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Physiology
Kidney-specific chloride channel, OmClC-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney
*Ocean Research Institute, University of
Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo
164-8639, Japan Asia; and
Homeostasis Medicine and Nephrology, Graduate
School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,
Tokyo 113-8519, Japan Asia
Communicated by Howard A. Bern, University of California, Berkeley, CA and approved October 9, 2002 (received for review June 19, 2002)
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Abbreviations: TALH, thick ascending limb of Henle's loop
Nishimura et al. (2) measured water and ion transport in nephron tubules isolated from freshwater-adapted rainbow trout, Oncorhynchus mykiss. They perfused nephron tubules and measured water and ion transport in the distal tubule. These results suggest that in freshwater-adapted rainbow trout, the distal tubule acts as a diluting segment, which is equivalent to the early distal segment of the frog kidney and the mammalian thick ascending limb of Henle's loop (TALH) (3, 4).
The extraction of NaCl from urine in the TALH is crucial to urinary concentration and dilution, which is accomplished by the active reabsorption of NaCl in the water-impermeable epithelia. Major advances have been made during the past few years in our understanding of NaCl transport mechanisms in the TALH. This understanding has been fueled by the molecular identification of Na-K-2Cl cotransporter (NKCC2) (5, 6), K+ secretory channel (ROMK) (7, 8), and basolateral Cl– channel (ClC-K2) (9–12) expressed in the TALH of mammalian kidney.
In teleost species, however, the ion-transport mechanisms in the kidney are less understood, and ion transporters of the fish nephron have not been identified thus far. In the present study, to investigate the molecular basis of ion transport in the teleost kidney, we have cloned a chloride channel from Mozambique tilapia, Oreochromis mossambicus. We named it OmClC-K, because it is a homologue of the mammalian kidney-specific chloride channels, ClC-K1 or -K2. To evaluate further the function of OmClC-K, immunohistochemical techniques were applied to the kidney of freshwater- and seawater-adapted tilapia. Our results indicate that OmClC-K is involved in the transepithelial Cl– reabsorption at the distal tubule of the kidney of hyperosmoregulating, freshwater tilapia.
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Materials and Methods |
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RT-PCR. Freshwater tilapia were anesthetized in 0.05% 2-phenoxyethanol. They were killed by decapitation, and the kidneys were immediately dissected out. Total RNA was extracted from the kidney by using ISOGEN Reagent (Nippon Gene, Tokyo). For RT-PCR, we made degenerate primers corresponding to the conserved sequences in the ClC-K family [rat ClC-K1 (GenBank accession no. D13927), K2 (D26111), Xenopus ClC-K (AJ011385) and partial sequence of zebra fish (Danio rerio) similar to mammalian ClC-K (BM776551)]: sense strand, 5'-CT(A/G)AA(C/T)GC(C/G)CAC(A/C)(A/G)(A/G)TGG-3'; antisense strand, 5'-GCCCA(C/G)TTT(G/T)CC(A/C/T)AG(A/G)AA-3'. These primers contained 5' extensions of eight nucleotides with restriction sites for BamHI and EcoRI, respectively. One microgram of total RNA was reverse-transcribed by using SuperScript II reverse transcriptase (Invitrogen) at 42°C for 60 min and then heated at 94°C for 5 min. The synthesized cDNA was used for subsequent PCR with synthetic primers in the following profile: 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles. The PCR products were cut with EcoRI and BamHI on both ends, ligated into EcoRI and BamHI-cut pSPORT1 (Invitrogen), and then sequenced.
Library Construction and Screening.
A freshwater tilapia kidney cDNA library in 
TriplEx2 expression
vector (CLONTECH) was constructed by the SMART cDNA Library
Construction Kit (CLONTECH). A PCR product that has a 150-bp insert
homologous to mammalian ClC chloride channel was screened from the
library. The full-length clone (2.9 kbp) obtained from the cDNA library
was subcloned into pTriplEx2 (CLONTECH), and nested deletion clones
prepared with Erase-A-Base System (Promega) were sequenced.
RNase Protection Assay. For detection of the expression of this clone, the PCR clone used for the library screening was linearized and used to prepare a radiolabeled antisense RNA probe. This probe (1 x 105 cpm per sample) was mixed with 20 µg of total RNA from the gills, kidney, urinary bladder, intestine, stomach, liver, spleen, skeletal muscle, and brain of freshwater- and seawater-adapted tilapia. After hybridization and subsequent RNase digestion (RPA II Kit, Ambion, Austin, TX), protected fragments were analyzed by electrophoresis in a 6% denaturing polyacrylamide gel.
Electrophysiological Analysis of OmClC-K in Xenopus Oocytes.
OmClC-K was cloned into the pSPUTK cloning vector (Stratagene)
with the SP6 promoter and Xenopus 
-globin 5' UTR
sequences. By using an in vitro capped RNA transcription kit
(mMESSAGE mMACHINE SP6 kit, Ambion), capped cRNA was transcribed from
the construct after linearization. For expression studies, oocytes were
defolliculated manually after collagenase digestion. Two hours after
defolliculation, stage V/VI oocytes were injected with either 20 nl
of nuclease-free water or 20 nl of 1.0 ng/nl cRNA solution as prepared
above with a 10-µl Digital microdispenser (Drummond Scientific,
Broomall, PA). The injected oocytes were incubated in modified Barth's
solution (88.0 mM NaCl/1.0 mM KCl/2.4 mM
NaHCO3/5.0 mM Tris•HCl/0.82 mM
MgSO4/0.33 mM
Ca(NO3)2/0.41 mM
CaCl2, pH 7.5). After 2–3 days at 16°C, they
were mounted in a recording chamber and impaled with microelectrodes
for measurement of current and voltage. Data acquisition and analysis
were performed by using commercially available software
(CLAMPEX PCLAMP8, Axon Instruments, Union City, CA).
The oocytes were clamped at –30 mV holding potential, and 1-s voltage
steps from –80 to +100 mV in 20-mV increments were applied. All
recordings were performed at room temperature in ND96 buffer (96 mM
NaCl/1.8 mM CaCl2/1 mM
MgCl2/5 mM Hepes, pH 7.4).
Generation and Affinity Purification of Anti-OmClC-K Serum. A specific antiserum was raised against a synthetic oligo peptide of OmClC-K in rabbits by Sawady Technology (Tokyo). The antigen was the C terminus of OmClC-K: Cys-Pro-Glu-Met-Lys-Arg-Ile-Leu-Glu-Asp-Leu-Ala-Lys-Glu-Ile. The amino acid sequence of the synthetic peptide had low homology with the corresponding sequence of other tilapia ClC chloride channels cloned in our previous study (13). The antigen was emulsified with complete Freund's adjuvant and immunization was performed in two New Zealand White rabbits. Boost injections were made into the rabbits with the peptide in incomplete Freund's adjuvant at days 14, 42, and 56 after the first injection. A specific antiserum from the rabbit with the highest titer was collected and the specific antibody was affinity-purified by using the antigen.
Western Blotting.
The specificity of the antibody against OmClC-K was confirmed by the
molecular mass of the endogenous protein by using Western
blot analysis. Membrane fractions were prepared from the kidney of
freshwater- and seawater-adapted tilapia. The kidney was homogenized on
ice in homogenization buffer [20 ml of 25 mM Tris (pH 7.4), 0.25 M
sucrose containing Complete Protein Inhibitor Mixture, Roche
Diagnostics]. The homogenate was first centrifuged at 8,000 x
g for 15 min, and the supernatant was subjected to higher
centrifugation at 400,000 x g for 1 h. The pellet
was resuspended in the homogenization buffer. All procedures were
performed at 4°C. Protein content of the sample was quantified by the
BCA Protein Assay kit (Pierce). The samples (20 µg) were solubilized
in a sample loading buffer (0.25 M Tris•HCl, pH 6.8/2%
SDS/10% -mercaptoethanol/30% glycerol/0.01%
bromophenol) and heated at 70°C for 15 min. They were separated by
SDS/PAGE using 7.5% polyacrylamide gels. As a molecular length
marker, prestained SDS/PAGE standards (188–7.3 kDa, Bio-Rad) were
electrophoresed in parallel. After electrophoresis, the protein was
transferred from the gel to a poly(vinylidene difluoride) (PVDF)
membrane (ATTO, Tokyo).
The membranes were preincubated in TBST [50 mM Tris-buffered saline (pH 7.6) containing 0.05% Triton X-100] and 2% skim milk at 4°C overnight and were then incubated with the primary antibody for 1 h at room temperature. The primary antibody was diluted at 1:500 with TBST. The specificity of the immunoreaction was also confirmed by incubating the membranes with preabsorbed antibody. After rinsing in TBST, the membranes were incubated with protein A gold conjugate (British Biocell International, Cardiff, U.K.) for 1 h at room temperature and then visualized by using silver-enhancing reagents (British Biocell International). The protein A was diluted at 1:500 with TBST.
Immunohistochemistry. Tilapia (15–20 g) adapted to freshwater or seawater were killed by decapitation, and the kidneys were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 h at 4°C. The kidney was then dehydrated in ethanol and embedded in Paraplast. Serial cross sections were cut at 4-µm thickness. Pairs of adjacent sections were mounted on separate slides for subsequent parallel examination and comparison; one section was stained with anti-Na+, K+-ATPase (14) and the other section was stained with an anti-OmClC-K serum. The sections were immunohistochemically stained with the Vectastain ABC kit (Vector Laboratories) as described by Uchida et al. (15). Some sections were also stained with hematoxylin and eosin to observe the general morphology of the kidney.
Electron Microscopic Observations. Small pieces of the kidney (3 mm each) were fixed in 2% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h at 4°C. The tissues were postfixed in 1% osmium tetroxide in the same buffer for 1 h. After dehydration in ethanol, the tissues were transferred to propylene oxide and embedded in Spurr's resin. Ultrathin sections were cut with a diamond knife and mounted on grids. The sections were stained with uranyl acetate and lead citrate, and examined a transmission electron microscope (Hitachi H-7100, Hitachi, Ibaraki, Japan).
Immunoelectron Microscopy. The kidneys were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 12 h, and then immersed in 20% sucrose in PBS (0.1 M) for 1 h at 4°C. Then, the tissues were embedded in OCT compound (Sakura Finetek, Torrance, CA), and frozen in a freezer. For immunoelectron microscopy, sections (20 µm thick) of the kidney were cut with a cryostat (Leica CM1100, Leica Microsystems, Wetzlar, Germany), and mounted on gelatin-coated slides. The sections were stained by the ABC method. After staining, the sections were washed in 0.1 M PBS, postfixed with 1% osmium tetroxide, and dehydrated in ethanol. Then, the tissues were embedded in Spurr's resin. Ultrathin sections were cut with a diamond knife and examined as described above without poststaining.
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280 bp) was
obtained. By using this PCR product as a probe, we screened the cDNA
library from freshwater-adapted tilapia kidney and obtained a 2.9-kbp
clone (GenBank accession no. AB075525). The ORF of this clone consisted
of 2,052 nucleotides, resulting in a 684-aa protein with a molecular
mass of 75 kDa (Fig. 1). The predicted
amino acid sequence of this clone was highly homologous to rat ClC-K1
(41%), ClC-K2 (43%), and a Xenopus ClC-K (xClC-K) (45%)
(Fig. 2A). Therefore, this
clone was designated as OmClC-K (O. mossambicus ClC-K). The
hydropathy analysis of the predicted protein (Fig.
2B) showed the presence of at least 13 hydrophobic
regions, which may represent transmembrane domains, as in other members
of the ClC family (16). The Asn-386 of OmClC-K located between
hydrophobic regions D8 and D9 (Fig. 1) was a highly conserved
glycosylation site among all ClC chloride channels identified to date.
A consensus sequence for phosphorylation by cAMP-dependent protein
kinase (Ser-546) also existed. To establish a possible evolutionary
relationship among the ClC chloride channels, a phylogenetic tree was
constructed by using the neighbor-joining method (Fig.
2A). A sequence comparison with other members of the
ClC family showed that OmClC-K is closest to the ClC-K subset.
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Renal Morphology of Tilapia, O. mossambicus. The kidney of tilapia is composed of numerous nephrons arranged in an irregular pattern. The nephron largely consists of a renal corpuscle and proximal and distal tubule segments (Fig. 4A). The distal segment is formed by the cuboidal cells with scanty microvilli and basally located nuclei (Fig. 4B). The epithelial cells of the proximal tubule are tall columnar cells with a centrally located nucleus and dense brush border on the apical side (Fig. 4C).
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ClC-K chloride channels belonging to the ClC chloride channel family are predominantly expressed in the kidney. Since the initial cloning of the ClC family, the identification of ClC-0 from the electric ray, nine mammalian ClCs have been cloned. ClC-1 is established as the skeletal muscle chloride channel that controls the excitability of the muscle fiber (25). The ubiquitously expressed ClC-2 can be activated by cell swelling in oocytes and possibly plays a role in the regulation of cell volume (26). ClC-3 (27), -4 (28), and -5 (29, 30) constitute a subbranch of this gene family. ClC-3 was recently shown to be a volume-regulated chloride channel (31), and loss-of-function mutations of human ClC-5 were shown to result in X-linked recessive nephrolithiasis (32). These channels have been cloned not only from mammals, but also from lower vertebrates, bacteria, and some plants. We have also cloned OmClC-3 and OmClC-5 (homologue of mammalian ClC-3 and ClC-5) from tilapia gills (13). The expression of OmCLC-5 is restricted to the osmoregulatory organs such as the gill, kidney, and intestine. It is likely that OmCLC-5 acts as a chloride channel and is responsible for osmoregulation of tilapia. Therefore, the ClC chloride channels are critical contributors of chloride transport in tissues of various species.
The present study demonstrated that the expression of OmClC-K mRNA was restricted to the kidney of freshwater-adapted tilapia (Fig. 3), as well as the protein expression (Fig. 5). Therefore, OmClC-K has an essential role for freshwater adaptation in tilapia. Moreover, the expression of OmClC-K protein is restricted to a specific nephron segment of freshwater tilapia (Fig. 6). These segments of the tilapia nephron are readily distinguishable as distal segments according to the cell size, shape, and presence of brush border in the apical surface of the epithelial cells. In immunoelectron microscopy, immunoreactive nephrons comprised low columnar cells with scanty microvilli on the apical surface. The basolateral membrane infoldings were extensive and were visualized by the immunoreaction of OmClC-K. These features concur with the general characteristics of the distal tubule segment in teleost kidney (33, 34). These results indicate that OmClC-K protein is expressed in the basolateral membrane of the distal tubules.
Nishimura et al. (2) found by a perfusion technique that the distal tubule of the freshwater-adapted rainbow trout was nearly impermeable to water, whereas net chloride absorption was demonstrated. This finding suggests that the distal tubule acts as a diluting segment that helps to excrete excess water as dilute urine by extracting only NaCl from the forming urine, as shown by the mammalian TALH. Recently, Kobayashi et al. (12) demonstrated that ClC-K2, a mammalian homologue of OmClC-K, was expressed in the TALH, where it is localized in the basolateral membrane. The localization of ClC-K2 to this nephron segment strongly implies that ClC-K2 confers the basolateral Cl– conductance in the TALH, where the Cl– is taken up by Na-K-2Cl cotransporter at the apical membrane (5).
We demonstrated that immunoreaction of Na+, K+-ATPase was observed on the same tubules of freshwater-adapted tilapia that were immunoreactive to OmClC-K (Fig. 6). Thus, the ultimate driving force for Cl– reabsorption across the epithelium may be Na+ electrochemical gradient established by Na+, K+-ATPase located in the same cell. It is most probable that Cl– enters the cell across the apical membrane by way of Na-K-2Cl cotransport as in other Cl– transporting epithelia, although the apical Na-K-2Cl cotransporter in the distal tubule has not yet been identified in the teleost. According to Nishimura et al. (2), furosemide, an inhibitor of Na-K-2Cl cotransport, added to the lumen of the distal tubule of freshwater trout markedly decreased transepithelial ion transport, whereas no change was noted when it was added to the basolateral side. These results suggest that the Cl– reabsorption mechanism of the distal tubule in freshwater tilapia is the same as that of the mammalian TALH.
To confirm the Cl–-transporting function of OmClC-K, we attempted expression of OmClC-K in Xenopus oocytes, but no Cl– current could be recorded. Several possible reasons exist to explain why OmClC-K cannot be functionally expressed as chloride channel. First, the expression system (Xenopus oocytes) may not be appropriate. Second, OmClC-K may reside in intracellular organelles, which are known to possess chloride channel as well (35–37). For OmClC-K, however, immunoreaction has been shown in the basolateral plasma membrane (Fig. 7). Finally, a possible explanation for the nonfunctional expression might relate to the multimeric assembly of ClC chloride channels. Biochemical and functional analyses have shown that many ClCs display a dimeric quaternary structure (38–41), suggesting that dimerization is probably a general feature of ClC's architecture. Therefore, functional expression of OmClC-K might depend on its association with another ClC subunit of as yet unknown nature.
Recently, Estevez et al. (42) identified barttin that is
mutated in a form of Bartter's syndrome and acts as an essential
-subunit for human ClC-Ka and ClC-Kb (human homologue of rat ClC-K1
and ClC-K2, respectively). Bartter's syndrome comprises several
closely related disorders of transepithelial transport in TALH.
The gene (43) encoding the integral membrane protein barttin is
mutated in a form of Bartter's syndrome that is associated with
congenital deafness and renal failure. When the barttin was coexpressed
with ClC-Ka or ClC-Kb in Xenopus oocytes, large
Cl– currents were observed. This result
indicates that barttin is the functional subunit for ClC chloride
channels. Although barttin has been identified only in mammals, tilapia
could also have a teleostean homologue of mammalian barttin.
The kidney of freshwater-adapted teleosts regulates the body fluid and salt balance in the aquatic environment and excretes dilute urine. Therefore, the distal tubule of teleost kidney acts as a diluting segment that helps in excreting an excess of water as dilute urine. The primitive forms of the nephron exemplified by those of the hagfish, a marine cyclostome, possess only renal corpuscles and the first segment of the proximal tubule, and their internal osmolality depends on the environmental media (44). However, in the river lamprey, a freshwater cyclostome, urine was hypoosmotic (45). Therefore, the diluting segment evolved during an early stage of vertebrate phylogeny and was likely essential for freshwater invasion and hyperosmoregulation. On the other hand, the kidney of mammals adapted to terrestrial life actively reabsorbs water to avoid dehydration. However, the diluting segment (mainly in the TALH) takes an essential role for urine concentration. Reabsorption of NaCl increases the NaCl concentration of the interstitium surrounding the TALH and that will drive water absorption from the descending limb of all nephrons (46). Consequently, the same properties of the diluting segment may be important in terms of the renal-functional evolution of the kidney from aquatic to terrestrial animals. The availability of homologous transport mediators such as ClC-K channels will be helpful in improving our understanding of molecular evolution of kidney function in relation to environmental adaptation.
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Acknowledgements |
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Footnotes |
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To whom correspondence should be addressed. E-mail:
hmiyazak{at}ori.u-tokyo.ac.jp. 
Data deposition: The sequence reported in this article has been deposited in the GenBank database (accession no. AB075525).
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, potential
N-glycosylation site.
