Clinical Endocrinology (1991)34, 85-89
Increased growth hormone response to growth hormone releasing hormone induced by erythropoietin in uraemic patients L. Cantaiamessa, L. Cremagnani, A. Orsatti, L. Vigna and G. Bucclanti Institute of Internal Medicine, Infectious Disease and Immunopathology, University of Milan, Milan, Italy (Received 5 March 1990; returned for revision 9 August 1990; finally revised 5 September 1990; accepted 25 September 1990)
Summary This study was designed to assess the response of growth hormone (GH) to growth hormone releasing hormone (GHRH) and the possible Interaction of acutely adminlstered recombinant human erythropoietin (rhEPO) on OH response to GHRH in a group of uraemic patients. Eight patients on malntenance haemodlalysis, not previously treated with rhEPO, and six healthy controls were tested with GHRH (100 pg i.v. in bolus), and with GHRH (100 pg i.v. in bolus) plus rhEPO (40 U/kg in constant infusion for 30 min) on different days. GHRH lnjectlon provoked a OH release in five out of eight uraemic patients; the overall mean response did not differ signiflcantiy from the OH response obtalned in controls (P = 0.30). Erythropoletln Infusion significantly increased GH release after GHRH (P 0.5). In thls study acutely administered rhEPO significantly potentiated the GH response to GHRH In uraemic patients whereas the same effect was not demonstrablein subjects with normal renal function.
Several disorders of growth hormone (GH) secretion have been described in patients with chronic renal failure (Wright et al., 1968; Feldman & Singer, 1974; Davidson et al., 1976; Lim et al., 1978). Elevated basal levels of plasma G H can be observed, associated with disordered responses to different Correspondence: Luigi Cantalamessa, Clinica Medica I (Pad. Granelli), Ospedale Maggiore Policlinico, via Francesco Sforza 35, 20122 Milano, Italy.
stimuli (Ramirez et af., 1978). Plasma GH rises paradoxically after administration of glucose, which normally suppresses GH levels (Samaan et al., 1966; Samaan & Freeman, 1970; De Fronzo, 1978) and after normally ineffective stimuli such as TRH (Gonzales-Barcena et al., 1973; Czernichow et al., 1976; Weissel et al., 1979). Moreover, exaggerated increase of plasma GH is caused by provocative stimuli such as insulin-induced hypoglycaemia and L-dopa, but no increases are observed in response to tolbutamide-induced hypoglycaemia (Lim et al., 1978; Ramirez et al., 1978; Krassas et al., 1981). Other aspects of GH secretion, e.g. diurnal rhythm of secretory pattern and responsiveness to physiologic stimuli such as exercise, sleep, and the specific hypothalamic releasing hormone (GHRH), remain to be clarified. Recombinant human erythropoietin (rhEPO) has recently been employed to treat anaemia in patients on chronic haemodialysis. The efficacy of the protein has been confirmed in several clinical studies (Winearls et al.. 1986; Eschbach et al., 1987; Lim et al., 1989). Moreover, some endocrine abnormalities have been reported to improve during the treatment with rhEPO (Kokot et al., 1989; Schaefer et al., 1989). The aim of the present study was to evaluate the response of GH to its physiological releasing hormone and the possible interference of acutely administered rhEPO on GH response to GHRH in a group of patients on maintenance haemodialysis. Patients and methods Subjects
Eight haemodialysed patients with chronic uraemia (age 3676 years, two men, six women) were studied. Informed consent was obtained prior to the study. No patient had a past history of diabetes mellitus or of endocrine disorders. All patients were on stable diet containing about 1.4 g/kg high biological value protein per day and 35 kcal/kg/day, without modifications during the study period. Body weight ranged between 44.6 and 72.3 kg, body mass index (BMI) being lower than 22. Interdialytic variations of body weight were 2-5f 1.2 kg. None had been treated before with rhEPO. The control group consisted of six healthy volunteers with normal renal function, matched for age, sex and weight. None was overweight, BMI being not higher than 22. 85
t. Cantalamessa eta/.
The study was carried out after an overnight fast on the day preceding a dialysis session. The patients were admitted to the Endocrinological Day Hospital and had an i.v. catheter introduced into an antecubital vein at 0830 h. Two basal blood samples were obtained a t a 30-min interval. Each subject received G H R H (Groliberin KabiVitrum) 100 pg i.v. in bolus, and blood samples were obtained at the intervals shown in Table I . In the experiment in which rhEPO 40 U/kg (erythropoietin, Boehringer Mannheim) was also administered, i t was infused at a constant rate for 30 min, starting 15 min before G H R H injection. A control test with rhEPO alone (40 U/kg in infusion for 30 min) was performed in four uraemic patients and in three normal controls, obtaining blood samples at the same intervals. In basal samples the following biochemical variables were measured: haemoglobin, haematocrit, plasma levels of creatinine, glucose, sodium, potassium, total calcium, growth hormone and prolactin. In the samples obtained after stimulation, growth hormone and prolactin plasma levels were determined. The tests with G H R H alone, rhEPO alone and G H R H plus rhEPO were performed on different days, with at least 5 days interval in randomized order.
Plasma G H and PRL were measured by double antibody RIA method (liso-phase Sclavo, Siena, Italy). All plasma samples obtained in different tests from individual patients and control subjects were analysed in duplicate in the same assay. The reference preparation for G H was HGH-MRC 66/217 (1st IRP); that for PRL was WHO IRP 75/504. The intra-assay coefficients of variation for G H were 5.7% for mean values of 6 mU/I, 3.3'%,for mean values of 14.4 mU/1 and 2.9% for mean values of 25.6 mU/I; the interassay coefficients of variation were 7.2% for mean values of 6.4 mU/I; 4.8%for mean values of 12.8 mU/I and 6.0%for mean values of24.8 mU/I. Normal values for G H ranged between 0 and 10.0 mU/I; for PRL between 57.5 and 345.0 mU/1 in males and 92.0 and 575.0 mU/l in females.
The pattern of G H response was evaluated using a mixed factorial analysis for repeated measurements of variance: (a) with GHRH test as a fixed factor within patients at two levels without and with rhEPO; (b) with time as a fixed factor within patients at seven levels (0. 15, 30, 45, 60, 90, 120); (c) with group as a fixed factor between patients at two levels (normal, uraemic subjects); (d) with subjects as a random
factor nested within the group factor. Analysis of covariance was also performed with the basal values as fixed covariates with respect to subsequent values. The significance level of the within factors and their interactions was adjusted according to the Geisser-Greenhouse procedure (Greenhouse & Geisser, 1959) in order to take into account the nonindependence of errors. Multiple comparisons have been performed according to the t-test for paired and unpaired data with Bonferroni's adjustment of the significance level (Winer, 1971).
Results Uraem ic pa tien ts
Basal plasma G H ranged between 3.00 and 27.16 mU/l with values clearly above normal range in two patients (nos 4 and 7, Table I). G H R H administration was followed by a clear elevation in plasma G H (Fig. la). As appears in Table 1, G H R H injection was followed by a clear G H increase in five out ofeight patients with chronic renal failure (patients l-5), while in the remaining three patients (nos 6-8), G H R H injection failed to induce significant variations in circulating GH. When G H R H was injected during EPO infusion, G H release was significantly higher than after administration of G H R H alone (P 0.5); the only difference between the two tests was a delayed pattern of response after G H R H plus rhEPO (Fig. 1 b). In the
Erythropoietin effect on GH in uraemia
Table 1 Plasma GH (mU/l) response t o GHRH and GHRH plus recombinant human erythropoietin (rhEPO) stimulation in uraemic patients and in controls Patient no sex
GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH+EPO GHRH GHRH + EPO
3.78 3-76 3.98 5.30 5.44 3.00 24.76 27.16 7.48 9.54 6.08 3.08 12.22 14.18 4.34 3.04
2.92 3.58 2.52 3.08 3.64 4.70 18.60 18.56 5.66 6.12 4.02 4.12 8.66 8.86 2.72 2.98
15.36 29.98 5.00 6.42 11.88 49.02 56.60 54.60 16.12 43.34 4.32 19.32 8.42 9.96 3.42 290
14.20 30.24 6.22 10.44 18.38 53.88 7240 75. 12 2 I .34 44.40 5.80 26. I8 5.94
10.00 2 1.68 9.44 25.58 19.72
3.74 6.12 17.82 27.62 6.70 29.24 33.60 52.12 8.78
3.24 3-38 18.84 19.74 5.54 12.78 23.80 28.60 6.42 6.98 2.98
14.02 30.60 4.48 32.20 4.80 7.04 2.82 3.10
5.72 12.56 12.86 39.04 I592 33.82 56.40 69.40 12.18 274 4.68 23.84 3.72 5.70 2.74 3.14
GHRH GHRH + EPO GHRH GHRH EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO GHRH GHRH + EPO
3.72 4.04 6.82 3.16 8.72 2.20 3.18 9.94 6.30 2.76
3-78 5.20 5.38 3.24 7.06 2.20 246 4.84 2.24 2.62 2.40 6.20
19.26 7.04 38.82 26.90 67.20 10.82
32.28 11.58 32.52 3 1.34 53.06 25.12 9.50 14.12 45-54 30. I6 I542 22.48
3796 22.76 22.94 20-98 27.56 45.62 6.88 10.10 37.40 45.68 2 1.86 33.70
27.58 27.20 10.44 19.64 17.46 75.04 10.70 5.52 36.48 3508 27.16 31.42
18.34 21.32 5.16 18.04 10.48 79.56 5.30 3.80 35.42 28.12 25.90 27.46
Uraemic patients 1
Normal controls 1 M 60 2 F 41 3 F 35 4 M 66 5
three normal controls rhEPO alone failed to induce significant variations in GH and PRL plasma levels.
Discussion We observed a potentiating effect of acutely administered rhEPO on GH response to GHRH in a group of patients with chronic renal failure on maintenance haemodialysis, but not in subjects with normal renal function. Among the GH secretion abnormalities reported in chronic renal failure, whereas elevated levels of GH in basal condition may be in part secondary to decreased catabolism and excretion by the kidney (Cameron et al., 1972; Feldman & Singer, 1974;
9.92 4 I .98 22.90 15.10 13.96
44.54 69.40 81.80
3.36 14.40 3.64 4.24 2.60 2.90
3.44 3.98 2.80 2.58
12.14 11.74 3.74 16.40 4.74 43.00 6.00 3.44 14.66 24.68 I594 14.02
Pimstone et ul., 1975), abnormal GH responsiveness to different stimuli is very likely due to disordered hypothalamic-pituitary regulation (Ramirez et al., 1978; Handelsman, 1985). The efficacy of acutely administered rhEPO in potentiating GH response to GHRH in uraemic patients, but not in controls, seems to indicate that an appropriate production of endogenous rhEPO may be involved in modulating somatotrophs’ responsiveness to GHRH. It must also be considered that rhEPO is one of the numerous growth factors identified in recent years (Russell & Van Wyck, 1989; Groopman et af., 1989); molecular characterization of these peptides and specific evaluation of their bioactivity, have revealed a broad spectrum of activities on
L. C a n f a l a m e s s a et a l .
I I i I l 0 15 30 45 60
l5 30 45 6o
Fig. 1. Plasma GH levels (mean +SE) after stimulation with 0 . GHRH and 0,GHRH plus recombinant human erythropoietin in a -group- of a, uraemic patients and b. in normal controls. * P < 0.01.
different cells and tissues for most of them (Dedhar et al., 1988; Bussolino et al., 1989; Berdel et al., 1989; Metcalf, 1989). In keeping with these observations, several effects of rhEPO administration in uraemic patients such as normalization of plasma prolactin, reduction of LH and FSH levels, elevation of testosterone, improvement of protein metabolism and C13 leucine retention (Kokot et al., 1989; Schaefer et al., 1989; Fisher et al., 1989), are hardly explained just by correction of anaemia. Impaired growth in uraemic children is commonly considered of multifactorial origin (Mehls et al., 1978). Disordered GH secretion apparently plays an important role. In fact, recombinant GH administration has been reported to increase growth both in experimental uraemia (Mehls et al., 1988a,b)and in children with chronic renal failure (Lippe et al., 1988; Tonshoff et a[., 1989). The available data on the use of synthetic erythropoietin in uraemic children are still limited (Burghard et al., 1988; Eschbach et a[., 1989). However, in a multicentre study, Scigalla et al. (1989) recently reported a distinct increase of body weight and in some children an acceleration in growth during rhEPO treatment. The potentiating effect of exogenous rhEPO on GHRH-induced GH release, observed in this study, might stimulate fuither investigations on the potential effects of rhEPO treatment on growth retardation of uraemic children.
De Fronzo, R.A. (1978) Pathogenesis of glucose intolerance in uremia. Metabolism, 27, 1866-1 880. Eschbach, J.W., Egrie, J.C., Downing, M.R., Browne, J.K.& Adamson, J.W. (1987) Correction of the anaemia of end-stage renal disease with recombinant human erythropoietin. Results of
Berdel, W.E., Danhausser-Riedl, S., Steinhauser, G. & Winton. E.F. (1989) Various human hematopoietic growth factors (interleukin3, GM-CSF, G-CSF) stimulate clonal growth of nonhematopoietic tumor cells. Blood, 73, 80-83. Burghard, R., Leititis, J., Pallacks, R., Scigalla, P. & Brandis. M. (1988) Treatment of a seven-year-old child with end-stage renal disease and hemosiderosis by recombinant human erythropoietin. Contributions to Nephrology, 66, 139- 148. Bussolino, F., Wang, J.M., Defilippi, P., Turrini, F., Sanavio. F., Edgell, C.-J. S., Aglietta. M., Arese, P. & Mantovani, A. (1989) Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature, 337,471473. Cameron, D.P., Burger, H.G.,Cavin, K.J., Gordon, E., Catt, K.J. & Watts, J. McK. (1972) Metabolic clearance of human growth hormone in patients with hepatic and renal failure, and in the isolated perfused pig liver. Metabolism, 21, 895-904. Czernichow, P., Dauzet, M.C., Broyer, M. & Rappaport. R. (1976) Abnormal TSH, PRL and GH response to TSH realising factor in chronic renal failure. Journal of Clinical Endocrinology and Metabolism, 43,630-637. Davidson, M.B., Fisher, M.B., DabiGVaziri, N. & Schaffer. M. (1976) Effect of protein intake and dialysis on the abnormal growth hormone, glucose. and insulin homeostasis in uremia. Metabolism, 25,455-464. Dedhar. S., Gaboury, L., Galloway, P. & Eaves, C. ( I 988) Human granulocytes-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhaemopoietic origin. Proceeding of the National Academy of Sciences, USA. 85,92539257.
The authors would like to thank Dr B. M. Cesana, Direzione Scientifica, Servizio di Biostatistica, Ospedale Maggiore di Milano, IRCCS, for performing statistical evaluation.
Erythropoietin effect on GH in uraemia
a combined phase I and 11 clinical trial. New England Journal of Medicine. 316,73-78. Eschbach, J.W. (1989) The anemia of chronic renal failure: pathophysiology and the effects of recombinant erythropoietin. Kidney International, 35, 134-148. Feldman, H.A. &Singer, 1. (1974) Endocrinology and metabolism in uremia and dialysis: a clinical review. Medicine, 54, 345-376. Fisher, Ch., Scigalla, P., Park. W., Becker, H., Schiller, R., Paust, H., Broesicke. H. & Kessel, M. (1989) Influence of rhEPO therapy on the protein metabolism of hemodialysis patients with terminal renal insufficiency. Contributions to Nephrology, 76,250-256. Gonzales-Barcena, D., Kastin, A.J., Schalch, D.S., Torres-Zamora, M., Perez-Pasten, E., Kato, A. & Schally, A.V. (1973) Responses to thyrotropin-releasing hormone in patients with renal failure and after infusion in normal men. Journal of Clinical Endocrinology and Metabolism, 36, 1 17- 120. Greenhouse, S.W. & Geisser, S.(1959) On methods in the analysis of profile data. Psychometrika, 24.95-1 12. Groopman, J.E., Molina, J.M. & Scadden, D.T. (1989) Hematopoietic growth factors. Biology and clinical applications. New England Journal of Medicine, 21, 1449- 1459. Handelsman, D.J. (1985) Hypothalamic-pituitary gonadal dysfunction in renal failure, dialysis and renal transplantation. Endocrine Review, 6, 151-182. Kokot, F., Wiecek, A., Grzesczack, W., Klepacka, J., Klin, M. & Lao, M. (1989) Influence of erythropoietin treatment on endocrine abnormalities in hemodialyzed patients. Contributions to Nephrology. 76,257-272. Krassas. G.E., Vergoulas, G., Evagelou, P.. Ziannas, I. & Faltsis, S. ( I 98 I ) The prolactin, TSH, GH and cortisol responses to TRH and insulin induced hypoglycaemia tests in patients with severe renal failure. Acta Endocrinologica (Suppl. 243), A 425. Lim, V.S., Kathpalia, S.C. & Henriquez C. (1978) Endocrine abnormalities associated with chronic renal failure. Medical Clinics of North America, 62, 1341-1361. Lim, V.S., De Gowin, R.L., Zavala, D., Kirchner, P.T., Abels, R.. Perry, P. & Fangman. J. (1989) Recombinant human erythropoietin treatment in pre-dialysis patients. A double-blind placebocontrolled trial. Annals of Internal Medicine, 110, 108-1 14. Lippe, B., Fine, R.N.. Koch, V.H. & Sherman, B.M. (1988) Accelerated growth following treatment of children with chronic renal failure with recombinant human growth hormone (Somatrem): a preliminary report. Aria Paediatrica Scandinauica (Suppl. 343), 127-131. Mehls, 0..Ritz, E., Gilli, G. & Kreusser, W. (1978) Growth in renal failure. Nephron, 21, 237-247. Mehls, 0.. Ritz, E., Hunziker, E.B., Tonshoff, B. & Heinrich, U. (1988a) Role of growth hormone in growth failure of uraemia.
Perspectives for application of recombinant growth hormone. Acta Paediatrica Scandinavica, 343 (Suppl.), 1 18-126. Mehls, 0..Ritz, E., Hunziker, E.B., Eggli, P., Heinrich, U. & Zapf, J. (1988b) Improvement of growth and food utilization by human recombinant growth hormone in uremia. Kidney International, 33, 45-52.
Metcalf, D. (1989) Haemopoietic growth factors 1. Lancer, i, 825827.
Pimstone, B.L., Le Roith, D., Epstein, S. & Kronheim, S. (1975) Disappearances rates of plasma growth hormone after intravenous somatostatin in renal and liver disease. Journal of Clinical Endocrinology and Metabolism, 4 1,392-395. Ramirez, G., ONeill, W.M., Bloomer, H.A. & Jubiz, W. (1978) Abnormalities in the regulation of growth hormone in chronic renal failure. Archives of Internal Medicine, 138, 267-271. Russell, W.E. & Van Wyk, J.J. (1989) Peptide growth factors. In Endocrinology (ed. L. J. De Groot), pp. 2504-2524. W. B. Saunders, Philadelphia. Samaan, N., Cumming, W.S., Craig, J.W. & Pearson, O.H. (1 966) Serum growth hormone and insulin levels in severe renal disease. Diabetes, IS, 546. Samaan, N.A.& Freeman, R. (1970) Growth hormone levels in severe renal failure. Metabolism, 19, 102-1 13. Schaefer, R.M., Kokot, F. & Heidland, A. (1989) Impact of recombinant erythropoietin on sexual function in hemodialysis patients. Contribuiions to Nephrology, 76, 273-282. Scigalla, P., Bonzel, K.E., Bulla, M., Bourhard, R., Dippel, J., Geisert, J., Leuman, E., v. Lilien, T., Muller-Wiefel, D.E., Offner, G., Pistor, K. & Zoellner, K. (1989) Therapy of renal anemia with recombinant human erythropoietin in children with end-stage renal disease. Contributions to Nephrology, 76, 227-241. Tonshoff, B., Schauer, A., Ranke, M., Blum, W., Heinrich, U. & Mehls, 0.(1989) Improvement of growth by recombinant human growth hormone in uraemic children. Acta Paediatrica Scandinavica 349 (Suppl.), 160. Weissel, M., Stummvoll, H.K., Kolbe, H. & Hoffer, R. (1979) Basal and TRH-stimulated thyroid and pituitary hormones in various degrees of renal insufficiency. Acra Endocrinologica, 90,23-32. Winearls, C.G., Oliver, D.O., Pippard, M.J., Reid, C., Downing, M.R. & Cotes, P.M. (1986) Effect of human erythropoietin derived from recombinant DNA on the anemia of patients maintained by chronic hemodialysis. Lancet, ii, I 175-1 178. Winer, B.J. (1971) Staristical Principles in Experimental Design. Mdjraw Hill, New York. Wright, A.D.. Lowy, C., Russel Fraser, T., Spitz, I.M., Rubenstein, A.H. & Bersohn, 1. (1968) Serum growth hormone and glucose intolerance in renal failure. Lancet, ii, 798-801.