ABSTRACT In Arapaima gigas , an obligate air-breather endemic to ion-poor Amazonian waters, a large complex kidney runs through the air-breathing organ (ABO). Previous indirect evidence suggested that the kidney, relative to the small gills, may be exceptionally important in ionoregulation and nitrogen (N) waste excretion, with support of kidney function by direct O 2 supply from the airspace. We tested these ideas by continuous urine collection and gill flux measurements in ∼700 g fish. ATPase activities were many-fold greater in kidney than gills. In normoxia, gill Na + influx and efflux were in balance, with net losses of Cl − and K + . Urine flow rate (UFR, ∼11 ml kg −1 h −1 ) and urinary ions (< 0.2 mmol l −1 ) were exceptional, with [urine]:[plasma] ratios of 0.02–0.002 for K + , Na + , and Cl − , indicating strong reabsorption with negligible urinary ion losses. Urinary [ammonia] was very high (10 mmol l −1 , [urine]:[plasma] ∼17) indicating strong secretion. The kidney accounted for 21–24% of N excretion, with ammonia dominating (95%) over urea-N through both routes. High urinary [ammonia] was coupled to high urinary [HCO 3 − ]. Aerial hypoxia (15.3 kPa) and aerial hyperoxia (>40.9 kPa) had no effects on UFR, but both inhibited branchial Na + influx, revealing novel aspects of the osmorespiratory compromise. Aquatic hypoxia (4.1 kPa), but not aquatic hyperoxia (>40.9 kPa), inhibited gill Na + influx, UFR and branchial and urinary ammonia excretion. We conclude that the kidney is more important than gills in ionoregulation, and is significant in N excretion. Although not definitive, our results do not indicate direct O 2 supply from the ABO for kidney function.

ABSTRACT The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas exchange and those that limit passive flux of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water flux. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus . Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In freshwater killifish, hypoxia exposure reduced branchial Na + /K + -ATPase and NEM-sensitive ATPase activities, as well as diffusive water flux rates. Unidirectional Na + influx and Na + efflux decreased during hypoxia in freshwater, but net Na + flux remained unchanged. Net loss rates of Cl − , K + and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na + flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia to that in freshwater. In summary, freshwater and seawater killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na + influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.

ABSTRACT The characteristics of the fish gill that maximize gas exchange are the same that promote diffusion of ions and water to and from the environment; therefore, physiological trade-offs are likely to occur. Here, we investigated how salinity acclimation affects whole-animal respiratory gas exchange during hypoxia using Fundulus heteroclitus , a fish that inhabits salt marshes where salinity and oxygen levels vary greatly. Salinity had marked effects on hypoxia tolerance, with fish acclimated to 11 and 35 ppt showing much longer time to loss of equilibrium (LOE) in hypoxia than 0 ppt-acclimated fish. Fish acclimated to 11 ppt (isosmotic salinity) exhibited the greatest capacity to regulate oxygen consumption rate ( Ṁ O 2 ) under hypoxia, as measured through the regulation index (RI) and P crit . At 35 ppt, fish had a higher routine metabolic rate (RMR) but a lower RI than fish at 11 ppt, but there were no differences in gill morphology, ventilation or blood O 2 transport properties between these groups. In contrast, 0 ppt-acclimated fish had the highest ventilation and lowest O 2 extraction efficiency in normoxia and hypoxia, indicating a higher ventilatory workload in order to maintain similar levels of Ṁ O 2 . These differences were related to alterations in gill morphology, where 0 ppt-acclimated fish had the smallest lamellar surface area with the greatest epithelial cell coverage (i.e. thicker lamellae, longer diffusion distance) and a larger interlamellar cell mass, contrasting with 11 ppt-acclimated fish, which had overall the highest respiratory surface area. The alteration of an array of physiological parameters provides evidence for a compromise between salinity and hypoxia tolerance in killifish acclimated to freshwater.

ABSTRACT Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while Ṁ O 2 initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O 2 debt repayment during recovery. Gill paracellular permeability (measured with [ 14 C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [ 14 C] PEG-4000) was 8-fold higher in seawater fish, but declined by ∼90% during hypoxia in both groups, with post-exposure overshoots to ∼270%. Gill diffusive water flux (measured with 3 H 2 O) was 1.9-fold higher in freshwater fish, and exhibited a ∼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve Ṁ O 2 , but nevertheless incurs a net water balance penalty in both freshwater and seawater.

ABSTRACT Ventilatory sensitivity to ammonia occurs in teleosts, elasmobranchs and mammals. Here, we investigated whether the response is also present in hagfish. Ventilatory parameters (nostril flow, pressure amplitude, velar frequency and ventilatory index, the last representing the product of pressure amplitude and frequency), together with blood and water chemistry, were measured in hagfish exposed to either high environmental ammonia (HEA) in the external sea water or internal ammonia loading by intra-vascular injection. HEA exposure (10 mmol l −1 NH 4 HCO 3 or 10 mmol l −1 NH 4 Cl) caused a persistent hyperventilation by 3 h, but further detailed analysis of the NH 4 HCO 3 response showed that initially (within 5 min) there was a marked decrease in ventilation (80% reduction in ventilatory index and nostril flow), followed by a later 3-fold increase, by which time plasma total ammonia concentration had increased 11-fold. Thus, hyperventilation in HEA appeared to be an indirect response to internal ammonia elevation, rather than a direct response to external ammonia. HEA-mediated increases in oxygen consumption also occurred. Responses to NH 4 HCO 3 were greater than those to NH 4 Cl, reflecting greater increases over time in water pH and P NH 3 in the former. Hagfish also exhibited hyperventilation in response to direct injection of isotonic NH 4 HCO 3 or NH 4 Cl solutions into the caudal sinus. In all cases where hyperventilation occurred, plasma total ammonia and P NH 3 levels increased significantly, while blood acid–base status remained unchanged, indicating specific responses to internal ammonia elevation. The sensitivity of breathing to ammonia arose very early in vertebrate evolution.

ABSTRACT A recent study demonstrated that in response to a feeding-induced metabolic acidosis, goldfish ( Carassius auratus ) adjust epithelial protein and/or mRNA expression in their kidney tubules for multiple transporters known to be relevant for acid–base regulation. These include Na + /H + exchanger (NHE), V-type H + -ATPase (V-ATPase), cytoplasmic carbonic anhydrase, HCO 3 − transporters and Rhesus proteins. Consequently, renal acid output in the form of protons and NH 4 + increases. However, little is known about the mechanistic details of renal acid–base regulation in C. auratus and teleost fishes in general. The present study applied the scanning ion-selective electrode technique (SIET) to measure proton flux in proximal, distal and connecting tubules of goldfish. We detected increased H + efflux into the extracellular fluid from the tubule in fed animals, resulting from paracellular back-flux of H + through the tight junction. By applying inhibitors for selected acid–base regulatory epithelial transporters, we found that cytosolic carbonic anhydrase and HCO 3 − transporters were important in mediating H + flux in all three tubule segments of fed goldfish. Contrastingly, V-ATPase seemed to play a role in H + flux only in proximal and distal tubules, and NHE in proximal and connecting tubules. We developed working models for transport of acid–base relevant equivalents (H + , HCO 3 − , NH 3 /NH 4 + ) for each tubule segment in C. auratus kidney. While the proximal tubule appears to play a major role in both H + secretion and HCO 3 − reabsorption, the distal and connecting tubules seem to mainly serve for HCO 3 − reabsorption and NH 3 /NH 4 + secretion.

ABSTRACT We used respirometric theory and a new respirometry apparatus to assess, for the first time, the sequential oxidation of the major metabolic fuels during the post-prandial period (10 h) in adult zebrafish fed with commercial pellets (51% protein, 2.12% ration). Compared with a fasted group, fed fish presented peak increases of oxygen consumption (78%), and carbon dioxide (80%) and nitrogen excretion rates (338%) at 7–8 h, and rates remained elevated at 10 h. The respiratory quotient increased slightly (0.89 to 0.97) whereas the nitrogen quotient increased greatly (0.072 to 0.140), representing peak amino acid/protein usage (52%) at this time. After 48-h fasting, endogenous carbohydrate and lipid were the major fuels, but in the first few hours after feeding, carbohydrate oxidation increased greatly, fueling the first part of the post-prandial specific dynamic action, whereas increased protein/amino acid usage predominated from 6 h onwards. Excess dietary protein/amino acids were preferentially metabolized for energy production.

ABSTRACT Ureotelic elasmobranchs require nitrogen for both protein growth and urea-based osmoregulation, and therefore are probably nitrogen-limited in nature. Mechanisms exist for retaining and/or scavenging nitrogen in the gills, kidney, rectal gland and gut, but as yet, the latter are not well characterized. Intestinal sac preparations of the Pacific spiny dogfish shark ( Squalus acanthias suckleyi ) incubated in vitro strongly reabsorbed urea from the lumen after feeding, but mucosal fluid ammonia concentrations increased with incubation time. Phloretin (0.25 mmol l −1 , which blocked urea reabsorption) greatly increased the rate of ammonia accumulation in the lumen. A sensitive [ 14 C]urea-based assay was developed to examine the potential role of microbial urease in this ammonia production. Urease activity was detected in chyme/intestinal fluid and intestinal epithelial tissue of both fed and fasted sharks. Urease was not present in gall-bladder bile. Urease activities were highly variable among animals, but generally greater in chyme than in epithelia, and greater in fed than in fasted sharks. Comparable urease activities were found in chyme and epithelia of the Pacific spotted ratfish ( Hydrolagus colliei ), a ureotelic holocephalan, but were much lower in ammonotelic teleosts. Urease activity in dogfish chyme was inhibited by acetohydroxamic acid (1 mmol l −1 ) and by boiling. Treatment of dogfish gut sac preparations with acetohydroxamic acid blocked ammonia production, changing net ammonia accumulation into net ammonia absorption. We propose that microbial urease plays an important role in nitrogen handling in the elasmobranch intestine, allowing some urea-N to be converted to ammonia, which is then reabsorbed for amino acid synthesis or reconversion to urea.

ABSTRACT P crit – generally defined as the P O 2 below which the animal can no longer maintain a stable rate of O 2 consumption ( Ṁ O 2 ), such that Ṁ O 2 becomes dependent upon P O 2 – provides a single number into which a vast amount of experimental effort has been invested. Here, with specific reference to water-breathers, I argue that this focus on the P crit is not useful for six reasons: (1) calculation of P crit usually involves selective data editing; (2) the value of P crit depends greatly on the way it is determined; (3) there is no good theoretical justification for the concept; (4) P crit is not the transition point from aerobic to anaerobic metabolism, and it disguises what is really going on; (5) P crit is not a reliable index of hypoxia tolerance; and (6) P crit carries minimal information content. Preferable alternatives are loss of equilibrium (LOE) tests for hypoxia tolerance, and experimental description of full Ṁ O 2 versus P O 2 profiles accompanied by measurements of ventilation, lactate appearance and metabolic rate by calorimetry. If the goal is to assess the ability of the animal to regulate Ṁ O 2 from this profile in a mathematical fashion, promising, more informative alternatives to P crit are the regulation index and Michaelis–Menten or sigmoidal allosteric analyses.

ABSTRACT Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes.

ABSTRACT In rainbow trout, the dominant site of Na + uptake ( J Na,in ) and ammonia excretion ( J amm ) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, J Na,in and J amm occur via loose Na + /NH 4 + exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, J Na,in by the yolk sac skin of PH trout occurs independently of J amm . Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l −1 NH 4 HCO 3 ; 600 µmol l −1 Na + ; pH 8), J amm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in J Na,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in J Na,in [Na + /H + -exchanger-2 (NHE2) and H + -ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na + /K + -ATPase) in putative peanut lectin agglutinin-positive (PNA + ) ionocytes in gill sections. Similar Na + /K + -ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of J Na,in and J amm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different.

ABSTRACT Nitrogen (N) appears to be a limiting dietary resource for elasmobranchs, required not only for protein growth but also for urea-based osmoregulation. Building on recent evidence that the toxicant ammonia can be taken up actively at the gills of the shark and made into the valuable osmolyte urea, we demonstrate that the uptake exhibits classic Michaelis–Menten saturation kinetics with an affinity constant ( K m ) of 379 µmol l −1 , resulting in net N retention at environmentally realistic ammonia concentrations (100–400 µmol l −1 ) and net N loss through stimulated urea-N excretion at higher levels. Ammonia-N uptake rate increased or decreased with alterations in seawater pH, but the changes were much less than predicted by the associated changes in seawater P NH 3 , and more closely paralleled changes in seawater NH 4 + concentration. Ammonia-N uptake rate was insensitive to amiloride (0.1 mmol l −1 ) or to a 10-fold elevation in seawater K + concentration (to 100 mmol l −1 ), suggesting that the mechanism does not directly involve Na + or K + transporters, but was inhibited by blockade of glutamine synthetase, the enzyme that traps ammonia-N to fuel the ornithine–urea cycle. High seawater ammonia inhibited uptake of the ammonia analogue [ 14 C]methylamine. The results suggest that branchial ammonia-N uptake may significantly supplement dietary N intake, amounting to about 31% of the nitrogen acquired from the diet. They further indicate the involvement of Rh glycoproteins (ammonia channels), which are expressed in dogfish gills, in normal ammonia-N uptake and retention.

ABSTRACT Hagfish skin has been reported as an important site for ammonia excretion and as the major site of systemic oxygen acquisition. However, whether cutaneous O 2 uptake is the dominant route of uptake remains under debate; all evidence supporting this hypothesis has been derived using indirect measurements. Here, we used partitioned chambers and direct measurements of oxygen consumption and ammonia excretion to quantify cutaneous and branchial exchanges in Pacific hagfish ( Eptatretus stoutii ) at rest and following exhaustive exercise. Hagfish primarily relied on the gills for both O 2 uptake (81.0%) and ammonia excretion (70.7%). Following exercise, both O 2 uptake and ammonia excretion increased, but only across the gill; cutaneous exchange was not increased. When branchial O 2 availability was reduced by exposure to anteriorly localized hypoxia (∼4.6 kPa O 2 ), cutaneous O 2 consumption was only slightly elevated on an absolute basis. These results refute a major role for cutaneous O 2 acquisition in the Pacific hagfish.

ABSTRACT Dissolved organic matter (DOM) is both ubiquitous and diverse in composition in natural waters, but its effects on the branchial physiology of aquatic organisms have received little attention relative to other variables (e.g. pH, hardness, salinity, alkalinity). Here, we investigated the effects of four chemically distinct DOM isolates (three natural, one commercial, ranging from autochthonous to highly allochthonous, all at ∼6 mg C l −1 ) on the physiology of gill ionoregulation and nitrogenous waste excretion in zebrafish acclimated to either circumneutral (7.0–8.0) or acidic pH (5.0). Overall, lower pH tended to increase net branchial ammonia excretion, net K + loss and [ 3 H]PEG-4000 clearance rates (indicators of transcellular and paracellular permeability, respectively). However, unidirectional Na + efflux, urea excretion and drinking rates were unaffected. DOM sources tended to stimulate unidirectional Na + influx rate and exerted subtle effects on the concentration-dependent kinetics of Na + uptake, increasing maximum transport capacity. All DOM sources reduced passive Na + efflux rates regardless of pH, but exerted negligible effects on nitrogenous waste excretion, drinking rate, net K + loss or [ 3 H]PEG-4000 clearance, so the mechanism of Na + loss reduction remains unclear. Overall, these actions appear beneficial to ionoregulatory homeostasis in zebrafish, and some may be related to physico-chemical properties of the DOM sources. They are very different from those seen in a recent parallel study on Daphnia magna using the same DOM isolates, indicating that DOM actions may be both species and DOM specific.

ABSTRACT Relative to the gills, the mechanisms by which the kidney contributes to ammonia and acid–base homeostasis in fish are poorly understood. Goldfish were exposed to a low pH environment (pH 4.0, 48 h), which induced a characteristic metabolic acidosis and an increase in total plasma [ammonia] but reduced plasma ammonia partial pressure ( P NH3 ). In the kidney tissue, total ammonia, lactate and intracellular pH remained unchanged. The urinary excretion rate of net base under control conditions changed to net acid excretion under low pH, with contributions from both the NH 4 + (∼30%) and titratable acidity minus bicarbonate (∼70%; TA–HCO 3 − ) components. Inorganic phosphate (P i ), urea and Na + excretion rates were also elevated while Cl − excretion rates were unchanged. Renal alanine aminotransferase activity increased under acidosis. The increase in renal ammonia excretion was due to significant increases in both the glomerular filtration and the tubular secretion rates of ammonia, with the latter accounting for ∼75% of the increase. There was also a 3.5-fold increase in the mRNA expression of renal Rhcg-b (Rhcg1) mRNA. There was no relationship between ammonia secretion and Na + reabsorption. These data indicate that increased renal ammonia secretion during acidosis is probably mediated through Rhesus (Rh) glycoproteins and occurs independently of Na + transport, in contrast to branchial and epidermal models of Na + -dependent ammonia transport in freshwater fish. Rather, we propose a model of parallel H + /NH 3 transport as the primary mechanism of renal tubular ammonia secretion that is dependent on renal amino acid catabolism.

ABSTRACT The osmorespiratory compromise, the trade-off between the requirements for respiratory and ionoregulatory homeostasis at the gills, becomes more intense during environmental hypoxia. One aspect that has been previously overlooked is possible change in transepithelial potential (TEP) caused by hypoxia, which will influence branchial ionic fluxes. Using the euryhaline killifish, we show that acute hypoxia reduces the TEP across the gills by approximately 10 mV in animals acclimated to both freshwater (FW) and seawater (SW), with a higher P O 2 threshold in the former. TEP becomes negative in FW, and less positive in SW. The effects are immediate, stable for at least 3 h, and reverse immediately upon return to normoxia. Hypoxia also blocks the normal increase in TEP that occurs upon transfer from FW to SW, but does not reduce the fall in TEP that occurs with transfer in the opposite direction. These effects may be beneficial in FW but not in SW.

ABSTRACT Larval rainbow trout ( Oncorhynchus mykiss ) were reared from hatch under control ([Na + ]=0.60 mmol l −1 ) or high NaCl ([Na + ]=60 mmol l −1 ) conditions to elucidate the driving force for the ontogeny of branchial Na + /NH 4 + exchange, one of the earliest gill functions. We hypothesized that if Na + uptake is the driving force, then in high NaCl there would be a delay in the skin-to-gill shift in ammonia excretion ( J amm ) and/or an elevation in whole-body total ammonia ( T amm ). In both groups, however, the skin-to-gill shift for J amm , determined using divided chambers, occurred at the same time (13 days post-hatch; dph) and whole-body T amm was unchanged. Moreover, high NaCl larvae displayed elevated whole-body [Na + ] relative to controls by 18 dph, suggesting that maintaining branchial J amm occurs at the expense of Na + balance. Overall, these results support the ‘ammonia hypothesis’, which posits that ammonia excretion, probably as Na + /NH 4 + exchange, is the primary function of the early fish gill.

In teleosts, a branchial metabolon links ammonia excretion to Na + uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs ( Rhag, Rhbg and Rhp2 ) and evaluated gill ammonia handling in Squalus acanthias . Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol −1 NH 4 HCO 3 ) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24–48 h. Control Δ P NH 3 (the partial pressure gradient of NH 3 ) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH 4 + electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na + /K + -ATPase ( NKA ) increased at 24 h and that of H + -ATPase decreased at 48 h, while expression of the potential metabolon components Na + /H + exchanger2 ( NHE2 ) and carbonic anhydrase IV ( CA-IV ) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.

Increased renal ammonia excretion in response to metabolic acidosis is thought to be a conserved response in vertebrates. We tested the hypothesis that Rhesus (Rh) glycoproteins in the kidney of the freshwater common carp, Cyprinus carpio , play a crucial role in regulating renal ammonia excretion during chronic metabolic acidosis. Exposure to water pH 4.0 (72 h) resulted in a classic metabolic acidosis with reduced plasma arterial pH and [HCO 3 − ], no change in P CO 2 and large changes in renal function. Urine [NH 4 + ] as well as [titratable acidity-HCO 3 − ] rose significantly over the acid exposure, but the profound reduction (fivefold) in urine flow rates eliminated the expected elevations in renal ammonia excretion. Low urine flow rates may be a primary strategy to conserve ions, as urinary excretion rates of Na + , Cl − and Ca 2+ were significantly lower during the acid exposure relative to the control period. Interestingly, renal Rhcg1 mRNA and protein levels were elevated in acid-exposed relative to control groups, along with mRNA levels of several ion transporters, including the Na + /H + exchanger, H + -ATPase and Na + /K + -ATPase . Immunofluorescence microscopy showed a strong apical Rhcg1 signal in distal tubules. Taken together, these data show that renal Rh glycoproteins and associated ion transporters are responsive to metabolic acidosis, but conservation of ions through reduced urine flow rates takes primacy over renal acid–base regulation in the freshwater C. carpio . We propose that an ‘acid/base–ion balance’ compromise explains the variable renal responses to metabolic acidosis in freshwater teleosts.

The influence of swimming activity on gill paracellular permeability has not been measured previously in fishes. We critically assessed the use of tritium-labeled polyethylene glycol ([ 3 H]PEG-4000) for this purpose, a substance that is also a classic marker for extracellular fluid volume, glomerular filtration rate and drinking rate. Tests (8 h) on resting freshwater trout showed that when measuring [ 3 H]PEG-4000 clearance from the plasma in the efflux direction, correction for a large excretion via glomerular filtration was essential, necessitating urinary catheterization. When measuring [ 3 H]PEG-4000 clearance from the water in the influx direction, correction for a significant uptake by drinking was essential, necessitating terminal gut removal, whereas glomerular filtration losses were minimal. After correction for these alternate routes of loss and uptake, [ 3 H]PEG-4000 clearance rates by efflux from the plasma and by influx from the water were identical, showing that gill paracellular permeability is not rectified, and can be measured in either direction. The influx technique with terminal gut removal was used to assess gill paracellular permeability in trout without urinary catheters freely swimming at 1.2 body lengths s −1 for 8 h. Branchial [ 3 H]PEG-4000 clearance rate (by influx from the water) increased significantly by ~80% in accord with a similar measured increase in O 2 consumption rate. Thus in trout, gill paracellular permeability does increase during exercise, in accord with the traditional concept of the osmorespiratory compromise.