From the Physiologisches Institut der Universität Würzburg, 97070 Würzburg, Germany
Received for publication, August 4, 2000, and in revised form, February 2, 2001
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ABSTRACT |
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The organic anion transport system in the
proximal tubule of the kidney is of major importance for the excretion
of a variety of endogenous and potentially toxic exogenous substances.
Furthermore, the clearance of model substrates (e.g.
para-aminohippurate) of this system is used for the
determination of renal blood flow. We investigated regulation of
organic anion secretion in a way that allowed us to examine
simultaneously regulation of overall transepithelial secretion and to
estimate the separate contributions of regulation of the basolateral
and apical transport steps to this overall regulation. The data were
verified by measurement of initial basolateral uptake rate and initial
apical efflux rate. Opossum kidney cells were used as a suitable model
system for proximal tubule cells, and
[14C]para-aminohippurate was utilized as an
organic anion. Stimulation of protein kinase C inhibited
transepithelial secretion because of inhibition of both apical efflux
and basolateral uptake. Inhibition of the mitogen-activated protein
kinase (MAPK) kinase MEK reduced transepithelial secretion via
inhibition of basolateral uptake and apical efflux. Epidermal growth
factor (EGF) enhanced transepithelial secretion via stimulation of
basolateral uptake but did not affect apical efflux. EGF induced
stimulation of basolateral uptake was abolished by inhibition of MEK.
EGF led to phosphorylation of ERK1/2, which was also abolished by
inhibition of MEK. Thus, EGF stimulated basolateral uptake of organic
anions via MAPKs. Transepithelial organic anion secretion can be
regulated at two sites, at least: basolateral uptake and apical efflux.
Both steps are under control of protein kinase C and MAPK. The
pathophysiologically relevant growth factor EGF enhances
transepithelial secretion via stimulation of basolateral uptake. EGF
stimulates basolateral uptake via MEK and ERK1/2. Thus, renal organic
anion extraction may be modulated, especially under pathophysiological conditions.
The organic anion transport system of the renal proximal tubule
plays a crucial role in the excretion of a variety of potentially toxic
compounds (1). This system consists of a basolaterally located organic
anion exchanger and a less well characterized transport step at the
apical membrane (2). The basolateral organic anion exchanger is a
tertiary active transport system, dependent on an inward directed
Na+ gradient to drive the uptake of
Very little is yet known about the modulation of the secretory organic
anion transport system. Nagai and co-workers (17) showed an inhibition
of basolateral uptake and transepithelial secretion of organic anions
in OK cells by parathyroid hormone via a staurosporine sensitive
mechanism. Inhibition of basolateral organic anion transport during
stimulation of protein kinase C (PKC) was reported in isolated tubules
of kilifish (18). The basolateral exchanger for organic anions and
dicarboxylates in isolated proximal tubules of rabbit kidney was shown
to be sensitive to inhibition of
Ca2+/calmodulin-dependent protein kinase II,
tyrosine kinase, phosphatidylinositol-3-kinase, and mitogen-activated
protein kinases (MAPKs) (19). Furthermore, inhibition of OAT1 by
bradykinin and phenylephrine via PKC in isolated rabbit proximal
tubules has been described (20). In most of the studies, only
regulatory events at the basolateral membrane were investigated. The
study of Nagai and co-workers investigated transepithelial transport,
but they used the rather unspecific kinase inhibitor staurosporine.
Inhibition of net secretory transport (21) of organic anions by
bradykinin and phenylephrine has been reported for isolated perfused
rabbit proximal tubules. Recently, Henderson and co-workers (22) showed
that PKC inhibits murine OAT without direct phosphorylation of the
transport protein itself. Taken together, these studies give no
detailed information concerning the contribution of the single
transport steps to the regulation of transcellular
secretion. To address this problem, we investigated transcellular
secretion in combination with measurements of initial basolateral
uptake rate and initial apical efflux rate.
In the present study, we determined the effect of epidermal growth
factor (EGF) and MAPKs on PAH transport in OK cells. EGF and its
receptor are known to be expressed in proximal tubular cells (23). EGF
has been suggested as a mediator of normal tubulogenesis and tubular
regeneration after injury. A reduction of renal EGF expression and/or
urinary excretion has been reported during acute and chronic tubular
injury (24). Additionally, EGF led to an increase in PAH excretion in
rats (25), indicating an influence of EGF on proximal tubular organic
anion transport. MAPKs are known to be involved in renal stress
response and represent an important downstream signal of the EGF
pathway (26).
Our data show that activation of PKC inhibits both the basolateral and
the apical step of PAH secretion. Moreover, we show that MAPK activity
is required for a proper activity of basolateral uptake step and the
apical exit of PAH. EGF stimulates transepithelial secretion via
stimulation of the basolateral uptake but does not affect the apical
transport step. EGF leads to successive activation of the MAPKs, ERK
kinase (MEK), and extracellular regulated kinase 1/2 (ERK1/2).
Cell Culture
OK cells were obtained from Dr. Biber (Department of Physiology,
University of Zurich). Cells were maintained in culture at 37 °C in
a humidified 5% CO2, 95% air atmosphere. The growth
medium was minimal essential medium, pH 7.4, supplemented with Earl's salts, nonessential amino acids, 10% (v/v) fetal calf serum (Biochrom KG, 12213 Berlin, FRG), and 26 mmol/liter NaHCO3. Cells
were cultured on permeable supports (3-µm pore diameter; Falcon,
Becton Dickinson Labware, Franklin Lakes, NJ) for transport
measurements. The effective growth area on one permeable support was
4.3 cm2/filter. All studies were performed between passages
60 and 100. The seeding density was 0.4·106
cm
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate, which is then exchanged for organic anions
(1-4). The basolateral exchanger for organic anions and dicarboxylates
was cloned by three independent groups (5-7) in 1997, and called OAT1
(rat), ROAT1 (rat), or fROAT1 (winter flounder). Only recently, the
homologous protein was cloned from human kidney and called hOAT1 or
hPAHT (8, 9). A number of mechanisms have been described for the apical efflux of organic anions (10), which differ with species and experimental setup used. For example, a
PAH1/dicarboxylate exchanger,
a PAH/anion exchanger, and a membrane potential-dependent
mechanism have been described (11). Furthermore, there is evidence for
the involvement of oatp, OAT-K1, and OAT-K2 (10, 12-16). Thus,
secretion of organic anions is mediated by a well described, tertiary
active transport step at the basolateral membrane and a not yet
settled, apical transport step.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2. The medium was changed every third day, and the
monolayers were used for experiments at day 10 after seeding. The
effect of 10
7 M phorbol 12-myristate
13-acetate or 5 × 10
5 M DOG on PAH
secretion was determined in nonquiescent and quiescent cells, as shown
in Fig. 1. OK cells were made quiescent
by cultivating them in serum depleted cell culture medium for 24 h
before the experiments. In both cases, secretion (Fig. 1A)
was reduced to a greater extent than cellular content (Fig.
1B), indicating that apical transport may be affected by
PKC. Moreover, the effects were more pronounced in quiescent (means
serum depleted for 24 h) cells (Fig. 1, right panel) as
compared with nonquiescent cells (Fig. 1, left panel). In
nonquiescent cells EGF showed no effect on cellular PAH content or
transepithelial PAH secretion (data not shown). Because fetal calf
serum contains a variety of chemokines, it is evident that
investigation of the effect of an isolated chemokine is only possible
in the absence of serum. As shown for PKC and mentioned for EGF, the
effects on organic anion transport in OK cells were more pronounced or
only apparent when cells were made quiescent. Thus, quiescent OK cells
were used for all subsequent experiments.
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Fig. 1.
Effect of PKC stimulation in nonquiescent
(left panel) and quiescent (right
panel) OK cells on secretion (A) and
cellular content (B) after 10 min. Phorbol
12-myristate 13-acetate (10 7 M) and DOG
(5 × 10
5 M) were added to the transport
buffer and were present throughout the 10 min of the transport process.
1.5 × 10
6 M [14C]PAH was
used as a substrate. Nonquiescent cells were cultivated in medium
containing 10% (v/v) fetal calf serum until the cell epithelia were
experimentally used. For quiescent cells, experimental medium was
replaced with medium without fetal calf serum 24 h prior to the
experiments. n for every bar is shown in
parentheses. *, p < 0.05 versus
control.
Transport Measurements
The volumes of the apical and basolateral compartment were 1.3 and 2.5 ml to avoid hydrostatic pressure differences. Before each
experiment, the cells were washed three times with phosphate-buffered Ringer (138 mmol/liter NaCl, 1 mmol/liter
NaH2PO4, 4 mmol/liter Na2HPO4, 4 mmol/liter KCl, 1 mmol/liter
MgCl2, 1 mmol/liter CaCl2, 5 mmol/liter
glucose, pH 7.4). Transport measurements were performed in
phosphate-buffered Ringer at pH 7.4 and 37 °C. The concentrations of
the radiolabeled substrates applied to the basolateral bath were:
1.5 × 106 mol/liter or 15 × 10
6
mol/liter [14C]PAH, and 55 × 10
9
mol/liter [3H]mannitol or 55 × 10
10
mol/liter [3H]mannitol. [3H]Mannitol was
used to correct secretion for paracellular fluxes and to determine
extracellular water space. At the end of the experiment, the apical and
basolateral solutions were collected. Subsequently, the filters were
washed twice with ice-cold PBS and cut from the supports. Radioactivity
of the solutions and the cells was measured using a liquid
scintillation counter (Packard Instruments, Frankfurt, Germany). Counts
of cells on filters were corrected for nonspecific binding on filters
by subtraction.
To investigate PAH efflux, the cells were incubated with 15 × 106 mol/liter [14C]PAH for 60 min. After
washing, the efflux was determined during the first minute. Apical and
basolateral solutions and the cellular compartment were collected
separately. Radioactivity in the solutions and the cells was measured
by liquid scintillation counting. The total amount of counted
[14C]PAH was set as amount of [14C]PAH in
the cells at time 0 of the efflux experiments.
Western Blot Analysis
OK cells were rinsed three times with PBS followed by a 10-min
incubation with EGF and/or PD98059. Subsequently cells were washed with
ice-cold PBS three times and lysed in ice-cold Triton X-100 lysis
buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM
-glycerophosphate, 200 µM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin A, 1% Triton X-100) for 25 min at 4 °C.
Insoluble material was removed by centrifugation at 12,000 × g for 15 min at 4 °C. The protein content was determined
using a microbicinchoninic acid assay (Pierce) with bovine serum
albumin as a standard. Cell lysates were matched for protein, separated
by 12% SDS-polyacrylamide gel electrophoresis, and transferred to a
polyvinylidene difluoride microporous membrane. Subsequently, membranes
were blotted with rabbit anti-pERK1/2 (p42/p44) antibody (New England
Biolabs). The primary antibody was detected using alkaline
phosphatase-conjugated goat anti rabbit IgG visualized by ECL Western
blotting reagents and Hyperfilm ECL (Amersham Pharmacia Biotech).
According to the manufacturer's handbook Hyperfilm ECL exhibits a
linear response to the light produced from enhanced chemiluminescence.
Additionally, linearity was verified for our experimental conditions by
a dilution series with increasing amounts of total cell protein.
Western blotting was performed with protein from five independent
extractions from five independent cell culture passages. Blots were
analyzed using SigmaGel 2000 Software (Jandel Scientific).
Processing of Experimental Data
10-min transport--
According to our measurements (Fig.
2A) secretory transport of
organic anions in the OK clone used is detectable after 3-4 min and is
then linear for at least 1 h (data not shown). Thus, secretory
transport after 10 min represents the linear phase of secretion.
Therefore, the amount of radiolabeled PAH in the apical compartment
after 10 min was used to measure PAH secretion. The quantity of PAH in
the cells is denominated cellular content. Summing up the values for
PAH secretion and for cellular PAH content gives the basolateral uptake
of PAH, i.e. the total amount of PAH transported across the
basolateral membrane during 10 min. Additionally, we calculated the
ratio of secretion to cellular content of PAH (the secretion-to-content
ratio). Introducing these parameters enabled us to gain information
regarding the contribution of basolateral and/or the apical transport
step(s) to the overall secretion of PAH.
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Basolateral Uptake during the First Minute-- Under our experimental conditions uptake of PAH is linear at least during the first 1.5 min as shown in Fig. 2B (right panel), and no net secretion of PAH occurs (data not shown). Thus, the cellular PAH content after 1 min represents the initial basolateral uptake rate. Thus, the predictions concerning the basolateral part of PAH transport derived from the 10-min transport experiments were verified by the 1-min data.
Apical Efflux of PAH during the First Minute-- After preloading the cells as described above, we determined the apical efflux of [14C]PAH during the first min as percentage of cellular PAH content at time 0. Because the volume of the apical compartment is about 1000 times that of the cells, there is a large outward gradient during the entire experimental period. According to Fig. 2C (right panel), efflux into the apical compartment is linear at least during 1.5 min, and, thus, the gradient is not collapsed in the time frame investigated. Therefore, the apical efflux during 1 min represents the initial apical efflux rate.
Data Analysis
Data are presented as the means ± S.E. n is given in the text or in the figures. n represents the number of culture plates or filters used. Statistical significance was determined by unpaired Student's t test or analysis of variance as appropriate. Results were considered statistically different at p < 0.05. Significant differences are indicated by asterisks.
Materials
[14C]PAH (55 mCi/mmol) and
[3H]mannitol (15 mCi/mmol) were purchased from American
Radiolabeled Chemicals Inc. (St. Louis, MO). PD98059 was from Alexis
Corp. (Läufelfingen, Switzerland). U0126 was from Promega Corp.
(Madison, WI). Antibody against the phosphorylated form of ERK1/2
(pERK1/2) was from New England Biolabs Inc. If not stated otherwise,
all other chemicals were from Sigma. EGF from Sigma was used as human,
recombinant substance.
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RESULTS |
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Validation of the Cell Clone Used-- We wanted to investigate regulation of organic anion secretion in a way that would allow us to examine regulation of overall transepithelial secretion and the separate contributions of the basolateral and apical transport steps to this overall regulation, simultaneously. For this purpose, we chose the proximal tubule-derived OK cell line cultured on permeable supports, a well characterized model system to investigate organic anion secretion (27-29). Probenecid (10 mM), the classical inhibitor of organic anion transport, and a 1000-fold excess of unlabeled PAH, both inhibited uptake and secretion of [14C]PAH by more than 95% (data not shown). Thus, our particular cell clone transports organic anions in accordance to the published data mentioned in the introduction.
PKC Affects Basolateral and Apical Transport--
Stimulation of
PKC by 5 × 105 M DOG reduced secretion
of PAH (Fig. 3A), whereas the
cellular content (Fig. 3B) was not significantly different
from control. Adding up secretion and cellular content of PAH gives the
amount of PAH transported across the basolateral membrane (Fig.
3C). Transport across the basolateral membrane was reduced
by DOG, in agreement with other studies (18-20). Furthermore, the
decreased secretion-to-content ratio (Fig. 3D) indicates
that the apical transport step is also inhibited by PKC. However, there is another possible explanation. The apical transport is carrier mediated and thus has a hyperbolic relationship to substrate
concentration. A decrease in intracellular concentration could reduce
this ratio, without inhibition of the apical step itself. Thus,
experiments on initial transport rates will have to decide what kind of
explanation applies to these data.
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Simultaneous inhibition of apical and basolateral transport was
confirmed by measurement of initial apical efflux rate and initial
basolateral uptake rate. As shown in Fig.
4A, activation of PKC reduced
the initial apical efflux rate, thereby leading to a decreased relative
amount of PAH in the apical bath as compared with control. In addition,
Fig. 4B shows that stimulation of PKC reduced the initial
basolateral uptake rate of PAH. By contrast, inhibition of PKC with
107 M BIM increased basolateral uptake of PAH
(data not shown). Thus, the basolateral organic anion transport in OK
cells is regulated by PKC in agreement with data published previously
(19, 20).
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EGF Stimulates Transepithelial Secretion--
EGF increased
cellular content (Fig. 5B) and
transepithelial secretion (Fig. 5A) of PAH. However, EGF did
not affect secretion-to-content ratio for PAH (Fig. 5D) at
all, indicating that EGF stimulates basolateral PAH transport in OK
cells but does not affect the apical transport step. Initial efflux
experiments showed no change in PAH efflux across the apical membrane
in EGF treated cells (Fig.
6A). These data confirm the
predictions derived from the 10-min transport experiments with EGF
(Fig. 5). As shown in Fig. 6B, EGF stimulates the initial
basolateral uptake rate of PAH in agreement with the prediction derived
from the 10-min experiments.
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MAPKs Affect Basolateral and Apical Transport--
It is known
that EGF uses the MAPK pathway also in OK cells (30). Therefore, we
investigated the effect of substances that inhibit MAPK activation.
Transepithelial secretion (Fig.
7A) and basolateral uptake
(Fig. 7C) of PAH were reduced by PD98059 (inhibitor of MEK),
whereas cellular PAH content (Fig. 7B) was increased 3-fold
as compared with control. The secretion-to-content ratio (Fig.
7D) was dramatically decreased in the presence of PD98059. These data indicate a strong inhibitory effect of PD98059 on the apical
transport step of PAH secretion. Furthermore, it is possible that the
increased cellular content of PAH results solely from the strong
inhibition of the apical exit step of PAH, whereas the basolateral
transport step remains unchanged. This is also true for the observed
reduction of basolateral uptake in Fig. 7. In fact, even an increase in
basolateral transport activity would be in agreement with these data.
Thus, in this particular configuration, it is impossible to draw a
final conclusion concerning the basolateral transport step. As Fig.
8A clearly shows, the prediction concerning the apical step is confirmed by the data obtained
from efflux experiments. PD98059 inhibited initial apical efflux rate
significantly. Initial basolateral uptake rate of PAH (Fig.
8B) is also reduced by PD98059. These data show that basolateral uptake of PAH in OK cells is inhibited by inhibition of
MEK. Increasing the concentration of PD98059 to 50 µM
(Fig. 8B), which represents the maximal effective
concentration, leads to an increased inhibition of initial basolateral
uptake rate of PAH, indicating a dose-dependent action of
PD98059 on the basolateral transport.
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In summary, transepithelial secretion of PAH in OK cells is under the
stimulatory control of the mitogen-activated protein kinase kinase MEK.
Increased secretion when MEK is active results from a stimulation of
both basolateral and apical transport (Fig. 9A). Similar results were
obtained with another, structurally different, MEK inhibitor U0126
(data not shown).
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EGF Acts on Basolateral Uptake via Successive Activation of MEK and
ERK--
As already mentioned, it is known that the MAPK pathway is
stimulated by EGF in OK cells (30). Thus, we investigated whether the
stimulatory effect of EGF on basolateral PAH transport is mediated by
MAPK. As shown in Fig. 10, inhibition
of the MAPK MEK by PD98059 or U0126 completely abolishes the
stimulatory effect of EGF on initial basolateral PAH uptake. These data
indicate that the stimulatory effect of EGF on basolateral uptake is
mediated by the activation of MEK.
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As shown in Fig. 11, EGF leads to
increased phosphorylation of ERK1/2 in OK cells within 10 min. PD98059
alone, slightly but significantly, decreased the amount of pERK1/2 as
compared with controls. These data are in good agreement with the
effects on PAH transport. However, EGF increased phosphorylation of
ERK1/2 is reduced by inhibition of MEK with PD98059, whereas
EGF-stimulated uptake of PAH is totally abolished by PD98059. We
explain this apparent discrepancy with the existence of intermediate
signaling steps between ERK and basolateral organic anion transport, as discussed in detail later on.
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Finally we tested whether preincubation for 10 min with EGF or PD98059
affects glutarate uptake in OK cells. Glutarate is a nonmetabolizable
analogue of the dicarboxylate -ketoglutarate. EGF and PD98059 did
not affect basolateral uptake of glutarate (control: 1.2 ± 0.1, n = 3; 10 ng/ml EGF: 1.4 ± 0.1, n = 3; 5 µM PD98059: 1.3 ± 0.3, n = 6; in pmol·cm
2·10 min
1). Furthermore,
EGF did not affect apical glutarate uptake (control: 0.9 ± 0.1, n = 3; 10 ng/ml EGF: 1.1 ± 0.1, n = 3; in pmol·cm
2·10 min
1). Thus,
availability of intracellular counterions for PAH uptake is not altered
by EGF or PD98059. Previously published data showed no effect of
PD98059 on basolateral glutarate uptake (19). Taken together, these
data present strong evidence that EGF stimulates exchange of
dicarboxylates and organic anions via stimulation of the ERK1/2 (Fig.
9C).
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DISCUSSION |
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The Experimental Setup-- The purpose of the present study was (a) to gain more information concerning the overall regulation of the proximal tubule organic anion transport and (b) to investigate the contribution of the basolateral and apical transport steps to this regulation. As shown for EGF, we were able to determine their effects on the basolateral and apical transport steps simultaneously with their effects on transepithelial secretion. However, inhibition of MEK by PD98059 led to a configuration (reduced secretion, increased content, reduced uptake, and reduced secretion-to-content ratio) where determination of the contribution of the basolateral transport step simultaneously with secretion was not possible, indicating the limitations of the method. This is also true for the DOG effect presented. However, with the help of initial apical efflux experiments and determination of the initial basolateral transport rate it is possible to investigate apical and basolateral transport steps separately. Nevertheless, we show that it is possible to estimate the site of action of a chemokine on the secretory transport simultaneously with transepithelial transport, although transport occurs across two membranes (and the cytosol). Thus, the particular processing of the 10-min transport data presented here is suitable to obtain a first estimate regarding the site of action of a given modulator of secretory transport.
Stimulation of PKC inhibits basolateral uptake rate and inhibition of MEK decreases basolateral uptake rate of PAH (Fig. 9A). These data are in agreement with the action of PKC (18-20) and MAPK (19) in isolated proximal tubules and again show the suitability of the OK cell system used. Additionally, we show for the first time that PKC inhibits and MEK stimulates the apical step of organic anion secretory transport in a proximal tubule derived cell line (Fig. 9A). Because the specific PKC inhibitors calphostin C and bisindolylmaleimide stimulate basolateral uptake of PAH, the observed inhibitory action of DOG is due to an interaction with regulatory cascades and not due to nonspecific or even toxic action. Furthermore, the calphostin C-induced stimulation of PAH uptake was prevented by both phorbol 12-myristate 13-acetate and DOG (data not shown). Systematic changes in mannitol flux were not observed with any of the substances used. Thus, no changes in epithelial tightness were induced, and the measured changes in PAH transport are not due to altered paracellular flux. The unchanged epithelial tightness, together with the short exposure time (10 min) and the moderate concentrations used, make it highly unlikely that any observed effect is due to unspecific toxic actions.
EGF Stimulates Organic Anion Secretion-- As the above mentioned maneuvers act directly on intracellular signaling pathways, we investigated the effect of more physiologically or pathophysiologically relevant stimuli, namely EGF. We show for the first time that EGF increases the secretion of PAH in OK cells by a stimulation of the basolateral uptake step. EGF does not affect the apical transport step (Fig. 9B).
The effect of EGF on basolateral uptake is mediated by MEK, because two specific, structurally distinct inhibitors of MEK (PD98059 and U0126) completely abolished EGF induced stimulation of initial basolateral PAH uptake. As shown in Fig. 8B, 5 µM PD98059 led to a slight decrease of initial PAH uptake. This is in parallel with the decrease of ERK1/2 phosphorylation induced by 5 µM PD98059. However, the same concentration of PD98059 completely prevented the EGF-induced stimulation of initial basolateral PAH uptake. This was also the case for the structurally distinct MEK inhibitor U0126. The fact that inhibition of MEK decreased uptake only slightly but abolished EGF-stimulated uptake completely is strong evidence for the fact that EGF acts on basolateral transport via stimulation of MEK. Moreover, as already mentioned above, no secretion of organic anions in OK cells was detected during the first min. Thus, alterations of the apical transport step should not influence initial basolateral uptake rate.
Inhibition of MEK prevented EGF-induced stimulation of basolateral PAH uptake completely; however, it reduced EGF induced ERK1/2 activation only partially. We consider this difference of action as evidence that ERK1/2 does not act directly on basolateral organic anion uptake but via one or more intermediate signaling steps. In fact, preliminary data from our laboratory indicate that phospholipase A2 is involved in downstream signaling following ERK1/2 activation (31). These intermediate steps (as e.g. phospholipase A2) possibly modulate the signal downstream of ERK1/2 in such a way that a partial inhibition of ERK1/2 activation leads to a complete inhibition of PAH uptake stimulation. This could be explained by a certain ERK1/2 activation threshold that has to be exceeded to stimulate the downstream signals. Thus, we hypothesize that EGF stimulates basolateral organic anion uptake via the successive activation of MEK, ERK, and additional downstream signaling steps (Fig. 9C). However, additional experiments will be necessary to clarify the downstream signal transduction and amplification network with respect to organic anion uptake in more detail.
As seen in the control lane of Fig. 11B, OK cells show a certain intrinsic ERK1/2 activation, although they were serum depleted for 24 h. Our observations indicate that OK cells with higher intrinsic ERK1/2 activation show higher control levels of basolateral PAH uptake as compared with those with a lower ERK1/2 activation (data not shown). These data again support the hypothesis that ERK1/2 activity regulates organic anion transport in OK cells.
Because the effects of EGF were studied after 10 min of exposure, it can be excluded that the regulatory events observed resulted from changes in protein synthesis. However, up to now we can only speculate on the molecular events involved in the observed regulatory phenomena. Possible mechanisms include an increased insertion of preformed transport protein by fusion of vesicles with the basolateral membrane in response to EGF. Of course EGF could also lead to stimulation of transport proteins in the cell membrane, by e.g. phosphorylation. Future experiments will investigate the effects of EGF on the affinity (Km) and the maximum transport rate (Vmax) of basolateral organic anion uptake in more detail.
By contrast to the events at the basolateral membrane, stimulation of MEK by EGF did not lead to a stimulation of the apical transport step, although PD98059 data indicate a regulatory role of MEK on the apical transport step, too. This apparent discrepancy could be explained by the fact that basal MEK activity already induces maximal stimulation of apical transport. Consequently, reduction of basal ERK1/2 activity by PD98059 (Fig. 11) leads to reduced apical and basolateral transport, whereas activation of MEK by EGF affects only the basolateral step. Another possible explanation is the involvement of additional signal pathways downstream of EGF, which might antagonize the MEK effect on apical transport but not on basolateral transport. Finally, there is the possibility of basolateral signaling microdomains, which confine the effect of EGF. Future experiments will have to explain this apparent discrepancy.
PAH clearance is routinely used for the determination of renal blood flow, based on the assumption of a high and constant renal PAH extraction. Because PAH extraction would vary according to the regulatory state of its transport system, it is conceivable that the determination of renal blood flow from PAH clearance may lead to under- or overestimation. Corrigan et al. (32), for example, described a decreased renal PAH extraction after postischemic acute renal failure in humans, leading to severe underestimation of renal blood flow. After renal injury, a rapid fall of EGF mRNA in the kidney was measured (26). We could show that the basolateral step of PAH secretory transport is under stimulatory control of EGF, which is in agreement with excretion data from rat (25) and could explain, at least in part, the above mentioned data from Corrigan.
In summary, we were able to determine the site of regulation of the
secretory transport of organic anions. The results obtained were
confirmed by measurements of initial basolateral uptake rate and
initial apical efflux rate. Thus, we could show that activation of PKC
inhibits not only the basolateral but also the apical step of organic
anion secretion. We also showed that MEK stimulates not only the
basolateral but also the apical transport step of organic anion
secretion. Additionally, we presented data indicating a stimulation of
the organic anion secretion by EGF. This stimulation resulted from an
increase of the basolateral uptake rate only. Furthermore, we showed
that EGF stimulates the basolateral uptake of organic anions via
successive activation of MEK and ERK. In conclusion, the excretion of
organic anions in proximal tubular cells seems to be a regulated and
therefore variable process. This may be particularly important under
pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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We thank Prof. Dr. Stefan Silbernagl for stimulating and helpful discussions. We thank Prof. Dr. William H. Dantzler for proofreading the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Ge 905/3-4.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Physiologisches
Inst., Universität Würzburg, Röntenring 9, 97070 Würzburg, Germany. Tel.: 49-931-31-2724; Fax:
49-931-31-2741; E-mail: christoph.sauvant@mail.uni-wuerzburg.de.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M007046200
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ABBREVIATIONS |
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The abbreviations used are: PAH, para-aminohippurate; DOG, 1; 2-dioctanoyl-sn-glycerol, EGF, epidermal growth factor; ERK1/2, extracellular regulated kinase (isoforms) 1 and 2; MAPK, mitogen activated protein kinase; MEK, mitogen-activated/extracellular-signal regulated kinase kinase; OK, opossum kidney; PKC, protein kinase C; PBS, phosphate-buffered saline.
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