ADRENALECTOMIZED rats drink less than normal rats when 2 per cent saline. daily by stomach tube and water to drink freely, died quickly but such

Transcription

1 THE EFFECT OF PROLONGED INTRAGASTRIC INFUSIONS OF ISOTONIC AND HYPERTONIC SALINE ON WATER AND SODIUM EXCRETION AND ON EXCHANGEABLE BODY SODIUM IN NORMAL AND ADRENALECTOMIZED RATS. By C. J. EDMONDS. From the Department of Pharmacology and Therapeutics, University of Sheffield. (Received for publication 2nd October 1959) A method is described for implanting stomach tubes into rats. This has been used to study the effects on sodium metabolism of prolonged infusions of saline in normal and adrenalectomized rats. Adrenalectomy resulted in a delay in the excretion of sodium and water, but the excess fluid was excreted within 12 hr. of stopping the infusion. This fluid retention was associated with a considerable reduction in voluntary fluid intake. Adrenalectomized rats infused for 4 days with either 55 ml. of 2 per cent saline or 95 ml. of 9 per cent saline daily showed no increase of total exchangeable sodium when this was measured 12 hr. after stopping the infusion. In normal rats infused with 2 per cent saline, GFR rose immediately and remained elevated throughout the infusion. When 9 per cent saline was given, a rise did not occur until the 5th hr. of infusion. The adrenalectomized rats showed no significant changes in GFR with either solution. The delay in dosium excretion after adrenalectomy appears to be due, in part, to an alteration in renal tubular function. ADRENALECTOMIZED rats drink less than normal rats when 2 per cent saline is the only drinking fluid provided [Edmonds, 196 a]. It was possible that this change in drinking behaviour protected the adrenalectomized animals from harmful effects of excessive salt intake. Some support for this hypothesis comes from the work of Anderson, Joseph and Herring [194] who found that adrenalectomized rats when given 5 per cent saline four times daily by stomach tube and water to drink freely, died quickly but such animals remained in excellent health if given 1 per cent saline only; they suggested that the excess salt was harmful to adrenalectomized rats. The most striking change in sodium metabolism which follows adrenalectomy is increased urinary sodium loss when the salt intake is normal; but there is evidence that the adrenal cortex plays some part in sodium excretion when this is provided in excess [Reforzo-Membrives, Power and Kepler, 1945; Roemmelt, Sartorius and Pitts, 1949; Gross and Dettbarn, 1956]. Most of this work has demonstrated delay in the excretion of sodium and water after the acute administration of sodium loads intravenously or by stomach tube. Only occasionally has an attempt been made to differentiate the relative parts played by glomerular filtration and renal tubular reabsorption. In a previous paper, it has been shown that increasing the sodium chloride intake of adrenalectomized rats by giving them 2 per cent saline to drink, produced 171

2 172 Edmnonds a slight rise of serum sodium concentration but no significant increase in exchangeable sodium, findings similar to those obtained with normal rats [Edmonds, 196 a]. There is, therefore, an apparent paradox between the effects of acute and chronic high salt intake. The object of the present work was to determine whether, in fact, large amounts of sodium chloride were toxic to adrenalectomized rats and whether salt and water retention occurred when intake was raised considerably above the level attainable by giving 2 per cent saline to drink. To do this, a method of implanting stomach tubes was developed which allowed prolonged infusions of saline to be carried out without disturbing the rat. Kellog and his associates [1954] described such a method but the present technique is simpler and, moreover, it is not necessary to restrain the rat during the infusion. METHODS Male albino rats weighing 25-3 g. were used. Their care, the operative procedures and the method of measurement of exchangeable sodium were as previously described [Edmonds, 196 a]. Implantation of stomach tubes was carried out under ether anaesthesia. Approach to the stomach was by a left transverse subcostal incision. A small pouch of stomach was formed by using a silk-purse string suture. The apex of the pouch was opened and a polyvinyl chloride tube (diameter 2 mm.) was introduced to about 1 cm. into the stomach. The purse string was tightened, care being taken not to interfere with the blood supply. The stomach pouch was then sutured to the abdominal wall, the apex of the pouch being outside the peritoneal cavity. The muscular abdominal wall was closed around the pouch and the whole area dusted with an antibiotic and sulphonamide powder. The distal end of the tube was threaded beneath the skin and brought out through an incision in the back of the neck. Two anchoring sutures were placed at intervals along the subcutaneous course of the tube to prevent its removal by the rat. The implanted tubes were tolerated well by both the normal and adrenalectomized rats. They remained in place for several weeks and the animals gained weight normally. After the stomach tube implantation, the rats were kept in individual cages. Technique of Intragastric Infusions.-Infusions in the control rats were started 12 days after implanting the stomach tubes. Adrenalectomy was performed 1 days after the tube implantation and 3 days later the infusions were begun. The infusions were carried out daily for four successive days. Each morning the rat was placed in a small circular cage and the end of the implanted tube connected by way of a narrow bore polyvinyl tube to a funnel. The latter was filled hourly with 5-5 ml. of 2 per cent saline or 9-5 ml. of 9 per cent saline. The height of the funnel and the length of the polyvinyl resistance tubing were adjusted by preliminary trial so that the saline dripped slowly into the stomach during the following hour. In this way it was possible to infuse for many hours without provoking diarrhea. The rats were undisturbed by the procedure. They were unrestricted in their movements but generally slept for most of the time. No food or fluid were allowed during the period of infusion. At night, the rats infused with 2 per cent saline were given 1-5 per cent saline to drink; those receiving 9 per cent saline infusions were given 1 per cent saline. On the morning of the 5th day, the rats were injected intraperitoneally with 24Na for estimation of exchangeable sodium. Infusion was not carried out on this day and the rats were allowed only 2 ml. of 1 per cent saline for drinking during the following 24 hr. Measurement of Glomerular Filtration Rate (GFR) and Sodium Excretion during the

3 Intragastric Infusions in Rats 173 Intragastric Infusions.-Twenty-four rats were used. Stomach tubes were implanted 1 days before adrenalectomy or mock-operation and the infusion was carried out 7 days after the latter operation. All the rats remained fit and were eating well: they received 1 per cent saline to drink from the time of stomach tube implantation. At 9 A.M. on the day of infusion, the rat's bladder was emptied by gentle supra-pubic pressure and the rat then placed in a metabolic cage provided with 1 per cent saline for drinking but no food. All the urine passed subsequently was collected in graduated cvlinders. At 2 P.M. the bladder was emptied again and the urine obtained added to the morning's collection. This 5 hr. urine collection provided baseline excretion rates of sodium and creatinine. The stomach tube was then connected to a constant infusion pump and the infusion of saline begun. Urine collections were made at intervals, on each occasion the bladder being emptied by gentle supra-pubic pressure. The infusions of 2 per cent saline were continued for 4 hr. and those of 9 per cent saline for 5 hr. At the end of the infusion, a final urine collection was made, the rat was immediately anaesthetized with ether and blood taken from the common carotid artery for serum creatinine and sodium determinations. Chemical Methods.-Sodium and potassium determinations were made by flame photometry. Creatinine was estimated in plasma and urine using the method described by Muntwyler and Griffen [1953]. The results are given as means ± standard error of the mean. RESULTS The Effect of Intragastric Infusions upon Exchangeable Body Sodium Infusion with -9 per cent Saline.-The changes in weight and in urine output for a typical day of infusion showed that the adrenalectomized rats excreted substantially less urine than the normal rats and gained weight (Table I). Overnight the rats had access to 1 per cent saline and the adrenalectomized animals drank much less than the controls. Without infusion, the overnight this was reduced fluid intake of the adrenalectomized rats was 35 ± 7-5 ml.; by the infusion to ml. By the following morning, the weights of both the normal and adrenalectomized rats had returned to the pre-infusion level. After 4 consecutive days of infusion, serum sodium concentration and exchangeable sodium were similar in the two groups (Table II). The serum potassium concentration was 4-2 +±-12 meq/l. in the control group compared with 5.5 ± -8 meq/l. in the adrenalectomized rats. Infusion with 2 per cent Saline.-In this study on eight adrenalectomized rats, three developed diarrhcea and had to be withdrawn from the experiment. This diarrhcea did not occur during the infusion but developed during the following night. It apparently resulted from excessive drinking since the affected rats took more than 12 ml. of 1'5 per cent saline overnight. The five adrenalectomized rats from which the results given in the tables were obtained, remained fit throughout. Table I shows the changes in weight and the urine output on a typical day of infusion. The adrenalectomized rats excreted a smaller volume of urine than the normal rats and this was reflected in the weight changes. Both the normal and the adrenalectomized rats had free access to 1.5 per cent saline overnight and by the following morning the weight of all the rats had returned to the pre-infusion level. When at the end of 4 days of infusion, serum sodium concentration and

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5 Intragastric Infusions in Rats exchangeable sodium were measured no significant difference between the groups was found (Table II). The serum potassium concentration was higher in the adrenalectomized rats at 6-7 ±-17 meq/l. than the value 4.4 ±35 meq/l. found in the controls. TABLE II.-EFFECT OF INTRAGASTRIC INFUSION OF 95 ML. OF -9 PER CENT OR 55 ML. OF 2 PER CENT SALINE DAILY FOR 4 DAYS ON THE EXCHANGEABLE SODIUM OF NORMAL AND ADRENALECTOMIZED RATS. THE TOTAL DAILY FLUID AND SODIUM INTAKE INCLUDES THE OVERNIGHT FLUID CONSUMPTION. The body weights of the rats are given in the last column of Table I No. Infused Total daily Total daily Serum Exchangeable of solution fluid intake Na intake Na Na rats per cent ml. meq meq/l. meq/1 g. body weight Control ±t ± ±.1 Adrenalectomized ± ±* ±-7 4-6± 11 Thus, with 2 per cent as with 9 per cent saline infusions, excretion of water and sodium was delayed in the adrenalectomized rats when compared with the normal animals but within 12 hr. of stopping the infusion, the retained fluid was excreted and body water and sodium restored to normal. The Effect of Intraqastric Infusions on GFR and Sodium Excretion.-The experiments were carried out to investigate the changes in renal function during the infusions, responsible for the delay in excretion after adrenalectomy. GFR was calculated from the endogenous creatinine clearance. Infusions with.9 per cent Saline.-The rate of sodium excretion before the infusion was higher in the adrenalectomized rats than in the normal rats, and it rose in both groups during the infusion (Table III). The normal rats, however, increased their rate of sodium excretion more rapidly than the adrenalectomized rats so that during the last hour they excreted twice as much sodium as the latter. TABLE III.-EFFECT OF INTRAGASTRIC INFUSION OF -9 PER CENT SODIUM CHLORIDE SOLUTION AT 17 ML./MIN. ON WATER AND SODIUM EXCRETION AND ON GFR or NORMAL AND ADRENALECTOMIZED RATS. Infusion was carried out over time -5 hr. (Rate of sodium infusion=25-7 1Eq/min.) Na No. Time Water Na GFR concentraratfs hr. l./min.,eq/min. ml./min. tion meq/ml. Control ± ± ± ±1*1 1-3 ±1-21 2*16 ± ± ±2-5 2*16 ± ±1* ± Adrenalectomized ± ± ± ± ± ± ± ± i ± ±

6 176 Edmonds The GFR was similar in the two groups before the infusion. No significant change occurred in GFR during the course of the infusion in the adrenalectomized rats, and the normal rats showed a significant increase only during the last hour of collection. There was little change in urinary sodium concentration during the infusion in the normal rats, the rates of water and sodium excretion increasing in parallel. The sodium concentration in the urine of the adrenalectomized rats fell during the infusion to a value approximating to that of the infused fluid. Infusion with 2 per cent Saline.-This resulted in a rise of sodium excretion in both the normal and adrenalectomized rats, but the rate of increase was greater in the former (Table IV). During the first hour, there was no increase in sodium excretion by the adrenalectomized rats although the normal rats nearly trebled their excretion, and a considerable difference between the groups was maintained throughout the period of study. TABLE IV.-EFFECT OF INTRAGASTRIC INFUSION OF 2 PER CENT SODIUM CHLORIDE SOLUTION AT 11 ML./MIN. ON WATER AND SODIUM EXCRETION AND ON GFR OF NORMAL AND ADRENALECTOMIZED RATS. Infusion was carried out over the time -4 hr. (Rate of sodium infusion = 37.6,iEq/min.) No. Time Water Na GFR concentrarats hr cl./min. ueq/min. ml./min. tion meq/ml. Control i ± ± i *54 ± ± ± ± ± ±F1-8 2f32 ± Adrenalectomized ± 5. 7 ± ± ± 4.9 5f27 ±1f3 2f4 ± ± ± ± ± ±2* ± Before the infusion, GFR was similar in the two groups but subsequently while that of the adrenalectomized rats did not change significantly during the infusion, that of the normal rats underwent a substantial rise. This was seen in the first hour and the GFR remained elevated during the following 3 hr Ṫhe changes in water excretion followed fairly closely the changes in sodium excretion. The urinary sodium concentration rose during the infusion in both the normal and the adrenalectomized rats and eventually approximated to the sodium concentration of the infused fluid. Serum Sodium Concentration.-This was not measured before the infusions but in groups of normal and adrenalectomized rats maintained drinking 1 per cent saline, there was no significant difference in serum sodium level. The infusion of 9 per cent saline produced no significant increase in the serum sodium when this was measured at the end of the 5 hr. infusion. At the end of the 2 per cent infusion, however, there was a considerable increase in

7 Intragastric Infusions in Rats serum sodium concentration in both the normal and adrenalectomized rats; in the controls, it rose from 144 ±-76 to 158 ±3-8 meq/l. while in the adrenalectomized animals the rise was from 141 ± 1 3 to *2 meqil. DISCUSSION The explanation of the apparent discrepancy between the effects of acute salt loads and chronic high salt intake after adrenalectomy is now apparent. Sodium and water excretion was, in fact, delayed compared with the normal state, but the excess was excreted during the 12 hr. following the infusion. The adrenalectomized rats were as capable as the control rats of restoring the volume and sodium content of the extracellular fluid to the normal level, after the administration of large amounts of hypertonic or isotonic saline, although they did this less quickly. Rats consume nearly all their daily fluid intake during the night whether the drinking fluid is water or saline [Dicker and Nunn, 1957]. It would be expected that the adrenalectomized rats would tend to accumulate sodium and water during the night when drinking saline, so that in the early morning, they would have a higher body water and sodium content than the normal rats drinking saline in the same quantity. During the course of the day the excess would be excreted and body water and sodium restored to the normal level. There is some support for this hypothesis in the observation that sodium excretion during the morning before infusion was considerably greater in the adrenalectomized than in the normal rats although their intake of 1 per cent saline was similar (Tables III and IV). The cause of the delay in excretion after adrenalectomy may lie either in slower absorption of the infused solution from the gastrointestinal tract or in impairment in the response of the kidneys to the expansion of the body fluids. Gastrointestinal absorption of sodium has been shown to be somewhat delayed after adrenalectomy [Dennis and Woods, 1942], but it seems unlikely that this was an important factor in the present experiments since diarrhoea was not produced although a large volume of saline was given. Consideration of the second possibility, namely an alteration in renal function, led to a closer study of the changes in GFR and sodium excretion during the infusions. The rise of GFR which occurred when hypertonic saline was given to normal rats probably contributed to the rapid rise of sodium excretion. But when isotonic saline was infused sodium excretion rose almost as rapidly although GFR tended to fall slightly. The differences between the normal and adrenalectomized rats infused with isotonic saline were so small, however, that it seems unlikely that the considerable difference in sodium excretion could be entirely related to this. It is probable that the increased sodium excretion during isotonic saline infusion was due, in part, to a change in renal tubular reabsorption of sodium and that adrenalectomy interfered with this response. Some evidence for this possibility has been previously obtained by intravenous hypertonic saline infusions in rats [Edmonds, 196 b] and has been suggested by others [Roemmelt, Sartorius and Pitts, 1949; Gross and 177

8 178 Edmonds Dettbarn, 1957]. Probably in rats, adrenalectomy impairs sodium excretion during oral saline loading in two ways. First by affecting renal tubular function so that decrease in sodium reabsorption is delayed, and secondly, by eliminating the rise of GFR. Some patients with adrenal insufficiency are unable to excrete salt satisfactorily when on a high salt intake and they may become cedematous [Willson and Sunderman, 1939; Reforzo-Membrives, Power and Kepler, 1945]. In the present experiments, the adrenalectomized rats did not develop cedema despite the large amount of saline infused, but the reason for this was that the excess retained was excreted during the overnight hours after the infusion. The normal rat has a much greater ability to excrete salt than man in whom an addition of only 2 g. of sodium chloride (about 5 meq of sodium/kg. body weight) daily to the normal diet leads to definite increase in weight and fluid and salt retention [Grant and Reischsman, 1946]. Evidently adrenal deficient rat maintains a superiority in this respect over adrenal deficient man. Burnett [1951] showed by means of intravenous hypertonic saline infusions in patients with adrenal insufficiency that the maximum urinary/plasma sodium ratio was less than in normal subjects. A similar finding in dogs has been described [Roemmelt, Sartorius and Pitts, 1949]. It has been postulated that this might be responsible for the inability of adrenalectomized animals to excrete sodium as rapidly as normal animals during salt loading. However, no impairment of this character was found in the present experiments. Adrenalectomy did not affect the maximum urinary sodium concentration attained during hypertonic infusions, and during the isotonic infusions changes in urinary sodium concentration occurred in a way essentially similar to those seen in normal animals. ACKNOWLEDGMENT My thanks are due to Professor G. M. Wilson for his generous advice and encouragemenit. REFERENCES ANDERSON, E., JOSEPH, M. and HERRING, V. V. (194). Proc. Soc. exp. Biol., N.Y. 44, 477. BURNETT, C. H. (1951). Transactions of the 2nd Conference on Renal Function. Josiah Macey Jr. Foundation. Page 16. DENNIS, C. and WOOD, E. H. (194). Amer. J. Physiol. 129, 182. DICKER, S. E. and NUNN, J. (1957). J. Physiol. 136, 235. EDMONDS, C. J. (196 a). Quart. J. exp. Physiol. 45, 163. EDMONDS, C. J. (196 b). J. Endocr. [In the press.] GRANT, H. and REISCHSMAN, F. (1946). Amer. Ht. J. 32, 74. GROSS, F. and DETTBARN, W. D. (1957). Acta Endocr. 22, 335. KELLOG, R. H., BTURACK, W. R. and ISSELBACHER, K. J. (1954). Amer. J. Physiol. 177, 27. MUNTWYLER, E. and GRIFFEN, G. E. (1953). Amer. J. Physiol. 173, 145. REFORZO-MEMBRIVES, J., POWER, M. H. and KEPLER, E. J. (1945). J. clin. Endocr. 5, 76. ROEMMELT, J. C., SARTORIUS,. W. and PITTS, R. F. (1949). Amer. J. Physiol. 159, 124. WILLSoN, D. M. and SUNDERMAN, F. W. (1939). J. clin. Invest. 18, 35.