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Drinking by anuric rats in excess of apparent need
Physiology' and Behavior. Vol. 5, pp. 86i-871. Pergamon Press, 1970. Printed in Great Britain
Drinking by Anuric Rats in Excess of Apparent Need' STEPHEN
L. B L A C K A N D E D W A R D
M. S T R I C K E R
Department of Psychology, McMaster University, Hamilton, Ontario, Canada (Received 4 D e c e m b e r 1969) BLACK, S. L. AND E. M. STRICKER. Drinking by anuric rats in excess of apparent need. PHYSIOL.BEHAV.5 (8) 867-871, 1970.--Nephrectomized and ureterally ligated rats were found to drink in excess of apparent body fluid requirements as measured by external water exchange and by depression of plasma sodium concentration. The nature of the stimulus initiating this behavior is obscure. A renal hormone is unlikely to be a primary cause of the drinking because the fluid intakes of nephrectomized and ureterally ligated rats do not differ. While anuric rats are hypovolemic, this stimulus may not entirely account for the observed drinking response. Instead, the increased plasma concentrations of urea and potassium observed may cause drinking through cerebral dehydration and consequent shrinkage of hypothalamic osmoreceptors, despite hydration elsewhere in the body, because these substances penetrate the blood-brain barrier with difficulty. Alternatively, the drinking may be in response to the accumulation of toxic substances during anuria and may function to dilute them. Whatever the cause of drinking, the results suggest that anuric rats are inappropriate subjects for many experiments on thirst, unless the duration of anuria is brief. Anuria
Blood-brain barrier
Drinking
Nephrectomy
Thirst
Urea
Ureteral ligation
Procedure
THE STUDY of thirst in relation to body fluids is complicated by the existence of a renal mechanism for maintaining fluid balance. Consequently, nephrectomized or ureteraUy ligated animals are frequently used to ensure that a renal response will not occur. Although prolonged anuria results in progressive changes in body fluids [4, 21], it is generally assumed that such alterations have no effect on drinking. In this regard, Peters [19] has noted: "The anuric animal drinks only enough to maintain a constant effective osmotic pressure; that is, to keep the concentration of sodium in the serum from rising. It does not increase its drinking as the blood urea r i s e s . . . it drinks only enough to keep the volume of fluid and the effective osmotic pressure of this fluid in the body normal". The experiment reported here does not support this conclusion and suggests instead that anuric rats drink in excess of apparent body needs.
All operations were performed between 9.00 and 11.30 a.m., using ether anesthesia. Anuria was induced by bilateral nephrectomy (n = 31) through a dorsal approach, or by ligation of both ureters (n = 38) through a small ventral incision. Sham operations (n = 52) included all steps except removal of kidneys or ureteral ligation. Each operation lasted approximately 15 min. Immediately following surgery, the rats were weighed ( + 0.1 g) and replaced in their home cages without food. A funnel and graduated urine collection tube ( ± 0.1 ml) were placed beneath the cage of each sham operated rat. The rats were either deprived of fluid or allowed to drink water or 0.15 M NaCI solution. The treatment schedule for each group is presented in Table 1. Fluid intakes were recorded at variable time intervals, usually within one hour of the previous reading but less frequently overnight (the maximum interval between readings was 11 hr), and cumulative volumes of fluid ingested were later interpolated graphically. The total volume of urine excreted by each sham operated rat was recorded at the end of the experiment. Either 12 or 30 hr following surgery, each animal was deeply anesthetized with sodium pentobarbital (Nembutal ; 80 mg/kg), weighed, and bled from the abdominal aorta. An additional group of six intact rats was bled to provide control values. Plasma was analyzed for protein (by refractometer), potassium and sodium concentrations (by flame photometry), and osmolality (by freezing point depression). The water content of the plasma was determined by drying samples for 48 hr at 100°C.
METHOD
Subjects and Maintenance Male albino rats, weighing between 200 and 325 g, were individually housed in wire-mesh metabolism cages. Purina food pellets and water were available ad lib except during testing. In addition, the groups tested with 0.15 M NaCI solution were allowed continuous access to this fluid, beginning three days prior to testing. The fluids were presented in graduated tubes (d: 0.2 ml) attached to the front of each cage.
XThis study was supported, in part, by Research Grant APA-248 to Dr. E. M. Stricker from the National Research Council of Canada. 867
BLACK A N I) S IR ICI,.Llit
868
TABLE 1
Furthermore, the intake of water or saline by each anuric group did not differ from that of the appropriate sham operated group (all p's > 0.5). Individual anuric rats differed considerably in fluid intakes during the 30 hr test period. Water intakes ranged from 1.0 to 13.8 mill00 g body weight, while saline intakes ranged from 5.0 to 15.2 ml/100 g body weight. The volumes of fluid ingested by sham operated rats were equally variable. Standard errors of the mean across all treatments ranged from 0.9 to 2.1 ml/100 g body weight.
TREATMENT SCHEDULE
Group A. Sacrificed at 12 hr. Nephrectomized Sham nephrectomized Ureterally ligated Sham ureterally ligated B. Sacrificed at 30 hr Nephrectomized
Sham nephrectomized
Ureterally ligated
Sham ureterally ligated
n
Fluid Available
5 4 4 10 5 7
water none water water none water
13 5 4 13 5 3 14 5 4 12 5 3
water saline none water saline none water saline none water saline none
Water Exchange Table 2 presents the external water exchanges of anuric and sham operated rats during the experimental period. ABW, the change in body weight, was used to estimate net
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RESULTS
Cumulative volumes of fluid ingested by anuric and sham operated rats are presented in Fig. 1. Anuric rats began to drink within 5 hr following the induction of anuria and continued drinking throughout the experiment. Nephrectomized rats did not differ from ureterally ligated rats in the total volumes of water or saline ingested (both p's > 0.1).
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FIG. 1. Cumulative mean intakes of water and 0.15 M NaCI by ureterally ligated, nephrectomized and sham operated rats, The number in each group is given in Table lB.
TABLE 2 ESTIMATED WATER EXCHANGE DURING A N U R I A FOR 3 0 HR
Group
Intake minus Urine* Fluid Available Deprived
Ureterally ligated Sham ureterally ligated Nephrectomized Shamnephrectomized
---2.6±0.1 ---3.2±0.1
Water
0.15MNaCI
IWL*t Fluid Available Deprived
6.7i0.9§
8.3:kl.6
5,54-0.3
0.3±0.3 6.6±0.8 0.2±0.2
0.5 LI.1 10.4±1.3 2.3il.3
8.1±0.4 6.1 t0.5 9.1±0.1
*g/lO0 g initial body weight. finsensible weight loss. :~finalbody weight minus initial body weight. §mean ± standard error of the mean.
Water 7.2±0.3
ABW**+ Fluid Available
0.15MNaCI 5.9~0.4
11.8±0.5 9.5 ~z0.7 7.3 zE0.6 6.64-0.2 10.1±0.4 10.6±0.2
Deprived --5.5±0.3
Water --0.5:!1.0
0.15MNaC1 2.4±1.6
--10.7±0.3 --11.5±0.4 --9.0±0.8 --6.1±0.5 --0.7±0.8 3.8tl.6 --12.3±0.2 --9.9i0.3 --&3±0.9
DRINKING BY ANURIC RATS
869
fluid retention. Insensible weight loss, calculated as IWL = ABW----(intake--urine), provided an estimate of insensible water loss [31]. Fecal losses were small and therefore omitted from the calculations. Sham operated rats lost weight whether fluids were present (bothp's < 0.01 ; drinking water or saline)or absent (p < 0.05). Anuric rats lost weight only when deprived of fluid (p < 0.01); they did not lose weight while drinking water (p > 0.2), and gained weight while drinking saline (p < 0.05). These results occurred because the sham operated rats excreted as urine a volume of fluid nearly equal to that ingested, while the anuric rats, of course, were unable to urinate. Moreover, the IWL of the sham operated rats was greater than that of the anuric rats (all p's < 0.001; drinking water or saline, or deprived of fluid).
Blood Analysis Mean values of plasma osmolality, and plasma sodium and potassium concentrations are presented in Fig. 2. The results demonstrate that ureteral ligation produces a body fluid disorder similar to that resulting from nephrectomy, both
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when fluid is present and when it is absent. Consequently, both anuric groups have been combined for the following comparisons. Mean plasma osmolality of anuric rats deprived of fluid was increased 73 m0sm/kg at 30 hr (p < 0,001 compared with values for sham operated rats). This increase in osmolality is presumably due to an increase in plasma urea concentration ([21]; calculations from data reported by Whang [29] indicate that the rise in blood urea nitrogen in nephrectomized rats deprived of food and fluid increases the plasma osmotic pressure by 50 m0sm/kg in 24 hr). Plasma potassium was also elevated in anuric rats deprived of fluid for 30 hr (p < 0.001 compared with values for sham operated rats), as was plasma protein [~: = 6.4 =k 0.1 (standard error) vs. 5.4 i 0.1 g per cent; p < 0.006]. However, plasma sodium of anuric rats deprived of fluid was not significantly different from that of sham operated rats (p > 0.1 at 30 hr). But when water was present, anuric rats drank enough to sharply depress plasma sodium concentrations (p < 0.001 at 12 and 30 hr). Because potassium concentration and osmolality rapidly increased during anuria, note that the water ingestion was insufficient to lower these concentrations to within the range of control values (both p's < 0.001 at 30 hr). When saline was present, drinking by anuric rats did not change plasma sodium concentrations (p > 0.1 at 30 hr).
I
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12
30
(hr)
FIG. 2. Mean values of plasma sodium and potassium concentrations and osmolality of ureterally ligated, nephrectomized and sham operated rats, deprived, or with access to water or 0.15 M NaCl. Electrolyte concentrations are expressed as mEq/l of plasma water. Values from intact rats (X) are given at 0 hr.
Previous studies have either reported [14, 28] or denied [13] that anuric rats drink in response to endogenous stimuli, but did not investigate whether the response is an appropriate one in relation to body fluid balance. Other studies have measured body fluids in anuric rats with access to water and reported depression of plasma sodium [1, 4], but failed to relate this finding to the ingestion of water by the anuric animals. The present results confirm that anuric rats drink; in addition, they demonstrate that the ingested water causes the depression of plasma sodium, because plasma sodium is not depressed in anuric rats deprived of fluid or given saline to drink. Since depression of plasma sodium indicates overhydration of body tissues [12], it appears that anuric rats drink in excess of body fluid requirements. The conclusion that anuric rats drinking water are overhydrated may seem paradoxical since their body weights remain unchanged throughout the experiment. However, this observation may be explained as a result of a redistribution of body fluids. As gut contents diminish during food deprivation, the large pool of fluid normally contained within the alimentary canal is slowly released to other body fluid compartments. Intact rats excrete this excess fluid as dilute urine [17] and by insensible loss of water, and therefore lose weight without becoming dehydrated. The smaller loss of body weight of anuric rats (Table 2) indicates that they do not excrete an equivalent volume of fluid, because they cannot urinate and because they lose water insensibly at a slower rate than sham operated rats. Thus, anuric rats have available almost enough fluid to replace insensible water losses without drinking (as indicated by the plasma sodium concentrations of anuric rats deprived of fluids; Fig. 2). Nevertheless, these animals drink, and drink sufficient water to sharply depress plasma sodium concentrations. Wynn [30] may have been referring to a similar phenomenon in humans when he reported that "many water intoxicated patients complain of thirst, and this is especially so in acute anuria". The cause of drinking by anuric rats despite apparent
870
BLACK AND STRICKER
hydration remains obscure. Several stimuli are known to elicit drinking in the intact animal, and one or more of these may be present in anuric rats. Renin is a potent stimulus of drinking in the rat [8], and may be released by back pressure caused by ureteral ligation [26]. However, a renal hormone is unlikely to be a primary cause of the drinking observed in anuric rats because the fluid intakes of nephrectomized and ureterally ligated rats do not differ. A more reasonable hypothesis is that hypovolemia mayelicit the drinking response. Hypovolemia induced by injection of polyethylene glycol is an effective stimulus for drinking in nephrectomized as well as in intact rats [7, 25]. Moreover, hypovolemia has been reported to occur as a consequence of anuria [3, 4, 10]. In the present study, plasma protein concentrations were somewhat elevated in anuric rats deprived of fluid for 30 hr, suggesting that these animals were, in fact, hypovolemic. (Note that the hypovolemia must result from an internal fluid shift rather than from hemorrhage, because in the latter case the concentration of plasma protein would be decreased [16].) However, the increase in plasma protein was small and suggests a moderate decrease in plasma volume [23], which would not elicit sufficient drinking to result in the observed degree of dilution of plasma sodium [24]. Thus, while hypovolemia may contribute to drinking in anuric rats, there is likely to be an additional stimulus. It is improbable that anuric rats are responding to general dehydration of cells, because the elevated osmolality of these rats results from an increase in blood urea [21, 29] which rapidly enters cells [15] and is therefore ineffective in causing intracellular dehydration. However, urea may still be responsible for the drinking response. Urea passes through the blood-brain barrier with difficulty in the rat, and thus withdraws water osmotically from the brain [20]. Therefore, the high levels of plasma urea in anuric rats may stimulate drinking through dehydration of the brain and consequent shrinkage of hypothalamic osmoreceptors [2], despite hydration elsewhere in the body. This hypothesis is supported by the observation that a nephrectomized rat will drink when injected
with hypertonic urea [6]. It is opposed by evidence that injected urea is ineffective in stimulating thirst in intact dogs [9, i 1], and that urea does not initiate the antidiuretic response characteristic of a stimulus causing dehydration [27]. But urea is rapidly excreted by intact animals, and it is therefore improbable that a high concentration of plasma urea resulted in the above experiments. Moreover, urea enters the brain of the dog relatively quickly [5]. Thus, the osmotic gradient between blood and brain necessary for stimulation of thirst would be unlikely to occur in these experiments. A cerebral dehydration hypothesis also implicates potassium as a stimulus for drinking in anuric rats, because (a) the concentration of potassium is elevated during anuria, (b) potassium passes with difficulty through the blood-brain barrier of the rat [18], and (c) potassium causes drinking in nephrectomized rats [6]. Alternatively, the drinking observed may be in response to the accumulation of toxic substances during anuria, and may function to dilute them. However, the drinking response is not very effective if its function is dilution of toxic substances, because plasma potassium and osmolality in anuric rats ingesting fluid still remain clearly elevated. Moreover, nephrectomized rats do not survive longer when allowed to drink [14]. Thus, this hypothesis appears to be unwarranted. Whatever the cause of drinking, the conclusion that anuria leads to water ingestion in excess of apparent need suggests that nephrectomized and ureterally ligated rats are inappropriate subjects for many experiments on thirst. The difficulties resulting from the ingestion of water by anuric animals cannot be avoided by the use of control animals because the drinking response may not simply add to the drinking induced by a given experimental procedure. Quantitative comparisons of drinking behaviour are particularly uncertain when anuria is prolonged because overhydration may cause inhibition of thirst [24]. Therefore, experiments on thirst using anuric animals should be terminated early in the development of anuria, and should be supplemented by measures of body fluids.
REFERENCES
1. Aach, R. D., D. Rolf and H. L. White. Water losses and gains in fasting and nephrectomized rats. Am. J. Physiol. 188: 156-158, 1957. 2. Andersson, B. The effect of injection of hypertonic NaCI solutions into different parts of the hypothalamus of goats. Acta physiol, scand. 28: 188-201, 1953. 3. Castro Mendoza, H., C. Jiminez Diaz and J. M. Linazasoro. Cambios en la distribution de liquidos despues de la nefrectomia. Revta clin. esp. 37: 78-80, 1950. 4. Coats, D. A. and M. L. Wellby. Acute anuria in rats. Aust. J. exp. Biol. reed. Sci. 30: 21-31, 1953. 5. Coxon, R. V. The blood-brain barrier system in various species with special reference to urea. In: Comparative Neurochemistry, edited by D. Richter. Oxford: Pergamon Press, 1964, pp. 261-274. 6. Fitzsimons, J. T. Drinking by nephrectomized rats injected with various substances. J. Physiol., Lond. 155: 563-579, 1961. 7. Fitzsimons, J. T. Drinking by rats depleted of body fluid without increase in osmotic pressure. J. PhysioL, Lond. 159: 297-309, 1961. 8. Fitzsimons, J.T. Hypovolaemic drinking and renin. J. Physiol., Lond. 186: 130P-131P, 1966. 9. Gilman, A. The relation between blood osmotic pressure, fluid distribution and voluntary water intake. Am J. Physiol. 120: 323-328, 1937.
10. Harris, H., L R. McDonald and W. Williams. The electrolyte pattern in experimental anuria. Aust. J. exp. Biol. reed. ScL 30: 33-51, 1952. 11. Holmes, J. H. and M. I. Gregersen. Observations on drinking induced by hypertonic solutions. Am. J. Physiol. 162: 326-337, 1950. 12. Leaf, A. The clinical and physiological significance of the serum sodium concentration. New Engl. J. Med. 267: 24-30, 1962. 13. Linazasoro, J. M., C. Jiminez Diaz and H. Castro Mendoza. The kidney and thirst regulation. Bull. Inst. Med. Res. Univ. Mad. 7: 53-61, 1954. 14. Mack, I. and S. Rodbard. Voluntary fluid intake after nephrectomy. J. Urol. 62: 446-447, 1949. 15. McCance, R. A. and E. M. Widdowson. A method of breaking down the body weights of living persons into terms of extracellular fluids, cell mass and fat, and some applications of it to physiology and medicine. Proc. R., Soe. Lond., 138: 115-130, 1951. 16. Moore, F. D. Common patterns of water and electrolyte change in injury, surgery and disease. New Engl. J. Med. 258: 277-285, 1958. 17. Morrison, S. D., C. Mackay, E. Hurlbrink, J. K, Wier, M, S. Nick and F. K. Mlllar. The water exehanse a n d ~ l y u r i a of rats deprived of food. Q. J! exp. Physiol $2: 51-67, 1967,
D R I N K I N G BY A N U R I C RATS 18. Noonan, T. R., W. O. Fenn and L. Haege. The distribution of injected radioactive potassium in rats. Am. J. Physiol. 132: 474--488, 1941. 19. Peters, J. P. The problem of cardiac edema. Am. J. Med. 12: 66--76, 1952. 20. Reed, D. J. and D. M. Woodbury. Effect of hypertonie urea on cerebrospinal fluid pressure and brain volume. J. Physiol., Lond. 164: 252-264, 1962. 21. Schriener, G. E. and J. F. Maher. Uremia: Biocbemistry, Pathogenesis and Treatment. Springfield, III: Charles C. Thomas, 1961. 22. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. Toronto: McGraw-Hill, 1956. 23. Stricker, E. M. Some physiological and motivational properties of the hypovolemic stimulus for thirst. Physiol. Behav. 3: 379-385, 1968. 24. Stricker, E. M. Osmoregulation and volume regulation in rats: Inhibition of hypovolemic thirst by water. Am. J. Physiol. 217: 98-105, 1969.
871 25. Stricker, E. M. and G. Wolf. Behavioral control of intravascular fluid volume: Thirst and sodium appetite. Annls N. Y. Aead. Sci. 157: 553-568, 1969. 26. Vander, A. J. and R. Miller. Control of renin secretion in the anesthetized dog. Am. J. Physiol. 207: 537-546, 1964. 27. Verney, E. B. The antidiuretic hormone and the factors which determine its release. Proc. R. Soc., Lond. 135: 25-106, 1947. 28. Wayner, M. J. and J. J. Burger. A comparison of drinking in the normal and nephrectomized hooded rat. Psychonom. Sci. 6: 99-100, 1966. 29. Whang, R. and R. Reyes. The influence of alkalinization on the hyperkalemia and hypermagnesmia in uremic rats. Metabolism 16" 941-948, 1967. 30. Wynn, V. Water intoxication and serum hypotonicity. Metabolism 5: 490-499, 1956. 31. Zak, E. R. and G. C. Leiner. Studies on insensible loss of water. Expl Med. Surg. 2: 339-351, 1944.
Drinking by Anuric Rats in Excess of Apparent Need' STEPHEN
L. B L A C K A N D E D W A R D
M. S T R I C K E R
Department of Psychology, McMaster University, Hamilton, Ontario, Canada (Received 4 D e c e m b e r 1969) BLACK, S. L. AND E. M. STRICKER. Drinking by anuric rats in excess of apparent need. PHYSIOL.BEHAV.5 (8) 867-871, 1970.--Nephrectomized and ureterally ligated rats were found to drink in excess of apparent body fluid requirements as measured by external water exchange and by depression of plasma sodium concentration. The nature of the stimulus initiating this behavior is obscure. A renal hormone is unlikely to be a primary cause of the drinking because the fluid intakes of nephrectomized and ureterally ligated rats do not differ. While anuric rats are hypovolemic, this stimulus may not entirely account for the observed drinking response. Instead, the increased plasma concentrations of urea and potassium observed may cause drinking through cerebral dehydration and consequent shrinkage of hypothalamic osmoreceptors, despite hydration elsewhere in the body, because these substances penetrate the blood-brain barrier with difficulty. Alternatively, the drinking may be in response to the accumulation of toxic substances during anuria and may function to dilute them. Whatever the cause of drinking, the results suggest that anuric rats are inappropriate subjects for many experiments on thirst, unless the duration of anuria is brief. Anuria
Blood-brain barrier
Drinking
Nephrectomy
Thirst
Urea
Ureteral ligation
Procedure
THE STUDY of thirst in relation to body fluids is complicated by the existence of a renal mechanism for maintaining fluid balance. Consequently, nephrectomized or ureteraUy ligated animals are frequently used to ensure that a renal response will not occur. Although prolonged anuria results in progressive changes in body fluids [4, 21], it is generally assumed that such alterations have no effect on drinking. In this regard, Peters [19] has noted: "The anuric animal drinks only enough to maintain a constant effective osmotic pressure; that is, to keep the concentration of sodium in the serum from rising. It does not increase its drinking as the blood urea r i s e s . . . it drinks only enough to keep the volume of fluid and the effective osmotic pressure of this fluid in the body normal". The experiment reported here does not support this conclusion and suggests instead that anuric rats drink in excess of apparent body needs.
All operations were performed between 9.00 and 11.30 a.m., using ether anesthesia. Anuria was induced by bilateral nephrectomy (n = 31) through a dorsal approach, or by ligation of both ureters (n = 38) through a small ventral incision. Sham operations (n = 52) included all steps except removal of kidneys or ureteral ligation. Each operation lasted approximately 15 min. Immediately following surgery, the rats were weighed ( + 0.1 g) and replaced in their home cages without food. A funnel and graduated urine collection tube ( ± 0.1 ml) were placed beneath the cage of each sham operated rat. The rats were either deprived of fluid or allowed to drink water or 0.15 M NaCI solution. The treatment schedule for each group is presented in Table 1. Fluid intakes were recorded at variable time intervals, usually within one hour of the previous reading but less frequently overnight (the maximum interval between readings was 11 hr), and cumulative volumes of fluid ingested were later interpolated graphically. The total volume of urine excreted by each sham operated rat was recorded at the end of the experiment. Either 12 or 30 hr following surgery, each animal was deeply anesthetized with sodium pentobarbital (Nembutal ; 80 mg/kg), weighed, and bled from the abdominal aorta. An additional group of six intact rats was bled to provide control values. Plasma was analyzed for protein (by refractometer), potassium and sodium concentrations (by flame photometry), and osmolality (by freezing point depression). The water content of the plasma was determined by drying samples for 48 hr at 100°C.
METHOD
Subjects and Maintenance Male albino rats, weighing between 200 and 325 g, were individually housed in wire-mesh metabolism cages. Purina food pellets and water were available ad lib except during testing. In addition, the groups tested with 0.15 M NaCI solution were allowed continuous access to this fluid, beginning three days prior to testing. The fluids were presented in graduated tubes (d: 0.2 ml) attached to the front of each cage.
XThis study was supported, in part, by Research Grant APA-248 to Dr. E. M. Stricker from the National Research Council of Canada. 867
BLACK A N I) S IR ICI,.Llit
868
TABLE 1
Furthermore, the intake of water or saline by each anuric group did not differ from that of the appropriate sham operated group (all p's > 0.5). Individual anuric rats differed considerably in fluid intakes during the 30 hr test period. Water intakes ranged from 1.0 to 13.8 mill00 g body weight, while saline intakes ranged from 5.0 to 15.2 ml/100 g body weight. The volumes of fluid ingested by sham operated rats were equally variable. Standard errors of the mean across all treatments ranged from 0.9 to 2.1 ml/100 g body weight.
TREATMENT SCHEDULE
Group A. Sacrificed at 12 hr. Nephrectomized Sham nephrectomized Ureterally ligated Sham ureterally ligated B. Sacrificed at 30 hr Nephrectomized
Sham nephrectomized
Ureterally ligated
Sham ureterally ligated
n
Fluid Available
5 4 4 10 5 7
water none water water none water
13 5 4 13 5 3 14 5 4 12 5 3
water saline none water saline none water saline none water saline none
Water Exchange Table 2 presents the external water exchanges of anuric and sham operated rats during the experimental period. ABW, the change in body weight, was used to estimate net
15
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l ,' J A .~d,"'"
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Statistical analysis was carried out for selected data by the Mann-Whitney U-test [22].
I
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i /
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RESULTS
Cumulative volumes of fluid ingested by anuric and sham operated rats are presented in Fig. 1. Anuric rats began to drink within 5 hr following the induction of anuria and continued drinking throughout the experiment. Nephrectomized rats did not differ from ureterally ligated rats in the total volumes of water or saline ingested (both p's > 0.1).
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(hr)
FIG. 1. Cumulative mean intakes of water and 0.15 M NaCI by ureterally ligated, nephrectomized and sham operated rats, The number in each group is given in Table lB.
TABLE 2 ESTIMATED WATER EXCHANGE DURING A N U R I A FOR 3 0 HR
Group
Intake minus Urine* Fluid Available Deprived
Ureterally ligated Sham ureterally ligated Nephrectomized Shamnephrectomized
---2.6±0.1 ---3.2±0.1
Water
0.15MNaCI
IWL*t Fluid Available Deprived
6.7i0.9§
8.3:kl.6
5,54-0.3
0.3±0.3 6.6±0.8 0.2±0.2
0.5 LI.1 10.4±1.3 2.3il.3
8.1±0.4 6.1 t0.5 9.1±0.1
*g/lO0 g initial body weight. finsensible weight loss. :~finalbody weight minus initial body weight. §mean ± standard error of the mean.
Water 7.2±0.3
ABW**+ Fluid Available
0.15MNaCI 5.9~0.4
11.8±0.5 9.5 ~z0.7 7.3 zE0.6 6.64-0.2 10.1±0.4 10.6±0.2
Deprived --5.5±0.3
Water --0.5:!1.0
0.15MNaC1 2.4±1.6
--10.7±0.3 --11.5±0.4 --9.0±0.8 --6.1±0.5 --0.7±0.8 3.8tl.6 --12.3±0.2 --9.9i0.3 --&3±0.9
DRINKING BY ANURIC RATS
869
fluid retention. Insensible weight loss, calculated as IWL = ABW----(intake--urine), provided an estimate of insensible water loss [31]. Fecal losses were small and therefore omitted from the calculations. Sham operated rats lost weight whether fluids were present (bothp's < 0.01 ; drinking water or saline)or absent (p < 0.05). Anuric rats lost weight only when deprived of fluid (p < 0.01); they did not lose weight while drinking water (p > 0.2), and gained weight while drinking saline (p < 0.05). These results occurred because the sham operated rats excreted as urine a volume of fluid nearly equal to that ingested, while the anuric rats, of course, were unable to urinate. Moreover, the IWL of the sham operated rats was greater than that of the anuric rats (all p's < 0.001; drinking water or saline, or deprived of fluid).
Blood Analysis Mean values of plasma osmolality, and plasma sodium and potassium concentrations are presented in Fig. 2. The results demonstrate that ureteral ligation produces a body fluid disorder similar to that resulting from nephrectomy, both
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DISCUSSION
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when fluid is present and when it is absent. Consequently, both anuric groups have been combined for the following comparisons. Mean plasma osmolality of anuric rats deprived of fluid was increased 73 m0sm/kg at 30 hr (p < 0,001 compared with values for sham operated rats). This increase in osmolality is presumably due to an increase in plasma urea concentration ([21]; calculations from data reported by Whang [29] indicate that the rise in blood urea nitrogen in nephrectomized rats deprived of food and fluid increases the plasma osmotic pressure by 50 m0sm/kg in 24 hr). Plasma potassium was also elevated in anuric rats deprived of fluid for 30 hr (p < 0.001 compared with values for sham operated rats), as was plasma protein [~: = 6.4 =k 0.1 (standard error) vs. 5.4 i 0.1 g per cent; p < 0.006]. However, plasma sodium of anuric rats deprived of fluid was not significantly different from that of sham operated rats (p > 0.1 at 30 hr). But when water was present, anuric rats drank enough to sharply depress plasma sodium concentrations (p < 0.001 at 12 and 30 hr). Because potassium concentration and osmolality rapidly increased during anuria, note that the water ingestion was insufficient to lower these concentrations to within the range of control values (both p's < 0.001 at 30 hr). When saline was present, drinking by anuric rats did not change plasma sodium concentrations (p > 0.1 at 30 hr).
I
,I
12
30
(hr)
FIG. 2. Mean values of plasma sodium and potassium concentrations and osmolality of ureterally ligated, nephrectomized and sham operated rats, deprived, or with access to water or 0.15 M NaCl. Electrolyte concentrations are expressed as mEq/l of plasma water. Values from intact rats (X) are given at 0 hr.
Previous studies have either reported [14, 28] or denied [13] that anuric rats drink in response to endogenous stimuli, but did not investigate whether the response is an appropriate one in relation to body fluid balance. Other studies have measured body fluids in anuric rats with access to water and reported depression of plasma sodium [1, 4], but failed to relate this finding to the ingestion of water by the anuric animals. The present results confirm that anuric rats drink; in addition, they demonstrate that the ingested water causes the depression of plasma sodium, because plasma sodium is not depressed in anuric rats deprived of fluid or given saline to drink. Since depression of plasma sodium indicates overhydration of body tissues [12], it appears that anuric rats drink in excess of body fluid requirements. The conclusion that anuric rats drinking water are overhydrated may seem paradoxical since their body weights remain unchanged throughout the experiment. However, this observation may be explained as a result of a redistribution of body fluids. As gut contents diminish during food deprivation, the large pool of fluid normally contained within the alimentary canal is slowly released to other body fluid compartments. Intact rats excrete this excess fluid as dilute urine [17] and by insensible loss of water, and therefore lose weight without becoming dehydrated. The smaller loss of body weight of anuric rats (Table 2) indicates that they do not excrete an equivalent volume of fluid, because they cannot urinate and because they lose water insensibly at a slower rate than sham operated rats. Thus, anuric rats have available almost enough fluid to replace insensible water losses without drinking (as indicated by the plasma sodium concentrations of anuric rats deprived of fluids; Fig. 2). Nevertheless, these animals drink, and drink sufficient water to sharply depress plasma sodium concentrations. Wynn [30] may have been referring to a similar phenomenon in humans when he reported that "many water intoxicated patients complain of thirst, and this is especially so in acute anuria". The cause of drinking by anuric rats despite apparent
870
BLACK AND STRICKER
hydration remains obscure. Several stimuli are known to elicit drinking in the intact animal, and one or more of these may be present in anuric rats. Renin is a potent stimulus of drinking in the rat [8], and may be released by back pressure caused by ureteral ligation [26]. However, a renal hormone is unlikely to be a primary cause of the drinking observed in anuric rats because the fluid intakes of nephrectomized and ureterally ligated rats do not differ. A more reasonable hypothesis is that hypovolemia mayelicit the drinking response. Hypovolemia induced by injection of polyethylene glycol is an effective stimulus for drinking in nephrectomized as well as in intact rats [7, 25]. Moreover, hypovolemia has been reported to occur as a consequence of anuria [3, 4, 10]. In the present study, plasma protein concentrations were somewhat elevated in anuric rats deprived of fluid for 30 hr, suggesting that these animals were, in fact, hypovolemic. (Note that the hypovolemia must result from an internal fluid shift rather than from hemorrhage, because in the latter case the concentration of plasma protein would be decreased [16].) However, the increase in plasma protein was small and suggests a moderate decrease in plasma volume [23], which would not elicit sufficient drinking to result in the observed degree of dilution of plasma sodium [24]. Thus, while hypovolemia may contribute to drinking in anuric rats, there is likely to be an additional stimulus. It is improbable that anuric rats are responding to general dehydration of cells, because the elevated osmolality of these rats results from an increase in blood urea [21, 29] which rapidly enters cells [15] and is therefore ineffective in causing intracellular dehydration. However, urea may still be responsible for the drinking response. Urea passes through the blood-brain barrier with difficulty in the rat, and thus withdraws water osmotically from the brain [20]. Therefore, the high levels of plasma urea in anuric rats may stimulate drinking through dehydration of the brain and consequent shrinkage of hypothalamic osmoreceptors [2], despite hydration elsewhere in the body. This hypothesis is supported by the observation that a nephrectomized rat will drink when injected
with hypertonic urea [6]. It is opposed by evidence that injected urea is ineffective in stimulating thirst in intact dogs [9, i 1], and that urea does not initiate the antidiuretic response characteristic of a stimulus causing dehydration [27]. But urea is rapidly excreted by intact animals, and it is therefore improbable that a high concentration of plasma urea resulted in the above experiments. Moreover, urea enters the brain of the dog relatively quickly [5]. Thus, the osmotic gradient between blood and brain necessary for stimulation of thirst would be unlikely to occur in these experiments. A cerebral dehydration hypothesis also implicates potassium as a stimulus for drinking in anuric rats, because (a) the concentration of potassium is elevated during anuria, (b) potassium passes with difficulty through the blood-brain barrier of the rat [18], and (c) potassium causes drinking in nephrectomized rats [6]. Alternatively, the drinking observed may be in response to the accumulation of toxic substances during anuria, and may function to dilute them. However, the drinking response is not very effective if its function is dilution of toxic substances, because plasma potassium and osmolality in anuric rats ingesting fluid still remain clearly elevated. Moreover, nephrectomized rats do not survive longer when allowed to drink [14]. Thus, this hypothesis appears to be unwarranted. Whatever the cause of drinking, the conclusion that anuria leads to water ingestion in excess of apparent need suggests that nephrectomized and ureterally ligated rats are inappropriate subjects for many experiments on thirst. The difficulties resulting from the ingestion of water by anuric animals cannot be avoided by the use of control animals because the drinking response may not simply add to the drinking induced by a given experimental procedure. Quantitative comparisons of drinking behaviour are particularly uncertain when anuria is prolonged because overhydration may cause inhibition of thirst [24]. Therefore, experiments on thirst using anuric animals should be terminated early in the development of anuria, and should be supplemented by measures of body fluids.
REFERENCES
1. Aach, R. D., D. Rolf and H. L. White. Water losses and gains in fasting and nephrectomized rats. Am. J. Physiol. 188: 156-158, 1957. 2. Andersson, B. The effect of injection of hypertonic NaCI solutions into different parts of the hypothalamus of goats. Acta physiol, scand. 28: 188-201, 1953. 3. Castro Mendoza, H., C. Jiminez Diaz and J. M. Linazasoro. Cambios en la distribution de liquidos despues de la nefrectomia. Revta clin. esp. 37: 78-80, 1950. 4. Coats, D. A. and M. L. Wellby. Acute anuria in rats. Aust. J. exp. Biol. reed. Sci. 30: 21-31, 1953. 5. Coxon, R. V. The blood-brain barrier system in various species with special reference to urea. In: Comparative Neurochemistry, edited by D. Richter. Oxford: Pergamon Press, 1964, pp. 261-274. 6. Fitzsimons, J. T. Drinking by nephrectomized rats injected with various substances. J. Physiol., Lond. 155: 563-579, 1961. 7. Fitzsimons, J. T. Drinking by rats depleted of body fluid without increase in osmotic pressure. J. PhysioL, Lond. 159: 297-309, 1961. 8. Fitzsimons, J.T. Hypovolaemic drinking and renin. J. Physiol., Lond. 186: 130P-131P, 1966. 9. Gilman, A. The relation between blood osmotic pressure, fluid distribution and voluntary water intake. Am J. Physiol. 120: 323-328, 1937.
10. Harris, H., L R. McDonald and W. Williams. The electrolyte pattern in experimental anuria. Aust. J. exp. Biol. reed. ScL 30: 33-51, 1952. 11. Holmes, J. H. and M. I. Gregersen. Observations on drinking induced by hypertonic solutions. Am. J. Physiol. 162: 326-337, 1950. 12. Leaf, A. The clinical and physiological significance of the serum sodium concentration. New Engl. J. Med. 267: 24-30, 1962. 13. Linazasoro, J. M., C. Jiminez Diaz and H. Castro Mendoza. The kidney and thirst regulation. Bull. Inst. Med. Res. Univ. Mad. 7: 53-61, 1954. 14. Mack, I. and S. Rodbard. Voluntary fluid intake after nephrectomy. J. Urol. 62: 446-447, 1949. 15. McCance, R. A. and E. M. Widdowson. A method of breaking down the body weights of living persons into terms of extracellular fluids, cell mass and fat, and some applications of it to physiology and medicine. Proc. R., Soe. Lond., 138: 115-130, 1951. 16. Moore, F. D. Common patterns of water and electrolyte change in injury, surgery and disease. New Engl. J. Med. 258: 277-285, 1958. 17. Morrison, S. D., C. Mackay, E. Hurlbrink, J. K, Wier, M, S. Nick and F. K. Mlllar. The water exehanse a n d ~ l y u r i a of rats deprived of food. Q. J! exp. Physiol $2: 51-67, 1967,
D R I N K I N G BY A N U R I C RATS 18. Noonan, T. R., W. O. Fenn and L. Haege. The distribution of injected radioactive potassium in rats. Am. J. Physiol. 132: 474--488, 1941. 19. Peters, J. P. The problem of cardiac edema. Am. J. Med. 12: 66--76, 1952. 20. Reed, D. J. and D. M. Woodbury. Effect of hypertonie urea on cerebrospinal fluid pressure and brain volume. J. Physiol., Lond. 164: 252-264, 1962. 21. Schriener, G. E. and J. F. Maher. Uremia: Biocbemistry, Pathogenesis and Treatment. Springfield, III: Charles C. Thomas, 1961. 22. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. Toronto: McGraw-Hill, 1956. 23. Stricker, E. M. Some physiological and motivational properties of the hypovolemic stimulus for thirst. Physiol. Behav. 3: 379-385, 1968. 24. Stricker, E. M. Osmoregulation and volume regulation in rats: Inhibition of hypovolemic thirst by water. Am. J. Physiol. 217: 98-105, 1969.
871 25. Stricker, E. M. and G. Wolf. Behavioral control of intravascular fluid volume: Thirst and sodium appetite. Annls N. Y. Aead. Sci. 157: 553-568, 1969. 26. Vander, A. J. and R. Miller. Control of renin secretion in the anesthetized dog. Am. J. Physiol. 207: 537-546, 1964. 27. Verney, E. B. The antidiuretic hormone and the factors which determine its release. Proc. R. Soc., Lond. 135: 25-106, 1947. 28. Wayner, M. J. and J. J. Burger. A comparison of drinking in the normal and nephrectomized hooded rat. Psychonom. Sci. 6: 99-100, 1966. 29. Whang, R. and R. Reyes. The influence of alkalinization on the hyperkalemia and hypermagnesmia in uremic rats. Metabolism 16" 941-948, 1967. 30. Wynn, V. Water intoxication and serum hypotonicity. Metabolism 5: 490-499, 1956. 31. Zak, E. R. and G. C. Leiner. Studies on insensible loss of water. Expl Med. Surg. 2: 339-351, 1944.