Pediatr Nephrol (1991) 5:552-555 �9 IPNA 1991

Endocrine changes

Pediatric Nephmlogy

Original article

Recombinant human growth hormone overcomes the growth-suppressive effect of methylprednisolone in uraemic rats

Gabbr Kovhcsl, Richard N. Fine2, Stefan Worgalll, Franz Schaefer1, Ernst B. HunzikerS, and Otto Mehlsl

a Division of Paediatric Nephrology, University Children's Hospital, Im Neuenheimer Feld 150, W-6900 Heidelberg, Federal Republic of Germany 2 Department of Pediatrics, SUNY at Stony Brook, New York, USA 3 M, E. Miiller-Institute for Biomechanics, University of Berne, Bernes Switzerland

Received December 20, 1990; accepted February 25, 1991

Abstract. Paediatric renal allograft recipients frequently manifest growth retardation because of suboptimal graft function and/or concomitant corticosteroid treatment. To determine if the growth-suppressive effects of methylpred- nisolone (MP) could be counterbalanced by concomitant treatment with recombinant human growth hormone (rhGH) under conditions of normal and reduced renal func- tion, the following animal model was set up. Female uraemic Sprague-Dawley rats (140 g) together with pair- fed and ad libitum-fed control animals were treated with 6 mg/kg per day MP with or without 10 IU/kg per day rhGH. MP suppressed linear growth and weight gain by 43% and 63%, respectively in ad libitum-fed normal con- trol animals; the suppression was more pronounced in uraemic animals (57% and 107%, respectively). The sup- pressive effects were independent of food intake. The food conversion ratio (weight gain/food intake) was diminished to one-third in control and to more than one-tenth in uraemic animals. Concomitant treatment with rhGH completely reversed the suppression of length gain and weight gain (total body and muscle) and normalized the food conversion ratio. We concluded that rhGH can completely reverse the catabolic effects of corticosteroids under conditions of normal or reduced renal function. The data provide a reasonable rationale for prospective control- led studies on the use of rhGH treatment in paediatric kidney transplant recipients with growth failure.

Key words: Corticosteroids - Growth retardation - Growth hormone - Rats - Kidney transplantation - Uraemia

Introduction

Growth retardation is a significant complication in children with chronic renal failure (CRF) [1, 2]. The pathogenesis

Offprint requests to: O. Mehls

of the growth failure is muldfactorial; inadequate cal- orie/protein intake, acidosis and multiple hormonal distur- bances have been implicated. The identification and correction of these factors has not uniformly resulted in therapeutic success [3]. It had been anticipated that the growth velocity would improve dramatically after renal transplantation; however, many recipients fail to manifest catch-up growth or even normal growth because of subop- timal graft function and/or the need for concomitant immu- nosuppressive therapy with corticosteroids.

Since recombinant human growth hormone (rhGH) is now available for use in children with various growth disorders, we speculated that rhGH would be efficacious for the treatment of children following renal transplanta- tion. Specifically, we questioned if the growth-retarding effects of corticosteroid treatment could be overcome by rhGH in both healthy control animals and uraemic rats.

Methods

Animals. Female Sprague-Dawley rats (Ivanovas, Kisslegg/Allg~iu, FRG), weighing 120 g, were used for the investigation. Prior to the study, the animals had free access to food [Altromin C 1000 diet (13 800 kJ/kg, 0.95% Ca, 0.8% P, 500 IU/kg vitamin D3, 18% protein wt/wt) Altromin, Lage/Lippe, FRG] and deionized water. They were maintained in indi- vidual cages under constant room temperature (24~ and humidity (70%) conditions on a 12 h ordl2 h off light cycle. The rats were sub- jected to a two-stage nephrectomy as described previously [4]. Following removal of the second kidney and establishment of uraemia, mean body weight was about 140 g. Controls were sham-operated (renal decapsnla- tion) 1 day later; therefore, the mean body weight at the start was about 148 g.

Experimental protocol. The animals were randomized for division into three groups: group A received solvent twice daily s.c.; group B was treated with methylprednisolone (MP) at 6 mg/kg per day s.c.; group C received MP at 6 mg/kg per day s.c. and 5 IU/kg rhGH twice daily s. c. Each group (A, B, C) was further divided into three subgroups of five animals each: uraemic animals, sham-operated pair-fed controls and sham-operated controls fed ad libitum. The duration of the experiment was 14 days. Doses of MP and rhGH required for maximal catabolic and anabolic effects, respectively, had been previously determined.

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30 E E

t- �9 ~ 20

Y, r 10

_. . I

0 Ad tibitum controt Uraemia Pair-fed contro[

Fig. 1. Effects of methylprednis~176 (MP, N ) and MP recombinant human growth hormone (rhGH) ( O ) on length gain in uraemic rats, controls fed ad libitum and pair-fed controls. Results were compared with those obtained in solvent-treated ( �9 ) rats. The differences between MP-treated group solvent group, and MP + rhGH-treated group were significant (P _< 0.01)

o) r

~3

t -

A d tibitum contro[ Uraemia Pair-fed controt

Fig. 2. Effect of MP (@) and MP and rhGH ( O ) on weight gain in uraemic rats, controls fed ad libitum and pair-fed controls. Results were compared with those obtained in solvent-treated ( � 9 rats. The differ- ences between the MP-treated group solvent group, and MP + rhGH- treated group were significant (P _< 0.0!)

Measurements, Body weight was measured in non-fasted animals in the morning. Nose-tail tip distances were measured in ether-anaesthetized rats under conditions of deep musc le relaxation as previously described [4, 5]. All organs were weighed before and after desiccation performed at 80 ~ C in the presence of a desiccant for 24 h. Blood was obtained by aor- tic puncture of non-fasted animals uPon termination of the investigation. Serum analytes were measured us ing a multichannel Technicon Antoan- alyzer (Technicon Instruments, Tarrytown, New York). Blood pressure was measured by tail plethysmography as previously described [4].

For histological measurements of the proximal tibia growth rate, animals were given the fluorescent marker calcein [6] in a single injec- tion (10 mg/kg body weight s. c.) 1 week prior to sacrifice. The proximal tibiae were cut in frontal slices and fixed in 40% (vol/vol) ethanol at +4 ~ C for 24 h. After dehydration in a graded series of ethanol at +20 ~ C, tissue slices were infiltrated and embedded in methylmetacrylate. After completion of polymerization at 30 ~ C, 5-gin-thick sections were cut in the frontal plane. Sections were subsequently mounted on gelatin-coated slides and stained by a combined van Kossa-McNeils Tetrachrome-pro- tocol for examination by light microscopy and measurement of growth plate height [7, 8]. In addition, ] 0 -gm sections f rom each tibia were mounted unstained for measurement of growth rate using incident light fluorescence microscopy [8].

Statistics. Data are given as mean • SD. Results were evaluated using the Kruskall Wallis test.

R e s u l t s

The results obtained in the three experimental groups (uraemic animals, pair-fed controls and controls fed ad libitum each of which was receiving either solvent, MP, or MP together with rhGH, are reported in Table 1 and Fig. 1 and 2.

In control animals fed ad libitum, MP reduced cumula- tive weight and length gains by 63% and 43%, respectively (Fig. 1, 2), this effect being paralleled by a reduced daily growth rate, calculated from tibial growth (Table 1). Con- comitant treatment with rhGH counterbalanced the growth-depressing effects of MP completely. Weight and length gains were even slightly higher than in solvent- treated animals.

Similar effects of MP and rhGH were seen in pair-fed control animals. Weight and length gains were reduced by 69% and 51%, respectively. This response was reversed in the presence of rhGH in MP-treated animals. As indicated below, the lower absolute growth rate found in pair-fed rats was a consequence of the reduced food intake.

The growth inhibiting-effect o f MP was more obvious in uraemic animals than in ad libitum fed controls: weight gain was not only reduced but became negative, and length

Table 1. Growth and biochemical data in sub-totally nephrectomized rats (U) and control rats (CO) treated with methylprednisolone (MP) and recombinant human growth hormone (rhGH) s.c.

Group Initial Final Initial Final Growth rate Cumulative Weight gain Blood Serum Serum weight weight length length (metaphysis) food intake pressure pressure creatinine urea (g) (g) (ram) (ram) (gin/day) (g) (g/g) (ram Hg) (mg/dl) (mg/dl)

Usolvent 136• 6 157+10 351+ 5 373_+ 8 102• 7* 147• 5** 0.14_+0.03"* 147_+15" 0.69_+0.05"* 96• U+MP 140• 4 139+ 5 352+ 7 361_+ 8 53• 6*** 133• 5 -0.01• 149• 0.71+0.03 107+11 U+MP+rhGH 142_+10 162+ 7 354_+11 377_+ 8 110_+10 156_+ 9 0.13• 146+ 9 0.65_+0.05 104•

CO pair-fed solvent 149+_ 4 167+ 8 364+_ 9 387+_10 113+10 147+- 5 0.12_+0.03 103_+17 0.23_+0.06 24+- 4 CO+MP 148_+ 7 153+ 4 365-+ 9 376• 7 78+ 7*** 133+ 4 0.04-+0.03**** 121• 6 0.29+0.05 36+_ 7 CO+MP+rhGH 148_+ 9 176_+ 7 366_+12 390_+12 105_+ 8 156_+ 9 0.18-+0.04 121_+ 6 0.27_+0.01 32• 3

CO ad libitum-fed solvent 147• 5 182_+ 9 355_+13 383_+14 125_+ 6 173-+13 0.21_+0.03 113• 0.26_+0.05 31+_ 5 CO+MP 149_+12 157_+13 358_+ 6 373_+ 8 80_+14"** 153• 0.08+_0.02**** 111• 0.30_+0.04 32_+ 4 CO+MP+rhGH 148• 6 193_+13 360• 5 389• 137_+I3 182-+29 0.25_+0.03 110+-15 0.34_+0.06 26+_ 3

* P _ 0.05 U solvent vs CO solvent; ** P <0.01 U solvent vs CO solvent; *** P _< 0.05 MP vs MP + rhGH; **** P <0.01 MP vs MP + rhGH

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gain was reduced by 57% (Fig. 1, 2). Despite the severe catabolic action of MP in uraemic animals, rhGH counter- balanced these effects completely.

Serum creatinine and urea levels were similar in all uraemic animals, irrespective of treatment with MP or rhGH. Serum urea levels tended to be higher in animals receiving MP. This difference was not significant.

Uraemic animals had a significantly lower food con- sumption than control animals fed ad libitum. MP had only a slightly negative effect, upon cumulative food intake in uraemic and control animals, this effect being reversed by rhGH.

The catabolic effects of MP are illustrated by the signif- icantly lower food conversion ratio (weight gain food in- take, Table 1) in all animals receiving this drug. Treatment with rhGH normalized of even ameliorated changes in this parameter in both control and uraemic animals.

Uraemic animals had a slightly higher blood pressure than control animals (P _< 0.05), but this was not signifi- cantly influenced by either MP or rhGH.

Creatinine clearance was not affected by MP treatment, either in uraemic (solvent 353 _+_ 61 pl/min per 100 g, MP 313--+51 gl/min per 100 g) or pair-fed control animals (solvent 815_+79 gl/min per 100 g, MP 915+75 gl/min per 100 g). Treatment with rhGH significantly (P <0.05) increased this parameter in controls (1128 + 86 pl/min per 100 g) but not in uraemic animals (333 ___68 pl/min per 100 g).

Discussion

Several factors contribute to uraermc growth failure, in- cluding catabolism, inadequate protein and calorie intake, acidosis, disturbed vitamin D metabolism and hormonal changes. Irrespective of the mode of treatment (e. g. con- servative, dialysis or renal transplantation) growth in one- third of uraemic adolescents remains stunted when final adult height has been attained [3, 9]. At one time, it was believed that successful renal transplantation would reverse stunting, but this has not been the case [10-12]. One cause of these unsatisfactory results lies in the im- paired function of the renal transplant due to primary injury during transplantation or subsequent rejection crises. It is well known from CRF patients undergoing conservative treatment that a reduced growth rate may be evident long before terminal renal failure occurs. Even in children with excellent renal transplant function, catch-up growth is only exceptionally seen, and then primarily in prepubertal patients [10]. Treatment with corticosteroids is believed to be responsible for these effects. Low-dose steroid treat- ment combined with cyclosporine A improves, but does not normalize, growth [ 13]. Improvement of growth veloc- ity following alternate-day corticosteroid treatment has been unsatisfactory [14], whereas preliminary results sug- gest elimination of corticosteroids is beneficial [ 15]. How- ever, the latter has the risk of precipitating a rejection episode.

Treatment with corticosteroids has a strong catabolic effect on the organism [16] this does not lead to weight

loss in man because reduction in muscle mass is compen- sated for by an increase in fat mass. In rats, however, diminished weight gain or weight loss is associated with corticosteroid treatment since the increase in fat mass is minimal. The growth-depressing effects of corticosteroids may be explained by inhibition of insulin-like growth fac- tor-I (IGF-I) transcription [17] and by inhibitory effects on collagen metabolism and bone matrix formation [18]. Whether the latter are direct or are mediated by their effect on local IGF-I prodnction or by modulating local IGF-I effects [19-21] or by suppression of endogenous GH secretion [22, 23], is unknown. In the present study, MP treatment resulted in reduced weight gain and reduced length gain in healthy control animals. Interestingly, not only was the weight gain reduced, but also the food conver- sion ratio was decreased (e. g. weight gain per food intake). This indicates catabolism and/or reduced anabolism. The negative effects of MP on growth were higher in uraemic than in control animals, suggesting that this response is more marked in an organism with a chronic disease.

Pharmacological doses of rhGH improve growth in var- ious conditions [24-26], increase muscle mass [27], and are responsible for positive nitrogen balance [28], but the mechanisms underlying its anabolic action are not fully understood, rhGH may well have a direct effect on chon- drocytes although it also acts via an effect on peripheral and local IGF-I levels [29]. This latter effect may be the principal mechanism by which rhGH is able to compete with corticosteroids. In the present study, treatment with rhGH significantly increased gains in length and weight and the food conversion ratio, in both uraemic and control animals treated with MP. At the same time, this drug fully compensated for the catabolic effects of MP.

Both the catabolic effects of MP and the anabolic ef- fects of rhGH were independent of food intake; this param- eter was only slightly reduced by steroid's and not signifi- cantly increased by rhGH treatment. The lower food intake for controls fed in pairs compared with those fed ad libitum resulted in a reduction of absolute growth rates, but the relative MP-induced suppression of weight and length gains were not influenced. In both groups, reduction in body growth was fully compensated for by treatment with rhGH.

These studies suggest that GH will fully reverse the catabolic situation associated with reduced graft function and/or concomitant treatment with corticosteroids in chip dren with kidney grafts. Our dose-dependency experiments (data not given) provide strong evidence that a low corti- costeroid dose requires only a low rhGH dose to reverse the corticosteroid-induced catabolic effects, whereas the effects of high corticosteroid doses can only be reversed by a higher daily dose of rhGH.

There is a potential risk when giving MP and rhGH to children with transplants. If rhGH reverses the catabolic effects of MP it might also modulate the immune-suppres- sive effects of MP. This requires investigation by carefully controlled prospective clinical trials. Concern has also been raised that rhGH could exaggerate hyperfiltration and accelerate progression of CRF. However, as in previous experiments using the inulin clearance technique for meas- uring glomerular filtration rate [30], in the current study

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rhGH produced hyperfi l tration in control but not in uraemic rats.

In summary, the present experimental data document that both corticosteroids and reduced renal funct ion have a negative effect on growth. The catabolic effects of corti- costeroids are more evident in CRF. rhGH completely reverses the catabolic effects of CRF and corticosteroids. Prospective controlled studies in growth-retarded paediat- ric renal allograft recipients are therefore justified. In such studies, not only the effect of rhGH on growth and cata- bolism, but also the potential modula t ing effects of rhGH on the i m m u n e system require investigation.

Acknowledgements. This work was undertaken during Dr. Kovhcs's stay as an exchange student in Heidelberg and Dr. Fine's tenure as a "Senior, US Scientist" of the Alexander von Humboldt-Foundation in Heidelberg, FRG. The study was supported by Kabi Company, Stockholm, Sweden who kindly provided rhGH (Genotropin). The authors are indebted to Tanj a Durrer, Berne, for her excellent technical assistance. The excellent secretarial help of Mrs. R. Greiffenhagen is kindly acknowledged.

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