Seasonal acclimatization in water flux rate, urine osmolality and kidney water channels in free-living degus: molecular mechanisms, physiological processes and ecological implications
1 Center for Advanced Studies in Ecology & Biodiversity and Departamento
de Ecología, Facultad de Ciencias Biológicas, Pontificia
Universidad Católica de Chile, Santiago, Chile, 6513677
2 Laboratorio de Fisiopatología Molecular, Programa de
Fisiopatología, ICBM, Facultad de Medicina, Universidad de Chile,
Santiago, Chile, 6513677
3 Centre for Isotope Research, Nijenborgh 4, 9747 AG Groningen, The
Netherlands
4 Zoological Laboratory, PO Box 14, 9750 AA Haren, The
Netherlands
5 Departamento de Biología, Facultad de Ciencias Universidad de La
Serena, Casilla 599, La Serena Chile
* Author for correspondence (e-mail: fbozinov{at}genes.bio.puc.cl)
Accepted 20 May 2003
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Summary |
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Key words: acclimatization, arid environment, water economy, rodent, aquaporin, degu, Octodon degus
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Introduction |
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Maintaining a positive water economy is a difficult task for
desert-dwelling animals (Walsberg,
2000). Rodents from arid and semi-arid habitats live under
conditions where the spatial and temporal availability of free water is
limited, or scarce, so these rodents are faced with the problem of water
conservation (Degen, 1997
).
Nevertheless, the physiology of water regulation among desert rodents appears
to show remarkable flexibility in both time and space. The response of small
mammals to unproductive desert environments and water deficits has been
intensively investigated (e.g.
Schmidt-Nielsen, 1964
).
However, current understanding of the cellular, systemic and organismal
physiology of water economy relies heavily on short-term, laboratory-oriented
experiments, which usually focus on responses at isolated levels of biological
organization.
Here, we examined the phenotypic flexibility of water flux rate, urine
osmolality and expression of kidney aquaporins (AQP), or water channels, in a
South American desert-dwelling rodent (see below) and, as far as we know, for
the first time through a field study integrating cellular, systemic and
organismal levels. Rates of water flux in the field represent the loss of
water, via excretion and evaporative water loss, and the simultaneous
input of water, via metabolic water production, and pre-formed water,
via food and drink (Nagy and
Costa, 1980; Nagy and
Peterson, 1988
; Speakman,
1997
). In this context, labeled water could be used to estimate
water flux (Speakman, 1997
).
In addition, for small mammals, urine osmolality represents the capacity of
the kidneys to efficiently conserve water (e.g.
Schmidt-Nielsen, 1964
;
Prosser, 1991
;
Willmer et al., 2000
;
McNab, 2002
;
Seldin and Giebisch, 2000
).
Finally, studies at cellular levels of biological organization have now shown
that the urine-concentrating mechanism associated with water reabsorption
relies on the activities of a series of NaCl co-transporters, ion channels,
Na+/K+-ATPase and AQP water channels
(Seldin and Giebisch, 2000
).
AQP water channels are probably central structures leading to the mechanisms
responsible for the changes in water balance. Here, we studied AQP-1 and AQP-2
water channels. In the kidneys, AQP-1 channels are constitutively active water
channels that allow rapid transmembrane osmotic water flux and are involved in
the reabsorption of filtered water in proximal tubules and thin descending
limbs (Nielsen et al., 2002
).
On the other hand, AQP-2 is the vasopressin-regulated water channel expressed
in apical membranes of connecting and principal cells of the distal nephron.
AQP-2 is localized in the apical cell membrane but it also resides in a pool
of membrane vesicles within the cytoplasm and can be regulated by the animal's
state of water balance. Through apical AQP-2 and basolateral AQP-3, principal
cells and inner medullary collecting duct cells are able to efficiently
reabsorb free water (Nielsen et al.,
2002
).
Since small desert mammals regularly encounter seasonal variation in both their physical and biotic habitats, we hypothesize that they should change their physiology through phenotypic flexibility to cope with desert conditions. We studied seasonal acclimatization of Octodon degus (degu; see below) to its habitats. We determined, under field conditions, the effect of seasonal changes in environmental water availability on the distribution and regulation of AQP-1 and AQP-2, on urine osmolality and on total water flux.
Because during the dry summer months the plants consumed by degus (Vulpea sp. and Erodium sp.) contained nearly 5.7% and 2.8% of water, respectively, while during the wet winter these plant species contained 70.6% and 76.9% of water, respectively (C. Veloso, personal communication), we predict that rates of water flux will be higher during winter than during summer; thus, urine osmolality should be significantly higher during the dry season, and, consequently, we also predict a constitutive activity of AQP-1 and an increase in the insertion of AQP-2 into apical kidney cell membrane during the dry summer period.
We used the degu, Octodon degus (Molina) (Rodentia: Octodontidae),
as the study organism. The degu is endemic to Chile and is a good model
because it is diurnal, allowing for field observations. Furthermore, the degu
inhabits the seasonal, semi-arid and Mediterranean environments of northern
and central Chile, where summers are hot and dry and winters are cold and
rainy. Degus are efficient at water conservation
(Cortés et al., 1990)
but have a low capacity for evaporative thermolysis (Bozinovic et al.,
1995
,
2000
).
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Materials and methods |
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We selected a representative area within the semi-arid matorral of San Carlos de Apoquindo that encompassed an extensive open space with sparse shrub and tree cover. In this area, we constructed an enclosure measuring 70 mx50 m, with a 1.8 m high fence that was buried 40 cm into the substrate and covered with chicken wire. Conditions within the enclosure can be considered natural. In the enclosure, we released four families of adult degus that were previously captured from another population. A total of 32 degus were released in the enclosure.
We measured the mean rate of water flux and field metabolic rate during
winter and summer using double-labeled water (DLW) mixture provided by The
Center for Isotope Research in The Netherlands (CIO)
(http://www.cio.phys.rug.nl).
The water was enriched in both 2H and 18O (34.4% and
64.1%, respectively). Following the protocol established by CIO and Kenagy et
al. (1989), we injected the
DLW quantitatively (0.8 ml in a 1 ml insulin syringe). We always noted if some
DLW was spilled. For equilibration times of the DLW, we also followed the
protocol outlined by Kenagy et al.
(1989
). That is, after 1 h at
equilibrium, a first blood sample was taken from the orbital sinus using 80
µl heparinized microhematocrit capillary tubes; the tubes were subsequently
flame-sealed and stored at 5°C until analysis. Animals were always
anaesthetized prior to measurements. Each capillary tube contained
approximately 15 µl of blood. We used a propane torch with a very fine
pointed flame for flame sealing. Degus were weighed with a portable electronic
balance (±0.01 g; Sartorius, Gottingen, Germany). The ease of trapping
degus in the enclosure allowed us to resample individuals after 48 h and to
collect a second series of blood samples. Samples were mailed to CIO for
further triplicate analysis. Background samples were obtained from degus
inhabiting the same enclosure. Water fluxes were calculated for each
individual on the basis of the individual-specific size of the body water pool
(based on the plateau value of the 2H dilution;
Visser et al., 2000
) and the
individual-specific fractional 2H turnover rate. We took isotope
fractionation effects into account, assuming that 50% of the water efflux was
lost through evaporative pathways, following Kenagy et al.
(1989
). In three animals, the
sizes of their body water pools could not be assessed due to minor leakage of
the DLW dose. In these cases, we used the mean percentage of body water to
estimate the individual-specific sizes of the body water pool. Observed water
flux rates were compared against the standard water flux rates (in ml
day-1) predicted for small herbivorous mammals
(Nagy and Peterson, 1988
)
using the following equation: water flux
rate=0.708xMb0.795, where
Mb is body mass in g.
Field urine osmolality
We conducted field measurements of urine osmolality for individuals
captured in Quebrada de las Vacas, Fray Jorge National Park, Northern Chile
(30°38' S, 71°40' W) during winter and summer sampling
occasions. These measurements were made during the winter and summer of 1994.
Briefly, the biotic and abiotic habitat in Fray Jorge is
semi-arid-Mediterranean, similar to our enclosure at San Carlos de Apoquindo.
Rodents were captured using Sherman live traps during a period of five
consecutive days. A total of 30 adult animals was captured for this study. To
avoid resampling, all rodents were ear-tagged. Urine samples were obtained
each morning, at the same hour, using microhematocrit capillary tubes, which
were subsequently sealed with parafilm. Immediately after collection, we
measured urine total solids (S; in g per 100 g), following the method
of Cortés and Rosenmann
(1988), using a field
refractometer (AO TS Meter; Scientific Instruments, Lakewood, NJ, USA). Our
refractometer measurements (urine samples) were continuously calibrated
against a freezing-point osmometer (Advanced Instruments, Norwood, MA, USA).
Finally, we estimated urine osmotic concentration (U; in mosmol
kg-1) using the following equation:
U=140xS0.984
(Cortés and Rosenmann,
1988
).
Seasonal changes in AQP distribution and regulation
To test for the effect of seasonal changes in water availability on the
distribution and regulation of AQP-1 and AQP-2, three adult male degus were
live-caught with Sherman traps at San Carlos de Apoquindo during winter and
summer collecting trips and transferred to the laboratory. Immediately
following capture, animals were sacrificed via an overdose of
pentobarbital sodium (60 mg kg-1; i.p.). In the laboratory, both
kidneys were removed, placed in ice-cold phosphate-buffered saline (PBS; pH
7.4) and processed for immunocytochemistry and immunoblot.
Localization of AQPs was carried out via immunocytochemistry.
Immunocytochemical studies were carried out in paraplast-embedded tissue
sections (6 µm thick), previously fixed in Bouin solution. The steps for
immunocytochemistry are described in Gallardo et al.
(2002). The primary antibodies
used were anti-AQP-1 and anti-AQP-2 (diluted 1:200; kindly provided by M.
Knepper, NIH, USA). Immunoreactive sites were revealed using biotinylated
swine anti-rabbit IgG (Dako, Carpinteria, CA, USA) followed by
streptavidin-conjugated horseradish peroxidase; the chromogens used were
3,3'-diaminobenzidine (Dako Liquid DAB plus kit) or Vector SG substrate
(Vector, Burlingame, CA, USA), in the presence of hydrogen peroxide. Tissue
sections were observed and photographed on a Nikon Optiphot microscope.
Variations in AQP-2 protein abundance were studied through immunoblot. Membranes were prepared from renal medulla of both groups by differential centrifugation. The tissue was homogenized in buffer containing 250 mmol l-1 sucrose, 10 mmol l-1 triethanolamine, 21 µmol l-1 leupeptin, 57.4 µmol l-1 phenylmethyl sulfoxide (PMSF), 0.1 mg ml-1 aprotinin, pH 7.5 and centrifuged at 3000 g for 10 min at 4°C; the supernatant was centrifuged at 100 000 g for 1 h at 4°C. The final pellet was resuspended in the same buffer, and protein concentration was determined spectrophotometrically with Bradford reagent (BioRad, Hercules, CA, USA). For immunoblotting, 15 µg of protein was solubilized in Laemmli buffer and heated at 65°C for 15 min. Immunoblotting was performed in 12% SDS-PAGE mini-gels and run on a mini-gel system (BioRad Mini-Protean III). Proteins were blotted onto nitrocellulose membranes, blocked for 1 h, washed with TBS-T pH 7.4 and incubated for 18 h at 4°C with the primary antibody. The secondary antibody was goat anti-rabbit IgG coupled to horseradish peroxidase. Immunoreactivity in the membrane was detected by enhanced chemiluminescence. In addition, we measured plasma and urine osmolality in animals used for AQP determinations, using a freezing-point osmometer (Advanced Instruments). Plasma was obtained by centrifugation of a venous sample collected from the inferior cava vein. Urine samples were obtained from a single bladder puncture.
Statistics
Statistical analyses were performed using the STATISTICA® for Windows
(2001; version 6.0) statistical package. Data were analyzed using analysis of
covariance (ANCOVA), with body mass (Mb) as a covariable.
Data fulfilled the assumptions of the tests. Results are reported as means
± 1 S.D.
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Results |
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The mean rates of water flux in free-living degus for each season are shown in Fig. 1B. ANCOVA revealed that season was the only factor that had a significant effect on water flux rate. As predicted, rates of water flux were higher during winter than during summer (Fig. 1B; Table 1). Observed water influx rate during summer was 10.3±2.3 ml day-1, which was 22.8% of the expected value (45.2 ml day-1) based on Mb.. In winter, the rate of water influx (40.4±9.1 ml day-1) was similar to the expected value, making up 88.8% of the value expected from Mb (45.5 ml day-1). No significant effects of sex or interactions among factors were observed on the rate of water flux (Table 1). In addition, rates of water influx and efflux were not significantly different (Table 1).
In a second group of individuals, we seasonally measured field urine
osmolality, U (Fig.
1C). The mean Mb of degus used for field
U were not significantly different, being 124.79±6.99 g during
summer (nine females and 10 males) and 119.72±9.19 g during winter
(three females and eight males) (see Table
2). Interestingly, the same statistical pattern and results
observed for the mean rates of water flux were also obtained for field
U (Fig. 1C;
Table 2). As expected,
U was significantly higher during summer than during winter
(Fig. 1C). The observed mean
U during summer was 3137±472 mosmol kg-1, which is
27.7% lower than the maximum U ever recorded in degus [Cortés
et al. (1988) registered a
mean U of 4338 mosmol kg-1]. Our winter U
(1123±472 mosmol kg-1) was 74.1% lower than the maximum
recorded U for degus.
Seasonal changes in AQPs and urine:plasma concentration ratio
The study of seasonal changes in AQP distribution and regulation was
carried out using three degus (mean Mb=164.07±24.25
g) captured during summer and three degus (mean
Mb=170.9±12.9 g) captured during winter. Body mass
was not significantly different between seasons
(F1,3=1.202, P=0.353).
AQP-2 immunolabeling was observed in connecting tubules and cortical and medullary segments of collecting ducts. At the subcellular level, AQP-2 immunostaining was evident in apical plasma membrane as well as subapical cytosol. AQP-2 medullary immunolabeling was more abundant in kidneys of degus captured during the dry season (summer; Fig. 2A) than the rainy season (winter; Fig. 2B). This immunoreactivity was higher in apical cell membranes of medullary collecting ducts of summer degus in comparison with those of winter degus. AQP-1 immunostaining did not differ between groups (Fig. 2C,D). Consistently, AQP-2 protein levels were higher in the medulla of the summer individuals compared with the winter individuals, as judged by the density of the 29 kDa band on the immunoblot (Fig. 2E).
|
Degus used for AQP determinations exhibited significantly higher U values during summer (2223.0±117.6 mosmol kg-1) than in winter (794.0±166.06 mosmol kg-1) (F1,3=36.623, P=0.009). As expected, plasma osmolality (p) was constant throughout seasons, being 310.0±14.5 mosmol kg-1 in summer and 293.3±3.71 mosmol kg-1 in winter (F1,3=1.202, P=0.353). Consequently, and as expected, the U/p ratio was significantly higher during summer (summer=7.58±0.31; winter=2.52±0.4; F1,3=71.913, P=0.003).
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Discussion |
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Degus live in open-scrub habitats subject to summer drought. Geographical
and seasonal dietary differences in the relative proportions of shrub and
grass foliage occur between localities. At the most arid and semi-arid
localities, degus feed primarily on shrub foliage, seeds and conductive
tissue. In Mediterranean environments, they feed primarily on foliage of forbs
and grasses and on seeds. Thus, degus experience geographical and seasonal
changes in the availability of water and nutrients in their food
(Meserve, 1981; Meserve et al.
1983
,
1998
). Bozinovic
(1995
) indicated that in the
dry summer months the plants consumed by degus (Vulpea sp. and
Erodium sp.) contained nearly 60% neutral detergent fiber with a
range of water content from approximately 3% to 6% (C. Veloso, personal
communication), while during the wet winter these plant species contained 37%
neutral detergent fiber and 70-80% water
(Bozinovic and Torres-Contreras,
1998
; C. Veloso, personal communication). Although small mammals
such as degus may select sparsely distributed, high-quality plants or use
coprophagy to recycle water and nutrients
(Kenagy et al., 1999
), during
nutritional-water bottlenecks they must consume low-quality food out of
necessity rather than choice (Bozinovic,
1995
,
1997
;
Bozinovic et al., 1997
;
Torres-Contreras and Bozinovic,
1997
). Indeed, the differences in seasonal water flux in O.
degus are probably a product of differences in consumption of preformed
water in plants. A similar phenomenon was reported in fat sand rats by Degen
et al. (1991
). Seasonal
differences in water flux as a result of dietary shifts from a dry diet in
summer to a relatively humid diet in winter and spring are common in degus,
and such fluxes have also been reported in several Negev desert rodents
(Degen et al., 1997
). Also,
water influx and efflux rates reflecting dietary preformed water were reported
in two diurnal species, the pocket gopher Thomomys bottae and the
antelope ground squirrel Ammospermophilus leucurus, by Gettinger
(1984
) and Karasov
(1983
), respectively. As
observed in degus, the dietary items consumed by these rodents exhibited
seasonal availability, thus influencing the homeostasis of the consumers.
Classical studies have traditionally viewed the capacity to concentrate
urine as an indicator of the efficiency of water regulation
(Schmidt-Nielsen, 1964;
Abbott, 1971
;
Prosser, 1991
;
McNab, 2002
), as well as an
advantage for colonization and survival in deserts
(Degen, 1997
). According to
MacMillen and Hinds (1983
),
this view is probably correct for rodents that lose body mass when deprived of
water and are kept on a dry diet. Under such conditions, rodents should
exercise maximal capabilities of water conservation, reflected in maximal
urine osmolalities. Bozinovic et al.
(1995
) reviewed studies dealing
with renal performance and maximal urine osmolality among rodent species
inhabiting semixeric and xeric regions of Chile, southwestern USA and
Australia. For the five species inhabiting semixeric and xeric habitats in
Chile, the maximal capacity to concentrate urine (recorded under laboratory
conditions) ranged from
3.3 mosmol kg-1 in the fossorial
octodontid rodent Spalacopus cyanus to nearly 4.5 mosmol
kg-1 in the sigmodontine rodent Phyllotis darwini. The
mean value for the species inhabiting the matorral was 4.138 mosmol
kg-1, a value similar to that found for nine western North American
rodent species, including the wood rat Neotoma albigula (3.930 mosmol
kg-1). In the laboratory, the maximum U attained by degus
is 4.338 mosmol kg-1
(Cortés et al., 1988
).
Our observed mean U during summer was 27.7% lower than the maximum
U recorded in degus, probably because the diets used in experimental
laboratory conditions were drier than those consumed by degus in the
field.
What are the cellular mechanisms that allow degus to concentrate urine and
to be able to save water during the dry season? AQP water channels are
membrane-integral proteins that mediate facilitated water transport across
cell membranes of a wide variety of cells, including epithelia. In the renal
tubule, several AQPs are expressed with a very specific pattern. AQP-1 is
expressed in apical and basolateral membranes of proximal tubule and thin
descending limb cells (Nielsen et al.,
1993a). AQP-2 is expressed in apical membrane of connecting,
principal and inner medullary collecting duct cells of the distal nephron
(Nielsen et al., 1993b
). AQP-3
and AQP-4 are basolateral aquaporins expressed in the same cells that express
AQP-2 (Ecelbarger et al.,
1995
; Terris et al.,
1995
). These water channels play a key physiological role in the
urine-concentrating mechanism (Ma et al.,
2000
; Schnermann et al.,
1998
; Promeneur et al.,
2000
; Chou et al.,
1998
). The role of vasopressin in the urine-concentrating
mechanism is related to the regulation of AQP-2 and AQP-3. Vasopressin
regulates AQP-2 through an acute and long-term two-part mechanism. In the
first part, vasopressin stimulates AQP-2 insertion in the apical membrane. In
the second part, vasopressin increases AQP-2 gene transcription
(Nielsen et al., 2002
).
In O. degus, AQP-1 is expressed in apical and basolateral
membranes of surface absorptive and crypt epithelium of the distal colon
(Gallardo et al., 2002).
Studies of water absorption in the distal colon of this rodent have shown that
absorption is dramatically decreased by the presence of the mercurial agent, a
known inhibitor of water channels. Thus, AQP-1 may be involved in water
absorption and fecal dehydration (Gallardo
et al., 2002
), allowing water conservation in degus
(Cortés et al., 1988
).
As far as we know, not much information is available concerning the regulation
of AQPs in desert-dwelling rodents; in fact, this appears to be the first
field study reporting AQP-2 acclimatization, but see Huang et al.
(2001
).
The degu kidney expresses several AQPs involved in the urine-concentrating mechanism: AQP-1, AQP-2 and AQP-3. Their distribution is quite similar to that described for the laboratory rat (P. A. Gallardo, unpublished results). Indeed, in this study, we observed that degus captured during the winter (i.e. with free access to water) do not express a high capacity for renal water reabsorption and excrete a dilute urine. On the other hand, degus captured during summer (i.e. during the dry season) have a limited supply of free water, feed on dry food (see above) and therefore must conserve water. Hence, during the summer, degus excrete a hyperosmotic urine.
The functional state of the urine-concentrating mechanism can be easily associated with the urine using the U/p ratio. The main concept of water balance is the regulation of plasma osmolality, which is achieved by the mechanism of thirst and renal water reabsorption. This mechanism is vasopressin dependent, and its activation results in the excretion of hypertonic urine. In fact, in degus captured during the dry season the U/p ratio was significantly higher than in winter degus. This increased U/p ratio also suggests that vasopressin levels are higher in animals from the dry season compared with those of the rainy season. This hormone should be measured in plasma and under field conditions.
At the molecular stage, high levels of vasopressin imply short- and long-term mechanisms of action. In fact, AQP-2 immunolabeling in the apical membrane, as well as AQP-2 protein levels, was increased in degus captured during summer; conversely, winter animals had less AQP-2 immunoreactivity and lower protein levels. Thus, the shift in vasopressin secretion, initially triggered by an increase in plasma osmolality, allows (together with other water-conserving mechanisms) the maintenance of the water balance through changes in the subcellular distribution and abundance of AQP-2. Although animals captured during the dry season did not attain the maximal urine osmolality recorded under artificial laboratory conditions, our results clearly demonstrate a degree of flexibility for the kidney, which allows degus to conserve water.
What are the implications of the flexibility of osmoregulation and AQP
expression on survival? As mentioned before, since the semi-arid areas of
Chile are characterized by a seasonal regime of temperature and precipitation,
small mammals experience a strongly seasonal environment, with warm dry
summers and cool wet winters. In this habitat, the populations of rodent
species exhibit numerical fluctuations associated with high and low rainfall
years (Yunger et al., 2002).
Indeed, climate-related variables (mainly rainfall) exhibited more influence
on reproductive variables than on survival.
From our study, we can suggest that the observed in-field phenotypic flexibility of water `economy and management' at organismal, systemic and cellular levels of desert-dwelling rodents is critically important and may allow animals to successfully overcome the physiological challenges of arid environments. Consequently, the observed molecular and physiological flexibility reported here (which allow for a high water economy) may explain values of desert survival and colonization at the individual, as well as the population, level during low rainfall seasons and years in semi-arid habitats. In summary, the observed acclimatization for dealing with water economy may serve as a way to budget survival in the face of the hot, arid and unpredictable habitat of desert rodents.
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Acknowledgments |
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