(Received for publication, August 8, 1996, and in revised form, January 2, 1997)
From the Renal and Genetics Divisions, Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115
We have identified a gene product (NKT) encoding an apparently novel transcript that appears to be related to the organic ion transporter family and is expressed almost exclusively in the kidney. Analysis of the deduced 546-amino acid protein sequence indicates that NKT is a unique gene product which shares a similar transmembrane domain hydropathy profile as well as transporter-specific amino acid motifs with a variety of bacterial and mammalian nutrient transporters. Nevertheless, the overall homology of NKT to two recently cloned organic ion transport proteins (NLT and OCT-1) is significantly greater; together these three gene products may represent a new subgroup of transporters. The NKT was characterized further with respect to its tissue distribution and its expression during kidney development. A 2.5-kilobase transcript was found in kidney and at much lower levels in brain, but not in a number of other tissues. Studies on the embryonic kidney indicate that the NKT transcript is developmentally regulated with significant expression beginning at mouse gestational day 18 and rising just before birth, consistent with a role in differentiated kidney function. Moreover, in situ hybridization detected specific signals in mouse renal proximal tubules. NKT was mapped by linkage disequilibrium to mouse chromosome 19, the same site to which several mouse mutations localize, including that for osteochondrodystrophy (ocd). Although initial experiments in a Xenopus oocyte expression system failed to demonstrate transport of known substrates for OCT-1, the homology to OCT-1 and other transporters, along with the proximal tubule localization, raise the possibility that this gene may play a role in organic solute transport or drug elimination by the kidney.
The proximal tubule of the kidney is a major site of transport of small organic molecules, including glucose, amino acids, and uric acid. The proximal tubule also plays a key role in drug elimination by the kidney. Xenobiotics and their metabolites are transported mainly by the organic anion and organic cation transport systems. These two transport systems share common substrates, and many functional features (2-9), and it is likely that these transport systems may also resemble each other at the molecular level.
A complementary DNA from rat kidney (OCT-1), which has the functional characteristics of organic cation uptake in the basolateral membrane of renal proximal tubules has been recently isolated (8). At the present time, only one nucleotide sequence (NLT) with significant homology to OCT-1 has been reported (9). NLT is a transporter protein of unknown substrate(s) present in the sinusoidal (basolateral) domain of hepatocytes. Increased expression of NLT at the time of birth correlates with the maturation of enterohepatic circulation. NLT is also present in the kidney although at a lower level than in liver. Organic anions, such as bilirubin and bromosulfophthalein, have been postulated as potential substrates for NLT, although this remains to be determined.
We report here the cloning and the molecular characterization of a transcript encoding a novel protein (NKT) apparently related to the recently identified OCT-1 and NLT. The gene product is almost exclusively expressed in kidney.
We have previously reported a method to selectively represent mammalian protein-coding regions based on statistically designed primer sets (1). This method is based on the distribution frequency of nucleotide combinations (k-tuples) in certain genetic subsets, and the combined ability of primer pairs, based on these oligonucleotides, to detect genes. Total RNA was prepared from various mouse tissues (brain, heart, placenta, lung, liver, spleen, kidney, and stomach) using the guanidinium thiocyanate-cesium chloride method (10). First strand cDNA was synthesized using a commercial kit (Life Technologies, Inc., Gaithersburg, MD). A 50-µl reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM each dNTP, 10 µg/ml oligo(dT)12-18, 5 µg total RNA, 200 units of Moloney murine leukemia virus (reverse transcriptase) was incubated 60 min at 37 °C, followed by PCR amplification. In hot start PCR microcentrifuge tubes (Fisher Scientific, Pittsburg, PA) a 20-µl reaction mixture containing 2 µl of solution from the first reaction (the final concentration of buffer components was 50 mM KCl, 1.425 mM MgCl2), 1 µM of each primer (Life Technologies, Inc.), 12.5 µM of each dNTP, 0.5 µM [35S]dATP, and 1 unit of Taq DNA polymerase (Perkin-Elmer) was used. The reaction mixture was subjected to 35 PCR thermocycles at 94 °C for 30 s to denature, 50 °C for 30 s for annealing, and 72 °C for 30 s for extension, followed by 5 min at 72 °C. For analysis of the PCR products, the samples were electrophoresed in 6% sequencing-grade gels, DNA bands were visualized by autoradiography.
Cloning and SequencingBands from these gels that were only present in the kidney were cut using a razor blade and DNA was dissolved in water and subsequently precipitated in a solution of 0.3 M sodium acetate (pH 6) and 2.5 volumes of ethanol. DNA in the pellet was reamplified using the same primer pair and PCR conditions. The amplified material was examined in a low-melting point 2% agarose gel, and a commercial kit (TA Cloning[Trade], Invitrogen, San Diego, CA) was employed to clone the PCR products. Positive clones (screened by blue-white changes) were grown in 1 ml of LB broth and plasmid DNA was isolated and then sequenced on an ABI 373A DNA fluorescent automated sequencer. Sequence homology searches were performed at the National Center for Biotechnology Information (NCBI) using the BLAST network service (11, 12).
RNA Blot AnalysisTotal RNA was extracted from several
mouse tissues (see above), as well as from mouse embryonic kidney from
several developmental stages as has been previously described (10).
Total RNA was electrophoresed on a 1% agarose/formaldehyde gel and
transferred to a nylon membrane. In addition, human multiple tissue
Northern blots I and II were purchased from Clontech (Palo Alto, CA).
The probe used for hybridization was the 332-base pair fragment from the NKT cDNA clone originally isolated from the differential
display gels. The probe was labeled with [32P] using a
random oligonucleotide labeling kit (Pharmacia). The final washes were
carried out at 65 °C. Blots were exposed to x-ray film with an
intensifying screen for 3 days at 80 °C.
Adaptor-ligated mouse kidney double-stranded cDNA
ready for use as template in 5- and 3
-RACE was purchased from
Clontech. Gene-specific primers for 5
- and 3
-RACE reactions were
designed based on the sequence of the 332-base pair fragment from the
NKT cDNA originally obtained from the differential display gels.
RACE reactions were performed using Clontech's Advantage[Trade]
KlenTaq Polymerase Mix, 0.5 ng of template, 50 µM of each
dNTP, 0.2 µM of the adapter primer (API), and 0.2 µM of either the 5
or 3
gene-specific primer (5
- and
3
-RACE respectively). The PCR products obtained were cloned and
sequenced as has been previously described.
Mouse kidney was collected, rinsed in
phosphate-buffered saline, and then fixed in ice-cold freshly prepared
4% paraformaldehyde/phosphate-buffered saline for 1 h. They were
then rinsed in 0.9% NaCl and dehydrated through a graded series of
ethanol and embedded in paraffin. 7-µm sections were cut, mounted on
slides, dewaxed, pretreated, and prehybridized as described in Wedden
et al. (13). Antisense RNA probes labeled with
[-35S]UTP (Amersham) were produced with T7 RNA
polymerase and HindIII-linearized PCR II-NKT. Hybridization
was done overnight at 50 °C. Post-hybridization treatments were as
follows: (i) two washes in 50% formamide, 2 × SSC, 20 mM mercaptoethanol (FSM) at 60 °C for 30 min, (ii)
digestion with 10 µg/ml RNase A in 4 × SSC, 20 mM
Tris-HCl (pH 7.6), 1 mM EDTA at 37 °C for 30 min, and
(iii) two washes in FSM at 60 °C for 45 min. Slides were dipped in
Kodak NTB-2 emulsion and exposed for 10 days. Slides were then stained
in 5 µg/ml Hoechst 33258 dye in water for 2 min, followed by rinsing
2 min in water. The slides were viewed under epifluorescence
optics.
Primers were designed to amplify a
region corresponding to the 3-untranslated region of NKT in order to
test for single strand conformation polymorphisms (SSCPs) between mouse
strains. These were analyzed as described previously (14). Briefly,
oligonucleotides were radiolabeled with [32P]ATP using
polynucleotide kinase and genomic DNAs from a series of mouse strains
were amplified using standard protocols (anneal at 55 °C for 1 min,
extend at 72 °C for 2 min, and denature at 94 °C for 1 min for 40 cycles, with a final extension at 72 °C). 2 µl of the amplified
reaction was added to 8.5 ml of U. S. Biochemical Corp. stop solution,
denatured at 94 °C for 5 min, and immediately placed onto ice. 2 µl of each reaction was loaded on a 6% nondenaturing acrylamide
sequencing gel and electrophoresed in 0.5 × TBE buffer for 2-3 h
at 40 watts in a 4 °C cold room. The primer pair with the sequences
CGGAGCCTGCCATTCAGAGAAAT (forward) and CTTGCAATGTCCTGGAGGTGGAA (reverse)
identified polymorphisms between C57BL/6J and Mus spretus, and was used to analyze DNA prepared from the BSS backcross (15) (Fig.
7). The strain distribution pattern was analyzed using the Map Manager
Program (16).
Xenopus Oocyte Microinjection and Transport Measurement
Xenopus oocyte expression was performed as described previously (28). Manually defolliculated oocytes were injected with 40-50 ng of rat kidney medulla mRNA or NKT cRNA. Five days after injection, the uptake of radioisotope-labeled substrates was determined. For analyzing urea transport (positive control), 2.74 µCi of [14C]urea/ml and 1 mM urea were added to the uptake solution containing 200 mM mannitol, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM Tris (pH 7.4). Uptake was stopped by washing the oocytes with ice-cold uptake solution containing unlabeled urea. Washed oocytes were dissolved in 10% SDS and radioactivity was counted in a scintillation counter. For organic anion and cation uptake, the same procedure was followed except that the uptake solution contained 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM Tris (pH 7.4). The uptake was stopped by washing the oocytes with ice-cold choline solution (100 mM choline, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM Tris, pH 7.4) and the radioactivity was counted as described above.
Using a new approach to selectively
represent mammalian protein-coding regions (1), we identified a novel
cDNA with a kidney-specific pattern of expression (Fig.
1). This clone is referred to as NKT cDNA.
NKT cDNA Nucleotide and Primary Amino Acid Sequence
The
NKT cDNA is 2161 nucleotides in length and contains both a
consensus polyadenylation signal (AATAAA), and a nucleotide poly(A)
tract defining the 3 end of the clone (Fig. 2). The
open reading frame is 1638 nucleotides long and encodes a protein of 546 amino acids. The deduced primary amino acid sequence of NKT is
shown in Fig. 2. The deduced amino acid sequence was separately confirmed from a cDNA clone amplified from mouse kidney mRNA. The AUG located in nucleotide position 182 has the strongest
translation initiation consensus sequence according to Kozak's rules
and was tentatively assigned as the first codon (17). An analysis of the primary amino acid sequence using the Kyte and Doolittle algorithm predicts 11
-helical transmembrane spanning domains (18) (Fig. 3). These same domains were identified as likely
transmembrane domains using the Eisenberg algorithm (19). The rather
large 100-amino acid loop between putative transmembrane regions one and two is presumably located extracellularly. This loop contains four
N-linked glycosylation consensus sites
(Asn-X-Ser/Thr) at positions Asn-56, Asn-86, Asn-91, and
Asn-107, as well as four cysteine residues Cys-49, Cys-78, Cys-99, and
Cys-122 that may be involved in the formation of disulfide bridges. In
addition, two hydroxyl amino acids (Ser-265 and Ser-270) located in the large intracellular loop between putative transmembrane domains six and
seven represent potential targets for kinase C phosphorylation (20,
21).
Computer Searches and Conserved Motifs
Comparison of the
deduced peptide sequence of this protein with those found in available
data banks revealed that NKT is a novel gene product related to the
family of nutrient transport proteins from eukaryotes and bacteria,
including, the mammalian facilitated glucose transporters, the yeast
transporters for maltose, lactose, and glucose, and the proton driven
bacterial transporters for arabinose, xylose, and citrate.
Computer-based homology searches of GenBank, EMBL, and SwissProt data
bases indicated that our cloned cDNA has not been previously
described. The data base searches indicated the NKT cDNA clone
shares the greatest homology with the rat organic cation transporter
(OCT-1) and a rat liver-specific transporter (NLT) of still
undetermined substrate specificity. These transporters were found to be
30 and 35% identical to NKT at the amino acid level (Fig.
4). These proteins are also homologous to a group of
sugar transport proteins including the human glucose transporters, the
Escherichia coli xylose-proton symporter, yeast low affinity
glucose transporter, and the yeast high affinity glucose transporter.
Although the sugar transporter family is a fairly diverse group,
members share several common structural features including, the
presence of 11 transmembrane spanning domains. Also, three short
sequence motifs (22) present in many transport proteins including
bacterial sugar/H+ cotransporters, mammalian facilitated
glucose transporters, bacterial citrate/H+ transporters,
and some drug resistance proteins. All of these motifs are within the
sequence of NKT clone. The first is a
Gly-(Xaa3)-Asp-(Arg/Lys)Xaa-Gly-Arg(Arg/Lys) motif, which
is conserved between the second and third transmembrane spanning
domains. The second set of conserved motifs includes the sequences
PESPRXL and PETK located after the predicted sixth and
eleventh transmembrane domains, respectively. A final conserved
sequence is a motif passing through transmembrane domains four and
five. The conserved sequence and amino acid spacing are
Arg-Xaa3-Gly-Xaa3-(Gly/Ala)-Xaa8-Pro-Xaa-Tyr-Xaa2-Glu-Xaa6-Arg-Gly-Xaa6-Gln-Xaa5-Gly.
The overall sequence homology, the presence of 11 putative transmembrane domains, and the presence of the specific transporter motifs, all strongly suggest that NKT is a member of this family of
proteins.
Tissue Distribution and Expression of NKT mRNA
A single
transcript of about 2.5 kb was observed in a mouse multiple tissue RNA
blot (Fig. 5A). The transcript was most
abundant in the kidney, but was also detected in very low levels in the brain. NKT transcript was not present in mouse heart, placenta, lung,
liver, spleen, or stomach. In human mRNA blots, a single transcript
of similar size (2.5 kb) was also observed in kidney. No signal was
detected in a large number of human non-kidney tissues (Fig.
5A).
In order to determine the temporal pattern of expression of the NKT gene during kidney development, we carried out RNA blot analysis of total RNA extracted from mouse kidneys at various stages of development. As shown in Fig. 5C, NKT transcripts appeared shortly before birth (fetal day 18) and were present at relatively high levels in the adult. Apparently the NLT gene becomes transcriptionally active close to the time of birth and remains active throughout adulthood.
In situ hybridization using sense and antisense cRNA on
mouse kidney paraffin sections showed that the most intense signal was
present in kidney cortex, following a pattern characteristic of
proximal tubular localization (Fig. 6). There was no
detectable signal in the glomeruli, distal tubules, or collecting
ducts. RNA blot analysis done in mouse microdissected kidney also
showed an intense signal in the cortex, a moderate signal in outer
stripe of the outer medulla, a faint signal in inner stripe, and no
signal in inner medulla (Fig. 5B), once more consistent with
a proximal tubular distribution.
Chromosomal Localization of the NKT Gene
SSCP analysis was
used to map the chromosomal localization of NKT (14, 23). Two primer
pairs corresponding to non-overlapping regions of the 3-untranslated
region of NKT were analyzed and found to identify SSCPs between mouse
species (see "Materials and Methods" and Fig. 7).
The BSS interspecific backcross was genotyped and the strain
distribution pattern, which were identical for the two primer pairs,
was analyzed using the Map Manager program. NKT was found to map to
chromosome 19 with a LOD likelihood score of 27.1. No recombinants were
found between NKT and D19Mit32 in 94 progeny; NKT is
therefore the most proximal gene mapped on chromosome 19 on the
BSS cross. This is the very site to which a number of unknown
murine mutations have been mapped (see "Discussion").
Since TEA and PAH are the prototype substrates
for organic cation and organic anion transporters, respectively (8,
24), and the organic anion transporter was reported to transport PAH by
exchanging with intracellular -ketoglutarate (30), we examined these
possibilities with NKT cRNA-injected oocytes under different conditions. The uptake of [14C]urea into rat medulla
mRNA-injected oocytes was used as a positive control. Uptake of
[14C]urea (1 mM) into rat medulla
mRNA-injected oocytes resulted in a
4-fold increase above that
of water control level. This is consistent with a previous study
reported by You and co-workers (29). Nevertheless, NKT cRNA injected
oocytes did not demonstrate a significant amount of transport of either
PAH and TEA under 100 µM concentration (Fig.
8a). When the concentration of these substrates (and, in addition, cimetidine) were increased to 1 mM (Fig. 8b), still no transport was activity
observed. Next, we preincubated 100 µM
-ketoglutarate
for 30 min before the uptake of 100 µM PAH was measured
(Fig. 8c); however, we were not able to show any transport
activity. Our results suggest that NKT cRNA-injected oocytes do not
demonstrate significant transport for the substrates tested, at least
under the conditions employed here (see "Discussion").
Renal tubular cells are responsible for the reabsorption and secretion of numerous substrates. These cells are highly polarized with unique species of transporters localized to either the basolateral or the apical domains of their plasma membranes. Using a new approach to selectively represent mammalian protein-coding regions (1), we have identified a novel transport protein which, by Northern analysis, is almost exclusively expressed in the kidney.
The sequence analyses of NKT suggest that it belongs to a recently identified subgroup of transport proteins. One member of this subgroup (OCT-1) has been shown to translocate hydrophobic and hydrophilic organic cations of different structures over the basolateral membrane of renal proximal tubules and hepatocytes (8). OCT-1 is currently considered a new prototype of polyspecific transporters likely to be important in drug elimination, although presently little is known of its specific role in various tissues. Xenobiotics and their metabolites are transported mainly by the organic anion (PAH) and organic cation transport systems, and there exist substrates that interact with both the transporter for organic anions and that for organic cations (2, 3). Neither transporter appears to detect the degree of ionization in bulk solution, and they also accept nonionizable substrates (4). Since these two transport systems (cationic and anionic) share so many common functional features, it is possible that they may also resemble each other at the molecular level. Functional expression of renal organic anion transport in Xenopus laevis oocytes injected with rat kidney poly(A)+ RNA has shown that the active species with respect to PAH transport was in the range of 1.8 to 3.5 kb (24). The size of NKT (2.5 kb) is within this range. Deduced amino acid sequence analysis showed that four cysteine residues are conserved among NKT, NLT, and OCT-1. Previous studies of the effect of N-ethylmaleimide (NEM), an irreversible sulfhydryl modifying reagent, on the transport of organic cations in the renal basolateral membrane imply that inactivation involves the binding of at least four molecules of N-ethylmaleimide per active transport unit. This is most consistent with the presence of four sulfhydryl groups at this site. The capability of organic cations to alter the susceptibility to sulfhydryl modification suggests that these groups may have a dynamic role in the transport process (25). For these and other reasons already discussed, NKT was considered to be a strong candidate for the important task of drug elimination by the kidney, a major function of the organ.
PAH and TEA are the prototype substrates of organic cation and organic
anion transporters. Therefore, we tested these possibilities by
measuring the uptake of the radiolabeled PAH, TEA as well as cimetidine
into Xenopus oocytes. However, under the conditions we
employed (including measurements in the presence of -ketoglutarate (30)), we were unable to show any transport activity. At present it is
unclear whether this negative result was due to suboptimal conditions
for NKT transport (despite robust transport in the positive control),
poor expression or insertion of an inactive (incompletely
processed) transporter protein, or because NKT might transport
other substrates than those we have examined so far. Expression of NKT
protein in other mammalian cells such as COS-7 cells may be required to
answer these questions.
Assuming NKT is a transporter, the forementioned data raises the possibility that it may have significantly different substrates from OCT-1. The low overall homology of the NKT sequence to the hexose transporters argues against its participation in sugar transport. When considering possible substrates for NKT, it is of importance to keep in mind that the expression of NKT appears to be kidney-specific, or at least relatively so. Both NLT and OCT-1 are located within the basolateral membrane. Other less closely related members of the family of nutrient transport proteins (e.g. SV2) are expressed in intracellular organelles or the membrane of synaptosomes rather than the plasma membrane (26). The subcellular location of NKT awaits the development of antibodies to carry out indirect immunofluorescent detection of the protein in kidney sections. In situ hybridization in mouse kidney showed that NKT is expressed in proximal tubules, but not in distal tubules, collecting ducts, or glomeruli (a pattern similar to that observed for OCT1). Confirmatory evidence for proximal tubular distribution was also obtained by Northern blot analysis with positive signals obtained in cortex and outer stripe, but not inner stripe and inner medulla. But unlike OCT-1 and NLT, transcripts were also detected in brain (mouse but not human), while no signal was found in liver (NLT and OCT-1) or small intestine (OCT-1). Of special interest is that NKT is expressed preferentially in the kidney and that its expression is developmentally regulated. The NKT transcripts appear shortly before birth. Studies of gene expression during kidney development have shown that genes appearing late in kidney development or at birth represent markers for highly differentiated kidney tubular cells, and these markers are often lost during neoplastic transformation. Therefore, NKT cDNA in addition to being related to organic ion transporters represents a new molecular marker for the terminally differentiated nephron.
SSCP analysis was used to localize NKT to mouse chromosome 19, tightly linked to D19Mit32. The human homologs of genes in this region such as Gstp1 and Adrbk1 map to 11q13 (27). Since subchromosomal linkage relationships are conserved in many cases between mouse and man, this result suggests that the human homolog of NKT will be found in this region. A number of interesting mouse mutations have been mapped to the proximal portion of chromosome 19, including several that affect neurological function or development (neuromuscular degeneration (nmd), muscle deficient (mdf), Dancer (Dc), deafness (dn)) or bone development (osteochondrodystrophy (ocd), osteosclerosis (oc)). Whether NKT plays a role in these murine mutations awaits further analyses.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U52842[GenBank].
We thank Christine Miller and Duane Hinds for expert technical assistance, and Lucy Rowe for help with data analysis.