Fluid balance, electrolyte profiles and plasma parathyroid hormone concentrations in ewes treated with epidermal growth factor C. B.

Gow, M. Wilkinson, M. J. Silvapulle and G. P. M. Moore

School of Agriculture, La Trobe University, Bundoora, Victoria, 3083 Australia *Endocrine Laboratory, The Royal North Shore Hospital, St Leonards, New South Wales, 2065 Australia tCSIRO, Division of Animal Production, P.O. Box 239, Blacktown, New South Wales, 2148 Australia received

19 December 1991

ABSTRACT

The infusion of low doses of epidermal growth factor (EGF) into lactating ewes stimulates water intake and urine volume. The plasma concentrations and daily output of various electrolytes in milk and urine are also affected. We have investigated this further by recording the effects of EGF infusion on fluid balance, electrolyte profiles and plasma concentrations of glucose and parathyroid hormone (PTH) in non-pregnant, non-lactating ewes. Twenty-four animals (n= 8 per group) received infusions of 100 ml saline/day into the jugular vein for 10 days (days 1\p=n-\10)followed by EGF at a dose rate of either 1 (low dose), 5 (medium dose) or 10 (high dose) \g=m\g/kgliveweight per All ewes day in 100 ml saline for 5 days (days 1 \p=n-\15). then received an infusion of 100 ml saline/day for 10

days (days 16\p=n-\25). Most plasma and

urine samples had undetectable concentrations of EGF-immunoreactive material during the periods of saline infusion. During EGF infusion, the highest amounts of EGF infusate excreted in urine were 1\m=.\6,5\m=.\9 and 5\m=.\6% for ewes in low, medium and high dose groups respectively. Water intake increased by 17% (0\m=.\5kg), 88% (2\m=.\5kg) and urine volumeincreased by 29% 89% (2\m=.\3kg) and and 134% (2\m=.\1kg) for the (0\m=.\5kg), 108% (2\m=.\2kg) three groups respectively. Fluid balance and feed

INTRODUCTION

The kidney is a major site of synthesis for the epider¬ mal growth factor (EGF) precursor (Rail, Scott, Bell et al. 1985) and EGF itself (Kanda, Saha, Nomata et al. 1991). Specific renal receptors for EGF have been identified in vitro (Norman, Badie-Dezfooly, Nord et al. 1987) and it has also been shown that EGF is

intake

were

not

affected

output of faecal dry

by

matter

EGF was

infusion, but the reduced in

ewes

higher doses of EGF. All levels of EGF resulted in hypocalcaemia, increased plasma PTH concentrations and hypermagreceiving the

two

nesaemia. There was no effect of EGF on plasma concentrations of K+ and glucose or on daily urinary excretion of K+ and Mg2+. The only response to the low dose was a reduced plasma concentration of Na+ and an increased daily urinary urate excretion. The two higher doses increased the daily urinary excretion of Na+, PO3\m=-\4and urate, but had no effect on the respective concentrations in plasma. Urinary Ca2+ excretion was reduced only during infusion of the medium dose of EGF. The responses of most variables were similar during infusion of the medium and high doses of EGF. All three doses of EGF induced polydipsic and diuretic responses in ewes, and infusions of 5\p=n-\10\g=m\gEGF/kg liveweight per day affected renal excretion of Ca2+, Na+ and PO3\m=-\4.We interpret the responses of the kidney and plasma PTH concentrations as a means of maintaining the homeostasis of plasma profiles of

electrolytes. Journal of Endocrinology (1992) 135,

91\p=n-\101

potent growth stimulator in renal tubular cells (Norman et al. 1987; Kanda et al. 1991). In addition, Breyer, Jacobson & Breyer (1988) demonstrated that EGF can alter electrolyte and water transport in iso¬ lated perfused rabbit cortical collecting ducts. How¬ ever, the direct in-vivo role of EGF in the kidney still remains to be elucidated (Fisher, Salido & Barajas, 1989). Sack & Talor (1988) suggested a physiological a

role for EGF in the proximal tubules and medullary portions of the rabbit nephron, due to the presence of low- and high-affinity binding sites for EGF. During daily i.v. infusions of 500 pg mouse EGF into early lactating ewes, profound increases in water intake and urine volume (Gow & Moore, 1992), in conjunction with changes in electrolyte profile (Gow, Silvapulle & Moore, 1992), were observed throughout a 4-day period of treatment. In order to investigate these responses further, we have measured the effects of three doses of EGF on renal function through changes in fluid balance and the excretion of Na+, K+, Ca2+, Mg2+, PO|~ and urate in+ non-pregnant, non-lactating ewes. As plasma Ca concentrations are regulated by parathyroid hormone (PTH) (Rasmussen, 1985), circulating concentrations of this hormone were measured during the experiment. Plasma glucose concentrations were also estimated as the induction of hypoglycaemia in rats injected with insulin concurrently increased water intake (LopezSela, Brime, Diaz et al. 1989).

NaCl/1) to give a stock concentration of 50 pg EGF/g and was stored in aliquots at —17 °C until infusion.

MATERIALS AND METHODS

University. Blood samples (10 ml) were collected into centri¬ fuge tubes coated with heparin each day at 09.00 h, and immediately cooled in ice. Following the separa¬ tion and removal of the red blood cells, the plasma 17 °C until assayed for EGF, Na+, was stored at K+, Ca2+, Mg2+, , urate, glucose and PTH. Urine was collected daily in plastic buckets contain¬ ing sufficient 10 mol HC1/1 to keep the pH below 3. Aliquots (100 ml) were stored at -17 °C until assayed for EGF and electrolytes. Urine volumes were calcula¬ ted as described previously (Gow & Moore, 1992). Faeces were collected and weighed every day; dupli¬ cate samples (approximately 90 g) were dried over¬ night at 65 °C for the determination of faecal dry

Experimental animals Twenty-four 6-year-old adult crossbred (BorderLeicester x Merino) ewes were selected from a single flock for these experiments. They were shorn, weighed and assigned to one of the three groups (n 8 per group) designated to receive a low dose (LD), medium dose (MD) or high dose (HD) of EGF. Their liveweights (LW; meanis.E.M.) were 54-3±2-2kg, 52-6±2-3kg and 51-4± 1-8 kg respectively. At the end of the experiment, the mean LW of each group had not altered significantly. The experiment was con¬ ducted in three trials, with six, eight and ten ewes in the first, second and third trials respectively. Ewes were housed randomly with continuous light in a tem¬ perature-controlled ( 17-22 °C) room. Each ewe was kept in an individual metabolism cage that facilitated the separation and collection of urine and faeces. =

Water was available ad libitum and each ewe was of¬ fered a calculated maintenance diet of 50:50 lucerne chaff:crushed oats (11-5 MJ metabolizable energy/ kg, air dry). The ration was offered in two halves at 11.00 h and 18.00 h daily. The ewes were accustomed to handling during the 3 weeks before the treatment

period.

Preparation

of EGF

The EGF for infusion was purified from mouse submandibular glands using the procedure of Savage & Cohen (1972). This was dissolved in saline (9 g

Experimental procedures Two polyvinyl chloride catheters (1-0 mm inner diameter by 1 5 mm outer diameter ; Durai Plastics, Sydney, NSW, Australia) were placed in the jugular veins of each ewe 4-5 days before infusion. One catheter was used for the infusion of saline or EGF and the other for the collection of blood. The ewes received a continuous infusion of 100 ml saline/day for 10 days (days 1-10), followed by 1 (LD), 5 (MD) or 10 (HD) pg EGF/kg LW per day in 100 ml saline for 5 days (days 11-15). Subsequently all ewes were infused with 100 ml saline/day for 10 days (days 1625). Infusâtes were kept in an iced-water bath and delivered through a filter (0-22 pm; Millipore Corp., Bedford, MA, U.S.A.) using a peristaltic pump (Gilson, Minipuls 2; John Morris Scientific Pty Ltd, Balwyn, Victoria, Australia). All procedures on ani¬ mals were carried out with the prior approval of the Animal Experimentation Ethics Committee, La Trobe ·

—

matter.

Radioimmunoassays for EGF

and PTH

Concentrations of EGF were measured in plasma and urine by the method of Panaretto, Moore & Robert¬ son (1982). The lower limit of sensitivity of this assay was 0- 8 pg/1 and the intra-assay coefficients of varia¬ tion (C.V.) were 4-5-7-3%. Plasma PTH was measured by an in-house radioim¬ munoassay using a polyclonal antiserum specific for the intact and N-terminal fragments of human PTH. There was no cross-reaction with the mid-molecule or C-terminal fragments of human PTH (McElduff, Wilkinson, Lackmann et al. 1989). The lower limit of detectability was 0-10 pg/1 and the intra-assay C.V. was 4-5%.

Concentrations of electrolytes and

glucose

The concentrations of Na+ and K+ in plasma and urine samples were measured by emission flame pho¬ tometry (IL flame photometer, Model 943; Instru¬ mentation Laboratories, Wilmington, MA, U.S.A.). The Ca2+ and Mg2+ concentrations were determined by atomic absorption spectrophotometry (IL model 457 Apparatus; Instrumentation Laboratories). The concentrations of P04~ and urate in plasma and urine, as well as plasma glucose, were measured with an Olympus Multianalyser (Model AU 5031;

Olympus Optical Ltd, Tokyo, Japan). The following style of abbreviation has been used : plasma concentrations of Na+ are denoted by PNa and urine concentrations of Na+ by UNa The daily renal excretion of Na+ (UNaV) was calculated by multi¬ plying urine volume with UNa ·

Statistical

analyses

Differences between the effects of treatment with saline and EGF were compared by using the Wilcoxon signed rank test (Devore & Peck, 1986; section 9.5, pp. 393-401). Observations on the first day of the experiment were not included as these samples were prepared incorrectly. Following an analysis of vari¬ ance to compare the three doses, Bonferroni confi¬ dence intervals were used for pair-wise comparison of dose means, whenever such further analysis was considered relevant (Devore & Peck, 1986; section 13.3, pp. 577-580). Observations recorded for the first day of the second period of saline infusion (day 16) were not included in these analyses because many of the ewes were exhibiting carry-over effects from the previous EGF infusion. RESULTS

EGF concentrations in

plasma

Immunoreactive EGF was not detected in the plasma of ewes during the saline infusion periods. However, during EGF treatment, individual plasria concentra¬ tions of EGF ranged from 0-8 to 9-3 pg/1 (LD group), 0-8 to 14-1 pg/1 (MD group) and 0-8 to 20-7 pg/1 (HD group). The protein was not detected over at least 2 consecutive days in 7, 2 and 2 ewes of each group

respectively.

Feed intake and output of faecal dry matter Throughout the experiment the mean voluntary daily feed intake was l-18±0-03 (s.e.m.) kg, l-15±0-03 kg and l-12±0-03 kg for the ewes in the LD, MD and HD groups respectively. One ewe in each of the LD

and HD groups voluntarily reduced its feed consump¬ tion during days 15-17. Both ewes consumed all of the daily ration thereafter (10 kg each). There was a reduction in the output of faecal dry matter during the period of EGF infusion in all groups (Fig. la), but this attained significance only for ewes in the MD (P