University of Edinburgh.)

Transcription

1 THE ACTION OF DIURETICS. BY ARTHUR R. CUSHNY AND C. G. LAMBIE. (From the Pharmacological Laboratory, University of Edinburgh.) IN the study of the action of diuretics', the first question to determine is whether the drug acts directly on the kidney itself or indirectly through changes in the blood or in the circulation. The changes in the renal circulation cannot be satisfactorily determined by taking the carotid bloodpressure since the local circulation is influenced by the local vasomotor mechanism; nor can it be determined by the use of an oncometer unless the secretion rate remains constant. Direct measurement of the renal blood-flow, introduced by B arcroft and Brodie, appears to be the only method which can be employed in the study of diuresis, if we except the difficult experiments of Richards and Plant on the excised kidney maintained in apparently normal activity by perfusion. In the method of B arc r o f t and B ro di e the renal circulation remains intact, but at intervals the renal venous blood is diverted downwards into the lower vena cava and escapes into a measuring vessel which permits the amount of blood passing in a given time to be measured. The blood is turned into the measuring pipette by placing a small clamp on the vena cava above the renal vessels and removing one below them. We have followed their method in principle, but have modified their method of collecting the blood in estimating the rate of flow. We may mention that the blood cannot be collected in an ordinary pipette for this purpose since the narrow bore offers too great resistance to the flow and it is difficult to keep the inclination of the pipette uniform in successive measurements. The measuring cannula we have used is represented in Fig. 1. The internal diameter was 10 mm. tapering to 3-5 mm.; it had a side tube, S, which in use was clamped in a vertical position, and was closed at its end by the cork C. The cannula, apart from the side tube, usually contained 3.5 c.c.; it was paraffined inside and generally washed out with leech extract; sometimes leech extract was injected intravenously. After 1 The work on diuresis has been discussed in detail by the senior author in his monograph on The Secretion of the Urine, 1917, so that it need not be given here.

2 ACTION OF DIURETICS. an estimation had been made, the blood was blown back into the vena cava through a rubber tube attached to the side tube. The application and removal of ordinary clamps disturbed the position of the vessels, and in addition was difficult to time exactly, and this was avoided by the use of clamps devised by Mr N. E. Condon of this A 277 Fig. I. Fig. 2. Fig. 1. Cannula for vena cava. In use the side tube S was fixed vertically, and not as in the figure. A, B position of the clamps on the vena cava. Fig. 2. Condon's pneumatic clamps. laboratory (Fig. 2). Each of these consists of a tube in which a piston is moved up and down by means of a small rubber air cushion which is inflated by pressure on a larger bulb. At the opposite end the piston carries a small plate which forms one jaw of the clamp, the other jaw being attached to the end of the tube. In A, inflation of the air cushion

3 278 A. R. CUSHNY AND C. G. LAMBIE. depresses the piston and closes the clamp. In B, the inflation raises the piston and opens the clamp. When the air cushion is not inflated, the piston is kept in a fixed position by an indiarubber band, b, which raises the piston in A and depresses it in B. Both clamps were placed on the vena cava, A above and B below the entrance of the left renal vein. The time taken to divert the blood-flow, fill the cannula and drive back the blood into the vein was usually less than half a minute. Rabbits of 2-3 kg. body weight, anaesthetised with urethane 1-5 g. per kg., were employed. Cannulae were placed in the trachea, jugular vein (for injection) and carotid artery, and the normal blood-pressure was recorded. The abdomen was then opened and a cannula placed in the bladder. The suptrior mesenteric artery was tied and the whole of the intestine was ligatured en masse and removed. A small clamp was placed temporarily on the vena cava below the left renal vein and the aorta was ligatured at the same point. All the branches of the vena cava were tied for 2-3 cm. below the renal vein and for 1 cm. above it except the suprarenals; the suprarenal blood was thus measured along with the renal, but the error is insignificant. The measuring cannula was now inserted in the vena cava and Condon's, clamps were put on and carefully adjusted so as to avoid disturbing the alignment of the vein. During the operation the blood-pressure had often fallen considerably, and in this case, it was brought back to the normal by Bayliss' gum-ringer solution. The urine secretion was recorded by an attendant who marked the fall of each drop by an electric signal. Several observations of the normal rate of renal blood-flow were now made, one of us compressing the bulbs of the clamps and the other taking the interval before the blood filled the cannula by a stop-watch. When this had been ascertained, the drug was injected into the jugular vein and the rate of flow was taken at suitable intervals thereafter. Caffeine. The changes induced by caffeine in the renal circulation have been investigated by many authors(l) by means of the oncometer. Landergren and Tigerstedt employed the "stromuhr" but no diuresis followed in their experiments. Schwarz measured the blood-flow directly in rabbits and found some increase in the urine under caffeine, while the blood-flow was unchanged, but the latter was so small throughout that the kidney must have been asphyxiated. In Barcroft and Straub's experiments, by the Barcroft and Brodie method, there was no definite diuresis, and the same is true of R o b e rt s o n' s work on dogs'. Richards and Plant kept the kidney alive by a special form 1 In the dog and cat, caffeine has very little effect on the secretion of the urine com-

4 ACTION OF DIURETICS. of perfusion, and found that caffeine may cause diuresis without any change in the blood-flow through the organ. It seemed desirable to confirm this by experiment on the living animal. After our experiments were completed, Miwa and Tamura(2) published a paper on the renal metabolism and blood-flow during caffeine diuresis, employing a method which was essentially that introduced by B arc ro ft and B ro die. They found that in diuresis from caffeine and its allies' there is no increase in the blood-flow through the kidney and no change in the 02 taken up from the blood. In our experiments caffeine was injected in doses of 20 mg. in about j c.c. of warmed Ringer or gum solution into the jugular vein. In several experiments no diuresis followed, probably owing to faulty preparation, and these have been discarded. In others diuresis set in soon after the injection. The course of the experiment may be followed from the tracings in Figs. 3 and 4. In these the injection of caffeine was followed by a sharp fall in the blood-pressure which rose again to the normal height or beyond it in about one minute. During the return of the blood-pressure or immediately after it, there followed a transient acceleration of the renal blood-flow. In Fig. 3 this acceleration was from 6 seconds to 5*1 secs. at the maximum, or 15 p.c., wbile in Fig. 4 the time required to fill the cannula fell from 4 secs. to 3-1 secs., an acceleration of 22*5 p.c. This acceleration lasted 4-5 minutes, and the rate of flow then returned to the normal or became slower. The flow of urine began to increase about one minute after the injection, as the bloodpressure was recovering and rather before there was any definite dilatation of the renal vessels; this is well seen in Fig. 4 in which we succeeded in measuring the rate of flow between these two points. The rate of secretion increased for 4-5 minutes and then slowly declined, reaching the normal in about minutes after the injection. The fall in blood-pressure has been noted by many observers and is geilerally attributed to direct action on the heart muscle; it lasts a very short time and may be followed by a slight. rise above the normal, which may probably be due to the vasoconstriction in compensation for the previous fall in pressure. The acceleration of the blood-flow through the kidney occurs as the blood-pressure rises or after it has completely recovered, and the two features are quite distinct in time. It seems not unlikely that they have pared with that in the rabbit. Only one of B a r c rof t and S t ra u b's experiments was done on the rabbit and here there was very little effect. Diuretin increases the 02 absorption owing to its content of salicylate. 279

5 280 A. R. CUSHNY AND C. G. LAMBIE. a similar origin, however, and that the action on the heart muscle which reduces the blood-pressure is followed by depression of the vascular wall which leads to dilataition and an acceleration of the current. In both T $n'i Fig. 3. M INWS Fig. 4. Figs. 3 and 4. Blood-pressure, B.P.; number of seconds required to fill the measuring cannula, R; drops of urine per minute, U; Caffeine injection (0-02 grm.), C. cases the muscle is weakened by direct action of the caffeine in high concentration, but as soon as the concentration in the blood falls, the muscle recovers; the heart muscle is affected sooner than the renal

6 ACTION OF DIURETICS. 281 vessels, and the short fall in blood-pressure therefore precedes the acceleration in blood-flow. The dilatation of vessels when caffeine is perfused through them is a well-known phenomenon and has led to the use of caffeine as a vasodilator in disease of the coronary arteries; it does not seem to have been realised how very short this effect is in the living animal. This acceleration of the blood-flow appears at first sight to give an adequate explanation of the diuresis. But in the experiment from which Fig. 4 is drawn, the increase in the urine began rather before the accelerated flow, and in all cases the diuresis continued long after the blood-flow had returned to the normal rate or was even slower. Unless the short dilatation of the vessels be conceived to have a prolonged effect on the rate of secretion, this seems to be fatal to the idea that the increased blood-flow and diuresis under caffeine are correlated. In several experiments a second injection of 0-02 grm. caffeine was made after the first diuresis had passed off. This was generally followed by the same transient changes in the blood-pressure and renal blood-flow, but the urine generally ceased and in no case was it increased. The poisonous action of caffeine on the kidney was apparently induced by the second dose Ṁiwa and Tamura observed no change in the renal blood-flow under caffeine during diuresis, but this is easily explained by the fact that they never took the rate of flow until minutes after the injection. At this stage the blood-flow has long returned to the normal, while the diuresis may persist. We thus agree with them that the caffeine diuresis is independent of the rate of blood-flow through the kidney, and must arise from a direct action on the kidney cells. Pituitary extract. The diuresis from the injection of extract of the posterior lobe of the pituitary body has been examined by means of the oncometer by Sc haifer and others, who found that the kidney volume generally increases while the blood-pressure rises from the constriction of the vessels in other regions; but the diuresis may occur without any change in the blood-pressure or in the volume of the kidney. Ro b e r t so n estimated the outflow in the renal vein and found no increased blood supply under pituitary extract. In our experiments we used Duncan and Flockhart's "pituitary fluid" generally in doses of 0x2 c.c. The intravenous injection was followed in every instance by a considerable acceleration in the rate of flow through the kidney and by a rise in the general blood-pressure (Fig. 5). These changes?ersisted for minutes. The blood-pressure

7 282 A. R. CUSHNY AND C. G. LAMBIE. reached its highest point rather sooner than the maximum acceleration as a general rule, and both then slowly fell to the normal. The diuresis was small compared with that from caffeine, while the acceleration of the blood-flow was more marked and persistent. There was in some experiments a short period of 3-4 minutes after the injection during which the secretion was smaller than before the injection or ceased altogether. After this the urine increased in amount and then slowly fell off again, reaching the normal point about the same time as the return of the bloodpressure to its former level. In a number of experiments under pituitary the change in the blood-pressure and rate of flow was unaccompanied s.~~~~~g mu% Hs l0 I so10 s, 30 as 4 omins *0 Fig. 5. Blood-pressure, B.P.; number of seconds required to fill the measuring cannula, R; drops of urine recorded on the base line, U. Injection of pituitary' fluid 0 2 c.c., P.P. by any significant change in the amount of urine secreted. But when diuresis was induced by pituitary, its course ran so nearly parallel with the blood-flow through the kidney, that it suggests that diuresis is the result of the larger supply of blood to the kidney and not of any action on the secretory cells. Strophanthin. The effects of the digitalis series in increasing the urine, an early clinical observation, have been examined repeatedly by means of the oncometer, but not by any more direct method of determining the renal blood supply. In our experiments a specimen of crystalline strophanthin prepared by Sir Thomas Fraser from Strophanthus

8 ACTION OF DIURETICS. 283 glaber was used in doses of mg. This was sufficient to cause a slight increase in the urine of a rabbit in which cannulae had been placed in the jugular vein and bladder, without further operative procedure. But in a series of five experiments in which the animal was prepared as described above for the measurement of the rate of blood-flow through the kidney, strophanthin in these doses had no diuretic effect and gave no appreciable change in the blood supply. Larger quantities increased the blood-pressure and reduced the flow through the kidney, probably from a direct constricting action on the renal vessels. Normal animals often show no diuresis after the administration of members of the digitalis series, and even in successful experiments the increase in the urine is small compared with that from caffeine and its allies. The severity of the operation may also have contributed to the absence of effect in our experiments, and especially the exclusion of the large intestinal area. It is still unproved that the diuresis in cases of dropsy arises from changes in the renal circulation under this series, for it may be argued that the improved nutrition of other tissues leads to the reabsorption of effused fluid into the blood, and that the consequent hydraemia is the efficient factor in the increased secretion of urine. This could be disproved only by the measurement of the plasma percentage in case of dropsy before and after digitalis treatment. If the diuresis arises indirectly from the absorption of fluid, hydraemia would be present, while if it is the result of changes in the renal circulation, digitalis ought to reduce the fluid of the blood. Whether the change in the plasma attains dimensions measureable by our present methods may be regarded as questionable. Sodium sulphate. Barcroft and Brodie, and Barcroft and St r a u b found that the renal blood-flow was sometimes increased under saline diuresis, but that this is transient when present and is not proportional to the urine. This was confirmed by Lamy and Mayer, who further pointed out that the diuresis was accompanied by acceleration only when large quantities of salt solution were injected, while smaller quantities increased the urine without change in the blood-flow. Robertson observed acceleration of the flow during diuresis, while Richards and Plant state that salt solution may cause diuresis without any change in the blood supply to the kidney. We have measured the blood-flow under the injection of sodium sulphate in a number 'of animals, generally at the end of experiments in which other diuretics had been injected. The solution was 20 p.c. and 5 c.c. were injected. Marked diuresis followed at once and the rate of flow through the kidney rose, but soon returned to the normal, while the

9 284 A. R. CUSHNY AND C. G. LAMBIE. urine was increased for long afterwards. Our results thus confirm those of the previous workers-that the diuresis is independent of the increased blood supply to the kidney. One of us has already pointed out, that the efficient factors in the saline diuresis are the dilution of the colloids of the blood, and the osmotic resistance to the reabsorption of the filtrate in the tubules owing to the salts themselves being absorbed to only a limited extent. Urea. The injection of urea (1 grm. in 2 c.c. Ringer solution) was followed in two experiments by diuresis 9 'Re-t 6.a with acceleration of the blood-flow, but : : the latter was short-lived, while the urine 8 *. remained high after the normal rate of blood-flow had returned (Fig. 6). The urea diuresis is thus not dependent on ULT the blood-flow only, but on the same f factors as have been mentioned under sulphate diuresis. X GGum solution. In several experiments, injections of Bayliss' 6 p.c. solution of acacia in warm Ringer were made, gener- Fig. 6. Number of seconds required to fill measuring cannula, R; drops ally when the blood-pressure had fallen of urine per minute, U; injection of greatly and the flow urea 1-0 grin., X.gralflwtouhhekdy through the kidney was slow. In some of these, the bloodpressure rose to the normal, the blood-flow was quickened and the urine increased. In other experiments in which the blood-pressure was satisfactory before the gum was injected, there was a smaller rise in blood-pressure, the rate of flow was accelerated, but no significant change in the urine followed. The difference in these results may perhaps be explained by the mechanical effects of the gum solution: when the blood-pressure is low from the exhaustion of the animal, the urine ceases from the failure of the pressure in the glomerular capillaries and the gum in raising the pressure restores the secretion, even though it does not itself supply a readily filtrable fluid. On the other hand, when the blood-pressure and renal circulation are normal, this mechanical action in increasing the bulk of the blood and raising the blood-pressure by a slowly diffusing fluid has comparatively little effect on the amount of urine. The gum solution increases the filtrate by improving the blood-pressure in the glomerulus, not by diluting the colloids; the salts act by diluting the colloids without necessarily changing the blood-pressure or the renal circulation time.

10 ACTION OF DIURETICS. 285 Adrenaline. Several experiments were performed with adrenaline 005 c.c. 0.1 p.c. solution rather as a test of the method than with the idea of ascertaining any new facts. Fig. 7 gives the blood-pressure, and the measurement of the rate of blood-flow and of the urine has been inserted beneath. The parallelism between the two upper curves is remarkable: the urine practically ceased during the high pressure and returned as it passed off. Section of the splanchnic nerve. In two experiments the left splanchnic nerve was cut in the course of the experiment. This was followed by a short slowing in the rate of the renal blood-flow, and after 1-2 minutes by acceleration. Unfortunately no record of the secretion was made in these two experiments, but many writers have found it increased by Fig. 7. Blood-pressure tracing, B.P., xi; time required to fill measuring cannula, R. Drops of urine recorded on U. Time in 10 seconds. A, injection of adrenaline. splanchnic section; the increased blood-flow has also been recorded by Burton-Opitz and Lucas (1908) by means of a "stromuhr." DISCUSSION AND SUMMARY. Among the diuretics examined, the increase in the urine appeared to depend directly on the blood-flow through the kidney in only one instance-the pituitary extract. Here the diuresis set in with the acceleration in the venous flow and passed off as it disappeared, and the action may thus be attributed to the improved circulation in the kidney; according to the Heidenhain school this may act by improving the nutrition in the organ, while others would ascribe the increase in the urine to a higher pressure in the glomerular capillaries and to a more rapid replacement of the plasma; the concentration of the colloids by the drainage of the fluid through the glomerular filter is thus counteracted through the more rapid replacement of the blood.

11 286 A. R. CUSHNY AND C. G. LAMBIE. Under caffeine the diuresis begins before the accelerated flow and lasts long after it has become normal. The action is thus not dependent on changes in the blood supply but on changes in the renal cells. This. has been assumed to be the action by a number of investigators, who have generally located it in the epithelium of the tubules. But the chief effect of the purine diuretics is by common consent an increase in the water of the urine, while the solids are relatively slightly augmented. This seemg to point to an action on the glomerular capsule rather than on the tubules, whatever view be held of the division of labour among the renal elements. The simplest view which reconciles the observations is that caffeine causes diuresis by reducing the resistance to filtration through the glomerular capsule by a specific action on its cells. The diuresis from salts and urea is not necessarily accompanied by acceleration of the renal blood-flow, though this may accompany it in the beginning, and presumably favours its occurrence. One of the chief factors is the hydrsemia, which reduces the osmotic resistance to filtration by diluting the colloids, the other the difficulty of reabsorption of these bodies in their course through the tubules. Adrenaline lessens the blood supply and thus the secretion of urine, while section of the splanchnic nerve has the opposite effect in increasing the blood-flow through the kidney and the amount of urine. Strophanthin had no diuretic action in our experiments, nor did it alter the circulation through the kidney in moderate doses, while larger ones increased the blood-pressure, slowed the renal blood-flow and lessened the secretion of urine. REFERENCES. (1) Cushny. Secretion of Urine, pp (2) MittheiL d. Med. Fak. d. K. Univ. zu Tokyo, 23. p