Starling2); for the blood-pressure is insufficient to overcome any

Transcription

1 ON DIURESIS AND THE PERMEABILITY OF THE RENAL CELLS. BY ARTHUR R. CUSHNY. (Five Figures in Text.) (From the Pharmacological Laboratory oj the University of Michigan.) THE process of secretion in the renal cell may be resolved into two distinct phases-the entrance into the cell of the molecule to be secreted, and its extrusion. Any molecule penetrating the cell is extruded by it, and the force employed in executing this is the question at issue between Ludwig and Heidenhain, which has engaged the attention of most subsequent workers in this field. An eqlially important subject is the relative tendency of different molecules to enter the cells, or the permeability of the latter. And in fact if this could be determined for the glomerulus, the question of the force employed in extruding the molecule would also be decided, since the followers of Ludwig admit that unless the glomerulus is permeated with practically no resistance by the non-proteid constituents of the plasma, the theory of Ludwig must be abandoned (Tammann1, Starling2); for the blood-pressure is insufficient to overcome any osmotic resistance such as would oppose itself if the capsule resisted the passage of any of the non-proteid constituents. On the other hand, the Bowman-.Heidenhain theory, that only water and the inorganic salts are excreted thr ugh the glomerulus, involves the conclusion that the capsule is only partially permeable, permitting the entrance of salts but not of urea, sugar, &c. The glomerular fluid according to this view contains the salts in the relative proportion in which they occur in the urine. In the tubules the reabsorption of certain constituents of the glomerular fluid postulated by the mechanical theory requires the partial permeability of the cells, which permit the entrance of certain solids and of water, while rejecting others, such as urea. Some of the PH. XXVII. I Zeitschr. f. physikal. Chemie, xx. p This Journa7l, xxiv. p

2 430 A. R. CUSINY. glomerular fluid doubtless escapes without coming into intimate contact with the walls of the tubules, and this would reach the ureter in the same state as it left the glomerulus, were it not burdened with the rejected constituents of that part which came into more immediate connection with the absorbing cells. The larger the fraction escaping the selective influence of these cells, the more closely must the urine come to resemble the glomerular fluid'. According to the theory of vital secretion, the cells of the tubules resemble those of the capsule in being partially permeable, but here urea can penetrate, while the salts of the plasma are rejected. Heidenhain, it is true, is not quite clear in his account here, for he ascribes the excess of fluid to the tubules in the diuresis induced by urea and salts2, and this seems to suggest that the salts also are excreted here when present in such excess as to induce diuresis. Both theories thus admit the partial permeability of the cells of the tubules, for while the mechanical view requires that a fluid corresponding to the plasma in all points except in its content of proteids shall pass along the exterior of the cells and there be subjected to a selective diffusion, the vital theory involves the passage of the lymph along the internal surface of the cells and the permeation of certain of its constituents. As regards the behaviour of the individual constituents the two views are diametrically opposed, Ludwig holding that the urea fails to diffuse into the cells, while the chloride finds ready entrance; Heidenhain supposing that the urea penetrates and that the chlorides are rejected. The relative amount of any two solids in the urine, e.g. of chloride and urea, is determined by two factors if the mechanical theory be correct, their relative amount in the blood and the resistance each meets in entering the cells of the tubules. According to the theory of vital secretion, it depends upon (1) the proportion in which they exist in the blood, (2) the degree in which they are capable of permeating the cells by which they are severally excreted, and (3) the activity of these cells. My attention was directed to the relation of the salts to the renal epithelium by the observation that the chlorides of the urine were considerably augmented in the glycosuria induced by phloridzin. Katsuyama3 noted a similar increase in the sodium and chloride in caffeine 1 Cf. Starling. This Journal, xxiv. p Hermann's Handbuch, v. p Zeitschr. f. physiol. Chenmie, xxvin. p. 587; xlxii. p. 235.

3 DIURESIS OF RENAL CELLS. 431 and urea diuresis. This cannot easily be explained on the prevailing Bowman-Heidenhain theory, and seemed to indicate some relation between the chlorides and the renal cells different from that of the other urinary constituents. The excretion of the salts by the kidney has recently been investigated by Magnus1 and Gottlieb, who compared the behaviour of sodium chloride solution inijected intravenously in one series of animals with that of the sulphate in another series. In order to eliminate differences in the degree of hydraemia induced, Magnus used solutions of equal osmotic pressure, his hemaglobin estimations showing that in this way approximately the same dilution of the blood was obtained. More recently Sollman2 has confirmed and extended Magnus's observations by a similar method. The actual amounts of chloride and sulphate were not equal in these experiments, and in fact approximated the proportion of 3: 2. Before I became acquainted with Magnus's results I had adopted a method which eliminates the difference in the degree of hydraemia induced by the salts and at the same time permits the injection of equal quantities-namely, the simultaneous injection of the two bodies under examination. And as the difference in the behaviour of two such bodies as the chloride and sulphate of sodium3 are obviously due to the acid radicles, the solution injected was made up so as to contain an equal number of these. The standard was a normal solution of sodium chloride (Cl = 3-55 per cent.) and the sodiuim sulphate solution was made up to contain an equal number of sulphate radicles; i.e. SO4 = 9-6 per cent. (or sodium sulphate 14,2 per cent.). Similarly a solution of sodium phosphate was formed, neutralized with phosphoric acid and diluted until it contained 9'5 per cent. of P04. The urea solution ordinarily used was 6 per cent. When equal quantities of two of these solutions were mixed, the resulting fluid contained an equal number of the bodies under consideration, the anions in the case of the salts, or urea. Of course when such a mixture was injected into the blood no such equality was maintained, but the excess of chloride and of sulphate in the body was at any rate equal, and the results of this excess were the first objects of investigation. The rabbits used in my experiments were anaesthetised with I Arch. f. exp. Pathologie u. Pharmakol, XLIV. p. 68 (I.); XLIV. p. 396 (II.); my. p. 210 (III.); Gottlieb. u. Magnus, Ibid. XLV. p. 223 (IV.) and p. 248 (V.). 2 Arch. f. exp. Path. u. Pharm. XLVI. p Most of my experiments were done with these two salts, as their behaviour towards other organs suggested that the kidney would react very differently towards them, and at the same time the sulphate is only slightly poisonous. 28-2

4 432 A. R. CUSHNY. urethane (1-5 g. per kg.), or where the urea was to be estimated, with paraldehyde (1 g. per kg.). A Naunyn-Pfaff cannula was bound in the bladder and a ligature tied round the bladder between the orifices of the ureters and of the urethra. The animal was kept warm and the urine collected for 2-3 hours in order to ascertain the normal rate of secretion and where possible to obtain enough for chemical examination. When, as not infrequently happened, the secretion was insufficient for this purpose, some of the urine obtained from the bladder at the operation was added to it. After the normal secretion had been ascertained, a cannula was tied in the jugular vein and attached to a burette containing a warmed mixture of the two solutions under examination, which was allowed to flow in at the rate of 1-4 c.c. per minute according to the toxicity of the salts used. The first 2-4 c.c. of urine collected after the infusion commenced was not used for analysis as it contained some normal urine from the bladder and cannula c.c. of solution was injected and the urine was collected every fifteen minutes during the diuresis and afterwards every half-hour or hour. The first experiments were performed with chloride and sulphate of sodium, the mixture actually injected containing 1P775 gr. of Cl and 4'8 gr. of SO4 in 100 c.c. The chloride of the urine was determined by Volhard's method, the total sulphate by precipitation with barium chloride and weighing. Exp. I. A rabbit of 1500 grms. weight was anststhetised with 3 grms. urethane p.m urine collected 4c.c infusion into the jugular vein of 50c.c. of the solution of chloride and sulphate of sodium. Chloride Chloride Sulphate Water Chloride Chloride equiva- in Sulphate Sulphate equiva- Sulphate Time infused in c.c. Urine in c.c. infused (Cl) in urine lents in urine urine per cent. infused (S04) in urine lents in urine in urine per cent *0031 0'078 - '0012 '031 (per 15') - 0' ' ' '639 '258 '0078 *373 1'728 *349 '0036 * '2485 '1039 '0029 *3248 0'672 '352 ' '5 - '0214 '0006 ' '201 ' _ ' ' '0026 2' *338 ' '5 -J J Total X5 '8875 '3900 '011 2' * In the above analysis, as in all others in this paper, the chloride and sulphate are stated in terms of Cl and SO4, neglecting the base. In

5 DIURESIS OF RENAL CELLS. 433 the columns marked " chloride equivalents " and " sulphate equivalents " the total amount in the urine is divided by the atomic weight of chlorine and the molecular weight of SO4 in order to permit of a comparison of the number of chloride and sulphate radicles present in the urine. In the accompanying curves of excretion the amounts are those of these two columns =l ~~~~ = Fig. 1. Curve of excretion in Exp. 1. The heavy unbroken line represents the urine, the light line the sulphate equivalents ( =-9-64)' The broken line the chloride equivalents = 5. along the base.. The duration of the injection is indicated by the heavy line This experiment was repeated a number of times with practically the same results, the only difference of importance being that the chloride did not generally disappear entirely from the urine as in Exp. I. It always fell to a mere fraction of that present in the earlier part of the experiment, however, and the percentage also diminished. The uniform result in this series was a marked diuresis, the fluid excreted much exceeding that injected; the diuresis began within a few minutes of the commencement of the infusion and diminished after

6 4.34. A. B. CUSHNY. its termination, but the secretion did not fall to the normal during the observation, i.e. within 2v hours after the injection began. The chloride of the urine rose with the injection, not only in absolute amount but also per cent., attained its maximum with the maximal diuresis, and then rapidly fell in absolute amount and per cent. In the course of 2-2j hours the percentage reached that of the normal urine or below it, and in some cases towards the end of the experiment no reaction was obtained on the addition of silver nitrate. The sulphate of the urine rose in absolute amount immediately after the injection began and reached its maximum about the same time as the chloride; but it fell more slowly and never disappeared from the urine within 2-3 hours of the injection. The percentage of sulphate rose during the injection and continued rising throughout the time of observation, the urine finally containing 2-3 per cent. of sulphate. The whole of the chloride and sulphate injected was not excreted when the experiments were broken off. This is the more remarkable because there must have been in Exp. I. an excess of chloride in the tissues at a time when there was no chloride in the urine. Magnus, noted similarly that after an infusion of dilute salt solution there may be a large surplus of water and salt in the tissues, while the urine has retuirned to its normal quantity. The total weight of chloride excreted from the beginning of the injection to the end of the experiment was less than the total weight of sulphate, this agreeing with the result of Magnus. Even when the quantities were reduced to equimolecular values the sulphate radicles were considerably more numerous than the chlorides in experiments lasting two hours or more. In the earlier stages of the experiment however (marked diuresis), the chloride ions exceeded the sulphate. Thus in Exp. I. between 2.35 and 3.20 the equimolecular ratio of the total chloride and sulphate excreted was 108: 94. In the course of the next 1 hours it changed to The great mass of the chloride thus appears early in the experiment, while the sulphate is more evenly distributed throughout the time of observation. Magnus' statement that the sulphate is more readily excreted by the kidney than the chloride (harnfahiger) thus seems to require some qualification, for in the stage of marked diuresis the chloride appears in larger quantities. An invariable feature in my experiments was the parallelism of the water anid chloride of the urine; they rose and reached their maximum I. p. 83.

7 DIURESIS OF RENAL CELLS. together and then fell together, though in some cases the chloride disappeared while there was still a small secretion of fluid. The sulphate also ran parallel to the others in the first stage of marked diuresis, but then fell much more slowly than either, as is shown by the graphic representation in Fig. 1. The variation in the relative amounts of chloride and sulphate in the urine in different phases of the diuresis suggested the determination of the amounts in the blood at different stages. In the rabbit sufficient blood for examination cannot be drawn repeatedly without vitiating the results, and a series of experiments was accordingly performed in the same way as the first series except that some of the animals were bled to death-durina the diuresis and others at later stages. The urine was examined for chloride and sulphate each 15 minutes. The blood was defibrinated, the serum obtained by the centrifuge, and the proteids precipitated by boiling with acetic acid. The filtrate and washwater were neutralised, evaporated, and charred, and the salts repeatedly extracted with boiling water and estimated by the methods adopted in the examination of the urinie. The changes in the urine corresponded to those observed in Exp. I., so that it is unnecessary to give them in detail. Exp. II. Rabbit of 1660 gr. weight antesthetised with 2 5 gr. of urethane at 11 a.m. From p.m. 25 c.c. of a mixture of equal parts of 5'85 /o NaCl and /0 Na2SO4 was infused into the jugular vein. The animal was bled to death at From the urine amounted to 28 c.c., so that the animal was killed at the height of the diuresis. In the urine the chloride amounted to gr. ( 373 per cent.), the sulphate to gr. (-546 per cent.). The serum contained per cent. of chloride and per cent. of sulphate. ExP. III. A rabbit of 2000 gr. weight anesthetised with 3 gr. of urethane p.m. 40 c.c. of the same mixture was infused into the jugular vein. The course of the experiment resembled that of Exp. I. From p.m. 12 c.c. of urine were collected and the animal was then bled from the carotids. The urine last collected (12 c.c.) contained *0114 gr. chloride (0 095 per cent.) and 0-24 gr. of sulphate (2-0 per cent.), while the serum contained per cent. of chloride and per cent. of sulphate. Comparing these results, Chloride per cent Sulphate per cent Exp. II. (diuresis) Serum ,259 Urine Exp. III. Serum (l) Urine (1) Three hours after the infusion of sulphate in the dog, Magnus found none of it in the serum, but he states that dogs excrete it much more rapidly than rabbits. Thus the kidney secretes in the first stage a urine in which the chloriide stands to the chloride of the plasma in the ratio of about 2: 3,

8 436 A. R. CUSHNY. while in the last phase it bears the proportion of 1: 5. The sulphate of the urine in the first phase bears the ratio of 2 :1 to that of the plasma, in the last phase of 10: 1. The relative amounts of the salts in the blood thus do not appear to be the chief determining factor in their excretion. Nor do these experiments support the theory that the kidney recognising the presence in excess of some constituent in the blood proceeds to excrete it, for the excess of chloride was the same as of sulphate, yet they were not equally eliminated. The same failure of the kidney to excrete the useless while retaining the valuable constituents of the blood is evident in many of Magnus' and Sollmann's results, and was well exemplified in several of my experiments in which sodium sulphate was injected alone and in which there was therefore no excess of chloride in the plasma. Exp. IV. A rabbit of 2100 gr. weight anmsthetised with 3 gr. urethane. Urine collected from =14 c.c. (normal) , infusion of 30 c.c. sodium sulphate solution (14.2 per cent.). Chlloride in Chloride Sulphiate SulPhate Water Urine Chloride urine in in urine Sulpate Sulphaate in urine in in urine Time injected in c.c. in urine equivalents per cent. injected in urine equivalents per cent. I ' '083 - '021 * 149 in 1imins ' '0021 ' c.c. 50 '1171 '0033 * gr. ' ' *5057 '0088 '778 In this animal the chloride available for excretion by the kidney must have been small in amount, since the normal urine contained only 0'08 per cent., yet the injection of chloride-free sulphate was followed at first by a larger excretion of chloride equivalents than of sulphate. This lasted but a short time, it is true, but it is sufficient to demonstrate that the excess over the normal of one salt in the blood does not lead to a corresponding excess of. that salt in the urine. In some of the other experiments in which only sulphate was injected the chloride increased to a marked extent but did not actually exceed the sulphate units in the urine. In these experiments the diuresis was not so marked as in Exp. IV. A similar result will, however, be noted in Exp. VII. The relative amounts of chloride and sulphate in the urine thus vary independently of their proportions in the plasma and also of the degree in which each is present in excess. In Bowman and Heidenhain's scheme the proportion of sulphate and chloride in the urine is that of the glomerular fluid and is determined by the amount of each entering the secretory cells of the capsule and the activitv of these cells. The secretory activity of these cells can scarcely be

9 DI URESIS OF REATAL CELLS. 437 held to differentiate between the two salts in solution, but can only tend to expel impartially those constituents which enter its field of action. So that the proportion in which they are secreted must be determined by the proportion in which they enter the cells, that is by their relative concentration in the lymph and their ability to diffuse into the cells. As the proportions in the urine are not dependent on the concentration in the plasma, the adherents of the theory of special secretion must resort to the explanation that the permeability of the cells of the capsule varies during the course of the experiment, being greater for chloride in the beginning and for sulphate in the later phases. Further, the special secretion may act to the disadvantage of the organism (Exp. IV.), and may in fact, as in some of Magnus' and Sollmann's experiments, lead to the retention in the body of the cause of disturbance and the elimination of the remedy. The changes in the urine described may be explained much more simply on the view that the tubules alter the glomerular fluid by absorbing some of its constituents. Sollmiann has already discussed this possibility, but rejects it because he believed that if absorption occurred the organic and inorganic constituents of the urine would be equally affected by it, which does not appear to me to be a necessary corollary. To explain my results the only assumption necessary is that the sulphates are absorbed by the tubules with greater difficulty than the chlorides. In the first stage of hydraemia (Magnus) and diuresis both pass into the lumen of the capsule in large quantity along with the fluid, and the whole passes through the tubules so rapidly that much of it fails to come into intimate contact with the lining epithelium. The urine thus resembles the glomerular fluid as far as these salts are concerned. In the later stages, as the hydraemia passes off (Magnus), the flow from the glomerulus becomes slower and, the fluid remaining longer in the tubules, each molecule has an opportunity to enter the cells. Much of the chloride follows this route along with some of the water and probably some sulphate, while most of the sulphate, failing to diffuse into the lining epithelium, appears in the urine. The diffusible constituents of the glomerular fluid therefore decrease in the urine together, both in percentage and absolute amount; the sulphate diffusing with greater difficulty, decreases in absolute amount owing to the smaller percentage in the blood and also probably owing to some of it being absorbed, but increases in percentage because the fluid in which it was originally dissolved escapes in large part through the epithelium. An analogy may be drawn with the behaviour of the intestinal mucous

10 438 A. R. CUSHNY. membrane, which ordinarily absorbs almost all the chlorides of the food, but which allows some to escape in the faeces when the movement of the bowel is accelerated by a purge and less time is allowed for the chlorides to lie in contact, with the walls. The slow permeation of the sulphates through the walls of the renal tubules is in accord with their behaviour towards 'other cells. Their slow absorption in the intestine has led to their use as saline cathartics and appears to be due to their failure to peinetrate the epithelial cells. The red blood corpuscles similarly are pernmeated much more slowly by the sulphate than by the chloride of ammoniuiml, and gelatin discs take up less fluid from sulphate than from chloride solutions2. Many colloids, such as the globulins, are thrown out of solution more readily by the sulphates than by the chlorides3, and some crystalline substances resemble them in this4. On the other hand, certain membranes seem to absorb sulphate solutions as readily as chloride, as was shown for the pleura by Leathes and Starling5, and was rendered probable for the capillary walls by Magnus6 and Sollmann. The slow absorption of the sulphate in the renal tubules, which I have assumed, is therefore not without analogy, and certainly explains the changes in the relations between the chlorides and sulphates of the urine more easily than is possible on the view of special secretion. Another feature in the urinary secretion of the rabbit may also be explained in this way-the appearance of large amounts of precipitate which is a characteristic of the normal urine. These salts were originally excreted in solution according to both Ludwig and Heidenhain's views, and it is much more simple to explain their precipitation by the reabsorption of the solvent in the tubules than by any other hypothesis. Heidenhain attempts to account for the presence of particles of indigo bluie in the tubules by their precipitation by the salts of the glomerular secretion, but an even more doubtful theory would have to be formed to explain the precipitation of these carbonates without absorption of some constituent by the tubules. It is needless to state that this absorption is not a simple diffusion such as Ludwig at first I Gryns, Arch.f. d. ges. Physiol. LXIII. p. 86. Hedin, Ibid. LXVIII. p. 229, and LXX. p Hofmeister, Arch.f. exp. Path. u. Pharm. xxviii. p Lewith, Arch. f. exp. Path. it. Pharm. xxiv. p. 1. Hofmeister, Ibid. p Pohl, Zeitschr. f. phys. Chem. xiv. p Young, This Journal, xxi. p. xvi. 5 This Journal, xviii. p II. Mittheilutng.

11 DIURESIS OF RENAL CELLS. 439 assumed. Some unknown force causing a current from the lumen towards the blood must be assumed here exactly as in the intestine. Which constituents of the glomerular fluid shall be subjected to this force, however, is determined by their diffusibility into the cells'. The further question as to the diffusibility of the chloride and sulphate through the glomerular capsule could be determined absolutely only by a comparison of the serum with the glomerular filtrate, but my results afford some indirect evidence on the point. During diuresis, less absorption taking place in the tubules, the urine resembles the glomerular fluid more closely than usual, but even here absorption probably occurs. In Exp. II. the 28 c.c. of urine last secreted contained ar. of sulphate and gr. of chloride. This amount of sulphate corresponds to that contained in 59 c.c. of serum, which would carry also gr. chloride. Thus ifs there had been no absorption, and all the constituents of the plasma except the proteids had passed through the capsule, the urine would have amounted to 59 c.c. with gr. chloride and gr. sulphate. The two alternative interpretations are that sulphate permeates the capsule more readily than either water or chloride, or that 31 c.c. of water and gr. chloride were reabsorbed in the tubules. In view of the absorption of chloride and water in the later phases and the inherent improbability that sulphate diffuses more readily than water, the latter view is certainly the more plausible -that some absorption takes place even during the diuresis; but no inference can be drawn from this experiment as to the relative permeation of chloride and sulphate through the capsule. Some evidence however may be gathered from the curves of excretion of the chloride and sulphate. The infusion of a strong salt solution induces Hydraemia, followed by diuresis and deficient absorption of water and chloride, and the chloride of the urine must thus increase with the hydrnemia and diuresis. If the sulphate permeates the capsule as readily as the chloride and water, its amount in the glomerular fluid and urine must vary with the amount in the blood, that is in this case with the amount injected and the bydraemia. The maximum of sulphate, chloride and water excreted, must thus correspond in time, if they pass the capsule equally readily, while if the chloride penetrates 1 Magnu s (II) and Sollmann injecting dilute solutions of sulphate intravenously in dogs did not observe the marked increase in the chlorides of the urine which I have described. But the sulphate was present in much higher percentage in the urine than in the serum (2-3 /0 Magnu s), which indicates a large absorption of fluid and with it of chloride in the tubules.

12 440 A. R. CUSHNY. less rapidly its maximum may be expected to occur somewhat later than those constituents which pass without resistance. In some of my experiments performed in the same way as Exp. I. the maximum sulphate excretion actually appeared to occur somewhat later than the maximum chloride and water, so that I supposed at first that the sulphate met some opposition in its passage through the capsule. But the periods during which the urine was collected were too long, and in several experiments, therefore, the urine was collected for every five minutes during the diuresis and each sample was analysed sepaxately. Exp. V. A rabbit of 1670 gr. weight anesthetised with 2 5 gr. of urethane , urine= 2'7 c.c , infusion into jugular vein of 30 c.c. of a mixture of equal parts of 14'2 per cent. Na2SO4 solution and 5'85 NaCl solution. Some urine found in the bladder was added to the normal urine to permit of analysis. Water Time inj ected t ' Urine in c.c. 2'7 7 14'2 2A '8 2' o9j Cliloride injected '1686 '1065 '142 '1154 Cliloride in urine '0036 '024 ' '077 '026 '007 '005 Clhloride equivalents m urine '0001 '0007 *0014 *0028 '0022 '0007 '0002 '0001 not analysed for chloride, t Chloride in urine per cent. '133 *343 *355 '390 '387 '343 '2536 '1775 Sulphate injected '456 ' '312 Sulphate in urine '026 '0712 ' '143 '0732 '0406 '0481 Sulphate equivalents in urine '0002 '0007 '0011 '0018 '0015 '0008 ' Sulphate in urne per cent ' '699) '715 '963 1' '0742 '0008 2'06 - '0538 '0006 2'24 - '0528 '0005 2' i IS Fig. 2. The curve of excretion corresponding to Exp. 5. The lines represent the same constituents as in Fig. 1.

13 DIURESIS OF RENAL CELLS. 441 Exp. V. and the corresponding curve show that the maximum sulphate excretion falls at the same time as the maximum chloride and water excretion and also that the rise of each constituent begins together, and these experiments thus all fail to indicate any difference in the rate of diffusion through the capsule of the sulphates, chlorides or water. I had anticipated some difficulty in the permeation of sulphate from its behaviour in the intestine and elsewhere, but apparently the capsular membrane resembles the endothelium of the pleura rather than the epithelium of the intestine in its relation to sulphates and chlorides. That is the capsular membrane is unable to differentiate between sulphate and chloride, which penetrate it equally readily. The greater height of the chloride curve, which is especially marked in Exp. I., might at first sight be taken to indicate a greater diffusibility of the chloride, but really is explained by the larger amount of chloride in the blood, as will be shown in the experiments on urea. It was impossible to inject sulphate in large enough quantities to equal the chlorides of the blood owing to its poisonous action. The question as to the relative permeability of the water and the chloride of the plasma through the capsule presents the same difficulty as that of the chloride and sulphate. In all my experiments the chloride and water ran parallel during the diuretic stage, and both fell together in the later phases, though the chloride often decreased more rapidly than the water, for reasons which will be discussed later. The chloride of the urine in the diuresis never was higher in percentage than that of the serum, but approached very near to it occasionally. Thus in one experiment the urine at the height of diuresis contained 0408 per cent. of chloride, the serum 0419 per cent., the two being thus practically identical in content of chloride. If the filtration hypothesis be correct, the salts must diffuse through the glomerulus with the same ease as the fluid, resistance being offered only to the proteids. The results obtained in my experiments show no facts incompatible with this, although it must be admitted that they do not offer any indisputable evidence in its favour. The second salt examined was the phosphate of sodium. A solution of disodic-hydric phosphate was neutralised with phosphoric acid and the percentage of phosphate ascertained by titration with uranium acetate with cochineal as an indicator. The solution was then diluted until it contained 95 gr. of phosphate (P04) per litre and mixed with an equal amount of sodium chloride 5 85 per cent. solution. The mixture containing gr. Cl and 4-75 gr. PO4 per cent. was inftused more

14 442 A. R. CUSHNY. slowly than the chloride-sulphate mixture, as phosphate is much more poisonous. The phosphate of the urine was determined by titration with uranium acetate. Exp. VI. A rabbit of 1600 gr. weight ansesthetised with 2-5 gr. urethane , normal urine = 32 c.c , infusion into the jugular vein of 50 c.c. of the phosphate-chloride mixture. Chloride Chloride Phlosphate Phosphate Water Chloride Chloride equivalents in urine Phosphate Phosphiate equivalents in urine Time injected Urine injected in urine in urine per cent injected in urine in urine per cent in 15' * * a ) _2_ ) Totals If Exp. VI. is compared with Exp. I. the-excretion of chloride is found to bear practically the same relation to the sulphate of the latter and the phosphate of the former. The increase in fluid is accompanied by a rapid increase in the chloride and phosphate; all three reach their maximum together and then fall, but the phosphate excretion diminishes more slowly than the others. The phosphate falls somewhat short of the sulphate excretion of Exp. I. but the close analogy between the two permits of the same inferences as were drawn from the sulphate excretion. In order to permit of a comparison between the elimination of the phosphate and sulphate directly, two experiments were performed in which equal parts of the phosphate and sulphate solutions were mixed and injected. This experiment confirms the inferences drawn from a comparison of Exp. I. and VI., that the phosphate resembles the sulphate very closely in its excretion by the kidney. Rather more sulphate than phosphate occurs in the later stages, indicating that the latter is the more readily absorbed by the tubules, or that it is deposited or eliminated from the blood in some other organ. It is to be remarked that during the diuresis more chloride equivalents are excreted than sulphates or phosphates although no chloride was injected; in fact

15 DI URESIS OF RENAL CELLS. 443 C) C) - c- eq eq eq 17 c. v c v m 0 Co 0 0 C) C) c6e co o 10 VD o co 8Co Co 0) 0U 0) 0) e o> r 0 Co eq '-4 0 Ca a) 0 o. 10,-4 F4 0 :- Co eq as Co o *g 0 10 eq P-,-4 10 Co 0 a) ds OD C),a C 0 0 CC o Co co co I? r eq -,b cc o to Co Co 10 eq eq ao Ca) eq eq oco eq U: km eqs 710 a) o *E 0 4 8Io 0) r- Co Q a)a eq *' eq ca* Co I:-. 10 r l Co t' O 10 Co rli v m 0o 0,c.S CQ t- 10 C) a)a C 0) eq Co '-4 eq '-4 eqco 10 t Co 1 0) 0 cq 0c1 40 Co CO 00 0 co 1t '- Co 4 14 " u 101 t

16 444 A. R. CUSHNY. at the height of diuresis (3.45) almost as many chloride ions appeared as both of the other constituents together. As the diuresis passed off the chloride fell to a mere trace, while the sulphate and phosphate lessened more slowly in absolute amount and increased in percentage. A similar increase in the chloride of the urine withouit any injection of this salt was discussed in reference to Exp. IV. Time for 15' Totals I , Fig. 3. The curve of excretion of Exp. 7. The lines correspond to those of Fig. 1. The phosphate excretion runs so near that of the sulphate that it was impossible to reproduce it. In a number of experiments a normal solution of urea (6 per cent.) was mixed with an equal quantity of normal sodium chloride solution (5855 per cent.). The urea of the urine was estimated by the Morner- Sjokvist method. Exp. VIII. A rabbit of 1300 gr. ansesthetised with 2-0 gr. of paraldehyde , 4 c.c. of urine collected' , infusion of 50c.c. of the urea and chloride mixture C1 Cl in Urea Urea Fluid Cl Cl in equivalents urine Urea in in urine injected Urine injected urine m urine per cent. injected urine equivalents 4 '0011 '028 - '108 '0018 '4 '0001 ' * i 30 '6834 '1416 *004 '472 1'155 '1197 '002 11* 48 '2041 '2573,0072 '536 '345 ' '0758 '0021 '541 - '0672 ' '5 '0687 '0019 *55 - ' '0122 *0003 *358 - '0692 ' '8875 '5556 ' '6378 '0107 Some urine found in the bladder was added to this for analysis. Urea in urine per cent '423 '48 1'43 2'04

17 Time per 15' Totals DIURESIS OF RENAL CELLS. 445 Exp. IX. A rabbit of 1325 gr. weight was anesthetised with 2 gr. paraldehyde p.m. the urine amounted to 6 c.c., to which some urine previously collected was added for analysis , infusion of 60 c.c. of a mixture of equal parts of solution of urea (20 per cent.) and sodium chloride (normal). Cliloride, Chloride Fluid Chloride Chloride equivalents in uirine Urea Urea injected Urine injected in urine in urine per cent. inj ected in urine 6 '0036 '0001 '06 '032 2 '0012 '06 ' '5325 '1470 '0041 *387 3'0 ' '5325 '5670 ' '0 1'38 22 '1015 *0029 '4615 '31 15 '0495 *0014 *33 '294 8 '0128 '0004 *16 '206 5i '0074 '0002 *135 '085 * '5 1'065 *8852 '0250 6' '0447 Urea equivalents in urine '0005 * '0051 *0049 '0034 Trea in urine per cent. ' '098 1'21 1'41 1'96 2' (?) 1XfNXI'C - -t r -I-' 111'iZ0t;i I l ' _ I _ UiiiAi -- _ Fig. 4. Curve of excretion of Exp. VIMI. The heavy line represents the fluid, the light the urea equivalents, and the broken the chloride equivalents; i.e. the urea of the urine divided by 60, and the chloride by In Exp. VIII. the urea and chloride of the urine run parallel during the diuresis, attain their maximum with it and commence their descent together. The fall in the urea soon becomr.5 slower than thabt of the P11, XXVII. 29

18 446 A. R. CUSHNY. chloride however and the former remains at a higher level. The percentage of chloride undergoes changes similar to those seen in Experiment I. except that it is unusually high for some time after the diuresis had practically disappeared, a point noted in several other experiments in which urea was injected with chloride. The percentage of urea was high in the normal urine and fell abruptly during the diuresis, to rise again gradually as the quantity of urine returned to the normal. The urea thus resembles the sulphates and phosphates, apparently passing through the capsule readily but failing to penetrate the epithelium of the tubules so readily as the chloride. Urea is generally regarded as one of the most readily diffusing constituents of the body, and it seemed improbable that it wotuld be rejected by these cells; but it has been shown by Overton that urea permeates vegetable cells1 only very slowly and that when tadpoles are put in a weak solution, it passes into the blood and tissues so slowly that hours or more are required to establish equilibrium between the animal and its environment2. Somewhat stronger solutions actually withdraw water from the tadpole. So that the behaviour of the epithelium of the renal canals towards urea is not without analogy in other living cells. In the ureter, bladder, and urethra the nmucous membrane must possess a similar lack of affinity for urea, otherwise much of it would be absorbed from the urine. I have tacitly assumed above that the urea is not secreted by the cells of the tubules, but escapes from the glomerulus along with the water and salts. The complete parallelism between it and the chloride during the diuresis and the resemblance its course throughout bears to that of the sulphate and phosphate certainly suggest this view. And I am not aware of any evidence whatever that the urea diverges from the salts in its excretion. Bowman appears to have suggested that the specific constituents of the urine required a special excretory apparatus, and Heidenhain and his followers accept this without examination. There is however no inhereint quality in urea, as far as is known, which wouild require it to be differentiated from the salts in its excretion, and such evidence as can be drawn from my experiments indicates a common method of excretion for all. Exp. IX. differed from No. VIII. in the chloride and urea not being injected in equimolecular solution but in the proportion of 3: 10. If the rabbit's blood be taken at A of the body weight, the whole blood of 1 Zeitschr. f. physik. Chem. xxii. p Studien iiber die Narkose, p. 120, Jena

19 DI URESIS OF RENAL CELLS. 447 the animal in this experiment would contain *388 gr. of chloride', and probably the whole available fluids of the body would scarcely contain /r' ta : g To i a d T L0 _~ g ~~ zr 39\ ~~- - = Fig. 5. Curve of excretion in Exp. 9. The interpretation is the same as in Fig. 4. Fig. 5 is reduced to i the height it would occupy on the same scale as Figs more than 1-2 gr. at a liberal estimate. To this was added 6 gr. of urea anid about 1 gr. of Cl. The urea must obviously have exceeded the chloride in the blood and lymph. The only essential difference between the results of Exp. VIII. and IX. is however the excess of urea molecules over the chloride, which is present in the stage of diuresis as well as later. This experiment and another performed in precisely the same way are the only ones in which the chloride did not exceed the other constituent injected in the stage of diuresis. They are also the only ones in which another body was present in the blood in larger amounts than the chloride. The inference seems justifiable that the excess of chloride in the urine during the diuresis in other experiments is due not to any special affinity between it and the renal cells, but to its preponderance in the serum. Doubtless if sulphate could be injected in such quantity that it exceeded the chloride in the blood there would prove to be more sulphate than chloride in the urine during the diuresis. In other words, provided the absorption of the tubules is sufficiently in abeyance, the relative amount of the constituents of the urine is determinied by their relative proportions in the blood. When, I Abderhalden, Zeitschr.f. physiol. Chemie, xxv.. p. 65, 29-2

20 448 A. R. CUSHINY. however, as in the later stages, the absorption is active, the constitution of the blood has comparatively little effect upon the constituents of the urine. As the excretion of urea seemed to resemble that of the sulphate and phosphate in its general character, the standard solutions of sulphate and of urea were mixed in equal parts and injected in four experiments. In these the urea and sulphate of the urine reached the maximum together and then decreased in absolute quantity while the percentage of each rose. The urea equivalents actually excreted were fewer than the sulphate at each stage, so that at the end of the experiment only about one-half of the urea injected had reappeared in the urine, while two-thirds to three-fourths of the sulphate had been excreted. The sulphate thus appeared to be more readily eliminated (harnfihiger) than the urea. The explanation probably is that the urea disappears into the tissues from the blood more readily than the sulphate, so that though the number of molecules injected was the same, the blood at any moment actually contains fewer of urea than of sulphate. The fact that the excretion runs parallel indicates that each permeates the capsule equally readily, and the high percentages in the later phases shows that they are absorbed with difficulty by the tubules. The relative permeability of the two could be determined only by injecting such quantities that the urea in the blood would equal the sulphate throughout the experiment, which is difficult if not impossible to attain in the intact animal. Attempts to compare the excretion of potassium, magnesium, and ammonium with that of sodium failed owing to the poisonous action of these bases. Starling regards the diuresis following the injection of salts as the resultant of two factors, (1) the hydrwmic plethora and consequent rise in the pressure and velocity of the blood in the kidneys, (2) a direct dilator effect of the injected substance on the renal vessels. Magnus brings forward several objections to this view and concludes that the hydramia changes the activity of the secretory cells. Among the grounds for his rejection of the mechanical explanation, the weightiest seems to be that sodium sulphate causes a greater diuresis than the chloride although in his experiments each induced an equal degree of hydraemia. The sulphate diuresis also lasted longer than the chloride, the urine returning more slowly to its normal amount. He infers from this that there must be some local factor apart from the hydraemia. This local factor is, I think, supplied by the more difficult absorption of the

21 DI URESIS OF RENAL CELLS. 449 sulphate by the tubules. For if the sulphate remain unabsorbed the epithelium must meet a greater opposition to the absorption of fluid than if a readily permeating salt such as the chloride were present alone in the tubules, and if the injection of each causes the same amount of glomerular fluid, less would be absorbed in the tubules and more reach the ureters in the case of the sulphate than of the chloride. The greater diuresis after sulphate injection then does not indicate any stimulation of the renal cells, and is quite consistent with the idea that the diuresis is a direct result of the hydraemia. The slow return of the urine to its normal amount may be explained in the same way, and is analogous to the continued diuresis in my experiments (No. I.) which persisted after all the chloride had disappeared from the urine. The work demanded of the renal tubules in reducing the fluid leaving the glomerulus to a solution containing 2-3 per cent. of SO4 must be enormous, and even the opportunity offered by the slow passage through the tubules is insufficient to admit of further concentration. Sulphates are not suitable in practice as diuretics because they are only slowly absorbed from the intestine. Otherwise, their failure to return to the blood from the tubules would indicate their use in therapeutics for this purpose. Urea however seems admnirably adapted as a " saline diuretic " for it enters the blood readily from the intestine and appears to be absorbed by the renal tubules with difficulty. The question may arise however whether it induces as great movement in the fluids of the body and hydramia as some of the inorganic salts. That it is of value as a diuretic bas been shown by numerous clinical observers. My results may be shortly explained as follows in conformity with the mechanical theory of renal secretion modified by the acceptation of active absorption in the tubules:-strong solutions of sulphate, chloride or phosphate of sodium, or of urea induce hydremia, which results in an increase in the fluid passing through the capsule. This fluid appears to carry with it the salts and urea in the proportions in which they occur in the plasma, there being no evidence that any of them permeate the capsule more readily than the others. The rapid flow through the tubules permits of only imperfect absorption, but a certain amount of the readily diffusing bodies (water and chloride) return to the blood; the less diffusible sulphate, phosphate and urea escape into the ureters with less loss by absorption, so that they may be present in the urine in a more concentrated form than in the blood. The chloride being present in the plasma in larger quantities than the other salts, however, more of it is present

22 450 A. R. CUSHNY. in the glomerular fluid, and although some is doubtless reabsorbed in the tubules, enough remains in the urine to exceed the other salts during the diuresis if all be reduced to equivalent terms. If more urea than chloride be present in the blood, then the urea of the urine exceeds the chloride during the diuiresis. As the hydrwmia and diuresis pass off, the flow through the tubules assumes more normal dimensions, and, more time being allowed for absorption, the diffusible bodies are taken up by the epithelium, while the indiffusible increase in percentage in the urine owing to the absorption of the water. The presence of indiffusible substances in the tubules retards the absorption of the water, so that sulphate induces a larger and more prolonged diuresis than chloride. It may be added that the same phenomenon is seen in the bowel on comparing the behaviour of a weak sulphate solution with that of a weak chloride one; the fluid fails to be absorbed from the former, while that of the latter disappears rapidly with the salt dissolved. The epithelium lining the renal tubules is often compared to that of a true secreting gland such as the salivary, but it resembles that of the intestine as closely histologically, and in its reaction to chlorides, phosphates and sulphates is analogous from a physiological point of view.