Laboratory of Pharmacology and Chemistry, National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina
27709
The organic anion
transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a
function associated with the basolateral membrane in rat proximal
tubule. To directly establish the subcellular localization of rROAT1 in
renal epithelia, we made a rROAT1-green fluorescent protein (GFP)
fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence
was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal
microscopy. Uptake of 3H-labeled
p-aminohippurate (PAH) increased
2.5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was
abolished by 1 mM probenecid. Thus the construct was capable of
specific organic anion transport. Cultured renal epithelial cell lines
(MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of
rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish (Fundulus
heteroclitus) renal tubules transfected with
pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse
cytoplasmic fluorescence. Function of the rROAT1-GFP construct was
demonstrated in transfected killifish tubules by fluorescein transport
assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new
technique for localizing cloned transporters within intact renal tubules.
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INTRODUCTION |
A MAJOR FUNCTION of the renal organic anion secretory
system in renal proximal tubule is to limit the toxicity of anionic xenobiotics, xenobiotic metabolites, and waste products of metabolism. Studies with intact kidneys, tissue slices, isolated renal tubules, and
membrane vesicles clearly demonstrate this organic anion transport is
driven by basolateral and apical membrane transporters and that these
transporters have separate and distinct physiological characteristics.
Movement across the basolateral membrane is a tertiary active process
that indirectly taps the energy of the sodium gradient (for review, see
Ref. 16). Once within the cell, substrates may be bound or sequestered
within vesicles (5, 13), followed by transport across the luminal
membrane, apparently by anion exchange or facilitated diffusion (1, 9,
27).
Although the physiological role of the kidney in the elimination of
charged xenobiotic compounds from the body has been investigated for
decades, it was only recently that we began to understand such
transport at the molecular level. Through the application of molecular
biology techniques and the Xenopus
oocyte expression assay system, cDNAs encoding some of these
transporters have been cloned (21), allowing their transport properties
to be examined in isolation, under controlled conditions. On the basis
of such physiological analyses, transporter positions within the renal transport model, and thus their subcellular localization, could be
assigned. However, there are examples in the recent literature in which
subsequent analysis has yielded conflicting data as to subcellular
distribution. When the kidney-specific organic anion transporter
rOAT-K1 was expressed and characterized in
LLC-PK1 cells, it was concluded
that rOAT-K1 was a basolateral membrane transporter in proximal tubule
(18). However, when a rOAT-K1 antiserum was used to examine kidney
basolateral and brush-border membrane preparations by Western blot, an
immunoreactive band was detected in the brush border, but none was
found in the basolateral membranes (10). Similarly, when the organic
cation transporter, rOCT2, was characterized in
Xenopus oocytes, it met the
physiological criteria of a basolateral transporter in proximal tubule
(14). Yet, when the pig OCT2 homolog was cloned from
LLC-PK1 cells and subsequently
expressed and characterized in human embryonic kidney 293 cells, it was
concluded that pOCT2 had the predicted physiological profile of an
apical membrane cation transporter (3). Additionally, the human
homolog, hOCT2, was detected in the apical membranes of distal (not
proximal) tubule cells in human kidney by in situ hybridization and
immunocytochemistry (2).
Therefore, to investigate the issue of subcellular localization from a
different perspective, we combined the technology of green fluorescent
protein (GFP) with the unique assay system of isolated teleost renal
proximal tubules. Each of these components offers several advantages
for this type of study. Since GFP fluorescence has been documented in a
wide variety of organisms, either it does not require any cofactors,
substrates, or additional gene products from the source organism
(Aequorea victoria), or the factors
it requires are ubiquitous (20, 26). GFP is ideal for use in
comparative studies, because it is stable when expressed in various
organisms and its fluorescence is species independent. There is also
evidence that GFP provides greater sensitivity and resolution than
staining with fluorescently labeled antibodies and that it is more
resistant to photobleaching (25). Perhaps one of the greatest
attributes of GFP is that detection can be performed in living cells
and tissues, e.g., cultured cells and isolated renal tubules. Teleost
fish represent a particularly good tissue source of proximal tubules,
because their nephrons contain a very high proportion of proximal
segment. Teleost proximal tubules are easily obtained, remain viable
for long periods of time, and broken ends rapidly reseal, maintaining a
fluid-filled lumen that only communicates with the medium through the
tubular epithelium (15). Additionally, the mechanisms of transport that exist in teleost renal tubules appear to be the same as those in
mammalian proximal tubules (15).
For the present study, we made a fusion construct of rat renal organic
anion transporter 1 (rROAT1, Ref. 24) and GFP, which allows direct visual observation of the subcellular
localization of the fusion protein (rROAT1-GFP). Function of rROAT1-GFP
was confirmed by Xenopus oocyte
expression assay. The fusion construct was transfected into cultured
renal cell lines, and a plasma membrane localization of the
fluorescent fusion protein was demonstrated with confocal microscopy.
Moreover, we report here, for the first time, the successful
transfection of intact, viable renal proximal tubules and the
subsequent detection of the rROAT1-GFP fusion protein in the
basolateral membrane.
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MATERIALS AND METHODS |
Plasmid construction. The coding
region of the rROAT1 gene was amplified from a full-length cDNA clone
(23) using PCR and the primers
Cycle
parameters were as follows: denature at 94°C for 4 min; followed by
10 cycles of 94°C denature for 1 min, 35°C anneal for 30 s, and
72°C extension for 2 min; followed by 30 cycles of 94°C
denature for 1 min, 55°C anneal for 30 s, 72°C extension for 2 min; with a final 10-min extension at 72°C. The PCR product was
digested with the restriction enzyme
Kpn I, gel isolated, and ligated into
the vector pEGFP-C3 (Clontech Laboratories, Palo Alto, CA) cut with
Kpn I. The resulting expression
vector, pEGFP-C3/rROAT1, contains an in-frame fusion of rROAT1 to the
carboxy terminus of green fluorescent protein (rROAT1-GFP) under the
control of the human cytomegalovirus promoter. The nucleotide
sequence of rROAT1-GFP was confirmed by fluorescent
sequencing on a DNA sequencer (model 377; Applied Biosystems, Foster
City, CA).
For Xenopus oocyte expression studies,
the DNA fragment containing the rROAT1-GFP fusion coding region was
removed from pEGFP-C3/rROAT1 by restriction enzyme digestion with
SnaB I and
BamH I. The fragment was gel purified
and ligated into pSPORT1 (GIBCO-BRL; Life Technologies, Gaithersburg,
MD) cut with SnaB I and
BamH I. The resulting plasmid, pSPORT1/rROAT1-GFP was linearized and used as template for in vitro
cRNA synthesis.
The organic cation transporter 2 (rOCT2)-GFP fusion construct was made
as follows: pSPORT1/OCT2, which contains the full-length rOCT2 cDNA
(22), was cut with the restriction enzymes
BamH I and
Kpn I; the fragment containing the
rOCT2 cDNA was gel isolated and ligated into pEGFP-C3 cut with
BamH I and
Kpn I, resulting in the plasmid
pEGFP-C3/rOCT2, which contains an in-frame fusion of rOCT2 to the
carboxy terminus of GFP (rOCT2-GFP). For
Xenopus oocyte expression studies, the
exact same strategy that was used to make pSPORT1/rROAT1-GFP was
employed, resulting in the plasmid pSPORT1/rOCT2-GFP.
The plasmid pCMV/myc/mito/GFP was purchased from Invitrogen (Carlsbad,
CA), and the ER-GFP plasmid was a gift from Dr. Richard McKay.
Xenopus oocyte expression assay.
Oocyte isolation procedures and uptake assay were performed as
described previously (24). Briefly, adult female
Xenopus laevis (Xenopus One, Ann
Arbor, MI) were anesthetized by hypothermia and decapitated. Stage V and stage VI oocytes were manually dissected free of the ovary, and the
follicles were removed by treatment with collagenase A. Oocytes were
maintained at 18°C in Barth's medium containing 0.05 mg/ml gentamicin sulfate, 2.5 mM sodium pyruvate, and 5%
heat-inactivated horse serum. Overnight recovery was allowed before injection.
An in vitro transcription kit (mMessage mMachine; Ambion, Austin, TX)
was used to synthesize capped cRNA from plasmid DNA linearized with
BamH I. The cRNA products were
quantitated in a spectrophotometer and diluted prior to injection to
allow delivery of 20-60 ng of cRNA/oocyte in 17 nl with a 10-s injection.
Six days after injection, the oocytes were divided into experimental
groups (containing 10 oocytes each) and incubated at 18-22°C
for 60 min in oocyte Ringer 2 (OR-2) containing 50 µM 3H-labeled
p-aminohippurate
([3H]PAH, 4 µCi/ml)
in the absence or presence of 1 mM probenecid. After uptake, oocytes
were rapidly rinsed three times with ice-cold OR-2 and placed into
individual scintillation vials containing 0.5 ml 1 M NaOH, incubated at
65°C for 20 min, and neutralized with 0.5 ml 1 M HCl. Finally, 4.7 ml of Ecolume (ICN Biomedical, Cleveland, OH) was added, and oocyte
radioactivity measured in disintegrations per minute in a Packard
1600TR liquid scintillation counter with external quench correction.
Tissue culture. The MDCK (established
cell line derived from renal distal tubules of an adult female cocker
spaniel) and LLC-PK1 (established
cell line derived from renal proximal tubules of a 3- to 4-wk old male
pig) cell lines were obtained from the American Type Culture Collection
(ATCC, Manassas, VA). Cell lines were negative for
mycoplasma upon receipt from ATCC. The MDCK line was retested before
publication and found to be negative for mycoplasma. Both cell lines
were maintained in Eagle's modified essential medium supplemented with
10% fetal bovine serum in a humidified incubator at 37°C with 5%
CO2. Cultures were split 1:20
approximately every 3-4 days.
Killifish (Fundulus heteroclitus)
were collected near the Duke University Marine Laboratory (Beaufort,
NC) and maintained in tanks with recirculating artificial sea water at
the National Institute of Environmental Health Sciences. After
decapitation, renal tubular masses were isolated, adherent
hematopoietic tissue was removed, and individual killifish proximal
tubules were dissected. After transfection, the tubules were maintained
in a confocal chamber containing 2 ml of medium 199 at 12°C.
Transfection. One day prior to
transfection, 1.5 × 105
cells were plated into
poly-D-lysine-treated
glass-bottomed confocal chambers (~4.9
cm2). Cells were transfected
with 8 µg plasmid DNA for 1 h at 37°C using SuperFect Reagent (2 µl SuperFect/µg DNA; Qiagen, Chatsworth, CA). Transfected cells
were washed with PBS, given fresh medium, and maintained at 37°C
with 5% CO2 for the remainder of
the experiment. Cells were examined by confocal fluorescence microscopy
~24 and 48 h after transfection.
Groups of killifish tubules were transfected with 8 µg plasmid DNA
for 1 h at 12°C using SuperFect Reagent (2 µl SuperFect/µg DNA). The tubules were subsequently rinsed with fresh medium 199 and
placed in a confocal chamber containing 2 ml of medium 199 and
maintained at 12°C for the remainder of the experiment. Tubules were examined by confocal fluorescence microscopy ~24 and 48 h after
transfection. Additionally, rROAT1-GFP transport function was
demonstrated in transfected tubules by fluorescein (FL) transport assay. Two days after transfection with either pEGFP-C3 or
pEGFP-C3/rROAT1, tubules were exposed to 1 µM FL in their culture
medium. After 30 min, confocal images were acquired.
Confocal fluorescence microscopy.
Cultured cells and tubules were imaged using a Zeiss model 410 inverted
laser-scanning confocal microscope fitted with a ×40
water-immersion objective (NA 1.2). Fluorescent images were collected
by illuminating samples with an Ar-Kr laser at 488 nm. A 510-nm
dichroic filter was positioned in the light path and a 515-nm long-pass
emission filter was positioned in front of the detector. Confocal
images (512 × 512 × 8 bits) were acquired as single 8- or
16-s scans, saved to an optical disk or high-capacity floppy disk (Jaz,
Iomega), and analyzed on a Power Macintosh 9600 computer
using NIH Image 1.61 software. For the FL transport studies, cellular
and luminal fluorescence intensities were measured from the stored
confocal images as described previously (11, 12). Briefly, for each
tubule, two to three adjacent cellular and luminal areas were selected.
After background subtraction, the average pixel intensity for each area
was calculated, and the values used for each tubule were the means for
all selected areas. Measurements were made from 9-12 tubules in
each group, and mean values were determined.
Statistics. Uptake data are presented
as means ± SE. Differences in mean values were considered to be
significant when P < 0.05, as
determined by unpaired Student's
t-test.
Chemicals.
[3H]PAH (3.7 Ci/mmol)
was obtained from Dupont-NEN (Boston, MA). FL and the fluorescent
Golgi-specific marker
6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine (NBD C6-ceramide) were purchased
from Molecular Probes (Eugene, OR). All other chemicals were obtained
from commercial sources and were of the highest grade available.
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RESULTS |
Xenopus oocytes. Oocytes expressing
rROAT1-GFP exhibited a low but detectable level of plasma
membrane-associated fluorescence, and water-injected oocytes showed no
such signal (Fig. 1). Transport function of
the rROAT1-GFP fusion protein was confirmed by
Xenopus oocyte expression assay (Fig.
2). Uptake of 50 µM
[3H]PAH by oocytes 6 days after injection with rROAT1-GFP cRNA was significantly higher than
in water-injected oocytes (2.5-fold increase,
P < 0.01), or in oocytes injected
with rOCT2-GFP cRNA. Treatment with 1 mM probenecid reduced uptake to
the level in water-injected oocytes, demonstrating that rROAT1-GFP
mediated specific PAH transport.

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Fig. 1.
Confocal micrographs of Xenopus
oocytes. Oocytes used in the p-aminohippurate (PAH)
transport experiment were examined by confocal microscopy prior to the
uptake assay. A: water-injected
oocytes show no plasma membrane-associated fluorescence.
B: oocytes injected with rROAT1-green
fluorescent protein (GFP) fusion construct (rROAT1-GFP) cRNA have a
distinct plasma membrane-associated fluorescence.
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Fig. 2.
Transport function of rROAT1-GFP expressed in
Xenopus oocytes. Six days after
injection with water or 60 ng of cRNA, oocytes were exposed to 50 µM
[3H]PAH for 60 min in
absence or presence of 1 mM probenecid. Oocytes injected with
rROAT1-GFP cRNA showed probenecid-sensitive PAH uptake. Neither
water-injected oocytes nor oocytes injected with rOCT2-GFP cRNA showed
any mediated PAH uptake. Data are means ± SE (10 oocytes/treatment). PAH uptake in absence of inhibitor was
significantly greater in oocytes injected with rROAT1-GFP cRNA than in
oocytes injected with water or rOCT2-GFP
(** P < 0.01).
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Renal cell lines.
LLC-PK1 cells were transfected
with pEGFP-C3 (expresses cytoplasmic GFP), pCMV/myc/mito/GFP (expresses
mitochondrial-targeted GFP), ER-GFP (expresses endoplasmic
reticulum-targeted GFP), or pEGFP-C3/rROAT1 (expresses rROAT1-GFP).
Confluent monolayers were examined 24 and 48 h after transfection by
confocal microscopy. Cells transfected with pEGFP-C3 had a diffuse,
evenly distributed signal over the entire cell including both cytoplasm
and nucleus (Fig.
3A). No
membrane-localized fluorescence could be detected. Cells expressing the
mitochondrial-targeted GFP had a distinctive, punctate fluorescence
found exclusively in the cytoplasm (Fig. 3B). In contrast, cells transfected
with pEGFP-C3/rROAT1 exhibited strong plasma membrane fluorescence, but
no nuclear fluorescence (Figs. 3C and
4). Additionally, a perinuclear region of
high fluorescence, believed to be in the area of the Golgi complex, was
detected (see below). When rROAT1-GFP-expressing cells were examined in cross section, the extensive labeling of the lateral membranes was
evident, as was the absence of a nuclear signal (Fig.
4B). However, whether there was
labeling of the basal and/or apical membranes was not readily
discerned. A weak reticulate pattern of cytoplasmic fluorescence was
also observed that was similar to the pattern seen in
ER-GFP-transfected cells (Fig. 5).

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Fig. 3.
Confocal images of transfected
LLC-PK1 cells in culture.
Transfection was carried out as described in MATERIALS
AND METHODS, and confluent monolayers were examined by
confocal microscopy. A: pEGFP-C3
transfection. Cells show a diffuse signal that fills the cytoplasm and
nucleus. B: pCMV/myc/mito/GFP
transfection. Cells show a punctate staining in the cytoplasm
(mitochondria) with no nuclear or plasma membrane-localized
fluorescence. C: pEGFP-C3/rROAT1
transfection. Note the strong plasma membrane-localized fluorescence
and exclusion of signal from the nucleus.
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Fig. 4.
Cross-sectional image analysis of rROAT1-GFP-expressing
LLC-PK1 cells in culture.
Transfection was carried out as described in MATERIALS
AND METHODS, and confluent monolayers were examined by
confocal microscopy. Microscope was focused at bottom of the chamber,
and 8-s scans were collected 0.5 µm apart (i.e., up through the
cell). Individual images were then compiled to render a
three-dimensional reconstruction of the tissue.
A: fluorescence image (single plane)
of pEGFP-C3/rROAT1-transfected cells. Diagonal line designates the axis
used for a cross section through the image stack.
B: cross-sectional projection
corresponding to line in A. Note
strong labeling of lateral plasma membranes and absence of signal in
nucleus.
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Fig. 5.
Cross-sectional image analysis of ER-GFP-transfected
LLC-PK1 cells in culture.
Transfection was carried out as described in MATERIALS
AND METHODS, and the stack of confocal images was
obtained as described in Fig. 4. A:
fluorescence image (single plane) of ER-GFP-transfected cells clearly
showing the reticulate pattern of endoplasmic reticulum staining. There
is no labeling of plasma membrane and no signal in nucleus. Diagonal
line designates the axis used for a cross section through the image
stack. B: cross-sectional projection
corresponding to line in A. Again,
note absence of plasma membrane-localized fluorescence.
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The plasmid constructs were also transfected into MDCK cells. As was
observed in the LLC-PK1 cells,
pEGFP-C3-transfected MDCK cells exhibited an intense, evenly
distributed cytoplasmic fluorescence that penetrated the nucleus, but
was not associated with the plasma membrane (not shown). Again,
rROAT1-GFP was highly localized to the plasma membrane, but was
excluded from the nucleus (Fig.
6B). The
perinuclear region of high fluorescence was seen, as well (Fig.
6B). Examination of a
cross-sectional view through an image stack again showed the
localization of rROAT1-GFP to the lateral, and perhaps basal, plasma
membranes, as well as its exclusion from the nucleus (Fig.
7B).
Targeting of rROAT1-GFP to the apical membrane could not be ruled out
by these images.

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Fig. 6.
Confocal images of pEGFP-C3/rROAT1-transfected MDCK cells in culture.
Transfection was carried out as described in MATERIALS
AND METHODS. A:
Transmitted light image showing confluent monolayer of cells.
B: corresponding fluorescence image to
A showing rROAT1-GFP fluorescence
localized to plasma membrane.
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Fig. 7.
Cross-sectional image analysis of rROAT1-GFP-expressing MDCK cells in
culture. Image stack was obtained as described in Fig. 4.
A: fluorescence image (single plane)
of pEGFP-C3/rROAT1-transfected cells. Diagonal line designates the axis
used for a cross section through the image stack.
B: cross-sectional projection
corresponding to line in A. Note
intense fluorescent signal localized to lateral plasma membrane.
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To confirm that the region of highly localized perinuclear fluorescence
was indeed Golgi, the fluorescent Golgi-specific dye NBD
C6-ceramide was used (Fig.
8). While observing a field of cells
containing both pEGFP-C3/rROAT1-transfected and
nontransfected cells, NBD
C6-ceramide was added to the
culture. The subsequent appearance of NBD
C6-ceramide-induced Golgi
fluorescence correlated with the perinuclear fluorescence seen in the
rROAT1-GFP-expressing cells (Fig. 8), suggesting that the localized
perinuclear GFP fluorescence represented some step in the Golgi
processing of rROAT1-GFP.

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Fig. 8.
Confirmation of rROAT1-GFP labeling of Golgi apparatus. Transfection
was carried out as described in MATERIALS AND
METHODS. A: field of
cells showing an isolated pair of rROAT1-GFP-expressing cells.
B: same field of view as in
A, 10 min after addition of 1.25 µM
NBD C6-ceramide, showing
correlation of the region of perinuclear fluorescence observed in
pEGFP-C3/rROAT1-transfected cells with Golgi-specific dye.
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Renal proximal tubules. Although
targeting of rROAT1-GFP to the lateral plasma membrane of renal cells
in culture was clear, evidence of insertion into the basal plasma
membrane was not as convincing, and insertion into the apical membrane
could not be ruled out. Therefore, we attempted to determine whether
intact renal tubules could be transfected and, if so, whether an
unambiguous localization could be obtained. Dissected renal tubules
from killifish were transfected with the pEGFP-C3 and pEGFP-C3/rROAT1
plasmids and examined by confocal microscopy at 24 and 48 h after
transfection. Untransfected control tubules had a low level of
autofluorescence (Fig.
9A). As
with the cultured cells, pEGFP-C3-transfected tubules showed a strong,
evenly distributed cytoplasmic and nuclear signal (Fig.
9B). In contrast, tubules
transfected with pEGFP-C3/rROAT1 showed specific localization to the
basal region of tubule cells (Fig. 9,
C and
D). No signal was observed in the
nucleus or above the nucleus at the apical pole of the cells. Cross
sections made through an image stack of a rROAT1-GFP-expressing tubule
clearly demonstrated the basal localization (Fig.
10).

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Fig. 9.
Confocal images of transfected killifish renal proximal tubules. Groups
of killifish tubules were transfected as described in
MATERIALS AND METHODS and
examined by confocal microscopy. A:
untransfected tubule showing low level of autofluorescence.
B: pEGFP-C3-transfected tubule showing
diffuse signal throughout cytoplasm and within nuclei.
C: low-magnification micrograph of
pEGFP-C3/rROAT1-transfected tubules showing intense basolateral
membrane fluorescence. D: higher
magnification micrograph of a pEGFP-C3/rROAT1-transfected tubule. Note
absence of signal from apical pole of tubule cells. Each bar = 40 µm.
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Fig. 10.
Cross-sectional image analysis of rROAT1-GFP-expressing killifish renal
proximal tubules. Image stack was obtained as described in Fig. 4.
A: pEGFP-C3/rROAT1-transfected tubule.
Lines b and
c designate the axes used for cross
sections through the image stack (shown in
B and
C, respectively). Note strong signal
in basal and lateral membranes of tubule cells and absence of signal
from nucleus and apical region.
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Active transport of FL by cytoplasmic GFP-expressing tubules, 2 days
after transfection, demonstrated the long-term viability of killifish
tubules in culture (Figs. 11 and
12). Function of the expressed rROAT1-GFP
fusion protein in transfected killifish tubules was demonstrated by
their increased capacity to accumulate the organic anion FL, over that
of pEGFP-C3-transfected tubules (Fig. 11). At steady state, the luminal
concentration of FL in tubules from the pEGFP-C3/rROAT1-GFP-transfected
group exceeded that observed in the pEGFP-C3-transfected
control group (Fig. 11). The average fluorescence intensities of
both the cells (2-fold increase, P < 0.05) and lumina (3-fold increase, P < 0.0001) of pEGFP-C3/rROAT1-transfected tubules were significantly
higher than in pEGFP-C3-transfected tubules, indicating increased
transport capacity in the rROAT1-GFP-expressing tubules (Fig.
12).

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Fig. 11.
Fluorescein (FL) transport in transfected killifish renal proximal
tubules. Two days after transfection with either pEGFP-C3 or
pEGFP-C3/rROAT1, tubules were exposed to 1 µM FL in their culture
medium. After 30 min, confocal images were acquired.
A: pEGFP-C3-transfected tubule showing
intracellular concentration of FL above the medium concentration and
fluorescence intensity of lumen higher than cells.
B: pEGFP-C3/rROAT1-transfected tubule,
also showing intracellular concentration of FL above the medium
concentration. Note the increase in luminal fluorescence intensity over
the tubule in A.
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Fig. 12.
Transport function of rROAT1-GFP in transfected killifish renal
proximal tubules. Two days after transfection with either pEGFP-C3 or
pEGFP-C3/rROAT1, tubules were exposed to 1 µM FL in their culture
medium. After 30 min, confocal images were acquired, and cellular and
luminal fluorescence intensities were measured from the stored confocal
images. Each bar represents mean fluorescence intensity for 9-12
tubules ± SE. Cellular and luminal fluorescence intensities were
significantly greater in pEGFP-C3/rROAT1-transfected tubules than in
pEGFP-C3-transfected tubules
(* P < 0.05 and
*** P < 0.0001, respectively).
Transport assay was performed on 3 separate occasions, and a
representative experiment is shown.
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DISCUSSION |
The cells of the renal proximal tubule are highly polarized with
respect to structure and function, and the transport properties of the
basolateral and apical membranes reflect this polarity (16). For the
"classic" organic anion transport system, the entry step is
mediated by organic anion/dicarboxylate exchange. Recently, organic
anion transporters were cloned from rat (rROAT1; rOAT1), mouse (mNKT,
also known as mROAT1), and winter flounder (fROAT) (7, 19,
24, 28). When expressed in Xenopus
oocytes, rROAT1 possessed all of the known functional properties of the basolateral organic anion/dicarboxylate exchanger (24). Here we
confirmed directly the precise subcellular localization of rROAT1 in
renal epithelia through observation of a rROAT1-GFP fusion protein.
rROAT1-GFP was found to be inserted into the plasma membrane of
Xenopus oocytes (Fig. 1) and shown to
possess transport function in those oocytes by expression assay (Fig.
2). Although rROAT1-GFP transport function was detected, its activity
was clearly less than the native gene. Determining whether this is an
indication that the GFP construct is not as efficient at transport, or
that the mRNA is less stable or not as efficiently transcribed, will require additional studies.
Initially, standard transfection techniques were used to examine the
expression of rROAT1-GFP in renal epithelial cell lines (LLC-PK1 and MDCK cells), and
rROAT1-GFP localization to the plasma membrane was observed (Figs. 3,
4, 6, and 7). Analysis of cross-sectional image projections from stacks
of confocal slices provided direct evidence of rROAT1 targeting to the
lateral membranes (Figs. 4 and 7). However, localization to the basal
membrane and exclusion from the apical membrane were not as certain.
Also, a reticulate cytoplasmic signal was observed in
rROAT1-GFP-expressing cells that resembled the pattern observed with an
endoplasmic reticulum-specific GFP construct (Fig. 5). We believe that
this could reflect the trafficking of rROAT1-GFP protein as it is
processed in the endoplasmic reticulum and transported through the
Golgi for packaging and subsequent insertion into the plasma membrane.
The Golgi-associated fluorescence detected in the rROAT1-GFP-expressing
cells, which was absent from cytoplasmic or mitochondrial
GFP-expressing cells, is consistent with this explanation (Fig. 8).
Since the polarity of rROAT1-GFP expression in the cell culture-based
system was inconclusive, we investigated whether the GFP constructs
could be successfully transfected into intact renal tubules.
Transfected renal proximal tubules from a teleost fish (killifish)
showed cytoplasmic GFP distribution similar to that in renal cell lines
in culture (Fig. 9B). In the intact
renal tubules, rROAT1-GFP was clearly expressed in a polarized manner, with fluorescence localized to the basal and lateral membranes (Figs.
9, C and
D, and 10). There appeared to be some
internal fluorescence in the cells of rROAT1-GFP-transfected tubules.
If this represented diffuse cytoplasmic fluorescence, then at some point it would have been visible in the region above the nucleus. This
was not the case. Therefore, this eliminated the possibility of a free,
diffuse cytoplasmic signal. It is well established that the basal and
lateral membranes of proximal tubule cells are not flat sheets, but
rather they have large infoldings that extend into the interior of the
cell (4). Labeling of these infoldings with rROAT1-GFP would certainly
make it appear as if the construct were localized to the basal
cytoplasm. Thus the labeling patterns are consistent with basolateral localization.
The FL uptake studies were indicative of
1) the long-term viability of
teleost proximal tubules in culture and
2) enhanced basolateral transport in
rROAT1-GFP-transfected tubules (Figs. 11 and 12). The increased
cellular fluorescence, relative to cytoplasmic GFP-expressing cells, is
consistent with greater uptake across the basolateral membrane. The
stronger luminal fluorescence is surely a consequence of the greater
free cytoplasmic pool of FL available for secretion in rROAT1-GFP
tubules, due to the increased uptake. These data confirm rROAT1-GFP
retains transport function in an intact tubule and also suggest a
basolateral localization.
The ability to use GFP technology in intact teleost tubules represents
a significant technical advance. By using intact renal tubules the
polarity and function of the native epithelium is preserved. Similar,
but less conclusive, results were obtained with mammalian (rabbit)
proximal tubules (not shown). However, the teleost system provides
several distinct advantages. The teleost tubules are maintained at
12°C, vs. 37°C for rabbit, and it has been documented that GFP
fluorescence is stronger at lower temperatures (6). Teleost tubules can
also be easily maintained in culture for up to 5 days without any
special treatment or equipment (8), whereas the rabbit tubules were
losing their integrity within 48 h. Perhaps most significant is the
ability to make functional measurements of secretory transport with the
teleost tubules.
Thus, through the combination of a comparative model (isolated
killifish proximal tubules) and GFP technology, we have confirmed the
basal and lateral plasma membrane localization of rROAT1 (Figs. 9 and
10). These are the first data demonstrating the usefulness of a
powerful new technique with which to establish a correlation between
the predicted position of a cloned transporter within the transport
model, based on physiology, and its actual subcellular distribution
within an intact, functional renal tubule.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
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Address for reprint requests and other correspondence: J. B. Pritchard, Laboratory of Pharmacology and Chemistry, NIH/NIEHS, Mail
Drop F1-03, P.O. Box 12233, Research Triangle Park, NC 27709 (E-mail: pritchard{at}niehs.nih.gov).