Principles of General Physiology

corpuscles. K, Spiral tubule. d, Descending limb of Henle's loop. All the above are left white. H, Bend of the loop, a, Ascending limb of Henle's loop. p.e, Proximal convoluted tubule. j, Junctional tubule. These last lour parts are grey. c, Collecting tubule. B, Duct of Bellini, receiving a number of collecting We have found sufficient evidence to show that the first stage is a mere filtration from the blood. It is clear that this, if copious enough, would be able to get rid as far as necessary, of all the non-colloidal metabolic products. But, since these are present in very small concentration in the blood, a large amount of water must be filtered with them.

In the case of water animals, this would not be a serious matter. In the fish, Lophiu* pixcatorius, Denis (1913) found that the urine in the bladder had a specific gravity of 1 •()!»; and contained only 0*083 per cent, of total nitrogen, but 1'08 per cent, of chlorine. Contrast these figures with those of one of the higher land vertebrates. In man, the percentage of nitrogen is about 1, and that of chlorine about 0'6 per cent. It is obvious that the sea fish has no need to be careful as regards water and chlorides.

In land animals, where water is often difficult to obtain, its loss would be serious to the organism. Salts are also of importance, so that we find that arrangements have been evolved to diminish losses of both kinds. According to the theory put forward by Ludwig (1844), water is absorbed by the cells lining the tubules, as the dilute glomerular filtrate passes over them. The evidence for this must be examined. Further, we have to remember that mere concentration by removal of water will not account for the fact that the concentration of urea goes up very much more than that of sodium chloride, to take the two chief constituents. To do this, cither sodium chloride must be absorbed, or urea must be excreted in the tubules It will be noticed that these two compounds represent two distinct classes of substances which air present in the glomerular filtrate, which is an indiscriminate mixture of all the diffusible substances in the blood. Urea represents the various metabolic products which require removal as far as possible; sodium chloride represents valuable food-stuffs, inclusive of glucose, which should not be lost more than is avoidable and are present in the glomerular filtrate because they cannot help being there, if one may use the phrase. Sodium chloride itself is probably chiefly of importance for the maintenance of the correct osmotic pressure of the blood. This could be done by other salts, but, as we have seen above, those of sodium are the least toxic. Where they are not to be obtained from earth or sea, there is great desire for them, especially in animals taking a diet in which vegetable matter preponderates, since the food does not contain sufficient. The tubules of an ideal kidney, therefore, would absorb, together with water, the useful substances of the filtrate, leaving the metabolites untouched, or even adding to them by active secretion.

In contradistinction to the theory of Ludwig, Bowman (1842), while also regarding the glomerular function as that of filtration, believed that the cells of the tubules secreted the specific contents, such as urea, uric acid, etc., while the filtrate itself, which would be much less copious than that required by Ludwig's theory, contained only water and salts. On p. 75, Bowman speaks of the " escape of water " from the blood, and, from the description given, it seems evident that he regarded the process as a filtration effected by the blood pressure. In any case, the total process must involve work on the part of the cells, since the osmotic pressure of the urine is higher than that of the blood.

Absorption of Water. — If we calculate the amount of glomerular filtrate which must be concentrated in order to give the daily output of urea, as is done by Starling (1912, p. 1288), we find that 28 litres of water must be reabsorbed by the tubules from 30 litres of filtrate. While it is not impossible for so large a quantity of fluid to be filtered by the glomeruli, it seems a wasteful process. On the other hand, if we confine our attention to the sodium chloride, and assume that the excess of urea is secreted by the tubules, only 6 litres of filtrate would be necessary, since 1 litre of blood contains about 2-7 g. of sodium chloride and 15 g. are excreted daily. These 6 litres would only need concentrating down to 1*5 litres. It seems to be forgotten by some opponents of the reabsorption of water, as is pointed out by Cushny, that the cells of the tubules are not comparable to those of a secreting gland which elaborate new substances, and that their function consists in the separation of urea, etc., from the blood. Since, therefore, the urea is only present in a very small definite amount in

the blood, the quantity of liquid to be dealt with by the cells of the kidney is the same, whether it comes to the cells from the blood or from the lumen of the tubules. Perhaps the following a priori considerations may assist the argument. If there is no absorption of water by the tubules, it is necessary to assume that not only are urea and similar metabolites secreted by the tubule cells, but also useful substances like glucose and sodium chloride. Now it is difficult to understand why a wasteful, or at any rate useless, process should have been produced in the course of evolution. If we confine our attention to the higher land animals, it might seem an inefficient process to filter off water and solutes from the blood, only to be in great part reabsorbed. The ancestral excretory organs, however, were probably merely filters, like the glomerulus, and the process was a satisfactory one, since there was no need to preserve either water or salts on account of their abundance in the ocean. As regards organic, diffusible food materials which would escape with the filtrate, they might be kept back in great part by adsorption on colloidal surfaces in the cells of the organism. In the course of evolution on land, the saving of water and salts became more and more advantageous, so that the power of reabsorption began to show itself, while the necessity of so large a volume of filtrate was decreased by the development of active secretory cells for elimination of the waste products.

We must not forget, moreover, that the filtration process involves no expenditure of energy on the part of the kidney itself, however large the amount filtered. The energy comes from the heart and is but a small fraction of that used in other ways. Ribbert (1883) believes that he has positive evidence of the absorption of water by the tubules in the results of removing the medulla of the rabbit's kidney, which takes away the greater part of the tubules. It was found that a much more dilute urine was excreted. But the kidney is a very sensitive organ, and the procedure a somewhat violent one, so that too much stress must not be laid on these experiments.

Absorption of water, in the mammal entirely performed in the renal tubules themselves, appears to take place also in the cloaca, or posterior part of the alimentary canal, in the bird. Sharpe (1912) finds that the urine is a clear liquid in the ureter and only attains its well-known semi-solid nature after leaving the ureter. If we grant the process of glomerular filtration, and the evidence for this is overwhelming, the fact that the urine contains a larger percentage of sodium chloride than the blood does, is an indirect proof of absorption of water. For, as we shall see later, there is every reason to believe that no secretion of sodium chloride occurs in the tubules.

So far, then, we may state what appears to be the most probable view thus : The glomeruli filter from the blood sufficient fluid to contain the whole of the sodium chloride excreted and probably more; part of the water together with a part of the valuable solutes, such as sodium chloride, glucose and amino-acids, is reabsorbed in the tubules. But this process would not sufficiently get rid of the urea produced by the organism, unless a great excess of sodium chloride were also excreted ; accordingly, urea and similar substances are actively secreted by the tubule cells, and turned into the fluid as it passes over them. We have then to see further what evidence there is that these two processes of secretion by the tubules and of absorption of certain solutes by them actually take place.

Secretion by Tubules. — Bauer (1898) injected intravenously a solution of uric acid in piperazine ; considerable diuresis resulted — the kidneys, on microscopic examination twenty to sixty minutes afterwards, showed no uric acid in the glomeruli, probably because the filtrate was too dilute. But in the lumen of the convoluted tubules there were considerable masses of uric acid deposit. This, however, might have been produced by absorption of water. That it was not so, and was probably due to secretion by the cells, was shown by the presence of uric acid particles within them. The cells of the medulla were free from uric acid, which was only present in the lumen of the tubules in this situation. It seems impossible to believe that so much could have been filtered off by the glomeruli in the time, even if we reject the evidence of the deposit inside the cells.

Histological evidence in other animals shows all stages of extrusion of granules from cells of the tubules (see the observations of Metzuer in Altmann's paper, 1894, p. 133, in the case of the embryo chick, and in the kitten, 1907, 2, p. 221 and footnote on p. 222. Also Todaro, 1902, in Salpa). It is obviously more difficult to prove the secretion of urea, but possibly the formation in the cells of vacuoles which discharge into the lumen of the tubules, as described by Gurwitsch (1902), is connected with the process. No direct evidence has been found, so far as I am aware, of excretion of glucose or sodium chloride by the tubules. On the contrary, as we shall see, there is evidence that they are absorbed.

Absorption of Solutes by Tubules. — In the frog, owing to the fact that the glomeruli are supplied by blood directly from the aorta, while the tubules are supplied from a separate renal portal vein, it is possible to investigate the two systems separately. I do not propose to describe the earlier experiments of Nussbaum and others, since tlu-v were to a certain extent inconclusive, on account of neglect of the fact that, while the ivnal portal blood supplies the tubules alone, the arterial blood from the aorta, aft IT passing through the glomeruli, supplies the tubules with oxygenated blood, so that cutting off the glonuTu hi circulation at the same time caused death of the tubules from asphyxia. Those interested will find a description of the experiments in the work of Starling (1912), or of Met/.nn

The later experiments of Bainbridge, Collins, and Menzies (1913) have brought out some points of interest, to which brief reference may be made. The urine of the frog is normally of a lower osmotic pressure than the blood, or, if the kidney is perfused with Ringer's solution, the salt concentration of the urine is lower than that of the Ringer's solution. This state of affairs is brought about by the tubules, since, when they are poisoned, the urine is always isotonic with the solution perfused, that is, it is a pure glomerular filtrate. Now this activity of the tubules may be due either to secretion of water or to absorption of salts. No evidence could be obtained of the former except, perhaps, under the influence of some diuretic agent such as urea. Sodium chloride must therefore be absorbed. The frog, being essentially a water animal, is under no necessity of hoarding water and, in fact, it has been stated that the urine secreted in twenty-four hours may exceed the total weight of the body. It would seem possible, then, that the whole of its excretory products could be got rid of by mere filtration ; but it is important that the valuable substances, like sodium chloride and glucose, also in the filtrate, should be retained.

Experiments made by Cushny (1901) point in the same direction. In the later stages of the diuresis brought about by injection of a mixture of sodium chloride and sulphate, the proportion of chloride to sulphate in the blood was 0'493 to 0*191, whereas in the urine it was 0'094 to 2'0. The sulphate is much less readily absorbed by the tubule cells than the chloride is, as by cells in general, and it is evident that the fact favours the reabsorption of the valuable chloride. It is possible that the foreign sulphate may actually be excreted by the tubules, but there is no direct evidence of the fact. During the maximum of diuresis, the concentrations of the two salts in the urine approach much more closely to tln»i' in the blood, although that of the sulphate is higher than in the blood, while that of the chloride is lower. It is clear that the faster the liquid passes along the tubules, the less opportunity is there for the activity of the cells of the tubules effect changes in its composition, so that the more rapidly the urine is produced, the more nearly is it isotonic with the blood. It is important to notice that, in Cushny's experiments, the percentage of chloride in the urine was never higher than in the blood. It would appear from some experiments by Loewi (1902) that mere diffusibility is not the only controlling factor when poisonous salts are concerned, since sodium iodide is excreted as effectively as sodium sulphate.

Cushny also performed some experiments in which the kidney was caused to secrete under an increased pressure in the ureter, so that the glomerular filtrate remained longer in contact with the tubules. The results showed a greater absorption of sodium chloride than of sulphate and urea. Of course, the total amount of filtrate is less under the increased ureter pressure, so that one can only compare the proportions of the different constituents and the experiments do not show that there was in fact any absorption of sulphate or urea.

If an animal receives no sodium chloride in the food for several days, the serum still contains nearly the whole of its normal amount, but the urine practically none. Very nearly the whole of that filtered through in the glomeruli must be reabsorbed in the tubules. As already pointed out, the filtration process tends to cause a loss of food materials, so far as these are non-colloidal, as indeed those of the blood are. Although a part of these may be held in adsorption equilibrium, even glucose itself, as pointed out above (page 57), a certain quantity must escape in proportion to the amount of the filtrate. In fact, small amounts of glucose and amino-acids are normally present in the urine. Nishi (1910), however, brings evidence that there is absorption of sugar in the tubules of the cortex. Even when excess of glucose is present in the blood, it is found that the medulla of the kidney contains none, although it is present in the cortex. If diuresis is produced, glucose is present in both parts. The obvious explanation of the results is that most of that present in the glomerular filtrate is reabsorbed in the tubules, except when the current is too rapid to allow sufficient time Some experiments by Easier (1906) support this view. Sugar solution was run into the ureter of one side under a pressure of 26 mm. of water and was found to be present in the urine of the opposite side.

In experiments of this kind, however, it must be remembered that unless we assume complete impermeability of the tubule cells to the particular substance in question, diffusion must take place to some extent, if the concentration is greater in the lumen of the tubules than in the blood vessels. For the reason last mentioned, most of the earlier experiments with dyes are capable of interpretation either on the hypothesis of absorption or of secretion. This objection does not seem to hold for those of Ghiron (1913), who injected a small amount of aniline-blue or Congo-red into a vein, while observing with the microscope the surface of the living kidney of the mouse (for the method, see Ghiron's paper of 1912). It was seen that a pale blue or red glomerular filtrate first appeared in the convoluted tubules. This would have the same concentration in dye as that of the blood, so that no dye would pass through the cells of the tubules by mere diffusion, since the concentration would be the same on both sides. But it was seen that the border of the cells next the lumen was the first to become filled with particles of dye, which gradually passed towards the side of the capillaries. So that the cells evidently absorbed material from the lumen and passed it back to the blood.

The, Normal Process. — We arrive then at the following conception of the normal process of renal activity in the higher land animals, as was sketched in outline above. By a retention of the pure filtration process of the lower animals, a filtrate is first made, which contains all the non-colloidal constituents of the blood in the same concentration as therein. But, if this were to be sufficient to carry away the whole of the waste products, which are present in very low concentration in the blood, an enormous loss, both of water and of valuable constituents, would be entailed. To meet this, a mechanism has been developed, by which not only a great part of the water is reabsorbed, but also a large proportion of the valuable salts, such as sodium chloride, and also organic foodstuffs, such as glucose and amino-acids. At the same time there is an active secretion of waste products, such as urea and uric acid, by the cells of the tubules, into the glomerular filtrate bathing them. With these, foreign salts injurious to the organism are excreted. The existence of this latter process renders the filtration of such large quantities of water unnecessary; but it is important to remember that the actual work done is measured by the difference between the osmotic pressures of the constituents of the blood and urine, irrespective of the way in which the actual concentration is brought about.

Since the rate of filtration in the glomerulus depends on the difference between the blood pressure in it and that in the tubules, it is clear that any process increasing the difference will increase the rate of flow. Rise of general blood pressure, produced by means to be described in Chapter XXIII., is one of these. Dilatation of the arterioles of the kidney on the heart side of the glomeruli themselves is another means, and, clearly, a combination of the two would be most effective. Conversely, a diminution of general blood pressure, or a constriction of renal arterioles, decreases the rate of flow. The kidney is, in fact, copiously supplied with vasoconstrictor nerves, and to some extent with vaso-dilator nerves, so that the requisite mechanism is not wanting.

We have seen further that the cells of the tubules intervene by active processes requiring the consumption of energy, so that it does not seem improbable that secretory nerves may exist, similar to those of the salivary or sweat glands. Histologists have described nerve fibres ending in the cells of the tubules especially the work of Smirnov, 1901, one of whose figures is reproduced in Fig. 474 on p. 374 of Schafer's " Essentials of Histology ").

Certain experimental evidence has been brought forward by Rohde and Ellinger (1913) that the splanchnic nerve contains fibres which inhibit the activity of the tubule cells. The chief fact in support of this view seems to be that the diuretic effects of section of the renal nerves. due in the first place to removal of tonic vaso-constrictor impulses, lasts for several months, by which time it is supposed that the renal arterioles have recovered from the immediate effect of the section. It is to be remembered that vaso-constrictor reflexes are probably hrini; sent to the intact gland during the time of observation, which are the cause of a diminished secretion on this side; on the side of the section, of course, they would be absent. Si me other evidence, with regard to the solid constituents of the urine, seems to me to be explicable by the vaso -motor change, without the necessity of assuming secretory nerves. It must be confessed, however, that there are many difficulties in the way of deciding the question. Asher and Pearce (1913) believe that they have evidence that there are secretory nerves to the kidney contained in the vagus nerve, but the evidence that all vaso-motor action was excluded is not altogether satisfactory. Some observers had previously stated that this nerve contains inhibitory fibres for the secretion of urine (see Bradford, 1889, p. 395). Bradford himself \v a^ unable to find any vaso-motor fibres in the nerve.

An interesting morphological point was made out by Bradford (1889) in his investigation of the nerve roots by which the renal nerves leave the spinal cord. The area is a very extensive one, from the 4th thoracic to the 4th lumbar, although the largest number are contained in the llth, 12th, and 13th thoracic. This long area is of interest in connection with the ancestral origin of the kidney from a series of segmental organs extending over a considerable number of segments.

Diuretics. — All substances, such as salts, sugar, etc., which raise the osmotic pressure of the blood, bring about the passage of water from the tissues into the blood and thus decrease the osmotic pressure of the colloids of the blood. The pressure necessary to separate the glomerular filtrate is thus reduced, or, if it remains constant, the rate of filtration is increased. In addition to this effect, a salt foreign to the organism, such as sodium sulphate, excites an active process in the tubules. This is shown by the experiments of Barcroft and Straub (1910), in which injections of isotonic sodium chloride produced, by mere dilution of the blood, a diuresis unaccompanied by any increase of oxygen consumption. The diuresis produced by sodium sulphate, on the contrary, showed a considerable extra consumption of oxygen.

Urea causes diuresis by dilatation of the renal arterioles, without any considerable effect on the general blood pressure. The diuretic effect of glucose lasts longer than its effect on the concentration of the blood plasma (" hydrsemic plethora"), so that it seems to bring about a local dilatation of the kidney arterioles, in addition to its dilution effect. It has been shown by Cushny (1902) that if the increased blood flow through the kidney, produced by injection of 3 per cent, sodium chloride, be brought back to its initial rate by an adjustable clamp on the renal artery, the diuresis ceases ; so that the vascular change is the responsible factor and no specific action on the cells is present.

Certain evidence indicates that such specific diuretics as the purine derivatives, caffeine, etc., may have a paralytic effect on the absorption by the tubules. We have seen above that, although an animal may be deprived of chlorides in the food, the blood continues to preserve nearly its normal concentration (0*7 per cent.) in sodium chloride, while the urine may contain as little as OO8 per cent., owing to the almost complete reabsorption of this important salt by the tubules. Under these conditions, if one of the diuretic drugs referred to be administered, the amount of the urine is increased and the sodium chloride goes up to 0-64 per cent., as shown by Pototsky (1902). Such an increase is considerably greater than would be accounted for by the lessened absorption of sodium chloride .011 account of the more rapid passage along the tubules.

An interesting specific diuretic action is exerted by a hormone formed by the pituitary gland, as described by Magnus and Schiifer (1901) and by Schiifer and Herring (1906). Extracts of this organ cause a rise of blood pressure together with vaso-dilatation of the kidney and increased flow of urine. The diuresis and kidney dilatation last longer than the rise of general blood pressure, so that there must be a specific effect on the kidney itself. The fact is suggestive in connection with the view taken by Gaskell (1908, pp. 215 and 321) of the origin of the pituitary body from the coxal glands of the invertebrate ancestor, which were excretory in function and remain the chief excretory organ in Limulus. One is reminded also of the effect of saliva in producing activity of the submaxillary gland, as described by Demoor (1913)'.

Some special products of secretory activity may be referred to briefly in order to show the great variety of products which different organisms are able to manufacture. Acid and Alkali. — In the large mollusc, Dolium galea, a kind of salivary gland exists, which produces sulphuric acid of the strength of 4 to 5 per cent. (Preyer, 1866), apparently used for attacking the calcareous shells and spines of starfish, and other echinoderms used as food. The same purpose is probably served by the large percentage of aspartic acid produced by some related molluscs. It seems desirable that the fact of secretion of 5 per cent, sulphuric acid should be reinvestigated.

The production of hydrochloric acid in the stomach, of decimolar or even higher concentration, has not yet received a satisfactory explanation. It is clear that a large amount of osmotic work must be done in the process, and it is difficult to suggest a possible chemical reaction by which it might be obtained under the conditions compatible with cell life. Miss Fitzgerald (1910) gives some hypotheses on the question. In a mixture of chlorides and acid phosphates, there will be present both H' and Cl' ions, so that if the cell membrane is permeable to these and not to other ions of the cell contents, it seems possible that the secretion may be explained/

A theory has been suggested by Koeppe (1900) on the hypothesis that the gland membrane is impermeable to Cl' ions and permeable to H' and Na- ions, but experimental facts obtained by Benrath and Sachs (1905) do not support the view. The fact of the production of an acid reaction when an electro-negative colloid, such as arsenious sulphide, is thrown down by neutral salts of barium, etc., as mentioned above (page 94), may have some connection with the phenomenon. It is, perhaps, most likely that the surface action of colloids may ultimately afford a satisfactory explanation, when taken in connection with special arrangements of the cell membrane as regards permeability.

Similar remarks apply to the production of a secretion of alkaline reaction, such as the pancreatic juice. The cuttle-fish, Sepia, as is well known, produces an inky fluid to cover its retreat from enemies. The pigment contained in this secretion is used by artists as a pleasant warm black or brown paint. It is one of those black or brown compounds known as melanins, and, according to von Fiirth (1903, p. 372), is formed in the cuttle-fish by the action of an oxidising enzyme, tyrosinase, on tyrosine.