(From the Physiology Institute, Cardiff.) (Received May 10, 1935.)

Transcription

1 458 6I2 463 THE ELIMINATION OF XYLOSE; CREATININE AND UREA BY THE PERFUSED MAMMALIAN KIDNEY. BY A. HEMINGWAY. (From the Physiology Institute, Cardiff.) (Received May 10, 1935.) THE method of estimating the rate of glomerular filtration by following the elimination in the urine of a substance which is supposed to be filtered through the glomerulus and to suffer no change in amount while passing along the tubules has been applied by several workers [Mayrs, 1922; White, 1923 (sulphate); Rehberg, 1926 (creatinine)], and has recently received fresh impetus from the claim of Jolliffe, Shannon and Smith [1932] that "non-metabolized sugars" and, in particular, xylose, can be used for such a purpose. Since no two substances appear in the urine concentrated to the same extent, it is certain that only one, if any, can be present solely as a result of filtration. No rigid proof has been given that any one of the substances examined is eliminated only by filtration. In an endeavour to obtain further evidence regarding the site and method of elimination of these substances, the output of creatinine, xylose and urea has been measured and compared in experiments with the perfused kidney where the renal circulation and blood flow can be controlled and measured. The rate of elimination of a substance may be expressed in terms of the "clearance rate" [M6ller, McIntosh and van Slyke, 1928] given by the expression UVIP, where U=concentration in the urine, P = concentration in the plasma and V = rate of urine flow. The clearance rate becomes a measure of the glomerular filtration rate if the substance is eliminated only through the glomerulus. The rate of filtration calculated in this way should be independent of the plasma concentration provided the vascular conditions are unaffected by change in concentration. While fulfilment of this condition does not necessarily mean that a substance is eliminated solely by

2 EXCRETION BY PERF USED KIDNEY. 459 glomerular filtration, it may be applied as a preliminary test; and in the series of experiments to be described, the clearance rates for xylose, creatinine and urea have been measured at different plasma concentrations. A comparison of the behaviour of the perfused kidney with that in the normal animal has also been made by examining the ratios between the clearance rates for these substances in relation to similar measurements made by other workers on the intact animal. METHODS. The pump-lung-kidney preparation was made by the methods previously described [Hemingway, 1931, 1933]. Xylose, creatinine and urea were made up as isotonic solutions and added to the perfusing blood to give desired concentrations in the plasma. Collection of samples was begun when the flow of urine was becoming constant, usually mi. after commencing perfusion. Blood samples were collected at the mid-point of periods of urine collection. Blood was centrifuged and a protein-free filtrate prepared, using the modification of Folin and Wu's tungstic acid procedure due to van Slyke and Hawkins [1928]. Xylose was estimated in the filtrate by the method of van Slyke and Hawkins [1929] using the Somogyi-Shaffer-Hartman reagent for reduction. Factors for calculation were read from a curve prepared from analyses of known xylose solutions. In common with Jolliffe, Shannon and Smith [1932] it was observed that a portion of the xylose disappeared during the yeast treatment. This loss amounted to p.c. of the total, depending on the sample of yeast, but no allowance was made for it since it did not influence the calculation of the concentration ratio. Estimations were performed in triplicate and the results averaged. Creatinine was estimated by Folin's method and urea by the urease method. Similar methods were applied to urine after diluting to give a concentration equivalent to that of the corresponding plasma filtrate. Perfusion pressure was kept constant in each experiment except where otherwise described, and measurements of pressure and rate of flow were made from tracings. RESULTS. Preliminary experiments in which suitable amounts of creatinine were added to the blood perfusing a normal kidney and also one treated with cyanide, showed no significant change in the renal circulation or

3 460 A. HEMINGWAY. output of urine following the addition. Addition of xylose caused a slight vaso-constriction but it was not regarded as sufficient to hinder the course of the experiments. (a) The clearance of xylose at different plasma concentrations. The results of eleven successful and consecutive experiments in which the clearance for xylose was calculated at two different plasma levels in each experiment are given in Table I. The addition of xylose necessary to give the higher concentration was made immediately following the first experimental period, and 8-10 min. were allowed for complete mixing of the blood and washing of the urinary passages before samples were collected in a second period. Jolliffe, Shannon and Smith [1932] have already reported that xylose clearance is independent of plasma concentration, and the present results support their findings since following increases in the xylose plasma concentration there was an average percentage change in clearance rate, compared with the original, of only This small change is not regarded as significant in view of the large average deviation which is no doubt partly due to the difficulties associated with accurate estimation of xylose and the slight vascular disturbance already referred to. The range of xylose clearance rates in twenty-three determinations where the perfusion pressure was between 100 and 120 mm. Hg was 0*25-0*94 c.c./kg. dog/min. with an average of 0-47 c.c. These figures are one-third to one-half of those given by Jolliffe, Shannon and Smith [1932] for intact and unanesthetized dogs, which have a range, halving their figures to obtain the clearance for one kidney, of *20 c.c./kg./min., although the urine flows in the perfusion experiments are higher than those recorded by Jolliffe, Shannon and Smith. The lower clearances found in the present experiments may be due to partial failure of the water reabsorption mechanism [Starling and Verney, 1925; Verney, 1929] with a consequent rise in tubular back pressure which diminishes the effective filtration pressure. A similar suggestion with regard to tubular resistance has already been put forward by Ekehorn [1931] to explain the diminished rate of elimination of certain substances which Starling and Verney found to occur after treating the kidney with cyanide. This possibility is being explored and will be discussed in a subsequent paper.

4 EXCRETION BY PERFUSED KIDNEY. 461 obo I~~.++ ~ 111 ~~1 Ii Ca Ca Ca _r_ ~~ -- 0 ~ ~ ~ ~ ~ ~ ~ ~~ 8_o; o g s X m > X o X.o O m avoo o m * X x8 I"=-- a soex > or - -C)eq4 0 Q_ocxoa Ca~~~~~~~~~~~~~~~~~0 a *0 c; t:.d 4:: l (ZO C5> Ct-s X e Oq 000o q, H V-4 ac UC0 0 C O t- > o~~~~a xo X m a o n. P oo 0 g B-d bo- 00 = CD -4 = xo = oo o to N oo o c o o o Cci o H~~~~~t - qx 00R00 r-00 oo_--n-- w m : c;--b cq0 q Ca to Y ono o P O+ - o - QOC*H oit xo N r- =-_ I _I o aq cr m xo o. = * - "~~ ~ ~~~~~~~a,oo_.on > - -~~~~~~t lna c) x<eoo lo s_q. tl *m c o E c Pq exoa -4 E_ = Q <.o,o t_o 0g o e ~Ca P) ) c 0= C 0 c -% C9 0>oo Qa 0-20 to Ca o o0m, a) o lo : o,d o Ca~ bo_, CO C:b xo :O bo 0 -tetc H ~ ~ ~ ~ C t ce tm~~~~~~~~~~~~~

5 462 A. HEMINGWAY. (b) The clearance of creatinine at different plasma concentrations. Table II shows the results of five consecutive experiments in which plasma creatinine concentrations were changed. The course of each experiment was similar to that described in the previous section, and in each the clearance calculated during the second phase with raised creatinine concentration was less than the original. The average percentage diminution compared with the original was 19-8, and a rise in clearance rate following the addition of creatinine has never been observed in any experiment in which the rate of urine flow has remained steady. Thus, unlike xylose, the clearance falls with increasing plasma concentration and this was observed particularly with high creatinine concentrations. With low ones the fall in clearance did not always occur. For instance, in one experiment (not shown in the table) the plasma concentration was increased from 5-15 mg. per 100 c.c. to 6-50; the clearance, 7-3 c.c./min., was the same in both periods. Such variation in clearance with plasma concentration is opposed to the results of Shannon, Jolliffe and Smith [1932] on the dog; MacKay and Cockrill [1930], Cope [1934] on the rabbit; Cope [1931], Jolliffe and Chasis [1933] on man; but agrees with those of Kay and Sheehan [1933] on the rabbit, Shannon [1934] on the elasmobranchs and Marshall and Grafflin [1932] on the toadfish. Therefore creatinine cannot be used as a measure of glomerular filtration rate in the perfused kidney, and unless the eliminating mechanisms are quite different in the two cases, this must also hold for the normal kidney. It has been suggested [Shannon, Jolliffe and Smith, 1932] that a portion of the urinary creatinine is due to tubular secretion. If this is so, then a failure of the secretory mechanism with rising plasma concentration would account for the results which have been obtained. Examination of the mitochondria of the proximal convoluted tubule of the perfused kidney suggests that the cells are approaching exhaustion [Go ugh and Hemingway, 1934], and this might explain the relative failure of the kidney to eliminate creatinine by active secretory processes at high plasma concentrations. In a similar fashion, a failure of secreting cells due to a deficient blood supply following experimental interference may account for the results of Kay and Sheehan. On the other hand, the tubules of the intact mammal may be able to keep pace with increasing plasma concentration over a greater range, and so the linear relationship between the plasma concentration and the clearance rate in the normal animal would not be incompatible with the present results.

6 EXCRETION BY PERFUSED KIDNEY. 463 (c) Clearance rates after simultaneous increase of creatinine and xylose plasma concentrations. Table III gives the results of an experiment in which creatinine and xylose plasma concentrations were increased simultaneously. The xylose clearance remained almost constant, but the creatinine clearance diminished when the plasma concentration of both substances was increased. Xylose A A l-a TABLEBm Creatinine Urine Plasma Urine Plasma Urine Blood flow (V) con0. (P) conc. (U) conc. (P) conc. (U) Blood- flow c.c./ mg./ mg./ mg./ mg./ pressure c.c./ min. 100 c.c. 100 c.c. UV/P 100 c.c. 100 c.c. UV/P mm. Hg min mg. of creatinine and 500 mg. of xylose added to 450 c.c. of circulating blood (d) The ratio between creatinine and xylose clearances. Thirteen simultaneous measurements of creatinine and xylose clearances have been made and the ratios between corresponding clearances are given in Table IV. The average value of the ratio, creatinine clearance/xylose clearance, 1-72, is slightly higher than the figure of given by Shannon, Jolliffe and Smith [1932] and that of 1-68 obtained by White and Monaghan [1933] from the intact and unaneasthetized dog. The present experiments also demonstrate that the ratio is not constant. Moreover, in any one experiment the ratio falls with increasing plasma creatinine concentration as is shown in Table III, and as indeed is to be inferred from the effects, which have already been Xylose TABLE IV. Creatinine clearance clearance Creatinine clearance Exp. c.c./min. c.o./min. Xylose clearance Average 1-72 PH. LXXXIV. 30

7 464 A. HEMINGWAY. described, of changes in the plasma concentrations of xylose and creatinine on their respective clearances. As will be shown later, the ratio also changes with variations in the renal arterial pressure. (e) Effect of changes in perfusion pressure on creatinine and xylose clearances. The clearance for both creatinine and xylose varies directly with perfusion pressure, and Fig. 1 depicts an experiment in which the Perfusion pressure mm. Hg 130 o 12 ~ 0 g 16r 4I 2F _ 100 _.90 L I Time (min.) ig creatinine clearance was determined at different pressures. This result confirms those of Medes and Bellis [1934], who, however, regarded the creatinine clearance as a measure of glomerular filtration rate.

8 EXCRETION BY PERFUSED KIDNEY. 465 mgo m~~~~~~0~.' CO) C5 0 d Ow 0 COt- Cq C t'. - Cg O OD zo N M * _0 _* _t00 _4 _I00 _I _l".4:c; )4C~O1 Obo xst0o 0 X di I COlt Cq 0 Om O o 0 aq aq eq aq aq -400 C cq C1 - - P-m CO 10 A, d C9 c = XOH -- o oc -- o > m oo 0 m c ao ao o w t g8st p i p"~0 F oo ooomt oot- t- oo oo g Eo o 0 t - CO (j4 m w Tc10 to 10 odoo -- -_ ~~~~~~~~~c0c a0 0 qa 2 ~ ~ ~~~~~0 0z co_ O10P4t 99 *8-9w4C0 COC t- CO-O0 x xo cqcq 0 6 S "4 C. p1 i. ~ t' O ot'. 6 - H ~00 6 CO c Co C - oc 9b9 < ~~~, Cl -)t- O0O -t 0 COCO0100oL0 00( _ 0 8 s 0 O COt1 0 Ooq X0 t in t-a *Coo o t'.4 L-O(Z - 0 o8c toco eo6 2 t 6R -g 8 t t X > s > 3 8wtz A_o~~ ~~~~~~0 Mo o. o0t 0 co 0 -=o CO~~~~~~mC 0 O t CO CO C Ico E- P o o mm ie - --I co CH _O~ 10CO0 CtcO 00o rzl -

9 466 A. HEMINGWAY. When simultaneous determinations of creatinine and xylose clearances were made on the same preparation at two different perfusion pressures, both clearances were found to have increased when measured at the higher perfusion pressure, but that for creatinine was increased relatively more than that for xylose. If some of the creatini e is secreted by the tubules the change in the ratio of the clearances may be due to an increased blood supply to the tubule cells. On the other hand, if creatinine were eliminated solely by filtration, it might be expected with reason that the ratio would have fallen with a rise in perfusion pressure. The findings in two experiments are given in Table V. (f) Urea clearance and its relation to xylose clearance. Simultaneous measurements of the clearances for xylose and urea have been made in experiments with normal blood and also with the urea plasma concentration artificially raised by the addition of urea. Eighteen determinations from such experiments are given in Table VI. The average of the ratios, urea clearance/xylose clearance was Reference to Table VII shows that this figure is slightly higher than that obtained by other workers, whose individual results however exhibit considerable variation. TABLE VII. Urea clearance Authors Kidney Xylose clearance Jolliffe, Shannon and Smith [1932] Dog Jolliffe and Chasis [19331 Man 0-69 Ohasis, Jolliffe and Smith [1933] Man Keith, Power and Peterson [1934] Man 081 In several experiments attempts were made to compare the clearances for urea at two different plasma concentrations by adding urea to the blood after taking the first samples. But although the urine and blood flows were not greatly affected by the addition of urea the results were not regarded as satisfactory because of the large changes in urea clearance (see Table VI). In so far as any conclusion could be drawn from them they indicated that the urea clearance was independent of the plasma concentration. This is in accordance with the findings of van Slyke, Rhoads, Hillier and Alving [1934] on the dog, and Drury [1923] on the rabbit. Any change in the urea clearance was accompanied by a change in the xylose clearance in the same direction but the changes were not proportional.

10 EXCRETION BY PERFUSED KIDNEY. 467 DIscussION. The close agreement in the ratios of the clearances of creatinine, xylose and urea between the present results and those obtained by other workers on intact unanaesthetized animals, suggests that the mode of elimination of these substances by the perfused kidney is not very different from that in the normal organ and that the main difference is in the rate of elimination of water. Apart from this agreement the results must be examined for evidence regarding the claims that creatinine, xylose or urea are substances which may be used to measure glomerular filtration rate. Since the clearance for creatinine falls with increasing plasma concentration under conditions which do not appear to lead to any failure of glomerular filtration, while the clearances for xylose and urea, although lower than for creatinine, are independent of the plasma concentration, the experiments would appear to support the theory of glomerular origin for either urinary xylose or urea. If so, then a portion of the creatinine, varying with the plasma concentration, might be eliminated by tubular activity. That the kidney in the intact animal is capable of maintaining a constant clearance independent of the creatinine plasma concentration is beside the point. The tubular mechanism which preserves the linear relationship between tubular secretion and plasma concentration in the normal animal may well be disturbed by the conditions of a perfusion experiment. With regard to urea, the clearance for which is less than for xylose, although the general trend of the experiments suggests that the clearance is independent of the plasma concentration, yet the extent of the variations in clearance in adjacent periods coupled with the observations of Kay and Sheehan [1933] and van Slyke, Rhoads, Hillier and Alving [1934] who noticed release of urea from the kidney into the renal vein, lead to the opinion that a varying proportion of the urea filtered through the capsule is reabsorbed or diffuses through the tubule walls. The evidence presented is too inconclusive to be taken as proof of the glomerular origin of xylose and the question whether tubular secretion, reabsorption or diffusion plays any part in the elimination of this substance must be left open. If such activities do occur they are, however, so far as the present experiments are concerned, conditioned by the plasma concentration, and the xylose clearance if not an absolute measure may provisionally be regarded as proportional to the glomerular filtration rate.

11 468 A. HEMINGWAY. SUMMARY. 1. The plasma clearances for creatinine, xylose and urea have been measured in the isolated perfused kidney. 2. The average creatinine clearance/xylose clearance ratio is The average urea clearance/xylose clearance ratio is 0O The ratios are within the range of similar figures obtained from normal animals. 5. The clearance for creatinine falls with increasing plasma concentration while the clearances for xylose and urea are independent of the plasma concentration. The xylose clearance is regarded as being proportional to the glomerular filtration rate. The expenses of this research were defrayed in part by a grant from the Government Grant Committee of the Royal Society. REFERENCES. Chasis, H., Jolliffe, N. and Smith, H. W. (1933). J. clin. Invest. 12, Cope, C. L. (1931). Quart. J. Med. 24, 567. Cope, C. L. (1933). J. Phy8iol. 80, 238. Drury, D. R. (1923). J. biol. Chem. 55, 113. Ekehorn, G. (1931). "On the principles of renal function." Acta med. 8cand. Suppl. 36. Gough, J. and Hemingway, A. (1934). Unpublished observations. Hemingway, A. (1931). J. Physiol. 71, 201. Hemingway, A. (1933). Ibid. 77, 14 P. Jolliffe, N. and Chasis, H. (1933). Amer. J. Phy8iol. 104, 677. Jolliffe, N., Shannon, J. A. and Smith, H. W. (1932). Ibid. 100, 301. Kay, W. W. and Sheehan, H. L. (1933). J. Physiol. 79, 359. Keith, N. M., Power, M. H. and Peterson, R. D. (1934). Amer. J. Phy8iol. 107, 221. MacKay, E. M. and Cockrill, J. R. (1930). Ibid. 94, 220. Marshall, E. K. and Grafflin, A. L. (1932). J. cell. comp. Phy8iol. 1, 161. Mayrs, E. B. (1922). J. Physiol. 56, 58. Medes, G. and Bellis, C. J. (1934). Amer. J. Phy8iol. 107, 227. Moller, E., McIntosh, J. F. and van Slyke, D. D. (1928). J. clin. Inve8t. 3, 427. Rehberg, P. B. (1926). Biochem. J. 20, 447. Shannon, J. A. (1934). J. cell. comp. Phy8iol. 4, 211. Shannon, J. A., Jolliffe, N. and Smith, H. W. (1932). Amer. J. Physiol. 102, 534. Starling, E. H. and Verney, E. B. (1925). Proc. Roy. Soc. B, 97, 321. Van Slyke, D. D. and Hawkins, J. A. (1928). J. biol. Chem. 79, 739. Van Slyke, D. D. and Hawkins, J. A. (1929). Ibid. 83, 51. Van Slyke, D. D., Rhoads, C. P., Hillier, A. and Alving, A. S. (1934). Amer. J. Phy8iol. 109, 336. Verney, E. B. (1929). Lancet, 1, 539. White, H. L. (1923). Armer. J. Phy&iol. 65, 537. White, H. L. and Monaghan, B. (1933). Ibid. 106, 16. CAMBRIDGE: PRINTED BY WALTER LEWIS, M.A., AT THE UNIVERSITY PRESS