INTRODUCTION

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RENAL FUNCTION HAS BEEN reported to be altered following return to Earth from spaceflight (6, 12, 14, 18, 22). In humans, urine flow rate is decreased or not changed, and concentrating ability is reduced (8-10, 12-14, 18-20). These changes are postulated to be related to a decrease in sensitivity to vasopressin and a decrease in the concentration gradient in the renal medulla. Reports of rats following spaceflight suggest that urine flow rate is not changed, but the renal handling of solutes is altered (6, 7, 18, 19). Postflight, there is an increase in the percent of filtered sodium and potassium loads excreted; animals in one of these studies, however, had a period of limited access to water and were not evaluated over the first 48 h after flight (6, 7, 19).

Assessment of renal function in humans during spaceflight has been compromised by a variety of factors, including motion sickness, subject compliance, and the use of countermeasures such as exercise (9, 13, 15). At the onset of spaceflight, there is a cephalic shift of fluids in humans (31). This redistribution is purported to induce a diuresis; in-flight measurements, however, have not demonstrated an increase in urine output (8, 10, 12, 14). Furthermore, the decrease in total body water appears to be the result of a reduction in intake (14, 31). In response to a saline infusion during spaceflight, excretion of the fluid load was delayed and attenuated (21). These changes would affect the ability to regulate fluid and electrolyte homeostasis in response to redistribution of body fluids on return to Earth.

The rat hindlimb suspension model has been used to study many of the changes associated with spaceflight, including fluid and electrolyte homeostasis (3, 4, 28, 32). Tucker and Mendonca (26, 27) have evaluated the effects of this model on renal function, assessing the effects of reloading (simulating return to Earth) on animals after 14 and 30 days of suspension. Over the first 24 h of reloading, no change in the urine flow rate occurs despite an increase in glomerular filtration rate.

The present study was undertaken to 1) evaluate renal responses in rats following spaceflight and 2) identify specifically the changes in handling of solutes and the contributions of solutes to observed changes in urine flow rate. We hypothesized that, following spaceflight, there would be an antidiuresis in the rats primarily due to reduction in osmolal clearance. Animals were evaluated for 14 days following a 14-day spaceflight.

	METHODS

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Studies were conducted on specific pathogen-free Sprague-Dawley-derived rats (Harlan Laboratories, Prattsville, AL) received 8 days before launch when the animals were 30 days of age and weighed ~100 g. All animal protocols were approved by the appropriate National Aeronautics and Space Administration (NASA) Animal Care Committees and adhere to the National Institutes of Health guidelines for the humane care and use of animals.

Experimental procedures. Animals were arranged by descending weight and randomly assigned to three groups so that each group had the same initial mean body weight. The groups were flight, flight control, and vivarium control. Flight animals were individually housed and flown in a Research Animal Holding Facility, and flight control animals were individually housed in flight-simulation cages (~4 in. high by 4 in. wide by 12 in. deep; the height of these cages does not allow the animals to rear on their hind legs). All animals were maintained on the same 12:12-h light-dark cycle to which they were entrained before selection for the study. Both groups were fed food bars [Teklad (Madison, WI) NASA Experimental Rodent Diet no. TD 88179 extruded into food bars, dipped in 15% sorbate to retard mold growth, radiation sterilized, sealed in polyethylene bags, and stored at 4°C until use] and provided water ad libitum. Vivarium control animals were individually housed two animals per cage in standard shoe box cages (8 in. high by 10.5 in. wide by 19 in. deep) with a Plexiglas divider running parallel to the long axis of the cage to separate the two rats; vivarium controls were fed pieces of flight food bars (Teklad) and provided water ad libitum. Flight animals were flown aboard the Space Shuttle Columbia (STS-58) for 14 days. Initially there were 12 animals per group. On landing of the shuttle (R + 0), the animals underwent an examination by a veterinarian to assess their health and were turned over to the investigator within 6 h of landing. One-half of the animals from each group were then anesthetized with Metofane, and blood samples were taken. The remaining six animals in each group were placed in metabolic cages for the next 14 days. One vivarium control rat was removed from the study because it failed to gain weight when transitioned to the metabolic cage. Animals were provided a powdered diet (Teklad diet no. TD 88179) and water ad libitum. The animals were maintained on a 12:12-h light-dark cycle [lights on at 5:00 AM, lights off at 5:00 PM, Pacific Standard Time (PST)]. For the first week, the body weight of each animal was measured daily using an electronic balance; during the second week, the rats were weighed twice (day 9 and day 14). Food consumption and water consumption were determined by weighing the food and water containers on an electronic balance. Urine volumes were measured with a graduated cylinder. Over the first 4 days postflight, urine collections were taken twice a day at 5:30 AM (AM) and 3:30 PM (PM), PST. The AM collection covered the active period for rats. On all subsequent days, collections occurred at 3:30 PM. Daily collections were taken on days 5-7. Pooled 2-day collections were obtained on days 9, 11, and 13. A final 24-h collection was made on day 14. Data in the text are corrected for collection duration. On day 14, the animals were anesthetized by inhalation of Metofane until sedated, and blood samples were taken.

Sample handling. Blood was taken from the descending aorta of anesthetized animals at recovery (R + 0) and 14 days after landing (R + 14), six animals per group per time period. Blood was placed in serum separator tubes, refrigerated, and allowed to clot for 2 h. Samples were then centrifuged in a refrigerated (4°C) centrifuge, and aliquots of serum were frozen at 20°C for later analysis. Urine was collected at room temperature in vials containing 50 µl of Traysol (aprotinin; Bayer, West Haven, CT); Traysol was added at the request of other investigators who shared the samples. Aliquots of the daily urine sample were taken and frozen at 20°C. Serum and urine concentrations of creatinine, sodium, potassium, and calcium were measured using an automated analytical system (COBAS; Roche Diagnostic Systems, Somerville, NJ). Serum and urine osmolalities were measured by freezing-point depression (Advanced Instruments, Needham Heights, MA). Urine aldosterone concentration was measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA) following extraction.

Calculations and statistics. Urine flow rate (V) was calculated as the volume collected divided by the time duration of the collection. The rate of excretion of a substance was the urine concentration times V. Because blood samples were not obtained on the animals in the metabolic cages, the mean serum concentration obtained from animals during postflight procedures were used to calculate clearance rates. Osmolal clearance (C_osm) was calculated as the rate of osmotic excretion divided by the mean serum osmolality. Free water clearance was equal to V  C_osm. Glomerular filtration rate (GFR) was estimated from the clearance rate of creatinine (creatinine excretion rate divided by serum concentration). The percent of the filtered load excreted for a substance was calculated as the rate of excretion × 100 divided by the serum concentration × GFR.

A two-way ANOVA was used to compare the blood samples between groups, and immediately postflight versus after 14 days of recovery. A two-way ANOVA adjusted for repeated measures over time was used in the analysis of the urine data. Differences between means were determined by the Tukey-Kramer test on individual data. A probability of <0.05 was accepted as being significant. Values in the text are means ± SE.

	RESULTS

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Serum osmolality and serum concentrations of creatinine, sodium, potassium, and calcium were not significantly different among the groups of animals (Table 1), nor were there differences in osmolality and concentrations of sodium, potassium, or calcium determined immediately postflight compared with those determined after 14 days of recovery. Serum creatinine levels for flight animals, although not different among groups on R + 0, were elevated compared with those obtained for flight animals following recovery on R + 14. 

Body weight was not different among groups before flight (flight animals: 137 ± 1.8 g; flight controls: 137 ± 2.1 g; vivarium controls: 136 ± 2.9) and immediately on recovery (Table 2). Weight gain (the daily change in body weight) decreased in flight animals within 24 h after landing compared with both control groups (Table 2; Ref. 29). Water intake did not differ between groups or over time (Table 2). The decrease in weight gain in flight animals was associated with a significant reduction in food consumption over the first 2 days of recovery compared with both control groups (Table 2). The decrease in mean food consumption of the flight group compared with control groups during this 48-h period, ~10 g, could not solely account for the 20-g difference in body weight gain of flight animals compared with flight controls or 31 g compared with vivarium controls. The other factor contributing to the reduction in body weight of flight animals was an increase in urine volume of 31 ml over the first 48 h of recovery compared with the mean excretion rate for flight control animals. The increase in V of flight animals persisted for at least 3 days postspaceflight (Fig. 1, Table 2).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Urinary flow (A) and creatinine excretion (B) rates of rats for 14 days following spaceflight for flight (F; n = 6), flight control (FC; n = 6), and vivarium control (VC; n = 5) animals. * Significant difference between groups (F vs. FC or VC).

The daily creatinine excretion rate of flight animals was increased postflight for 2 days (Fig. 1). In the flight animals, osmotic excretion was increased above both flight and vivarium controls for 3 days postflight (Fig. 2). The daily rate of sodium excretion of flight animals was significantly increased only on day 1 of recovery, whereas potassium excretion of flight animals was elevated for 2 days (Fig. 2). The only significant group effect was for daily potassium excretion, with flight animals having a greater excretion rate compared with both groups of controls. Calcium excretion showed an effect of caging during the flight period, as both the flight and flight control animals had reduced initial values compared with vivarium controls (Fig. 2). Flight animals were thus compared directly with flight control animals. There was an increase in daily calcium excretion in both flight and flight control animals compared with vivarium control animals over the subsequent 2 days, with flight animals increasing more than flight controls on day 5. The calcium excretion rates of flight animals returned to levels comparable to vivarium controls, whereas flight control values remained elevated for the next 3 days. An increase in the excretion rate of aldosterone in flight animals was noted for 2 days postflight (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Sodium (A), potassium (B), calcium (C), and osmolality (D) excretion rates of rats for 14 days following spaceflight for F (n = 6), FC (n = 6), and VC (n = 5) animals. * Significant difference between groups (F vs. FC or VC).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Aldosterone excretion rates of rats for 14 days following spaceflight for F (n = 6), FC (n = 6), and VC (n = 5) animals. * Significant difference between groups (F vs. VC).

Clearance rates were calculated for the first 24 h using the mean serum concentration [no difference was noted in serum concentrations immediately postflight among groups (Table 1)] and in the urine excretion rate for a group (Fig. 2). C_osm was increased in the flight animals compared with both control groups (Fig. 4). Free water clearance was more negative in the flight animals compared with the control groups.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Osmolal clearance (Cosm), urine flow (V), and free water clearance (CH2O) rates of rats for 24 h following spaceflight for F (n = 6), FC (n = 6), and VC (n = 5) animals. * Significant difference between groups (F vs. FC or VC).

The differences in excretion in flight animals may be due to increases in GFR (Fig. 5) as indicated by the increase in the creatinine excretion rate (Fig. 1). The clearance of creatinine was assumed to be indicative of GFR. The ratio of urinary concentration of solutes to creatinine concentration was compared to correct for possible differences in filtration rates thus reflecting renal handling of the solute as serum levels were not changed. On day 1, the urinary sodium-to-creatinine ratio was increased for flight animals (0.9 ± 0.06) compared with the ratios for flight and vivarium controls (0.4 ± 0.17 and 0.4 ± 0.12, respectively), indicative of an increase in the percent of the fluid load excreted. Differences were not observed in the ratios of potassium, calcium, or osmolality to creatinine.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Creatinine clearance as an estimation of glomerular filtration rates of rats for 24 h following spaceflight for F (n = 6), FC (n = 6), and VC (n = 5) animals. * Significant difference between groups (F vs. FC or VC).

During recovery, diurnal variations in renal function in all groups were noted (Table 3). There were increases in excretion rates during the period from 3:30 PM to 5:30 AM (AM), which included the time when the lights were off. In the flight animals, V was increased for 3 days postflight during the AM period compared with both control groups. V in flight animals was increased during the PM period only on days 1 and 2. There were increases in creatinine, potassium, and osmotic excretion rates of flight animals during the AM period, with smaller increases noted over the PM period. However, the magnitude of the diurnal changes for flight animals compared with flight control values was similar for AM and PM. Calcium excretion showed no diurnal effect. The increase in sodium excretion on day 1 was predominately due to a fivefold increase in excretion during the PM period, but a twofold increase was also noted in the AM.

	DISCUSSION

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In the conduct of research on the effects of spaceflight, there are severe constraints: a limited number of specimens are available and access to the specimens during flight is restricted. At present, there is no method for collecting urine samples from rats during spaceflight that would allow the study of renal function during flight. In addition, numerous investigators are usually studying the same specimen, which, although permitting integration of data among studies, may cause one study to impact another. In the present study, the animals and samples were shared with other investigators. The amount of urine and blood samples available to us was limited. Access to future spaceflights to confirm the present findings is years off. The present study, although not optimal, allowed the effects of reloading on return to Earth on renal function of rats to be evaluated.

The data from this experiment strongly suggest that changes in renal function of growing rats occur immediately after spaceflight. Urine flow, creatinine clearance (i.e., GFR), C_osm, sodium excretion, potassium excretion, and aldosterone excretion increase in the first 24 h postflight.

Following spaceflight, rats exhibited an increase in V that persisted for over 48 h. This increase coupled with a reduction in food intake resulted in a decrease in body weight. The increase in V appears to be the result of an increase in the clearance of osmotic substances, specifically sodium in the first 24 h and potassium over the first 48 h. When corrected for creatinine clearance, only sodium remained elevated, suggesting that the other solutes showed differences due to changes in the rate of filtration rather than renal tubular handling.

Earlier studies of postspaceflight rats showed no difference in V, but the data were not collected until 40 h after spaceflight (6, 7, 18). However, a decrease in the ability to handle a water load was noted (6, 7, 18). In humans, a decrease in V following spaceflight was reported; however, these individuals had an increase in plasma osmolality indicative of dehydration (18, 20). In the present study, no differences were noted in serum osmolality, yet V in flight animals was increased approximately twofold for 2 days following spaceflight compared with both control groups. The increase in V was in contrast to hypothesized changes. In spaceflight, blood volume and total body water have been reported to be reduced in humans (24, 29). On return to the gravity of Earth, fluids move from the core of the body to the extremities (31). This shift, coupled with the reduction of body water during flight, would be expected to initiate the conservation of fluids by the kidneys. In rats, this does not appear to occur and may be the result of changes in the sensitivity of volume receptors or the sequestration of fluids in the periphery (31). Furthermore, in quadrupeds such as the rat, fluid loss may not occur in spaceflight.

An increase in GFR may contribute to an increase in V. The clearance of creatinine is often used as an indicator of GFR. In the present study, the increase in the excretion of creatinine may be indicative of an increase in GFR, as serum concentrations are not significantly different postflight between groups. However, plasma creatinine concentrations of rats are reported to be increased when measurements are obtained 11-40 h postlanding (11, 16). Furthermore, there are indexes of muscle damage immediately postflight that would contribute to an increase in plasma and urine creatinine concentrations (25). In the present study, serum creatinine concentrations were increased in flight animals immediately postflight compared with levels obtained after 14 days of recovery (Table 1). There are limitations to interpretation of the present data because plasma samples for determination of creatinine concentrations came from one group of animals and urine levels were measured in another. Although our data are characteristic of an increase in GFR, which could contribute to the increase in V, this increase in filtration has to be confirmed by direct measurement.

The increase in V in the first 24 h is due to an increase in C_osm. This increase in C_osm appears to be the result of a natriuresis with a secondary kaluresis. The increase in the excretion of sodium appears to be rectified within 24 h, possibly due to the increase in aldosterone as indicated by an increase in its excretion in urine. The conservation of sodium by the actions of aldosterone may contribute to the excretion of potassium. Flight animals had a greater total excretion of potassium over the 14-day recovery period compared with both control groups. However, this may also be the product of muscle damage (25). Of note is that the increase in the excretion of both creatinine and potassium occurred in the AM, lights-off period, which would have been the active weight-bearing period for the rats. The increase in the excretion of potassium may also result from a change in the concentrating mechanisms in the kidney. Gazenko et al. (6, 7) found a decrease in the ability of flight rats to handle a potassium load and postulated that this was the result of the observed decrease of potassium concentration in the outer zone of the renal medulla (23). These investigators also noted a postflight difference in the handling of sodium following a water load. Sodium retention was decreased in the flight animals. The reason for the increase in solute excretion following spaceflight in the present study is not clear, but solute excretion appears to be the primary component of the increase in V.

The increased excretion of sodium in the presence of an elevated excretion of aldosterone may appear paradoxical. In response to an increased sodium loss via the kidneys following landing, circulating aldosterone concentrations would be increased and a concomitant increase in urinary aldosterone levels would be observed. However, the actions of increased aldosterone concentration may have been delayed for hours because of the latency time necessary for protein synthetic responses to be initiated (17). Furthermore, the increase in aldosterone persisting for 2 days suggests a possible role in the correction of the renal handling of sodium, but the actions of aldosterone may have been delayed initially.

Natochin et al. (18-20) have reported decreased renal concentrating ability in both rats and humans postspaceflight. In rats, they found a disruption of tubular reabsorption of water; specifically, the ability to produce a concentrated urine was altered due to a decrease in the sensitivity of the kidney to vasopressin (antidiuretic hormone). In contrast, we found free water clearance to be decreased postflight. That is, there was an increase in the tubular reabsorption of water. This suggests that the flight rats still had the ability to form a concentrated urine and the differences in V were predominantly due to a decreased reabsorption (increased excretion) of osmotically active substances.

In both flight and flight control animals, the excretion of calcium was reduced compared with that of vivarium controls, suggesting an effect of the limited cage space. Recent studies show that there is a difference in response of the muscles of animals flown in space depending on how they are housed (25). For these reasons, we compared the calcium excretion rates of the flight animals only with the calcium excretion rates of flight controls. In both of these groups, there was a time effect. Following the transfer into the larger metabolic cages, both groups increased the excretion of calcium for 3 days. After this period, the flight animals dramatically reduced their calcium excretion while the flight control animals had a slow decrease. There was a group effect noted, with the flight animals having a reduced calcium excretion over the 14 days of recovery compared with the flight controls. The renal conservation of calcium postflight would agree with an increase in the remodeling of bone during this period (2).

Over the first 4 postflight days, urine samples were taken twice a day. In all groups, a distinct diurnal pattern in urine excretion rate is observed: an increase in urine flow during the AM lights-off collection period compared with the PM lights-on collection period. Rats are nocturnal animals, and the AM collection period includes their active period. Although recovery from spaceflight appeared to disrupt renal function, it did not affect the diurnal cycle. Others have shown changes in the diurnal temperature cycles of animals resulting from spaceflight, but the change in diurnal cycle does not appear to be reflected in renal function changes (4). However, renal function cycles are less sensitive than temperature cycles to diurnal changes.

Our findings of alterations of renal function postflight are in contrast to those previously reported. These differences may be due to the earlier access to our animals (within 3 h) compared with the delays in access to animals in previous studies. Our animals also had continuous availability of food and water. In other studies, animals have had periods of no access to food and water, a condition that, while also mimicked in the control groups, could have produced a different response in subsequent evaluations.

In summary, on return of rats to Earth following 14 days of spaceflight, there are significant alterations in renal function. The initial diuresis appears to be due to an increased osmolal clearance coupled to a natriuresis and kaluresis. The natriuresis is attenuated within 24 h, probably due to increased aldosterone. There is an increase in water reabsorption, compensating in part for obligatory losses associated with the reduction in solute reabsorption. However, there is still a net increase in the renal loss of fluids and electrolytes, contributing to a decrease in body weight (30). The alteration in the renal handling of fluid and electrolytes, resulting in a net loss of fluids in rats on return to Earth, is in conflict with existing theories of fluid shifts during spaceflight (31). It is possible that rats do not lose fluids during flight and may, in fact, retain fluids. In a recent experiment, the body weight of rats appeared to be increased when measured during exposure to spaceflight (1). This increase in body weight could be related to an increase in total body water. Our observations suggest possible changes in other homeostatic mechanisms of rats during and after spaceflight that need further investigation if this animal continues to be a primary model for study.