J. Physiol. (1975), 246, pp. 695-707 With 6 text-figtures Printed in Great Britain
695
THE RELATIONSHIP BETWEEN KALLIKREIN AND WATER EXCRETION AND THE CONDITIONAL RELATIONSHIP BETWEEN KALLIKREIN AND SODIUM EXCRETION
By IVOR H. MILLS AND P. E. WARD From the Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ
(Received 29 August 1974) SUMMABRY
1. The renal kallikrein-kinin system has previously been linked with renal control of sodium and water excretion. The present investigations were carried out to examine more closely these relationships. 2. In physiological studies with rabbits, urinary kallikrein was measured by a modification of the [3H]TAME method. 3. With rabbits on free sodium and water intake, urinary kallikrein was positively correlated with both sodium and water excretion. Kallikrein excretion was also negatively correlated with urinary osmolality. 4. In rabbits on chronic high and low sodium diets, urinary kallikrein was positively correlated with urinary volume but not with sodium excretion. 5. In rabbits held to a constant fluid intake but with sodium intake changed, urinary kallikrein was not correlated with sodium excretion. 6. These results indicate that the positive correlation of kallikrein excretion with sodium excretion under conditions of free sodium and water intake may be only secondary to the positive relationship of kallikrein excretion with urinary volume. 7. The results ofthe present investigations do not support the hypothesis that the renal kallikrein-kinin system is necessarily involved in renal control of sodium excretion under normal conditions but it is where a change in sodium intake leads to a change in fluid intake and consequently of urinary volume. 8. In the above experiments, urinary kallikrein was always positively correlated with urinary volume and negatively correlated with urinary osmolality. This may indicate a functional relationship between renal kallikrein and water excretion.
696
IVOR H. MILLS AND P. E. WARD INTRODUCTION
Urinary kallikrein is a proteolytic enzyme which releases the peptides kallidin and bradykinin from their alpha-2-globulin substrate in plasma (Webster & Pierce, 1963). It is biochemically indistinguishable from renal kallikrein (Nustad, 1970; Pierce & Nustad, 1972) which is located primarily in the cortex, but is different from plasma kallikrein (Webster & Pierce, 1961). Kallikrein excretion is thought to represent an active turnover of renal kallikrein. Several investigators have shown that the infusion of kinins into the renal artery produces a natriuresis (Webster & Gilmore, 1964; Barraclough & Mills, 1965; Gill, Melmon, Gillespie & Bartter, 1965; Willis, Ludens, Hook & Williamson, 1969; Stein, Congbalay, Karsh, Osgood & Ferris, 1972). Various workers have looked at kallikrein excretion in relation to the control of sodium and water excretion and have reported significant correlations of urinary kallikrein with both sodium excretion and urine volume in man (Adetuyibi & Mills, 1972), dogs (Marin-Grez, Cottone & Carretero, 1972) and rats (Marin-Grez & Carretero, 1971; Croxatto, Roblero, Garcia, Corthorn & San Martin, 1973). However, Geller, Margolius, Pisano, & Keiser (1972) have reported that kallikrein excretion is unrelated to sodium excretion and urinary volume and have suggested that kallikrein excretion is related to mineralocorticoid level. Escape from the effects of sodium-retaining steroidal hormones is associated with increased urinary kallikrein in patients (Abetuyibi & Mills, 1972; Edwards, Adetuyibi & Mills, 1973). Kallikrein excretion is elevated in deoxy-corticosterone acetate-salt hypertensive rats (Margolius, Geller, de Jong, Pisano & Sjoerdsma, 1972 a, b) and in patients with primary aldosteronism. Adetuyibi & Mills (1972) have shown that the excretion of a water load in patients is associated with a significant increase in urinary kallikrein while sodium excretion remained unchanged. In the present study we have looked at the relationship of urinary kallikrein with sodium and water excretion in rabbits. In view of the relationship of urinary kallikrein excretion with the excretion of a water load (Adetuyibi & Mills, 1972), we have specifically examined the relationship of urinary kallikrein to sodium excretion under conditions where changes in sodium excretion were unrelated to changes in urinary volume. METHODS
Rabbits used in the study were bred and reared within the department and housed in identical fashion. Twenty-four hour urine samples were collected in glass beakers from rabbits in metabolic cages and stored at 40 C until assayed. All samples were assayed within 20 days and sequential samples from any one rabbit were assayed at the same time. Urine samples from rabbits on high and low sodium diets were
KALLIKREIN, WATER AND Na EXCRETION
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collected after the animals had established sodium balance (a minimum of 2 weeks on their respective diets). Two rabbits were followed continuously for 17 days during which time their fluid intake was sequentially changed from 100 ml. 2 % saline to 100 ml. 1 % saline to 100 ml. water.
Diets Control rabbits were fed their normal rabbit feed ad lib. The low sodium diets were made with the commercial low sodium diet Edosol. Edosol contains 0-03 % by weight sodium chloride in powdered form. The final low salt diet contained 800 g Edosol, 200 g cellulose, 4 0 g vitamin supplement and sufficient distilled water to produce a paste. This was then dried to pellet form. Rabbits on the low salt diet received distilled water ad lib. Rabbits on the high salt diet received the same Eclosol mixture to which was added 5 0 g sodium chloride. In addition, rabbits on the high sodium diet received 1 % saline ad lib. The two rabbits monitored continuously for 17 days received their normal rabbit feed. Sodium, pota88ium and o8molality determinations Urinary osmolality was determined as a function of freezing point depression on an Advanced Osmometer (Advanced, Instruments Inc.). Sodium and potassium concentrations were determined on an Eppendorf flame photometer.
Urinacj kaflikrein assay Kallikrein excretion was determined by Dr N. A. A. Macfarlane's modification of the Beaven, Pierce & Pisano (1971) method using 3H-labelled p-tosyl-arginine methyl ester as a synthetic substrate (TAME). Each rabbit urine was incubated in the following manner. Reaction incubate. 200 ,ud. 0-2 M Tris-HCl, pH 8-5, 5 id. urine, and 50 1ud. [3H]TAME (30,000 d/min). Blank incubate. 200 gul. 0-2 M Tris-HCl, pH 8-5, 5 etl. urine, 50 gl. [3H]TAME (30,000 dlmin), and 100 gil. ethanol (00 C). The reaction was started with the addition of the [3H]TAME and immediate mixing with a vortex mixer. The incubate was then maintained at 370 C for 60 min in a water-bath. The reaction was then stopped by the addition of 100 ,l. ethanol (00 C) and placing the reaction tube on ice. The blank incubate was handled in exactly the same manner except that the ethanol was added at the same time as the [3H]TAME and placed immediately on ice. All column measurements and transfers were done with Hamilton precision syringes. During the 60 min incubation, Bio-rex 70 columns were prepared. Approximately 20 g Bio-rex 70 resin (200-400 mesh, sodium form) were washed five to seven times with approximately 200 ml. 0 5 M Tris-HCl buffer, pH 8f5. With each wash the resin was allowed to settle out partially and the supernatant buffer and 'fines' from the resin were removed by suction. After equilibration, 0 5 ml. of the resin-buffer slurry was injected with a 1 ml. syringe into a Pasteur pipette (14.5 x 0 5 cm) with a cotton plug. The ratio of resin to buffer was adjusted to give a final column height of about 1 cm. After the column had settled, about 10 ml. 0-2 M Tris-HCl buffer, pH 8-5 was washed through the column. Columns were used within 30 min and were discarded after being used once. After the reaction incubate had been put on ice, 250 gzl. of the reaction and blank incubates were applied to individual Bio-rex columns and washed successively through with 100, 200 and 100 1ld. 0-2 M Tris-HCl buffer, pH 8-5. The eluants were collected directly into counting vials to which was added 10 ml. Unisolve. After
698
IVOR H. MILLS AND P. E. WARD
mixing for 5 sec on a vortex mixer, the vials were counted overnight (10 min/sample) in a Packard Tri-Carb Liquid Scintillation Spectrometer. The amount of [3H]methanol released through the action of urinary kallikrein was computed as: kallikrein activity d/min = incubate d/min - blank d/min spontaneous hydrolysis d/min. Spontaneous hydrolysis was determined by incubating all components except the urine for 60 min and then treating as with the reaction incubate. Kallikrein activity is expressed as kallikrein esterase units (E.U.) per 24 hr urine. One esterase unit is equivalent to 4-8 /%mole tosyl-arginine-methyl ester hydrolysed per hour at 370 C at pH 8-5. Standard curves were prepared by incubating purified pancreatic kallikrein (Bayer; 218 E.U./mg) in place of urine in the assay just described. The amount of [3H]methanol released was plotted against the esterase units incubated. RESULTS
The degree of reproducibility of the [3H]TAME technique was determined by assaying six aliquots from a pooled urine sample. For the six samples the mean activity (E.U./ml.) was 0-33, the standard deviation was 00 1, the S.E. of the mean was 0-005 and the coefficient of variation was
4-4%. The effect of neat urine on the recovery of added kallikrein (Bayer; 218 E.U./mg) was determined by addition of a known amount of kallikrein to neat urine and comparing the activity of the added kallikrein alone, the urine alone and the kallikrein-urine mixture. The recovery of added kallikrein was determined by the equation:
U2-U1 U3 where U1 is the enzyme activity of the urine sample alone, U2 is the activity in the kallikrein-urine mixture and U3 is the activity of the kallikrein added. Recovery of added kallikrein activity was approximately 90 %. Differences in urine osmolality and ion concentration did not affect the recovery. Up to 40 % hydrolysis of substrate, the release of [3H]methanol was directly proportional to the incubation time and to the amount of urine added to the incubate. Soya bean trypsin inhibitor (SETI) is a potent inhibitor of plasma kallikrein, trypsin and plasmin, but not of urinary kallikrein (Back & Steger, 1968). In the present experiments, rabbit urine esterase hydrolysis was only slightly inhibited (- 10 %) by SBTI (Sigma; 10 jag per 5 jpl. urine). The percentage inhibition did riot significantly vary between different urine samples. Free sodium and water intake Thirty-one 24 hour urine samples were collected from fifteen rabbits on a free salt (i.e. free food) and water intake. The mean excretion (+ s.E.)
KALLIKREIN, WATER AND Na EXCRETION 699 of sodium, kallikrein and urine volume were respectively: 7-3 + 0 55 mequiv/day, 19-6 + 1P8 E.U./day and 78 + 6-0 ml./day. A significant positive correlation was found between kallikrein excretion and urinary volume (Fig. 1, r = 0-838, P < 0.001) and between urinary kallikrein and sodium excretion (Fig. 2, r = 0 577, P < 0.001). Additionally, a significant negative correlation (Fig. 3, r -00 574, P < 0 001) was found between urinary kallikrein and urinary osmolality.
High and low sodium intake With rabbits on a free sodium and water intake, sodium excretion was highly correlated with urinary volume (r = 0-673, P < 0.001) in the above group. Conversely, in fifteen rabbits on a constant high (n = 9) or low (n = 6) sodium diet (see Methods) of at least 2 weeks duration, no significant correlation existed between sodium excretion and urinary volume (r = 0.136) for the group as a whole (n = 15). Mean sodium excretion for rabbits on the high salt diet was 19-9 + 4-8 m-equiv/day while for those on the low salt diet it was 0-18 + 0-02 m-equiv/day. Mean urinary volume for these same rabbits was 146 + 16 ml. for the high salt diet rabbits and 182+26 ml. for the rabbits on the low salt diet. However it must be remembered that the rabbits on the high salt diet also had 1 % saline to drink so that an increase in fluid intake also caused an increase in sodium intake. Under these conditions, the group as a whole showed no significant correlation between urinary kallikrein and sodium excretion (r = 0.243). The correlation between urinary kallikrein and urinary volume remained significant (Fig. 4; r = 0-689, P < 0.01) as did the inverse correlation between urinary kallikrein and urinary osmolality (Fig. 5; r = -0k635, P < 0.05).
Constant fluid intake Table 1 shows the mean excretion (± S.E.) of sodium, potassium and kallikrein and urinary volume in the two rabbits which were given 2 % saline for 5 days followed by 1 % saline for 6 days and then tap water for 6 days with fluid intake kept constant at 100 ml./day. Plasma sodium, potassium and osmolality and body weight values for the three periods are shown in Table 2. Under these conditions ordinary volume fell significantly when the rabbits were changed from 1 % saline to water but the osmolality remained relatively constant (3152 + 193, 3221 + 156 and 3186 + 89 m-osmole/kg in the three periods) while sodium, potassium and the sodium/potassium ratio varied. In both animals the fall in the sodium/ potassium ratio accompanying a change from high to a lower sodium intake was complete within the first 24 hr collection period after each change.
700 IVOR H. MILLS AND P. E. WARD Urinary kallikrein was not related to sodium excretion either within or between the three stages of the experiment. Nevertheless, kallikrein excretion did show a significant positive correlation with daily urinary volume (Fig. 6; r = 0491, P < 0.01). so
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Fig. 3 Fig. 1. The fitted regression line for kallikrein excretion plotted against urinary volume in thirty-one collections of 24 hr urine from fifteen rabbits on free salt and water intake. Fig. 2. The fitted regression line for kallikrein excretion plotted against sodium excretion in thirty-one collections of 24 hr urine from fifteen rabbits on free salt and water intake. Fig. 3. The fitted regression line for kallikrein excretion plotted against urinary osmolality in thirty-one collections of 24 hr urine from fifteen rabbits on free salt and water intake.
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Fig. 4. The fitted regression line for kallikrein excretion plotted against urinary volume in fifteen collections of 24 hr urine from rabbits on low sodium diet (-) and high sodium diet (A) with free fluid intake. Fig. 5. The fitted regression line for kallikrein excretion plotted against urinary osmolality in fifteen collections of 24 hr urine from rabbits on low sodium diet (0) and high sodium -diet (A) with free fluid intake. TABLE 1. Mean (± S.E.) excretions in rabbits on constant 100 ml./day fluid intake.
Fluid intake 2% saline 1 % saline Water
Sodium Urinary volume (m-equiv/ day) (ml./day) 33-0+ 13 39.4+119 37 0 + 0-9 22-7 + 0.3*** 31-7 + 1.4** 11-5 + 0-4***
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*P < 0 05 **P < 0*01 ***P < 0*001 P is for the difference from the value in the line immediately above. TABLE 2. Mean levels (±s.E.) of plasma sodium (m-equiv 1.-i), potassium (mequiv 1.-i) and osmolality (m-osmole kg-1) and body weight (g) during three periods of sequentially lower sodium intake with fluid intake held constant
Fluid intake r
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Fig. 6. The fitted regression line for kallikrein excretion plotted against urinary volume in two rabbits held to a constant fluid intake but with sodium intake changed. DISCUSSION
The use of [3H]TAME as a substrate for the assay of urinary kallikrein has several advantages. The assay is quick and reproducible and does not require concentration of the urine. Beaven et al. (1971) have shown that the determination of urinary esterase activity by the [3H]TAME assay is highly correlated with titration and colorimetric assays of the esterase activity. In our own department the [3H]TAME assay has been shown to be highly correlated with the residual esterase assay of urinary kallikrein of Roberts (1958) (Adetuyibi, 1972). Esterase activity has been used in numerous studies as a measure of urinary kallikrein. This activity parallels kinin-generating activity (Pierce, 1970). The TAME esterase activity of urinary kallikrein is highly correlated with kinin formation as shown by radioimmunoassay (Macfarlane, Adetuyibi & Mills, 1973). Margolius, Pisano, Geller & Sjoerdsma (1971) and Margolius et al. (1972a) have shown that urinary hydrolysis of [3H]TAME is highly correlated with kinin formation as shown by bioassay. The positive correlation of urinary kallikrein excretion with sodium excretion and urinary volume in the control groups of rabbits in the present study is in agreement with the results ofprevious studies in man (Adetuvibi & Mills, 1972; Edwards et al. 1973), in dogs (Marin-Grez et al. 1972) and in rats (Marin-Grez & Carretero, 1971). Since the excretion of sodium was
KALLIKREIN, WATER AND Na EXCRETION 703 correlated with urinary volume, it left uncertain which factor was primarily related to kallikrein excretion. The positive correlation of kallikrein excretion with urinary volume was demonstrated in rabbits under conditions of free sodium and water intake (Fig. 1), sodium depletion and sodium load with free water intake (Fig. 4) and variable sodium intake with constant fluid intake (Fig. 6). Contrary to this, the positive relationship of kallikrein excretion to sodium excretion could only be shown in rabbits under conditions of free sodium and water intake (Fig. 2). Kallikrein excretion was not correlated with sodium excretion in rabbits on chronic high or low sodium intake nor with rabbits with variable sodium intake with fluid intake held constant (Table 1). It may be concluded that the positive correlation of kallikrein excretion with sodium excretion under conditions of free sodium and water intake is only secondary to the positive relationship of kallikrein excretion with urinary volume. During free sodium and water intake, sodium excretion is correlated with kallikrein excretion only when sodium excretion is correlated with urinary volume. This indicates that under conditions of free sodium and water intake, renal kallikrein may not be physiologically involved in the control of sodium excretion. Alternatively, kallikrein excretion may be involved in the control of sodium excretion only when sufficient fluid intake is available to allow for the expansion of some critical fluid volume. This could explain the lack of a change in kallikrein excretion in the two rabbits with variable sodium intake while fluid intake was held constant. However, if this were the case, the renal kinin system could not be involved in the control of sodium excretion where fluid intake was reduced. Stimulation of the renal kinin system can occur without fluid loading. Mills, Macfarlane & Ward (1974) and Macfarlane, Mills & Ward (1974) have shown that infusion of the polypeptide substance P results in increased kallikrein excretion in association with increased excretion of sodium and urinary volume. In these experiments, changes in fluid intake could not account for stimulation of the renal kinin system. Infusion of substance P in the rat has been shown to produce a marked reduction in the reabsorption of fluid from the proximal tubule (Arendshorst, Cook & Mills, 1974). Reabsorption of sodium and water in the proximal tubule occur together, so if the effect of substance P is due to the kallikrein released it would not be surprising that the urinary excretion of both sodium and water were related to kallikrein excretion. Decreased proximal tubular reabsorption also occurs during saline infusion (Dirks, Cirksena & Berliner, 1965) and during the escape from the sodium retaining effect of steroids (Wright, Knox, Howards & Berliner, 1969). In both these circumstances there is also an increased
704 IVOR H. MILLS AND P. E. WARD excretion of kallikrein (Marin-Grez et al. 1972; Adetuyibi & Mills, 1972; Marin-Grez, Oza & Carretero, 1973; Geller et al. 1972). Increased kallikrein excretion was also produced by infusion of natriuretic doses of angiotensin. This was associated with an increased excretion of sodium and urinary volume and a lowering of urinary osmolality (Macfarlane, Adettiyibi & Mills, 1974). These conclusions do not, however, rule out the renal kallikrein system as an important factor in the excretion of a saline load under conditions of reduced GFR and high mineralocorticoid activity (de Wardener, Mills, Clapham & Hayter, 1961; Mills, de Wardener, Hayter & Clapham, 1961). Saline infusion has been reported to lead to an increased excretion of kallikrein in the urine (Marin-Grez et al. 1972) but it is not known if this is a response to volume expansion or whether it is related to the sodium chloride content of the infusion. The renal kallikrein system may play a role in the excretion of sodium where mineralocorticoid level is high (low sodium/potassium ratio) or too slow to change to maintain sodium balance. These are the conditions under which the involvement of a factor other than GFR and aldosterone was first indicated as related to sodium excretion (de Wardener et al. 1961; Mills et al. 1961). Elevated kallikrein has been reported in patients escaping from the effects of a sodium retaining steroid (Adetuyibi & Mills, 1972; Edwards et al. 1973), in patients with primary hyper-aldosteronism and in deoxycorticosterone acetate-salt hypertensive rats (Margolius et al. 1972 a, b). Acute saline loading in dogs also resulted in increased kallikrein in renal lymph (de Bono & Mills, 1974). It is significant that in the two rabbits with varied sodium excretion unrelated to kallikrein excretion (Table 1), the absolute excretion of potassium rose significantly from 16 + 1 to 20 + 1 m-equiv/day and the sodium/potassium ratio fell from 2-1 to 0-6. This is compatible with a rising mineralocorticoid level and is in agreement with observations of Edwards et al. (1973) that changes in sodium excretion can occur without an associated change in urinary kallikrein when there is a change in mineralocorticoid level. Thus the evidence of the present study and others would indicate that renal kallikrein is probably not physiologically involved in the control of sodium excretion under conditions in which the levels of mineralocorticoid activity can change unless rapid blood volume expansion occurs but the relationship of urinary kallikrein to sodium excretion under conditions of high mineralocorticoid activity may have important physiological significance. However, the results of this study also demonstrate that in order to show a physiological relationship of kallikrein excretion to sodium excretion, such a relationship should be shown to be independent of changes in urinary volume. A simple positive correlation of kallikrein
705 KALLIKREIN, WATER AND Na EXCRETION excretion to sodium excretion may be secondary to the relationship to urinary volume. Several studies have shown kallikrein excretion to be correlated with sodium excretion in a manner that could not be accounted for by volume changes alone. It is significant that these studies involved either high mineralocorticoid level or acute saline loading. The rise in kallikrein excretion reported during acute saline loading in dogs (MarinGrez et al. 1972) was greater than could be accounted for by urinary volume changes alone. Until the mechanisms relating the renal kallikrein/kinin system to excretion of water and sodium are more precisely worked out it is not possible to be certain that the increased release of renal killikrein with increased sodium excretion (when it does occur, e.g. with arterial infusions of substance P or angiotensin) does not inevitably affect urinary osmolality and hence urinary volume. The positive correlation of kallikrein excretion with urinary volume and its negative correlation with osmolality suggests a role for the renal kallikrein/bradykinin system in the regulation of water excretion which is opposite to the action of vasopressin. Furtado (1971) has shown that bradykinin can inhibit the increase in water permeability brought about by vasopressin in the toad bladder. Barraclough & Mills (1965) showed that the unilateral lowering of urinary osmolality by infusion of bradykinin into one renal artery was not prevented by simultaneous infusion of vasopressin. Adetuyibi & Mills (1972) have shown a marked rise in kallikrein excretion in individuals given a water load. It seems, therefore, that the excretion of water is associated not only with a decrease in vasopressin secretion but also with an increase in renal kallikrein excretion. This work was supported by the National Kidney Research Fund. REFERENCES
ADETUYIBI, A. (1972). Some studies on kallikrein in urine. Ph.D. Thesis, University of Cambridge. ADETUYIBI, A. & MIlLS, I. H. (1972). Relation between urinary kallikrein and renal function, hypertension, and excretion of sodium and water in man. Lancet ii, 203-207. ARENDSHORST, W. J., COOK, MARGARET A. & MILLS, I. H. (1974). A micropuncture study of the natriuresis produced by arterial infusions of substance P in the rat. J. clin. Invest. 53, 2a. BACK, N. & STEGER, R. (1968). Effect of inhibitors on kinin-releasing activity of proteases. Fedn Proc. 27(1), 96-99. BARRACLOUGH, M. A. & MILLS, I. H. (1965). Effect of bradykinin on renal function. Clin. Sci. 28, 69-74. BEAVEN, V. H., PIERCE, J. V. & PISANO, J. J. (1971). A sensitive isotopic procedure for the assay of esterase activity: measurement of human urinary kallikrein. Clinic chin. Acta 32, 67-73.
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CROXATTO, H. R., ROBLERO, J., GARCIA, R., CORTHORN, J. & SAN MARTIN, M. L. (1973). Effect of furosemide upon urinary kallikrein excretion. Agents & Actions 3, 267-274. DE BONO, E. & MILLS, I. H. (1974). Simultaneous increases in kallikrein in renal lymph and urine during saline infusion. J. Physiol. 241, 127-128 P. 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, 249--258. DIRKS, J. H., CIRKSENA, W. J. & BERLINER, R. W. (1965). The effect of saline infusion on sodium reabsorption by the proximal tubule of the dog. J. cdin. Invest. 44, 1160-1170. EDWARDS, 0. M., ADETUYIBI, A. & MILLS, I. H. (1973). Kallikrein excretion during the 'escape' from the sodium retaining effect of fluorocortisone. J. Endocr. 59, xxxiv. FUIRTADO, M. R. F. (1971). Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: effects of bradykinin, kallidin, eledoisin, and physalaemin. J. Membrane Biol. 4, 165-178. GELLER, R. G., MARGOLIUS, H. S., PISANO, J. L. & KEISER, H. R. (1972). Effects of mineralocorticoids, altered sodium intake and adrenalectomy on urinary kallikrein in rats. Circulation Res. XXXI, 857-861. GILL, J. R., MELMON, K. L., GILLESPIE, L. Jr. & BARTTER, F. C. (1965). Bradykinin and renal function in normal man: effects of adrenergic blockade. Am. J. Physiol. 209, 844-848. MACFARLANE, N. A. A., ADETUYIBI, A. & MILLS, I. H. (1973). The radioimmunoassay of bradykinin in the measurement of kallikrein in urine. J. Endocr. 58, xxv. MACFARLANE, N. A. A., ADETUYIBI, A. & MILLS, I. H. (1974). Changes in kallikrein excretion during arterial infusion of angiotensin. J. Endocr. 61, lxxii. MAcFARLANE, N. A. A., MILLS, I. H. & WARD, P. E. (1974). The diuretic and natri. uretic effects of arterial infusions of substance P and their relationship to kallikrein excretion. J. Physiol. 239, 28-30 P. MARGOLIUS, H. S., GELLER, R. G., DE JONG, W., PISANO, J. J. & SJOERDSMA, A. (1972a). Altered urinary kallikrein excretion in rats with hypertension. Circulation Res. XXX, 358-263. MARGOLIUS, H. S., GELLER, R. G., DE JONG, W., PISANO, J. J. & SJOERDSMA, A. (1972 b). Urinary kallikrein excretion in hypertension. Suppl. II to Circulation Res. XXX and XXXI, 125-131. MARGOLIUS, H. S., PISANO, J. J., GELLER, R. G. & SJOERDSMA, A. (1971). Altered urinary kallikrein excretion in human hypertension. Lancet ii, 1063-1065. MARIN-GREZ, M. & CARRETERO, 0. A. (1971). Urinary kallikrein excretion in rats under low and high sodium intake. Physiologist, Wash. 14, 189. MARIN-GREZ, M., COTTONE, P. & CARRETERO, 0. A. (1972). Evidence for an involvement of kinins in regulation of sodium excretion. Am. J. Physiol. 223, 794-796. MARIN-GREZ, M., OZA, N. B. & CARRETERO, 0. A. (1973). The involvement of urinary kallikrein in the renal escape from the sodium retaining effect of mineralocorticoids. Henry Ford Hosp. Med. J. 21, 85-90. MILLS, I. H., MACFARLANE, N. A. A. & WARD, P. (1974). Increase in kallikrein excretion during the natriuresis produced by arterial infusion of substance P. Nature, Lond. 247, 108-109. MILLS, I. H., DE WARDENER, H. E., HAYTER, C. J. & CLAPHAM, W. F. (1961). Studies on the afferent mechanism of the sodium chloride diuresis which follows intravenous saline in the dog. Clin. Sci. 21, 259-264. NUSTAD, K. (1970). Relationship between kidney and urinary kininegenase. Br. J. Pharmac. 39, 73-86.
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PIERCE, J. V. (1970). Purification of mammalian kallikreins, kininogens and kinins. In Handbook of Experimental Pharmacology, vol. xxv, ed. ERDOS, E., p. 21. Berlin, Heidelberg, New York: Springer-Verlag. PIERCE, J. V. & NUSTAD, K. (1972). Purification of human and rat urinary kallikreins. Fedn Proc. 31, 623. ROBERTS, P. S. (1958). Measurement of the rate of plasmin action on synthetic substrates. J. biol. Chem. 232, 285. STEIN, J. H., CONGBALAY, R. C., KARSH, D. L., OSGOOD, R. W. & FERRIS, T. F. (1972). The effect of bradykinin on proximal tubular sodium reabsorption in the dog: evidence for functional nephron heterogeneity. J. cdin. Invest. 51, 1709-1721. WEBSTER, M. E. & GILMORE, J. P. (1964). Influence of kallidin-10 on renal function. Am. J. Physiol. 206, 714-718. WEBSTER, M. E. & PIERCE, J. V. (1961). Action of the kallikreins on synthetic ester substrates. Proc. Soc. exp. Biol. Med. 107, 186-191. WEBSTER, M. E. & PIERCE, J. V. (1963). Nature of the kallidins released from human plasma by kallikreins and other enzymes. Ann. N.Y. Acad. Sci. 104, 91-107. WILLIS, C. R., LUDENS, J. H., HooK, J. B. & WILLIAMSON, H. E. (1969). Mechanism of the natriuretic action of bradykinin. Am. J. Physiol. 217, 1-5. WRIGHT, F. S., KNOX, F. G., HOWARDS, S. S. & BERLINER, R. W. (1969). Reduced sodium reabsorption by the proximal tubule of DOCA-escaped dogs. Am. J. Physiol. 216, 869-875.
695
THE RELATIONSHIP BETWEEN KALLIKREIN AND WATER EXCRETION AND THE CONDITIONAL RELATIONSHIP BETWEEN KALLIKREIN AND SODIUM EXCRETION
By IVOR H. MILLS AND P. E. WARD From the Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ
(Received 29 August 1974) SUMMABRY
1. The renal kallikrein-kinin system has previously been linked with renal control of sodium and water excretion. The present investigations were carried out to examine more closely these relationships. 2. In physiological studies with rabbits, urinary kallikrein was measured by a modification of the [3H]TAME method. 3. With rabbits on free sodium and water intake, urinary kallikrein was positively correlated with both sodium and water excretion. Kallikrein excretion was also negatively correlated with urinary osmolality. 4. In rabbits on chronic high and low sodium diets, urinary kallikrein was positively correlated with urinary volume but not with sodium excretion. 5. In rabbits held to a constant fluid intake but with sodium intake changed, urinary kallikrein was not correlated with sodium excretion. 6. These results indicate that the positive correlation of kallikrein excretion with sodium excretion under conditions of free sodium and water intake may be only secondary to the positive relationship of kallikrein excretion with urinary volume. 7. The results ofthe present investigations do not support the hypothesis that the renal kallikrein-kinin system is necessarily involved in renal control of sodium excretion under normal conditions but it is where a change in sodium intake leads to a change in fluid intake and consequently of urinary volume. 8. In the above experiments, urinary kallikrein was always positively correlated with urinary volume and negatively correlated with urinary osmolality. This may indicate a functional relationship between renal kallikrein and water excretion.
696
IVOR H. MILLS AND P. E. WARD INTRODUCTION
Urinary kallikrein is a proteolytic enzyme which releases the peptides kallidin and bradykinin from their alpha-2-globulin substrate in plasma (Webster & Pierce, 1963). It is biochemically indistinguishable from renal kallikrein (Nustad, 1970; Pierce & Nustad, 1972) which is located primarily in the cortex, but is different from plasma kallikrein (Webster & Pierce, 1961). Kallikrein excretion is thought to represent an active turnover of renal kallikrein. Several investigators have shown that the infusion of kinins into the renal artery produces a natriuresis (Webster & Gilmore, 1964; Barraclough & Mills, 1965; Gill, Melmon, Gillespie & Bartter, 1965; Willis, Ludens, Hook & Williamson, 1969; Stein, Congbalay, Karsh, Osgood & Ferris, 1972). Various workers have looked at kallikrein excretion in relation to the control of sodium and water excretion and have reported significant correlations of urinary kallikrein with both sodium excretion and urine volume in man (Adetuyibi & Mills, 1972), dogs (Marin-Grez, Cottone & Carretero, 1972) and rats (Marin-Grez & Carretero, 1971; Croxatto, Roblero, Garcia, Corthorn & San Martin, 1973). However, Geller, Margolius, Pisano, & Keiser (1972) have reported that kallikrein excretion is unrelated to sodium excretion and urinary volume and have suggested that kallikrein excretion is related to mineralocorticoid level. Escape from the effects of sodium-retaining steroidal hormones is associated with increased urinary kallikrein in patients (Abetuyibi & Mills, 1972; Edwards, Adetuyibi & Mills, 1973). Kallikrein excretion is elevated in deoxy-corticosterone acetate-salt hypertensive rats (Margolius, Geller, de Jong, Pisano & Sjoerdsma, 1972 a, b) and in patients with primary aldosteronism. Adetuyibi & Mills (1972) have shown that the excretion of a water load in patients is associated with a significant increase in urinary kallikrein while sodium excretion remained unchanged. In the present study we have looked at the relationship of urinary kallikrein with sodium and water excretion in rabbits. In view of the relationship of urinary kallikrein excretion with the excretion of a water load (Adetuyibi & Mills, 1972), we have specifically examined the relationship of urinary kallikrein to sodium excretion under conditions where changes in sodium excretion were unrelated to changes in urinary volume. METHODS
Rabbits used in the study were bred and reared within the department and housed in identical fashion. Twenty-four hour urine samples were collected in glass beakers from rabbits in metabolic cages and stored at 40 C until assayed. All samples were assayed within 20 days and sequential samples from any one rabbit were assayed at the same time. Urine samples from rabbits on high and low sodium diets were
KALLIKREIN, WATER AND Na EXCRETION
697
collected after the animals had established sodium balance (a minimum of 2 weeks on their respective diets). Two rabbits were followed continuously for 17 days during which time their fluid intake was sequentially changed from 100 ml. 2 % saline to 100 ml. 1 % saline to 100 ml. water.
Diets Control rabbits were fed their normal rabbit feed ad lib. The low sodium diets were made with the commercial low sodium diet Edosol. Edosol contains 0-03 % by weight sodium chloride in powdered form. The final low salt diet contained 800 g Edosol, 200 g cellulose, 4 0 g vitamin supplement and sufficient distilled water to produce a paste. This was then dried to pellet form. Rabbits on the low salt diet received distilled water ad lib. Rabbits on the high salt diet received the same Eclosol mixture to which was added 5 0 g sodium chloride. In addition, rabbits on the high sodium diet received 1 % saline ad lib. The two rabbits monitored continuously for 17 days received their normal rabbit feed. Sodium, pota88ium and o8molality determinations Urinary osmolality was determined as a function of freezing point depression on an Advanced Osmometer (Advanced, Instruments Inc.). Sodium and potassium concentrations were determined on an Eppendorf flame photometer.
Urinacj kaflikrein assay Kallikrein excretion was determined by Dr N. A. A. Macfarlane's modification of the Beaven, Pierce & Pisano (1971) method using 3H-labelled p-tosyl-arginine methyl ester as a synthetic substrate (TAME). Each rabbit urine was incubated in the following manner. Reaction incubate. 200 ,ud. 0-2 M Tris-HCl, pH 8-5, 5 id. urine, and 50 1ud. [3H]TAME (30,000 d/min). Blank incubate. 200 gul. 0-2 M Tris-HCl, pH 8-5, 5 etl. urine, 50 gl. [3H]TAME (30,000 dlmin), and 100 gil. ethanol (00 C). The reaction was started with the addition of the [3H]TAME and immediate mixing with a vortex mixer. The incubate was then maintained at 370 C for 60 min in a water-bath. The reaction was then stopped by the addition of 100 ,l. ethanol (00 C) and placing the reaction tube on ice. The blank incubate was handled in exactly the same manner except that the ethanol was added at the same time as the [3H]TAME and placed immediately on ice. All column measurements and transfers were done with Hamilton precision syringes. During the 60 min incubation, Bio-rex 70 columns were prepared. Approximately 20 g Bio-rex 70 resin (200-400 mesh, sodium form) were washed five to seven times with approximately 200 ml. 0 5 M Tris-HCl buffer, pH 8f5. With each wash the resin was allowed to settle out partially and the supernatant buffer and 'fines' from the resin were removed by suction. After equilibration, 0 5 ml. of the resin-buffer slurry was injected with a 1 ml. syringe into a Pasteur pipette (14.5 x 0 5 cm) with a cotton plug. The ratio of resin to buffer was adjusted to give a final column height of about 1 cm. After the column had settled, about 10 ml. 0-2 M Tris-HCl buffer, pH 8-5 was washed through the column. Columns were used within 30 min and were discarded after being used once. After the reaction incubate had been put on ice, 250 gzl. of the reaction and blank incubates were applied to individual Bio-rex columns and washed successively through with 100, 200 and 100 1ld. 0-2 M Tris-HCl buffer, pH 8-5. The eluants were collected directly into counting vials to which was added 10 ml. Unisolve. After
698
IVOR H. MILLS AND P. E. WARD
mixing for 5 sec on a vortex mixer, the vials were counted overnight (10 min/sample) in a Packard Tri-Carb Liquid Scintillation Spectrometer. The amount of [3H]methanol released through the action of urinary kallikrein was computed as: kallikrein activity d/min = incubate d/min - blank d/min spontaneous hydrolysis d/min. Spontaneous hydrolysis was determined by incubating all components except the urine for 60 min and then treating as with the reaction incubate. Kallikrein activity is expressed as kallikrein esterase units (E.U.) per 24 hr urine. One esterase unit is equivalent to 4-8 /%mole tosyl-arginine-methyl ester hydrolysed per hour at 370 C at pH 8-5. Standard curves were prepared by incubating purified pancreatic kallikrein (Bayer; 218 E.U./mg) in place of urine in the assay just described. The amount of [3H]methanol released was plotted against the esterase units incubated. RESULTS
The degree of reproducibility of the [3H]TAME technique was determined by assaying six aliquots from a pooled urine sample. For the six samples the mean activity (E.U./ml.) was 0-33, the standard deviation was 00 1, the S.E. of the mean was 0-005 and the coefficient of variation was
4-4%. The effect of neat urine on the recovery of added kallikrein (Bayer; 218 E.U./mg) was determined by addition of a known amount of kallikrein to neat urine and comparing the activity of the added kallikrein alone, the urine alone and the kallikrein-urine mixture. The recovery of added kallikrein was determined by the equation:
U2-U1 U3 where U1 is the enzyme activity of the urine sample alone, U2 is the activity in the kallikrein-urine mixture and U3 is the activity of the kallikrein added. Recovery of added kallikrein activity was approximately 90 %. Differences in urine osmolality and ion concentration did not affect the recovery. Up to 40 % hydrolysis of substrate, the release of [3H]methanol was directly proportional to the incubation time and to the amount of urine added to the incubate. Soya bean trypsin inhibitor (SETI) is a potent inhibitor of plasma kallikrein, trypsin and plasmin, but not of urinary kallikrein (Back & Steger, 1968). In the present experiments, rabbit urine esterase hydrolysis was only slightly inhibited (- 10 %) by SBTI (Sigma; 10 jag per 5 jpl. urine). The percentage inhibition did riot significantly vary between different urine samples. Free sodium and water intake Thirty-one 24 hour urine samples were collected from fifteen rabbits on a free salt (i.e. free food) and water intake. The mean excretion (+ s.E.)
KALLIKREIN, WATER AND Na EXCRETION 699 of sodium, kallikrein and urine volume were respectively: 7-3 + 0 55 mequiv/day, 19-6 + 1P8 E.U./day and 78 + 6-0 ml./day. A significant positive correlation was found between kallikrein excretion and urinary volume (Fig. 1, r = 0-838, P < 0.001) and between urinary kallikrein and sodium excretion (Fig. 2, r = 0 577, P < 0.001). Additionally, a significant negative correlation (Fig. 3, r -00 574, P < 0 001) was found between urinary kallikrein and urinary osmolality.
High and low sodium intake With rabbits on a free sodium and water intake, sodium excretion was highly correlated with urinary volume (r = 0-673, P < 0.001) in the above group. Conversely, in fifteen rabbits on a constant high (n = 9) or low (n = 6) sodium diet (see Methods) of at least 2 weeks duration, no significant correlation existed between sodium excretion and urinary volume (r = 0.136) for the group as a whole (n = 15). Mean sodium excretion for rabbits on the high salt diet was 19-9 + 4-8 m-equiv/day while for those on the low salt diet it was 0-18 + 0-02 m-equiv/day. Mean urinary volume for these same rabbits was 146 + 16 ml. for the high salt diet rabbits and 182+26 ml. for the rabbits on the low salt diet. However it must be remembered that the rabbits on the high salt diet also had 1 % saline to drink so that an increase in fluid intake also caused an increase in sodium intake. Under these conditions, the group as a whole showed no significant correlation between urinary kallikrein and sodium excretion (r = 0.243). The correlation between urinary kallikrein and urinary volume remained significant (Fig. 4; r = 0-689, P < 0.01) as did the inverse correlation between urinary kallikrein and urinary osmolality (Fig. 5; r = -0k635, P < 0.05).
Constant fluid intake Table 1 shows the mean excretion (± S.E.) of sodium, potassium and kallikrein and urinary volume in the two rabbits which were given 2 % saline for 5 days followed by 1 % saline for 6 days and then tap water for 6 days with fluid intake kept constant at 100 ml./day. Plasma sodium, potassium and osmolality and body weight values for the three periods are shown in Table 2. Under these conditions ordinary volume fell significantly when the rabbits were changed from 1 % saline to water but the osmolality remained relatively constant (3152 + 193, 3221 + 156 and 3186 + 89 m-osmole/kg in the three periods) while sodium, potassium and the sodium/potassium ratio varied. In both animals the fall in the sodium/ potassium ratio accompanying a change from high to a lower sodium intake was complete within the first 24 hr collection period after each change.
700 IVOR H. MILLS AND P. E. WARD Urinary kallikrein was not related to sodium excretion either within or between the three stages of the experiment. Nevertheless, kallikrein excretion did show a significant positive correlation with daily urinary volume (Fig. 6; r = 0491, P < 0.01). so
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Fig. 4. The fitted regression line for kallikrein excretion plotted against urinary volume in fifteen collections of 24 hr urine from rabbits on low sodium diet (-) and high sodium diet (A) with free fluid intake. Fig. 5. The fitted regression line for kallikrein excretion plotted against urinary osmolality in fifteen collections of 24 hr urine from rabbits on low sodium diet (0) and high sodium -diet (A) with free fluid intake. TABLE 1. Mean (± S.E.) excretions in rabbits on constant 100 ml./day fluid intake.
Fluid intake 2% saline 1 % saline Water
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*P < 0 05 **P < 0*01 ***P < 0*001 P is for the difference from the value in the line immediately above. TABLE 2. Mean levels (±s.E.) of plasma sodium (m-equiv 1.-i), potassium (mequiv 1.-i) and osmolality (m-osmole kg-1) and body weight (g) during three periods of sequentially lower sodium intake with fluid intake held constant
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The use of [3H]TAME as a substrate for the assay of urinary kallikrein has several advantages. The assay is quick and reproducible and does not require concentration of the urine. Beaven et al. (1971) have shown that the determination of urinary esterase activity by the [3H]TAME assay is highly correlated with titration and colorimetric assays of the esterase activity. In our own department the [3H]TAME assay has been shown to be highly correlated with the residual esterase assay of urinary kallikrein of Roberts (1958) (Adetuyibi, 1972). Esterase activity has been used in numerous studies as a measure of urinary kallikrein. This activity parallels kinin-generating activity (Pierce, 1970). The TAME esterase activity of urinary kallikrein is highly correlated with kinin formation as shown by radioimmunoassay (Macfarlane, Adetuyibi & Mills, 1973). Margolius, Pisano, Geller & Sjoerdsma (1971) and Margolius et al. (1972a) have shown that urinary hydrolysis of [3H]TAME is highly correlated with kinin formation as shown by bioassay. The positive correlation of urinary kallikrein excretion with sodium excretion and urinary volume in the control groups of rabbits in the present study is in agreement with the results ofprevious studies in man (Adetuvibi & Mills, 1972; Edwards et al. 1973), in dogs (Marin-Grez et al. 1972) and in rats (Marin-Grez & Carretero, 1971). Since the excretion of sodium was
KALLIKREIN, WATER AND Na EXCRETION 703 correlated with urinary volume, it left uncertain which factor was primarily related to kallikrein excretion. The positive correlation of kallikrein excretion with urinary volume was demonstrated in rabbits under conditions of free sodium and water intake (Fig. 1), sodium depletion and sodium load with free water intake (Fig. 4) and variable sodium intake with constant fluid intake (Fig. 6). Contrary to this, the positive relationship of kallikrein excretion to sodium excretion could only be shown in rabbits under conditions of free sodium and water intake (Fig. 2). Kallikrein excretion was not correlated with sodium excretion in rabbits on chronic high or low sodium intake nor with rabbits with variable sodium intake with fluid intake held constant (Table 1). It may be concluded that the positive correlation of kallikrein excretion with sodium excretion under conditions of free sodium and water intake is only secondary to the positive relationship of kallikrein excretion with urinary volume. During free sodium and water intake, sodium excretion is correlated with kallikrein excretion only when sodium excretion is correlated with urinary volume. This indicates that under conditions of free sodium and water intake, renal kallikrein may not be physiologically involved in the control of sodium excretion. Alternatively, kallikrein excretion may be involved in the control of sodium excretion only when sufficient fluid intake is available to allow for the expansion of some critical fluid volume. This could explain the lack of a change in kallikrein excretion in the two rabbits with variable sodium intake while fluid intake was held constant. However, if this were the case, the renal kinin system could not be involved in the control of sodium excretion where fluid intake was reduced. Stimulation of the renal kinin system can occur without fluid loading. Mills, Macfarlane & Ward (1974) and Macfarlane, Mills & Ward (1974) have shown that infusion of the polypeptide substance P results in increased kallikrein excretion in association with increased excretion of sodium and urinary volume. In these experiments, changes in fluid intake could not account for stimulation of the renal kinin system. Infusion of substance P in the rat has been shown to produce a marked reduction in the reabsorption of fluid from the proximal tubule (Arendshorst, Cook & Mills, 1974). Reabsorption of sodium and water in the proximal tubule occur together, so if the effect of substance P is due to the kallikrein released it would not be surprising that the urinary excretion of both sodium and water were related to kallikrein excretion. Decreased proximal tubular reabsorption also occurs during saline infusion (Dirks, Cirksena & Berliner, 1965) and during the escape from the sodium retaining effect of steroids (Wright, Knox, Howards & Berliner, 1969). In both these circumstances there is also an increased
704 IVOR H. MILLS AND P. E. WARD excretion of kallikrein (Marin-Grez et al. 1972; Adetuyibi & Mills, 1972; Marin-Grez, Oza & Carretero, 1973; Geller et al. 1972). Increased kallikrein excretion was also produced by infusion of natriuretic doses of angiotensin. This was associated with an increased excretion of sodium and urinary volume and a lowering of urinary osmolality (Macfarlane, Adettiyibi & Mills, 1974). These conclusions do not, however, rule out the renal kallikrein system as an important factor in the excretion of a saline load under conditions of reduced GFR and high mineralocorticoid activity (de Wardener, Mills, Clapham & Hayter, 1961; Mills, de Wardener, Hayter & Clapham, 1961). Saline infusion has been reported to lead to an increased excretion of kallikrein in the urine (Marin-Grez et al. 1972) but it is not known if this is a response to volume expansion or whether it is related to the sodium chloride content of the infusion. The renal kallikrein system may play a role in the excretion of sodium where mineralocorticoid level is high (low sodium/potassium ratio) or too slow to change to maintain sodium balance. These are the conditions under which the involvement of a factor other than GFR and aldosterone was first indicated as related to sodium excretion (de Wardener et al. 1961; Mills et al. 1961). Elevated kallikrein has been reported in patients escaping from the effects of a sodium retaining steroid (Adetuyibi & Mills, 1972; Edwards et al. 1973), in patients with primary hyper-aldosteronism and in deoxycorticosterone acetate-salt hypertensive rats (Margolius et al. 1972 a, b). Acute saline loading in dogs also resulted in increased kallikrein in renal lymph (de Bono & Mills, 1974). It is significant that in the two rabbits with varied sodium excretion unrelated to kallikrein excretion (Table 1), the absolute excretion of potassium rose significantly from 16 + 1 to 20 + 1 m-equiv/day and the sodium/potassium ratio fell from 2-1 to 0-6. This is compatible with a rising mineralocorticoid level and is in agreement with observations of Edwards et al. (1973) that changes in sodium excretion can occur without an associated change in urinary kallikrein when there is a change in mineralocorticoid level. Thus the evidence of the present study and others would indicate that renal kallikrein is probably not physiologically involved in the control of sodium excretion under conditions in which the levels of mineralocorticoid activity can change unless rapid blood volume expansion occurs but the relationship of urinary kallikrein to sodium excretion under conditions of high mineralocorticoid activity may have important physiological significance. However, the results of this study also demonstrate that in order to show a physiological relationship of kallikrein excretion to sodium excretion, such a relationship should be shown to be independent of changes in urinary volume. A simple positive correlation of kallikrein
705 KALLIKREIN, WATER AND Na EXCRETION excretion to sodium excretion may be secondary to the relationship to urinary volume. Several studies have shown kallikrein excretion to be correlated with sodium excretion in a manner that could not be accounted for by volume changes alone. It is significant that these studies involved either high mineralocorticoid level or acute saline loading. The rise in kallikrein excretion reported during acute saline loading in dogs (MarinGrez et al. 1972) was greater than could be accounted for by urinary volume changes alone. Until the mechanisms relating the renal kallikrein/kinin system to excretion of water and sodium are more precisely worked out it is not possible to be certain that the increased release of renal killikrein with increased sodium excretion (when it does occur, e.g. with arterial infusions of substance P or angiotensin) does not inevitably affect urinary osmolality and hence urinary volume. The positive correlation of kallikrein excretion with urinary volume and its negative correlation with osmolality suggests a role for the renal kallikrein/bradykinin system in the regulation of water excretion which is opposite to the action of vasopressin. Furtado (1971) has shown that bradykinin can inhibit the increase in water permeability brought about by vasopressin in the toad bladder. Barraclough & Mills (1965) showed that the unilateral lowering of urinary osmolality by infusion of bradykinin into one renal artery was not prevented by simultaneous infusion of vasopressin. Adetuyibi & Mills (1972) have shown a marked rise in kallikrein excretion in individuals given a water load. It seems, therefore, that the excretion of water is associated not only with a decrease in vasopressin secretion but also with an increase in renal kallikrein excretion. This work was supported by the National Kidney Research Fund. REFERENCES
ADETUYIBI, A. (1972). Some studies on kallikrein in urine. Ph.D. Thesis, University of Cambridge. ADETUYIBI, A. & MIlLS, I. H. (1972). Relation between urinary kallikrein and renal function, hypertension, and excretion of sodium and water in man. Lancet ii, 203-207. ARENDSHORST, W. J., COOK, MARGARET A. & MILLS, I. H. (1974). A micropuncture study of the natriuresis produced by arterial infusions of substance P in the rat. J. clin. Invest. 53, 2a. BACK, N. & STEGER, R. (1968). Effect of inhibitors on kinin-releasing activity of proteases. Fedn Proc. 27(1), 96-99. BARRACLOUGH, M. A. & MILLS, I. H. (1965). Effect of bradykinin on renal function. Clin. Sci. 28, 69-74. BEAVEN, V. H., PIERCE, J. V. & PISANO, J. J. (1971). A sensitive isotopic procedure for the assay of esterase activity: measurement of human urinary kallikrein. Clinic chin. Acta 32, 67-73.
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