known about the reversal of this process. In order to discover how extracellular

Transcription

1 585 J. Physiol. (I957) 136, THE RECOVERY OF KIDNEY SLICES FROM ANOXIA IN DIFFERENT MEDIA BY J. R. ROBINSON From the Department of Experimental Medicine, University of Cambridge (Received 18 January 1957) Although it is established that the percentage of water in surviving slices of a variety of tissues increases when their respiration is interrupted, not much is known about the reversal of this process. In order to discover how extracellular ions affected the swelling of rats' renal cortical slices deprived of oxygen, and their ability to recover a normal water content when oxygen was supplied again, slices were incubated at 380 C without oxygen in a model extracellular fluid in which kidney slices had been found to respire satisfactorily (Robinson, 1949), or in modifications made by replacing sodium chloride by choline chloride, sodium sulphate or choline sulphate, and then re-incubated in each of the same four media in the presence of oxygen. Slices which had first been in the aqueous media without oxygen were also incubated in oxygenated liquid paraffin in an attempt to minimize the contribution of external osmotic pressure to the movement of water from the cells. It was found that external sodium and chloride increased the uptake of water when oxygen was absent, and hindered its removal when oxygen was supplied. There was also a remarkably consistent loss of water from slices incubated in oxygenated paraffin. METHODS Media. The media were prepared exactly as described by Robinson (1956), and the extraordinary media have again been designated by the names of their principal dissolved constituents. Technique. Slices were cut as previously described from the renal cortex of normal young adult male rats of the black and white hooded Lister strain maintained in the Department of Experimental Medicine. The slices cut from the cortical tissue of two kidneys were washed to remove debris in the ordinary saline solution (without glucose), and then gently picked out with forceps and drained on filter paper. The drained slices were next placed in about 30 ml. of one of the four media (with glucose) in a boiling-tube, and a stream of nitrogen (hydrogen in four experiments) was passed through the solution at a rate of about 2 bubbles/sec for 30 min during which the tube was kept in a thermostat at 380 C. The slices were then gently drained, spread out on filter paper and lightly blotted. Some of the blotted slices were transferred at once to small weighed tubes for drying to determine the percentage of water in them; the remainder were distributed between five boiling-tubes, one containing each of the four media and one

2 586 J. R. ROBINSON containing liquid paraffin. Each tube contained 4-6 slices in about 10 ml. of liquid. The five tubes were placed in the thermostat, and oxygen was bubbled through them for a further min, after which the slices were removed, drained and blotted as before, and transferred to small weighed tubes for drying. Constant weight was reached in an oven at 105 C in 5 hr. The dried residues were extracted three times with ether and three times with petroleum ether and then re-weighed after a further 30 min in the oven to find the percentage of water in the fat-free portion of the tissue. The results of all experiments in aqueous media are reported as percentages of water in the whole tissue; but figures based on fat-extracted tissue have been given for slices which had been immersed in paraffin, because paraffin adhering to them would otherwise have led to spuriously low figures for the water content of these slices. The average percentages of water in fat-free tissue were greater than those in whole tissue for slices which had been incubated in aqueous media, and greater in the case of slices which had been incubated in paraffin. In order to discover how far prior deprivation of oxygen impaired the oxygen consumption of the tissue, some of the aerobic experiments were carried out in the flasks of Barcroft manometers instead of in boiling-tubes, and the rate at which the slices took up oxygen during the first 45 min after it was made available was measured. There appeared to be no difference in water content between slices incubated in the manometric vessels and those incubated in boiling-tubes, and results from both sets of experiments were pooled in computing the average figures for water content. The general plan of the experiments may be more readily gathered from the accompanying scheme. About 30 slices incubated ANAEROBICALLY SCHEME OF EXPERIMENTS About 5 slices transferred after blotting, to for 30 min (1) Weighed tubes for drying at 38 C (2) Oxygenated ordinary in: medium Ordinary medium (3) Oxygenated choline At 380 C in boiling or chloride medium tubes or manometric Choline chloride (4) Oxygenated sodium flasks for min; medium sulphate medium then blotted and or (5) Oxygenated choline transferred to weighed Sodium sulphate sulphate medium tubes for drying medium (6) Oxygenated liquid or Choline sulphate medium paraffin RESULTS The percentage of water in the slices The results are displayed in a diagram (Fig. 1) which falls into four main sections, A, B, C and D, separated by horizontal lines. The upper five horizontal bars in each section show the 'recovery' in oxygen of slices which had previously been warmed without oxygen in one of the four aqueous media, and the solid block at the right of each bar indicates the mean percentage of water in the whole tissue and the range of twice the standard error on either side of the mean. The lower two bars in each section show corresponding means and ranges for the fat-free portion of slices incubated anaerobically in the aqueous media and then aerobically in paraffin. The number of observations contributing to each average is indicated to the right of the corresponding bar.

3 IONS, ANOXIA AND TISSUE SWELLING 587 The compositions of the media are shown in labels on the bars as well as by a shading convention. For purposes of comparison, the vertical band crossing the horizontal bars in each section shows the range of twice the standard error above and below the mean percentage of water in fresh slices which had been incubated in the same media without previous anaerobic treatment (Robinson, 1956). Comparison of the uppermost bars in the four main sections of Fig. 1 with the vertical bands representing the earlier results shows that lack of oxygen was associated with the uptake of more water when the medium contained ordinary concentrations of sodium and chloride than when only one of these ions was present; when both were absent slices incubated without oxygen contained less water than those which had been freshly incubated with oxygen. Section A considered by itself shows that the water content of slices which had first been incubated without oxygen in the ordinary medium fell 3-4% when oxygen was supplied again in the same medium, a little over 4 % in the choline chloride medium, and about 5% in the two chloride-free media. The water content of the slices incubated in paraffin fell about 4 %. In no case was the water content restored to that of slices which had been incubated aerobically in the ordinary medium without previous deprivation of oxygen; their water content was more than 7 % less than that of the slices which were incubated anaerobically. Section B of Fig. 1 shows that the water content of slices which had been incubated without oxygen in the choline chloride medium fell 1-2% in the same medium with oxygen, a little less, about 1 %, in the ordinary medium, and rather more, about 2% and 2-3% respectively, in the sodium sulphate and choline sulphate media. The water content of these slices fell about 3% in oxygenated paraffin. Section C shows that the water content of slices incubated without oxygen in the sodium sulphate medium was about 3 % greater than that of fresh slices incubated aerobically in the same medium. Hardly any water was lost when oxygen was supplied in the choline medium, and the water content only fell about 1% in the other aqueous media, but the water content of the slices incubated in paraffin fell about 3 %. Section D shows that the water content of slices incubated without oxygen in the choline sulphate medium was about 1X5% less than that of fresh slices in the same medium with oxygen. These slices did not lose water when supplied with oxygen in the same medium, and their water content actually increased about 1% in oxygenated media containing sodium, chloride or both, but it fell 3% in paraffin. Since it seemed possible that the reduction in water content of the slices immersed in paraffin might be caused by blotting these slices twice without

4 588 J. R. ROBINSON Water content (%) I I I I I I I I _ Ordinary. No oxygen 141.Ordinary. Oxygen. 2_4 50_Choi. chlor. -Oxygen ZQf0 m12a &\\xsod. sufe.~ ~Oxygen Q\& 12 Chol. s-ulph-.--- Oxygen ~12 Oriay No oxyen Oxygen 18 0ho. chlor. No oxygen Ordinary. Oxygen 8m *2 Chol. chlor. Oxygen7jM Sod. suiph. Chol. suiph. Ch11IF-Chol. chlor. Paraffin. Oxygen Oxygen No oxygen Oxygen S-od. sulpjh. No oxygen '-'----' riay Oxygen3 2 1 I c. Z C oi chor, xyrn3; Z% O e I * it a I I I I 2020 *18 Mn12 14 B C Cholsulph. Oxygen 12 - mw- J'g - Sod. sulph. No oxygen Paraffin. Ia Oxygen_:,. a *.* 17 K. i. N% A.k 1-20 %Fk..A A Chol. chior. Ox*en D Chol. suiph. Oxy en 14 Chol. sulph. No ox;en 20 Paraffin. en i. I. - I - : I u Water content (%) For legend see opposite.

5 IONS, ANOXIA AND TISSUE SWELLING 589 intermediate exposure to an aqueous medium, or be due to other physical causes unrelated to the metabolic effects of oxygen, slices which had been in the ordinary medium wvithout oxygen were immersed in liquid paraffin and treated exactly as in the aerobic experiments except that hydrogen or nitrogen was passed through the oil instead of oxygen. Thirteen sets of slices from the ordinary medium had an average initial water content of 82-1 % (S.E %) in terms of fat-free tissue. Seventeen groups of corresponding slices showed an average final water content of 81F6 % (S.E %) after exposure to hydrogen or nitrogen in paraffin. The difference between the means was not statistically significant (t=1-73; P=0.1), so that the effect of oxygen is unlikely to have been an artifact. Changes in the actual amount of water in the slices Changes in the percentage water content do not adequately express the actual amounts of water lost by the slices. To reduce its water content from 84 to 80%, 100 mg of tissue would have to lose 20 mg of water; for the percentage of dry matter would have to increase from 16 to 20, so that the 16 mg of dry matter contained in the original 100 mg of tissue became 20% of a new total weight of 80 mg. Actual exchanges of water are better shown by expressing the water content of the tissue in terms of unit weight of dry matter. Thus, the total weight of tissue per kg of dry matter is 100/(100 - % water content) kg. If this is denoted by T, the total tissue water is (T - 1) 1./kg. If extracellular water always contributed one-quarter of the weight of the moist tissue (Robinson, 1950; Whittam, 1956), the amount of intracellular water per kg of dry matter would be (T-1)-T/4=3T/ Table 1 shows values calculated in this way from the averages of Fig. 1, and also the differences, in ml./kg dry matter, produced by oxygen in the aqueous media. Table 2 gives corresponding figures, calculated on a basis of fat-free instead of total dry matter, for experiments in paraffin. Fig. 1. Water content of slices after anaerobic incubation in aqueous media followed by aerobic incubation in the same media and in liquid paraffin (see text). jjwater content of Anaerobic experiment fresh slices incubated Sodium in medium in oxygen Chloride in medium Paraffin L lcholine sulphate medium Water content of fatfree portion of Water content of tissue tissue

6 590 J. R. ROBINSON The uptake of oxygen by the slices The results of the manometric experiments were not directly comparable with those in the same media reported by Robinson (1956) because the slices weighed into the manometric vessels contained different proportions of water on account of their previous anoxic treatment. When allowance was made for these differences in initial water content, by comparing results in terms of initial dry weight, the rates of oxygen consumption of slices in the aqueous TABLE 1. Effect of oxygen in aqueous media on intracellular water of slices previously incubated without oxygen Intracellular water, per kg dry matter After incubation with oxygen in Medium in which Ordinary medium Choline chloride Sodium sulphate Choline sulphate slices were A, incubated without Initial Diff. Diff. Diff. Diff. oxvygen (1.) (1.) (ml.) (1.) (ml.) (1.) (ml.) (1.) (ml.) Ordinary * Choline chloride Sodium sulphate Choline sulphate TABLE 2. Effect of oxygen in liquid paraffin on intracellular water of slices previously incubated in aqueous media without oxygen Intracellular water per kg fat-free dry matter After aerobic incubation Initial in paraffin Difference Aqueous medium (1.) (1.) (ml.) Ordinary Choline chloride Sodium sulphate Choline sulphate media were found to be 10-20% less than those of fresh slices incubated aerobically in the same media without prior deprivation of oxygen. No satisfactory figures were obtained for slices in paraffin, because the precautions recommended by Rodnight & Mcllwain (1954) were not taken, and the periods were too short to allow the oil to come into equilibrium with the gas phase. The apparent rates ranged from 1-6 to 2-5l./hr/mg; but they were still increasing. Volume of paraffin adhering to the slices The average weight of 'fat' removed by extracting the dried residues from 293 groups of slices which had been in aqueous media was 0-81 mg/100 mg moist tissue, with a S.E. of mg. Sixty-seven groups of slices which had been immersed in paraffin gave an average 'fat' content of 2-01 mg/100 mg with S.E mg. No loss of weight occurred when tubes containing small amounts of the liquid paraffin were left in the drying oven during the night.

7 IONS, ANOXIA AND TISSUE SWELLING 591 Hence about 1-2 mg of paraffin adhered to each 100 mg of the slices: and since the specific gravity of the paraffin was found to be g/ml. at 200 C, the volume of paraffin remaining on the slices after draining and blotting was about 1.4,u1./mg of moist tissue. DISCUSSION Slices deprived of oxygen might take up water because the entry of previously extracellular ions or the retention of products of autolysis increased the amount of osmotically active material in the cells; possibly also because active extrusion of water ceased for lack of energy. No appreciable shift of water should follow the entry of sodium, unaccompanied by anions, in exchange for internal potassium lost because of anoxia. The entry of water with external ions should vary with the concentration of sodium and freely permeant anions, but in assessing the amount of swelling to be expected from this source it must be remembered that renal cells normally contain more sodium and chloride than muscle cells and neurones, which are usually regarded as typical. Each minute the mammalian tubular epithelium secretes into the blood about half its own volume of an extracellular fluid, and the composition of its cells must reflect this. Analytical figures assembled by Harris (1956) suggest that the concentrations of sodium, potassium and chloride in mammalian renal tissue are of the same order of magnitude; hence if about one-quarter of the tissue is extracellular fluid, about one-half of the sodium and the chloride must be inside cells. Besides showing that sodium and chloride promoted the uptake of water during anoxia and opposed its removal when oxygen was supplied again, Fig. 1 illustrates the low water content of slices from the choline sulphate medium, which had presumably lost their normal quota of intracellular sodium and chloride. The fact that the water content of slices in the choline sulphate medium was unaffected by oxygen suggests that the loss with potassium escaping during anoxia balanced gains arising from autolytic changes and the cessation of possible active extrusion. The small increases in water content in other oxygenated media were probably due to the entry of sodium and chloride in addition to potassium. Although the somewhat greater oxygen consumption in choline media of slices which had not previously been immersed in them might suggest that some choline entered the cells and was metabolized, much greater swelling should have occurred in these media if choline entered freely. Indeed the low water content of slices in the choline sulphate medium argues against the free penetration of choline and sulphate into the cells either aerobically or anaerobically. Limited comparisons may be made of the effects, in ordinary media, of anoxia and of interference with respiration by other means. The slices deprived of oxygen contained 83-3 % of water, compared with 83-6 % from the regression

8 592 J. R. ROBINSON line calculated (Robinson, 1956) for results on slices poisoned with cyanide, and 81-6 % for slices chilled to 0-4 C (Robinson, 1950). The water content had returned further towards normal when cyanide was removed from the medium than it did when oxygen was supplied again in the present experiments, although the rate of oxygen consumption had recovered more completely after anoxia than after poisoning with cyanide. Hence it seems that their somewhat low oxygen consumption would only partly explain the failure of slices, which had been deprived of oxygen in media containing sodium, ever to regain the water content of fresh slices incubated aerobically in the same media. Moreover, retention of sodium in the cells could hardly be responsible, for the higher water content persisted in sodium-free media (Fig. 1). The extent to which potassium and other ions are retained in the cells of slices in the various media is being investigated. Tlhe behaviour of the slices in paraffin From the rate at which inulin occupied its volume of distribution in similar slices in the cold (Robinson, 1950), the aqueous media probably permeated throughout the interstitial water of the tissue and effectively bathed the cells in an unlimited extracellular phase of constant composition and osmotic pressure. If all the cells in the slices kept in osmotic equilibrium with their surroundings, their volume should have been determined by the quantity of intracellular solutes, and they should have lost water during recovery from anoxia simply because the internal osmotic pressure fell below that of the medium when products of autolysis were destroyed and intruding ions expelled. The osmotic gradient responsible for the movement of water out of the cells should disappear if the external aqueous phase could be removed; a reduction in the osmotic pressure of the cells' contents should not then lead to a loss of water, and active processes transporting water might be unmasked. Unpublished preliminary experiments by 0. D. Batt and Robinson suggested that the swelling of kidney slices which had been deprived of oxygen in ordinary media could be reversed by oxygen in liquid paraffin. Although it is now clear that the aqueous phase surrounding the cells was not completely removed, since paraffin entered so small a fraction of the extracellular space of the slices, the 'osmotic reservoir' bathing the cells was reduced to a small pool of the order of one-third of the cells' volume, trapped round the slices by the oil. Fluid leaving the cells of a slice under paraffin was presumably first added to this pool, and subsequently removed when the slice was blotted. Loss of water from slices which had been in ordinary media without oxygen need not imply active secretion of water, for the expulsion of sodium from the cells could raise the concentration of the little extracellular pool and shift water out by osmosis. Fig. 1 shows, however, that the water content of slices which had been in relatively sodium-free anoxic media (B and D), and whose

9 IONS, ANOXIA AND TISSUE SWELLING 593 cells presumably contained little sodium to be extruded, was reduced almost as much by oxygen in paraffin as that of slices from the ordinary medium (A). Since Table 2 shows further that the actual loss of water from the sodiumdepleted slices was about half as great, extrusion of sodium could probably not account for more than one-half of the aerobic loss of water from slices swollen in the ordinary medium. It remains to explain the curious fact that the slices immersed in paraffin lost so much more water to the tiny pools trapped by the oil than to the far larger volumes which surrounded slices suspended in the aqueous media; for the small pools presumably had the same initial composition as the media in which the slices had been incubated without oxygen. An important effect of restricting the extracellular aqueous phase to the small oil-enclosed pool must have been severely to limit the amount of potassium available to be re-accumulated by the cells. The greater net loss of water by slices under paraffin might therefore be explained by the smaller gain of water in association with potassium which could offset losses arising from the reversal of autolytic changes, and, possibly, from active extrusion of water. Another effect of restricting the extracellular aqueous phase would be, that when the total concentration was reduced inside the cells by aerobic removal of products of autolysis, the small enclosed pools would soon be diluted by water from the cells, so that slices in paraffin should lose less water during osmotic equilibration than slices in aqueous media. It can easily be calculated that one-quarter to one-third as much water should leave the cells to a pool of one-third of their volume as to an unlimited extracellular phase of the same initial osmotic pressure. Hence both the uptake of water with potassium and the loss of water from repair of autolytic changes should have been minimized by restricting the available volume of extracellular fluid. But a special transporting system capable of actively expelling water from the cells should have been unaffected, and might have been responsible for a considerable part of the water lost from slices which had been incubated in sodium- and chloride-free media. It is hoped to secure unequivocal evidence on this point by examination of the fluid trapped round slices in non-aqueous media. SUMMARY 1. Rats' kidney slices which had first been incubated anaerobically at 380 C in an ordinary medium, or in modifications made by substituting choline chloride, sodium sulphate or choline sulphate for sodium chloride, were supplied with oxygen in the aqueous media and in liquid paraffin. 2. Previous anoxia did not greatly impair the oxygen consumption measured in aqueous media. 3. External sodium and chloride increased the amount of water taken up when oxygen was lacking, and opposed the reduction in water content which occurred when oxygen was supplied again. 38 PHYSIO. CXXXVI

10 594 J. R. ROBINSON 4. Slices from all of the oxygen-free media lost considerable amounts of water when supplied with oxygen in paraffin. Hence, although movements of common ions were important, they were not the sole cause of shifts of water between the cells and their medium. REFERENCES HARRIS, E. J. (1956). Tran8port and Accumulation in Biological Systems. London: Butterworth. RoBINsoN, J. R. (1949). Some effects of glucose and calcium upon the metabolism of kidney slices from adult and newborn rats. Biochem. J. 45, ROBINsoN, J. R. (1950). Osmoregulation in surviving slices from the kidneys of adult rats. Proc. Roy. Soc. B, 137, ROBrNSON, J. R. (1956). The effect of sodium and chloride ions upon swelling of rat kidney slices treated with a mercurial diuretic. J. Physiol. 134, RODNIGHT, R. & McILwAIN, H. (1954). Techniques in tissue metabolism. 3. Study of tissue fragments with little or no added aqueous phase, and in oils. Biochem. J. 57, WHITTAM, R. (1956). The permeability of kidney cortex to chloride. J. Physiol. 131,