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First published online March 17, 2006
Journal of Experimental Biology 209, 1207-1216 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02123
Effect of osmotic stress on expression of a putative facilitative urea transporter in the kidney and urinary bladder of the marine toad, Bufo marinus
1 Department of Biology, Faculty of Science, University of Toyama, 3190
Gofuku, Toyama, 930-8555, Japan
2 Laboratory of Physiology, Ocean Research Institute, University of Tokyo,
1-15-1 Minamidai, Nakano, Tokyo, 164-8639, Japan
* Author for correspondence (e-mail: uchiyama{at}sci.toyama-u.ac.jp)
Accepted 24 January 2006
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55 kDa) was detected in extracts
from plasma membrane fractions of the kidney and urinary bladder. When toads
were acclimated to dry and hyper-saline environments for 7 days, UT mRNA
expression was upregulated in the kidney and urinary bladder and there was an
elevated plasma urea concentration and osmolality. Immunohistochemistry showed
that the UT was specifically localized on the apical membrane of the early
distal tubule, known to be the diluting segment, in the kidney and the
epithelial cells of urinary bladder. Immunoreactive cells were not detected
along the late distal tubule, the connecting tubule or the collecting duct in
the kidney. The present findings suggest that the Bufo UT probably
contributes to urea transport in the kidney and urinary bladder in response to
hyperosmotic stresses such as body fluid hypertonicity and dehydration.
Key words: urea transporter (UT), kidney, diluting segment, Bufo marinus, osmoregulation
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Introduction |
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;
Withers, 1998). When frogs and
toads are acclimated to dry and hyper-saline environments, they accumulate
urea in their body fluids as a result of increases in hepatic urea production
and/or decreases in renal excretion
(Funkhouser and Goldstein,
1973; Balinsky,
1981). In conditions of osmotic stress, dry and hyper-saline
acclimation, urea accumulation contributes to retention of body water and
helps avoid dehydration. This osmotic strategy is known to be used by
desiccating and aestivating toads, such as the desert spadefoot toad
Scaphipus couchi (McClanahan,
1967) and the urodele Siren lacertian
(Etheridge, 1990), and by
hyper-saline-acclimated anurans such as Rana cancrivora and
Bufo sp. (Gordon et al.,
1961; Katz,
1973).
In short-term acclimation to a hyper-saline environment, urea retention is
associated with a decrease in urine production and a modest increase in the
rate of urea synthesis (McBean and
Goldstein, 1970a; McBean and
Goldstein, 1970b). By contrast, long-term acclimation is
associated with accelerated urea synthesis and increased activity of urea
cycle enzymes (Balinsky, 1981).
Renal retention is also an important mechanism for accumulation of urea in
anuran amphibians (Schmidt-Nielsen and
Lee, 1962; Shpun and Katz,
1995). Urea clearance is probably influenced by the glomerular
filtration rate (GFR) and by renal tubule function, secretion and reabsorption
in the kidney. It has been reported that in anurans, urea is reabsorbed across
the renal tubule in the kidney and also in the urinary bladder
(Schmidt-Nielsen and Lee,
1962; Shpun and Katz,
1995).
Previously, although urea was proposed to cross cell membranes by
lipid-phase permeation, it has been found that passive urea movement occurs
through facilitative urea transporters, which have been identified and
characterized in cell membranes from mammalian kidney
(Smith and Rousselet, 2001;
Bagnasco, 2005), amphibian
urinary bladder (Couriaud et al.,
1999) and fish gill and kidney
(Smith and Wright, 1999;
Mistry et al., 2001,
2005). In mammals,
facilitative urea transporters are the renal urea transporter (UT-A) and the
erythrocyte urea transporter (UT-B). The renal urea transporter UT-A has four
isoforms: UT-A1 (Shayakul et al.,
1996), UT-A2 (Smith et al.,
1995), UT-A3 (Karakashian et
al., 1999) and UT-A4
(Karakashian et al., 1999).
Mammalian UT- As are expressed in the renal medulla of the kidney and
stimulated by several hormones such as arginine-vasopressin (AVP),
glucocorticoids and mineralocorticoids
(Wade et al., 2000;
Shayakul et al., 2000;
Peng et al., 2002;
Gertner et al., 2004), and
also in various physiological and pathological states
(Shayakul et al., 2000;
Sands, 2004).
In amphibians, the presence of a facilitative urea transporter has been
previously demonstrated in studies of functional urea transporters, which were
found to be expressed after microinjection of Xenopus oocytes with
total or fractionated poly(A)+ RNA isolated from amphibian urinary
bladder epithelial cells (Martial et al.,
1991). In addition, a cDNA encoding frog UT has been cloned from
the urinary bladder of Rana esculenta
(Couriaud et al., 1999). The
frog UT protein has high identity to rat UT-A2 and UT-B1, and the urea uptake
mediated by the protein is inhibited by both phloretin and the mercurial
reagent para-chloromercuribenzene sulfonate (pCMBS). However, to our
knowledge, there has been no report of the expression and localization of UTs
in the amphibian kidney, which, as well as the urinary bladder, is an
important osmoregulatory organ in anurans.
In this study, we identified a full-length cDNA encoding the urea transporter from the kidney of the marine toad Bufo marinus, and investigated changes in the expression of UT mRNA in the kidney and urinary bladder of the toad under hyperosmotic stress. In addition, we clarified cellular localization of Bufo UT along the nephron segments of the kidney.
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Materials and methods |
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Osmotic treatments and analysis of plasma and urine components
Experimental protocols were described in detail in our previous paper
(Konno et al., 2005). Briefly,
toads in the control group were maintained on moist soil and allowed free
access to water, and toads in two experimental groups were subjected to
osmotic treatments (dry and hyper-saline acclimation). In one group, toads
were kept on dried sponge fragments, whereas in the other, toads were immersed
in 300 mOsmol kg–1 H2O NaCl solution. Toads in
each group were starved during the 7-day experiment, and their body mass was
measured every day. Blood samples were collected by cardiac puncture using
heparinized 1-ml syringes and centrifuged at 2000 g for 20 min
at 4°C. Urine samples were collected from the urinary bladder using
syringes. Osmolality, Na+, Cl–, K+ and
urea concentration were measured in plasma and urine samples.
Molecular cloning of Bufo UT cDNA
Total RNA was extracted from the Bufo kidney using acid
guanidinium thiocyanate–phenol–chloroform extraction, as described
previously (Chomczynski and Sacchi,
1987). Poly(A)+ RNA was purified from the total RNA
with an Oligotex-dT30 super mRNA purification kit (TaKaRa, Otsu, Japan).
Adaptor-ligated double-strand kidney cDNA was synthesized with a SMART cDNA
Library Construction Kit (Clontech, Mountain View, CA, USA). Degenerate
primers for the urea transporter (UT) were designed based on the UT cDNA
sequences of the dogfish, Triakis scyllium
(Hyodo et al., 2004), the
edible frog, Rana esculenta
(Couriaud et al., 1999), and
mammalian UT-A2 sequences. The primer sequences were
5'-GTNCARAAYCCNTGGTGGRC-3' (sense) and
5'-CCANGGRTTRTCRCANCCRTA-3' (antisense). PCR was performed using
high-fidelity Ex-Taq DNA polymerase (TaKaRa) using the following schedule:
94°C for 2 min, 35 cycles of 94°C for 1 min, 55°C for 30 s,
72°C for 1 min and finally 72°C for 10 min. The PCR products were
separated electrophoretically on 1% agarose gel containing ethidium bromide
(0.5 µg ml–1), and the major band of the expected size was
cut from the gel. The cDNA fragment purified from the sliced gel was ligated
into pT7Blue T-Vector (Novagen, San Diego, CA, USA) and the resulting plasmid
was transformed into the competent cell (XL1-Blue, Invitrogen, Carlsbad, CA,
USA). Blue/white screening on Luriabroth (LB) ampicillin–agar plates
identified white colonies with the potential insert, and the identified
colonies were liquid cultured. The plasmid DNA was isolated by a modified
alkaline/SDS method (Rapid Plasmid Purification Systems, Marligen Bioscience,
Ijamsville, MD, USA). The sequencing reaction was performed with a BigDye
Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
The nucleotide sequence was determined using an ABI Prism 310 or 3100 DNA
sequencer (Applied Biosystems). The full-length Bufo UT cDNA was
obtained by 5'- or 3'-rapid amplification of cDNA ends (RACE) with
adaptor primers (Clontech) and UT gene-specific primers, which were designed
on the basis of the sequences of cDNA fragments obtained by degenerate PCR.
The specific primers were 5'-AAGCAGTGGTATCAACGCAGAGT-3' (5'
adaptor primer), 5'-CTGTCAGCGTGGACACTACAGT-3' (antisense),
5'-GCTCACCTGGCAAACTCAC-3' (sense), and
5'-ATTCTAGAGGCCGAGGCGGCCGACA-d(T)30N-1N-3'
(3' adaptor primer).
Tissue distribution of Bufo UT mRNA
Tissue expression of UT mRNA was examined by RT-PCR. Total RNA was isolated
from various tissues (brain, tongue, heart, lung, liver, stomach, spleen,
small intestine, large intestine, kidney, urinary bladder, testis, ovary,
ventral pelvic skin and dorsal skin) using Isogen (Nippon gene, Tokyo, Japan).
To prepare the first-strand cDNA, 1 µg of total RNA was reverse-transcribed
with a First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel,
Switzerland). The specific PCR primers (sense:
5'-ACCTGGTCTGATGTCCACATAC-3' and antisense:
5'-GCAGCTCCTAGGTAGGCACA-3') were synthesized based on sequences
637–658 and 955–974 of the Bufo UT cDNA sequence (GenBank
accession no. AB212932). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
was used as an internal standard. The GAPDH primer pair (sense:
5'-CTGGCTCCTCTTGCAAAGGT-3' and antisense:
5'-GTGTATCCCAGGATTCCCTTC-3') was designed based on the GAPDH
sequences of the marsh frog, Rana ridibunda (GenBank accession no.
AY072703), and the African clawed frog, Xenopus laevis (GenBank
accession no. U41753). PCR was performed with 27 cycles (UT) and 24 cycles
(GAPDH) of denaturation (94°C, 1 min), annealing (55°C, 30 s) and
extension (72°C, 1 min). The PCR products were separated
electrophoretically in 3% agarose gel containing ethidium bromide, and
detected by a gel photograph instrument (Printgraph, Atto, Tokyo, Japan). Band
densities were analyzed using Scion Image software (Scion Corporation,
Frederick, MD, USA).
Preparation of urea transporter polyclonal antibody
A polyclonal antibody against the Bufo UT was raised by immunizing
Japanese white rabbits subcutaneously with a synthetic peptide that included
cysteine at the COOH terminus, NH2-LSKVTYPEC-COOH, corresponding to
amino acids 364–370 of the Bufo UT. The antiserum was collected
and purified using an affinity column bearing the immobilized synthetic
peptide with affinity gel beads (Affi-Gel 10, Bio-Rad, Hercules, CA, USA).
Immunoblotting
Tissue samples from the kidney, urinary bladder, heart and liver were
homogenized in ice-cold membrane isolation solution (250 mmol
l–1 sucrose, 10 mmol l–1 triethanolamine,
containing 1 µg ml–1 leupeptin, and 0.1 mg
ml–1 phenylmethyl sulfonyl fluoride, adjusted to pH 7.6)
using a tissue homogenizer (Physcotron NS-310E, Nition, Chiba, Japan). The
homogenates were centrifuged at 2000 g for 20 min at 4°C
to remove whole cells, nuclei and mitochondria, and the supernatant was
centrifuged at 17,000 g for 1 h at 4°C to collect a pellet
containing the plasma membrane fractions. The pellet was suspended in ice-cold
membrane isolation solution including 1% Triton X-100. Total protein
concentration in the samples was measured with a Bio-Rad Protein Assay reagent
(Bio-Rad, Japan) using the Bradford method
(Bradford, 1976). The samples
were solubilized at 60°C for 15 min in Laemmli buffer. SDS–PAGE was
performed on 12.5% polyacrylamide gel, and the proteins were then transferred
from the gel to a nitrocellulose membrane (Hybond-C, Amersham Biosciences,
Piscataway, NJ, USA). To prevent non-specific binding, the membranes were
blocked with 5% skimmed milk for 2 h at room temperature, and then probed
overnight at 4°C with the Bufo UT polyclonal antibody (dilution
1:2000 with 1% BSA–PBS) raised against Bufo UT. After washing
with TBS–Tween 20, the membranes were incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (ECL plus Western Blotting Detection
System, Amersham Biosciences, USA) for 2 h at room temperature. After further
washing of the membranes with TBS–Tween 20, secondary antibody binding
was visualized using an enhanced chemiluminescence kit (ECL plus Western
Blotting Detection System, Amersham Biosciences, USA).
Immunohistochemistry
Kidney and urinary bladder were perfusion-fixed in situ via the
dorsal aorta with Bouin's fixative, and then removed and post-fixed in the
same solution overnight at 4°C. The tissues were dehydrated and embedded
in paraffin, and then serial sections (6 µm) were cut and stained using the
immunoperoxidase technique (Vectastain ABC Kit, Vector Laboratories,
Burlingame, CA, USA). The sections were incubated overnight at 4°C with
the Bufo UT antibody (dilution 1:4000 with 1% BSA–PBS).
Adjacent sections were stained with vacuolar type H+-ATPase
antiserum (dilution 1:8000 with 1% BSA–PBS) that identifies specifically
intercalated cells of the late distal tubule and the collecting duct
(Uchiyama and Yoshizawa,
2002). Immunoreactivity for UT was visualized with DAB solution
(3,3'-diaminobenzidine, Sigma-aldrich, St Louis, MO, USA) containing
0.02% H2O2. Sites showing immunoreactivity for UT or
vacuolar-type H+-ATPase were confirmed by omitting the primary
antibodies, and replacing the respective antibodies with rabbit preimmune
sera, and immunoreabsorption of antibodies with the synthetic antigens (5
µg ml–1) for 24 h at 4°C. All control preparations
were negative for immunostaining.
Statistical analysis
Data are presented as means ± s.e.m. Statistical significance was
assessed by one-way analysis of variance (ANOVA) followed by Bonferroni's
multiple comparison test and Student's paired t-test. Differences at
P<0.05 were considered statistically significant.
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Molecular cloning of Bufo UT
A full-length cDNA sequence for Bufo UT is 1363 bp long, and has a
polyadenylation signal (AATAAA) at position 1290–1295. A putative open
reading frame (109–1278) encodes a protein of 390 amino acid (GenBank
accession no. AB212932; Fig.
1A). Hydropathy analysis using the Kyte–Doolittle algorithm
predicted 10 putative transmembrane regions with an N terminus and a C
terminus located in the cytoplasm (Fig.
1B). There was one putative N-glycosylation site at amino
acids 211–213 (NIT) in the putative central extracellular loop
(Fig. 1A). The Bufo UT
protein has high identity to Rana UT (80%) and also has 76% and 68%
amino acid identity with rat UT-A2 and UT-B2, respectively.
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Effects of dry and hyper-saline acclimation to UT mRNA expression in the kidney and the urinary bladder
We performed semi-quantitative RT–PCR to clarify whether UT mRNA
expression in the kidney and urinary bladder is regulated in response to dry
and hyper-saline environments. The levels of UT mRNA expression in the kidney
and urinary bladder were normalized to the expression of GAPDH mRNA. The
expression of UT mRNA in the kidney and urinary bladder was significantly
increased by both dry and hyper-saline acclimation for 7 days
(Fig. 3). There was no
significant difference in UT mRNA expression in the kidney and urinary bladder
between toads acclimated to dry conditions and toads acclimated to
hyper-saline environment.
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Identification of immunoreactive Bufo UT proteins
To detect expression of UT protein in the kidney and urinary bladder, and
to confirm specificity of Bufo UT antibody, we performed immunoblot
analysis using extracts from both organs. In the membrane fractions from the
kidney and urinary bladder, the affinity-purified antibody demonstrated
prominently labeled a single band at 52 kDa
(Fig. 4, lanes 1 and 2).
However, this immunopositive band was not detected in the extracts from the
heart and liver (Fig. 4, lanes
3 and 4). The molecular mass of the Bufo UT protein was similar to
that of mammalian UT-A2 (55 kDa). As shown in
Fig. 4 (lanes 5 and 6), no
immunopositive band was observed when the antibody was preincubated with an
excess of the immunizing peptide (5 µg ml–1).
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). The vacuolar H+-ATPase antibody sharply labeled
the intercalated cells along the late distal tubule, connecting and collecting
tubules (Fig. 5D), whereas
immunolabeling for Bufo UT was not observed in these tubules. The
scheme in Fig. 5E,F summarizes
the distribution of Bufo UT and vacuolar H+-ATPase
proteins in the marine toad, Bufo marinus.
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In our acclimation study, we found that urine volume in the urinary bladder
decreased and increased in response to dry and hyper-saline acclimations,
respectively. Similar responses have been reported in Bufo viridis
(Katz, 1986;
Shpun and Katz, 1989). It is
known that in conditions of dehydration, the whole kidney glomerular
filtration rate decreases and a large fraction of the filtered fluid is
reabsorbed by the renal tubule and the urinary bladder
(Shoemaker and Bickler, 1979).
Hyper-saline-acclimated anurans such as the crab-eating frog, Rana
cancrivora (Chew et al.,
1972), and Bufo viridis
(Katz, 1986) develop severe
hypernatremia resulting in saliuresis, and store a large volume of bladder
urine but do not void it. This indicates reabsorption of water and urea by
recycling bladder urine. Urea is freely filtered in the glomerulus and its
excretion is determined by filtration and varying degrees of reabsorption in
the kidney and urinary bladder. It seems that urea reabsorption from tubular
to extracellular fluid occur through facilitative urea transporters, following
a urea gradient between plasma and the renal tubular fluid. In the urinary
bladder, urine is stored and a substantial amount of urea is facilitatively
reabsorbed (Katz and Ben-Sasson.,
1984; Shpun and Katz,
1989). In Rana ridibunda, the U/Purea ratios
were 2.29 and 0.62 in fresh water and 300 mOsm in saline, respectively. Renal
handling of urea may change from tubular secretion to reabsorption during
hyper-saline acclimation (Shpun and Katz,
1995). In the present study, the U/Purea in bladder
urine was 2.49, 2.31 and 1.07 in toads under control, dry and hyper-saline
conditions, respectively. This result shows that toads in a hyper-saline
environment increased their reabsorption of urea. It is also assumed that net
urea reabsorption is stimulated in the kidney, considering that GFR is
decreased and water reabsorption is increased significantly in desiccated
toads under dry conditions.
Although specific urea transporters are thought to be present in the
amphibian kidney and urinary bladder (see
Jorgensen, 1997), very little
is known about the molecular mechanism of the urea transport process. In the
present study, the Bufo UT protein was found to have 80% amino acid
identity with the frog UT and 76% with rat UT-A2 and a similar membrane
topology (Couriaud et al.,
1999; Smith et al.,
1995). The Bufo UT protein lacks the ALE domain, which is
considered to be a signature sequence for UT-B. These data suggest that
Bufo UT belongs to the facilitative UT-A2 family. In immunoblot
analysis, Bufo UT protein was identified as a single band of 52 kDa
in extracts from plasma membrane fractions of the kidney and urinary bladder,
but not of the heart and liver. Similar to the glycosylated forms of mammalian
UT-A2 (molecular mass 55 kDa), Bufo UT probably has one
potential N-glycosylation site.
Under dry and hyper-saline acclimation, UT mRNA expression in the kidney
and urinary bladder as well as plasma urea concentrations was significantly
increased relative to that in control toads. In mammals and fish, changes in
UT mRNA expression under a variety of osmotic environments have been reported.
Smith et al. (Smith et al.,
1995) and Bagnasco (Bagnasco,
2000) demonstrated that dehydration increases the expression of
UT-A2 mRNA in rat kidney. Morgan et al.
(Morgan et al., 2003) found
that renal osmolytes and urea concentration were decreased and that renal UT
transcripts (SkUT) were significantly diminished in response to environmental
dilution in a marine elasmobranch. These data support the possibility that UT
expression in the kidney and urinary bladder of Bufo is regulated in
response to hyperosmosis of body fluid, and that up-regulation of UT
expression may contribute to urea accumulation in extracellular fluid.
The sites of passive urea absorption and the amounts reabsorbed have never
been clearly defined in the nephron of the amphibian kidney. In the present
immunohistochemical study, we showed that Bufo UT is localized in a
limited segment of the nephron known as the early distal tubule, or diluting
segment, in the ventral zone of the kidney. The UT was present on the apical
membrane but not on the basolateral membrane of epithelial cells in the early
distal tubule. In addition, no Bufo UT immunoreactivity was observed
along the late distal tubule, the connecting tubule and collecting tubule,
where vacuolar-type H+-ATPase is expressed in the intercalated
cells (Uchiyama and Yoshizawa,
2002). In a renal micropuncture study in Rana
catesbeiana, Long (Long,
1973) suggested that the amphibian collecting tubule has a very
low permeability to urea, so that essentially no urea is secreted into or
reabsorbed from the tubule fluid in this segment. Accordingly, our results
suggest that urea may be transported from the tubule to intracellular fluid by
passive permeation through the facilitative urea transporter located on the
apical cell membrane of the early distal tubule in the amphibian kidney.
However, movement of urea from the intracellular fluid to blood and/or the
extracellular space across the basolateral membrane has yet to be demonstrated
clearly. Kato and Sands (1998)
and Sands (1999) suggested
that an absorptive sodium/urea counter-transporter is located in the
basolateral membrane of the rat inner medullary collecting duct
(IMCD1). In addition, Walsh and Smith
(Walsh and Smith, 2001) have
pointed out two hypothetical components for active urea transport in the
elasmobranch kidney; an apical sodium/urea cotransporter and a basolateral
sodium/urea counter-transporter. In these models, an inwardly directed sodium
gradient in the epithelial cell, established by the basolateral
Na+,K+-ATPase, provides the driving force for urea
movement through the transporters. The early distal tubule of the amphibian
nephron has been demonstrated to show extremely low water permeability and
significant solute absorption (Stoner,
1977; Stoner,
1985; Oberleithner et al.,
1982). In this nephron segment,
Na+,K+-ATPase is located in the basolateral membranes of
cells (Uchiyama and Yoshizawa,
2002; Dantzler,
2003). Thus, in this segment expressing
Na+,K+-ATPase, movement of urea across the basolateral
membrane may occur through other urea transporters such as sodium/urea
counter-transporter. However, with regard to urea transport in the amphibian
urinary bladder, which has high water permeability, there may be not only a
facilitative urea transporter, but also several candidates such as
aquaglyceroporins, including aquaporin 3, and active urea transporters.
In this study, we found that Bufo UT mRNA was abundantly expressed
in the kidney and urinary bladder, but not in the ventral and dorsal skin. It
has been reported that the skins of frogs and toads develop inwardly directed
urea transport when the animals are adapted to hyperosmotic saline solutions
or to dehydration (Katz et al.,
1981; Garcia-Romeu et al.,
1981; Rapoport et al.,
1988). However, kinetic and pharmacological studies, and
investigations of hormonal sensitivity, have shown that urea transport in the
skin of amphibians differs from that in the urinary bladder (see
Ehrenfeld, 1998). Further study
of UT in the kidney and urinary bladder may help to characterize differences
in urea transport between the urinary system and the skin.
In conclusion, we have demonstrated that the UT cloned from the kidney of Bufo belongs to the UT-A2 family of facilitative urea transporter proteins. Immunohistochemically, we showed for the first time that the UT is located in the apical membrane of epithelia along the early distal tubule or the diluting segment of the amphibian kidney. Upon acclimation to dry and hyper-saline conditions, the plasma urea concentration and osmolality were significantly elevated, and these physiological changes were correlated with significant increases in the levels of Bufo UT mRNA in both the kidney and urinary bladder. Thus, the putative facilitative urea transporter expressed in the kidney and urinary bladder probably plays an important role in the urea retention response to hyperosmotic stress in the marine toad.
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Acknowledgments |
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