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Effect of endothelin on renal function in rats
European Journal of Pharmacology, 163 (1989) 187-189
187
Elsevier EJP 20353
Short communication
Effect of endothelin on renal function in rats A n t o n i o L 6 p e z - F a r r r , I n m a c u l a d a M o n t a h r s , I n m a c u l a d a Millfis a n d Jos6 M. L 6 p e z - N o v o a * Renal Physiopathology Laboratory, Medical Research Institute, Fundacibn Jirn~nez Diaz-Consejo Superior de Inoestigaciones Cientlfieas, Avda. Reyes Catblicos 2, 28040 Madrid, Spain Received 31 January 1989, accepted 14 February 1989
The effect of synthetic porcine endothelin on glomerular filtration rate, renal plasma flow and electrolyte excretion was studied in rats. Endothelin, 1 nmol/kg body weight given as a bolus, induced a transient decrease in glomerular filtration rate (72%) and in renal plasma flow (76%) as well as in sodium excretion, accompanied by a sustained increase in renal vascular resistance. This dose had no sustained effect on mean arterial pressure. It is concluded that endothelin induces a marked decrease in glomerular filtration rate and renal perfusion. This peptide could play a role in the alterations in renal function observed after renal injury. Endothelium; Endothelin; Glomerulus; Glomerular filtration rate; Mesangial cells; Renal plasma flow; (Contraction)
1. Introduction Vasoconstriction dependent on or enhanced by intact endothelium has been observed in response to various chemical and physical stimuli (Vanhoutte, 1988). This has been demonstrated in coronary and in cerebral arteries (Rosemblum and Nelson, 1988; Vanhoutte and Katusic, 1988). A potent vasoconstrictor peptide was recently isolated from the culture supernatant of porcine aortic endothelial cells (Yanagisawa et al., 1988). This peptide has received the name of endothelin, and its sequence does not belong to any previously known peptide family. However, it shows local homologies to a certain group of neurotoxins (Yanagisawa et al., 1988). Several studies have demonstrated a profound effect of endothelin on the renal vasculature (Lippton et al., 1988; Wright and Fozard, 1988;
* To whom all correspondence should be addressed: Fundaci6n Jimrnez Diaz, Avda. Reyes Catblicos 2, 28040Madrid, Spain.
Yanagisawa et al., 1988). This led us to analyze the effect of endothelin on renal function.
2. Materials and methods We used eight male Wistar rats weighing about 306 + 6 g. The rats were surgically prepared for clearance studies as previously reported (L6pezN o v o a et al., 1982). In brief, under sodium phenobarbital anesthesia, PE-50 catheters were placed in the femoral artery and vein and in the bladder. [3H]Inulin and [14C]PAH were continuously i.v. infused in order to determine the glomerular filtration rate and renal plasma flow respectively. The mean arterial pressure was continuously monitored. After a period for equilibration and hemodynamic stabilization, two 20 rain basal clearance periods were allowed. Endothelin (porcine, Peptide Institute Inc., Osaka, Japan), 1 n m o l / k g body weight dissolved in 0.25 ml of isotonic saline was then infused i.v. as a bolus, and three consecutive 20 rain clearance periods were observed, with blood sampling at the beginning
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
188
also caused an increase in packed cell volume but not in total plasma proteins.
and end of each period. Packed cell volume was measured in triplicate in each blood sample. Total plasma proteins were measured by refractometry in a temperature-compensated refractometer (American Optical, Buffalo, NY, USA). Urine was collected in preweighed plastic vials containing 0.2 ml of water-equilibrated mineral oil. Sodium and potassium in plasma and urine were measured by means of selective electrodes (Astra 4, Beckmann, USA). Changes with respect to basal values were analyzed by paired Student's t-test.
4. Discussion
The present study demonstrated that endothelin, at non-pressor doses, induced a significant decrease in glomerular filtration rate and renal plasma flow. This decrease seems to be based, at least in part, on the contraction of the renal arteries a n d / o r arterioles, because endothelin induced an increase in renal vascular resistance. Yanagisawa et al. (1988) have reported that endothelin induces a potent contraction in arterial strips of various origins, including rabbit renal arteries. Tomobe et al. (1988) has also reported that endothelin contracts rat renal artery. The small but significant increase in filtration fraction observed in the present experiment suggests a preferential postglomerular action. Wright and Fozard (1988) have shown that a dose of endothelin similar to that used here induced a transient decrease in mean arterial pressure in anesthetized, ganglion-blocked, spontaneously hypertensive rats, followed by an increase to slightly supranormal values. These results were very similar to those obtained in the present ex-
3. Results
The data for renal function are shown in table 1. Endothelin induced a sharp decrease in urine flow, glomerular filtration rate, renal plasma flow and sodium excretion, accompanied by an increase in renal vascular resistance. These values recovered partially in the second clearance period and returned to values similar to those of the basal period in the third period (40-60 rain after endothelin injection). Endothelin induced an acute but transient (30 s-1 rain) decrease in mean arterial pressure, followed by return to values not significantly different from the basal ones. Endothelin
TABLE 1 Effect of endothelin (1 n m o l / k g ) on renal function in rats. The data are m e a n s ± S.E.M. a Statistically significant values (P < 0.05; paired t-test) with respect to basal values. Abbreviations: MAP: mean arterial pressure; U.V.: urinary flow; GFR: glomerular filtration rate; RPF: renal plasma flow; FF: filtration fraction; RVR: renal vascular resistance; UNaV: urinary sodium excretion; UKV: urinary potassium excretion; PCV: packed cell volume; TPP: total plasma protein concentration. Basal
Endothelin 0-20
MAP (nun Hg) U.V.(bd/min) GFR (ml/min) RPF ( m l / m i n ) FF(%) RVR (mm H g . m i n / m l ) UNaV ( m E q / m i n ) UKV ( m E q / m i n ) PCV (%) TPP(g/1)
128 ±4 7.2 +0.6 1.99±0.17 5.85±0.44 34 ±2 11.8 ± 1.4 0.11 ± 0.02 0.07 ± 0.08 46.1 ±0.8 53.3 ±0.5
129 ± 4.1 ± 0.56± 1.39± 38 ± 47.1 ± 0.08 ± 0.07 ± 49.8 ± 55.3 ±
5 1.4 a 0.18 a 0.38 a 3a 10.8 a 0.03 a 0.01 0.8 a 0.7
20-40
40-60
130 ±5 6.7 ±1.4 1.60±0.16 a 3.64±0.57 a 40 ±3 a 17.8 ± 5.2 a 0.06 + 0.02 a 0.10 ± 0.03 49.8 ±1.5 a 56.4 ±0.8
125 8.8
±5 ~_0.5
a
1.74±0.16 4.37±0.27 a 38 ±2 a 14.8 ± 2.4 a 0.08 ± 0.02 a 0.13 ± 0.04 a 47.9 ±1.3 53.0 ±1.2
189
periments. Lippton et al. (1988) have reported a transient (< 1 min) systemic and renal vasodilatation induced by low doses of endothelin, but no data on more prolonged effects were reported. In the study of Yanagisawa et al. (1988), the injection to chemically denervated animals of a dose of endothelin similar to that we now used induced a significant and long-lasting increase in arterial pressure. It must be noted that the compensatory hemodynamic changes that must have occurred in our rats are not present in the chemically denervated animals. Another interesting observation is the increase in packed cell volume induced by endothelin infusion. The mechanism of this increase cannot be easily elucidated from the data available. A first possibility is that endothelin induces a trapping of blood in the peripheral capillaries temporary closed to the circulation by the potent constrictor effect of endothelin. As capillary blood has a decreased packed cell volume, the systemic blood-packed cell volume should increase. A second possibility is that endothelin induces an escape of fluid out of the circulation, thus increasing the erythrocyte concentration. As the total plasma protein does not change, the escaped fluid should be composed of whole plasma. However this accumulation of protein-rich fluid in the interstitium is not easy to remove, whereas in our experiments the packed cell volume returned to normal values in the third experimental period, thus making this second possibility unlikely. Although the physiological meaning of these data is not clear, because the plasma levels of this peptide are unknown, their physiopathological relevance is evident. It has been suggested that endothelin could be released by endothelial cells during hypoxia or reperfusion injury (Vanhoutte and Katusic, 1988; Yanagisawa et al., 1988). In fact, hypoxia has been reported to produce endothelium-dependent contraction of several large arteries or veins in vitro (Dheins et al., 1987; Vanhoutte and Miller, 1985). Thus, it can be hypothesized that, after an ischemic or toxic injury to the kidney, endothelin released by endothelial vascular cells will reach high concentrations in the
glomerular area, inducing glomerular arteriole constriction, and thus reduction of renal plasma flow and glomerular filtration rate. This mechanism of reduction of glomerular filtration rate after renal injury, although very plausible, needs further studies to be adequately demonstrated.
Acknowledgements This study was partially supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 1873/88) and Comisi6n Asesora para la Investigaci6n Cientifica y Ttcnica (594/84). A. L6pez-Farr6 is a Fellow of the Fundaci6n Conchita Rhbago de Jimenez Diaz.
References Dheins, S., F. Essens, A. Salameh, R. Rosen and W. Klaus, 1987, Evidence for a new endothehum-derived vasoconstricting factor, Med. Sci. Res. 15, 573. Lippton, H., J. Goff and A. Hyman, 1988, Effects of endothelin in the systemic and renal vascular beds in vivo, European J. Pharmacol. 155, 197. Lopez-Novoa, J.M. and M. Martinez-Maldonado, 1982, Impaired renal response to splanchnic infusion of hypertonic saline in conscious cirrhotic rats, Am. J. Physiol. 242, F390. Rosemblum, W.I. and G.H. Nelson, 1988, Endothelium-dependent contraction demonstrated in vivo in mouse cerebral arterioles, Circ. Res. 63, 837. Tomobe, Y., A. Miyauchi, A. Saito, M. Yanagisawa, S. Kimura, K. Goto and T. Masaki, 1988, Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats, European J. Pharmacol. 152, 373. Vanhoutte, P.M., 1988, The endothelium, modulator of vascular smooth muscle tone, N. Engl. J. Med. 319, 512. Vanhoutte, P.M. and Z.S. Katusic, 1988, Endothelium-derived contracting factor, endothelin a n d / o r superoxide anion, Trends Physiol. Sci. 9, 229. Vanhoutte, P.M. and V.M. Miller, 1985, Heterogeneity of endothelium-dependent responses in mammalian blood vessels, J. Cardiovasc. Pharmacol. 7 (Suppl. 3), S12. Wright, C.E. and J.R. Fozard, 1988, Regional vasodilation is a prominent feature of haemodynamic response to endothelin in anasthetized, spontaneously hypertensive rat, European J. Pharmacol. 155, 201. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.
187
Elsevier EJP 20353
Short communication
Effect of endothelin on renal function in rats A n t o n i o L 6 p e z - F a r r r , I n m a c u l a d a M o n t a h r s , I n m a c u l a d a Millfis a n d Jos6 M. L 6 p e z - N o v o a * Renal Physiopathology Laboratory, Medical Research Institute, Fundacibn Jirn~nez Diaz-Consejo Superior de Inoestigaciones Cientlfieas, Avda. Reyes Catblicos 2, 28040 Madrid, Spain Received 31 January 1989, accepted 14 February 1989
The effect of synthetic porcine endothelin on glomerular filtration rate, renal plasma flow and electrolyte excretion was studied in rats. Endothelin, 1 nmol/kg body weight given as a bolus, induced a transient decrease in glomerular filtration rate (72%) and in renal plasma flow (76%) as well as in sodium excretion, accompanied by a sustained increase in renal vascular resistance. This dose had no sustained effect on mean arterial pressure. It is concluded that endothelin induces a marked decrease in glomerular filtration rate and renal perfusion. This peptide could play a role in the alterations in renal function observed after renal injury. Endothelium; Endothelin; Glomerulus; Glomerular filtration rate; Mesangial cells; Renal plasma flow; (Contraction)
1. Introduction Vasoconstriction dependent on or enhanced by intact endothelium has been observed in response to various chemical and physical stimuli (Vanhoutte, 1988). This has been demonstrated in coronary and in cerebral arteries (Rosemblum and Nelson, 1988; Vanhoutte and Katusic, 1988). A potent vasoconstrictor peptide was recently isolated from the culture supernatant of porcine aortic endothelial cells (Yanagisawa et al., 1988). This peptide has received the name of endothelin, and its sequence does not belong to any previously known peptide family. However, it shows local homologies to a certain group of neurotoxins (Yanagisawa et al., 1988). Several studies have demonstrated a profound effect of endothelin on the renal vasculature (Lippton et al., 1988; Wright and Fozard, 1988;
* To whom all correspondence should be addressed: Fundaci6n Jimrnez Diaz, Avda. Reyes Catblicos 2, 28040Madrid, Spain.
Yanagisawa et al., 1988). This led us to analyze the effect of endothelin on renal function.
2. Materials and methods We used eight male Wistar rats weighing about 306 + 6 g. The rats were surgically prepared for clearance studies as previously reported (L6pezN o v o a et al., 1982). In brief, under sodium phenobarbital anesthesia, PE-50 catheters were placed in the femoral artery and vein and in the bladder. [3H]Inulin and [14C]PAH were continuously i.v. infused in order to determine the glomerular filtration rate and renal plasma flow respectively. The mean arterial pressure was continuously monitored. After a period for equilibration and hemodynamic stabilization, two 20 rain basal clearance periods were allowed. Endothelin (porcine, Peptide Institute Inc., Osaka, Japan), 1 n m o l / k g body weight dissolved in 0.25 ml of isotonic saline was then infused i.v. as a bolus, and three consecutive 20 rain clearance periods were observed, with blood sampling at the beginning
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
188
also caused an increase in packed cell volume but not in total plasma proteins.
and end of each period. Packed cell volume was measured in triplicate in each blood sample. Total plasma proteins were measured by refractometry in a temperature-compensated refractometer (American Optical, Buffalo, NY, USA). Urine was collected in preweighed plastic vials containing 0.2 ml of water-equilibrated mineral oil. Sodium and potassium in plasma and urine were measured by means of selective electrodes (Astra 4, Beckmann, USA). Changes with respect to basal values were analyzed by paired Student's t-test.
4. Discussion
The present study demonstrated that endothelin, at non-pressor doses, induced a significant decrease in glomerular filtration rate and renal plasma flow. This decrease seems to be based, at least in part, on the contraction of the renal arteries a n d / o r arterioles, because endothelin induced an increase in renal vascular resistance. Yanagisawa et al. (1988) have reported that endothelin induces a potent contraction in arterial strips of various origins, including rabbit renal arteries. Tomobe et al. (1988) has also reported that endothelin contracts rat renal artery. The small but significant increase in filtration fraction observed in the present experiment suggests a preferential postglomerular action. Wright and Fozard (1988) have shown that a dose of endothelin similar to that used here induced a transient decrease in mean arterial pressure in anesthetized, ganglion-blocked, spontaneously hypertensive rats, followed by an increase to slightly supranormal values. These results were very similar to those obtained in the present ex-
3. Results
The data for renal function are shown in table 1. Endothelin induced a sharp decrease in urine flow, glomerular filtration rate, renal plasma flow and sodium excretion, accompanied by an increase in renal vascular resistance. These values recovered partially in the second clearance period and returned to values similar to those of the basal period in the third period (40-60 rain after endothelin injection). Endothelin induced an acute but transient (30 s-1 rain) decrease in mean arterial pressure, followed by return to values not significantly different from the basal ones. Endothelin
TABLE 1 Effect of endothelin (1 n m o l / k g ) on renal function in rats. The data are m e a n s ± S.E.M. a Statistically significant values (P < 0.05; paired t-test) with respect to basal values. Abbreviations: MAP: mean arterial pressure; U.V.: urinary flow; GFR: glomerular filtration rate; RPF: renal plasma flow; FF: filtration fraction; RVR: renal vascular resistance; UNaV: urinary sodium excretion; UKV: urinary potassium excretion; PCV: packed cell volume; TPP: total plasma protein concentration. Basal
Endothelin 0-20
MAP (nun Hg) U.V.(bd/min) GFR (ml/min) RPF ( m l / m i n ) FF(%) RVR (mm H g . m i n / m l ) UNaV ( m E q / m i n ) UKV ( m E q / m i n ) PCV (%) TPP(g/1)
128 ±4 7.2 +0.6 1.99±0.17 5.85±0.44 34 ±2 11.8 ± 1.4 0.11 ± 0.02 0.07 ± 0.08 46.1 ±0.8 53.3 ±0.5
129 ± 4.1 ± 0.56± 1.39± 38 ± 47.1 ± 0.08 ± 0.07 ± 49.8 ± 55.3 ±
5 1.4 a 0.18 a 0.38 a 3a 10.8 a 0.03 a 0.01 0.8 a 0.7
20-40
40-60
130 ±5 6.7 ±1.4 1.60±0.16 a 3.64±0.57 a 40 ±3 a 17.8 ± 5.2 a 0.06 + 0.02 a 0.10 ± 0.03 49.8 ±1.5 a 56.4 ±0.8
125 8.8
±5 ~_0.5
a
1.74±0.16 4.37±0.27 a 38 ±2 a 14.8 ± 2.4 a 0.08 ± 0.02 a 0.13 ± 0.04 a 47.9 ±1.3 53.0 ±1.2
189
periments. Lippton et al. (1988) have reported a transient (< 1 min) systemic and renal vasodilatation induced by low doses of endothelin, but no data on more prolonged effects were reported. In the study of Yanagisawa et al. (1988), the injection to chemically denervated animals of a dose of endothelin similar to that we now used induced a significant and long-lasting increase in arterial pressure. It must be noted that the compensatory hemodynamic changes that must have occurred in our rats are not present in the chemically denervated animals. Another interesting observation is the increase in packed cell volume induced by endothelin infusion. The mechanism of this increase cannot be easily elucidated from the data available. A first possibility is that endothelin induces a trapping of blood in the peripheral capillaries temporary closed to the circulation by the potent constrictor effect of endothelin. As capillary blood has a decreased packed cell volume, the systemic blood-packed cell volume should increase. A second possibility is that endothelin induces an escape of fluid out of the circulation, thus increasing the erythrocyte concentration. As the total plasma protein does not change, the escaped fluid should be composed of whole plasma. However this accumulation of protein-rich fluid in the interstitium is not easy to remove, whereas in our experiments the packed cell volume returned to normal values in the third experimental period, thus making this second possibility unlikely. Although the physiological meaning of these data is not clear, because the plasma levels of this peptide are unknown, their physiopathological relevance is evident. It has been suggested that endothelin could be released by endothelial cells during hypoxia or reperfusion injury (Vanhoutte and Katusic, 1988; Yanagisawa et al., 1988). In fact, hypoxia has been reported to produce endothelium-dependent contraction of several large arteries or veins in vitro (Dheins et al., 1987; Vanhoutte and Miller, 1985). Thus, it can be hypothesized that, after an ischemic or toxic injury to the kidney, endothelin released by endothelial vascular cells will reach high concentrations in the
glomerular area, inducing glomerular arteriole constriction, and thus reduction of renal plasma flow and glomerular filtration rate. This mechanism of reduction of glomerular filtration rate after renal injury, although very plausible, needs further studies to be adequately demonstrated.
Acknowledgements This study was partially supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 1873/88) and Comisi6n Asesora para la Investigaci6n Cientifica y Ttcnica (594/84). A. L6pez-Farr6 is a Fellow of the Fundaci6n Conchita Rhbago de Jimenez Diaz.
References Dheins, S., F. Essens, A. Salameh, R. Rosen and W. Klaus, 1987, Evidence for a new endothehum-derived vasoconstricting factor, Med. Sci. Res. 15, 573. Lippton, H., J. Goff and A. Hyman, 1988, Effects of endothelin in the systemic and renal vascular beds in vivo, European J. Pharmacol. 155, 197. Lopez-Novoa, J.M. and M. Martinez-Maldonado, 1982, Impaired renal response to splanchnic infusion of hypertonic saline in conscious cirrhotic rats, Am. J. Physiol. 242, F390. Rosemblum, W.I. and G.H. Nelson, 1988, Endothelium-dependent contraction demonstrated in vivo in mouse cerebral arterioles, Circ. Res. 63, 837. Tomobe, Y., A. Miyauchi, A. Saito, M. Yanagisawa, S. Kimura, K. Goto and T. Masaki, 1988, Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats, European J. Pharmacol. 152, 373. Vanhoutte, P.M., 1988, The endothelium, modulator of vascular smooth muscle tone, N. Engl. J. Med. 319, 512. Vanhoutte, P.M. and Z.S. Katusic, 1988, Endothelium-derived contracting factor, endothelin a n d / o r superoxide anion, Trends Physiol. Sci. 9, 229. Vanhoutte, P.M. and V.M. Miller, 1985, Heterogeneity of endothelium-dependent responses in mammalian blood vessels, J. Cardiovasc. Pharmacol. 7 (Suppl. 3), S12. Wright, C.E. and J.R. Fozard, 1988, Regional vasodilation is a prominent feature of haemodynamic response to endothelin in anasthetized, spontaneously hypertensive rat, European J. Pharmacol. 155, 201. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.