J. Physiol. (1979), 294, pp. 223-238 With 6 text-ffiguree Printed in Great Britain
223
RENAL CALCIUM AND MAGNESIUM EXCRETION DURING VASOPRESSIN ADMINISTRATION INTO SHEEP WITH ACID OR ALKALINE URINE
BY A. M. BEAL* From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 28 November 1978) SUMMARY
1. The proposition that changes in renal calcium excretion during vasopressin administration are positively correlated with concurrent changes in urine hydrogen ion concentration was tested by administration of vasopressin into twelve conscious diuresing sheep receiving either alkalinizing or acidifying infusions. 2. Vasopressin-induced antidiuresis in sheep with alkaline urine was associated with significant increases in urinary pH and decreases in the rate of calcium excretion whereas antidiuresis in sheep with acid urine was associated with significant decreases in urinary pH and no consistent effect on calcium excretion. 3. Magnesium excretion increased during vasopressin administration in most experiments regardless of urinary pH changes. 4. Vasopressin administration did not significantly alter the rates of excretion of sodium, potassium, chloride and phosphate or the rates of sodium, potassium, chloride, inulin, para-aminohippurate and osmolal clearance in sheep with either acid or alkaline urine. Potassium excretion and clearance in sheep with alkaline urine was higher than that of sheep with acid urine during vasopressin infusion. 5. The results support the hypothesis that changes in renal tubular hydrogen ion concentration or bicarbonate concentration caused by water reabsorption from the collecting duct and possibly the late distal tubule could be part of the explanation for changes in renal calcium excretion which occur during vasopressin-induced antidiuresis. INTRODUCTION
In 1960, Thorn reported that intravenous injection of lysine-vasopressin into hydrated dogs caused an increase in the renal excretion of calcium which was correlated with the onset and duration of the antidiuretic effect of the injection. Subsequently increased calcium excretion during antidiuresis, induced by administration of lysine and arginine vasopressins, oxytocin and synthetic analogues of these hormones, was found to occur in hydrated rat, dog and man (Thorn, 1961; Dicker & Eggleton, 1961; Nielsen, 1964) although in both rat and man increased * Present address: University of New South Wales, School of Zoology, P.O. Box 1, Kensington, New South Wales, 2033.
0022-3751/79/4880-0872 $01.50 © 1979 The Physiological Society
224
A. M. BEAL
calcium excretion failed to occur in some experiments with vasopressin (Thorn, 1961; Nielsen, 1964). When this phenomenon was investigated in water-loaded sheep using hormone dose rates closer to 'physiological' than those of the previous authors, Kuhn (1966) found that antidiuresis produced by the administration of vasopressin or oxytocin was always associated with significant decreases in renal calcium excretion whereas Beal, Clark, Cross & French (1976) reported significant decreases in excretion after vasopressin only in sheep with calcium excretion rates less than 1 #smole/min. At higher rates of calcium excretion, vasopressin infusion did not alter calcium excretion although antidiuresis still occurred. Because of these reported differences in vasopressin's action on calcium excretion any explanation of the effect should attempt to accommodate this variability of response. Acidosis is associated with increased renal calcium excretion (Lamb & Evvard, 1919; Farquharson, Salter, Tibbetts & Aub, 1931; Williamson & Freeman, 1957; Reidenbert & Sevy, 1967). This calciuria occurs partly as a result of impaired reabsorption of calcium in the renal tubule (Lemann, Litzow & Lennon, 1966) particularly in the distal regions of the nephron (Sutton, Wong & Dirks, 1975). In acidotic dogs calcium reabsorption was restored to normal by administration of sodium bicarbonate to correct the acidosis and the fractional excretion of calcium reduced when bicarbonate was given to non-acidotic dogs (Sutton et al. 1975) which suggests that bicarbonate concentrations or more probably hydrogen ion concentrations are a determinant of the rate of calcium reabsorption in the distal segments of the nephron. Further evidence of the dependence of calcium reabsorption on tubular pH was provided by the observation that elevated urinary pH during hyperkalaemia caused by infusion of the neutral salt, potassium chloride, was accompanied by depressed renal calcium excretion in the sheep (Beal, Budtz-Olsen, Clark, Cross & French, 1973, 1974). Thus the rate of renal calcium excretion may partially depend on localized changes in pH in the renal tubule. Since vasopressin increases water reabsorption from the distal nephron without causing proportionate increases in reabsorption of most tubular solutes, the hydrogen ion concentration of the reabsorbate will tend to move towards that of distilled water (100 n-mole/l.). Thus the concentrating effect of vasopressin in the collecting duct region and possibly in the late distal tubule of some species should result in alkaline tubular fluid becoming more alkaline and acid tubular fluid becoming more acid. Under these conditions, vasopressin administration should enhance renal calcium excretion when the urine is acid and depress calcium excretion when the urine is alkaline. The following series of experiments were performed to test the hypothesis. A preliminary account of this work has been published (Beal, 1976). METHODS
Experimental procedures The experiments were performed on four Merino-crossbred ewes (39-5-46-5 kg) and eight Clun Forest ewes (36*0-57*0 kg), each of which had one carotid artery exteriorized in a loop of skin. Two sheep of each breed were splenectomized. The sheep were maintained on a diet of hay chaff (1000 g/day) and had unrestricted access to water and to a mineral salt supplement. During the afternoon before experiment, a vinyl cannula (1.4 mm i.d., 2-0 mm o.d.; Portex
ADH AND URINE pH ON Ca AND Mg EXCRETION
225
Limited) was inserted under local anaesthesia into one jugular vein using the technique of Seldinger (1953), a disposable plastic cannula (Braunula, size 0-5; Armour Pharmaceutical Company Limited) was introduced into the exteriorized carotid artery and the urinary bladder was catheterized with a short 14 F.G. catheter (Foley, 5 ml. balloon; Warne Surgical Products). At 18.00 hr any uneaten food and the mineral supplement were withdrawn, a blood sample (control 1) was taken and an infusion of either an acidifying solution (2.5 mM-CaCl2, 1 mM-MgCl2, 3 5 mm-KCl, 1 or 10 mn-HCl, 91 or 100 mm-NaCl; -220 m-osmole/l.) or an alkalinizing solution (2-5 mM-CaCl2, 1 mM-MgCl2, 3.5 mM-KCl, 30 mM-Na lactate, 15 or 30 mM-NaHCO3, 41 or 56 mM-NaCl; _ 220 m-osmole/l.) was commenced at 3 ml./min into the jugular cannula. Fifteen hours later the sheep were transferred to and restrained on canvas stretchers in a normal upright position with their feet just off the floor. After taking a blood sample (control 2) from the carotid artery, the overnight acidifying or alkalinizing infusate was replaced with a solution of similar composition but incorporating inulin (10 g/l.) and sodium paraaminohippurate (5 g/l.) with the sodium chloride content being reduced to maintain the osmolarity at 220 mosmole/l. A priming injection of 50 ml. of the inulin/PAH solution was given intravenously and thereafter this solution was infused at 3 ml./min to the end of the experiment. When the sheep had received a further 2 hr infusion, two serial 1 hr collections of diuretic urine under paraffin oil were made. Arginine vasopressin was then infused intravenously at 250 Fuu./min and after a 30 min transition period two serial 1 hr collections of antidiuretic urine were obtained. Vasopressin administration was then terminated and when the water diuresis recommenced, 45-90 min later, a further two serial 1 hr samples of diuretic urine were collected. Blood samples (10 ml.) were taken in syringes heparinized with 1 drop of heparin (5000 i.u./ml.) at the beginning and end of each hourly urine collection. Alkaline urine samples for use in calcium, magnesium and phosphate determinations were acidified with glacial acetic acid to prevent precipitation.
Analytical procedures The pH of urine and arterial blood was estimated immediately after collection under anaerobic conditions at 39 'C using thermostated Radiometer micro-electrodes. Microhaerratocrit determinations were made in triplicate within 15 min of sampling on blood spun at 12,000 g for 10 min in a microhaematocrit centrifuge (Hawksley). The remainder of each blood sample was centrifuged in glass tubes at 2750 g for 15 min to obtain plasma for analysis. Duplicate estimations of sodium and potassium in urine and plasma were made simultaneously by emission flame photometry in an oxygen-propane flame (Autotechnicon) with 15 m-mole/l. lithium as an internal reference and using mixed standards of sodium and potassium in the appropriate ranges to correct for mutual interference. Calcium and magnesium in urine and plasma were estimated in duplicate by atomic absorption in an air-acetylene flame (Pyelinicam SP- 191) using disodium ethylenediaminetetraacetic acid to overcome suppression of absorption by phosphate and protein. Total inorganic phosphate concentrations in urine and plasma were estimated in duplicate by the colorimetric method of Paginski, Foa & Zak (1967) adapted for automated analysis on the Technicon autoanalyser. The chloride concentrations of urine and plasma were measured in duplicate using a Radiometer chloride titrator (model CMT 10). The osmolality of urine and plasma was estimated in duplicate by freezing-point depression using a Fiske osmometer. Duplicate estimations of inulin in urine and plasma were made by the calorimetric method of Heyrovsky (1956) adapted to the Technicon autoanalyser by Dawborn (1965). PAH was measured in duplicate by the method of Bratton & Marshall (1939) as adapted for the Technicon autoanalyser by Harvey & Brothers (1962). Statistical procedures The differences in plasma electrolyte concentration and in urinary water and electrolyte excretion between the period of vasopressin infusion and the periods of diuresis both before and after vasopressin infusion were tested for statistical significance by t test of differences between correlated means (paired t test). The data for corresponding time periods of the alkaline and acid experiments were then compared by analysis of variance. To ensure that no effect of vasopressin was obscured by differences in electrolyte status which had been present before 8
PHYY 294
A. M. BEAL
226
or had occurred during the alkalinizing or acidifying infusions the data were also analysed after being adjusted to eliminate these differences by use of the following procedures. Blood and plasma data were subjected to analysis of covariance using the values for each variable in the blood samples taken at 17 hr (control 1) and 2 hr (control 2) before urine collection as covariates. Urinary data were subjected to analysis of variance after being expressed as ratios of the mean value of each variable for the 4 hr of diuresis. Since the use of a large number of statistical tests is likely to increase the frequency of erroneous rejection of the null hypothesis no result was considered to be statistically significant unless the probability level was less the 0-02.
RESULTS
The alkalinizing infusate contained either 15 or 30 mM-sodium bicarbonate and the acidifying infusate contained either 1 or 10 mm hydrochloric acid. As there were no significant differences between the results obtained with the two strengths of alkalinizing solution, the data for the two levels of alkalinizing infusion have been ADH
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Fig. 1. Urine flow rate, blood pH, urine pH and hydrogen ion excretion rate before, during and after i.v. administration of vasopressin at 250 ,uu./min into sheep receiving an alkalinizing infusion (n = 12; means + s.E. of mean).
ADH AND URINE pH ON Ca AND Mg EXCRETION 227 grouped together and are presented as combined mean results of the twelve alkalinizing experiments. Similarly there were no differences between the results for the two strengths of acidifying solution and the data for the acidifying infusions are presented as the mean results of the twelve acidifying experiments. ADH
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=
Blood The haematocrit, plasma osmolality and the plasma concentrations of hydrion, calcium, magnesium, sodium, potassium, chloride and inorganic phosphate were estimated for arterial blood samples taken before, during and after vasopressin administration into sheep receiving alkalinizing or acidifying infusions (Figs. 1-6). Alkalinizing infusion. Blood pH and plasma calcium concentration of sheep receiving the alkalinizing infusion were reasonably stable throughout the period of urine collection (Figs. 1 and 2) whereas haematocrit, plasma osmolality and plasma 8-2
A. M. BEAL 228 magnesium, sodium, potassium, chloride and phosphate concentrations fell during vasopressin infusion (Figs. 5 and 6). By the end of vasopressin administration the concentrations of magnesium, sodium and phosphate in the plasma and plasma osmolality were significantly less than the mean plasma concentrations during the 2 hr before vasopressin was given (magnesium, P < 0001; sodium, P < 0001; ADH
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#su./min
phosphate, P < 0-001; osmolality, P < 0-01). After termination of vasopressin infusion the haematocrit, plasma osmolality and plasma concentrations of magnesium, sodium, potassium and chloride increased so that, by the end of the experiment, only plasma phosphate was significantly depressed below pre-vasopressin levels (P < 0-001). Acidifying infusion. When the sheep were given the acidifying infusion, blood pH fell slightly throughout the period of urine collection (Fig. 3). This change in plasma hydrogen ion concentration was not statistically significant. During vaso-
ADH AND URINE pH ON Ca AND Mg EXCRETION 229 pressin administration the haematocrit, plasma osmolality and plasma calcium, magnesium, sodium, potassium, chloride and phosphate were decreased below prevasopressin levels (Figs. 4, 5 and 6). At the end of the vasopressin infusion, all of the above parameters except plasma potassium and chloride were significantly reduced below the mean values for blood taken during the 2 hr before vasopressin ADH
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Fig. 4. Plasma calcium and magnesium concentrations and urinary calcium and magnesium excretion rates before, during and after intravenous administration of vasopressin at 250 Asu./min into sheep receiving an acidifying infusion (n = 12; means + s.E. of mean).
infusion (haematocrit, P < 0-01; calcium, P < 0-01; magnesium, P < 0-001; sodium, P < 0-01; phosphate, P < 0-001; osmolality, P < 0.001). After termination of vasopressin infusion the haematocrit, plasma osmolality and plasma electrolyte concentrations increased so that only the phosphate concentration remained significantly below pre-vasopressin values by the end of the experiment (P < 0-01). Comparison of alkaline and acid experiments. Analysis of variance of corresponding
A. M. BEAL
230
blood samples from the two experiments showed that the sheep had significantly higher plasma hydrogen ion, calcium and chloride concentrations during the acidifying infusions than during the alkalinizing infusions (Table 1). Other possible differences in plasma electrolyte concentrations were not statistically significant. When the data was adjusted by analysis of covariance to eliminate differences in electrolyte concentration which had occurred before urine collection was comTABLE 1. Comparison of the electrolyte concentrations in plasma samples from sheep receiving alkalinizing infusions to those of corresponding plasma samples from sheep receiving acidifying infusions using analysis of variance and analysis of covariance techniques. Plasma samples were taken before, during and after vasopressin (A.DH) administration. Every plasma variable on Figs. 1-6 was tested and omission of a comparison from this Table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 002; ** - P < 0-01; *** = P < 0-001; - = not significant)
Blood sample A
Control Variable
(d.f. = 22) Hydrogen ion concn. Calcium concn. Chloride concn.
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menced, no significant differences were found between the two experiments with the exception of plasma chloride which remained significantly higher in the acidifying infusion experiment (Table 1).
Urine Hourly urine samples taken before, during and after vasopressin administration into sheep receiving either alkalinizing or acidifying infusions were analysed for hydrogen ion, calcium, magnesium, sodium, potassium, chloride, inorganic phosphate and osmolal concentrations. Alkalinizing infusions. The periods of high urine flow rate which preceded and followed vasopressin infusion were associated with positive values for free-water clearance (Fig. 5). Vasopressin administration caused a significant decline in urine flow rate (P < 000 1) and development of negative free-water clearance (P < 0-001). The mean rate of osmolal clearance declined slightly throughout the experiment and this pattern was not altered significantly by vasopressin infusion. No consistent changes in either inulin clearance or PAH clearance were observed throughout the experiment (Fig. 5). Urinary pH (Fig. 1) was higher during vasopressin infusion than during either the pre- or the post-vasopressin periods (P < 0-001) whereas urinary hydrogen ion excretion was lower at this time (P < 0.001). The rate of
ADH AND URINE pH ON Ca AND Mg EXCRETION 231 calcium excretion during the periods of diuresis was reasonably stable within experiments but varied between experiments from 0-5 to 5-3 ,tmole/min. Calcium excretion (Fig. 2) always decreased markedly during vasopressin administration (P < 0.001). Urinary magnesium excretion (Fig. 2) rose in some experiments during vasopressin infusion and was always decreased during the post-vasopressin period to values well below the pre-vasopressin excretion rates (P < 0-001). The mean 400)
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rates of excretion and clearance of sodium, potassium and chloride and the rate of excretion of phosphate fell throughout the period of urine collection. The infusion of vasopressin did not cause any significant alterations in the rates of excretion or clearance of these electrolytes (Fig. 6). Acidifying infusion. During the periods of high urine flow before and after vasopressin administration the sheep had positive free-water clearances (Fig. 5). Infusion
232 A. M. BEAL of vasopressin depressed urine flow rate significantly (P < 0 001) and caused the animals to have negative free water clearance values (P < 0-001). Mean osmolal clearance was reasonably stable throughout the experiment and was not altered by vasopressin. No significant changes in inulin clearance or PAH clearance (Fig. 5) Alkaline infusion
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2
45
were observed throughout the duration of the experiment. Urinary pH (Fig. 3) during vasopressin administration was lower than that of urine produced during the pre- and post-vasopressin periods (P < 0 001 and P < 0.01) and hydrogen ion excretion rate was lowest during vasopressin infusion (P < 0.01).
ADH AND URINE pH ON Ca AND Mg EXCRETION 233 Although large differences in the rate of calcium excretion were found between experiments (range 1-9-12-3 ,umole/min) the mean rate of calcium excretion was reasonably constant throughout the period of urine collection (Fig. 4). Any changes in calcium excretion during vasopressin administration were usually small and varied in direction. Magnesium excretion rate was increased significantly during TABLE 2. Comparison of renal electrolyte concentrations excretion rates and clearance rates of sheep receiving alkalinizing infusions to those of sheep receiving acidifying infusions using analysis of variance of the ra-w data and analysis of veriance of the data after each variable had been expressed as a ratio of the mean diuretic value for that variable. Urine collections were made before, during and after vasopressin (ADH) administration. Every renal variable on Figs. 1-6 was tested and omission of a comparison from this table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 0-02; ** = P < 0-01; *** = P < 0001; - = not significant) Clearance
During AB)H
Before ADH
Variable (d.f. = 22) Potassium excretion Potassium clearance Chloride excretion Chloride clearance Phosphate excretion Calcium excretion Calcium excretion/inulin clearance Hydrogen ion concentration Hydrogen ion excretion
period After ADH 2-1 2 1 2 2
1 2 Analysis of variance ** **
**
**
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** *
**
-
-
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Analysis of variance of ratios
Potassium excretion Potassium clearance Chloride excretion Chloride clearance Calcium excretion Calcium excretion/inulin clearance Hydrogen ion concentration Hydrogen ion excretion
-
-
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vasopressin infusion (P < 0.01). The rates of excretion and clearance of chloride and the excretion of phosphate fell slightly during the course of the experiment whereas the excretion rates and clearances of sodium and potassium had not decreased by the end of the experiment (Fig. 6). Infusion of vasopressin did not cause any statistically significant alteration in the rates of excretion or clearances of these electrolytes. Comparison of alkaline and acid experiments. Using analysis of variance no significant differences were found between the two types of experiment in the rates of urine flow, magnesium excretion, sodium excretion and clearance, osmolal and free-water clearances, inulin clearance and PAH clearance. Throughout the period of urine collection, urine hydrogen ion concentration and excretion rate, calcium
A. M. BEAL 234 excretion rate and chloride excretion and clearance rates were significantly higher during the acidifying infusion than during the alkalinizing infusion (Table 2). Urine potassium excretion and clearance rates were higher during vasopressin administration and phosphate excretion was higher following vasopressin administration into sheep with alkaline urine. The urine data were also subjected to analysis of variance after each urinary parameter had been expressed as a ratio of the mean value for that parameter during the periods of diuresis. Under these conditions the differences in hydrogen ion concentration and excretion rate, calcium excretion rate and potassium excretion and clearance rates between the alkaline and acid experiments remained statistically significant during vasopressin administration only (Table 2). DISCUSSION
Plasma calcium concentrations of sheep receiving the acid infusion were significantly higher than those of sheep given the alkaline infusate. Since this difference existed before the alkalinizing or the acidifying infusions were given (Figs. 2 and 4; Table 1), it cannot be attributed to these infusions. During the acidifying infusion, renal calcium and chloride excretions were higher and potassium excretion was lower than those of the alkalinizing experiment. The higher rate of calcium excretion throughout the acidifying experiment presumably stems from higher plasma calcium concentrations and from the calciuretic effect of acidosis and acid urines. The differences in chloride and potassium excretions can be attributed to differences in acid/base balance since elevation of plasma and urinary hydrogen ion concentrations depresses potassium secretion into the tubular fluid and stimulates bicarbonate reabsorption from it (Brenner & Berliner, 1973). As predicted the hydrogen ion concentration of the urine was lowered when tubular water reabsorption was stimulated by vasopressin administration into sheep with alkaline urine and elevated during antidiuresis in sheep with acid urine. During antidiuresis the fall in hydrogen ion concentration in alkaline urines was always associated with a marked fall in urinary calcium excretion whereas the increase in hydrogen ion concentrations of acid urines was not associated with any consistent alteration in calcium excretion. Vasopressin increases water reabsorption in the collecting duct. It also acts on rat distal tubules (Wirz, 1956; Gottschalk & Mylle, 1959; Darmandy, Durant, Matthews & Stranack, 1960) but not on the distal tubules of dog, monkey and Meriones (Clapp & Robinson, 1966; Bennett, Brenner & Berliner, 1968; Rouffignac, Lechene, Guinnebault & Morel, 1969). Vasopressin does not alter the water permeability of the proximal tubule (Clapp, Watson & Berliner, 1963; Gertz, Kennedy & Ullrich, 1964; Ullrich, Rumrich & Fuchs, 1964) or the loop of Henle of dogs or rats (Wirz, 1956; Gottschalk, 1961; Morgan & Berliner, 1968). Thus the region of the sheep nephron where vasopressin alters the pH of tubular fluid must be the collecting duct and possibly the late distal tubule. If urinary calcium excretion during antidiuresis depends on changes in tubular pH, the site at which calcium reabsorption has been modified by vasopressin must coincide with these distal segments of the nephron. The fact that the rate of sodium excretion in both alkaline and acid experiments showed no consistent change
ADH AND URINE pH ON Ca AND Mg EXCRETION 235 during vasopressin infusion may also indicate modification of calcium reabsorption at a distal site. In the proximal tubule and loop of Henle, the tubular fluid/plasma concentration ratios of sodium and calcium are similar which indicates parallel
reabsorption of the two elements whereas in the distal tubule and collecting duct, sodium and calcium reabsorptions are dissociated (Sutton & Dirks, 1975). Estimation of the calcium filtration rate was not attempted in the experiments for two reasons. It was not possible to measure calcium in the glomerular filtrate directly as the animals were conscious. Also the errors inherent in the calculation of calcium filtration rate using ultrafilterable calcium measured in an artificial membrane system and glomerular filtration rate measured by inulin clearance would be much larger than the observed changes in calcium excretion. However, it was noted that vasopressin administration was not associated with any consistent changes in inulin clearance in either the acid or the alkaline urine experiments; that during vasopressin administration plasma calcium concentrations fell significantly in the acid experiment but not in the alkaline experiment; and that in both experiments, vasopressin caused dilution of the plasma without any changes in hydrogen ion concentration which would favour increased ionization of calcium. These observations suggest that alterations in the rate of calcium filtration were unlikely to be the major cause of the fall of calcium excretion found during vasopressin infusion into sheep with alkaline urine. At low urine flow rates, the concentration of citrate, phosphate, sulphate and other anions which form complexes with calcium are increased in the collecting duct and, by reducing the amount of calcium available for reabsorption, should increase calcium excretion (Walser, 1961). In sheep with alkaline urine, vasopressin infusion caused a fall in calcium excretion under conditions which should have caused the greatest degree of association of calcium with anions (high concentration and high pH). Thus increased formation of complexed calcium was not a major influence on reabsorption in the collecting duct. Parathyroid hormone appears to enhance calcium reabsorption in the distal segments of the nephron (Buerkert, Marcus & Jamison, 1972; Agus, Chiu & Goldberg, 1975; Sutton, Wong & Dirks, 1976). However, increased parathyroid hormone levels were unlikely to have been the cause of the vasopressin-induced fall in calcium excretion by sheep with alkaline urine for two reasons. (1) Calcium excretion was not reduced by vasopressin infusion into sheep with acid urine although these sheep suffered essentially the same changes in plasma electrolyte concentrations. (2) In most experiments magnesium excretion rose during vasopressin administration which also indicates that increased parathyroid hormone levels did not cause the fall in calcium excretion because infusion of parathyroid hormone into sheep causes a fall in renal magnesium excretion as well as calcium excretion (Clark, French, Beal, Cross & Budtz-Olsen, 1975). The original hypothesis predicted that calcium excretion would increase during vasopressin administration into sheep with acid urine but no difference in excretion rate was observed between diuresis and antidiuresis for this condition. This can be explained by postulating that calcium reabsorption in the collecting duct varies with tubular hydrogen ion concentration over a limited range of hydrogen ion concentrations close to that of extracellular fluid. Thus, if the hydrogen ion concentration
A. M..RA BEAL 236 A of diuretic urine exceeds the range which influences calcium reabsorption, further increases in hydrogen ion concentration during vasopressin administration will not alter calcium excretion. Attempts to test this postulate by reducing the rate of acid loading were unsuccessful. Because sheep reabsorb most of the filtered phosphate their urine is poorly buffered at acid pH values near pH 7 and consequently, it is extremely difficult to maintain urine pH in this range. In manl vasopressin administration increased renal excretion of magnesium as well as calcium (Nielsen, 1961) which led this author to suggest that these two ions might share a common transport mechanism. In sheep, vasopressin usually increased magnesium excretion regardless of whether the urine was acid or alkaline or whether calcium excretion fell or was unaltered. These observations do not suggest any linkage of calcium and magnesium transport in the distal nephron. These experiments on sheep provide support for the hypothesis that vasopressin alters calcium excretion by causing changes in tubular hydrogen ion concentration (or possibly, tubular bicarbonate concentration). All previously published data for the effect of vasopressin on renal calcium excretion in sheep are consistent with and can be explained by this hypothesis. 236
I am indebted to Mr P. V. Burrow for skilful technical assistance.
REFERENCES AGUS, Z. S., CHIU, P. J. S. & GOLDBERG, M. (1975). Role of the terminal nephron in regulation of urine calcium and sodium excretion: Site of action of volume expansion and parathyroid hormone. Clin. Res. 23, 428A. BAGINSKI, E. S., FOA, P. P. & ZAK, B. (1967). Microdetermination of inorganic phosphate, phospholipids and total phosphate in biologic materials. Clin. Chem. 13, 326-332. BEAL, A. M., BUDTZ-OLSEN, 0. E., CLARK, R. C., CROSS, R. B. & FRENCH, T. J. (1973). Renal and salivary responses to infusion of potassium chloride, bicarbonate and phosphate in Merino sheep. Q. Ji exp. Physiol. 58, 251-265. BEAL, A. M., BUDTZ-OLSEN, 0. E., CLARK, R. C., CROSS, R. B. & FRENCH, T. J. (1974). Renal function and salivary potassium secretion during potassium chloride infusion into sodiumdeficient sheep. Q. Ji exp. Physiol. 59, 141-151. BEAL, A. M. (1976). The effect of vasopressin on renal calcium excretion; a possible explanation. J. Physiol. 263, 253-254P. BEAL, A. M., CLARK, R. C., CROSS, R. B. & FRENCH, T. J. (1976). The effect of vasopressin upon the excretion of calcium by the sheep. Q. Ji exp. Physiol. 61, 121-125. BENNETT, C. M., BRENNER, B. M. & BERLINER, R. W. (1968). Micropuncture study of nephron function in the Rhesus monkey. J. clin. Invest. 47, 203-216. BRATTON, A. C. & MARSHALL, E. K. (1939). A new coupling component for sulphanilamide determination. J. biol. Chem. 128, 537-550. BRENNER, B. M. & BERLINER, R. W. (1973). The transport of potassium. In Handbook of Physiology; section 8, Renal Physiology, ed. ORLOFF, J. & BERLINER, R. W., pp. 514-515. Washington: American Physiological Society. BUERKERT, J., MARCUS, D. & JAMISON, R. L. (1972). Renal tubule calcium reabsorption after parathyroidectomy. J. cdin. Invest. 51, 17a. CLAPP, J. R., WATSON, J. F. & BERLINER, R. W. (1963). Osmolality, bicarbonate concentration and water reabsorption in proximal tubule of the dog nephron. Am. J. Physiol. 205, 273280. CLAPP, J. R. & ROBINSON, R. R. (1966). Osmolality of distal tubular fluid in the dog. J. clin. Invest. 45, 1847-1853.
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CLARK, R. C., FRENCH, T. J., BEAL, A. M., CROSS, R. B. & BUDTZ-OLSEN, 0. E. (1975). The acute effects of intravenous infusion of parathyroid hormone on urine, plasma and saliva in the sheep. Q. Ji exp. Physiol. 60, 95-106. DARMADY, E. M., DURRANT, J., MATTHEWS, E. R. & STRANACK, F. (1960). Location of 131I Pitressin in the kidney by autoradiography. Clin. Sci. 19, 229-241. DAWBORN, J. K. (1965). Application of Heyrovsky's inulin method to automatic analysis. Clinica Chim. Acta 12, 63-66. DICKER, S. E. & EGC.LETON, M. G. (1961). Renal excretion of hyaluronidase and calcium in man during the anti-diuretic action of vasopressin and some analogues. J. Physiol. 157, 351-361. FARQUHARSON, R. F., SALTER, WV. T., TIBBETTS, D. M. & AUB, J. C. (1931). Studies of calcium and phosphorus metabolism. XII. The effect of the ingestion of acid-producing substances. J. din. Imnest. 10, 221-249. GERTZ, K. H., KENNEDY, G. C. & ULLRICH, K. J. (1964). Mikropunction-untersuchungen uber die Flussigkeitsriickresorption aus den einzelnen Tabulusabschnitten bei Wasserdiurese (diabetes insipidus). Pfliigers Arch. ges. Physiol. 278, 513-519. GOTTSCHALK, C. W. (1961). Micropuncture studies of tubular function in the mammalian kidney. Physiologist, Waash. 4, 35-55. GOTTSCHALK, C. WV. & MYLLE, M. (1959). Micropuncture study of the mammalian urinary concentration mechanism: evidence for the countercurrent hypothesis. Am. J. Physiol. 196, 927-936. HARVEY, R. D. & BROTHERS, A. J. (1962). Renal extraction of para-aminohippurate and creatiiune measured by continuous in vit'o sampling of arterial and renal vein blood. Ann. N.Y. Acad. Sci. 102, 46-54. HEYROVSKY, A. (1956). A new method for the determination of inulin and creatinine. Clinica Chim. Acta 1, 470-474. KUHN, E. (1966). Influence de l'antidiurese obtenue par infusion de l'arginine-vasopressine (AVP) de la lysine-vasopressine (LVP) et de l'ocytocine sur l'exeretion du calcium chez la brebis. Archs int. Pharmacodyn. Their. 160, 480-484. LAMB, A. R. & EVVARD, J. Al. (1919). The acid-base balance in animal nutrition. II. Metabolism studies on the effects of certain organic and mineral acids on swine. J. biol. Chem. 37, 329342. LEMANN, J., LITZOwN, J. R. & LENNON, E. J. (1966). The effects of chronic acid loads in normal man: Further evidence for the participation of bone mineral in the defence against chronic metabolic acidosis. J. clin. Invest. 45, 1608-1614. MORGAN, T. & BERLINER, R. WV. (1968). Permeability of the loop of Henle, vasa recta and collecting duct to water, urea and sodium. Am. J. Physiol. 215, 108-115. NIELSEN, B. (1964). Correlation between antidiuretic hormone effect and the renal excretion of magnesium and calcium in man. Acta endocr. 45, 151-160. REIDENBERG, M. M. & SEV-Y, R. XV. (1967). Mechanism of calciuria in acidosis in intact and thyroparathyroidectomized rats. Fedn Proc. 26, 368. ROUFFIG.NAC, C. D. E., LECHENE, C., GUINNEBAULT, M. & MOREL, F. (1969). Etude par microponction de l'e'laboration de l'urine. III Chez le me'rion non diure'tique et en diure'se par le mannitol. Nephron 6, 643--666. SELDINGER, S. I. (1953). Catheter replacement of the needle in percutaneous arteriography. Acta radiologica 39, 368-376. SUTTON, R. A. L. & DIRKS, J. H. (1975). The renal excretion of calcium: a review of micropuncture data. Can. J. Physiol. Pharmacol. 53, 979-988. SUTTON, R. A. L., WONG, N. L. M. & DIRKS, J. H. (1975). The hypercalciuria of metabolic acidosis - a specific impairment of distal calcium reabsorption. Clin. Res. 23, 434A. SUTTON, R. A. L., WoONG, N. L. M. & DIRKS, J. H. (1976). Effects of parathyroid hormone on sodium and calcium transport in the dog niephron. Clin. Sci. 51, 345-351. THORN, N. A. (1960). An effect of antidiuretic hormone on renal excretion of calcium in dogs. Dan. med. Bull. 7, 110-113. THORN, N. A. (1961). Correlation between antidiuretic hormone effect and changes in renal excretion of calcium in rats and dogs. Acta endocr. 38, 563-570. ULLRICH, K. J., RUMRICH, G. & FUCHS, G. (1964). W\asserpermeabilitat und transtubularer
238
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Wasserfluss corticaler Nephronabschnitte bei verschiedener Diuresezustanden. Pfluiger8 Arch ge8. Phy8iol. 280, 99-119. WALSER, M. (1961). Calcium clearance as a function of sodium clearance in the dog. Am. J. Phy8iol. 200, 1099-1104. WILLIAMSON, B. J. & FREEMAN, S. (1957). Effect of acute changes in acid-base balance on renal calcium excretion in dogs. Am. J. Phy8iol. 191, 384-387. WIRZ, H. (1956). Der osmotische Druck in den corticalen Tubuli der Ratteniere. Helv. phyjiol. pharmac. Acta 14, 353-362.
223
RENAL CALCIUM AND MAGNESIUM EXCRETION DURING VASOPRESSIN ADMINISTRATION INTO SHEEP WITH ACID OR ALKALINE URINE
BY A. M. BEAL* From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 28 November 1978) SUMMARY
1. The proposition that changes in renal calcium excretion during vasopressin administration are positively correlated with concurrent changes in urine hydrogen ion concentration was tested by administration of vasopressin into twelve conscious diuresing sheep receiving either alkalinizing or acidifying infusions. 2. Vasopressin-induced antidiuresis in sheep with alkaline urine was associated with significant increases in urinary pH and decreases in the rate of calcium excretion whereas antidiuresis in sheep with acid urine was associated with significant decreases in urinary pH and no consistent effect on calcium excretion. 3. Magnesium excretion increased during vasopressin administration in most experiments regardless of urinary pH changes. 4. Vasopressin administration did not significantly alter the rates of excretion of sodium, potassium, chloride and phosphate or the rates of sodium, potassium, chloride, inulin, para-aminohippurate and osmolal clearance in sheep with either acid or alkaline urine. Potassium excretion and clearance in sheep with alkaline urine was higher than that of sheep with acid urine during vasopressin infusion. 5. The results support the hypothesis that changes in renal tubular hydrogen ion concentration or bicarbonate concentration caused by water reabsorption from the collecting duct and possibly the late distal tubule could be part of the explanation for changes in renal calcium excretion which occur during vasopressin-induced antidiuresis. INTRODUCTION
In 1960, Thorn reported that intravenous injection of lysine-vasopressin into hydrated dogs caused an increase in the renal excretion of calcium which was correlated with the onset and duration of the antidiuretic effect of the injection. Subsequently increased calcium excretion during antidiuresis, induced by administration of lysine and arginine vasopressins, oxytocin and synthetic analogues of these hormones, was found to occur in hydrated rat, dog and man (Thorn, 1961; Dicker & Eggleton, 1961; Nielsen, 1964) although in both rat and man increased * Present address: University of New South Wales, School of Zoology, P.O. Box 1, Kensington, New South Wales, 2033.
0022-3751/79/4880-0872 $01.50 © 1979 The Physiological Society
224
A. M. BEAL
calcium excretion failed to occur in some experiments with vasopressin (Thorn, 1961; Nielsen, 1964). When this phenomenon was investigated in water-loaded sheep using hormone dose rates closer to 'physiological' than those of the previous authors, Kuhn (1966) found that antidiuresis produced by the administration of vasopressin or oxytocin was always associated with significant decreases in renal calcium excretion whereas Beal, Clark, Cross & French (1976) reported significant decreases in excretion after vasopressin only in sheep with calcium excretion rates less than 1 #smole/min. At higher rates of calcium excretion, vasopressin infusion did not alter calcium excretion although antidiuresis still occurred. Because of these reported differences in vasopressin's action on calcium excretion any explanation of the effect should attempt to accommodate this variability of response. Acidosis is associated with increased renal calcium excretion (Lamb & Evvard, 1919; Farquharson, Salter, Tibbetts & Aub, 1931; Williamson & Freeman, 1957; Reidenbert & Sevy, 1967). This calciuria occurs partly as a result of impaired reabsorption of calcium in the renal tubule (Lemann, Litzow & Lennon, 1966) particularly in the distal regions of the nephron (Sutton, Wong & Dirks, 1975). In acidotic dogs calcium reabsorption was restored to normal by administration of sodium bicarbonate to correct the acidosis and the fractional excretion of calcium reduced when bicarbonate was given to non-acidotic dogs (Sutton et al. 1975) which suggests that bicarbonate concentrations or more probably hydrogen ion concentrations are a determinant of the rate of calcium reabsorption in the distal segments of the nephron. Further evidence of the dependence of calcium reabsorption on tubular pH was provided by the observation that elevated urinary pH during hyperkalaemia caused by infusion of the neutral salt, potassium chloride, was accompanied by depressed renal calcium excretion in the sheep (Beal, Budtz-Olsen, Clark, Cross & French, 1973, 1974). Thus the rate of renal calcium excretion may partially depend on localized changes in pH in the renal tubule. Since vasopressin increases water reabsorption from the distal nephron without causing proportionate increases in reabsorption of most tubular solutes, the hydrogen ion concentration of the reabsorbate will tend to move towards that of distilled water (100 n-mole/l.). Thus the concentrating effect of vasopressin in the collecting duct region and possibly in the late distal tubule of some species should result in alkaline tubular fluid becoming more alkaline and acid tubular fluid becoming more acid. Under these conditions, vasopressin administration should enhance renal calcium excretion when the urine is acid and depress calcium excretion when the urine is alkaline. The following series of experiments were performed to test the hypothesis. A preliminary account of this work has been published (Beal, 1976). METHODS
Experimental procedures The experiments were performed on four Merino-crossbred ewes (39-5-46-5 kg) and eight Clun Forest ewes (36*0-57*0 kg), each of which had one carotid artery exteriorized in a loop of skin. Two sheep of each breed were splenectomized. The sheep were maintained on a diet of hay chaff (1000 g/day) and had unrestricted access to water and to a mineral salt supplement. During the afternoon before experiment, a vinyl cannula (1.4 mm i.d., 2-0 mm o.d.; Portex
ADH AND URINE pH ON Ca AND Mg EXCRETION
225
Limited) was inserted under local anaesthesia into one jugular vein using the technique of Seldinger (1953), a disposable plastic cannula (Braunula, size 0-5; Armour Pharmaceutical Company Limited) was introduced into the exteriorized carotid artery and the urinary bladder was catheterized with a short 14 F.G. catheter (Foley, 5 ml. balloon; Warne Surgical Products). At 18.00 hr any uneaten food and the mineral supplement were withdrawn, a blood sample (control 1) was taken and an infusion of either an acidifying solution (2.5 mM-CaCl2, 1 mM-MgCl2, 3 5 mm-KCl, 1 or 10 mn-HCl, 91 or 100 mm-NaCl; -220 m-osmole/l.) or an alkalinizing solution (2-5 mM-CaCl2, 1 mM-MgCl2, 3.5 mM-KCl, 30 mM-Na lactate, 15 or 30 mM-NaHCO3, 41 or 56 mM-NaCl; _ 220 m-osmole/l.) was commenced at 3 ml./min into the jugular cannula. Fifteen hours later the sheep were transferred to and restrained on canvas stretchers in a normal upright position with their feet just off the floor. After taking a blood sample (control 2) from the carotid artery, the overnight acidifying or alkalinizing infusate was replaced with a solution of similar composition but incorporating inulin (10 g/l.) and sodium paraaminohippurate (5 g/l.) with the sodium chloride content being reduced to maintain the osmolarity at 220 mosmole/l. A priming injection of 50 ml. of the inulin/PAH solution was given intravenously and thereafter this solution was infused at 3 ml./min to the end of the experiment. When the sheep had received a further 2 hr infusion, two serial 1 hr collections of diuretic urine under paraffin oil were made. Arginine vasopressin was then infused intravenously at 250 Fuu./min and after a 30 min transition period two serial 1 hr collections of antidiuretic urine were obtained. Vasopressin administration was then terminated and when the water diuresis recommenced, 45-90 min later, a further two serial 1 hr samples of diuretic urine were collected. Blood samples (10 ml.) were taken in syringes heparinized with 1 drop of heparin (5000 i.u./ml.) at the beginning and end of each hourly urine collection. Alkaline urine samples for use in calcium, magnesium and phosphate determinations were acidified with glacial acetic acid to prevent precipitation.
Analytical procedures The pH of urine and arterial blood was estimated immediately after collection under anaerobic conditions at 39 'C using thermostated Radiometer micro-electrodes. Microhaerratocrit determinations were made in triplicate within 15 min of sampling on blood spun at 12,000 g for 10 min in a microhaematocrit centrifuge (Hawksley). The remainder of each blood sample was centrifuged in glass tubes at 2750 g for 15 min to obtain plasma for analysis. Duplicate estimations of sodium and potassium in urine and plasma were made simultaneously by emission flame photometry in an oxygen-propane flame (Autotechnicon) with 15 m-mole/l. lithium as an internal reference and using mixed standards of sodium and potassium in the appropriate ranges to correct for mutual interference. Calcium and magnesium in urine and plasma were estimated in duplicate by atomic absorption in an air-acetylene flame (Pyelinicam SP- 191) using disodium ethylenediaminetetraacetic acid to overcome suppression of absorption by phosphate and protein. Total inorganic phosphate concentrations in urine and plasma were estimated in duplicate by the colorimetric method of Paginski, Foa & Zak (1967) adapted for automated analysis on the Technicon autoanalyser. The chloride concentrations of urine and plasma were measured in duplicate using a Radiometer chloride titrator (model CMT 10). The osmolality of urine and plasma was estimated in duplicate by freezing-point depression using a Fiske osmometer. Duplicate estimations of inulin in urine and plasma were made by the calorimetric method of Heyrovsky (1956) adapted to the Technicon autoanalyser by Dawborn (1965). PAH was measured in duplicate by the method of Bratton & Marshall (1939) as adapted for the Technicon autoanalyser by Harvey & Brothers (1962). Statistical procedures The differences in plasma electrolyte concentration and in urinary water and electrolyte excretion between the period of vasopressin infusion and the periods of diuresis both before and after vasopressin infusion were tested for statistical significance by t test of differences between correlated means (paired t test). The data for corresponding time periods of the alkaline and acid experiments were then compared by analysis of variance. To ensure that no effect of vasopressin was obscured by differences in electrolyte status which had been present before 8
PHYY 294
A. M. BEAL
226
or had occurred during the alkalinizing or acidifying infusions the data were also analysed after being adjusted to eliminate these differences by use of the following procedures. Blood and plasma data were subjected to analysis of covariance using the values for each variable in the blood samples taken at 17 hr (control 1) and 2 hr (control 2) before urine collection as covariates. Urinary data were subjected to analysis of variance after being expressed as ratios of the mean value of each variable for the 4 hr of diuresis. Since the use of a large number of statistical tests is likely to increase the frequency of erroneous rejection of the null hypothesis no result was considered to be statistically significant unless the probability level was less the 0-02.
RESULTS
The alkalinizing infusate contained either 15 or 30 mM-sodium bicarbonate and the acidifying infusate contained either 1 or 10 mm hydrochloric acid. As there were no significant differences between the results obtained with the two strengths of alkalinizing solution, the data for the two levels of alkalinizing infusion have been ADH
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ADH AND URINE pH ON Ca AND Mg EXCRETION 227 grouped together and are presented as combined mean results of the twelve alkalinizing experiments. Similarly there were no differences between the results for the two strengths of acidifying solution and the data for the acidifying infusions are presented as the mean results of the twelve acidifying experiments. ADH
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Blood The haematocrit, plasma osmolality and the plasma concentrations of hydrion, calcium, magnesium, sodium, potassium, chloride and inorganic phosphate were estimated for arterial blood samples taken before, during and after vasopressin administration into sheep receiving alkalinizing or acidifying infusions (Figs. 1-6). Alkalinizing infusion. Blood pH and plasma calcium concentration of sheep receiving the alkalinizing infusion were reasonably stable throughout the period of urine collection (Figs. 1 and 2) whereas haematocrit, plasma osmolality and plasma 8-2
A. M. BEAL 228 magnesium, sodium, potassium, chloride and phosphate concentrations fell during vasopressin infusion (Figs. 5 and 6). By the end of vasopressin administration the concentrations of magnesium, sodium and phosphate in the plasma and plasma osmolality were significantly less than the mean plasma concentrations during the 2 hr before vasopressin was given (magnesium, P < 0001; sodium, P < 0001; ADH
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phosphate, P < 0-001; osmolality, P < 0-01). After termination of vasopressin infusion the haematocrit, plasma osmolality and plasma concentrations of magnesium, sodium, potassium and chloride increased so that, by the end of the experiment, only plasma phosphate was significantly depressed below pre-vasopressin levels (P < 0-001). Acidifying infusion. When the sheep were given the acidifying infusion, blood pH fell slightly throughout the period of urine collection (Fig. 3). This change in plasma hydrogen ion concentration was not statistically significant. During vaso-
ADH AND URINE pH ON Ca AND Mg EXCRETION 229 pressin administration the haematocrit, plasma osmolality and plasma calcium, magnesium, sodium, potassium, chloride and phosphate were decreased below prevasopressin levels (Figs. 4, 5 and 6). At the end of the vasopressin infusion, all of the above parameters except plasma potassium and chloride were significantly reduced below the mean values for blood taken during the 2 hr before vasopressin ADH
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infusion (haematocrit, P < 0-01; calcium, P < 0-01; magnesium, P < 0-001; sodium, P < 0-01; phosphate, P < 0-001; osmolality, P < 0.001). After termination of vasopressin infusion the haematocrit, plasma osmolality and plasma electrolyte concentrations increased so that only the phosphate concentration remained significantly below pre-vasopressin values by the end of the experiment (P < 0-01). Comparison of alkaline and acid experiments. Analysis of variance of corresponding
A. M. BEAL
230
blood samples from the two experiments showed that the sheep had significantly higher plasma hydrogen ion, calcium and chloride concentrations during the acidifying infusions than during the alkalinizing infusions (Table 1). Other possible differences in plasma electrolyte concentrations were not statistically significant. When the data was adjusted by analysis of covariance to eliminate differences in electrolyte concentration which had occurred before urine collection was comTABLE 1. Comparison of the electrolyte concentrations in plasma samples from sheep receiving alkalinizing infusions to those of corresponding plasma samples from sheep receiving acidifying infusions using analysis of variance and analysis of covariance techniques. Plasma samples were taken before, during and after vasopressin (A.DH) administration. Every plasma variable on Figs. 1-6 was tested and omission of a comparison from this Table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 002; ** - P < 0-01; *** = P < 0-001; - = not significant)
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Urine Hourly urine samples taken before, during and after vasopressin administration into sheep receiving either alkalinizing or acidifying infusions were analysed for hydrogen ion, calcium, magnesium, sodium, potassium, chloride, inorganic phosphate and osmolal concentrations. Alkalinizing infusions. The periods of high urine flow rate which preceded and followed vasopressin infusion were associated with positive values for free-water clearance (Fig. 5). Vasopressin administration caused a significant decline in urine flow rate (P < 000 1) and development of negative free-water clearance (P < 0-001). The mean rate of osmolal clearance declined slightly throughout the experiment and this pattern was not altered significantly by vasopressin infusion. No consistent changes in either inulin clearance or PAH clearance were observed throughout the experiment (Fig. 5). Urinary pH (Fig. 1) was higher during vasopressin infusion than during either the pre- or the post-vasopressin periods (P < 0-001) whereas urinary hydrogen ion excretion was lower at this time (P < 0.001). The rate of
ADH AND URINE pH ON Ca AND Mg EXCRETION 231 calcium excretion during the periods of diuresis was reasonably stable within experiments but varied between experiments from 0-5 to 5-3 ,tmole/min. Calcium excretion (Fig. 2) always decreased markedly during vasopressin administration (P < 0.001). Urinary magnesium excretion (Fig. 2) rose in some experiments during vasopressin infusion and was always decreased during the post-vasopressin period to values well below the pre-vasopressin excretion rates (P < 0-001). The mean 400)
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rates of excretion and clearance of sodium, potassium and chloride and the rate of excretion of phosphate fell throughout the period of urine collection. The infusion of vasopressin did not cause any significant alterations in the rates of excretion or clearance of these electrolytes (Fig. 6). Acidifying infusion. During the periods of high urine flow before and after vasopressin administration the sheep had positive free-water clearances (Fig. 5). Infusion
232 A. M. BEAL of vasopressin depressed urine flow rate significantly (P < 0 001) and caused the animals to have negative free water clearance values (P < 0-001). Mean osmolal clearance was reasonably stable throughout the experiment and was not altered by vasopressin. No significant changes in inulin clearance or PAH clearance (Fig. 5) Alkaline infusion
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_
I 11I
I LI I III I I I I 75 -17-2 0 2 45 75 Time (hr) Fig. 6. Plasma sodium, potassium, chloride and phosphate concentrations and urinary sodium, potassium, chloride and phosphate excretion rates before, during and after I.v. administration of vasopressin at 250 jelu./min into sheep receiving either an alkalinizing or an acidifying infusion (n = 12; means + 5.E. of mean). -17-2 0
2
45
were observed throughout the duration of the experiment. Urinary pH (Fig. 3) during vasopressin administration was lower than that of urine produced during the pre- and post-vasopressin periods (P < 0 001 and P < 0.01) and hydrogen ion excretion rate was lowest during vasopressin infusion (P < 0.01).
ADH AND URINE pH ON Ca AND Mg EXCRETION 233 Although large differences in the rate of calcium excretion were found between experiments (range 1-9-12-3 ,umole/min) the mean rate of calcium excretion was reasonably constant throughout the period of urine collection (Fig. 4). Any changes in calcium excretion during vasopressin administration were usually small and varied in direction. Magnesium excretion rate was increased significantly during TABLE 2. Comparison of renal electrolyte concentrations excretion rates and clearance rates of sheep receiving alkalinizing infusions to those of sheep receiving acidifying infusions using analysis of variance of the ra-w data and analysis of veriance of the data after each variable had been expressed as a ratio of the mean diuretic value for that variable. Urine collections were made before, during and after vasopressin (ADH) administration. Every renal variable on Figs. 1-6 was tested and omission of a comparison from this table means that no significant differences were found. Differences are indicated as levels of significance (* = P < 0-02; ** = P < 0-01; *** = P < 0001; - = not significant) Clearance
During AB)H
Before ADH
Variable (d.f. = 22) Potassium excretion Potassium clearance Chloride excretion Chloride clearance Phosphate excretion Calcium excretion Calcium excretion/inulin clearance Hydrogen ion concentration Hydrogen ion excretion
period After ADH 2-1 2 1 2 2
1 2 Analysis of variance ** **
**
**
**
** *
**
-
-
-
**
**
**
**
Analysis of variance of ratios
Potassium excretion Potassium clearance Chloride excretion Chloride clearance Calcium excretion Calcium excretion/inulin clearance Hydrogen ion concentration Hydrogen ion excretion
-
-
~**
**
**
** _
***
_
**
**
**
**
*
vasopressin infusion (P < 0.01). The rates of excretion and clearance of chloride and the excretion of phosphate fell slightly during the course of the experiment whereas the excretion rates and clearances of sodium and potassium had not decreased by the end of the experiment (Fig. 6). Infusion of vasopressin did not cause any statistically significant alteration in the rates of excretion or clearances of these electrolytes. Comparison of alkaline and acid experiments. Using analysis of variance no significant differences were found between the two types of experiment in the rates of urine flow, magnesium excretion, sodium excretion and clearance, osmolal and free-water clearances, inulin clearance and PAH clearance. Throughout the period of urine collection, urine hydrogen ion concentration and excretion rate, calcium
A. M. BEAL 234 excretion rate and chloride excretion and clearance rates were significantly higher during the acidifying infusion than during the alkalinizing infusion (Table 2). Urine potassium excretion and clearance rates were higher during vasopressin administration and phosphate excretion was higher following vasopressin administration into sheep with alkaline urine. The urine data were also subjected to analysis of variance after each urinary parameter had been expressed as a ratio of the mean value for that parameter during the periods of diuresis. Under these conditions the differences in hydrogen ion concentration and excretion rate, calcium excretion rate and potassium excretion and clearance rates between the alkaline and acid experiments remained statistically significant during vasopressin administration only (Table 2). DISCUSSION
Plasma calcium concentrations of sheep receiving the acid infusion were significantly higher than those of sheep given the alkaline infusate. Since this difference existed before the alkalinizing or the acidifying infusions were given (Figs. 2 and 4; Table 1), it cannot be attributed to these infusions. During the acidifying infusion, renal calcium and chloride excretions were higher and potassium excretion was lower than those of the alkalinizing experiment. The higher rate of calcium excretion throughout the acidifying experiment presumably stems from higher plasma calcium concentrations and from the calciuretic effect of acidosis and acid urines. The differences in chloride and potassium excretions can be attributed to differences in acid/base balance since elevation of plasma and urinary hydrogen ion concentrations depresses potassium secretion into the tubular fluid and stimulates bicarbonate reabsorption from it (Brenner & Berliner, 1973). As predicted the hydrogen ion concentration of the urine was lowered when tubular water reabsorption was stimulated by vasopressin administration into sheep with alkaline urine and elevated during antidiuresis in sheep with acid urine. During antidiuresis the fall in hydrogen ion concentration in alkaline urines was always associated with a marked fall in urinary calcium excretion whereas the increase in hydrogen ion concentrations of acid urines was not associated with any consistent alteration in calcium excretion. Vasopressin increases water reabsorption in the collecting duct. It also acts on rat distal tubules (Wirz, 1956; Gottschalk & Mylle, 1959; Darmandy, Durant, Matthews & Stranack, 1960) but not on the distal tubules of dog, monkey and Meriones (Clapp & Robinson, 1966; Bennett, Brenner & Berliner, 1968; Rouffignac, Lechene, Guinnebault & Morel, 1969). Vasopressin does not alter the water permeability of the proximal tubule (Clapp, Watson & Berliner, 1963; Gertz, Kennedy & Ullrich, 1964; Ullrich, Rumrich & Fuchs, 1964) or the loop of Henle of dogs or rats (Wirz, 1956; Gottschalk, 1961; Morgan & Berliner, 1968). Thus the region of the sheep nephron where vasopressin alters the pH of tubular fluid must be the collecting duct and possibly the late distal tubule. If urinary calcium excretion during antidiuresis depends on changes in tubular pH, the site at which calcium reabsorption has been modified by vasopressin must coincide with these distal segments of the nephron. The fact that the rate of sodium excretion in both alkaline and acid experiments showed no consistent change
ADH AND URINE pH ON Ca AND Mg EXCRETION 235 during vasopressin infusion may also indicate modification of calcium reabsorption at a distal site. In the proximal tubule and loop of Henle, the tubular fluid/plasma concentration ratios of sodium and calcium are similar which indicates parallel
reabsorption of the two elements whereas in the distal tubule and collecting duct, sodium and calcium reabsorptions are dissociated (Sutton & Dirks, 1975). Estimation of the calcium filtration rate was not attempted in the experiments for two reasons. It was not possible to measure calcium in the glomerular filtrate directly as the animals were conscious. Also the errors inherent in the calculation of calcium filtration rate using ultrafilterable calcium measured in an artificial membrane system and glomerular filtration rate measured by inulin clearance would be much larger than the observed changes in calcium excretion. However, it was noted that vasopressin administration was not associated with any consistent changes in inulin clearance in either the acid or the alkaline urine experiments; that during vasopressin administration plasma calcium concentrations fell significantly in the acid experiment but not in the alkaline experiment; and that in both experiments, vasopressin caused dilution of the plasma without any changes in hydrogen ion concentration which would favour increased ionization of calcium. These observations suggest that alterations in the rate of calcium filtration were unlikely to be the major cause of the fall of calcium excretion found during vasopressin infusion into sheep with alkaline urine. At low urine flow rates, the concentration of citrate, phosphate, sulphate and other anions which form complexes with calcium are increased in the collecting duct and, by reducing the amount of calcium available for reabsorption, should increase calcium excretion (Walser, 1961). In sheep with alkaline urine, vasopressin infusion caused a fall in calcium excretion under conditions which should have caused the greatest degree of association of calcium with anions (high concentration and high pH). Thus increased formation of complexed calcium was not a major influence on reabsorption in the collecting duct. Parathyroid hormone appears to enhance calcium reabsorption in the distal segments of the nephron (Buerkert, Marcus & Jamison, 1972; Agus, Chiu & Goldberg, 1975; Sutton, Wong & Dirks, 1976). However, increased parathyroid hormone levels were unlikely to have been the cause of the vasopressin-induced fall in calcium excretion by sheep with alkaline urine for two reasons. (1) Calcium excretion was not reduced by vasopressin infusion into sheep with acid urine although these sheep suffered essentially the same changes in plasma electrolyte concentrations. (2) In most experiments magnesium excretion rose during vasopressin administration which also indicates that increased parathyroid hormone levels did not cause the fall in calcium excretion because infusion of parathyroid hormone into sheep causes a fall in renal magnesium excretion as well as calcium excretion (Clark, French, Beal, Cross & Budtz-Olsen, 1975). The original hypothesis predicted that calcium excretion would increase during vasopressin administration into sheep with acid urine but no difference in excretion rate was observed between diuresis and antidiuresis for this condition. This can be explained by postulating that calcium reabsorption in the collecting duct varies with tubular hydrogen ion concentration over a limited range of hydrogen ion concentrations close to that of extracellular fluid. Thus, if the hydrogen ion concentration
A. M..RA BEAL 236 A of diuretic urine exceeds the range which influences calcium reabsorption, further increases in hydrogen ion concentration during vasopressin administration will not alter calcium excretion. Attempts to test this postulate by reducing the rate of acid loading were unsuccessful. Because sheep reabsorb most of the filtered phosphate their urine is poorly buffered at acid pH values near pH 7 and consequently, it is extremely difficult to maintain urine pH in this range. In manl vasopressin administration increased renal excretion of magnesium as well as calcium (Nielsen, 1961) which led this author to suggest that these two ions might share a common transport mechanism. In sheep, vasopressin usually increased magnesium excretion regardless of whether the urine was acid or alkaline or whether calcium excretion fell or was unaltered. These observations do not suggest any linkage of calcium and magnesium transport in the distal nephron. These experiments on sheep provide support for the hypothesis that vasopressin alters calcium excretion by causing changes in tubular hydrogen ion concentration (or possibly, tubular bicarbonate concentration). All previously published data for the effect of vasopressin on renal calcium excretion in sheep are consistent with and can be explained by this hypothesis. 236
I am indebted to Mr P. V. Burrow for skilful technical assistance.
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