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UBC Theses and Dissertations

The osmotic and ionic regulatory capacities of the kidney of the harbor seal, Phoca vitulina Tarasoff, Frederick John 1968

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THE OSMOTIC AND IONIC REGULATORY CAPACITIES OF THE KIDNEY OF THE HARBOR SEAL,.PHOCA VITULINA by FREDERICK JOHN TARASOFF A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of . Zoology We accept t h i s t h e s i s as conforming to the re q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia J u l y , 1968 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha11 make i t f ree ly , a v a i l a b l e for reference and Study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by hlis representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of ^ O C U O G f  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada Date i i ABSTRACT The mechanisms of osmotic and i o n i c r e g u l a t i o n i n marine mammals are of i n t e r e s t because of the apparent l a c k of " f r e s h " water i n t h e i r environment. Previous i n v e s t i g a t i o n on the harbor s e a l , (pjhoca v i t u l i n a , L.) / g e n e r a l l y i n d i c a t e d that the s e a l can o b t a i n a l l the water i t r e q u i r e s from i t s food. However, some dispute s t i l l e x i s t s as to whether the s e a l may i n g e s t sea water along w i t h i t s food and conserve water by concentrating ions and e x c r e t i n g them w i t h a net water g a i n . The e f f e c t s of a 16-hour p e r i o d w i t h no f l u i d s and a l s o of i n t u b a t i o n w i t h v a r y i n g amounts of d i s t i l l e d water and var y i n g amounts and concentrations of sea water were determined. The concentrations of sodium, c h l o r i d e and potassium ions as w e l l as the osmotic pressures of plasma and u r i n e were measured f o r the periods before and a f t e r i n t u b a t i o n . The r e s u l t s of t h i s study are discussed w i t h respect to published data and proposed mechanisms of osmotic and i o n i c r e g u l a t i o n by the kidney. The f i n d i n g s i n d i c a t e , as suggested by others, that the s e a l does not gain any s u b s t a n t i a l amount of water from sea water i n g e s t i o n . i i i TABLE OF CONTENTS Page INTRODUTION 1 MATERIALS AND METHODS 6 (i) Animals 6 ( i i ) Sampling procedure 6 ( i i i ) A n a l y t i c a l procedure 8 RESULTS 10 DISCUSSION 29 SUMMARY 40 LITERATURE CITED 41 i v LIST OF TABLES Table Page I Percentage of input of volume, c h l o r i d e , sodium and potassium excreted i n 13 hours. 26 I I Maximum values recorded f o r u r i n e o s m o l a l i t y and c h l o r i d e , sodium and potassium i o n concentrations. 27 LIST OF FIGURES Figure Page 1 Changes i n plasma o s m o l a l i t y (mOsm./l.), wit h time. 11 2 Changes i n ur i n e o s m o l a l i t y (mOsm./l.) and s p e c i f i c g r a v i t y w i t h time. 12 2 Changes i n u r i n e o s m o l a l i t y (mOsm./l.) and (b) s p e c i f i c g r a v i t y w i t h time. 13 3 Changes i n plasma c h l o r i d e concentration (mEq./l.) w i t h time. 16 4 Changes i n plasma sodium concentration (mEq./l.) w i t h time. 17 5 Changes i n ur i n e volume (ml.) w i t h time. 19 6 Changes i n ur i n e c h l o r i d e concentration (mEq./l.) w i t h time. 20 7 Changes i n ur i n e sodium i o n concentration (mEq./l.) w i t h time. 22 8 Changes i n ur i n e potassium i o n concentration (mEq./l.) w i t h time. 24 v i ACKNOWLEDGMENT I wish to express my thanks to Dr. H.D. F i s h e r who has a s s i s t e d and supervised t h i s p r o j e c t . My g r a t i t u d e i s a l s o extended to Drs. P.A. Dehnel, J.E. P h i l l i p s and D.T. Suzuki f o r the use of equipment and l a b o r a t o r y f a c i l i t i e s . I would a l s o l i k e to thank Mr. Dan Toews, Miss E l s p e t h McGowan and my w i f e , Mary f o r t e c h n i c a l a s s i s t a n c e . INTRODUCTION The p h y s i o l o g i c a l adaptations of marine mammals to t h e i r environment have been of i n t e r e s t to b i o l o g i s t s f o r some time and have been r e c e n t l y reviewed by Ha r r i s o n and Tomlinson (1961), L i l l y (1964), Scholander (1964) and Andersen (1966). The question of osmotic and i o n i c r e g u l a t i o n has been of p a r t i c u l a r i n t e r e s t because of the apparent lack of " f r e s h " water a v a i l a b l e to these animals. With reference to water balance and osmotic r e g u l a t o r y mechanisms, the most e x t e n s i v e l y s t u d i e d marine mammal has been the harbor s e a l , phoca v i t u l i n a . This s e a l has been found considerable distances up r i v e r s and i n e s t u a r i e s although the vast m a j o r i t y l i v e s e n t i r e l y i n a marine environment (Fisher, 1952 and Sc h e f f e r , 1958). Those harbor se a l s i n f r e s h and est u a r i n e h a b i t a t s have an abundant supply of f r e s h water. However, to renew i t s body water, a s e a l which l i v e s i n sea water would have to migrate to r i v e r mouths p e r i o d i c a l l y to dri n k f r e s h water, d r i n k sea water and excrete a hypertonic u r i n e , or o b t a i n water from the metabolic breakdown of i t s food. P e r i o d i c m i g r a t i o n to f r e s h water seems u n l i k e l y i n view of observations by the author of some se a l s s u r v i v i n g without f r e s h water on i s l a n d s i n a marine s e t t i n g , f o r example P r i b i l o f I s l a n d s . I r v i n g , et a l . (1935) examined the c h l o r i d e content of u r i n e and f e c a l matter from twenty young harbor s e a l s kept i n sea water and determined that the c h l o r i d e content was comparable to that of t e r r e s t r i a l mammals. They 2 concluded that the s e a l d i d not d r i n k sea water and that the s e a l ' s kidney was not unique i n i t s a b i l i t y to excrete s a l t . From c a l c u l a t i o n s i n v o l v i n g the amount of water a v a i l a b l e from metabolism of h e r r i n g and the p o s s i b l e p h y s i o l o g i c a l uses of t h i s water, they concluded that the harbor s e a l could manage adequately on the water from ingested f i s h . Smith (1936) examined the i n o r g a n i c composition of the u r i n e and the composition of s a l t s of r e c t a l washings of one c a p t i v e harbor s e a l . Smith concluded t h a t the s e a l d i d not swallow any considerable q u a n t i t y of sea water. He assumed, on the b a s i s of I r v i n g ' s work and h i s own r e s u l t s , t h a t body water and water f o r u r i n e formation are d e r i v e d from water i n the food.and from metabolism. A l b r e c h t (19 50) i n t u b a t e d harbor s e a l s w i t h sea water and a f t e r observing the r e a c t i o n s of the animals, vomiting and d i a r r h e a , concluded that they do not d r i n k sea water. She a l s o dehydrated the animals and observed t h a t the s e a l s could not be induced to d r i n k sea water. Fresh water d r i n k i n g has been reported by I r v i n g , et a l . (1935) and A l b r e c h t (1950) and has been observed i n newly a r r i v e d s e a l s of the U.B.C. colony. In a l l cases, dehydration of the s e a l s had occurred p r i o r to d r i n k i n g . However, these s e a l s have never been observed to d r i n k sea water. I f i t can be assumed that the s e a l does not d r i n k sea water, the question of how i t conserved i t s supply of water should be considered. I r v i n g , e_t a_l. (193 5) c a l c u l a t e d that of the 1100 grams of water y i e l d e d by 1250 grams of h e r r i n g , 300 3 grams would be used f o r p e r s p i r a t i o n , r e s p i r a t i o n and the formation of feces, and the remaining 800 grams would be a v a i l a b l e f o r u r i n e formation. Although the s e a l does have sweat glands, I r v i n g , e_t al_. (193 5) assumed that evaporated water from p e r s p i r a t i o n i s not necessary f o r thermal r e g u l a t i o n since the animal r e s i d e s i n a c o o l a q u a t i c h a b i t a t . Smith (1936), H i a t t and H i a t t (1942) and Page, et  a l . (19 54) observed an increase i n u r i n e volume i n the s e a l a f t e r a meal of h e r r i n g and a decrease i n u r i n e f l o w between feedings. The H i a t t s suggested t h a t the kidney a c t i v i t y i ncreases when there i s water to spare f o r e x c r e t i o n and decreases between meals i n order to conserve water. The H i a t t s f u r t h e r suggested t h a t post-feeding r e n a l v a s o d i l a t i o n occurs o n l y when water i s a v a i l a b l e , v a s o c o n s t r i c t i o n and the a s s o c i a t e d r e d u c t i o n i n glomerular f i l t r a t i o n l i m i t i n g water e x c r e t i o n at other times. They p o s t u l a t e d that i t was t h i s r e d u c t i o n of blood flow i n the kidneys, and not the change i n the number of a c t i v e g l o m e r u l i , that was r e s p o n s i b l e f o r the reduced u r i n e output. D i v i n g and i t s a s s o c i a t e d r e d u c t i o n of blood f l o w to various organs has been shown to p l a y an important r o l e i n conserving body water. I r v i n g , e t a l , (1935) i n i t i a l l y demonstrated t h a t during d i v i n g there was a decrease i n p e r i p h e r a l blood f l o w i n the s e a l . Furthermore, Bradley' and Bing (1942) found t h a t during d i v i n g there was marked v a s o c o n s t r i c t i o n of a r t e r i e s l e a d i n g to the kidneys w i t h almost complete c e s s a t i o n of glomerular f i l t r a t i o n and u r i n e 4 formation. They concluded that the i n t r a - r e n a l v a s o c o n s t r i c t i v e response to apnea appears to i n v o l v e both a f f e r e n t and e f f e r e n t . a r t e r i o l e s i n a r e l a t i v e l y uniform manner. Bradley, e_t al_. (1954) observed t h a t sodium, potassium and water e x c r e t i o n decreased g r e a t l y w i t h r e d u c t i o n i n f i l t r a t i o n during apnea. Lowrance, et a l . (1956) compared the e f f e c t s of anoxia and apnea i n the harbor s e a l and concluded that they have comparable e f f e c t s on r e n a l a c t i v i t y . During d i v i n g there i s a stoppage of breathing and consequently no water l o s t by evaporation from the lungs. A h i s t o l o g i c a l examination of the nephrons of the kidney of the harbor s e a l has not been attempted. However, Sperber (1944) has examined nephrons of two c l o s e l y r e l a t e d pinnipeds, Phoca barbata and Phoca h i s p i d a . The length of the loops of Henle and medullary thickness i n these two s e a l s were comparable to those of t e r r e s t r i a l carnivores, thereby supporting the p h y s i o l o g i c a l data of I r v i n g , e_t al_. (193 5) t h a t these a q u a t i c animals can form a hyperosmotic u r i n e , but not as concentrated as would be expected i f they drank sea water. Several i n v e s t i g a t o r s have answered, i n p a r t , the question whether the s e a l can conserve water from sea water by forming a hyperosmotic u r i n e . A l b r e c h t (1950) recorded the highest u r i n e c h l o r i d e i o n concentration a f t e r i n t u b a t i n g sea water. From her graph the value would be approximately 500 m i l l i e q u i v a l e n t s per l i t e r (mEq./l.),. The highest u r i n e sodium and potassium values reported i n the l i t e r a t u r e are 5 480 and 370 mEq.l., r e s p e c t i v e l y (Bradley, et a l , 1954). These values i n d i c a t e t h a t the s e a l cannot excrete a l l of these ions i n ur i n e a t concentrations higher than those of open sea w a t e r ( c h l o r i d e : 548.3; sodium: 470.2; pottasium: 9.9 (Barnes, 1954)). In the harbor s e a l n e i t h e r the osmotic and i o n i c r e g u l a t o r y c a p a c i t i e s of the kidney, nor the excre t o r y patterns f o l l o w i n g i n g e s t i o n of sea water have been f u l l y examined. In t h i s study the e f f e c t s of dehydration of the s e a l and i n t u b a t i o n of d i s t i l l e d water and of sea water i n var y i n g volumes and concentrations were examined and the f o l l o w i n g p o i n t s considered: 1. the u r i n e volumes and the concentrations of sodium, c h l o r i d e and potassium i n the plasma and u r i n e over a p e r i o d of t h i r t e e n hours a f t e r i n t u b a t i n g d i s t i l l e d water and sea water. 2. the maximum u r i n e osmotic pressures and the maximum sodium, c h l o r i d e and potassium concentrations that the kidney of the harbor s e a l i s capable of producing. 3. the source of water, e i t h e r ingested or produced from body-water rese r v e s , which i s used to e l i m i n a t e the ions ingested. C o n s i d e r a t i o n of these problems w i l l help determine whether the kidney has any s p e c i a l osmotic or i o n i c r e g u l a t o r y adaptations that enable the s e a l to su r v i v e i n the marine environment. MATERIALS AND METHODS 6 ANIMALS: Two y e a r l i n g female harbor s e a l s captured o f f Northern Vancouver I s l a n d were s t u d i e d over a one year p e r i o d . The se a l s were kept i n wood-stave tanks, nine f e e t i n diameter, which contained two f e e t of running f r e s h water. Over the t e s t p e r i o d they were each fed about f i v e pounds of thawed h e r r i n g d a i l y and maintained t h e i r weights at 9 5 + 8 pounds. SAMPLING PROCEDURE: To ensure t h a t the se a l s were i n a post-absorptive s t a t e , they were starved f o r 3 6 hours p r i o r to an experiment. One hour a f t e r removing the animal from i t s tank and r e s t r a i n i n g i t , the f i r s t u r i n e sample was taken and one-half hour l a t e r the f i r s t blood sample was taken. Over the next 16 hours, the presence of f l u i d around the eyes, mouth and n o s t r i l s were recorded hourly. The room temperature was maintained a t 67 t 2°F. The f o l l o w i n g f l u i d s were intubated i n i n d i v i d u a l experiments two hours a f t e r the i n i t i a l u r i n e c o l l e c t i o n : 500 ml. and 1000 ml. of d i s t i l l e d water; 500 ml., 750 ml. and 1000 ml. of sea water; and 750 ml. of sea water which had been evaporated to give 1.5 times the normal sea water concentrations of the ions to be s t u d i e d {1-h x S.W.). In a c o n t r o l experiment, to determine the e f f e c t s of dehydration and s t r e s s during the 16 hour c o l l e c t i o n p e r i o d , the animal was r e s t r a i n e d and sampled i n the above manner, but no f l u i d was intubated. The c o n t r o l experiment was done once f o r each animal and was the f i r s t t e s t c a r r i e d out. 7 a. Urine C o l l e c t i o n : The hind f l i p p e r s of the s e a l were strapped to the r e s t r a i n i n g board. A p l e x i g l a s s speculum (3.5" i n length and 1.5" i n diameter) was placed i n t o the u r o g e n i t a l v e s t i b u l e as f a r as the u r i n a r y p a p i l l a . A s t e r i l e X-ray catheter was then i n s e r t e d i n t o the bladder and the u r i n e was c o l l e c t e d i n a s t e r i l e polyethylene bag (Whirlpac Co.). A f t e r r e c o r d i n g the volume, the u r i n e was p i p e t t e d i n t o containers f o r a n a l y s i s . Between c o l l e c t i o n s the f l i p p e r s were f r e e to move. A g l a s s d i s h was placed to catch any u r i n e or f e c a l m a t e r i a l voided between sampling times. b. Blood C o l l e c t i o n : The h a i r above two lumbar vertebrae was removed and the s k i n scrubbed w i t h an a n t i s e p t i c (Phisohex) and w i t h 70% ethanol. In t h i s r e g i o n a l o c a l a n e s t h e t i c (10 ml. of 1% xylocaine) was i n j e c t e d i n t r a m u s c u l a r l y . An 18-gauge t h i n - w a l l e d needle w i t h f i t t e d s t y l e t was i n s e r t e d between two n e u r a l spines of the vertebrae i n t o the i n t r a v e r t e b r a l - e p i d u r a l v e i n . The s t y l e t was r e p l a c e d w i t h a s t e r i l e polyethylene canula (Intramedic PE 50) and then the needle was removed l e a v i n g the canula i n p l a c e . To prevent blood from c l o t t i n g i n the canula, i t was f i l l e d w i t h a 0.1% sodium heparin s o l u t i o n . Blood was withdrawn i n a h e p a r i n i z e d 10 ml. disposable syringe from which the l i q u i d heparin had been f o r c i b l y removed. A 21-gauge needle was used to connect the syringe to the canula. A f t e r each 8 10 ml. blood sample was c o l l e c t e d , the canula was f i l l e d w i t h heparin. The whole blood was c e n t r i f u g e d f o r 25 minutes a t 3 500rpm and the plasma p i p e t t e d i n t o containers f o r a n a l y s i s . ANALYTICAL PROCEDURES: a. Osmotic Pressure: Two m i l l i l i t e r a l i q u o t s of each u r i n e and plasma sample were p i p e t t e d i n t o osmometer tubes. The number of m i l l i o s m o l e s was : determined on a F i s k e Osmometer (Model G-62) which had been c a l i b r a t e d using prepared sodium c h l o r i d e standards so that d i r e c t readings ranging from 0 to 3 000 m i l l i o s m o l e s could be taken. The samples were analysed w i t h i n four hours a f t e r the completion of each experiment. b. C h l o r i d e Ion Determination: The c h l o r i d e i o n concentration i n the u r i n e and plasma samples was determined using a Buchler-Cotlove chloridometer. S o l u t i o n s were prepared by combining 0.1 ml. of sample w i t h 4 ml. of a n i t r i c - a c e t i c reagent (0.1 N HNO3 and 10% g l a c i a l a c e t i c a c i d ) . Standards were prepared f o r both HIGH and LOW r a t e s of t i t r a t i o n by adding 0.1 ml. of 104 mEq./l. and 0.1 ml. of 1 mEq./l. sodium c h l o r i d e s o l u t i o n , r e s p e c t i v e l y , > t o 4 ml. of the n i t r i c - a c e t i c a c i d reagent. Method blanks of 4 ml. of n i t r i c - a c e t i c a c i d reagent were a l s o prepared. Before t i t r a t i o n 0.2 ml. of g e l a t i n pH-indicator s o l u t i o n was added to each v i a l . c. Sodium Ion Determination: Samples of u r i n e and plasma were prepared by d i l u t i n g 1 of sample i n 5 ml. of d i s t i l l e d , demineralized water i n polyethylene v i a l s 9 (Nalgene Co.). Prepared samples were frozen and sto r e d a t -10°c. u n t i l the sodium i o n concentration could be determined. Sodium c h l o r i d e standards were prepared by d i l u t i n g 1 j u l . of stock s o l u t i o n s i n 5 ml. of d i s t i l l e d , demineralized water and were used to c a l i b r a t e the Unicam flame photometer (Model SP9 00) and to o b t a i n a standard curve. Samples were analyzed on the flame photometer and the sodium i o n concentration (mEq./l.) was determined from the standard curve. d. Potassium Ion Determination: Samples of u r i n e and plasma were prepared by d i l u t i n g 50 /xl. of sample i n t o 5 ml. of d i s t i l l e d , demineralized water. The prepared samples were fro z e n and s t o r e d at -10°C. u n t i l the concentrations could be determined. The Unicam flame photometer was c a l i b r a t e d using 50 / x i . of KCl standards d i l u t e d i n 5 ml. of water. Potassium i o n concentrations i n the samples were determined from the standard curve which ranged from 0 to 275 mEq./l. " L a b - t r o l " (Dade Reagents I n c . ) , a c o n t r o l serum i n l i q u i d form c o n t a i n i n g known concentrations of substances found i n the blood, was used i n a l l c h l o r i d e , sodium and potassium i o n determinations as a standard. 10 RESULTS I n i t i a l l y , three s e a l s were chosen f o r study, however, one animal was unable to take the s t r e s s of the experimental c o n d i t i o n s and gave very e r r a t i c r e s u l t s or became i l l during the experiments. Thus, i n the f i n a l a n a l y s i s , only two s e a l s could be used. Although the q u a l i t a t i v e responses of the two se a l s are s i m i l a r , there are obvious q u a n t i t a t i v e d i f f e r e n c e s . The changes i n plasma o s m o l a l i t y p r i o r to and f o l l o w i n g i n t u b a t i o n of f l u i d s are shown i n Figures l a - l g . Each of the i n t u b a t i o n experiments was compared to the c o n t r o l i n which no f l u i d was given. I t can be noted t h a t the p r e i n t u b a t i o n values v a r i e d between 318 and 33 0 mOsm./l. f o r s e a l #1 and between 318 and 343 mOsm./l. f o r s e a l #2. Int u b a t i o n of 500 ml. of d i s t i l l e d water and 500, 750 and 1000 ml. of sea water does not appear to have any great e f f e c t on the o s m o l a l i t y of the plasma i f one considers the changes from the p r e i n t u b a t i o n values and the changes observed i n the c o n t r o l . However, 1000 ml. of d i s t i l l e d water decreased the o s m o l a l i t y of the plasma to as low as 305 mOsm./l. (Seal #1) and 308 mOsm./l. (Seal #2). With 750 ml. of 1^ x sea water the o s m o l a l i t y of the plasma increased f o r 3 - 4 hours a f t e r i n t u b a t i o n and then returned g r a d u a l l y to the p r e i n t u b a t i o n values. In a n a l y s i n g the u r i n e o s m o l a l i t y (Figure 2a-2g), i t can be seen that during the c o n t r o l t e s t s , S e a l #2 had a much higher osmotic pressure than Seal #1. A f t e r d i s t i l l e d water i n g e s t i o n , the se a l s responded s i m i l a r l y w i t h the FIGURE 1: Changes i n plasma o s m o l a l i t y (mOsm./l.). Intubation at 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 7 50 ml. sea water f. 1000 ml. sea water g. 750 ml. 1% x sea water Seal #1 Seal #2 11 350 325k a 300LJ . I i i i s i i i i i i i i • 0 4 8 . 1 2 350r 325 3 0 0 L , i L _1 i i— 350 325 300 12 12 E co O <b L. =3 CO CO <D Q. U -t-J O CO o Time -(hours) 350r 325b 300 350r 6 325 300 • i . . . i i , » 1 I I L _ 0 12 350r 325 300Li L ' i I 1 I I I 1 I I L _ 12 350r g 325 30QI . i l . . — i — -0 4 —1 1 1 I I ' ' 8 12 FIGURE 2: Changes i n u r i n e o s m o l a l i t y (mOsm./l.) and s p e c i f i c g r a v i t y . I ntubation a t 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water Seal #1 Seal #2 12 1900r 1 . 3 5 5 0 1 . 3 4 5 0 a 1.3350 ' ' Q 1 1 1 ^  8 12 d) L. ZJ I/) (D L. 1900 1200h U o E CO O 5001 +-> > a U) 1.3550! 1.3450 0 12 1900r 1200h 500 L 12 1.3550r 1.3450 1.335 01 0 12 Ti m e ( h o u r s ) FIGURE 2 (b): Changes i n ur i n e o s m o l a l i t y (mOsm./l.) and s p e c i f i c g r a v i t y . I ntubation at 0 time. d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 750 ml. lJg x sea water Seal #1 Seal #2 -O s m o t i c p r e s s u r e ( m O s m . / l . ) 00 U> CO OJ OJ W OJ C O W OJ OJ OJ O J ^ . cn OJ cn co (j, co ^ Cn 01 Cn Cn Cn Cn Cn <-n Cn Cn Cn Cn Cn O O O O O O O O O O O O 14 o s m o l a l i t y d e c r e a s i n g w i t h i n one hour f o l l o w i n g i n t u b a t i o n . In both the t e s t s w i t h 500 ml. and 1000 ml. o f d i s t i l l e d water, the o s m o l a l i t y reached the minimum value 5 hours a f t e r i n t u b a t i o n and then s t a r t e d to i n c r e a s e immediately i n the former case and a f t e r another 5 hours i n the l a t t e r . One hour f o l l o w i n g the i n g e s t i o n of sea water, the two s e a l s gave d i f f e r e n t r e s u l t s f o r the 500 ml. t e s t . W i t h S e a l #1 there was no o v e r a l l change i n o s m o l a l i t y f o r 500 ml. of sea water, S e a l #2 i n c r e a s e d i t s u r i n e o s m o l a l i t y throughout the experiment. However, f o l l o w i n g i n t u b a t i o n of 7 50 ml. and 1000 ml. of sea water, and 750 ml. of 1^ x sea water experiments the r e s u l t s were q u a l i t a t i v e l y s i m i l a r . For the 7 50 and 1000 ml. sea water t e s t s there i s an o v e r a l l i n c r e a s e i n o s m o l a l i t y above the p r e i n t u b a t i o n value w i t h S e a l #2 i n c r e a s i n g e a r l i e r and to a higher v a l u e . In the 1% x sea water t e s t s there was l e s s o v e r a l l change i n o s m o l a l i t y (Seal #1 i n c r e a s e d 100 and S e a l #2 i n c r e a s e d 200 mOsm./lJ, whereas i n the 750 and 1000 ml. sea water t e s t s there was an i n c r e a s e o f at l e a s t 150 mOsm./l. f o r S e a l #1 and 600-700 mOsm./l. f o r S e a l #2. Although there were i n d i v i d u a l v a r i a t i o n s , the r e s u l t s i n d i c a t e d no l a r g e o v e r a l l change i n plasma o s m o l a l i t y . F i g u r e 2a-2g a l s o show the r e l a t i o n s h i p between s p e c i f i c g r a v i t y of the u r i n e a t d i f f e r e n t times and the o s m o l a l i t y . In the c o n t r o l and d i s t i l l e d water experiments, the trends i n these two values are very s i m i l a r . With sea water t e s t s , however, there i s a drop i n the s p e c i f i c g r a v i t y w i t h i n the second hour a f t e r i n t u b a t i o n , but no corresponding drop i n 15 the o s m o l a l i t y . By the end of the sampling time, the s p e c i f i c g r a v i t y had increased and the u r i n e of Seal #2 had the higher s p e c i f i c g r a v i t y and a l s o the higher osmotic pressure. The s p e c i f i c g r a v i t y values as a r e f l e c t i o n of t o t a l s o l i d s i n the u r i n e may help account f o r the o s m o l a l i t y changes observed. The c h l o r i d e concentrations i n the plasma samples were determined and are presented i n F i g u r e 3a-3g. In comparing the patterns of changes i n concentrations to that of the c o n t r o l , i t can be seen t h a t no obvious d i f f e r e n c e s occur f o l l o w i n g d i s t i l l e d water i n t u b a t i o n . However, w i t h i n g e s t i o n of sea water, the c h l o r i d e i o n concentration appears to be increased over the p r e i n t u b a t i o n values i n both s e a l s . Changes i n plasma sodium concentrations are shown i n F i g u r e 4a-4g. Again, the p r e i n t u b a t i o n values vary (between 152 and 172 mEq./l.). There appear to be no measurable e f f e c t s on the sodium concentrations f o l l o w i n g i n t u b a t i o n w i t h d i s t i l l e d water. Further sampling i s necessary to determine i f the peaks i n the concentration a f t e r sea water i n g e s t i o n are s i g n i f i c a n t and repeatable. Figures 4e-4g i n d i c a t e t h a t the sodium i o n concentration f l u c t u a t e s as much as i t does i n the c o n t r o l and d i s t i l l e d water experiments. Plasma potassium concentrations were measured, but d i d not change g r e a t l y i n any of the t e s t s , v a r y i n g between 4.3 and 5.0 mEq./l. No obvious p a t t e r n of v a r i a t i o n was detected. The more i n t e r e s t i n g r e s u l t s were obtained w i t h a n a l y s i s of u r i n e volume and the concentration of sodium, c h l o r i d e and potassium i n the urine f o l l o w i n g i n t u b a t i o n . FIGURE 3: Changes i n plasma c h l o r i d e concentration". (mEq./l.) w i t h time. Intubation a t 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 7 50 ml. sea water f. 1000 ml. sea water g. 750 ml. lh x sea water Seal #1 Seal #2 16 FIGURE 4: changes i n plasma sodium concentration (mEq./l-) w i t h time. I n t u b a t i o n a t 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 750 ml. 1^ x sea water Seal #1 Seal #2 18 The u r i n e volumes are presented i n Figu r e 5a-5g. The average r a t e of volume output (ml./hour) i n the c o n t r o l , i n which no f l u i d was intubated, was 14.8 f o r Seal #1 and 13v8 f o r Seal #2. Intubation of 500 ml. of d i s t i l l e d water increased the average e x c r e t i o n r a t e to 26.9 and 19.1 ml./hour, and 1000 ml. increased i t to 38.1 and 27.7 ml./hour i n se a l s #1 and #2, r e s p e c t i v e l y . The pa t t e r n was a gradual o v e r a l l increase to the maximum value i n the d i s t i l l e d water experiments o c c u r r i n g at 4 or 8 hours f o r 500 ml. and 10 or 11 hours f o r 1000 ml. In the sea water t e s t s , however, d i u r e s i s occurred w i t h i n the f i r s t two hours a f t e r i n t u b a t i o n , reaching a maximum i n the t h i r d hour i n a l l cases (Figures 5d-5g), and then decreasing to p r e i n t u b a t i o n values. In the sea water t e s t s , the maximum e x c r e t i o n r a t e v a r i e d i n d i r e c t p r o p o r t i o n w i t h the volume ingested and the s a l t l o a d . F i g u r e s 5b and 5c show that w i t h no s a l t load, the volume intubat e d i s a f a c t o r i n determining the maximum e x c r e t i o n r a t e and Figures 5e and 5g i n d i c a t e the e f f e c t of i n c r e a s i n g the s a l t l o ad without changing the volume i n p u t . The concentration of c h l o r i d e i o n i n the u r i n e , shown i n Figures 6a-6g, v a r i e d from 0 to 508 mEq./l. i n the experiments. The lowest values were obtained during the c o n t r o l t e s t s and a f t e r d i s t i l l e d water i n t u b a t i o n , and the highest values, a f t e r sea water i n t u b a t i o n . In the c o n t r o l , the c h l o r i d e i o n concentration decreased g r a d u a l l y from 13.1 to 5.2 mEq./l. f o r Seal #1 and from 44.6 to 2.8 mEq./l. f o r Se a l #2. Intubation w i t h 500 ml. r e s u l t e d i n decreases from FIGURE 5: Changes i n u r i n e volume (ml.) w i t h time. Intubation a t 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 750 ml. 1^ x sea water Seal #1 Seal #2 FIGURE 6: Changes i n u r i n e c h l o r i d e concentration (mEq./l.) w i t h time. Intubation a t 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 750 ml. 1^ x sea water Seal #1 Seal #2 20 a 21 44.2 to 1.1 mEq./l. i n Seal #1, and from 20.3 to 1.0 mEq./l. i n Seal #2. S i m i l a r l y , there were decreases a f t e r the i n t u b a t i o n of 1000 ml. of d i s t i l l e d water (24.1 to 0.0 and 9.5 to 0.1 mEq./l. i n Seals #1 and #2, r e s p e c t i v e l y ^ The i n t e r e s t i n g e f f e c t s were obtained a f t e r sea water i n t u b a t i o n . The maximum c h l o r i d e concentration i s obtained w i t h i n the f i r s t three to f i v e hours f o r normal sea water and i n the ei g h t h hour f o r % x sea water and i s independent of the amount of f l u i d i n t u b a t e d or s a l t load (Figures 6d-6g). The shape of the graph v a r i e d s l i g h t l y depending on the amount and concentration of sea water ingested. The c h l o r i d e i o n concentration decreased, but d i d not r e t u r n to the p r e i n t u b a t i o n values at any time during the 13 hours. The changes i n sodium i o n concentrations (mEq./l.) are shown i n Figures 7a-7g. These are very s i m i l a r to those obtained f o r c h l o r i d e i o n concentration (Figures 6a-6g). The lowest values occurred i n the c o n t r o l t e s t s and f o l l o w i n g d i s t i l l e d water i n t a k e . The range of values over the 13 hours i n the c o n t r o l , 500 ml. and 1000 ml. d i s t i l l e d water are as f o l l o w s : 72.0 to 9.5 (#1) and 34.0 to 13.0 (#2); 49.0 to 8.0 (#1) and 75.0 to 24.5 (#2); 33.0 to 6.0 (#1) and 65.0 to 6.0 (#2), r e s p e c t i v e l y . F o l l o w i n g sea water i n t u b a t i o n , the maximum concentration of sodium excreted was obtained w i t h i n the f i r s t three to four hours a f t e r i n t u b a t i o n and t h i s value was approximately the same (470 to 500 mEq./l.) r e g a r d l e s s of the amount of concentration ingested. A l s o , as i n the c h l o r i d e i o n analyses, there are FIGURE 7: Changes i n u r i n e sodium i o n concentration (mEq./l.). Intubation at 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 7 50 ml. 1% x sea water Seal #1 — Seal #2 — 22 23 some d i f f e r e n c e s i n the shapes o f the c u r v e s , b u t on the b a s i s o f the t e s t s done, i t i s n o t known whether t h e s e a r e s i g n i f i c a n t . F o l l o w i n g 1*2 x sea water i n t u b a t i o n , b o t h the c h l o r i d e and sodium c o n c e n t r a t i o n s . r e a c h e d t h e i r maximum c o n c e n t r a t i o n s l a t e r and remained c l o s e r t o t h e i r maximum v a l u e s f o r t h e l a t t e r p a r t o f the t e s t p e r i o d t h a n the o t h e r sea water e x p e r i m e n t s . The r e s u l t s o f p o t a s s i u m a n a l y s e s a r e p r e s e n t e d i n F i g u r e s 8a-8g. I t i s r e a d i l y seen t h a t t h e y d i f f e r from t h o s e p a t t e r n s o b t a i n e d i n the sodium and c h l o r i d e a n a l y s e s . As i n the case o f u r i n e o s m o l a l i t y , S e a l #2 appears t o have h i g h e r v a l u e s than S e a l #1 i n the c o n t r o l . However, i t i s n o t p o s s i b l e t o know w i t h o u t d o i n g f u r t h e r e x p e r i m e n t s whether the c o n t r o l v a l u e s a r e r e p e a t a b l e . The average v a l u e o f p o t a s s i u m i o n c o n c e n t r a t i o n d i d n o t appear t o change d r a s t i c a l l y i n the d i s t i l l e d water t e s t s when compared t o those o f the c o n t r o l f o r S e a l #1, whereas, f o r S e a l #2 the c o n t r o l v a l u e s were v e r y much h i g h e r than i n the d i s t i l l e d water t e s t s . The i n g e s t i o n o f 500 ml. o f sea water r e s u l t e d i n an i n c r e a s e i n ^potassium i o n c o n c e n t r a t i o n a f t e r 4 h o u r s . T h i s c o u l d be r e l a t e d t o the d e c r e a s e d sodium i o n c o n c e n t r a t i o n ( F i g u r e 7 d ) . I n the o t h e r sea water e x p e r i m e n t s , the p o t a s s i u m i o n c o n c e n t r a t i o n tended e i t h e r t o remai n c o n s t a n t o r t o de c r e a s e s l i g h t l y 4 hours a f t e r i n t u b a t i o n , e x c e p t w i t h S e a l #1 a f t e r 750 m l . sea water i n g e s t i o n . I n t h i s s t u d y , one q u e s t i o n o f i n t e r e s t i s whether the f u n c t i o n i n g o f the k i d n e y and o f t h e body e x c r e t i o n systems EIGURE 8: Changes i n u r i n e potassium i o n concentration (mEq./l.) w i t h time. I n t u b a t i o n at 0 time. a. C o n t r o l b. 500 ml. d i s t i l l e d water c. 1000 ml. d i s t i l l e d water d. 500 ml. sea water e. 750 ml. sea water f. 1000 ml. sea water g. 750 ml. 1% x sea water Seal #1 Seal #2 24 25 are overtaxed by the i n g e s t i o n of s a l t water. Does the kidney f u n c t i o n e f f i c i e n t l y enough to r i d the body of the ingested ions without t a x i n g the reserves of body water? Table I presents the t o t a l output of f l u i d , c h l o r i d e , sodium and potassium as a percentage of the t o t a l i n p u t . The volume output f o l l o w i n g d i s t i l l e d water i n g e s t i o n i s from 42-79%. When sea water i s intubated, the amount of water r e q u i r e d to e l i m i n a t e a l l of the sodium and c h l o r i d e ions i s greater than t h a t made a v a i l a b l e by sea water alone. With 1% x sea water, more than 100% of the volume ingested i s excreted w i t h i n 13 hours, although not a l l of the sodium and c h l o r i d e ions are (94 and 63 - 80%, r e s p e c t i v e l y . ) In a l l cases two to f i v e times the amount of potassium input i s excreted w i t h i n the 13 hours, whereas the percent output of the sodium and c h l o r i d e ions i s between 3 5 and 69% and 28 and 55%, r e s p e c t i v e l y f o r sea water experiments. The i n g e s t i o n of 1% x sea water i s found to r e s u l t i n the e x c r e t i o n w i t h i n 13 hours of 1% .to .'-12 times the i o n output as compared to the t e s t s using normal sea water. Table I I l i s t s the osmotic and i o n i c concentrating c a p a c i t i e s that have been obtained f o r the kidneys of the two sea l s s t u d i e d . Seal #2 had higher values f o r o s m o l a l i t y and e l e c t r o l y t e concentrations. The maximum o s m o l a l i t y was a t t a i n e d i n Seal #1 two hours a f t e r 1000 ml. of sea water were intubated, and i n Seal #2, 13 hours a f t e r 750 ml. of sea water were intubated, w i t h values of 1700 and 2050 mOsm./l., r e s p e c t i v e l y . The maximum sodium i o n concentration occurred i n Seal #1 during the f i f t h hour of the 1000 ml. 26 TABLE I: Percentage of input of volume, c h l o r i d e , sodium and potassium excreted i n 13 hours. Water Ch l o r i d e Sodium potassium output output output output S e a l #1 (%) (%) (%) (%) 500 ml. D.W. 79 1000 ml. D.W. 55 500 ml. S.W. 73 50 63 313 750 ml. S.W. 60 49 61 205 1000 ml. S.W. 63 55 69 256 750 ml. ±H x S.W. 128 80 94 380 Water output Seal #2 ( % ) 500 ml. D.W. 58 1000 ml. D.W. 42 500 ml. S.W. 50 750 ml. S.W. 48 1000 ml. S.W. 35 750 ml. Ik x S.W. 13 2 Chl o r i d e Sodium Potassium output output output (%) (%) :•{%) 41 44 549 39 49 317 28 35 193 63 94 335 TABLE I I : Maximum v a l u e s r e c o r d e d f o r u r i n e o s m o l a l i t y and c h l o r i d e , sodium and p o t a s s i u m i o n c o n c e n t r a t i o n s . O.P. Max.(mosm./l.) U/P S.W. U/S.W. S e a l #1 1700 5.07 1099 1.87 S e a l #2 2050 5.93 1099 2.25 N a + Max.(mEq./l.) U/P S.W. U/S.W. S e a l #1 496 3.08 447 1.11 S e a l #2 523 3.02 447 1.17 C l Max.(mEq./l.) U/P S.W. U/S.W. S e a l #1 472 3.37 448 1.05 S e a l #2 508 3.90 448 1.12 K + Max.(mEq./l.) U/P S.W. U/S.W. S e a l #1 172 34.4 10 17.2 S e a l #2 263 52.6 10 26.3 OPEN SEA WATER (Barnes, 19 54) C l " : 548.30 N a + : 470.20 K + : 9.96 28 of sea water t e s t and i n Seal #2 during the f o u r t h hour of the 750 ml. sea water t e s t , w i t h values of 496 and 523 mEq./l., r e s p e c t i v e l y . Tne maximum c h l o r i d e i o n - co n c e n t r a t i o n was reached i n Seal #1 nine hours a f t e r i n t u b a t i o n of 750 ml. of 1% x sea water and i n Seal #2 four hours a f t e r i n t u b a t i o n of 500 ml. of sea water. The potassium values were at t h e i r maximum 13 hours a f t e r 500 ml. of sea water were intubated i n Seal #1 and 12 hours a f t e r the same i n Seal #2. The urine/plasma (U/P) r a t i o s are presented i n Table I I . The r a t i o s of o s m o l a l i t y and e l e c t r o l y t e concentrations i n the u r i n e to that of sea water are a l s o presented i n Table I I . These r a t i o s i n d i c a t e that the c h l o r i d e and sodium excreted i n the ur i n e are present a t about the same concentr a t i o n as i n sea water, whereas the potassium i o n concentration and o s m o l a l i t y are higher i n the ur i n e than i n sea water. For comparisons, the i o n i c concentrations of "open" sea water are l i s t e d . 29 DISCUSSION I n t e s t i n a l absorption i n man and dog has been shown to be complete w i t h i n 12 hours a f t e r feeding.(Davenport, 1961). Although the g a s t r o - i n t e s t i n a l physiology of the s e a l has not been examined and experiments to determine the r a t e of passage of food were not attempted, i t was assumed that the s e a l , a ca r n i v o r e , would be i n a post-absorptive s t a t e by 36 hours a f t e r i t s l a s t meal. i f so, then., changes i n the plasma and u r i n e a f t e r t h i s time would be due p r i m a r i l y to the f l u i d s ingested and.absorbed during the experiments r a t h e r than by any food remaining i n the stomach and i n t e s t i n e . G a s t r i c evacuation (Hunt, et. a_l., 1951) and absorption i n the small i n t e s t i n e (Parsons and Wingate, 1961) have been shown to be slower f o r hypertonic s o l u t i o n s than f o r hypo- and i s o t o n i c s o l u t i o n s . Therefore, i t i s probable t h a t d i s t i l l e d water i s absorbed more r a p i d l y than the sea water s o l u t i o n s . Retarded absorption of the sea water s o l u t i o n s occurs a l s o because of the presence of magnesium and s u l f a t e i o n s , which according to Davenport (1961), "... are slowly absorbed, and t h e i r presence i n i n t e s t i n a l contents prevents water absorption." Changes i n plasma o s m o l a l i t y and e l e c t r o l y t e composition r e f l e c t the absorption of intubated s o l u t i o n s . In t h i s p r o j e c t , plasma changes a f t e r f l u i d i n t u b a t i o n were apparent only a f t e r the i n g e s t i o n of the higher volumes and concentrations of e l e c t r o l y t e s p o s s i b l y because of the larg e blood volume of marine mammals as compared w i t h t e r r e s t r i a l mammals and the d i l u t i o n e f f e c t . 30 Smirk, (1933) using human sub j e c t s , demonstrated that one l i t e r of d i s t i l l e d water was absorbed w i t h i n 22 to 25 minutes. K l i s i e c k e , et a l . . (1933) found that absorption of d i s t i l l e d water i n dogs took s l i g h t l y longer. I t therefore seems l i k e l y t hat i f plasma changes occurred, they should be observed i n the s e a l 1^ hours a f t e r i n t u b a t i o n . The absence of d i a r r h e a may i n d i c a t e complete or near-complete absorption. Magnesium and s u l f a t e ions i n concentrations found i n sea water are emetics f o r humans (Futcher, e t a l . , 1943), although they can s a f e l y i n g e s t and completely absorb small q u a n t i t i e s (Goodman ..and Gilman, 1965) . These s e a l s d i d not appear to have any t r o u b l e absorbing these ions s i n c e no di a r r h e a was observed. Any f e c a l matter voided was t h i c k , slimy and c h a r a c t e r i s t i c of the s e a l . In a l l mammals there are s e v e r a l s i t e s of f l u i d e x c r e t i o n , such as the eyes, nose, s a l i v a r y glands and kidneys. The eyes (Thaysen and Thorn, 19 54) and s a l i v a r y glands (Thaysen, et a l . , 1954) produce f l u i d s w i t h a potassium i o n concentration that i s hypertonic to the blood plasma. However, i t i s l i k e l y that n e i t h e r these, nor the nose, played a s i g n i f i c a n t r o l e i n e x c r e t i n g excess water or e l e c t r o l y t e s . Observations during experiments i n which d i s t i l l e d water was intubated i n d i c a t e d no increase of f l u i d from these areas, whereas i n the sea water experiments, f l u i d output from these areas decreased. E x t r a r e n a l s i t e s f o r water and e l e c t r o l y t e e x c r e t i o n , comparable to those of marine b i r d s and r e p t i l e s , have not been reported i n mammals. The main 31 s i t e of osmotic and i o n i c r e g u l a t i o n i n the harbor s e a l can, th e r e f o r e , be viewed as o c c u r r i n g predominantly i n the kidneys. The volume and composition of the body f l u i d s of mammals are r e g u l a t e d w i t h i n narrow l i m i t s . The r e g u l a t o r y c a p a c i t i e s of the kidney can be determined by s u b j e c t i n g experimental animals to such c o n d i t i o n s as dehydration, hydration and s a l t - l o a d i n g . The osmotic and i o n i c r e g u l a t o r y c a p a c i t i e s of the kidney r e f e r to the a b i l i t y of the kidney, as d i c t a t e d by changes i n body f l u i d s , to maintain the qu a n t i t y and q u a l i t y of substances which are u l t i m a t e l y excreted. When body f l u i d s change i n pH, osmotic pressure, i o n i c composition or volume, mechanisms are i n i t i a t e d by the nervous and endocrine systems which act on the r e n a l system to r e g a i n the e q u i l i b r i u m s t a t e . Changes i n kidney processes, f o r the most p a r t , i n v o l v e a l t e r a t i o n s i n the rea b s o r p t i o n and secretion!.processes, and a l s o i n the glomerular f i l t r a t i o n r a t e (GFR). Changes i n the GFR are seldom detected i n man, but i n se a l s they are fequent and e s p e c i a l l y marked a f t e r feeding ( H i a t t and H i a t t , 1942) and during asphyxia (Lowrance, e t a l . , 1956). The o v e r a l l r e s u l t of these processes i s the production of a ur i n e varying i n volume and composition which r e s u l t s i n the maintenance of w e l l - r e g u l a t e d body f l u i d s . Periods of dehydration (the c o n t r o l experiments) r e s u l t e d i n r e l a t i v e l y low r a t e s of u r i n e production and e l e c t r o l y t e e x c r e t i o n (sodium and c h l o r i d e ) . An average of 14 to 15 ml. 32 of u r i n e per hour was produced by both s e a l s . These were s i m i l a r to the q u a n t i t i e s determined f o r periods between meals by Smith (1935), Bradley and Bing (1942) and Schmidt-Nielsen, et a l . (1959). Man produces on the average 30 ml. of u r i n e per hour, i n cases of moderate hydropenia and can drop to 12 to 18 ml. per hour i n cases of extreme dehydration ( P i t t s , 1963). During moderate dehydration the decreased u r i n a r y output i s due to changes i n water r e a b s o r p t i o n and not to a l t e r a t i o n s of the GFR. In more extreme dehydration, the changes i n the f i l t r a t i o n r a t e are caused by a reduced blood volume. In the s e a l , low u r i n e volume during dehydration and s t a r v a t i o n has been a t t r i b u t e d to i ncreased e l e c t r o l y t e and water r e a b s o r p t i o n (Bradley, et a l . , 1954) and to v a s o c o n s t r i c t i o n w i t h the r e s u l t a n t r e d u c t i o n i n the GFR ( H i a t t and H i a t t , 1942). I t was proposed by the H i a t t s that the reduced u r i n e f l o w between meals enables the s e a l to conserve i t s water supply. With low u r i n e output there was a l s o a low e l e c t r o l y t e e x c r e t i o n and a high u r i n a r y osmotic pressure. The high osmotic pressure was probably due to a high urea output, which i s the major o s m o t i c a l l y a c t i v e substance i n the u r i n e during f a s t i n g (Smith, 1935). With high a n t i d i u r e t i c hormone (ADH) and aldosterone l e v e l s the r e s u l t i s a high p e r m e a b i l i t y of water and r e a b s o r p t i o n of e l e c t r o l y t e s i n the nephrons and a concentrated u r i n e w i t h a high osmotic pressure during dehydration and s t a r v a t i o n . Maximum rea b s o r p t i o n i s a l s o a s s i s t e d by reduced GFR which decreases f i l t r a t i o n 33 and f i l t r a t e p e r f u s i o n i n a l l nephrons, r e s u l t i n g i n a longer p e r i o d of contact between f i l t r a t e and reabsorbing tubular c e l l s . With the r e d u c t i o n of f i l t r a t e , the r e a b s o r p t i v e mechanisms may operate more e f f i c i e n t l y i n removing s o l u t e s . Smith (1936) found t h a t considerable q u a n t i t i e s of ammonia, c r e a t i n i n e and c r e a t i n e were a l s o present i n the u r i n e i n a d d i t i o n to urea, 24 to 36 hours a f t e r the l a s t meal, and c o n t r i b u t e d to the o s m o l a l i t y . In the c o n t r o l t e s t s , u r i n e volumes were between 14 and 15 ml. per hour and the concentrations of sodium and c h l o r i d e ions were low, i n d i c a t i n g a r e l a t i v e l y constant output of o s m o t i c a l l y a c t i v e p a r t i c l e s r e q u i r i n g a c e r t a i n q u a n t i t y of water, and a l s o a near-maximum re a b s o r p t i o n of f i l t e r e d water and these two e l e c t r o l y t e s . The d i f f e r e n c e s between the two s e a l s i n Figure 2a may be accounted f o r by d i f f e r e n t kidney s i z e s , l e v e l s of organic substance e x c r e t i o n , u r i n e concentrating a b i l i t i e s or d i f f e r e n t degrees of dehydration before the t e s t s . The concentrating a b i l i t i e s of the two s e a l s are shown i n F i g u r e 2a, i n which Seal #1 concentrated i t s u r i n e to a maximum of 1550 mOsm./l., whereas rSeal #2 concentrated i t s u r i n e as high as 2050 mOsm./l. Urine potassium i o n concentrations i n the c o n t r o l were r e l a t i v e l y higher than other e l e c t r o l y t e s and were probably due to the movement of potassium out of c e l l s as a r e s u l t of water movement from i n t r a c e l l u l a r to e x t r a c e l l u l a r f l u i d s , r e q u i r e d to maintain a constant volume of e x t r a c e l l u l a r f l u i d during periods of dehydration. The e f f e c t s of s t r e s s 34 on potassium s e c r e t i o n ( p i t t s , 1963) and p o s s i b l e t i s s u e damage may, i n p a r t , account f o r the r e l a t i v e l y high concentrations of t h i s i o n . When f l u i d s were intubated, there was a d i u r e s i s w i t h i n two hours, a water d i u r e s i s w i t h d i s t i l l e d water and an osmotic d i u r e s i s w i t h sea water. The p a t t e r n of u r i n e volume changes a f t e r d i s t i l l e d water i n t u b a t i o n was s i m i l a r to the excretory p a t t e r n i n man and dog a f t e r i n g e s t i o n of an i s o t o n i c s o l u t i o n ( P i t t s , 1963), i n that the increase i n ur i n e volume was gradual. An abrupt change normally occurs i n man and dog a f t e r i n g e s t i o n of hypotonic:.', J s o l u t i o n s . A l b r e c h t (1950) and Bradley, e t a l . (1954) r e p o r t a d i u r e t i c p a t t e r n f o r the s e a l s i m i l a r to those of the dog and man. No measurements were made to determine whether the GFR changed during any of the t e s t s . However, Bradley, et a l . (1954), Page, et a l . (1954) and Schmidt-Nielsen, et a l . (1959) found th a t the GFR d i d not change during e l e c t r o l y t e and water l o a d i n g . On the b a s i s of t h e i r r e s u l t s , i t seems probable that i f changes i n f i l t r a t i o n r a t e d i d not occur, then the changes i n e l e c t r o l y t e concentrations and i n u r i n e volume were due to a l t e r a t i o n s i n the re a b s o r p t i v e and se c r e t o r y processes i n the kidney. The increase i n ur i n e volume could be a t t r i b u t e d to a decreased l e v e l of a n t i d i u r e t i c hormone which would increase water output. The se a l s may have been dehydrated, even though they were i n f r e s h water up to the time of the experiment and had the opportunity to increase t h e i r body water, s i n c e they r e t a i n e d from 21 to 35 50% of the intubated water over 13 hours. The reduced GFR that occurs i n the s e a l during s t a r v a t i o n may a l s o have c o n t r i b u t e d to the low water output. The i n t u b a t i o n of hypertonic s o l u t i o n s r e s u l t e d i n an increased u r i n e volume up to 210 ml. per hour w i t h i n three hours. The patterns of volume change f o r sea water and hypertonic s a l t s o l u t i o n s were s i m i l a r to those reported by A l b r e c h t (1950) and Bradley, et. a l . (1954), r e s p e c t i v e l y . The t o t a l e l e c t r o l y t e e x c r e t i o n i n the sea water experiments increased w i t h the e l e c t r o l y t e load. The increased volume or u r i n e and e l e c t r o l y t e concentrations could be accounted f o r by decreased aldosterone l e v e l s during the e a r l y stages which would r e s u l t i n decreased sodium re a b s o r p t i o n and an osmotic d i u r e s i s . In the l a t t e r stages of the experiments the decreased e l e c t r o l y t e and u r i n e volume can be explained by increased aldosterone l e v e l s which would increase e l e c t r o l y t e and water r e a b s o r p t i o n . S i m i l a r ; e f f e c t s due to v a r y i n g l e v e l s of aldosterone have been demonstrated i n the dog (Roemmelt, e_t a l . , 1949). In mammals, as the u r i n e volume incr e a s e s , the osmotic pressure decreases i f there i s no increase i n e x c r e t i o n of o s m o t i c a l l y a c t i v e p a r t i c l e s . With a decreased urine volume, there i s an increased p r o p o r t i o n of s o l u t e s i n the u r i n e and thus the o s m o l a l i t y i n c r e a s e s . The changes i n osmotic pressure during the experiments can thus, i n p a r t , be explained i n terms of u r i n e volume and the proportion of o s m o t i c a l l y a c t i v e p a r t i c l e s present. Figure 2 presents 36 a comparison of the s p e c i f i c g r a v i t y and osmotic pressure values obtained i n the d i s t i l l e d water experiments. I t can be seen t h a t there i s a great s i m i l a r i t y i n the changes o c c u r r i n g i n these values. The changes i n sodium, c h l o r i d e and potassium i o n concentration discussed p r e v i o u s l y are not great enough to account f o r the l a r g e change i n o s m o l a l i t y a f t e r i n t u b a t i o n of d i s t i l l e d water. I t i s t h e r e f o r e probable that the amount of t o t a l s o l i d s (urea being the major substance, Koch, 1965) i n the u r i n e i s the predominant f a c t o r determining the osmotic pressure. Schmidt-Nielsen, et. _ a l . (1959) found that the concentration of urea decreased a f t e r i n f u s i o n w i t h d i s t i l l e d water and the u r i n e volume increased. A corresponding change between s p e c i f i c g r a v i t y and u r i n e volume occurred i n these experiments a l s o . The osmotic pressure of the u r i n e decreased i n the d i s t i l l e d water experiments, but at no time was i t hypoosmotic to the plasma. The f a i l u r e of the u r i n e to become hypoosmotic i n the s e a l may be accounted f o r by assuming that even though there was a decreased e l e c t r o l y t e e x c r e t i o n , the other major c o n s t i t u e n t s of the u r i n e (urea, ammonia, c r e a t i n e and c r e a t i n i n e ) were present i n s u f f i c i e n t concentration to maintain the recorded osmotic pressure. A l s o i n f l u e n c i n g the o s m o l a l i t y was the gradual increase i n the u r i n e volume, w i t h i n which there was a l a r g e r p r o p o r t i o n of o s m o t i c a l l y a c t i v e p a r t i c l e s than there would 37 have been i f an abrupt increase had occurred. I f the pa t t e r n of water d i u r e s i s was s i m i l a r to that observed i n the dog ( P i t t s , 1963) then the p r o p o r t i o n of p a r t i c l e s i n the u r i n e perhaps would have been low enough to make the u r i n e hypoosmotic to the plasma. In the sea water experiments, the u r i n e o s m o l a l i t y values remained r e l a t i v e l y constant f o r Seal #1, but increased f o r Seal #2 over the 13 hours f o l l o w i n g i n t u b a t i o n (Figures 2d-2g). Figures 6d-6g and 7d-7g showed that the c h l o r i d e and sodium concentrations i n c r e a s e d r a p i d l y a f t e r 1 to 2 hours and then remained constant or decreased. Therefore, there does not appear to be an obvious major c o n t r i b u t i o n to o s m o l a l i t y by these ions s i n c e a l a r g e increase i n i o n concentration should have been r e f l e c t e d by a s i m i l a r i n c rease i n osmotic pressure. However, i f the s p e c i f i c g r a v i t i e s of the ur i n e samples are p l o t t e d (Figure 2d-2g) and r e l a t e d to i o n concentrations and u r i n e volumes, the osmotic pressure trends can be explained. With an increase i n u r i n e volume, the concentration of s o l i d s decreases, as r e f l e c t e d by the decrease i n s p e c i f i c g r a v i t y . This statement i s based on the f a c t t h a t the time of the gre a t e s t decrease i n s p e c i f i c g r a v i t y occurs a t the same time as d i u r e s i s , and a l s o that Seal #1 w i t h a greater u r i n e volume, has the lower s p e c i f i c g r a v i t y . However, a t t h i s time, the i o n concentrations g r e a t l y i n c r e a s e , thus counteracting the decrease i n osmotic pressure expected by a decrease i n 38 the concentration of s o l i d s excreted. The net r e s u l t i s an osmotic pressure t h a t does not drop, but increases w i t h time. This l a t t e r increase i s probably due to the f a c t that the i o n concentrations remain maximal or decrease s l i g h t l y w h i l e the s p e c i f i c g r a v i t y i n c r e a s e s . The increases i n s p e c i f i c g r a v i t y could be due to an increased water r e a b s o r p t i o n i n the l a t t e r p a r t of the experiments, as r e f l e c t e d i n the decreased u r i n e volume. The s p e c i f i c g r a v i t y values a l s o provide a p o s s i b l e explanation f o r the d i f f e r e n c e s between the osmotic pressure values of the two sea l s at the end of the experiment, since the s p e c i f i c g r a v i t y values are d i f f e r e n t i n the same d i r e c t i o n as the osmotic pressure values, whereas the i o n concentrations are very s i m i l a r . The concentrating c a p a c i t y of the kidney i n d i c a t e s how the s e a l i s able to meet i t s water requirements, whether, i n f a c t , i t can dri n k sea water and remove the e l e c t r o l y t e s without body dehydration or whether i t gains a l l of i t s water from i t s food. The s e a l can concentrate sodium and c h l o r i d e to approximately the same concentrations found i n open sea water (Table I I ) , t h i s i n d i c a t e s that the s e a l can gain l i t t l e or no water by d r i n k i n g sea water. In f a c t , t h i s i s the case, as can be seen i n Table I. In order to e l i m i n a t e a l l of the sodium and c h l o r i d e introduced w i t h sea water the s e a l w i l l have to draw on body water. Further evidence to i n d i c a t e t h a t t h i s s e a l cannot b e n e f i t from d r i n k i n g l a r g e volumes of sea water i s found 39 i n the i n a b i l i t y of the animal to e l i m i n a t e both the water and e l e c t r o l y t e s i n sea water. Bradley, e t a l . (1954) s t a t e t h a t i n the periods between meals, f i l t r a t i o n as w e l l as water and e l e c t r o l y t e e x c r e t i o n decreases, i n f a c t , "... tubular a c t i v i t y may f a i l to operate i n accordance w i t h body needs, i . e . , water and s a l t r e t e n t i o n occurs despite the need to e l i m i n a t e loads imposed expe r i m e n t a l l y . 11 The r e s u l t s of the present experiments support t h i s suggestion i n t hat the s e a l r e t a i n e d from 27 to 65% of .the water, from 45 to 72% of the c h l o r i d e and from 31 to 65% of the sodium a f t e r t h i r t e e n hours. The r e s u l t s suggest that by continued d r i n k i n g of sea water, the animal may become edemic and increase i t s body e l e c t r o l y t e concentration to t o x i c l e v e l s . I f the animal takes i n small q u a n t i t i e s of sea water, as i t may do when feeding, there would be s u f f i c i e n t i s o r r h e i c water provided, and w i t h post-feeding h y p e r f i l t r a t i o n the body would be able to excrete the excess e l e c t r o l y t e s from the sea water. However, i f the animal i n g e s t s sea water and does not feed, the v a s o c o n s t r i c t i o n and reduced GFR would cause the water and e l e c t r o l y t e s to be r e t a i n e d i n the body. Since t h i s animal spends o n e - t h i r d of i t s time submerged (Schmidt-Nielsen, e t a l , , 1959), during which the GFR,water and e l e c t r o l y t e output, and r e s p i r a t o r y water l o s s are reduced, and a l s o during which there i s l i t t l e water l o s s by evaporation and i n the feces, then the p o s s i b i l i t y of body hydration seems j u s t as great a hazard as e l e c t r o l y t e i n t o x i c a t i o n f o l l o w i n g frequent i n g e s t i o n of la r g e q u a n t i t i e s of sea water. 40 SUMMARY 1. The osmotic and i o n i c r e g u l a t o r y c a p a c i t i e s of the kidney have been determined f o r two female harbor seals during periods of dehydration and water and s a l t l o a d i n g . 2. The maximum values obtained f o r u r i n a r y osmotic pressure, and sodium and c h l o r i d e and potassium i o n concentrations are 2050 mOsm./l., 523 mEq.Na+/l.» 508 mEq.Cl~/l. and 263 mEq.K +/l., r e s p e c t i v e l y . 3. D i s t i l l e d water i n g e s t i o n r e s u l t s i n an increase i n uri n e volume and decrease i n osmotic pressure and i o n concentrations. 4. F o l l o w i n g sea water i n g e s t i o n , a l a r g e increase i n u r i n e volume occurs w i t h i n two hours, u r i n e o s m o l a l i t y remains constant or increases', the sodium and c h l o r i d e i o n concentrations increase to t h e i r maximum values w i t h i n 3 to 5 hours, and the potassium i o n concentration behaves d i f f e r e n t l y from these i o n s . 5. The concentrations of sodium and c h l o r i d e i n the ur i n e are approximately equal to those of f u l l s t r e n g t h sea water, i n d i c a t i n g t h a t no water can be gained from i t s i n g e s t i o n . In order f o r the s e a l to e l i m i n a t e these e l e c t r o l y t e s i t must draw on i t s body water. 4 1 LITERATURE CITED Albrecht, Carolyn B., 1950. Tox i c i t y of sea water i n mammals. Amer. J. Physiol. 163: 370-385. Andersen, Harald T., 1966. Phsyiological adaptations i n diving vertebrates. Physiol. Rev. 46: 212-243. Barnes, H., 1954. Some tables for the i o n i c composition of sea water. J. exp. B i o l . 3_1: 582-588. Bradley, S.E. and R.J. Bing, 1942. Renal function i n the harbor seal (phoca v i t u l i n a , L.) during asphyxial ischemia and pyrogenic hyperemia. J. C e l l , and Comp. Physiol. 19: 229-237. Bradley, S.E., G.H. Mudge and W.D. Blake, 1954. The renal excretion of sodium, potassium, and water by the harbor seal (phoca v i t u l i n a L.): E f f e c t of apnea; sodium, potassium, and water loading; p i t r e s s i n ; and mercurial d i u r e s i s . J. C e l l , and Comp. Physiol. 43_: 1-22. Davenport, H.W., 1961. Physiology of the Digestive Tract. Year Book Medical Publishers, Inc., Chicago. Fisher, H.D., 1952. The status of the harbour seal i n B r i t i s h Columbia, with p a r t i c u l a r reference to the Skeena River. B u l l . F i s h . Res. Can. No. 93_: 1-58. Futcher, P.H., W.V. Consolazio and Nello Pace, 1943. The e f f e c t of drinking unmodified sea water, and a comparison with the eff e c t s of drinking D-S (GOETZ) water and a li m i t e d supply of "fresh water. U.S. Naval Medical Research Institute, project Number X-100 (General 15), 1-21. Goodman, L. and A. Gilman, 1965. The Pharmacological Basis of Theropeutics. 3rd ed., MacMillan, New York. Harrison, R.J. and J.D.W. Tomlinson, 1963. Anatomical and physiological adaptations i n diving mammals. In "Viewpoints i n Biology". Eds. Carthy and Duddington, Butterworths, London. Hiatt, E.P. and R.B. Hiatt, 1942. The e f f e c t of food on the glomerular f i l t r a t i o n rate and renal blood flow i n the harbor seal (phoca v i t u l i n a , L . ). J. C e l l , and Comp. Physiol. 19_: 221-227. 42 Hunt, J.N., I . Macdonald and W.R. S p u r r e l l , 1951. The g a s t r i c response to p e c t i n meals of high osmotic pressure. J . P h y s i o l . 115: 185-195. I r v i n g , L., O.M. Solandt, D.Y. Solandt and K.C. F i s h e r , 1935. The r e s p i r a t o r y metabolism of the s e a l and i t s adjustment to d i v i n g . J . C e l l and Comp. P h s y i o l . 2: 137-151; I r v i n g , L., K.C. F i s h e r and F.C. Mclntoch, 1935. The water balance of a marine mammal, the s e a l . J . C e l l , and Comp. P h y s i o l c j6: 387-391. K l i s i e k e , A., M. P i c k f o r d , P. R o t h s c h i l d and E.B. Verney, 1933. The absorption and e x c r e t i o n of water by the mammals. P t . 1. The r e l a t i o n between absorption of water and i t s e x c r e t i o n by the innervated and denervated kidney. Proc. Roy. S o c , B, 112: 496-521. Koch, A., 1965. Kidney f u n c t i o n and body f l u i d s . In "Physiology and Biophysics," 19th e d i t i o n . Eds. Ruch and Patton. Saunders, P h i l a d e l p h i a pp. 843-870. L i l l y , John C , 1964. Animals i n aqua t i c environments: adaptation of mammals to the ocean. Handbook of Physiology. Sect. 4: Adaptation to the c Environment. Amer. P h y s i o l . S o c , Wash. D.C. Lowrance, P.B., J.F. N i c k e l , C. M c Smythe and S.E. Bradley, 19 56. Comparison of the e f f e c t of anoxic anoxia and apnea on r e n a l f u n c t i o n i n the harbor s e a l (Phoca v i t u l i n a , L . ) . J . C e l l , and Comp. P h y s i o l . 48: 35-49. Page, Lot B., J.C. Scott-Baker, G.A. zak, E.L. Becker and C.F. Baxter, 19 54. The e f f e c t of v a r i a t i o n i n f i l t r a t i o n r a t e on the u r i n a r y concentrating mechanism i n the s e a l , phoca v i t u l i n a L . J . C e l l , and Comp. P h y s i o l . 43_: 257-269. Parsons, D.S. and D.L. Wingate, 1961. The e f f e c t of osmotic gradients on f l u i d t r a n s f e r across r a t i n t e s t i n e i n v i t r o . Biochem. biophys. a c t a , 46_: 170-183. P i t t s , R.F., 1963. physiology of the Kidney and Body F l u i d s . Year Book Medical P u b l i s h e r s , Inc., Chicago. Roemmelt, J . C , O.W. S a r t o r i u s and R.F. P i t t s , 1949. E x c r e t i o n and r e a b s o r p t i o n of sodium and water i n the adrenalectomized dog. Am. J . P h y s i o l . 159: 124-136. 43 Sc h e f f e r , V.B., 1958. Sea l s , Sea-Lions and Walruses: A Review of the P i n n i p e d i a . Stanford Univ. Press, Stanford, C a l i f o r n i a . Schmidt-Nielsen, B o d i l , H.V. Murdaugh, J r . , Roberta O'Dell and J . Bacsanyi, 19 59. Urea e x c r e t i o n and d i v i n g i n the s e a l (Phoca v i t u l i n a , L . ) . J . C e l l , and Comp. P h y s i o l . 53_: 393-411. Scholander, P.F., 19 64. Animals i n aquatic environments: d i v i n g mammals and b i r d s . Handbook of physiology, Sect. 4: Adaptation to the Environment. Amer. P h y s i o l . S o c , Wash., D.C. Smirk, F.H., 1933. The e f f e c t of water d r i n k i n g on the blood composition of human subjects i n r e l a t i o n to d i u r e s i s . J . P h y s i o l . £8: 127-146. Smith, H.W., 1936. The composition of ur i n e i n the s e a l . J . C e l l , and Comp. P h y s i o l . l_i 465-474. Sperber, I . , 1944. Studies on the mammalian kidney. Zoologiska Bidrag Fran Uppsala 22} 249-43 2. Thaysen, J.H. and N.A.S. Thorn, 1954. E x c r e t i o n of urea, sodium, potassium and c h l o r i d e i n human t e a r s . Amer. J . P h y i o l . 178: 160-164. Thaysen, J.H., N.A.S. Thorn and I.L. Schwartz, 1954. E x c r e t i o n of sodium, potassium, c h l o r i d e and carbon d i o x i d e i n human p a r o t i d s a l i v a . Amer. J . P h y s i o l . 178: 155-159. 

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