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Renal uptake and excretion of epidermal growth factor from plasma in the rat
Regulatory Peptides, 28 (1990) 273-280 Elsevier
273
REGPEP 00908
Renal uptake and excretion of epidermal growth factor from plasma in the rat Per E. Jorgensen
1,3,4, Torben N. Rasmussen z, Peter Skov Olsen 2, Lasse
Raaberg 3, Steen Seier Poulsen 3 and Ebba Nexo 4 1Department of Urology D and 2Department of Gastroenterology C, Rigshospitalet, 31nstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen and 4Department of Clinical Chemistry, Central Hospital, Hillerod (Denmark)
(Received 31 July 1989; revised version received 25 January 1990; accepted 31 January 1990) Key words: Epidermal growth factor; Rat; Kidney; Blood; Urine; Metabolism
Summary The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary E G F might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous E G F from plasma. I.v. injected ~zsI-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12 ~o of the 125I-EGF. The kidneys seemed to degrade most of the ~25I-EGF which they accumulated from blood, as only 4~o of the injected label was excreted as intact ~25I-EGF in the urine. The amount of endogenous E G F in plasma was under the detection limit of our enzymelinked immunosorbent assay (0.03nmol/1) and it remained so after bilateral nephrectomy. Even if plasma E G F was 0.03 nmol/1 excretion of E G F from plasma could account for less than 5 ~o of the urinary EGF. This study shows that the kidneys are able to accumulate E G F from plasma and excrete a part of it as intact E G F in the urine. However, excretion of immunoreactive E G F from plasma can only account for a minor part of the urinary EGF.
Introduction Epidermal growth factor (EGF) is a growth-promoting peptide with a molecular weight of approximately 6 k D a [ 1 ]. In the digestive tract of the rat, E G F has been Correspondence: E. Nexo, Department of Clinical Chemistry, Hillerod Central Hospital, DK-3400 Hillerod, Denmark.
0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
274 localized to the submandibular glands, the duodenal Brunner's glands, and the Paneth cells of the small intestine [2]. E G F is secreted from these glands to the lumen of the digestive tract and E G F has been detected in saliva and in gastric and duodenal juice [3,4]. Rat urine contains nanomolar amounts of EGF. The kidneys synthesize E G F [2,5,6], but it is not known whether part of urinary E G F is derived from the circulation. This is a possibility since the kidneys accumulate a considerable part of i.v. injected E G F [7] and since the luminal reabsorption of E G F in the tubules is small [8,9]. The purpose of this study was to investigate the renal uptake and excretion of i.v. injected homologous E G F and to examine whether renal excretion of endogenous E G F from plasma contributes significantly to the urinary output of EGF.
Materials and Methods
Laboratory animals Male Wistar rats weighing 300-400 g were used. The rats were anaesthetized by intraperitoneal injection of 50 mg/kg methohexital (Brietal ®, Lilly, U.S.A.).
Experimental procedure Elimination of i. v. injected leSI-EGF from the circulation.
7 rats underwent bilateral nephrectomy and 7 controls were sham operated. Immediately after the operation all rats received a bolus injection in the inferior vena cava of 0.18 ml 125I-EGF (10 pmol) over a 3 s period. Blood samples of 0.30 ml were taken from the inferior vena cava 0.5, 1, 1.5, 2, 3 and 5 rain after the injection. Then the rats were sacrificed. The radioactivity of the blood samples was examined. Renal uptake of i.v. injected EGF. 19 rats were used. The rats were anaesthetized, the abdomen was opened through a midline incision, and the bladder was emptied. The rats received an injection of 0.30 ml saline with E G F into the inferior vena cava. 8 rats received 1 pmol 125I-EGF, and 7 rats received 1 pmol ~25I-EGF plus 440pmol unlabelled homologous submandibular EGF. 5 rain after the injection the rats were connected to a respirator and perfused through the left cardiac ventricle with isotonic saline at a rate of 100 ml/min for 2 min in order to remove blood from the circulation. For every rat the amount of perfusion fluid used was measured and 1 ml was investigated in order to estimate the radioactivity in the blood. Also the kidneys, the ureters, the bladder and the urine were removed for measurement of radioactivity. 4 rats were used for autoradiographic studies. These rats underwent the same procedure as described above. 2 rats received 6 pmol ~25I-EGF and 2 rats received 6 pmol 125I-EGF plus 440 pmol unlabelled EGF. 5 min after the injection, the rats were perfused with isotonic saline for 20 s followed by 4~o glutaraldehyde for 2 min. Autoradiographs from the kidneys were made as previously described [7]. Renal excretion ofi. v. injected leSI-EGF. In 6 rats a midline incision was made. The bladder was emptied and the urethra ligated. Then the rats received a bolus injection of 0.30 ml ~25I-EGF (1 pmol) into the inferior vena cava. The incision was sutured and the rats regained consciousness. 2 h later the rats were anaesthetized again and perfused with isotonic saline as described above. The kidneys, the bladder with the ureters, and
275 the urine produced during the 2 h were removed and kept at - 20 ° C for later measurement of radioactivity and the amount of intact 125I-EGF in the urine. Endogenous EGF in plasma and daily excretion of EGF in urine. 8 rats were placed in metabolic cages with free access to water and food. The 24 h volume of urine was measured, and the urine was stored at - 20 °C for later measurement of EGF. Then a blood samples of 2 ml was taken from the inferior vena cava and bilateral nephrectomy was performed. The blood sample was heparinized (50 IU/ml blood). Plasma was collected after centrifugation (3000 rpm, 10 min, 4 °C) and stored at - 20 °C for later measurement of EGF. 24 h later, a new blood sample was taken before the rats were sacrificed. In 8 other rats a blood sample was taken from the aorta close to the renal arteries. Plasma was obtained and treated as described above.
Preparation of EGF and z25I-EGF E G F was purified from rat submandibular glands [4] and iodinated as previously described employing the chloramine-T method [ 7 ]. The specific activity of the iodinated peptide was 450,000-850,000 cpm per pmol EGF.
Laboratory analyses The radioactivity of ~25I-EGF was measured in a V-spectrometer (Riacounter type 1200, MolsgSxd Medical, Denmark). Gel filtration was performed on a 11 × 450 mm G-50 Sephadex column (Pharmacia, Sweden) run in 0.1 M phosphate buffer, 0.1 ~o human albumin (Behringwerke, F.R.G.), 0.9~o NaC1 (pH 8.0) at a flow rate of 0.6 ml/min. Quantitation of endogenous EGF. E G F was quantitated with an enzyme-linked immunosorbent assay (ELISA) developed by us. EGF, purified from urine and quantirated by aminoacid analysis [10], was employed as calibrator in a concentration between 2.00 and 0.03 nmol/1 diluted in 0.1 M phosphate (pH 8.0), 1 ~o albumin. The IgG fraction from rabbit antiserum against rat submandibular E G F [4] was purified by precipitation with 2 M ammonium sulphate followed by ion exchange chromatography on DEAE Sephadex [ 11 ]. 0.3/~g IgG (antibody no 3123) in 50 #1 0.05 M carbonate (pH 9.6) was added to micro-titer plates with 96 wells (Nunc, Denmark). The plates were shaken at 700 rpm for 1 min, incubated for 20 h at 4 °C and washed twice with 200 #1 washing buffer (sodium chloride 0.5 M, sodium phoshate 0.008 M (pH 7.2), Triton X-100 10 ml/l). After addition of 200/A ethanolamin 1 M (pH 8.0) to each well, the plate was shaken at 500 rpm for 1 min, and incubated at 4 °C for 20 h. The plates were stored at - 20 °C until employed. For preparation of biotinylated IgG (antibody no 3124), 9 mg IgG in 3 ml 0.1 M phosphate, 0.154 M sodium chloride (pH 8.6) was mixed with 90 ~g biotin (Sigma, U.S.A.) dissolved in 45/~1 dimethylformamide (Merck, F.R.G.). After incubation for 4 h at room temperature the sample was dialyzed against phosphate buffer (pH 7.2) for 20 h at room temperature. Sodium azide was added (final concentration 0.02~o) and the biotinylated IgG was stored at 4 ° C. Prior to use, the plates were thawed and washed 4 times with washing buffer (standard washing procedure). Calibrators and samples (50 #1) were applied and the
276
plates were shaken at 500 rpm for 1 min (this shaking procedure was performed each time a new reagent was added) and incubated at 4 °C for 20 h. After washing, 50/A of biotinylated antibody dissolved in 0.1 M phosphate containing 0.1 ~o human albumin (pH 8.0) (corresponding to approximately 0.5 ~zg IgG) was added, and the plates were incubated for 1 h at room temperature. After washing 50/A peroxidase conjugated avidin (Dakopatts, Denmark) diluted 1:1000 in ethanolamin 0.5 M (pH 8.7) was added, and the plates were incubated in the dark for 1 h at room temperature. After washing, 50#1 1,2-phenylendiamin, dihydrochloride was added (2OPD-tablets (Dakopatts) dissolved in 6 ml citric acid phosphate buffer 0.1 M (pH 5.0) and 3/~1 perhydrol 30~o H 2 0 2 (Merck)), and the plates were incubated in the dark at room temperature for 10-30 min. The reaction was stopped with 150 #1 1 M sulphoric acid. The plates were measured at 492 nm employing 620 nm as reference on an EAR 400 SLT-labinstrument (Austria) provided with a Panasonic KX-P1091 printer (Japan). Serum samples were analysed undiluted. Urine was diluted 1 + 19 with 0.1 M phosphate (pH 8.0) prior to analysis. Detection limit of the assay was 0.03 nmol/l. The assay was linear for quantitation of urinary E G F in a concentration up to 1.1 nmol/1. Quantitation of a diluted urine pool sample measured in 19 runs over a period of 2 months was used for calculation of the precision. The coefficient of variation was 0.07 for a mean of 0.43 nmol/1. The assay recognized 1/~mol/l mouse E G F as 0.08 nmol/1, 0.6/~mol/1 human E G F as less than 0.03 nmol/l and 1/~mol/1 rat TGF~ as less than 0.03 nmol/1. Calculations and statistics
Results are given as medians and total ranges unless otherwise stated. The Mann-Whitney test was used for statistical evaluation. Values of P < 0.05 were considered significant. Results
After an i.v. bolus injection of iodine-labelled E G F to the rat, the radioactivity disappeared within a few minutes from the circulation (Fig. 1). The amount of radioactivity in the blood in the bilaterally nephrectomized rats was approximately 25~'o higher than in the controls. The amount of radioactivity present in the kidneys, the lower urinary tract (ureters, bladder and urine) and the blood in intact rats 5 min after the injection of ~25I-EGF and ~25I-EGF plus an excess of unlabelled E G F is shown in Table I. When an excess of unlabelled E G F was injected together with 125I-EGF the radioactivity of the kidneys decreased by approximately 50 ~o, but there was no decrease of the radioactivity in the lower urinary tract. The radioactivity in the blood did not change. The autoradiographic study showed the label in the kidneys to be located in the proximal tubules (Fig. 2). When ~25I-EGF was injected together with an excess of unlabelled EGF, no change in the distribution of label in the kidney could be identified by autoradiography. 2 h after an i.v. injection of ~25I-EGF to intact rats 13~o (8-16~o) of the label was recovered in the kidneys and in the lower urinary tract including the urine. Of the injected label 1.9'~, (1.7-2.0~o) was found in the kidneys, 0.1~o (0.0-0.4~o) in the
277 Cpm x 1 0 3 in 0.3 ml blood
40
•
30
C~ C o n t r o l group
Bilateral
nephrectorny group
20
10 e-
6-
4-
2" II M i n u t e s
Fig. 1. The effect of bilateral nephrectomy on the removal of homologous ~25I-EGFfrom blood in rats after an i.v. bolus injection. The values shown are the medians and quartiles. b l a d d e r tissue and the ureters, and 11~o (6-14~o) in the urine. U p o n gel filtration, the label present in the urine eluted as one p e a k c o r r e s p o n d i n g to native 125I-EGF a n d one p e a k c o r r e s p o n d i n g to d e g r a d e d 125I-EGF (Fig. 3). The first p e a k represented 42~o ( 3 4 - 5 9 ~o) of the label in the urine. Relative to the injected a m o u n t of radioactivity 4~o ( 2 - 8 ~ o ) was recovered as l z s I - E G F in the urine. W e quantitated the endogenous a m o u n t o f E G F present in urine and p l a s m a employing our E L I S A method. The a m o u n t o f E G F in the 24 h urine was 2.2 n m o l ( 1 . 5 - 3 . 2 nmol). F o r all rats studied (n = 8) the concentration o f E G F in venous p l a s m a was below the detection limit o f the E L I S A (0.03 nmol/1) both before and 24 h after bilateral nephrectomy. The arterial p l a s m a samples from 8 other rats was also below 0.03 nmol/1. TABLE I The distribution of 125I-EGFin the kidneys, the lower urinary tract (bladder, ureters, urine) and the blood of the rat 5 min after an i.v. bolus injection of ~25I-EGFand 125I-EGFplus 440 pmol unlabelled homologous EGF
Kidneys Lower urinary tract Blood
1 pmol JasI-EGF
1 pmo1125I-EGF + 440 pmol EGF
12~o (10-17%) 0.9% (0.1-3.4%) 7% (5-10%)
6~o b (2-6 ~/o) 2.4% a (1.2-4.6%) 7% a (6-10%)
a Not significantly different from the 1 pmol ~251-EGFgroup. b Significantly different from the 1 pmol ~25I-EGFgroup (P < 0.005). The values shown are the percentages of the injected amount of label (medians and total ranges).
279 Cpm Vo
VEG~
Vt
3000
2000.
1000,
0
5'o
16o m Frmction
Fig. 3. Sephadex G-50 gel filtration of urine produced during the first 2 h after an i.v. bolus injection of homologous ~2SI-EGFin rats. Of the label in the urine 4290 eluted as native ~25I-EGF.
Discussion
In the present study we find i.v. injected homologous 12~I-EGF to be removed from the circulation within a few minutes in the rat, and we thereby confirm studies performed in dogs with heterologous E G F [ 12,13]. 125I-EGF is removed quickly from the circulation also in bilaterally nephrectomized rats, indicating that other organs than the kidneys are able to eliminate E G F from blood. This is in accordance with studies which show that the liver has a high capacity for removal of E G F from the circulation [ 14]. In rats the kidneys accumulate 12~o of i.v. injected trace amounts of 125I-EGF. Approximately 40~o of the accumulated label is recovered in the urine as intact ~25I-EGF. This relatively high amount of accumulated E G F recovered in urine might be explained by the unique handling of E G F by the nephrone. Having a molecular weight of 6 kDa, E G F is filtrated in the glomeruli. Unlike most other small peptides which are reabsorbed in the proximal tubules, E G F seems to remain in the ultrafiltrate [8,9]. The renal accumulation of ~25I-EGF decreases when trace amounts of ~2SI-EGF is injected together with excess of unlabelled EGF. This supports the existence of saturable E G F receptors in the kidneys [15]. In vitro studies have demonstrated E G F receptors in the basolateral membranes of kidney cells from rabbit and dog [ 16,17], and in the rabbit a transtubular transport of E G F from the peritubular compartment to the luminal compartment has been demonstrated [9]. Thus the renal uptake of E G F from plasma seems to consist of both glomerular filtration and a saturable receptor dependent uptake. A renal vascular constriction might contribute to the reduced renal accumulation of ~25I-EGF when trace amounts of 12SI-EGF are injected together with 400 pmol unlabelled EGF. In a recent study on rats, a constant intrarenal arterial administration of approximately 20 pmol EGF/min reduced the renal plasma flow by 20 ~/o and the
280
glomerular filtration rate by 40~o [18]. However, such a vascular constriction is not likely to be the only explanation of the reduced renal accumulation of 125I-EGF in our study because we find no decrease of the radioactivity in the lower urinary tract when 12SI-EGF is injected together with excess of unlabelled EGF. In human venous plasma, only small amounts of endogenous E G F have been found, whereas serum may contain E G F released from the platelets during coagulation [ 19-21 ]. The level of endogenous E G F in venous plasma in the rat is below 0.03 nmol/l. This low level of E G F in venous plasma is not caused by an effective renal clearance of EGF, as venous plasma E G F remains below 0.03 nmol/1 after bilateral nephrectomy, and as arterial plasma E G F is also below 0.03 pmol/ml. In order to calculate the contribution of plasma E G F to the total urinary output of the peptide one needs information about the urinary output of E G F and information about the amount of E G F reaching the kidneys by the circulation. The urinary output of E G F per 24 h is around 2.2 nmol. As the concentration of E G F in arterial plasma is below 0.03 nmol/l the maximal amount of E G F reaching the kidneys by the circulation can be calculated to be 222 pmol per 24 h. In this calculation we have used the following values for the rat: plasma E G F 0.03 pmol/ml, cardiac output 50 ml/min, fraction of cardiac output to the kidneys 19~o, and packed cell volume 46~o [22,23]: 222 pmol/24 h = 50 mlblood/min x 0.19 x 0.54 mlplasma/ml blood x 0.03 pmol/ml x 1440 min/24 h. If the 222 pmol E G F is entirely accumulated in the kidneys and 42~o of the 222 pmol E G F is excreted as intact E G F in the urine it will account for 93 pmol or 4~o of the daily urinary output of EGF. In conclusion this study shows that the kidneys are able to accumulate homologous E G F from plasma and excrete part of it as intact E G F in the urine. However, excretion of immunoreactive E G F from plasma can only account for a minor part of the E G F found in urine.
Acknowledgements This study was supported in part by a grant from the Danish Cancer Society (88-090). The technical assistance of Marianne R. Hansen, Kamma Velin and Henny Falsing is warmly acknowledged.
References 1 Marti, U., Burwen, S.J. and Jones, A.L., Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: An update, Hepatology, 9 (1989) 126-138. 2 Poulsen, S. S., Nexo, E., Skov Olsen, P., Hess, J. and Kirkegaard P., Immunohistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 3 Gregory, H., Walsh, S. and Hopkins, C. R., The identification ofurogastrone in serum, saliva, and gastric juice, Gastroenterology, 77 (1979) 313-318.
273
REGPEP 00908
Renal uptake and excretion of epidermal growth factor from plasma in the rat Per E. Jorgensen
1,3,4, Torben N. Rasmussen z, Peter Skov Olsen 2, Lasse
Raaberg 3, Steen Seier Poulsen 3 and Ebba Nexo 4 1Department of Urology D and 2Department of Gastroenterology C, Rigshospitalet, 31nstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen and 4Department of Clinical Chemistry, Central Hospital, Hillerod (Denmark)
(Received 31 July 1989; revised version received 25 January 1990; accepted 31 January 1990) Key words: Epidermal growth factor; Rat; Kidney; Blood; Urine; Metabolism
Summary The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary E G F might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous E G F from plasma. I.v. injected ~zsI-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12 ~o of the 125I-EGF. The kidneys seemed to degrade most of the ~25I-EGF which they accumulated from blood, as only 4~o of the injected label was excreted as intact ~25I-EGF in the urine. The amount of endogenous E G F in plasma was under the detection limit of our enzymelinked immunosorbent assay (0.03nmol/1) and it remained so after bilateral nephrectomy. Even if plasma E G F was 0.03 nmol/1 excretion of E G F from plasma could account for less than 5 ~o of the urinary EGF. This study shows that the kidneys are able to accumulate E G F from plasma and excrete a part of it as intact E G F in the urine. However, excretion of immunoreactive E G F from plasma can only account for a minor part of the urinary EGF.
Introduction Epidermal growth factor (EGF) is a growth-promoting peptide with a molecular weight of approximately 6 k D a [ 1 ]. In the digestive tract of the rat, E G F has been Correspondence: E. Nexo, Department of Clinical Chemistry, Hillerod Central Hospital, DK-3400 Hillerod, Denmark.
0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
274 localized to the submandibular glands, the duodenal Brunner's glands, and the Paneth cells of the small intestine [2]. E G F is secreted from these glands to the lumen of the digestive tract and E G F has been detected in saliva and in gastric and duodenal juice [3,4]. Rat urine contains nanomolar amounts of EGF. The kidneys synthesize E G F [2,5,6], but it is not known whether part of urinary E G F is derived from the circulation. This is a possibility since the kidneys accumulate a considerable part of i.v. injected E G F [7] and since the luminal reabsorption of E G F in the tubules is small [8,9]. The purpose of this study was to investigate the renal uptake and excretion of i.v. injected homologous E G F and to examine whether renal excretion of endogenous E G F from plasma contributes significantly to the urinary output of EGF.
Materials and Methods
Laboratory animals Male Wistar rats weighing 300-400 g were used. The rats were anaesthetized by intraperitoneal injection of 50 mg/kg methohexital (Brietal ®, Lilly, U.S.A.).
Experimental procedure Elimination of i. v. injected leSI-EGF from the circulation.
7 rats underwent bilateral nephrectomy and 7 controls were sham operated. Immediately after the operation all rats received a bolus injection in the inferior vena cava of 0.18 ml 125I-EGF (10 pmol) over a 3 s period. Blood samples of 0.30 ml were taken from the inferior vena cava 0.5, 1, 1.5, 2, 3 and 5 rain after the injection. Then the rats were sacrificed. The radioactivity of the blood samples was examined. Renal uptake of i.v. injected EGF. 19 rats were used. The rats were anaesthetized, the abdomen was opened through a midline incision, and the bladder was emptied. The rats received an injection of 0.30 ml saline with E G F into the inferior vena cava. 8 rats received 1 pmol 125I-EGF, and 7 rats received 1 pmol ~25I-EGF plus 440pmol unlabelled homologous submandibular EGF. 5 rain after the injection the rats were connected to a respirator and perfused through the left cardiac ventricle with isotonic saline at a rate of 100 ml/min for 2 min in order to remove blood from the circulation. For every rat the amount of perfusion fluid used was measured and 1 ml was investigated in order to estimate the radioactivity in the blood. Also the kidneys, the ureters, the bladder and the urine were removed for measurement of radioactivity. 4 rats were used for autoradiographic studies. These rats underwent the same procedure as described above. 2 rats received 6 pmol ~25I-EGF and 2 rats received 6 pmol 125I-EGF plus 440 pmol unlabelled EGF. 5 min after the injection, the rats were perfused with isotonic saline for 20 s followed by 4~o glutaraldehyde for 2 min. Autoradiographs from the kidneys were made as previously described [7]. Renal excretion ofi. v. injected leSI-EGF. In 6 rats a midline incision was made. The bladder was emptied and the urethra ligated. Then the rats received a bolus injection of 0.30 ml ~25I-EGF (1 pmol) into the inferior vena cava. The incision was sutured and the rats regained consciousness. 2 h later the rats were anaesthetized again and perfused with isotonic saline as described above. The kidneys, the bladder with the ureters, and
275 the urine produced during the 2 h were removed and kept at - 20 ° C for later measurement of radioactivity and the amount of intact 125I-EGF in the urine. Endogenous EGF in plasma and daily excretion of EGF in urine. 8 rats were placed in metabolic cages with free access to water and food. The 24 h volume of urine was measured, and the urine was stored at - 20 °C for later measurement of EGF. Then a blood samples of 2 ml was taken from the inferior vena cava and bilateral nephrectomy was performed. The blood sample was heparinized (50 IU/ml blood). Plasma was collected after centrifugation (3000 rpm, 10 min, 4 °C) and stored at - 20 °C for later measurement of EGF. 24 h later, a new blood sample was taken before the rats were sacrificed. In 8 other rats a blood sample was taken from the aorta close to the renal arteries. Plasma was obtained and treated as described above.
Preparation of EGF and z25I-EGF E G F was purified from rat submandibular glands [4] and iodinated as previously described employing the chloramine-T method [ 7 ]. The specific activity of the iodinated peptide was 450,000-850,000 cpm per pmol EGF.
Laboratory analyses The radioactivity of ~25I-EGF was measured in a V-spectrometer (Riacounter type 1200, MolsgSxd Medical, Denmark). Gel filtration was performed on a 11 × 450 mm G-50 Sephadex column (Pharmacia, Sweden) run in 0.1 M phosphate buffer, 0.1 ~o human albumin (Behringwerke, F.R.G.), 0.9~o NaC1 (pH 8.0) at a flow rate of 0.6 ml/min. Quantitation of endogenous EGF. E G F was quantitated with an enzyme-linked immunosorbent assay (ELISA) developed by us. EGF, purified from urine and quantirated by aminoacid analysis [10], was employed as calibrator in a concentration between 2.00 and 0.03 nmol/1 diluted in 0.1 M phosphate (pH 8.0), 1 ~o albumin. The IgG fraction from rabbit antiserum against rat submandibular E G F [4] was purified by precipitation with 2 M ammonium sulphate followed by ion exchange chromatography on DEAE Sephadex [ 11 ]. 0.3/~g IgG (antibody no 3123) in 50 #1 0.05 M carbonate (pH 9.6) was added to micro-titer plates with 96 wells (Nunc, Denmark). The plates were shaken at 700 rpm for 1 min, incubated for 20 h at 4 °C and washed twice with 200 #1 washing buffer (sodium chloride 0.5 M, sodium phoshate 0.008 M (pH 7.2), Triton X-100 10 ml/l). After addition of 200/A ethanolamin 1 M (pH 8.0) to each well, the plate was shaken at 500 rpm for 1 min, and incubated at 4 °C for 20 h. The plates were stored at - 20 °C until employed. For preparation of biotinylated IgG (antibody no 3124), 9 mg IgG in 3 ml 0.1 M phosphate, 0.154 M sodium chloride (pH 8.6) was mixed with 90 ~g biotin (Sigma, U.S.A.) dissolved in 45/~1 dimethylformamide (Merck, F.R.G.). After incubation for 4 h at room temperature the sample was dialyzed against phosphate buffer (pH 7.2) for 20 h at room temperature. Sodium azide was added (final concentration 0.02~o) and the biotinylated IgG was stored at 4 ° C. Prior to use, the plates were thawed and washed 4 times with washing buffer (standard washing procedure). Calibrators and samples (50 #1) were applied and the
276
plates were shaken at 500 rpm for 1 min (this shaking procedure was performed each time a new reagent was added) and incubated at 4 °C for 20 h. After washing, 50/A of biotinylated antibody dissolved in 0.1 M phosphate containing 0.1 ~o human albumin (pH 8.0) (corresponding to approximately 0.5 ~zg IgG) was added, and the plates were incubated for 1 h at room temperature. After washing 50/A peroxidase conjugated avidin (Dakopatts, Denmark) diluted 1:1000 in ethanolamin 0.5 M (pH 8.7) was added, and the plates were incubated in the dark for 1 h at room temperature. After washing, 50#1 1,2-phenylendiamin, dihydrochloride was added (2OPD-tablets (Dakopatts) dissolved in 6 ml citric acid phosphate buffer 0.1 M (pH 5.0) and 3/~1 perhydrol 30~o H 2 0 2 (Merck)), and the plates were incubated in the dark at room temperature for 10-30 min. The reaction was stopped with 150 #1 1 M sulphoric acid. The plates were measured at 492 nm employing 620 nm as reference on an EAR 400 SLT-labinstrument (Austria) provided with a Panasonic KX-P1091 printer (Japan). Serum samples were analysed undiluted. Urine was diluted 1 + 19 with 0.1 M phosphate (pH 8.0) prior to analysis. Detection limit of the assay was 0.03 nmol/l. The assay was linear for quantitation of urinary E G F in a concentration up to 1.1 nmol/1. Quantitation of a diluted urine pool sample measured in 19 runs over a period of 2 months was used for calculation of the precision. The coefficient of variation was 0.07 for a mean of 0.43 nmol/1. The assay recognized 1/~mol/l mouse E G F as 0.08 nmol/1, 0.6/~mol/1 human E G F as less than 0.03 nmol/l and 1/~mol/1 rat TGF~ as less than 0.03 nmol/1. Calculations and statistics
Results are given as medians and total ranges unless otherwise stated. The Mann-Whitney test was used for statistical evaluation. Values of P < 0.05 were considered significant. Results
After an i.v. bolus injection of iodine-labelled E G F to the rat, the radioactivity disappeared within a few minutes from the circulation (Fig. 1). The amount of radioactivity in the blood in the bilaterally nephrectomized rats was approximately 25~'o higher than in the controls. The amount of radioactivity present in the kidneys, the lower urinary tract (ureters, bladder and urine) and the blood in intact rats 5 min after the injection of ~25I-EGF and ~25I-EGF plus an excess of unlabelled E G F is shown in Table I. When an excess of unlabelled E G F was injected together with 125I-EGF the radioactivity of the kidneys decreased by approximately 50 ~o, but there was no decrease of the radioactivity in the lower urinary tract. The radioactivity in the blood did not change. The autoradiographic study showed the label in the kidneys to be located in the proximal tubules (Fig. 2). When ~25I-EGF was injected together with an excess of unlabelled EGF, no change in the distribution of label in the kidney could be identified by autoradiography. 2 h after an i.v. injection of ~25I-EGF to intact rats 13~o (8-16~o) of the label was recovered in the kidneys and in the lower urinary tract including the urine. Of the injected label 1.9'~, (1.7-2.0~o) was found in the kidneys, 0.1~o (0.0-0.4~o) in the
277 Cpm x 1 0 3 in 0.3 ml blood
40
•
30
C~ C o n t r o l group
Bilateral
nephrectorny group
20
10 e-
6-
4-
2" II M i n u t e s
Fig. 1. The effect of bilateral nephrectomy on the removal of homologous ~25I-EGFfrom blood in rats after an i.v. bolus injection. The values shown are the medians and quartiles. b l a d d e r tissue and the ureters, and 11~o (6-14~o) in the urine. U p o n gel filtration, the label present in the urine eluted as one p e a k c o r r e s p o n d i n g to native 125I-EGF a n d one p e a k c o r r e s p o n d i n g to d e g r a d e d 125I-EGF (Fig. 3). The first p e a k represented 42~o ( 3 4 - 5 9 ~o) of the label in the urine. Relative to the injected a m o u n t of radioactivity 4~o ( 2 - 8 ~ o ) was recovered as l z s I - E G F in the urine. W e quantitated the endogenous a m o u n t o f E G F present in urine and p l a s m a employing our E L I S A method. The a m o u n t o f E G F in the 24 h urine was 2.2 n m o l ( 1 . 5 - 3 . 2 nmol). F o r all rats studied (n = 8) the concentration o f E G F in venous p l a s m a was below the detection limit o f the E L I S A (0.03 nmol/1) both before and 24 h after bilateral nephrectomy. The arterial p l a s m a samples from 8 other rats was also below 0.03 nmol/1. TABLE I The distribution of 125I-EGFin the kidneys, the lower urinary tract (bladder, ureters, urine) and the blood of the rat 5 min after an i.v. bolus injection of ~25I-EGFand 125I-EGFplus 440 pmol unlabelled homologous EGF
Kidneys Lower urinary tract Blood
1 pmol JasI-EGF
1 pmo1125I-EGF + 440 pmol EGF
12~o (10-17%) 0.9% (0.1-3.4%) 7% (5-10%)
6~o b (2-6 ~/o) 2.4% a (1.2-4.6%) 7% a (6-10%)
a Not significantly different from the 1 pmol ~251-EGFgroup. b Significantly different from the 1 pmol ~25I-EGFgroup (P < 0.005). The values shown are the percentages of the injected amount of label (medians and total ranges).
279 Cpm Vo
VEG~
Vt
3000
2000.
1000,
0
5'o
16o m Frmction
Fig. 3. Sephadex G-50 gel filtration of urine produced during the first 2 h after an i.v. bolus injection of homologous ~2SI-EGFin rats. Of the label in the urine 4290 eluted as native ~25I-EGF.
Discussion
In the present study we find i.v. injected homologous 12~I-EGF to be removed from the circulation within a few minutes in the rat, and we thereby confirm studies performed in dogs with heterologous E G F [ 12,13]. 125I-EGF is removed quickly from the circulation also in bilaterally nephrectomized rats, indicating that other organs than the kidneys are able to eliminate E G F from blood. This is in accordance with studies which show that the liver has a high capacity for removal of E G F from the circulation [ 14]. In rats the kidneys accumulate 12~o of i.v. injected trace amounts of 125I-EGF. Approximately 40~o of the accumulated label is recovered in the urine as intact ~25I-EGF. This relatively high amount of accumulated E G F recovered in urine might be explained by the unique handling of E G F by the nephrone. Having a molecular weight of 6 kDa, E G F is filtrated in the glomeruli. Unlike most other small peptides which are reabsorbed in the proximal tubules, E G F seems to remain in the ultrafiltrate [8,9]. The renal accumulation of ~25I-EGF decreases when trace amounts of ~2SI-EGF is injected together with excess of unlabelled EGF. This supports the existence of saturable E G F receptors in the kidneys [15]. In vitro studies have demonstrated E G F receptors in the basolateral membranes of kidney cells from rabbit and dog [ 16,17], and in the rabbit a transtubular transport of E G F from the peritubular compartment to the luminal compartment has been demonstrated [9]. Thus the renal uptake of E G F from plasma seems to consist of both glomerular filtration and a saturable receptor dependent uptake. A renal vascular constriction might contribute to the reduced renal accumulation of ~25I-EGF when trace amounts of 12SI-EGF are injected together with 400 pmol unlabelled EGF. In a recent study on rats, a constant intrarenal arterial administration of approximately 20 pmol EGF/min reduced the renal plasma flow by 20 ~/o and the
280
glomerular filtration rate by 40~o [18]. However, such a vascular constriction is not likely to be the only explanation of the reduced renal accumulation of 125I-EGF in our study because we find no decrease of the radioactivity in the lower urinary tract when 12SI-EGF is injected together with excess of unlabelled EGF. In human venous plasma, only small amounts of endogenous E G F have been found, whereas serum may contain E G F released from the platelets during coagulation [ 19-21 ]. The level of endogenous E G F in venous plasma in the rat is below 0.03 nmol/l. This low level of E G F in venous plasma is not caused by an effective renal clearance of EGF, as venous plasma E G F remains below 0.03 nmol/1 after bilateral nephrectomy, and as arterial plasma E G F is also below 0.03 pmol/ml. In order to calculate the contribution of plasma E G F to the total urinary output of the peptide one needs information about the urinary output of E G F and information about the amount of E G F reaching the kidneys by the circulation. The urinary output of E G F per 24 h is around 2.2 nmol. As the concentration of E G F in arterial plasma is below 0.03 nmol/l the maximal amount of E G F reaching the kidneys by the circulation can be calculated to be 222 pmol per 24 h. In this calculation we have used the following values for the rat: plasma E G F 0.03 pmol/ml, cardiac output 50 ml/min, fraction of cardiac output to the kidneys 19~o, and packed cell volume 46~o [22,23]: 222 pmol/24 h = 50 mlblood/min x 0.19 x 0.54 mlplasma/ml blood x 0.03 pmol/ml x 1440 min/24 h. If the 222 pmol E G F is entirely accumulated in the kidneys and 42~o of the 222 pmol E G F is excreted as intact E G F in the urine it will account for 93 pmol or 4~o of the daily urinary output of EGF. In conclusion this study shows that the kidneys are able to accumulate homologous E G F from plasma and excrete part of it as intact E G F in the urine. However, excretion of immunoreactive E G F from plasma can only account for a minor part of the E G F found in urine.
Acknowledgements This study was supported in part by a grant from the Danish Cancer Society (88-090). The technical assistance of Marianne R. Hansen, Kamma Velin and Henny Falsing is warmly acknowledged.
References 1 Marti, U., Burwen, S.J. and Jones, A.L., Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: An update, Hepatology, 9 (1989) 126-138. 2 Poulsen, S. S., Nexo, E., Skov Olsen, P., Hess, J. and Kirkegaard P., Immunohistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 3 Gregory, H., Walsh, S. and Hopkins, C. R., The identification ofurogastrone in serum, saliva, and gastric juice, Gastroenterology, 77 (1979) 313-318.