original article http://www.kidney-international.org & 2008 International Society of Nephrology Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney Tamara Castro1, Marcela Low1, Katterine Salazar1, Hernán Montecinos1, Manuel Cifuentes2, Alejandro J. Yáñez3, Juan Carlos Slebe3, Carlos D. Figueroa4, Karin Reinicke1, Marı́a de los Angeles Garcı́a1, Juan Pablo Henriquez1 and Francisco Nualart1 1 Departamento de Biologı́a Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile; 2Departamento de Biologı́a Celular, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain; 3Instituto de Bioquı́mica, Facultad de Ciencias Biológicas, Universidad Austral de Chile, Valdivia, Chile and 4Instituto de Anatomı́a, Histologı́a y Patologı́a, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane. Kidney International (2008) 74, 1278–1286; doi:10.1038/ki.2008.329; published online 9 July 2008 KEYWORDS: SVCT1; vitamin C; proximal tubule; PHA lectin; FBPase; immunohistochemistry Correspondence: Francisco Nualart, Departamento de Biologı́a Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla, Concepción 160-C, Chile. E-mail: frnualart@udec.cl Received 28 December 2007; revised 2 April 2008; accepted 29 April 2008; published online 9 July 2008 1278 Vitamin C transport is essential in all mammalian cells. The biological value of ascorbic acid (AA) rests in its ability to serve as a reductant for ferric and cupric ions. Additionally, ascorbate is involved in collagen, steroid, and neuropeptide biosynthesis.1 As an aqueous-phase antioxidant concentrated within the cell cytosol, vitamin C appears to play an important role in protecting DNA from oxidative damage linked to mutagenesis and the initiation of carcinogenesis.2 The ingested vitamin C is absorbed mainly in the duodenum and proximal jejunum. For instance, in different species, the absorption of L-ascorbate is linked to a pHdependent saturable active transport system that relies on the presence of a carrier and sodium ions.3,4 L-ascorbate is reabsorbed in the renal tubules in a similar manner using an active and saturable process, an essential mechanism that maintains homeostasis of circulating vitamin C. Molecular cloning studies have identified two different isoforms of high-affinity sodium-dependent vitamin C co-transporters (SVCT1 and SVCT2).5–10 SVCT1 is a 604 amino acid protein mediating AA transport in the kidney, intestine, and liver.5,6,8 SVCT2 is a 592 amino acid protein that has been detected in brain cells (neurons, choroid plexus cells, and cultured astrocytes) and different cells from peripheral organs.7,8,11–14 SVCT1 functional expression has been observed in rat kidney.15,16 Enhanced green fluorescence protein-tagged SVCT1 expression showed apical distribution in polarized cultures of kidney-derived Madin–Darby canine kidney cells.17 Additionally, high expression of SVCT1 has been demonstrated in the straight segment (S3) proximal tubule by in situ hybridization,8 and further confirmed by immunohistochemistry.18 Even though previous data suggest that vitamin C is efficiently reabsorbed in the S3 segment, we hypothesized that a resourceful AA absorption mechanism in the kidney may also involve segments S1 and S2. In this study, we have carried out a detailed experimental analysis of SVCT1 Kidney International (2008) 74, 1278–1286 original article T Castro et al.: SVCT1 distribution in renal proximal tubule expression (1) in the three separate segments of the renal proximal tubule of mouse and human kidneys by optical and ultrastructural immunohistochemistry and (2) by reverse transcription-PCR (RT-PCR), immunoblotting, and functional kinetic analyses in polarized LLC-PK1 cells. RESULTS SVCT1 distributes as a gradient of expression in the apical domain of proximal tubule cells We used a standard nomenclature to describe the gross morphology of the kidney.19 The kidney cortex consists of renal corpuscle, convoluted portions of the proximal and distal tubules, and short lengths of the straight portions of the tubules (Figure 1a, cortex). The medulla is divided into Immunohistochemistry DCT C Anti-SVCT1 PCT S1–S2 In situ hybridization Anti-sense-SVCT1 Cortex Cortex S3 OS OM IS M CT OS/OM IM OS/OM IS/OM an outer and an inner medulla; the outer medulla is further divided into outer and inner stripes (Figure 1a). The medulla consists of varying lengths of the straight portions of proximal and distal tubules, the loops of Henle, collecting ducts, as well as the vasa recta, which are unusually straight blood vessels. Using immunohistochemistry and in situ hybridization, we carried out a detailed expression analysis of the SVCT1 transporter in the different cells and structures of the kidney (Figure 1b and c). This approach showed that SVCT1 expression of both mRNA (Figure 1c and d) and protein (Figure 1b, g, i, and j) was mainly associated with the straight segment (S3) of proximal tubules located in the outer strip of the outer medulla. However, SVCT1 expression was also detected in the renal cortex by in situ hybridization and immunohistochemical experiments (Figure 1e–g and k, respectively). SVCT1 immunoreactivity was clearly concentrated in the apical region of the epithelial cells (Figure 1k, arrows). To obtain the precise localization of the transporter, we increased the sensitivity of our immunodetection method by performing immunostaining in 40-mm-thick free-floating sections (Figure 2). A clear anti-SVCT1 immunoreaction was IS/OM Cortex Cortex S1 S1 Anti-SVCT1 OS/OM Inner cortex S1 Merge Anti-FBPasa Outer cortex Cortex Medulla Medulla S2 Medulla S2 S2 Anti-SVCT1 OS/OM S3 Anti-SVCT1 Anti-FBPase Cortex Control Figure 1 | Immunohistochemical detection and in situ hybridization of SVCT1 mRNA expression in the adult mouse kidney. (a) Schematic representation of adult kidney nephrons. Histological sections of the adult mouse kidney were subjected to immunohistochemistry using the specific anti-SVCT1 antisera (b, g–k) and to in situ hybridization using a specific 657 bp digoxigenin-labeled riboprobe for SVCT1 mRNA (c–f). High expression of SVCT1 was observed in the proximal tubules (S3 segment) present in the outer strip of the outer medulla (c–d and g, i, and j), whereas low expression was detected in segments S2 and S1 at the inner and outer cortex (e, f and g, k). SVCT1 was specifically localized in the brush border of epithelial proximal cells (i–k, arrows). Control experiments were performed incubating the sections with antibodies pre-absorbed with an excess of the peptide used for immunization (h). C, cortex; CT, collecting tube; DCT, distal convoluted tubule; IS, inner stripe; IM, inner medulla; M, medulla; OS, outer stripe; OM, outer medulla; PCT, proximal convoluted tubule. Scale bars ¼ 100 mm (b, c, and g); 20 mm (d–f, h–k). Kidney International (2008) 74, 1278–1286 S3 OS/OM S3 Merge Medulla Relative fluorescensce OS/OM 1.0 0.8 0.6 0.4 0.2 0.0 Cortex Medulla Figure 2 | Brush-border membrane polarization of SVCT1 in the mouse kidney cortex and medulla. Thick floating cryosections of mouse kidney cortex (a–c) and medulla (d–f) were double stained with anti-SVCT1 (a, d, g, and h) and antiFBPase (b, e) antibodies and secondary antibodies conjugated to Cy3 and Cy2, respectively. Merged images are shown in panels c and f. SVCT1 was detected in the apical region of epithelial cells in segments S3, S2, and S1, whereas FBPase only stained cells in segments S1 and S2. OS, outer strip; OM, outer medulla. (g–h) Pseudocolor images of SVCT1 immunostaining kidney cortex (g) and medulla (h). (i) Semiquantitative analysis plot showing relative fluorescence of SVCT1 immunocytochemical reaction in the renal cortex and medulla. Scale bars ¼ 20 mm. 1279 original article visualized in the epithelial cells of the cortical proximal tubules (Figure 2a). The tubule segments were identified on the basis of their cellular structure and differential staining for FBPase, an enzyme involved in glycolitic metabolism. A similar sequence of expression was observed with antiSVCT1 (Figure 2a) and anti-FBPase (Figure 2b). In the inner cortex, SVCT1 was expressed in segments S1 and S2, as evidenced by positive FBPase staining (Figure 2a–f), whereas straight tubules of the outer medulla (segment S3), with high SVCT1-expression, were FBPase-negative (Figure 2d–f). A semiquantitative analysis of the staining reaction was obtained using confocal pseudocolor function, with yellow and red depicting the highest intensity values based on color scale (1500–2500) (Figure 2g and h). As shown in Figure 2i, SVCT1 staining in the apical region of the tubule cells is about 4.5 times higher in the medulla (segment S3) than in the cortex. To further characterize SVCT1 localization in the apical domain of proximal cells of the cortex and outer medulla, we carried out phaseolus vulgaris (PHA) lectin histochemical analysis. This lectin displays an elevated affinity for sugar moieties concentrated in the apical brush border of straight proximal tubules; however, it binds also to the brush border of convoluted proximal tubules. As shown in Figure 3, SVCT1 transporter showed an extensive colocalization with PHA lectin in the brush border of cortical and medullary proximal cells (Figure 3d–i). However, in the glomerulus, reduced binding of PHA lectin was associated with an absence of SVCT1 staining (Figure 3f, asterisk). By viewing the ultrastructural images, we confirmed that SVCT1 is distributed within the apical brush border of proximal tubules of the outer medulla (Figure 4a and b); however, we were unable to observe the transporter in the cortical region. In the human kidney, an intense signal of the immunolabeled SVCT1 was visualized in the brush border of proximal tubules situated in the medullary rays and cortex (Figure 5a, d and e). Even though a high intensity for SVCT1 was detected in straight proximal tubules of the outer medulla (Figure 5e, inset), the immunostaining of cortical proximal tubules was higher than that observed in the mouse (Figure 5b and c). The glomerulus and distal convoluted tubules remained unstained for SVCT1 (Figure 5b and c). Kinetic characterization of the AA transport in LLC-PK1 cells To determine the physiological importance and function of SVCT1 transporter in renal proximal tubule epithelial cells, a series of in vitro experiments were conducted using the LLCPK1 kidney-derived cell line. RT-PCR and western blot analyses demonstrated the expression of SVCT1 in this cell line (Figure 6a and b). Mouse kidney extracts were used for positive controls (Figure 6a and b). Immunocytochemistry showed that SVCT1 was mainly distributed intracellularly in pre-confluent LLC-PK1 cells (Figure 6c); however, in postconfluent cultures, endogenous SVCT1 transporter was detected within the apical domain (Figure 6d). Considering this, post-confluent LLC-PK1 cell cultures were used to carry 1280 T Castro et al.: SVCT1 distribution in renal proximal tubule PHA Lectin Cortex SVCT1 Cortex OS/OM IS/OM Merge Cortex OS/OM OS/OM IS/OM IS/OM Cortex Cortex Cortex OS/OM OS/OM OS/OM Figure 3 | Colocalization of SVCT1 and PHA lectin binding in the mouse adult kidney. Thick floating cryosections of the mouse kidney were stained with PHA lectin (a, d, and g) and immunostained with anti-SVCT1 antibody and a secondary Cy3-conjugated antibody (b, e, and h). Merged images are shown in panels c, f, and i. Low-magnification images (a–c) show strong SVCT1 and PHA lectin staining in the outer stripe (OS) of the outer medulla (OM); however, high-magnification images show extensive colocalization of PHA lectin and SVCT1 in the brush border of proximal tubules at the cortex (d–f, arrows) and at the OS of the OM (g–i, arrows). IS/OM, inner stripe of the outer medulla. Asterisk (*), glomerulus. Scale bars ¼ 50 mm (a–c); 12 mm (d–i). out a detailed kinetic characterization of AA transport. The transport was linear for the first 30 min (Figure 7a), with an initial uptake velocity of 20±3 pmol  106 cells per min. The total AA uptake after 30 min was 440±45 pmol per 106 cells (Figure 7a). Dose-response studies using 5 min assays revealed that AA transport was saturated at concentrations above 250 mM (Figure 7c). Data analysis suggested the presence of one kinetic component for ascorbate transport. The Eadie–Hofstee transformation showed an apparent Km of 180 mM and a maximum velocity (Vmax) of 12±3 pmol  106 cells per min (Figure 7d). To test the sodium dependence of AA transport, we replaced NaCl with choline chloride in the assay. Transport of AA by LLC-PK1 cells was decreased by at least 90% in the absence of sodium and the uptake reached a plateau of 40±2 pmol per 106 cells after 20 min (Figure 7a). These data strongly suggest that vitamin C is incorporated into LLC-PK1 cells using an SVCT-like transporter. Furthermore, inhibition experiments revealed that cytochalasin B Kidney International (2008) 74, 1278–1286 original article T Castro et al.: SVCT1 distribution in renal proximal tubule RT-PCR bp 1 2 3 WB 1 4 500 bp 400 bp 439 bp 400 bp 300 bp 333 bp 2 3 kDa 68 β-Actin Anti-SVCT1 x–y m x–z Figure 4 | Ultrastructural immunogold labeling of SVCT1 in proximal epithelial cells of the mouse kidney. (a) Proximal tubule cells (apical region) observed with low magnification. (b) Brush-border membrane observed with high magnification. Arrows indicate gold particles on the microvilli. Original magnification were (a)  17,000; (b)  35,000. G Cortex G Cortex PCT Cortex DCT PCT Cortex DCT MR PST Figure 6 | RT-PCR, western blot, and immunocytochemistry for SVCT1 in LLC-PK1 cells. (a) mRNAs isolated from adult human (lane 1) and mouse (lane 2) kidneys as well as from LLC-PK1 cells (lane 3) were subjected to RT-PCR with specific primers against SVCT1 transporter. Lane 4, reverse transcriptase-negative reaction for LLC-PK1 cells mRNA. As controls, RT-PCRs were performed with specific primers against b-actin (lower panel). Arrows in the right of each panel indicate the expected RT-PCR product size for mouse SVCT1 (439 bp) and b-actin (333 bp). (b) Western blot analysis using specific anti-SVCT1 antibodies detected a 68 kDa band in membrane protein extracts from the mouse kidney and LLC-PK1 cells (lanes 1 and 2, respectively). As controls, western blots were developed avoiding primary antibodies (lane 3). (c) Immunocytochemical analysis of SVCT1 (green) in pre-confluent (c) and post-confluent (d) LLC-PK1 cells. Cell nuclei in panel c were stained with propidium iodide. Inset in panel c shows negative controls and primary antibodies were avoided. Lower (x–z) panel in panel d show confocal z-sections of the plane indicated with white line in panel d. Scale bar ¼ 20 mm. PCT MR MR Figure 5 | Immunohistochemical detection of SVCT1 expression in the adult human kidney. Histological sections of the adult human kidney were subjected to immunohistochemistry using the specific anti-SVCT1 antisera and a peroxidaseconjugated secondary antibody. (a) A low-magnification image shows intense immunostaining for SVCT1 in the proximal tubules situated in the medullary ray (MR) and cortex. High-magnification images show that SVCT1 immunostaining is strongly concentrated at the brush-border membrane of epithelial cells of the proximal convoluted (b–d) and straight (e) tubules and MRs (d, e). Low-to-absent expression was detected in the cells of the distal convoluted tubes (DCT). PCT, proximal convoluted tubule; PST, proximal straight tubule; G, glomerulus. Scale bars ¼ 20 mm (a–e). failed to affect AA transport, indicating that the transporters expressed by LLC-PK1 cells are functionally unrelated to the dehydroascorbic acid transporters. However, ouabain at a concentration of 20 mM inhibited transport by 50%; a similar Kidney International (2008) 74, 1278–1286 effect was observed when we replaced the NaCl with choline chloride (Figure 7b). These results strongly suggest that AA is co-transported with sodium ions in LLC-PK1 cells. Analysis of sodium ion effect on AA transport at 100 mM AA revealed that this transporter was strongly activated by sodium in a cooperative fashion, as indicated by the slightly sigmoidal shape of the concentration/velocity curve (Figure 7e). This conclusion was corroborated by the Hill plot of the data, which displays a straight line with a slope (Hill coefficient) of 3.1 (Figure 7f). When the effect of temperature in the uptake of AA was analyzed, we observed that transport was almost at basal levels at 4 1C, but when temperature raised to 22 1C, the velocity of transport increased to 58±3 pmol  106 cells per min (Figure 7g). AA transport was almost three times higher than that recorded at 4 1C when the uptake was carried out at 37 1C (145±45 pmol  106 cells per min) (Figure 7g). Finally, we observed that AA uptake was also regulated by pH (Figure 7h). 1281 original article V (pmol AA×106 cells per min) 80 % Control 400 300 ∗ 60 200 40 100 20 ∗ 0 10 8 6 4 2 2 0 B in 0 0.00 0.02 0.04 0.06 0.08 V/S 100 200 300 400 in ba 6 4 ha la s AA (µM) 25 0.8 20 15 10 n H= 3.1 0.6 0.4 0.2 0.0 5 –0.2 0 –0.4 0 30 60 90 120 150 180 Sodium concentration (mM) 40 200 0.4 0.8 1.2 1.6 2.0 2.4 2.8 Log [Na+] V (pmol×106 cells per min) 1.0 V (pmol×106 cells per min) 30 Log v /(V–v) V (pmol×106 cells per min) C yt oc Time (min) 8 0 lin e 5 10 15 20 25 30 O ua 0 C ho 0 12 100 V Sodium Choline 500 AA (pmol per 106 cells) T Castro et al.: SVCT1 distribution in renal proximal tubule 160 120 80 40 0 0 5 10 15 20 25 30 35 40 Temperature (°C) 35 30 25 20 15 10 5 0 5 6 7 8 9 pH Figure 7 | Kinetic analysis of L-ascorbate uptake in LLC-PK1 cells. (a) Time course of 50 mM AA uptake at 37 1C in the presence of NaCl (open circles) or replacing NaCl with choline chloride (solid circles). (b) Uptake at 10 min of 50 mM AA in LLC-PK1 cells that were treated with choline chloride, 10 mM ouabain for 1 h, and 25 mM cytochalasin B for 10 min. Results are expressed as percentage of the control (*Po0.001). (c) Dose-response curve of L-ascorbic acid at 37 1C. (d) Eadie–Hofstee transformation of the data in panel c. (e) Velocity of 50 mM AA uptake at 10 min as a function of the concentration of extracellular sodium at 37 1C. (f) Hill plot of the data in panel e. (g) Uptake of 50 mM AA at 5 min between 4 and 37 1C. (h) pH dependence of 50 mM AA uptake at 5 min. Data represent the mean±s.d. of at least two experiments performed in triplicate. AA regulates SVCT1 apical distribution and uptake in LLC-PK1 cells Our results revealed a gradient of expression for SVCT1 in the apical domain of proximal tubules. On the basis of this observation we hypothesized that this pattern of expression may be regulated through changes in local concentrations of ascorbate. Thus, the increasing gradient of SVCT1 expression toward distal regions of the proximal tubule could correlate with an inverted decreasing gradient of ascorbate. To experimentally test this concept, we examined the expression of SVCT1 in post-confluent LLC-PK1 cells that were previously incubated with increasing concentrations of AA. As a control, we used LLC-PK1 cells cultured with a low AA concentration known to be present in the serum added to the culture medium. Confocal z-sections showed that the strong apical pattern of SVCT1 expression, observed under control conditions, dropped off in parallel with the increase in ascorbate used to pretreat the cells (Figure 8a). Additionally, we performed transport assays in LLC-PK1 cells exposed to AA. Consistent with our immunolabeling data, the uptake of 100 mM 14C-AA was reduced by 50% on cells pretreated with high concentrations (50–100 mM) of AA (Figure 8b). These 1282 results show that LLC-PK1 cells displaying a reduced apical distribution of SVCT1, still retain the capacity to transport AA. To further analyze the extent to which these cells efficiently transport AA, the uptake of increasing concentrations of AA was studied in cells pretreated with increasing AA concentrations (Figure 9). Our results show that cells pretreated with 100 mM AA are able to transport significantly more 14C-AA than cells having comparatively more SVCT1 in their apical membrane, a condition induced by incubating the cells with a low AA concentration (C and 10–25 mM) (Figure 9). These results suggest that despite changes in the apical distribution of SVCT1 in LLC-PK1 cells, the uptake of AA is driven by the availability of 14C-AA in the extracellular space. DISCUSSION Previous reports have shown that the mRNA for the sodium–vitamin C co-transporter SVCT1 is expressed in epithelial absorptive cells of the intestine, kidney, and liver,5,6,8 as well as in primary cultures and cell lines derived from these tissues.20–22 After in situ hybridization and immunolabeling experiments, we demonstrated that SVCT1 Kidney International (2008) 74, 1278–1286 original article T Castro et al.: SVCT1 distribution in renal proximal tubule AA treatment 10 µM Control 25 µM 50 µM 100 µM 40 V (pmol AA×106 cells per min) x–y x–z V (pmol AA×106 cells per min) 80 30 20 10 70 60 0 AA treatment 50 Uptake 40 30 20 10 0 AA treatment (µM) C 10 Uptake 25 50 100 100 µM AA Figure 8 | SVCT1 detection and function in LLC-PK1 cells treated with different ascorbic acid concentration. (a) Immunocytochemical analysis of SVCT1 in post-confluent LLCPK1 cells incubated with different concentrations of AA (C, 10, 25, 50, and 100 mM). Lower panel (x–z) show confocal z-sections of the planes indicated with white lines. (b) Uptake at 10 min of 100 mM AA in LLC-PK1 cells that were treated with different concentrations of the vitamin. As a control (c), we used LLC-PK1 cells cultured with a low AA concentration (probably lesser than 10 mM) present in the serum added to the culture medium. Data represent the mean±s.d. of at least two experiments performed in triplicate. Scale bar ¼ 10 mm (a). is preferentially expressed in proximal tubules of the outer strip of the external medulla, specifically in the S3 segment. These results are consistent with the previous work showing the expression of SVCT1 mRNA in rat kidney tissue,9 isolated chick proximal tubules, and cultured cells22 and SVCT1 by immunolabeling experiments in the mouse kidney.18 Nonetheless, our detailed analysis of SVCT1 distribution showed that this transporter is not restricted to the S3 segment, but it is expressed all along the proximal tubule where it can be visualized as a gradient of expression. Indeed, semiquantitative data obtained by confocal pseudocolor function showed that the levels of expression of SVCT1 in the outer strip of the outer medulla were about 4.5-fold higher than in the cortex. The localization of SVCT1 at segments S1 and S2 present in the inner cortex was further confirmed in thick floating sections stained for FBPase. In these experiments, epithelial cells at the S3 segment showed high SVCT1 expression, but no reactivity for FBPase. Interestingly, a similar SVCT1 distribution was expressed in the adult human kidney. Kidney International (2008) 74, 1278–1286 C 10 10 10 25 25 50 100 µM 50 100 µM Figure 9 | L-Ascorbic acid uptake in LLC-PK1 cells pretreated and incubated with different concentrations of vitamin C. LLC-PK1 cells were cultured with 10, 25, 50, and 100 mM AA and the vitamin C uptake was performed using the same concentration in each point. As a control (C), we incubated LLC-PK1 cells with a low AA concentration (probably lesser than 10 mM) present in the serum added to the culture medium. Data represent the mean±s.d. of at least two experiments performed in triplicate. SVCT1 localization in the brush border of proximal tubules followed the gradient of expression similar to that observed in the mouse kidney. These results suggest a conserved mechanism of vitamin C absorption in the kidney. In experiments to determine the precise cellular expression, SVCT1 was visualized in the brush-border membrane of mouse and human kidney absorptive cells. The specific subcellular localization of SVCT1 was confirmed using the PHA lectin as a marker of the apical domain and ultrastructural immunocytochemistry. In both cases, SVCT1 immunoreactivity was localized within the brush-border membrane and the apical domain of proximal epithelial cells, suggesting that SVCT1 reabsorps AA from the glomerular filtrate. Previous work, using isolated proximal tubules, have demonstrated that immunoreactivity of SVCT118 and AA transport22 is restricted to the apical membrane. Also, enhanced green fluorescence protein-tagged constructs of SVCT1 have been shown to be confined to the apical domain.17 To analyze the kinetic behavior of SVCT1 we used LLCPK1 cells, an epithelial cell line derived from pig proximal tubule cells that expresses SVCT1,5 an observation that we confirmed through RT-PCR and western blot analyses. Similar to its distribution in vivo, post-confluent LLC-PK1 cell cultures distributed SVCT1 within the apical domain, suggesting that these cells are an appropriate in vitro model to study renal SVCT1 function. Ascorbate dose-response curves showed a Michaelis–Menten curve, whereas the Eadie–Hofstee transformation showed one kinetic component with an apparent Km of 180 mM. These results 1283 original article corresponded with Km values obtained when human SVCT1 was overexpressed in COS-1 cells23 and in mRNA microinjection studies in Xenopus laevis oocytes.9,10 Moreover, comparable Km values were obtained for the incorporation of AA into vesicles derived from the apical membrane of proximal tubules.15,24 Our kinetic analyses also showed that ascorbate transport is activated by the presence of sodium in the assay medium. Ouabain inhibition decreased ascorbate transport velocity by about 50%, which correlates with data obtained from Xenopus oocytes microinjected with human SVCT1 mRNA8 and from CHO and Madin–Darby canine kidney cells overexpressing human SVCT1.17 Additionally, we demonstrated that the velocity of ascorbate uptake is directly affected by the temperature and the pH. The expression of SVCT1 in epithelial cells of renal proximal tubules is consistent with the function of these structures in the sodium-dependent clearance of different biomolecules. We reasoned that most of the AA in the glomerular filtrate is reabsorbed at the segment S1, as it is well known for sodium ions and other solutes. Thus, our observation that SVCT1 is expressed in an increasing gradient toward distal regions of the proximal tubule (from S1 to S3 segments) could be linked with an inverted decreasing gradient of ascorbate. Of particular interest in this regard, is the fact that intestinal epithelial cells exposed to high concentrations of ascorbate display reduced SVCT1 expression.21 To analyze whether such a mechanism regulates SVCT1 expression and function in renal proximal tubules, we incubated post-confluent LLC-PK1 cells with different concentrations of AA. Our data show that preincubation with high concentrations of AA results in diminished apical distribution of SVCT1 and AA uptake. On the basis of these results, we speculated that epithelial cells at the S3 segment upregulate SVCT1 expression to reabsorb efficiently the comparative low concentration of AA present in this region. As an in vitro approach to test this idea, we studied the uptake of increasing concentrations of AA in cells displaying decreasing apical distribution of SVCT1. Our data show that control LLC-PK1 cells uptake comparatively less AA than cells having decreased apical distribution of SVCT1. In the context of our previous data, these results suggest that the low affinity of SVCT1 for AA is not compensated by the upregulation of the transporter in the apical membrane of cells. In conclusion, SVCT1 is expressed in the apical brushborder membrane of the epithelial cells throughout segments S1–S3 of proximal tubules of both human and mouse kidneys. Our kinetic data led us to conclude that SVCT1 is a functional transporter for AA uptake in kidney epitheliumderived LLC-PK1 cells. Finally, we postulate that AA regulates the apical membrane distribution of SVCT1 in polarized LLC-PK1 cells. MATERIALS AND METHODS Animals and experimental procedures The Universidad de Concepcion Ethics Committee approved all experimental procedures carried out during this study. Healthy male 1284 T Castro et al.: SVCT1 distribution in renal proximal tubule normotensive C57BL/J6 mice (aged 6–12 weeks, weighing 32±3 g) were obtained from our University Breeding Center. Animals were maintained on a 12-h light–dark cycle at constant room temperature with free access to normal sodium rat chow and water. Immunohistochemistry Mouse kidneys were dissected and fixed directly by immersion in Bouin’s solution, or fixed in situ by vascular perfusion.25 Samples were dehydrated in graded alcohol solutions and embedded in paraffin. Sections (5 mm) were obtained and mounted on poly-Llysine-coated glass slides. Before immunostaining, the sections were treated with absolute methanol and 3% hydrogen peroxide to inactivate endogenous peroxidase activity.26 We used anti-SVCT1 antibodies raised against a synthetic peptide from human and rat sodium–vitamin C co-transporters (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-SVCT1 (D-19) was used to analyze samples from mouse and rat tissues. Anti-SVCT1 (N-20) was used to analyze samples from human tissues. Anti-SVCT1 (C-15) was used to analyze samples isolated from LLC-PK1 cells. Sections were incubated with anti-SVCT1 polyclonal antibody (1:100) overnight at room temperature in a humid chamber. For double labeling, we also used anti-FBPase (1:100), an antibody that recognizes the gluconeogenic enzyme fructose-1-6-bisphosphatase. The immunohistochemistry technique has been previously described.27 Alternatively, after incubation with the primary antibody, some slides were incubated for 2 h at room temperature with Cy2- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and analyzed by confocal laser microscopy.28 For double labeling, we used 25 mg/ml Alexa 488-labeled PHA lectin (Molecular Probes, Eugene, OR, USA). As controls, we used both primary antibodies pre-absorbed with an excess of the cognate peptides used for immunization or preimmune serum. Ultrastructural immunohistochemistry Mouse kidney tissues were immersed for 2 h in fixative containing 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M phosphate buffer, and pH 7.4. The samples were dehydrated in dimethylformamide and embedded in London Resin Gold (Electron Microscopy Science, Washington, DC, USA). Ultrathin sections were mounted on uncoated nickel grids and processed for immunohistochemistry.27 The anti-SVCT1 antibody (1:100) was diluted in incubation buffer Tris-HCl (pH 7.8).29 In situ hybridization A cDNA of approximately 0.6 kb subcloned in pCR-4-Blunt-TOPO and encoding a human SVCT1 fragment was used to generate sense and antisense digoxigenin-labeled riboprobes. The human probe for SVCT1 used for in situ hybridization has high-sequence homology with mouse SVCT1 (87%). RNA probes were labeled with digoxigenin-UTP by in vitro transcription with SP6 or T7 RNA polymerase following the manufacturer’s procedure (Boehringer Mannheim, Mannheim, Germany). In situ hybridization was performed on mouse sagital kidney sections mounted on poly-Llysine-coated glass slides. The hybridization methodology has been previously described.29 After hybridization, the slides were rinsed in 4  saline-sodium citrate (SSC) and washed twice for 30 min at 42 1C. The slides were washed at 37 1C for 30 min each in 2  SSC, 1  SSC, and 0.3  SSC. Visualization of digoxigenin was performed by incubation with a monoclonal antibody coupled to alkaline phosphatase.29 Kidney International (2008) 74, 1278–1286 original article T Castro et al.: SVCT1 distribution in renal proximal tubule Reverse transcription-PCR The following samples were used for RT-PCR analysis: (i) cortical and medullar kidney regions of adult C57BL/J6 mice; (ii) mRNA from adult human kidney (Clontech, Palo Alto, CA, USA); and (iii) mRNA from LLC-PK1 cells. The poly(A) RNA was isolated using the Oligotex direct kit (Qiagen, Valencia, CA, USA). For RTPCR, 0.5–1 mg of RNA was incubated in 20 ml reaction volume containing 10 mM Tris (pH 8.3), 50 mM KCl, 5 mM MgCl2, RNase inhibitor 20 U, 1 mM dNTPs, 2.5 mM of random hexanucleotides, and 50 U of MuLV reverse transcriptase (Perkin Elmer, Branchburg, NJ, USA) for 10 min at 23 1C, followed by 30 min at 42 1C and 5 min at 94 1C. Parallel reactions were performed in the absence of reverse transcriptase to control for the presence of contaminant DNA. For amplification, a cDNA aliquot in a volume of 12 ml containing 20 mM Tris, pH 8.4, 50 mM KCl, 1.6 mM MgCl2, 0.4 mM dNTPs, 0.04 U of Taq DNA polymerase (Gibco-BRL, Rockville, MD, USA), and 0.4 mM primers was incubated at 94 1C for 4 min, 94 1C for 50 s, 55 1C for 50 s, and 72 1C for 135 s for 35 cycles. PCR products were separated by 1.2% agarose gel electrophoresis and visualized by staining with ethidium bromide. The following primers (based on human sequence AF170911) were used to analyze the expression of SVCT1-like transporter: forward primer, 50 -TGGTTCCAGCCAAA GCCATAC-30 and reverse primer, 50 -ATGGCCAGCATGATAGGA AA-30 (expected product 439 bp). Cell culture LLC-PK1 cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. For immunocytochemistry, pre- or post-confluent cells were fixed in 4% paraformaldehyde diluted in phosphatebuffered saline for 30 min. Western blot LLC-PK1 and mouse kidney membrane proteins were obtained by homogenizing the cells in 0.3 mM sucrose, 3 mM dithiothreitol, 1 mM EDTA, 100 mg/ml phenylmethanesulfonylfluoride, pepstatin A 1 mg/ ml, and aprotinin 2 mg/ml. Total membranes were collected by highspeed centrifugation. For immunoblotting, 30 mg of membrane protein was loaded in each lane and fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, transferred to nitrocellulose membranes, and probed against antiSVCT1 antibodies or pre-absorbed antibodies (1:100–1:500).30 The secondary antibody was rabbit anti-goat IgG coupled to peroxidase (1:5000). The reaction was developed with enhanced chemiluminescence according to the ECL western blotting analysis system (Amersham Corporation, Arlington Heights, IL, USA). Vitamin C uptake analyses LLC-PK1 cells were carefully selected under the microscope to ensure that only plates showing uniformly growing cells were used at 200,000 cells per well. The cells were incubated in buffer containing 15 mM HEPES (N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, and 0.8 mM MgCl2 at room temperature for 30 min. Uptake assays were performed in 500 ml of incubation buffer containing 0.1–0.4 mCi of 1-14C-AA (specific activity 8.2 mCi/mmol) to a final concentration of 5–300 mM. The Michaelis constant, Km, was calculated using the Lineweaver–Burk analysis. Data represent means±s.d. of three experiments with each determination performed in duplicate. In inhibition experiments, statistical comparison between two or more groups of data was carried out using analysis of variance (followed Kidney International (2008) 74, 1278–1286 by Bonferroni post-test). Po0.05 was considered to be statistically significant. 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