Invertebrate Mechanisms for Diluting Urine

Prévia do material em texto

INVERTEBRATE MECHANISMS FOR DILUTING 
AND CONCENTRATING THE URINEI 
By BODIL SCHMIDT-NIELSEN 
Departments of Zoology and Physiology, Duke University, Durham, North Carolina 
IN COLLABORATION WITH 
DONALD F. LAWS 
Department of Zoology, University of Adelaide, Adelaide, South Australia 
INTRODUCTION 
The present review deals with the function of excretory organs in a num­
ber of invertebrate phyla. For a few of these phyla the volume of available 
information is considerable; for others it is virtually nil. The only reason for 
this gap in knowledge appears to be lack of interest since the physiology and 
anatomy can easily be investigated with modern techniques. For this reason, 
one of the primary purposes of this review is to stimulate interest in the un­
explored phyla by demonstrating the considerable structural and functional 
similarities between the excretory organs of different phyla and between 
invertebrates and vertebrates. 
The ability to produce a urine that is hypoosmotic to the "plasma" is 
found in the fresh-water and terrestrial forms of almost every phylum in the 
animal kingdom. The ability to make a hyperosmotic urine is much more 
confined and is, according to our present knowledge, found only in insects, 
birds, and mammals, and to a slight degree in crustaceans. In birds and 
mammals the urine is concentrated through the countercurrent multiplier 
system, which has been comprehensively treated in recent excellent reviews 
by Scholander (161), Ullrich et al. ( 193), and Morel & Guinnebault (108) . 
The concentration mechanism of insects and crustaceans which is not com­
pletely understood will be discussed here. 
The specialization of the countercurrent mUltiplier mechanism in the 
mammalian kidney is based on the existing ability of distal tubular cells of 
invertebrates and lower vertebrates to dilute the fluid inside the tubule by 
reabsorbing electrolytes and leaving water behind. For this reason the dilut­
ing mechanism as it has evolved in lower animals is of considerable interest 
for the understanding of the mammalian kidney function. 
The kidney plays an" important role in the osmoregulation of fresh-water 
animals where its major function appears to be the excretion of excess water. 
Since fresh-water animals are hyperosmotic to their environment, there is a 
net influx of water by diffusion through their gills and body surface. To 
avoid dilution of the body fluids, excess water must be excreted with a min­
imum of salt. 
1 The survey of literature pertaining to this review was concluded in July 1962. 
631 
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632 SCHMIDT-NIELSEN AND LAWS 
In marine invertebrates and lower vertebrates the kidney assumes a 
different role. The majority of these animals are isosmotic to their environ­
ment. There is no osmotic influx of water, and the kidney does not serve an 
osmoregulatory function. In some of these animals urine flow may be reduced 
to one tenth, or less, that of related fresh-water forms, while others may have 
no excretory organs at all (67, 86). It appears that the excretory organs, when 
present in marine forms, serve mainly in ion and volume regulation (173). 
Some marine organisms are hypoosmoregulators; i.e., their body fluids 
have a lower osmotic concentration than the surrounding sea water. In some 
hypoosmotic regulators, such as most crustaceans, the urine is not more 
concentrated than the blood and the kidney's role in osmoregulation is 
negligible. Excess sodium chloride is excreted by such organs as the gills and 
rectal gland (20, 85), while divalent ions are frequently excreted by the 
kidneys (134). 
In brackish water, estuaries, and saline pools, animals must often adapt 
to a great variety of environmental concentrations. Some forms are always in 
osmotic equilibrium with their environment. Others show hyperosmotic but 
not hypoosmotic regulation. Still others show both hyper- and hypoosmotic 
regulation and are able to maintain a fairly constant osmotic concentration of 
the body fluids over a wide range of salinities (77, 78, 164, 173). Artemia 
salina is a beautiful example of an animal belonging to this latter group (28). 
In terrestrial animals the need for water conservation makes it advan­
tageous to excrete solutes with a minimum of water. Many terrestrial insects, 
for example, excrete a urine that is practically dry (202) . The role of osmoreg­
ulation in terrestrial insects is taken over entirely by the excretory organs. 
The mechanisms for urine production vary greatly among insects as do the 
composition and osmolality of this urine (26). 
The various renal structures associated with the production of urines 
with different osmolalities relative to the blood appear to be characteristic 
and to show certain similarities when compared in widely different groups of 
animals. Hence, even in animals where physiological data are lacking, the 
structure of the kidney may reveal basic information concerning its function. 
PROTOZOA 
Structure.-Contractile vacuoles are characteristic of fresh-water Proto­
zoa and parasitic and marine ciliates but are lacking in other marine and 
parasitic groups. The primary function of the vacuole is believed to be that of 
removing water entering the cell by osmosis (85), but there is considerable 
uncertainty as to how the vacuole functions. 
In most protozoans the vacuole appears to be an impermanent structure, 
formed anew after each contraction. In the ciliates, however, it occupies a 
fixed position with one or several canals leading to it. In the amoeba the 
vacuole is surrounded by a membrane which according to Mercer ( 106) is 
identical to the external membrane of the animal. Electron micrographs 
show that the membrane consists of two dense zones about 20 A thick sepa-
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URINARY DILUTING AND CONCENTRATING MECHANISMS 633 
rated by a light zone (total thickness 50 to 70 A). The mature vacuole is sur­
rounded by a crowd of mitochondria which is not yet present in the small 
growing vacuole (119) . A vast number of tiny vesicles, which presumably 
burst into the main vacuole, are found between the layer of mitochondria and 
the membrane. Bairati & Lehmann (3) observed in addition an intermediate 
fibrillar layer between the mitochondria and vacuolar membrane. They sug­
gest that this layer may be the contractile part of the vacuole. 
The fixed contractile vacuole of the ciliates frequently has a permanent 
canal or elaborate structure connecting it with the outside (135, 156) . Sur­
rounding the vacuole are vesicles, a dense reticulum of anastomosing tubules, 
and mitochondria (36, 156) . 
Function.-Considerable interest is beginning t o center around the func­
tion of the contractile vacuole. It is generally agreed that it is osmoregula­
tory, and until recently it was believed that water was actively secreted into 
the vacuole (79, 85) . However, the improbability of active water transport on 
thermodynamic grounds ( 15) as well as from a comparative physiological 
point of view makes this unlikely. 
Fresh-water protozoans are distinctly hyperosmotic to their environment 
and there is suggestive evidence that even some marine forms are slightly 
hyperosmotic. The osmoticpressure of protozoans has been estimated by 
various indirect methods, such as disintegration in distilled water (95), 
osmotic pressure at which the contractile vacuole ceases to function (145), 
and volume changes at various concentrations of the medium (65). The 
results differ widely. This may result partly from variation between species 
but also partly from erroneous assumptions. Thus, Hopkins (65), disregard­
ing the lag in function of the contractile vacuole (79), has argued: 
When the amoebae are placed in dilute sea water of a given strength with a resulting 
decrease in volume, we can safely conclude that the osmotic pressure is higher ex­
ternally than internally. If, on the other hand, the volume of the amoebae increases 
the osmotic pressure of the protoplasm is greater than that of the medium. 
On this basis he found the osmotic pressure of the protoplasm of amoebae to 
be close to that of the medium. L6vtrup & Pig6n (95), on the other hand, 
found the osmotic pressure of the amoeboid Pelomyxa to be about 90 mOs. 
The ionic composition of protozoans has been determined by direct 
analysis and by equilibration with radioisotopes (22, 33, 82). In Tetrahymena 
in very dilute media, potassium and a small amount of sodium are maintained 
intracellularly (33) . Chloride concentration is much lower than the cation 
concentration. Increases in cellular potassium or sodium are not accompanied 
by increases in chloride. In more concentrated media the intracellular potas­
sium concentration is always higher than that of the medium while intra­
cellular sodium is lower. Thus, in a medium of 4.75 mM potassium and 
36.5 mM sodium the cellular concentrations were 32 mM potassium and 
12.7 mM sodium. The authors suggest that potassium is specifically ac­
cumulated and retained by a system of internal binding sites with a satura-
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634 SCHMIDT-NIELSEN AND LAWS 
tion level, while sodium is extruded by an active mechanism. An adenosine 
triphosphate-splitting enzyme has been found to be localized in the cellular 
membrane of Amoeba proteus (65) , which may be involved in the ion trans­
port. 
Klein (82) similarly found in A canthamoeba a higher intracellular concen­
tration of potassium than of sodium. Carter (22) measured the concentrations 
of potassium, sodium, and bromide in the ciliate Spirostomum ambiguum by 
equilibration with radioactive media. He found much lower sodium and 
potassium concentrations inside the cell than did the other authors, 10 to 12 
m M potassium and about 1 m M sodium. These concentrations are probably 
too low because of the inexchangeability of a large fraction of the intracellu­
lar potassium (82). Carter (22) determined the time of half exchange for 
potassium to be two to three hours in Spirostomum. Klein (82) found that 
only 40 per cent of the potassium in A ncanthamoeba is exchangeable and that 
the exchangeable fraction consists of a rapid and slow component. The half 
lives of the fast and slow components are three minutes and forty-six min­
utes, respectively. In Spirostomum, sodium is exchanged in a matter of a few 
minutes (22) . In Acanthamoeba (82), sodium exchange was found to be 
similar to potassium exchange with approximately the same half lives for the 
fast and slow components. 
Since fresh-water protozoans are hyperosmotic to their environment and 
highly permeable to water (37, 65, 81) , it is obvious that water continuously 
diffuses into them. The finding that the rate of vacuolar output varies in­
versely with the salinity of the medium (25, 51, 65) agrees with the notion 
that the vacuole serves the function of bailing out inflowing water. The rate 
of output, however, also varies with the rate of catabolism (65) and with 
temperature (80). 
Cosgrove & Kessel (25) found a linear relation between the estimated 
osmotic gradient across the cellular wall and the rate of output of the vacuole 
in media concentrations ranging from 10 to 100 m M sodium chloride. The 
water must be removed as a solution that is either isosmotic or hypoosmotic to 
the protoplasm of the cell. Disregarding active water transport as a possible 
mechanism, we are left with the following possibilities. A solute could be 
transported into the vacuole with water following passively . The solute 
could either remain in the vacuole and be expelled to the outside when the 
vacuole empties, or it could be reabsorbed into the cell leaving a hypoosmotic 
solution behind. It is conceivable that the fluid in the vesicles surrounding 
the main vacuole is isosmotic to the protoplasm while solutes are actively 
reabsorbed in the main vacuole. This, however, would require an exceedingly 
low degree of water permeability of the vacuolar wall, since the small size of 
the vacuole (0.0001 mJ.tl) gives a very large surface-to-volume ratio. 
COELENTERATA 
Most of the coelenterates are marine, but fresh- and brackish-water 
species are known (116, 152). No excretory organs are known in these ani-
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URINARY DILUTING AND CONCENTRATING MECHANISMS 635 
mals. The ionic composition of marine forms differs from that of the sea 
waterj and fresh-water forms are distinctly hyperosmotic to their environ­
ment. Lenhoff & Bovaird (89) found that a trace amount of sodium is essen­
tial for the growth of fresh-water hydra. They have essentially no extracellu­
lar fluid, and the body potassium-to-sodium ratio is about 9.8. Kinne (77, 78) 
found the optimum salt concentration for maximal growth of hydra to be 
1.67 per cent. 
The osmolality of the fresh-water medusa Craspedacusta sowerbyi cor­
responds to about 3 per cent sea water and is more than fifteen times that of 
the river water in which it lives (116). How water gained by osmosis is 
excreted is unknown. 
ECHINODERMATA 
All echinoderms are aquatic; none can live in fresh water and only a few 
in brackish water (12, 191). They have no special excretory organs and are 
unable to osmoregulate (134). Ion exchange takes place through tube feet, 
respiratory tree, etc. 
PLATYHELMINTHES 
Structure.-The phylum includes parasitic as well as free-living fresh­
water and marine species. The excretory system comprises a pair of longi­
tudinal, long coiled tubules, one on each side of the body. These are connected 
near the posterior end by a transverse tube or by a large bladder (115). The 
longitudinal tubes give off numerous branches ramifying through the entire 
body and ending in flame cells. The animals have no coelom, anus, circula­
tory, or respiratory system. The flame cells and tubules are thus surrounded 
by connective tissue and not by spaces containing fluid (124). There are some 
rather interesting reports on the structure of the flame cells in trematodes. 
Willey (205) examined the flame cells with a light microscope and found the 
flame to be longitudinally striated and composed of long cilia with a canal 
extending through the center of the flame. Electron microscopy (166) 
revealed that the flame cells consist of bundles of about eighty cilia en­
closed by a single double-layered membrane with a series of stiffening 
members arranged in stockade fashion around the bundle of cilia. The de­
velopment of the excretory system in a parasitictrematode has been de­
scribed by Kuntz (88). 
Kromhout (86) has made a very interesting comparison of the proto­
nephridial system of fresh-water, brackish-water, and marine forms of the 
Turbellarian Gyratrix hermaphroditus which clearly demonstrates the impor­
tance of various parts of the excretory system in osmoregulation. In the fresh­
water form the protonephridial system is most extensive and complex. The 
parts of the tubules closest to flame cells are enveloped by paranephrocytes, 
cells measuring from 50 to 60 p. each. After many convolutions the tubule 
runs into a contractile region, the "ampulla", which is a continuation of the 
main tubule surrounded by an enlargement of the syncytial tissue. The 
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636 SCHM IDT-NIELSEN AND LAWS 
posterior end of the tubule enlarges into a bladder which opens to the outside 
through the nephridiopore. 
In the brackish-water form the ampulla is entirely lacking, as are the 
paranephrocytes. In the marine form neither ampullae, paranephrocytes, nor 
tubules are found ; i.e., the marine form has no excretory system. 
Function.� The actual function of the flame cells is unknown. Senft et al. 
( 167) postulated that the membrane surrounding the flame is freely per­
meable to water. They found no specialized secretory cells in the flame cell 
system and suggested, in agreement with Martin (104) and Kummel (87), 
that the beating action of cilia causes a distal movement of fluid within the 
flame cell lumen. This motion is thought to produce enough pressure differ­
ential to allow filtration through the flame cell membrane. However, since 
there are no fluid spaces around them (124) , this seems somewhat unlikely. 
Coil (24) has found a relatively high activity of alkaline phosphatase in the 
flame cells and in the fine tubules leading from them, which may suggest 
active sodium transport possibly followed by diffusion of water and solutes 
into the tubules, Beaver (9) found that the flame cells can be stimulated by 
saline. No work has been done on the function of the rest of the tubule or 
the paranephrocytes. 
Kromhout's findings (86) that the ampulla is found only in fresh-water 
forms strongly suggest that it is concerned with the production of hypo­
osmotic fluid through reabsorption of salt. This is also supported by Hyman's 
observation (66) that ampullae seem to occur only in fresh-water representa­
tives of marine Turbellaria. 
ASCHELMINTHES 
Structure.� The more recent experimental work done on animals from 
this phylum is almost exclusively limited to the nematodes. However, de­
spite the lack of physiological data, certain deductions concerning the func­
tion of the excretory organs can be made by correlating their structure (49, 
67) with the habitat of the animals. 
In the class Rotifera there are both fresh-water and marine forms. While 
the fresh-water forms have long, coiled protonephridia with flame cells and 
urinary bladder, the marine forms lack excretory organs. The same is true of 
the class Gastrotricha. 
It appears quite general, according to Goodrich's (49) description, that 
the protonephridia of fresh-water Aschelminthes are differentiated into a 
posterior glandular region opening at the excretory pore and an anterior 
"capillary" region ending in the flame cells. 
The class Kinorhyncha is exclusively marine (67). Here there is an excre­
tory organ, but as ill other marine animals (compare annelids, crustaceans, 
fish, etc.) the excretory tube is very short. The protonephridium is described 
as a multinucleated flame cell, the flame of which consists of one long flagel­
lum. A short tube provided with driving flagella leads directly from each 
flame cell to its own nephridiopore. In Priapulida the protonephridial part of 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 637 
Ie urogenital system consists of large clusters of solenocytes (protonephrid-
11 bulbs provided with flagella instead of tufts of cilia) opening into the uro­
Jcnital duct by short collecting canals. 
The Nematoda have a peculiar excretory system, entirely different from 
that of other classes of the phylum. Although the cla:;>:;> includes marine, 
fresh-water, and parasitic forms, the primitive stem nematode was evi­
dently marine (21, 39, 67, 162). The excretory system appears to have 
originated de novo (199) from the renette cell found in marine nematodes and 
not from a protonephridial system as suggested by Chitwood & Chitwood 
(23). Two types of excretory organs, glandular and tubular, have developed. 
In the marine nematodes (Adenophorea) the excretory system consists of a 
single glandular renette cell provided with a short duct which opens through 
the excretory pore. In the fresh-water and parasitic forms (Secernentea) a 
lateral canal system is attached to the renette gland cell (1,64). Some authors 
say that the excretory canals are simply channels within what appears to be 
a single gland cell (199). Others (64) claim that they are composed of numer­
ous cells. It is of particular interest that the terminal duct which leads to the 
excretory pore is lined by cuticle in the fresh-water forms but not in the 
marine forms (64). This cuticle-Hned duct is acidophilic (199). A cuticle­
lined terminal duct of the excretory organs appears to be present in a number 
of fresh-water animals. 
Functiort.-In all the fresh-water forms we shall assume that the excre­
tory system serves in an osmoregulatory capacity, excreting the water enter­
ing the body through the water-permeable cuticle (14). In the fresh-water 
forms, which all have long, dit'Iet'entiated protonephridia, it seems likely that 
a hypoosmotic fluid is produced by solute reabsorption in the posterior 
glandular region of the protonephddium. Experimental data are entirely 
lacking, but should be rather easy to obtain through micropuncture studies. 
In the fresh-water nematodes it has been shown that the renette gland 
cell has an excretory function (200) and that it participates in osmoregulation. 
An inver;;e relation between pulsation mte of the excretory ampUlla and the 
solute concentration of the media was shown by \VeinsteiI\ (198). In a larva 
of Nippostrongylus muris placed in distilled water, 530 }J.8 per min were dis­
charged from the excretory pore, whereas when it was placed in saJine the 
rate of discharge decreased to 246 JLa per min. 
It i;; possible that the cuticle-lined, acidophilic portion of the terminal 
duct is the part in which the fluid is made hypoosmotic since the terminal 
duct is not cuticle-lined in the marine forms (compare Annelida and Insecta). 
The function of the excretory organs in the marine forms is not well 
understood. Since the organs are missing in several species, they do not seem 
to be essential. The so!enocytc8 in Priapulida (107) have been shown to 
concentrate ammoniacal carmine. The results of dye studies on the excretory 
�ells of nematodes have been reviewed by Weinstein (199) j in Ascaridina 
(five species studied) the potassium salts of fluorescein and erythro5in were 
concentrated and excreted through the excretory pore alter Q,a! in.jection. 
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638 SCHMIDT-NIELSEN AND LAWS 
Injected ammoniacal carmine concentrated in the lateral lines adjacent to 
the canal. Nitrogenous compounds are apparently excreted through this 
organ but it is also quite possible that they play an important part in ion 
regulation. 
ANNELIDA 
Structure.-This phylum includes fresh-water, marine, and terrestrial 
forms. The excretory system consists of sets of paired excretory tubules, or 
nephridia, located in each body segment. Each nephridium opens into the 
coelomic cavity by means of a ciliated funnel, or nephridiostome. In some 
annelids the nephridium forms a long coiled tubule, differentiated into several 
parts, which opens at an external nephridiopore. In others it is short and 
undifferentiated. Grobben (54) pointed out that the length of the nephridial 
canal in annelids and crustaceans varies with habitat, being short in marine 
forms and long in fresh-water and terrestrial forms.2 
The nephridium of the polychaete Nereis vexillosa has been described in 
detail by Jones (71). It is a long, convoluted tubule the outer surface of 
which is covered by a single thin layer of squamous coelomic epithelium. 
Interestingly enough, at the terminal end of the nephridial canal, the wall of 
the lumen thins and becomes lined with i nvaginated cuticle [ef. cestodes (94), 
nematodes, and insects]. The mean diameter of the nephridium is 24 JL and 
the over-all length 1.7 mm. According to Jones (71) no blood vessels occur 
within the nephridial mass, and in only two places is the nephridial system 
approached by vascular elements. In contrast to this, Krishnan (84) has 
indicated that in Lycastis indica, Nereis chilkaensis, and Perinereis nuntia, 
blood vessels are found in close association with the distal part of the nephrid­
ium. He also has pointed out that the extent of nephridial vascularization, 
as well as the length of the nephridium, is inversely related to the salinity of 
the environment. Furthermore, he found that the blood supply to the 
nephridia undergoes a diminution when Lycastis is acclimatized to sea water. 
Young Nereis have sman short protonephridia. During growth some of 
these degenerate while others acquire a coiled canal and a nephridiostome 
(49). Thus, the originally marine polychaetes appear to be able to adapt their 
excretory system for osmoregulation in fresh water. It is i nteresting that the 
viviparous Neanthes can reproduce in fresh water, while Nereis diversicolor, 
which is oviparous, cannot (179). It may be that the nephridia of young 
Neanthes are more fully developed at birth and therefore are able to produce 
a hypoosmotic urine. This, then, could explain the success of fresh-water 
reproduction of Neanthes. 
A description of the nephridium of the earthworm Lumbricus terrestris has 
been given by Meisenheimer (105). The nephridium is differentiated into 
several parts. Following the nephridiostome is a narrow tube ciliated in the 
first part, then a wider middle tube, and finally a wide tube which is further 
2 Terrestrial annelids are essentially fresh-water forms, since they live in moist 
soil. 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 639 
differentiated into three parts before it finally opens into a muscular duct or 
bladder. The nephridium is very richly vascularized. 
Function.-Osmoregulation has been shown for some Nereid polychaetes 
(72,179). Nereis diversicolor can tolerate fresh water with 1 to 2 meq Cl per 
liter, and Nereis virens can also tolerate lowered salinities, although only to 
67 meq Cl per liter (72). The permeability to water and chloride is not the 
same in the two species. In N. virens 50 per cent of the body chloride is ex­
changed per hour in a medium containing 65 meq Cl per liter, while in N. 
diversicolor the exchange is only 25 per cent per hour. It is not known whether 
the ability to dilute the urine differs in these two species. Smith (179) found 
that the effect of temperature on adaption to low salinities by N. diversicolor 
and Neanthes litthi differs somewhat. Nereis osmoregulates below 1.soC, 
while at this temperature the osmoregulation in Neanthes breaks down. 
Ramsay (136) determined the osmotic pressure and chloride concentra­
tions of coelomic fluid, blood, and urine of earthworms kept in various saline 
media. The blood was slightly hypoosmotic to the coelomic fluid. As the 
concentration of the medium was increased, the osmotic pressure of the two 
body fluids also increased and was always greater than that of the medium. 
The urine was always strongly hypoosmotic to the body fluids except in the 
most concentrated media, where the osmotic pressure of coelomic fluid cor­
responded to a salt solution of 1.4 per cent and that of the urine to 1.37 
per cent. This finding shows that the nephridium of the earthworm, like the 
distal tubule of a vertebrate, can produce either a hypoosmotic urine or a 
urine that is essentially isosmotic to the blood. Other fresh-water annelids 
have been shown to tolerate higher salinities, e.g., two species of leech (97, 
154) tolerate salinities up to 1.3 to 1.5 per cent. No measurements of urine 
osmolality were made. 
By means of micropuncture studies together with microdeterminations 
of freezing point, Ramsay (137) was able to locate the tubular zone in which 
the fluid becomes hypoosmotic. This proved to be the so-called middle tube. 
Samples from the more distal part of the nephridium where the tube becomes 
wider were still more hypoosmotic. Apparently, solute reabsorption takes 
place against an osmotic gradient over an extended zone stretching from the 
end of the thin tubule to the muscular duct or bladder. Whether or not 
reabsorption takes place in the bladder cannot be seen from Ramsay's data. 
The sipunculids, a marine group closely related to the Annelida, appar­
ently have no ability to osmoregulate, but they do show volume control. The 
volume increases initially in dilute sea water (80 to 90 per cent), then gradu­
ally returns to normal. The worm is permeable to salt mostly through the gut 
or nephridiopores or both, while the body wall is highly permeable to water. 
When the nephridiopores were plugged, volume control failed (55), indi­
cating to us that any excess salt is normally eliminated with water by the 
nephridia. 
Experiments by Greif (53) have shown that excised nephridia from the 
sipunculid Phascolosoma gouldi accumulate far more Hg20a from a solution of 
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640 SCHMIDT-NIELSEN AND LAWS 
labeled chlormerodrin than do other tissues of this animal. The uptake of 
mercury could be inhibited by cyanide, penicillin, and cyanine dye and 
partly inhibited by probenecid. 
In the annelids another tissue in addition to the nephridia, the chlorogo­
cytes, had been considered as a kidney of storage. However, these cells, which 
appear to be modified peritoneal cells (48), have no such function (48, 194, 
195). Their primary function is to store lipids and glycogen( 153). 
MOLLUSCA 
Structure.-Aquatic as well as terrestrial forms are found among the 
Mollusca. The aquatic molluscs were originally marine, but many have 
invaded brackish and fresh. water and have become secondarily adapted to 
these environments. From the existing literature we have not been able to 
detect any characteristic structural variations of the excretory organs in 
molluscs from different habitats. 
The molluscan excretory system consists of a central cavity, the peri­
cardium, surrounding the heart and intestine and of either one or two kidneys 
(often called Bojanus organs) into which the pericardial cavity opens through 
a narrow aperture. Each kidney is basically a tube connecting the peri­
cardium with the exterior, but its structure may be complicated in various 
ways (49, 187). Frequently it consists of a glandular spongy section followed 
by a thin-walled, nonglandular part called the bladder or posterior renal 
coelom. The bladder opens to the exterior by a small aperture.3 
In the oyster (40, 41) the paired kidneys seem to consist mostly of 
"bladder", bordered medially and posteriorly by blind tubules which 
diminish in size with distance from the bladder. These tubules are bathed in 
blood from the adductor muscle region, which, in turn, is supplied by a blood 
vessel coming directly from the ventricle. 
In the garden snail the wall of the glandular portion of the kidney forms 
many permanent folds. The cells are tall and columnar, containing coarse 
acidophilic granules and many crystalline concretions. In the bladder portion 
there are low columnar cells showing vertical striations from the lum,inal 
surface to the basement membrane (118) . Electron microscopy of the kidney 
of the snail Helix pomatia (13) has shown that the wall of the bladder consists 
of two cell types. Certain cells, small and few in number, are ciliated and 
located in the luminal corners between the other , more numerous cells. The 
other cells are described as follows : "Practically the entire height of the cell 
is occupied by a system of cytomembranes, between which lie intercalated 
numerous oval mitochondria. These cells are also ciliated at their apical 
end." As Bouillon himself points out, these cells, except for the ciliation. are 
I Another structure called Keber's organ, which consists of specialized cells 
lining the anterior part of the pericardium, had been thought to have an excretory 
function and to discharge its products into the pericardial cavity. Kato (76). working 
on' certain JameJlibranchs, has shown that the excreta from this organ are discharged 
from the mantIe and gills and not through the Bojanus organ. 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 641 
remarkably similar in ultrastructure to those of the mammalian renal distal 
tubule. 
Function.-Most molluscs can tolerate wide variations in the salinity of 
their environment (40, 4 1, 46, 207). This tolerance appears to be mostly on 
the cellular level since they remain isosmotic to their environment over a wide 
range of salinities and only become hyperosmotic in fresh water or very dilute 
brackish water (31, 40, 41, 45, 46, 109, 111, 114, 129, 155, 197, 207). When 
mussels are exposed to sudden changes in salinity they close their valves and 
thus delay the exchange with the environment (45, 109). 
Weak osmoregulatory ability appears to be widespread among molluscs 
(197). It is found in bivalves (40, 4 1, 43, 45, 46, 117, 126, 130, 146, 155) as 
well as in snails (13, 104, 111, 126, 157). Kidneys of bivalves and snails can 
elaborate a hypoosmotic urine and it is possible that active uptake of salt 
by the gills also plays a role in hyperosmotic regulation. 
The osmotic concentration of the body fluids in fresh-water forms is 
extremely low. In the mussel Anodonta the plasma chloride concentration 
ranges from 3 to 20 meq per liter when the surrounding water has a total salt 
concentration of 1 meq per liter (43). Potts (129) determined the sodium 
concentration in Anodonta plasma to be about 15.5 meq per liter and Picken 
( 126) found that the osmotic concentration corresponded to 0.10 per cent 
sodium chloride which is about 34 mOs. All these values agree essentially . 
The snail Theodoxus fiuviatilis is hyperosmotic in fresh water and main­
tains a blood concentration of 80 to 90 mOs ( 111) . In another snail Viviparus 
fasciatus, the blood concentration was 113 mOs in fresh water (114). 
The renal function has been most carefully investigated in Anodonta. A 
filtrate of the blood accumulates in the pericardium. This fluid was found to 
be isosmotic (126) and to have the same chloride, phosphate, and calcium 
concentration as the blood (43), and thus it appears to be a true ultrafiltrate. 
During the passage of the filtrate through the two parts of the Bojanus 
organ, salt is reabsorbed and protein and nonprotein nitrogenous waste 
products are added. The chloride concentration of the urine collected from 
the bladder is about one half that of the blood, and the nitrogen concentra­
tion is about three- to fourfold higher (43). The urine osmolality is about 0.6 
that of the blood (126). This means that an osmotic gradient of about 15 
mOs is created across the membrane of the bladder. From data obtained by 
Potts ( 130) it appears that the filtration rate approximately equals the urine 
flow, being about 1 to 2 ml per hr per 100 g; thus water does not appear to 
be reabsorbed along with the salt. 
Snails are likewise able to produce a hypoosmotic urine. In the African 
snail Achatina fulica, the osmolality of the blood was 250 mOs and that of 
the urine 153 mOs (104). Terrestrial snails apparently can adapt to water 
conservation as well as to excretion of excess water ( 13, 157). During estiva­
tion the vector snail Oncomelania nosophora can tolerate a 40 per cent loss of 
body water (83). Kidney function continues during estivation, and nitroge­
nous waste (uric acid) is stored in the bladder. During such periods water is 
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642 SCHMIDT-NIELSEN AND LAWS 
probably reabsorbed along with salt from the bladder (13). To our knowledge 
no determinations of the osmolality of the "stored urine" have been made. 
Bouillon suggests that the highly specialized cells with many "cytomem­
branes" and mitochondria found in the wall of the bladder of the snail Helix 
pomatia are characteristic of adaptation to terrestrial life, i.e. , to water con­
servation. To the present authors it seems likely that their specialized struc­
ture is related to their function of producing a hypoosmotic as well as an 
isosmotic urine similar to the cells in the vertebrate distal tubule. 
Investigations by Jullien et al. (73,74,75) indicate that a sodium load is 
not readily excreted in the snail Helix pomatia. Injections of NaCl, KCI, or 
CaCl2 resulted in marked increases of these ions in the hemolymph. Injected 
calcium disappeared from the hemolymph quite rapidly in about two days. 
Sodium or potassium injection resulted in a compensatory fall in the calcium 
concentration while the hemolymph concentration of sodium or potassium 
remained elevated for up to seven days . Apparently the kidney conserves 
sodium and excretes divalentions (d. crustaceans, fish, and birds). 
The io nic regulation in marine molluscs has been little investigated. I n 
the bivalve Mytilus edulis the ionic composition o f the blood closely resembles 
that of sea water, but shows somewhat greater concentrations of calcium, 
postassium, and total carbon dioxide. The ionic composition of the urine of 
the octopus has been determined and compared with sea water, but not with 
the blood (35). Potassium and sulfate concentrations appear to be signifi­
cantly higher in the urine than in sea water. 
Three independent facts suggested that the ability to osmoregulate in 
fresh water may not be present in young bivalves. (a) The embryos of 
A nodonta after hatching in the fall remain in the gills of the mother all 
winter. The following spring they attach themselves to the gills of fish and 
lead a parasitic life for 3 to 12 weeks (63). (b) In the fresh-water mussel 
Lamellidens marginalis, the osmotic pressure of the blood increases with age, 
rising from about 20 to 35 mOs (117). (c) In the Japanese marsh clam 
Corbicula japonica, it has been shown that the adults osmoregulate but the 
young do not (207). It is possible that the kidney in the young bivalve has 
not developed to the point where it can produce a hypoosmotic urine, and 
that this is related to the fact that bivalves have invaded fresh water from a 
marine habitat. 
Finally, it should be mentioned that Potts (132) has made determinations 
of sodium fluxes in the muscle fibers of the marine mussel Mytilus and the 
fresh-water mussel Anodonta. From these measurements he calculated the 
energy required for sodium extrusion, assuming that it is entirely an active 
process. The values he arrived at were 0.26 cal per g per hr for Mytilus and 
0.046 cal per g per hr for A nodonta. He suggests on this basis that a fresh· 
water animal, with a low osmotic concentration of its tissues, 
may perform less ionic work than a marine animal; for although it has to perform a 
certain amount of ionic work at the body surface it may be saved a large amount of 
ionic work at the surface of each cell. Conversely, the many marine animals which 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 643 
maintain a salt concentration in the blood which is less than that of sea water, for 
e.'{ample: teleosts, selachians, lampreys, sturgeons, grapsoid crabs and many shrimps, 
may be more efficient than otherwise appears. 
These considerations may explain the observation (96, 180) that the 
oxygen consumption of certain molluscs increases when the salinity of their 
environment is increased; i.e . , the increased energy demand for ionic regula­
tion may more than balance the decreased energy demand for osmoregula­
tory purposes. 
ARTHROPODA 
This phylum is the largest in the animal kingdom, but of its eight classes 
only two, the crustaceans and the insects, have been studied in any detail. 
These two classes will therefore occupy all of the present discussion. 
CRUSTACEA 
Structure.-Fresh-water, brackish-water, marine, and terrestrial forms 
are found. In the majority of crustaceans the excretory organs consist of a 
pair of "antennary glands" or "green glands" located in the head (54, 125). 
Each gland has (a) a closed coelomic sac; (b) a canal, the "labyrinth" or 
"green body"; (c) a nephridial canal (absent in marine and brackish-water 
forms); and (d) a bladder. 
A single layer of epithelial cells surrounds the coelomic sac, which is 
richly supplied with hemolymph through small vessels or lacunae. The 
labyrinth is a highly involuted canal, composed of a single layer of large cells 
in contact with a thin basement membrane. The cells, like those of the verte­
brate proximal tubule, show basal striation and an apical brush border (2, 
98, 99, 100). Electron microscopy (2) has shown simple basal infoldings of 
the plasma membrane and mitochondria scattered throughout the cyto­
plasm. Cytoplasmic inclusions of unknown derivation were also observed. 
The cells in the nephridial canal have much in common with the distal 
tubular cells Of vertebrates (8). Electron micrographs demonstrate a series 
of lamellae or infoldings of the plasma membrane at the base of the cells. The 
cytoplasmic compartments between the lamellae contain rows of mitochon­
dria (d. mollusca). Maluf (99) noted large apical vacuoles, which he associ­
ated with water secretion; however, these were not mentioned by Beams, 
Anderson & Press (8). 
It is of interest that distinct morphological and functional differences are 
found between the kidneys of fresh-water crustaceans on the one hand, and 
secondary invaders of fresh or brackish water and marine forms, on the other 
hand. Grobben (54) noticed that the nephridial canal is present only in fresh­
water forms. Peters (125) found that the lobster, which lacks the nephridial 
canal, can make isosmotic urine, while the crayfish, which possesses a long 
nephridial canal, can make hypoosmotic urine. He further showed that the 
fluid in the coelomic sac and labyrinth has the same chloride concentration as 
the hemolymph, but that the fluid along the nephridial canal becomes pro-
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644 SCHMIDT-NIELSEN AND LAWS 
gressively more dilute as it approaches the bladder. Schwabe (163) found that 
the fresh-water crustacean Palaemonites varians microgenitor does not have a 
nephridial canal. It was later shown (121, 122) that the urine is always is­
osmotic to the hemolymph in various species of Palaemonites adapted to fresh 
water. In other cases also, where fresh-water crustaceans have been found to 
lack a nephridial canal, it has been shown that they lack the ability to pro­
duce a hypoosmotic urine (158). The degree to which the urine can be made 
hypoosmotic appears to be related to the length of the nephridial canal in 
Gammarus (68, 92). 
It is quite evident that the nephridial canal is essential for the production 
of a hypoosmotic urine. However, the significance of this ability is not clear, 
since successful adaptation to fresh water has frequently been accomplished 
without it (121, 158, 163). 
Osmoregulation.-The crustaceans show a wide range of osmoregulatory 
ability (85). Some, like the crayfish Asellus aquaticus (91) and the shore crab 
Carcinus maenas (174), are hyperosmotic in dilute sea water but are not able 
to maintain a lower osmotic concentration than the medium. Others show 
hypo- as well as hyperosmotic regulation (4, 56, 57, 134, 148), the most 
extreme example being Artemia salina, which can osmoregulate successfully 
in salinities ranging from 0.25 per cent sodium chloride to crystallizing salt 
brine (27, 28,127). Still others may have no osmoregulation (57). 
Three factors are involved in crustacean osmoregulation: (a) the active 
uptake or secretion of sodium and chloride by the gills, (b) the permeability 
of the body surface (gills primarily) to salt and water, and (c) the excretion of 
salt and water by the kidneys. Potts (131) estimated the theoretical min­
imum osmotic work required by a crustacean in fresh water to produce an 
isosmotic or a hypoosmotic urine. His calculations were based on the assump­
tion that the animal is permeable to water but not to electrolytes.They 
showed that an animal producing a urine hypoosmotic to the hemolymph and 
isosmotic to the medium can maintain its body salt concentration with only 
10 per cent of the osmotic work required by an animal making a urine is­
osmotic to the hemolymph. 
That this conclusion is based on incomplete assumptions was shown by 
several investigators (16, 17,18,28, 29, 121, 168 to 172, 174,175) who found 
high rates of sodium and chloride in- and outfluxes in various crustaceans . 
Shaw (175), on the basis of flux determinations, suggested that adaptation to 
. fresh water is not primarily a renal matter, but involves two main factors: 
(a) a gradual reduction in permeability of the body surface to salt, and (b) 
the acquisition of an active uptake mechanism with a high affinity for the 
ions which it is transporting. 
Shaw's suggestions are supported by good experimental evidence. The 
mechanism in the gills for active uptake of sodium and chloride ions is fully 
saturated at lower external concentrations in fresh-water forms than in 
brackish-water forms (175, 176). Also, the rate of sodium loss, measured as 
the 22Na outflux, was much lower in fresh- than in brackish-water forms. In 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 645 
Carcinus maenas (174) it was 890 ,uM per hr per SO g, while it was only 7 .S 
,liM per hr per SO g in the fresh-water crayfish A stacus papillipes (168), 
indicating a reduced permeability in the fresh-water form. 
The urinary sodium loss is quite low compared to the over-all loss even in 
Carcinus maenas whose urine is isosmotic to the blood and whose urine flow 
increases up to tenfold when it is adapted to fresh water (174). This loss 
ranges from only 2.7 per cent of the total loss, when the animal is in sea 
water, to 21.1 per cent when it is in 40 per cent sea water (174). Loss of 
sodium in the urine, however, may be a little more significant than appears at 
first sight, since part of the total outflux might be accounted for by "ex­
change diffusion", and therefore might not represent actual loss of sodium. 
Bryan (17) estimates that under normal conditions 30 per cent of the sodium 
outflux in Astacus fluviatilis is caused by exchange diffusion in connection 
with the active uptake of sodium by the gills. Similarly, Croghan (29) found 
extremely high sodium fluxes between the hemolymph of Artemia salina and 
the medium, with a marked increase in more concentrated media where 
active excretion of salt by the gills takes place. He concluded that rapid 
exchange diffusion of sodium and chloride takes place across the gills. 
Kidney function.-The question of filtration into the coelomic sac has 
been reinvestigated in the lobster (19) and in the crayfish (150). Both studies 
indicate that filtration does indeed occur here. The argument is based on the 
facts that inulin is excreted, that glucosuria can be induced by increasing the 
plasma glucose concentration, and that phlorizin poisoning causes glucose to 
appear in the urine. Maluf ( 101) earlier arrived at the conclusion that inulin 
is actively secreted in the coelomic sac of the crayfish because he found that 
the UjB (urine : blood) ratios decreased toward unity when the inulin con­
centration of the blood was increased. However, Riegel & Kirschner (ISO) 
suggest that this conclusion is erroneous, because in their experiments the 
inulin UjB ratios remained nearly constant when the range of blood concen­
trations was extended down to less than one milligram per cent inulin. 
Malui's data may, furthermore, be differently interpreted (104). 
The function of the labyrinth can best be dealt with through a discussion 
of the marine and brackish-water crustaceans which do not possess a nephrid­
ial canal. In all of these the urine is either isosmotic or slightly hyperosmotic 
to the blood. In the lobster, the inulin U jB ratio is unity (19) . In Carcinus 
maenas and in other crabs, the inulin U jB ratio may be as high as 2 to 3 
(lS l), indicating that water is reabsorbed in the labyrinth. 
In the lobster, the sodium concentrations in the urine and hemolymph 
are equal, but in various crabs, namely, Uca pugnax and U. pugilator (52), 
Carcinus maenas (lS l) , Pachygrapsus crassipes (60, 133), Palaemon serratus 
(120), and Potamon niloticus ( 169), the sodium concentration in the urine is 
lower than in the hemolymph, and sodium must therefore be actively re­
absorbed in the labyrinth. 
Changes in sodium and magnesium excretion in the urine are both 
involved in adaptation of crustaceans to varying salinities of their environ-
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646 SCHMIDT-NIELSEN AND LAWS 
ment, and, in many, an i nverse relationship between the excretion of these 
two ions is found. Magnesium is usually present in the urine in considerably 
greater concentrations than in the hemolymph and must be actively secreted 
(59) . When a lobster (19) is placed in dilute sea water, the magnesi u m VIB 
ratio decreases to values below unity indicating reabsorption of magnesium, 
while in full strength sea water, magnesium is secreted, giving a V IB ratio 
of 2 to 3. In Palaemon serratus (120) the urinary magnesium concentration is 
always higher than that of the hemolymph and increases considerably with 
increasing salinity of the medium, even though the hemolymph concentra­
tion of magnesium remains almost unchanged. The hemolymph sodium con­
centration, on the other hand, increases with increasing salinity. In the 
lobsters (19) Palaemon serratus (1 20) and Potamon niloticus (169), the urine 
sodium concentration varies in the same direction as the hemolymph sodium 
concentration, and the sodium VIB ratio remains constant. In others, Uca 
pugnax (52), Carcinus maenas (1S1), Pachygrapsus crassipides (58, 133), 
however, the sodium concentration in the urine decreases significantly with 
increasing salinity of the medium or with dehydration. Thus, in Pachygrapsus 
(133) the sodium VIB ratio was 1.1 when the animal was in 50 per cent sea 
water, 0.68 in 100 per cent sea water, and 0.38 in 170 per cent sea water. In 
these same crabs the magnesium concentration increased considerably with 
increasing salinity. 
It has further been shown that the concentration of magnesium in the 
urine is dictated by the salinity of the medium and not by the magnesium 
content (60) because during brief periods of submersion i n salt solutions 
corresponding to SO per cent, 100 per cent, and 1 75 per cent sea water the 
magnesium concentration of urine of Pachygrapsus reflected the salinity of 
the medium regardless of whether magnesiu m was present or absent in 
abnormally high concentrations, while the urine sodium concentration de­
creased with increasing salinity. Prosser et at. (133) , who submerged the 
crabs for longer periods of time, found that, in the absence of MgS04 in the 
medium, urine sodium increased with i ncreasing salinity. The difference in 
findings could be a result of the difference in lengths of submersion. 
Another extremely interesting finding in the crustaceans is that a urine 
slightly hyperosmotic to the hemolymph has been observed independently 
by several investigators in various crabs. In Carci1tus the urine osmolality 
exceeded thehemolymph osmolality by about 3 per cent after the crab had 
been dehydrated for four days (151). In Uca (52) , hyperosmotic urine (10 to 
20 per cent) was produced when the crab was placed in 175 per cent sea 
water. In Ocypode albicans and Goniopsis cruentatus the chloride V IB ratio 
reached values of 1 .6 to 1.8 when the crabs were placed in full strength sea 
water (42) . Flemister suggests active water reabsorption, but it seems more 
likely to us that the hyperosmolality is brought about by the secretion of 
magnesium, perhaps in the most distal, and possibly relatively water­
impermeable part of the labyrinth. The secretion of divalent ions such as 
magnesium and sulfate into the urine and the conservation of sodium are 
found in many other groups of animals. 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 647 
As mentioned earlier, the nephridial canal is necessary for the production 
of a hypoosmotic urine and is present only in true fresh-water forms. Its func­
tion appears to be similar to that of the vertebrate distal tubule. From 
Peter's ( 125) finding that chloride concentration decreases along the canal 
and from analogy with the mammalian tubule, we can assume that active 
reabsorption of electrolytes takes place against an osmotic gradient leaving 
water behind. The urinary osmotic concentration may be as low as 4 to 5 
mM sodium chloride. Some water reabsorption, however, also takes place in 
the nephridal canal. The average inulin U IB ratio in the crayfish is 3 to 4 but 
values as high as 28 have been recorded ( 150). Such high values have so far 
not been recorded for crustaceans without a nephridial canal. 
The renal response to a salt load is quite different in the fresh-water 
crayfish with a nephridial canal from that in crabs without a nephridial 
canal. Thus, when the sodium concentration in the hemolymph of the cray­
fish A stacus ( 18) was increased by placing the animal in saline media exceed­
ing 200 mM per liter, the urine sodium concentration increased almost to the 
level of the hemolymph. The urine flow, moreover, increased. An increase in 
the sodium concentration in the urine could in theory be brought about 
either by a decreased sodium reabsorption in the nephridial canal or an 
increased permeability to (hence reabsorption of) water, but the increase in 
urine volume may suggest that the first explanation is correct. The renal 
response to water loads of another species of crayfish Orconectes virilis was 
studied by Riegel ( 149), who found that severe water loads caused large and 
pronounced increases in the inulin clearance, urine flow, and total sodium 
excretion. Urine flow nearly doubled and sodium excretion increased fourfold 
while the inulin VIB ratios decreased. 
INSECTA 
Structure.-Insects have been adapted to a terrestrial life since the early 
Pennsylvanian Period. Some have secondarily become adapted to fresh 
water in the larval stage or in the larval and adult stages. A few have 
migrated to salt marshes and some inhabit the crystallizing brine of salt 
lakes (188) . 
Because of their unique ability to produce not only a hypoosmotic urine 
but also a highly hyperosmotic urine, the excretory organs in insects play a 
much more important role in osmoregulation than they do in other inverte­
brates. In the majority of insec"ts they consist of the Malpighian tubules and 
the hindgut.4 These parts are intimately concerned with the formation of 
urine. 
The Malpighian tubules vary greatly in number, from two to over one 
hundred, in the different orders of insects. They are long, slender, blind 
tubules derived as an outgrowth from the intestine. One end opens into the 
4 The Collemboia and Aphids are exceptions in that they do not have Malpighian 
tubules. In the primitive wingless Collembola, excretion takes place through labial 
nephridia, middle intestinal epithelium, hypodermis, and fat bodies (38). 
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648 SCHMIDT-NIELSEN AND LAWS 
junction between midgut and hindgut ; the other, the blind end, either lies 
free in the hemocoele or is "cryptonephric", i .e . , attached to the wall of the 
posterior hindgut (69) and enveloped in the peritoneal membrane. Lison 
(90) has given a detailed description of this attachment in Tenebrio (the flour 
beetle). The surface of the Malpighian tubules toward the intestine is 
smooth, while the surface toward the hemococle is swollen. Openings in the 
membrane of the swollen surface have been postulated, but according to 
Lison, the membrane is continuous. Poll ( 128) has suggested filtration 
through this surface. 
The Malpighian tubules consist of several histologically distinct segments 
which vary in number and structure in the different orders of insects. In some 
a segment is concerned with mucous secretion (47) or secretion of silk (70) or 
brochosomes (30, 1 77) . Only two segments appear to be present in most 
orders. Wiggleworth (201) has described these parts in Rhodnius prolixus 
which possesses four tubules, each of which consists of an upper segment 
(including the closed end) with granular epithelium and with clear fluid in the 
lumen, and a lower segment with almost granular-free epithelium and with 
crystalline spheres of uric acid filling the lumen. The description for Corcyra 
cephalonica ( 182) is almost identical except that the tubules of Corcyra are 
cryptonephric which they are not in Rhodnius. For this reason Srivastava 
(182) divides the tubule into three segments. However, the cryptonephric 
and the middle segments are histologically identical to the upper segment as 
described in Rhodnius. Both upper and lower segments have a striated border 
toward the lumen. The striated border of the upper segment is of the honey­
comb type, while in the lower segment it is a brush border with well separated 
filaments (201) . Electron micrographs of these segments in Rhodnius (204) , 
in Macrosteles ( 1 78), in Melanoplus (7), and in Gryllus (10, 1 1 ) have shown 
that in the upper segment the mitochondria are most conspicuous in the 
apical part of the cells. The prominent honeycomb border is 3 to 4 JJ. long, 
each filament being about 1 JJ. wide and sometimes densely packed with mito­
chondria. The basal zone of the cells has only a few mitochondria. Crystals of 
uric acid are found in the cells (182, 204). The cells of the lower segment differ 
in that the maximum density of mitochondria is found between the invagi­
nated cytomembranes at the base of the cells. Crystalline spheres of uric acid 
are found in the lumen between the filaments of the brush border. 
The hindgut with the rectum and rectal gland constitutes an important 
part of the excretory system because the fluid emerging from the Malpighian 
tubules is modified as it passes through this part. Wigglesworth (202) made a 
detailed comparison of the hindgut in all main orders of insects and suggested 
that the rectal gland and the rectal epithelium reabsorb water from the urine 
before it is discharged. In most orders the hindgut consists of two or three 
main segments : (a) a long thin region with comparatively small epithelial 
cells ; (b) a capacious sac, "the rectum", surrounded by a muscular coat and, 
in some, followingthe rectum: (c) a muscular canal with low epithelium. The 
rectum and anal canal are usually lined with cuticle. The "rectal glands", 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 649 
usually six in number, are situated in the anterior part of the rectum. They 
are composed of large cuboidal cells, very richly supplied by tracheae. Often 
the terminal tracheoles can be seen ramifying throughout the cells. Electron 
microscopy of the hindgut of insects reveals some interesting structures 
( 1 13, 1 78). In Macrosteles fascifrons Stal (Homoptera) ( 178), the entire 
rectum is lined by a uniform columnar epithelium which, according to 
Wigglesworth (202), is of the same type as that composing the rectal gland. 
These cells possess lamellae filled with mitochondria on the luminal side. 
Similar cells have been described in the rectum of termites by Noirot & 
Noirot-Timothee (113) who state, "At the apical part of the cells the mem­
brane folds irregularly toward the interior forming cytoplasmic compart­
ments with many mitochondria, giving a striated appearance to the cell which 
is visible in the light microscope." According to the authors, these cells are 
remarkably similar to the cells of the distal tubule of mammals, but with the 
polarity reversed, since in the distal tubule it is the basal part of the cell 
which exhibits the cytoplasmic compartments filled with mitochondria. 
Ramsay ( 138) has studied the structure and function of the hindgut in 
two species of mosquito larvae Aedes aegypti and Aedes detritus. The first 
inhabits fresh water and can produce a urine hypoosmotic or isosmotic to the 
hemolymph. The other, A . detritus, lives in brackish water and can, in addi­
tion, make a hyperosmotic urine. He associates the ability to form a hyper­
osmotic urine with a region in the anterior part of the rectum lined with an 
epithelium distinctly different from that in the remainder of the rectum. This 
anterior region was not found in A . aegypti. It is not clear from Ramsay's 
description whether or not this anterior region in A . detritus is identical to the 
"rectal glands" described by Wigglesworth (202). Further investigations 
comparing cellular specialization in the rectum with ability or inability to 
produce a hyperosmotic urine in various insects would be highly desirable. 
Osmoregulation.-Some aquatic insects are able to osmoregulate success­
fully in fresh (110) and salt water (93, 112), maintaining the hemolymph 
osmotic concentration relatively constant over wide ranges of salinities. 
Thus, in Chironomus salinarius (112) the freezing point depression of the 
hemolymph is tl = 0.63°C (350 mOs) when the larvae are in a medium of 
tl = 0.05°C and rises to only tl =0.94°C when the medium is tl = 1.5°C. A 
relativel y large fraction of the total osmotic concentration of insect hemo­
lymph is attributable to amino acids (32, 203) . When the larvae of Libellula 
and Aeschna are placed in distilled water, the osmotic concentration of the 
hemolymph is maintained through a rise in amino acid concentration in spite 
of a fall in the chloride concentration ( 159, 160). In caddis larvae (Trichop­
tera) (190), the regulation is of a different type. The salt concentration of the 
hemolymph is maintained at a relatively constant level, lower than that of 
the medium, while the total osmotic concentration of the hemolymph re­
mains equal to that of the medium over a wide range of media concentrations. 
Insects are not impermeable to salt and water. The permeability of the 
cuticle of a number of aquatic insect larvae and terrestrial insects has been 
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650 SCHMIDT-NIELSEN AND LAWS 
determined by Beament and others (5, 6, 102) . A considerable variation in 
permeability of the cuticle is found among the aquatic insects. The greatest 
permeability is evident in the anal papillae or "gills". The exchange of labeled 
sodium between the external medium and the hemolymph and whole body in 
Aedes aegypti was found to be half complete in 60 hours ( 184, 192) with 90 
per cent of the exchange taking place through the anal papillae ( 184) . The 
function of the anal papillae apparently is that of active uptake of ions from 
a dilute medium (141 , 184, 1 85). As in the crustaceans, it is quite possible that 
such exchange studies do not indicate the true permeability since "exchange 
diffusion" may take place at the site of active ion uptake. 
When insect larvae are placed in dilute media or in media having extreme 
deficiencies of chloride, alkali, or cations ( 186), the papillae hypertrophy, 
whereas when the larvae are placed in more concentrated media the papillae 
atrophy (62) . Thus, the papillae appear to be of no importance in hypo­
osmoregulation. Insect larvae in hyperosmotic environments osmoregulate 
primarily with the excretory organs. 
Function of Malpighian tubules and hindgut.-It was suggested by Wig­
glesworth (201) on the basis of his experiments with Rhodnius that potassium 
and urate ions are secreted into the upper segment of the Malpighian tubules, 
the segment in which the cells were later found to contain urate crystals 
(204). Ramsay (140) confirmed these findings and showed that the fluid in 
the upper segment is slightly hyperosmotic to the hemolymph and has a 
potassium concentration around 120 meq per liter while that of the hemo­
lymph has only 5 to 10 meq-per liter. The sodium concentration, on the other 
hand, was lower in the upper segment than in the hemolymph. Electrical 
potential measurements also indicated active potassium secretion in this 
segment ( 142) . Not all insects, however, secrete uric acid in the upper seg­
ment of the Malpighian tubule. I n Sitophilus granaritts (L.) (61) and in 
Periplaneta americana ( 183), uric acid and urates are secreted in the hindgut. 
In many insects a large fraction or all of the uric acid is stored in the fat 
bodies ( 181 , 183) . 
Patton & Craig ( 123) and Srivastava ( 182) on the basis of their findings 
from insects with cryptoncphric tubules, Tenibrio molitor (L.) ( 123) and 
Corcyra cephalonica Stainton ( 182), suggest that filtration takes place in the 
so-called middle tube, the part of the upper segment immediately following 
the cryptonephric part. The hemolymph apparently moves into this middle 
portion leaving only larger molecules behind. Thus, glycogen has been shown 
to enter the lumen. Such a movement into the tubule is quite likely in view of 
the findings by Ramsay (140) that the tubular fluid is hyperosmotic to the 
hemolymph. Ramsay and I<iegel (144) have recently confirmed these find­
ings and shown that inulin as well as glucose, fructose, and sucrose enters the 
urine of the stick insect Dixippus morosus through the wall of the Malpighian 
tubule. However, the UjB ratio for inulin is much lower (0.046) than the 
ratios for the sugars (0.75 , 0.58, and 0.58 respectively) . 
The function of the lower segment is, according to Wigglesworth (201) , 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 65 1 
that of reabsorption of alkali and water, thus acidifying the content of the 
tubule with resulting precipitation of uric acid. The results by Ramsay 
( 140) have confirmed this hypothesis. In the lower segment the osmotic 
concentration is slightly (6 to 39 mM) lower than that of the hemolymph 
( 143). On the basis of this finding Ramsay ( 140) suggested that water is 
secreted into the tubule ; however, it is much more likely that the hypoosmo­
lality is brought about through active salt reabsorption. Ramsay ( 140) 
showed that the potassium concentration decreases in the lower segment 
while the sodium concentration increases. Presumably some passive water 
reabsorption also takes place in this segment. 
The fluid entering the hindgut from the Malpighian tubules is thus 
always slightly hypoosmotic to the hemolymph regardless of the final concen­
tration of the urine ( 139, 140, 141). Final changes in osmotic concentration 
take place in the hindgut where the urine may remain isosmotic or become 
hypoosmotic or hyperosmotic to the hemolymph. 
As · mentioned earlier, the fresh-water insect larva Aedes aegypti can 
produce a hypoosmotic urine when placed in a dilute medium, whereas the 
urine becomes isosmotic to the hemolymph when the larva is placed in a 
concentrated medium. In contrast to this, the brackish-water mosquito 
larva A . detritus can, in addition, produce a hyperosmotic urine in concen­
trated media (138). The same is true of the fly larva Coelopa jrigida which 
can concentrate the urine up to six times the concentration of hemolymph 
(189). Another brackish-water fly larva Ephydra riparia cannot make the 
urine hypoosmotic but can make it either isosmotic or highly hyperosmotic, 
up to ten times the concentration of the hemolymph ( 189) . Hyperosmotic 
urine has also been demonstrated in the adult bloodsucking insect Rhodnius 
(140). Most adult insects and terrestrial insect larvae make a urine that is 
practically dry (202). The osmolality of this urine has, to our knowledge, not 
been determined. 
The mechanisms through which the osmolality of the urine is changed are 
still quite obscure. The production of hypoosmotic urine is probably accom­
plished by active electrolyte reabsorption by epithelium with a low per­
meability to water. The epithelium concerned with this operation presum­
ably can change permeability to water since the urine can also be made 
isosmotic. Thus, the insect hindgut in certain respects appears to function 
much the same as the bladder of molluscs, the nephridium of the earthworm, 
or the distal tubule of vertebrates. 
The "dry urine", if isosmotic to the hemolymph, could likewise be made 
through passive water reabsorption, secondary to electrolyte reabsorption. 
Wigglesworth (202) associated the presence of a well-developed rectal gland 
with the ability to reabsorb water from the urine but he also points out that 
part of the solidification takes place in the elongated thin region of the 
hindgut. 
Hyperosmolality of the urine could be brought about through two pos­
sible mechanisms, ion secretion into the hindgut fluid or active water reab-
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652 SCHMIDT-NIELSEN AND LAWS 
sorption. The unlikelihood of the latter leaves us with the first possibility. 
In Rhodnius (140) it seems likely that potassium is secreted in the rectum 
since Ramsay (140) found the osmolality of the urine to increase in direct 
proportion to the potassium concentration but to be independent of the 
sodium concentration. 
Whether or not sodium is secreted in the rectum of brackish-water 
species is not known. In the fresh-water larvae of Aedes aegypti the sodium 
concentration of the intestinal fluids is only about one half the sodium 
concentration of the hemolymph ; however, when the larvae are kept in solu­
tions with high sodium chloride concentrations, the sodium concentration in 
intestinal fluid never exceeds that of the hemolymph. To our knowledge the 
urine of brackish-water species has not been analyzed for sodium. 
The correlation between structure and function in the excretory system 
of the insects is particularly intriguing, but needs to be explored much fur­
ther. It appears that ions are secreted in the upper segment of the Malpighian 
tubule as well as in the rectum and reabsorbed in the lower segment and in 
some unidentified part of the hindgut. The epithelial cells in the upper seg­
ment and rectal gland are peculiar in that they have lamellae with many 
mitochondria in the apical part of the cell. We would suggest that this partic­
ular feature may be characteristic for ion secretion. The cells of the lower 
segment resemble the cells of the proximal tubule of most other species in 
that the mitochondria are situated more in the basal and middle parts of the 
cell. They also appear to function like those of a proximal tubule. 
A COMPARISON B ETWEEN I NVERTEBRATE 
AN D VERTEB RATE KIDN EYS 
The excretory mechanism of invertebrates (except insects) is in many 
ways quite similar to that of vertebrates. The functional renal unit in 
vertebrates and in most invertebrates consists of (a) a part into which secre­
tion or filtration takes place, (b) a proximal tubule with a high permeability 
to water, and (c) a distal tubule in which the permeability to water is usually 
low but can increase in response to the need for water conservation. 
Filtration or secretion.-In most vertebrate nephrons an ultrafiltrate of 
the blood is formed by filtration through the glo�erular capillaries. In the 
aglomerular fish the tubular fluid apparently is formed through secretion of 
solutes into the tubule with water following passively ( 103) . 
In many of the invertebrates the question of whether the tubular fluid is 
formed by secretion or filtration has not yet been answered. In the Malpig­
hian tubules of insects a combination of the two seems to be involved : 
solute is secreted into the tubule, creating an osmotic gradient which causes 
bulk flow into the tubule of an ultrafiltrate of the hemolymph ( 123, 182). The 
function of the flame cells in Platyhelminthes and Aschelminthes may be of a 
similar nature, with the beating action of the cilia enhancing the flow through 
the tubules ( 167) . Filtration is evident in molluscs, crustaceans, and annelids. 
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URINARY DILUTING AND CONCENTRATING MECHANISMS 653 
In the latter the coelomic fluid entering through the nephrostome is also an 
ultrafiltrate of the blood. 
Proximal tubule.-The proximal tubular cells of the vertebrate nephron 
are characteristically cuboidal with brush borders on the luminal side. At the 
basal end of the cell, the membrane is thrown into folds which reach a short 
distance into the cell, giving it a striated appearance. Mitochondria are 
scattered throughout the cytoplasm with a few being found between the 
cytomembranes. 
For most invertebrates electron-microscopical studies of the renal unit 
have not been made. In crustaceans, however, the description given by 
Anderson & Beams (2) of the cells of the labyrinth is almost identical to the 
description