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The effects of chronic administration of epidermal growth factor (EGF) to rats on the levels of endogenous EGF in the submandibular glands and kidneys
Regulatory Peptides 67 Ž1996. 179–185
The effects of chronic administration of epidermal growth factor ž EGF/ to rats on the levels of endogenous EGF in the submandibular glands and kidneys Lars Vinter-Jensen a
a,b,)
, Per E. Jørgensen a , Steen Seier Poulsen c , Ebba Nexø
a
Department of Clinical Biochemistry, KH Aarhus UniÕersity Hospital, Nørrebrogade 44, DK-8000 Aarhus, Denmark b Department of Pathology, Aalborg Hospital, Aalborg, Denmark c Institute of Medical Anatomy, The Panum Institute, UniÕersity of Copenhagen, Copenhagen, Denmark Received 5 February 1996; revised 26 August 1996; accepted 26 August 1996
Abstract Epidermal growth factor ŽEGF. is mainly produced in the submandibular glands ŽSMG. and in the kidneys. It has recently been reported that EGF-related ligands may induce their own biosynthesis Žautoinduction. in vitro. In the present paper, we investigated whether chronic systemic treatment with EGF influenced the amount of endogenous EGF in the SMG and kidneys. Eight-week-old female Wistar rats were treated with subcutaneous injections of placebo Ž n s 16. or human recombinant EGF Ž150 mgrkg per day, n s 8. for 4 weeks. Urine was sampled the last 24 h of the study period. At the time of killing, the SMG and the kidneys were removed. The SMG was larger in the EGF-treated animals, 229.8 " 35.5 Žmean " SD. mg than in the control animals, 181.7 " 18.1 mg Ž P - 0.01.. The total EGF content was smaller Ž0.51 " 0.15 vs. 1.12 " 0.40 nmol EGFrSMG, P - 0.001.. The kidneys were larger in the EGF-treated animals Ž1.38 " 0.08 vs. 1.28 " 0.08 g, P - 0.05., but the EGF content and urinary excretions were not changed. In conclusion, chronic systemic treatment with EGF causes growth of the SMG with concomitantly reduced contents of EGF, and growth of the kidneys with unchanged content and excretion of EGF. These findings suggest that EGF may play a part in the regulation of the growth of the SMG and in EGF biosynthesis. Keywords: Keywords please
1. Introduction Epidermal growth factor ŽEGF. and related ligands ŽEGF agonists. exert their action via binding to the EGF receptor w1x. Intestinal cells in culture upregulate the expression of EGF agonists when EGF or one of the EGF agonists activates the EGF receptor w2x. The production of these ligands may therefore partly be autoregulated by autocrine or paracrine mechanisms. Systemic treatment with EGF prevents tissue damage and accelerates healing of experimentally induced damage to various organs w3–5x, and treatment with EGF may become a future option in human therapeutics. From a pharmacologic point of view, it is therefore interesting to know how EGF administration in vivo affects EGF biosynthesis. EGF is synthesized by the granular convoluted tubular )
Corresponding author. Fax: Ž45-89. 493-060.
cells ŽGCT. of the submandibular gland ŽSMG. w6x, and by the cells of the thick ascending limb of Henle and early part of the distal convoluted tubules in the kidney w7x. EGF is secreted in an exocrine manner to saliva w8x and to urine w9x and EGF probably exerts trophic effects on the epithelia with which is in contact w10,11x. In the present paper, we have investigated the effects of chronic systemic administration of EGF on organ size and tissue EGF content at the two principal sites of EGF biosynthesis, namely, the SMG and the kidneys.
2. Materials and methods 2.1. Study animals Twenty-four female 8-week-old Wistar rats from our own breed ŽDepartment of Pathology, Aalborg Hospital,
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L. Vinter-Jensen et al.r Regulatory Peptides 67 (1996) 179–185
Denmark., were used. The animals were housed individually in cages on white special spanwall bedding Žtemperature 218C, humidity 55 " 5%, darkrlight cycle 12-h shift.. They were fed a standard laboratory diet, ŽAltromin 1314.. The animals were kept in the same room throughout the study and handled by the same two technicians, only. The study complied with Danish regulations for care and use of laboratory animals. 2.2. Study design The 24 animals were randomly allocated into 2 groups. They received either solvent Žisotonic saline. Ž n s 16. or human recombinant EGF, 150 mgrkg per day Ž n s 8; Upstate Biotechnology, New York, NY, USA. by two daily subcutaneous injections for 4 weeks. The total body weight was determined once a week. Urine was collected in metabolic cages for the last 24 h of the study period. After 28 days of treatment, the animals were anaesthetized with Ketamine ŽKetalar w , Parke Davis, Barcelona, Spain. 100 mgrkg and xylosin ŽRompun w , Bayer, Leverkusen, Germany. 15 mgrkg intramuscularly. Blood samples from the retrobulbar venous plexus were collected through heparinized capillary tubes. One SMG Žincluding the sublingual gland. and the kidneys were removed, weighed, snap-frozen in liquid nitrogen, and stored at y808C for later analysis. The other SMG was fixed in phosphate-buffered paraformaldehyde Ž4%, pH 7.0. The animals were killed thereafter. The procedures described above were carried out on a blinded basis. For some of the procedures described below, only a subgroup of the control animals was included. The control animals in the subgroups were randomly chosen. 2.3. Tissue preparation The SMG was homogenized in 10 volumes of distilled water. The homogenate was centrifuged Ž20 000 = g, 40 min, 48C., and supernatant 1 was stored at y208C. The pellet was resuspended and homogenized in 2 ml 50 mM acetic acid. This homogenate was also centrifuged Ž20 000 = g, 40 min, 48C. and supernatant 2 was stored at y208C. The total contents of EGF and protein in SMG were calculated as the sum of the contents in the two supernatants. In the SMG, EGF is present as soluble EGF in secretory granules. This EGF is released by homogenization, and it is therefore possible to measure the amount of EGF per gland. In the kidney, EGF is present in the form of a big membrane bound precursor, and it is not released by homogenization. It is, however, possible to release the renal EGF from a membrane preparation by trypsinization and the renal EGF content per mg membrane protein can thereby be calculated. Kidney membranes were isolated by differential centrifugation, essentially as described w12x. In
brief, kidneys were homogenized on ice in 0.25 M sucrose, 25 mM Tris-HCl ŽpH 7.4., 5 ml per g kidney. The homogenate was centrifuged to remove intact cells Ž600 = g, 10 min, 48C., and the resulting supernatant was recentrifuged Ž18 000 = g, 30 min, 48C.. The membrane fraction of the pellet was resuspended in the supernatant and adjusted to 0.1 M in NaCl and 0.2 mM in MgSO4 before centrifugation Ž35 000 = g, 40 min, 48C.. The pellet was washed three times by resuspension with 25 mM Tris-HCl ŽpH 7.4., and harvested by centrifugation. The membranes were suspended in 25 mM Tris-HCl ŽpH 7.4.. 2.4. Biochemical analyses 2.4.1. Rat EGF Rat EGF was measured by ELISA either without ŽSMG. or after pretreatment with trypsin Žurine and kidneys. as previously described w13x. The urine samples were trypsinized with 3.13 mg trypsinrml whereas the renal membrane preparations were diluted in 25 mM Tris ŽpH 7.4. so that the total protein concentration was 3 mgrml prior to trypsinization with 31.3 mg trypsinrml. Pretreatment with trypsin converts the various molecular weight forms of rat urinary EGF to a single molecular form that corresponds to the 48 amino acid form of EGF present in the rat submandibular glands w14x. This ensures that the variety of molecular weight forms of EGF in urine and kidney tissue are measured with equimolar potency w13x. Human EGF does not cross-react with rat EGF in these assays w13x. 2.4.2. Other biochemical analysis The total protein concentration was measured with a kit, BCA Protein Assay Reagent ŽPIERCE.. Human EGF in urine and in plasma was measured by a previously described ELISA for human EGF w15x. Creatinine in urine was analysed on a Kodak, Ektachem 500. 2.5. Immunohistochemistry for endogenous EGF in the SMG After finding reduced concentrations and contents of endogenous EGF in the SMG, a semiquantitative immunohistochemical analysis of the GCT cells was performed on sections from 5 controls and 6 EGF-treated animals. Fivemm thick paraformaldehyde-fixed, paraffin-embedded sections were employed. The deparaffinized sections were preincubated with 10% porcine serum ŽDakopatt, Copenhagen, Denmark. in phosphate-buffered albumin for 30 min. The serum was removed and sections were incubated over-night with rabbit serum against rat submandibular EGF ŽNo. 813 raised in our lab.. The immunoreactions were visualized by means of the peroxidase-antiperoxidase technique, using swine-antirabbit IgG and peroxidase-anti-
L. Vinter-Jensen et al.r Regulatory Peptides 67 (1996) 179–185
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peroxidase ŽDakopatt. and diaminobenzidine as chromogen. The number of immunostained GCT cells and the volume fraction of the GCT cells were estimated by means of an Olympus BH-2 microscope with projection to a monitor giving a total magnification X1550. An area of submandibular tissue Žexcluding sublingual tissue. of 22 mm2 Žmean. was defined and the GCT cells were counted in 140 Žmean. randomly chosen fields of vision of 0.0195 mm2 . The number of cells per mm2 was thereafter calculated. GCT cells appear very often in clusters and are therefore difficult to delineate from each other. The fraction of the tissue occupied by the immunoreaction for rat EGF was therefore also determined in the same fields of vision by means of point counting Ža mean of 32 000 points was employed per animal.. 2.6. Statistical analyses Results are expressed as means and standard deviations ŽSD.. Mann-Whitneys non-parametric test was employed to test differences between groups. The level of significance chosen was 0.05.
3. Results 3.1. General obserÕations All animals thrived throughout the study. The animals increased in weight from 172 " 5 to 203 " 11 g in the control group and from 172 " 3 g to 213 " 8 g in the EGF-treated group. The total body weight was significantly larger in the EGF-treated group at the end of study Ž P s 0.02.. 3.2. The submandibular gland (SMG) The right SMG was increased in weight from 181.7 " 18.1 mg in the control animals to 229.8 " 35.5 mg in the EGF-treated animals Ž P s 0.004., Fig. 1. This increase was proportionately larger than the increase in total body weight Ž P s 0.02.. The total protein content increased from 11.4 " 1.5 mg in the controls to 13.5 " 1.9 mg in the EGF animals Ž P s 0.010., whereas the total rat EGF content decreased from 1.12 " 0.40 to 0.51 " 0.15 nmol Ž P s 0.0003.. The amount of rat EGF per amount of protein was consequently reduced from 97 " 30 to 38 " 12 nmol rat EGFrg protein Ž P s 0.002.. The density of GCT cells was reduced from 54 cellsrmm2 in the controls to 23 cellsrmm2 in the EGF animals Ž P - 0.02., Fig. 2. The volume fraction of immunostaining for rat EGF was accordingly reduced from 1.4% to 0.5% Ž P - 0.02.. Based on the visual impression, the intensity of the immunostaining of the GCT cells in the EGF-treated animals was also reduced.
Fig. 1. Scatterplots of total weight and rat EGF contents in submandibular glands from female Wistar rats treated with placebo Ž`. or human recombinant EGF Ž150 mgrkg per day. Žv . for 4 weeks. The P-value for the Mann-Whitney test is indicated.
3.3. The kidneys The kidneys Žright plus left. were proportionately increased in weight from 1.28 " 0.08 g in the control group to 1.38 " 0.08 g in the EGF-treated group Ž P s 0.018.. The renal EGF content was not increased significantly Ž3.5 " 1.1 nmol EGFrg protein in the controls Ž n s 8. vs. 4.1 " 0.7 nmol EGFrg protein in the EGF-treated animals Ž n s 8. Ž P s 0.074... 3.4. Urinary excretion of EGF, creatinine and protein (Table 1) There were no changes in the total urine volume per 24 h. The total excretions of rat EGF, protein and creatinine were not increased significantly in the EGF-treated group. The EGFrcreatinine ratio and the EGFrprotein ratio were identical in the 24 h urinary samples in the two groups: 24 " 5 and 24 " 2 nmol EGFrmmol creatinine Žcontrols and EGF-treated group. and 24 " 6 and 23 " 2 nmol EGFrg protein Žcontrols and EGF-treated group.. Table 1 Urinary excretions of rat EGF, protein and creatinine in rats treated with isotonic saline Žcontrols. and rats treated with human recombinant EGF, 150 mgrkg per day, for 4 weeks ŽEGF group. Controls Ž ns12. Total volume Žmlr24 h. EGF Žnmolr24 h. Protein Žmgr24 h. Creatinine Žmmolr24 h.
9.8"2.9 1.80"0.31 78.7"15.2 76.0"16.4
EGF group Ž ns8. NS NS NS NS
Values are mean"SD. NS, no significant difference ŽMann-Whitney test..
10.9"1.2 2.00"0.21 87.5"7.2 83.0"10.3
Fig. 2. Immunostaining for rat EGF in a control rat ŽA, C. and in a rat treated for 4 weeks with EGF 150 mgrkg per day ŽB, D.. The reduced density and intensity of the immunostaining of the GCT cells are seen in the EGF animal. Magnification: A,B =90; C,D =360.
182 L. Vinter-Jensen et al.r Regulatory Peptides 67 (1996) 179–185
L. Vinter-Jensen et al.r Regulatory Peptides 67 (1996) 179–185
A small fraction of parenterally administered EGF is excreted in urine w16,17x. Even though human EGF do not cross-react with rat EGF in the assays used to quantitate rat EGF w13x, we wanted to confirm that the urinary excretion of systemically administered human EGF was negligible compared to that of rat EGF. The mean concentration of human EGF was 1.2 nmolrl compared to 190 nmol rat EGFrl, i.e., less than 1%. 3.5. Human recombinant EGF in plasma There was no detectable human recombinant EGF in plasma at killing – week 12 h after the last injection of EGF. Human recombinant EGF was, however, excreted to urine and therefore absorbed from the site of injection. 4. Discussion The major finding of the present study is that prolonged systemic treatment with EGF increases the weight of the SMG and reduces the EGF content of the SMG. Interestingly, no change was observed in the EGF concentration within and EGF excretion of the other major EGF producing organ, the kidney. In the SMG, we found reduced concentration and content of EGF and reduced density, volume fraction and intensity of the immunostaining of the GCT cells. EGF biosynthesis was therefore most likely reduced. The EGF content of the SMG changes with age and is influenced by various hormones. In female Wistar rats, the EGF content increases gradually from birth until 8 weeks of age w18x. The EGF content then increases rapidly and reaches a plateau after week 12. The SMG therefore, might be especially sensitive to stimuli from week 8 to 12. We intervened with systemic EGF treatment in exactly this period of life. The EGF content in the SMG differ in the two sexes, being larger in males. This difference is attributed to male sex hormones. In female rats, testosterone treatment increases SMG EGF content to male levels w18x; in males gonadectomy reduces SMG EGF to female levels and treatment with testosterone reverses the change w18x. The influence of female sex hormones on EGF biosynthesis is less clear. Neither ovariectomy nor estrogen treatment in female rats influences the SMG EGF content w18x. In mice, however, ovariectomy increases SMG EGF content and estrogen treatment reduces the EGF content w19x. Progesterone like testosterone increases the EGF content. The thyroid hormones and cortisone also increase the SMG EGF content in neonatal and mature rats and mice w20–22x, and hypothyroidism may reduce SMG EGF content in mice w23x. Reduced EGF content of the SMG has also been reported in senescence of mice w24x, in streptozotocin-induced and genetic diabetes mellitus in mice w25x, and in experimental sialodacryoadenitis in female Wistar rats w26x. EGF might, on systemic administration exert a plethora of effects and influence the levels of the above
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mentioned hormones. We did not measure these hormones. In sheep, EGF administration increases the serum concentration of progesterone w27x, decreases the estrogen concentration w27x, reduces thyroid hormones w23x, and increases the cortisone concentration w28x. We have measured thyroid hormones in serum in a similarly treated set of male rats Žunpublished results. and in Goettingen minipigs w29x and found unchanged T4 and increased serum concentrations of T3. As discussed above, thyroid hormones increase the SMG EGF content, and one might have expected therefore that the SMG EGF content was increased in the EGF-treated animals. The fact that the SMG content of EGF was reduced suggests that systemic EGF exerts a direct effect on the SMG. This is supported by the observation that small amounts of EGF seems to be taken up by the SMG after intravenous administration w30x. Anticipating that systemic EGF exerts its effect via binding to EGF receptors in the SMG, the findings suggest that EGF receptor activation is involved in the growth and EGF biosynthesis of the gland. Such an effect should, however, not necessarily be confined to endogenous EGF in the gland but could as well be an effect of another locally produced EGF agonist Žfor example transforming growth factor alpha ŽTGFa ... SMG EGF most likely is not affected by circulating EGF under physiological circumstances, since the plasma contains either no or only very small concentrations of EGF in rats and other species of higher order w15,17x. The changes in the kidneys were more modest. The kidneys increased in weight in proportion to the increase in total body weight with no changes in the endogenous renal EGF production. The EGF production in the kidneys is regulated quite differently than in the SMG. For example, in rats and mice testosterone decreases and estrogens increase renal EGF, thyroid hormones increase and adrenal hormones either reduce or do not affect renal EGF w20,31,32x. As reported in most of the cited papers and corroborated in the present study, the endogenous EGF is much more responsive to external stimuli in the SMG than in the kidneys. The weight increases of the two organs were also different, the SMG increased disproportionately and the kidneys proportionately. EGF treatment during this period of life does not exert a generel trophic effect on the organism but influences different organ systems in different manners. We did not dissect and weigh all organs of the rats in the present study. We have previously reported data on male rats w33x treated for 4 weeks with EGF and have data from a treatment series with female rats very similar to the rats of the present paper Ždata not shown.. In brief, EGF treatment caused disproportionate growth of the gastrointestinal and urogenital tracts, as compared with the heart and kidneys, and relative atrophy of perirenal fat. In conclusion, prolonged systemic treatment with heterologous recombinant human EGF in the rat affects the two organs with the largest endogenous EGF production differ-
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ently. EGF causes enlargement of the SMG with reduced EGF content, and enlargement of the kidneys with unchanged endogenous EGF content and excretion.
Acknowledgements This study was supported by The Danish Medical Research Council Ž12-1317.. The recombinant EGF was a generous gift from Professor Esam Z. Dajani, International Drug Development Consultants, a Division of Mid Gulf, USA Inc. ŽLong Grove, IL, USA.. The technicians Ole Sørensen, Torben Madsen, Department of Pathology Aalborg Hospital Denmark, and the laboratory assistants Jette Hansen, Inge Marie Jensen and Lisbeth Jensen, Department of Clinical Biochemistry, KH Aarhus University Hospital are thanked for their handling of the animals and the biochemical procedures, respectively.
References w1x Prigent, S.A. and Lemoine, N.R., The type 1 ŽEGFR-related. family of growth factor receptors and their ligands, Prog. Growth Factor Res., 4 Ž1992. 1–24. w2x Barnard, J.A., Graves Deal, R., Pittelkow, M.R., DuBois, R., Cook, P., Ramsey, G.W., Bishop, P.R., Damstrup, L. and Coffey, R.J., Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family, J. Biol. Chem., 269 Ž1994. 22817– 22822. w3x Juhl, C.O., Vinter-Jensen, L., Jensen, L.S., Nexø, E., Djurhuus, J.C. and Dajani, E.Z., Recombinant human epidermal growth factor accelerates healing of sclerotherapy-induced esophageal ulcers and prevents esophageal stricture formation in pigs, Dig. Dis. Sci., 39 Ž1994. 2671–2678. w4x Humes, H.D., Cieslinski, D.A., Coimbra, T.M., Messana, J.M. and Galvao, C., Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure, J. Clin. Invest., 84 Ž1989. 1757–1761. w5x Barrow, R.E., Wang, C.Z., Evans, M.J. and Herndon, D.N., Growth factors accelerate epithelial repair in sheep trachea, Lung, 171 Ž1993. 335–344. w6x Gresik, E.W., Gubits, R.M. and Barka, T., In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse, J. Histochem. Cytochem., 33 Ž1985. 1235–1240. w7x Salido, E.C., Lakshmanan, J., Fisher, D.A., Shapiro, L.J. and Barajas, L., Expression of epidermal growth factor in the rat kidney. An immunocytochemical and in situ hybridization study, Histochemistry, 96 Ž1991. 65–72. w8x Olsen, P.S., Kirkegaard, P., Poulsen, S.S. and Nexø, E., Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor, Gut, 25 Ž1984. 1234–1240. w9x Jørgensen, P.E., Hilchey, S.D., Nexø, E., Poulsen, S.S. and Quilley, C.P., Urinary epidermal growth factor is excreted from the rat isolated perfused kidney in the absence of plasma, J. Endocrinol., 139 Ž1993. 227–234. w10x Olsen, P.S., Poulsen, S.S., Kirkegaard, P. and Nexø, E., Role of submandibular saliva and epidermal growth factor in gastric cytoprotection, Gastroenterology, 87 Ž1984. 103–108. w11x Messing, E.M., Hanson, P., Ulrich, P. and Erturk, E., Epidermal growth factor – interactions with normal and malignant urothelium: in vivo and in situ studies, J. Urol., 138 Ž1987. 1329–1335.
w12x Jørgensen, P.E., Nexø, E. and Poulsen, S.S., The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes, Biochim. Biophys. Acta, 1074 Ž1991. 284–288. w13x Jørgensen, P.E., Vinter-Jensen, L. and Nexø, E., An immunoassay designed to quantitate different molecular forms of rat urinary epidermal growth factor with equimolar potency: application on fresh rat urine, Scand. J. Clin. Lab. Invest., 56 Ž1996. 25–36. w14x Nexø, E., Jørgensen, P.E., Thim, L. and Roepstorff, P., Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine, Biochim. Biophys. Acta, 1037 Ž1990. 388–393. w15x Nexø, E., Jørgensen, P.E. and Hansen, M.R., Human epidermal growth factor - on molecular forms present in urine and blood, Regul. Pept., 42 Ž1992. 75–84. w16x Vinter-Jensen, L., Frøkiær, J., Jørgensen, P.E., Marqversen, J., Rehling, M., Dajani, E.Z. and Nexø, E., Tissue distribution of 131 I-epidermal growth factor in the pig visualized by dynamic scintigraphy, J. Endocrinol., 144 Ž1995. 5–12. w17x Jørgensen, P.E., Rasmussen, T.N., Skov Olsen, P., Raaberg, L., Seier Poulsen, S. and Nexø, E., Renal uptake and excretion of epidermal growth factor from plasma in the rat, Regul. Pept., 28 Ž1990. 273–281. w18x Hiramatsu, M., Kashimata, M., Takayama, F. and Minami, N., Developmental changes in and hormonal modulation of epidermal growth factor concentration in the rat submandibular gland, J. Endocrinol., 140 Ž1994. 357–363. w19x Pascall, J.C., Saunders, J., Blakeley, D.M., Laurie, M.S. and Brown, K.D., Tissue-specific effects of castration and ovariectomy on murine epidermal growth factor and its mRNA, J. Endocrinol., 121 Ž1989. 501–506. w20x Schaudies, R.P., Grimes, J., Davis, D. and Koldovsky, O., Modulation of immunoreactive epidermal growth factor levels in the submandibular gland, pancreas, liver, kidney and gastrointestinal tract of suckling rats by cortisone and tri-iodothyronine, J. Endocrinol., 131 Ž1991. 95–100. w21x Perheentupa, J., Lakshmanan, J. and Fisher, D.A., Epidermal growth factor in neonatal mouse urine: maturative effect of thyroxine, Pediatr. Res., 18 Ž1984. 1080–1084. w22x Fujieda, M., Murata, Y., Hayashi, H., Kambe, F., Matsui, N. and Seo, H., Effect of thyroid hormone on epidermal growth factor gene expression in mouse submandibular gland, Endocrinology, 132 Ž1993. 121–125. w23x Corcoran, J.M., Waters, M.J., Eastman, C.J. and Jorgensen, G., Epidermal growth factor: effect on circulating thyroid hormone levels in sheep, Endocrinology, 119 Ž1986. 214–217. w24x Dagogo Jack, S., Age related changes in immunoreactive epidermal growth factor concentrations in murine thyroid and submaxillary glands, Endocrinol. Regul., 25 Ž1991. 221–224. w25x Kasayama, S., Ohba, Y. and Oka, T., Epidermal growth factor deficiency associated with diabetes mellitus, Proc. Natl. Acad. Sci. USA, 86 Ž1989. 7644–7648. w26x Percy, D.H., Hayes, M.A., Kocal, T.E. and Wojcinski, Z.W., Depletion of salivary gland epidermal growth factor by sialodacryoadenitis virus infection in the Wistar rat, Vet. Pathol., 25 Ž1988. 183–192. w27x Murray, J.F., Downing, J.A., Evans, G., Findlay, J.K. and Scaramuzzi, R.J., Epidermal growth factor acts directly on the sheep ovary in vivo to inhibit oestradiol-17beta and inhibin secretion and enhance progesterone secretion, J. Endocrinol., 137 Ž1993. 253–264. w28x Singh Asa, P. and Waters, M.J., Stimulation of adrenal cortisol biosynthesis by epidermal growth factor, Mol. Cell Endocrinol., 30 Ž1983. 189–199. w29x Vinter-Jensen, L., Juhl C.O., Frystyk, J., Dajani, E.Z., Oksbjerg, N., Flyvbjerg, A., The effect of epidermal growth factor ŽEGF. on circulating levels of insulin-like growth factor ŽIGF. and IGF binding proteins ŽIGFBPs. in adult Goettingen minipigs, J. Endocrinol., In press.
L. Vinter-Jensen et al.r Regulatory Peptides 67 (1996) 179–185 w30x Jørgensen, P.E., Poulsen, S.S. and Nexø, E., Distribution of i.v. administered epidermal growth factor in the rat, Regul. Pept., 23 Ž1988. 161–169. w31x Gubits, R.M., Shaw, P.A., Gresik, E.W., Onetti Muda, A. and Barka, T., Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland, Endocrinology, 119 Ž1986. 1382–1387. w32x Tuomela, T., Miettinen, P., Viinikka, L. and Perheentupa, J., Estro-
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gen-androgen antagonism in the regulation of epidermal growth factor in mouse submandibular salivary gland and kidneys, Life Sci., 47 Ž1990. 1925–1932. w33x Vinter-Jensen, L., Smerup, M., Jørgensen, P.E., Juhl, C.O., Ørntoft, T., Poulsen, S.S. and Nexø, E., Chronic treatment in the rat with epidermal growth factor stimulates growth of the urinary tract, Urol. Res., 24 Ž1996. 15–21.
The effects of chronic administration of epidermal growth factor ž EGF/ to rats on the levels of endogenous EGF in the submandibular glands and kidneys Lars Vinter-Jensen a
a,b,)
, Per E. Jørgensen a , Steen Seier Poulsen c , Ebba Nexø
a
Department of Clinical Biochemistry, KH Aarhus UniÕersity Hospital, Nørrebrogade 44, DK-8000 Aarhus, Denmark b Department of Pathology, Aalborg Hospital, Aalborg, Denmark c Institute of Medical Anatomy, The Panum Institute, UniÕersity of Copenhagen, Copenhagen, Denmark Received 5 February 1996; revised 26 August 1996; accepted 26 August 1996
Abstract Epidermal growth factor ŽEGF. is mainly produced in the submandibular glands ŽSMG. and in the kidneys. It has recently been reported that EGF-related ligands may induce their own biosynthesis Žautoinduction. in vitro. In the present paper, we investigated whether chronic systemic treatment with EGF influenced the amount of endogenous EGF in the SMG and kidneys. Eight-week-old female Wistar rats were treated with subcutaneous injections of placebo Ž n s 16. or human recombinant EGF Ž150 mgrkg per day, n s 8. for 4 weeks. Urine was sampled the last 24 h of the study period. At the time of killing, the SMG and the kidneys were removed. The SMG was larger in the EGF-treated animals, 229.8 " 35.5 Žmean " SD. mg than in the control animals, 181.7 " 18.1 mg Ž P - 0.01.. The total EGF content was smaller Ž0.51 " 0.15 vs. 1.12 " 0.40 nmol EGFrSMG, P - 0.001.. The kidneys were larger in the EGF-treated animals Ž1.38 " 0.08 vs. 1.28 " 0.08 g, P - 0.05., but the EGF content and urinary excretions were not changed. In conclusion, chronic systemic treatment with EGF causes growth of the SMG with concomitantly reduced contents of EGF, and growth of the kidneys with unchanged content and excretion of EGF. These findings suggest that EGF may play a part in the regulation of the growth of the SMG and in EGF biosynthesis. Keywords: Keywords please
1. Introduction Epidermal growth factor ŽEGF. and related ligands ŽEGF agonists. exert their action via binding to the EGF receptor w1x. Intestinal cells in culture upregulate the expression of EGF agonists when EGF or one of the EGF agonists activates the EGF receptor w2x. The production of these ligands may therefore partly be autoregulated by autocrine or paracrine mechanisms. Systemic treatment with EGF prevents tissue damage and accelerates healing of experimentally induced damage to various organs w3–5x, and treatment with EGF may become a future option in human therapeutics. From a pharmacologic point of view, it is therefore interesting to know how EGF administration in vivo affects EGF biosynthesis. EGF is synthesized by the granular convoluted tubular )
Corresponding author. Fax: Ž45-89. 493-060.
cells ŽGCT. of the submandibular gland ŽSMG. w6x, and by the cells of the thick ascending limb of Henle and early part of the distal convoluted tubules in the kidney w7x. EGF is secreted in an exocrine manner to saliva w8x and to urine w9x and EGF probably exerts trophic effects on the epithelia with which is in contact w10,11x. In the present paper, we have investigated the effects of chronic systemic administration of EGF on organ size and tissue EGF content at the two principal sites of EGF biosynthesis, namely, the SMG and the kidneys.
2. Materials and methods 2.1. Study animals Twenty-four female 8-week-old Wistar rats from our own breed ŽDepartment of Pathology, Aalborg Hospital,
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Denmark., were used. The animals were housed individually in cages on white special spanwall bedding Žtemperature 218C, humidity 55 " 5%, darkrlight cycle 12-h shift.. They were fed a standard laboratory diet, ŽAltromin 1314.. The animals were kept in the same room throughout the study and handled by the same two technicians, only. The study complied with Danish regulations for care and use of laboratory animals. 2.2. Study design The 24 animals were randomly allocated into 2 groups. They received either solvent Žisotonic saline. Ž n s 16. or human recombinant EGF, 150 mgrkg per day Ž n s 8; Upstate Biotechnology, New York, NY, USA. by two daily subcutaneous injections for 4 weeks. The total body weight was determined once a week. Urine was collected in metabolic cages for the last 24 h of the study period. After 28 days of treatment, the animals were anaesthetized with Ketamine ŽKetalar w , Parke Davis, Barcelona, Spain. 100 mgrkg and xylosin ŽRompun w , Bayer, Leverkusen, Germany. 15 mgrkg intramuscularly. Blood samples from the retrobulbar venous plexus were collected through heparinized capillary tubes. One SMG Žincluding the sublingual gland. and the kidneys were removed, weighed, snap-frozen in liquid nitrogen, and stored at y808C for later analysis. The other SMG was fixed in phosphate-buffered paraformaldehyde Ž4%, pH 7.0. The animals were killed thereafter. The procedures described above were carried out on a blinded basis. For some of the procedures described below, only a subgroup of the control animals was included. The control animals in the subgroups were randomly chosen. 2.3. Tissue preparation The SMG was homogenized in 10 volumes of distilled water. The homogenate was centrifuged Ž20 000 = g, 40 min, 48C., and supernatant 1 was stored at y208C. The pellet was resuspended and homogenized in 2 ml 50 mM acetic acid. This homogenate was also centrifuged Ž20 000 = g, 40 min, 48C. and supernatant 2 was stored at y208C. The total contents of EGF and protein in SMG were calculated as the sum of the contents in the two supernatants. In the SMG, EGF is present as soluble EGF in secretory granules. This EGF is released by homogenization, and it is therefore possible to measure the amount of EGF per gland. In the kidney, EGF is present in the form of a big membrane bound precursor, and it is not released by homogenization. It is, however, possible to release the renal EGF from a membrane preparation by trypsinization and the renal EGF content per mg membrane protein can thereby be calculated. Kidney membranes were isolated by differential centrifugation, essentially as described w12x. In
brief, kidneys were homogenized on ice in 0.25 M sucrose, 25 mM Tris-HCl ŽpH 7.4., 5 ml per g kidney. The homogenate was centrifuged to remove intact cells Ž600 = g, 10 min, 48C., and the resulting supernatant was recentrifuged Ž18 000 = g, 30 min, 48C.. The membrane fraction of the pellet was resuspended in the supernatant and adjusted to 0.1 M in NaCl and 0.2 mM in MgSO4 before centrifugation Ž35 000 = g, 40 min, 48C.. The pellet was washed three times by resuspension with 25 mM Tris-HCl ŽpH 7.4., and harvested by centrifugation. The membranes were suspended in 25 mM Tris-HCl ŽpH 7.4.. 2.4. Biochemical analyses 2.4.1. Rat EGF Rat EGF was measured by ELISA either without ŽSMG. or after pretreatment with trypsin Žurine and kidneys. as previously described w13x. The urine samples were trypsinized with 3.13 mg trypsinrml whereas the renal membrane preparations were diluted in 25 mM Tris ŽpH 7.4. so that the total protein concentration was 3 mgrml prior to trypsinization with 31.3 mg trypsinrml. Pretreatment with trypsin converts the various molecular weight forms of rat urinary EGF to a single molecular form that corresponds to the 48 amino acid form of EGF present in the rat submandibular glands w14x. This ensures that the variety of molecular weight forms of EGF in urine and kidney tissue are measured with equimolar potency w13x. Human EGF does not cross-react with rat EGF in these assays w13x. 2.4.2. Other biochemical analysis The total protein concentration was measured with a kit, BCA Protein Assay Reagent ŽPIERCE.. Human EGF in urine and in plasma was measured by a previously described ELISA for human EGF w15x. Creatinine in urine was analysed on a Kodak, Ektachem 500. 2.5. Immunohistochemistry for endogenous EGF in the SMG After finding reduced concentrations and contents of endogenous EGF in the SMG, a semiquantitative immunohistochemical analysis of the GCT cells was performed on sections from 5 controls and 6 EGF-treated animals. Fivemm thick paraformaldehyde-fixed, paraffin-embedded sections were employed. The deparaffinized sections were preincubated with 10% porcine serum ŽDakopatt, Copenhagen, Denmark. in phosphate-buffered albumin for 30 min. The serum was removed and sections were incubated over-night with rabbit serum against rat submandibular EGF ŽNo. 813 raised in our lab.. The immunoreactions were visualized by means of the peroxidase-antiperoxidase technique, using swine-antirabbit IgG and peroxidase-anti-
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peroxidase ŽDakopatt. and diaminobenzidine as chromogen. The number of immunostained GCT cells and the volume fraction of the GCT cells were estimated by means of an Olympus BH-2 microscope with projection to a monitor giving a total magnification X1550. An area of submandibular tissue Žexcluding sublingual tissue. of 22 mm2 Žmean. was defined and the GCT cells were counted in 140 Žmean. randomly chosen fields of vision of 0.0195 mm2 . The number of cells per mm2 was thereafter calculated. GCT cells appear very often in clusters and are therefore difficult to delineate from each other. The fraction of the tissue occupied by the immunoreaction for rat EGF was therefore also determined in the same fields of vision by means of point counting Ža mean of 32 000 points was employed per animal.. 2.6. Statistical analyses Results are expressed as means and standard deviations ŽSD.. Mann-Whitneys non-parametric test was employed to test differences between groups. The level of significance chosen was 0.05.
3. Results 3.1. General obserÕations All animals thrived throughout the study. The animals increased in weight from 172 " 5 to 203 " 11 g in the control group and from 172 " 3 g to 213 " 8 g in the EGF-treated group. The total body weight was significantly larger in the EGF-treated group at the end of study Ž P s 0.02.. 3.2. The submandibular gland (SMG) The right SMG was increased in weight from 181.7 " 18.1 mg in the control animals to 229.8 " 35.5 mg in the EGF-treated animals Ž P s 0.004., Fig. 1. This increase was proportionately larger than the increase in total body weight Ž P s 0.02.. The total protein content increased from 11.4 " 1.5 mg in the controls to 13.5 " 1.9 mg in the EGF animals Ž P s 0.010., whereas the total rat EGF content decreased from 1.12 " 0.40 to 0.51 " 0.15 nmol Ž P s 0.0003.. The amount of rat EGF per amount of protein was consequently reduced from 97 " 30 to 38 " 12 nmol rat EGFrg protein Ž P s 0.002.. The density of GCT cells was reduced from 54 cellsrmm2 in the controls to 23 cellsrmm2 in the EGF animals Ž P - 0.02., Fig. 2. The volume fraction of immunostaining for rat EGF was accordingly reduced from 1.4% to 0.5% Ž P - 0.02.. Based on the visual impression, the intensity of the immunostaining of the GCT cells in the EGF-treated animals was also reduced.
Fig. 1. Scatterplots of total weight and rat EGF contents in submandibular glands from female Wistar rats treated with placebo Ž`. or human recombinant EGF Ž150 mgrkg per day. Žv . for 4 weeks. The P-value for the Mann-Whitney test is indicated.
3.3. The kidneys The kidneys Žright plus left. were proportionately increased in weight from 1.28 " 0.08 g in the control group to 1.38 " 0.08 g in the EGF-treated group Ž P s 0.018.. The renal EGF content was not increased significantly Ž3.5 " 1.1 nmol EGFrg protein in the controls Ž n s 8. vs. 4.1 " 0.7 nmol EGFrg protein in the EGF-treated animals Ž n s 8. Ž P s 0.074... 3.4. Urinary excretion of EGF, creatinine and protein (Table 1) There were no changes in the total urine volume per 24 h. The total excretions of rat EGF, protein and creatinine were not increased significantly in the EGF-treated group. The EGFrcreatinine ratio and the EGFrprotein ratio were identical in the 24 h urinary samples in the two groups: 24 " 5 and 24 " 2 nmol EGFrmmol creatinine Žcontrols and EGF-treated group. and 24 " 6 and 23 " 2 nmol EGFrg protein Žcontrols and EGF-treated group.. Table 1 Urinary excretions of rat EGF, protein and creatinine in rats treated with isotonic saline Žcontrols. and rats treated with human recombinant EGF, 150 mgrkg per day, for 4 weeks ŽEGF group. Controls Ž ns12. Total volume Žmlr24 h. EGF Žnmolr24 h. Protein Žmgr24 h. Creatinine Žmmolr24 h.
9.8"2.9 1.80"0.31 78.7"15.2 76.0"16.4
EGF group Ž ns8. NS NS NS NS
Values are mean"SD. NS, no significant difference ŽMann-Whitney test..
10.9"1.2 2.00"0.21 87.5"7.2 83.0"10.3
Fig. 2. Immunostaining for rat EGF in a control rat ŽA, C. and in a rat treated for 4 weeks with EGF 150 mgrkg per day ŽB, D.. The reduced density and intensity of the immunostaining of the GCT cells are seen in the EGF animal. Magnification: A,B =90; C,D =360.
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A small fraction of parenterally administered EGF is excreted in urine w16,17x. Even though human EGF do not cross-react with rat EGF in the assays used to quantitate rat EGF w13x, we wanted to confirm that the urinary excretion of systemically administered human EGF was negligible compared to that of rat EGF. The mean concentration of human EGF was 1.2 nmolrl compared to 190 nmol rat EGFrl, i.e., less than 1%. 3.5. Human recombinant EGF in plasma There was no detectable human recombinant EGF in plasma at killing – week 12 h after the last injection of EGF. Human recombinant EGF was, however, excreted to urine and therefore absorbed from the site of injection. 4. Discussion The major finding of the present study is that prolonged systemic treatment with EGF increases the weight of the SMG and reduces the EGF content of the SMG. Interestingly, no change was observed in the EGF concentration within and EGF excretion of the other major EGF producing organ, the kidney. In the SMG, we found reduced concentration and content of EGF and reduced density, volume fraction and intensity of the immunostaining of the GCT cells. EGF biosynthesis was therefore most likely reduced. The EGF content of the SMG changes with age and is influenced by various hormones. In female Wistar rats, the EGF content increases gradually from birth until 8 weeks of age w18x. The EGF content then increases rapidly and reaches a plateau after week 12. The SMG therefore, might be especially sensitive to stimuli from week 8 to 12. We intervened with systemic EGF treatment in exactly this period of life. The EGF content in the SMG differ in the two sexes, being larger in males. This difference is attributed to male sex hormones. In female rats, testosterone treatment increases SMG EGF content to male levels w18x; in males gonadectomy reduces SMG EGF to female levels and treatment with testosterone reverses the change w18x. The influence of female sex hormones on EGF biosynthesis is less clear. Neither ovariectomy nor estrogen treatment in female rats influences the SMG EGF content w18x. In mice, however, ovariectomy increases SMG EGF content and estrogen treatment reduces the EGF content w19x. Progesterone like testosterone increases the EGF content. The thyroid hormones and cortisone also increase the SMG EGF content in neonatal and mature rats and mice w20–22x, and hypothyroidism may reduce SMG EGF content in mice w23x. Reduced EGF content of the SMG has also been reported in senescence of mice w24x, in streptozotocin-induced and genetic diabetes mellitus in mice w25x, and in experimental sialodacryoadenitis in female Wistar rats w26x. EGF might, on systemic administration exert a plethora of effects and influence the levels of the above
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mentioned hormones. We did not measure these hormones. In sheep, EGF administration increases the serum concentration of progesterone w27x, decreases the estrogen concentration w27x, reduces thyroid hormones w23x, and increases the cortisone concentration w28x. We have measured thyroid hormones in serum in a similarly treated set of male rats Žunpublished results. and in Goettingen minipigs w29x and found unchanged T4 and increased serum concentrations of T3. As discussed above, thyroid hormones increase the SMG EGF content, and one might have expected therefore that the SMG EGF content was increased in the EGF-treated animals. The fact that the SMG content of EGF was reduced suggests that systemic EGF exerts a direct effect on the SMG. This is supported by the observation that small amounts of EGF seems to be taken up by the SMG after intravenous administration w30x. Anticipating that systemic EGF exerts its effect via binding to EGF receptors in the SMG, the findings suggest that EGF receptor activation is involved in the growth and EGF biosynthesis of the gland. Such an effect should, however, not necessarily be confined to endogenous EGF in the gland but could as well be an effect of another locally produced EGF agonist Žfor example transforming growth factor alpha ŽTGFa ... SMG EGF most likely is not affected by circulating EGF under physiological circumstances, since the plasma contains either no or only very small concentrations of EGF in rats and other species of higher order w15,17x. The changes in the kidneys were more modest. The kidneys increased in weight in proportion to the increase in total body weight with no changes in the endogenous renal EGF production. The EGF production in the kidneys is regulated quite differently than in the SMG. For example, in rats and mice testosterone decreases and estrogens increase renal EGF, thyroid hormones increase and adrenal hormones either reduce or do not affect renal EGF w20,31,32x. As reported in most of the cited papers and corroborated in the present study, the endogenous EGF is much more responsive to external stimuli in the SMG than in the kidneys. The weight increases of the two organs were also different, the SMG increased disproportionately and the kidneys proportionately. EGF treatment during this period of life does not exert a generel trophic effect on the organism but influences different organ systems in different manners. We did not dissect and weigh all organs of the rats in the present study. We have previously reported data on male rats w33x treated for 4 weeks with EGF and have data from a treatment series with female rats very similar to the rats of the present paper Ždata not shown.. In brief, EGF treatment caused disproportionate growth of the gastrointestinal and urogenital tracts, as compared with the heart and kidneys, and relative atrophy of perirenal fat. In conclusion, prolonged systemic treatment with heterologous recombinant human EGF in the rat affects the two organs with the largest endogenous EGF production differ-
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ently. EGF causes enlargement of the SMG with reduced EGF content, and enlargement of the kidneys with unchanged endogenous EGF content and excretion.
Acknowledgements This study was supported by The Danish Medical Research Council Ž12-1317.. The recombinant EGF was a generous gift from Professor Esam Z. Dajani, International Drug Development Consultants, a Division of Mid Gulf, USA Inc. ŽLong Grove, IL, USA.. The technicians Ole Sørensen, Torben Madsen, Department of Pathology Aalborg Hospital Denmark, and the laboratory assistants Jette Hansen, Inge Marie Jensen and Lisbeth Jensen, Department of Clinical Biochemistry, KH Aarhus University Hospital are thanked for their handling of the animals and the biochemical procedures, respectively.
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gen-androgen antagonism in the regulation of epidermal growth factor in mouse submandibular salivary gland and kidneys, Life Sci., 47 Ž1990. 1925–1932. w33x Vinter-Jensen, L., Smerup, M., Jørgensen, P.E., Juhl, C.O., Ørntoft, T., Poulsen, S.S. and Nexø, E., Chronic treatment in the rat with epidermal growth factor stimulates growth of the urinary tract, Urol. Res., 24 Ž1996. 15–21.