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ETA receptor-mediated role of endothelin in the kidney of doca-salt hypertensive rats
Life Scieaas. Vol. 58. NO. 1. pp. K 1-7. 19% copyri& 0 1995 Elacvia science Inc. Printed in the USA. All rights maewed 0024-32051% $15.00 + a0
ELSEVIER
0024-3205(95)02259-7
Pi%4Rh#2COLOGYLET Accelerated Communicadon ETA RECEPTOR-MEDIATED ROLE OF ENDOTHELIN IN THE KIDNEY DOCA-SALT HYPERTENSIVE RATS
OF
Katsuya Fujita, Yasuo Matsumura, Yohko Miyazaki, Norio Hashimoto, Masanori Takaoka and Shiro Morimoto* Department of Pharmacology, Osaka University of Pharmaceutical Sciences, 2-10-65 Kawai, Matsubara, Osaka 580, Japan (Submitted August 9, 19995;accepted August 31, 1995; received in final form October 16, 1995)
AI&& Renal effects of FRl39317, an endothelin ETA receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; -13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; -12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; -26%) accompanied by a potent renal vasodilation (RVR; -33%). FR139317 significantly increased renal blood flow only in the DOCAsalt rats. In contrast, FR1393 17 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-l) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-l is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-l and ETA receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-l production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension. Key Words: endothelin-1, ETA receptor, DOCA-salt hypertension, renal function
Endothelin-1 (ET-l) is a potent vasoconstrictor peptide isolated from the porcine vascular endothelial cells (1). ET-l produces contraction of vascular smooth muscle by activation of ETA or ETu receptors in various tissues (2,3). Recently, several ET receptor antagonists have been developed and utilized to evaluate the physiological and/or pathophysiological roles of endogenous ET-l and its receptor subtypes (4,5,6). FR139317 is a selective ETA receptor antagonist and inhibits ET-l-induced vasoconstrictive effect in vitro and in vivo (5). Previously, we and others reported that an intravenous bolus injection of FRl39317 produced a potent hypotensive effect in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (7,8). These results indicate that ET-l and ETA receptors make an important contribution to the maintenance of DOCA-salt hypertension, but the precise mechanism involved has remained unclear. In DOCA-salt hypertensive rats, a marked elevation in renal vascular resistance (RVR) and a decrease in renal blood flow (RBF) often occur, and these abnormalities in renal hemodynamics are thought to play an important role in the development and maintenance of the hypertension (9,lO). We compared the effect of ETA receptor blockade on renal hemodynamics and function in normotensive and DOCA-salt hypertensive rats, the objective being to evaluate the possible involvement of ET-l and the ETA receptor in renal hemodynamic abnormalities in DOCA-salt hypertensive rats. * To whom correspondence should be addressed
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Endothelin
and DOCA-salt
Hypertension
Vol. 58, No. 1. 1996
DOCA-salt treatment Male Sprague-Dawley rats, weighing 160-180 g were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the right kidney was removed via a right flank incision. After a l-week postsurgical recovery period, the rats were treated twice weekly with DOCA suspended in corn oil , which was administered subcutaneously (15 mg/kg) and 1% NaCl added to their tap water for drinking. Control rats were uninephrectomized but not given DOCA or salt. After 4 weeks of this treatment, the rats were used in clearance study or Northern blot analysis. Animal preparation for clearance study Animals were anesthetized with sodium thiobutabarbital (Inactin, 100 mg/kg, i.p.) and placed on a heated surgical tray that maintained a rectal temperature between 37 “C and 38 ‘C. After tracheotomy, the right femoral vein was cannulated for infusion of 0.9% saline containing 0.5% inulin. The right and left femoral arteries were also cannulated to measure mean arterial pressure (MAP) and for blood sampling, respectively. After an abdominal midline incision was made, the left kidney was exposed, and the renal artery was carefully stripped of connective tissue, followed by the application of 5% phenol in 70% ethanol to exclude the influence of renal sympathetic nerves. An electromagnetic flow probe (1 .O mm in diameter, Nihon Kohden, Tokyo, Japan) connected to a square-wave flowmeter (MFV-2100, Nihon Kohden) was positioned on the renal artery to measure RBF. A polyethylene cannula was inserted into the left ureter for urine collection. MAP and RBF were continuously recorded on a polygraph (RM 6000, Nihon Kohden), throughout all the experiment. A 60-90 min period was allowed for stabilization of MAP, RBF and urine flow (UF). Experimental protocol After the equilibration period, urine samples were collected during two 20 min control clearance periods. Following the control periods, FRl39317 (10 mg/kg) was administered intravenously by slow bolus injection. The dose of FR 1393 17 used in this study has been shown to produce complete inhibition of ET-l-induced pressor action (5). During the first 5 min after injection, urine was not collected in order to take into account for the dead space in the collection system. Following this, urine samples were collected during four consecutive 20 min periods (El-E4). Blood samples (0.2 ml each) were obtained at 20 min before drug injection and at 25 min and 65 min after the injection, respectively. The blood loss was replaced by giving an equal volume of blood from donor rats. Plasma was immediately separated by centrifugation. In the control experiment, the vehicle solution was administered. In this case, MAP, renal hemodynamics and function remained unchanged through the experiment. Results in the second control period served as the basal values of renal hemodynamics and function. Analyticalprocedure Urine and plasma inulin levels were measured by spectrofluorometry (Hitachi 650-50) according to the method of Vurek and Pergram (11). Glomerular filtration rate (GFR) was calculated from the inulin clearance. Urine and plasma sodium concentrations were determined using a flame photometer (Hitachi 205D). RNA extraction and Northern blot analysisfor prepro ET-l mRNA Total RNA from rat kidney was prepared by selective precipitation in 3 mol/l LiCl and 6 mol/l urea (12). Total RNA (20 @lane) was separated in formaldehyde-l.1 % agarose gel electrophoresis, and transferred to a nylon membrane (Hybond-N+, Amersham, Tokyo, Japan) in the presence of 20 x SSC (3 mol/l sodium chloride, 0.3 mol/l sodium citrate, pH 7.2). The membrane was hybridized in hybridization buffer (6 x SSC, O.Olmol/l EDTA, 5 x Denhardt’s solution, 0.5% SDS, 100 yg/ml sheared, denatured salmon sperm DNA) at 67 “C, overnight with ran prepro ET-l cDNA probe (a gift from Dr K. Goto, University of Tsukuba, Japan) labeled with [(%-32P]dCTP using a Random Primer DNA labeling Kit (Takara Shuzo, Osaka, Japan). -4utoradiography was then carried out by exposure to X-Omat AR film (Kodak, New York, USA)
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with intensifying screens at -80 ‘C for 12 h. The autoradiograms of ET-l were quantified by densitometric analysis, and the signals of ET-1 mRNA were normalized for each sample with respect to the 28s rRNA density in agarose gel stained by ethidium bromide.
FRl39317 was a kind gift from Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan. FRl39317 was dissolved in IN NaOH and diluted with saline. Other chemicals were purchased from Nacalai Tesque, Inc (Kyoto, Japan). Statistical analysis All values were expressed as mean + SEM. For statistical analysis, we used the unpaired Student’s t test for two-group comparisons and one-way analysis of variance combined with Dunnett’s multiple range test for multiple comparison. Differences were considered significant at P
TABLE
I.
Basal renal hemodynamics and function in anesthetized normotensive rats and DOCA-sait hypertensive rats.
normotensive control rats (n=6) MAP (mmHg) RBF (ml/g.min) RVR (mmHgfml/g.min) GFR (ml/g.min)
123+5 4.65 f 0.22 26.7 k 1.4 0.72 + 0.05
control
DOCA-salt hypertensive rats (n=6) 160 +_7** 2.34 + 0.3 1** 77.2 + 14.2** 0.70 f 0.09
UF (pl/g.min)
10.8 + 2.6
14.0 + 3.2
UNaV (@q/g.min)
2.47 + 0.27 2.39 f 0.26 365 I!Z4 1.91 * 0.03
2.18 f 0.54 2.28 f 0.67 318f. 8** 2.79 f 0.17**
ENa
(%)
Body wt. (g) Left kidney wt. (g)
Each value represents the mean + SEM. MAP; mean arterial pressure, RBF; renal blood flow, GFR; glomerular filtration rate, RVR; renal vascular resistance, UF; urine flow, UNaV; urinary excretion of sodium, FENa; fractional excretion of sodium. **P
Table I summarizes the body weight, the left kidney weight and the basal renal hemodynamics and function in anesthetized normotensive control rats and DOCA-salt hypertensive rats. The MAP, RVR and left kidney weight were significantly higher in DOCA-salt rats than in control rats, RBF was significantly decreased in DOCA-salt rats and was about half the value of that of control rats. The body weight was lower in DOCA-salt rats than in control rats. There were no significant differences in GFR and urine formation between the two groups.
Vol.58, No. 1, 1996
Endothelinand DOCksalt Hypertension
PL-4
W
4 160 CL2 ‘40 gE 120 C. 100 OfE
6
kE E$
5 !z.
OL 100
2
‘e
80
T
40 E 20 55!z. 600 kl_bTlk
**
** **
*+
“2
(pl/g!iin)
10.78 11.99 7.56 6.26 5.64* k2.61 f3.42 fl.61 kl.26 fl .12
UNaV (pEq/g.min)
2.47 2.72 1.97 1.37’+1.27** HI.27 20.20 a.31 HI.35 f0.29
FENa (“A)
UNaV (pEq/g.min) FENa
2.39 2.63 2.23 1.79 1.61 fo.26 M.36 fo.44kO.56 ztO.47 control
El
E2
E3
(“A)
2.18 f0.54 2.28 500.67 control
E4
Fig.
12.74 9.12 10.55 11.34 f3.01 f2.22 f3.10 i3.22 2.79 2.15 2.48 1.99 fl.08f0.85 fl.02 M.66 2.49 2.27 2.79 2.16 No.79fo.81 fl.40 fo.70 El
E2
E3
E4
1
Renal hemodynamic and excretory response to intravenous injection of FR139317 (IO mg/kg) in normotensive control (A) and DOCA-salt hypertensive (B) rats. Values are mean f SEM for six rats. MAP; mean arterial pressure, RBF; renal blood flow, GFR; glomerular filtration rate, RVR; renal vascular resistance, UF; urine flow, UNaV; urinary excretion of sodium, FENa; fractional excretion of sodium. Statistically significant change from control period (*P
Figure 1A) shows the changes in MAP, renal hemodynamics and function following the intravenous injection of FR1393 17 in normotensive control rats. FR 1393 17 produced a slight but slow-onset and significant decrease in MAP. A significant decrease in MAP was observed in E2-E4 (about lo-15mmHg decrease from the control value). There was no significant alteration in RBF after the injection of FR139317. A moderate but significant decrease in RVR was observed in E2 and E4. FR139317 produced a significant antinatriuretic effect. After the injection of FR1393 17, UF and urinary excretion of sodium (UNaV) decreased gradually over the experimental period. In E4, UF and UNaV decreased to about 50% of each control value. GFR and fractional excretion of sodium (FENa) fell gradually over the experimental period, but the change was not statistically significant
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Endothelinand DOCA-saltHypertension
Vol.58, No. 1. 1996
Figure 1B) shows the change in MAP, renal hemodynamics and function following the intravenous injection of FR139317 in DOCA-salt hypertensive rats. Administration of FR139317 produced a marked reduction of MAP. The maximum response (about 40 mmHg decrease form the control value) was observed in E2 and this decrease was sustained throughout the experimental periods. In DOCA-salt-treated group, FRl39317 produced an increase in RBF. After the administration of FR139317, RBF was time-dependently increased and the levels were significantly higher in E3 and E4 than that in control period (control value of 2.34 + 0.31 to 2.71 + 0.42 and 2.83 f 0.43 ml/g.min, respectively). Simultaneously, marked decreases in RVR were apparent after the drug injection. UF and UNaV were not significantly changed throughout the experimental period, in contrast to these findings in the normotensive rats. There were no significant alterations in GFR and FENa after the injection of FR 1393 17.
W
A)
1.2 -
‘-1 mIRNA 1.0 -
2.3 kb
0.8 -
0.6 -
28s
18s
control
DOCA-salt
DOCA-salt
Fig. 2 Northern blot analysis of prepro endothelin-1 (ET-l) mRNA in the kidney of normotensive control and DOCA-salt hypertensive rats. A): typical example of ET-l gene expression in the kidney of the control rat and DOCA-salt hypertensive rat. B): prepro ET-I mRNA level in the kidney normalized 28s rRNA density. Values are mean f. SEM (control; n=4, DOCA-salt; n=3). Statistically significant change from control (*P
A typical example of Northern blot analysis is shown for the levels of prepro ET-l mRNA in the kidney of control rats and DOCA-salt hypertensive rats (Fig. 3A). The autoradiogram from Northern blot analysis was scanned by a densitometer, and the levels of prepro ET- 1 mRNA were normalized by 28s rRNA density in the agarose gel stained with ethidium bromide (Fig. 3B). In DOCA-salt rats, pre.pro ET-l mRNA expression in the kidney was significantly increased compared with findings in control rats.
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Endothelin
and DOCA-salt
Hypertension
Vol. 58, No. 1, 1996
Our observations mean that ETA receptors play an important role in the maintenance of DOCA-salt hypertension and that, at least in part, ET-l and the ETA receptor are responsible for the marked renal vasoconstriction which occurred in DOCA-salt hypertensive rats. Since renal function is considered to play the central role in the long-term control of arterial pressure and the pathogenesis of hypertension (13), the abnormalities in renal hemodynamics may participate in the development and maintenance of DOCA-salt hypertension. Furthermore, we have found that prepro ET-I gene expression by kidney homogenates from animals with DOCA-salt treatment significantly increased as compared to control animals, hence, increased production of renal ET- 1 may be related to the marked renal vasoconstriction in DOCA-salt hypertensive rats. In normotensive control rats, FR139317 produced a significant decrease in UF and UNaV. One study using BQ123, an ETA receptor antagonist, showed that the antagonist decreased UF, UNaV and FENa without affecting systemic and renal hemodynamics in conscious rats. These results suggest that ET-l may function as a natriuretic hormone in rats (14). Since ET-l is known to stimulate the release of endogenous factors, such as atrial natriuretic peptide and nitric oxide (NO), which increase urine formation, these factors may be involved in the ET- 1-induced natriuretic effect (15). One interesting observation in the present study is that FR 1393 17 did not decrease urine formation in anesthetized DOCA-salt hypertensive rats, despite the agent-induced marked hypotension. If the functional role of ET-l via ETA receptor in urine formation is similar between DOCA-salt hypertensive and normotensive rats, there should be a pronounced decrease in urine formation by the agent-induced marked hypotension, in the DOCA-salt hypertensive rats. Although it is difficult to explain these data because of a complex action of the drug on systemic and renal hemodynamics, our results suggest that the regulation mechanism of urine formation via ETA receptor is altered in DOCA-salt hypertensive rats. In addition, it has been shown that ETB receptors are involved in the renal effects of ET-l in the rat (16). Further studies are required to determine whether ETB receptors are responsible for the abnormalities of renal function of DOCAsalt hypertensive rats. In the present study, Northern blot analysis revealed that prepro ET-l mRNA is slightly but significantly increased in the kidney of DOCA-salt hypertensive rats. To our knowledge, this is the first evidence for up-regulation of renal ET-l gene expression in experimental hypertension. Previous report has shown that renal ET-l gene expression is significantly decreased in spontaneously hypertensive rats (SHRs) (17). These results indicate that regulation of ET-l gene expression in the kidney differs among experimental models of hypertension, and increased renal ET-l gene expression in DOCA-salt hypertensive rats is not merely due to the consequence of hypertension. Furthermore, our results suggest that this change in renal ET-l gene expression may contribute to the marked renal vasoconstriction in DOCA-salt hypertensive rats. In a previous study, vascular ET-l mRNA level was increased in DOCA-salt hypertensive rats (18). Our present study using an ETA receptor antagonist showed that the antagonist produced a potent renal vasodilation in DOCA-salt hypertensive rats, observations which suggest that vascular tissues may be the origin of the increased renal ET- 1 mRNA levels in DOCA-salt hypertensive rats. However, the possibility that ET-l mRNA is increased in tubular cells or mesangial cells cannot be ruled out, since these cells synthesize and secrete ET-I (19,20). The role of ET-l in blood pressure regulation in human is still unclear. However, several studies using an ET receptor antagonist in experimental animals revealed the importance of ET-l in the pathogenesis of DOCA-salt hypertension (2 1,22). On the other hand, administration of an ET receptor antagonist produced a slight lowering effect on blood pressure of SHRs (21,22), thereby suggesting that the contribution of ET- 1 in the pathogenesis of the genetic hypertensive rat is minor. We noted that FR139317 treatment to anesthetized 2-kidney l-clip renal hypertensive rats or NO synthase-blocked hypertensive rats produced only a moderate hypotensive effect (23). Thus, it seems likely that the contribution of ET-l is specific in DOCA-salt hypertension among several models of experimental hypertension. Most recently, we found that long-term (2 weeks) treatment with FR139317 in DOCA-salt hypertensive rats efficiently suppressed the development of hypertension and cardiovascular hypertrophy (24). Taken together, ET-l seems to be involved in both the development and maintenance of DOCA-salt hypertension.
Vol. 58, No. 1, 1996
Endothelin and DOCA-salt Hypertension
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In conclusion, an endogenous ET-l is responsible for the development and maintenance of DOCA-salt-induced hypertension. Our results also suggest that at least in part, ET-l and ETA receptors are involved in the renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-l production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.
This study was supported in part by the Science Research Promotion Fund of Japan Private School Promotion Foundation. We thank M. Ohara for critical comments.
BeferenceS 1. M. YANAGISAWA, H. KURIHARA, S. KIMURA, Y. TOMOBE, M. KOBAYASHI, Y. MITSUI, K. GOT0 and T. MASAKI, Nature s 411-415 (1988). 2. M. CLOZEL, G.A. GRAY, V. BREU, B.-M. LOFFLER and R. OSTERWALDER, Biochem. Biophys. Res. Commun. .I&j 867-873 (1992). 3. J.P. CRISTOL, T.D. WARNNER, C. THIEMERMANN and J.R. VANE, Br. J. Pharmacol. 108 776-779 (1993). 4. M. IHARA, K. NOGUCHI, T. SAEKI, T. FUKURODA, S. TSUCHIDA, S. KIMURA, T. FUKAMI, K. ISHIKAWA and M. YANO, Life Sci. 50 247-255 (1992). 5. K. SOGABE, H. NIREI, M. SHOUBO, A. NOMOTO, S. AO, Y. NOTSU and T. ONO, J. Pharmacol. Exp. Ther. 264 1040-1046 (1993). 6. M. CLOZEL, V. BREU, G.A. GRAY, B. KALINA, B.-M. LGFFLER, K. BURRI, J.-M. CASSAL, G. HIRTH, M. MULLER, W. NEIDHART and H. RAMUZ, J. Pharmacol. Exp. Ther. 270 228-235 (1994). 7. K. FUJITA, Y. MATSUMURA, Y. MIYAZAKI, S. KITA, K. HISAKI, M. TAKAOKA and S. MORIMOTO, Br. J. Pharmacol. 114 925-930 (1995). 8. P.D. STEIN, J.T. HUNT, D.M. FLOYD, S. MORELAND, K.E.J. DICKINSON, C. MITCHELL, E.C.-K. LIU, M.L. WEBB, N. MURUGESAN, J. DICKEY, D. MCMULLEN, R. ZHANG, V.G. LEE, R. SERAFINO, C. DELANEY, T.R. SCHAEFFER and M. KOZOLOWSKI, J. Med. Chem. 3:329-331 (1994). 9. M. HUANG, R.L. HESTER, T.G. COLEMAN, M.J. SMITH and A.C. GUYTON, Hypertension 20 828-833 (1992). 10. R.J. ROMAN, M.L. KALDUNSKI, D.L. MATTSON, M. MISTRY and A. NASJLETTI, Hypertension 12 287-294 (1988). 11. G.G. VUREK and SE. PERGRAM, Anal. Biochem. 16 409-419 (1966). 12. Y. MATSUMURA, S. MURATA, K. TAKADA, M. TAKAOKA and S. MORIMOTO, Clin. Exp. Pharmacol. Physiol. 3 99 l-996 ( 1994). 13. A.C. GUYTON, Hypertension lo l-6 (1987). 14. M. GELLAI, M. JUGUS, T. FLETCHERI, R. DEWOLF and P. NAMBI, J. Clin. Invest. 93 900-906 (1994). 15. G.M. RUBANYI and M.A. POLOKOFF, Pharmacol. Rev. 46 325-415 (1994). 16. D.M. POLLOCK and T.J. OPGENORTH, Am. J. Physiol. 264 R222-R226 (1993). 17. A.K. HUGHES, R.C. CLINE and D.E. KOHAN, Hypertension 20 666-673 (1992). 18. R. LARIVIERE, R. DAY and E.L. SCHIFFRIN, Hypertension 2l_ 916-920 (1993). 19. D.E. KOHAN and F.T. FIEDOREK, JR. J. Am. Sot. Nephrol. 2. 150-155 (1991). 20. H. SAKAMOTO, S. SASAKI, Y. HIRATA, T. IMAI, K. ANDO, T. IDA, T. SAKURAI, M. YANAGISAWA, T. MASAKI and F. MARUMO, Biochem. Biophys. Res. Commun. 169 462-468 (1990). 2 1. E.L. SCHIFFRIN, Hypertension 25. 1135 I 143 (1995). 22. J.E. BIRD, S. MORELAND, T.L. WALDRON and J.R. POWELL, Hypertension 25. 11911195 (1995). 23. K. FUJITA, Y. MATSUMURA, Y. MIYAZAKI, M. TAKAOKA and S. MORIMOTO, Eur. J. Pharmacol. 280 311-316 (1995). 24. Y. MATSUMURA, K. FUJITA, Y. MIYAZAKI, M. TAKAOKA and S. MORIMOTO, J. Cardiovasc. Pharmacol. 26 (SUDD~. 3) S456-S458 (1995).
ELSEVIER
0024-3205(95)02259-7
Pi%4Rh#2COLOGYLET Accelerated Communicadon ETA RECEPTOR-MEDIATED ROLE OF ENDOTHELIN IN THE KIDNEY DOCA-SALT HYPERTENSIVE RATS
OF
Katsuya Fujita, Yasuo Matsumura, Yohko Miyazaki, Norio Hashimoto, Masanori Takaoka and Shiro Morimoto* Department of Pharmacology, Osaka University of Pharmaceutical Sciences, 2-10-65 Kawai, Matsubara, Osaka 580, Japan (Submitted August 9, 19995;accepted August 31, 1995; received in final form October 16, 1995)
AI&& Renal effects of FRl39317, an endothelin ETA receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; -13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; -12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; -26%) accompanied by a potent renal vasodilation (RVR; -33%). FR139317 significantly increased renal blood flow only in the DOCAsalt rats. In contrast, FR1393 17 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-l) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-l is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-l and ETA receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-l production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension. Key Words: endothelin-1, ETA receptor, DOCA-salt hypertension, renal function
Endothelin-1 (ET-l) is a potent vasoconstrictor peptide isolated from the porcine vascular endothelial cells (1). ET-l produces contraction of vascular smooth muscle by activation of ETA or ETu receptors in various tissues (2,3). Recently, several ET receptor antagonists have been developed and utilized to evaluate the physiological and/or pathophysiological roles of endogenous ET-l and its receptor subtypes (4,5,6). FR139317 is a selective ETA receptor antagonist and inhibits ET-l-induced vasoconstrictive effect in vitro and in vivo (5). Previously, we and others reported that an intravenous bolus injection of FRl39317 produced a potent hypotensive effect in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (7,8). These results indicate that ET-l and ETA receptors make an important contribution to the maintenance of DOCA-salt hypertension, but the precise mechanism involved has remained unclear. In DOCA-salt hypertensive rats, a marked elevation in renal vascular resistance (RVR) and a decrease in renal blood flow (RBF) often occur, and these abnormalities in renal hemodynamics are thought to play an important role in the development and maintenance of the hypertension (9,lO). We compared the effect of ETA receptor blockade on renal hemodynamics and function in normotensive and DOCA-salt hypertensive rats, the objective being to evaluate the possible involvement of ET-l and the ETA receptor in renal hemodynamic abnormalities in DOCA-salt hypertensive rats. * To whom correspondence should be addressed
PL-2
Endothelin
and DOCA-salt
Hypertension
Vol. 58, No. 1. 1996
DOCA-salt treatment Male Sprague-Dawley rats, weighing 160-180 g were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the right kidney was removed via a right flank incision. After a l-week postsurgical recovery period, the rats were treated twice weekly with DOCA suspended in corn oil , which was administered subcutaneously (15 mg/kg) and 1% NaCl added to their tap water for drinking. Control rats were uninephrectomized but not given DOCA or salt. After 4 weeks of this treatment, the rats were used in clearance study or Northern blot analysis. Animal preparation for clearance study Animals were anesthetized with sodium thiobutabarbital (Inactin, 100 mg/kg, i.p.) and placed on a heated surgical tray that maintained a rectal temperature between 37 “C and 38 ‘C. After tracheotomy, the right femoral vein was cannulated for infusion of 0.9% saline containing 0.5% inulin. The right and left femoral arteries were also cannulated to measure mean arterial pressure (MAP) and for blood sampling, respectively. After an abdominal midline incision was made, the left kidney was exposed, and the renal artery was carefully stripped of connective tissue, followed by the application of 5% phenol in 70% ethanol to exclude the influence of renal sympathetic nerves. An electromagnetic flow probe (1 .O mm in diameter, Nihon Kohden, Tokyo, Japan) connected to a square-wave flowmeter (MFV-2100, Nihon Kohden) was positioned on the renal artery to measure RBF. A polyethylene cannula was inserted into the left ureter for urine collection. MAP and RBF were continuously recorded on a polygraph (RM 6000, Nihon Kohden), throughout all the experiment. A 60-90 min period was allowed for stabilization of MAP, RBF and urine flow (UF). Experimental protocol After the equilibration period, urine samples were collected during two 20 min control clearance periods. Following the control periods, FRl39317 (10 mg/kg) was administered intravenously by slow bolus injection. The dose of FR 1393 17 used in this study has been shown to produce complete inhibition of ET-l-induced pressor action (5). During the first 5 min after injection, urine was not collected in order to take into account for the dead space in the collection system. Following this, urine samples were collected during four consecutive 20 min periods (El-E4). Blood samples (0.2 ml each) were obtained at 20 min before drug injection and at 25 min and 65 min after the injection, respectively. The blood loss was replaced by giving an equal volume of blood from donor rats. Plasma was immediately separated by centrifugation. In the control experiment, the vehicle solution was administered. In this case, MAP, renal hemodynamics and function remained unchanged through the experiment. Results in the second control period served as the basal values of renal hemodynamics and function. Analyticalprocedure Urine and plasma inulin levels were measured by spectrofluorometry (Hitachi 650-50) according to the method of Vurek and Pergram (11). Glomerular filtration rate (GFR) was calculated from the inulin clearance. Urine and plasma sodium concentrations were determined using a flame photometer (Hitachi 205D). RNA extraction and Northern blot analysisfor prepro ET-l mRNA Total RNA from rat kidney was prepared by selective precipitation in 3 mol/l LiCl and 6 mol/l urea (12). Total RNA (20 @lane) was separated in formaldehyde-l.1 % agarose gel electrophoresis, and transferred to a nylon membrane (Hybond-N+, Amersham, Tokyo, Japan) in the presence of 20 x SSC (3 mol/l sodium chloride, 0.3 mol/l sodium citrate, pH 7.2). The membrane was hybridized in hybridization buffer (6 x SSC, O.Olmol/l EDTA, 5 x Denhardt’s solution, 0.5% SDS, 100 yg/ml sheared, denatured salmon sperm DNA) at 67 “C, overnight with ran prepro ET-l cDNA probe (a gift from Dr K. Goto, University of Tsukuba, Japan) labeled with [(%-32P]dCTP using a Random Primer DNA labeling Kit (Takara Shuzo, Osaka, Japan). -4utoradiography was then carried out by exposure to X-Omat AR film (Kodak, New York, USA)
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with intensifying screens at -80 ‘C for 12 h. The autoradiograms of ET-l were quantified by densitometric analysis, and the signals of ET-1 mRNA were normalized for each sample with respect to the 28s rRNA density in agarose gel stained by ethidium bromide.
FRl39317 was a kind gift from Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan. FRl39317 was dissolved in IN NaOH and diluted with saline. Other chemicals were purchased from Nacalai Tesque, Inc (Kyoto, Japan). Statistical analysis All values were expressed as mean + SEM. For statistical analysis, we used the unpaired Student’s t test for two-group comparisons and one-way analysis of variance combined with Dunnett’s multiple range test for multiple comparison. Differences were considered significant at P
TABLE
I.
Basal renal hemodynamics and function in anesthetized normotensive rats and DOCA-sait hypertensive rats.
normotensive control rats (n=6) MAP (mmHg) RBF (ml/g.min) RVR (mmHgfml/g.min) GFR (ml/g.min)
123+5 4.65 f 0.22 26.7 k 1.4 0.72 + 0.05
control
DOCA-salt hypertensive rats (n=6) 160 +_7** 2.34 + 0.3 1** 77.2 + 14.2** 0.70 f 0.09
UF (pl/g.min)
10.8 + 2.6
14.0 + 3.2
UNaV (@q/g.min)
2.47 + 0.27 2.39 f 0.26 365 I!Z4 1.91 * 0.03
2.18 f 0.54 2.28 f 0.67 318f. 8** 2.79 f 0.17**
ENa
(%)
Body wt. (g) Left kidney wt. (g)
Each value represents the mean + SEM. MAP; mean arterial pressure, RBF; renal blood flow, GFR; glomerular filtration rate, RVR; renal vascular resistance, UF; urine flow, UNaV; urinary excretion of sodium, FENa; fractional excretion of sodium. **P
Table I summarizes the body weight, the left kidney weight and the basal renal hemodynamics and function in anesthetized normotensive control rats and DOCA-salt hypertensive rats. The MAP, RVR and left kidney weight were significantly higher in DOCA-salt rats than in control rats, RBF was significantly decreased in DOCA-salt rats and was about half the value of that of control rats. The body weight was lower in DOCA-salt rats than in control rats. There were no significant differences in GFR and urine formation between the two groups.
Vol.58, No. 1, 1996
Endothelinand DOCksalt Hypertension
PL-4
W
4 160 CL2 ‘40 gE 120 C. 100 OfE
6
kE E$
5 !z.
OL 100
2
‘e
80
T
40 E 20 55!z. 600 kl_bTlk
**
** **
*+
“2
(pl/g!iin)
10.78 11.99 7.56 6.26 5.64* k2.61 f3.42 fl.61 kl.26 fl .12
UNaV (pEq/g.min)
2.47 2.72 1.97 1.37’+1.27** HI.27 20.20 a.31 HI.35 f0.29
FENa (“A)
UNaV (pEq/g.min) FENa
2.39 2.63 2.23 1.79 1.61 fo.26 M.36 fo.44kO.56 ztO.47 control
El
E2
E3
(“A)
2.18 f0.54 2.28 500.67 control
E4
Fig.
12.74 9.12 10.55 11.34 f3.01 f2.22 f3.10 i3.22 2.79 2.15 2.48 1.99 fl.08f0.85 fl.02 M.66 2.49 2.27 2.79 2.16 No.79fo.81 fl.40 fo.70 El
E2
E3
E4
1
Renal hemodynamic and excretory response to intravenous injection of FR139317 (IO mg/kg) in normotensive control (A) and DOCA-salt hypertensive (B) rats. Values are mean f SEM for six rats. MAP; mean arterial pressure, RBF; renal blood flow, GFR; glomerular filtration rate, RVR; renal vascular resistance, UF; urine flow, UNaV; urinary excretion of sodium, FENa; fractional excretion of sodium. Statistically significant change from control period (*P
Figure 1A) shows the changes in MAP, renal hemodynamics and function following the intravenous injection of FR1393 17 in normotensive control rats. FR 1393 17 produced a slight but slow-onset and significant decrease in MAP. A significant decrease in MAP was observed in E2-E4 (about lo-15mmHg decrease from the control value). There was no significant alteration in RBF after the injection of FR139317. A moderate but significant decrease in RVR was observed in E2 and E4. FR139317 produced a significant antinatriuretic effect. After the injection of FR1393 17, UF and urinary excretion of sodium (UNaV) decreased gradually over the experimental period. In E4, UF and UNaV decreased to about 50% of each control value. GFR and fractional excretion of sodium (FENa) fell gradually over the experimental period, but the change was not statistically significant
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Endothelinand DOCA-saltHypertension
Vol.58, No. 1. 1996
Figure 1B) shows the change in MAP, renal hemodynamics and function following the intravenous injection of FR139317 in DOCA-salt hypertensive rats. Administration of FR139317 produced a marked reduction of MAP. The maximum response (about 40 mmHg decrease form the control value) was observed in E2 and this decrease was sustained throughout the experimental periods. In DOCA-salt-treated group, FRl39317 produced an increase in RBF. After the administration of FR139317, RBF was time-dependently increased and the levels were significantly higher in E3 and E4 than that in control period (control value of 2.34 + 0.31 to 2.71 + 0.42 and 2.83 f 0.43 ml/g.min, respectively). Simultaneously, marked decreases in RVR were apparent after the drug injection. UF and UNaV were not significantly changed throughout the experimental period, in contrast to these findings in the normotensive rats. There were no significant alterations in GFR and FENa after the injection of FR 1393 17.
W
A)
1.2 -
‘-1 mIRNA 1.0 -
2.3 kb
0.8 -
0.6 -
28s
18s
control
DOCA-salt
DOCA-salt
Fig. 2 Northern blot analysis of prepro endothelin-1 (ET-l) mRNA in the kidney of normotensive control and DOCA-salt hypertensive rats. A): typical example of ET-l gene expression in the kidney of the control rat and DOCA-salt hypertensive rat. B): prepro ET-I mRNA level in the kidney normalized 28s rRNA density. Values are mean f. SEM (control; n=4, DOCA-salt; n=3). Statistically significant change from control (*P
A typical example of Northern blot analysis is shown for the levels of prepro ET-l mRNA in the kidney of control rats and DOCA-salt hypertensive rats (Fig. 3A). The autoradiogram from Northern blot analysis was scanned by a densitometer, and the levels of prepro ET- 1 mRNA were normalized by 28s rRNA density in the agarose gel stained with ethidium bromide (Fig. 3B). In DOCA-salt rats, pre.pro ET-l mRNA expression in the kidney was significantly increased compared with findings in control rats.
PG6
Endothelin
and DOCA-salt
Hypertension
Vol. 58, No. 1, 1996
Our observations mean that ETA receptors play an important role in the maintenance of DOCA-salt hypertension and that, at least in part, ET-l and the ETA receptor are responsible for the marked renal vasoconstriction which occurred in DOCA-salt hypertensive rats. Since renal function is considered to play the central role in the long-term control of arterial pressure and the pathogenesis of hypertension (13), the abnormalities in renal hemodynamics may participate in the development and maintenance of DOCA-salt hypertension. Furthermore, we have found that prepro ET-I gene expression by kidney homogenates from animals with DOCA-salt treatment significantly increased as compared to control animals, hence, increased production of renal ET- 1 may be related to the marked renal vasoconstriction in DOCA-salt hypertensive rats. In normotensive control rats, FR139317 produced a significant decrease in UF and UNaV. One study using BQ123, an ETA receptor antagonist, showed that the antagonist decreased UF, UNaV and FENa without affecting systemic and renal hemodynamics in conscious rats. These results suggest that ET-l may function as a natriuretic hormone in rats (14). Since ET-l is known to stimulate the release of endogenous factors, such as atrial natriuretic peptide and nitric oxide (NO), which increase urine formation, these factors may be involved in the ET- 1-induced natriuretic effect (15). One interesting observation in the present study is that FR 1393 17 did not decrease urine formation in anesthetized DOCA-salt hypertensive rats, despite the agent-induced marked hypotension. If the functional role of ET-l via ETA receptor in urine formation is similar between DOCA-salt hypertensive and normotensive rats, there should be a pronounced decrease in urine formation by the agent-induced marked hypotension, in the DOCA-salt hypertensive rats. Although it is difficult to explain these data because of a complex action of the drug on systemic and renal hemodynamics, our results suggest that the regulation mechanism of urine formation via ETA receptor is altered in DOCA-salt hypertensive rats. In addition, it has been shown that ETB receptors are involved in the renal effects of ET-l in the rat (16). Further studies are required to determine whether ETB receptors are responsible for the abnormalities of renal function of DOCAsalt hypertensive rats. In the present study, Northern blot analysis revealed that prepro ET-l mRNA is slightly but significantly increased in the kidney of DOCA-salt hypertensive rats. To our knowledge, this is the first evidence for up-regulation of renal ET-l gene expression in experimental hypertension. Previous report has shown that renal ET-l gene expression is significantly decreased in spontaneously hypertensive rats (SHRs) (17). These results indicate that regulation of ET-l gene expression in the kidney differs among experimental models of hypertension, and increased renal ET-l gene expression in DOCA-salt hypertensive rats is not merely due to the consequence of hypertension. Furthermore, our results suggest that this change in renal ET-l gene expression may contribute to the marked renal vasoconstriction in DOCA-salt hypertensive rats. In a previous study, vascular ET-l mRNA level was increased in DOCA-salt hypertensive rats (18). Our present study using an ETA receptor antagonist showed that the antagonist produced a potent renal vasodilation in DOCA-salt hypertensive rats, observations which suggest that vascular tissues may be the origin of the increased renal ET- 1 mRNA levels in DOCA-salt hypertensive rats. However, the possibility that ET-l mRNA is increased in tubular cells or mesangial cells cannot be ruled out, since these cells synthesize and secrete ET-I (19,20). The role of ET-l in blood pressure regulation in human is still unclear. However, several studies using an ET receptor antagonist in experimental animals revealed the importance of ET-l in the pathogenesis of DOCA-salt hypertension (2 1,22). On the other hand, administration of an ET receptor antagonist produced a slight lowering effect on blood pressure of SHRs (21,22), thereby suggesting that the contribution of ET- 1 in the pathogenesis of the genetic hypertensive rat is minor. We noted that FR139317 treatment to anesthetized 2-kidney l-clip renal hypertensive rats or NO synthase-blocked hypertensive rats produced only a moderate hypotensive effect (23). Thus, it seems likely that the contribution of ET-l is specific in DOCA-salt hypertension among several models of experimental hypertension. Most recently, we found that long-term (2 weeks) treatment with FR139317 in DOCA-salt hypertensive rats efficiently suppressed the development of hypertension and cardiovascular hypertrophy (24). Taken together, ET-l seems to be involved in both the development and maintenance of DOCA-salt hypertension.
Vol. 58, No. 1, 1996
Endothelin and DOCA-salt Hypertension
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In conclusion, an endogenous ET-l is responsible for the development and maintenance of DOCA-salt-induced hypertension. Our results also suggest that at least in part, ET-l and ETA receptors are involved in the renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-l production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.
This study was supported in part by the Science Research Promotion Fund of Japan Private School Promotion Foundation. We thank M. Ohara for critical comments.
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