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WATER AND ELECTROLYTE HOMEOSTASIS
1Max-Planck-Institut für Molekulare Physiologie, Abteilung Epithelphysiologie, Dortmund, Germany; and 2Mount Desert Island Biological Laboratory, Salisbury Cove, Maine
Submitted 1 July 2006 ; accepted in final form 19 February 2007
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ABSTRACT |
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sodium glucose cotransporter 1; intrarenal localization; cloning; fish; elasmobranch
In early vertebrates like fish, sodium-dependent D-glucose transport has been observed in brush-border membrane fractions derived from hagfish, toadfish, flounder, trout, shark, and skate kidney (8). The latter studies revealed that in the skate kidney D-glucose transport exhibits a 2:1 stoichiometry (Na+:D-glucose) and a high D-glucose affinity (0.12 mM). Therefore, it was suggested that in the skate kidney, only an SGLT1-type transporter is operating. These observations differed from those with spiny dogfish shark (Squalus acanthias) kidney brush-border membrane vesicles, in which a 1:1 stoichiometry, a lower affinity to sugars, and phlorizin and different substrate specificity were observed (9).
In addition, it was found that in the closely related shark renal D-glucose reabsorption involves exclusively an SGLT2-type transporter (1). This prompted the current investigation to seek genomic evidence for the differences in the two transporters, as exhibited in the functional studies in vitro.
Furthermore, we found in the shark some striking differences in the intrarenal distribution of the transporter compared with the mammalian kidney (1). Thus, the second aim of the study was to create a specific antibody for use in immunohistochemistry to find out whether this phenomenon is restricted to the shark or whether it also occurs in the skate.
The current study demonstrates that the sodium-D-glucose cotransporter in the skate kidney belongs indeed to the SGLT1 subfamily. Skate SGLT1 is mainly expressed in the PIIa segment and the early distal tubule, which constitute 
60–65% of the nephron mass but also in the collecting tubule. Thus, its intrarenal distribution differs significantly both from that found in the shark and in the mammalian kidney. The higher stoichiometry of transport and the high abundance may also explain that in the skate, but not in the shark, D-glucose is completely reabsorbed even at high glucose loads.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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RNA extraction and cDNA synthesis. Total RNA was extracted with Tri Reagent (TRIzol) from Sigma (Deisenhofen, Germany) following the manufacturer's protocol. The tissue was homogenized using either a Polytron homogenizer (Kinematica AG, Switzerland) or a loose and a tight Dounce homogenizer. Possible DNA contamination was removed with DNaseI digestion with the DNA-free kit (Ambion) before use of the extracted RNA. RNA integrity was checked by formaldehyde agarose gel electrophoresis (1.2% agarose). To synthesize cDNA from a total RNA template, we used oligo-dT15 primer (Promega, Mannheim, Germany) and SuperScript II reverse transcriptase (GIBCO BRL, Inchinnan, UK). After first-strand synthesis, all samples were treated with RNase H (GibcoBRL) to remove the RNA template.
Preparation of cDNA library.
cDNA library was prepared with the SuperScript kit for cDNA libraries from GIBCO BRL. For integration into plasmid pSPORT1, first strands were synthesized from total RNA with oligo-dT15 with Not I adapter and labeled with [
32P]dCTP. To introduce asymmetry, Sal I adapters were ligated to the cDNA after second-strand synthesis. Subsequent digestion with Not I exposed the 5' extensions of Not I sites for ligation. cDNA was purified by size fractionation by column chromatography. The size-fractionated cDNA was then ligated into pSPORT1 vectors and transformed into Escherichia coli using electroporation.
Southern blot analysis. Probes for Southern blot (dot-blot) were labeled with DIG-dUTP in a random priming process with the DIG DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). High stringent screening included hybridization of the probe with DIG Easy Hyb Granules (Boehringer) at 68°C for 16 h, washing once with 2x SSC + 0.1% SDS for 5 min at room temperature, two times with 0.2x SSC + 0.1% SDS at 50°C for 15 min and once with 0.2x SSC + 0.1% SDS at 60°C for 15 min. Low-stringency conditions meant incubation at 40°C and the last two washing steps with 0.2x SSC at 50°C. Signals were detected with the CSPD chemiluminescence phosphatase system from Boehringer.
Cloning of SGLT from the kidney of L. erinacea. We previously reported the successful cloning of an SGLT2 from the kidney of S. acanthias by screening a cDNA library with a probe derived from trout (O. mykiss) kidney SGLT (1). In a similar approach, primers against conserved regions of mammalian sglt1 genes were designed and used in RT-PCR on trout kidney mRNA. A combination of 5'-AAT AAT GGC ACA GTG GCA GTC AAC-3' and 5'-ACC TCA TTG AAG GCA TAA CCC ATG-3' gave rise to a fragment of 650 bp. As this fragment showed similarity to sglt1 genes, it was named sglt1tk. The fragment was labeled and used as a probe to screen a library derived from L. erinacea kidney. Thereby, we isolated several clones that were subsequently subjected to restriction mapping, subcloning, and sequencing. Analysis showed that they were carrying a cDNA with high homology to sglt1 genes, as revealed by BLASTn search in the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) (2) nucleotide database. In a screen with the 1,573 bp probe containing sglt2tk (1), we detected the same 2,468 bp cDNA as with sglt1tk.
Plasmid preparation. To isolate plasmids from E. coli, we used the Plasmid kit from Qiagen (Hilden, Germany).
Sequencing. DNA was sequenced by labeling with BigDye terminator and reading on an ABI PRISM 3100 (Applied Biosystems).
Real-time PCR. Quantitative real-time PCR was performed in a GeneAmp 5700 Sequence Detection System from Applied Biosystems (Weiterstadt, Germany). Applied Biosystems also supplied all reagents used, including primers, probes, and disposables for real-time PCR. Fifty-microliter reactions contained 2x TaqMan Universal PCR Master Mix, 10 pmol TaqMan probe (labeled with carboxyfluorescein-reporter and carboxytetramethylrhodamine-quencher), 45 pmol of forward and reverse primer and 0–2 µl cDNA (20 ng) or plasmid solution as template. The standard thermal protocol was as following: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. The threshold of fluorescence signal was set to 0.06; the baseline was defined during seven or eight cycles in a range from cycle 7 to 17. Four replicates for each value were made. According to a standard curve derived from 7.50 pg, 1.88 pg, 0.47 pg, 0.12 pg, and 0.03 pg plasmid (141000 copies/pg) cloned from skate SGLT1, the control software of the GeneAmp 5700 calculated the quantity (in picograms) from the measured Ct-value in each well. Replicates with the highest deviation were taken out of the analysis. Statistics and conversion to the number of copies were calculated with Microsoft Excel. All values are presented as number of start copies in 20 ng cDNA with SE.
Topology model. On the basis of hydrophobicity [as predicted by TMHMM2.0; www.cbs.dtu.dk (10, 14)], a tentative topology model was generated. Furthermore, we used the PROSITE database (www.expasy.ch) (4) to predict possible sites for N-glycosylation and phosphorylation.
Uptake studies on Xenopus laevis oocytes. The library vector pSPORT is also suitable for in vitro transcription. Thus, the vector was linearized by HindIII digestion, and cRNA was synthesized using Ambion's T7 mMESSAGE mMACHINE kit. cRNA coding for skate kidney SGLT or a corresponding volume of water as negative control was injected into oocytes on the same or one day after preparation. To allow expression from the injected cRNA oocytes were incubated in Barth's solution supplemented with gentamycin under standard conditions for 3–5 days. Before uptake, the oocytes were washed 2x with Barth's solution and subsequently preincubated in Na+-free uptake solution for 3 min. Uptake solution for Na+-dependent uptake was composed of 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris, pH 7.5, 10 µM cytochalasin B, D-glucose in the indicated concentration, and 40 µCi/ml D-[3H]glucose. In the solution for Na+-independent uptake NaCl was replaced with 100 mM N-methylglucamine. Oocytes were placed in the respective uptake media for 20 min at room temperature after which uptake of D-[3H]glucose was terminated by removing the medium and washing 3x with cold stop solution (composition as for Na+-independent uptake). To measure the content of D-[3H]glucose, each oocyte was placed in a single vial and lysed in 10% SDS for 1 h. After this, ReadyProtein+ scintillation solution (Beckman Coulter, Fullerton, CA) was added, and counting was performed in a Beckman Coulter LS 6500. For each condition, 8–10 oocytes were tested, and experiments were performed twice with independent sets of oocytes. For analysis, data of the uptake experiments were pooled and analyzed employing Microsoft Excel and Microcal Origin 6.0 (nonlinear regression).
Generation of antibodies. As loop 13 has a quite unique primary structure for each isoform of SGLT, amino acids 553–634 from this region were selected as epitope for antigen production. The respective DNA fragment was amplified from the cloned cDNA and tagged with recombination adaptors for Invitrogen's Gateway cloning system (Karlsruhe, Germany). The sequence-specific parts of the primers were as follows: 5'-GAG GAT AAG CAC CTC CAC CGA-3' and 5'-TGA GAT GTC GGT CAG CTT CTT CTG-3'. After integration of the PCR product into the pGEX-5x-3 expression vector, the sequence information was checked by DNA sequencing. The peptide was produced in BL21 E. coli as glutathione S-transferase fusion protein, extracted, purified with glutathione-sepharose (Amersham Pharmacia Biotechnology, Piscataway, NJ), and sent to Biotrend (Köln, Germany) for immunization of rabbits. The immune serum was purified by Biotrend with protein A to obtain the IgG fraction.
Immunohistochemistry.
Immediately after the animals were killed and the kidneys were exposed, the tissue was fixed in situ by dripping ice-cold fixation fluid (4% paraformaldehyde in 3x PBS; 900 mOsmol/l) onto the samples. The tissue was dissected under a constant drip of fixation fluid. Small pieces from the middle part of the organ were transferred into fresh fixative and incubated overnight at 4°C. Afterward, the tissue was washed overnight at 4°C in PBS. The specimens were embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA) and frozen in liquid nitrogen; sections of 5–7 µm were obtained with a Leica Microsystems cryostat (Bensheim, Germany). After thawing and briefly air-drying the sections on glass slides, the sections were stored at –80°C until use. Unspecific immunoreactive binding sites were blocked with 3% nonfat dry milk in PBS during a 1-h incubation at room temperature. Subsequently, the respective primary antibodies were diluted 1:300 in PBS + 1% Triton X-100 and incubated on the slides for 1 h at room temperature. In antigen competition experiments, 50 µg of antigenic peptide were added to the antibody dilution before application. Immuno signals were detected with Alexa-Fluor 488 donkey anti-rabbit IgG or Alexa-Fluor 555 goat anti-rabbit IgG secondary antibodies (Molecular Probes, Eugene, OR) diluted 1:300, and incubated for 1 h at room temperature. The following fluorescein-labeled lectins were used to stain distinct nephron segments: Concanavalin A (Con A), Jacalin (JAC), Lens culinaris agglutinin (LCA), Lycopersicon esculentum lectin, Pisum sativum agglutinin (PSA), Peanut agglutinin (PNA), Phaseolus vulgaris erythroagglutinin (PHA-E), Ricinus communis agglutinin I, Solanum tuberosum lectin (STL), and succinylated wheat germ agglutinin (SWGA) (Vector Laboratories, Burlingame, CA). The dilution was 1:150 in PBS, and the incubation was performed for 20 min at room temperature. Nuclei were counterstained with the DNA dye 4',6-diamidino-2-phenylindole, which was included in the ProLong antifade Gold solution (Molecular Probes) used for mounting the sections. Multichannel analysis of the immunostainings was performed with an Axiovert 200M fluorescence microscope equipped with Plan-Neofluar lenses 16x/0.5, 25x/0.8 and F-Fluar 40x/1.3. Images were acquired with an Axiocam MR CCD camera Apotome (multidimensional images).
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RESULTS |
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Identification of renal structures and localization of SGLT1 protein in the skate kidney by immunohistochemistry. In the kidney of elasmobranch fish like L. erinacea, three different zones can be distinguished: 1) The lateral bundle zone with the countercurrent system, 2) a small region where the glomeruli are located, and 3) the zone of mesial tissue (3, 6, 7). Fig. 4 gives a schematic overview of the meandering course of a single nephron. It also shows the binding sites of an antibody raised against the predicted loop 13 of skate SGLT1 used for immunostainings, as described below.
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Proximal tubule segment PII. The later part of the proximal tubule, which is located in the mesial tissue, is called PII. It is further differentiated into PIIa and PIIb. PIIa has large high prismatic cells with a central round nucleus but only a small lumen. A strong fluorescence signal for SGLT1 was observed in the brush-border membrane of this segment. Single cells also reacted with PNA. Overlay of the two signals resulted in a yellow color (Fig. 6D). In PIIb the cells are smaller while lumen of the tubule gets wider. Here, binding of neither lectins nor the antibody against SGLT1 was observed (Fig. 7A).
Intermediate segment. The intermediate segment (IS) connects the proximal tubule to the distal tubule. The cells are cubic to flat with large nuclei. Interspersed are multicilliary cells. Thus, it is similar to the neck segment. Usually, the intermediate segment starts in the zone of the glomeruli and extends into the lateral bundle, forming part of the countercurrent system. As shown in Fig. 7A, JAC labeled parts of the low epithelium of the IS, but no reaction with the anti-SGLT1 antibody was found.
Early distal tubule. The early distal tubule segment (EDT) is found only in the lateral bundle zone. The EDT was stained by several lectins: LCA (Fig. 6C), JAC (Fig. 7A), and STL (Fig. 7B). There was also a strong signal for SGLT1 at the apical side of the cells, as evident from the yellow staining of the lumen of the early distal tubule in Fig. 6C and Fig. 7, A and B.
Late distal tubule. On the way to the mesial tissue zone the early distal tubule changes to the LDT. Out of the tested lectins, none labeled the LDT, and no labeling with the anti-SGLT1 antibody was found (Fig. 6B).
Collecting tubule-collecting duct system. After another turn in the mesial tissue back to the lateral bundles the cells of the late distal tubule flatten, spread more, and evolve into the collecting tubule. Back in the bundle zone, the collecting tubule eventually joins the collecting duct. JAC (Fig. 7A), and PHA-E (Fig. 7C) marked this nephron segment. Fig. 7, A and C shows also that in the CT, there was again strong colocalization with the antibody against SGLT1 at the luminal membrane.
Table 1 gives a semiquantitative comparison of the intensity of the immunoreactivity observed. It is noteworthy that the two segments that comprise the major mass of the tubule, the late proximal tubule PIIa with 50% of abundance and the early distal tubule (10–15% abundance), show strong to very strong signals. In addition the collecting tubule is clearly stained. These results suggest a very high expression of SGLT1 in the late tubular segments of the skate kidney.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Southern blot analysis screen of a skate kidney mRNA library resulted in the isolation of a cDNA that showed high homology to the mammalian SGLT1. The high homology to SGLT1 also extended to the derived amino acid sequence with 662 residues forming 14 transmembrane domains, a typical feature of this class of transporters (16) and containing sodium-symporter motifs. In a dendrogram of the comparison of the amino acid sequences, the skate transporter is sorted into the branch leading to the mammalian SGLT1 and SGLT3. One can now speculate that SGLT3 evolved after the separation of the chondrichthyes from the main path of evolution leading to the higher vertebrates.
There are several motifs (20) and single amino acids that are either conserved between the different members of the slc5a family or have been directly connected to the function of the protein (1). All of them are conserved in similar positions in the skate. The fact that all of these residues already appear in the early vertebrates underlines their functional relevance. However, some diversity among the SGLT sequences can be found. The NH2-terminus and the larger loops, especially loop 13, show more variation. Thus, this might explain the differences observed in the functional properties.
There are, however, some major differences between the skate and the shark protein: for example, the sequence of loop 13 is very different. This might be the reason for the different interaction of the skate and the shark transporter with phlorizin and alkylglucosides observed previously (9). However, further studies similar to ones performed with the isolated loop 13 of the rabbit SGLT1 (11, 12) are required to substantiate this assumption.
With regard to posttranslational modifications, there is also much homology (1). Computer prediction also found several of the possible phosphorylation sites in Leucoraja SGLT1 (Ser51, Ser304, Thr419, and Thr563). The threonine at 419 is no PKA site, as in the human SGLT1; instead, there is one at threonine 631. Several other sites are potential motifs for phosphorylation by casein kinase (four in loop 13). Recently, position Ser418 has been shown to be involved in the regulation of the transport activity of mammalian SGLT1 (S. Subramanian and R. K. Kinne, unpublished data). Investigation of the elasmobranch transporters might give additional information about which of these sites are of functional importance.
To prove the functionality of the cloned sequence and to investigate kinetic properties, cRNA was transcribed in vitro, injected into Xenopus laevis oocytes and uptake of D-[3H]glucose was measured. Oocytes injected with cRNA coding for skate kidney SGLT showed a sodium-dependent uptake of glucose with an apparent affinity of 0.11 mM. This is in agreement with the results obtained in vesicle studies where a Km of 0.12 mM had been determined (9) and is also in the range of mammalian SGLT1 (i.e., human SGLT1 Km 
0.8 mM; Ref. 5).
The expression of skate SGLT1 in different tissues is similar to mammalian tissues, too. The transporter from Leucoraja was cloned from and is expressed in kidney. But more than twice, the cellular concentration of transcripts can be found in the intestine. Furthermore, expression of SGLT1 in this organ was confirmed by immunostaining of the tissue with the anti-skate SGLT1 antibody used in this study (13).
Despite the many similarities between the mammalian and the elasmobranch SGLT1 in regard to the molecular features, transport characteristics, and tissue distribution, we found marked differences in the expression pattern along the nephron. In the mammalian nephron, a serial system of SGLT2 and SGLT1 exists. That is, in the early segments of the proximal tubule (S1), the low-affinity but high-capacity SGLT2 performs the bulk reabsorption of D-glucose. In the later segments of the proximal tubule (S3), SGLT1 can remove small residual amounts of sugar, because of its high affinity (17). The later parts of the nephron like the early and late distal tubule and the collecting duct then serve for fine-tuning the water inorganic electrolyte content of the urine but no longer absorb organic compounds.
In L. erinacea, we detected SGLT1 predominantly in the later proximal tubule PIIa, and only a weak signal was observed in the early proximal tubule PIa. This corresponds to the situation observed in the mammalian kidney. The presence of SGLT1 in the early distal tubule and in the collecting tubule of skate kidney is, however, not observed in the nephron of mammals.
Compared to Squalus acanthias, in which we detected SGLT2 in the proximal tubule PIa, PIb, PIIb, and LDT (1), it seems as if the place of D-glucose reabsorption moved one segment closer to the glomerulus. A comparable phenomenon has been observed for the NaPi-II transporters. In fish like flounder and zebrafish, NaPi is found in the late nephron segments. In contrast, the rat expresses NaPi only in the early segments. The reason for this shift during evolution could be the much higher glomerular filtration rate of mammals that requires, in return, use of the full reabsorptive potential of the proximal tubule (18). Whether this holds true for skate and shark remains to be determined.
According to personal observations the final urine of Leucoraja is free of glucose, even after D-glucose infusion. Thus, the one-transporter system in the skate kidney seems to be as effective as the two-transporter system in the mammals in reabsorbing glucose. This is probably achieved by expressing SGLT1 for a longer distance along the nephron and using the full reabsorptive potential of the proximal tubule. Whether the SGLT is involved in D-glucose reabsorption in the collecting tubule, too, is not clear; it might be necessary to compensate for the generally low capacity of SGLT1 transporters to achieve complete glucose reabsorption. But an involvement of SGLT1 in the transport of urea and/or water is also possible (20).
To conclude, we have reported the isolation and characterization of an SGLT1-type transporter from the kidney of the early vertebrate L. erinacea. In immunohistochemistry, the intrarenal distribution of the transporter revealed a localization of the transporter partly differing from the mammalian homologs.
On the basis of the current data, it seems unlikely that the skate expresses another SGLT-type transporter. Although we cannot exclude the existence of an SGLT2 in skate kidney completely, the vesicle studies, the lack of cross reactivity of the antibody, as well as PCR experiments, indicate a homogenous population of SGLT1.
These findings are quite in contrast to those obtained in shark kidney, raising some interesting questions on the relation between the two species.
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ACKNOWLEDGMENTS |
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Parts of the data were presented in form of abstracts in the Bulletin of the Mount Desert Island Biological Laboratory in Maine. Parts of the data are part of the diploma thesis of Thorsten Althoff at Philipps-Universität Marburg, Germany, supervised by Rolf K.-H. Kinne and Jürgen Kunz.
Present address for T. Althoff: Max-Planck-Institut für Biophysik, Abteilung Strukturbiologie, Frankfurt, 60438, Germany.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and absence (passive uptake 
) of Na+; Na+-dependent uptake (
) is the difference between both. Michaelis-Menten plot fitted with nonlinear regression (microcal origin) for skate kidney SGLT; Vmax = 207 ± 16 pmol/20 min; Km = 0.11 ± 0.04 mM. Values are means from 2 experiments (8–10 oocytes each condition).
