technique by Hemingway [1931] makes it possible to

Transcription

1 6I2.464 THE ACTION OF CYANIDE ON THE ISOLATED MAMMALIAN KIDNEY. BY L. E. BAYLISS' AND E. LUNDSGAARD2 (Copenhagen). (From the Department of Physiology and Biochemistry, University College, London.) THE behaviour of the isolated mammalian kidney when perfused with blood containing cyanide was fully investigated and discussed by Starling and Verney [1924]. We considered ourselves justified in reopening this question for the following reasons: (a) the development of the pumplung-kidney technique by Hemingway [1931] makes it possible to perfuse the kidney with cyanided blood for as long as may be desired, instead of for only about 10 min.; (b) the development of the conception that under the influence of tubular poisons, such as mercuric chloride and cyanides, the tubule walls may become permeable to the fluid within them [Richards, 1929], introduces a new approach to the interpretation of the experimental results; (c) the development by Rehberg [1926a] of the conception that loss by reabsorption and/or diffusion is less for creatinine than for any other urinary constituent, and may be zero, makes it possible, in certain circumstances, to measure changes in this rate of leakage; (d) complete suppression of osmotic activity (as indicated by the concentrations of creatinine, glucose and chlorides, for example, becoming the same in the urine as in the blood) would provide a fluid which was essentially an ultra-filtrate of blood, and hence would make it possible to estimate the concentration of diffusible calcium in the serum. METHODS. The pump-lung-kidney preparation as described by Hemingway [1931] was used with certain modifications. This consisted, essentially, in a Brodie-Dixon pump which pumped liquid paraffin into and out of ' Beit Memorial Medical Research Fellow. 2 Rockefeller TraveUing Fellow. PH. LXXIV. 18

2 280 L. E. BAYLISS AND E LUNDSGAARD. a vessel of large diameter containing both paraffin and blood, so that the movements of the paraffin were communicated to the blood without permitting any blood to come into contact with the piston of the pump: communicating with the lower end of this oil-blood vessel were a pair of rubber valves of the Bunsen pattern. So long as the excursion of the surface of separation was small, we had no trouble with undue mixing of the oil and the blood. The pump took blood from a vacuum-jacketed reservoir, and delivered it to the kidney, from which it flowed by gravity to a pair of lungs taken from the dog used to provide the blood necessary to fill the apparatus. These were enclosed in a water-jacketed funnel, from which the blood flowed to the reservoir. The blood was circulated through the lungs for at least 20 min. before connecting in the kidney. The lungs were ventilated by positive pressure with room air, and the preparation was thus acapnic. 1 g. of urea and 150 mg. of creatinine hydrochloride were added to the blood as soon as the preliminary circulation was established. At least 5 c.c. of urine were collected and discarded before the first sample was taken for analysis. Immediately after this sample, sufficient sodium cyanide, dissolved in a few c.c. of 0 9 p.c. sodium chloride, to make a concentration of about M/1500 in the circulating blood were added to the reservoir. At least 5 min. were then allowed for the concentration to become uniform throughout the circuit, and then urine and blood samples were taken as requisite. In some experiments further additions of cyanide were made, the greatest concentration reached being M/500. The following substances were estimated in the urine and serum: creatinine by the method of Folin as described by Rehberg [1926 a]; calcium by the method of Kramer and Tisdall [1921]; glucose by the method of Hagedorn and Jensen [1923]; phosphates by the method of Briggs as modified by Eggleton and Eggleton [1929]; chlorides by the method described by Volhard and Becher [1929] (these analyses were kindly performed for us by Dr Rothschild); vapour pressure by the method of A. V. Hill [1930] (these determinations were kindly performed for us by Miss Hetherington); and protein (in the urine only) by the method of Kerridge [1931]. The serum concentrations were expressed in mg. (or millimols) per 100 g. water, the water content of the serum being measured by determining the loss of weight on drying at 1100C.

3 ACTION OF CYANIDE ON KIDNEY. 281 RESULTS. Figs. 1, 2, and 3 show the results of Exps. 19,17 and 21, which we have selected as demonstrating most clearly the points to which we wish to draw attention. In Figs. la, lb, 2 and 3 are represented quantities a2 F *S 6 5." 0$3 0-' 0z ;2:.5 c; 05 Time Fig. 1 a. Exp. 19. Weight of kidney 53 g. Volume of blood in circulation 500 c.c. Excretion rates in millimols per 100 min. (chlorides in millimols per 10 min.) and urine flow in c.c. per min. directly estimated; in Fig. lc are represented quantities derived by means of the hypothesis which will be developed in the discussion. All three experiments show the typical marked increase in urine flow immediately following the addition of cyanide-confirming the earlier 18-2

4 282 L. B. BAYLISS AND E. LUNDSGAARD. observations of Starling and Verney [1924]. Later, however, the rate of flow slowly falls off, and may, eventually, almost cease, although with a good preparation, and a relatively low concentration of cyanide, recovery (as indicated by an increase in the absolute excretion rates of one ~~~~~~~~~~~~~ N S. 1*6 0 0 s1i ce ~ ~ ~ ~ Tm Fig. lb. Exp. 19. Concentration ratios (urine/serum) and urine flow in c.c. per min. Or more substances) may set in after some 1 to 12 hours, presumably owing to the removal of the cyanide in the lungs. This recovery began to take place in Exp. 21 before the second addition of cyanide. The next point to be noticed is that each substance estimated is affected by the addition of cyanide in a manner which is independent of the behaviour of the others. The behaviour was not entirely uniform in all

5 ACTION OF CYANIDE ON KIDNEY. 283 experiments, and the mechanisms responsible for the control of the excretion of water, chlorides and glucose, for example, were affected by cyanide with different rapidity, both absolutely and relatively, in different kidneys. 1 2 Water Chlorides per 10 mints. Le cc/m.. 2 Glucose 10.~9.8 7 cc/mm. Calcu 3 Phospates. 2 1 ~~~~~~~~~Chlorides per 10 mins. Creatininer M/1550 NaCN ' Time Fig. 1 c. Exp. 19. Reabsorption rates in millimols per 100 min. (chlorides in mnillimols per 10 min. and water in c.c. per min.) and urine flow in c.c. per min. Creatinine. Serum and urine were analysed for creatinine in seventeen experiments. Initially, the concentration ratio (urine/serum) was usually about 20: addition of cyanide to the perfusing blood resulted in an immediate fall, but in no case did the ratio actually reach unity (i.e. there was never a complete suppression of secretion of creatinine-or

6 284 L. E. BAYLISS AND E. LUNDSGAARD. reabsorption of water). As the rate of urine flow diminished, moreover, this concentration ratio increased. In one experiment in which the rate of urine flow remained more or less steady at about 0 5 c.c. per min. after an initial fall from a peak of 1 1 c.c. per min. immediately following the addition of cyanide, the perfusion pressure was raised from 120 mm. Hg to 140 mm. Hg, Ca 0 o1c 1200' Time Fig. 2. Exp. 17. Weight of kidney 24 g. Volume of blood in circulation 600 c.c. Concentration ratios (urine/serum) and urine flow in c.c. per min. with a concomitant increase in urine flow to 1 c.c. per min.: the creatinine concentration ratio feu from 1*48 to 1*45. Subsequent lowering of the perfusion pressure to 100 mm. Hg reduced the urine flow to 0*4 c.c. per min. and raised the concentration ratio to We, therefore, interpret this secondary rise in the creatinine concentration ratio as the result of a diminished rate of flow down the tubules,

7 ACTION OF CYANIDE ON KIDNEY. 285 the absolute rate of secretion of creatinine (or of reabsorption of water) either remaining constant, or being reduced (or increased, respectively) to a smaller extent P ca cz 0 Cz 6 8 k bo Time Fig. 3. Exp. 21. Weight of kidney41 g. Volume of blood in circulation 600c.c. Concentration ratios (urine/serum), urine flow in c.c. per min. and protein concentration in urine in mg. of protein nitrogen per 100 c.c. urine. The figures on the protein concentration curve represent the absolute rates of excretion in mg. protein nitrogen per min. Phosphates. The initial phosphate concentration of the urine was always less than one-tenth of that of the serum. After the addition of cyanide, however, the concentration ratio rose to a value greater than unity, and became, in most preparations (seven out of nine), equal to, or somewhat greater than, the creatinine concentration ratio.

8 286 L. E. BAYLISS AND E. LUNDSGAARD. Glucose was estimated in fourteen experiments. In six the glufcose concentration ratio rose, after the addition of cyanide, to a value greater than unity, and in four experiments reached the same value as that of creatinine. The rise, however, took place much more slowly than that of the phosphates, and three experiments lasted too short a time (owing to faulty technique) to allow the concentration ratio to rise above 1. When making these observations we observed that the rate of disappearance of glucose from the circulating blood after the addition of cyanide was surprisingly large (of the order of 20 mg. per min.). The resulting rapid changes in the blood-glucose concentration made some of the values for the concentration ratio somewhat uncertain, since it is impossible to determine at what moment during the collection of any given urine sample, the blood sample should be taken. The very low concentrations of sugar at the end of the experiments might, also, have affected the behaviour of the kidney in some way. In some experiments, therefore, the blood-sugar concentration was maintained, or in some cases, slowly raised, by allowing a 50 p.c. solution of glucose to drip into the blood reservoir: no significant difference was observed between these experiments and the others. In no case, of course, was it possible to maintain the blood-sugar concentration absolutely constant. It was assumed, therefore, that the rate of disappearance of glucose at any moment was proportional to the glucose concentration at that moment, and the concentration at the time when half of any given urine sample had been secreted (chosen arbitrarily to represent the mean during the period of secretion) could be calculated accordingly when necessary. In two experiments, the concentration ratio failed to rise above 1, and in two others failed to exceed We have no explanation for these anomalies except the obvious one of postulating very high reabsorptive activities in these kidneys. In one experiment, in which the blood-sugar concentration was raised by the addition of glucose, the concentration ratio reached the value of 1 and remained there in spite of a considerable increase in the rate of urine flow. By this time, however, the blood contained between 300 and 350 mg. glucose per 100 g. water, owing to an excessive rate of addition. Chlorides. As is well known, the chloride concentration ratio in isolated kidneys is less than 1. In all six experiments in which analyses were made the concentration ratio rose, after the addition of cyanide, to about unity (slightly over in three experiments, slightly under in three), and then fell steadily, reaching, in some cases, a value as low as 0-5, even though there were no indications that the kidney was recovering from the action of cyanide. Further addition of cyanide in one experiment resulted in the ratio rising once more to 0 9. Calcium. Initially, the calcium concentration ratio was about Immediately after the addition of cyanide it rose to about 0 7 and re-

9 ACTION OF CYANIDE ON KIDNEY. mained at this value irrespective of changes in the urine flow or the values of any other concentration ratios. Vapour pressure. Measurements of the vapour pressure of the blood and urine samples taken some time after the addition of cyanide were made in four experiments. In every case the urine was less concentrated than the blood, the ratios of vapour pressures being about 09. In two experiments the ratio fell from 09 to 0O85 as the experiment was continued, in one experiment it fell from 094 to 090, and in one experiment it rose from 090 to 095. These determinations are not in disagreement with those of the chloride concentration by chemical analysis shown in Fig. lb, since all the urine samples analysed for vapour pressure were taken some time after the addition of cyanide when the chloride concentration was also low. Protein. The concentration of protein in the urine was only measured in two experiments, but rough estimates were made in many others during the course of the estimations of creatinine and phosphates, when the protein was precipitated by picric acid and trichloracetic acid respectively. The urine obtained before the addition of cyanide contained small amounts of protein in almost every case (see Fig. 3). In the early stages of cyanide poisoning the absolute quantity of protein excreted remained constant, and equal to that before the addition of cyanide, i.e. when there was an increase in the rate of urine flow (as there usually was) the concentration of protein decreased. In the terminal stages, when the urine flow had decreased, the absolute rate of excretion may also have increased in some experiments. Proteinuria, therefore, does not necessarily result from simple asphyxia. DISCUSSION. Before the administration of cyanide, the concentration of creatinine in the urine is greater than that in the serum. After the addition of cyanide, the rate of excretion of creatinine (Fig. la) is reduced progressively; this might be interpreted as a progressive inhibition of secretory activity. Normal urine from the isolated kidney, on the other hand, contains chlorides, glucose, phosphates and calcium in lower concentration than in the serum; the excretion rates of these substances are increased by the addition of cyanide, and this might equally be interpreted as an inhibition of reabsorptive activity. These last excretion rates, however, do not increase progressively, but fall during the later stages of cyanide poisoning; this fall results, presumably, from a reduction in the rate of supply of glomerular fluid to the tubules, since it is 287

10 288 L. E. BAYLISS AND E. LUNDSGAARD. unlikely that the rate of reabsorption would increase, at any rate, so quickly. In Exp. 19 the final excretion rates were of the order of 1/10th of those immediately after the addition of cyanide, so that the rate of supply of glomerular fluid must have been reduced to about 1/10th of its initial value. Now in the circumstances obtaining in our experiments, the rate of formation of glomerular fluid will be determined largely, if not solely, by the glomerular pressure. In our experiments, moreover, the arterial pressure was kept constant, while the rate of blood flow remained unchanged throughout the period of cyanide poisoning (it was increased in most experiments some 30 p.c. as a result of the addition of cyanide). In these circumstances we find it difficult to believe that the glomerular pressure really falls sufficiently to account for so large a reduction in the rate of formation of glomerular fluid, and we prefer to imagine that a progressively increasing fraction of the fluid produced in the glomeruli leaks out of the outer walls of the capsules of Bowman and/or the proximal tubules, and so never reaches the cells engaged in reabsorption. It is possible to account for the observed effects by postulating a leakage from the distal tubules. Such an hypothesis, however, does not lend itself so readily to quantitative treatment, and requires that the kidney should do considerably more osmotic work during the excretion of a given quantity of urine of a given composition than the hypothesis preferred above. It is possible, on the other hand, that an cedematous swelling of the tubule cells may occur. This would have the effect of partially blocking the lumen of the tubules and so increasing the intracapsular pressure. There would thus be a real reduction in the rate of glomerular filtration, and the effects observed in these experiments might be accounted for without postulating any leakage from the capsules or the tubules. Although artificially perfused organs are well known to be subject to cedema, we have no evidence that the tubule cells in our experiments really were sufficiently swollen to account for the effects observed, but the conception of leakage may well have to be rejected in favour of that of cedema when further evidence has been obtained. The fact that the concentration ratio of creatinine (Fig. lb) was always greater than 1 shows that either (a) some secretion of creatinine was still proceeding, or (b) that water was continually being reabsorbed. If (a) is true, then cyanide must not only stop the tubules reabsorbing phosphates (the initial ratio being less than 1), but also start them secreting it (the concentration ratio becoming greater than 1): and, moreover, they must secrete it at just the right rate to make the phosphate concentration ratio approximately the same as the creatinine concentration ratio at all

11 ACTION OF CYANIDE ON KIDNEY. rates of urine flow. We, therefore, prefer the second hypothesis and assume that creatinine is neither secreted nor reabsorbed: it is only necessary, then, to assume that the reabsorption of phosphates is entirely suppressed, while that of water is only partly suppressed, in order to account for the approximate identity of the concentration ratios at a value greater than 1. The fact that the rate of excretion of creatinine (Fig. la) falls progressively after the addition of cyanide must then be the result of the diminished supply of glomerular fluid to the active parts of the tubules. Multiplication of the creatinine concentration ratio by the urine flow shows that in Exp. 19 before the addition of cyanide, the rate of supply of glomerular fluid was 13 c.c. per min., that immediately after the addition of cyanide the rate fell to 6 c.c. per min., and that during the collection of the last sample it was 045 c.c. per min. The rate of leakage, therefore, must have steadily increased with time after the addition of cyanide: in some experiments there were indications of a diminution after about 41 to 2 hours. If we may assume that there is no secretion or reabsorption of creatinine, we can calculate the rates of reabsorption of the other substances. The calculations are made as follows: (1) Rate of supply of glomerular fluid to the active parts of the tubules = Ga- [creatinine]urine x(urine flow), [creatinine]ersm square brackets indicating concentrations. (2) Rate of active reabsorption of water = W = G - U, where U is the urine flow. (3) Rate of active reabsorption of any substance, A = RA = G X[Ax].m - U x [A].ri. 289 These rates are shown in Fig. 1 c. The rate of reabsorption of water, and of all solutes estimated, progressively decreases, while the rate of leakage progressively increases (the assumption that there is no leakage before the addition of cyanide is arbitrary, since there is at present no means of estimating its value). Reabsorption of phosphate is stopped completely almost at once. Reabsorption of glucose is stopped more slowly but, eventually, almost completely. Reabsorption of chlorides proceeds fairly rapidly even at the end; it must be remembered in this connection that the initial rate of reabsorption of chlorides is very much larger than that of any other substance. The rate of reabsorption of calcium is a constant fraction of the rate of reabsorption of water; this, of course, is an expression of the constant concentration ratio.

12 290 L. E. BAYLISS AND E. LUNDSGAARD. Table I presents some calculations as to the quantity and composition of the fluid reabsorbed by the tubules in the five successful experiments in which the chlorides were estimated. The following points are brought out in this table: (1) The reabsorption rates fall steadily after the addition of cyanide in all experiments except Nos. 17 and 21. In No. 21 there is reason to suppose that some recovery was setting in, but in No. 17 this explanation of the rise in reabsorption rate is hardly warranted, since the subsequent fall took place without any further addition of cyanide. (2) There is no necessary correlation between the concentration ratio and the reabsorption rate. For example, between the last two samples of TABLE I. Reabsorption rates Chloride concentration Cyanide concentration Chloride - millimols/100 g. water and time concen- Chloride Exp., tration Water millimols Reabsorbed min. ratio c.c./min. per 100 min. fluid Serum M/ ,,J M/ ,, M/ M/ M/ M/ NOTE. "Time" indicates the time intervening between the addition of cyanide and the middle of the collection of the urine sample. The chloride concentration of the urine before the addition of cyanide has been assumed; a considerable error in this value would have very little effect on the calculated concentration ratio, or the reabsorption rate. Exp. 17, the concentration ratio fell markedly in spite of a big reduction in the rate of reabsorption of chloride: this resulted from the even bigger reduction in the rate of reabsorption of water. It is not justifiable, therefore, to infer an increase in the rate of reabsorption of chloride whenever the concentration ratio diminishes. (3) It might appear surprising that the chloride concentration of the reabsorbed fluid is of the same order after the addition of cyanide as it was before, in spite of the very large change in the concentration ratio. It

13 ACTION OF CYANIDE ON KIDNEY. 291 will be remembered that Rehberg [1926b] found that in man the chloride concentration of the reabsorbed fluid remained remarkably constant so long as the concentration in the plasma remained approximately normal. The large change in the concentration ratio is accounted for by the assumption that before the addition of cyanide, practically the whole of the glomerular fluid was reabsorbed by the tubules, so- that a relatively small difference between the chloride concentration of the reabsorbed fluid and that of the glomerular fluid would have a large effect on the chloride concentration of the fully formed urine. In the presence of cyanide, however, a much smaller proportion of the glomerular fluid is reabsorbed, with a correspondingly smaller effect on the composition of the urine. Thus, if 1000 c.c. of glomerular fluid containing 100 millimols of chloride were supplied to the tubules in a given time, and 900 c.c. of a fluid containing 110 millimols of chloride per litre were reabsorbed, there would be left 100 c.c. of urine containing 1 millimol of chloride, and the concentration ratio would be 0'1. If, on the other hand, after the addition of cyanide, 200 c.c. of glomerular fluid were supplied to the tubules in the same time, and 100 c.c. of fluid of the same composition as before were reabsorbed, there would again be left 100 c.c. of urine, but it would contain 9 millimols of chloride, and the concentration ratio would be 0*9. It would thus appear that the reabsorption of chloride and water are inhibited to approximately the same extent by the concentrations of cyanide used in these experiments, although an inspection of the figures in the table will show that the chloride reabsorptive mechanism appears to be somewhat more resistant than the water reabsorptive mechanism. It will be noticed that we were unable to suppress completely the osmotic activity of the kidney, as indicated by the chemical analyses, but also, more conclusively, by the measurements of vapour pressure. This recalls the work of Dixon [1929], in which he found that cyanide, in any concentration, was only able to inhibit some p.c. (depending on the animal) of the oxygen uptake of sliced kidney tissue. The independence of the calcium concentration ratio both of the other concentration ratios and of the urine flow seems to indicate that the walls of the tubules become permeable to calcium, after the addition of cyanide, to the same extent as are the glomerular membranes. The concentration ratio of about 0-7 is thus a measure of the ratio of diffusible calcium to total calcium in the serum (the mean of twenty determinations in ten different experiments is 0x72 with a maximum of 0-83 and a minimum of 0.59).

14 292 L. E. BAYLISS AND E. LUNDSGAARD. According to Hunter [1931], the accepted value for the fraction of total calcium not combined with protein in human plasma is 55 p.c. Figures of this order were obtained by ultra-filtration through collodion of dog serum by Hertz [1930], but the literature (see Reed [1928]) contains many reports of values of the order of 70 p.c. Rona and Takahashi [1911], and, more recently Rona and Melli [1925], using the compensation dialysis method, regularly found values of the order of 70 p.c.: the last-named authors obtained lower values, of the order of 50 p.c. when they added lecithin to the collodion membranes used for dialysis. Since there was nearly always some protein in the urine obtained in our experiments, it is probable that our estimate of the concentration of diffusible calcium is somewhat too large, but it is improbable that this concentration could be raised 50 p.c. by the relatively small amounts of protein obtained. We were unable to observe, moreover, any correlation between the protein concentration in the urine and the calcium concentration ratio. Our experiments, therefore, indicate that the concentration of diffusible calcium lies between 60 and 70 p.c. of the total calcium concentration. The close approximation of the phosphate concentration ratio to the creatinine concentration ratio indicates that the whole of the phosphate in the serum is diffusible. This conclusion is at variance with that of Eicholtz and Starling [1925], who also observed the increase in phosphate concentration ratio from almost zero to approximately unity, after the addition of cyanide to the blood perfusing a kidney. They, however, ascribed this increase to an increase in the permeability of the glomerular membranes, so that colloidal calcium phosphate could pass through them, it being assumed that practically the whole of the phosphate in the serum was in this form. If this were the case, the ratio of extra calcium excreted after the addition of cyanide to extra phosphorus (in mg.) should be 1*94, the ratio in Ca3(PO4)2: actually, thirteen determinations in seven experiments gave ratios varying between 0 5 and 0 95, and in the majority of cases the whole quantity of calcium excreted after the addition of cyanide was less than that of phosphorus. The hypothesis of Eicholtz and Starling is further negatived by the fact that the concentration ratio of calcium is independent of the rate of urine flow, while that of phosphates varies inversely with it: and by the fact that no increase in the excretion of protein occurs at a time when the excretion of phosphate has increased to its maximum, so that no very great increase in the permeability of the glomerular membranes can have taken place.

15 ACTION OF CYANIDE ON KIDNEY. 293 SUMMARY. 1. Perfusion of an isolated kidney with blood containing cyanide in concentrations up to M/500 results in the composition of the urine approximating to that of an ultra-filtrate. 2. It is not possible, however, entirely to suppress the activity of the kidney, and so long as any urine is obtained it all, it differs appreciablyin composition from that of an ultra-filtrate. 3. The behaviour of the kidney in these circumstances appears to be most easily and completely accounted for by a combination of the filtration-reabsorption hypothesis with the conception that appreciable quantities of glomerular fluid may be lost from the tubules by diffusion. 4. The excretion of each substance analysed is affected by cyanide in a manner which appears to be independent of the behaviour of the others. 5. Evidence is presented to show that some 60 to 70 p.c. of the total calcium in the serum, and the whole of the phosphates, can pass through the glomerular membranes. We are indebted especially to Miss Mary Hetherington and Dr Paul Rothschild for making the analyses of vapour pressure and chlorides respectively, to Dr F. R. Winton for many valuable suggestions during the course of the work and the presentation of the results, and to Prof. E. B. Verney and Prof. C. Lovatt Evans for their help in the preparation of the manuscript. REFERENCES. Dixon, M. (1929). Bio-Chem. J. 23, 812. Eggleton, G. P. and Eggleton, P. (1929). J. Phy8iol. 68, 193. Eiclfoltz, F. and Starling, E. H. (1925). Proc. Roy. Soc. B, 98, 93. Hagedorn and Jensen (1923). Biochem. Z. 135, 46. Hemingway, A. (1931). J. Physiol. 71, 201. Hertz, W. (1930). Biochem. Z. 217, 337. Hill, A. V. (1930). Proc. Roy. Soc. A, 127, 9. Hunter, D. (1931). Quart. J. Med. 24, 393. Kerridge, P. M. T. (1931). Lancet, 221, 21. Kramer, B. and Tisdall, F. F. (1921). J. Biol. Chem. 47, 475. Reed, C. I. (1928). J. Biol. Chem. 77, 547. Rehberg, P. B. (1926a). Bio-Chem. J. 20, 447. Rehberg, P. B. (1926b). Bio-Chem. J. 20, 461 Richards, A. N. (1929). Methods and Results of Direct Investigations of the Function of the Kidney. Baltimore. Rona, P. and Melli, G. (1925). Biochem. Z. 168, 242. Rona, P. and Takahashi, D. (1911). Biochem. Z. 31, 336. Starling, E. H. and Verney, E. B. (1924). Proc. Roy. Soc. B, 97, 321. Volhard F. and Becher E. (1929). Handb. biol. ArbMet. Abt. iv, Teil 5, 2. Halfte, Heft 3, 427 and 474.