*Department of Paediatrics, University of Melbourne, Australia
†Department of Gastroenterology, Alfred Hospital, Prahran, Australia
‡Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia
§University of Otago, Dunedin, New Zealand.
Received 3 November, 2010
Accepted 19 January, 2011
Address correspondence and reprint requests to Winita Hardikar, MBBS, FRACP, PhD, Head of Hepatology, Department of Gastroenterology, Royal Children's Hospital, Flemington Rd, Parkville VIC 3051, Australia (e-mail: email@example.com).
The authors report no conflicts of interest.
Urotensin II (U-II) is an 11-amino acid somatostatin-like vasoactive peptide, originally isolated from the caudal neurosecretory system of the goby, Gillichthys mirabilis (1). In humans, U-II and U-II receptor mRNA has been found in a number of organs including the kidney (2), liver (3,4), heart (5), and the endothelial and smooth muscle layers of various blood vessels (6).
U-II is the most potent vasoconstrictor discovered to date (6) and is 16 times more potent than endothelin-1. U-II acts via its receptor, GPR-14 (6–8), and binding results in an increase of intracellular calcium (9).
Its vascular actions are thought to be species and vascular bed specific (10). A study by Bohm and Pernow (11) found a vasoconstrictor response following a U-II infusion into the brachial artery of healthy volunteers. In contrast, U-II has been shown to be a strong vasodilator of human small muscular pulmonary arteries and human abdominal resistance arteries (12).
Elevated levels of U-II have been found in adults with cirrhosis (13–15). Our group has shown a relation in adults between the severity of liver disease as determined by the Child-Pugh score and U-II levels (14). The role that U-II plays in the pathophysiology of cirrhosis and/or portal hypertension remains to be elucidated. Recent studies indicate that U-II may influence splanchnic blood flow and therefore contribute to portal venous inflow (4). Furthermore, U-II may have a profibrotic effect upon the liver (16). Data on U-II levels in children are limited to those with cardiac (17) or renal dysfunction (18,19), with no studies having examined U-II levels in children with liver disease. In the present study, we aimed to measure U-II levels in healthy children ages 0 to 15 years to determine whether U-II levels are ontogenically regulated. We then examined the relation between U-II, liver disease, and portal hypertension in a well-characterised cohort of children. In addition, we examined the relation between U-II levels and clinical outcomes.
MATERIALS AND METHODS
The paediatric control group comprised sera from a previous seroprevalence study of GBV-C virus in healthy children (20). The sera were obtained from October 1997 to January 1998 from otherwise healthy children who were undergoing minor procedures at the Royal Children's Hospital Day Surgery unit. These children had not had previous invasive surgeries, blood transfusions, or major illnesses. Blood was taken at the time of induction of anesthetic for the minor procedure. The sera from healthy children who did not have GBV-C were stored at −80°C in 200-μL aliquots. Because a minimum of 500 μL of sera were needed for U-II testing, 3 aliquots from age- and sex-matched participants were pooled and treated as 1 sample. The adult control group consisted of 80 healthy subjects (men/women = 74/6 and age range 52.5 ± 10.1) without liver disease used in a previously published study (14).
Liver Disease Group
Participants for the liver disease group were recruited from the liver clinic at the Royal Children's Hospital, Melbourne. Patients with both cirrhosis and portal hypertension were enrolled. Portal hypertension was diagnosed on the basis of compatible radiological and/or endoscopic findings or the presence of ascites. Patients with concurrent systemic hypertension, diabetes, heart, and renal failure were excluded because these conditions have been shown to increase U-II levels. After obtaining informed consent, the following data were collected for each participant: age, sex, height, weight, aetiology of liver disease, features of portal hypertension, Child-Pugh score, and paediatric end-stage liver disease (PELD) score for children 12 years old or younger. Two millilitres of blood, collected in a serum gel tube, was centrifuged at 3000 rpm for 10 minutes. The serum was aliquoted into separate tubes and frozen at −80°C.
Long-term clinical outcome was recorded at 2 years. Patients were categorised into 3 groups: clinically stable, variceal bleeding, and death/transplantation.
A radioimmunoassay was used to measure U-II levels. This was performed at Endolab (Christchurch, New Zealand). Two millilitres of serum was extracted on C18 Sep-Pak cartridges (Waters, Milford, MA) and eluted with 80% isopropanol in 0.1% trifluoroacetic acid. The eluant was dried and reconstituted with a 0.5-mL assay buffer. The extract was preincubated with U-II antiserum (cat. no. T4736, Bachem, Bubendorf, Switzerland) for 21 to 24 hours, before addition of 125I-labelled U-II and incubation for another 22 to 24 hours. Bound and free U-II were separated using a solid-phase second antibody (Sac-Cel, IDS, Boldon, UK) and counted in a gamma counter. Recoveries of U-II were 113% at 30 pmol/L. The assay detection limit was 0.35 pmol/L. Within-assay coefficients of variation were 6.8% at 1 to 3 pmol/L and 4.7% at >3 pmol/L. Between-assay coefficients of variation were 8.5% at 3.5 pmol/L and 13.6% at 0.90 pmol/L. The normal reference range (100 healthy adult subjects) is 0.5 to 1.7 pmol/L (21).
Analysis was performed using the statistical package SPSS 13.0 (SPSS Inc, Chicago, IL). Data are expressed as median ± standard deviation unless otherwise stated. U-II data were not uniformly normally distributed; therefore, statistical results were validated using nonparametric testing where appropriate. For continuous data, comparisons between groups were assessed using Wilcoxon rank sum/Mann-Whitney U test or Kruskal-Wallis test as appropriate. Correlations between variables used Spearman correlation. A 2-sided P ≤ 0.05 was considered to be statistically significant. Ethics approval was obtained from the Royal Children's Hospital ethics in research committee.
Sera from 129 children were pooled as above to give a total of 43 samples. There were 23 samples between 0 to 4 years of age, 14 samples between 5 to 9 years of age, and 6 samples between 10 to 15 years of age.
The median peripheral U-II concentration was 1.35 ± 0.96 pmol/L (interquartile range 25–75; 0.99–1.77). There was no correlation between age and U-II levels in healthy children (r2 = 0.0, P = 0.8, Fig. 1). There was no difference in U-II levels between the paediatric and the adult control populations (1.35 ± 0.96 vs 1.25 ± 0.78, P = 0.8) (Fig. 2). There was also no significant difference between sex and U-II levels in healthy children (P = 0.5).
Liver Disease Group
There were 20 participants (8 boys and 12 girls) with mean age of 7.17 years (range 6 months–15 years). There were 9 participants in the 0 to 4 age group, 3 in the 5 to 9 age group, and 8 in the 10–15 age group. Biliary atresia was the aetiology of the liver disease in 11 patients, whereas other diagnoses included primary sclerosing cholangitis, congenital hepatic fibrosis, Wilson disease, bile acid–transport disorder, cystic fibrosis, and myofibroblastic tumour. Two patients had portal hypertension secondary to cirrhosis after liver transplantation. The median peripheral U-II concentration was 3.56 ± 2.21 pmol/L (interquartile range 25–75; 2.03–5.26). U-II was significantly higher in children with liver disease compared with controls (P < 0.001) (Fig. 3). There was a weak negative correlation between U-II concentration and age amongst the liver disease population (r2 = 0.33, P < 0.01).
U-II levels correlated strongly with severity of liver disease as assessed by Child-Pugh score (r = 0.7, P = 0.001) (Fig. 4) and PELD score (Fig. 5) for the 13 children who were 12 years or younger. There was no correlation between sex and U-II levels (P = 0.6) or between U-II and creatinine levels (P = 0.2).
During the follow-up period, 1 patient died and 5 patients underwent liver transplantation. Baseline U-II concentration was significantly higher in the death/transplantation group (5.50 ± 2.38) compared with the clinically stable group (2.36 ± 1.34, P < 0.01) and the variceal bleeding group (2.45 ± 1.54, P = 0.02) (Fig. 6). Five of 6 (83%) of the patients who reached the endpoint of death/transplantation had baseline U-II levels ≥4.0 pmol/L (Fig. 7). The sensitivity, specificity, and positive and negative predictive values of different U-II levels to predict adverse outcomes are shown in Table 1.
Amongst cirrhotic individuals, vasoactive mediators are known to be important in the development and propagation of portal hypertension. Previous studies by our group and others have implicated U-II as a relevant vasoactive mediator in the adult chronic liver disease population (22,23). To our knowledge, this is the first study to examine U-II in a paediatric liver disease population.
There are several novel findings arising from the present study. First, U-II levels do not appear to be influenced by age within a healthy paediatric population. Of interest, the U-II levels observed in the healthy paediatric population do not differ from the levels we observed in a healthy adult population (14) (mean age 52 [range 34–58], 1.52 vs 1.55, P = 0.8). Second, as in the adult population, children with chronic liver disease demonstrate a significant increase in circulating U-II levels, with levels correlated to the degree of liver dysfunction as determined by the Child-Pugh score and PELD score. Finally, U-II levels are correlated with disease outcomes.
Because of the limited data pertaining to U-II in a paediatric population, several aspects of our study are worthy of further discussion. The present study is consistent with Simpson's data (17) in demonstrating that age does not influence U-II levels in a healthy control population; however, in terms of establishing a normal level of U-II in healthy children, our mean ± SEM U-II level of 1.52 ± 0.15 pmol/L was significantly higher than the 0.85 pmol/L reported by Simpson et al. This discrepancy could result from a number of factors. Our study had almost double the number of samples. Of importance, marked heterogeneity in serum U-II concentration has been recorded between different studies (10,24). Different testing techniques and laboratories may therefore account for the discrepancy. In the present study, we used a previously validated U-II assay (21) with minimal cross-reactivity with other vasoactive mediators (0% cross-reactivity seen with angiotensin II and endothelin 1). Cross-reactivity with U-II–related protein was 1.43% at 50% displacement in the assay (data not shown). Finally, it is unlikely that the freezing of the sera that constituted our control group contributed to the discrepancy because electrolyte levels checked in 2 of the frozen samples before the commencement of the present study were normal and stability of urotensin in frozen specimens has been described by our group previously (14).
The pathophysiological implications of elevated circulating U-II in chronic liver disease requires further evaluation. Heller et al (13) initially demonstrated increased levels of U-II in adult subjects with chronic liver disease. Since then, U-II concentrations have been found to be positively correlated with not only the severity of the underlying liver disease but also the degree of portal hypertension (14). Recent data suggest that U-II can directly modulate portal pressure and induce hepatic fibrosis (16,4). Consistent with data from the adult populations, our data confirm that the observed correlation between liver disease severity and U-II exists in the paediatric population. Therefore, it is not surprising that we observed a relation between U-II and clinical outcomes. Demonstrating that U-II is increased in children with cirrhosis and portal hypertension may indicate that this peptide has a role in the pathogenesis of portal hypertension in this group of patients. Improved understanding of U-II and its role in liver disease may therefore have clinical and potentially therapeutic relevance.
The site of overproduction of U-II in chronic liver disease is unknown. Heller and colleagues' article (13) suggested hepatic overproduction based on a differential in the U-II concentration between portal venous and central venous blood in a subgroup of subjects undergoing transjugular intrahepatic portosystemic shunt. Conversely we found increased concentrations of U-II in peripheral blood compared with hepatic venous blood, implying a degree of systemic overproduction (14). This is consistent with the absence of increased expression in U-II–expressing cells in liver cirrhosis (25). The present study was not able to further define the site of U-II overproduction.
The implications of the increased circulating U-II levels observed in the paediatric population require further study. It is unknown whether U-II is overproduced as a homeostatic response to the haemodynamic changes that occur in cirrhosis or is itself implicated in the pathophysiology of these haemodynamic changes. There are no human studies using U-II antagonists, and these studies are required to help answer these important questions. The U-II antagonist Palosuran has shown promise in inhibiting the action of U-II (26). It was shown to decrease contraction of rat aortic strips, reduce renal ischaemia, and inhibit renal failure. It also has greater binding potency to the U-II receptor than to U-II itself. Use of this agent in a bile duct ligation murine model showed an effect in reducing portal pressure, thought to be mediated by an increase in splanchnic vascular resistance (4). The use of U-II antagonists with therapeutic intent is an area of further research (27).
In conclusion, our study is the first to examine U-II levels in children with liver disease. Peripheral U-II levels are significantly increased in children with chronic liver disease compared with healthy controls. Furthermore, the magnitude of the U-II increase is related to the severity of the liver disease and there appears to be a relation with baseline U-II levels and long-term liver disease outcomes. The later relation may be a useful clinical predictive tool and is worthy of further evaluation in larger studies. The expanding knowledge and research regarding U-II has real potential to translate into clinical benefit.
1. Bern HA, Pearson D, Larson BA, et al. Neurohormones from fish tails: the caudal neurosecretory system. I. “Urophysiology” and the caudal neurosecretory system of fishes. Recent Prog Horm Res 1985; 41:533–552.
2. Matsushita M, Shichiri M, Imai T, et al. Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 2001; 19:2185–2190.
3. Coulouarn Y, Lihrmann I, Jegou S, et al. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci U S A 1998; 95:15803–15808.
4. Trebicka J, Leifeld L, Hennenberg M, et al. Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats. Hepatology 2008; 47:1264–1276.
5. Totsune K, Takahashi K, Arihara Z, et al. Role of urotensin II in patients on dialysis. Lancet 2001; 358:810–811.
6. Ames RS, Sarau HM, Chambers JK, et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 401:282–286.
7. Liu Q, Pong SS, Zeng Z, et al. Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem Biophys Res Commun 1999; 266:174–178.
8. Nothacker HP, Wang Z, McNeill AM, et al. Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. Nat Cell Biol 1999; 1:383–385.
9. Maguire JJ, Davenport AP. Is urotensin-II the new endothelin? Br J Pharmacol 2002; 137:579–588.
10. Kemp W, Roberts S, Krum H. Urotensin II: a vascular mediator in health and disease. Curr Vasc Pharmacol 2005; 3:159–168.
11. Bohm F, Pernow J. Urotensin II evokes potent vasoconstriction in humans in vivo. Br J Pharmacol 2002; 135:25–27.
12. Stirrat A, Gallagher M, Douglas SA, et al. Potent vasodilator responses to human urotensin-II in human pulmonary and abdominal resistance arteries. Am J Physiol Heart Circ Physiol 2001; 280:H925–H928.
13. Heller J, Schepke M, Neef M, et al. Increased urotensin II plasma levels in patients with cirrhosis and portal hypertension. J Hepatol 2002; 37:767–772.
14. Kemp W, Krum H, Colman J, et al. Urotensin II: a novel vasoactive mediator linked to chronic liver disease and portal hypertension. Liver Int 2007; 27:1232–1239.
15. Kemp W, Roberts S, Krum H. Increased circulating urotensin II in cirrhosis: potential implications in liver disease. Peptides 2008; 29:868–872.
16. Kemp W, Kompa A, Phrommintikul A, et al. Urotensin II modulates hepatic fibrosis and portal hemodynamic alterations in rats. Am J Physiol Gastrointest Liver Physiol 2009; 297:G762–G767.
17. Simpson CM, Penny DJ, Stocker CF, et al. Urotensin II is raised in children with congenital heart disease. Heart 2006; 92:983–984.
18. Balat A, Karakok M, Yilmaz K, et al. Urotensin-II immunoreactivity in children with chronic glomerulonephritis. Ren Fail 2007; 29:573–578.
19. Balat A, Pakir IH, Gok F, et al. Urotensin-II levels in children with minimal change nephrotic syndrome. Pediatr Nephrol 2005; 20:42–45.
20. Siebert DJ, Bowden DS, Tracy SL, et al. Prevalence of hepatitis g/GBV-C in a healthy paediatric population. J Paediatr Child Health 2002; 38:423.
21. Richards AM, Nicholls MG, Lainchbury JG, et al. Plasma urotensin II in heart failure. Lancet 2002; 360:545–546.
22. Kemp W, Colman J, Thompson K, et al. Norfloxacin treatment for clinically significant portal hypertension: results of a randomised double-blind placebo-controlled crossover trial. Liver Int 2009; 29:427–433.
23. Kemp W, Roberts S, Komesaroff PA, et al. Urotensin II in chronic liver disease: in vivo effect on vascular tone. Scand J Gastroenterol 2008; 43:103–109.
24. Ong KL, Lam KS, Cheung BM. Urotensin II: its function in health and its role in disease. Cardiovasc Drugs Ther 2005; 19:65–75.
25. Leifeld L, Clemens C, Heller J, Trebicka J, Sauerbruch T, Spengler U. Expression of urotensin II and its receptor in human liver cirrhosis and fulminant hepatic failure. Dig Dis Sci 2010;55:1458–64.
26. Clozel M, Binkert C, Birker-Robaczewska M, et al. Pharmacology of the urotensin-II receptor antagonist palosuran (ACT-058362; 1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt): first demonstration of a pathophysiological role of the urotensin system. J Pharmacol Exp Ther 2004; 311:204–212.
27. Krum H, Kemp W. Therapeutic potential of blockade of the urotensin II system in systemic hypertension. Curr Hypertens Rep 2007; 9:53–58.