Commonwealth Scientific and Industrial Research Organization,

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1 J. Phy8iol. (1966), 184, pp With 5 text-ftgures Printed in Great Britain THE EFFECT OF AN INTRAVENOUS INFUSION OF HYPER- TONIC SALINE ON RENAL MECHANISMS AND ON ELECTROLYTE CHANGES IN SHEEP BY B. J. POTTER From the Division of Nutritional Biochemistry, Commonwealth Scientific and Industrial Research Organization, Kintore Avenue, Adelaide, South Australia (Received 26 July 1965) SUMMARY 1. The ability of the sheep to tolerate excess sodium chloride has been investigated by subjecting sheep to an intravenous infusion of a 1% solution of sodium chloride. 2. Inulin and diodrast clearances failed to show any consistent changes in glomerular filtration rate but the effective renal plasma flow was slightly more. Plasma levels of sodium and chloride increased by 2-25% and potassium decreased by 3 %. Urinary levels for sodium and chloride showed a corresponding increase and potassium excretion was reduced. 3. The rates of re-absorption of sodium and chloride from the renal tubules were found to be proportional to their rates of filtration at the glomerulus, but this ratio was reduced after the hypertonic saline infusion. No such correlation could be established for potassium. 4. Osmolar clearances indicated that continued re-absorption of osmotically free water from the kidney tubular fluid occurred during and after the hypertonic saline. Excretion of urine, hyperosmotic to plasma, was thus maintained and water conservation supported. 5. Possible renal mechanisms associated with these effects are discussed. INTRODUCTION Sheep possess the ability to drink relatively high concentrations of salt water without suffering in any adverse manner (Heller, 1933; Peirce, 1957). Potter (1961, 1963) permitted sheep to ingest a solution of 1-3% sodium chloride as their only source of drinking water for periods of 6 months or more: renal adjustments occurred which assisted in the elimination of this excess salt and indicated that the sheep kidney may be unusually tolerant to excess sodium chloride. McDonald & Macfarlane (1958) and Macfarlane, Morris, Howard, McDonald & Budtz-Olsen (1961) have shown that the kidneys of Merino

2 66 B. J. POTTER sheep, subjected to conditions of dehydration in tropical climates, assume an important role in the conservation of water. However, the peculiar gastric anatomy of the ruminant, with its large rumen (or forestomach) containing up to 4 1. of fluid, may exert a significant influence on the salt and water metabolism of the sheep. Stacy & Brook (1964) have given evidence of this in a report which indicated that events in the rumen of sheep, after feeding, result in concurrent renal changes. The renal response to an increased load of sodium chloride given as an intravascular hypertonic solution has been extensively studied in recent years with attention being mainly directed to changes in filtration, tubular re-absorption and osmotic effects (Green & Farah, 1949; Selkurt & Post, 195; Wesson & Anslow, 1955; O'Connor, 1962; Maude & Wesson, 1963; Giebisch, Klose & Windhager, 1964). The observations of Stacy & Brook (1964) suggest that some of the renal findings reported for sheep by Potter (1961, 1963) might be affected by activities of the rumen. To ascertain whether this inference is correct and as a means of restricting possible influences from the rumen, it was decided to subject the kidneys of sheep to a sodium chloride load delivered as a hypertonic solution by continuous intravenous infusion. In this way it might be possible to determine whether sheep are tolerant to hypertonic sodium chloride and whether the renal response differs from those reported for other mammalian species. METHODS Experimental animals. The sheep used were all Merino ewes aged 2-4 yr (weighing kg) which were kept in pens for a period of 6 months and fed a diet of 7 g chaffed wheaten hay and 3 g chaffed lucerne. Water for drinking was allowed ad libitum. Before each clearance period the animals were installed in metabolism cages for several weeks to allow them to become placid and accustomed to experimental treatment. Experimental procedure. One hour before the commencement of the experimental period, food and water were removed from the sheep and withheld for the duration of the infusion: at the same time 1 1. of water was given by mouth to facilitate initial urine sampling. Plastic tubing was then inserted and secured into each extermal jugular vein for blood sampling and infusion purposes, and an indwelling Foley catheter placed into position for urine samples from the bladder. Aln initial blood sample was then taken through one of the plastic tubes for inulin and diodrast blanks and for initial plasma electrolyte concentrations. The bladder was drained and washed with 2 ml. distilled water: 2 ml. of air was injected to complete the expulsion of urine. A priming injection of inulin (-8 ml. 5 % inulin/kg body weight) was given immediately and followed 2 min later by diodrast (-5 ml. 35 % diodrast/kg body weight). On the completion of these priming injections a continuous intravenous infusion of an isotonic solution of NaCl (-9 g/1 ml.) containing 1 g/1 ml. of inu.lin and 7 g/1 ml. of diodrast was commenced and maintained at a rate of 4 ml./min for a period of 1 hr by means of a constant infusion pump. During this and the subsequent periods of infusion, blood and urine samples were taken at 2 min intervals of time, the blood sample being taken at the mid-point of each urine collection period. It was anticipated that plasma concentration and urinary

3 H YPERTONIC SALINE INFUSIONS IN SHEEP 67 excretion of inulin and diodrast would have stabilized sufficiently at the end of 1 hr to allow reasonably consistent values for glomerular filtration rate (G.F.R.) and effective renal plasma flow (E.R.P.F.) to be calculated. For the next hour the isotonic saline solution was replaced by a hypertonic solution of NaCl (1 g/1 ml.) containing 5 g/1 ml. of inulin and 35 g/1 ml. of diodrast. The quantity of inulin and diodrast injected was maintained at the same previous constant rate by increasing the speed of the infusion pump so that the rate of administration was increased to 8 ml./min. The sheep, in this way, therefore received a total of 48 g NaCl (816 m-equiv Na+) during this period of 1 hr. The infusion of this hypertonic solution was then discontinued and the original isotonic solution re-infused at a rate of 4 ml./min for the next 2 hr. Infusion of all solutions then ceased and food and water were restored to the sheep. For the next 2 hr blood samples and urine were collected at one hourly intervals instead of 2 min periods of time, and thereafter at 12 and 24 hr following the priming injection. The animals showed no ill effects at any time from the NaCl administration and consumed their food in the normal manner when it was given to them at the conclusion of the infusion. Chemical estimation. Blood samples were withdrawn through one of the plastic tubes by syringe and placed into dry heparinized 15 ml. centrifuge tubes which were then centrifuged to allow the separation of the plasma. Inulin in the plasma and the urine (after suitable dilutions) was determined by the method of Schreiner (195) and diodrast by the method of Alpert (1941). Sodium and potassium were estimated in the plasma and urine by flame photometry with an 'EEL' flame photometer, and chloride by the method of Van Slyke & Hiller (1947). Osmolalities were measured in plasma and urine by freezing point depression with a Fiske osmometer. RESULTS The investigations were performed on five animals which remained placid and showed no ill-effects throughout the 4-hourly infusion of the isotonic and hypertonic saline. The sheep were observed to ruminate frequently during the 1% saline infusion and only towards the end of the infusion period did they moisten their lips in an endeavour to overcome drying of the buccal mucosa. At the conclusion of the infusion they usually drank water within the first 1 hr and their food and water intakes for the next 2 hr were the same as those of a normal daily period. Responses of the sheep to intravenous NaCI Inulin and diodrast clearances. The values obtained for G.F.R., E.R.P.F. and filtration fraction (F.F.) were difficult to assess because of the extreme variations encountered. However, on no occasion did a consistent change in G.F.R. occur nor could any correlation be shown between the excretion of sodium and the clearance of inulin. The E.R.P.F. tended to rise slightly after the infusion of hypertonic saline and the F.F. was correspondingly slightly lower. Plasma clearances of sodium, potassium and chloride. Plasma values for sodium remained stable for the first hour while the isotonic sodium chloride was being injected, but increased steadily after commencement of the hypertonic saline infusion. This increase reached a maximum value 2-25 % 39 Physiol. 184

4 68 B. J. POTTER greater than the initial value after a period of 6-8 min: the value then decreased slightly but remained elevated after cessation of the hypertonic saline and during the remainder of the isotonic saline infusion. The values, thereafter, gradually decreased and were almost reduced to normal after 12 hr. The concentrations of chloride in the plasma were found to agree closely with those observed for sodium: the return to normal however was less rapid and only after 24 hr had elapsed was the pre-injection concentration of chloride again obtained. Plasma potassium values fell slightly during the initial isotonic infusion, but with the onset of the hypertonic saline this fall became much more pronounced and at the end of the latter infusion the value of potassium had decreased by about 3 %. With the re-introduction of the isotonic saline, the values increased sharply during the next 2-4 min to values just less than the pre-hypertonic figures and these remained reasonably constant until the end of the infusion. A steady increase then occurred until pre-injection, and sometimes higher, values were obtained at the end of 12 hr. The typical response for sodium, potassium and chloride concentrations observed in the plasma of sheep after intravenous saline is shown in Fig. 1. Urine flow and excretion rates of sodium, potassium and chloride. The rate of urine flow was not greatly affected during the first hour while isotonic saline was being infused, but the introduction of the hypertonic saline was accompanied by the onset of a profound diuresis during which the urine flow increased from a mean figure of (s.e.) ml./min. to 18* (s.e.) ml./min when the diuresis was at its peak. This maximum rate of urine excretion occurred during the last 2 min of the hypertonic saline, or immediately following this period when the hypertonic infusion had been replaced with isotonic saline. The diuresis then showed an appreciable decline but was still evident 2 hr after all infusions had ceased: the rate of urine flow was completely normal again 12 hr after the initial saline infusion. The excretion patterns for sodium and chloride were remarkably similar in their behaviour, and were not unlike that observed for the urine flow rate. Thus little change in the rates of excretion of sodium and chloride occurred during the initial isotonic saline infusion, but the hypertonic solution caused the rate of excretion of sodium to rise from a mean initial figure of (s.e.) m-equiv/min to 3* (S.E.) m-equiv/mi;n: at the same time the chloride values rose from a mean initial rate of excretion of (s.e.) m-equiv/min to a maximum value of (S.E.) m-equiv/min. The peak values for sodium and chloride excretory rates occurred within 1-2 hr of the commencement of the hyper-

5 HYPERTONIC SALINE INFUSIONS IN SHEEP 69 tonic saline infusion and at the same time in any one animal. The rates thereafter progressively declined and returned to normal values within hr. z; NaCI _Io.9/41dY/A 2-9% X E& I I I I I I I I I I Time (hr) Fig. 1. The effect of intravenous sodium chloride infusion on sodium, chloride and potassium concentrations in the plasma of one sheep which is typical of all the others. The rates of excretion of potassium were invariably increased for the first hour during which the isotonic saline was infused, but fell steadily following the advent of the hypertonic saline. From a mean initial value of (S.E.) m-equiv/min the value rose to (S.E.) m-equiv/min and then fell to (S.E.) m-equiv/min: this minimum rate of excretion was reached about 1 hr after replacement of the hypertonic saline with the isotonic solution. The potassium excretion then underwent a slight increase and remained relatively stable for about 8 hr after the withdrawal of all infusions: within the next 12 hr the potassium excretion returned to normal. Figure 2 is an indication of the rates of urine flow and sodium, potassium and chloride excretion in one of the sheep subjected to the intravenous infusion of sodium chloride. Tubular effects of sodium chloride and potassium. The rates of re-absorption of sodium and chloride from the renal tubules were calculated by the subtraction of the excretion rates of these electrolytes from their filtration 39-2

6 61 B. J. POTTER rates. However, as variations in glomerular filtration rates will necessarily introduce variations in tubular activity, the rates of tubular re-absorption of sodium for all animals were plotted against sodium filtration rates and are shown in Fig. 3. It is apparent that for sodium the rate of re-absorption is proportional to the glomerular filtration rate and that the re-absorption rates may be arranged into two distinct groups. One of these groups, NaCI.9/413/.9/. 3 [ 9 o$o O-% 2 - I1oL o 4oI_ E 4H L l l l l l l l l l l l I Time (hr) Fig. 2. The rates of excretion of urine and of sodium, chloride and potassium in one of the sheep subjected to intravenous sodium chloride infusion. representing the values obtained after the introduction of the hypertonic saline infusion, is displaced to a lower level than that of the values obtained before the hypertonic saline and thereby indicates that the ratio of tubular re-absorption to glomerular filtration is reduced with salt load. As the plasma concentrations and excretion rates of chloride closely paralleled those of sodium (see Figs. 1 and 2), the ratio of chloride reabsorbed from the renal tubules to chloride ifiltered at the glomeruli must have also decreased with the introduction of hypertonic saline.

7 HYPERTONIC SALINE INFUSIONS IN SHEEP 611 The tubular re-absorption rates for potassium were plotted against the filtration rates but no correlation could be shown to exist. In fact, as indicated in Fig. 4, both tubular re-absorption and secretion occurred during the saline infusion. 2 - E18 _ E *: 17 _ OO o o 17 ~~~~~ 16 E 15 o 14 o m13 ' 12 o 1 _ Ca 9 : 8 o Sodiulm filtration rate (m-equiv/min) Fig. 3. The influence of the filtered load of sodium on tubular re-absorption in all sheep which received intravenous saline. The open circles represent the period before hypertonic saline infusion. Changes in osmolality in plasma and urine. The plasma osmolality did not change during the first hour of isotonic saline infusion but increased as soon as the hypertonic salt solution was infused. Conversely, the osmolality of the urine decreased with hypertonic saline. However, as a result of the increased urine flow the osmolar clearances were increased. The osmolal clearance, Cosm = Uosm V/Posm, where Cosm is the osmolal clearance Uosm and Po.m are the urine and plasma osmolality (m-osmol/ (ml./min), g H2) and V is the rate of urine flow (ml./min), is a measure of the volume ofwater required to keep the excreted osmotically active constituents at the same osmolality as the blood plasma. If the osmolal clearance is less than the urine flow, the difference represents the clearance of solute-free water. With all the sheep, however, the osmolal clearances for each sample in general exceeded the urine flow, and therefore indicated that the excretion of solute-free water was negative or that the urine was hyperosmotic to the plasma. The magnitude of this negative solute-free water clearance, designated TcH2O, is an indication of water re-absorption in excess of

8 612 B. J. POTTER solute, and is shown together with the corresponding values for osmolal clearance in Fig A 1 Tt.: ~~~ I *~~~ Re-absorption *.* O > > _ :> -----e~. O Secretion O ; o o o A -i o _ -2 e 4 *3 _ o ~~~~~~ ~ H5 _ -6 1 l Potassium filtration rate (m-equiv/min) Fig. 4. The influence of the filtered load of potassium on tubular re-absorption and secretion in all sheep which were infused with intravenous saline. The open circles represent the period before hypertonic saline infusion. DISCUSSION Salt solutions supplied as drinking water to sheep have led to the suggestion by Potter (1961, 1963) that the kidneys of these animals may be unusually efficient in their ability to eliminate excess sodium chloride. Functional tests applied to the kidney indicated that increased glomerular filtration rates invariably occurred in sheep which drank a 1-3 % solution of sodium chloride as their only source of drinking water for periods of six months or more. Although re-absorption of sodium and chloride, when calculated as a percentage of filtered load, was reduced, the absolute amounts re-absorbed in the kidney tubules were increased. The effect on the sheep kidney of an intravenous load. of sodium chloride, given as a 1 % solution for 1 hr, has now been investigated to see whether intravenous salt is handled as effectively by the sheep as ingested saline. From the results obtained it appears that the sheep is relatively tolerant

9 HYPERTONIC SALINE INFUSIONS IN SHEEP also to intravenous salt, but the renal changes involved differ in some respects from those observed for ingested salt. Although increased G.F.R. was invariably increased when sheep received oral saline, the same response was not evoked after intravenous NaCl: the infusion of the latter produced variable results without a consistent increase or decrease in G.F.R. At the same time an increased excretion of sodium and chloride in the urine of the sheep confirmed the observations of de Wardener, Mills, Clapham & Hayter (1961) for the dog, and substantiated their claim that sodium excretion is virtually independent of changes in G.F.R. and E.R.P.F. NaCI 6 I.9/41/4.9o/ _ ^ S Time (hr) Fig. 5. The effect of intravenous infusion of saline on the osmolar clearance (C.m) and the re-absorption of water in excess of solute (TCH2) in one of the sheep. The literature on sodium diuresis contains numerous conflicting reports on the responses of G.F.R. and E.R.P.F. to hypertonic saline. Although filtration is undoubtedly important in the elimination of excess sodium, the influence of tubular re-absorption may be of greater significance, particularly in view of the active transport of solute, presumably sodium ions, in concentrating urine by means of the hairpin countercurrent multiplier system (Wirz, Hargitay & Kuhn, 1951; Hargitay & Kuhn, 1951). Tubular re-absorption of sodium as a result of hypertonic saline loading has been investigated in dogs by Bressler (196), Kamm & Levinsky (1964) and Stein, Bercovitch & Levitt (1964). Bressler (196) found that for a

10 614 B. J. POTTER given rate of glomerular filtration, tubular re-absorption of sodium chloride was increased as the levels of plasma sodium and chloride rose. Kamm & Levinsky (1964) observed progressive increases in tubular re-absorption as plasma sodium was elevated, independently of changes in G.F.R. They postulated that increased tubular re-absorption may result from the increased filtered sodium which occurs when plasma sodium is elevated. Stein et al. (1964), however, on the basis of their experiments, have suggested that saline, apart from its effects on filtered load, progressively inhibits sodium re-absorption by decreasing the percentage of sodium load re-absorbed. The observations reported here for sheep provide further support for the suggestions of Stein et al. (1964). WVhen isotonic saline was being infused the rates of re-absorption of sodium were directly proportional to the filtered load of sodium at the glomeruli. The advent of the hypertonic saline with concurrently increased plasma sodium levels, was followed again by a direct linear relation, but the ratio given by the rate of tubular re-absorption divided by the filtered load was less. In other words the hypertonic saline produced a reduction in the percentage of the filtered load of sodium which was re-absorbed in the tubules. A similar relation existed for chloride. The linear relation which was found to exist between tubular re-absorption of sodium (and chloride) and the ifitered load, indicates that when the latter is increased, primarily as a result of raised plasma levels of sodium (and chloride), the re-absorption of sodium (and chloride) will increase proportionately. In this respect the results agree with those reported by Bressler (196). However, when considered as a percentage of the filtrate which is re-absorbed in the tubules, it is apparent that the percentage re-absorbed is less with an increased salt load. Stein et al. (1964) have postulated that a net increase in sodium re-absorption might result from a pronounced increase in filtered sodium co-existent with a slight reduction in the percentage of sodium load re-absorbed. The converse might apply to a net reduction in sodium re-absorption and this could account for some of the conflicting reports on tubular re-absorption in dogs. Re-absorption may then be increased in both sheep and dogs by intravenous hypertonic saline, but the increase in sheep may not be as great as in dogs because of the reduction in the percentage of filtered sodium which is re-absorbed. The influence of hypertonic saline on the tubular movement ofpotassium is more difficult to explain. Although plasma levels for potassium were inversely related to sodium, the urinary excretion of potassium fell to its lowest level some time later than that of-the maximal excretion of sodium. Tubular re-absorption and secretion bore no relation to filtered load and

11 HYPERTONIC SALINE INFUSIONS IN SHEEP 615 the results indicated that secretion predominated over re-absorption. This is not unreasonable in view of the report of Vander (1961) in which, using stop-flow techniques in dogs, he found that potassium re-absorption and secretion may occur at the same moment in both the collecting ducts and the distal tubules. Berliner (1961) concluded that potassium secretion is related to the rate of exchange of sodium for potassium in the distal tubules and collecting ducts. In the sheep, however, tubular secretion of potassium was not directly related to potassium load and it is impossible therefore to conclude that secretion of potassium was reduced during the infusion of hypertonic saline. It is also difficult to ascertain whether tubular secretion of potassium is related to sodium re-absorption in the tubules. It must be concluded that although tubular secretion of potassium may continue in sheep subjected to intravenous hypertonic saline, this secretion, if increased, is not sufficient to outweigh the reduction in filtered load resulting essentially from reduced plasma levels of potassium, and the final result therefore is reduced excretion of potassium in the urine of these animals. The osmolal clearances of the sheep clearly showed that water re-absorption exceeded that of solute and resulted in the excretion of urine which was hyperosmotic to plasma. Water conservation was thus maintained throughout. The re-absorption of osmotically free water increased during the control period of isotonic saline infusion but began to decrease soon after commencement of the hypertonic saline. After reaching its lowest level it again increased and stabilized before returning to a level just less than the initial value obtained. Since Gottschalk & Mylle (1959) have shown by micro-puncture techniques that concentrated urine results from the diffusion of water from isosmolar fluid in the collecting ducts into the hypertonic medulla of the kidney, it is reasonable to assume that the rate of this transfer of water will be determined by the rate of fluid delivery into the collecting ducts. This could then account for the fact that TcH2 is increased during the control period, when active transport of solute, presumably sodium, takes place from the ascending limb of the loop of Henle to maintain hypertonicity in the medulla. Under the possible influence of the antidiuretic hormone of the pituitary, water would then pass from the hypotonic fluid as it enters the distal tubules and from the fluid in the collecting tubules to equilibrate with the hypertonic medulla. However, the increased urine flow, coinciding with the infusion of the hypertonic saline, is indicative of an increased rate of fluid delivery to the loops of Henle. This might result in a reduction in active transport from the ascending limb and reduced hypotonicity in the fluid entering the distal tubules with less re-absorption of free water from the collecting ducts. An explanation could then be afforded for both the reduction in

12 616 B. J. POTTER the percentage of filtered sodium which is re-absorbed, as well as the reduction in free water re-absorption from the collecting ducts. It would appear that the sheep may differ from the dog in its ability to excrete salt under high sodium load, but the results reported for dogs are too confused to permit any categorical differentiation. However, the renal mechanism responsible for the elimination of excess sodium chloride in the sheep certainly varies according to the route of administration. With intravenous sodium chloride no consistent increase in glomerular filtration rate was observed: this differs from previously reported observations on sheep drinking sodium chloride solutions. Urinary excretion of sodium and chloride was increased despite a reduction in the percentage ofthese electrolytes re-absorbed from the filtered load; and concentration of renal tubular fluid was maintained to produce urine which was hyperosmotic to plasma. REFERENCES ALPERT, L. K. (1941). A rapid method for the determination of diodrast-iodine in blood and urine. Johns Hopkins Hosp. Bull. 68, BERLINER, R. W. (1961). Renal mechanisms for potassium excretion. Harvey Lect. 55, BRESSLER, E. H. (196). Reabsorptive response of renal tubules to elevated sodium and chloride concentrations in plasma. Am. J. Physiol. 199, GIEBISCH, G., KLosE, R. M. & WINDRAGER, E. E. (1964). Micropuncture study of hypertonic sodium chloride loading in the rat. Am. J. Phy8iol. 26, GOTTSCHALK, C. W. & MYLLE, M. (1959). Micropuncture study of the marumalian urinary concentrating mechanism; evidence for the countercurrent hypothesis. Am. J. Phyaiol. 196, GREEN, D. M. & FARAH, A. (1949). Influence of sodium load on sodium excretion. Am. J. Physiol. 158, HARGITAY, B. & KuHN, W. (1951). Das Multiplikationsprinzip als Grundlage der Harnkonzentrierung in der Niere. Z. Elektrochem. 55, HETLER, V. G. (1933). The effect of saline and alkaline waters on domestic animals. Bull. Okla. agric. Exp. Stn, no KCA, D. E. & LEVINSKY, N. G. (1964). Effect of plasma sodium elevation on renal sodium reabsorption. Am. J. Phy8iol. 26, MCDONALD, J. & MACFARLANE, W. V. (1958). Renal function of sheep in hot environments. Amst. J. agric. Res. 5, MACFARLANE, W. V., MoRRis, R. J. H., HOWARD, B., MCDONALD, J. & BUDTZ-OLSEN,. E. (1961). Water and electrolyte changes in tropical Merino sheep exposed to dehydration during summer. Aust. J. agric. Res. 12, MAuDE, D. L. & WESSON, L. G. Jnr. (1963). Renal water reabsorption during saline and urea osmotic diuresis in the dog. Am. J. Phy8iol. 25, O'CoNNoR, W. J. (1962). Renal Function, p. 72. London: Edward Arnold. PEIRCE, A. W. (1957). The tolerance of sheep for sodium chloride in the drinking water. Aust. J. agric. Re8. 8, POTTER, B. J. (1961). The renal response of sheep to prolonged ingestion of sodium chloride. Aut. J. agric. Res. 12, POTTER, B. J. (1963). The effect of saline water on kidney tubular function and electrolyte excretion in sheep. Aust. J. agric. Res. 14, SCHREINER, G. E. (195). Determination of inulin by means of resorcinol. Proc. Soc. exp. Biol. Med. 74, SELKURT, E. E. & POST, R. S. (1 95). Renal clearance ofsodium in the dog: effect ofincreasing sodium load on reabsorptive mechanism. Am. J. Phy8iol. 162,

13 HYPERTONIC SALINE INFUSIONS IN SHEEP 617 STACY, B. D. & BROOK, A. H. (1964). The renal response of sheep to feeding. Aust. J. agric. Res STEIN, R. M., BERCOVITCH, D. D. & LEVITT, M. F. (1964). Dual effects of saline loading on renal tubular sodium reabsorption in the dog. Am. J. Physiol. 27, VANDER, A. J. (1961). Potassium secretion and reabsorption in distal nephron. Am. J. Physiol. 21, VAN SLYKE, D. D. & HILLER, A. (1947). Application of Sendroy's iodometric chloride titration to protein-containing fluids. J. biol. Chem. 167, DE WARDENER, H. E., MILLS, I. H., CLAPHAM, W. F. & HAYTER, C. J. (1961). Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin. Sci. 21, WEssoN, L. G. Jnr. & ANSLOW, W. P. Jnr. (1955). Relationship of changes in glomerular filtration, plasma chloride and bicarbonate concentrations and urinary osmotic load to renal excretion of chloride. Am. J. Physiol. 18, WIRZ, H., HARGITAY, B. & KUHN, W. (1951). Lokalisation des Konzentrierungsprozesses in der Niere durch direkte Kryoskopie. Helv. physiol. pharmac. Acta, 9,