Visscher(6) that the oxygen consumption of the heart-lung preparation

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1 BY A. R. FEE1 AND A. HEMINGWAY. (From the Department of Physiology and Biochemistry, University College, London.) SEVERAL investigations of the metabolism of the kidney have been made to ascertain the mechanism and efficiency of that organ. B arcroft and Brodie(l), Barcroft and Straub(2), Tamura and Miwa(3) and others have measured renal oxygen consumption in the whole animal, and Bainbridge and Evans(4) made measurements upon the heartlung-kidney preparation (the oxygen consumption of the heart and lungs, after separate determination, was deducted from that of the whole preparation). None of these methods, however, admitted of a continuous series of measurements over an extended period of time. Recently the writers(5) confirmed the observation of Starling and Visscher(6) that the oxygen consumption of the heart-lung preparation bears, within reasonable limits, a direct linear relationship to the diastolic volume of the ventricles, and also showed that a similar relationship existed between the diastolic volume of the whole heart and the oxygen consumption of the preparation. An experiment was also given which demonstrated that this relationship was not destroyed if the heart-lung preparation were used to perfuse an isolated kidney. This led to the suggestion that if the (diastolic volume): (oxygen consumption) ratio of a heart-lung preparation were determined before inserting a kidney into the circuit, subsequent measurements of diastolic volume and the total oxygen consumption of the preparation would afford data from which the oxygen usage of the perfused organ could be determined. In this paper the experimental details of such a method of determining renal oxygen usage will be given along with the results which have already been obtained by its use. EXPERIMENTAL DETAILS. (A) The closed-circuit. Heart-lung-kidney preparation. The heart-lung-kidney preparation as originally described by Bainbridge and Evans(4) and modified by Starling and Verney(7) was Exhibition Scholar-now Beit Memorial Research Fellow.

2 101 used with a few minor alterations. In Fig. 1 ABCD is the heart-lung circuit and is similar in all essentials to the closed circuit described by M xi Y Fig. 1. Diagram of the closed circuit heart-lung-kidney apparatus. A, B. Heart-lung preparation and resistance. C, M. Heating coils. D. Venous reservoir. E. Water bath. K. Kidney. 0. Water-jacketed funnel. P. Measuring vessel. X, Y. Rubber bags of pump. V1, V2. Valves of pump. Starling and Visscher(6). The kidney (K) which is placed on a waterjacketed funnel (0) is connected to the arterial side of the heart-lung circuit by a cannula in the renal artery and to the venous reservoir (D) by a cannula in the renal vein. In order to obtain the maximum advantage of the arterial pressure and to minimise the work of the heart in overcoming the resistance due to gravity, the kidney was placed as far as possible below the level of the heart. This necessitated the use of a pump to return the blood from

3 102 A. R. FEE AND A. HEMINGWAY. the kidney to the venous reservoir, a pump of such a type that would accommodate itself to variations in the renal blood flow. The device used consisted of two rubber bags having a capacity of 50 c.c. (Fig. 1, X and Y). As blood left the kidney it passed through the first bag (X) into the second (Y) from which it was expelled by a flat striker actuated by a crank shaft. The flap valves V1 and V2 directed the flow of blood. Any back pressure which might have developed on the renal side of the pump during the closure of V1 was not transmitted to the kidney owing to the distensibility of the walls of the first bag (X). Renal blood flow was measured approximately by diverting the blood leaving the kidney into the graduated tube P by means of a three-way tap. As the tube P filled with blood there was a gradual, although slight, rise in the renal venous pressure; consequently this method of measurement did not give the unobstructed blood flow through the kidney, although changes in the rate were easily discernible. The preparation of the closed heart-lung-kidney circuit was essentially the same as in the description given by Starling and Verney(7) with the exception that a cannula was placed in the renal vein as well as in the renal artery and ureter. The cardiometer used for measuring the diastolic volume of the heart was attached as described by Hemingway and Fee(5) after the heart-lung circuit had been completed. The only other modifications in technique necessary were those required to reduce the gradual leakage of blood which normally occurs in such a preparation. The cesophagus was tied off and several ligatures were placed around the thoracic aorta since this procedure was found to reduce the possibility of loss of blood through anastomotic channels. It was also necessary to ligature the perinephric tissue to avoid haemorrhage after transferring the kidney to the heart-lung circuit. It is worthy of note that the actual transference of the kidney to the heart-lung circuit could be effected in half a minute, the cannula being inserted into the renal vein immediately before the excision of the kidney. This was the only period during which the renal blood flow was arrested. (B) The measurement of oxygen consumption. Oxygen determinations were made by allowing a known volume of pure oxygen to enter a closed circuit in connection with the lungs and measuring the time taken for its consumption. Since carbon dioxide was continually removed with soda lime, any reduction in the volume of this closed circuit was an index of the amount of oxygen absorbed

4 103 by the blood in passing through the lungs. Such changes in extrapulmonary gas volume were measured with a modified Krogh spirometer. In Fig. 2, B is a water-jacketed burette capable of delivering accurately y;l -1 t Iv lap, a Mpt. w a I LUNGS & PUMP Fig. 2. Diagram of respiratory circuit. A. Aspirator. B. Gas burette with bulbs X and Y. a. Fine adjustment for volume of gas in B. D. Spirometer. E. Bell. F. Signal marking on kymograph. and quickly 20, 40, and 50 c.c. volumes of oxygen. By closing tap 1 and opening taps 2 and 3 oxygen will pass from the aspirator A into the burette expelling water from X into Y. When approximately the required volume has been forced in, tap 3 is closed and the water level in the burette allowed to settle. Before delivering the oxygen into the lung circuit this level is accurately adjusted by means of a clamp on a length of rubber tubing (C), and tap 2 is closed. Delivery is effected by opening tap 1. The absolute volume delivered will depend upon the temperature and barometric pressure as well as the difference in water level in the two arms of the burette. Corrections for these factors are necessary and also for the tension of aqueous vapour. Air leaving the lungs passes through the respiration pump,.#&the

5 104 A. R. FEE AND A. HEMINGWAY. "ideal" type as described by Starling(s)) and enters the spirometer D where the carbon dioxide is removed by soda lime. As oxygen is removed by the blood circulating through the lungs the spirometer gradually falls and a lever bearing two contact points magnifies this change in level. When a certain point is reached the first contact closes a circuit which rings the bell E. This serves as a warning signal and shortly afterward the second contact is made and the signal F records on a kymograph. Since the resistance of this circuit is less than the first the bell also stops ringing. At this instant a stopwatch is started and the burette full of oxygen emptied into the circuit. While this oxygen is being used the burette is refilled in preparation for the following signal. The error of the gas delivery system is less than 1 p.c. The main objection to measurements of oxygen usage by this system is that any change in lung volume is reflected in the movements of the spirometer. Extreme care must be taken in preparing the heart-lung circuit as rough handling of the lungs causes them to leak appreciably. It is advisable to avoid increasing the stroke of the respiration pump beyond the absolute minimum until all operative manipulations have been completed. The slightest cedema of the lungs vitiates the readings, but is easily detected by the marked disagreement of consecutive readings taken under the same conditions. The development of any leak in the gas circuit during the perfusion of the kidney is readily detected by determining the (diastolic volume): (oxygen consumption) ratio of the heart-lung preparation alone at the end of the experiment and comparing it with that determined at the beginning. EXPERIMENTAL RESULTS. (A) Chloride excretion. Starling and Verney(7) have shown that the isolated kidney perfused by the heart-lung preparation gradually approaches a condition similar to that found in diabetes insipidus, inasmuch as large amounts of a markedly hypotonic urine are excreted. Whereas the urinary chlorides in the normal intact animal are usually above 0-6 grm. p.c. as NaCl, at the end of an hour's perfusion the isolated kidney often produces a urine containing as little as 0*03 grm. p.c. The absolute output of chloride usually falls also, but this is not always so owing to the diuresis which occurs. The renal oxygen consumption, as measured by the method just described, increases as this condition is attained. The results of a typical

6 experiment are given in Table I. There is a fall both in the percentage and absolute amounts of chloride excreted per unit time, an increase in the rate of urine flow and also in the renal oxygen consumption. TABLE I. Urinary chloride as Blood flow 02 usage Urine NaCl Time c.c./min. c.c./min. Time c.c./10 min. grm. p.c * * * trace Weight of kidney 37 grm. 105 The average oxygen consumption in the above experiment is c.c. per grm. per min. with a minimum consumption of c.c. and a maximum of c.c. In most experiments performed the oxygen consumption fell between a minimum oxygen consumption of 0 03 c.c. and a maximum of 0-20 c.c. per grm. per min. Bainbridge and Evans(4) found an average renal oxygen consumption of c.c., while Barcroft and Straub(2) obtained values between 0 03 c.c. and 0'10 c.c. per grm. per min. (B) The effect of changes in arterial pressure. Barcroft and Straub (loc. cit.) in their investigation of changes in oxygen consumption during different types of diuresis made no inquiry into the relationship between the arterial pressure and the gaseous metabolism of the kidney, only noting that an increase in arterial pressure resulted in an increased urine flow. Starling and Verney(7) also showed the dependence of urine flow upon the arterial pressure, and Richards and Plant(9) demonstrated that the rate of blood flow was of little importance in the rate of formation of urine when compared to the arterial pressure. By altering the resistance of the heart-lung circuit we were able to alter the pressure at which the kidney was perfused. Any alterations in diastolic volume of the heart which occurred as a result were minimised by suitable adjustments of the venous inflow in order to obviate any error which might be introduced in the calculation of the cardiac oxygen consumption from the (diastolic volume): (oxygen consumption) ratio. The results of such an experiment are given in Table II. It can be seen that there is a distinct parallelism between the blood pressure and oxygen usage. It must also be remembered that the gradual increase in renal oxygen consumption during the perfusion of an isolated kidney

7 106 A. R. FEE AND A. HEMINGWAY. Oxygen consumption 02 usage Time c.c./min X Arterial pressure mm./hg TABLE II. Time 2.1X Urine analysis Urine c.c./10 min X Chloride as NaCl grm. p.c First urine sample rejected to allow for "dead space" in renal pelvis and cannula. Weight of kidney approx. 25 grm. which has just been described is also taking place, consequently the rate of oxygen consumption towards the end of the experiment will be relatively higher than at the beginning. (C) The effect of pituitary extract. Before the effect of pituitary extract upon the kidney could be measured it was necessary to determine if the addition of the extract to the circulating blood would in any way alter the (diastolic volume): (oxygen consumption) ratio of the heart. Four experiments were carried out upon the heart-lung preparation to control this possible source of error, and in none of them could any appreciable change in the ratio be detected except immediately after the injection of the pituitrin. One of the experiments is given in Table III. Moreover in an experiment TABLE III. Diastolic volume Oxygen usage Time of heart c.c./min x plus 12-7 c.c. 12X ,, s ,, 12.5 c.c Heart weight 80 grm. Approx. 800 c.c. blood in circulation. Blood flow 400 v.0. per mm. A quarter unit of B.D.H. pituitrin added at

8 107 given in a previous paper(5) the oxygen usage of a heart-lung preparation was redetermined after it had been used to perfuse an isolated kidney for two and a half hours. During this period three quarter-unit amounts of pituitary extract had been added to the circulating blood. The observed cardiac oxygen consumption was 8-10 c.c. per min. and the value calculated from the preliminary data 7-92 c.c. per min. The effect of pituitary extract upon the renal oxygen consumption is illustrated in Table IV. A quarter unit of B.D.H. extract was added TABLE IV. Urine analysis Oxygen consumption U anay Blood Chloride as 0 usage flow Urine NaCl Time c.c./min. c.c./min. Time c.c./10 min. grm. p.c * * Weight of kidney 19 grm. Approx. 1 litre of blood in circulation. A quarter unit of B.D.H. pituitrin added at 1.53 and Blood-pressure throughout experiment 122 mm. Hg. at the times indicated. Approximately a litre of blood was in circulation. There was an immediate increase in the percentage and absolute excretion of chlorides and a diminution in the urine flow and renal oxygen consumption. The arterial pressure was constant throughout the experiment and practically no change in blood flow occurred after the addition of such a small amount of the extract. SUMMARY. 1. A method of measuring the oxygen consumption of the perfused isolated kidney is described. 2. The oxygen consumption of such a kidney varies between 0-03 and 0-20 c.c. per grm. of kidney tissue per min. 3. Associated with the gradual change in the volume of and chloride concentration in the urine produced by the isolated perfused kidney there is a gradual rise in the renal oxygen consumption. 4. An increase in arterial pressure causes a rise in the renal oxygen consumption. 5. Following the addition of small amounts of pituitary extract to the blood circulating through an isolated kidney the changes in the

9 108 A. R. FEE AND A. HEMINGWAY. chloride concentration and volume of urine are associated with a fall in the renal oxygen consumption. This investigation was begun under the supervision of the late Professor E. H. Starling who was largely responsible for the successful development of the experimental technique employed. The expenses of the research were defrayed by a grant from the Royal Society. REFERENCES. 1. Barcroft and Brodie. This Journ. 32. p Barcroft and Straub. Ibid. 41. p Tamura and Miwa. Mitt. med. Fak. Kaiser. Univ. Tokio, 23. p Bainbridge and Evans. This Journ. 48. p Hemingway and Fee. Ibid. 63. p Starling and Visscher. Ibid. 62. p Starling and Verney. Proc. Roy. Soc. B, 97. p Starling. This Journ. Proc. Physiol. Soc. 61. p. xiv Richards and Plant. Amer. Journ. Physiol. 59. p