![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands (S.N., L.H.K., P.S., F.G.M.R., R.M.); Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (D.S.M.); and Mount Desert Island Biological Laboratory, Salisbury Cove, Maine (S.N., D.S.M., L.H.K.)
Received May 4, 2005; accepted August 4, 2005.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Terlouw et al., 2001; Notenboom et al., 2002, 2004). Here, we determined the long-term effects on Mrp2-mediated transport (luminal fluorescein methotrexate accumulation) of short-term (30 min) exposure to ET-1 and the aminoglycoside antibiotic, gentamicin. Our data show that over the 3 h following exposure, proximal tubules recovered fully from the initial decrease in Mrp2-mediated transport and that transport activity was not changed 9 h later. However, 24 h after exposure, luminal accumulation of an Mrp2 substrate had increased by 50%. Increased transport at 24 h was accompanied by an increased transporter protein content of the luminal plasma membrane as measured by immunostaining. Blocking ET-1 signaling at the ETB receptor or downstream at NOS or guanylyl cyclase abolished both stimulation of transport and increased transporter expression. Thus, regardless of whether signaling was initiated by a short exposure to ET-1 or to a nephrotoxicant, the time course of Mrp2 response to ETB signaling was the same and was multiphasic. Finally, when tubules were exposed to gentamicin for 30 min and removed to gentamicin-free medium for 24 h, they were less sensitive to acute gentamicin toxicity than paired controls not initially exposed to the drug. Thus, short-term exposure to ET-1 or gentamicin resulted in long-term protection against a second insult.
; Schinkel and Jonker, 2003). In the vertebrate renal proximal tubule, Mrp2 has been localized to the luminal pole of the epithelial cells (Schaub et al., 1997), the correct location to provide the final step in transport of many anionic xenobiotics from blood to urine. The efflux pump handles a wide range of substrates, and its activity and expression level in the kidney is influenced by hormones, local mediators, nuclear receptors, protein kinases, and disease conditions (Terlouw et al., 2003). As with drug-metabolizing enzymes, e.g., cytochrome P450s and transferases (Rushmore and Kong, 2002; Korashy et al., 2004), pump activity is regulated in response to exposure to substrates and physiological state.
We previously showed in intact killifish renal proximal tubules that Mrp2 activity, as measured by luminal accumulation of a fluorescent methotrexate (MTX) derivative [fluorescein (FL)-MTX], is rapidly reduced by subnanomolar to nanomolar concentrations of ET-1 acting through an ETB receptor, NOS, cGMP, and PKC. Surprisingly, acute exposure of tubules to several nephrotoxicants, i.e., aminoglycoside antibiotics, radiocontrast agents, and heavy metal salts, also reduces FL-MTX transport, and blocking ET signaling at any point in the chain abolishes the nephrotoxicant effects on transport (Terlouw et al., 2001; Notenboom et al., 2002, 2004). From these experiments, it was also clear that the nephrotoxicants caused Ca2+-dependent release of ET from the tubules and that released ET activated intracellular signaling by an autocrine mechanism. In contrast, after long-term, continuous exposure (6-24 h) to the nephrotoxic heavy metal salt CdCl2, transport activity and immunostaining of Mrp2 at the luminal membrane of the proximal tubules had increased (Terlouw et al., 2002). This long-term induction of Mrp2 may function as a compensatory mechanism for the initially reduced efflux of potentially toxic compounds, serving a protective route.
The present study addresses the issue of whether short-term signaling through the ET-activated pathway has longer term consequences to tubular function. Our results show that after 30 min of exposure to ET-1 or the aminoglycoside antibiotic, gentamicin, Mrp2-mediated transport initially declined; this is in agreement with previous studies (Terlouw et al., 2001; Notenboom et al., 2002). When tubules were removed to ET-1- and gentamicin-free medium, transport recovered over the next several hours. Twenty-four hours after exposure, Mrp2-mediated transport and Mrp2 protein expression were significantly higher than controls. These increases in transport and Mrp2 expression were abolished when ET signaling was disrupted. Finally, short-term gentamicin exposure and subsequent recovery for 24 h was protective against acute gentamicin tubular toxicity. Thus, short-term signaling has long-term consequences with regard to transport function and nephrotoxicant resistance.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, 2002). All other chemicals used were obtained at the highest purity available commercially.
Animals and Tissue Preparation. Killifish (Fundulus heteroclitus) were collected by local fishermen in the vicinity of Mount Desert Island, Maine and maintained at the Mount Desert Island Biological Laboratory in tanks with natural flowing seawater. Renal tubular masses were isolated in a marine teleost saline based on that of Forster and Taggart (1950) containing 140 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, and 20 mM Tris at pH 8.0. Isolation and short-term exposure were carried out at 18 to 20°C. Under a dissecting microscope, each mass was teased with fine forceps to remove adherent hematopoietic tissue. Individual killifish proximal tubules from several fish were dissected, pooled, and transferred to a well plate containing 3 ml of marine teleost saline in the presence or absence of 10 µM gentamicin. After 30 min of exposure, tubules were washed in marine teleost saline and transferred to recovery medium, i.e., modified medium 199 with Earle's salts supplemented with 30.0 mM NaCl, 4.2 mM NaHCO3, 1.0 mM L-glutamine, 25.0 mM HEPES, 14.75 mM NaOH (pH 7.5, 347 mosmol/kg H2O), 20 mg/l tetracycline, 10 µg/ml insulin, 5 µg/ml hydrocortisone, and 10% flounder serum (Renfro et al., 1999) with added effectors. The viability of teleost tubules is not preserved at 18 to 20°C past 6 h but can be extended to 48 h by reducing the incubation temperature to 13.5°C (D. S. Miller, unpublished data). After 1.5, 3, 12, or 24 h of recovery at 13.5°C, tubules were washed in marine teleost saline and transferred to a foil-covered Teflon chamber containing 1 ml of marine teleost saline with 1 µM FL-MTX. The chamber floor was a 4- x 4-cm glass coverslip to which the tubules adhered lightly and through which the tissue could be viewed by means of an inverted microscope. Tubules were incubated at room temperature for 30 min until steady state was reached for FL-MTX. Analysis of tubule extracts by high-pressure liquid chromatography showed no metabolic degradation of FL-MTX when incubated with killifish proximal tubules for periods of at least 1 h (Schramm et al., 1995; Masereeuw et al., 1996).
In toxicity experiments, isolated killifish tubules were transferred to a well plate containing marine teleost saline in presence or absence of 10 µM gentamicin. After 30 min of exposure, tubules were washed in marine teleost saline and transferred to the recovery medium. After 24 h of recovery at 13.5°C, tubules were exposed to 100 µM gentamicin for 2 h and subsequently washed in marine teleost saline and transferred to a foil-covered Teflon chamber containing 1 ml of marine teleost saline with an indicator of tubular function. We used transepithelial transport of the fluorescent agent fluorescein and the mitochondrial marker, MitoTracker Red CM-H2Xros, as two functional indicators of tubule viability. Both were measured using confocal microscopy and quantitative image analysis.
Confocal Microscopy. The chamber containing renal tubules was mounted on the stage of an Olympus FluoView inverted confocal laser scanning microscope and viewed through a 40x water immersion objective (numerical aperture 1.15). Excitation was provided by the 488-nm line of an argon ion laser. A 510-nm dichroic filter and a 515-nm long-pass emission filter were used. Neutral density filters and low laser intensity were used to avoid photobleaching. With the photomultiplier gain set to give an average luminal fluorescence intensity of 1500 to 3000 (on a scale of 0-4096), tissue autofluorescence was undetectable. To obtain an image, dye-loaded tubules in the chamber were viewed under reduced, transmitted light illumination, and a single proximal tubule with well defined lumen and undamaged epithelium was selected. The plane of focus was adjusted to cut through the center of the tubular lumen, and an image was acquired by averaging four scans. The confocal image was viewed on a high-resolution monitor and saved to an optical disk. In previous studies, it has been shown that there is a linear relationship between fluorescence intensity and dye concentration (Miller and Pritchard, 1991). However, because of the many uncertainties in relating cellular fluorescence to actual compound concentration in cells and tissues with complex geometry, data are reported here as a percentage of average measured pixel intensity compared with control rather than estimated dye concentration. Fluorescence intensities were measured from stored images using Scion Image version 1.8 for Windows as described previously (Masereeuw et al., 1996; Miller et al., 1996). Briefly, two or three adjacent cellular and luminal areas were selected from each tubule, and the average pixel intensity for each area was calculated for measurement of fluorescein and FL-MTX transport. The values used for that tubule were the means of all selected areas.
Measurement of Mitochondrial Function. MitoTracker Red CM-H2Xros was used for determination of mitochondrial functional integrity. We used the optical sectioning capabilities of the confocal microscope to measure the dye fluorescence intensity inside tubular cells. Essentially all of the cellular fluorescence was in discrete structures, suggesting mitochondrial accumulation (Pendergrass et al., 2004).
Immunohistochemistry. Isolated killifish proximal tubules were exposed to 10 µM gentamicin or 10 nM ET-1 for 30 min and transferred to gentamicin- and ET-1-free medium for a 24-h recovery period. Subsequently, tubules were processed in phosphate-buffered saline for immunostaining: fixation with 2% (v/v) formaldehyde/0.1% (v/v) glutaraldehyde, permeabilization with 1% (v/v) Triton X-100, 90-min exposure to primary antibody (k78 1:50 for Mrp2, or anti-Mrp4, 1:10), and 60-min exposure to secondary antibody (Alexa488-labeled goat anti-rabbit IgG, 1:20). Antibody binding was detected with the Zeiss confocal laser scanning microscope using a 20x objective, and staining was quantified using ImageJ version 1.30 (National Institutes of Health, Bethesda, MD). A grid, consisting of 655.02-µm2 squares, was placed on top of the confocal images to semiquantify the fluorescent staining. Only there where the grid crossed were luminal staining fluorescence intensities measured. The average of the fluorescence intensities measured for that tubule was used for quantitation.
Data Analysis. Data are given as mean percentage of control fluorescence ± S.E., unless indicated otherwise. Mean values were considered to be significantly different when P < 0.05 by use of the unpaired Student's t test or by a one-way analysis of variance followed by Bonferroni's multiple comparison test. Software used for statistical analysis was GraphPad Prism (version 3.00 for Windows; GraphPad Software Inc., San Diego, CA).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Unlike mammalian proximal tubules, the broken ends of teleost proximal tubules reseal after isolation, thus, forming a closed fluid filled luminal compartment. This enables study of cellular uptake and luminal accumulation of fluorescent compounds using imaging techniques. As in mammalian proximal tubules, killifish express high levels of Mrp2 in the luminal membrane of renal proximal tubule cells. Moreover, killifish tubules exhibit Mrp2-mediated transport of a number of fluorescent substrates, e.g., FL-MTX, that can be visualized and measured in intact individual tubules using confocal microscopy (Masereeuw et al., 1996, 2000; Miller et al., 1996). Figure 1, A and B, show a typical confocal image of a killifish tubule after 30 min (steady state) incubation in medium with 1 µM FL-MTX following 24 h of recovery. The fluorescence distribution pattern is the same as shown previously after short-term exposure, i.e., fluorescence intensity in lumen > cells > medium (Masereeuw et al., 1996, 2000). We have demonstrated that this pattern is indicative of a two-step process, involving uptake at the basolateral membrane mediated by an as-yet uncharacterized transporter for large organic anions and secretion into the lumen mediated by a teleost form of Mrp2 (for data on substrate and inhibitor specificities as well as immunostaining with Mrp2 antibodies, see Masereeuw et al., 1996, 2000; Terlouw et al., 2001). Using an Sf9 overexpression system, we previously showed that FL-MTX is a substrate for MRP2 (Terlouw et al., 2002). Although MRPs are known to share many substrates, interference of other members of the MRP family with FL-MTX transport in this model is unlikely. Mrp5 and Mrp6 are located in the basolateral membrane and not in the apical membrane of renal proximal tubules, whereas Mrp1 and Mrp3 are not expressed in renal proximal tubules (Russel et al., 2002). Furthermore, we can exclude the contribution of Mrp4 because results from our group show that FL-MTX is not a substrate for MRP4 (P. H. E. Smeets and F. G. M. Russel, unpublished data), and MRP4-mediated transport is insensitive to leukotriene C4 (Van Aubel et al., 2002), which is an excellent inhibitor of FL-MTX secretion in killifish proximal tubules (Masereeuw et al., 1996, 2000).
|
To determine whether short-term ET-1 signaling has long-term effects, we exposed tubules to ET-1 for 30 min, removed them to ET-free medium, and assayed Mrp2 transport function at several times after transfer. Figure 1C shows that 1.5 h after transfer, the tubules exhibit significantly reduced transport. This result is in agreement with previous experiments from this laboratory where tubules were exposed continuously to ET-1 in transport experiments lasting up to 90 min (Masereeuw et al., 2000). However, with time in the ET-free medium, transport increased, and 3- and 12-h values were nearly identical to tubules not exposed to ET-1 (controls). Moreover, 24 h after ET-1 exposure, luminal accumulation of FL-MTX had increased by about 50% (P < 0.001; Fig. 1). Neither short-term exposure to ET-1 nor short-term exposure followed by recovery affected cellular accumulation of FL-MTX. Thus, both the short- and long-term effects of 30-min ET-1 exposure on luminal accumulation were evident with FL-MTX as substrate.
We previously demonstrated that several tubular nephrotoxicants (aminoglycoside antibiotics, radiocontrast agents, and heavy metal salts) were capable of mimicking the effects of ET-1 on FL-MTX transport. Each of these activated the ET signaling pathway by a Ca2+-dependent mechanism causing ET release from the tubules. None of them interacted with Mrp2 directly (Terlouw et al., 2001; Notenboom et al., 2002). When we incubated tubules in medium containing the aminoglycoside antibiotic, gentamicin (10 µM), transferred them to gentamicin-free medium, and monitored FL-MTX transport, we found the same pattern of effects as observed with ET-1 (Fig. 2). Thus, both ET-1 and gentamicin had a triphasic effect on FL-MTX: short-term reduction, followed by recovery, and finally significantly increased transport. Note that when FL was used as a substrate, there was no initial decrease in transport with 10 µM gentamicin (Fig. 3A) and no increase 24 h after gentamicin exposure (Fig. 3B). However, with short-term exposure to higher concentrations of gentamicin (Fig. 3A) and with 24-h continuous exposure to 10 µM gentamicin (preliminary data not shown), a decrease in FL transport was seen, indicating toxicity.
|
|
; Terlouw et al., 2001, 2002; Notenboom et al., 2002, 2004). Thus, at a minimum, the long-term effects of short-term exposure to ET-1 or gentamicin appear to be a consequence of signaling through the ETB receptor, NOS, and cGMP.
|
The increase in luminal accumulation of FL-MTX 24 h after short-term exposures to ET-1 or gentamicin may have resulted from increased transporter expression or increased transport activity with no increase in expression. To determine whether the expression level was altered, we used immunofluorescence to examine the long-term effects on Mrp2. Figure 5 shows representative confocal images of killifish tubules immunostained for Mrp2. In agreement with previous studies in mammalian and killifish renal tissue, this transporter localized to the luminal (brush border) membrane of the tubule epithelial cells (Schaub et al., 1997, 1999; Masereeuw et al., 2000; Terlouw et al., 2001, 2002). Figure 5 also shows representative confocal images of tubules exposed to gentamicin or ET-1 for 30 min and then assayed 24 h later. In these tubules, Mrp2 immunofluorescence appears to be more intense than in controls (no ET-1 or gentamicin exposure). Quantitation of Mrp2 immunofluorescence bears out this impression. Both ET-1 and gentamicin increased luminal membrane immunofluorescence by about 25% (P < 0.01). In contrast, no such increase was found when tubules were stained for Mrp4, which, as in mammalian renal proximal tubules (Van Aubel et al., 2002), is also localized to the luminal membrane of killifish renal proximal tubule cells (Fig. 5E). Consistent with the transport data presented above, inhibiting NOS with L-NMMA blocked the increase in Mrp2 expression caused by 30-min exposure to ET-1 or gentamicin (Fig. 6).
|
|
Thus, one consequence of short-term exposure to ET-1 or gentamicin is an increase in Mrp2 expression and function 24 h later. To determine whether the response to nephrotoxicants was also affected, we used MitoTracker Red CM-H2XRos in tubules exposed to 100 µM gentamicin to indirectly assess mitochondrial functional integrity. This concentration of nephrotoxicant is an order of magnitude higher than that which alters Mrp2-mediated transport though ET signaling and was found to affect tubular viability (Fig. 3). The advantage of using MitoTracker Red CM-H2XRos is that the probe is highly stable. It is oxidized within cells and then selectively sequestered in mitochondria of actively respiring cells. Probes often used for measuring mitochondrial membrane potential, e.g., JC-1 and Safranine O, cannot be used in our model because they are substrates for efflux carriers present in the apical membrane of the tubular cells. In this experiment, tubules were pre-exposed to 0 (controls) or 10 µM gentamicin for 30 min and then incubated in gentamicin-free medium for 24 h. Subsequently, they were challenged with a 2-h exposure to 100 µM gentamicin, and changes in mitochondrial integrity were measured using 500 nM MitoTracker Red CM-H2XRos and confocal imaging after 30-min incubation with the probe. In tubules not pre-exposed to the low concentration of gentamicin (controls), 100 µM gentamicin significantly decreased mitochondrial integrity (P < 0.05). This effect was roughly half of that seen when control tubules were exposed to 1 mM NaCN for 2 h (Fig. 7). In contrast, no such gentamicin-induced alteration in mitochondrial functional integrity was observed in tubules that had been pre-exposed to 10 µM gentamicin. Thus, at least for one nephrotoxicant and one measure of toxicity, gentamicin pre-exposure and subsequent recovery were protective.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Terlouw et al., 2001, 2002). These inhibitory effects on Mrp2-mediated transport were observed within 15 min of exposure and were maximal within 60 min. After 60 min of exposure, the Mrp2 protein content of the luminal plasma membrane was not changed, suggesting that transporter activity in the luminal plasma membrane was modified, or transporter was removed to a subapical compartment that was not resolved at the light microscope level (Terlouw et al., 2001). In the present study, we investigated the effects of exposing renal proximal tubules to ET-1 or to gentamicin for a short time (30 min) and then returning them to ET-1- and gentamicin-free medium. The data show that over the 3 h following exposure, tubules recovered fully from the initial decrease in Mrp2-mediated transport and that transport activity was not changed 9 h later. However, 24 h after exposure, luminal accumulation of FL-MTX had increased by 50%. Increased transport at 24 h was accompanied by an increased transporter protein content of the luminal plasma membrane measured by immunostaining. This finding could not be confirmed using immunoblots. Immunoblot analysis is not feasible in this situation because of the small amount of membrane protein isolated from killifish tubules. A recent direct comparison of quantitative immunostaining with immunoblot analysis, however, indicates that the results obtained with the two methods are comparable (Bauer et al., 2004). Furthermore, preliminary data in rats treated with gentamicin in vivo showed a clear up-regulation of Mrp2 in the apical membrane of renal proximal tubule confirming the findings of our killifish experiment in a different species (S. Notenboom, F. G. M. Russel, and R. Masereeuw, unpublished data).
Blocking ET-1 signaling at the ETB receptor, or downstream at NOS, abolished both stimulation of transport and increased transporter expression. Thus, regardless of whether signaling was initiated by a short exposure to ET-1 or by a nephrotoxicant, the time course of the Mrp2 response to ETB signaling was the same. It was multiphasic, involving reduced Mrp2-mediated transport, recovery to control levels, and a delayed increase over control levels 24 h after exposure. Nonspecific leakage of the fluorescent dye can be excluded because luminal FL-MTX accumulation is concentrative with respect to medium, and leakage of the dye at the tight junctions would decrease the luminal concentrating ability of the tubules. Note that FL transport in killifish renal tubules was constant over the entire exposure/recovery time course (Fig. 3) as observed previously (Terlouw et al., 2002). This fluorescent organic anion is avidly transported from bath to tubular lumen by an organic anion transport system that does not include Mrp2 (Masereeuw et al., 1996). Fluorescein may be transported by members of the SLC superfamily of transporters, organic anion transporters and organic anion-transporting polypeptides. However, the finding that fluorescein transport was unchanged argues for the lack of an effect on other carriers. In addition, in agreement with the zebrafish genome, unpublished findings demonstrate that killifish tubules express only one organic anion transporter that is localized to the basolateral membrane (J. B. Pritchard and D. S. Miller, personal communication). Concerning the organic anion-transporting polypeptide carriers, a large species difference exists in this subfamily of transporters, and it is unknown whether an isoform is present in killifish renal proximal tubules. Furthermore, 24 h after exposure, we did not detect any qualitative change in luminal plasma membrane content of Mrp4. This suggests no increase in expression of luminal membrane transporters in general or in the expression of MRP subfamily members in particular.
The increase in Mrp2 transport function (
50%) 24 h after exposure to ET-1 or gentamicin was accompanied by a less than proportional increase in transporter content in the plasma membrane (25%). Assuming a one-to-one correspondence between transport activity and transporter content, this difference suggests that multiple mechanisms contribute to the increase in transporter function. These might include de novo synthesis of Mrp2, increased insertion of Mrp2 into the apical membrane, reduced Mrp2 retrieval from the apical membrane, and functional activation of membrane-bound transporter. Mrp2 activity is known to be modulated by transcriptional and post-transcriptional mechanisms. For example, several ligand-activated nuclear receptors have been shown to transcriptionally regulate the activity of xenobiotic-metabolizing enzymes and xenobiotic transporters, including Mrp2, in liver, intestine, and blood-brain barrier (Kast et al., 2002; Kullak-Ublick and Becker, 2003; Bauer et al., 2004). These include the pregnane X nuclear receptor (PXR) and the constitutive androstane receptor, both of which are activated by a wide range of xenobiotics. Teleost fish do express a PXR homolog (Moore et al., 2002; Maglich et al., 2003), but it is not known to what extent (if any) gentamicin or ET-1 affect PXR activity in any species. In addition, in mammalian hepatocytes, both PKC and PKA have been implicated in bidirectional, regulated trafficking of Mrp2 between intracellular stores and the canalicular membrane (Roelofsen et al., 1998; Beuers et al., 2001; Kubitz et al., 2001). Furthermore, Hegedus et al. (2003) suggested that PKC is involved in the Mrp2 targeting and recycling through phosphorylation of the PDZ domain, which influences the interaction between Mrp2 and its anchoring PDZ proteins and thereby its transport function. PKA activation does not appear to be involved in short-term exposure, since we previously found no effect of a PKA-selective inhibitor on ET-1 signaling (Masereeuw et al., 2000). Preliminary data using a canine kidney cell line overexpressing MRP2 pointed to an increased insertion of the transport protein in the luminal membrane after 1 h of gentamicin exposure and 24-h recovery or 24-h gentamicin exposure, whereas overall MRP2 expression remained unchanged (S. Notenboom, F. G. M. Russel, and R. Masereeuw, unpublished data). Additional experiments are needed to further characterize in the killifish renal proximal tubule model the mechanisms that link ET-NOS-cGMP-PKC signaling and Mrp2 up- and down-regulation.
Up-regulation of Mrp2 may be interpreted as part of a mechanism called preconditioning. Thus, an up-regulation of Mrp2 and other adaptive mechanisms after recovery protects the tissue against a second exposure to gentamicin. This phenomenon has been observed for ischemia in heart, liver, and kidney (Andreucci et al., 2003; Jaeschke, 2003; Park et al., 2003; Juhaszova et al., 2004). Mrp2 serves a protective function through the elimination of potentially harmful chemicals. Indeed, Mrp2 up-regulation was also observed after long-term exposure to cadmium (Terlouw et al., 2002) and after ischemia (Laouari et al., 2001). Since Mrp2 handles many potentially toxic compounds, like xenobiotics and their metabolites (Russel et al., 2002), up-regulation of the transport protein may be part of a protective pathway of proximal tubules following harmful events.
Finally, use of gentamicin and other aminoglycoside antibiotics has been associated with severe proximal tubular nephrotoxicity, which limits their clinical use. Although such irreversible toxicity has been historically associated with multiple administration of high doses (Bennett, 1989), gentamicin can alter renal function even at low dose levels (Foster et al., 1992). The present data show for the first time that low-level, short-term, gentamicin exposure could have beneficial side effects, possibly by triggering a survival/protective pathway. Certainly, ET signaling and gentamicin preconditioning affect more than Mrp2 expression and function. It will be important to determine which of these changes in gene expression or enzyme/transporter function confers increased resistance to aminoglycoside antibiotic toxicity. Note that the present experiments, in which Mrp2 transport function was the endpoint, provide an additional example of context-dependent signaling, as demonstrated recently for protein kinases (Bhalla et al., 2002; Ingolia and Murray, 2002). In this regard, we have been able to document three patterns of effects following tubule exposure to gentamicin (and other nephrotoxicants): inhibition of transport with short-term exposure (Terlouw et al., 2001, 2002; Notenboom et al., 2002, 2004); toxicity with long-term, continuous exposure (S. Notenboom and D. S. Miller, unpublished data), and increased transport with long-term exposure to low concentrations of nephrotoxicants (Terlouw et al., 2002) or with short-term exposure followed by a period of recovery (present study).
In conclusion, renal proximal tubules exposed to gentamicin or ET-1 and allowed to recover for 24 h display an increase in Mrp2-mediated transport and Mrp2 expression in the luminal membrane. These increments are a result of intracellular signaling consequences caused by short-term nephrotoxicant exposure in the renal proximal tubule. Several signaling molecules were identified as participants in this pathway leading to the following sequence of events: Short-term exposure to gentamicin triggers ET-1 release, ET-1 binds to the ETB receptor, and, subsequently, NOS is stimulated, resulting in the activation of soluble guanylyl cyclase. The produced cGMP might cause de novo synthesis of Mrp2, stimulation of Mrp2 insertion in the luminal membrane, and/or inhibition of Mrp2 retrieval from the luminal membrane, eventually leading to increased Mrp2-mediated transport through increased Mrp2 insertion in the luminal membrane in the renal proximal tubule. Thus, short-term signaling (nephrotoxicant exposure) has long-term consequences for renal proximal tubule functioning.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Mrp, multidrug resistance protein; MTX, methotrexate; FL, fluorescein; ET, endothelin; NOS, nitric-oxide synthase; PKC, protein kinase C; L-NMMA, NG-methyl-L-arginine acetate salt; RES701-1, cyclo(-Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp)-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp-OH; ODQ, oxadiazole quinoxalin; PXR, pregnane X nuclear receptor.
Address correspondence to: Dr. Rosalinde Masereeuw, Department of Pharmacology and Toxicology, 233 Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: r.masereeuw{at}ncmls.ru.nl
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Andreucci M, Michael A, Kramers C, Park KM, Chen A, Matthaeus T, Alessandrini A, Haq S, Force T, and Bonventre JV (2003) Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK(1) cells result in phosphorylation of FKHR and FKHRL1. Kidney Int 64: 1189-1198.[CrossRef][Medline]
Bauer B, Hartz AM, Fricker G, and Miller DS (2004) Pregnane X receptor up-regulation of P-glycoprotein expression and transport function at the blood-brain barrier. Mol Pharmacol 66: 413-419.
Bennett WM (1989) Mechanisms of aminoglycoside nephrotoxicity. Clin Exp Pharmacol Physiol 16: 1-6.[Medline]
Beuers U, Bilzer M, Chittattu A, Kullak-Ublick GA, Keppler D, Paumgartner G, and Dombrowski F (2001) Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 33: 1206-1216.[CrossRef][Medline]
Bhalla US, Ram PT, and Iyengar R (2002) MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network. Science (Wash DC) 297: 1018-1023.
Forster RP and Taggart JV (1950) Use of isolated renal tubules in the estimation of metabolic processes associated with active cellular transport. J Cell Comp Physiol 36: 251-270.[CrossRef]
Foster JE, Harpur ES, and Garland HO (1992) An investigation of the acute effect of gentamicin on the renal handling of electrolytes in the rat. J Pharmacol Exp Ther 261: 38-43.
Hegedus T, Sessler T, Scott R, Thelin W, Bakos E, Varadi A, Szabo K, Homolya L, Milgram SL, and Sarkadi B (2003) C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins. Biochem Biophys Res Commun 302: 454-461.[CrossRef][Medline]
Ingolia NT and Murray AW (2002) Signal transduction: history matters. Science (Wash DC) 297: 948-949.
Jaeschke H (2003) Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol 284: G15-G26.
Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, et al. (2004) Glycogen synthase kinase-3
mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Investig 113: 1535-1549.[CrossRef][Medline]
Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, and Edwards PA (2002) Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor and constitutive androstane receptor. J Biol Chem 277: 2908-2915.
Korashy HM, Elbekai RH, and El Kadi AO (2004) Effects of renal diseases on the regulation and expression of renal and hepatic drug-metabolizing enzymes: a review. Xenobiotica 34: 1-29.[CrossRef][Medline]
Kubitz R, Huth C, Schmitt M, Horbach A, Kullak-Ublick G, and Haussinger D (2001) Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells. Hepatology 34: 340-350.[CrossRef][Medline]
Kullak-Ublick GA and Becker MB (2003) Regulation of drug and bile salt transporters in liver and intestine. Drug Metab Rev 35: 305-317.[CrossRef][Medline]
Laouari D, Yang R, Veau C, Blanke I, and Friedlander G (2001) Two apical multidrug transporters, P-gp and MRP2, are differently altered in chronic renal failure. Am J Physiol 280: F636-F645.
Maglich JM, Caravella JA, Lambert MH, Willson TM, Moore JT, and Ramamurthy L (2003) The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily. Nucleic Acids Res 31: 4051-4058.
Masereeuw R, Russel FGM, and Miller DS (1996) Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy. Am J Physiol 271: F1173-F1182.
Masereeuw R, Terlouw SA, Van Aubel RA, Russel FG, and Miller DS (2000) Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol Pharmacol 57: 59-67.
Miller DS, Letcher S, and Barnes DM (1996) Fluorescence imaging study of organic anion transport from renal proximal tubule cell to lumen. Am J Physiol 271: F508-F520.
Miller DS and Pritchard JB (1991) Indirect coupling of organic anion secretion to sodium in teleost (Paralichthys lethostigma) renal tubules. Am J Physiol 261: R1470-R1477.
Moore LB, Maglich JM, McKee DD, Wisely B, Willson TM, Kliewer SA, Lambert MH, and Moore JT (2002) Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol Endocrinol 16: 977-986.
Notenboom S, Miller DS, Smits P, Russel FGM, and Masereeuw R (2002) Role of NO in endothelin-regulated drug transport in the renal proximal tubule. Am J Physiol 282: F458-F464.
Notenboom S, Miller DS, Smits P, Russel FGM, and Masereeuw R (2004) Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule. Am J Physiol 287: F33-F38.
Park KM, Byun JY, Kramers C, Kim JI, Huang PL, and Bonventre JV (2003) Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney. J Biol Chem 278: 27256-27266.
Pendergrass W, Wolf N, and Poot M (2004) Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry 61: 162-169.[Medline]
Pritchard JB and Miller DS (1991) Comparative insights into the mechanisms of renal organic anion and cation secretion. Am J Physiol 261: R1329-R1340.
Renfro JL, Maren TH, Zeien C, and Swenson ER (1999) Renal sulfate secretion is carbonic anhydrase dependent in a marine teleost, Pleuronectes americanus. Am J Physiol 276: F288-F294.
Roelofsen H, Soroka CJ, Keppler D, and Boyer JL (1998) Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets. J Cell Sci 111: 1137-1145.[Abstract]
Rushmore TH and Kong AN (2002) Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr Drug Metab 3: 481-490.[CrossRef][Medline]
Russel FGM, Masereeuw R, and Van Aubel RAMH (2002) Molecular aspects of renal anionic drug transport. Annu Rev Physiol 64: 563-594.[CrossRef][Medline]
Schaub TP, Kartenbeck J, Konig J, Spring H, Dorsam J, Staehler G, Storkel S, Thon WF, and Keppler D (1999) Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal cell carcinoma. J Am Soc Nephrol 10: 1159-1169.
Schaub TP, Kartenbeck J, Konig J, Vogel O, Witzgall R, Kriz W, and Keppler D (1997) Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J Am Soc Nephrol 8: 1213-1221.[Abstract]
Schinkel AH and Jonker JW (2003) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 55: 3-29.[CrossRef][Medline]
Schramm U, Fricker G, Wenger R, and Miller DS (1995) P-glycoprotein-mediated secretion of a fluorescent cyclosporin analogue by teleost renal proximal tubules. Am J Physiol 268: F46-F52.
Terlouw SA, Graeff C, Smeets PHE, Fricker G, Russel FGM, Masereeuw R, and Miller DS (2002) Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule. J Pharmacol Exp Ther 301: 578-585.
Terlouw SA, Masereeuw R, and Russel FGM (2003) Modulatory effects of hormones, drugs and toxic events on renal organic anion transport. Biochem Pharmacol 65: 1393-1405.[CrossRef][Medline]
Terlouw SA, Masereeuw R, Russel FGM, and Miller DS (2001) Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux. Mol Pharmacol 59: 1433-1440.
Van Aubel RAMH, Smeets PHE, Peters JGP, Bindels RJM, and Russel FGM (2002) The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 13: 595-603.
Van Aubel RAMH, van Kuijck MA, Koenderink JB, Deen PMT, van Os CH, and Russel FGM (1998) Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells. Mol Pharmacol 53: 1062-1067.
This article has been cited by other articles:
|
|
 |
|
  V. Reichel, R. Masereeuw, J. J. M. W. van den Heuvel, D. S. Miller, and G. Fricker Transport of a fluorescent cAMP analog in teleost proximal tubules Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2382 - R2389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Miller, J. R. Shaw, C. R. Stanton, R. Barnaby, K. H. Karlson, J. W. Hamilton, and B. A. Stanton MRP2 and Acquired Tolerance to Inorganic Arsenic in the Kidney of Killifish (Fundulus heteroclitus) Toxicol. Sci., May 1, 2007; 97(1): 103 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Wever, R. Masereeuw, D. S. Miller, X. M. Hang, and G. Flik Endothelin and calciotropic hormones share regulatory pathways in multidrug resistance protein 2-mediated transport Am J Physiol Renal Physiol, January 1, 2007; 292(1): F38 - F46. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Notenboom, A. C. Wouterse, B. Peters, L. H. Kuik, S. Heemskerk, F. G. M. Russel, and R. Masereeuw Increased Apical Insertion of the Multidrug Resistance Protein 2 (MRP2/ABCC2) in Renal Proximal Tubules following Gentamicin Exposure J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 1194 - 1202. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
