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Effect of water deprivation, desmopressin (DDAVP) infusion, and oral loads of water, Na+ and NH4+ on urinary excretion of epidermal growth factor in the rat
Regulatory Peptides, 44 (1993) 17-24 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
17
REGPEP 01268
Effect of water deprivation, desmopressin (DDAVP) infusion, and oral loads of water, Na ÷ and N H ~ on urinary excretion of epidermal growth factor in the rat Per E. Jorgensen a,b,c, Steen S. Poulsen b, Ebba Nexo a,c and Sten Christensen d aDepartment of Clinical Chemistry, KH University Hospital of Aarhus, Aarhus (Denmark), bInstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen (Denmark), ¢Department of Clinical Chemistry, Central Hospital, Hiller~d (Denmark) and a Department of Pharmacology, University of Copenhagen, Copenhagen (Denmark) (Received 8 July 1992; revised version received 5 November 1992; accepted 25 November 1992)
Key words: Renal function; Fluid and electrolyte physiology; Dehydration; Urine; Kidney; Desmopressin
Summary Epidermal growth factor (EGF) is synthesized in the kidneys and excreted in urine. Administration of exogenous EGF modulates the reabsorption of Na ÷ and the vasopressin stimulated reabsorption of water in the collecting tubules. In order to clarify whether this reflects a physiological role for urinary EGF we examined the effects of changes in the oral loads of water, Na ÷ and NH~ as well as the effect of infusion of the vasopressin analogue, desmopressin (DDAVP) on the endogenous urinary EGF excretion in the rat. Water deprivation for 48 h reduced the urinary excretion of EGF by 25 ~o and the urinary EGF/creatinine ratio by 8 ~o. Also, urinary volume, Na ÷ excretion, and urinary pH were reduced by water deprivation. Infusion of DDAVP, low plasma vasopressin induced by polydipsia, and changes in the renal excretion ofNa ÷ and H ÷ did not affect the urinary excretion of EGF. In conclusion: it seems unlikely that nephrogenous EGF excreted in the urine plays a physiological role in the regulation of the renal excretion of Na ÷ and H + and in the vasopressin stimulated reabsorption of water in the rat. However, since water deprivation reduced the urinary excretion of EGF it remains possible that urinary EGF plays a role in the complex physiological response to dehydration.
Correspondence to." P.E. Jorgensen, Department of Clinical Chemistry, KH University Hospital of Aarhus, Norrebrogade 44, DK-8000 Aarhus C, Denmark.
18 Introduction
Epidermal growth factor (EGF) is a 6 kDa peptide that is known to produce a number of biological effects both in vitro and in vivo [ 1-3]. High amounts of E G F are present in urine and almost all urinary E G F is of renal origin [4-7]. E G F is synthesized as a 130 kDa precursor in the thick ascending limb of Henle and in the early part of the distal convoluted tubule [ 8-11 ]. The E G F precursor is thought to be localized as a membrane spanning peptide in the luminal cell membrane with the E G F moiety orientated into the tubular lumen [10,12,13]. E G F seems to be excreted into the urine after proteolytic cleavage of the precursor [14,15]. The physiological role of urinary E G F is unknown. It has been suggested that E G F might be involved in the renal handling of salt and water. Thus, administration of exogenous E G F modulates the renal excretion of water and electrolytes in intact animals and in isolated perfused collecting tubules [ 16-21]. If these pharmacological effects reflect a physiological role for E G F in the kidneys, one should expect the urinary excretion of E G F to be affected by changes in the salt and water excretion. In order to test this possibility, we examined the effects of changes in the oral load of water, Na ÷ and NH4+ as well as the effect of infusion of the vasopressin analogue, desmopressin (DDAVP) on the urinary excretion of E G F in the rat.
Materials and Methods
Experimental groups Groups of female wistar rats (Wist:Pan) weighing 180-220 g were kept in metabolic cages (Techniplast, model 1700 (Italy)) in a temperature (21°C) and moisture (45-70~o) controlled room with a 12h light-dark cycle. The rats had free access to rat chow (Altromin No. 1311 (Germany)) and drinking water (distilled water). Unless otherwise indicated, the Na ÷ content in the rat chow was 114 mmol/kg (control chow). The renal excretions of water, Na + and
H + as well as plasma vasopressin were manipulated by the following procedures. Water deprivation for 15 h. Nine rats were acclimatized to the metabolic cages for 3 days with free access to control chow and drinking water. The drinking water was removed and 3 h later the collection of urine started. The urine was collected during the next 12 h (the dark period). Nine control rats were treated similarly except they were not deprived of drinking water. Water deprivation for 48 h. 18 rats had free access to control chow and drinking water for 3 days. The drinking water was removed and the 24 h urine samples were collected during day 5 corresponding to the 24-48 h period of water deprivation. Control group. This group consisted of 18 rats with free access to control chow and drinking water. The 24 h urine samples collected on day 5 served as control material for the 48 h water deprivation group. Nine of the rats were then killed. In addition, the 24 h urine samples of the remaining nine rats were collected on day 7. These 24 h urine samples served as control material for rats with acidic urine, for rats with high and low Na + excretion, and for rats with polyuria (see below). Rats with acidic urine. These rats had free access to drinking water and control chow. Powdered NH4C1 was added to the chow (377 mmol NH4C1/kg chow) from day 4. The 24 h urine samples were collected on day 7. Only rats with a urinary pH lower than 6.5 were included in the study (nine rats). Rats with a low urinary excretion of Na + Nine rats had free access to drinking water and rat chow with a low amount of Na + (27 mmol Na ÷/kg chow). The 24 h urine samples were collected on day 7. Rats with a high urinary excretion of Na +. In this group nine rats had free access to drinking water and rat chow to which Na + was added (1027mmol Na+/kg chow). The 2 4 h urine samples were collected on day 7. Rats with polyuria and low plasma vasopressin. These rats had free access to control chow and
19 drinking water. Glucose (Merck, Germany), 5 ~o, was added to the drinking water from day 4. The 24 h urine samples were collected on day 7. Only rats with a 24 h urinary volume exceeding 75 ml were included in the study (nine rats). The addition of 5 ~o glucose to the drinking water caused polydipsia and polyuria, presumably due to the pleasant taste of glucose [22]. The polyuric state was not caused by osmotic diuresis, since no glucose was detected in the urine (Redia-Test, Boehringer-Mannheim (Germany)).
diluted urine was stored at - 2 0 °C for later measurement of EGF, creatinine, pH and Na + . The rats were anaesthetized by an intraperitoneal injection of 50 mg/kg methohexital. The abdomen was opened and a slice of kidney tissue was fixated in ice cold Bouin's fixative for immunohistochemical examination.
Rats treated with the vasopressin analogue DDA VP. Nine rats were anaesthetized by an intraperitoneai injection of 50 mg/kg methohexital (Brietal, Lilly, USA). An Alzet mini-osmotic pump (model 2001, Alza Corporation, USA) filled with 0.4 mg/ml of DDAVP (deamino-Cysl,D-Arg8-vasopressin), (desmopressin, Sigma, USA) was implanted under the neck skin. DDAVP is a potent vasopressin analogue with specific activity on the renal vasopressin receptor and no effect on the vascular smooth muscle vasopressin receptor [23]. The pumps delivered a constant subcutaneous infusion of 1/~l/h corresponding to 0.4/ag/h of desmopressin. This dosage has been shown to cause antidiuresis in control rats and even in rats with vasopressin resistant polyuria induced by lithium treatment [24]. The rats were placed in metabolic cages on the first postoperative day. They had free access to control chow and drinking water. The 24 h urine samples were collected on day 6. This was done in order to perform the study well within the time limit of the optimal function of the pumps (7 days). Mini-osmotic pumps filled with isotonic saline were implanted in 9 pump control rats and their 24 h urine samples were collected on day 6.
Experimental procedure At the end of the 24 h urine collection period, the urinary volume was determined and the sampling device of the metabolic cage was rinsed thoroughly with distilled water in order to obtain all the urine produced. The rinsing water was added to the urine and the total volume was measured. A sample of the
A
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Fig. 1. Urinary excretion of E G F and creatinine in 24 h urine samples from control rats and from rats that were deprived of drinking water for 48 h. The 24 h urine samples were obtained during the 24-48 h of water deprivation. (A) 24 h urinary excretion of EGF. (B) 24 h urinary excretion of creatinine. (C) E G F / creatinine ratio in the 24 h urine samples. For one of the rats in the control group, some urine was spilled during the collection period. This rat is excluded with regard to the absolute amount ot E G F and creatinine in the urine.
20
Laboratory analyses Quantitation of EGF, creatinine and Na ÷ in urine. EGF was quantitated with an enzyme-linked immunosorbent assay (ELISA) [5]. Creatinine in urine was measured with automatic equipment (SMAC, Technicon, USA). Na + was measured by flame photometry (IL Model 243 Flame Photometer, Instrumentation Laboratory, USA) and the urinary pH was measured by a digital acid-base analyser (model PHM 72, Radiometer, Denmark). Immunohistochemical studies. After fixation, the kidney slices were dehydrated, embedded in paraffin and cut into sections of 7-10/~m. Immunoreactions were visualized by means of the unlabelled peroxidase-antiperoxidase technique [25]. Porcine antirabbit immunoglobulin G and peroxidase-rabbitantiperoxidase complexes (Dakopatts, Denmark) were used with diaminobenzidine for staining. After preincubation for 30 min with normal porcine serum, diluted 1:10, sections were incubated for 20 h at room temperature with antiserum 3124 [8]. The antiserum was raised against rat submandibular EGF in arab-
bit [26]. Kidney tissue from three animals selected at random from each group was examined. Calculations. Results are given as medians and total ranges. The Mann-Whitney test was used unless otherwise stated. Values of P<0.05 were considered significant.
Results
Water deprivation for 15 h reduced the excretion of EGF by 20~o (from 1.5 nmol/12 h (1.3-1.9 nmol/ 12 h) to 1.2 nmol/12 h (0.9-1.7 nmol/12 h)), P<0.05 and the excretion of creatinine by 12~o (from 50.6 #mol/12 h (41.9-59.1 #mol/12 h) to 44.5 #mol/ 12 h (39.1-50.0 #mol/12 h)), P<0.02. The median EGF/creatinine ratio in the urine was not statistically changed (28pmol EGF//~mol creatinine versus 30 pmol EGF/#mol creatinine), P = 0.67. The urinary volume was reduced from 11.0 ml/12 h (5.519.9ml/12 h) to 3.6ml/12h (2.5-5.9 ml/12 h), P<0.001 and the urinary pH was reduced from 8.4
TABLE I Volume, pH and amount of Na ÷ in 24 h urine samples obtained from rats during water deprivation as compared with the same parameters in 24 h urine samples obtained from rats treated with the vasopressin analogue DDAVP or given different diets in order to manipulate plasma vasopressin and the renal excretion of water, Na* and H ÷ Group
Control I 48 h water deprivation Control II DDAVP Polydipsia Low Na ÷ diet High Na + diet NH4+ diet
Urine volume (ml/24 h)
17.8 (2.8-31.3) 2.9 (1.2-6.2) 17.3 (12.4-24.6) 8.8 (6.5-10.3) 105.7 (77.8-116.5) 13.3 (10.5-20.3) 39.4 (29.3-59.4) 19.6 (13.6-24.7)
Urinary pH
8.2 (6.8-8.9) 6.6 (6.3-7.8) 8.5 7.7 7.9 7.8 7.9 5.8
(8.0-8.9) (6.9-8.1) (7.3-8.8) (7.5-8.2) (7.7-8.6) (5.4-6.3)
Urinary Na ÷ excretion (retool/24 h) 1.7 (0.4-2.1) 0.6 (0.4-1.0) 1.5 (1.0-2.3) 1.6 (0.3-2.2) 1.4 (0.7-1.6) 0.2 (0.1-0.6) 12.5 (8.4-16.8) 1.6 (1.2-2.7)
The values shown are medians and ranges. Both the 48 h water deprivation group and its control group (control I) consisted of 18 rats. Their 24 h urine samples were collected on day 5. Each of the other groups consisted of nine rats and their 24 h urine samples were collected on day 7 (DDAVP group on day 6). Nine randomly selected rats from the first control group (control I) were kept in the cages for 7 days and used as control II.
21 A
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Fig. 2. Urinary excretion of EGF and creatinine in 24 h urine samples from groups of rats that were treated with the vasopressin analogue DDAVP or offered different diets in order to manipulate plasma vasopressin and the excretion of Na ÷ and H + . (A)24 h urinary excretion of EGF. (B) 24 h urinary excretion of creatinine. Control, control group; low pH, rats with low urinary pH caused by NH4C1 in the diet; polyuria, rats with polyuria and low plasma vasopressin caused by polydipsia; low Na + , rats with low Na +
( 8 . 1 - 8 . 6 ) to 8.2 (7.2-8.6), P < 0 . 0 4 . T h e m e d i a n urinary excretion o f N a ÷ , 1.2 a n d 1.3 m m o l / 1 2 h respectively, was not affected by water deprivation for 15 h, P = 1.0. T h e differences were m o r e p r o n o u n c e d after 48 h o f w a t e r deprivation. T h e excretions o f b o t h E G F a n d creatinine were r e d u c e d by 2 0 - 2 5 ~ o , P < 0.001 (Fig. 1A a n d B). The urinary E G F / c r e a t i n i n e ratio w a s r e d u c e d by 8 ~ , P < 0.05 (Fig. 1C). The m e d i a n urinary volume was r e d u c e d from 17.8 ml/24 h to 2.9 ml/24 h, P < 0 . 0 0 1 , the m e d i a n urinary p H from 8.2 to 6.6, P < 0 . 0 0 1 , a n d the m e d i a n excretion o f N a ÷ from 1 . 7 m m o l / 2 4 h to 0.60 m m o l / 2 4 h, P < 0 . 0 0 1 , T a b l e I. The next set o f experiments were designed in o r d e r to establish whether infusion o f D D A V P , low p l a s m a vasopressin, a n d changes in the renal excretion of N a ÷ a n d H ÷ did affect the urinary excretion o f E G F . O n e group o f rats w a s treated with D D A V P , while other groups were offered different diets in o r d e r to cause low p l a s m a v a s o p r e s s i n a n d to m a n i p u l a t e the renal excretion o f N a ÷ a n d H ÷. Distinct differences in the excretion o f water a n d N a + as well as in the urinary p H were o b t a i n e d (Table I). T h e changes in urinary p H a n d in the excretion o f N a + were in fact m o r e p r o n o u n c e d t h a n the changes o b s e r v e d during 48 h o f w a t e r deprivation (Table I). F o r each group o f rats there was a c o n s i d e r a b l e variation in the urinary E G F excretion (Fig. 2A). H o w e v e r , there was no significant difference between the control group, the group with acidic urine, the group with polyuria, a n d the two groups with high a n d low excretion of N a + , neither with respect to the excretion o f E G F , P = 0.23 ( K r u s k a l l - W a l l i s test) n o r with respect to the excretion o f creatinine, P = 0.46 (Kruskall-WaUis test) (Fig. 2 A a n d B). Similarly, there was no significant difference in the excretion o f E G F , P = 0.30, excretion caused by low amount of Na ÷ in the diet; high Na ÷, rats with high Na ÷ excretion caused by high amount of Na ÷ in the diet; DDAVP, rats treated with continuous infusion of the vasopressin analogue DDAVP from a subcutaneously placed mini-osmotic pump; pump control, control group with miniosmotic pumps infusing isotonic saline.
22 and in the excretion of creatinine, P = 1.0, between the DDAVP group and its control group (Fig. 2A and B). The immunohistochemical examination localized the E G F immunoreactivity in all the groups to the luminal parts of the distal tubular cells as previously described [8] (data not shown). No quantitative differences in the amount of renal E G F could be demonstrated by immunohistochemistry. No tubular damage demonstrable by light microscopy was found in the water deprivation groups.
Discussion Recent in vitro studies have shown that exogenous administration of E G F modulates the reabsorption o f N a + and the vasopressin stimulated reabsorption of water in the collecting tubules [ 16,18,20,21 ]. Similar results have been obtained in vivo after i.v. infusion of EGF: in the sheep E G F caused an increase in urinary volume and in Na + and K + excretion despite a fall in the glomerular filtration rate (GFR) [19], and in the rat E G F reduced both G F R and Na + and K + excretion [ 17]. If these pharmacological effects of E G F reflect a physiological role for E G F in the kidneys, one should expect the renal E G F metabolism and the urinary excretion of E G F to be modified by changes in the renal excretion of salt and water. However, the present study showed that infusion of DDAVP, low plasma vasopressin induced by polydipsia, and changes in the renal excretion of Na + and H + did not affect the urinary excretion of EGF. In order to interpret our results and the results from studies with exogenously administered EGF, it is necessary to consider the localization of E G F and its receptor in the kidney. E G F is synthesized in the kidneys as a 130 kDa membrane bound precursor that is located in the luminal cell membrane of the cells of the thick ascending limb of Henle and the early part of the distal convoluted tubule, with the E G F moiety orientated into the tubular lumen
[ 9-11,13 ]. Presumably, urinary E G F is excreted after intraluminal, extracellular proteolytic cleavage of the precursor [9,14,15]. E G F receptors have been demonstrated in mesangial ceils, in medullary interstitial cells, and in the basolateral cell membrane of the proximal tubules and the collecting tubules [ 17,2729]. Studies on isolated perfused collecting tubules from rabbits showed that application of E G F to the basolateral side of the tubules inhibited Na ÷ reabsorption and vasopressin stimulated water reabsorption [ 16,18,20,21 ]. No effect has been observed when E G F is applied to the luminal side [16,18,20,21]. The renal E G F receptors could be activated by E G F in blood. However, blood E G F is confined to the platelets [30-32] and the concentration of E G F in plasma is very low [2,5,33]. It might therefore be relevant to consider whether E G F synthesized in the kidneys could be the physiological ligand for the renal E G F receptors. The unanswered question is whether renal E G F is able to reach these receptors and thus have a paracrine function. It has been speculated that urinary E G F might be transported from the tubular lumen to the peritubular space or that basolateral secretion of E G F might occur despite the apparent apical localization of E G F in the distal tubule [3]. The present study does not exclude the possibility of such a basolateral secretion of E G F in the kidneys. However, the results from rats treated with DDAVP and from rats given glucose in the drinking water or NHaC1 in the rat chow indicate that the renal excretion of E G F is independent of the urine flow and the urinary pH. These characteristics are in accordance with absence of tubular reabsorption by non-ionic back diffusion. Water deprivation reduced the urinary excretion of both E G F and creatinine. After a more prolonged water deprivation (48 h), the excretion of E G F was reduced more than the excretion of creatinine. The reason for this reduced urinary E G F excretion is unknown. The urinary E G F excretion is known to be reduced by kidney diseases with impaired renal function such as diabetic nephropathy, glomerulonephritis and acute tubulointerstitial nephritis [6,34,35].
23
Thus, one possible explanation for the reduced urinary excretion of EGF during water deprivation could be a tubular dysfunction in the dehydrated rat. The fact that we found no tubular damage visible by light microscopy do not exclude this possibility. Another possible explanation for the reduced urinary excretion of EGF during water deprivation could be a coupling between the renal handling of salt and water and the excretion of EGF. It is unlikely that the reduced EGF excretion during water deprivation is coupled to the reduced excretion of Na ÷ and H ÷ because the EGF excretion was unaffected when more pronounced changes in the excretion of these electrolytes were obtained by giving the rats different diets. In addition, after 15 h of water deprivation the reduced EGF excretion was not accompanied by a decrease in the Na ÷ excretion. It should be noted, however, that the urine volume was lower during water deprivation than could be induced by DDAVP infusion (median values 2.9 ml/24 h and 8.8 ml/24 h). The decreased EGF excretion during dehydration might be related to this or to the extracellular volume contraction, which causes a fall in the GFR [36]. It is also possible that the reduced urinary EGF excretion observed during water deprivation could be caused by high amounts of endogenous plasma vasopressin. Dehydration is a strong stimuli to vasopressin release and endogenous vasopressin activates both the renal and the vascular smooth muscle vasopressin receptor, whereas DDAVP only activates the renal vasopressin receptor. The infusion of DDAVP showed that activation of the renal vasopressin receptor did not affect the urinary EGF excretion. However, endogenous vasopressin might affect the urinary EGF excretion due to a decrease in GFR mediated by the vascular smooth muscle vasopressin receptor. The physiological response to dehydration is very complex and clarification of the mechanism behind the reduced urinary EGF excretion during water deprivation awaits further studies. In conclusion: it seems unlikely that nephrogenous EGF excreted in the urine plays a physiological role in the regulation of the renal excretion of Na + and
H ÷ and in the vasopressin stimmulated reabsorption of water in the rat. The mechanism of the reduced EGF excretion during water deprivation is unclear, and it cannot be excluded that urinary EGF plays a role in the complex physiological response to dehydration.
Acknowledgements This study was supported by the foundation of Helen and Ejnar Bjornow, by the foundation of Ruth I.E. Konig-Petersen, by the Danish Cancer Society (91-026), and by the Danish Medical Research Council (12-9312). The technical assistance of Mette Wolf, Marianne R. Hansen and Jette Schousboe is warmly acknowledged.
References 1 Carpenter, G. and Wahl, M.I., The epidermal growth factor family. In Sporn, M.B. and Roberts, A.B. (Eds.), Peptide growth factors and their receptors I, Handbook Experimental Pharmacology 95/1, Springer Verlag, Berlin, 1990, pp. 69171. 2 Fisher, D.A., Salido, E.C. and Barajas, L., Epidermal growth factor and the kidney, Annu. Rev. Physiol., 51 (1989) 6780. 3 Harris, R.C., Potential physiological roles for epidermal growth factor in the kidney, Am. J. Kidney Dis., 17 (1991) 627-630. 4 Callegari, C., Lahorde, N.P., Buenaltor, G., Nascimento, C.G., Brasel, J.A. and Fisher, D.A., The source of urinary epidermal growth factor in human, Eur. J. Appl. Physiol., 58 (1988) 26-31. 5 Jorgensen, P. E., Rasmussen, T.N., Olsen, P. S., Raaberg, L., Poulsen, S.S. and Next, E., Renal uptake and excretion of epidermal growth factor from plasma in the rat, Regul. Pept., 28 (1990) 273-281. 6 Mattila, A.-L., Pasternack, A., Viinikka, L. and Perheentupa, J., Subnormal concentrations of urinary epidermal growth factor in patients with kidney disease, J. Clin. Endocrinol. Metab., 62 (1986) 1180-1183. 70lsen, P.S., Next, E., Poulsen, S.S., Hansen, H.F. and Kirkegaard, P., Renal origin of rat urinary epidermal growth factor, Regul. Pept., 10 (1984) 37-45.
24 8 Poulsen, S. S., Next, E., Olsen, P. S., Hess, J. and Kirkegaard, P., Immunohistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 9 Rail, L.B., Scott, J., Bell, G.I., Crawford, R.J., Penschow, J. D., Niall, H. D. and Coghlan, J. P., Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues, Nature, 313 (1985) 228-231. 10 Salido, E.C., Fisher, D.A. and Barajas, L., Immunoelectron microscopy of epidermal growth factor in mouse kidney, J. Ultrastruct. Res., 96 (1986) 105-113. 11 Salido, E.C., Yen, P.H., Shapiro, L.J., Fisher, D.A. and Barajas, L., In situ hybridization of prepro-epidermal growth factor mRNA in the mouse kidney, Am. J. Physiol., 256 (1989) F632-F638. 12 Bell, G.I., Fong, N.M., Stempien, M.M., Wormsted, M.A., Caput, D., Ku, L., Urdea, M.S., Rall, L.B. and SanchezPescador, R., Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization, Nucleic Acids Res., 14 (1986) 8427-8446. 13 Mroczkowski, B., Reich, M., Chen, K., Bell, G.I. and Cohen, S., Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity, Mol. Cell. Biol., 9 (1989) 2771-2778. 14 Jorgensen, P. E., Nexo, 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 (1991) 284-288. 15 Jorgensen, P. E., Raaberg, L., Poulsen, S. S. and Nexe, E., The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor, Regul. Pept., 31 (1990) 115-124. 16 Breyer, M.D., Jacobson, H.R. and Breyer, J.A., Epidermal growth factor inhibits the hydroosmotic effect of vasopressin in the isolated perfused rabbit cortical collecting tubule, J. Clin. Invest., 82 (1988) 1313-1320. 17 Harris, R.C., Hoover, R.L., Jacobson, H. R. and Badr, K. F., Evidence for glomerular actions of epidermal growth factor in the rat, J. Clin. Invest., 82 (1988) 1028-1039. 18 Muto, S., Furuya, H., Tabei, K. and Asano, Y., Site and mechanism of action of epidermal growth factor in rabbit cortical collecting duct, Am. J. Physiol., 260 (1991) F163-F169. 19 Scoggins, B. A., Butkus, A., Coghlan, J. P., Fei, D.T.W., McDougall, J. G., Niall, H. D., Walsh, J. R. and Wang, X., In vivo cardiovascular, renal and endocrine effects of epidermal growth factor in sheep. In Labile, F. and Proulx, L. (Eds.), Endocrinology, Elsevier, Amsterdam, 1984, pp. 573-576. 20 Vehaskari, V.M., Hering-Smith, K.S., Moskowitz, D.W., Weiner, I.D. and Hamm, L.L., Effect of epidermal growth factor on sodium transport in the cortical collecting tubule, Am. J. Physiol., 256 (1989) F803-F809.
21 Vehaskari, V.M., Hemdon, J. and Harem, L.L., Mechanism of sodium transport inhibition by epidermal growth factor in cortical collecting ducts, Am. J. Physiol., 261 (1991) F896F903. 22 Christensen, S., Effects of water deprivation in rats with polydipsia and polyuria due to long-term administration of lithium, Acta Pharmacol. Toxicol., 35 (1974) 201-211. 23 Manning, M. and Sawyer, W.H., Development of selective agonists of vasopressin and oxytocin. In Schrier, R.W. (Ed.), Vasopressin, Raven Press, New York, 1985, pp. 131-144. 24 Christensen, S., DDAVP (1-desamino-8-o-arginine-vasopressin) treatment of lithium-induced polyuria in the rat, Scand. J. Clin. Lab. Invest., 40 (1980) 151-157. 25 Sternberger, L., Immunocytochemistry, Wiley & Sons, New York, 1974, pp. 104-169. 26 Olsen, P.S. and Next, E., Quantitation of epidermal growth factor in the rat, identification and partial characterization of duodenal EGF, Scand. J. Gastroenterol., 18 (1983) 771-776. 27 Breyer, J. and Harris, R., EGF binds to specific EGF receptors and stimmulates mitogenesis in renal medullary interstitial cells, Kidney Int., 33 (1988) 255. 28 Harris, R.C., Response ofrat inner medullary collecting duct to epidermal growth factor, Am. J. Physiol., 256 (1989) Fll17-Fl124. 29 Harris, R.C. and Daniel, T. O., Epidermal growth factor binding, stimmulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule, J. Cell. Physiol., 139 (1989) 383-391. 30 Ben-Ezra, J., Sheibani, K., Hwang, D.L. and Lev-Ran, A., Megakaryocyte synthesis is the source of epidermal growth factor in human platelets, Am. J. Pathol., 137 (1990) 755-759. 31 Oka, Y. and Orth, D.N., Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets, J. Clin. Invest., 72 (1983) 249-259. 32 Savage, A. P., Chatterjee, V. K., Gregory, H. and Bloom, S. R., Epidermal growth factor in blood, Regul. Pept., 16 (1986) 199-206. 33 Mattila, A.-L., Viinikka, L., Saario, I. and Perheentupa, J., Human epidermal growth factor: renal production and abscence from plasma, Regul. Pept., 23 (1988) 89-92. 34 Safirstein, R., Zelent, A.Z. and Price, P.M., Reduced renal prepro-epidermal growth factor mRNA and decreased EGF in excretion in ARF, Kidney Int., 36 (1989) 810-815. 35 Mathiesen, E.R., Nexo, E., Hommel, E. and Parving, H.-H., Reduced urinary excretion of epidermal growth factor in incipient and overt diabetic nephropathy, Diab. Med., 6 (1989) 121-126. 36 Gellai, M., Edwards, B.R. and Valtin, H., Urinary concentrating ability during dehydration in the absence ofvasopressin, Am. J. Physiol., 237 (1979) FI00-F104.
17
REGPEP 01268
Effect of water deprivation, desmopressin (DDAVP) infusion, and oral loads of water, Na ÷ and N H ~ on urinary excretion of epidermal growth factor in the rat Per E. Jorgensen a,b,c, Steen S. Poulsen b, Ebba Nexo a,c and Sten Christensen d aDepartment of Clinical Chemistry, KH University Hospital of Aarhus, Aarhus (Denmark), bInstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen (Denmark), ¢Department of Clinical Chemistry, Central Hospital, Hiller~d (Denmark) and a Department of Pharmacology, University of Copenhagen, Copenhagen (Denmark) (Received 8 July 1992; revised version received 5 November 1992; accepted 25 November 1992)
Key words: Renal function; Fluid and electrolyte physiology; Dehydration; Urine; Kidney; Desmopressin
Summary Epidermal growth factor (EGF) is synthesized in the kidneys and excreted in urine. Administration of exogenous EGF modulates the reabsorption of Na ÷ and the vasopressin stimulated reabsorption of water in the collecting tubules. In order to clarify whether this reflects a physiological role for urinary EGF we examined the effects of changes in the oral loads of water, Na ÷ and NH~ as well as the effect of infusion of the vasopressin analogue, desmopressin (DDAVP) on the endogenous urinary EGF excretion in the rat. Water deprivation for 48 h reduced the urinary excretion of EGF by 25 ~o and the urinary EGF/creatinine ratio by 8 ~o. Also, urinary volume, Na ÷ excretion, and urinary pH were reduced by water deprivation. Infusion of DDAVP, low plasma vasopressin induced by polydipsia, and changes in the renal excretion ofNa ÷ and H ÷ did not affect the urinary excretion of EGF. In conclusion: it seems unlikely that nephrogenous EGF excreted in the urine plays a physiological role in the regulation of the renal excretion of Na ÷ and H + and in the vasopressin stimulated reabsorption of water in the rat. However, since water deprivation reduced the urinary excretion of EGF it remains possible that urinary EGF plays a role in the complex physiological response to dehydration.
Correspondence to." P.E. Jorgensen, Department of Clinical Chemistry, KH University Hospital of Aarhus, Norrebrogade 44, DK-8000 Aarhus C, Denmark.
18 Introduction
Epidermal growth factor (EGF) is a 6 kDa peptide that is known to produce a number of biological effects both in vitro and in vivo [ 1-3]. High amounts of E G F are present in urine and almost all urinary E G F is of renal origin [4-7]. E G F is synthesized as a 130 kDa precursor in the thick ascending limb of Henle and in the early part of the distal convoluted tubule [ 8-11 ]. The E G F precursor is thought to be localized as a membrane spanning peptide in the luminal cell membrane with the E G F moiety orientated into the tubular lumen [10,12,13]. E G F seems to be excreted into the urine after proteolytic cleavage of the precursor [14,15]. The physiological role of urinary E G F is unknown. It has been suggested that E G F might be involved in the renal handling of salt and water. Thus, administration of exogenous E G F modulates the renal excretion of water and electrolytes in intact animals and in isolated perfused collecting tubules [ 16-21]. If these pharmacological effects reflect a physiological role for E G F in the kidneys, one should expect the urinary excretion of E G F to be affected by changes in the salt and water excretion. In order to test this possibility, we examined the effects of changes in the oral load of water, Na ÷ and NH4+ as well as the effect of infusion of the vasopressin analogue, desmopressin (DDAVP) on the urinary excretion of E G F in the rat.
Materials and Methods
Experimental groups Groups of female wistar rats (Wist:Pan) weighing 180-220 g were kept in metabolic cages (Techniplast, model 1700 (Italy)) in a temperature (21°C) and moisture (45-70~o) controlled room with a 12h light-dark cycle. The rats had free access to rat chow (Altromin No. 1311 (Germany)) and drinking water (distilled water). Unless otherwise indicated, the Na ÷ content in the rat chow was 114 mmol/kg (control chow). The renal excretions of water, Na + and
H + as well as plasma vasopressin were manipulated by the following procedures. Water deprivation for 15 h. Nine rats were acclimatized to the metabolic cages for 3 days with free access to control chow and drinking water. The drinking water was removed and 3 h later the collection of urine started. The urine was collected during the next 12 h (the dark period). Nine control rats were treated similarly except they were not deprived of drinking water. Water deprivation for 48 h. 18 rats had free access to control chow and drinking water for 3 days. The drinking water was removed and the 24 h urine samples were collected during day 5 corresponding to the 24-48 h period of water deprivation. Control group. This group consisted of 18 rats with free access to control chow and drinking water. The 24 h urine samples collected on day 5 served as control material for the 48 h water deprivation group. Nine of the rats were then killed. In addition, the 24 h urine samples of the remaining nine rats were collected on day 7. These 24 h urine samples served as control material for rats with acidic urine, for rats with high and low Na + excretion, and for rats with polyuria (see below). Rats with acidic urine. These rats had free access to drinking water and control chow. Powdered NH4C1 was added to the chow (377 mmol NH4C1/kg chow) from day 4. The 24 h urine samples were collected on day 7. Only rats with a urinary pH lower than 6.5 were included in the study (nine rats). Rats with a low urinary excretion of Na + Nine rats had free access to drinking water and rat chow with a low amount of Na + (27 mmol Na ÷/kg chow). The 24 h urine samples were collected on day 7. Rats with a high urinary excretion of Na +. In this group nine rats had free access to drinking water and rat chow to which Na + was added (1027mmol Na+/kg chow). The 2 4 h urine samples were collected on day 7. Rats with polyuria and low plasma vasopressin. These rats had free access to control chow and
19 drinking water. Glucose (Merck, Germany), 5 ~o, was added to the drinking water from day 4. The 24 h urine samples were collected on day 7. Only rats with a 24 h urinary volume exceeding 75 ml were included in the study (nine rats). The addition of 5 ~o glucose to the drinking water caused polydipsia and polyuria, presumably due to the pleasant taste of glucose [22]. The polyuric state was not caused by osmotic diuresis, since no glucose was detected in the urine (Redia-Test, Boehringer-Mannheim (Germany)).
diluted urine was stored at - 2 0 °C for later measurement of EGF, creatinine, pH and Na + . The rats were anaesthetized by an intraperitoneal injection of 50 mg/kg methohexital. The abdomen was opened and a slice of kidney tissue was fixated in ice cold Bouin's fixative for immunohistochemical examination.
Rats treated with the vasopressin analogue DDA VP. Nine rats were anaesthetized by an intraperitoneai injection of 50 mg/kg methohexital (Brietal, Lilly, USA). An Alzet mini-osmotic pump (model 2001, Alza Corporation, USA) filled with 0.4 mg/ml of DDAVP (deamino-Cysl,D-Arg8-vasopressin), (desmopressin, Sigma, USA) was implanted under the neck skin. DDAVP is a potent vasopressin analogue with specific activity on the renal vasopressin receptor and no effect on the vascular smooth muscle vasopressin receptor [23]. The pumps delivered a constant subcutaneous infusion of 1/~l/h corresponding to 0.4/ag/h of desmopressin. This dosage has been shown to cause antidiuresis in control rats and even in rats with vasopressin resistant polyuria induced by lithium treatment [24]. The rats were placed in metabolic cages on the first postoperative day. They had free access to control chow and drinking water. The 24 h urine samples were collected on day 6. This was done in order to perform the study well within the time limit of the optimal function of the pumps (7 days). Mini-osmotic pumps filled with isotonic saline were implanted in 9 pump control rats and their 24 h urine samples were collected on day 6.
Experimental procedure At the end of the 24 h urine collection period, the urinary volume was determined and the sampling device of the metabolic cage was rinsed thoroughly with distilled water in order to obtain all the urine produced. The rinsing water was added to the urine and the total volume was measured. A sample of the
A
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Fig. 1. Urinary excretion of E G F and creatinine in 24 h urine samples from control rats and from rats that were deprived of drinking water for 48 h. The 24 h urine samples were obtained during the 24-48 h of water deprivation. (A) 24 h urinary excretion of EGF. (B) 24 h urinary excretion of creatinine. (C) E G F / creatinine ratio in the 24 h urine samples. For one of the rats in the control group, some urine was spilled during the collection period. This rat is excluded with regard to the absolute amount ot E G F and creatinine in the urine.
20
Laboratory analyses Quantitation of EGF, creatinine and Na ÷ in urine. EGF was quantitated with an enzyme-linked immunosorbent assay (ELISA) [5]. Creatinine in urine was measured with automatic equipment (SMAC, Technicon, USA). Na + was measured by flame photometry (IL Model 243 Flame Photometer, Instrumentation Laboratory, USA) and the urinary pH was measured by a digital acid-base analyser (model PHM 72, Radiometer, Denmark). Immunohistochemical studies. After fixation, the kidney slices were dehydrated, embedded in paraffin and cut into sections of 7-10/~m. Immunoreactions were visualized by means of the unlabelled peroxidase-antiperoxidase technique [25]. Porcine antirabbit immunoglobulin G and peroxidase-rabbitantiperoxidase complexes (Dakopatts, Denmark) were used with diaminobenzidine for staining. After preincubation for 30 min with normal porcine serum, diluted 1:10, sections were incubated for 20 h at room temperature with antiserum 3124 [8]. The antiserum was raised against rat submandibular EGF in arab-
bit [26]. Kidney tissue from three animals selected at random from each group was examined. Calculations. Results are given as medians and total ranges. The Mann-Whitney test was used unless otherwise stated. Values of P<0.05 were considered significant.
Results
Water deprivation for 15 h reduced the excretion of EGF by 20~o (from 1.5 nmol/12 h (1.3-1.9 nmol/ 12 h) to 1.2 nmol/12 h (0.9-1.7 nmol/12 h)), P<0.05 and the excretion of creatinine by 12~o (from 50.6 #mol/12 h (41.9-59.1 #mol/12 h) to 44.5 #mol/ 12 h (39.1-50.0 #mol/12 h)), P<0.02. The median EGF/creatinine ratio in the urine was not statistically changed (28pmol EGF//~mol creatinine versus 30 pmol EGF/#mol creatinine), P = 0.67. The urinary volume was reduced from 11.0 ml/12 h (5.519.9ml/12 h) to 3.6ml/12h (2.5-5.9 ml/12 h), P<0.001 and the urinary pH was reduced from 8.4
TABLE I Volume, pH and amount of Na ÷ in 24 h urine samples obtained from rats during water deprivation as compared with the same parameters in 24 h urine samples obtained from rats treated with the vasopressin analogue DDAVP or given different diets in order to manipulate plasma vasopressin and the renal excretion of water, Na* and H ÷ Group
Control I 48 h water deprivation Control II DDAVP Polydipsia Low Na ÷ diet High Na + diet NH4+ diet
Urine volume (ml/24 h)
17.8 (2.8-31.3) 2.9 (1.2-6.2) 17.3 (12.4-24.6) 8.8 (6.5-10.3) 105.7 (77.8-116.5) 13.3 (10.5-20.3) 39.4 (29.3-59.4) 19.6 (13.6-24.7)
Urinary pH
8.2 (6.8-8.9) 6.6 (6.3-7.8) 8.5 7.7 7.9 7.8 7.9 5.8
(8.0-8.9) (6.9-8.1) (7.3-8.8) (7.5-8.2) (7.7-8.6) (5.4-6.3)
Urinary Na ÷ excretion (retool/24 h) 1.7 (0.4-2.1) 0.6 (0.4-1.0) 1.5 (1.0-2.3) 1.6 (0.3-2.2) 1.4 (0.7-1.6) 0.2 (0.1-0.6) 12.5 (8.4-16.8) 1.6 (1.2-2.7)
The values shown are medians and ranges. Both the 48 h water deprivation group and its control group (control I) consisted of 18 rats. Their 24 h urine samples were collected on day 5. Each of the other groups consisted of nine rats and their 24 h urine samples were collected on day 7 (DDAVP group on day 6). Nine randomly selected rats from the first control group (control I) were kept in the cages for 7 days and used as control II.
21 A
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Fig. 2. Urinary excretion of EGF and creatinine in 24 h urine samples from groups of rats that were treated with the vasopressin analogue DDAVP or offered different diets in order to manipulate plasma vasopressin and the excretion of Na ÷ and H + . (A)24 h urinary excretion of EGF. (B) 24 h urinary excretion of creatinine. Control, control group; low pH, rats with low urinary pH caused by NH4C1 in the diet; polyuria, rats with polyuria and low plasma vasopressin caused by polydipsia; low Na + , rats with low Na +
( 8 . 1 - 8 . 6 ) to 8.2 (7.2-8.6), P < 0 . 0 4 . T h e m e d i a n urinary excretion o f N a ÷ , 1.2 a n d 1.3 m m o l / 1 2 h respectively, was not affected by water deprivation for 15 h, P = 1.0. T h e differences were m o r e p r o n o u n c e d after 48 h o f w a t e r deprivation. T h e excretions o f b o t h E G F a n d creatinine were r e d u c e d by 2 0 - 2 5 ~ o , P < 0.001 (Fig. 1A a n d B). The urinary E G F / c r e a t i n i n e ratio w a s r e d u c e d by 8 ~ , P < 0.05 (Fig. 1C). The m e d i a n urinary volume was r e d u c e d from 17.8 ml/24 h to 2.9 ml/24 h, P < 0 . 0 0 1 , the m e d i a n urinary p H from 8.2 to 6.6, P < 0 . 0 0 1 , a n d the m e d i a n excretion o f N a ÷ from 1 . 7 m m o l / 2 4 h to 0.60 m m o l / 2 4 h, P < 0 . 0 0 1 , T a b l e I. The next set o f experiments were designed in o r d e r to establish whether infusion o f D D A V P , low p l a s m a vasopressin, a n d changes in the renal excretion of N a ÷ a n d H ÷ did affect the urinary excretion o f E G F . O n e group o f rats w a s treated with D D A V P , while other groups were offered different diets in o r d e r to cause low p l a s m a v a s o p r e s s i n a n d to m a n i p u l a t e the renal excretion o f N a ÷ a n d H ÷. Distinct differences in the excretion o f water a n d N a + as well as in the urinary p H were o b t a i n e d (Table I). T h e changes in urinary p H a n d in the excretion o f N a + were in fact m o r e p r o n o u n c e d t h a n the changes o b s e r v e d during 48 h o f w a t e r deprivation (Table I). F o r each group o f rats there was a c o n s i d e r a b l e variation in the urinary E G F excretion (Fig. 2A). H o w e v e r , there was no significant difference between the control group, the group with acidic urine, the group with polyuria, a n d the two groups with high a n d low excretion of N a + , neither with respect to the excretion o f E G F , P = 0.23 ( K r u s k a l l - W a l l i s test) n o r with respect to the excretion o f creatinine, P = 0.46 (Kruskall-WaUis test) (Fig. 2 A a n d B). Similarly, there was no significant difference in the excretion o f E G F , P = 0.30, excretion caused by low amount of Na ÷ in the diet; high Na ÷, rats with high Na ÷ excretion caused by high amount of Na ÷ in the diet; DDAVP, rats treated with continuous infusion of the vasopressin analogue DDAVP from a subcutaneously placed mini-osmotic pump; pump control, control group with miniosmotic pumps infusing isotonic saline.
22 and in the excretion of creatinine, P = 1.0, between the DDAVP group and its control group (Fig. 2A and B). The immunohistochemical examination localized the E G F immunoreactivity in all the groups to the luminal parts of the distal tubular cells as previously described [8] (data not shown). No quantitative differences in the amount of renal E G F could be demonstrated by immunohistochemistry. No tubular damage demonstrable by light microscopy was found in the water deprivation groups.
Discussion Recent in vitro studies have shown that exogenous administration of E G F modulates the reabsorption o f N a + and the vasopressin stimulated reabsorption of water in the collecting tubules [ 16,18,20,21 ]. Similar results have been obtained in vivo after i.v. infusion of EGF: in the sheep E G F caused an increase in urinary volume and in Na + and K + excretion despite a fall in the glomerular filtration rate (GFR) [19], and in the rat E G F reduced both G F R and Na + and K + excretion [ 17]. If these pharmacological effects of E G F reflect a physiological role for E G F in the kidneys, one should expect the renal E G F metabolism and the urinary excretion of E G F to be modified by changes in the renal excretion of salt and water. However, the present study showed that infusion of DDAVP, low plasma vasopressin induced by polydipsia, and changes in the renal excretion of Na + and H + did not affect the urinary excretion of EGF. In order to interpret our results and the results from studies with exogenously administered EGF, it is necessary to consider the localization of E G F and its receptor in the kidney. E G F is synthesized in the kidneys as a 130 kDa membrane bound precursor that is located in the luminal cell membrane of the cells of the thick ascending limb of Henle and the early part of the distal convoluted tubule, with the E G F moiety orientated into the tubular lumen
[ 9-11,13 ]. Presumably, urinary E G F is excreted after intraluminal, extracellular proteolytic cleavage of the precursor [9,14,15]. E G F receptors have been demonstrated in mesangial ceils, in medullary interstitial cells, and in the basolateral cell membrane of the proximal tubules and the collecting tubules [ 17,2729]. Studies on isolated perfused collecting tubules from rabbits showed that application of E G F to the basolateral side of the tubules inhibited Na ÷ reabsorption and vasopressin stimulated water reabsorption [ 16,18,20,21 ]. No effect has been observed when E G F is applied to the luminal side [16,18,20,21]. The renal E G F receptors could be activated by E G F in blood. However, blood E G F is confined to the platelets [30-32] and the concentration of E G F in plasma is very low [2,5,33]. It might therefore be relevant to consider whether E G F synthesized in the kidneys could be the physiological ligand for the renal E G F receptors. The unanswered question is whether renal E G F is able to reach these receptors and thus have a paracrine function. It has been speculated that urinary E G F might be transported from the tubular lumen to the peritubular space or that basolateral secretion of E G F might occur despite the apparent apical localization of E G F in the distal tubule [3]. The present study does not exclude the possibility of such a basolateral secretion of E G F in the kidneys. However, the results from rats treated with DDAVP and from rats given glucose in the drinking water or NHaC1 in the rat chow indicate that the renal excretion of E G F is independent of the urine flow and the urinary pH. These characteristics are in accordance with absence of tubular reabsorption by non-ionic back diffusion. Water deprivation reduced the urinary excretion of both E G F and creatinine. After a more prolonged water deprivation (48 h), the excretion of E G F was reduced more than the excretion of creatinine. The reason for this reduced urinary E G F excretion is unknown. The urinary E G F excretion is known to be reduced by kidney diseases with impaired renal function such as diabetic nephropathy, glomerulonephritis and acute tubulointerstitial nephritis [6,34,35].
23
Thus, one possible explanation for the reduced urinary excretion of EGF during water deprivation could be a tubular dysfunction in the dehydrated rat. The fact that we found no tubular damage visible by light microscopy do not exclude this possibility. Another possible explanation for the reduced urinary excretion of EGF during water deprivation could be a coupling between the renal handling of salt and water and the excretion of EGF. It is unlikely that the reduced EGF excretion during water deprivation is coupled to the reduced excretion of Na ÷ and H ÷ because the EGF excretion was unaffected when more pronounced changes in the excretion of these electrolytes were obtained by giving the rats different diets. In addition, after 15 h of water deprivation the reduced EGF excretion was not accompanied by a decrease in the Na ÷ excretion. It should be noted, however, that the urine volume was lower during water deprivation than could be induced by DDAVP infusion (median values 2.9 ml/24 h and 8.8 ml/24 h). The decreased EGF excretion during dehydration might be related to this or to the extracellular volume contraction, which causes a fall in the GFR [36]. It is also possible that the reduced urinary EGF excretion observed during water deprivation could be caused by high amounts of endogenous plasma vasopressin. Dehydration is a strong stimuli to vasopressin release and endogenous vasopressin activates both the renal and the vascular smooth muscle vasopressin receptor, whereas DDAVP only activates the renal vasopressin receptor. The infusion of DDAVP showed that activation of the renal vasopressin receptor did not affect the urinary EGF excretion. However, endogenous vasopressin might affect the urinary EGF excretion due to a decrease in GFR mediated by the vascular smooth muscle vasopressin receptor. The physiological response to dehydration is very complex and clarification of the mechanism behind the reduced urinary EGF excretion during water deprivation awaits further studies. In conclusion: it seems unlikely that nephrogenous EGF excreted in the urine plays a physiological role in the regulation of the renal excretion of Na + and
H ÷ and in the vasopressin stimmulated reabsorption of water in the rat. The mechanism of the reduced EGF excretion during water deprivation is unclear, and it cannot be excluded that urinary EGF plays a role in the complex physiological response to dehydration.
Acknowledgements This study was supported by the foundation of Helen and Ejnar Bjornow, by the foundation of Ruth I.E. Konig-Petersen, by the Danish Cancer Society (91-026), and by the Danish Medical Research Council (12-9312). The technical assistance of Mette Wolf, Marianne R. Hansen and Jette Schousboe is warmly acknowledged.
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