First published online December 10, 2003
Journal of Experimental Biology 207, 347-356 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00773
A facilitative urea transporter is localized in the renal collecting tubule of the dogfish Triakis scyllia
Susumu Hyodo1,*,
Fumi Katoh2,
Toyoji Kaneko2 and
Yoshio Takei1
1 Laboratory of Physiology, Ocean Research Institute, University of Tokyo,
1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan
2 Center for International Cooperation, Ocean Research Institute, University
of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan
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Fig. 1. Primary structure of the urea transporter (UT) isolated from the kidney of
Triakis scyllia. The deduced amino acid sequence is aligned with
those of the Squalus acanthias UT (accession number AF257331), rat
UT-A2 (U09957) and rat UT-B2 (U81518) using the Clustal algorithm. Amino acid
residues identical to the Triakis UT are indicated in black. A
putative N-glycosylation site and polypeptides used as antigens to
generate antibodies are indicated with horizontal bars.
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Fig. 2. Expression of the urea transporter (UT) in various tissues of Triakis
scyllia as examined by (A) RT-PCR and (B,C) northern blotting. (A) PCR
was performed using specific primers for Triakis UT (U) and
Triakis GAPDH (G) for three fishes (a-c). Lane 1, brain; 2, gill; 3,
kidney; 4, intestine; 5, rectal gland; 6, liver; 7, kidney without reverse
transcription. (B,C) 20 µg of poly-A+ RNA from the
Triakis kidney (lane 1), brain (2), liver (3) and gill (4) were
electrophoresed and hybridized with the 32P-labelled UT cDNA (B)
and GAPDH cDNA (C) in high stringent condition.
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Fig. 3. (A-C) Immunohistochemical localization of the urea transporter (UT; stained
in brown colour) in the Triakis kidney using antibody raised against
the C-terminal peptide of UT. Sections are counterstained with haematoxylin.
(A,B) Immunoreactive signal was detected in tubules in the bundle zone (BZ)
but not in tubules in the sinus zone (SZ) and in renal corpuscles (RC). (C)
Magnified view of five tubular segments in the bundle zone. Only one segment
(the collecting tubule) was immunoreactive to UT. (D,E) Confocal laser
scanning micrographs of immunoreactive tubules stained with
fluorescein-labelled secondary antibody. Cross-sectional view (D) and sagittal
view (E) of immunoreactive tubules. Scale bars: A, 200 µm; B, 100 µm;
C-E, 20 µm.
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Fig. 4. Immunohistochemical localization of Na+/K+-ATPase
(stained in brown colour) in the Triakis kidney. Sections are
counterstained with haematoxylin. (A) Low power micrograph. Large renal
corpuscles (RC) are situated between sinus and bundle zones. The
anti-Na+/K+-ATPase antibody stained numerous tubules in
both bundle and sinus zones (arrows and arrowheads). (B) Magnified view of
five tubular segments in the bundle zone. Intense signal to
Na+/K+-ATPase was detected in the ascending limb of the
third loop (arrow) and the convoluted early distal segment of the third loop
(arrowheads). Na+/K+-ATPase was not detected in the
collecting tubule (CT). (C) Magnified view of the convoluted early distal
segment at the distal end of the bundle zone. (D) Magnified view of the sinus
zone. Intense signal was observed in the basolateral membrane of the
intermediate segment (arrows). Arrowheads show the proximal II segment of the
second loop. Scale bars: A, 500 µm; B, 20 µm; C,D, 100 µm.
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Fig. 5. Identification of the nephron segment immunoreactive to the cloned urea
transporter (UT) using antibody raised against the C-terminal peptide of UT.
(A) Immunoreactive tubules in the bundle zone representing the straight
portion (arrowhead) and the convoluted portion (arrows) of the collecting
tubule. (B) Immunoreactive tubules (arrows) are connected directly to the
collecting duct (CD) in the bundle zone. (C) Magnified view showing the
connection between the immunoreactive tubule (arrow) and the collecting duct
(CD). (D) Schematic drawing of the course of a single nephron and of the
segment expressing the UT. UT was expressed only in the collecting tubule, the
final segment in the bundle zone, in the kidney of Triakis scyllia.
Each nephron makes four turns (1-4) and traverses repeatedly between the sinus
zone (SZ) and the bundle zone (BZ). The resulting five tubular segments in the
bundle zone are enclosed in a peritubular sheath. RC, renal corpuscle. Scale
bars: A,B, 100 µm; C, 20 µm.
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Fig. 6. Specificity check of the urea transporter (UT) antibodies by absorption
test. (A,B) The same nephron segments were stained with antibodies raised
against the C-terminal peptide (anti-UTC) and N-terminal peptide (anti-UTN) of
the UT. Treatment of the anti-UTC antibody with the UTN peptide did not affect
immunoreactivity (C), while preabsorption with the UTC peptide resulted in
complete extinction of the immunoreactive signals (arrows in D). Scale bar,
200 µm.
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Fig. 7. Western blot analysis using the anti-UTC antibody. The antibody recognized
a single band at  55 kDa (lane 1). The signal was not affected by
preincubation with the UTN peptide (lane 2), whereas the band disappeared
after preabsorption with the UTC peptide (lane 3).
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© The Company of Biologists Ltd 2004
