University College, London.) kidney for perfusion. It therefore seemed advisable to re-investigate the

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1 6I2* STUDIES ON WATER DIURESIS. Part III. A comparison of the excretion of urine by innervated and denervated kidneys perfused with the heart-lung preparation. > BY L. E. BAYLISS AND A. R. FEE. (Beit Memorial Research Fellows.) (From the Department of Physiology and Biochemistry$>\. University College, London.) IN the previous paper of this series(2), as a result of experiments upon the excretion of urine from innervated and denervated kidneys in hypophysectomized and decerebrated dogs, one of us suggested that under normal conditions the pituitary body is not concerned with the regulation of the renal excretion of chlorides and water; and that the "ingravescent polyuria" characteristic of the isolated kidney perfused by the heartlung preparation is not entirely due to the disappearance of pituitrin or pituitrin-like substances from the circulating blood, but rather the result of complete denervation consequent upon the removal of the kidney for perfusion. It therefore seemed advisable to re-investigate the excretion of urine from the isolated kidney, in order to determine to what extent denervation could be held responsible for the increased urine flow and reduced chloride output. Experimental technique. The only fundamental difference in the final procedure adopted in these experiments and that employed in the preparation of a heart-lung circuit as described by Starling and Verney(4) was that, instead of completely excising the kidney, it was perfused in situ by means of a cannula placed in the aorta distal to the renal artery; mixing of the perfused blood with that of the animal being prevented by appropriately placed ligatures. As the blood left the kidney, through a slit in the renal vein, it was collected from the abdominal cavity by a tube piercing the dorsal wall of the animal, and returned to the venous reservoir of the heart-lung preparation by the automatic venous return pump described by us elsewhere (1). Adequate precautions were taken to avoid the blood

2 136 L. E. BAYLISS AND A. R. FEE. of the heart-lung-kidney circuit coming into contact with that of the kidneyr dog, and the renal nerves were left undisturbed as far as was possible. We wish to emphasize the following points of our technique: (1) The establishment of the heart-lung circuit from 30 to 60 minutes before the kidney was switched into the circuit. (2) The fact that no saline or urea were added to the blood circulating through the kidney. (3) At no period was the renal blood flow cut off or the renal bloodpressure lowered during the transfer of the kidney from its own circulation to that of the heart-lung preparation. (a) The heart-lung circuit. Details of the heart-lung circuit were altered to ensure greater stability in the temperature of the blood, reduction in the volume of blood in circulation, and greater facility in recording blood flows and blood-pressure. The heating coil in the main circuit was eliminated and the remainder of the apparatus, including the venous reservoir, enclosed in a continuously stirred water bath, the temperature of which was accurately controlled by an electric thermo-regulating system. The venous blood leaving the kidney was passed through a heating coil in this bath before being returned to the main venous reservoir. These slight modifications ensured a regular and easily adjustable blood temperature. The usual precaution of respiring the animal with warmed air to avoid heat loss from the blood circulating through the lungs was also observed. It was usual to so time the completion of the heart-lung circuit that 30 to 60 minutes were available before shuinting the kidney into the circuit. This allowed time for the removal of any " vasotonins " in the freshly defibrinated blood, and also for the disappearance of any posterior pituitary substance which may have been normally present. (b) The kidney circuit. After chloralose anesthesia a mid-line incision was made in the abdomen of the kidney dog and all of the intestines, from the rectum to the junction of the tail of the pancreas with the duodenum, carefully removed between double ligatures. The spleen was also excised and the right kidney ligatured off but left in situ. The fascia binding the left kidney to the dorsal wall of the peritoneal cavity was then slit at its lateral attachment and the kidney itself gently retracted towards the right side. This allowed free access to the aorta and exposed the renal nerves. The aorta was then cleared and lifted and all branches within 2 cm. above and

3 WATER DIURESIS. 137 below the origin of the left renal artery tightly ligatured. Three ligatures were then loosely placed around the aorta, one proximal and two distal to the renal artery, care being taken to avoid including any of the small nerves which frequently pass along the sheath of the aorta to the kidney, via the renal artery. Another loose ligature was placed about the renal vein about 1*5 cm. from its origin from the renal pelvis. Finally a ureteral cannula was inserted and the drainage tube of the venous return system thrust through the dorsal wall of the abdominal cavity lateral to the kidney. The kidney was then returned to its normal position, and the carotid blood-pressure and urine flow recorded before switching over to the heart-lung circuit. The transference of the kidney to the heart-lung circuit was effected without reduction in the arterial pressure or stoppage of the renal blood flow in the following manner. A clip was placed over the aorta distal to the renal artery and a cannula, connected with the arterial side of the heart-lung circuit, inserted. The pressure at the head of this cannula was then adjusted until it was approximately that recorded from the carotid artery of the kidney dog. After placing a small amount of heparin in the abdominal cavity the renal vein was quickly ligatured and slit in such a manner to allow free exit of blood from the kidney. Upon removal of the aortic clip blood was capable of entering the kidney either from the heart-lung circuit or from the kidney dog itself. The transfer was then completed by tying the aortic ligature " cephalad " to the renal artery. Complete isolation of the kidney circuit can only be obtained when all haemorrhage is carefully controlled and all branches leaving the aorta in the region of the renal artery tightly ligatured. It was usual to increase the kidney perfusion pressure above that in the animal in order to ensure that no blood should find its way through anastomotic channels into the heart-lung-kidney circuit. RESULTS. (a) A comparison of the renal blood flow and urine excretion before and after denervation. In Table I are given the renal blood flow, renal blood-pressure and urine excretion of an innervated kidney perfused by a heart-lung preparation, as well as the carotid blood-pressure of the kidney dog. For the sake of comparison these factors are also given before perfusion and after denervation. After evisceration and other preparations for the switch-over had been completed 065 to 053 c.c. of urine were being

4 138 L. B. BAYLISS AND A. R. FEE. TABLE I. Heart-lung preparation finished at Perfusion of innervated kidney started at Denervation of kidney at 4.59, 0-75 unit Parke Davis "Pitressin" into kidney circulation at Kidney weight 38-0 g. Temperature of perfusion blood 37-5 to C. Blood Urine Carotid - A,_A blood- Flow Pressure Flow Cl. Absolute Time pressure c.c./min. mm./hg. c.c./lomin. mg.p.c. CL 348A Fore period 4"& Slight - - Innervated per fusion period Denervated 51C perfusion period Recovery from unit "Pi- 5U tressin" (Parke Xt56 - Davis) excreted per 10 minutes; the carotid blood-pressure being 97 mm. Hg. During perfusion with the renal nerve supply intact little or no excretion took place during the first 10 minutes, after which 0-25 c.c. per 10 minutes were excreted during the next 20 minute period; the renal perfusion pressure being 110 to 120 mm. Hg. and the renal blood flow 98 c.c. per minute. The kidney was then decapsulated and the adventitia removed from the artery. As this operation was in progress the urine flow began to increase to a notable extent, as did also the renal blood flow, so that within 20 minutes 15-6 c.c. of urine were being excreted per 10 minutes at a blood-pressure of 102 mm. of Hg. and a renal blood flow of 200 c.c. per minute of a unit of Parke Davis's " Pitressin " was then added to the circulating blood. The urine flow was at once inhibited, but gradually increased during the next 35 minutes to 12-8 c.c. per 10 minutes. This experiment illustrates that until denervation is effected no polyuria is observable, but immediately after complete isolation of the kidney the rate of excretion rises steadily and at a rapid rate, until it may be as much as 30 times above that during the period of innervation; the blood flow is also noticeably increased. Another experiment illustrating these observations is given in Fig. 1. In this case after denervation, although the renal perfusion pressure dropped by nearly 20 mm. of Hg., the rate of excretion of urine was increased approximately six times. Similar results have been obtained in the ten experiments in which denervation was carried out. The action of pituitrin upon the polyuria resulting from denervation

5 .WATER DIURESIS is similar in all respects to that described by Starling and Verney(4) for the heart-lung-kidney preparation. The behaviour of the chlorides in our experiments will be discussed in relation to this aspect of the problem later. bo Perfusion Started I 1.ri Time in minutes Fig. 1. Effect of denervation and "Pitressin" on renal blood flow and urine flow. Kidney weight 34*0 g. Perfusion temperature *O0 C. (b) Reflex alterations in the blood and urine flow of the innervated kidney perfused by the heart-lung preparation.- A steady urine flow was not always obtained during the period of perfusion in which the nerve supply to the kidney was undisturbed. In Figs. 1 and 2 the carotid blood-pressure of the kidney dog, the renal perfusion pressure and rate of blood flow, and urine excretion are compared. Fig. 1 shows that a gradual rise in the carotid pressure of the kidney dog takes place and is associated with a reflex, increase in the renal blood flow and output of urine. The probable reason for this rise in blood-pressure in the kidney animal is that (as the result of inserting the aortic cannula for the renal perfusion) the circulation to the hind

6 140 L. E. BAYLISS AND A. R. FEE. limbs of the animal is occluded, and this gives rise to a reflex increase in the general blood-pressure of the animal. Another possible factor is the gradual leakage of blood from the heart-lung-kidney circuit, which was usually run at a higher pressure than that in the kidney animal. The Spla.nchnic C and E anaeethesia Stimulatel Animal in F. Ee ofsplanchnic stim d h a on off fon o rea bw ~ Perfusion pre_sure\115m. Hg.Prfusontm ~ 70\/ 90 loo o0 o detryebydenretnofeflow whln0p o 0~~~~~~~~~~~~~~~ " 90 0 '30 60 Fig. 2. Time in minutes Fig. 3. Fig. 2. Effect of splanhnic stimulation andhoemorrhage on renal blood flow and urine flow. Kidney weight 57-0 g. Perfusion pressure 115 mm. Hg. Perfusion temperature C. Fig. bybledn 3. Effect of oraaetesa aniesthesia on renal blood flow and urine flow. Kidney weight 34.0 g. Perfusion pressure 115 mm. Hg. Perfusion temperture n50C. relationship between the blood-pressure of the kidney animal and the blood flow and urine output of its perfused kidney is, however, always destroyed by denervation of the kidney, when the typical polyuria already described occurs whether the animal is left alive or killed either by bleeding or anesthesia. In the second experiment (Fig. 3) the blood-pressure of the kidney animal was reduced by chloroform-ether anaesthesia. As can be readily observed this produced a reflex lowering of the renal blood flow and output of urine. The series, of experiments represented by Figs. 1 and 3 definitely indicate that alterations in the general blood-pressure of the kidney animal are capable of producing reflex alterations in the calibre of the

7 WATER DIURESIS. 141 renal blood vessels, and that the rate of urine excretion follows the blood flow even when the renal blood-pressure remains constant. Further reflex alterations in urine and blood flow are shown in Fig. 2. Appropriate faradic stimulation of the splanchnicus major inhibited the urine flow and markedly reduced the renal blood flow. This confirms earlier work on the whole animal. In the same experiment it is shown that excessive haemorrhage of the kidney dog causes a similar reduction of renal blood flow and urine output, but that after denervation, although the blood-pressure of the animal was still low, an increased renal blood flow and urine output immediately occurred. The reflex inhibition of blood flow following haemorrhage is very marked. In one experiment, as a result of extensive heemorrhage, the renal blood flow was stopped entirely and the kidney became asphyxiated before denervation could be completed. In general, therefore, a rise in blood-pressure in the kidney animal will produce reflexly an increased output of urine and an increased blood flow through the kidney. Reduction of the general blood-pressure, either by further ansesthesia or by haemorrhage, has the opposite effect. Appropriate stimulation of the great splanchnic nerve will imitate the latter effect; section of the renal nerves the former. Similar reflex changes in the size of the renal blood vessels have recently been described by Heymans (6). (c) The excretion of chlorides. Urinary chlorides were estimated in several experiments. It was found that usually the amount present in the urine was low both during the fore-period and during perfusion with the renal nerves intact (50 to 100 mg. p.c.). After denervation, although the percentage output decreased concomitantly with the increased excretion of urine, the absolute rate of excretion remained practically constant. Upon reducing the denervation polyuria with commercial "pitressin," however, the absolute output was invariably increased two to three times. The addition of blood fresh from the kidney dog had a similar effect. In the one experiment during which the splanchnic nerve was stimulated such an increased absolute output of chlorides was not manifest, although the percentage amount increased with the decrease in urine flow which fell from 14-5 c.c. to an average of 3 c.c. per 10 minutes.

8 142 L. B. BAYLISS AND A. R. FEE. DISCUSSION. The above experiments are of interest, primarily because they indicate another factor which must be taken into account in interpreting the behaviour of the isolated kidney perfused by the heart-lung preparation. Starling and Verney4 suggested that the characteristic increase of urine flow in such cases was due to a gradual disappearance of pituitrin or pituitrin-like substances from the circulating blood. In some of our experiments, however, we have perfused an innervated kidney for 50 minutes or more without obtaining any evidence of such a lack being evinced by an increased output of urine. Moreover, in all experiments, the heart-lung preparation was completed some time before shunting the kidney into the circuit; consequently, if the gradual disappearance of pre-existing post-pituitary principles is to be held responsible for the normal "ingravescent polyuria," such disappearance would, in our experiments, have been completed before the kidney was introduced into the circuit, and the polyuria should have been immediate. Actually there was no polyuria at all. Possible blood leakages from the animal into the heart-lung-kidney circuit were, in the majority of cases, prevented by keeping the renal blood-pressure higher than that of the kidney dog. If then simple denervation will increase the output of urine of the isolated kidney 30 times or more, it is extremely improbable that a further polyuria resulting from the absence of pituitary hormones could be ascertained in such a preparation. In this connection it is worthy of note that part of the routine technique employed by Starling and Verney in the preparation of a heart-lungkidney circuit is the addition of 1 to 5 g. of urea to the circulating blood for the purpose of counteracting the vasotonic effects of freshly defibrinated blood. We have, by adding similar amounts of urea to our preparations, obtained a marked diuresis (from 7*2 to 13-0 c.c. per 10 minutes). This practice is therefore, in our opinion, inadvisable, particularly since the same ends may be achieved by preparing the heart-lung preparation a few minutes earlier, and allowing the blood to circulate for a longer period. The effect of innervation and denervation upon chloride excretion is not clear. In none of our experiments did we obtain an increase in the absolute output of chlorides as the result of a reflex reduction in the urine flow. Usually the absolute rate of excretion remained unchanged. Commercial pituitary extracts, however, invariably increased the absolute output. In this connection it is interesting that, in the protocols

9 WATER DIURESIS.14 given by Starling and Verney(4), the addition of pituitrin always resulted in an increased chloride output, whereas in the experiments of Verney(5), although the addition of a head to a heart-lung-kidney circuit reduced the output of urine, the absolute excretion of chlorides remained practically unaltered. We feel that this subject requires very careful investigation before alterations in the renal output of chlorides may be taken as evidence for or against the direct intervention of secretions of the pituitary in renal excretion, particularly after the unusual deviations in chloride output recorded in the whole animal by Magnus (3). The reflex alterations of renal blood flow and urine excretion are of interest in suggesting a direct nervous control of renal function, apart from any changes which may follow directly upon changes in bloodpressure during haemorrhage, shock, etc. SUMMARY AND CONCLUSIONS. 1. A method of perfusing an innervated kidney with a heart-lung preparation is described. 2. Such a preparation, unlike the isolated kidney perfused by the heart-lung preparation, shows no signs of excreting large amounts of hypotonic urine until denervation is effected. 3. With a constant perfusion pressure and blood composition a reflex rise in the renal blood flow produces an increase in urine flow. 4. Alterations in the blood-pressure of the kidney animal by heemorrhage or shock produce reflex changes in the blood flow and urine output of the perfused kidney. When the blood-pressure is high the renal blood flow may be reflexly increased and the rate of urine excretion will then rise; and vice versa. Stimulation of the splanchnicus major (one experiment) of course reduced the blood flow and the excretion of urine, i.e. the same effects as were induced reflexly by a fall of blood-pressure in the kidney animal. The expenses of this research were defrayed by a grant from the Royal Society to one of us (A.R.F.). REFERENCES. 1. Bayliss and Fee. This Journ. Proc. Physiol. Soc. Jan. 18, Fee. This Journ. 68. p Magnus. Arch. exp. Path. Pharmak. 44. pp. 68, Starling and Verney. Proc. Roy. Soc. B, 97. p Vernev. Ibid. 99. p Heymans. Le Sinus carotidien. Louvain,