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Figures
- Fig. 1.
Transcript quantification with real-time RT-PCR. mRNA expression levels are shown for (A,E) Na+/K+ ATPase subunit α1 (NKAα1), (B,F) Na+-K+-Cl− cotransporter 2 (NKCC2), (C,G) Na+-Cl− co-transporter (NCC) and (D,H) urea transporter (UT) in bull shark (A–D) and houndshark (E–H) kidneys. Open bars represent seawater (SW; control) individuals, whereas filled bars show freshwater (FW)- or 30% SW-acclimated fish. Data are expressed as means±s.e.m. of n=4 SW bull sharks, n=3 FW bull sharks, n=5 (SW houndsharks) and n=4 (30% SW houndsharks). Statistically significant differences (t-test) are shown with asterisks (*P<0.05; **P<0.001; bull shark NKAα1, P=0.0192; bull shark NCC, P=0.000004).
- Fig. 2.
Representative kidney sections showing the sinus zone (SZ) and the bundle zone (BZ) of the bull shark kidney. Sections were stained with hematoxylin and periodic acid–Schiff (A–C). B and C are magnified views of the SZ and BZ, respectively. Open and filled arrows represent the second loop and the late distal tubule (LDT; fourth loop), respectively. Scale bars=500 µm (A) and 50 µm (B,C). (D) Schematic representation of the bull shark nephron.
- Fig. 3.
In situ hybridization analyses of mRNA encoding NKAα1, NKCC2, NCC and UT in the kidneys of bull sharks acclimated to SW and FW. NKAα1 (A–F), NKCC2 (G–L), NCC (M–R) and UT (S–X) mRNAs in SW- (A–C,G–I,M–O,S–U) and FW-acclimated (D–F,J–L,P–R,V–X) bull sharks. B,E,H,K,N,Q,T,W and C,F,I,L,O,R,U,X are magnified views of the SZ and BZ, respectively. The localization of each mRNA in the nephron is schematically illustrated (i–viii). Open arrows, filled arrows and open arrowheads represent the transitional segment from the early distal tubule (EDT) to the LDT, the LDT and the EDT, respectively. Filled arrowheads represent the transition segment from the LDT to the collecting tubule (CT) in the SZ, and the CT in the bundle zone. Note that NCC mRNA was intensely expressed in the LDT and the anterior part of the CT in FW-acclimated bull shark kidney. Sections were counterstained with Nuclear Fast Red. Scale bars=400 µm (A,D,G,J,M,P,S,V) and 50 µm (B,C,E,F,H,I,K,L,N,O,Q,R,T,U,W,X).
- Fig. 4.
Co-localization of mRNA signals in the kidney of bull shark in FW. NKAα1 (A,B), NKCC2 (C,D), NCC (E,F) and UT (G,H) mRNA in the SZ (A,C,E,G) and BZ (B,D,F,H). Filled arrows and open arrows in the sinus zone represent the LDT and the transitional segment from the EDT to the LDT, respectively. Filled arrowheads and open arrowheads in the bundle zone represent the CT and the EDT, respectively. Sections were counterstained with Nuclear Fast Red. Scale bar=50 µm.
- Fig. 5.
In situ hybridization analyses of mRNA encoding NKAα1, NKCC2, NCC and UT in the kidneys of houndsharks acclimated to SW and 30% SW. NKAα1 (A–F), NKCC2 (G–L), NCC (M–R) and UT (S–X) mRNAs in SW- (A–C,G–I,M–O,S–U) and 30% SW-acclimated (D–F,J–L,P–R,V–X) houndsharks. B,E,H,K,N,Q,T,W and C,F,I,L,O,R,U,X are magnified views of the SZ and BZ, respectively. The localization of each mRNA in the nephron is schematically illustrated (i–viii). Open arrows, filled arrows and open arrowheads represent the transitional segment from the EDT to the LDT, the LDT and the EDT, respectively. Filled arrowheads represent the transitional segment from the LDT to the CT in the SZ, and the CT in the BZ. Sections were counterstained with Nuclear Fast Red. Scale bars=400 µm (A,D,G,J,M,P,S,V) and 50 µm (B,C,E,F,H,I,K,L,N,O,Q,R,T,U,W,X).
- Fig. 6.
Immunohistochemistry of NCC in the kidney of FW bull shark. Signals were localized on the apical membrane of the LDT (A, open arrows). The use of preimmune serum (B) and pre-absorbed antiserum (C) resulted in the disappearance of the immunoreactive signals. Signals were not observed in the kidney of SW individual (D). (E) In situ hybridization of NCC mRNA in the kidney of FW bull shark showing the co-localization of immunoreactive signals (A) and mRNA signals (E) in adjacent sections. Sections were counterstained with hematoxylin (A–D) or Nuclear Fast Red (E). (F) Double-labeling fluorescence immunohistochemistry with anti-NCC antibody (magenta; indicated by arrows) and anti-NKA antibody (green). Scale bars=20 µm.
- Fig. 7.
Schematic diagram showing changes in kidney function between SW- and FW-acclimated bull sharks. (A) SW; (B) FW. Note that reabsorption of NaCl is enhanced in FW-acclimated bull shark kidney by the upregulated expression of NCC and NKAα1 in the LDT and the anterior part of the CT (shown in black). The enhanced reabsorption of NaCl in the CT may contribute to urea retention.
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