Comp. Physiol.

Having looked at strategies of retaining water and salt balance we will now briefly review morphology and function of organs involved in these processes.

Evolution of excretory organs (primarily invertebrates)

Water channels – a key to water transport and its regulation

Excretion of nitrogenous waste products – a good example of don’t believe everything you read!

EVOLUTION OF EXCRETORY SYSTEMS

Overhead: Withers 17-7 (nephridia and coelomoducts)

1. Nephridia: ectodermal origin

Protonephridia: Blind ending ducts. Primitive. Fluid drawn into the tube by terminal cilia or flagella. Solenocytes are monocililate; Flame cells are multiciliate

Metanephridia: Have direct connection to the coelomic cavity. Open into coelomic space via a ciliated funnel-like opening. Fluid passes down the metanephridial tubule and is eliminated from the nephridopore. Present in larger, more complex animals.

2. Coelomoduct: mesodermal origin

Coelomic fluid is drawn into the ciliated coelomostome, passes through the coelomoduct, and is eliminated from the coelomopore. Original function of coelomoducts was likely as an exit route for gametes.

Protonephromixium and mixonephridium are combinations of nephridia and coelomoducts.

Malpighian tubules: endodermal origin, develop from the gut, empty into the hindgut – not directly to the exterior

Overhead: Withers 17-8 (functional classification)

A more recent (1988) classification of tubular excretory organs uses functional rather than structural/embryological basis.

Protonephridia: Blind-ended excretory tubules of animals with single body cavity (coelom). Entry of fluid into the tubules by ciliary/flagellar beating. Inside the protonephridial tubules the pressure is negative relative to the coelom. Generally present in primitive and small animals.

Metanephridia: In animals with coelomic and vascular space. A primary urine is formed by hydrostatic pressure filtration of vascular fluid into the coelomic space. The primary urine is drawn into the metanephridial duct where the secondary urine is formulated. According to this classification, metanephridia include embryologically defined metanephridia and coelomostomes. Generally present in larger and more advanced animals.

Overhead: Withers 17-9 (Principal functions of tubular excretory organ)

4 processes involved in urine formation:

- Filtration

- Secretion

- Reabsorption

- Osmoconcentration (in nephrons of mammals and some birds)

Flame cell in the end of a protonephridium creates a negative hydrostatic pressure inside the tubule

B. In metanephridia (functional definition) the blood is filtered under positive hydrostatic pressure. Colloid osmotic pressure in blood acts against the hydrostatic pressure, but the net filtration pressure will be in the direction from the blood to either the coelom or the duct directly.

C. Malpighian tubules of insects do not have filtration. Urine is formed by secretion processes.

ANNELIDA

Overhead: Withers 17-14 (Lumbricus nephridium)

The earthworm, Lumbricus, and well and the medicinal leech, Hirudo, produce a markedly hypoosmotic urine. The metanephridium of the earth worm is a complex multicoiled tubule with a nephridiostome of varying morphology and a bladder before the nephridiopore. Fluid, which may be filtered from the vascular system into the coelom space near the nephridiostome, enters the tubule through the ciliated nephridiostome. By the distal portion of the tubule, the osmotic concentration has declined to about 20% of the initial filtrate concentration. The osmodilution is a consequence of solute reabsorption, especially active Na⁺ and passive Cl^- reabsorption without passive comigration of water.

MOLLUSCA

Overhead: Withers 17-15 (Mytilus nephridium)

The excretory system of mollusks includes protonephridia in larval stages and metanephridia (coelomoducts) in adults. In bivalve molluscs, like the blue mussel, Mytilus, the primary excretory filtrate is formed by ultrafiltration from the atria into the pericardial sac via the pericardial glands and flows down the renopericardial canal, through the pericardial funnel into the kidney. After renal modification, the urine exits via the excretory pore. The excretory systems of prosobranchs is similar, but that of opisthobranchs and pulmonates is reduced to a single kidney, and in pulmonates a secondary urether forms to drain the urine.

The filtration rate varies dramatically between different mollusks. The general trend is from low filtration rates in marine species to high rates in FW mollusks. Terrestrial forms fall somewhere between.

The primary filtrate is not much different in composition from the blood, but the final urine is. The urine is isoosmotic for marine mollusks, but varies for FW and terrestrial mollusks from isoosmotic to markedly hypoosmotic.

ARTHROPODA

Overhead: Withers 17-16 (Crustacean anterior [green] gland)

There is considerable variation among arthropods is their excretory organs. Crustaceans heave coelomoduct-type antennal glands and maxillary glands and also gut-derived rectal and cephalic glands. The antennal gland of the crayfish has a terminal coelomosac, a labyrinth, a nephridial tubule, and a bladder terminating at the nephridiopore. The cells of the coelomosac (called podocytes) rest on a basement menbrane with a series of interdigitating "foot" structures (pedicells); this resembles the arrangement of the glomerular filtration apparatus in vertebrate nephrons. The coelomosac recieves arterial blood supply and its structure suggests a filtration mechanism for formation of primary urine.

In the FW crayfish, 95 – 99% of the Na⁺ and Cl^- is reabsorbed. Water is reabsorbed from the labyrinth and from the proximal tublule. Virtually all glucose is reabsorbed.

Overhead: Withers 17-17 (Excretory systems of mygalomorph spider)

The principal excretory organ for arachnids are the coxal glands or the Malpighian tubules or both. Coxal glands are thin-walled, spherical sacs that collect wastes from the hemolymph and eliminate urine through a duct opening onto the coxa.

The coxal gland has a thin-walled saccule, to which coxal gland muscles and a tubular organ with two discrete segments are attached. Hemolymph is filtered through the thin saccule wall, probably by hydrostatic pressure difference due to contractions in the coxal muscles. Contraction of these muscles establish a negative pressure inside the saccule in comparison with the outside. Relaxation of the muscles probably forces fluid along the excretory tubule.

Large molecules, such as casein and albumin, are not filtered, although haemoglobin is. The excretory tube is thick-walled, has a peculiar internal chitin skeleton, and is highly tracheate; it probably reabsorbes solutes and water.

The coxal gland secretions of various terrestrial arachnids can be anything from hypoosmotic to hyperosmotic.

Overhead: Withers 17-18 (in vitro prep of Malpighian tubule)

The primary excretory organs of insects are the Malpighian tubules and the rectum. Most insects have a variable number of Malpighian tubules (2 – 250), but the primitive collembolans (and aphids) lack them. There is no filtration and the primary urine is formed by secretion of solutes and water. In most insects, K⁺ is actively pumped, followed by passive movements of Cl^-, other solutes, and water.

The function of the Malpighian tubules has been extensively studies by the in vitro preparation shown here. The preparation consists of an isolated tubule immersed in paraffin.

The fluid secreted from the distal end of the tubule (would normally open into the gut) is always isoosmotic with the hemolymph (or the bathing medium in the in vitro preparation. The rate of secretion, however, is inversely proportional to the medium osmotic concentration.

Overhead: Withers 17-19 (Excretory system of insects; M. tubule + rectum)

The Malpighian tubules open into the gut at the junction of the midgut and the hindgut or in the rectum. In many species there is further modification of the urine in the hindgut.

The rectum is the most important site for modification of the tubular urine by major bulk reabsorption of ions and other solutes (Na⁺, K⁺, Cl^-, amino acids, glucose, trehalose) and water. The excreta of insects can be markedly hyperosmotic to the hemolymph, by 500 to 5,500 mOsm for dehydrated terrestrial species and by 700 to 2,900 mOsm for saltwater species.

Overhead: Withers 17-20 (Osmoconcentration in blowfly)

The absorption of hypoosmotic fluid by the rectal epithelium of certain insects, such as the blowfly, is thought to occur by local osmosis coupled to solute reabsorption. The mechanism for rectal reabsorption of water in many insects, including the blowfly, involves specialized folds of the rectal epithelium, the ‘rectal pads.’

Model: The papillate rectum is thought to absorb fluid by osmosis in response to active ion transport from the rectal lumen into the epithelial cells. Ions are then transported and water follows into intercellular spaces between the epithelial cells. The absorbed solutes together with water move along the intercellular channels towards the hemocoel. Ions are reabsorbed into the epithelial cells for subsequent return to the intercellular spaces and for continued osmotic absorption of water from the lumen.

Direct measurements of ion concentrations in the rectal epithelium of the blowfly support this general model. The fluid in lateral intercellular sinuses near the lumen is hyperosmotic in dehydrated blowflies, but the total ion concentration declines markedly along the intercellular channel towards the hemocoel.

Overhead: Withers 17-6 (Standing gradient model for water transport)

The same as above, but diagrammed.

Overhead: Withers 17-21 (Evolution of the vertebrate nephron)

The vertebrate nephron is derived from a coelomoduct draining the coelomic cavity. Coelomic fluid was probably formed by a glomerulus-like arrangement of capillaries near the coelomic wall but not at the coelomostome. The ‘glomerulus’ then became associated with the tubule, as a more direct means o forming primary urine, and the glomerular blood supply then contributed to the tubular capillary network; the coelomostome remained as an adjunct for urine formation and to drain coelomic fluid into the nephron. The coelomostome was later lost.

Amphibians cannot concentrate urine above blood.

One reptile, the Salt Lake dragon, can excrete a concentrated urine (671 mOsm; U/P 1.5)

Birds can produce a moderately concentrated urine (459 mOsm; U/P 1.4 to 2,000 mOsm; U/P 5.8).

Most mammals can concentrate the urine to 3,000 – 5,000 mOsm/kg, but the range is from less than 1,000 mOsm/kg in FW mammals to more than 9,000 mOsm/kg in some desert mammals.

WATER CHANNELS

Ability to concentrate urine and to effectively transport water across epithelia in general is dependent upon a family of proteins that have become known as aquaporins, or water channels.

Overhead: Verkman et al., 1996 (Mammalian water channels)

6 mammalian aquaporins have been cloned, AQP-1 – 5 and hKID. All belong to the same family and are related to a prototype molecule, major intrinsic protein (MIP) of lens fibre. The function of MIP is not clear. Several of these water channel proteins have a wide tissue distribution, whereas some such as AQP-2 and hKID have only been found in the kidney. Only two of them, AQP-2 and AQP-3 are known to be inducible at the transcriptional level.

Overhead: Verkman et al., 1996 (High homology between membrane-spanning segments)

Amino acid sequence alignment of the proteins shows high homology in hydrophobic putative membrane-spanning segments (boxes) as well as absolutely conserved sequences including two NPA (asp-pro-ala) motifs. These NPA motifs sit on either side of the cell membrane and have been postulated to play a role in the formation of a water pore.

7 membrane-associated a -helical domains based on hydrophobicity.

Proposed transmembrane topology of mature AQP-1. Domains 3 and 4 are creating one passage through the membrane è 6 membrane-spanning domains.

Intermediate topology of AQP-1 as synthesized in ER

In membranes, AQP-1 forms tetramers. However, each AQP-1 monomer appears to be a functional unit – the 4 subunits function independently as water channels.

Overhead: CH (localization of aquaporins in nephrons)

Proximal tubule: AQP-1 (CHIP-28)

Thin descending limb of Henle: AQP-1 (CHIP 28)

Collecting duct: AQP-2, AQP-3, and AQP-4

Overhead: Knepper, 1997, Fig. 5 (AQP-distribution in principal cells of IMCD)

This figure summarizes the proposed mechanism of short-term, vasopressin-mediated, regulation of water permeability in the collecting duct. AQP-2 is present both in the apical membrane and in intracellular vesicles. When needed these vesicles can fuse with the apical membrane, in a process identical to exocytosis, and thereby inserting more water channels into the membrane. There is also an endocytosis going on, during which AQP-2 molecules are removed from the membrane by endocytosis. Vasopressin binds to V₂ receptors in the basolateral cell membrane. V₂ receptors act via the cAMP pathway to trigger a redistribution of AQP-2 from intracellular vesicles to the apical plasma membrane. The intracellular vesicles fuse with the apical membrane adding the water channels to the apical side. At the same time removal of apical AQP-2, by formation of edocytotic vesicles, may be inhibited in the presence of elevated intracellular cAMP.

The addition of water channels to the apical membrane increases its water permeability. The apical transfer is the rate-limiting step for water transport, presumably because of a high level of expression of AQP-3 and AQP-4 in the basolateral membrane. Hence, by inserting AQP-2 into the apical membrane, the water permeability of the epithelium as a whole is increased.

Overhead: text (vesicular trafficking is regulated by SNAREs and VAMPs)

So, how does cAMP stimulate exocytosis and insertion of AQP-2 in the apical membrane?

Vesicle targeting receptors (SNAREs) may mediate a specific interaction between the vesicles and its target membrane.

Two classes:

Syntaxins

2. SNAP-25 and its homologues

Targeting receptors in vesicles, VAMPs (vesicle associated membrane proteins; or "synaptobrevins").

Overhead: Knepper, 1997, Fig. 6 (Vesicle Targeting Receptors)

The presence of a VAMP and a syntaxin have recently been demonstrated in chief cells of IMCD (through which water transport occurs). VAMP-2 sit on AQP-2 vesicles and Syntaxin-4 has been localized to the apical membrane. VAMP-2 and syntaxin-4 specifically bind to each other, but the functional role for these targeting proteins remains to be demonstrated.

Overhead: text (Long-term regulation of transepithelial water permability)

Research in the 50s showed that the ability to concentrate the urine in response to vasopressin administration was markedly increased in human subjects after 48-h water restriction. Results indicated long-term conditioning effect.

In vitro preparations of rat IMCD showed that several days of thirsting increased the water permeability of the IMCD even if no vasopressin was present in the surrounding medium. Results again indicated conditioning effect.

It has now been shown that long-term thirsting causes increased transcription of both AQP-2 and AQP-3. Furthermore, vasopressin is probably responsible for this long-term conditioning as well as for the vesicle trafficking discussed earlier.

Draw: (transcriptional regulation)

The 5’ flanking regions of both AQP-2 and AQP-3 have cis-elements that are sensitive to increased cAMP.

AQP-2 (WCH-CD)

(CRE = cAMP-responsive element)

AQP-3 (GLIP)

EXCRETION OF NITROGENOUS WASTE

Overhead: Withers 17-39 (Strategies of N excretion)

This is the generalized view of how the animal kingdom is divided into ammonia excretors, urea excretors, and excretors of uric acid. Traditionally, fish have been considered solely ammonotelic, birds and reptiles uricotelic, and mammals ureotelic. In reality, the borders between these groups are diffuse and most animals excrete all of these three N-waste compounds to some extent.

Overhead: composite (ammonia, urea, and uric acid)

In general, aquatic vertebrates excrete ammonia. Ammonia is highly water soluble, but due to the high toxicity of ammonia it needs to be diluted which brings with it large volumes of urine and poor water conservation.

Urea is also readily dissolved in water but is far from as toxic as ammonia and each urea molecule contains 2 N which makes urea excretion better suited for water conservation and terrestrial life. A disadvantage with urea excretion is that each urea molecule costs 2ATP to produce.

Uric acid is also expensive to produce, but is superior to urea if you want to preserve water. Uric acid contains 4 N, which mean that for each mol of uric acid excreted you get rid of 4 N, as opposed to 2 N for each urea and 1 N for each ammonia. Furthermore, uric acid is insoluble in water which allows production of ‘dry urine.’

Overhead: text (N-Excretion in Chondrichtyes)

Urea osmolyte but expensive excretion product. Only 4 species investigated, 2 in FW and 2 in SW. Whereas all species did excrete significant amounts of ammonia, urea was the major nitrogenous waste product for all but the Amazon stingray, Pomatotrygon.

Overhead: Wood, 1993, Table 3 (N-Excretion in Osteichtys)

NH₃ cheapest alternative for N-excretion and for most aquatic organisms including fish it is easy to get rid of è the nitrogenous waste product of choice for most bony fish, in FW as well as SW. Indeed, many adult fish do not express all enzymes in the UOC, the most efficient synthesis pathway for urea. Resent studies show, however, that the genes for the enzymes are there and they are typically expressed and urea is excreted during a brief window of their development (right after hatching).

Exceptions are conditions that impair ammonia excretion:

high pH

air exposure

Only in those air-breathing species that have a functioning OUC is there a strong activation of urea synthesis and excretion in response to air exposure. In African (Protopterus sp.) and South American (Lepidosiren paradoxa) lung fish ammonia production ceases during aestivation while urea production continues at a low rate. After a year’s aestivation, plasma [urea-N] levels may exceed 400 mM and ammonia toxicity is completely avoided.

Note the gulf toadfish.

Overhead: Wood, 1993, Fig. 3 (mechanism of branchial N-excretion)

Ammonia is voided via gills and kidney. The former excretory route dominates in most osteichtys species. Ammonia can relatively readily diffuse across cell membranes, whereas the ammonium ion has to pass the gill epithelium either via carrier mediated transport or across paracellular pathways. Because gills of SW fish are leaky and those of FW fish are not, paracellular movement of ammonium is of much higher significance in SW species (21% of total) than in FW fish (~5% of total). There is still controversy as to which of these routes is the most important but at least for FW fish it seems to be diffusion of ammonia that is the most significant.

pKa for the NH₃ + H⁺ è NH₄⁺ reaction is 9.1

Means that at the normal pH values in the body (~7.2), NH₄⁺ will constitute >95% of T_Amm. It also means that NH₃ that is leaving the gills is rapidly proteonated when it enters the water, thereby maximizing the diffusion gradient for NH₃. Indeed, in waters of high pH (>9) the diffusion gradient for NH₃ is greatly compromized. For fish species that do not have a functional OUC a high pH leads to build-up of ammonia in the plasma. Urea, produced via alternative pathways (primarily from breakdown of purines and/or direct cleavage of arginine to urea and ornithine), is also accumulating in the plasma during such conditions.

The Lake Magadi tilapia, from Lake Magadi (pH 10) in Kenya’s Rift Valley has solved the problem by being 100% ureotelic. The Lake Magadi tilapia produces urea via the OUC and it consequently has all the necessary enzymes.

Overhead: Wood, 1993, Fig. 6 (OUC in fish)

A couple of interesting differences in the OUC of most fish as compared with OUC enzymes from other vertebrates. Most fish have a different carbamoyl phosphate synthetase isozyme than that you’ll find in mammals. We have CPS I, which uses ammonia directly as substrate; fish has CPS III, which strongly prefers glutamine as N over free ammonia. CPS III as well as CPS I is present in the mitochondria. The requirement of CPS III for glutamine as N donor dictates that a second vital enzyme glutamine synthetase (GSase) must be located upsteam in the pathway to supply CPase III with glutamine. The activity of this enzyme is much higher in livers (primary site of urea synthesis) of OUC fish than in non-OUC fish and its affinity for ammonia is much higher than that in higher vertebrates. Furthermore, in most fish GSase occur in mitochondria where it can directly feed CPS III with glutamine.

Overhead: Withers 17-36 (Evolution of OUC: CPS I and CPS III)

Of fish species, only lungfish has CPS I. Amphibians, reptiles, birds, and mammals all have CPS I. Coelacanths have CPS III like other fish. CPS III is also found in invertebrate and is believed to be a more primitive isoenzyme. The fact that the coelacanth has the basic fish pattern, while the lungfish has the pattern of higher vertebrates, brings into question the pivotal role of the coelacanth in tetrapod evolution, and suggests that the lungfish led the way to higher verterates. This view has recently been supported by sequencing of mitochondrial DNA.

Overhead: Wood et al., 1995, Fig. 1 (urea pulse in toadfish)

-Opsanus beta facultatively ureotelic

-Switches from ammonotely to ureotely in response to confinement, crowding, or air-exposure.

-Voids all urea in a single pulse/day

-Urea transporters present in gill. May retain urea and then let go when it’s time for a pulse.

Overhead: text (N-excretion in birds)

N excretory product analysed in chicken, mallard, pigeon, turkey vulture, emu, greylag goose, and Anna’s hummingbird

Overhead: Preest & Beuchat, 1997 (ammonotely in hummingbirds)

Fed sucrose at 10, 20, and 40°C. Consumption the same at 20 and 40°C, but significantly elevated at 10°C. At 10°C the birds were consuming fluid at an amount of 27% of body mass per hour.