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SHORT COMMUNICATION

DIGITAL FLUORESCENCE IMAGING OF ORGANIC CATION TRANSPORT IN FRESHLY ISOLATED RAT PROXIMAL TUBULES

Department of Nephrology, School of Medicine, University Hospital, Essen, Germany

(Received July 14, 2005; accepted December 2, 2005)

	Abstract

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The secretion of cationic drugs and endogenous metabolites is a major function of the kidney. This is accomplished by organic cation transport systems, mainly located in the proximal tubules. Here, we describe a model for continuous measurement of organic cation (OC) transport. In this model, organic cation transport in individual freshly isolated rat proximal tubules is investigated by use of digital fluorescence imaging. To directly measure organic cation transport across the basolateral membrane, the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP⁺) is used with a customized perfusion chamber. ASP⁺ uptake in this model displayed the characteristics of organic cation transport. Over the tested range of 1 to 50 µM, it showed a concentration-dependent uptake across the basolateral membrane. In the presence of competitive inhibitors of OC transport such as N¹-methylnicotinamide⁺, tetraethylammonium⁺, and choline⁺, a concentration-dependent and reversible inhibition of ASP⁺ uptake could be documented. In conclusion, continuous measurement of organic cation transport in freshly isolated rat proximal tubules by digital fluorescence imaging using ASP⁺ is a useful tool for investigation of drug transport and interactions and, furthermore, may be helpful for investigation of organic cation transport under pathophysiological conditions.

Secretion of OCs by the kidney occurs from the blood into the tubular lumen. Therefore, OCs have to be taken up from the blood across the basolateral membrane into the proximal tubular cells. For most of these OCs, this entry involves an electrogenic transport driven by the electrochemical gradient generated by the inside negative membrane potential (Koepsell et al., 2003; Wright and Dantzler, 2004). This transport across the basolateral membrane can also function as an electroneutral antiport in exchange for other OCs (Dantzler et al., 1991; Budiman et al., 2000). This transport is enabled by several polyspecific organic cation transporters, which all belong to the solute carrier drug transporter family 22 (SLC 22) (Koepsell and Endou, 2004).

Investigations of OC transport previously have been performed in cellular substructures, such as cell culture. However, these models possess particular limitations. Cultured cells, during their adaptations necessary to survive in vitro, undergo a variety of phenotypic changes. These changes are particularly problematic for proximal tubular cells, which change from their physiological dependence on oxidative metabolism to glycolysis under culture conditions. In contrast, freshly isolated proximal tubules retain the biochemical properties of the in vivo state. In addition, they retain a high degree of structural integrity, as well as the highly polarized and fully differentiated functions of the normal proximal tubular epithelium (Lieberthal and Nigam, 2000). In fact, the model of freshly isolated proximal tubules ideally combines properties of in vitro as well as in vivo preparations in that external manipulations comparable to in vitro studies can be applied while many of the in vivo characteristics of proximal tubules are retained.

In isolated proximal tubules, OC transport has been estimated by the radiolabeled substrates N¹-methylnicotinamide⁺ (NMeN⁺) or tetraethylammonium⁺ (TEA⁺) (Schali et al., 1983; Tarloff and Brand, 1986; Besseghir et al., 1990; Dantzler et al., 1991; Groves et al., 1994; Goralski and Sitar, 1999). By using radiolabeled substrates, investigations are limited to distinct time points and cannot be followed continuously. Fluorescent substrates may serve as an advantageous alternative, by allowing for a continuous measurement of OC transport.

We identified 4-(4-dimethylamino-styryl)-N-methylpyridinium (ASP⁺) as a fluorescent substrate for the organic cation transport systems in the kidney with an apparent inhibition constant (K_i) of 0.10 ± 0.02 mmol/l for the luminal N-methyl-4-phenylpyridinium transport, and of 0.28 ± 0.12 mM for the contraluminal NMeN⁺ transport (Pietruck and Ullrich, 1995). Using the fluorescent substrate ASP⁺, together with digital fluorescence microscopy, enables a continuous real-time monitoring of OC transport.

To selectively investigate the transport of organic cations in freshly isolated rat proximal tubules, we used a customized microperfusion chamber, in which peritubular conditions can be varied. The aim of the study was to validate this model by investigation of organic cation transport under different conditions, including reversible transport inhibition by different inhibitors.

	Materials and Methods

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Freshly Isolated Proximal Tubules. Rat proximal tubules were freshly isolated as recently described (Kribben et al., 2003; Pietruck et al., 2003). In brief, after perfusion of the renal arteries with an oxygenated buffer containing collagenase and hyaluronidase, cortices of male Sprague-Dawley rat kidneys (220–300 g body weight) were minced on ice. Tissue separation with hyaluronidase and collagenase was continued at 37°C. Proximal tubules were obtained by Percoll gradient centrifugation, using 45% and 90% Percoll (Pharmacia, Uppsala, Sweden). After washing, tubules were suspended in an oxygenated solution, containing 106 mM NaCl, 20 mM NaHCO₃, 5 mM KCl, 1 mM CaCl₂, 2 mM NaH₂PO₄, 1 mM MgSO₄, 5 mM glucose, 10 mM HEPES, 2 mM glutamine, 10 mM sodium butyrate, 4 mM sodium lactate, adjusted to pH 7.05 at 4°C, and gassed on ice with 95% O₂/5% CO₂.

Microperfusion Chamber and Transport Measurements of the Organic Cation Transport with ASP⁺ in Freshly Isolated Individual Tubules. To investigate individual tubules, a microperfusion chamber was used, customized on the basis of a Leiden chamber, with a complete exchange of the fluid in the chamber in less than 30 s, as described previously (Pietruck et al., 2003). An aliquot of the tubule suspension was pipetted onto a Cell-Tak (Collaborative Biomedical Products, Bedford, MA)-coated cover slip, mounted to the base of the microperfusion chamber. The chamber was then fixed to the stage of an epifluorescence microscope (Zeiss Axiovert; Zeiss, Jena, Germany). The tubule suspension inside the chamber was gassed for 10 min with 95% O₂/5% CO₂ at room temperature. Meanwhile, the tubules settled down and attached to the cover slip. Afterward, the chamber was perfused continuously with 2 ml/min 95% O₂/5% CO₂ oxygenated buffer containing 106 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 1 mM CaCl₂, 2 mM NaH₂PO₄, 1 mM MgSO₄, 5 mM glucose, 2.5 mM HEPES, 1 mM glutamine, 1 mM sodium butyrate, and 1 mM sodium lactate, pH 7.35–7.40. During this period the temperature was gradually increased with an air stream incubator to 37°C.

After an accommodation period of 10 min, transport experiments were started, by switching to a buffer additionally containing different concentrations of the fluorescent organic cation ASP⁺ (Molecular Probes, Leiden, The Netherlands). Initial uptake of ASP⁺ was quantified as the increase of fluorescence between 30 and 60 s after switching to perfusion with ASP⁺ (Stachon et al., 1996).

Digital Video Imaging. Images were acquired by a 12-bit cooled slow-scan-frame-transfer CCD camera (Imago; T.I.L.L. Photonics, Planneg, Germany) and processed by using image processing software (VisIon; T.I.L.L. Photonics). Light with an excitation wavelength of 470 nm (bandwidth of 10 nm) was generated by a mesh monochromator (T.I.L.L. Photonics) and was inserted in the light path of an inverted microscope (Axiovert 100; Zeiss) via a fluorescence objective with 20-fold magnification (Fluar; Zeiss). Emitted light, collected through a dichroic mirror, was transmitted through an emission filter (>605 nm) to the camera. To prevent photo bleaching, the exposure time was limited to 30 ms every 10 s. Two to five tubules were individually analyzed in each experiment. Fluorescence of the tubules is expressed as relative fluorescence intensity.

Reversible Inhibition of ASP⁺ Transport by NMeN⁺, TEA⁺, and Choline⁺. NMeN⁺, TEA⁺, and choline⁺ were used to investigate competitive inhibition of ASP⁺ transport. In the first experiments, inhibitors and ASP⁺ were superfused together. This resulted in a constant tubular fluorescence, reflecting a complete inhibition of ASP⁺ transport. Therefore, we later superfused the tubules first with 5 µM ASP⁺ only, and after 1 min, switched to a perfusion buffer containing inhibitors in addition to ASP⁺. For testing the reversibility of inhibition, after 8 min of superfusion with ASP⁺ together with inhibitor, the buffer was again switched to a buffer containing ASP⁺ without inhibitor.

Two-Photon Microscopy for Imaging Tubules. To acquire images of freshly isolated proximal tubules envisioning the uptake of ASP⁺ into the tubular cells, two-photon microscopy was applied. Freshly isolated tubules were investigated in a glass-bottom dish coated with Cell-Tak. After settling of the tubules and attaching to the glass bottom of the dish, ASP⁺ was added to the oxygenated buffer at different concentrations. Images were generated over a time period of at least 10 min.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Time-dependent increase of tubular fluorescence in freshly isolated rat proximal tubules under different concentrations of ASP+. Tubules were superfused with perfusion buffer containing ASP+ between 1 and 50 µM (n = 3–6) as indicated.

	Results

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Concentration Dependence of ASP⁺ Transport. After starting perfusion of the chamber with buffer containing ASP⁺, fluorescence of the tubules increased. The increase of ASP⁺ fluorescence was time- and concentration-dependent over the tested range between 1 and 50 µM ASP⁺ (Fig. 1). In this concentration range, a linear increase of tubular fluorescence was observed within the first 2 to 3 min, whereas the uptake rates decreased in the later phase of the experiment, approaching equilibration (Fig. 1). We additionally evaluated the initial uptake rates of ASP⁺ because these values better reflect the transport across the basolateral membrane, whereas the maximal fluorescence in the later phase could also be influenced by other factors, such as extrusion of ASP⁺ from the cells, intracellular compartmentalization, or bleaching of ASP⁺. As can be seen from the concentration-response curve in Fig. 2, the initial rate of ASP⁺ uptake was not saturated at 50 µM, the maximal ASP⁺ concentration used. Due to the experimental setup, no higher concentrations were used.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Concentration dependence of ASP+ uptake in freshly isolated proximal tubules. Values are means ± S.E.M. of initial fluorescence increase with n = 3 to 6 experiments.

View larger version (74K): [in this window] [in a new window]   FIG. 3. Freshly isolated proximal tubule, imaged by two-photon microscopy, 1 min (left) and 5 min (right) after addition of 50 µM ASP+. The length of the bar corresponds to 20 µm.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Effect of different concentrations of NMeN+ on ASP+ uptake in freshly isolated proximal tubules. Perfusion was started with perfusion buffer containing 5 µM ASP+ without NMeN+ and after 1 min (arrow) was switched to a perfusion buffer also containing different concentrations of NMeN+ as indicated. Concentrations of 10 mM NMeN+ (n = 4, p < 0.005) and 5 mM NMeN+ (n = 4, p < 0.005) resulted in a significant inhibition of ASP+ uptake, whereas 1 mM NMeN+ did not significantly inhibit ASP+ transport (n = 4, p = N.S.).

Effect of Choline⁺ and TEA⁺ on ASP⁺ Transport. The addition of 10 mM choline⁺, as well as of 10 mM TEA⁺, 1 min after starting ASP⁺ perfusion showed an inhibition curve similar to that observed with 10 mM NMeN⁺. All inhibitors led to a rapid loss of incremental ASP⁺ fluorescence. In the presence of inhibitors, tubular fluorescence, after reaching its maximum, even decreased, possibly reflecting an outwardly directed transport or leakage (Fig. 5).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effect of NMeN+, TEA+, and choline+ on ASP+ uptake, and its reversibility. At 1 min (arrow) after starting perfusion with 5 µM ASP+, perfusion buffer was switched to a buffer containing a 10 mM concentration of either NMeN+, TEA+, or choline+ and after 8 min (arrow) was again switched to buffer without inhibitors (n = 3–5). Perfusion with 5 µM ASP+ served as control (n = 5).

	Discussion

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The aim of the present study was to establish a model for continuous measurement of organic cation transport in individual, freshly isolated rat proximal tubules. This was accomplished by using the fluorescent organic cation ASP⁺ together with digital fluorescence microscopy and video image analysis.

We have proven different variables of ASP⁺ transport reflecting the physiological function of OC transport in this model. We first investigated the concentration dependence of ASP⁺ uptake. In the tested range between 1 and 50 µM, intracellular fluorescence of ASP⁺ reflected a concentration-dependent transport across the basolateral membrane, without reaching saturation at 50 µM, the highest feasible concentration. These findings indicate that the K_m for ASP⁺ in this model must be much higher than 50 µM. This is consistent with data determined for ASP⁺ uptake across the basolateral membrane of rat proximal tubules in situ (280 µM) (Pietruck and Ullrich, 1995). The time pattern in general was similar to that of earlier experiments with radiolabeled TEA⁺ in rabbit proximal tubules (Groves et al., 1994). For further experiments, we chose an ASP⁺ concentration of 5 µM, which showed no saturation in the first 5 min, when ASP⁺ transport was quantified.

Second, we investigated whether organic cation transport in freshly isolated proximal tubules could be inhibited by N¹-metyhlnicotinamide, a well known inhibitor of organic cation transport (Besseghir et al., 1990; Ullrich et al., 1991). When NMeN⁺ was administered before or simultaneously with ASP⁺, no relevant increase in fluorescence could be registered, reflecting a complete inhibition of ASP⁺ uptake (data not shown). Therefore, in the subsequent experiments we started ASP⁺ perfusion without inhibitor for a short period of time (1 min), so that an initial tubular uptake of ASP⁺ occurred, and then immediately switched to a perfusion buffer also containing the inhibitor NMeN⁺. Shortly after switching to the inhibitor, a strong inhibition of ASP⁺ transport could be observed with the higher concentrations of 5 and 10 mM NMeN⁺, whereas 1 mM NMeN⁺ did not change intracellular fluorescence, reflecting the dose dependence of the inhibition of OC transport.

Third, to prove the specificity of this inhibition by NMeN⁺, we tested the inhibitory effect of two structurally different inhibitors of OC transport, TEA⁺ and choline⁺ (Ullrich et al., 1991). Both inhibitors showed a similar pattern of ASP⁺ transport inhibition with an immediate effect after switching to the inhibitor perfusion. Especially in the presence of TEA⁺ and choline⁺, and for NMeN⁺ in lower concentrations, tubular fluorescence, after reaching its maximum, thereby demonstrating complete transport inhibition, decreased during the course of the experiment, reflecting the loss of some of the intracellular ASP⁺. These data are consistent with an outwardly directed transport of ASP⁺ at the basolateral membrane, whereas at the outside of the cell membrane, a complete inhibition still persisted. A quantitatively relevant secretion into the luminal space is rather unlikely, because in this model, the lumen of the proximal tubules is collapsed.

Fourth, we demonstrated the reversibility of ASP⁺ transport inhibition by switching from the perfusion buffer with inhibitor back to a perfusion buffer containing ASP⁺ alone, which resulted in another strong increase in ASP⁺ fluorescence, reflecting OC transport across the basolateral membrane (Fig. 5).

In conclusion, continuous measurement of organic cation transport in freshly isolated rat proximal tubules by digital fluorescence imaging using ASP⁺ is a useful tool for investigation of drug transport and interactions; furthermore, it may allow continuous measurement of organic cation transport under pathophysiological conditions in the future.

	Acknowledgments

 
We gratefully acknowledge the excellent technical assistance of Simone Blaschke.

	Footnotes

 
This study was supported by the Deutsche Forschungsgemeinschaft Grant Kr 1202-2.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.006403.

ABBREVIATIONS: OC, organic cation; NMeN⁺, N¹-methylnicotinamide⁺; TEA⁺, tetraethylammonium⁺; ASP⁺, 4-(4-dimethylamino-styryl)-N-methylpyridinium.

Address correspondence to: Frank Pietruck, Klinik für Nieren- und Hochdruckkrankheiten, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. E-mail: frank.pietruck{at}uni-essen.de

	References

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