Pharmacological Laboratory, University College, London

Transcription

1 87 J. Physiol. (I939) 96, 87-IO3 6I2.46:6I2.26 THE INFLUENCE OF UREA AND OF CHANGE IN ARTERIAL PRESSURE ON THE OXYGEN CONSUMP- TION OF THE ISOLATED KIDNEY OF THE DOG BY K. KRAMER AND F. R. WINTON From the Physiological Laboratory, Cambridge and the Pharmacological Laboratory, University College, London (Received 31 March 1939) CHANGES in the oxrygen consumption of the kidney have been regarded as important indicators of renal function since the classical researches of Barcroft & Brodie [1904, 1905] and Barcroft & Straub [1910]. An increase in oxygen consumption was found to accompany diuresis due to urea or sulphate, but no increase occurred during Ringer diuresis. This has been adduced as important evidence distinguishing between the former tubular type of diuresis and the latter mechanical or glomerular type. Some later workers appear on the whole to confirm these observations, e.g. Gremels [1929] on the heart-lung-kidney preparation and Glaser, Laszlo & Schiirmeyer [1932] on anaesthetized dogs, though the latter report a fourfold increase in oxygen consumption during Ringer diuresis compared with a small and doubtful increase during sulphate diuresis. Other workers have found essentially the opposite relation to that described by Barcroft and his colleagues, e.g. Hayman & Schmidt [1928] and Fee [1929] who found no increase in oxygen consumption during sulphate diuresis unless accompanied by an increase in blood flow, the latter finding, on the other hand, a rise in oxygen consumption with rise in arterial pressure. Van Slyke, Rhoads, Hiller & Alving [1934] discovered no essential relation between urea diuresis and the oxygen consumption of the kidney in conscious dogs. The two current views of the significance of changes in oxygen consumption in distinguishing between glomerular and tubular types of diuresis may be contrasted in the quotations "The oxygen consumption of the kidney increases in proportion to the blood flow, but is not related to the urine flow" [Smith, 1937], and "Certain measures such as injection of saline or the elevation

2 88 K. KRAMER AND F. R. WINTON of blood pressure cause profuse diuresis but little or no increase in oxygen consumption. This is further evidence that physical processes are concerned, rather than secretion; the latter, of course, would entail oxidation processes and the expenditure of energy" [Best & Taylor, 1937]. The recent introduction of an accurate and convenient photoelectric method of determining the oxygen content of flowing blood [Kramer, 1935] seems to us to justify a re-examination of the conflicting evidence mentioned above. This method can be adapted to give a continuous record of the arteriovenous oxygen difference, and this, together with a continuous record of the blood flow, provides a technique which meets the criticisms of the Barcroft & Brodie technique raised by Hayman & Schmidt [1928]. Previous observations on the oxygen consumption of the isolated kidney, initiated by Bainbridge & Evans [1914], were made in detail by Fee & Hemingway [1928], who subtracted the oxygen consumption of a heart-lung preparation from that of the heart-lung-kidney preparation, obtaining that of the kidney as a small difference between two relatively large quantities. In its application to the influence of changes in arterial pressure, this method depended on the assumption that the oxygen usage of the heart would be exactly the same at two different pressures if its diastolic volume were kept constant by appropriate adjustment of the inflow to the heart. The recent observations of Kiese & Garan [1937] have raised doubts about the validity of this assumption, nevertheless, as will be seen, we have been able to confirm the conclusion of Fee & Hemingway that increase in arterial pressure increases the oxygen consumption of the kidney. Sulphate was employed as a diuretic by Fee & Hemingway since urea increases the efficiency of the heart; we employed urea in our experiments in preference to sulphate because sulphate changes the light absorption of blood. METHODS The pump-lung-kidney preparation and apparatus were as used by Bickford & Winton [1937], with two differences. First, the oxygen unit was included as described below, and secondly, owing to the difficulty experienced in keeping the oxygen saturation of the defibrinated arterial blood up to within a normal range, the blood flow through the lung was augmented by a second Dale-Schuster pump which transferred blood from the reservoir in which blood leaving the pulmonary veins collected to the pulmonary arterial cannula. Doubling the pulmonary blood flow

3 OXYGEN USAGE AND DIURESIS in this way brought it to about 800 c.c./min. passing through lungs isolated from kg. dogs, and raised the arterial saturation with a single kidney in the circuit from about 80 to over 90 %. Even before the kidney was put on to the perfusion circuit, however, the blood (about 1 1.) failed to approach complete saturation when the lung was ventilated with air by positive pressure from an " Ideal " pump; and to ensure the complete saturation necessary for immediate calibration of the oxygen unit, temporary ventilation with a 95 % oxygen-5 % carbon dioxide mixture 89 FRig. 1. Diagram of unit for measuring the oxrygen content of flowing blood, and of the circuit for recording the galvanometer deflegions on the smoked drum. Lamp (L) illuminates photocell (PC 1) through the film of blood. The resulting current deflects galvanometers G1 and G2, affecting a second photocell (PC2) attached to the grid of a triode operating the milliammeter which records on a smoked drum. was arranged. Ventilation with oxygen throughout an experiment was impracticable with the colorimetric estimation, as the amount of oxygen in physical solution in the blood then became large and indeterminate. Ventilation with air, containing 5 % carbon dioxide, was, therefore, employed during the latter stages of the experiment. The oxygen unit (Fig. 1) was a modification of the "sauerstoffuhr" previously described by one of us [Kramer, 1935]. Its operation depends on the fact that reduced haemoglobin absorbs more red light than oxyhaemoglobin, the percentage oxygen saturation in blood being a logarithmic function of the transmitted light. In the unit, blood flowed along a glass tube of which a portion 30xll1 mm. had been flattened, the blood

4 90 K. KRAMER AND F. R. WINTON forming a film about 1-2 mm. thick. The most uniformly thick and parallel-sided portion of this film was exposed through a mask 16 x 5 mm. to light from a 6 V. flashlamp bulb (L) run at red heat by applying only 3 V. The light transmitted through the film of blood impinged on a rectifier type of photocell (PCI) (selenium oxide) which has its maximum sensitivity at the red end of the spectrum. No colour filter was employed. The cell was connected to a relatively insensitive mirror galvanometer (GI) (1000 Q. Cambridge "Pot"). The unit, made of ebonite and brass ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Galvanometer (G 1) scale reading (mm.) Fig. 2. Calibration curves of oxygen unit. --o -- Tube I, van Slyke estimations by J. R. Pappenheimer. -*- Tube II, estimations by Dr T. Gostev. The film of blood was thinner in tube II than in tube I. so as to exclude adventitious light, can be used in bright daylight, and has overall dimensions of 34 mm. in the direction of blood flow, 44 mm. in the direction of the beam of light, and 29 mm. across. With an arrangement of six T-pieces, either the arterial blood going to the kidney, or the venous blood coming from the kidney, could be transmitted through the unit in the same direction by transferring three clamps from one to another of a set of rubber tubes. Calibration of the instrument was performed by drawing samples of blood from the rubber tubes at the same time as the corresponding galvanometer reading was taken. The oxygen in the samples was estimated by van Slyke's method or by the colorimetric method [Kramer,

5 OXYGEN USAGE AND DIURESIS 1935]. Fig. 2 shows the result of two such calibrations; the logarithm of the galvanometer deflexion from the unit is accurately a linear function of the oxygen content of the blood as estimated by the van Slyke method. The accuracy of the method, especially in the range of high oxygen content which is relevant to these experiments, appears to be at least as high as that of the van Slyke method. With a galvanometer deflexion of 340 mm. corresponding with saturated blood, a difference of 1 c.c. 02/ 100 c.c. blood produces about a 50 mm. deflexion in the arterial range, and since self-consistent results can easily be obtained within 1 mm., differences of 0-02 c.c. 02/100 c.c. blood can be detected. Changing the velocity of blood flowing through the unit has no detectable effect on the galvanometer deflexions within the range of blood flow relevant to renal perfusion. Complete arrest of flow, however, reduces the deflexion by about 5 mm., and this effect can be seen in Figs. 3 and 4 whenever a change of the unit from arterial to venous blood or back involved momentarily clamping the tubes and stopping the flow through the unit. The effect of differences in the haemoglobin content of different bloods is to produce different calibration curves, which if plotted logarithmically as in Fig. 2 are parallel. Since the slope of the calibration curve of the unit is known, and it is unaffected by changes in the intensity of the light or by changes in galvanometer sensitivity, one or two estimations of oxygen in blood samples by the van Slyke method sufficed to determine the particular calibration curve for the given blood. The effective duration of a perfusion experiment was normally set by the period during which variations in the haemoglobin content of the blood was negligible, as indicated by measurement of oxygen capacity, or in some cases by haematocrit determinations. If, however, changes in the concentration of the blood did occur, these only introduced errors into the measurement of the absolute oxygen content of the blood, but not into the determination of the arterio-venous oxygen difference with which we were mainly concerned, since such changes in the blood do not affect the slope of the calibration curve. Records were taken on the smoked drum, as shown in Figs. 3 and 8, of blood flow and urine conductivity [Winton, 1936a] and of urine flow [Winton, 1936 b]. In Figs. 4 and 6 the blood flow was recorded by Gaddum's [1929] method. The oxygen content of the blood was recorded by the following methods (Fig. 1). A second galvanometer (G2) of the same type was connected in series with the galvanometer giving scale readings from the oxygen unit. This second galvanometer, together with the light illuminating it (6 V. 24 W. headlamp heated by a large car 91

6 92 K. KRAMER AND F. R. WINTON battery) and a gas-filled type photocell (PC 2) (G.E.C. CMG 8), were placed in a light-tight box about 2 ft. long. Light from the filament of the headlamp was reflected from the galvanometer mirror and focused on to the cell, so that deflexion of the galvanometer increased or decreased the length of the image of the filament impinging on the light-sensitive surface. The photocell, in series with 5MQ., was polarized with 105 V. from a dry battery, the voltage of which remained steady, in spite of variations of resistance of the photocell, if a loading resistance of 0 5 or 1MQ2. was placed across it. The connexion of the cell to the P.X. 4 valve is shown in Fig. 1, and the anode current, supplied from accumulators, passed through the milliammeter (modified Weston relay) recording on the drum [Winton, 1936 a]. With a suitable filament in the headlamp, the relation between galvanometer input and anode current of the valve was about as accurately linear as can be seen on ordinary graph paper. The photocell could be moved from outside the box, and the second galvanometer was provided with a variable shunt, so that the pointer on the drum could be arranged to score only the relevant portion of the total oxygen scale, giving larger deflexions for the relatively small arteriovenous oxygen difference than would have been possible with an arrangement insensitive enough to cover the whole range of oxygen content. RESULTS The effiects of change of arterial pressure Two sets of effects of pressure changes on the kidney may be distinguished: (1) transient effects which are prominent within a few seconds of changing the pressure, (2) relatively permanent effects following immediately after the transient effects. The transient effects of a rise in arterial pressure which are shown in Fig. 3 are an immediate rise in the oxygen content of the venous blood accompanied by an overshoot of the increase in the inflow of blood. There is no question that these effects are genuine properties of the kidney, that they are of considerable magnitude, and that the corresponding opposite changes occur after a fall in arterial pressure. Since our recording instruments have not been calibrated under rapidly changing conditions we can, however, for the moment, only describe the phenomena qualitatively. The increase in urine flow occurs within the period of a drop and may be regarded as immediate in contrast with the delayed diuresis due to urea described below. The overshoot in blood flow and the associated diminution in arterio-venous oxygen difference

7 OXYGEN USAGE AND DIURESIS have never failed to follow a rise in arterial pressure in a great number of observations, though they differ in the degree to which they are manifested in different kidneys, Fig. 3 representing a kidney in which the effects are prominent, and Fig. 4 representing one in which they are not prominent. The outflow recorder illustrated in Fig. 4 was not suitable for showing short-lived changes in blood flow. 93 Fig. 3. Record of the effects of a change in arterial pressure on the oxygen content of blood in the renal artery and vein, on the blood flow through the isolated kidney and on the urine flow. The large rises in the " oxygen " tracing, lasting over a minute, are due to switching the oxygen unit over from the venous to the arterial blood. Note the increase in arterio-venous oxygen difference with rise in pressure, preceded by a transient fall in the difference, and the reversibility of these phenomena. The relatively permanent effects of changes in arterial pressure are recorded in Fig. 4. The arterio-venous oxygen difference, which remains practically constant during the series of increases in arterial pressure, falls slightly during the series of decreases. The changes in blood flow are

8 94 K. KRAMER AND F. R. WINTON much smaller than would have been produced by corresponding changes in arterial pressure in other organs as already described [Winton, 1932, 1937]. The increase in oxygen consumption with arterial pressure, thus shown, is characteristic of the kidney and has never failed to appear in Fig. 4. Record of the effects of changes in arterial pressure between 80 and 190 mm. Hg on the oxygen content of renal arterial and venous blood, and on the urine and blood flows from the isolated kidney. The temporary rise in the oxygen curve in the middle of each period at a different pressure level represents the change from venous to arterial blood. Note (1) the relative constancy of the arterio-venous oxygen difference, (2) the characteristically small change in blood flow with large changes in arterial pressure. any of about eighty observations on eight kidneys. Its order of magnitude, and its reversibility are illustrated in Fig. 5, which also shows the associated changes in the amount and composition of the urine secreted, and, as a contrast, the consequence of two doses of urea. The rise in oxygen consumption with rise in arterial pressure is a peculiar property of the kidney; there is no corresponding relation in the hindlimb perfused under exactly the same conditions. This might suggest that the rise in oxygen consumption in the kidney is due to the increased urine formation at higher pressures. But this, it seems, is not

9 OXYGEN USAGE AND DIURESIS 95 Minutes Fig. 5. Observations on a pump-lung-kidney preparation showing the contrast in the effects of rises in arterial pressure and of urea on (from below upwards) the blood flow, arterio-venous oxygen difference, oxygen consumption, urine flow, chloride concentration in the urine, and the creatinine urine/plasma concentration ratio. Abscissa represents time after the beginning of perfusion of the kidney.

10 96 K. KRAMER AND F. R. WINTON so, for the effect is quite as well marked in kidneys which for various reasons, e.g. after transference to the perfusion circuit, are secreting no urine as in kidneys which are secreting freely. The arterio-venous oxygen difference in the kidney is usually remarkably constant within the physiological range of arterial pressure, as illustrated in Fig. 4, and differs strikingly in this respect from that in Fig. 6. Record of the effects of changes in arterial pressure on the isolated kidney, showing in contrast with Fig. 4 that below about mm. Hg the changes in arterio-venous oxygen difference and in blood flow for a given pressure change are much greater than they are at higher levels of pressure. the hindlimb in which an increase in pressure always decreases the arteriovenous oxygen difference whether the limb be resting or active. If the arterio-venous oxygen difference in the kidney varies with arterial pressure it is almost always in the sense opposite to that in the hindlimb, Fig. 4 representing a typical kidney in which there is a barely detectable reduction in arterio-venous oxygen difference with fall in pressure but no change during the rise in pressure, and Fig. 3 illustrating a particularly sensitive kidney in which the reversible increase in arteriovenous oxygen difference with increase in arterial pressure is well marked.

11 OXYGEN USAGE AND DIURESIS The relative constancy of the arterio-venous oxygen difference in the kidney breaks down, however, if the arterial pressure be lowered too far, as illustrated in Fig. 6. When the arterial pressure is lowered in steps the Minutes Fig. 7. Observations on a pump-lung-kidney preparation showing the contrasts in the effects of changes in arterial pressure above and below a critical value of about 70 mm. Hg on the blood flow, the arterio-venous oxygen difference, and the oxygen consumption of the kidney. arterio-venous oxygen difference remains about constant down to 80 mm. or even to 60 mm. as in Fig. 7, but below this the kidney resembles a limb in that the arterio-venous oxygen difference rises with fall in arterial pressure. The contrast between the small changes in the blood PH. XCVI. 7

12 98 K. KRAMER AND F. R. WINTON flow due to change in pressure at normal pressures, and the much larger corresponding changes in flow at arterial pressures below about 70 mm. Hg are well seen by comparing Figs. 4 and 6, and again in the data charted in Fig. 7. While the relation of the arterio-venous oxygen difference to pressure seems to be essentially different above and below a critical pressure of about 70 mm. Hg, the relation of oxygen consumption to pressure does not seem to be very different as shown in Fig. 7, and it may well be that the former difference is a derivative of the difference in the relation between blood flow and pressure in the two ranges of pressure. Many kidneys when transferred from a relatively low to a higher arterial pressure show traces of the lower metabolic rate for some time, the arterio-venous oxygen difference gradually increasing during the persistence of the high pressure. A substantial fall in pressure may show the opposite effect, the metabolism gradually declining further after the initial changes due to the fall. This effect is seen in exaggerated form if after a period of abnormally low pressure, e.g mm., the pressure be raised; the arterio-venous oxygen difference at first falls considerably below its final value, which it approaches in the course of a few minutes. Anaemia of the kidney is, therefore, followed by a slowing of its metabolism rather than by an augmentation such as might have been expected if it had incurred an oxygen debt. The effects of administration of urea If urea in a dose of 5 c.c. of 20 % solution be added to the 1000 c.c. of blood in the reservoir of the pump-lung-kidney apparatus, the changes that follow can be described (1) as transient changes which are over in 2-3 min., and (2) as permanent changes which continue for half an hour or more. The transient changes following administration of urea are illustrated in Fig. 8. There is first an approximately simultaneous increase in venous oxygen content and in blood flow, accompanied by an inhibition of urine flow which is often more striking than in Fig. 8. After half a minute or so, both venous oxygen and blood flow begin to fall again, the former falling more quickly and reaching its original value just after the onset of diuresis. The blood flow also returns to its original value but more slowly. The oxygen consumption falls transiently, for the reduction in arteriovenous oxygen difference is usually greater than the rise in blood flow. Since, however, a small rise in blood flow persists for 1 or 2 min. after the arterio-venous oxygen difference has returned to its original value, the

13 OXYGEN USAGE AND DIURESIS level of oxygen consumption is slightly raised during this period. This group of changes can regularly be demonstrated whenever urea be added whatever the dose so long as it be sufficient to induce diuresis. Successive 99 -~ Fig. 8. Record showing the transient effects of a dose of urea (about 1 g./l. of blood) on the isolated kidney. Note (1) a transient fall in the arterio-venous oxygen difference, (2) a rather more prolonged rise in the blood flow, (3) a transient fall in oxygen consumption, (4) a delay in the onset of diuresis preceded by a small reduction in urine flow, (5) reduction in the conductivity of the urine accompanying diuresis. additions of urea each produce qualitatively similar effects. Exactly the same set of effects are observed if, in a double pump-lung-kidney preparation, the kidney is switched over from a circuit containing blood with less urea to one containing blood with more urea, the arterial pressure remaining unchanged. 7-2

14 100 K. KRAMER AND F. R. WINTON The relatively permanent effects of urea on the kidney may be seen to follow the transient effects in Fig. 8, and are contrasted with the consequences of changing the arterial pressure in Fig. 5. During this period the urea diuresis continues, the characteristic fall in both creatinine and chloride concentrations contrasting with the fall in creatinine but rise in chloride concentrations during pressure diuresis as shown in Fig. 5; during the urea diuresis, however, the blood flow and arterio-venous oxygen difference, and therefore the oxygen consumption are, within the limits of experimental error, the same as before the administration of urea. Owing to the theoretical importance of the observation that no detectable change in oxygen consumption accompanies urea diuresis, and the interest attaching to this conclusion from the photometric method of estimation of the oxygen content, the details of ten observations are given in Table I. In the circumstances of these experiments, TABLE I. Urea diuresis and oxygen consumption Urine Blood Oxygen Urea flow flow consumption dose increase increase increase g./l. % % % * Mean S.E. of mean i.e. with only small changes in blood flow, we believe that systematic changes in the oxygen consumption of about 2 % should be detectable. It will be seen from the table that, for a complex biological system like the kidney, the change in oxygen consumption is remarkably uniformly near zero although the urine flow is more than doubled. DISCUSSION The transient changes in blood flow and oxygen consumption immediately following changes in arterial pressure or administration of urea to the kidney require measurement with more quickly responding

15 OXYGEN USAGE AND DIURESIS 101 apparatus and observation on additional variables before an attempt at their interpretation can profitably be made. With reference to the more lasting influence of change in arterial pressure, there are three peculiarities of the kidney which are manifested at pressures exceeding a critical value of mm. Hg, but disappear at pressures below this value, so that the kidney resembles other organs in respect to them. These peculiarities are (1) the increase in oxygen consumption with pressure such that a change in the arteriovenous oxygen difference may be barely detectable within the range of mm. Hg, (2) the well-marked increased resistance to blood flow with increase in pressure, and (3) the secretion of urine. It would be tempting to relate these three to a single mechanism, but the relation cannot be quite a simple one for the first two peculiarities can be observed in kidneys which are secreting no urine either because they have only just been transferred from the dog to the perfusion circuit, or because after many hours of perfusion with no diuretic in the blood the urine flow has dried up. It is possible that in these circumstances urine formation has not ceased although no urine emerges from the ureter, but that the urine leaks back into the venules as previously suggested for quite other reasons [Winton, 1934]. An hypothesis that the relative constancy of the arterio-venous oxygen difference within the physiological range of arterial pressure implies that increase in pressure increases the proportion of vascular channels patent in the kidney without augmenting the blood flow through the individual channels, is rendered unattractive (1) by the considerable increase in resistance to blood flow through the kidney which is produced by the increase in pressure, (2) by the accurately reversible nature of these effects seen when the arterial pressure is correspondingly diminished, and (3) by the difficulty such an hypothesis would encounter in explaining even the small increase in arterio-venous oxygen difference which occasionally accompanies a rise in arterial pressure (Fig. 3). The problem whether the rise in metabolic rate with rise in arterial pressure is more directly concerned with the change in pressure or with the accompanying change in blood flow has not been solved; our only observations bearing on it are those showing that during spontaneous changes in blood flow, such as the gradual increase often occurring during the early period of perfusion of a kidney, the oxygen consumption usually hardly changes. This is illustrated in Fig. 5. The oxygen consumption of the kidney appears to be remarkably insensitive to changes in the oxygen content of the arterial blood, for

16 102 K. KRAMER AND F. R. WINTON considerable diminution of the oxygen content produced by intermission of ventilation of the lungs, or by ventilation with nitrogen produced so little change in either the arterio-venous oxygen difference or in the blood flow that we could not detect it in preliminary experiments. SUMMARY 1. In view of the conflict of evidence concerning the relation between oxygen consumption of the kidney and the various forms of diuresis, and in view of the recent development of relatively precise means (Figs. 1, 2) of continuous measurement of the oxygen content of flowing blood, some factors affecting the oxygen consumption of the kidney have been reinvestigated using the pump-lung-kidney preparation in which the blood flow through the organ can also be continuously recorded and directly measured. 2. An increase in arterial pressure produces a transient decrease in arterio-venous oxygen difference accompanied by an overshoot in the increase of the inflow of blood (Fig. 3); the onset of diuresis is immediate. Thereafter the arterio-venous oxygen difference resumes about the same value as before the change in pressure, despite the increase in blood flow, the oxygen consumption being substantially increased (Figs. 4, 5). 3. While the arterio-venous oxygen difference is substantially constant between 70 and 200 mm. Hg, this difference varies with pressure at lower arterial pressures, decreasing with increase in pressure (Fig. 6) as it does over the whole range of pressures in other organs such as the hind limb. Even at these lower pressures, however, the oxygen consumption increases with increase in pressure (Fig. 7). 4. An increase in the urea content of the perfusion fluid produces (Fig. 8) a transient reduction in arterio-venous oxygen difference, proportionately greater than an accompanying increase in blood flow; during this period there is an inhibition of urine flow. The arterio-venous oxygen difference returns accurately to its former value just after the onset of diuresis; the blood flow also returns accurately to its former value a minute or two later. The changes in oxygen consumption are, therefore, first a reduction, then an increase, and finally a return to the prediuretic value which remains unchanged throughout the diuresis (Fig. 5, Table I). 5. The relations of the oxygen consumption of the kidney to the blood flow and urine formation are discussed, and it is concluded that urine formation and oxygen consumption are not simply related.

17 OXYGEN USAGE AND DIURESIS 103 REFERENCES Bainbridge, F. A. & Evans, C. L. [1914]. J. Phy8iol. 48, 278. Barcroft, J. B. & Brodie, T. G. [1904]. J. Phy8iol. 32, 18. Barcroft, J. B. & Brodie, T. G. [1905]. J. Physiol. 33, 52. Barcroft, J. B. & Straub, H. [1910]. J. Physiol. 41, 145. Best, C. H. & Taylor, N. B. [1937]. The Phy8iological Basi8 of Medical Practice. London: Bailli6re, Tindall and Cox. Bickford, R. G. & Winton, F. R. [1937]. J. Phy8iol. 89, 198. Fee, A. R. [1929]. J. Phy8iol. 67, 14. Fee, A. R. & Hemingway, A. [1928]. J. Phy8iol. 65, 100. Gaddum, J. H. [1929]. J. Phy8iol. 67, 16P. Glaser, H., Laszlo, D. & Schurmeyer, A. [1932]. Arch. exp. Path. Pharmak, 168, 139. Gremels, H. [1929]. Arch. exp. Path. Pharmak, 140, 205. Hayman, J. M. & Schmidt, C. F. [1928]. Amer. J. Phy8iol. 83, 502. Kiese, M. & Garan, R. S. [1937]. Klin. W8chr. p Kramer, K. [1935]. Handb. Biol. Arb. Meth. 5-8, Smith, H. [1937]. The Physiology of the Kidney. N.Y.: Oxford Univ. Press. Van Slyke, D. D., Rhoads, C. P., Hiller, A. & Alving, A. S. [1934]. Amer. J. Physiol. 109, 336. Winton, F. R. [1932]. Trans. XI VthaCongresso Internaz. di Fisiol. p Winton, F. R. [1934]. J. Physiol. 83, 38P. Winton, F. R. [1936a]. J. Physiol. 87, 65P. Winton, F. R. [1936b]. J. Physiol. 87, 20P. Winton, F. R. [1937]. Physiol. Rev. 17, 408.