Renal Function in Unanaesthetized River Lampreys (Lampetra Fluviatilis L.): Effects of Anaesthesia, Temperature and Environmental Salinity

The euryhaline river lamprey spawns in fresh water and experiences the same problems of osmotic and ionic regulation as freshwater teleosts, i.e. a large osmotic influx of water and a diffusional and urinary efflux of ions. The mechanisms of freshwater osmoregulation in lampreys are similar to those of teleosts (Morris, 1960) and include the excretion of large volumes of dilute urine. However, estimates of urine flow rate in lampreys vary widely (from 60–400 ml kg⁻¹ day⁻¹; Wikgren, 1953; Hardisty, 1954; Morris, 1956, 1960; Bentley & Follett, 1963; Malvin, Carlson, Legen & Churchill, 1970; Logan, Moriarty & Rankin, 1980a) and, although part of this variation is due to the highly variable flow rates which may be found in individual fish (Bentley & Follett, 1963), much of it probably reflects the different temperatures and variety of methods, all more or less stressful, which have been employed. Recent micropuncture experiments (Logan et al. 1980a; Logan, Morris & Rankin, 1980b), performed on anaesthetized animals, have yielded new data on the functioning of the lamprey kidney, but the relationship of these results to the normal physiology of lampreys would be clarified by studies on unanaesthetized, minimally-stressed lampreys. The present work set out to use chronically catheterized lampreys, which had been allowed a long period to recover from the stress of the operation, to identify factors possibly affecting urine production (temperature, anaesthesia, respiration, environmental calcium ion concentration) and to use the preparation to study effects of different environmental salinities on renal function.

The present work used fresh-run lampreys which had been adapted to a range of seawater dilutions. However, migrating lampreys rapidly lose the capability for marine osmoregulation as a result of breakdown of extra-renal osmoregulatory mechanisms (Morris, 1956, 1958; Pickering & Morris, 1970) and experiments performed on fish in 40% and 50% sea water were limited to the small numbers of fish which successfully adapted to these salinities.

River lampreys were trapped on the River Severn during their autumnal and spring migrations, transported to Bangor with as little delay as possible after capture, and after slow adjustment of the water temperature to 10 °C, were transferred to large tanks of aerated, copper-free, dechlorinated water maintained at 10 °C. Batches of fresh-run fish were transferred to progressively increased seawater concentrations to provide lampreys adapted to a range of salinities up to 50% sea water (500 mosmol l⁻¹ ). All lampreys were adapted to particular salinities for at least 10 days before use.

Lampreys were weighed after immobilization in a solution of MS222 (ethyl-m-aminobenzoate methane sulphonic acid salt, Sigma, 55 mgl⁻¹ water). Any water retained in the gill pouches or on the body surface was carefully removed before weighing. Body weights ranged from 27–82 g.

After weighing, the fish was laid on its back in a Perspex trough so that its head, gill region and dorsal surface were immersed in a circulating solution of MS222 (275 mg in 51) maintained at 10°C. The exposed body surface was covered in most tissue paper to prevent dehydration. A catheter was constructed from approximately 40 cm of polypropylene tubing (PP60, Portex Ltd) by drawing out the tubing to provide a constriction about 1–2 mm from the tip. The catheter was filled with distilled water, carefully inserted into the urinogenital papilla of the lamprey, and a tie made around the papilla and tubing constriction, to prevent any forward or backward movement of the catheter. The catheter was secured by further ties to the tail approximately 1 cm and 2 cm posterior to the papilla.

The fish was then removed and transferred to a Perspex box (size 40 × 4 × 3 cm) which was connected to an anaesthetic-free, water-circulating system. The catheter was led to a fraction collector and urine was collected under water-saturated paraffin oil in tared plastic tubes. The system was covered in black polythene sheeting to reduce the risk of disturbance.

Ventilation rates were determined in some experiments using an impedance pneumograph (George Washington). A small silver electrode was inserted under the skin below the 3rd gill aperture of the lamprey and tied into position. A disc earth electrode was tied to the body about 1 cm anterior to the urinogenital papilla. Recordings were taken for 10–15 s at suitable intervals.

When required, 150–200 μl of blood was taken from the caudal vein of the fish and centrifuged immediately. Plasma samples were suitably diluted with double distilled water for analysis.

Urine was collected from unanaesthetized lampreys in circulating fresh water, at 10 °C, and MS222 was then added to give a range of concentrations of 0–80 mg l⁻¹. Urine was collected at each concentration for 20–30 min intervals for periods of up to 8 h.

The effect of rapid (approximately 6°Ch⁻¹) changes in temperature on urine flow rates was determined over a range of 2–18 °C. The water was held at the required temperature and urine collected for intervals of 20–60min for periods of up to 4h.

Urine was collected from unanaesthetized lampreys in fresh water and in salinities of up to 50% sea water. Water temperature was maintained at 10 °C, and urine was collected for 60–90 min intervals for up to 48 h.

The apparatus was thoroughly washed in distilled water and urine was then collected from lampreys held in 101 of circulating distilled water. Calcium chloride salt (Analar) was then added to the water to give a calcium concentration approximately equivalent to that of 100% sea water (10·5 mm). After urine collection, the water was drained and replaced with 0·3% or 0·6% sodium chloride (Analar) solutions with osmolarities (90, 206mosmoll⁻¹) equivalent to those of 10% and 20% sea water respectively. Urine was collected for 90 min intervals over 20–30 h.

Electrolyte concentrations of suitably diluted samples of plasma and urine were measured by emission (sodium) or atomic absorption (potassium, calcium and magnesium) spectrophotometry (EEL 240 Atomic Absorption Spectrophotometer). Sulphate was measured by indirect atomic absorption spectrophotometry (Logan et al. 1980b). Osmolarities of plasma, urine and water samples were measured using a semi-micro osmometer (Knauer).

An initial ‘settling down’ period (6–8 h) was allowed for recovery from surgery. Urine flows were expressed as ml urine kg⁻¹ body weight day⁻¹. Statistical analysis was by Student’s t-test.

The rate of urine flow in unanaesthetized lampreys in fresh water was 200·7 ± 14·3 ml kg⁻¹ day⁻¹ (Table 1) and decreased rapidly as the salinity of the external medium was increased (Fig. 1), so that the urine flow rate of 30% seawater-adapted lampreys was only 7·6% that of freshwater fish. This rapid decrease was associated with the approach of isotonicity between the body fluids and the environment. Urine was expelled at least once during each collection period when lampreys were in fresh water, 10%, 20% and 30% sea water, but there were long anuric periods in 40% and 50% sea water (Fig. 2). Urine flow was highly variable in fish in all salinities studied, and in individual freshwater lampreys it correlated with spontaneous changes in gill ventilation rate (Fig. 3).

Urine collected from freshwater lampreys had an osmolarity of 28·9 ± 7·0 mosmol l⁻¹ (Table 1) and there was little change in urine osmolarity with increasing water salinity up to 30% sea water when there was a large increase (P < 0·001). Urinary ion concentrations were generally increased in these fish, but only those of magnesium (P < 0·001) and sulphate (P < 0·001) were significantly higher compared to freshwater fish. Urine osmolarity was further increased in lampreys adapted to higher salinities, and in a small number of 50% sea water fish, the urine was slightly hyperosmotic to the plasma (Table 1).

The osmolarity of plasma from freshwater lampreys was 217·3 ± 15·6 mosmol l⁻¹ (Table 1) and was significantly (P< 0·001) higher in fish in 30% sea water. Plasma osmolarity was further increased with increasing water salinity so that lampreys adapted to 50% sea water had a plasma osmolarity of 361·8 ± 9·5 mosmol l⁻¹.

Urine: plasma electrolyte concentration ratios were less than one in freshwater lampreys, but those of magnesium and sulphate ions rapidly increased with increasing water salinity, and very high urine: plasma concentration gradients for these ions were established in fish in 20–50% sea water (Table 1) and the total daily excretion ions was greatly increased (Fig. 4). Sodium excretion was greatly reduced in fish in 50% sea water compared to all other groups.

The presence of calcium ions in the water did not have a significant effect on urine flow rate in lampreys. The urine flow rate (206·7 ± 42·3 ml kg⁻¹ day⁻¹, N = 5) of lampreys held in a 10·5 mm-calcium chloride solution (osmolarity 30–33 mosmol l⁻¹) was not significantly different from that (223·7 ± 40·3 mlkg⁻¹ day⁻¹, N = 5) of fish in distilled water alone. Transferring lampreys to 0·3% (osmolarity 90mosmoll⁻¹) and 0·6% (osmolarity 206 mosmol l⁻¹) sodium chloride solutions (i.e. calcium-free) resulted in new urine flowratesof 194·3 ± 33·8(N = 4) and50·4 ± 8·6(N = 4) mlkg⁻¹ day⁻¹, respectively, but although the urine flow rate of fish in 0·6% sodium chloride solution was significantly (P< 0·001) lower than that of fish in distilled water, it was not significantly different from that of fish in 20% sea water (osmolarity 205 mosmol l⁻¹, calcium = 2·1 mm, Table 1).

Concentrations of MS222 anaesthetic (50–55 mg l⁻¹), sufficient to induce anaesthesia, had little effect on urine flow rates of freshwater lampreys (Fig. 5), and no noticeable effect on gill ventilation rate was observed.

The effects of higher concentrations of MS222 were investigated in two lampreys, and urine flow rate was markedly decreased in both fish. Decreased gill ventilation rates were also observed in these fish.

Urine flow rates of freshwater lampreys were very sensitive to rapid temperature changes (Fig. 6). Q₁₀ (6–16°C) value was approximately 5. Gill ventilation rates were observed to increase as water temperature increased.

The present study confirms that the rate of urine production by freshwater lampreys is very high. Although the plasma osmolarity of lampreys is lower than that of teleosts (e.g. Hunn & Willford, 1970; Schmidt-Nielsen & Renfro, 1975) the resultant lower osmotic gradient between the body fluids and the environment is probably offset by the very high water permeability of freshwater lampreys (Wikgren, 1953; Bentley, 1962), resulting in a higher rate of urine flow. Urine flow rate in freshwater lampreys was positively correlated with gill ventilation rate, which indicates a high influx of water at high ventilation rates.

Urine flow rates of freshwater lampreys were extremely sensitive to changes in water temperature, and the Q₁₀ (6–16 °C) value was approximately 5. Teleosts acclimated to 20–25 °C exhibited an increased water permeability when compared with fish acclimated to 5–10 °C (Mackay & Beatty, 1968; Isaia, 1972; Motais & Isaia, 1972). However, when eels which were acclimated to one temperature were rapidly transferred to water at a higher temperature, the change in urine flow was greater than when fish were allowed to acclimate to the higher temperature (Gaitskell & Chester-Jones, 1971; Motais & Isaia, 1972), and this indicates that the increase in water permeability which follows an increase in water temperature is greater when the changes in temperature are rapid, than when fish are allowed to acclimate to the new temperature. Similar changes in branchial water permeability, coupled with the observed increase in gill ventilation rate, could account for the higher Q₁₀ value for urine flow found in lampreys in the present study compared with that found in temperature-acclimated lampreys (Wikgren, 1953).

High concentrations (>55 mg l⁻¹) of MS222 anaesthetic caused large decreases in urine flow and gill ventilation rates in freshwater lampreys but both parameters were little affected at concentrations (50–55 mg l⁻¹) sufficient to induce anaesthesia. Furthermore the osmolarity of urine from unanaesthetized freshwater lampreys was similar to that found in anaesthetized animals (Logan et al. 1980a): this indicates that renal tubular function is not impaired in lampreys under light MS222 anaesthesia.

The plasma and environment were isotonic in lampreys in salinities of between 20–30% sea water, and in these salinities lampreys showed a greatly decreased urine flow rate when compared with freshwater fish. Similar changes have been found in anaesthetized lampreys and resulted from a decrease in single nephron (SNGFR) and whole kidney (GFR) glomerular filtration rates (Rankin, Logan & Moriarty, 1980), with tubular water reabsorption essentially unchanged. The present data indicate that changes in renal function in these fish were in response to an increase in environmental osmotic pressure and not to calcium-mediated decreases in water permeability and hence water influx.

Lampreys in salinities hyperosmotic to the plasma (i.e. greater than 30% sea water) replace osmotic losses by drinking large volumes of the medium (Morris, 1958; Pickering & Dockray, 1972) and by increasing tubular reabsorption of water (Logan et al. 1980b; Rankin et al. 1980). Excess magnesium and sulphate ions, absorbed in the gut as a consequence of ingesting sea water, are excreted renally and the urinary concentrations of these ions were very high in anaesthetized 50% and 100% seawater-adapted lampreys (Logan et al. 1980b). The SNGFR of anaesthetized lampreys in 50% sea water was not significantly different from that of fish in 20% (i.e. hypo-osmotic) sea water (Rankin et al. 1980). It is of significance that, in the present study, marked changes in tubular function occurred in unanaesthetized lampreys in salinities above 20–30% sea water. Lampreys in 50% sea water had very high magnesium and sulphate ion excretion rates, indicating that a marked tubular secretion of ions had occurred. The excretory rate for sodium ions was lower in these lampreys than in fish in any other salinity, indicating that tubular sodium reabsorption was enhanced.

Unanaesthetized lampreys in 50% sea water excreted a urine which was slightly hyperosmotic to the plasma, largely due to very high urinary concentrations of magnesium and sulphate ions. A hyperosmotic urine has previously been reported in anaesthetized lampreys adapted to 100% sea water, but not in those adapted to 50% sea water (Logan et al. 1980b).

Generally, the present data obtained from unanaesthetized lampreys compare favourably with data obtained from anaesthetized lampreys. Renal function is minimally affected by light MS222 anaesthesia, and the intrarenal mechanisms recently determined in anaesthetized animals (Logan et al. 1980a,b; Rankin et al. 1980; Rankin, Griffiths, McVicar & Gilham, 1982), probably relate to the normal renal physiology of lampreys.

This work was supported by SRC (SERC) research grant GR/B 38179. A. J. McVicar was supported by an SRC (SERC) research studentship. We thank Miss V. Griffiths for technical assistance, and Mr P. J. Gaskins of Tirley for supplying us with lampreys. The manuscript was typed by Miss L. Hancocks.