Department of Physiology, College of Medicine, University of
Arizona, Tucson, Arizona 85724
fluorescein; sodium-dicarboxylate cotransporters; transepithelial
transport in real time
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INTRODUCTION |
a wide variety of organic anions (or weak organic acids
that exist as anions at physiological pH), for which
p-aminohippurate (PAH) is a prototype,
are secreted by the proximal tubules of mammals and most other
vertebrates (12, 13). In the S2 segment of mammalian renal proximal
tubules, transepithelial secretion of organic anions (OA) involves
transport into the cells against an electrochemical gradient at the
basolateral membrane and movement from the cells into the lumen down an
electrochemical gradient (12). Transport into the cells at the
basolateral membrane is a tertiary active process, the final step of
which is the transport of OA into the cells against its electrochemical
gradient in exchange for a dicarboxylate (DC) [physiologically,
-ketoglutarate (
KG)] moving down its electrochemical
gradient through an OA/DC exchanger (9, 16). The outwardly directed
gradient for
KG appears to be maintained through a combination of
intracellular metabolism and
Na+-coupled secondary active
uptake of
KG across the basolateral membrane. This basic model,
first based on studies with renal basolateral membrane vesicles (BLMV)
(9, 16), has now been shown to function in intact renal proximal
tubules from mammals and reptiles (1, 2, 17, 24). A transporter that
mediates OA/DC exchange has now been cloned from mammalian renal tissue (15, 21).
Most studies that have attempted to determine the role of uptake of
exogenous
KG via
Na+-dicarboxylate (Na-DC)
cotransport in this OA secretory process have either involved
preloading tubules with abnormally high concentrations of
KG to
stimulate uptake (2, 19) or have involved nonphysiological buffer
solutions and temperature (11). Recently, Welborn et al. (24) used
physiological concentrations of
KG (~10 µM) (14) in the bathing
medium to examine the role of the basolateral Na-DC cotransporter in
establishing and maintaining the outwardly directed
KG gradient for
basolateral uptake of OA [using fluorescein (FL)] in
isolated renal tubules. Although the medium was as close to physiological as possible in this study, the tubules were not perfused.
Therefore, the degree to which basolateral Na-DC cotransport actually
functioned to support net transepithelial secretion was still unclear.
Moreover, filtered
KG is also reabsorbed by a luminal Na-DC
cotransporter, which has been cloned and sequenced (8) and shows a
higher capacity than the basolateral Na-DC cotransporter (27). It
appeared possible that filtered
KG taken up from the lumen by this
transporter also could contribute to the outwardly directed
KG
gradient for the basolateral uptake of OA. Indeed, in a previous study
with perfused rabbit tubules, we demonstrated that the addition of
KG to the lumen could increase net transepithelial PAH secretion
(5). However, this only occurred with abnormally high concentrations of
KG in the lumen and with reduced PAH secretion in the absence of
bicarbonate (5). Therefore, we were not certain that this process was
of physiological significance.
To determine more rigorously the roles of the luminal and basolateral
Na-DC cotransporters in establishing and maintaining net
transepithelial secretion of OA, we developed a system whereby we could
measure the net steady-state transepithelial secretion of FL in
isolated, perfused renal tubules in real time. Using S2 segments of
rabbit renal proximal tubules, we demonstrated that such secretion of
FL was a saturable, inhibitable process that occurred via the classic
OA (PAH) transport pathway. We also examined and quantified the roles
of the luminal and basolateral Na-DC cotransporters in this
transepithelial process under conditions as close to physiological as
possible. The results clearly indicate that the basolateral Na-DC
cotransporter plays a significant role in recycling
KG at that
membrane even in the absence of exogenous
KG. Although the luminal
cotransporter appeared to be markedly less important than the
basolateral cotransporter, the data indicate that it, as well as the
basolateral Na-DC cotransporter, can contribute to basolateral uptake
and net transepithelial secretion of FL under physiological conditions
when appropriate levels of exogenous
KG are present in both the
perfusate and bathing medium.
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METHODS |
Chemicals. Spectral-grade FL and
neutral tetramethylrhodamine dextran (TMRD) (40,000 mol wt) were
purchased from Molecular Probes (Eugene, OR). All other chemicals were
purchased from commercial sources and were of the highest purity available.
Solutions. A modified rabbit Ringer
solution, used throughout the studies (unless otherwise indicated) as
dissection buffer, superfusion bathing buffer, and perfusing solution,
consisted of the following (in mM): 110 NaCl, 25 NaHCO3, 5 KCl, 2 Na2HPO4, 1.8 CaCl2, 1 MgSO4, 10 sodium acetate, 8.3 D-glucose, 5 L-alanine, 4 lactate, and 0.9 glycine; and was adjusted to pH 7.4 with HCl or NaOH. This solution was
gassed continuously with 95%
O2-5% CO2 to maintain the pH. The
bathing medium also contained 3 g/100 ml neutral dextran (40,000 ± 3,000 mol wt) to approximate the plasma protein concentration. The
osmolarity of the solution was ~290
mosmol/kgH2O.
Preparation of isolated tubules. New
Zealand White rabbits, purchased from Myrtle's Rabbitry (Thompson
Station, TN), were killed by intravenous injection of pentobarbital
sodium. The kidneys were flushed via the renal artery with an
ice-chilled solution containing 250 mM sucrose and 10 mM HEPES,
adjusted to pH 7.4 with Tris base. They were then gently removed and
sliced transversely using a single-edge razor. A kidney slice was
placed in a petri dish containing ice-chilled dissection buffer aerated
with 95% O2-5%
CO2. Dissection of tubules from a
slice was performed manually from the cortical zone without the aid of
enzymatic agents. All dissections were performed at 4°C, but all
experiments were performed at 37°C. We used only proximal S2
segments in this study, because the S2 segment of the rabbit proximal
tubule is the primary site of OA (e.g., PAH) secretion (25).
Perfusion of tubules. The in vitro
perfusion technique used in these studies was the same as that
described previously (3, 4) with some modification so that the
collecting pipette had a length of uniform diameter that could be
positioned parallel to the bottom of the bathing chamber to serve as a
flow-through cuvette (Fig. 1). The outside
diameter of the collecting pipette was ~120 µm, and the inside
diameter was ~100 µm. The design of the collecting pipette reduced
background fluorescence from the bath, which was caused by the addition
of FL during transepithelial secretion studies, sufficiently to permit
a simple correction. Each isolated tubule was transferred into a
custom-made, temperature-controlled chamber with a coverslip as the
bottom. Both tubule ends were held in glass micropipettes, and the
tubule was perfused through a micropipette with its tip centered in the
tubule lumen at a rate of ~10-15 nl/min. The chamber was
continuously superfused with bathing medium at ~3 ml/min and the
temperature of the incoming solution was controlled at 37°C as
described previously (24). During perfusion experiments, FL was added
to the superfusion bathing media and TMRD was added to the perfusion
solution as a volume marker.

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Fig. 1.
Instrumentation for real-time measurement of transepithelial secretion
of fluorescein (FL). TMRD, tetramethylrhodamine dextran. See
METHODS for details.
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Measurement of FL and TMRD in collected
perfusate. Figure 1 shows the instrument setup
diagrammatically. The perfusion chamber was mounted on the stage of an
inverted microscope (Olympus model IMT-2) fitted with epifluorescence
optics. A ×60 oil-immersion objective (1.4 numerical aperture,
Olympus) was used to focus excitation light from a 100-W mercury arc
lamp and to collect fluorescence emitted from the solution in the
collecting pipette. The intensity of excitation light was reduced by a
2.0 neutral density filter (Oreil, Stratford, CT). Both FL and TMRD
were excited at 490 ± 10 nm using a selective band-pass
filter (Oriel) for this wavelength. The excitation light was reflected
to the sample with a 490DRLP dichroic filter (Omega
Optical, Brattleboro, VT), which passed more than 90% of emitted light
above 505 nm. The emission fluorescence was first limited to an area of
50 µm diameter by an iris diaphragm and then separated into two beams
by a second dichroic mirror (540DRLP, Omega Optical). Each beam was
appropriately filtered (520 ± 10 nm for FL; 580 ± 30 nm for
TMRD; Oriel and Omega Optical, respectively), and the two beams were
simultaneously counted, each by a separate photomultiplier tube (model
HC120; Hamamatsu, Bridgewater, NJ) in photon-counting
mode. The fluorescence intensity was integrated at 1-s intervals and
saved for subsequent analysis with a MSC II data acquisition
microcomputer interface and software purchased from Oxford Instrument
(Oak Ridge, TN). Figure
2A shows a
fluorescence profile of FL and TMRD during transepithelial secretion
(in arbitrary units) by an S2 segment of proximal tubule after the
bathing medium was changed to one containing 250 nM FL as indicated. At
the beginning of the experiment (Fig.
2A), 30 mg/100 ml TMRD was added to
the perfusion solution as a volume marker without any interference with
the signal in the FL-detecting channel. During transepithelial
secretion of FL, however, fluorescence from FL interfered with
fluorescence from TMRD in the TMRD-detecting channel. The amount of
this interference of FL in the TMRD-detecting channel was determined
from standard curves (see below) and simply subtracted to obtain the
actual counts for TMRD in the collected perfusate.

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Fig. 2.
A: fluorescence profiles for FL and
TMRD recorded simultaneously from the center of a pipette collecting
fluid from an isolated perfused S2 segment of rabbit proximal tubules
during a transepithelial secretion study. At the beginning of the
experiment, TMRD (30 mg/100 ml) was added to the perfusion solution as
a volume marker. This resulted in fluorescence in the TMRD channel
without any detectable counts in the FL channel. When the bathing
medium was switched to one containing 250 nM FL as well as TMRD, there
was a small and rapid increase in background fluorescence within 30 s
(not clearly seen in the tracings) followed by an increase in
fluorescence from FL that was secreted into the perfused lumen. FL
fluorescence was detected in both channels. The larger signal for FL in
the TMRD channel than in the FL channel reflects the higher gain in the
TMRD channel required to detect the TMRD signal. However, a correction
for the contribution of FL fluorescence to the TMRD signal can be made
(see methods), thereby providing the
actual counts of TMRD in collected perfusate.
B and
C: actual concentrations of FL
(B) and TMRD
(C) in collected perfusate. These
were obtained after the original counts in the collected perfusate from
each channel were corrected and converted using standard curves
constructed at the end of the experiment (all settings and the position
of the collection pipette during generation of the standard curves were
the same as those during the experiment) (see
methods).
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Determination of FL and TMRD concentrations in
collected perfusate. Concentrations of FL and TMRD were
determined from standard curves constructed at the end of each
experiment by retrograde infusion of known concentrations of FL or TMRD
into the collecting pipette while the bathing solution contained either
only bathing solution or bathing solution plus the appropriate
concentration of FL. The background fluorescence of FL in the bathing
medium during transport studies was determined by infusing perfusion solution alone into the collecting pipette while the bathing solution contained the appropriate concentration of FL for that experiment. The
background reading usually averaged less than 1% of total fluorescence
during secretion studies. To construct standard curves, we averaged
twenty 1-s data points for each FL or TMRD concentration. The
interference of the FL fluorescence in the TMRD-detecting channel was
also determined during infusion of FL into the collecting pipette. The
autofluorescence and the appropriate FL background counts were
subtracted from the counts obtained during net secretion to yield the
actual FL or TMRD counts in the collecting pipette. The photon count
was then converted into concentration from the standard curves. Figure
2, B and
C, shows profiles of the FL and TMRD
signals from Fig. 2A after conversion
to concentrations. About 30 s after adding 250 nM FL to the bathing
medium, fluorescence began to rise and reached a steady state within
about 5 min. The concentration of FL in the collected perfusate was
~16 times higher than the concentration in the bath indicating
transepithelial transport against a concentration gradient. The
concentration of TMRD did not change much in this and subsequent
studies. Therefore, no measurements of volume change due to water
reabsorption were made, and TMRD was used simply as an indicator of
leaks in the tubule throughout the study.
Measurement of transepithelial secretion of
FL. The net transepithelial secretion of FL,
JFL (in
mol · min
1 · mm
1),
was determined from the following relationship
In
the equation, VC is the perfusion
rate (in nl/min) measured directly;
CC is the concentration of FL (in
mol/nl) in the collected perfusate in the collecting pipette during
steady-state net secretion; and L is
the length of the perfused tubule (in mm) measured with an ocular
micrometer. This equation is based on the assumption that there is
essentially no backflux of FL from lumen to bath, an assumption shown
to hold for PAH (4, 22). The perfusion rate was ~10-15 nl/min.
Following the addition of FL to the bath, its concentration at steady
state was determined by a 1-min average of data points.
Statistical analysis. Results are
summarized as means ± SE; n is the
number of experiments. One tubule from one animal was used for each
experiment. Replicates for each experiment in a single tubule were
averaged to represent a single value for that experiment. Differences
in steady-state transepithelial secretion rates were evaluated by
either a paired t-test, a one-way,
two-sample t-test, or an ANOVA
followed by a multiple contrast posttest employing the Dunnett method
as indicated in the legends to Figs. 1-9. Differences were assumed
to be significant when P < 0.05.
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RESULTS |
Kinetics of steady-state transepithelial secretion by
isolated perfused S2 segment of renal proximal tubules.
Initially, we examined the profile of steady-state transepithelial
secretion of FL and determined kinetic parameters of the transport
process by S2 segment of proximal tubules. Figure
3 represents a profile of transepithelial
secretion by an isolated perfused S2 segment that was incubated in a
continuously flowing bathing medium that was alternately switched from
a FL-free medium to one containing FL at concentrations
ranging from 250 nM to 10 µM. For each concentration of
FL, a steady-state secretion of FL was reached ~5 min after the FL
was added. This steady-state secretion of FL increased rapidly as the
FL concentration increased from 0.25 to 2 µM and then leveled off at
higher concentrations (Fig. 3), a typical characteristic of a
saturating transport process. Table 1
summarizes the transport rate of FL at different concentrations by
isolated perfused S2 segments of proximal tubules in which the
perfusion rate was held constant for each tubule but varied from
10-15 nl/min among this set of experiments. Also
shown in this table is the tubular fluid-to-bath ratio (TF/B) of 1-mm
tubule length (n = 10). This ratio is
greater than unity at all concentrations of FL added to the bath,
indicating that transepithelial secretion involves transport against a
concentration gradient. This ratio decreased gradually from ~5 to
~1 as the concentration of FL increased from 0.25 to 10 µM,
suggesting that the transepithelial secretion involved a
carrier-mediated process in which carriers were becoming saturated at
high concentrations of substrate. Indeed, four of the tubules showed
TF/B values of less than 1.0 with 10 µM FL in the bath, indicating
that the secretory process was already saturated in these tubules. The
kinetic profile of transepithelial secretion of FL by the S2 segment of
proximal tubules can be adequately described by the following equation
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(1)
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This
equation has the same form as the Michaelis-Menten relationship, where
JFL is net
transepithelial secretion rate at steady state,
Jmax is the
maximal transepithelial secretion rate, and
Kt is the FL
concentration at one-half
Jmax. Kinetic
analysis revealed a
Kt of ~4 µM
and a Jmax of
~280
fmol · min
1 · mm
1
(Fig. 3B). Subsequently, we used FL
as the OA substrate at a concentration of 1 µM, which is well below
the Kt,
throughout the rest of our studies.


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Fig. 3.
A: a sample tracing of FL
transepithelial secretion rate profile in
fmol · min 1 · mm 1
from an isolated perfused S2 segment of rabbit proximal tubules in
response to addition of various concentrations of FL to the bath.
Tracing was obtained after conversion of concentration profile (as in
Fig. 2) using perfusion rate and length of the tubule measured directly
in the experiment. A steady-state transepithelial secretion was reached
(usually in ~5 min) after FL was added. Then the bathing medium was
switched back to standard buffer containing no FL (wash).
B: effect of increasing FL
concentration (shown in µM on abscissa) on rate of transepithelial
secretion by perfused S2 segments of rabbit renal proximal tubules
(shown in
fmol · min 1 · mm 1
on ordinate). Each point represents mean ± SE
(n = 10). Kinetic parameters,
Kt and
Jmax, were
derived for each tubule with a nonlinear regression algorithm
(Enzfitter, Biosoft). Line fitted to data was calculated from
Eq. 1 using averaged kinetic
parameters obtained from each tubule.
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Table 1.
Steady-state tubular fluid-to-bath ratio and transepithelial
secretion rate of FL at different FL concentrations
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Inhibition of transepithelial secretion of FL by
PAH. In recent years, FL has been used as a model
substrate for the peritubular OA/DC exchanger on the assumption that
the basolateral uptake of FL utilizes the classic OA (PAH) transporter.
Increasing evidence supports the idea that basolateral uptake of FL by
proximal tubules involves and is limited to the PAH transporter (19,
23, 24). However, we wished to establish that transepithelial secretion of FL involves the same transport system as that for PAH. That this is
the case is indicated by the data shown in Fig.
4. The transport rate of FL was decreased
when PAH was added during the steady-state secretion of 1 µM FL. The
inhibition increased with increasing PAH concentration and was
completely reversed after removal of PAH. The kinetic profile suggested
competitive inhibition by PAH. The kinetic parameters were calculated
by a modification of the isotope dilution procedure of Malo and
Berteloot (7) according to the following equation
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(2)
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where
J,
Jmax, and
Kt are as
previously defined, [I] is the concentration of inhibitor
(PAH), Ki is the
Michaelis constant of the inhibitor, [*T] is the
concentration of FL, and D is a coefficient describing nonsaturable
transport (passive diffusion). Equation 2 can be rearranged to give the following
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(3)
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where
Japp is defined
as
(Ki/Kt)Jmax,
Ki app is
an apparent Ki
that is defined as
Ki(1 + [*T]/Kt),
and C is a constant derived from D and [*T]
reflecting passive transepithelial flux through or between tubule
cells. Therefore, knowledge of J,
[I], and [*T] permits the calculation of
Japp,
Ki app, and
C using nonlinear regression analysis. When [*T] is <<
Kt,
Ki app
Ki. Analysis of
12 separate experiments yielded a
Ki app
value for the inhibition of FL secretion by PAH of ~108 µM (Fig.
4B). This value is almost identical
to the concentration for half-maximum transport
(Kt) concentration of PAH of ~110 µM reported previously for S2 segments of rabbit proximal tubules (6, 17). The similarity between these
kinetic parameters strongly supports the idea that FL and PAH share the
same transport system. In addition, transepithelial secretion of 1 µM
FL decreased from 45 ± 6 to 4 ± 1 fmol · min
1 · mm
1
(91% inhibition) (Fig. 4B), when 5 mM PAH was added to the bathing medium. This finding further indicates
that transepithelial secretion of FL is essentially limited to the PAH
transport system. In other words, it indicates that less than 10% of
transepithelial secretion of FL is by some other pathways (e.g.,
passive paracellular flux).


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Fig. 4.
A: a sample tracing of FL
transepithelial secretion rate profile in
fmol · min 1 · mm 1
from an isolated perfused S2 segment of rabbit proximal tubules in
response to addition of various concentrations of
p-aminohippurate (PAH) to bath. FL
concentration was 1 µM. Addition of PAH to bath during steady
secretion of FL resulted in a concentration-dependent reversible
inhibition profile. B: effect of
increasing PAH concentration (shown in mM on abscissa) on steady-state
transepithelial secretion rate of 1 µM FL by perfused S2 segments of
rabbit renal proximal tubules (shown in
fmol · min 1 · mm 1
on ordinate). Each point represents mean ± SE
(n =12, except at PAH concentrations
of 0.02 and 0.05 mM, where n = 11).
Kinetic parameters,
Ki app and
Japp, were
derived for each tubule with a nonlinear regression algorithm
(Enzfitter, Biosoft). Line fitted to data was calculated by a
modification of isotope dilution procedure of Malo and Berteloot (7)
using averaged kinetic parameters obtained from each tubule.
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Influence of basolateral Na-DC cotransporter on
transepithelial secretion of FL in the absence of exogenous
KG. The basolateral Na-DC cotransporter plays an
important role in the tertiary transport model for peritubular uptake
of OA by helping to maintain the in > out gradient for
KG that
otherwise could be dissipated during exchange for peritubular OAs (9,
13, 16). However, the actual extent to which recycling of
KG by the
basolateral Na-DC cotransporter contributes to the transepithelial
secretion of OAs is not certain. To obtain a quantitative assessment of
the importance of reuptake by the basolateral Na-DC cotransporter of
KG that has been exchanged for FL, we inhibited this cotransporter with LiCl in the absence of exogenous
KG. We added 2 mM LiCl to the
bathing medium in the absence of exogenous
KG while measuring the
transepithelial secretion of 1 µM FL. This concentration of LiCl has
been shown to inhibit the Na-DC cotransporter (26). When LiCl was added
to the bathing medium, FL secretion decreased from the control value of
~54 to 41 fmol · min
1 · mm
1,
a decrease of ~23% (Fig. 5). This
inhibition was reversible upon removal of LiCl from the bathing medium.
In contrast, when the same concentration of LiCl was added to the
luminal perfusion solution, it had no effect on FL secretion (Fig. 5).
Taken together, these results indicate that the inhibitory effect of
LiCl when added to the bath is likely limited to inhibition of
KG
recycling by the basolateral Na-DC cotransporter rather than to other
metabolic effects of any LiCl that might have entered the cells via the Na-DC cotransporters located on either basolateral or luminal side.
Therefore, we conclude that recycling of
KG by the basolateral Na-DC
cotransporter contributes ~25% to net OA secretion.

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Fig. 5.
Effect of 2 mM LiCl added to bath or perfusion solution (lumen) on
steady-state transepithelial secretion of 1 µM FL by isolated
perfused S2 segments of rabbit renal proximal tubules. Values are means ± SE. Numbers in parentheses indicate numbers of experiments.
* Significantly different from control group
(P < 0.05) as determined by ANOVA.
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Influence of exogenous basolateral
KG on
transepithelial secretion of FL. Most previous studies
on mammalian proximal tubules used nonphysiologically high
concentrations (100 µM) of dicarboxylates,
KG or glutarate, for
preloading the cells to maximize the stimulatory effect on
OA transport in a dicarboxylate-free medium (2, 20). This protocol does
not take into consideration the effect of the physiologically available
KG in the plasma, ~10 µM (14), on OA transport. Although
Pritchard (11) showed that the addition of 10 µM
KG to the medium
bathing rat renal cortical slices caused a 30% increase in PAH uptake,
the experiments were performed under nonphysiological conditions (i.e.,
at room temperature, and in nutrient-free and bicarbonate-free
phosphate buffer), conditions that are likely to compromise cellular
metabolism and rates of OA transport. Therefore, we sought to evaluate
the extent to which exogenous
KG influences FL transport under
conditions resembling as closely as possible those to which proximal
tubules are exposed in vivo (i.e., nutrient-rich bicarbonate buffer at
37°C).
We first determined the influence of exogenous peritubular
KG on
steady-state transepithelial secretion of 1 µM FL by adding increasing concentrations of
KG to the bathing medium. Figure 6 is a typical profile of transepithelial
secretion of 1 µM FL by an isolated perfused S2 segment of proximal
tubules in response to exogenous peritubular
KG at concentrations
ranging from 5 µM to 1 mM. As shown in Fig. 6 and summarized in Table
2, the addition of
KG to the bathing
medium affected FL secretion in a biphasic manner. Concentrations of
KG below 200 µM significantly stimulated FL secretion;
concentrations of 500 µM and above significantly inhibited it in a
concentration-dependent manner. These findings support the idea that
high concentrations of dicarboxylates interact competitively with OAs
at the extracellular face of the OA/DC exchanger. Maximum stimulation
of secretion (~40-50%) was found at
KG concentrations
ranging from 10 to 100 µM. Upon removal of 1 mM
KG from the
bathing medium, there was an abrupt increase in FL secretion above the
control level, presumably due to the accumulation of
KG within the
cells following the incubation at high concentrations of
KG (i.e.,
the equivalent of preloading renal tubules with a high concentration of
KG). This increase was transient and gradually decreased to the
control level in the absence of exogenous
KG (Fig. 6). The finding
that 10 µM of peritubular
KG stimulated net secretion of FL
~40% (Table 2) indicates that under physiological conditions uptake
of exogenous
KG by the basolateral Na-DC cotransporter can support
~29% of net OA secretion; i.e., [(increase in secretion in
presence of 10 µM
KG in bath from the "basal" state)/(net
secretion in presence of 10 µM
KG in bath)] × 100%.
Although it appeared most likely that
KG that entered the tubule
cells via the basolateral Na-DC cotransporter stimulated
transepithelial FL secretion by countertransport at the basolateral
membrane, it was also possible that stimulation could have resulted
from metabolism of
KG. To be certain that this was not the case, in
another set of experiments, we examined the effects of glutarate or
KG in the bath on FL secretion by the same tubule. Glutarate is not
significantly metabolized by the renal cells (10) and is one of the few
dicarboxylates other than
KG that is exchanged for PAH at the
basolateral membrane (9). As shown in Fig.
7, the addition of 10 µM glutarate to the
bath stimulated transepithelial secretion of FL to an extent similar to
that of 10 µM
KG (~37% and ~24% of control, respectively). There was no significant difference between the stimulation produced by
glutarate and
KG. In addition, 1.0 mM glutarate, like 1.0 mM
KG,
inhibited the secretion (~57% and ~51% of control, respectively). Therefore,
KG that enters the renal tubule cells via the
basolateral Na-DC cotransporter apparently stimulates net FL secretion
by exchange for FL at the basolateral membrane.

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Fig. 6.
A representative tracing showing a biphasic effect of -ketoglutarate
( KG) in bath on transepithelial secretion rate of FL in
fmol · min 1 · mm 1
by an isolated perfused S2 segment of rabbit proximal tubules. FL
concentration was 1 µM. Upon removal of 1 mM KG from the bathing
medium, there was an abrupt increase in FL secretion above the control
level, presumably due to the accumulation of KG following the
incubation at a high concentration of KG; i.e., effect of preloading
renal tubules with a high concentration of KG. This increase was
transient and gradually decreased to the control level in absence of
exogenous KG.
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Fig. 7.
Effect of KG or glutarate added to bath on transepithelial secretion
of 1 µM FL by isolated perfused S2 segments of rabbit proximal
tubules. Concentrations of KG or glutarate are indicated above each
pair of bars. Preload group represents the result obtained after
removal of 1 mM KG or glutarate from the bath as described in Fig.
6. Values are means ± SE (n = 7).
No difference was observed between effects of KG and glutarate.
a Significantly different
from control group (P < 0.05).
b Significantly different
from other experimental groups (P < 0.05). Differences were determined by ANOVA.
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Influence of exogenous luminal
KG on
transepithelial secretion of FL. In rabbit renal
tissue, the luminal Na-DC cotransporter, which has been cloned and
sequenced (8), shows a higher capacity than the basolateral Na-DC
cotransport. Because
KG is also filtered, it can be readily
reabsorbed from the lumen by the luminal Na-DC cotransporter.
Therefore, we hypothesized that the luminal uptake of filtered
KG
could be as important as, or perhaps more important than, the uptake of
KG across the basolateral membrane in establishing the in > out
KG gradient for basolateral OA/DC exchange. To evaluate this
possibility, we explored the effects of
KG in the lumen on net FL
secretion by isolated, perfused S2 segments of proximal tubules in the
absence of
KG in the bathing medium. As shown in Fig.
8, addition of
KG to the perfusion
solution stimulated steady-state transepithelial secretion of 1 µM FL
at every concentration tested. However, at the presumed physiological
luminal
KG concentrations of ~25 to 50 µM, FL secretion was
stimulated by only ~15-20%. From these results, we suggest that
luminal uptake of
KG via the luminal Na-DC cotransporter can
contribute to transepithelial secretion of OAs by ~15%; i.e.,
[(the increase in secretion in the presence of 50 µM
KG in
the lumen from the "basal" state)/(net secretion in the presence
of 50 µM
KG in the lumen)] × 100%. The data also
indicate that at physiological exogenous
KG concentrations, the
basolateral Na-DC cotransporter is more effective than the luminal
Na-DC cotransporter in promoting OA secretion.

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Fig. 8.
Effect of KG added to perfusion solution on transepithelial
secretion of 1 µM FL by isolated perfused S2 segments of rabbit
proximal tubules. Values are means ± SE. Numbers in parentheses
indicate numbers of experiments.
a Significantly different
from control group (P < 0.05).
b Significantly different
from groups at a concentration of 0.2 mM and below
(P < 0.05). Differences were
determined by one-way, paired
t-test.
|
|
Influence of both basolateral and luminal exogenous
KG on transepithelial secretion of FL. Under normal
in vivo conditions, the renal tubule cells are always exposed to
dicarboxylates on both basolateral and luminal sides. Transepithelial
secretion of OAs, therefore, occurs under conditions where exchangeable dicarboxylates are distributed at steady state within the cells by
metabolism and continuous uptake from both sides of the tubule cells.
To evaluate the contribution of both basolateral and luminal Na-DC
cotransporters to transepithelial secretion of OAs, we examined the
effects of
KG in the lumen, in the bath, and in both lumen and bath
simultaneously on steady-state transepithelial secretion of 1 µM FL
by the same individual S2 segment of proximal tubules. The
concentrations of
KG added were 10 µM to the bath and 50 µM to
the perfusion solution, on the assumption that the latter concentration
could possibly be in tubular fluid reaching the S2 segment in vivo. The
results are shown in Fig. 9. Addition of
KG to either the luminal or the basolateral side produced an
increase in transepithelial secretion of 1 µM FL by ~13 or 15%,
respectively. Interestingly, when the tubule was exposed on both sides
to these same concentrations of
KG, the secretion increased to 28%.
These data indicate that
KG that enters the cells via the luminal
Na-DC cotransporter can further support transepithelial secretion of
OAs by increasing the cellular pool of exchangeable
KG at the
basolateral membrane.

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Fig. 9.
Effect of KG added to bath, perfusion solution (lumen), or both on
the transepithelial secretion of 1 µM FL by isolated perfused S2
segments of rabbit renal proximal tubules. Values are means ± SE
(n = 9). * Significantly
different from control group (P < 0.05). ** Significantly different from other experimental groups
in which KG was added either to bath or perfusion solution
(P < 0.05). Differences were
determined by ANOVA.
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 |
DISCUSSION |
In the present study, we evaluated the transepithelial secretion of the
organic anion FL by isolated, perfused S2 segments of rabbit renal
proximal tubules in real time by using a specially constructed
epifluorescence system in which the collecting pipette functioned as a
flow-through cuvette. The studies were also performed under conditions
that were as close to physiological as possible, i.e.,
nutrient-enriched, bicarbonate-buffered bathing and perfusing solutions
at 37°C. Initially, we demonstrated that the steady-state transepithelial secretion of FL saturated with an apparent
Kt of ~4 µM
and Jmax of
~280
fmol · min
1 · mm
1.
These values were similar to those reported by Sullivan et al. (19) for
the basolateral uptake of FL by nonperfused S2 segments of rabbit renal
proximal tubules
(Kt = 10 µM; Jmax = ~ 498 fmol · min
1 · mm
1).
Although these authors had doubts about the reliability of the value
for Jmax (19),
the similarity between the values for perfused tubules in the current
study and those for nonperfused tubules in the earlier study (19)
strongly suggests that basolateral transport into the cells is the
rate-limiting step for transepithelial transport. Our initial studies
also confirmed the assumption that FL transport occurs by the classic
OA (PAH) pathway, for PAH inhibited the transepithelial FL transport
with an apparent
Ki almost
identical to the
Kt reported
previously for PAH transport at the basolateral membrane of this same
rabbit tubule segment (6, 17). In the present study, we used the
parallel measurements of fluorescence from TMRD in the lumen only to
check for leaks during the perfusion. However, with higher
concentrations of TMRD in the perfusate, it would be possible to make
online measurements of volume change resulting from tubular
reabsorption of perfusate.
The use of epifluorescence microscopy to study transepithelial
secretion of FL in perfused tubules in real time also allowed us to
investigate directly the extent to which the activity of the Na-DC
cotransporters located on both the basolateral and luminal membranes
contributed to the net transepithelial secretion of OAs. The tertiary
active transport model for OA transport at the basolateral membrane
suggests that
KG is recycled through the parallel activity of the
OA/DC exchanger and the basolateral Na-DC cotransporter (12). The
recent study by Welborn et al. (24) on nonperfused rabbit S2 renal
proximal tubules indicates that reuptake by the basolateral Na-DC
cotransporter of
KG that has moved out of the cells in exchange for
FL accounts for ~25% of the initial rate of basolateral FL uptake in
the absence of exogenous
KG. Similarly, in the current study on
perfused tubules, inhibition of the basolateral Na-DC cotransporter
indicated that reuptake of
KG by this transporter accounts for
~25% of the steady-state transepithelial secretion of FL in the
absence of exogenous
KG. Therefore, the parallel activities of the
basolateral OA/DC exchanger and the basolateral Na-DC cotransporter
recycling
KG apparently account for the maintenance of ~25% of
the outwardly directed gradient for
KG and the corresponding
basolateral uptake and transepithelial secretion of OA in the absence
of exogenous
KG.
Under physiological conditions in vivo, renal tubules are exposed to
~10 µM
KG from the blood (bathing medium) side. In previous work
on nonperfused rabbit S2 renal proximal tubules, the addition of this
concentration of
KG to the bathing medium (which was identical to
that used in the present study) containing 1 µM FL led to an increase
of ~75% in the initial rate of FL uptake (24). In the present study
with perfused tubules, the addition of 10 µM
KG to the bathing
medium containing 1 µM FL led to an increase of ~15% to ~40% in
the steady-state transepithelial secretion of FL (Table 2; Figs. 7 and
9). The difference in degree of stimulation between our current study
on perfused tubules and the previous one on nonperfused tubules (24)
may reflect differences in: 1)
transport measured (steady-state transepithelial secretion vs. initial
rate of basolateral uptake); 2)
intracellular distribution of
KG taken up from the bath because of
differences in the metabolic state of perfused vs. nonperfused tubules;
and 3) the exchangeable intracellular pool of
KG in tubules from different rabbits. The variability between tubules from different rabbits was particularly marked in the current study with perfused tubules (compare data in
Table 2 and Figs. 7 and 9). These differences may indeed reflect differences in metabolic state and the available exchangeable intracellular pool of
KG produced by metabolism. This possibility is
lent some credence by the observation that glutarate, which is not
significantly metabolized (10), tended to produce a slightly higher
stimulation of FL transport at 10 µM and inhibition of FL transport
at 1 mM than
KG at the same concentrations (Fig. 7).
In the present study on perfused S2 segments of rabbit proximal
tubules, it was possible to evaluate the contribution of
KG transport into the cells by the luminal Na-DC cotransporter to the
transepithelial secretion of FL in real time. We found that the
addition of a concentration of
KG to the lumen that might be
expected to be present physiologically (assuming that normal fluid
absorption concentrates the filtered
KG before it reaches the S2
segment) produced a significant increase in steady-state net secretion
of FL in the absence of
KG in the bathing medium. This stimulation
was demonstrated in the presence of bicarbonate-buffered perfusing and
bathing solutions, which are essential to maintaining physiological
levels of tricarboxylic acid cycle intermediates, such as
KG, in the
cells and, thus, normal levels of OA transport (18). In a previous
study on perfused S2 segments, Dantzler and Evans (5) also showed that
the addition of
KG or glutarate to the lumen could stimulate net
transepithelial secretion of radiolabeled PAH in the absence of
KG
in the bathing medium. This stimulation was prevented if the luminal
Na-DC cotransporter was inhibited by the simultaneous inclusion of LiCl
in the perfusate with the
KG or glutarate. However, in this previous
study, the stimulation was apparent only with very high concentrations
of
KG or glutarate in the lumen and only when the control rate of net steady-state PAH secretion was depressed by using bicarbonate-free perfusate (5). In retrospect, it appears that relatively high concentrations of malate and citrate in the bicarbonate-buffered solutions may have prevented
KG uptake from the lumen by competing for the luminal Na-DC cotransporter. Indeed, we found during the present study that when the bathing solution contained malate and
citrate in the presence of a 10 µM concentration of
KG, the usual
stimulatory effect on FL secretion was reduced, presumably by
competition between malate, citrate, and
KG for the basolateral Na-DC cotransporter (unpublished observations). Moreover, the simple
removal of malate and citrate from the bathing medium produced an
increase in FL secretion (unpublished observations). In this case,
malate and citrate were probably inhibiting reuptake of
KG by the
basolateral Na-DC cotransporter.
Although kinetic data are not available for the transport of
KG
itself by either the luminal or basolateral Na-DC cotransporter, studies of succinate transport by brush-border membrane vesicles (BBMV)
and BLMV provide some information on the possible affinities and
capacities of these two transporters for
KG (27). In BBMV, the
Kt and
Jmax for
succinate are ~600 µM and ~90
nmol · min
1 · mm
1,
respectively; in BLMV, they are ~10 µM and ~5
nmol · min
1 · mm
1
(16). The inhibitory effects of
KG on succinate transport suggest
that the Kt
values for it may be similar to those for succinate (27). These data
suggest that physiological concentrations of
KG in the lumen should
certainly be taken up by the luminal Na-DC cotransporter to contribute
to the gradient for FL/
KG exchange at the basolateral membrane, as
clearly occurred in the present study. However, in general, 50 µM
KG in the lumen had less stimulatory effect on transepithelial FL
secretion (in the absence of
KG in the bath) than did 10 µM
KG
in the bath (in the absence of
KG in the lumen). This observation
might be related to the anatomic proximity of the basolateral Na-DC
cotransporter to the basolateral OA/DC exchanger. The
KG transported
into the cells via the basolateral Na-DC cotransporter might be
supplied relatively directly to the OA/DC exchanger, whereas the
KG
transported into the cells by the luminal Na-DC cotransporter might
contribute to both the intracellular pool of exchangeable
KG for the
OA/DC exchanger and to other cellular metabolic events.
In the previous study on the role of the luminal uptake of
KG in the
process of PAH secretion, the time course of the radioisotopic measurements markedly limited the sensitivity of the determinations of
PAH secretion, and it was not feasible to study the effects of
KG in
the lumen and bathing medium simultaneously (5). In the present study,
it was possible to determine the effect of
KG in the lumen alone,
the bath alone, and in both the lumen and bath simultaneously on
steady-state transepithelial FL secretion in the same individual
tubules. The data revealed a significant additive effect on FL
secretion when
KG was in both the lumen and the bath at
approximately physiological concentrations (Fig. 9). Therefore, the
luminal uptake of
KG can play a role in maintaining secretion of OA
under physiological conditions with
KG present at the basolateral
side. However, it should be noted that in the series of experiments
reported in Fig. 9, the degree of stimulation of FL secretion by 10 µM
KG in the bathing medium was substantially less than that in
other experiments (for example, Table 2). Therefore, although uptake of
KG from the lumen can contribute to OA secretion even with
KG in
the bath, the contribution may be less when the basolateral uptake is
more effective.
The present observations on the effects of
KG permit us to estimate
the extent to which the basolateral and luminal Na-DC cotransporters
may contribute to net transepithelial secretion of OAs under normal
physiological conditions. Recycling of
KG by the basolateral Na-DC
cotransporter supports ~25% of the "basal" secretion in the
absence of exogenous
KG. The basolateral Na-DC cotransporter can
also support a further ~30% (average value from all experiments) of
OA secretion by uptake of exogenous
KG from the physiological
basolateral concentration of 10 µM. Therefore, the activity of the
basolateral Na-DC cotransporter is responsible for supporting ~42%
of the transepithelial secretion of OAs; i.e., {[(increase
in secretion in presence of 10 µM
KG in bath from the
"basal" state) + (difference in secretion in presence of 2 mM
LiCl in bath from the "basal" secretion)]/(net secretion in presence of 10 µM
KG in bath)} × 100%. The luminal
Na-DC cotransporter can support an additional ~20% (average value
from all experiments) of OA secretion by uptake of
KG from the
luminal fluid, if the luminal concentration is 50 µM. As noted above,
the effect of
KG uptake by both the luminal and basolateral Na-DC
cotransporters on OA secretion can be additive with physiological
concentrations of
KG in lumen and bath. If this is the case and if
we use the average individual values obtained, we can conclude that the
luminal and basolateral Na-DC cotransporters together directly support ~50% of the net transepithelial OA secretion; i.e.,
{[(increase in secretion in presence of 10 µM
KG in
bath from the "basal" state) + (difference in secretion in
presence of 2 mM LiCl in bath from the "basal" secretion) + (increase in secretion in presence of 50 µM
KG in lumen from the
"basal" state)]/(net secretion in presence of 10 µM
KG
in bath and 50 µM
KG in lumen)} × 100%. We assume
that this occurs by these transporters establishing and maintaining
~50% of the outwardly directed gradient for
KG that is
responsible for driving the OA/DC exchange at the basolateral membrane.
The remaining ~50% would be maintained by metabolic production of
KG in the cells.
We thank the Royal Thai Government for support of A. Shuprisha
during the course of this work.
This study was supported in part by National Institutes of Health
Research Grant ES-06757, by Training Grants HL-07249, NS-07309, and
GM-08400, and by Southwest Environmental Health Science Center Grant
ES-06694.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. H. Dantzler,
Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ
85724-5051 (E-mail: dantzler{at}u.arizona.edu).