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Effect of sodium chloride supplementation on urinary endothelin-1 excretion in premature infants
Effect of sodium chloride supplementation on urinary endothelinexcretion in premature infants W, Rascher, PhD, Gyula Gy6di, MD, S, Worgall, MD, a n d Endre Sulyok, MD, DSc From County Children's Hospital, P6cs, Hungary, County Hospital Kaposv~, Hungary, and Department of Pediatrics, University Medical School, Giessen, Germany We investigated the role of endothelin-I in the renal adaptation to alterations in sodium b a l a n c e in premature infants. The postnatal course of urinary endothelin-I excretion, an estimate of renal endothelin-I production, was c o m p a r e d in premature infants receiving low or high sodium intake. Sodium supplementation was given in a dose of 3 to 5 mmol/kg per d a y and 1.5 to 2.5 mmol/kg per d a y at the postnatal ages of 8 to 21 and 22 to 35 days, respectively. Sodium bala n c e and urinary endothelin-I excretion were determined weekly up to the fifth week of life. Urinary endothelin-I concentration (expressed in picomoles per liter) and urinary endothelin-1 excretion (expressed either in terms of picomoles per square meter per d a y or picomoles per millimole creatinine) were significantly lower in infants receiving a high sodium intake c o m p a r e d with those receiving low sodium intake (p < 0.001) in weeks 2 through 5. We conclude that in sodium-depleted premature infants with high urinary sodium excretion, an angiotensin ll-mediated increase in renal endothelin-I production occurs, which acts in concert with angiotensin II to restore sodium balance. (J PEDIATR 1994;125:793-7)
Endothelin- 1, a potent vasoactive peptide, has been isolated from the culture medium of porcine aortic endothelial cells,1 and has been shown to participate in the control of several cardiovascular, renal, and endocrine functions.2-4 In the kidney its production is not confined to the vascular endothelium; it is also released by epithelial cells derived from different nephron segments. 57 The locally produced endothelin-1 acts as an autocrine/paracrine factor to modulate renal tubular transport processes, including the reabsorption of sodium and water, s l ° On the basis of these observations, we assumed that endothelin-1 might be involved in the renal adaptation to alterations in sodium balance. We designed a study to c o m Supported in part by the Hungarian Ministry of Welfare (grant No. ETT T-007/1990 and OTKA T5182) and by a grant from the Deutsche Forschungsgemeinschaft (Ra 326/3-1). Submitted for publication March 16, 1994;accepted May 27, 1994. Reprint requests: Endre Sulyok, MD, County Children's Hospital, P6cs, Hungary. Copyright © 1994 by Mosby-Year Book, Inc. 0022-3476/94/$3.00 + 0 9/23/57806
pare urinary endothelin-1 excretion, an estimate of renal endothelin-1 production,11-14 in premature infants receiving low or high sodium intakes. METHODS The studies were performed in two groups of healthy, premature male infants appropriate in size for gestational age. Group S consisted of nine infants with birth weights of 1280 to 1750 gm (mean, 1578 gin) and gestational ages of 29 to 33 weeks (mean, 30.4 weeks). These infants were given sodium supplementation in a dose of 3 to 5 and 1.5 to 2.5 mmol/kg per day at a postnatal age of 8 to 21 and 22 to 35 days, respectively, as previously described. 15 Group NS included nine infants with birth weights ranging from 1250 to 1810 gm (mean, 1537 gm) and gestational ages ranging from 28 to 34 weeks (mean, 30.8 weeks) who received no salt supplements. There were no significant differences in birth weight and gestational age between the two groups. The infants were randomly assigned on day 8 to either group. All infants were born vaginally after an uncomplicated pregnancyl No clinical and laboratory evidence of perinatal as-
793
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Rascher et al.
The Journal of Pediatrics November 1994
Table I. Sodium and fluid intake, urinary excretion of sodium and potassium, plasma sodium concentration, and weight gain in p r e m a t u r e infants with and without NaC1 supplementation during the first 5 weeks of life Postnatal a g e (wk) I Fluid intake (ml/kg per day) Group NS 142.8 ± 17.1 Group S 135.7 ± 15.7 NS Na intake (mmol/kg per day) Group NS 1.38 _+ 0.04 Group S 1.35 ± 0.05 NS Urinary Na excretion (mmol/kg per day) Group NS 2.7 + 0.6 Group S 2.8 ___0.6 NS Plasma Na concentration (retool/L) Group NS 138.3 _+ 3.5 Group S 137.5 ± 3.2 NS Urinary K excretion (mmol/kg per day) Group NS 0.7 + 0.2 Group S 0.6 + 0.2 NS Weight gain (gm/kg per day) Group NS -8.6 _+ 3.5 Group S -8.4 ± 3.9 NS
2
3
4
5
155.7 _+ 18.6 148.6 ± 16.8 NS
168.6 + 21.4 162.0 ± 20.1 NS
171.4 + 18.8 168.6 ± 22.2 NS
174.3 + 20.0 178.6 ± 23.4 NS
1.41 + 0.08 5.11 _+ 0.46 p <0.001
1.53 ± 0.31 4.95 ± 0.45 p <0.001
1.55 _+ 0.26 3.10 ± 0.30 p <0.01
1.60 ± 0.18 3.21 ± 0.22 p <0.01
2.2 ± 0.5 3.8 ± 0.7 NS
1.4 _+ 0.4 3.6 _+ 0.6 p <0.05
0.9 + 0.3 3.2 ± 0.5 p <0.01
0.9 _+ 0.4 3.1 ± 0.5 p <0.01
135.2 ± 2.8 138.2 + 3.1 NS
131.1 ± 2.1 139.1 _+ 3.4 p <0.05
132.3 ± 2.4 138.6 ± 2.7 p <0.05
135.0 ± 3.0 137.4 ± 3.2 NS
1.1 ___0.3 1.2 + 0.3 NS
1.3 + 0.4 1.2 _± 0.3 NS
1.4 + 0.4 1.4 _+ 0.3 NS
1.7 _+ 0.5 1.6 _+ 0.4 NS
8.0 + 3.1 11.6 ± 3.0 NS
14.6 _+ 2.9 19.2 +_ 2.4 NS
16.0 + 2.9 18.8 + 2.2 NS
16.5 _+ 2.3 18.2 + 2.1 NS
NS, Not significant.
phyxia, infection, or cardiopulmonary distress was noted, and the infants remained well during the course of the study. All infants were fed pooled h u m a n milk; the daily fluid intake approximated 150 to 180 m l / k g by the end of the second week. P l a s m a sodium concentration and daily excretion of sodium, potassium (flame photometry), creatinine (modified Jaff6 r e a c t i o n ) a n d endothelin-1 (radioimmunoassay m e t h o d ) were determined from urine collected for 24 hours on the seventh day of life and at weekly intervals thereafter up to the fifth week. For reasons of convenience and accurate urine collections, only male infants were selected for the studies. U r i n e was fractionally collected in plastic bags; the specimens were pooled and stored at - 2 0 ° C until analyzed. Collections were started after spontaneous voiding, and when t e r m i n a t e d mild suprapubic compression was applied to complete the procedure. For endothelin-1 measurements, 1 ml urine was acidified with 0.25 ml 2 m o l / L HC1 solution, mixed and centrifuged at 14,000g for 5 minutes. Extraction was performed by
C2 silica cartridges ( A m prep Ethyl C2, R P N 1913; A m e r s h a m , Braunschweig, G e r m a n y ) . The columns were prewashed with 2 ml 100% m e t h a n o l and 2 ml distilled water. Acid±fed urine samples were placed on the columns and washed twice with 2.5 ml 0.1% trifluoroacetic acid solution. T h e eluate (2 ml of 80% m e t h a n o l in 0.1% trifluoroacetic acid solution) was concentrated to dryness in a v a c u u m centrifuge (Hetovac; N u n c G m b H , Wiesbaden, G e r m a n y ) , redissolved in 0.1 m o l / L phosphate buffer, and assayed. T h e endothelin- 1 immunoreactivity was m e a s u r e d with a specific antibody ( R P A 535, A m e r s h a m ) . The detection limit was 1.0 p m o l / L , 50% binding at 4.0 p m o l / L . The intraassay and interassay coefficients of variation were 8.1% (n = 10) and 13.9% (n = 24), respectively. The results were expressed as picomoles per liter, picomoles per square m e t e r per day, a n d picomoles per mill±mole creatinine. 16 Statistical analysis was done by paired and unpaired S t u d e n t t tests. D a t a are expressed as m e a n + S E M .
The Journal o f Pediatrics Volume 125, Number 5, Part l
Table
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795
II. Effect of NaC1 supplementation on urinary endothelin-1 excretion in premature infants
Age (wk)
Urinary endothelin-1 concentration (pmol/L) Group NS
Group S
1
21.4 + 3.2
2 3 4 5
17.1 ± i6.3 ± 16.6 ± 16.7 ±
18.3 ± 1.7 10.5 ± 0.9* 9.6 _+ 0.6* 8.7 ± 0.6* 9.1 ± 0.6*
2.2 1.7 1.6 1.7
Urinary EI"-4 excretion (pmol/m a per day) Group NS 12.4 ± 16.2 + 17.9 + 22.9 ± 19.5 ±
0.7 3.6 3.2 2.2 2.2
Group S 11.4 ± 9.1 + 8.7 ± 8.3 ± 9.4 ±
1.1 0.8* 1.3" 3.9* 1.5"
(pmol/mmol creatinine) Group NS 17.0 ± 19.2 ± 20.4 ± 21.8 ± 18.2 ±
2.1 3.0 3.4 1.6 2.4
Group S 15.3 + 1.3 10.9 ± 0.8" 10.3 ± 0.4* 10.2 ___0.5* 9.7 ± 0.7*
*p <0.001.
Informed parental consent and approval of the institutional ethics committee were obtained for the study.
lin-1 excretion between the two groups were significant in weeks 2 to 5.
RESULTS
DISCUSSION
Table I shows that premature infants receiving supplemental sodium chloride had higher urinary sodium excretion, but retained more sodium, maintained plasma sodium concentration at normal levels, and gained weight at a slightly higher rate than those infants receiving a low sodium intake. Sodium supplementation did not cause hypernatremia, clinically apparent edema, or any other symptoms of fluid overload, and there was no significant difference in blood pressure. Urinary potassium excretion increased progressively at a low rate during the course of the study, and NaC1 supplementation had no apparent influence on its developmental pattern. The mean endothelin- 1 concentration declined slightly in groupNS, from 21.4 _+ 3.2 pmol/Lon week 1 to 16.3 + 1.7 pmol/L in week 3 and remained essentially unchanged thereafter. When supplemental sodium was given, urinary endothelin-1 concentration stabilized at about 9 pmol/L, which was significantly lower than that in group NS in weeks 2 to 5 (p < 0.001; Table II). Urinary endothelin-1 excretion corrected for body surface area increased gradually in group NS from the initial value of 12.4 _+ 0.7 pmol/m 2 per day in the first week to reach its maximum of 22.9 + 2.2 pmol/m 2 per day (p < 0.01) in the fourth week. By contrast, high dietary NaC1 intake prevented any rise in urinary endothelin-1 excretion in group S; it remained at about the same level throughout the study. As a result, the differences in urinary endothelin-1 excretion between the two groups were significant during weeks 2 to 5 (p < 0.001). Endothelin-1 excretion expressed per millimole creatinine increased slightly with advancing age in group NS, whereas after an initial decline from 15.3 _.+ 1.3 pmol/mmol creatinine in week 1 to 10.9 + 0.8 pmol/mmol creatinine in week 2, no consistent change could be detected in group S during the period of NaCI supplementation. Again, the differences in endothe-
This study demonstrates an association of sodium depletion with increased urinary endothelin-1 excretion, and reduction in endothelin- 1 excretion with restoration of sodium balance in low birth weight premature infants given low or high sodium intake. Furthermore, our results suggest that the adaptive increase in renal endothelin-1 production in sodium-depleted premature infants may contribute to more efficient renal sodium conservation. The role of endothelin-1 in the renal control of sodium and water homeostasis during the neonatal period has not been established. Bhat et al. 17 reported a dose-dependent decrease in urine flow and fractional sodium excretion in association with reduced renal blood flow and glomerular filtration rate in newborn piglets after administration of endothelin-1. Administration of endothelin-1 in newborn rabbits resulted in a significant increase in urine flow and sodium excretion rates independent of renal hemodynamic alterations.18; the authors proposed direct tubular effects of endothelin-1 because endothelin-1 has been demonstrated to inhibit Na+-K+-ATPase activity,8 as well as vasopressinstimulated osmotic water permeability and cyclic adenosine monophosphate accumulation in the inner medullary collecting ducts. 9' 10 In human neonates no data are available on the endothelin-l-related changes in renal sodium excretion. However, the postnatal development of urinary endothelin- 1 excretion in term and preterm infants and the urinary endothelin-1 response to perinatal asphyxia or infection and to dopamine therapy have been described. 16, 19, 20 Moreover, there was a significant positive correlation between urinary endothelin-1 excretion and diuresis. 16 The results of our study challenge the possibility that endothelin-I produced by the renal tubular cells may serve as a general inhibitor of tubular sodium transport, and are in agreement with clinical and experimental evidence favoring
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Rascher et al.
the concept that endothelin-1 enhances renal tubular sodium reabsorption during a sodium-depleted state. In response to sodium depletion the renin-angiotensin system is activated; angiotensin II simultaneously stimulates proximal tubular sodium reabsorption, aldosterone production, and the release of endothelin-l-like immunoreactivity in endothelial cells through immediate and dosedependent induction of the endothelin-1 gene expression. 21 Several observations suggest a physiologic role for endothelin- 1 in the proximal nephron. Endothelin- 1 binding sites have been identified,22,23 and endothelin-1 synthesis has been documented in the proximal tubules. 57 Endothelin-1 has been shown to increase the activity of the apical N a + / H + exchanger and the basolateral Na+/HCO3 cotransporter in membrane vesicles prepared from rabbit renal cortex. 24 Furthermore, a low salt intake in rats increases the activity of endogenous endothelin, and infusion of endothelin antiserum to block endogenous endothelin action causes a significant increase in urinary sodium excretion and fractional excretion of sodium,z5 Consistent with these findings, there have been reports of elevated or depressed plasma endothelin-1 levels in patients with depletion hyponatremia or in patients with the syndrome of inappropriate antidiuretic hormone secretion, respectively. Correction of sodium and water balance normalizes plasma endothelin-1 levels. 26 It is tempting to postulate that in low-volume states an angfotensin II-mediated increase in endothelin-1 production acts in concert with angiotensin II to stimulate proximal tubular sodium reabsorption and aldosterone productionS, 28 In high volume states endogenous endothelin-1 production is inhibited, most likely by atrial natriuretic peptidedependent mechanisms. In support of this contention, plasma endothelin-1 levels have been found to be suppressed by both a-human atrial natriuretic peptide and angiotensinconverting enzyme inhibition in normal men. 29 We conclude that in sodium-depleted premature infants with high urinary sodium excretion the renin-angiotensin-aldosterone system is excessively activated and the plasma atrial natriuretic peptide level is low. When positive sodium balance is restored by giving supplemental NaC1, the activity of the renin-angiotensin-aldosterone system is markedly reduced and the plasma atrial natriuretic peptide level becomes elevated. 15, 30 These endocrine reactions may account, at least in part, for the low and high rate of renal endothelin-1 production in premature infants with or without NaC1 supplementation, respectively. Consequently, the adaptive changes in renal endothelin-1 production can be regarded as a further attempt to maintain sodium balance. We thank Mrs. U. Jacobs for her skillful technical assistance.
The Journal of Pediatrics November 1994
REFERENCES
1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictorpeptide producedby vascular endothelialcells. Nature 1988;332:441-415. 2. Miller WL, Redfield MM, Burnett JC. Integrated cardiac, renal and endocrine actions of endothelin. J Clin Invest 1989; 83:317-20. 3. Lerman A, Hildebrand FL, Margulies KB, et al. Endothelin: a new cardiovascular regulatory peptide. Mayo Clin Proc 1990;65:1441-55. 4. Leppaluoto J, Ruskoaho H. Endothelin peptides: biological activities, cellular signalling and clinical significance. Ann Med 1992;24:153-61. 5. Kohan DE. Endothelin synthesis by rabbit renal tubule cells. Am J Physiol 1991;261:F221-6. 6. Ujiie K, Terada Y, Nonoguchi H, Shinohara M, Tomita K, Marumo F. Messenger RNA expression and synthesis of endothelin-1 along rat nephron segments. J Clin Invest 1992; 90:1043-8. 7. WilkesBM, Susin M, Mento PF, et al. Localization of endothelin-like immunoreactivity in rat kidneys. Am J Physiol 1991;260:F913-20. 8. Zeidel ML, Brady MR, Kone BC, Gullans SR, Brenner BM. Endothelin, a peptide inhibitor of Na+-K+ATPase in intact renal tubular epithelial cells. Am J Physiol 1989; 257:C1101-7. 9. Oishi R, Nonogouchi H, Tomita K, Marumo F. Endothelin-1 inhibits AVP-stimulated osmotic water permeability in rat inner medullary collecting duct. Am J Physiol 1991;261: F951-6. 10. Nadler SP, Zimpelmann JA, Hebert RL. Endothelin inhibits vasopressin stimulated water permeability in rat terminal inner medullary collecting duct. J Clin Invest 1992;90:145866. 11. Benigni A, Perico N, Gaspari F, et al. Increased renal endothelin production in rats with reduced renal mass. Am J Physiol 1991;260:F331-9. 12. Berbinschi A, Ketelslegers JM. Endothelin in urine. Lancet 1989;2:46. 13. Ando K, Hirata Y, Takei Y, Kawakami M, Marumo F. Endothelin-l-like immunoreactivity in human urine. Nephron 1991;57:36-9. 14. Ohta K, Hirata Y, Shichiri M, et al. Urinary excretion of endothelin-1 in normal subjects and patients with renal disease. Kidney Int 1991;39:307-11. 15. SulyokE, Nemeth M, Tenyi I, Csaba IF, Varga L, Varga F. Relationship between the postnatal developmentof the reninangiotensin-aldosteronesystem and electrolyte and acid-base status of the NaC1 supplementedpremature infants. In: Spitzer A, ed. The kidney during development:morphologyand function. New York: Masson, 1982:272. 16. SulyokE, Ertl T, Adamovits K, HovanyovszkyS, Rascher W. Urinary endothelin excretion in the neonate: influence of maturity and perinatal pathology. Pediatr Nephrol 1993; 7:881-5. 17. Bhat R, John E, Chari G, Fornell L, Raju T, Vidyasagar D. Endothelin-1 (ET-1) induced glomerular dysfunction: dose versus renal functions (RF) [Abstract]. Pediatr Res 1992; 31:329A. 18. Semama DS, Thonney M, Guignard J-P, Gouyon J-B. Effects of endothelinon renal function in newbornrabbits. Pediatr Res 1993;34:120-3.
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19. Iwata I, Takagi T, Yamaji K, Tanizawa O. Increase in the concentration of immunoreactive endothelin in human pregnancy. J Endocrinol 199l;129:301-7. 20. Sanchez ME, Reale MR, Girardi LM, Dweck HS. Endothelin (ET) in the urine of newborn infants [Abstract]. Pediatr Res 1992;31:342A. 21. Imai T, Hirata Y, Emori T, Yanagisawa M, Masald T, Marumo F. Induction of endothelin-1 gene by angiotensin and vasopressin in endothelial cells. Hypertension 1992;19: 753-7. 22. Kohzuki M, Johnston CL, Chai SY, Casley D, Mendelsohn FAO. Localization of endothelin receptors in rat kidney. Eur J Pharmacol 1989;160:193-4. 23. Waeber C, Hoyer D, Palacios J-M. Similar distribution of [t25I]sarafotoxin-6b and [12SI]endothelin 1,2,3, binding sides in the human kidney. Eur J Pharmacol 1990;176:233-6. 24. Eiham-Ong S, Hilden SA, King A J, Johns CA, Madias NE. Endothelin-i stimulates the Na+/H + and Na+HCO3 - transporters in rabbit renal cortex. Kidney Int 1992;42:18-24.
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25. Yamada K, Yoshida S. Role of endogenous endothelin on renal functions in rats. Am J Physiol 1991;260:F34-8. 26. Kamoi K, Ishibashi M, Yamaji T. Plasma endothelin-1 levels in patients with hyponatremia. Nephron 1992;62:469-70. 27. Rosolowski L J, Campbell WB. Endothelin enhances adrenocorticotropin-stimulated aldosterone release from cultured bovine adrenal cells. Endocrinology 1990; 126:1860-6. 28. Hinson JP, Vinson GP, Kapas S, Teja R. The role of endothelin in the control of adrenocortical function: stimulation of endothelin release by ACTH and the effects of endothelin-i and endothelin-3 on steroidogenesis in rat and human adrenocortical cells J Endocrinol 1991;128:275-80. 29. Uemasu J, Matsumoto H, Kitano M, Kawasaki H. Suppression of plasma endothelin-1 level by a-human atrial natriuretic peptide and angiotensin converting enzyme inhibition in normal men. Life Sci 1993;53:969-73. 30. Tulassay T, Rascher W, Seyberth HW, Lang RE, Toth M, Sulyok E. Role of atrial natriuretic peptide in sodium homeostasis in premature infants. J PEDIATR 1986;109:1023-7.
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Endothelin- 1, a potent vasoactive peptide, has been isolated from the culture medium of porcine aortic endothelial cells,1 and has been shown to participate in the control of several cardiovascular, renal, and endocrine functions.2-4 In the kidney its production is not confined to the vascular endothelium; it is also released by epithelial cells derived from different nephron segments. 57 The locally produced endothelin-1 acts as an autocrine/paracrine factor to modulate renal tubular transport processes, including the reabsorption of sodium and water, s l ° On the basis of these observations, we assumed that endothelin-1 might be involved in the renal adaptation to alterations in sodium balance. We designed a study to c o m Supported in part by the Hungarian Ministry of Welfare (grant No. ETT T-007/1990 and OTKA T5182) and by a grant from the Deutsche Forschungsgemeinschaft (Ra 326/3-1). Submitted for publication March 16, 1994;accepted May 27, 1994. Reprint requests: Endre Sulyok, MD, County Children's Hospital, P6cs, Hungary. Copyright © 1994 by Mosby-Year Book, Inc. 0022-3476/94/$3.00 + 0 9/23/57806
pare urinary endothelin-1 excretion, an estimate of renal endothelin-1 production,11-14 in premature infants receiving low or high sodium intakes. METHODS The studies were performed in two groups of healthy, premature male infants appropriate in size for gestational age. Group S consisted of nine infants with birth weights of 1280 to 1750 gm (mean, 1578 gin) and gestational ages of 29 to 33 weeks (mean, 30.4 weeks). These infants were given sodium supplementation in a dose of 3 to 5 and 1.5 to 2.5 mmol/kg per day at a postnatal age of 8 to 21 and 22 to 35 days, respectively, as previously described. 15 Group NS included nine infants with birth weights ranging from 1250 to 1810 gm (mean, 1537 gm) and gestational ages ranging from 28 to 34 weeks (mean, 30.8 weeks) who received no salt supplements. There were no significant differences in birth weight and gestational age between the two groups. The infants were randomly assigned on day 8 to either group. All infants were born vaginally after an uncomplicated pregnancyl No clinical and laboratory evidence of perinatal as-
793
794
Rascher et al.
The Journal of Pediatrics November 1994
Table I. Sodium and fluid intake, urinary excretion of sodium and potassium, plasma sodium concentration, and weight gain in p r e m a t u r e infants with and without NaC1 supplementation during the first 5 weeks of life Postnatal a g e (wk) I Fluid intake (ml/kg per day) Group NS 142.8 ± 17.1 Group S 135.7 ± 15.7 NS Na intake (mmol/kg per day) Group NS 1.38 _+ 0.04 Group S 1.35 ± 0.05 NS Urinary Na excretion (mmol/kg per day) Group NS 2.7 + 0.6 Group S 2.8 ___0.6 NS Plasma Na concentration (retool/L) Group NS 138.3 _+ 3.5 Group S 137.5 ± 3.2 NS Urinary K excretion (mmol/kg per day) Group NS 0.7 + 0.2 Group S 0.6 + 0.2 NS Weight gain (gm/kg per day) Group NS -8.6 _+ 3.5 Group S -8.4 ± 3.9 NS
2
3
4
5
155.7 _+ 18.6 148.6 ± 16.8 NS
168.6 + 21.4 162.0 ± 20.1 NS
171.4 + 18.8 168.6 ± 22.2 NS
174.3 + 20.0 178.6 ± 23.4 NS
1.41 + 0.08 5.11 _+ 0.46 p <0.001
1.53 ± 0.31 4.95 ± 0.45 p <0.001
1.55 _+ 0.26 3.10 ± 0.30 p <0.01
1.60 ± 0.18 3.21 ± 0.22 p <0.01
2.2 ± 0.5 3.8 ± 0.7 NS
1.4 _+ 0.4 3.6 _+ 0.6 p <0.05
0.9 + 0.3 3.2 ± 0.5 p <0.01
0.9 _+ 0.4 3.1 ± 0.5 p <0.01
135.2 ± 2.8 138.2 + 3.1 NS
131.1 ± 2.1 139.1 _+ 3.4 p <0.05
132.3 ± 2.4 138.6 ± 2.7 p <0.05
135.0 ± 3.0 137.4 ± 3.2 NS
1.1 ___0.3 1.2 + 0.3 NS
1.3 + 0.4 1.2 _± 0.3 NS
1.4 + 0.4 1.4 _+ 0.3 NS
1.7 _+ 0.5 1.6 _+ 0.4 NS
8.0 + 3.1 11.6 ± 3.0 NS
14.6 _+ 2.9 19.2 +_ 2.4 NS
16.0 + 2.9 18.8 + 2.2 NS
16.5 _+ 2.3 18.2 + 2.1 NS
NS, Not significant.
phyxia, infection, or cardiopulmonary distress was noted, and the infants remained well during the course of the study. All infants were fed pooled h u m a n milk; the daily fluid intake approximated 150 to 180 m l / k g by the end of the second week. P l a s m a sodium concentration and daily excretion of sodium, potassium (flame photometry), creatinine (modified Jaff6 r e a c t i o n ) a n d endothelin-1 (radioimmunoassay m e t h o d ) were determined from urine collected for 24 hours on the seventh day of life and at weekly intervals thereafter up to the fifth week. For reasons of convenience and accurate urine collections, only male infants were selected for the studies. U r i n e was fractionally collected in plastic bags; the specimens were pooled and stored at - 2 0 ° C until analyzed. Collections were started after spontaneous voiding, and when t e r m i n a t e d mild suprapubic compression was applied to complete the procedure. For endothelin-1 measurements, 1 ml urine was acidified with 0.25 ml 2 m o l / L HC1 solution, mixed and centrifuged at 14,000g for 5 minutes. Extraction was performed by
C2 silica cartridges ( A m prep Ethyl C2, R P N 1913; A m e r s h a m , Braunschweig, G e r m a n y ) . The columns were prewashed with 2 ml 100% m e t h a n o l and 2 ml distilled water. Acid±fed urine samples were placed on the columns and washed twice with 2.5 ml 0.1% trifluoroacetic acid solution. T h e eluate (2 ml of 80% m e t h a n o l in 0.1% trifluoroacetic acid solution) was concentrated to dryness in a v a c u u m centrifuge (Hetovac; N u n c G m b H , Wiesbaden, G e r m a n y ) , redissolved in 0.1 m o l / L phosphate buffer, and assayed. T h e endothelin- 1 immunoreactivity was m e a s u r e d with a specific antibody ( R P A 535, A m e r s h a m ) . The detection limit was 1.0 p m o l / L , 50% binding at 4.0 p m o l / L . The intraassay and interassay coefficients of variation were 8.1% (n = 10) and 13.9% (n = 24), respectively. The results were expressed as picomoles per liter, picomoles per square m e t e r per day, a n d picomoles per mill±mole creatinine. 16 Statistical analysis was done by paired and unpaired S t u d e n t t tests. D a t a are expressed as m e a n + S E M .
The Journal o f Pediatrics Volume 125, Number 5, Part l
Table
R a s c h e r et al.
795
II. Effect of NaC1 supplementation on urinary endothelin-1 excretion in premature infants
Age (wk)
Urinary endothelin-1 concentration (pmol/L) Group NS
Group S
1
21.4 + 3.2
2 3 4 5
17.1 ± i6.3 ± 16.6 ± 16.7 ±
18.3 ± 1.7 10.5 ± 0.9* 9.6 _+ 0.6* 8.7 ± 0.6* 9.1 ± 0.6*
2.2 1.7 1.6 1.7
Urinary EI"-4 excretion (pmol/m a per day) Group NS 12.4 ± 16.2 + 17.9 + 22.9 ± 19.5 ±
0.7 3.6 3.2 2.2 2.2
Group S 11.4 ± 9.1 + 8.7 ± 8.3 ± 9.4 ±
1.1 0.8* 1.3" 3.9* 1.5"
(pmol/mmol creatinine) Group NS 17.0 ± 19.2 ± 20.4 ± 21.8 ± 18.2 ±
2.1 3.0 3.4 1.6 2.4
Group S 15.3 + 1.3 10.9 ± 0.8" 10.3 ± 0.4* 10.2 ___0.5* 9.7 ± 0.7*
*p <0.001.
Informed parental consent and approval of the institutional ethics committee were obtained for the study.
lin-1 excretion between the two groups were significant in weeks 2 to 5.
RESULTS
DISCUSSION
Table I shows that premature infants receiving supplemental sodium chloride had higher urinary sodium excretion, but retained more sodium, maintained plasma sodium concentration at normal levels, and gained weight at a slightly higher rate than those infants receiving a low sodium intake. Sodium supplementation did not cause hypernatremia, clinically apparent edema, or any other symptoms of fluid overload, and there was no significant difference in blood pressure. Urinary potassium excretion increased progressively at a low rate during the course of the study, and NaC1 supplementation had no apparent influence on its developmental pattern. The mean endothelin- 1 concentration declined slightly in groupNS, from 21.4 _+ 3.2 pmol/Lon week 1 to 16.3 + 1.7 pmol/L in week 3 and remained essentially unchanged thereafter. When supplemental sodium was given, urinary endothelin-1 concentration stabilized at about 9 pmol/L, which was significantly lower than that in group NS in weeks 2 to 5 (p < 0.001; Table II). Urinary endothelin-1 excretion corrected for body surface area increased gradually in group NS from the initial value of 12.4 _+ 0.7 pmol/m 2 per day in the first week to reach its maximum of 22.9 + 2.2 pmol/m 2 per day (p < 0.01) in the fourth week. By contrast, high dietary NaC1 intake prevented any rise in urinary endothelin-1 excretion in group S; it remained at about the same level throughout the study. As a result, the differences in urinary endothelin-1 excretion between the two groups were significant during weeks 2 to 5 (p < 0.001). Endothelin-1 excretion expressed per millimole creatinine increased slightly with advancing age in group NS, whereas after an initial decline from 15.3 _.+ 1.3 pmol/mmol creatinine in week 1 to 10.9 + 0.8 pmol/mmol creatinine in week 2, no consistent change could be detected in group S during the period of NaCI supplementation. Again, the differences in endothe-
This study demonstrates an association of sodium depletion with increased urinary endothelin-1 excretion, and reduction in endothelin- 1 excretion with restoration of sodium balance in low birth weight premature infants given low or high sodium intake. Furthermore, our results suggest that the adaptive increase in renal endothelin-1 production in sodium-depleted premature infants may contribute to more efficient renal sodium conservation. The role of endothelin-1 in the renal control of sodium and water homeostasis during the neonatal period has not been established. Bhat et al. 17 reported a dose-dependent decrease in urine flow and fractional sodium excretion in association with reduced renal blood flow and glomerular filtration rate in newborn piglets after administration of endothelin-1. Administration of endothelin-1 in newborn rabbits resulted in a significant increase in urine flow and sodium excretion rates independent of renal hemodynamic alterations.18; the authors proposed direct tubular effects of endothelin-1 because endothelin-1 has been demonstrated to inhibit Na+-K+-ATPase activity,8 as well as vasopressinstimulated osmotic water permeability and cyclic adenosine monophosphate accumulation in the inner medullary collecting ducts. 9' 10 In human neonates no data are available on the endothelin-l-related changes in renal sodium excretion. However, the postnatal development of urinary endothelin- 1 excretion in term and preterm infants and the urinary endothelin-1 response to perinatal asphyxia or infection and to dopamine therapy have been described. 16, 19, 20 Moreover, there was a significant positive correlation between urinary endothelin-1 excretion and diuresis. 16 The results of our study challenge the possibility that endothelin-I produced by the renal tubular cells may serve as a general inhibitor of tubular sodium transport, and are in agreement with clinical and experimental evidence favoring
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Rascher et al.
the concept that endothelin-1 enhances renal tubular sodium reabsorption during a sodium-depleted state. In response to sodium depletion the renin-angiotensin system is activated; angiotensin II simultaneously stimulates proximal tubular sodium reabsorption, aldosterone production, and the release of endothelin-l-like immunoreactivity in endothelial cells through immediate and dosedependent induction of the endothelin-1 gene expression. 21 Several observations suggest a physiologic role for endothelin- 1 in the proximal nephron. Endothelin- 1 binding sites have been identified,22,23 and endothelin-1 synthesis has been documented in the proximal tubules. 57 Endothelin-1 has been shown to increase the activity of the apical N a + / H + exchanger and the basolateral Na+/HCO3 cotransporter in membrane vesicles prepared from rabbit renal cortex. 24 Furthermore, a low salt intake in rats increases the activity of endogenous endothelin, and infusion of endothelin antiserum to block endogenous endothelin action causes a significant increase in urinary sodium excretion and fractional excretion of sodium,z5 Consistent with these findings, there have been reports of elevated or depressed plasma endothelin-1 levels in patients with depletion hyponatremia or in patients with the syndrome of inappropriate antidiuretic hormone secretion, respectively. Correction of sodium and water balance normalizes plasma endothelin-1 levels. 26 It is tempting to postulate that in low-volume states an angfotensin II-mediated increase in endothelin-1 production acts in concert with angiotensin II to stimulate proximal tubular sodium reabsorption and aldosterone productionS, 28 In high volume states endogenous endothelin-1 production is inhibited, most likely by atrial natriuretic peptidedependent mechanisms. In support of this contention, plasma endothelin-1 levels have been found to be suppressed by both a-human atrial natriuretic peptide and angiotensinconverting enzyme inhibition in normal men. 29 We conclude that in sodium-depleted premature infants with high urinary sodium excretion the renin-angiotensin-aldosterone system is excessively activated and the plasma atrial natriuretic peptide level is low. When positive sodium balance is restored by giving supplemental NaC1, the activity of the renin-angiotensin-aldosterone system is markedly reduced and the plasma atrial natriuretic peptide level becomes elevated. 15, 30 These endocrine reactions may account, at least in part, for the low and high rate of renal endothelin-1 production in premature infants with or without NaC1 supplementation, respectively. Consequently, the adaptive changes in renal endothelin-1 production can be regarded as a further attempt to maintain sodium balance. We thank Mrs. U. Jacobs for her skillful technical assistance.
The Journal of Pediatrics November 1994
REFERENCES
1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictorpeptide producedby vascular endothelialcells. Nature 1988;332:441-415. 2. Miller WL, Redfield MM, Burnett JC. Integrated cardiac, renal and endocrine actions of endothelin. J Clin Invest 1989; 83:317-20. 3. Lerman A, Hildebrand FL, Margulies KB, et al. Endothelin: a new cardiovascular regulatory peptide. Mayo Clin Proc 1990;65:1441-55. 4. Leppaluoto J, Ruskoaho H. Endothelin peptides: biological activities, cellular signalling and clinical significance. Ann Med 1992;24:153-61. 5. Kohan DE. Endothelin synthesis by rabbit renal tubule cells. Am J Physiol 1991;261:F221-6. 6. Ujiie K, Terada Y, Nonoguchi H, Shinohara M, Tomita K, Marumo F. Messenger RNA expression and synthesis of endothelin-1 along rat nephron segments. J Clin Invest 1992; 90:1043-8. 7. WilkesBM, Susin M, Mento PF, et al. Localization of endothelin-like immunoreactivity in rat kidneys. Am J Physiol 1991;260:F913-20. 8. Zeidel ML, Brady MR, Kone BC, Gullans SR, Brenner BM. Endothelin, a peptide inhibitor of Na+-K+ATPase in intact renal tubular epithelial cells. Am J Physiol 1989; 257:C1101-7. 9. Oishi R, Nonogouchi H, Tomita K, Marumo F. Endothelin-1 inhibits AVP-stimulated osmotic water permeability in rat inner medullary collecting duct. Am J Physiol 1991;261: F951-6. 10. Nadler SP, Zimpelmann JA, Hebert RL. Endothelin inhibits vasopressin stimulated water permeability in rat terminal inner medullary collecting duct. J Clin Invest 1992;90:145866. 11. Benigni A, Perico N, Gaspari F, et al. Increased renal endothelin production in rats with reduced renal mass. Am J Physiol 1991;260:F331-9. 12. Berbinschi A, Ketelslegers JM. Endothelin in urine. Lancet 1989;2:46. 13. Ando K, Hirata Y, Takei Y, Kawakami M, Marumo F. Endothelin-l-like immunoreactivity in human urine. Nephron 1991;57:36-9. 14. Ohta K, Hirata Y, Shichiri M, et al. Urinary excretion of endothelin-1 in normal subjects and patients with renal disease. Kidney Int 1991;39:307-11. 15. SulyokE, Nemeth M, Tenyi I, Csaba IF, Varga L, Varga F. Relationship between the postnatal developmentof the reninangiotensin-aldosteronesystem and electrolyte and acid-base status of the NaC1 supplementedpremature infants. In: Spitzer A, ed. The kidney during development:morphologyand function. New York: Masson, 1982:272. 16. SulyokE, Ertl T, Adamovits K, HovanyovszkyS, Rascher W. Urinary endothelin excretion in the neonate: influence of maturity and perinatal pathology. Pediatr Nephrol 1993; 7:881-5. 17. Bhat R, John E, Chari G, Fornell L, Raju T, Vidyasagar D. Endothelin-1 (ET-1) induced glomerular dysfunction: dose versus renal functions (RF) [Abstract]. Pediatr Res 1992; 31:329A. 18. Semama DS, Thonney M, Guignard J-P, Gouyon J-B. Effects of endothelinon renal function in newbornrabbits. Pediatr Res 1993;34:120-3.
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19. Iwata I, Takagi T, Yamaji K, Tanizawa O. Increase in the concentration of immunoreactive endothelin in human pregnancy. J Endocrinol 199l;129:301-7. 20. Sanchez ME, Reale MR, Girardi LM, Dweck HS. Endothelin (ET) in the urine of newborn infants [Abstract]. Pediatr Res 1992;31:342A. 21. Imai T, Hirata Y, Emori T, Yanagisawa M, Masald T, Marumo F. Induction of endothelin-1 gene by angiotensin and vasopressin in endothelial cells. Hypertension 1992;19: 753-7. 22. Kohzuki M, Johnston CL, Chai SY, Casley D, Mendelsohn FAO. Localization of endothelin receptors in rat kidney. Eur J Pharmacol 1989;160:193-4. 23. Waeber C, Hoyer D, Palacios J-M. Similar distribution of [t25I]sarafotoxin-6b and [12SI]endothelin 1,2,3, binding sides in the human kidney. Eur J Pharmacol 1990;176:233-6. 24. Eiham-Ong S, Hilden SA, King A J, Johns CA, Madias NE. Endothelin-i stimulates the Na+/H + and Na+HCO3 - transporters in rabbit renal cortex. Kidney Int 1992;42:18-24.
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25. Yamada K, Yoshida S. Role of endogenous endothelin on renal functions in rats. Am J Physiol 1991;260:F34-8. 26. Kamoi K, Ishibashi M, Yamaji T. Plasma endothelin-1 levels in patients with hyponatremia. Nephron 1992;62:469-70. 27. Rosolowski L J, Campbell WB. Endothelin enhances adrenocorticotropin-stimulated aldosterone release from cultured bovine adrenal cells. Endocrinology 1990; 126:1860-6. 28. Hinson JP, Vinson GP, Kapas S, Teja R. The role of endothelin in the control of adrenocortical function: stimulation of endothelin release by ACTH and the effects of endothelin-i and endothelin-3 on steroidogenesis in rat and human adrenocortical cells J Endocrinol 1991;128:275-80. 29. Uemasu J, Matsumoto H, Kitano M, Kawasaki H. Suppression of plasma endothelin-1 level by a-human atrial natriuretic peptide and angiotensin converting enzyme inhibition in normal men. Life Sci 1993;53:969-73. 30. Tulassay T, Rascher W, Seyberth HW, Lang RE, Toth M, Sulyok E. Role of atrial natriuretic peptide in sodium homeostasis in premature infants. J PEDIATR 1986;109:1023-7.
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