INTRODUCTION

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NORTHERN ELEPHANT SEAL (Mirounga angustirostris) (NES) pups naturally endure postweaning fasts of 8-12 wk without any apparent deleterious effects on water and electrolyte balance (26, 29). During this fasting period, water balance in NES pups is maintained by the production of metabolically derived water from the oxidation of large fat stores (26). During the transition between nursing and fasting, the kidneys must switch between a state of almost constant excretion to a condition of chronic conservation of water and electrolytes to maintain fluid and electrolyte homeostasis.

Under normal conditions, water and electrolyte homeostasis is rigidly maintained through the mediation of a number of hormonal systems. Arginine vasopressin (AVP) stimulates the resorption of water from the collecting duct of the kidney (18, 42, 44). In pharmacological doses, AVP may act as a natriuretic factor (6, 43) and, in concert with urea, may stimulate an increase in glomerular filtration rate (GFR; see Refs. 2 and 3). Release of AVP is inhibited by plasma volume expansion (PVE) or stimulated by increased plasma osmolality (hyperosmolality; see Refs. 25 and 47). The renin-angiotensin-aldosterone system (RAAS) is the primary regulatory pathway by which renal Na⁺ resorption is achieved. Secretion of renin is inhibited by PVE and by an increase in plasma Na⁺ (11, 15). Renin may also be inhibited by atrial natriuretic peptide (ANP), which is stimulated by an increase in atrial pressure, or PVE (23).

In mammals, PVE and hyperosmolality induce a number of renal and hormonal alterations. PVE results in an increased diuresis and natriuresis associated with reduced levels of AVP, plasma renin activity (PRA) or ANG II, and aldosterone (10, 34, 36, 39). During PVE in most species, GFR usually remains constant (21, 34). After elevated dietary Na⁺ intake or hypertonic saline infusion, plasma AVP increases (7, 37, 47), whereas ANG II or PRA and aldosterone decrease (13, 20, 37). In humans, hyperosmolality is generally associated with a lack of a change in GFR and unchanged (12) or decreased (16) urine flow. However, in dogs, GFR and urine flow have been reported to increase only acutely and moderately (7, 37). Unfortunately, data are limited and inconclusive on the renal and hormonal responses to PVE and hyperosmolality in marine-adapted mammals (for review, see Ref. 28), which provide an ideal model for studies on the responsiveness and adaptation of mammalian kidneys to extreme conditions.

A decrease in urine output during the fast, accompanied by a reduction in excreted urea and an increase in urine osmolality, suggests that AVP may be elevated during this period. Previous studies in seals suggest that AVP has an antidiuretic role (8, 14, 33, 40). However, the antidiuretic function of AVP during the fast in NES pups remains inconclusive (29, 30). Aldosterone and PRA are highly correlated during the fast, with each exhibiting a peak in concentrations after ~5 wk postweaning, suggesting that RAAS is involved in the conservation of water and electrolytes and that the sensitivity may change over the course of the fast (30). The role of ANP in Na⁺ excretion in any marine mammal has yet to be examined.

Reduced GFR and urinary water output and increased resorption of electrolytes, which appears to be attributed to the increase in RAAS, are some of the physiological mechanisms NES pups employ to conserve water and electrolytes during the fast. However, the excretory capabilities of pups during their postweaning fast have yet to be examined. Therefore, the present study was conducted to provide an understanding of renal responses and hormonal regulation of a marine-adapted mammal when subjected to PVE and hyperosmolality. The objectives of the present study were 1) to quantify changes in renal function associated with PVE and hyperosmolality, 2) to quantify the hormonal responses to PVE and hyperosmolality, and 3) to provide insight to the adaptation of kidney function in seals as they transition between a state of chronic conservation to acute excretion of water and electrolytes. Because body water must be tightly regulated during fasting, we hypothesized that pups will excrete Na⁺ from an isotonic saline load quicker than an equimolar hypertonic saline load because more water is available for excretion with an isotonic load than with an hypertonic load. Insight into the renal excretory mechanisms in fasting NES pups will further our knowledge of the ability of these animals to exist in two diverse environments and conditions (fasting on land and feeding at sea).

	METHODS

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Animals. Fifteen pups were transported from Año Nuevo State Park (~30 km north of Santa Cruz, CA) to Long Marine Laboratory, University of California, Santa Cruz. Eight pups (100 ± 24 kg; 5 males, 3 females) were used for an isotonic saline infusion study and seven (109 ± 15 kg; 4 males, 3 females) for an hypertonic saline infusion study. Animals were brought to the laboratory one or two at a time over the course of 9 wk during their postweaning fast. Upon arrival at the marine laboratory, pups were weighed using a hanging-load cell and placed in a sand pit overnight. The next morning, a pup was sedated with 0.01 ml/kg tiletamine hydrochloride and zolazepam hydrochloride (Telazol; Fort Dodge Animal Health, Fort Dodge, IA), and a 110-cm catheter (5 french) was inserted in the extradural spinal vein. After the catheterization procedure, pups were allowed to recover for ~22 h before the collection of control data. The catheter served as the sole route by which materials were infused or blood was collected. A three-way stop cock at the exposed end of the catheter facilitated the dosing and flushing of materials into the pups and the collection of blood samples.

Isotonic saline infusion. On the day before infusion of sterile, isotonic saline (0.9%; Fisher Scientific), a series of control blood samples was taken in the same manner as on the day of the infusion. Control samples consisted of pre- and postdose samples of a sham dosing followed by 15-, 30-, 45-, 60-, 90-, and 120-min and 24-h samples. The 24-h sample also served as the preinfusion sample for the isotonic saline infusion experiment that was initiated at that time. On the day of the isotonic saline infusion, pups were infused with saline at a volume calculated to be ~33% 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; see Ref. 45). Warmed (32°C), sterile saline was drawn into sterile 60-ml syringes, and the syringes were placed on the stop cock for infusion. After the infusion (2 min), a postsample was taken, and subsequent samples were obtained at 15, 30, 45, 60, 90, and 120 min and 24 h postinfusion. Infusion time ranged between 40 and 75 min (12-15 ml/min), depending on the infusion volume. The mean ± SE Na⁺ load was 319 ± 25 mmol.

Hypertonic saline infusion. As with the isotonic saline study, animals in the hypertonic saline group served as their own control using the same blood sampling regime for control and postinfusion periods. Warmed (32°C) sterile saline (16.7%) was drawn in sterile 60-ml syringes, and the syringes were placed on the stop cock for infusion. Animals were infused with 1 ml/kg body mass at a rate of 4 ml/min (28 ± 1 min). The mean ± SE Na⁺ load was 310 ± 15.

Blood samples and plasma analyses. All blood samples were obtained from the indwelling catheter into 20-ml syringes. Before the collection of each blood sample, a 3-ml sample was drawn in a 20-ml syringe with 10 ml of sterile isotonic saline to clear the catheter line of any residual blood that could potentially contaminate the samples drawn. Blood was transferred into one prechilled lithium heparin and one prechilled EDTA-treated tube. After 30 s of 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 (1,500 g at 4°C), and plasma was collected and frozen at 20°C for later analyses.

Urine analyses. In each study, urine volume in the collection flask was measured and a 3-ml aliquot was filtered and frozen for later analyses. For the isotonic saline study, urine was collected on a 24-h basis after postcatheterization, control, and the postinfusion periods. Urine collections for the hypertonic saline study were taken on a 24-h basis for the postcatheterization and control periods. However, the following four collections were obtained postinfusion: 1) 30-90 min, 2) 120-180 min, 3) 320-450 min, and 4) 14.5-16.5 h. A urine sample was collected for each animal at each of the four postinfusion periods. A cumulative 24-h postinfusion value for each parameter measured was then calculated from the four collection periods. Therefore, the hypertonic saline study consisted of seven urinary excretion values (postcatheterization, control, 1 postinfusion, 2 postinfusion, 3 postinfusion, 4 postinfusion, and cumulative 24 h postinfusion).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Representative estimations of the percentage of cross-reactivity between urinary hormones from northern elephant seals (NES) and the commercial antibodies. A: example of an urinary peptide hormone. B: example of an urinary steroid. B/Bo, bound-to-free ratio.

Calculations. For all variables, excretion values were calculated by multiplying the urinary concentration by urine flow. GFR was estimated by creatinine clearance. For the calculation of GFR during both postcatheterization periods, the plasma creatinine (P_crt) value used was from the precontrol sample. During the control periods, the P_crt value used was the average of all the samples taken during that period. The same was done for the postinfusion period of the isotonic saline study. However, because multiple urine collections were taken during the hypertonic saline study, the P_crt value (or the mean of the values) used was that corresponding with the period of urine collection. When a plasma sample was not taken during a urine collection period, the mean of the most recent and the 24-h postinfusion sample was used.

Statistics. Means for plasma values during the postinfusion period were compared with those during the control period by two-way ANOVA adjusted for repeated measures over time. If significant group times time interactions were not observed, means during the postinfusion period were compared with preinfusion values by one-way ANOVA adjusted for repeated measures. Urine values were compared by one-way ANOVA adjusted for repeated measures. Means for the control period for isotonic saline and hypertonic saline were compared by two-way ANOVA adjusted for repeated measures over time to determine if any group effects existed. Fisher's protected least significant difference test was administered post hoc if significance was determined. Correlations were determined by simple regression of the means and were considered different at P < 0.05. Means ± SE were considered significantly different at P < 0.05. All statistical analyses were made using Statview (38).

	RESULTS

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Among all the parameters measured, a significant group effect during the control period existed for only Hct, plasma ANP, and excreted cAMP. Changes in Hct were reported as the percent change from baseline to correct for the difference in Hct (isotonic saline, 52 ± 1% and hypertonic saline, 58 ± 1%; P < 0.01) between the two groups.

Isotonic saline plasma data. After infusion, Hct and total proteins decreased significantly (Fig. 2). Osmolality (Fig. 2), Na⁺, and creatinine (Table 1) were not altered. BUN (3.1 ± 0.4 and 2.8 ± 0.4 mM) and K⁺ (4.1 ± 0.1 and 4.2 ± 0.2 mM) also did not change during the control and infusion periods, respectively. After infusion, AVP decreased significantly and remained reduced (Fig. 3). Cortisol and PRA also were reduced postinfusion and remained so after 24 h (Fig. 3). Aldosterone (895 ± 109 and 862 ± 94 pg/ml) and ANP (11.9 ± 3.3 and 9.3 ± 2.2 pg/ml) were not altered during the control and infusion periods, respectively. However, aldosterone was reduced 24 h after infusion (427 ± 62 pg/ml) compared with preinfusion concentrations by paired t-test.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Mean ± SE hematocrit (A), plasma total proteins (B), and plasma osmolality (C) in response to the infusion of either isotonic (0.9%; n = 8) or hypertonic (16.7%; n = 7) saline. Significantly different (P < 0.05) from control.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Mean ± SE plasma arginine vasopressin (AVP; A), plasma renin activity (PRA; B), and plasma cortisol (C) in response to infusion of either isotonic or hypertonic saline. Data taken immediately before (pre) and after (post) infusion. Significantly different (P < 0.05) from control. *Significantly different (P < 0.05) from pre.

Hypertonic saline plasma data. After infusion, osmolality, Na⁺, and Cl increased significantly, and Hct, creatinine, and total proteins decreased significantly (Fig. 2 and Table 1). K⁺ was reduced immediately after infusion (3.7 ± 0.2 mM), remained reduced after the first 15 min postinfusion (3.8 ± 0.1 mM), but returned to the preinfusion level (4.2 ± 0.1 mM) after 30 min. BUN did not change during the control (2.4 ± 0.1 mM) and infusion (2.5 ± 0.2 mM) periods. After infusion, AVP and cortisol increased significantly, and PRA decreased significantly (Fig. 3). Aldosterone (602 ± 120 and 669 ± 140 pg/ml) and ANP (3.6 ± 1.1 and 2.7 ± 0.2 pg/ml) were not altered during the control and infusion periods, respectively. However, aldosterone was reduced 24 h after infusion (366 ± 59 pg/ml) compared with preinfusion concentrations by paired t-test.

Isotonic saline excretion data. The increase in urine volume after the infusion of isotonic saline accounted for only 30 ± 5% of the infused volume. Correcting for control excretion values, pups excreted 19 ± 4 and 17 ± 5% of infused Na⁺ and Cl, respectively. Urine volume increased threefold after infusion compared with control (266 ± 29 and 816 ± 125 ml/day). C_osm was elevated postinfusion; however, GFR and C_H2O were not altered (Figs. 4 and 5). Excretion, excretion adjusted for excreted creatinine, and FE of Na⁺ and Cl increased after infusion (Tables 2 and 3). FE of K⁺ (8.5 ± 1.6 and 9.1 ± 1.2%) and urea FE (FE_urea; 37.6 ± 3.2 and 43.6 ± 2.9%) were not different between control and postinfusion periods, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Mean ± SE glomerular filtration rate (GFR) during postcatheterization (PC), control, and postinfusion (PI) periods of isotonic (0.9%; n = 8) and hypertonic (16.7%; n = 7) studies. Urine samples were collected at intervals between 1) 30 and 90 min, 2) 120 and 180 min, 3) 320 and 450 min, and 4) 14.5 and 16.5 h PI of hypertonic saline. Cumulative, 24-h postinfusion value was calculated from the 4 postinfusion samples. *Significantly different (P < 0.05) from control.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Mean ± SE urine flow (UF; A), osmotic clearance (COsm; B), and free water clearance (CH2O; C) during the PC, control, and PI periods of the isotonic (0.9%; n = 8) and hypertonic (16.7%; n = 7) studies. See Fig. 4 legend for details. *Significantly different (P < 0.05) from control.

Hypertonic saline excretion data. Urine volume increased fourfold 24 h after infusion compared with control (218 ± 47 and 883 ± 85 ml/day). GFR, urine flow rate, and C_osm increased and C_H2O decreased almost immediately (30-90 postinfusion) and remained elevated or reduced for the remainder of the collection periods (Figs. 4 and 5). When Na⁺ and Cl excretion postinfusion was corrected for control excretion levels, the percentage of Na⁺ and Cl infused that was excreted equaled 97 ± 4 and 94 ± 3%, respectively. Creatinine excretion increased postinfusion (Table 4). After infusion, osmotic and electrolyte excretion increased and remained elevated, as well as when excretion was adjusted for excreted creatinine (Table 4). FE of Na⁺ and Cl increased at 120-180 min postinfusion and remained elevated, whereas FE_urea was increased at periods 120-180 min postinfusion and at 320-450 min postinfusion compared with control (Table 3). Excretion of all measured hormones was increased at 30-90 min postinfusion and remained elevated (Table 5). AVP, aldosterone, and cortisol excretion remained elevated when excretion was adjusted for excreted creatinine (Table 5).

Correlated excretion data. Means from both isotonic saline and hypertonic saline studies for excreted AVP were significantly and positively correlated with osmotic excretion and significantly and negatively correlated with C_H2O (Fig. 6). Means of excreted cAMP for isotonic saline were significantly greater than those for hypertonic saline and therefore were not included in the regression between excreted AVP and excreted cAMP (Fig. 6). Means of excreted aldosterone for hypertonic saline were positively and significantly correlated with both excreted ANG II (excreted aldosterone = 9 + 71 excreted ANG II; R = 0.885; P < 0.002) and excreted cortisol (excreted aldosterone = 42 + 6 excreted cortisol; R = 0.979; P < 0.0001). Means of FE_urea for hypertonic saline were positively and significantly correlated with both excreted aldosterone (FE_urea = 20 + 0.1 excreted aldosterone; R = 0.702; P < 0.01) and excreted cortisol (FE_urea = 22 + 0.6 excreted cortisol; R = 0.794; P < 0.01). No relationships between excreted AVP and urea were observed.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Correlations between the means ± SE of excreted AVP and excreted cAMP (A), free CH2O (B), and osmotic excretion (C) from both the isotonic and hypertonic studies. Regression was considered significant at P < 0.05.

	DISCUSSION

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In general, the renal responses to PVE and hyperosmolality in humans and other terrestrial mammals have been well identified. Unfortunately, such is not the case for marine-adapted mammals. A better understanding of osmoregulation in these mammals could provide valuable insight on the evolution of renal function in mammals as they transitioned from an Na⁺-depleted, terrestrial habitat to a salt-rich, marine environment. Pups in both the isotonic saline and hypertonic saline studies received equivalent mass-specific amounts of Na⁺, with the underlying difference being the volume of water associated with this Na⁺ load. The stark contrasts in the renal and hormonal responses observed between isotonic saline and hypertonic saline suggest that the renal handling of Na⁺ depends on the volume of water associated with the salt load. Infusion of isotonic saline induced a condition of water and electrolyte retention. However, pups infused with hypertonic saline excreted excess Na⁺ within 24 h, suggesting that these marine mammals are highly sensitive to excess Na⁺, although they have adapted well to living in an Na⁺-rich environment.

The lack of significant differences (with the exceptions of Hct, plasma ANP, and excreted cAMP) in renal and hormonal parameters (i.e., GFR, urine flow, AVP, etc.) between the control periods of the isotonic saline and hypertonic saline groups reveals that the two groups of animals were in essentially the same state of renal development. However, pronounced differences in response to the infusion of equimolar quantities of Na⁺ as either an isotonic or hypertonic solution were observed (Table 6). The infusion of hypertonic saline induced a diuresis similar in magnitude to that induced by the infusion of isotonic saline. Although the two stimuli resulted in similar ends, the means by which they were achieved were mechanistically dissimilar. Hypertonic saline resulted in a twofold increase in GFR and C_osm and an almost fivefold decrease in C_H2O compared with isotonic saline postinfusion. Pups infused with hypertonic saline excreted 97 ± 4% of infused Na⁺ and 94 ± 3% of infused Cl within the first 24 h postinfusion. However, pups infused with isotonic saline only excreted 19 ± 4 and 17 ± 5% of Na⁺ and Cl, respectively, and 30 ± 5% of water within the first 24 h. Although infusion of isotonic saline resulted in retention of water and electrolytes, infusion of hypertonic saline did not induce retention of either electrolytes or water. In contrast, dogs switched to a high-salt diet exhibited a decrease in urine output in the first 24 h along with retention of Na⁺ and water (20). Fasting pups appear to retain Na⁺ and Cl when sufficient water is made available for subsequent and delayed excretion. The retained Na⁺ is probably stored interstitially as previously suggested (12), since plasma Na⁺ was not elevated. Retention of Na⁺ and water may closely resemble a condition comparable to when the animals are naturally feeding. Thus retention of water and electrolytes may be a mechanism by which marine-adapted animals avoid dehydration in a hyperosmotic environment.

Hypertonic saline resulted in an osmotic diuresis associated with an increase in GFR, despite a decrease in C_H2O that was comparable in magnitude to the increase in C_osm. Before the infusion of hypertonic saline, urinary excretion of water accounted for ~0.3% of filtration. After infusion, this value was only increased to 0.5%, suggesting that the efficiency in renal water resorption was only reduced by 0.2% in spite of the nearly fourfold increase in urine flow. To achieve an efficiency of renal water resorption after the infusion comparable to that before infusion, C_H2O had to be reduced in magnitude similar to that of the increase in C_osm. The net result was that the water required to maintain fluid homeostasis and support the increased filtration rate and subsequent natriuresis was recycled at nearly the same efficiency as before the infusion. Although the increase in urine output was statistically significant, this increase only accounted for ~2% of the pups' total body water pool (27), suggesting that the net loss of water to accomplish this diuresis was not physiologically significant. Therefore, seal pups can excrete excess Na⁺ rapidly (within 24 h) and efficiently without retention and at the expense of a small amount (2%) of body water, which does not appear to negatively impact the hydration state of the animal. Dogs provided with a high-Na⁺ diet that was more than two times the mass-specific salt load than that infused in the present study decreased urine volume and retained water and Na⁺ after the 1st day of treatment (20), indicating a stark contrast in renal handling of excess Na⁺ between a terrestrial and marine mammal. This contrast in renal handling of Na⁺ may reflect the differences in the adaptation to the environments in which terrestrial and marine mammals have evolved.

Increasing GFR reduces the transit time of Na⁺ in the filtrate through the proximal tubule and thus reduces the resorption of Na⁺. A number of factors such as blood pressure, AVP, and cortisol (stress) may have contributed to the observed GFR-induced natriuresis after the infusion of hypertonic saline. Although not quantified in the present study, an increase in blood pressure may have led to the observed elevation in GFR and produced a pressure natriuresis. Unfortunately, the measurement of blood pressure was not technically feasible in the present study; however, an elevation in mean arterial pressure (MAP) after Na⁺ loading in dogs has been observed (i.e., Ref. 7). If the increase in GFR is directly related to an elevation in MAP in the present study, then this increase in pressure was most likely induced by hypernatremia and not PVE. Both isotonic saline and hypertonic saline resulted in PVE and natriuresis, but the natriuresis was more pronounced in hypertonic saline, and only hypertonic saline induced an increase in GFR and a state of hypernatremia. Therefore, the hypertonic saline-induced natriuresis was the consequence of increased filtration likely associated with a pressure natriuresis. An increase in GFR in response to an Na⁺ insult is not common in terrestrial mammals (12). The mechanisms involved with the isotonic saline-induced natriuresis are not as evident and warrant further investigation.

AVP release is inhibited by an increase in baroreceptor stimulation, which could reflect an increase in blood pressure, and is stimulated by an increase in plasma osmolality (41, 48). The volume of isotonic saline infused was sufficient to produce PVE, as indicated by reductions in Hct and total proteins, which were sustained for at least 24 h. PVE was associated with a sustained reduction in plasma AVP without a change in plasma osmolality, suggesting that AVP in these animals responds to volume expansion and most likely increased blood pressure, similar to terrestrial mammals (46, 47). Pups infused with hypertonic saline exhibited a sustained increase in plasma osmolality and plasma AVP, suggesting an osmoreceptor-mediated response to increase AVP as in other mammals (41, 47, 48). The positive correlations between mean plasma AVP and plasma osmolality and Hct further corroborate the presence of baro- and osmoreceptor-mediated mechanisms of AVP modulation in NES pups. Also, in pharmacological doses, AVP has been shown to produce natriuresis (43), suggesting that AVP may have contributed to the natriuresis observed after the infusion of hypertonic saline.

Resorption of free water from the collecting duct is primarily mediated by the AVP-cAMP cascade in mammals (18, 19, 42). After the infusion of hypertonic saline, the increase in excreted AVP was significantly and positively correlated with an increase in excreted cAMP and osmotic excretion and significantly and negatively correlated with C_H2O (or increased free water resorption), providing the most conclusive data of an antidiuretic function of AVP in any marine mammal. The resorption of free water from renal collecting ducts in NES pups is most likely mediated via an AVP-cAMP mechanism as in terrestrial mammals (18, 42, 44). Therefore, the maintenance of relatively low and unchanged AVP concentrations during the fast observed previously (30) suggests that the tubular resorption of water in fasting NES pups is highly sensitive to AVP under normal conditions.

Both isotonic saline and hypertonic saline also resulted in an increase in FE of Na⁺ and Cl, with hypertonic saline exhibiting threefold greater excretions of both electrolytes. However, the natriuresis observed after both stimuli was not associated with an increase in excreted ANP (or ANP adjusted for excreted creatinine in the case of hypertonic saline) and/or reduced ANG II and aldosterone. Although Na⁺ loading generally increases ANP (i.e., Ref. 7), neither stimuli resulted in an increase in circulating or excreted ANP or excreted cGMP. The suppression of ANG II has been identified as a means by which natriuresis is achieved after Na⁺ loading (37, 39). However, excreted ANG II and excreted ANG II adjusted for excreted creatinine were not reduced despite sustained decreases in PRA, suggesting dynamic alterations in the renin-catalyzed conversion of angiotensinogen to ANG I in the current model. Therefore, the observed natriuresis in hypertonic saline was likely the result of a sustained increase in GFR and was independent of any hormonal cascade, which is a unique response among mammals. The moderate natriuresis induced after isotonic saline was also independent of increased ANP or reduced ANG II.

The adrenal steroids, aldosterone and cortisol, are altered in response to PVE and elevated dietary Na⁺ intake in terrestrial mammals. Infusion of isotonic saline (10, 39) and elevated dietary Na⁺ intake (9, 12, 13, 20) reduce circulating aldosterone, with the decrease occurring within 30 and 180 min in some cases (9, 10, 39). Plasma and excreted cortisol remained unaffected after the infusion of isotonic saline (10), whereas plasma cortisol decreased after increased dietary Na⁺ intake (13). In both isotonic saline and hypertonic saline, plasma aldosterone was not reduced until 24 h postinfusion, indicating the latency in the adrenal-glomerulosa response to PVE and hypernatremia in fasting pups. However, plasma cortisol decreased immediately after isotonic saline-induced PVE and increased immediately after hyperosmolality, indicating an increased responsiveness of the adrenal fasciculata. Regions of the adrenal gland responded differentially to PVE and hyperosmolality in the present study, suggesting independent mechanisms of regulation in response to alterations in plasma volume and osmolality in fasting pups.

Ironically, hypertonic saline resulted in an increase in excreted aldosterone adjusted for excreted creatinine, suggesting an increase in adrenal secretion. The sustained increase in plasma cortisol and the significant and positive correlation between the means of excreted aldosterone and excreted cortisol suggest that the infusion of hypertonic saline may have induced a neuroendocrine stress response (1), which may have masked any signal to inhibit aldosterone secretion. If excessive Na⁺ intake can be interpreted as an environmental stressor to these animals, then it is highly unlikely that these marine mammals voluntarily drink sea water, and large amounts of incidentally ingested salts would be effectively and efficiently excreted in short order, as discussed previously. Therefore, hypernatremia resulting from the infusion of hypertonic saline may have induced a stress response, which may have played a pivotal role in potentiating the subsequent natriuresis via an increase in blood pressure and thus GFR (4).

In humans and rats, AVP stimulates urea transporters (UTs) to enhance urea resorption into the renal medulla and thus decreases FE_urea (3, 17). Glucocorticoids have been shown to downregulate vasopressin-regulated UTs, resulting in an increase in FE_urea (17). In aldosterone-induced volume-expanded rats fed a high-salt diet, urea excretion increased associated with a decrease in UT-A1 and UT-A3, suggesting a possible correlation between hypernatremia and urea excretion (49). For 3-6 h after the infusion of hypertonic saline, FE_urea was elevated in the presence of increased excreted AVP and cortisol, suggesting that cortisol may have inhibited urea resorption and thus increased FE_urea. However, after isotonic saline, FE_urea was not significantly altered, and neither was excreted AVP nor cortisol altered. The potential interactions between AVP and adrenal steroids in renal urea handling in marine mammals warrants further investigation.

In summary, the renal responses to PVE and hyperosmolality in fasting NES pups appear to be different from those typically associated with such stimuli in humans and other terrestrial mammals. Both PVE and hyperosmolality induced an osmotic diuresis and natriuresis. Under natural fasting conditions, the conservation of Na⁺ appears to be the result of a reduced GFR and an increase in RAAS (30). However, natriuresis in response to hyperosmolality was achieved by an immediate and sustained elevation in GFR and independent of an increase in ANP and decreases in ANG II and aldosterone. Infusion of isotonic saline resulted in the retention of water and electrolytes; however, excessive Na⁺ is excreted rapidly and efficiently without retention and at the expense of a small amount of body water. The present study provides the most conclusive evidence that the secretion of AVP is regulated by both volume (pressure) and osmolality and that AVP has an antidiuretic role in marine mammals, which has been a point of contention for decades (22, 29, 40). The variable responses of adrenal steroids to both isotonic and hypertonic saline infusions indicate that the adrenal gland in fasting NES pups, and possibly all marine mammals, possesses dynamic, osmoregulatory functions, and suggest that the gland is differentially modulated, possibly by neural factors undefined by the present study. Elephant seal pups possess a refined kidney that is capable of exquisite regulation of Na⁺ during conditions of both chronic conservation during the fast and acute excretion during excessive Na⁺ intake.

Perspectives