Renal effects of fresh water-induced hypo-osmolality in a marine adapted seal

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1 J Comp Physiol B (2002) 172: DOI /s ORIGINAL PAPER R.M. Ortiz Æ C.E. Wade Æ D.P. Costa Æ C.L. Ortiz Renal effects of fresh water-induced hypo-osmolality in a marine adapted seal Accepted: 9 January 2002 / Published online: 21February 2002 Ó Springer-Verlag 2002 Communicated by L.C.-H. Wang R.M. Ortiz (&) Æ D.P. Costa Æ C.L. Ortiz A316 Earth and Marine Sciences, Department of Biology, University of California-Santa Cruz, Santa Cruz, CA 95064, USA rortiz@mail.arc.nasa.gov Tel.: Fax: C.E. Wade Neuroendocrinology Laboratory, Division of Life Sciences, NASA Ames Research Center, Moffett Field, CA Abstract With few exceptions, marine mammals are not exposed to fresh water; however quantifying the endocrine and renal responses of a marine-adapted mammal to the infusion of fresh water could provide insight on the evolutionary adaptation of kidney function and on the renal capabilities of these mammals. Therefore, renal function and hormonal changes associated with fresh water-induced diuresis were examined in four, fasting northern elephant seal (Mirounga angustirostris) (NES) pups. A series of plasma samples and 24-h urine voids were collected prior to (control) and after the infusion of water. Water infusion resulted in an osmotic diuresis associated with an increase in glomerular filtration rate (GFR), but not an increase in free water clearance. The increase in excreted urea accounted for 96% of the increase in osmotic excretion. Following infusion of fresh water, plasma osmolality and renin activity decreased, while plasma aldosterone increased. Although primary regulators of aldosterone release (Na +,K + and angiotensin II) were not significantly altered in the appropriate directions to individually stimulate aldosterone secretion, increased aldosterone may have resulted from multiple, non-significant changes acting in concert. Aldosterone release may also be hypersensitive to slight reductions in plasma Na +, which may be an adaptive mechanism in a species not known to drink seawater. Excreted aldosterone and urea were correlated suggesting aldosterone may regulate urea excretion during hypo-osmotic conditions in NES pups. Urea excretion appears to be a significant mechanism by which NES pups sustain electrolyte resorption during conditions that can negatively affect ionic homeostasis such as prolonged fasting. Keywords Aldosterone Æ Kidney Æ Marine mammals Æ Osmoregulation Æ Vasopressin Abbreviations ACTH adrenocorticotropin hormone Æ AII angiotensin II Æ ANP atrial natriuretic peptide Æ AVP vasopressin Æ BUN blood-urea nitrogen Æ C H2O free water clearance Æ FE fractional excretion Æ GFR glomerular filtration rate Æ Hct hematocrit Æ HTO tritiated water Æ NES northern elephant seal Æ PC postcatheterization Æ P crt plasma creatinine Æ P osm plasma osmolality Æ PRA plasma renin activity Æ TBW total body water Æ U osm urinary osmolality Æ V urine flow Introduction Animals may obtain fresh water: (1) by drinking free water, (2) from the metabolism of body stores and consumed food, and (3) from pre-formed water in the diet. Marine mammals without access to fresh water obtain water primarily from metabolism of body fat stores and from preformed water in their diet. Because many species of seals are adapted to extended (up to 12 weeks) periods of fasting as a natural component of their life histories (Riedman 1990), these mammals must possess robust physiological mechanisms for conserving metabolically derived water. For example, pups of the northern elephant seal (Mirounga angustirostris) (NES) naturally fast for 8 12 weeks after weaning, which is a period of extreme conservation of water and electrolytes in order to maintain ionic and osmotic homeostasis (Ortiz et al. 2000). Pups rely on water produced by the subsequent oxidation of body fat to maintain water balance (Ortiz et al. 1978).

2 298 Although marine mammals are not normally exposed to fresh water, each taxonomic order of marine mammals has at least one species that exists solely in a fresh water environment. The adaptation of closely related species to such divergent habitats provides an ideal opportunity to investigate the evolutionary adaptation of kidney function as mammals transitioned from a fresh water-accessible terrestrial habitat to a fresh water-devoid marine environment. Renal responses to the administration of saline in the Baikal seal (Phoca sibirica), a fresh water species, suggest that this seal, isolated from a marine environment for 0.5 million years, has retained the renal function of its marine counterparts (Hong et al. 1982). Whether or not a marine-adapted mammal isolated from fresh water for approximately 22 million years (Ray 1976) has a diminished renal capacity to handle excess water warrants investigation. Therefore, the present study will expand our understanding of the evolution of kidney function in marine mammals. Endocrine changes in marine mammals exposed to fresh water infusion (or oral loading) are very limited and inconclusive (Ridgway 1972; Hong et al. 1982; Skog and Folkow 1994). In grey seals (Halichoerus grypus), fresh water loading increased urine flow and decreased plasma osmolality associated with a decrease in urine osmolality, but there was no change in plasma vasopressin (AVP; Skog and Folkow 1994). Increased diuresis in response to water loading in Baikal and ringed (Phoca hispida) seals increased the fractional excretion of electrolytes and urea, and reduced urinary AVP and aldosterone concentrations (Hong et al. 1982). Urinary concentrations of these hormones were too low to accurately determine excretion rates, thus the involvement of AVP and aldosterone in the observed diuresis and saluresis could not be ascertained. fresh water loading in a bottlenose dolphin (Tursiops truncatus) increased diuresis and decreased natriuresis. However, aldosterone excretion was also reduced suggesting that aldosterone does not mediate renal-na + resorption during elevated diuresis in these marine mammals (Ridgway 1972). Therefore, to further elucidate hormonal regulation of water and electrolytes in marine mammals, fresh water was infused into a group of NES pups during their postweaning fast. The objectives of the present study were: (1) to quantify the alterations in renal function in response to water-induced diuresis in fasting NES pups, and (2) to examine the endocrine changes associated with this diuresis. We hypothesized that the infusion of fresh water induces a diuresis characterized by an increase in free water clearance (C H2O ) and a suppression of AVP, and a reduction in Na + excretion associated with an increase in aldosterone release. The infusion of fresh water allows insight into the renal and hormonal mechanisms involved with the renal handling of excess water and conservation of Na + in these seals, and thus expands our understanding of renal capabilities in these fascinating mammals. Materials and methods Animals Four NES pups (two males, two females; 95.3±9.9 kg; ±SD) were transported from An o Nuevo State Park (approximately 30 km north of Santa Cruz, Calif.) to Long Marine Laboratory, University of California, Santa Cruz. Animals were brought to the laboratory one or two at a time over the course of 3 weeks during their postweaning fast. Pups were held for 5 6 days from capture until the termination of an individual experimental trial. Upon arrival at the laboratory, pups were weighed using a hanging-load cell (±0.2 kg) and placed in a sand pit over night. The following morning, a pup was anesthetized with 0.01ml kg 1 body mass tiletamine HCl and zolazepam HCl (Telazol; Fort Dodge Animal Health, Fort Dodge, Iowa) and a 110-cm catheter (5 french) was inserted into the extradural vein. The catheter served as the sole route by which materials were infused or blood was collected. Administration of fluids and collection of blood samples was facilitated by a three-way stop cock at the exposed end of the catheter. Immediately following the catheterization and every day (4 days) until the catheter was removed, each pup received 1g cefazolin sodium (Fort Dodge Animal Health, Fort Dodge, Iowa). Total body water (TBW) pool size was estimated by infusing each pup with 0.3 mci tritiated water (HTO) in 3 ml saline on the 1st day. Estimate of TBW provided an indication of the magnitude of the infused water load when compared to the pups TBW pool size. The catheterized pup was placed in a m metabolic cage with a urine collection pan underneath attached to a collection flask. Infusion Prior to intravenous infusion of sterile, deionized fresh water, a series of control blood samples were taken in the same manner as on the day of the infusion. Control samples consisted of a pre-dose sample and post-dose samples, following a mock dosing, taken at 15, 30, 60, and 120 min. On the day of the infusion, pups were dosed with water at a volume calculated to be 15% of estimated plasma volume. Plasma volume was estimated by multiplying the averaged hematocrit (Hct) measured for each animal during the control period by the estimated blood volume (13% of body mass; Thorson 1993). Warmed (32 C) water was drawn into sterile 60-ml syringes and the syringes placed on the stop cock for infusion. Infused volumes (888±51 ml) amounted to 2.1±0.1% of the pups TBW pool size (42±3 l or 44±2%). Infusion rates ranged between 32 ml min 1 to 45 ml min 1. Two minutes after the infusion, a postinfusion sample was taken and subsequent samples were obtained at 15, 30, 60, and 120 min post-infusion. Blood samples and plasma analyses All blood samples were obtained from the indwelling catheter into 20-ml syringes. Blood was transferred into pre-chilled lithium heparin and pre-chilled EDTA-treated vacutainer tubes. After 30 s gentle rocking, duplicate aliquots of whole blood were removed in capillary tubes and spun in a microcentrifuge to determine Hct (%). The remaining blood was centrifuged for 15 min (1500 g at 4 C), and plasma collected and frozen at 20 C for later analyses. All assays were conducted on commercially available radioimmunoassay (RIA) kits validated previously (Zenteno-Savin and Castellini 1998; Ortiz et al. 2000, 2001, 2002). Aldosterone (Diagnostic Products, Los Angeles, Calif.), cortisol (Diagnostic Products), atrial natriuretic peptide (ANP; Phoenix Pharmaceuticals, Belmont, Calif.), and AVP (Phoenix Pharmaceuticals) were analyzed from heparinized plasma, and plasma renin activity (PRA; Dupont-NEN, Mass.) was determined from EDTA-treated plasma. ANP and AVP were extracted from the plasma prior to being assayed as previously described (Zenteno-Savin and

3 299 Castellini 1998). All samples were run in duplicate in each assays. Hormone assays displayed intra-assay percent coefficient of variation (% CV) of between 4% and 9% and interassay percent CV of between 5% and 12%. Electrolytes (Na +,K +,Cl ), creatinine, glucose, total proteins, and blood urea-nitrogen (BUN), were analyzed from heparinized plasma and were measured on a clinical auto-analyzer (Roche Diagnostics, Somerville, N.J.). Osmolality was determined using a freezing point osmometer (Fiske, Norwood, Mass.). For the analysis of HTO, the isotope was freezetrapped from duplicate 250-ll aliquots of plasma and counted in duplicate for 1h on a scintillation-counter (Ortiz et al. 1978). Urine analyses In each study, urine volume in the collection flask was measured and a 3-ml aliquot filtered and frozen for later analyses. Urine was collected on a 24-h basis following post-catheterization (PC), control, and the post-infusion periods. Urine samples were analyzed for electrolytes (Na +,K +,Cl ), creatinine, osmolality, total proteins, and urea-nitrogen using the same techniques as with the plasma samples. Aldosterone and cortisol were extracted as previously described (Ortiz et al. 1999). ANP, AVP, and angiotensin II (AII) were extracted in a similar way to that of the plasma samples, with the exception of the volume (0.5 ml). The same commercial assays used to measure the plasma hormones were used to measure the extracted urinary hormones. For the determination of camp and cgmp (Assay Designs, Ann Arbor, Mich.) concentrations, urine samples were diluted between 1:50 and 1:200 prior to being assayed by enzymatic immunoassay. Calculations For all variables, excretion values were calculated by multiplying the urinary concentration by urine flow. TBW was calculated from isotopic dilution space as previously described in fasting NES pups (Ortiz et al. 1978) and assumed 4% overestimation. Glomerular filtration rate (GFR) was estimated by standard creatinine clearance methods. For the calculation of GFR during the PC period, the plasma creatinine (P crt ) value used was the pre-control sample. During the control and post-infusion periods, the P crt value used was the average of all the samples taken during that period. Osmotic clearance (C osm ) was calculated as: C osm ml h 1 ¼ Uosm V=P osm ; where U osm and P osm represent urinary and plasma osmolality, respectively, and V is urine flow. The same conditions used to determine the P crt value for the estimation of GFR were also applied in the determination of the P osm value used to estimate C osm at each period. C H2O was calculated as the difference between V and C osm. A negative value indicates the volume of free water reabsorbed by the collecting duct (Vander 1995). Efficiency of tubular reabsorption from the filtrate was calculated as the percentage of 1 (V/ GFR). Fractional excretion (FE) was calculated as: FE ð% Þ ¼ U ½Š V=P ½Š GFR 100%; where U [ ] and P [ ] represent urinary and plasma concentrations, respectively. Statistics Means for plasma values during the post-infusion period were compared to those during the control period by two way analysis of variance (ANOVA) adjusted for repeated measures over time. If significant group time interactions were not observed, means during the post-infusion period were compared to pre-infusion values by one way ANOVA adjusted for repeated measures. Urine values were compared by one-way ANOVA adjusted for repeated measures. Fisher s PLSD test was administered post-hoc if significance was determined. Correlations were determined by simple regression of the means and considered different at P<0.05. Means (±SE) were considered significantly different at P<0.05. All statistical analyses were made using Statview (SAS 1998). Results Plasma osmolality (Fig. 1) and PRA (Fig. 2) were significantly reduced from pre-infusion and control levels following infusion, while plasma aldosterone (percent change from pre-infusion) increased significantly (Fig. 1). Plasma electrolytes, total proteins, glucose, and Hct were not significantly altered following the infusion (Table 1). Mean plasma osmolality was correlated with mean plasma aldosterone (plasma aldosterone= plasma osmolality, r=0.808, P=0.0026). Plasma cortisol was initially reduced following infusion, but was increased above pre-infusion and control concentrations at 60 min and 120 min (Fig. 2). Following the infusion, GFR increased 36% above control (Fig. 3) and urine output increased three fold above control levels (6.3±0.9 ml h 1 and 19.8±4.3 ml hr 1 ; Fig. 4). The difference in urine volume between control and post-infusion collection periods accounted for 34±6% of the infused water. Osmotic clearance increased 46% above control; however, C H2O was not significantly altered (Fig. 4). Efficiency of tubular water reabsorption decreased from 99.87±0.01% prior to infusion to 99.72±0.03% following infusion. Urine osmolality was diluted by half (1280±87 mosmol l 1 and 625±104 mosmol l 1 ) following the infusion. However, osmolal excretion was increased by 41% (Table 2). Osmolal excretion and excreted urea both adjusted for excreted creatinine were significantly and positively correlated (Fig. 5). Mean excreted urea accounted for 52% of the increase in mean osmolal excretion, but when excretion values were adjusted for excreted creatinine, the increase in urea excretion accounted for 96% of the increase in osmolal excretion. Excreted urea exhibited a logarithmic relationship with urine output (Fig. 6). Excretion of creatinine, urea, aldosterone and ANP were increased significantly following fresh water infusion (Table 2). Excreted urea and ANP remained elevated after values were adjusted for excreted creatinine. Excreted aldosterone adjusted for excreted creatinine and FE urea exhibited a significant logarithmic relationship (FE urea = ln excreted aldosterone:creatinine; r=0.581; P=0.047). However, excreted AVP and urea were not correlated. FE urea increased 22% following infusion. However, the fractional excretion of electrolytes did not significantly change (Table 3). Discussion Previous water-loading studies in marine mammals have reported on some of the renal changes associated with the subsequent diuresis (Bradley et al. 1954; Murdaugh

4 300 Fig. 1. Mean (±SE) A plasma osmolality and B plasma aldosterone (percent change from Pre) in response to the infusion of fresh water over time. Aldosterone Pre-control=1471±453 pg ml 1 ; Preinfusion = 1521±333 pg ml 1. Asterisk indicates significantly different (P<0.05) from Pre et al. 1961). However, data on the hormonal regulation of electrolyte conservation (Bradley et al. 1954; Ridgway 1972) or saluresis (Tarasoff and Toews 1972; Hong et al. 1982) during this diuresis remains extremely limited and inconclusive (Ridgway 1972; Hong et al. 1982; Skog and Folkow 1994). The present study provides a more comprehensive description of the renal and hormonal changes associated with water-induced hypo-osmolality in a marine mammal. Although mammals living in a strictly marine habitat are not normally exposed to fresh water, water infusion (loading) studies can provide valuable insight into the renal mechanisms these animals have evolved to handle excess water and conserve electrolytes. Representative species of each order of marine mammals inhabit a strictly fresh water habitat. A comparison of kidney function between closely related species inhabiting either a fresh water or a marine environment provides an ideal opportunity to investigate the evolutionary adaptation of kidney function as mammals transitioned from land to sea. For example, a Baikal seal, a fresh water species maintained on approximately 0.5 g Na + day 1, excreted 46% of a Na + -load equivalent to 3.8-fold of its daily ration within 24 h. This Na + excretion value was similar to that for ringed seals, a marine counterpart maintained on approximately 2.3 g Na + day 1, suggesting that the fresh water-adapted seal, isolated from a marine environment for 0.5 million years, retained renal excretory

5 301 Fig. 2. Mean (±SE) A plasma renin activity (PRA) and B plasma cortisol in response to the infusion of fresh water. significantly different (P<0.05) from control functions comparable to its marine counterparts (Hong et al. 1982). The question as to whether a marineadapted mammal isolated from fresh water for approximately 22 million years exhibits a diminished renal capacity to handle excess water can thus be raised. In general, a fresh water load in terrestrial mammals induces a diuresis characterized by an increase in C H2O and excretion of the load within hours (approximately 4 h; Rosas-Arellano et al. 1992; Ota et al. 1994). In the present study, the diuresis was not associated with an increase in C H2O, but rather an increase in osmotic diuresis. Also, seals only excreted approximately 34% of the infused water after 24 h indicating a condition of volume retention. We calculated that the fresh wateradapted Baikal seal retained approximately 31% of its fresh water load (assuming that urine flow from 9 24 h post-infusion was similar to pre-infusion levels, see Hong et al. 1982). Because Hct was not significantly altered, the infused volume was most likely sequestered by the extravascular space rapidly. This difference in water handling of excess water between terrestrial and marine mammals suggests that marine-adapted mammals may be insensitive to hypotonic cues of fluid regulation and more sensitive to the osmotic cues. This idea is further corroborated by the fact that pups infused with isotonic saline exhibited a similar degree of volume retention, however, pups infused with hypertonic saline (equimolar Na + content) did not exhibit retention of

6 302 Table 1. Means (±SE) of hematocrit (Hct) and plasma data from fasting elephant seal pups (n=4) taken immediately prior to (Pre) and after (Post) the infusion of fresh water, and subsequently at 15, 30, 60, and 120 min. For the control period, a mock infusion was substituted for an actual dosing of water Pre Post Recovery Hematocrit (%) Control 56±156±155±154±155±153±3 Infusion 56±156±155±2 53±157±157±1 Na + (mm) Control 146±1 144±2 142±2 146±1 147±1 144±2 Infusion 146±1 144±1 144±1 144±1 142±1 143±4 K + (mm) Control 4.3± ± ± ±0.14.0±0.14.4±0.1 Infusion 4.3±0.14.3± ± ± ±0.14.0±0.1 Cl (mm) Control 102±1 101±1 100±1 101±1 103±1 104±1 Infusion 103±1 100±1 100±1 100±1 102±1 104±3 Creatinine (lm) Control 75.6± ± ± ± ± ±2.0 Infusion 78.7± ± ±1.2* 71.4±1.7* 73.4±2.4* 73.8±3.4 Urea (mm) Control 2.3± ± ± ± ± ±0.1 Infusion 2.5± ± ± ± ± ±0.3 Total protein (g dl 1 ) Control 6.3±0.16.1± ± ±0.16.3±0.16.2±0.1 Infusion 6.2±0.16.1±0.16.2±0.16.2±0.16.1±0.16.4±0.3 Glucose (mm) Control 8.7± ± ± ± ± ±0.3 Infusion 8.3± ± ± ± ± ±0.1 ANP (pg ml 1 ) Control 4.9± ± ± ± ± ±1.0 Infusion 7.3± ± ± ± ± ±7.1 AVP (pg ml 1 ) Control 1.7± ± ± ± ± ±0.3 Infusion 3.5± ± ± ± ± ±1.4 * Significantly different (P<0.05) from PRE either water or solutes. Collectively, these scenarios would then suggest that freely feeding seals exposed to an excess water load would also experience volume and solute retention. Although, when compared to terrestrial mammals, NES pups appear to possess a diminished capacity to excrete an excess water load, the retention of fluid may be an evolved mechanism, which has allowed these mammals to inhabit a hyperosmotic environment, and thus avoid dehydration. Water-induced diuresis is not associated with a sustained increase in GFR in humans (Kimura et al. 1986; Ota et al. 1994), dogs (Bie et al. 1984; Kompanowska- Jezierska et al. 1998), goats (Olsson et al. 1982), rats (Bouby et al. 1996), and other seals (Bradley et al. 1954; Murdaugh et al. 1961; Hong et al. 1982). However, NES pups in the present study exhibited a 36% increase in GFR. Maximum urine flow during water diuresis in seals has been shown to represent between 10% and 12% of GFR, indicating that only 88 90% (efficiency of tubular reabsorption) of filtered water gets reabsorbed following fresh water infusions (Ladd et al. 1951; Hong et al. 1982). In the present study, tubular reabsorption decreases 0.15%, however, this decrease when compared to that observed in other seals does not appear to be physiologically significant. The increase in fractional clearance of water suggests that water resorption decreases as GFR increases (Ladd et al. 1951). However, in the present study, urine flow represented only 0.3% of GFR with no change in C H2O indicating that the increase in GFR did not negatively affect resorption as previously reported in harbor seals (Ladd et al. 1951). The difference in the relationship between GFR and C H2O between the present and previous studies could be attributed to the discrepancy in mass-specific infusion volumes, which were 2-fold (Hong et al. 1982) to 15-fold (Ladd et al. 1951) greater in those studies. In terrestrial mammals, the increase in C H2O following water loading (Olsson et al. 1982; Kimura et al. 1986) can account for as much as 95% of the diuresis (Ota et al. 1994) indicating that C osm plays a lesser role in the diuresis. However, infusion of water in NES pups resulted in a 41% increase in C osm and no change in C H2O. After adjusting excreted urea and osmolal excretion by excreted creatinine, at least 96% of the increase

7 303 Fig. 3. Mean (±SE) glomerular filtration rate (GFR) in response to the infusion of fresh water during the post-catheterization (PC), control and postinfusion periods. Asterisk indicates significantly different (P<0.05) from control Fig. 4. Mean (±SE) urine flow, osmotic clearance, and free water clearance in response to the infusion of fresh water during the PC, control, and post-infusion periods Asterisk indicates significantly different (P<0.05) from control in osmolal excretion can be accounted for by the increase in excreted urea suggesting that urea excretion is almost entirely responsible for the observed increase in osmotic diuresis in NES pups. Following the infusion, BUN remained constant during the post-infusion sampling period and FE urea increased, suggesting that the administration of fresh water resulted in medullary urea washout (Mudge et al. 1974; Sizeland et al. 1995). Urea washout may behoove marine mammals during conditions of extreme ionic conservation such as prolonged fasting by potentiating the reabsorption of electrolytes. Osmotic diuresis did not significantly increase the excretion of Na +,K +,orcl and, in fact, the fractional excretion of each electrolyte displayed a tendency to decrease suggesting that the degree of tubular reabsorption of electrolytes was at least maintained if not increased. Therefore, NES pups retained electrolytes at the expense of medullary urea to maintain ionic homeostasis and to rid itself of the excess water. Although the infusion of water resulted in hypoosmolality, circulating electrolytes were not significantly diluted suggesting that the renal reabsorption of ions was extremely efficient to maintain plasma electrolyte balance.

8 304 Table 2. Mean (±SE) urinary excretion data following (24 h) the post-catheterization (PC), control, and post-infusion periods. (AII angiotensin II, AVP vasopressin, ANP atrial natriuretic peptide) PC Control Post-infusion Creatinine (mmol h 1 ) 0.38± ± ±0.04* Osmolality (mosmol h 1 ) 7.6± ± ±1.5* Na + (mmol h 1 ) 0.75± ± ±0.10 K + (mmol h 1 ) 0.45± ± ±0.08 Cl (mmol h 1 ) 0.63± ± ±0.13 Urea (mmol h 1 ) 1.9± ± ±0.4* Total proteins (mg h 1 ) 62±4 70±4 70±6 camp (nmol h 1 ) 103±31 288±67 267±63 cgmp (nmol h 1 ) 139±76 41±17 32±14 AII (ng h 1 ) 1.3± ± ±0.2 AVP (ng h 1 ) 1.5± ± ±0.5 Aldosterone (ng h 1 ) 63±9 64±10 141±65* Cortisol (lg h 1 ) 1.4± ± ±0.3 ANP (pg h 1 ) 249±18 304±24 778±245* * Significantly different (P<0.05) from control Electrolyte balance may also be achieved by compartmentalizing or quickly excreting the excess water. Water loading usually results in hemodilution as indicated by a decrease in Hct and plasma proteins (Blair- West et al. 1987; Jimenez et al. 1999). However, the lack of a change in Hct and plasma total proteins in the present study indicates the lack of hemodilution. Hemodilution could have been avoided by: (1) rapidly sequestering the excess water into the extravascular space, or (2) filtering and excreting the excess water as quickly as it was infused. The increase in urine volume following infusion of water only accounts for a third of the infused volume indicating retention of water, which would support the former possibility and discount the latter possibility. Hormonal responses to water loading in humans (i.e., Kimura et al. 1986) and dogs (i.e., Bie et al. 1984) is well documented. However, very little data exists on the hormonal responses to fresh water infusion in marine mammals (Ridgway 1972; Hong et al. 1982; Skog and Folkow 1994). Unfortunately, this information is not sufficient to ascertain the involvement of hormones in the metabolism of water and electrolytes subsequent to water loading. Water intake increases blood pressure due to an increase in blood volume (Jordan et al. 2000), which stimulates a decrease in the secretion of renin (Weir and Dzau 1999). The observed decrease in PRA following the infusion of water may have resulted from volume-induced inhibition of renin release similar to that observed in NES pups infused with isotonic saline (Ortiz et al. 2002). As components of renin-angiotensinaldosterone system (RAAS), a decrease in PRA may be associated with a decrease in aldosterone (Funder 1993; Weir and Dzau 1999). However, plasma aldosterone (as a percent change from pre-samples) and subsequent excretion increased following infusion of water despite a decrease in PRA, suggesting that aldosterone secretion may have resulted independent of the renin-angiotensin pathway. Secretion of aldosterone also occurs in response to decreased circulating Na + or increased K +, Fig. 5. Correlation between osmolal excretion and excreted urea both adjusted for excreted creatinine following the PC, control, and post-infusion periods. Each symbol represents a different pup. Regression was considered significant at P<0.05 and increased adrenocorticotropin (ACTH; Funder 1993). Infused water did not result in hyponatremic or hyperkalemic conditions, which would nullify either of these possibilities. Plasma aldosterone was elevated even when plasma cortisol was reduced, and excreted aldosterone adjusted for excreted creatinine following infusion was greater than the control (when compared by paired t-test), while excreted cortisol adjusted for creatinine displayed a tendency to decrease, all of which strongly suggest that aldosterone secretion was not ACTH-stimulated, and thus, not a stress response. However, aldosterone secretion has been shown to respond to a decrease in plasma osmolality in vitro (Schneider et al. 1985) suggesting that the stimulation of aldosterone in the present study may have been induced by the decrease in plasma osmolality, which is supported by the negative correlation between the two variables. Alternatively, the non-significant decrease in Na + may have partially contributed to the increase in circulating aldosterone suggesting that these animals are hypersensitive to a decrease in circulating Na +, which would be advantageous in a fasting condition in order to help maintain electrolyte homeostasis. Interestingly, isotonic saline-induced plasma volume expansion and hypertonic saline-induced hypernatremia failed to reduce circulating aldosterone in the same time frame in fasting NES pups (Ortiz et al. 2002) suggesting that the adrenal glomerulosa may be more sensitive to stimulatory than inhibitory cues. If so, this idea would emphasize the importance of aldosterone in conserving Na + in these animals. Also, contrasting excretions of aldosterone and cortisol suggest that the adrenal glomerulosa and fasciculata were mediated independently in response to infused water in NES pups.

9 305 Fig. 6. Correlation between excreted urea and urine output following the PC, control, and post-infusion periods. Each symbol represents a different pup. Regression was considered significant at P<0.05 Following the infusion of water, BUN was not altered suggesting that urea was not involved in the osmotic stimulation of aldosterone secretion. Also, the increase in FE urea post-infusion indicates a decrease in tubular reabsorption of urea. The positive correlation between excreted aldosterone and FE urea suggests that aldosterone may play a role in urea excretion. Although this relationship is not causal, the possibility that aldosterone may help regulate renal urea handling in these animals warrants further investigation since glucocorticoids have been implicated in such a role (Klein et al. 1997). Increased blood pressure (via increased volume) is a potent stimulant of ANP (Lang et al. 1985; Synhorst and Gutkowska 1988). However, in the present study, plasma ANP was not significantly altered although each pup exhibited a burst secretion of ANP at different times during the blood sampling regime, which may have resulted in the cumulative increase in excreted ANP following the infusion. Also, ANP does not appear to be a significant natriuretic factor in NES pups as previously demonstrated in humans (Heer et al. 1993; Drummer et al. 1996) and dogs (Hildebrandt et al. 1992). The lack of a correlation between excreted ANP and urine flow would also suggest that increased diuresis occurred independent of ANP under conditions of the present study, despite the fact that both excreted ANP and urine flow were increased following the infusion of water. In summary, infusion of fresh water in NES pups resulted in an osmotic diuresis associated with an increase in GFR, but not an increase in C H2O, which appears to be a unique response since water-induced diuresis in other mammals is primarily attributed to an increase in free water (Olsson et al. 1982; Kimura et al. 1986; Ota et al. 1994). Therefore, resorption of water remains elevated despite an excess in body water and the resorption is independent of elevated filtration, which is Table 3. Mean (±SE) fractional excretion (FE; %) of electrolytes and urea following (24 h) the PC, control, and post-infusion periods FE PC Control Post-Infusion Na ± ± ±0.01 K ± ± ±0.17 Cl 0.12± ± ±0.01 Urea 17.5± ± ±1.7* * Significantly different (P<0.05) from control not the case in other water-loaded seals (Ladd et al. 1951; Hong et al. 1982). Renal medullary urea washout appears to be the sole contributing factor for the increase in osmotic diuresis following infusion of water. Urea washout allows the kidney to eliminate excess water gradually while sustaining, if not increasing, the reabsorption of electrolytes, which is important for maintaining ionic homeostasis during periods of extreme conservation such as prolonged fasting. After 24 h, pups retained approximately 66% of the infused water, which was probably sequestered by the extravascular space since Hct and concentrations of plasma total protein, glucose, and electrolytes were not reduced, indicating the lack of hemodilution. Infusion of water resulted in a decrease in PRA and an increase in excreted ANP. Although excreted ANP increased, it did not induce natriuresis in NES pups. Despite the decrease in PRA and the lack of a change in plasma Na + and K +, plasma and excreted aldosterone increased following infusion of water. Water-induced hypo-osmolality appears to be a significant stimulant of aldosterone secretion as described previously in vitro (Schneider et al. 1985). Alternatively, aldosterone secretion may be hypersensitive to small decreases in circulating Na +, which would behoove seals during periods of Na + conservation. Excreted aldosterone and urea were correlated suggesting

10 306 that aldosterone may regulate urea excretion during hypo-osmotic conditions in NES pups. Renal-urea handling appears to be a significant method of regulating ionic homeostasis during conditions of electrolyte conservation in fasting NES pups. The difference in renal handling of excess water between terrestrial mammals and NES pups suggests that the evolution of kidney function in these seals has afforded them an opportunity to inhabit a marine environment. Acknowledgements This research was conducted in partial fulfillment of the requirements for the doctorate degree in biology for RMO. We would like to thank A. Bigelow, A. Galarza, G. Guron, J. Lauzze, B. Litz, D. Noren, A. Ramirez, J. Ramirez, and T. Sierra for their assistance throughout the study. We also thank the G. Strachan and the rangers of An o Nuevo State Park (CA) for their assistance. This research was supported by M.A.R.C. grant GM (CLO), NASA grants and (CEW), NASA G.S.R.P. 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