/), urine flow rate ( V ) and urine osmolality (Uosm) in the animals pretreated with intravenous infusion of hexamethonium (HEX) prior to acute unilateral nephrectomy (AUN) (HEX AUN, n = 7). The arrows indicate the time of nephrectomy. Values are means ± SEM. * P < 0 . 0 5 , **P < 0.01 compared with the corresponding value in the control period.
nium blocks the efferent nerve traffic to the kidneys and can be used to differentiate renal afferent nerve activity from efferent activity [12]. The dose we used has been
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Fig. 4. The mean (±SEM) arterial blood pressure (MAP) in the animals pretreated with intravenous infusion of hexamethonium (HEX) prior to acute unilateral nephrectomy (AUN) (HEX AUN, n = 7). The arrow indicates the time of the nephrectomy.
36
w. Huang et al. / Neuroscience Letters 195 (1995) 33-36
shown to cause a total ganglionic blockade in rats [8]. Despite a fall in M A P after intravenous administration of hexamethonium, the renal functional adaptation of the remaining kidney to the loss of its partner still remained and the natriuresis was accompanied by a uniform transient rise in M A P immediately after A U N (Figs. 3 and 4). It has been assumed that the renorenal reflex and baroreceptor reflexes participate in the rapid adaptation of the remaining kidney after loss of its counterpart [5,14]. If this is true, the efferent limb of the reflex arc might involve not only the efferent renal nerves, but also a humoral effector, since i.v. hexamethonium did not sufficiently block the adaptation. This is supported by experiments performed on transplanted kidneys, in which postnephrectomy natriuresis persisted in newly transplanted kidneys [11]. In a previous study concerning the effects of neurohypophyseal antagonists in postnephrectomy natriuresis, evidence was obtained that circulating OT was such a humoral effector [3]. In an investigation involving the destruction of the paraventricular nucleus, Webb and Brody [17] pointed out the close relationship between the afferent renal projections and the paraventricular nucleus. The latter could be a site of integration of neuroendocrine and autonomic mechanisms [16]. Our results suggest that OT receptors may exist in the afferent renal signal route receiving the renal sensory signals. The contribution of the renal sympathetic nerves to hypertension has been summarized by DiBona [1]. The afferent renal nerves, conveying information from renal sensory receptors to the hypothalamus, are important modulators of integrative centres involved in the regulation of peripheral sympathetic nervous system activity. Thus, it seems that the kidneys may not only act under hypothalamic neurohumoral control, but also initiate stimuli to the hypothalamus to integrate the circulatory and endocrine responses to perturbation of fluid balance. Together with our previous observations [3,4], the present results indicate that oxytocin can function at sites both in the central nervous system and in the peripheral circulation, thus contributing to sodium homeostasis. The fact that the acute renal response to unilateral nephrectomy can be attenuated by intrathecal injection of the selective OT-receptor antagonist, but not by intravenous infusion of hexamethonium, implies that oxytocin receptors in the spinal cord may influence the acute renal adjustment of sodium excretion mainly through afferent mechanisms. We are grateful to Britta Isaksson for technical assistance, to Eva Jacobsson for help with the statistics, and to
Dr. Per Melin (Ferring) for supplying the OT-receptor antagonist. Financial support for this study was provided by the Swedish Medical Research Council project 00140, the Wiberg and the T. & R. S6derberg Foundation. [1] DiBona, G.F., Sympathetic neural control of the kidney in hypertension, Hypertension,19 (suppl. I) (1992) 128-I35. [2] Gilbey, M.P., Coote, J.H., Fleetwood-Walker, S. and Peterson, D.F., The influence of the paraventriculo-spinalpathway, and oxytocin and vasopressinon sympatheticpreganglionicneurones, Brain Res., 251 (1982) 283-290. [3] Huang, W., Lee, S.L. and Sj6quist, M., Effects of neurohypophyseal antagonists in postnephrectomynatriuresis in male rats, Kidney Int., 45 (1994) 692-699. [4] Huang, W., Lee, S.L. and Sj6quist, M., Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaCI, Am. J. Physiol.,268 (1995) R634-R640. [5] Humphreys, M.H., Lin, S.Y. and Wiedemann, E., Renal nerves and the natriuresisfollowing unilateral renal exclusion in the rat, Kidney Int., 39 (1991) 63-70. [6] Jenkins, J.S., Ang, V.T.Y., Hawthorn, J., Rossor, M.N. and Iversen, L.L., Vasopressin, oxytocin and neurophysins in the human brain and spinal cord, Brain Res., 291 (1984) 111-117. [7] Jones, P.M. and Robinson, I.C.A.F., Differential clearance of neurophysin and neurohypophysialpeptides from the cerebrospinal fluid in conscious guinea pigs, Neuroendocrinology, 34 (1982) 297-302. [8] Krueger, A.D., Lee, J.Y., Yang, P-C., Papaioannou, S.E. and Walsh, G.M., Selective vasodilation produced by renal denervation in adult spontaneously hypertensive rats., Hypertension, 8 (1986) 372-378. [9] Kuo, D.C., Nadelhaft, I., Hisamitsu, T. and de Groat, W.C., Segmental distributionand central projections of renal afferent fibers in the cat studied by transganglionictransport of horseradish peroxidase, J. Comp. Neurol., 216 (1983) 162-174. [10] Melin, P., Oxytocin antagonists in preterm labour and delivery, Bailli~res Clin. Obstet. Gynaecol.,7 (1993) 577-600. [11] Miiller-Suur,R., Norlen, B.J. and Persson, A.E.G., Resetting of tubuloglomerular feedback in rat kidneys after unilateral nephrectomy, Kidney Int., 18 (1980)48-57. [12] Osbom, Jr., J.W., Livingstone,R.H. and Schramm, L.P., Elevated renal nerve activity after spinal transection:effects on renal function, Am. J. Physiol.,253 (1987) R619-R625. [13] Pittman, Q.L, Riphagen, C.L. and Lederis, K., Release of immunoassayable neurohypophysealpeptides from rat spinal cord, in vivo, Brain Res., 300 (1984) 321-326. [14] Ribstein, J. and Humphreys, M.H., Renal nerves and cation excretion after acute reduction in functioning renal mass in the rat, Am. J. Physiol., 246 (1984) F260-F265. [15] Richard, P., Moos, F. and Freund-Mercier,M.-J., Central effects of oxytocin,Physiol. Rev., 71 ( 1991) 331-370. [16] Swanson, L.W. and Sawchenko, P.E., Paraventricularnucleus: a site for the integration of neuroendocrineand autonomic mechanisms, Neuroendocrinology,31 (1980) 410-417. [17] Webb, R.L. and Brody, M.J., Functional identification of the central projectionsof afferent renal nerves, Clin. Exp., A9 (suppl. 1.) (1987) 47-57. [18] Yaksh, T.L. and Rudy, T.A., Chronic catheterizationof the spinal subarachnoid space, Physiol. Behav. 17 (1976) 1031-1036.