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Circulating adrenomedullin is increased in relation with increased creatinine and atrial natriuretic peptide in liver-transplant recipients
Regulatory Peptides 114 (2003) 61 – 66 www.elsevier.com/locate/regpep
Circulating adrenomedullin is increased in relation with increased creatinine and atrial natriuretic peptide in liver-transplant recipients Bernard Geny a,*, Bernard Ellero b, Anne Charloux a, Gabrielle Brandenberger a, Ste´phane Doutreleau a, Francßois Piquard a a
Service de Physiologie Clinique et Equipe d’Accueil 3072, Institut de Physiologie, Faculte´ de Me´decine, 67085 Strasbourg Cedex, France b Service de Chirurgie Ge´ne´rale et Transplantations Multiorganes, Hoˆpitaux Universitaires de Strasbourg, Hoˆpital de Hautepierre, 67096 Strasbourg Cedex, France Received 10 September 2002; received in revised form 17 February 2003; accepted 1 March 2003
Abstract Objectives: Circulating adrenomedullin (ADM), a potent vasorelaxing and natriuretic peptide involved in cardiovascular homeostasis, is increased after cardiac and renal transplantation. ADM is also implicated in hemodynamic abnormalities during liver cirrhosis, but whether ADM is increased late after liver transplantation is unknown. Patients: A total of 18 subjects—10 liver-transplant patients (Ltx) and 8 healthy subjects—were enrolled in the study. Design and measurements: After a 15-min rest period in supine position, heart rate and systemic blood pressure were determined in all subjects. Then, venous blood samples were obtained in order to simultaneously determine the cyclosporine through levels, the biological (cyclosporine, renal and hepatic functions) and hormonal (ADM and atrial natriuretic peptide (ANP)) characteristics of the Ltx. Results: ADM (27.2F4.1 vs. 53.8F6.9 pmol/l, P=0.02), and ANP (5.9F0.9 vs. 12.8F1.4 pmol/l, P=0.001) were significantly increased in late, stable Ltx (35.4F9.6 months after transplantation). Furthermore, increased ADM correlated positively with elevated creatinine (r=0.76, P=0.01) and ANP (r=0.64, P=0.04) after liver transplantation. Conclusions: Liver-transplant patients exhibit a sustained increase in circulating ADM. Such an increase likely results from renal impairment associated with volume regulation abnormalities, suggesting a potential role for ADM in volume regulation after liver transplantation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Adrenomedullin; Atrial natriuretic peptide; Liver; Transplantation
1. Introduction Initially isolated from phaeochromocytoma, adrenomedullin (ADM) is a potent vasorelaxing and natriuretic peptide involved in cardiovascular homeostasis [1]. Primarily produced by vascular smooth muscle cells and endothelial cells, ADM is present in the plasma of normal humans and has been shown to change during situations modifying fluid and electrolyte balance, such as the menstrual cycle [2]. Increased during the onset of several endocrine diseases such as thyrotoxicosis and Addison’s disease [3,4], ADM is also elevated in case of heart and renal failure, hypertension and liver diseases [5 – 8]. In particular, ADM increases in proportion to the severity of liver disease and may be implicated in the hemodynamic * Corresponding author. Tel.: +33-3-90-24-34-35; fax: +33-3-90-2434-44. E-mail address: [email protected] (B. Geny).
alterations observed during liver cirrhosis [9 – 11]. In agreement with the data reported after cardiac and renal transplantation [12 13], one study demonstrated that circulating ADM is increased early—during the peri-operative period in liver transplantation [14]. However, unlike the hypothalamic – pituitary– testicular function, which has been shown to improve or to normalize after liver transplantation, there are no available data on the long-term response of ADM in liver-transplant patients (Ltx) [15,16]. Several factors involved in liver transplantation, such as systemic hypertension, increased volemia and or impaired renal function-related or not with the immunosuppressive therapies, might modulate the ADM level in Ltx. Thus, the calcineurin inhibitor cyclosporine (CsA) is well known to favor systemic hypertension through its direct or indirect vasoconstrictor and nephrotoxic effects. Furthermore, steroid therapy can induce fluid-volume expansion and/or directly stimulate ADM secretion [12,17 – 21].
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(03)00106-X
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B. Geny et al. / Regulatory Peptides 114 (2003) 61–66
The atrial natriuretic peptide family, composed mainly of four circulating molecular forms generated from pro-ANP (1 –126), can also be stimulated by the same factors [22 – 24]. Pro-ANP (l –98) has recently been shown to represent a useful marker of hemodynamic derangement during and after liver transplantation [23]. Other fragments such as proANP (1– 30 and 31– 67) are also affected by immunosuppression, hypertension, and renal failure in late renal transplant patients [25], and it will be interesting to investigate them further in the setting of liver transplantation. Nevertheless, in this study, we determined the C terminal peptide alpha-ANP (pro-ANP (99 –126)) in the liver transplant recipients. Indeed, importantly, such fragment—but not three others—has been shown to interact with ADM, enhancing its release in healthy humans [24]. Furthermore, alpha-ANP circulates and is increased when hypervolemia occurs after liver transplantation [26 – 28]. It is also largely implicated in blood pressure and body-fluid regulation, through its potent natriuretic, diuretic and vasodilatory properties, which are associated with its ability to enhance fluid extravasation from the intravascular to the interstitial space [22,26 – 28]. The aim of this study was to determine, for the first time, whether ADM is increased late after liver transplantation and to test the hypothesis of an eventual ADM’s clinical significance through potential relationships between ADM and renal and liver functions, hemodynamic and alpha atrial natriuretic peptide (ANP), implicated in blood pressure and body-fluid regulation in liver-transplant patients.
antecubital vein to simultaneously assess the cyclosporine through levels, the biological (renal and liver functions) and the hormonal (ADM and ANP) characteristics of the Ltx. Thus, plasma creatinine was used as an index of renal function. Serum glutamic oxalo-acetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), gammaglutamyl transpeptidase (gamma GT), lactate deshydrogenase (LDH), alcalin phosphatase, bilirubin, protidemia, albuminemia and prothrombin were used as index of the liver function. 2.3. Radioimmunoassays Blood for hormone assays was immediately placed on ice in EDTA-Aprotinin tubes to prevent proteolytic breakdown of peptides that might be present, centrifuged at 4 jC, and plasma was separated and stored at 80 jC for subsequent analysis, as previously reported [12]. Plasma ADM 1– 52 was assayed by radioimmunoassay (RIA) using kits from Peninsula Laboratories (Belmont, USA), after extraction by autonitrile on Sep. Pak C18 cartridges (Waters, Milford, MA). The recovery was 50%. Sensitivity was 1.6 fmol; the intraassay coefficient of variation (CV) was 15%. Plasma ANP was determined by RIA using kits from Amersham (Buckinghamshire, UK) after extraction by an ethanol– acetic acid solution on Sep. Pak C18 cartridges (Waters). The sensitivity of the method was 1.0 fmol. The intra-assay CV was 9% for the observed values. All samples were analysed in a single run. 2.4. Hemodynamic parameters
2. Material and methods 2.1. Population A total of 18 subjects, 10 stable liver-transplant patients and 8 healthy subjects, age- and weight-matched, gave informed consent and participated in this study, which was approved by the Institutional Review Board. None presented with cardiovascular symptoms. Ltx received the usual immunosuppressive therapy with prednisolone (7.1F1.4 mg/day, n=6), cyclosporine with whole blood trough level at 158F35 nmol/l (n=6; 5 with neoral and 1 with sandimmun), prograff (6.5F1.2 mg/day, n=4) and/or azathioprine (62.5F37.5 mg/day, n=2). Ltx were also under antihypertensive treatment either by nitrates (n=1), calcium antagonists (n=2), angiotensin conversion enzyme inhibitors (n=1) and/or furosemide (n=3). No subject presented any rejection episode. 2.2. Study design After an overnight fast and a 15-min rest period in supine position, heart rate and systemic blood pressure were determined in all subjects. Next, venous blood samples were obtained through an 18-gauge catheter inserted in an
Systemic blood pressure was determined with the oscillometric method with a tensiometer (Critikon, Paris, France). Heart rate was obtained simultaneously.
3. Statistical analysis All the results are expressed as meansFS.E.M. Differences between groups were assessed by one-way ANOVA. Table 1 Clinical, hemodynamic and biological characteristics of the subjects Controls (n=8) Age (years) Body mass index (kg/m2) Delay (months) Heart rate (beat/min) Systolic SBP (mm Hg) Diastolic SBP (mm Hg) Mean SBP (mm Hg) Creatinine (Amol/l)
45.6F2.9 26.3F1.5 69.8F3.5 127.4F6.6 73.9F5.5 91.3F5.6 93.4F5.2
Liver transplants (n=10) 53.6F3.0 27.4F1.7 35.4F9.6 75.3F2.3 142.0F6.1 74.0F2.2 96.7F2.6 138.0F17.0*
MeansFS.E.M. Delay, time since transplantation; SBP, systemic blood pressure. * Difference with controls: P<0.05.
B. Geny et al. / Regulatory Peptides 114 (2003) 61–66
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Table 2 Parameters characterizing the liver function of Ltx
SGOT (UI/l) SGPT (UI/l) Gamma GT (UI/l) LDH (UI/l) Alcaline phosphatase (UI/l) Total bilirubin (Amol/l) Total protein (g/l) Albumin (g/l) Prothrombin (%)
Normal range
Liver transplants (n=10)
11 – 35 5 – 43 8 – 75 210 – 443 30 – 90 5 – 18 70 – 80 30 – 50 100
29.7F4.4 32.7F7.6 87.4F35.3 488F25 109F12 15.7F2.2 74.6F0.9 39.7F0.6 97.0F3.20
MeansFS.E.M. SGOT, serum glutamic oxalo-acetic transaminase; SGPT, serum glutamic pyruvic transaminase; gamma GT, gammaglutamyl transpeptidase; LDH, lactate deshydrogenase.
The relationship between two variables was examined by regression analysis. The results were considered significant at a level of P<0.05.
4. Results The clinical characteristics and the creatinine plasma level of the two groups are presented in Table 1. Subjects were comparable concerning age, body mass index and heart rate. The delay since transplantation was 35.4F9.6 months. There was a trend towards an increase in systemic blood pressure after liver transplantation but it failed to reach statistical significance. Plasma creatinine was significantly higher in Ltx, as compared to controls ( P=0.02). Table 2 shows the Ltx liver function, as compared to the range of normal values. The mean values of the patients
Fig. 2. Relationships between plasma adrenomedullin and plasma ANP (bottom panel) and plasma creatinine (top panel) in liver transplant recipients, 35.4F9.6 months after transplantation.
were located either in, or just at the upper side of the range of normal values. Fig. 1 shows that circulating ADM was increased in Ltx, as compared to controls (27.2F4.1 vs. 53.8F6.9 pmol/l, P=0.024 in controls and Ltx, respectively). Similarly, ANP was increased in Ltx (5.9F0.9 vs. 12.8F1.4 pmol/l, P=0.001). Furthermore, positive and significant correlations were observed between plasma ADM and plasma creatinine (r=0.76, P=0.01) and plasma ADM and ANP (r=0.64, P=0.04) after liver transplantation (Fig. 2). ADM and systemic blood pressure only tended to be related in Ltx (r=0.572, P=0.07). In controls, no significant correlation was observed.
5. Discussion
Fig. 1. Plasma concentrations of adrenomedullin (ADM) and atrial natriuretic peptide (ANP) in controls (Ctrl) and liver transplant recipients (LTx), 35.4F9.6 months after transplantation. Differences with controls: *P<0.05; yP<0.01.
This study demonstrates that circulating ADM is increased late after liver transplantation and that such increase is statistically related to elevated plasma creatinine and atrial natriuretic peptide. The only previous report on ADM and liver transplantation was performed by Fabrega et al. [14] showing that ADM is increased early after liver transplantation. In further demonstration that ADM is elevated in case of liver cirrhosis, this team also demonstrated that after an initial increase at day 1 post-surgery, ADM declined to baseline levels at day 15 after transplantation. Like during major abdominal and cardiac surgeries, including transplantation, such transient increase might be mediated by the release of inflammatory cytokines occurring during the perioperative
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period [12,14]. To date, whether ADM remained increased thereafter in liver-transplant patients remains unknown. Our data demonstrate that ADM continues to be increased even as far as about 3 years after successful liver transplantation. This is consistent with previous reports in stable heart and renal transplant patients showing a similar increase in circulating ADM [12,13]. Such increase could be due to a decreased catabolism and/or an increased secretion rate. Concerning ADM clearance, there are relatively few data in the literature but ADM plasma half-life has been reported to be 22F1.6 min and ADM appears to be degraded in the human lungs [7,29,30]. To what extent the kidneys and the liver might participate in ADM degradation remains to be determined with precision in human being. Nevertheless, we observed a significant positive relationship between ADM and plasma creatinine, commonly used as an index of renal function. Thus, the circulating ADM level and the renal function are linked in Ltx. Although such a relationship was not always observed in liver cirrhosis with slightly impaired renal function [9], this is in agreement with previous data since ADM was positively correlated with serum creatinine and inversely correlated with glomerular filtration rate, in proportion to the severity of renal impairment, in patients with chronic renal or liver failure [6 – 8,10]. Alternatively, in view of ADM biological properties, it may be a physiological response to reduced kidney perfusion. According to previous data reported in cirrhosis patients and early post-liver transplantation, no relationship was observed between ADM and our Ltx’s liver function [10,14]. Although some parameters were located at the upper side of the normal range, it appears that taken together, Ltx’s liver function was similar to that of normal subjects and it is therefore unlikely that an impaired liver degradation of ADM participated significantly in the vasodilatory hormone increase after liver transplantation. Concerning ADM production, its source may be mainly systemic vasculature and several factors involved in liver transplantation such as systemic hypertension, increased ANP and hypervolemia—related or not with the immunosuppressive therapies—might play a modulatory role [17,18,27]. Systemic hypertension is known to enhance ADM release [6 –8]. Six patients were under antihypertensive therapy and although treated, one may consider them as hypertensive patients. However, their small blood pressure increase was not significant as compared to the controls. Furthermore, ADM and systemic blood pressure only tended to be related after liver transplantation (r=0.572, P=0.07). This suggests that if higher blood pressure would likely result in increased ADM after liver transplantation, in our group of Ltx, systemic blood pressure did not appear to play a key role in their ADM increase. Conversely, we observed a significant positive correlation between increased ANP and ADM after liver transplantation. ANP may act directly since its perfusion has
been shown to increase circulating ADM in healthy human [24]. On the other hand, increased ANP being considered as a good marker of increased body fluid volume both in healthy subjects and in cardiovascular, renal and livertransplant patients, ADM elevation might be related to such a volume increase [6,12,22,27,31]. Indeed, although plasma volume was not determined, the three Ltx needing a diuretic therapy presented with higher circulating ADM, as compared with other Ltx. Accordingly, increased ADM correlates with features associated with increased volemia, such as ascite in liver diseases and with hypervolemia after liver transplantation—further supporting the hypothesis that increased ADM may be related to increased volemia in our Ltx [5,9 – 11,27]. Considering the immunosuppressive therapies, both cyclosporine and corticoids could increase ADM production and/or decrease its elimination through their hypertensive, hypervolemia and deleterious renal effects. Nevertheless, we failed to find any relationship between ADM and cyclosporine or prednisolone after liver transplantation. Although caution should be used in view of the relatively small number of patients involved, this is consistent with previous data reported in heart-transplant patients and suggests that maintenance dose of immunosuppressive therapy might be too low to modulate importantly circulating ADM in Ltx [12]. Accordingly, dexamethasone-induced up-regulation of the gene encoding for ADM is dose-dependent and maintenance cyclosporine dose fails to result in acute renal dysfunction after heart transplantation [21,32]. Moreover, despite reaching elevated peak levels, cyclosporine therapy failed to acutely increase plasma ADM in heart-transplant recipients [33]. Whether or not increased ADM plays a pathophysiological role after liver transplantation still remains to be investigated. It has been proposed that the greater ADM increase observed in early Ltx presenting with vascular thrombosis might suggest that ADM acts against the excessive release of vasoconstrictor peptides occurring during thrombus formation [14]. Similarly, since ADM is natriuretic and increases local blood flow in several organs including the kidneys and the heart, it may be involved in the defense mechanisms against further development of renal and cardiovascular failure in Ltx. Accordingly, besides inhibiting the long-lasting vasoconstrictor endothelin production by vascular smooth muscle cells, ADM has been shown to be related to endothelin, the renin –angiotensin – aldosterone and the sympathetic systems [9 – 11,34,35]. On the other hand, plasma renin activity and aldosterone decreases in response to acute volume expansion were not associated with a concommitant decrease in ADM, suggesting that increased ADM was not solely a consequence of the activation of theses vasoconstrictor factors [10]. ADM has also been proposed to contribute to vasodilation, thus favoring hyperdynamic circulation and leading ultimately to volume expansion in patients with liver dis-
B. Geny et al. / Regulatory Peptides 114 (2003) 61–66
ease. Alternatively, ADM release could be secondary to an increased shear stress in the vasculature caused by the hyperdynamic circulation and might then exacerbate vasodilatation directly or indirectly through the eventual generation of nitric oxide [9– 11]. These hypotheses remain to be discussed and need further investigations. In summary, circulating ADM is significantly increased in late stable liver-transplant patients. The positive correlations observed between ADM and elevated plasma creatinine on one hand and ANP on the other support that increased ADM results likely, at least partly, from decreased renal clearance together with an increased production secondary to increased ANP, possibly related with hypervolemia. Elevated systemic blood pressure might also potentially increase ADM in Ltx. Knowing ADM’s biological properties, it is tempting to speculate that increased ADM might protect Ltx against further renal impairment, or may participate in volume regulation. Further larger-scale studies are warranted to investigate such important issue after liver transplantation.
[12]
[13]
[14]
[15]
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Acknowledgements [20]
We are grateful to Michele Simeoni, Isabelle Bentz and Fabienne Goupilleau for expert technical assistance. [21]
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et al. Changes in circulating levels of atrial natriuretic factor (ANF) during orthotopic liver transplantation in humans. Transpl Int 1994;7: S134 – 8. [32] Piquard F, Richard R, Hardy H, Doutreleau S, Charloux A, Brandenberger G, et al. Hormonal, renal, hemodynamic responses to acute neutral endopeptidase inhibition in heart-transplant patients. J Appl Physiol 2002;93:569 – 75. [33] Geny B, Hardy H, Lampert E, Charloux A, Charpentier A, Lonsdorfer J, et al. Short term effect of cyclosporine on circulating adrenome-
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Circulating adrenomedullin is increased in relation with increased creatinine and atrial natriuretic peptide in liver-transplant recipients Bernard Geny a,*, Bernard Ellero b, Anne Charloux a, Gabrielle Brandenberger a, Ste´phane Doutreleau a, Francßois Piquard a a
Service de Physiologie Clinique et Equipe d’Accueil 3072, Institut de Physiologie, Faculte´ de Me´decine, 67085 Strasbourg Cedex, France b Service de Chirurgie Ge´ne´rale et Transplantations Multiorganes, Hoˆpitaux Universitaires de Strasbourg, Hoˆpital de Hautepierre, 67096 Strasbourg Cedex, France Received 10 September 2002; received in revised form 17 February 2003; accepted 1 March 2003
Abstract Objectives: Circulating adrenomedullin (ADM), a potent vasorelaxing and natriuretic peptide involved in cardiovascular homeostasis, is increased after cardiac and renal transplantation. ADM is also implicated in hemodynamic abnormalities during liver cirrhosis, but whether ADM is increased late after liver transplantation is unknown. Patients: A total of 18 subjects—10 liver-transplant patients (Ltx) and 8 healthy subjects—were enrolled in the study. Design and measurements: After a 15-min rest period in supine position, heart rate and systemic blood pressure were determined in all subjects. Then, venous blood samples were obtained in order to simultaneously determine the cyclosporine through levels, the biological (cyclosporine, renal and hepatic functions) and hormonal (ADM and atrial natriuretic peptide (ANP)) characteristics of the Ltx. Results: ADM (27.2F4.1 vs. 53.8F6.9 pmol/l, P=0.02), and ANP (5.9F0.9 vs. 12.8F1.4 pmol/l, P=0.001) were significantly increased in late, stable Ltx (35.4F9.6 months after transplantation). Furthermore, increased ADM correlated positively with elevated creatinine (r=0.76, P=0.01) and ANP (r=0.64, P=0.04) after liver transplantation. Conclusions: Liver-transplant patients exhibit a sustained increase in circulating ADM. Such an increase likely results from renal impairment associated with volume regulation abnormalities, suggesting a potential role for ADM in volume regulation after liver transplantation. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Adrenomedullin; Atrial natriuretic peptide; Liver; Transplantation
1. Introduction Initially isolated from phaeochromocytoma, adrenomedullin (ADM) is a potent vasorelaxing and natriuretic peptide involved in cardiovascular homeostasis [1]. Primarily produced by vascular smooth muscle cells and endothelial cells, ADM is present in the plasma of normal humans and has been shown to change during situations modifying fluid and electrolyte balance, such as the menstrual cycle [2]. Increased during the onset of several endocrine diseases such as thyrotoxicosis and Addison’s disease [3,4], ADM is also elevated in case of heart and renal failure, hypertension and liver diseases [5 – 8]. In particular, ADM increases in proportion to the severity of liver disease and may be implicated in the hemodynamic * Corresponding author. Tel.: +33-3-90-24-34-35; fax: +33-3-90-2434-44. E-mail address: [email protected] (B. Geny).
alterations observed during liver cirrhosis [9 – 11]. In agreement with the data reported after cardiac and renal transplantation [12 13], one study demonstrated that circulating ADM is increased early—during the peri-operative period in liver transplantation [14]. However, unlike the hypothalamic – pituitary– testicular function, which has been shown to improve or to normalize after liver transplantation, there are no available data on the long-term response of ADM in liver-transplant patients (Ltx) [15,16]. Several factors involved in liver transplantation, such as systemic hypertension, increased volemia and or impaired renal function-related or not with the immunosuppressive therapies, might modulate the ADM level in Ltx. Thus, the calcineurin inhibitor cyclosporine (CsA) is well known to favor systemic hypertension through its direct or indirect vasoconstrictor and nephrotoxic effects. Furthermore, steroid therapy can induce fluid-volume expansion and/or directly stimulate ADM secretion [12,17 – 21].
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(03)00106-X
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The atrial natriuretic peptide family, composed mainly of four circulating molecular forms generated from pro-ANP (1 –126), can also be stimulated by the same factors [22 – 24]. Pro-ANP (l –98) has recently been shown to represent a useful marker of hemodynamic derangement during and after liver transplantation [23]. Other fragments such as proANP (1– 30 and 31– 67) are also affected by immunosuppression, hypertension, and renal failure in late renal transplant patients [25], and it will be interesting to investigate them further in the setting of liver transplantation. Nevertheless, in this study, we determined the C terminal peptide alpha-ANP (pro-ANP (99 –126)) in the liver transplant recipients. Indeed, importantly, such fragment—but not three others—has been shown to interact with ADM, enhancing its release in healthy humans [24]. Furthermore, alpha-ANP circulates and is increased when hypervolemia occurs after liver transplantation [26 – 28]. It is also largely implicated in blood pressure and body-fluid regulation, through its potent natriuretic, diuretic and vasodilatory properties, which are associated with its ability to enhance fluid extravasation from the intravascular to the interstitial space [22,26 – 28]. The aim of this study was to determine, for the first time, whether ADM is increased late after liver transplantation and to test the hypothesis of an eventual ADM’s clinical significance through potential relationships between ADM and renal and liver functions, hemodynamic and alpha atrial natriuretic peptide (ANP), implicated in blood pressure and body-fluid regulation in liver-transplant patients.
antecubital vein to simultaneously assess the cyclosporine through levels, the biological (renal and liver functions) and the hormonal (ADM and ANP) characteristics of the Ltx. Thus, plasma creatinine was used as an index of renal function. Serum glutamic oxalo-acetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), gammaglutamyl transpeptidase (gamma GT), lactate deshydrogenase (LDH), alcalin phosphatase, bilirubin, protidemia, albuminemia and prothrombin were used as index of the liver function. 2.3. Radioimmunoassays Blood for hormone assays was immediately placed on ice in EDTA-Aprotinin tubes to prevent proteolytic breakdown of peptides that might be present, centrifuged at 4 jC, and plasma was separated and stored at 80 jC for subsequent analysis, as previously reported [12]. Plasma ADM 1– 52 was assayed by radioimmunoassay (RIA) using kits from Peninsula Laboratories (Belmont, USA), after extraction by autonitrile on Sep. Pak C18 cartridges (Waters, Milford, MA). The recovery was 50%. Sensitivity was 1.6 fmol; the intraassay coefficient of variation (CV) was 15%. Plasma ANP was determined by RIA using kits from Amersham (Buckinghamshire, UK) after extraction by an ethanol– acetic acid solution on Sep. Pak C18 cartridges (Waters). The sensitivity of the method was 1.0 fmol. The intra-assay CV was 9% for the observed values. All samples were analysed in a single run. 2.4. Hemodynamic parameters
2. Material and methods 2.1. Population A total of 18 subjects, 10 stable liver-transplant patients and 8 healthy subjects, age- and weight-matched, gave informed consent and participated in this study, which was approved by the Institutional Review Board. None presented with cardiovascular symptoms. Ltx received the usual immunosuppressive therapy with prednisolone (7.1F1.4 mg/day, n=6), cyclosporine with whole blood trough level at 158F35 nmol/l (n=6; 5 with neoral and 1 with sandimmun), prograff (6.5F1.2 mg/day, n=4) and/or azathioprine (62.5F37.5 mg/day, n=2). Ltx were also under antihypertensive treatment either by nitrates (n=1), calcium antagonists (n=2), angiotensin conversion enzyme inhibitors (n=1) and/or furosemide (n=3). No subject presented any rejection episode. 2.2. Study design After an overnight fast and a 15-min rest period in supine position, heart rate and systemic blood pressure were determined in all subjects. Next, venous blood samples were obtained through an 18-gauge catheter inserted in an
Systemic blood pressure was determined with the oscillometric method with a tensiometer (Critikon, Paris, France). Heart rate was obtained simultaneously.
3. Statistical analysis All the results are expressed as meansFS.E.M. Differences between groups were assessed by one-way ANOVA. Table 1 Clinical, hemodynamic and biological characteristics of the subjects Controls (n=8) Age (years) Body mass index (kg/m2) Delay (months) Heart rate (beat/min) Systolic SBP (mm Hg) Diastolic SBP (mm Hg) Mean SBP (mm Hg) Creatinine (Amol/l)
45.6F2.9 26.3F1.5 69.8F3.5 127.4F6.6 73.9F5.5 91.3F5.6 93.4F5.2
Liver transplants (n=10) 53.6F3.0 27.4F1.7 35.4F9.6 75.3F2.3 142.0F6.1 74.0F2.2 96.7F2.6 138.0F17.0*
MeansFS.E.M. Delay, time since transplantation; SBP, systemic blood pressure. * Difference with controls: P<0.05.
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Table 2 Parameters characterizing the liver function of Ltx
SGOT (UI/l) SGPT (UI/l) Gamma GT (UI/l) LDH (UI/l) Alcaline phosphatase (UI/l) Total bilirubin (Amol/l) Total protein (g/l) Albumin (g/l) Prothrombin (%)
Normal range
Liver transplants (n=10)
11 – 35 5 – 43 8 – 75 210 – 443 30 – 90 5 – 18 70 – 80 30 – 50 100
29.7F4.4 32.7F7.6 87.4F35.3 488F25 109F12 15.7F2.2 74.6F0.9 39.7F0.6 97.0F3.20
MeansFS.E.M. SGOT, serum glutamic oxalo-acetic transaminase; SGPT, serum glutamic pyruvic transaminase; gamma GT, gammaglutamyl transpeptidase; LDH, lactate deshydrogenase.
The relationship between two variables was examined by regression analysis. The results were considered significant at a level of P<0.05.
4. Results The clinical characteristics and the creatinine plasma level of the two groups are presented in Table 1. Subjects were comparable concerning age, body mass index and heart rate. The delay since transplantation was 35.4F9.6 months. There was a trend towards an increase in systemic blood pressure after liver transplantation but it failed to reach statistical significance. Plasma creatinine was significantly higher in Ltx, as compared to controls ( P=0.02). Table 2 shows the Ltx liver function, as compared to the range of normal values. The mean values of the patients
Fig. 2. Relationships between plasma adrenomedullin and plasma ANP (bottom panel) and plasma creatinine (top panel) in liver transplant recipients, 35.4F9.6 months after transplantation.
were located either in, or just at the upper side of the range of normal values. Fig. 1 shows that circulating ADM was increased in Ltx, as compared to controls (27.2F4.1 vs. 53.8F6.9 pmol/l, P=0.024 in controls and Ltx, respectively). Similarly, ANP was increased in Ltx (5.9F0.9 vs. 12.8F1.4 pmol/l, P=0.001). Furthermore, positive and significant correlations were observed between plasma ADM and plasma creatinine (r=0.76, P=0.01) and plasma ADM and ANP (r=0.64, P=0.04) after liver transplantation (Fig. 2). ADM and systemic blood pressure only tended to be related in Ltx (r=0.572, P=0.07). In controls, no significant correlation was observed.
5. Discussion
Fig. 1. Plasma concentrations of adrenomedullin (ADM) and atrial natriuretic peptide (ANP) in controls (Ctrl) and liver transplant recipients (LTx), 35.4F9.6 months after transplantation. Differences with controls: *P<0.05; yP<0.01.
This study demonstrates that circulating ADM is increased late after liver transplantation and that such increase is statistically related to elevated plasma creatinine and atrial natriuretic peptide. The only previous report on ADM and liver transplantation was performed by Fabrega et al. [14] showing that ADM is increased early after liver transplantation. In further demonstration that ADM is elevated in case of liver cirrhosis, this team also demonstrated that after an initial increase at day 1 post-surgery, ADM declined to baseline levels at day 15 after transplantation. Like during major abdominal and cardiac surgeries, including transplantation, such transient increase might be mediated by the release of inflammatory cytokines occurring during the perioperative
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period [12,14]. To date, whether ADM remained increased thereafter in liver-transplant patients remains unknown. Our data demonstrate that ADM continues to be increased even as far as about 3 years after successful liver transplantation. This is consistent with previous reports in stable heart and renal transplant patients showing a similar increase in circulating ADM [12,13]. Such increase could be due to a decreased catabolism and/or an increased secretion rate. Concerning ADM clearance, there are relatively few data in the literature but ADM plasma half-life has been reported to be 22F1.6 min and ADM appears to be degraded in the human lungs [7,29,30]. To what extent the kidneys and the liver might participate in ADM degradation remains to be determined with precision in human being. Nevertheless, we observed a significant positive relationship between ADM and plasma creatinine, commonly used as an index of renal function. Thus, the circulating ADM level and the renal function are linked in Ltx. Although such a relationship was not always observed in liver cirrhosis with slightly impaired renal function [9], this is in agreement with previous data since ADM was positively correlated with serum creatinine and inversely correlated with glomerular filtration rate, in proportion to the severity of renal impairment, in patients with chronic renal or liver failure [6 – 8,10]. Alternatively, in view of ADM biological properties, it may be a physiological response to reduced kidney perfusion. According to previous data reported in cirrhosis patients and early post-liver transplantation, no relationship was observed between ADM and our Ltx’s liver function [10,14]. Although some parameters were located at the upper side of the normal range, it appears that taken together, Ltx’s liver function was similar to that of normal subjects and it is therefore unlikely that an impaired liver degradation of ADM participated significantly in the vasodilatory hormone increase after liver transplantation. Concerning ADM production, its source may be mainly systemic vasculature and several factors involved in liver transplantation such as systemic hypertension, increased ANP and hypervolemia—related or not with the immunosuppressive therapies—might play a modulatory role [17,18,27]. Systemic hypertension is known to enhance ADM release [6 –8]. Six patients were under antihypertensive therapy and although treated, one may consider them as hypertensive patients. However, their small blood pressure increase was not significant as compared to the controls. Furthermore, ADM and systemic blood pressure only tended to be related after liver transplantation (r=0.572, P=0.07). This suggests that if higher blood pressure would likely result in increased ADM after liver transplantation, in our group of Ltx, systemic blood pressure did not appear to play a key role in their ADM increase. Conversely, we observed a significant positive correlation between increased ANP and ADM after liver transplantation. ANP may act directly since its perfusion has
been shown to increase circulating ADM in healthy human [24]. On the other hand, increased ANP being considered as a good marker of increased body fluid volume both in healthy subjects and in cardiovascular, renal and livertransplant patients, ADM elevation might be related to such a volume increase [6,12,22,27,31]. Indeed, although plasma volume was not determined, the three Ltx needing a diuretic therapy presented with higher circulating ADM, as compared with other Ltx. Accordingly, increased ADM correlates with features associated with increased volemia, such as ascite in liver diseases and with hypervolemia after liver transplantation—further supporting the hypothesis that increased ADM may be related to increased volemia in our Ltx [5,9 – 11,27]. Considering the immunosuppressive therapies, both cyclosporine and corticoids could increase ADM production and/or decrease its elimination through their hypertensive, hypervolemia and deleterious renal effects. Nevertheless, we failed to find any relationship between ADM and cyclosporine or prednisolone after liver transplantation. Although caution should be used in view of the relatively small number of patients involved, this is consistent with previous data reported in heart-transplant patients and suggests that maintenance dose of immunosuppressive therapy might be too low to modulate importantly circulating ADM in Ltx [12]. Accordingly, dexamethasone-induced up-regulation of the gene encoding for ADM is dose-dependent and maintenance cyclosporine dose fails to result in acute renal dysfunction after heart transplantation [21,32]. Moreover, despite reaching elevated peak levels, cyclosporine therapy failed to acutely increase plasma ADM in heart-transplant recipients [33]. Whether or not increased ADM plays a pathophysiological role after liver transplantation still remains to be investigated. It has been proposed that the greater ADM increase observed in early Ltx presenting with vascular thrombosis might suggest that ADM acts against the excessive release of vasoconstrictor peptides occurring during thrombus formation [14]. Similarly, since ADM is natriuretic and increases local blood flow in several organs including the kidneys and the heart, it may be involved in the defense mechanisms against further development of renal and cardiovascular failure in Ltx. Accordingly, besides inhibiting the long-lasting vasoconstrictor endothelin production by vascular smooth muscle cells, ADM has been shown to be related to endothelin, the renin –angiotensin – aldosterone and the sympathetic systems [9 – 11,34,35]. On the other hand, plasma renin activity and aldosterone decreases in response to acute volume expansion were not associated with a concommitant decrease in ADM, suggesting that increased ADM was not solely a consequence of the activation of theses vasoconstrictor factors [10]. ADM has also been proposed to contribute to vasodilation, thus favoring hyperdynamic circulation and leading ultimately to volume expansion in patients with liver dis-
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ease. Alternatively, ADM release could be secondary to an increased shear stress in the vasculature caused by the hyperdynamic circulation and might then exacerbate vasodilatation directly or indirectly through the eventual generation of nitric oxide [9– 11]. These hypotheses remain to be discussed and need further investigations. In summary, circulating ADM is significantly increased in late stable liver-transplant patients. The positive correlations observed between ADM and elevated plasma creatinine on one hand and ANP on the other support that increased ADM results likely, at least partly, from decreased renal clearance together with an increased production secondary to increased ANP, possibly related with hypervolemia. Elevated systemic blood pressure might also potentially increase ADM in Ltx. Knowing ADM’s biological properties, it is tempting to speculate that increased ADM might protect Ltx against further renal impairment, or may participate in volume regulation. Further larger-scale studies are warranted to investigate such important issue after liver transplantation.
[12]
[13]
[14]
[15]
[16]
[17] [18] [19]
Acknowledgements [20]
We are grateful to Michele Simeoni, Isabelle Bentz and Fabienne Goupilleau for expert technical assistance. [21]
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