SHORT COMMUNICATION Clin. Drug Invest. 10(3): 183-1 87, 1995 1173-2563/95/0009-o183/S02.50/0

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Chronic Low Dose Growth Hormone Treatment Stimulates Both Hypertrophy and Hyperplasia of Remnant Kidneys in Uraemic Rats J. Paul Frindik,1 Watson C. Arnold 2 and Eileen Ellis l

Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

2 Department of Pediatric Nephrology, Cook-Fort Worth Children's Medical Center, Fort Worth, Texas, USA

Growth retardation in children with chronic renal insufficiency (CRI) may be due to malnutri­tion, vitamin D deficiency, acidosis or hormonal imbalances.[I ·2] Although recombinant human growth hormone (rhGH) has recently received FDA approval for the treatment of growth retarda­tion of CRI prior to transplantation, [3] the long term effects on residual renal tissue and renal function remain unclear. Growth hormone (GH) could be detrimental by stimulating disproportionate in­creases in somatic growth compared with renal capacity[4] or by promoting glomerular hyper­filtration,[S] hypertrophy[4.6] or sclerosisP]

Uraemic rats treated for g to 9 weeks with rhGH at doses approximately lOO-fold greater than those used in uraemic children have increased glomeru­lar hypertrophy[4] and sclerosis with reduced long­evity. [6] It is unknown whether renal changes occur as a function of time (duration) or dose of rhGH. This study was undertaken to examine the effects of low dose, chronic rhGH administration in rats with CRI.

Methods

Nineteen weanling female Sprague Dawley (SD) rats (Charles River Laboratory, Wilmington, MA) at an initial weight of 50 to 75g were made

uraemic via an initial two-thirds nephrectomy (upper and lower poles) followed 1 week later by a complete contralateral nephrectomy. For con­trols, sham-operated female SD rats had renal decapsulation performed only. Four groups were then studied: sham-operated controls (control, n = 5), sham-operated controls receiving rhGH (GH control, n = 6), nephrectomised controls (uraemic, n = 6), and nephrectomised rats receiving rhGH (GH uraemic, n = 7).

GH-treated rats received rhGH (somatropin, Nutropin®, lot number N9272A, Genentech, Inc., South San Francisco, CA) 0.5 mg/kg/day for 6 months, given as single daily injections to mimic common clinical practice. Per company protocol, somatropin was reconstituted with sterile, bac­teriostatic water, stored at 2° to goC and used within 14 days of reconstitution. The untreated rats received daily injections of sterile bacteriostatic water.

All rats were caged at constant room tempera­ture (22 ± 0.2°C) on a 12-hour light/dark cycle and allowed free access to food (Purina Rat Chow con­taining 24% protein) and water. This study was approved by the Animal Care and Use Committee of the University of Arkansas for Medical Sciences.

184

Renal Function

Serum for blood urea nitrogen (BUN) and se­rum creatinine (Cr) determination was obtained at 3 months by tail bleeding and at 6 months via aortic puncture (see below). BUN and Cr were quanti­tated using a Beckman Creatinine and BUN Ana­lyser (Beckman Instruments, Brea, California, USA). Creatinine clearances (CLcr), calculated from Cr and 18-hour timed urine samples obtained in metabolic cages, were expressed as III per min­ute per I ~Og bodyweight and used as a measure of glomerular filtration rate (GFR).

Tissue Analysis

After 6 months, the animals were weighed, measured, given methoxytlurane anaesthesia and exsanguinated by aortic puncture. Liver, heart, whole kidneys and all noninfarcted remnant renal tissues were removed and weighed.

Cytosolic protein per gram of kidney tissue was quantitated using a bovine serum albumin standard (Bio-Rad Protein Assay, Richmond, CA). ON A per gram of kidney tissue was measured using the rela­tive intensity of the chelated fluorochrome Hoechst 33528 as read by an Aminco-Bowman spectrophoto­fluorometer (Travenol, Deerfield, Illinois, USA}.IXI

For microscopy studies, kidney tissue was fixed in 2% glutaraldehyde in cacodylate buffer. 141 An haematoxylin- and eosin-stained slide from each rat kidney served as a random cross-section to measure glomerular volume (GY). The string polygon perimeter of 100 sequential glomeruli

Frilldik ct til.

per kidney were outlined using a Y 150 vidcometer (American Innovision, Inc., San Diego, CAl. Proximal tubular diameters and cross-sectional and glomerular areas were measured and mean GY determined for each animal. lYI

These measurements were made by an observer unaware of treatment group assignment.

Statistical Analysis

Differences between groups were evaluated us­ing Bartlett's test for homogeneity of variance. I 111 1

If variances were found to be homogeneous with a 95% confidence level, a one-way analysis of vari­ance (ANOYA) was used. If not homogeneous, the Kruskal- Wallis test for 2 groups was used. Thc level of significance accepted was a = 0.05, and all data are presented as the mean ± standard deviation.

Results

Six nephrectomised rats died in the immcdiate postoperative interval, prior to receiving rhGH. One GH control rat died at 4 months, and 2 uraemic rats died at 3 months (I death each in the GH­treated uraemic and untreated uraemic groups). Data from these expired animals were excluded from analysis.

Renal functions at 3 and 6 months are shown in table I. BUN and Cr values were significantly elevated and CLcr rates decreased in all uraemic animals. rhGH had no effect on renal function in any group. Mean bodyweights and lengths of both

Table I. Effects of 3 and 6 months of growth hormone (GH) administration (0.5 mg/kg/day) on renal function

Control GH control Uraemic GH uraemic (n = 5) (n = 5) (n = 5) (n = 6)

3 months

BUN (mmol/L) 5.0 ± 0.8 5.0 ± 1.0 9.4 ± 2.2 9.8 ±2.1

Cr(~moI/L) 39.9 ± 9.7 40.8 ± 10.6 62.1 ± 14.2 68.3 ± 22.1

CL.", (~l/min/100g bodyweight) 0.56 ± 0.25 0.63 ± 0.27 0.32 ± 0.09 0.34 ± 0.06

6 months

BUN (mmoI/L) 5.6 ± 0.6 5.3 ± 0.5 12.2 ± 6.0 10.1 ±3.1

Cr (~moI/L) 32.8 ± 4.4 30.2 ± 5.3 80.7 ± 35.5 77.8 ± 26.6

CL.", (~l/min/100g bodyweight) 0.48 ± 0.12 0.48 ± 0.08 0.22 ± 0.09 0.30 ± 0.09

Abbreviations: BUN = blood urea nitrogen; CL.", = creatinine clearance; Cr = serum creatinine.

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Chronic Low Dose Growth Hormone in Uraemic Rats 185

Table II. Effecls of 6 months of growth hormone (GH) therapy (0.5 mg/kg/day) on uraemic rats

Control (n = 5)

Length (em) 41.3± 1.1

Weight (g) 312.7 ± 31.7

Kidney weight (g) 2.14±0.29

Kidney weight (g/l00g bodyweight) 0.70 ± 0.09

Protein (mg/g kidney) 46.1 ±8.0

DNA (mg/g kidney) 0.20 ± 0.07

Glomerular volume (11m3 x 105) 7.2± 1.6

Proximal tubular area (11m2 x 105) 1.4±0.4

" p = 0.05 GH uraemic vs uraemic.

control groups were similar at all times. The GH uraemic rats were larger than the placebo-in­jected uraemic rats at the study 's conclusion.

The effects of 6 months of rhGH therapy are shown in table II. Heart and liver weights were similar for all groups. Mean remnant kidney weight, renal protein, DNA and GV of the GH uraemic animals were increased compared with the untreated uraemic animals (p = 0.05). Renal DNA content was increased in both groups of rhGH­treated animals as compared with their untreated counterparts (p = 0.05). rhGH had no effects on proximal tubular diameters or cross-sectional areas, which were increased in all uraemic animals compared with all control animals (p < 0.005).

Discussion

We and other investigators have utilised the nephrectomised uraemic rat model to study the short term (13 days to 9 weeks) effects of large (pharmacological) doses of rhGH (lmg 3 times weekly to 5 mglkg/day).f4,11-13] Such large doses of rhGH are often required for significant somatic growth in rats because growth-limiting anti-rhGH antibodies can develop after 2 to 3 weeks of exo­genous rhGH treatmentJl4] Extrapolating the re­sults of such studies to the treatment of children with CRI, who do not develop significant anti­rhGH antibodies and thus require much lower rhGH doses (0 .05 mg/kg/day) for growth, is problematical.

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GH control Uraemic GH uraemic (n =5) (n = 5) (n =6)

42.S ± 0.7 40.3 ± 1.1 42.S ± 1.2"

349.0 ±25.5 255.2 ± 38.1 306.6 ± 29.1"

2.21 ±0.21 1.61 ± 0.20 1.S7±0.W

0.62 ±0.04 0.58 ± 0.08 0.58 ± 0.05

41.3± 11 .9 41.7±10.9 66.9± 15.0"

0.30±0.07 0.23±0.03 0.32±0.OS"

7.4 ± 1.5 7.6± 4.3 12.0±4T

1.3 ± 0.4 3.9 ±0.8 3.0±0.7

By contrast, antibody-induced GH resistance may not occur in rats treated with lower doses of foreign GH.[I5] If such antibodies developed in our current study, they were not associated with so­matic rhGH resistance as no growth deceleration occurred in the rhGH-treated animals compared with their placebo-treated cohorts. Whether or not such antibodies interfere with localised effects of rhGH on remnant kidneys has not been deter­mined.

The present study was therefore designed to more closely approximate clinical practice and the hormonal milieu of the kidneys of children with CRI undergoing rhGH therapy. As in our previous study, we were primarily interested in the renal effects of rhGH therapy and thus chose not to con­trol or measure food intake or other influences on uraemia.[4]

Following reduction in renal mass, a compensa­tory renal hypertrophy (CRH) occurs that is not mirrored by hypertrophy in any other organ.[16] Attempts to explain this phenomenon by the identi­fication of potential kidney-specific growth factors have been unsuccessfuJ.l16] Alternatively, an exag­gerated response of the kidney to GH may occur, perhaps via increased end-organ sensitivity. If true, even a marginal increase in GH exposure might further stimulate such cells resulting in increased renal hypertrophy (i.e. increased renal protein synthesis), hyperplasia (i.e. increased renal DNA synthesis) or glomerular enlargement.

Clin. Drug Invest. \0 (3) 1995

186

Growth hormone could promote these changes directly or via localised GH-dependent autocrine or paracrine growth factors such as insulin-like growth factor I (lGF-I)[ 17] or epidermal growth fac­tor (EGF).lI8] GH administration can increase renal IGF-I synthesis nearly 3-fold, without a corre­sponding increase in serum IGF-I levels.[I9] EGF is also synthesised by the rat kidney[20] and stimu­lated by exogenous GH in animals[2I] and chil­dren.[I8] Recently, both time- and dose-dependent effects of exogenous rhGH on urinary EGF have been demonstrated.l I8] Markedly elevated urinary EGF concentrations have been reported in I patient with Donohue syndrome (Leprechaunism) who had renal hypertrophy with increased glomerular and mesangial volumes.[22]

Our findings are consistent with GH-dependent changes in that somatic size and renal weight, hypertrophy, hyperplasia and GV were all in­creased in the rhGH-treated uraemic animals com­pared with the placebo-injected uraemic rats. How­ever, other possibilities, such as sampling error due to the small number of animals studied, differences in protein intake or water gain, could explain some of our observations.

Growth hormone treatment of uraemic rats can improve the efficiency of food metabolism and may increase total body fluid content; either or both effects could result in total somatic or tissue weight gains.[IU3] We previously found no effects on water content or serum electrolytes with IO-fold greater rhGH doses in uraemic rats,[4] but did not assess these variables directly in the current study.

Other potential sources of error include difficul­ties in obtaining representative samples from hypertrophied remnant kidneys and variable blood pressures postnephrectomy.

Conclusion

In conclusion, low doses of exogenous rhGH, when administered in a manner similar to clinical practice, are associated with glomerular enlarge­ment and stimulation of hypertrophy and hyper­plasia of remnant kidneys from uraemic rats. Increased GV was also seen after short term, high

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Frilldik cl nt.

dose rhGH therapy in our previous study.141 The time interval required before increased GV devel­ops with low dose rhGH therapy and whether or not further rhGH exposure subsequently leads to worsening glomerular hypertrophy, glomerulo­sclerosis[7] and renal failure cannot be determined from the current study. Although we observed no significant changes in renal function with rhGH therapy, we recommend that children with CRI be carefully monitored to ensure that long term rhGH treatment has no such deleterious effects.

Acknowledgements

The authors wish to thank Belinda Shirkey, MD. and Shelly Ba]dwin, MD, for their expert assistance. and Genentech, Inc. for their generous gift of growth hormone.

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Clin. Drug Invest. 10 (3) 1995

Chronic Low Dose Growth Hormone in Uraemic Rats

13. Nakano M, KainerG, Foreman J, et al. The effects of exogenous rat growth hormone therapy on growth of uremic rats fed an 8% protein diet. Pediatr Res 1989; 26: 204-7

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15. Stred SE, Benedict MR, Kuehnling E, et al. Effect of growth hormone on growth and glucose tolerance of normal rats. Am J Dis Child 1987; 141: 502-5

16. Fine LG, Kurtz I, Woolf AS, et al. Pathophysiology and nephron adaptation in chronic renal failure. In: RW Schrier, CW Gottschalk, editors. Diseases of the kidney, 5th ed. Boston: Little, Brown and Co., 1993: 2703-41

17. Hirschberg R, Kopple JD. The growth hormone-insulin-Iike growth factor I axis and renal glomerular function. J Am Soc Nephrol 1992; 2: 1417-22

18. Frindik JP, Kemp SF, Keams GL. Epidermal growth factor con­centrations in growth hormone deficient children. Effects of single versus multiple injections of recombinant human growth hormone. Drug Invest 1994; 8 (5): 257-62

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19. Orlowski CC, Chemausek SD. Discordance of serum and tissue somatomedin levels in growth hormone-stimulated growth in the rat. Endocrinology 1988; 123: 44-9

20. Olsen S, Paulsen PS, Kirkegaard P, et al. Renal origin of urinary epithelial growth factor in the rat. Regul Pept 1984; 10: 37-47

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22. Ellis EN, Kemp SF, Frindik JP, et al. Glomerulopathy in patient with Donohue syndrome (Leprechaunism). Diabetes Care 1991; 14 (5): 413-4

Correspondence and reprints: Dr J. Paul Frindik, Arkansas Children's Hospital, 800 Marshall Street, Little Rock, AR 72202-3591, USA.

Clln. Drug Invest. 10 (3) 1995