Pediatr Nephrol (2003) 18:1157–1160 DOI 10.1007/s00467-003-1263-5
ORIGINAL ARTICLE
Polyxeni Nicolaidou · Helen Georgouli · Vasiliki Getsi · Helen Tsapra · Fotini Psychou · Yiannis G. Matsinos · Petros M. Zeis · Dimitris Gourgiotis
Urinary excretion of endothelin-1 in children with absorptive idiopathic hypercalciuria Received: 17 January 2003 / Revised: 17 June 2003 / Accepted: 20 June 2003 / Published online: 2 October 2003 IPNA 2003
Abstract Urinary excretion of endothelin-1 (ET-1) and plasma ET-1 were measured in 21 children with absorptive idiopathic hypercalciuria (AIH) and 22 controls. The absorptive type of idiopathic hypercalciuria was determined by a calcium loading test. Daily urinary excretion of ET-1 and urinary ET-1/creatinine ratio were significantly increased (P=0.005 and P=0.007, respectively) in patients with AIH (9,274€6,444 pg/24 h and 14.04€9.52 pg/mg, respectively) compared with controls (4,699€2,120 pg/24 h and 7.36€4.71 pg/mg, respectively). Plasma ET-1 levels were significantly lower in patients with AIH (0.84€0.64 pg/ml) than in controls (1.54€0.54 pg/ml, P=0.0001). In conclusion, patients with AIH had increased urinary ET-1 excretion and decreased plasma ET-1 levels. This is most likely due to the decreased reabsorption of ET-1 in the renal tubule and increased renal production. Keywords Endothelin · Calcium · Hypercalciuria P. Nicolaidou · F. Psychou First Pediatric Department, Athens University Medical School, “Aghia Sophia” Children’s Hospital, Athens, Greece H. Georgouli · H. Tsapra · P. M. Zeis · D. Gourgiotis Second Pediatric Department, Athens University Medical School, “Aglaia Kyriakou” Children’s Hospital, Athens, Greece V. Getsi Pediatric Department, “Chatzicosta” Hospital, Ioannina, Greece Y. G. Matsinos Department of Environmental Sciences, University of the Aegean, Mytilene, Greece P. Nicolaidou ()) First Pediatric Department, Athens University Medical School, “Aghia Sophia” Children’s Hospital, Thivon and Levadias str., 115 27 Athens, Greece e-mail:
[email protected] Tel.: +30-210-7467519 Fax: +30-210-7759167
Introduction Endothelin-1 (ET-1), the most important endothelin (ET), is a potent vasoconstrictor peptide produced mainly by endothelial cells [1]. The kidney is an important organ of ET-1 production [2]. ET-1 synthesis is stimulated by hypoxia, hemodynamic shear stress, and various humoral factors, such as angiotensin II and transforming growth factor [3, 4, 5, 6]. Intracellular calcium has also been shown to increase ET-1 production and secretion [7, 8, 9]. Idiopathic hypercalciuria (IH), the most common cause of urolithiasis in children and adults, is a genetic disease characterized by normal blood calcium (Ca) levels and increased urinary Ca excretion [10, 11]. Absorptive IH (AIH) is due to Ca hyperabsorption from the intestinal mucosa, resulting in a positive Ca balance, with a subsequent increase in renal Ca excretion [12]. Since intracellular Ca promotes ET-1 production and secretion, the positive Ca balance in AIH patients may stimulate ET-1 production and urinary excretion. There is only one study investigating urinary ET-1 excretion in nine IH patients, with no differentiation regarding their type (absorptive or renal), in which increased levels of urinary ET-1 were found [13]. The purpose of the current study was to investigate urinary and plasma ET-1 levels in children with AIH.
Patients and methods Twenty-one patients with AIH (12 boys, 9 girls, mean age 9.19€2.73 years) and 22 healthy children of similar age and sex (10 boys, 12 girls, mean age 9.34€2.36 years) were studied. IH was defined as a 24-h urinary Ca excretion greater than 4 mg/kg of body weight, in the face of normal blood Ca levels. Other known causes of secondary hypercalciuria, such as tubular acidosis and hypervitaminosis D, were excluded. Informed consent was obtained from the parents of both the patient and the control groups. The absorptive type of IH was determined by a Ca loading test, according to the protocol recommended by Santos et al. [14], which was based on a preload Ca/Cr (mg/mg) ratio 0.2 (0.08€0.05) and a postload ratio >0.2 (0.37€0.15). The clinical presentation of AIH was nephrolithiasis in 3 patients, gross hematuria in 9, microscopic
1158 hematuria in 5, and recurrent abdominal pain in 4 patients. Patients and controls had normal blood pressure. In all patients and controls plasma and urinary ET-1 concentrations (in a 24-h urine collection and in a spot urine sample) were evaluated. Serum intact parathyroid hormone (iPTH), serum creatinine (Cr), urinary Cr (in a 24-h urine collection and in a spot urine sample), and urinary Ca and sodium (Na) (in a 24-h urine collection) were also determined. The ET-1 urinary excretion (pg/ 24 h and pg/m2 per 24 h) and the ET-1/Cr ratio were calculated. The investigation in both patients and controls took place after at least 15 days on a diet containing 2,400–2,600 mg Na and 800– 900 mg Ca/day. The individual urine samples were stored at 4 C immediately after voiding and complete collections were kept at 70 C prior to analysis. Blood sampling was performed early in the morning under fasting conditions at the end of the 24-h urine collection period. A spot urine sample was obtained from the second urination, the same day as the blood sample was taken. Blood samples were collected in frozen plastic tubes containing EDTA (0.1 ml EDTA/ml of blood). The blood was promptly centrifuged at 2,500 rpm at 4 C for 20 min and the plasma was then stored in tubes containing aprotinin at 70C until assayed. Urinary and plasma ET-1 levels were determined by radioimmunoassay (RIA) (Nichols Institute Diagnostics ET I125, San Juan Capistrano, Calif., USA) after solid extraction in C18 silica columns (Nichols Institute Diagnostics). The lower limit of detection was 0.2 pg/ml. The intra- and inter-assay variability was 4.5% and 7%, respectively. The antibody had the following cross-reactions: ET-1 100%, ET-2 67%, ET-3 84%, and proendothelin 2.6%. Urinary Ca was determined by atomic absorption spectrophotometry. Serum iPTH levels were determined by RIA (Nichols Institute Diagnostics I125). Cr clearance (C Cr) and urinary flow rate (ml/m2 per min) were also calculated. Statistical analysis In order to compare groups for differences in mean levels of urinary ET-1, urinary ET/Cr ratio, plasma ET-1, serum PTH, urinary Ca and Na, C Cr, and urinary flow rate, independent pairwise t-tests were performed. The above variables were also tested for significant correlations. Normality of the variables was assessed using the Kolmogorov-Smirnov test (with Lilliefors’ correction) and the assumption of equal variance was also assessed using Levene’s test.
Results Blood and urinary biochemical data are given in Table 1. Urinary concentration of ET-1 was higher (P=0.010) in AIH patients (9.16€4.10 pg/ml) than in controls (5.89€3.84 pg/ml). The absolute amount (24-h excretion) of ET-1 in the patients (9,274€6,444 pg/24 h) was also
Table 1 Blood and urinary data in patients with absorptive idiopathic hypercalciuria and controls (mean€SD) (ET-1 endothelin-1, iPTH intact parathyroid hormone, Ca calcium, Na sodium, Cr creatinine)
Fig. 1 Relationship between 24-h urinary endothelin-1 (ET-1) excretion (pg/24 h) and urinary ET-1/creatinine ratio (pg/mg) in patients (a) and controls (b)
higher (P=0.005) than in controls (4,699€2,120 pg/24 h). Correction for body surface did not alter this result (P=0.001). A significant increase in ET/Cr ratio (P=0.007) was observed in AIH patients (14.04€9.52 pg/mg) compared with controls (7.36€4.71 pg/mg). Plasma ET-1
Urinary ET-1 concentration (pg/ml) Urinary ET-1 excretion (pg/24 h) Urinary ET-1 excretion (pg/m2 per 24 h) Urinary ET-1/Cr (pg/mg) Plasma ET-1 (pg/ml) Serum iPTH (pg/ml) Urinary Ca (mg/kg per 24 h) Urinary Na (mmol/kg per 24 h) Urinary Cr (mg/dl) Cr clearance (ml/min per 1.73 m2) Urinary Cr excretion (mg/kg per 24 h) Urinary flow rate (ml/m2 per min)
Patients (n=21)
Controls (n=22)
P
9.16€4.10 9,274€6,444 8,604€5,610 14.04€9.52 0.84€0.64 17.81€5.085 3.61€1.84 3.50€0.98 79.05€33.01 125.82€57.60 22.38€8.88 0.62€0.28
5.89€3.84 4,699€2,120 3,793€1,605 7.36€4.71 1.54€0.54 26.98€11.91 1.00€0.51 2.43€0.60 90.36€23.94 154.60€65.87 18.33€7.20 0.46€0.21
0.010 0.005 0.001 0.007 0.0001 0.02 0.0001 0.0001
0.04
1159
P=0.006) (Fig. 1a and b, respectively). A positive correlation was also found between urinary flow rate and ET-1 excretion in patients (r=0.59, P=0.004) and controls (r=0.48, P=0.02) (Fig. 2a and b, respectively). No relationship was found between urinary Na and urinary ET-1 excretion in either patients or controls.
Discussion
Fig. 2 Relationship between 24-h urinary ET-1 excretion (pg/m2 per 24 h) and urinary flow rate (ml/ /m2 per min) in patients (a) and controls (b)
levels were lower (P=0.0001) in patients (0.84€0.64 pg/ ml) than in controls (1.54€0.54 pg/ml). Urinary Ca and Na excretions were higher (P=0.0001) in patients (3.61€1.84 mg/kg per 24 h and 3.50€ 0.98 mmol/kg per 24 h, respectively) than in controls (1.00€0.51 mg/kg per 24 h and 2.43€0.60 mmol/kg per 24 h, respectively). Serum iPTH levels were lower (P=0.02) in AIH patients (17.81€5.08 pg/ml) than in controls (26.98€11.91 pg/ml). There was also a significant difference in urinary flow rate (P=0.04) between patients (0.62€0.28 ml/m2 per min) and controls (0.46€ 0.21 ml/m2 per min). No significant difference was found in urinary Cr excretion between patients and controls. We found a positive correlation between urinary Ca and urinary Na only in the control group (r=0.41, P=0.056). A significant relationship was found between 24-h urinary ET-1 excretion and urinary ET-1/Cr ratio in patients (r=0.76, P=0.0001) and in controls (r=0.56,
Our study shows that in children with AIH the urinary excretion of ET-1 calculated over 24 h was significantly higher than in controls. Correction for body surface did not alter this result. Moreover, the urinary ET-1/Cr ratio was higher in patients. Increased urinary ET-1 excretion may reflect high glomerular filtration, high tubular secretion, and/or disturbed tubular reabsorption. Our study, as well as other similar studies, indicates that the urinary excretion of ET-1 does not correlate with ET-1 plasma levels or glomerular filtration rate, suggesting that urinary ET-1 excretion reflects mainly intrarenal production [15, 16]. ET-1 is produced by renal endothelial, mesangial, and medullary collecting duct cells, and is excreted into the urine in much higher concentrations compared with plasma levels. Various humoral factors, such as thrombin, vasopressin, angiotensin II, and transforming growth factor, stimulate ET-1 secretion [3, 4, 5, 6]. In vitro studies have already shown the stimulatory effect of intracellular Ca on ET-1 secretion in cultured endothelial cells [7, 8]. Furthermore, Ca channel blockers inhibit ET-1 mRNA expression in cardiovascular tissues via endothelium-dependent mechanisms [9]. AIH patients are considered to be in a positive Ca balance. In these patients increased serum levels of 1,25-dihydroxyvitaminD [17, 18], low or normal serum concentrations of PTH, a finding confirmed by this study as well as by others [19], and excessive intestinal Ca absorption [20] are compatible with a state of Ca excess. Thus it could be expected that in AIH patients the renal ET-1 synthesis might be increased. However, ET-1 is a small peptide that could be reabsorbed in the proximal tubule. It seems that the decreased plasma levels of ET-1 and the increased ET-1 urinary excretion, findings of this study, may be attributed, at least in part, to decreased tubular reabsorption. The lack of correlation between plasma ET-1 and urinary ET-1 in our study suggests that the low reabsorption does not constitute the sole factor for increased renal excretion. Children with AIH in our study had a higher urine flow rate than the controls, and in both groups there was a positive correlation between the urinary excretion of ET-1 and urine flow rate. These findings indicate that urinary excretion of ET-1 depends to some extent on the urinary flow rate. However, under normal clinical conditions the high flow rate is not the cause but is rather the result of enhanced renal ET-1 production. There is strong evidence from experimental studies that local ET inhibits Na reabsorption in the inner medulla, the main site of ET-1 synthesis [21] and thereby induces increased diuresis as
1160
well as increased Na excretion [22, 23]. Thus, increased secretion of ET-1 could also explain the increased Na excretion in our patients, but the lack of correlation between these two parameters does not support this hypothesis. Alternatively, enhanced urinary Na excretion in AIH patients, a finding also reported by others [24], could be attributed to reduced blood renin-aldosterone levels, which is in accordance with the findings of a previous study [17]. The correlation between 24-h urinary ET-1 excretion and urinary ET-1/Cr ratio in patients and controls, in accordance with other studies [25], suggests that the ET1/Cr ratio could be used as a reliable index of urinary excretion of ET-1. There were no significant differences in sex or age between patients and controls. Similar results have been reported in healthy controls in other studies [13, 26]. In conclusion, urinary ET-1 excretion was higher and plasma ET-1 was lower in children with AIH than in healthy controls. These results could be attributed to decreased reabsorption of ET-1 in the renal tubule but also to increased renal production.
References 1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415 2. Morita S, Kitamura K, Yamamoto Y, Eto T, Osada Y, Sumiyoshi A, Koono M, Tanaka K (1991) Immunoreactive endothelin in human kidney. Ann Clin Biochem 28:267–271 3. Rakugi H, Tabuchi Y, Nakamaru M, Nagano M, Higashimori K, Mikami H, Ogihara T, Suzuki N (1990) Evidence for endothelin-1 release from resistance vessels of rats in response to hypoxia. Biochem Biophys Res Commun 169:973–977 4. Yoshizumi M, Kurihara H, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, Yazaki Y. (1989) Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells. Biochem Biophys Res Commun 161:859–864 5. Kohno M, Horio T, Ikeda M, Yokokawa K, Fukui T, Yasunari K, Kurihara N, Takeda T (1992) Angiotensin II stimulates endothelin-1 secretion in cultured rat mesangial cells. Kidney Int 42:860–866 6. Kurihara H, Yoshizumi M, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, Hamaoki M, Kato H, Yazaki Y (1989) Transforming growth factor- stimulates the expression of endothelin mRNA vascular endothelial cells. Biochem Biophys Res Commun 159:1435–1440 7. Brunner F (1995) Dependence of endothelin-1 secretion on Ca2+. Biochem Pharmacol 49:1785–1791 8. Stangl K, Dschietzig T, Alexiou K, Brunner F (1999) Antithrombin increases pulmonary endothelins: inhibition by heparin and Ca2+ channel antagonism. Eur J Pharmacol 370:57– 61 9. Naruze M, Tanabe A, Seki T, Adachi C, Yoshimoto T, Mishina N, Imaki T, Naruse K, Demura R, Demura H (1999) Effects of two calcium channel blockers on messenger RNA expression of
10. 11.
12.
13.
14. 15. 16. 17.
18.
19.
20. 21. 22. 23.
24.
25. 26.
endothelin-1 and nitric oxide synthase in cardiovascular tissue of hypertensive rats. J Hypertens 17:53–60 Pak CY, McGuire J, Peterson R, Britton F, Harrod MJ (1981) Familial absorptive hypercalciuria in a large kindred. J Urol 126:717–719 Nicolaidou P, Themeli S, Karpathios T, Georgouli H, Athanassaki K, Xaidara A, Messaritakis J (1996) Family pattern of idiopathic hypercalciuria and its subtypes. J Urol 155:1042– 1044 Breslau NA, Preminger GM, Adams BV, Otey J, Pak CY (1992) Use of ketoconazole to probe the pathogenetic importance of 1,25-dihydroxyvitamin D in absorptive hypercalciuria. J Clin Endocrinol Metab 75:1446–1452 Worgall S, Manz F, Kleschin K, Feth F, Rascher W (1994) Elevated urinary excretion of endothelin-like immunoreactivity in children with renal disease is related to urine flow rate. Clin Nephrol 41:331–337 Santos F, Suarez D, Malaga S, Crespo M (1987) Idiopathic hypercalciuria in children: pathophysiologic considerations of renal and absorptive subtypes. J Pediatr 110:238–243 Ando K, Hirata Y, Takei Y, Kawakami M, Marumo F (1991) Endothelin-1-like immunoreactivity in human urine. Nephron 57:36–39 Ohta K, Hirata Y, Shichiri M, Kanno K, Emori T, Tomita K, Marumo F (1991) Urinary excretion of endothelin-1 in normal subjects and patients with renal disease. Kidney Int 39:307–311 Nicolaidou P, Nyktari G, Georgouli H, Athanassaki K, Garoufi A, Papadimitriou A, Kavazarakis E, Karpathios T (2000) Atrial natriuretic peptide in children with idiopathic hypercalciuria. Pediatr Nephrol 14:853–855 Martinez ME, Villa E, Vazquez Martul M, Sanchez-Cabezudo MJ, Sanchez JA, Villa JR (1993) Influence of calcium intake on calcitriol levels in idiopathic hypercalciuria in children. Nephron 65:36–39 Coe FL, Favus MJ, Crockett T, Strauss AL, Parks JH, Porat A, Gantt CL, Sherwood LM (1982) Effects of low calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects. Am J Med 72:25–32 Duncombe VM, Watts RW, Peters TJ (1984) Studies on intestinal calcium absorption in patients with idiopathic hypercalciuria. Q J Med 53:69–79 Morita S, Kitamura K, Yamamoto Y, Eto T, Osada Y, Sumiyoshi A, Koono M, Tanaka K (1991) Immunoreactive endothelin in human kidney. Ann Clin Biochem 28:267–271 Sandgaard NC, Bie P (1996) Natriuretic effect of non-pressor doses of endothelin-1 in conscious dogs. J Physiol 494:809–818 Perico N, Cornejo RP, Benigni A, Malanchini B, Ladny JR, Remuzzi G (1991) Endothelin induces diuresis and natriuresis in the rat by acting on proximal tubular cells through a mechanism mediated by lipooxygenase products J Am Soc Nephrol 2:57–69 Polito C, La Manna A, Maiello R, Nappi B, Siciliano MC, Di Domenico MR, Di Toro R (2002) Urinary sodium and potassium excretion in idiopathic hypercalciuria of children. Nephron 91:7–12 Tsau YK, Tsai WS, Chen CH (1998) Urinary endothelin-1 in children with acute renal failure of tubular origin. J Formos Med Assoc 97:387–391 Peppa-Patrikiou M, Dracopoulou M, Dacou-Voutetakis C (1998) Urinary endothelin in adolescents and young adults with insulin-dependent diabetes mellitus. Relation to urinary albumin, blood pressure and other factors. Metabolism 47:1408–1412
