The Formation of Urine by the Prosobranch Gastropod Mollusc Viviparus Viviparus Linn

In general, the methods of analysis were the same as those described in a first paper on Viviparus (Little, 1965). Operational techniques are described in the text.

A hole was filed in the shell approximately 1 cm. from the aperture. This revealed the surface of the mantle and the ureter could be clearly seen. Urine was withdrawn in a glass pipette, diameter 50 μ at the tip.

The volumes of samples were measured in small self-adjusting pipettes, and samples were transferred to planchets. Spreading was assisted by dilution with distilled water, and samples were dried down under an infra-red lamp. ¹⁴C was counted using a Nuclear-Chicago gas-flow detector. The time for 1000 counts was taken, and results are expressed as counts per minute or c.p.m. Counts were taken three times and averaged. The background count was never more than 15 c.p.m.

The composition of the urine of snails living in stream water is given in Table 1, and is compared with the composition of the blood of the same individuals. The urine is markedly hypotonic to the blood. A of the urine appears to be lower than that expected from its salt content, but this is probably due to the fact that A measurements are inaccurate in this low range. Sodium and chloride make up most of the A of the urine, but some calcium is present. The pH of urine is slightly lower than that of blood. This may be related to the presence of uric acid ; the kidney contains much uric acid (Spitzer, 1937), and yellow crystals have at times been seen in the ureter.

Fig. 1 shows the relation between A of the blood and A of the urine of snails from stream water and from dilutions of sea water. The urine is hypotonic to the blood in concentrations up to 10% sea water, but above this it is isotonic with the blood.

A hole was filed in the shell to expose the mantle and a length of ureter. The animal was supported by the base and apex of the shell, and a small hole was cut in the distal end of the ureter to allow the insertion of a polythene tube (external diameter 1 mm., internal diameter 0·7 mm.). The tube was held in place by a small clip of polythene and beryllium-copper clamped across the ureter. The snail was covered with tap water and the system was tested for leaks using concentrated dyes.

The polythene tube was placed on a flat surface slightly below the level of the animal, thus causing a slight negative pressure. As urine was produced the meniscus advanced along the tube and its position was marked at intervals. The volume between the marks was later measured with an ‘Agla’ micrometer syringe. The temperature was maintained at 19° C. ( ± 1° C.).

It was found that in some cases the rate of production of urine decreased over the first 15 min. This was regarded as a kind of shock effect, and figures for the first 15 min. were discarded. The rate of urine production was measured over a period of approximately 1 hr. in most cases, although some animals were followed for several hours to check that the rate of production did not decline. Table 2 gives figures for ten animals taken from stream water.

M_c = concentration in external medium,

U_ν= volume of urine produced,

M_ν= volume of external medium.

In this instance inulin has been used; ¹⁴C-inulin (carboxyl-labelled) was employed because it can be estimated in small amounts.

The animal was supported by the base and apex of the shell, and a small hole was filed in the shell over the afferent branchial vein. About 20 μl. of Ringer solution containing ¹⁴C-inulin was injected into the afferent branchial, and the hole in the shell was sealed with wax. The animal was allowed to crawl about in running tap water for approximately 4 hr., to remove any traces of inulin left on the surface of the body, and to allow for any changes in the rate of urine production consequent upon the injection of liquid into the blood.

A sample of about 4 μl. of final urine was taken through a hole in the shell over the distal end of the ureter; the shell was sealed with wax and the animal was placed in exactly 80 ml. of tap water. Exactly 3 μl. of urine was transferred to a planchet for ¹⁴C-counting. At intervals of some hours, further samples of final urine were taken, together with 0· 5 or 1·0 ml. of the external medium. The rates of urine production of five animals, as calculated from measurements taken at intervals of 6, 9 and 15 hr., are given in Table 3.

The average rate of urine production as found by cannulating the ureter is 0· 25 μl./g./min. The average rate as found by the use of inulin is 0· 91 μl./min., or about four times greater. One of the most important factors contributing to this difference is that in the method using inulin the animals were crawling about during the experimental period, so that the maximum possible body surface was exposed to tap water. In the method using cannulation of the ureter part of the shell was cut away and much of the animal was exposed to tap water, but it was never fully extended. This could account for a considerable difference in the amount of water drawn in by the osmotic gradient, and hence for a difference in the volume of urine produced. For this reason it might be suggested that the rate of urine production is nearer 0· 91 μl./g./min. than 0· 25 μl./g./min. On the other hand it must be pointed out that the concentration of inulin as measured in final urine may be lower than that in urine actually eliminated. Since the urine is hypotonic to the blood, water will be drawn from the urine into the blood because of the difference in osmotic pressure, thus causing an increase in the concentration of inulin in the urine. It is probable that urine is ejected in pulses, and if urine is sampled well before a pulse is ejected, the inulin concentration will be lower than if ejected urine were sampled. The rate of urine production as calculated would then be higher than the true rate.

For these reasons, it is supposed that the true rate of production of urine, at 19° C., is between 0· 25 and 0· 91 μl./g./min.

The rate of urine production in these experiments has been determined using a cannula in the ureter.

When the tap water in which an animal was held was replaced by 5 % sea water (about 30 mM./l. NaCl), the rate of urine production decreased gradually (Fig. 2). When the 5 % sea water was again replaced by tap water, the rate of urine production rose rapidly; this effect was found _kin several individuals. It seems improbable that such a rapid increase could be activated by an increase in blood volume. The alternative would be that Viviparui can detect changes in salt concentration of the external medium and can adjust its rate of urine production accordingly.

The rates of production of urine by snails adapted to various dilutions of sea water are plotted against the amount by which the blood is hypertonic to the medium (in Fig. 3). The points fall approximately on a straight line passing through the origin, suggesting that all the water expelled in the urine is taken up osmotically.

Perrier (1889) has described the kidney, pericardium and ureter of Paludina ( = Viviparus). The situation of these structures is shown diagrammatically in Fig. 4. The pericardium is very large and in an animal weighing 1·0 g. (wet weight without shell) it may contain as much as 100 μl. of liquid. The reno-pericardial canal is short and muscular.

The kidney consists of a network of irregular lamellae containing cells arranged in groups round small spaces, with blood lacunae between the cell groups (Fig. 5). The small spaces open into large spaces between the lamellae, and these in turn open into a bladder situated just interior to the kidney papilla. This papilla has a slit-like opening into the ureter, controlled by a sphincter. The reno-pericardial canal opens into the bladder close to the papilla; but although the end of the reno-pericardial canal and the kidney papilla are very close together, Perrier considers that their sphincter muscles are separate, thus allowing independent contraction and relaxation of the two.

Blood passes into the kidney in the afferent renal vein, which originates in the rectal sinus. Inside the kidney it passes into narrow lacunae between the groups of kidney cells and leaves through the efferent renal veins.

The ureter is a long tube which runs from the side of the kidney, first under the rectum, and then between the rectum and the genital duct almost to the edge of the mantle. Here there is an opening controlled by a sphincter and situated on a small papilla.

In Table 4 the composition of blood is compared with that of pericardial fluid. The pH of both is the same, and the ionic content is similar except that A and the concentration of calcium are slightly lower in pericardial fluid. In a first paper on Viviparus (Little, 1965) it was suggested thad some calcium in the blood is bound, and if pericardial fluid is an ultrafiltrate of the blood, containing less protein, a lower concentration of calcium would be expected. The protein content of pericardial fluid does indeed appear to be lower than that of blood, although no direct measurements have been made. The viscosity is much lower than that of blood, and when sucked up into a pipette under liquid paraflin, the meniscus does not break as it does when blood is pipetted. The breaking of such a meniscus is thought to be due to the deposition of a thin protein layer on the walls of the capillary. Furthermore, pericardial fluid is never blue, while blood may be bright blue from the haemocyanin.

These data all suggest that pericardial fluid could be an ultra-filtrate of the blood.

The following experiments were carried out to demonstrate that filtration actually occurs.

A method was devised by which a cannula could be fitted into the reno-pericardial canal. A polythene capillary (external diameter 800 μ, internal diameter 400 μ) was drawn until the external diameter was 300 μ and the internal diameter about 120 μ. The end of the capillary was cut diagonally as is the end of a hypodermic needle. With the animal supported by the base and apex of the shell, and with a large hole filed in the shell to reveal the pericardium and the proximal end of the ureter, the ureter was opened and the rectum was folded back to expose the side of the kidney. The capillary described above was inserted through the kidney papilla, and by careful turning towards the posterior end of the kidney it was pushed through the reno-pericardial canal so that the end protruded into the pericardium. The area around the kidney papilla was covered with liquid paraffin and flooded with concentrated dye dissolved in Ringer solution to reveal any leaks. The inserted capillary was connected to a length of polythene capillary (internal diameter approximately 800 μ) which was placed on a flat surface a little below the level of the animal. Gentle suction was applied and liquid flowed along the tube until the pericardium was drained.

In order to be able to impose variations of blood pressure a cannula was inserted into the efferent branchial vein which leads directly to the heart. The cannula was a Pyrex tube, approximately 1 mm. in diameter, and tapering rapidly to a diameter of 120 μ at the tip. It was pressed through the wall of the vein towards the heart until the diameter of the inserted portion was sufficient to stretch the walls of the vein slightly. The end of the cannula was supported on soft plasticene to allow it to follow movements of the animal. It was attached to a tube leading to a reservoir of Ringer solution which could be set at different heights. A fine suspension of Indian ink was added to the Ringer solution to show that the flow of liquid from the heart into the pericardium was not due to any rupture of the wall of the heart.

Results obtained from a typical animal are given in Fig. 6. An increase in pressure applied through the cannula in the efferent branchial vein results in an increased rate of flow of pericardial fluid ; a decrease in pressure produces a fall in the rate of flow of pericardial fluid.

The rate of production of pericardial fluid at a blood pressure of 5 cm. of water is higher than the average rate of urine production (Tables 2 and 3), although pressures as high as 5 cm. have been recorded in the efferent branchial veins of some individuals. It must be remembered, however, that in this experiment there was no back pressure exerted on the heart by fluid in the pericardium, and since most of the liquid passing into the heart was Ringer solution, the colloid osmotic pressure of the blood was also much reduced.

The relation between blood pressure and rate of production of pericardial fluid is good evidence for a filtration mechanism

Inulin has not been reported to be secreted or reabsorbed by any kidney mechanism. To demonstrate the flow of liquid through the kidney complex of Viviparus, under conditions of normal blood pressure, ¹⁴C-inulin was injected into the blood and its concentrations in blood, pericardial fluid and in urine at the proximal end of the ureter were measured at intervals. The hole in the shell made for injection and for withdrawal of samples was sealed with wax, and the animal was placed in tap water.

The concentrations of inulin expressed as c.p.m., in the blood, pericardial fluid and initial urine of two individuals over periods of and 16 hr. respectively are shown in Fig. 7,a and b. Fig. 7,a shows that 15 min. after the injection of inulin had begun inulin was detectable in the pericardial fluid, and just detectable though in a lower concentration in the initial urine. After 30 min. the concentration in initial urine was higher than that in pericardial fluid, and after 3 hr. it was more concentrated than in the blood. The concentration in the pericardial fluid rose to a level slightly higher than that in the blood and stayed higher; this is best shown in Fig. 7,b. The difference between concentrations in blood and pericardial fluid can be accounted for by the decrease in concentration in the blood; i.e. at any one time the fluid in the pericardium would be equal in concentration to the blood from which it was filtered some time before. The very constant relation of concentrations of inulin in blood and pericardial fluid shown in Fig. 7 b strongly suggests that blood is filtered into the pericardium. The increase in concentration of inulin in initial urine is discussed later.

The anatomy of the kidney is such that there seems little opportunity for any filtrate passing from pericardium to ureter to come into contact with the kidney cells. However, urine collected from just outside the kidney papilla is markedly hypotonic to the pericardial fluid (Table 5). Either salt is reabsorbed very quickly in the ureter or in some way it is reabsorbed in the kidney. To collect liquid from the kidney papilla a small cannula was constructed, as shown in Fig. 8. Urine flowed easily along the tube in definite pulses, and analysis showed it to be hypotonic to the blood. Salt must therefore be reabsorbed in the kidney.

The way in which pericardial fluid comes into contact with the kidney cells was discovered while watching the kidney during experiments with raised blood pressure. At high blood pressures pulses of urine are associated with contractions of the kidney. When the pericardial fluid is dyed blue the cycle of events during the production of each pulse of urine can be followed : the kidney contracts, the non-vascular spaces in its interior are squeezed, and a pulse of urine is forced out of the kidney. The kidney then re-expands, rather more slowly, and the kidney spaces fill up with blue liquid. Examination of the reno-pericardial canal shows it to be stained blue.

These observations have been extended by opening the kidney and observing the lamellae ; they are contractile, and when they contract fluid is expressed from the pores on their surfaces, which are the openings of the small spaces enclosed by the groups of kidney cells.

To summarize, these activities have been interpreted as follows:

• (i) Expulsion phase : kidney muscles contract, forcing liquid from the small spaces into the larger spaces, and thence into the large bladder ; the bladder muscles probably also contract, and while the reno-pericardial canal stays closed the papilla muscle opens the sphincter of the kidney pore so that liquid is forced into the ureter.

• (ii) Intake phase : the papilla muscle closes the kidney pore, kidney muscles relax, and the reno-pericardial canal opens. Positive pressure in the pericardium forces liquid into the kidney and re-expands it, aided by the tension exerted on the contracted kidney by surrounding organs; pericardial fluid becomes distributed in the kidney spaces, in contact with the kidney cells.

When the blood pressure is normal very small contractions of the kidney can be seen to occur before pulses of urine are produced, and it is thought that the normal method of transference of liquid from pericardium to kidney to ureter is the same as that outlined above.

The interval between pulses in normal animals varies from 3 min. to 8 min., and does not seem to change when the blood pressure is altered. In fact the opening of the kidney papilla appears to be at least partly governed by a rhythm originating in the kidney itself, since when the connexions to all other organs are severed, the papilla continues to open at intervals. Two experiments have thrown some light on the possible factors involved in control of the frequency and volume of the urine pulses.

In order to examine the effect of pericardial pressure on the rate of urine production a cannula was placed in the ureter as described in the direct measurement of the rate of urine production, but in this case the cannula was pushed up the ureter until the tip was opposite the kidney papilla. With this arrangement the urine flow was recorded as a series of separate pulses. Ringer solution was injected into the pericardium through a pipette with a diameter at the tip of about 15 μ. This ensured that no fluid leaked out when the pipette was withdrawn.

Fig. 9 shows the overall rate of urine production and the frequency and volume of the pulses of urine. The injection into, and removal of liquid from, the pericardium have no consistent effect on the overall rate of urine production; but injection of liquid increases the volume and decreases the frequency of the pulses, while removal of liquid lowers the volume of individual pulses and increases their frequency. This inverse relationship between frequency and volume keeps the total urine production constant despite changes in pericardial pressure which may result from body movements.

The pericardial pressure has no effect on the overall rate of urine production, so that changes in blood pressure will have no effect on the rate of urine production by increasing the rate of filtration into the pericardium. Nevertheless, experiments in which the blood pressure was raised (Fig. 10) show that an increase in blood pressure produces, with some delay, an increase in the rate of urine production. This change is brought about by an increase in the volume, but not in the frequency, of the urine pulses. In some cases it was observed that the rate of urine production was so much increased that the volume of the pericardium was noticeably reduced, i.e. the rate of production of urine had exceeded the rate of filtration through the heart.

In experiments using inulin to show the flow of liquid from the blood through the pericardium and the kidney into the ureter it was found that the concentration of inulin in the ureter was higher than that in the pericardium (Figs. 7 a and b). This could be due to secretion of inulin by the kidney— a phenomenon not so far recorded—or to the absorption of water by the kidney. An attempt was made to show that water is reabsorbed in the kidney, by injecting inulin into the pericardium and following the concentrations in pericardial fluid and in initial urine, but for several technical reasons this experiment was not successful. It should be remarked, however, that since about 20 mM./l. NaCl are reabsorbed in the kidney some passive uptake of water might be expected.

Urine taken from just outside the kidney papilla (initial urine) is hypotonic to pericardial fluid by about 20 mM./l. NaCl (Table 5). Urine taken from the distal end of the ureter (final urine) is less concentrated than initial urine by about 5 mM./l. NaCl (Table 6). The epithelium of the ureter must therefore either secrete water or reabsorb sodium and chloride to produce the difference in concentrations shown. The ureter has been perfused to discover whether it reduces the concentration of the urine by reabsorption of salt, and whether it is capable of reducing this concentration further than it does in normal urine formation.

The ureter was drained and a cannula was placed in the distal end as described above. A similar cannula was clipped into the proximal end by cutting through the genital duct and sliding one half of the clip under the ureter. 10 μl. of Ringer solution was injected through the proximal cannula, and after a time the ureter was drained into a polythene capillary attached to the distal cannula. The position of the meniscus in this capillary was marked, a further 10 μl. of Ringer solution was injected, and the procedure was repeated. The volume of fluid drained from the ureter was later measured by measuring the volume of the polythene capillary with an ‘Agla ‘micrometer syringe.

Results obtained by this method are given in Table 7. In no case is the volume of fluid drained from the ureter greater than that injected, but in all experiments except those using very dilute Ringer solution sodium and chloride have been reduced in concentration. These must therefore be reabsorbed by the walls of the ureter. Calcium is not reabsorbed, and, indeed, actually appears to be increased in concentration after passage through the ureter. The reduction in the concentrations of sodium and chloride is approximately 4 mM./l., which is similar to the difference found between initial and final urine in normal animals. The amount of salt reabsorbed is not increased if liquid is left in the ureter for long periods of time.

The rate of production of urine by Viviparus is between 0·25 and 0·91 μl./g./min. At least two factors can apparently influence this rate : a change in blood pressure, and a change in salt concentration of the external medium which may act via external sensory receptors. The action of blood pressure may be compared with vertebrate systems, where changes in blood pressure are detected by stretch receptors located in various parts of the blood system ; but as yet there appears to be no description of any sensory structure which can certainly be termed an ‘osmoreceptor’.

Three processes may be involved in the formation of urine, namely filtration, reabsorption and secretion. In Viviparus there is reasonable evidence that the primary process is filtration through the wall of the heart. This would agree with the findings of Picken (1937) on Anodonta, and Harrison (1962) on Haliotis. Filtration is followed by the reabsorption of salt and water in the kidney, and to a lesser extent in the ureter. The latter process is interesting in that it forms a parallel with the terrestrial pumonates, in which almost all reabsorption occurs in the ureter (Vorvohl, 1961); but whereas the ureter in pulmonates is derived from the coelomoduct, the ureter of Viviparus is probably derived from the mantle surface (Johansson, 1950).

Secretion could occur from the pericardial glands, which are situated on the wall of the auricle (Perrier, 1889), but, more important, secretion is likely to take place in the kidney. Yellow crystals have at times been seen in the ureter, and Cuénot (1900) describes two types of cell in the kidney. He says that the most numerous (those having each a single vacuole) eliminate indigo, while the ciliated cells eliminate carmine.

The transfer of pericardial fluid into the kidney and then from the kidney into the ureter is effected by the rhythmic contraction of the kidney. There is in fact reason to suppose that contractility of the renal organ in molluscs may be a more widespread and important phenomenon than has been appreciated up till now. Joliet (1883) described rhythmic contraction of the renal sac of the marine heteropod Pterotrachea and of the pelagic opisthobranch Phylirhoë ; and the lamellae of many monotocardian molluscs contain muscles (Fretter & Graham, 1962). In any excretory organ where fluid passes along a more or less straight tube the efficiency with which salts may be re-absorbed will not be much increased by contractions of the organ; but if the surface of the kidney epithelium is increased by folding and by complex lamellae, or if the reno-pericardial canal opens into the distal part of the kidney near its opening to the ureter or to the exterior, as is the case in many gastropods, then rhythmic contractions of the kidney will greatly help in distributing fluid to the blind-ending cavities.

1. The urine of Viviparus is hypotonic to the blood by about 30 mM./l. NaCl in tap water, and remains hypotonic in concentrations of up to 10% sea water.

2. The rate of production of urine is between 0·25 and 0·91 μ./g./min. in tap water at 19° C. The rate decreases in proportion to the decrease in osmotic difference between blood and external medium. Viviparus may be able to detect changes in salt concentration of the external medium and alter its rate of urine production accordingly.

3. Pericardial fluid is similar to blood in composition; the rate of flow of pericardial fluid through the reno-pericardial canal is proportional to the blood pressure ; and when inulin is injected into the blood, concentrations in blood and pericardial fluid are approximately the same. For these reasons it is supposed that blood is filtered through the heart into the pericardium.

4. About 20 mM./l. NaCl, and probably some water, are reabsorbed in the kidney. Liquid is passed through the kidney by rhythmic contractions of the kidney musculature. Pericardial pressure does not influence the overall rate of urine production but blood pressure does have an effect.

5. About 5 mM./l. NaCl, and probably a little water, are reabsorbed in the ureter.

This paper forms part of a dissertation for the degree of Ph.D. at the University of Cambridge. It is a pleasure to thank Dr J. A. Ramsay once again for his many suggestions and general advice. The work was carried out under a grant from the Department of Scientific and Industrial Research.