Camp. Biochem. Physiol. Vol. 103A. No. 4, pp. 687-693, 1992 0300-9629192 f5.00 + 0.00 Printed in Great Britain 0 1992 Pergamon Press Ltd

EPIDERMAL GROWTH FACTOR ALTERS THE ELECTROLYTE PROFILE OF LACTATING EWES

(0 VIS ARIES)

C. B. Gow,*i M. J. SILVAPULLE* and G. P. M. MOORE~

*School of Agriculture, La Trobe University, Bundoora, 3083, Victoria. Australia (Tel. 03 479-2139; Fax 03 471-0224); fDivision of Animal Production, CSIRO, P.O. Box 239, Blacktown, 2148,

New South Wales, Australia

(Received 10 April 1992; accepted 15 May 1992)

Abstract-I. Lactating ewes were treated with mouse epidermal growth factor (EGF) at a dose rate of 0.5 mg/day for 4 days and its effects on the electrolyte profile were observed.

2. There was no effect of EGF on plasma concentrations of sodium or potassium, although urinary and total (in urine and milk) losses of both were reduced.

3. EGF-induced hypocalcaemia was associated with reduced milk calcium secretion and increased urinary calcium excretion whereas EGF-induced hypermagnesaemia was associated with reduced urinary and total magnesium losses.

4. Glomerular filtration rate was reduced during EGF infusion. 5. Chronic intravenous EGF infusion affects the electrolyte profile by altering electrolyte secretion by

the mammary gland and renal electrolyte excretion

INTRODUCTION

The polypeptide epidermal growth factor (EGF) has been reported to be involved in mammogenesis and lactogenesis (see Read, 1988). In recent years, in uiuo studies on the effects of continuous long-term intra- venous (i.v.) EGF infusions have been performed on late-pregnant (Gow et a/., 1991) and early-lactating (Gow and Moore, 1992) ewes (Ovis aries). During the earlier studies (Gow et al., 1991), it was noticed that water intake increased throughout the treatment period (unpublished data) but no measurements on urine volume were made. The latter studies (Gow and Moore, 1992) demonstrated that EGF reduced milk yield and concomitantly increased water intake and urine volume (by 60 and 164%, respectively). This was the first time that the profound responses of sustained polydipsia and diuresis during EGF infu- sion in conscious sheep were reported (Gow and Moore, 1992).

EGF has also been implicated in the normal func- tions of other organs (Burgess, 1989; Fisher et al., 1989; Kanda et (II., 1989). In addition it has been shown that the kidneys can synthesize both the EGF-precursor (Rall et al., 1985) and EGF itself (Kanda et ul., 1991) and that receptors for EGF occur in nephron segments of many species (see Muto et al., 1991). However, the direct physiological role of EGF in tYvo in the kidney still remains to be elucidated (Fisher et u/., 1989). In the present report, we have investigated the changes in electrolyte concentrations of the plasma, milk and urine samples of the lactating ewes studied by Gow and Moore (1992) during EGF-induced polydipsia and diuresis.

tAuthor to whom all correspondence should be addressed.

MATERIALS AND METHODS

Experimen!al animals

Six S-year-old twin-bearing crossbred ewes (Border- Leicester x Merino) were selected from a single flock; the treatment of the ewes until late pregnancy has been de- scribed previously (Gow and Moore, 1992). After parturi- tion; the ewes were shorn, weighed (58.1 _+ 1.5 kg liveweight, LW; mean + SEM) and housed in metabolism cages. Water was available ad lib. The diet consisted of chaff hay [lucerne:oaten, 50: 50; 6.5 MJ metabohzable energy (ME)/kg] and pellets (Barastoc, Pakenham, Victoria, Australia; 9.0 MJ ME/kg) mixed 2:l. The ration, fed to maintenance (lactation), was offered in two halves after each milking routine, at 1Oa.m. and 1Op.m. The ewes weighed 47.4 + I.0 kg at the end of the experiment (day 21 of lactation).

Experimental procedures

Two polyvinyl chloride catheters of 1.0 mm i.d. and 1.5 mm o.d. (Dural Plastics. Sydney, New South Wales, Australia) were placed in the jugular veins of all ewes 4-5 days before infusions began. One catheter was for the infusion of saline or EGF, the other for the collection of blood. On day 9 of lactation, all ewes received continuous infusions of 200ml saline/day over 4 days (days 14). followed bv a continuous infusion of 0.5 me EGFidav in 200 ml saline over 4 days (days 558). Subseqiently all ewes received a continuous infusion of 200ml saline/day over another period of 4 days (days 9-12). PI1 infusates were kept in an iced-water bath and delivered through a filter (0.22pm, Millipore Corp., Bedford, MA, U.S.A.) using a peristaltic pump (Gilson, Minipuls 2, John Morris Scientific, Balwyn, Victoria, Australia).

Ewes were machine-milked and hand-stripped at 9 a.m. and 9 p.m. daily. Oxvtocin (10 I.U.. Sigma Chemical Co.. St. Louis,. MO, U:S.A.) WEtS injected i.v. just before milking to facilitate milk ejection (Mellor and Murray, 1985). Blood samples (IO ml) were collected in centrifuge tubes coated with heparin each day before the morning milkings, and placed on ice; following preparation of the plasma. all

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samples were stored at -17°C. Urine was collected in buckets containing sufficient 10 N HCI to keep the pH below 3; aliquots (100 ml) were stored at - 17°C.

Blood, milk and urine measurement3

The concentrations of sodium and potassium in plasma, urine and milk were measured by flame photometer emis- sions (Corning 400). The calcium and magnesium concen- trations in plasma, urine and milk were determined by atomic absorption s~ctrophotometry (Varian AA-1475). The creatinine concentrations in plasma and urine were measured (Taussky, 19.56) and daily creatinine clearance was calculated as an approximation of the glomerular filtration rate (GFR).

Statistical analysis

To analyse the effect of EGF infusion, the mean during the EGF infusion period (days 5-8) was compared with that during the first period of saline infusion (days l-4) by using the Wilcoxon signed-rank test (Snedecor and Cochran, 1976). This was the case for most variables, unless stated otherwise. Observations recorded for the first day of the second period of saline infusion (day 9) were not included in these analyses because the ewes were still exhibiting carry-over effects from the EGF infusion.

RESULTS

During days 10-12, the levels of most variables ap- peared to have returned to values recorded during the first saline infusion period, unless stated otherwise.

Electrolyte projiles

The substantial increase in urine volume excreted by the ewes during EGF infusion was accompanied

by a marked lowering (P < 0.02) in the urinary concentrations of all electrolytes (Figs lb, 2b, 3c and 4c), with the most marked decline occurring on days 5 and 6. For these variables, the measurement on the final day of EGF infusion (day 8), rather than the mean during the period of EGF infusion, was com- pared with the pretreatment mean. This comparison was considered more appropriate since the urinary concentratjons fell progressively throughout the period of EGF infusion. The length of time required for the urinary electrolyte concentrations to recover after EGF infusion ranged from immediately (for magnesium), to 1 day (calcium) and 3 days (sodium and potassium).

Sodium. Plasma sodium concentration was not affected by infusion of EGF; the mean value on day 4 was 167.0 & 2.8 mmol/l. The milk sodium concentration increased during EGF infusion (Fig. la) and was higher on day 8 (P < 0.06); the value on the final day of EGF infusion was com- pared with the pretreatment mean (for explanation, see section on electrolyte profiles). However, there was no effect of EGF on the secretion of sodium in milk; the value on day 4 was 19.1 + 1.3 mmof. By contrast, the mean excretion of urinary sodium declined during the period of EGF infusion (Fig. Ic) and was lower (P < 0.04) on day 8 than the pretreat- ment mean. Because more sodium was excreted in urine than secreted in milk, the profile of total sodium loss (total output in milk and urine, Fig. Id) was similar to that of the urinary excretion of sodium.

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Fig. I. Concentrations of sodium in (a) milk and (b) urine; (c) urinary excretion of sodium and (d) total sodium loss of early-lactating ewes (N = 6) during saline and EGF infusions. Ewes received a continuous infusion of saline (200 ml/day) for 4 days (days IA), then 0.5 mg EGF/day for 4 days (days 5-8). followed by saline (200 ml~day) for 4 days (days 9-12). The open symbois refer to periods of saline infusion: the closed symbols refer to the period of EGF infusion. Plotted points represent mean values and standard

errors are shown as vertical bars.

EGF alters electrolyte profile of lactating ewes 689

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Fig. 2. (a) Secretion of potassium in milk; (b) concentrations of potassium in urine; (c) urinary excretion of potassium and (d) total potassium loss of early-lactating ewes (N = 6) during saline and EGF infusions. Ewes received a continuous infusion of saline (200 ml/day) for 4 days (days l-4), then 0.5 mg EGF/day for 4 days (days S-8), followed by saline (200 ml/day) for 4 days (days 9-12). The open symbols refer to periods of saline infusion; the closed symbols refer to the period of EGF infusion. Plotted points represent

mean values and standard errors are shown as vertical bars.

During days 9 and 10, daily urinary and total sodium losses continued to fall and remained lower (P -C 0.04) than the pretreatment means (Figs lc and d). Thereafter, these values returned to levels observed in the first period of saline infusion.

Potassium. The plasma concentrations of potass- ium declined during EGF infusion, but this was not statistically significant; the value on day 8 (4.3 + 0.3 mmol/l) was compared with the pretreat- ment mean. On day 4, the mean concentration of potassium in plasma was 4.5 f 0.1 mmol/l. The con- centration of potassium in milk on day 4 was 29.8 * 0.74 mmol/l and values were not affected by EGF Infusion. Similarly, the secretion of potassium in milk was reduced during EGF infusion but the effect was not significant (Fig. 2a). The urinary excretion of potassium fell during the EGF infusion period (Fig. 2c) and was lower (P < 0.04) on day 8 than the pretreatment mean. Total potassium loss (Fig. 2d) had a similar profile to that of urinary excretion of potassium.

Calcium. Plasma concentrations of calcium (Fig. 3a) fell (P < 0.02) during EGF infusion. The con- centration of calcium in milk on day 4 was 47.6 + 1.3 mmol/l and there was no effect of EGF infuston. By contrast, the secretion of calcium in milk was reduced during EGF infusion (Fig. 3b) and was lower (P = 0.036) on day 8 than the pretreatment mean. Mean urinary excretion of calcium was in- creased (P = 0.036) markedly throughout EGF infu- sion (Fig. 3d). As more calcium was secreted into mammary secretions than excreted into urine, the

profile of total calcium loss (Fig. 3e) was similar to that of the secretion of calcium in milk; however there was no effect of EGF infusion on the total calcium loss.

During days 10-12, the secretion of calcium in milk, and thus the total daily calcium loss, were lower (P < 0.04) than the pretreatment means respectively.

Magnesium. Hypermagnesaemia (P < 0.02) oc- curred during EGF infusion (Fig. 4a); the value on day 8 was compared with the pretreatment mean. Similarly, the concentration of magnesium in milk was increased (P = 0.036) during the treatment period (Fig. 4b). However, the secretion of mag- nesium in milk was not affected by EGF; the value on day 4 was 11.4 + 0.9 mmol. By contrast, the urinary excretion of magnesium was not affected on day 5, but was reduced (P = 0.07) for the remainder of the EGF infusion period (Fig. 4d). Total magnesium loss had a similar profile to that of the urinary excretion of magnesium (Fig. 4e).

During the second period of saline infusion, the urinary, and thus the total, magnesium loss was higher (P = 0.059) than the pretreatment means, respectively (Figs 4d and e).

Creatinine clearance

The plasma concentration of creatinine on day 4 was 0.066 + 0.003 mmol/l and there was no effect of EGF infuston. By contrast, the concentration of creatinine in urine was progressively reduced during EGF infusion (Fig. 5a). The excretion of creatinine in urine was not affected on day 5, but fell (P = 0.036)

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Fig. 3. (a) Concentrations of calcium in plasma; (b) secretion of calcium in milk; (c) concentrations of calcium in urine; (d) urinary excretion of calcium and (e) total calcium loss of early-lactating ewes (N = 6) during saline and EGF infusions. Ewes received a continuous infusion of saline (200 ml/day) for 4 days (days IA), then 0.5 mg EGF/day for 4 days (days 5-8) followed by saline (200 ml/day) for 4 days (days 9-12). The open symbols refer to periods of saline infusion; the closed symbols refer to the period of EGF

infusion. Plotted points represent mean values and standard errors are shown as vertical bars.

during days 6-8 of the experiment (Fig. 5b). Simi- larly, GFR declined (P < 0.03) during the last 3 days of EGF infusion (Fig. 5~).

DlSClJSSION

We have investigated the responses of the electro- lyte profile in early-lactating ewes to chronic i.v. EGF infusion (0.5 mg/day for 4 days) and have demon- strated that electrolyte secretion by the mammary glands and urinary excretion of electrolytes are altered during the infusion of EGF. Other results from the same experiment have been reported pre- viously and the most interesting responses to EGF infusion were the immediate and concurrent poly- dipsia and diuresis (Gow and Moore, 1992). Despite this, there was no great effect of EGF on mean voluntary feed intake, output of faecal dry matter or water retention, although milk yield was adversely affected (Gow and Moore, 1992).

The physiological role of EGF on the kidney in vivo remains to be elucidated (Fisher et al., 1989). Sack and Talor (1988) suggested a physiological role of EGF in the proximal tubules and medullary portions of the rabbit nephron, because of the presence of low and high affinity binding sites for EGF in these areas. In other in vitro studies, it was shown that microper- fusion of isolated rabbit cortical collecting ducts (CCD) with EGF reduces the AVP-stimulated water (Breyer et al., 1988; Breyer, 1991) and electrolyte transport (Vehaskari et al., 1988). In the present experiment, we have demonstrated that low dose i.v. infusion of EGF into conscious ewes has affected, in different directions, the excretion of various electro- lytes by the kidney.

Although the lactating ewes of this study excreted daily 15 times more sodium and 12 times more potassium into urine than that secreted into milk during the control period, the daily output of both via urine (and thus total loss), but not via milk, was reduced during EGF infusion. Our in vivo results

EGF alters electrolyte profile of lactating ewes 691

contrast with the report by Vehaskari et al. (1988) that active sodium reabsorption in perfused isolated rabbit CCD is inhibited by EGF. Indeed, Scoggins et al. (1984) reported a transient increase in urinary sodium and potassium excretion (within 0.5 hr after the start of infusion of 125 pg EGF/hr) by crossbred ewes, but normal values were recorded by 5 hr. The differences between the results reported here and these workers (Scoggins et al., 1984; Vehaskari et al., 1988) indicate possibly a dose response, physiological or methodological effect.

The present study demonstrates that low-dose EGF infusion has no effect on the plasma concen- trations of sodium or potassium. However, the main determinant of thirst is plasma osmolality (Thomp- son and Baylis, 1988) and that to maintain relatively constant plasma osmolality, there are changes in plasma concentrations of various hormones (e.g. arginine vasopressin, AVP) and water intake. For example, intracarotid infusions of hypertonic saline solutions into goats increase osmolality and concen-

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tration of sodium in the lateral ventricle cerebrospinal fluid (CSF), and result in a 2-8-fold increase in total volume of water drunk within a 25 min period (Thornton et al., 1985). These workers also suggested that osmotically induced drinking in the goat may be due to an osmoreceptor mechanism, as hypertonic infusion of urea also increased sodium concentration and osmolality in the CSF, but had no effect on drinking. It appears that chronic low-dose EGF treatment stimulates diuresis and polydipsia in con- scious sheep through different mechanisms, as plasma concentrations of sodium remained unchanged throughout the present experiment. The EGF- induced reduction in urinary excretion of both sodium and potassium was associated with homeo- stasis of the plasma concentrations of both electro- lytes. By contrast, Scoggins et al. (1984) reported that continuous infusion of much higher doses of EGF (125 pg/hr) into crossbred ewes reduced plasma con- centrations of potassium by 24 hr, but no details of fluid balance were given.

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Fig. 4. Concentrations of magnesium in (a) plasma; (b) milk; (c) urine; (d) urinary excretion of magnesium and (e) total magnesium loss of early-lactating ewes (N = 6) during saline and EGF infusions. Ewes received a continuous infusion of saline (200 ml/day) for 4 days (days 14). then 0.5 mg EGF/day for 4 days (days 5--Q, followed by saline (200 ml/day) for 4 days (days 9-12). The open symbols refer to periods of saline infusion; the closed symbols refer to the period of EGF infusion. Plotted points represent mean

values and standard errors are shown as vertical bars.

C. B. Gow et al.

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Fig. 5. (a) Concentration of creatinine in urine; (b) output of creatinine in urine and (c) GFR of early-lactating ewes during saline and EGF infusions. Ewes received a continu- ous infusion of saline (2OOml/day) for 4 days (days Ia), then 0.5 mg EGF/day for 4 days (days S-8), followed by saline (200 ml/day) for 4 days (days 9-12). The open sym- bols refer to periods of saline infusion; the closed symbols refer to the period of EGF infusion. Plotted points represent mean values and standard errors are shown as vertical bars.

The lactating ewes secreted 35 times more calcium in milk than in urine during the control infusion period and the secretion of calcium in milk was progressively reduced as the plasma concentration of calcium fell during EGF infusion. The urinary ex- cretion of calcium increased during EGF infusion in the lactating ewes, and this response could be due to the observed decrease in GFR, a decrease in renal tubular reabsorption of calcium (not measured) or a change in renal blood flow (not measured). Hypocal- caemia was also reported (Moore et al., 1986) in Merino wethers given a considerably higher dose of EGF i.v. (129 pg/kg LW over 24 hr), but this was

associated with increased parathyroid hormone (PTH) concentration in the serum and reduced uri- nary excretion of calcium, reportedly due to the accompanying anorexia and resultant lowering of the filtered load of calcium. In the present study, food intake was reduced slightly during EGF infusion (Gow and Moore, 1992) but plasma PTH concen- trations and calcium absorption from the gastrointes- tinal tract were not measured. The difference between the responses of urinary calcium excretion to different EGF doses used in these two experiments could be due to a dose response effect to EGF, methodology, physiology or breed. The kidneys excreted daily three times more magnesium than the mammary glands during the control infusion period. The EGF-induced hypermagnesaemia in the lactating ewes was associ- ated with reduced urinary excretion of magnesium (and total Mg losses) but no change in the secretion of magnesium in milk. Moore et al. (1986) also reported hypermagnesaemia associated with reduced urinary excretion of magnesium during infusion of much higher doses of EGF in Merino wethers.

The sites of action of EGF at the level of the kidney and subsequent biological effects have been reviewed recently (Fisher et al., 1989). The present study has demonstrated hitherto unreported changes in electro- lyte profile and secretion/excretion of lactating ewes that accompanies EGF-induced polydipsia and diure- sis (Gow and Moore, 1992). It still remains to be determined whether the polydipsia induced by EGF infusion in sheep (Gow and Moore, 1992) is the primary physiological response or a secondary re- sponse to the concomitant diuresis. Water and elec- trolyte homeostasis is a balance between intake and output and is under the influence of plasma concen- trations of AVP, renin, aldosterone and atria1 natri- uretic factor (Andersson, 1978; Thompson and Baylis, 1988; Rouffignac et al., 1991). Because of the additional effect of electrolyte uptake from plasma and resultant secretory activity of the mammary glands on kidney responses during EGF infusion in lactating ewes, more detailed experiments are being conducted on non-lactating crossbred ewes to evalu- ate the neuroendocrine and renal electrolyte re- sponses to similar EGF treatment. This is the first time that EGF infusion has been shown to reduce calcium secretion by the mammary glands and thus EGF may be involved in the calcium homeostasis of the lactating animal.

Results from the present study have further ramifi- cations as the physiological importance of growth factors in mammary secretions for the sucking neonate has not been fully realized (Read, 1988). Indeed, it has been shown that the concentrations of EGF found in milk from humans, ruminants, mice and marsupials (Read, 1988), insulin-like growth factor (IGF-I) in pigs (Simmen et al., 1988) and platelet-derived growth factors and IGF-I in rumi- nants and marsupials (Read, 1988) are high in early-mid lactation and decline in mid-late lactation, especially towards the time of weaning. In conjunc- tion with this, most of the milk-derived growth factors are available to the infant, as degradation in the stomach is minimal (Read, 1988). The effect of milk-derived EGF on the renal function of suckling neonates is unknown.

EGF alters electrolyte profile of lactating ewes 693

Acknowledgements-The authors thank A. F. Davidson, R. Fitzpatrick, L. Sayer, S. Smith and R. R. Vavala for skilled technical assistance.

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