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First published online February 29, 2008
Journal of Experimental Biology 211, 852-859 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.006395
The effect of water deprivation on the tonicity responsive enhancer binding protein (TonEBP) and TonEBP-regulated genes in the kidney of the Spinifex hopping mouse, Notomys alexis
School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia
* Author for correspondence at present address: Department of Physiology, University of Otago, PO Box 913, Dunedin, 9054, New Zealand (e-mail: ray.bartolo{at}otago.ac.nz)
Accepted 8 January 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: Notomys alexis, tonicity responsive enhancer binding protein (TonEBP), compatible osmolytes, kidney, water deprivation
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The excretion
of highly concentrated urine is dependent on the regulation of two processes:
ultrafiltration of plasma in the glomerulus and reabsorption of water in the
renal tubules. Accordingly, desert mammals have a reduced glomerular
filtration rate and enhanced tubular water reabsorption
(Degen, 1997). Water
reabsorption relies on the medullary osmotic gradient created by the loop of
Henle (Bankir and Rouffignac,
1985). Desert rodents, in particular, have long loops of Henle,
and routinely excrete urine in the range of 4000–7000 mOsm
l–1 (Degen,
1997). A high osmolality of the inner medulla and papilla of the
kidney exposes the cells in that region to a hypertonic environment, which can
severely compromise cell function and lead to DNA damage and apoptosis
(Burg et al., 2007;
Jeon et al., 2006). The most
abundant osmolytes in the renal medulla and papilla of the kidney are
Na+, Cl– and urea
(Garcia-Perez and Burg, 1991).
High NaCl causes hypertonicity and disturbances in cell volume, but urea has
no effect on tonicity as it readily permeates cell membranes
(Yancey et al., 1982).
In response to hypertonicity, renal cells will rapidly accumulate inorganic
ions, but in conjunction with elevated urea concentrations, this will
compromise the function of cellular proteins and macromolecules
(Yancey et al., 1982). The
intracellular ionic concentration is then lowered by the replacement of the
inorganic ions with compatible osmolytes, which occurs over 1–3 days
(Rauchman et al., 1997;
Jeon et al., 2006;
Burg et al., 2007). Since
compatible osmolytes do not contribute to ionic strength, the intracellular
ionic concentration remains within an optimal physiological range, while the
osmolality of the interstitium and intracellular fluid is the same
(Beck et al., 1998;
Garcia-Perez and Burg, 1991).
The accumulation of compatible osmolytes is reversible since rehydration
leading to diuresis, causes swelling of the medullary cells and the release of
compatible osmolytes into the interstitial fluid
(Beck et al., 1998;
Garcia-Perez and Burg,
1991).
Sorbitol (D-glucitol), myo-inositol, betaine and
taurine are compatible osmolytes that are found in abundance in the renal
medulla. In response to hypertonicity, sorbitol is produced from glucose by
the aldose reductase (AR) enzyme, whereas myo-inositol, betaine and
taurine are transported into cells by Na+- or
Na+/Cl–-dependent transporters
(Burg et al., 1996). The
transcription of AR and the myo-inositol (SMIT; also known as
SLC5A3), betaine/GABA (BGT-1; also known as SLC6A12) and taurine (TauT; also
known as SLC6A6) transporters is regulated by the tonicity-responsive enhancer
binding protein (TonEBP; also known as NFAT5)
(Burg et al., 1996). The AR,
SMIT, BGT-1 and TauT genes have osmotic response elements (ORE) in their
5' flanking regions that contain the tonicity-responsive enhancer (TonE)
consensus sequence (Takenaka et al.,
1994). The binding of TonEBP to OREs increases the transcription
of AR, SMIT, BGT-1 and TauT, which in turn leads to an increase in the
intracellular accumulation of the respective compatible osmolyte
(Woo and Kwon, 2002;
Woo et al., 2002). TonEBP has
a basal activity level under isotonic conditions that is decreased by
hypotonicity and increased by hypertonicity. An increase in TonEBP activity is
reflected by an increase in the expression of AR, SMIT, BGT-1 and TauT mRNAs;
mice lacking the TonEBP gene have a reduced expression of AR, SMIT, BGT-1 and
TauT (Lopez-Rodriguez et al.,
2004). The bidirectionality of TonEBP activity has been
demonstrated in Madin-Darby canine kidney (MDCK) cells
(Woo et al., 2000a). In MDCK
cells grown under isotonic conditions, TonEBP is distributed between the
nucleus and cytoplasm, but under hypertonic stress TonEBP increases in
abundance and translocates to the nucleus to act as a transcription factor of
osmoprotective genes. By contrast, when the MDCK cells are transferred to a
hypotonic medium, TonEBP translocates to the cytoplasm, its abundance
decreases, and compatible osmolytes move out of the cell
(Woo et al., 2000a).
The Spinifex hopping mouse, Notomys alexis, is a small rodent that
is highly adapted to survive in arid environments where it can live without
drinking water (MacMillen and Lee,
1969; Weaver et al.,
1994). N. alexis has been reported to produce the most
concentrated urine of any mammal (9370 mOsm l–1)
(MacMillen and Lee, 1969). In
the laboratory, N. alexis can tolerate water deprivation for 28 days
without changes in plasma osmolality, vasopressin or renin, as seen in other
species of desert rodent (Weaver et al.,
1994; Heimeier et al.,
2002). By contrast, water deprivation experiments using laboratory
rats and mice are short-term because of the inability of the animals to
survive without drinking, and the animals show a marked increase in plasma
osmolality and vasopressin levels (see
Degen, 1997). Thus, the
mechanisms underpinning survival during long-term water deprivation in desert
mammals may be different from mesic species. Given the extraordinary
urine-concentrating ability of N. alexis, we predicted that the
expression of TonEBP and the genes encoding proteins that regulate
intracellular compatible osmolytes would play an important role and be
upregulated in the renal medulla of N. alexis during periods of water
stress. Thus, the aim of the current study was to analyse the expression of
TonEBP, AR, BGT-1 SMIT and TauT mRNAs and determine the distribution of TonEBP
protein in the kidney of water-deprived N. alexis, in comparison to
mice with ad libitum access to water.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Water deprivation experiments
All experiments involving N. alexis were performed with approval
from the Deakin University Animal Welfare Committee (Project number A31/2003).
The water deprivation experiments were carried out as previously described
(Donald and Bartolo, 2003).
For each water deprivation time point, there was a water-replete (control)
group in which N. alexis had ad libitum access to water, and
a water-deprived (experimental) group in which N. alexis was
subjected to 3, 7 or 14 days without access to free water; this was termed
water deprivation. All mice were ear-tagged to enable identification, and were
approximately 6 months old. There were eight animals in each group and they
were housed in groups of four in sand-filled glass aquaria (W 100 cmxH
40 cmxL 50 cm), which allowed for communal sleeping burrows. The mice
were weighed and fed 20 g of millet seed per cage daily; a group of four mice
eat a maximum of 4.5 g seed per day during water deprivation (R.C.B.,
unpublished). The mass of the animals in the 14-day water deprivation
experiment was used for the analysis shown in
Fig. 1. At the end of the
respective water deprivation periods, the mice were anaesthetised by halothane
inhalation followed by cervical dislocation. The kidneys were dissected free,
and the left kidney was frozen in liquid nitrogen and stored at
–80°C until RNA was isolated, while the right kidney was fixed
overnight at 4°C in 4% formaldehyde (pH 7.4; 
1100 mOsm
kg–1), and then stored in 70% ethanol until processing for
immunohistochemistry.
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Collection of urine for the measurement of osmolality
A separate water deprivation experiment using six N. alexis was
performed to measure urine osmolality. The mice were water-deprived for 14
days and urine was collected on day 0, 3, 7 and 14. Urine was collected from
individual mice placed for 1 h in cylindrical containers (90 mm diameter and
150 mm high) that had a wire mesh floor. The containers were suspended above a
collection tray that contained light paraffin oil, thus preventing evaporation
of the urine. Urine osmolality was then measured using a Vapro® vapour
pressure osmometer (Wescor Model 5520, Logan, UT, USA).
Amplification, cloning and sequencing of putative cDNAs
Kidney total RNA was isolated using TRIzol (Invitrogen, Mount Waverley,
Victoria, Australia), which utilises the single step acid guanidinium
thiocyanate-phenol-chloroform extraction method
(Chomczynski and Sacchi, 1987).
The RNA concentration was determined by spectrophotometry at 260 nm. First
strand cDNA was synthesised from kidney total RNA using Superscript II
(Invitrogen) as per the manufacturer's protocol. Primers were designed based
on Mus musculus sequence data obtained from GenBank (National Centre
for Biotechnology, NCBI). The accession numbers for the M. musculus
sequences are as follows: TonEBP, AF453571; AR, NM_009658; BGT-1, NM_133661;
SMIT, AF220915; and TauT, AAB54039.
Primer sequences, annealing temperature, and the size of the predicted PCR
amplicons are shown in Table 1.
PCR was performed in a total volume of 20 µl with a final concentration of:
1x PCR buffer, 0.2 mmol l–1 dNTPs, 1 µmol
l–1 of each forward and reverse primer, 1.0 i.u. of Taq DNA
polymerase (Scientifix, Melbourne, Australia), 2.5 mmol l–1
MgCl2 and 1 µl of the cDNA synthesis reaction. Amplification of
the various cDNAs was performed as follows: initial denaturation of 300 s at
94°C, 35 cycles of 45 s at 94°C, 30 s at the annealing temperature
(Table 1), 45 s at 72°C,
and a final extension of 300 s at 72°C. The PCR products were purified and
cloned into a pCR2.1 vector, which was then transformed into One Shot TOP10
chemically competent Escherichia coli cells using a TA cloning®
Kit (Invitrogen). The cloned cDNAs were sequenced on an Applied Biosystems
automated sequencer (Australian Genome Research Facility, Brisbane,
Australia). The BLAST (Basic Local Alignment Search Tool) program on the NCBI
database was used to search GenBank for similar sequences
(Altschul et al., 1997).
Alignments of N. alexis and M. musculus nucleotide and amino
acid sequences were carried out to determine homology between cloned N.
alexis cDNAs and the sequences from which PCR primers were designed,
using ClustalW
(http://www.ebi.ac.uk/clustalw/).
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mRNA expression analysis
Reverse transcription PCR was used to detect changes in the expression of
TonEBP, AR, BGT-1, SMIT and TauT mRNAs in the kidney of water-replete and
water-deprived N. alexis. Total RNA isolation and cDNA synthesis were
performed as described above. For the analysis of mRNA expression, 2 µg of
total RNA was reverse transcribed, and then 1 µl of the cDNA reaction was
used in PCR. To quantify the level of mRNA expression between control and
water-deprived N. alexis, the expression of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal
control, as the expression of GAPDH does not change in response to
water deprivation (Heimeier et al.,
2002; Sturzenbaum and Kille,
2001). The GAPDH primers were as follows: forward
5'-GAAGGTCGGTGTGAACGGATTTG-3', and reverse
5'-TTACTCCTTGGAGGCCATGTAGG-3'; these primers generated a 999 base
pair amplicon.
In preliminary experiments, the linear amplification range of the genes of interest and GAPDH were determined by running PCR reactions for a varying number of cycles between 17 and 35. The linear range was also determined in samples from control and water-deprived mice to ensure that there was no variation between individual animals or sample groups. The PCRs for gene expression analysis were performed for varying numbers of cycles, depending on the gene: GAPDH, 20; TonEBP, 22; AR, 24; BGT-1, 28; SMIT, 24; TauT, 31. The parameters for the PCR cycles were the same as previously mentioned.
For the quantification of the PCR products, the reactions were spiked with
2.5 µCi of [
-32P]dCTP. As the radio-labelled dCTP is
randomly incorporated into the PCR products, the level of radiation emitted by
a PCR product is directly proportional to the amount of PCR product. Equal
aliquots of the GAPDH PCR and gene of interest PCR were mixed (to avoid gel
loading error) and subjected to electrophoresis on a 1.5% agarose gel with a
1x Tris–borate–EDTA (TBE) running buffer at 100 V. The gel
was incubated in 0.5 µg ml–1 of ethidium bromide,
visualised on an UV light box and the cDNA bands were excised, and placed in
microcentrifuge tubes. The amount of isotope [-32P]
incorporated into the PCR products was measured by placing the microcentrifuge
tubes in vials and counting in a scintillation counter (Tri-Carb 2000CA Liquid
Scintillation Counter, United Technology Packard, Downers Grove, IL, USA). The
expression of the various mRNAs was determined as a ratio of GAPDH mRNA
expression (gene of interest/GAPDH), and the difference in the ratios between
water-replete and water-deprived groups were analysed for statistical
significance. The mRNA expression data are expressed as a percentage of the
control where the mean values from control animals represent 100% for
illustrative purposes only.
TonEBP Immunohistochemistry
One kidney from all mice (N=8 for each time point) was analysed
for TonEBP immunoreactivity (TonEBP-IR). Fixed tissues were processed in a
Leica TP 1010 automated tissue processor (Wetzlar, Germany), which dehydrated
the tissue through a series of ethanol and xylene washes. The kidneys were
then embedded in ParaplastTM tissue embedding medium, and 5 µm
sections were placed on slides coated in 2% 3-aminopropyltiethoxysilane
(Sigma) and allowed to dry overnight. Sections were prepared for
immunohistochemistry by dewaxing in xylene and rehydration through a graded
series of ethanol to water. Endogenous peroxidase activity was quenched by
incubating the sections in 3% hydrogen peroxide for 10 min. The sections then
underwent heat-induced epitope retrieval; sections were incubated in 1.0 mmol
l–1 EDTA buffer (pH 8.0) for 10 min, heated for 3x5 min
in a 650 W microwave oven, cooled to room temperature and washed in
phosphate-buffered saline (PBS; pH 7.4; 2x5 min washes). Endogenous
biotinylated proteins were blocked by the use of an Avidin–Biotin
blocking kit (Vector Laboratories, Burlingame, CA, USA), which involved
incubating sections in Avidin D solution for 15 min, a 1 min rinse with PBS,
and incubation in Biotin solution for 15 min, followed by incubation with an
affinity-purified rabbit anti-mouse TonEBP antiserum for 2 h at room
temperature. The TonEBP antiserum was diluted (1:5000) with PBS, and was
kindly donated by Prof. Seung Kyoon Woo, University of Maryland, Baltimore,
USA (Miyakawa et al., 1999).
The sections were then washed in PBS for 2x10 min. A Vectastain ABC kit
(Vector Laboratories) was used for the detection of the TonEBP antiserum. The
sections were incubated with biotinylated secondary antibody solution (1:200)
for 30 min, washed in PBS for 2x10 min, and incubated with the
Vectastain ABC reagent (Vector Laboratories) for 45 min. Sections were then
washed in PBS for 10 min, rinsed in 0.1 mol l–1 Tris (pH 7.4)
and incubated in 0.02% diaminobenzidine tetrahydrochloride (DAB; in 0.1 mol
l–1 Tris, pH 7.4) for 10 min. The slides were examined under
a light microscope (Axioskop 20, Carl Zeiss, Göttingen, Germany) and
sections were photographed with a digital colour system (Spot 35 Camera
System, Diagnostic Instruments, Sterling Heights, MI, USA). The specificity of
staining was determined by running negative controls omitting primary and/or
secondary antibody.
Data analysis
To test the difference in mRNA expression between control and experimental
groups, a Student's t-test was performed. Changes in body mass during
the 14-day water deprivation experiment were analysed using a two-way ANOVA
and a Student's t-test, and urine osmolalities were analysed using a
one-way ANOVA; each used a Tukey's post-hoc test. All statistical
probabilities were calculated using SPSS for Windows 14, and
P
0.05 was considered significant
(Quinn and Keogh, 2002).
Materials
[-32P]dCTP (3000 Ci mmol l–1) was
purchased from GE Life Sciences (Rydalmere, NSW, Australia). The Vectastain
ABC kit was purchased from Abacus ALS, Brisbane, Australia. All other
chemicals were either reagent or molecular grade and were purchased from
Sigma-Aldrich (Castle Hill, NSW, Australia) or Scientifix (Melbourne,
Australia).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Effect of water deprivation on body mass and urine osmolality of N. alexis
Control (water-replete) N. alexis showed no significant change in
body mass over the course of the experiment
(Fig. 1). By contrast, N.
alexis subjected to water deprivation lost mass over the first 7 days,
after which it stabilised (Fig.
1, Table 3).
Compared to the body mass at the beginning of water deprivation, there was a
significant decrease in mass after 3, 7 and 14 days of water deprivation; body
mass at day 7 and day 14 was significantly less than that at day 0 and day 3,
respectively. Mean urine osmolality significantly increased in response to 3,
7 and 14 days of water deprivation, when compared to water-replete N.
alexis (Table 3). The
urine osmolality at day 14 was significantly higher than that at day 0, and 3
and 7 days of water deprivation (Table
3).
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TonEBP mRNA expression and protein immunolocalisation in the kidney
TonEBP mRNA expression was unaffected (P=0.806) by 3 days of water
deprivation, but there was a significant increase in its expression after 7
(P=0.013) and 14 (P<0.001) days of water deprivation
(Fig. 2).
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Effect of water deprivation on transcription of AR, BGT-1, SMIT and TauT mRNAs
The expression of AR, BGT-1, SMIT and TauT mRNAs was analysed in the kidney
of water-replete N. alexis, and compared to that in 3-, 7- and 14-day
water-deprived mice. The expression of each gene was significantly increased
(P<0.05) after each period of water deprivation
(Fig. 5).
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Jeon et al., 2006). We found
that water deprivation in N. alexis caused an upregulation of TonEBP
mRNA expression, translocation of TonEBP to the nucleus of renal medullary
cells, and an increased expression of AR, SMIT, BGT-1 and TauT mRNAs. The
response would be an essential physiological mechanism that protects renal
medullary cells and contributes to the ability of rodents such as N.
alexis to tolerate prolonged periods of water deprivation.
In order to study the expression of TonEBP, AR, BGT-1, SMIT and TauT mRNAs
in N. alexis, partial cDNAs encoding each gene were initially cloned
and sequenced. All N. alexis cDNAs sequenced in this study showed
greater than 94% nucleotide sequence homology to the M. musculus
sequences from which the respective PCR primers were designed, and at least
95% amino acid sequence homology to the respective M. musculus
protein sequences. Previous sequencing of hopping mouse cDNAs encoding
regulatory hormones and receptors has shown high sequence identity to M.
musculus (e.g. Donald and Bartolo,
2003; Heimeier et al.,
2002); both species are Old World rodents and members of the
family Muridae and the subfamily Murinae.
During water deprivation, N. alexis lost mass until day 7, at
which point body mass stabilised and began increasing up to day 14, which is
consistent with previous water deprivation studies with hopping mice
(Heimeier and Donald, 2006).
During water deprivation, body fat is metabolised to increase the production
of metabolic water, which will contribute to the maintenance of water balance
(Degen, 1997). In N.
alexis, the loss of body fat in the early stages of water deprivation is
the main component of the observed weight loss (R.C.B., unpublished). The
ability of desert rodents to produce extremely concentrated urine during water
deprivation is a critical factor that contributes to the conservation of body
water. In the current study, N. alexis was able to significantly
increase urine osmolality during water deprivation, reaching a maximum of 6348
mOsm kg–1 at 14 days. Although this is well below the
previously reported maximal value for N. alexis urine, of 9370 mOsm
kg–1, MacMillen and Lee
(MacMillen and Lee, 1969)
reported a mean value of 6550±510 mOsm kg–1 for
hopping mice water-deprived for 21 days. This is comparable to the mean of
5346±336 mOsm kg–1 from the 14-day water-deprived
animals in this study. In desert rodents, urine concentration can be
influenced by the length of the water deprivation period, the salt and protein
content of the diet (Gamble et al.,
1929; Gamble et al.,
1934) and the housing arrangement of the mice. In particular,
communal nesting of N. alexis has been shown to be an important
behavioural adaptation that leads to a lowering of metabolic rate (up to 18%)
and a reduction in pulmo-cutaneous water loss (up to 25%), compared to mice
housed individually (Baudinette,
1972). In the current study, the mice were housed in groups of
four, but MacMillen and Lee (MacMillen and
Lee, 1969) housed the mice individually, which may partly explain
the very high urine osmolalities recorded in their study.
The expression of TonEBP mRNA in the kidney of 3-day water-deprived N.
alexis was not significantly different from that of N. alexis
with access to water, which is consistent with the findings of a study
performed with rats that also found no change in the expression of TonEBP mRNA
after 3 days of water deprivation (Cha et
al., 2001). The results of the current study and that of Cha et
al. (Cha et al., 2001), would
suggest that the stimulus for increasing the transcription of TonEBP mRNA is
not present after 3 days of water deprivation. Interestingly, in both studies
the urine osmolality was significantly higher in 3-day water deprived animals
compared to water-replete animals. This is likely to be due to an increase in
the corticomedullary osmotic gradient and, as a consequence, the tonicity of
the renal interstitium in the papilla
(Knepper, 1982;
Knepper and Burg, 1983). In
areas where the tonicity is highest, such as the inner medulla of rats, TonEBP
mRNA is not affected by water deprivation despite the perceived increase in
osmolarity of the interstitial fluid (Cha
et al., 2001). Thus, hypertonicity may not necessarily lead to an
increase in TonEBP mRNA abundance. Recently it was reported that hypertonicity
in cultured renal cells resulted in an increase in TonEBP mRNA abundance, but
the increase was due to the stabilization of the TonEBP mRNA pool rather than
an increase in actual mRNA transcription
(Cai et al., 2005).
In contrast to the effect of water deprivation on TonEBP mRNA
transcription, 3 days of water deprivation in N. alexis resulted in
an increase in the intensity of TonEBP-IR in the nuclei of the collecting duct
cells in the outer and inner medulla and papilla. In addition, the area
immediately surrounding the nuclei of some of the collecting duct epithelial
cells in the renal papilla appeared to have very little TonEBP-IR, suggesting
that TonEBP had mostly translocated to the nucleus. These observations are
also consistent with immunohistochemistry in water-deprived rats
(Cha et al., 2001). In cultured
cells, nuclear translocation of TonEBP is the typical response when the
extracellular fluid is hypertonic (Woo et
al., 2000b). Therefore, it appears that nuclear translocation of
TonEBP can be induced without an increase in TonEBP mRNA abundance.
Unlike rats, desert rodents such as N. alexis can survive
long-term water deprivation without suffering dehydration due to the
production of metabolic water and a highly concentrated urine. Desert rodents,
therefore, provide a unique opportunity to examine the role of TonEBP in renal
function during long-term water deprivation. N. alexis subjected to
long-term water deprivation (7 and 14 days) in this study showed an increase
in the expression of TonEBP mRNA in the kidney. The increase in TonEBP mRNA
may be due to an increase in mRNA stability as observed in cultured kidney
cells (Cai et al., 2005), or an
upregulation in the transcription of TonEBP mRNA. Nuclear translocation of
TonEBP was also found in the kidney of 7- and 14-day water-deprived hopping
mice, with the intensity of TonEBP-IR in the inner medulla and papilla being
greatest after 14 days of water deprivation. This trend was particularly
evident in the nuclei of the epithelial cells lining the collecting ducts in
the papilla. The increase in TonEBP activity is consistent with the urine
osmolality data for 7- and 14-day water-deprived hopping mice, which indicates
that the cells in the papilla are bathed in extracellular fluid that has an
equivalent osmolality to the urine
(Knepper, 1982).
In addition to the renal papilla and inner medulla, the epithelial cells of the tubules in the outer medulla of N. alexis deprived of water for 7 and 14 days showed a greater intensity of TonEBP-IR than those of both the control and 3-day water-deprived hopping mice. This suggests that the interstitial fluid in this region of the kidney is hypertonic in comparison to that of control and 3-day water-deprived N. alexis, and that the corticomedullary osmotic gradient in the kidney of N. alexis may have increased during water deprivation.
In the renal cortex, the osmolality of the interstitium remains isotonic
with the plasma, and previous studies in N. alexis have shown that
plasma osmolality does not change in response to water deprivation
(Heimeier and Donald, 2006).
Rats deprived of water for 3 days showed no change in TonEBP-IR in the renal
cortex (Cha et al., 2001).
Similarly, TonEBP-IR in the cortex of N. alexis did not change in
response to the different periods of water deprivation, which is consistent
with the observations in rats (Cha et al.,
2001).
Cells exposed to a hypertonic environment over a long period of time
accumulate high concentrations of compatible osmolytes that, unlike inorganic
ions and urea, do not inhibit intracellular proteins, enzymes or
macromolecules (Kultz et al.,
1998; Yancey et al.,
1982). The increase in intracellular sorbitol, betaine,
myo-inositol and taurine in renal cells exposed to hypertonic
extracellular fluid has been shown to occur in response to an increase in the
transcription of AR (Garcia-Perez et al.,
1989; Smardo et al.,
1992), BGT-1 (Nakanishi et
al., 1990), SMIT (Kitamura et
al., 1997; Kwon et al.,
1992) and TauT (Ito et al.,
2004), respectively. Furthermore, it is well established that
TonEBP regulates the mRNA transcription of AR, BGT-1, SMIT and TauT by binding
to the respective TonE consensus sites in their promoter regions
(Woo and Kwon, 2002).
Therefore, an upregulation of the transcription of AR, BGT-1, SMIT and TauT
during water deprivation in N. alexis, will be an indication of the
effect of water deprivation on TonEBP activity.
In N. alexis, 3, 7 and 14 days of water deprivation increased the
mRNA expression of AR, BGT-1, SMIT and TauT mRNAs in the kidney. The mRNA data
are supported by the immunohistochemical observation of nuclear translocation
of TonEBP at each time point examined during water deprivation. These provide
indirect evidence that the intracellular accumulation of sorbitol, betaine,
myo-inositol and taurine is a key adaptation of the renal medulla of
N. alexis in which the interstitial osmolality is likely to be
between 5000 and 6000 mOsm after 14 days of water deprivation
(Knepper, 1982). Thus, in
desert rodents, TonEBP functionality is likely to be a key regulatory
mechanism protecting renal cells from the extreme variations in medullary
hypertonicity required to produce the highly concentrated urine necessary to
survive in xeric environments.
LIST OF ABBREVIATIONS
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Acknowledgments |
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