Principles of General Physiology

A subsidiary point of interest in this connection, which also gives support to the filtration theory, is that, if there were some special function of the cells of the glomerulus in the nature of selective secretion, the kidney would not continue to turn out an important salt, such as sodium chloride, when the organism has been deprived of it in the food. Cohnheim's experiments (1912, p. 80) show that the contrary is the case. On the filtration hypothesis, sodium chloride must appear in the liquid leaving the glomerulus as long as there is any of it free in the blood. We shall see, however, that it may be reabsorbed to a considerable extent in the tubules,

A certain difficulty must not be overlooked. The urine of the frog, and of water animals in general, as it leaves the kidney, is of a lower osmotic pressure than that of the serum minus its colloids. After copious drinking of watery fluids, this may also happen in man. Burian (1910) calls attention to this fact, and suggests that the explanation may lie in the possibility of the particular layer of blood plasma in immediate contact with the filtering surface being, under certain conditions, of a lower concentration than the rest. An adsorption process of some kind suggests itself. It is to be remembered that we have no direct knowledge of the composition of the solution as it leaves the glomerulus in such cases, and it is known that the tubules are able to absorb valuable stuffs from the glomerular filtrate as it passes over their cells ; although, when the flow is very rapid, there does not seem to be much time given for the process. It has been held that the tubules secrete water, but there does not seem to be any evidence for this. The functions of the tubules will be discussed later.

Brodie (1914) calculates that the pressure required to drive urine down along the tubules at the rate of diuresis is practically identical with the blood pressure in the glomeruli, so that only one or two millimetres at most would be available for a filtration pressure. The difficulty of accepting the calculation rests on the fact that the fourth power of the radius of the capillarv tube enters into Poiseuille's formula, which was used, so that a slight difference iu this value would make a large one in the result, and JJrodie's measurements are made on a hardened kidnev. Apart from this, it is very difficult to believe that a pressure of S.S nun. of mercury could exist in the glomerulus end of the tubule without causing great dilatation. If this distension \\ere prevented by the resistance of the capsule of the kidnev, it would surely ivMilt in obstruction of the capillaries and veins. There is no evidence of a strong muscular coat to the tubules, such as there is in the arterioles of the glomerulus. The conclusion drawn l>\ Krodie from his calculation is that the glomerulus must lie an actively secreting organ for water, and that the water is driven on in some way by the aid of blood pressure. But if we grant that the cells act as secreting organs, like those of the salivary glands, why ia it in-i-e—ary t" assume any further driving pressure?

Although the glomerular filtrate of the kidney has no higher osmotic pressure than that of the blood plasma, it is well known that the urine, as it leaves the kidney, has a much higher molar concentration. Work must therefore be done in the total process. This work can be calculated in the way indicated in a previous chapter (page 33), and will be described presently. Chemical Work. — In the production of the special constituents of any secretion, chemical work must be done by the gland cells. Moreover, the source of the energy required for their osmotic work has also its origin in chemieal reactions in the cell systems. We have no direct way of measuring this work, but the amount of oxygen consumed, or the carbon dioxide given off, by the gland in different states of activity, gives us valuable indirect information. The energy at the disposal of the cells is derived from the oxidation of substances of high chemical potential to substances of low chemical potential, such as carbon dioxide and water. In the process, a part of the free energy is degraded to heat and carried off by the blood current, so that it is impossible in practice, at all events as yet, to obtain an absolute measurement of the amount of work required to form a given amount of secretion.

Osmotic Work. — It is advisable to give some further details of the method of calculating this, in addition to those already given in a general foim when discussing the formula for the isothermal compression of a gas. We must recollect that we cannot look upon the urine as merely a concentrated glomerular filtrate, as was done by Dreser (1892) in the first approximate calculation of the renal work. The relative proportion of the constituents is not the same. For example, the ratio of sodium chloride to urea in the blood (or glomerular filtrate) is about 10 to 1, whereas in the urine it is reversed, and becomes 1 to 2. Thus, while the osmotic pressure of the sodium chloride has only to be raised from that of a 0'18 molar solution to that of a 0'36 molar one, or about doubled, that of the urea has to be raised from a O'Ol molar strength to that of a 0*4 molar strength, or increased forty times.

It will be best, however, to obtain first the work done, as Dreser did, on the supposition that we are dealing only with an increase of concentration, leaving for the present the fact that the various constituents are unequally affected. At the outset it is well to call attention to the fact that the results obtained by such methods of calculation are valid, whatever be the exact mechanism by which the process is brought about in the organism. On page 33 we saw that the expression which gives us the work done in compressing a gas isothermally from a volume vl to a volume v.2 is —

R, of course, can be expressed in any convenient units, and is — The same expression, as we saw before, applies to the alteration of the concentration of a solution when produced in any way. Of course, when electrolytes are concerned, changes in electrolytic dissociation must be taken into account. Incidentally, it may be remarked that, as regards calculations involving energy factors, living organisms have the advantage of their reactions being carried on in a practically isothermal system, so that the formulae are comparatively simple. Any change of temperature is so small as to have only a minimal effect on the results of the calculation.

To proceed, the total osmotic concentration of the blood is about 0-3 molar, and from this, under ordinary conditions, the kidneys produce a urine which is about molar, that is, the osmotic pressure is increased rather more than threefold. Instead of volumes in our formula, it is convenient to take concentrations, which are the reciprocals of the volumes in which 1 gram-molecule is dissolved. Further, since the osmotic pressure (IT) and the depression of the freezing point (A) are also in direct relation to one another, we can take, in place

Strictly speaking, the use of the last expression is only permissible at the temperature of the freezing points in question, since electrolytic dissociation may not be the same. But the experimental error of the freezing point measurements exceeds the very small errors possible on account of differences of dissociation. Accordingly, the minimal work which the kidney must do in order to produce, from a glomerular filtrate of Ap a urine of A2> ^n an amount which contains 1 gram-molecule at 37° is —

If n mols. are compressed instead of one, the work is increased rc-fold. The molar concentration, in practice, can be best obtained from the depression of the freezing point, to which it is related, as we have seen (page 155), and in the following way : — The depression of the freezing point of urine (A2) is between l°-5 and 2°, so that, for simplicity of calculation, we may take it as l°-85, and there are, in man, about 1'5 litres produced per day, hence: —

But, as Dreser points out, we must remember that to produce 1-5 litres of urine of A = l°-85 from blood of A = 0°'56, In the calculation, the difference between this quantity and that of the urine secreted, namely, 4-955 - 1'5 = 3'455 litres, has been reckoned as pure water. In point of fact, it is kept in the blood and at a A of 0°'56. We have thus made the work of the kidneys too great by the amount required to raise 3-455 litres to the osmotic pressure corresponding to a A of 0°-56. A A of 1°'85, as we saw (page 155), is equivalent to the osmotic pressure of a molar solution, that is 22*4 atmospheres ;

freezing point, or 7 '7 atmospheres at 37°. We have to subtract, then, 3'455 x 7'7 = 26*6 litre atmospheres, from our first value of 45'6, leaving 19 litre atmospheres as the correct value, on our simple assumption of mere total concentration. But, as already remarked, this is not all. We must take account of the relative concentrations of the different constituents of the urine, since they are by no means equally compressed. The urine is not merely a glomerular filtrate boiled down, as it were. This question is treated in the paper by von Rhorer (1905), to which the reader is referred for more details than can be given here. It will be clear that a completely accurate measurement of the total work done could only be obtained by taking each constituent of the urine for itself. As an illustration of the method, we may take the two chief constituents of the urine, sodium chloride and urea, as is done by von Rhorer (pp. 388-390), and, indeed, the osmotic concentration of the other constituents is comparatively small, so that our result will not be far wrong.

Instead of the complex glomerular filtrate, we imagine, in the first place, a pure solution of sodium chloride of the same concentration as that in which it exists in the blood, that is 0'18 molar, inclusive of ions. We have to concentrate this solution to that of the sodium chloride in urine, that is, to 0'36 molar. It will be instructive to treat the problem in the way done by van't Hoff, described in one form on page 157 above. We imagine a cylinder closed at the end, and containing a piston impermeable to sodium chloride, but permeable to all the other solutes of the glomerular filtrate and to water. We compress the filtrate until the concentration of the sodium chloride below the piston is raised to 0'36 molar. In the kidney the concentration is only raised from 0'18 to 0'36, while in our imaginary model no sodium chloride passes through the piston, but water does, so that the original concentration of 0' 18 molar above the piston is lowered; we must therefore add continuously sodium chloride to the solution above the piston in order to maintain its concentration constant at 0'18 molar. We keep thus the osmotic pressure above the piston unaltered at p0, while below it the pressure during the operation is a variable one, p, and is raised gradually from p0 to 2p0 (0'18 to 0-36). The work done consists, then, in raising the pressure of a volume of solution by a series of infinitesimal steps from p0 to a higher one, through the variable pressure differences of p -p0. That is : —

dA = (p-Po)dv. The integral of this expression consists of two members — where v is the initial volume and v the final one, the actual process being performed by the diminution of the volume from v to v. p0, being kept constant, is not subject to integration. Instead of -„ we can put (concentrations instead of dilutions), and since Putting these values in the integral, we have A = rcRT 2-3 log ~ - We now, in imagination, repeat the operation on this same solution, using a piston which is impermeable to urea, permeable to water and sodium chloride with the other solutes. The compression has to raise c of O'Ol to c' of 0'4 ; n is 0'4, and therefore A is

By the simple process of calculation of total concentration by which a glomerular nitrate of initial concentration of 0-18 + 0-01=0-19 molar ( = c) is raised to one of 0-36 + 0-40 = 0-76 (c') by aid of a piston impermeable to urea and sodium chloride, we have, since w = 0'76 — Thus, when we take account of the different partial pressures of urea and sodium chloride, we obtain 2*5 times as great an expenditure of work. It may be pointed out that the work calculated in this manner is simply that necessary to effect the change of molar concentrations, and is independent of any particular process by which it is effected. The actual work done by the cells depends on the efficiency, in the engineer's sense, of the machinery by which the energy is afforded. The method can therefore equally well be used to find the osmotic work necessary to secrete a liquid more dilute than blood, as is done by von Rhorer (pp. 383, 384).

Since the glomerular filtrate has a lower osmotic pressure than the blood plasma by the amount of that of the colloids in the blood, it is clear that some energy is required for the separation in question. This is small, on account of the low osmotic pressure of the colloids, and is afforded by the arterial pressure, that is, by the contraction of the heart muscle. Barcroft and Straub (1910) show that the increased flow of urine brought about by injection of Ringer's solution is not accompanied by increased consumption of oxygen by the kidney, and therefore, presumably, by no increased consumption of energy on the part of the renal cells.

Alkaline and Acid Secretions. — This process may be looked upon, from the point of view of the present section, as the change of concentration of hydroxyl or hydrogen ions of the blood into that of the secretion, or as one of the osmotic partial phenomena, as dealt with above in the case of urine. Von Liebermann (1911, p. 34) points out how, in the case of the alkaline pancreatic juice, diminution of the OH' ion concentration of the blood by intravenous injection of lactic acid causes a reduction in the rate of flow of the secretion under a constant stimulus. This might be explained as due to the greater work necessary to raise the OH' ion concentration in the juice to the same height from a lower level ; but there may, no doubt, be other factors in addition. The mechanism of secretion of acid and alkali will be referred to again later (page 359).

Mention has frequently been made of the use of intravenous injections for various purposes, so that it may interest the reader to leani that, according to Sprat's "History of the Royal Society" (1722, p. 317), it was Christopher Wren who was, as the author puts it, "the first Author of the Noble Anatomical Experiment of Injecting Liquors into the Veins of Animal-. An Experiment now vulgarly known ; but long since exhibited to the Meetings at Oxford, and t In-nee carried by some (Germans, and published abroad. By this operation divers Creatures \MTI- immediately purg'd, vomited, intoxicated, kill'd or reviv'd, according to the quality <>t the Liquor infected. Hence arose many new Experiments, and chiefly that of Transfusing i;i""<l, which the Society has prosecuted in sundry instances, that will probably end in extraordinary Success."

Secreting glands also require energy for the production of the chemical constituents of their secretions, whenever these substances are not already present in the blood. When we come into possession of more knowledge of the chemical changes involved, it is possible that we may, by the application of Nernst's new thermodynamic theorem (page 30 above), be able to calculate the energy changes involved. For the present we must be content with indirect measurements by determining the difference between the oxygen consumption of the resting and the active organ. This knowledge we owe chiefly to the work of Barcroft and his co-workers. The measurement is made by determining the oxygen content of the blood supplied to the gland, that is, the ordinary arterial blood, and the oxygen content of that leaving it by the vein, together with the amount of blood passing in a given time. It is in the accuracy of the last estimation that the chief difficulty lies, since the rate of flow increases in activity. The resting submaxillary gland of the cat consumes about 0*02 c.c. of oxygen per gram per minute. When excited to secretion, the consumption may rise to as much as l-9 in the same units (Barcroft and Piper, 1912, p. 362), that is, more than five times as much. By taking the difference between the oxygen consumption of the resting and that of the active gland, the same observers have calculated the oxygen necessary to form 0'30 c.c. of saliva to be 0'18 c.c. What this means in terms of energy naturally depends on what chemical substance is oxidised. Taking it as glucose, it would imply the use of 0*17 g., since 180 g. of glucose require 192 g'. of oxygen for complete combustion. A small part only of the energy is required for osmotic work, on account of the small volume of the saliva secreted.

A very important result as regards the mechanism of the process was obtained in the course of the experiments of Barcroft and Piper. When the time course of the oxygen consumption was determined in relation to that of the flow of saliva, it was found that the maximal rate of the former occurred considerably later than that of the latter, and that the increased consumption might last for as long as seven minutes after the formation of saliva had ceased. The length of this period of increased consumption of oxygen was found to depend on the degree of activity of the gland previously, and also on the functional capacity of the organ. We shall meet with a similar state of affairs in the case of voluntary muscle. It seems to imply that the chemical energy derived from oxidation is not used directly in the process of secretion, but that potential energy is stored in some physicochemical system, from which it is given out for use in the actual process itself.

It might perhaps be thought, by adherents of the "biogen" theory, that the oxygen itself is taken up in combination in an explosive-like giant molecule, analogous to potassium chlorate, for example. It seems possible that this view might be tested by simultaneous determination of the carbon dioxide given out together with the oxygen consumption. If the two were found to correspond, it would indicate that an oxidation process was giving energy to another independent chemical or physical reaction. Want of parallelism between the oxygen and carbon dioxide would not decide the question in either way. We shall find later, however, that there is no evidence for the existence of "intramolecular" oxygen in the sense of the biogen hypothesis, and \M have already seen reason for doubting the correctness of this point of view.

It may be called to mind that Chauveau and Kaufmann (1886) found a diminution of glucose in the blood after it had passed through the active salivary gland of the horse. This was also found to be the case by Asher and Karaulov (1910), so far as the period immediately succeeding the flow of saliva is concerned. The fact suggests the possibility that the energy required to form the system of high potential energy, which afterwards breaks down in the process of secretion, may be derived from the oxidation of glucose.

It is remarkable that the latter investigators found an increase of glucose in the venous blood of the gland during the process of secretion itself ; they hold that it may be a constituent ot some substance which breaks down in secretory activity. This does not seem a very probable thing to happen, as it would not be capable of affording much energy It is also difficult to understand why the glucose does not appear in the saliva. It seems, moreover that the concentration of the venous blood, due to loss of water into saliva and lymph, has not been sufficiently taken into account in the experiments referred to. For example, in experiment 1 (p. 40 of the paper), during the two minutes necessary for collection of the 10 '57 c c of venous blood for analysis, 4 c.c. of saliva were secreted ; adding this to the blood makes 14 -o7 c.c. and the percentage of glucose must be diminished in the same ratio, which makes it 0-148 per cent., a value practically equal to that in the arterial blood supplied to the gland, and no account has been taken of the lymph, which would make the value in the venous blood less than that in the arterial blood. From the results of Barcroft and Piper, it is not to be expected that there would be any very considerable consumption of glucose during the first period of the activity of the gland.

In Barcroft and Brodie's work (1905, p. 65) on the gaseous metabolism of the active kidney, it was found that, taking all the experiments together, the output of carbon dioxide was equivalent to that of the oxygen taken in. That is, the respiratory quotient (see above, page 279) is practically unity, as it would be from the oxidation of carbohydrate only. So far as it goes, this result suggests that the substance oxidised is of carbohydrate nature and that it is completely oxidised and its energy used for some process in connection with secretion. The oxygen, moreover, could not be combined up in an intramolecular form in an "explosible" substance, since, if this were the case, the respiratory quotient

\ O/ wou^ ke greater than unity during the period of formation of this substance and less than unity during its breaking up. The great sensitiveness of the salivary glands to slight diminution of oxygen supply, as found by Heidenhain (1868, pp. 88-101), by Jonescu (1909, p. 68), and by Liebermann (1911, p. 26), shows that the process of formation of the secretion itself requires free oxygen in addition to the stored energy just referred to. Ludwig, however (1851), obtained a slight secretion after the circulation had nearly ceased, so that a current of blood is not absolutely necessary. This consumption of oxygen during actual secretory activity suggests that the system of high potential energy, formed previously, does not contain in itself the oxygen necessary for its combustion, but chiefly consists of an oxidisable substance capable of affording energy when supplied with oxygen.

This immediate dependence on oxygen is shown still more strikingly by the higher nerve centres and makes it probable that, for proper functional work, oxygen must be supplied, not only in a certain amount, but at a tension not far below that in which it is present in the atmosphere and in arterial blood. An organ may suffer from want of oxygen even when the venous blood coming from it still contains oxygen, so that oxygen has passed the cells unused.

Formation of Heat. — It is scarcely to be supposed that the efficiency of the gland machinery is so high that no free chemical energy is degraded to heat in the secretory process. It was, in fact, found by Ludwig and Spiess (1857) that the temperature of the saliva coming from the submaxillary gland was higher than that of the blood in the carotid artery, but Bayliss and Hill (1894, 1) were unable to detect any difference in this sense, if care was taken to obtain the actual temperature of the blood in the flowing stream. Ludwig, himself, in a letter to Prof. Schilfer, appears to have been prepared to admit these negative results. Of course, the fact merely shows that, if heat was produced, the blood current was sufficiently rapid to carry it away as fast as it was formed, as is very probable from considerations of the actual amount of the combustion going on. It should be stated also, however, that we were unable to detect any formation of heat in the excised salivary glands of the grass snake, although a very delicate method was used (p. 352 of the paper quoted).

There are two different ways in which glands can be made to secrete. The one is by the agency of chemical substances contained in the blood with which they are supplied ; the other is by stimulation of nerve fibres which terminate in the secretory cells. It may, of course, ultimately be found that, in the actual cell system itself, the processes are identical in the two cases, so that the nerve may act by production of the same chemical substance which excites directly, or the chemical excitant may act on the same terminal mechanism as the excitatory process in the nerve fibre does.

It has long been known that certain drugs, of which pilocarpine is the most familiar, are capable of causing practically all glands to enter into activity. The fact that glands, such as the mucous glands of the air passages, which do not appear to be supplied with nerves, are excited seems to indicate that this effect is produced independently of nerve supply. On the other hand, adrenaline causes a powerful secretion of the submaxillary gland of the cat and we know that the action of this substance is exerted on the endings of the sympathetic nerves, wherever they are found. It appears, then, that a chemical substance may excite the cells of glands either directly, or through the medium of the nerve terminations on them. In the former case, it is probable that the drug may act on some definite part of the cell system, the "receptive substance" of Langley (1906). After administration of atropine, pilocarpine is ineffective in producing secretion, nor can it be produced by exciting the nerves of such glands as are supplied with them. Pilocarpine seems to be an abnormal excitant for gland cells, since its action is very violent and profound morphological changes are caused in the cells. In this respect it differs from a normal chemical excitant, such as secretin for the pancreas, the mechanism of which we will now consider. It was shown by Pavlov and his fellowworkers (1901, p. 132 of the English translation) that the presence of various substances in the duodenum, especially acids, causes pancreatic juice to be poured in. This excitation of the pancreas was looked upon as a reflex through the nervous system until Bayliss and Starling (1902, 1), in investigating the local nervous reflexes connected with the alimentary canal, found that it was still produced by acid in the duodenum after all accessible nervous communications had been divided.

This fact suggested that some chemical mechanism was at work, set going by the acid. The injection of acid into the blood current has no effect, as would be expected, so that some substance must be produced by the action of the acid on the mucous membrane of the intestine, which substance then diffuses into the blood and, arriving at the pancreas, excites it to action. The next step was to scrape off the mucous membrane and rub it up with sand and dilute hydrochloric acid. After neutralisation and filtration, this extract was injected into a vein and we were naturally delighted to find that a copious flow of pancreatic juice was the result. It may be pointed out that it is quite immaterial whether the whole of the nerves were actually cut in the previous part of the experiment, since it was the belief that they were cut that led to the search for a chemical mechanism.