Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724
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ABSTRACT |
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To examine the role of protein kinase C
(PKC) in organic anion (OA) secretion, we used epifluorescence
microscopy to study steady-state transepithelial secretion of 1 µM
fluorescein (FL) by isolated perfused S2 segments of rabbit renal
proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate
(PMA), a known PKC activator, to the bathing medium decreased
steady-state secretion of FL by ~30% after 25 min. This inhibition
was irreversible and, indeed, increased to ~40% at 25 min following
removal of PMA [10 µM 1,2-dioctanoyl-sn-glycerol (DOG)
produced a comparable inhibition]. The inhibition produced by PMA
was blocked when 100 nM of either staurosporine (ST) or
bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to
the bath for a 20-min preexposure followed by the addition of PMA. ST
or BIM alone had no significant effect on FL secretion, suggesting that
the basal FL secretion rate was not under influence of PKC. Addition of
1 µM of either the peptide hormone bradykinin (BK) or the
1-receptor agonist phenylephrine (PE), both of which
stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath
also inhibited FL secretion by ~22 and ~27%, respectively.
However, the inhibition was completely reversible after removal of BK
or PE. Pretreatment of tubules with 100 nM BIM eliminated the
inhibition of FL secretion produced by exposure to PE. We conclude that
PKC negatively regulates the net secretion of OAs in rabbit renal
proximal tubules. The data indicate that BK or catecholamines can play
a physiological role in regulating OA secretion via PKC activation.
fluorescein; organic anion/dicarboxylate exchanger; transepithelial transport in real time; bradykinin; phenylephrine; phorbol 12-myristate 13-acetate; 1,2-dioctanoyl-sn-glycerol; staurosporine; bisindolylmaleimide I
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INTRODUCTION |
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THE ORGANIC ANION (OA) and cation transport systems of
vertebrate kidneys are responsible for the excretion of a large number of potentially toxic substances, including endogenous metabolic waste
products, drugs, and xenobiotics. A wide variety of OAs (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 (14, 15). In the
S2 segment of mammalian renal proximal tubules, transepithelial
secretion of OAs 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 (14).
Transport into the cells at the basolateral membrane is a tertiary
active process, the final step in 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 (13, 18). 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 (13, 18), has now been
shown to function in intact renal proximal tubules from mammals and reptiles (2, 3, 19, 20, 25). A transporter that mediates OA/DC exchange
has now been cloned from mammalian renal tissue (11, 17, 21).
Although the mechanism responsible for the basolateral uptake of OAs is well understood, only recently has the cellular regulation of the OA transport system received attention. Studies with rabbit proximal tubules (7, 9), killifish proximal tubules (12), flounder proximal tubules in primary culture (8), opossum kidney (OK) cells in culture (22), and even transiently expressed human kidney PAH (hPAH) transporter in HeLa cells (11) all implicate protein kinase C (PKC) in the control of this transport system. Indeed, the finding of consensus sequences for PKC phosphorylation sites on the renal OA transporter (17, 21, 26) supports this suggestion. All these studies, with one exception (9), indicate that activation of PKC depresses basolateral OA uptake. However, no study has yet determined whether PKC regulates the transepithelial secretion of OAs in intact lumen-perfused mammalian proximal tubules.
The present study, employing a recently developed epifluorescence microscopy system whereby net steady-state transepithelial secretion of fluorescein (FL) can be measured in isolated, perfused renal tubules in real time, examined the regulatory role of PKC on net transepithelial secretion of OAs. Using S2 segments of rabbit renal proximal tubules, we demonstrated that activation of PKC inhibited the secretion of FL. Moreover, bradykinin (BK) and catecholamine appear to be possible candidates for physiological signals that trigger the PKC regulation of OA secretion.
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METHODS |
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Chemicals. Spectral grade FL and neutral tetramethylrhodamine-dextran (TMRD, 40,000 mol wt) were purchased from Molecular Probes (Eugene, OR). BK, bisindolylmaleimide I (BIM), 1,2-dioctanoyl-sn-glycerol (DOG), phenylephrine (PE), phorbol 12-myristate 13-acetate (PMA), and staurosporine (ST) were purchased from Sigma Chemical (St. Louis, MO). 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 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; 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 (27).
Perfusion of tubules. The in vitro perfusion technique used in these studies was the same as that described previously (4, 5) with some modification so that the collecting pipette had a length of uniform diameter that could be positioned parallel to the bottom of bathing chamber to serve as a flow-through cuvette as described previously (20). Briefly, 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 (20). During perfusion experiments, 1 µM FL was added to the bath, and TMRD was added to the perfusion solution as an indicator of leaks in the tubule. FL at this concentration is well below the Km value of the OA transport system for FL, as shown previously (20).
Determination of FL and TMRD in collected perfusate. As previously described (20), the perfusion chamber was mounted on the stage of an inverted microscope (Olympus 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 band-pass filter (Oriel) for this wavelength. The excitation light was reflected to the sample with a 490 DRLP 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 (540 DRLP, Omega Optical). Each emission beam was appropriately filtered (520 ± 10 nm for FL; 580 ± 30 nm for TMRD; Oriel and Omega Optical, respectively) and counted simultaneously by a separate photomultiplier tube (model HC 120-03; 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). 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 autofluorescence and the appropriate FL background counts were subtracted from the counts obtained during net secretion to yield the absolute FL or TMRD counts in the collecting pipette. The photon count was then converted into concentration from the standard curves.
Measurement of transepithelial secretion of FL. The net
transepithelial secretion of FL, JFL (in
mol · min1 · mm
1),
was determined from the following relationship.
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Statistical analysis. Results are summarized as means ± SE. The n value 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 measured for each tubule during control periods and following experimental interventions were evaluated by a paired t-test. Differences were assumed to be significant when P < 0.05.
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RESULTS |
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Effect of PKC activation by PMA on transepithelial secretion of
FL. Initially, we examined the time course profile of
transepithelial secretion of 1 µM FL during exposure to PMA in
isolated, perfused S2 segments of rabbit renal proximal tubules. Figure
1 shows the effect on transepithelial
secretion of FL by a single perfused tubule of the addition of 100 nM
PMA to the bathing medium. This PMA concentration has been reported to
affect OA transport in other systems (7, 9, 11, 12, 22). The
inset to Fig. 1 compares the average results obtained in
studies with 5 control tubules (from 5 different rabbits) and 14 tubules (from 14 different rabbits) exposed to PMA. The inhibitory
effect on FL secretion was seen ~10 min after the addition of PMA. As
summarized in Fig. 2, we chose the 1-min
average data points at 25 min after PMA was added to the bath for
statistical comparison. Stimulation of PKC by PMA reduced
transepithelial secretion of FL by ~30% from the control
steady-state secretion rate of 54.2 ± 3.2 to 38.5 ± 2.8 fmol · min1 · mm
1.
The inhibitory effect was still observed even 25 min after removal of
PMA from the bath. At this point, the FL secretion decreased to ~40%
of the control value without any observed tendency to recover. In
contrast, the time course profile for control tubules, which were
exposed to the bathing medium containing vehicular solvent DMSO at a
final concentration equal to that used in the PMA experiments
(1:20,000), showed nearly constant transepithelial secretion of FL
throughout the study (Fig. 1). DOG (10 µM), a membrane-permeant
analog of the physiological PKC activator diacylglycerol that has been
shown to inhibit the initial rate of basolateral FL uptake in isolated
nonperfused S2 segments of rabbit proximal tubules (7), also inhibited
transepithelial secretion of FL in a fashion similar to that of PMA (1 tubule, data not shown). These data indicate that direct stimulation of
PKC by PMA produced persistent inhibition of net FL secretion.
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Effect of PKC inhibition by BIM or ST on transepithelial secretion
of FL. To be certain that the inhibitory effect of PMA on FL
secretion occurred via stimulation of PKC, we used ST, a well-known PKC
inhibitor, and BIM, a synthesized PKC inhibitor shown to have higher
specificity for PKC than ST (23). As shown in Fig.
3, exposure of tubules alone to either of
the PKC inhibitors (100 nM) in the bath for 25 min produced virtually
no effect on the transepithelial secretion of 1 µM FL compared with
the control values (the tubules were exposed to DMSO at a final
concentration of 1:10,000). The data imply that under basal
physiological conditions the OA transport system is not significantly
under the influence of PKC. However, the inhibitory effect of 100 nM
PMA on transepithelial secretion of FL was completely abolished when it
was administered in the presence of 100 nM of either ST or BIM (Fig.
3). The results support the idea that in intact perfused proximal
tubules downregulation of OA secretion by PMA requires activation of
PKC.
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Effect of PKC stimulation by BK or PE on transepithelial secretion
of FL. To further investigate whether physiological stimulation of
PKC via ligand-receptor interaction also downregulated OA secretion, we
used BK and PE, both known to activate PKC in vivo via BK receptors and
1-receptors, respectively. As shown in Fig.
4A, either of the substances at a
concentration of 1 µM reversibly inhibited transepithelial secretion
of 1 µM FL to roughly the same extent (~22% by BK and ~27% by
PE). After removal of the substance from the bath, the transepithelial
secretion of FL returned to the control values in 25 min (Fig.
4A). This is in contrast to a direct stimulation of PKC by PMA
(Fig. 1) where inhibition of OA secretion was irreversible. The
inhibitory effect of PE (1 µM) was prevented when it was added in the
presence of 100 nM BIM (Fig. 4B). This result confirmed that
the inhibitory effect of PE on the transepithelial secretion of FL was
mediated through PKC stimulation.
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DISCUSSION |
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In the present study, we investigated the role of PKC in the transepithelial secretion of the OA FL by isolated, perfused S2 segments of rabbit renal proximal tubules in real time. 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. Our studies clearly demonstrated that activation of PKC inhibits the transepithelial secretion of OA in perfused S2 segments of rabbit renal proximal tubules. Regarding the inhibitory effect of PMA on OA transport, our results are consistent with the findings previously reported for studies with killifish renal tubules (12), OK cells in culture (22), flounder proximal tubule cells in primary culture (8), and hPAH transporter transiently expressed in HeLa cells (11).
In perfused S2 segments of rabbit renal proximal tubules our data
showed that: 1) 100 nM PMA profoundly inhibited transepithelial secretion of FL (Figs. 1 and 2); 2) the inhibition became
clearly observed after 10 min of incubation with PMA (Fig. 1);
3) the inhibitory effect of PMA was abolished by ST or BIM
(Fig. 3); and 4) ST or BIM alone had no effect on FL secretion
(Fig. 3). Hohage et al. (9) have studied the role of PKC in basolateral uptake of PAH by isolated nonperfused S2 segments of rabbit renal proximal tubules. They reported a biphasic dose-response curve of
basolateral uptake of PAH during 10-min incubation with maximum stimulation being at a PMA concentration of
107 M (equal to that we used in our
studies). Although they showed that the effect of PMA could be blocked
by ST, they also reported that ST alone inhibited basolateral uptake of
PAH (9). Their results are in contrast to those in our present studies.
The reasons for the difference between their studies and our current
ones are not certain. The components and temperature of the incubating buffer were similar in both studies. The difference is not likely due
to a difference in response between perfused and nonperfused tubules,
because recent studies from our laboratory with nonperfused tubules
showed results that are in good agreement with our current studies with
perfused tubules in regard to PMA administration (7); i.e., PMA
inhibited the initial rate of basolateral FL uptake. The difference in
results also seems unlikely to be due to the difference
between the OA model used (PAH vs. FL) in the different studies,
because we previously demonstrated that transepithelial secretion of FL
is limited to the transport system utilized by PAH (20). Miller (12)
has reported a stimulation of FL transport by PKC inhibitors in intact
secreting proximal tubules of killifish. This is in contrast to our
finding that the PKC inhibitors BIM and ST had no effect on
transepithelial FL secretion. The difference may reflect differences in
response to PMA and perhaps in intracellular signaling mechanisms
between species. Therefore, it appears likely that basal OA transport
in freshly prepared rabbit proximal tubules is not under the influence
of PKC.
It also appears likely that the inhibitory effect of PMA on the transepithelial secretion of FL is due to PKC activation and not to nonspecific effects of PMA. The findings in the present study support this conclusion. First, DOG, an analog of diacylglycerol known to stimulate PKC, also inhibited the secretion (data not shown). Second, ST and BIM completely blocked the inhibitory effect of PMA (Fig. 3). Taken together, these observations indicate that the inhibitory effect of PMA on the transepithelial secretion of OAs is limited to the activation of PKC.
With regard to the physiological stimulation of PKC, our present data
show that BK and PE, both known to stimulate PKC in renal proximal
tubules (1, 6, 10) by binding to their receptors (BK receptors and
1-receptors, respectively), inhibited transepithelial
secretion of FL (Fig. 4A). The inhibitory effect of PE is,
indeed, blocked by BIM (Fig. 4B), confirming that PE inhibits
OA secretion via a ligand-receptor-PKC coupling pathway. Unlike the
inhibition of FL secretion by direct stimulation of PKC with PMA, PKC
stimulation via the receptor-coupled pathway with BK or PE is
completely reversed in 25 min after removal of these substances. This
might be the result of rapid metabolism of BK and PE, but not PMA, by
the proximal tubule cells. However, we cannot rule out the
possibilities that the degree of PKC stimulation by the PMA
concentration (100 nM) used in the present studies might have been so
great that it also interfered with other metabolic events, resulting in
the observed further decrease in OA secretion. It seems reasonable that
PKC stimulation by BK and PE downregulates OA secretion to help
preserve cellular energy, because both BK and PE reduce blood flow to
the kidneys. However, determination of the physiological significance
of these ligand-receptor-PKC coupling pathways on the transepithelial
secretion of OAs in vivo requires further investigation.
Several possible mechanisms involving the inhibition of OA transport by PKC have been postulated. The most direct is a downregulation of OA/DC exchanger activity. Indeed, the finding of a consensus sequence of PKC phosphorylation sites on the cloned OA/DC exchanger from several species (17, 21, 26) makes it likely that PKC-stimulated inhibition of basolateral OA transport occurs by direct phosphorylation of the OA/DC exchanger. Although no direct evidence of OA/DC exchanger phosphorylation by PKC stimulation has yet been shown, studies in heterologous expression systems of the rat and human orthologs of the cloned OA/DC exchanger, OAT1, have shown that activation of PKC results in a downregulation of PAH transport (11). At least two other transport processes, the peritubular Na-DC cotransporter and "the luminal OA transporter," influence OA secretion and could be targets for PKC regulation. However, we consider it unlikely that either of these processes plays a significant role in the observed regulation of FL secretion by PKC. The peritubular Na-DC cotransporter in rabbit proximal tubules does not respond to PKC (16), and inhibition of Na-DC activity does not influence the downregulation of peritubular OA/DC exchanger activity influenced by activation of PKC (7). Luminal exit of FL in isolated teleost tubules is not affected by exposure to PMA (although peritubular FL accumulation is downregulated) (12). Some other mechanisms, such as an increase in a paracellular leak, internalization of the basolateral membrane containing the transporters and/or inhibition of insertion of newly synthesized transporters into the membrane may also be involved in the regulation of OA secretion by PKC. Indeed, Miller (12) has reported a translocation of PKC from the cytoplasm to the plasma membrane. Therefore, further studies are needed to identify the specific mechanisms involved in the regulation of renal OA transport.
In conclusion, we demonstrated that PMA inhibited the transepithelial secretion of OAs, most likely by the activation of PKC, in isolated perfused S2 segments of rabbit renal proximal tubules. At least two physiological hormones, BK and catecholamines, appear to be possible candidates for physiological signals that can downregulate the OA secretory system.
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ACKNOWLEDGEMENTS |
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We thank the Royal Thai Government for its support of Apichai Shuprisha during the course of this work.
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FOOTNOTES |
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This study was supported in part by National Institutes of Health Research Grant ES-06757; Training Grants HL-07249, NS-07309, and GM-08400; and 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: S. H. Wright, Dept. of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724-5051 (E-mail: shwright{at}u.arizona.edu).
Received 23 April 1999; accepted in final form 17 August 1999.
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