INTRODUCTION

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RIBOFLAVIN (RF), a water-soluble vitamin, is involved in key metabolic reactions, including carbohydrate, lipid, and amino acid metabolism, and therefore is essential for normal cellular functions, growth, and development (4, 17, 18). RF deficiency leads to a variety of clinical abnormalities including degenerative changes in the nervous system, anemia, endocrine dysfunction, and skin disorders (4, 11). Deficiency of RF has also been suggested to lead to an increase in the susceptibility to carcinogens (19). Normal RF body homeostasis depends on intestinal absorption and renal recovery of the filtered RF by renal tubule epithelial cells (5, 12-14). Animal and cell line studies have suggested the involvement of a carrier-mediated system for RF uptake by the renal epithelial cells (1, 16, 25). However, very little is known about the mechanism of RF uptake in humans, and nothing is known about the intracellular regulation of the uptake process. In this study, we used the human normal renal proximal tubule epithelial cell line HK-2 as a model to address these issues. HK-2 cells are well-differentiated cells that possess functional characteristics similar to those of native renal proximal tubule epithelial cells [e.g., they take up methyl--D-glucopyranoside by the well-characterized Na⁺-dependent and phlorizin-inhibitable process (22)]. Furthermore, these cells are well suited to the study of intracellular regulation of uptake processes in renal epithelial cells because of their homogenous nature (i.e., they are unlike the diverse types of kidney cells). Our results have demonstrated the existence of a specialized, carrier-mediated uptake system for RF by these cells. Furthermore, this system appears to be under the regulation of Ca²⁺/calmodulin-mediated pathways and level of the substrate in the growth medium.

	MATERIALS AND METHODS

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[³H]RF (sp act 48 Ci/mmol, radiochemical purity >97%) was purchased from Moravek Biochemicals (Brea, CA). Methyl--D-[U-¹⁴C]glucopyranoside (AMG; sp act 292 mCi/mmol) was from Amersham International (Arlington Heights, IL). Keratinocyte serum free medium (K-SFM) was from GIBCO-BRL (Grand Island, NY). Isoriboflavin (isoRF) and 8-amino-riboflavin (8-amino-RF) are generous gifts from Dr. Vincent Massey of the Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), trypsin, and other cell culture ingredients were from Sigma Chemical (St. Louis, MO). All other chemicals were of analytical grade and were purchased from commercial sources.

HK-2 cells obtained from American Type Culture Collection (Rockville, MD) were grown in K-SFM and supplemented with epidermal growth factor (5 ng/ml) and bovine pituitary extract (40 µg/ml). The cells were grown in 75-cm² plastic flasks (Costar) at 37°C in a 5% CO₂-95% air atmosphere with media changes every 3 days. HK-2 cells were subcultured by trypsinization with 0.05% trypsin and 0.9 mM EDTA in Ca²⁺-free and Mg²⁺-free phosphate-buffered saline solution and plated onto 12-well plates at a concentration of 5 × 10⁵ cells/well. Uptake of RF was studied 3-6 days after confluence. Cell growth was observed by periodic monitoring with an inverted microscope. Cell viability was tested by the trypan blue dye exclusion method and found to be >95%.

To examine the effect of growing HK-2 cells in an RF-oversupplemented medium, the cells were grown 48 h before the uptake study in a growth medium containing 50-fold (2.0 µg/ml) the amount of RF provided under control conditions (0.04 µg/ml). Cells were grown in RF-free medium for RF-deficient conditions.

Uptake experiments were performed at 37°C, unless otherwise specified. The incubation buffer was Krebs-Ringer-phosphate buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO₄, 0.85 CaCl_2, 5 glucose, 5 glutamine, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 10 2-(N-morpholino)ethanesulfonic acid, pH 6.5. [³H]RF was added to the incubation buffer at the start of the uptake experiment, and uptake was terminated after 3 min of incubation (unless otherwise specified) by the addition of 1 ml of ice-cold buffer followed by immediate removal by aspiration. The monolayers were rinsed twice with ice-cold buffer and digested with 1 ml of 1 N NaOH, neutralized by HCl, and then counted for radioactivity in a liquid scintillation counter. Protein contents of cell digests were estimated on parallel wells or on the same wells, using a protein assay kit from Bio-Rad Laboratories (Hercules, CA), based on the method of Lowry et al. (15), with bovine serum albumin as the standard. Data presented here are means ± SE of multiple separate monolayers performed on at least two different occasions and are expressed in picomoles or femtomoles per milligram protein per unit time. P values were calculated, using the Student's t-test. Kinetic parameters of RF uptake, i.e., maximal velocity (V_max) and the apparent Michaelis constant (K_m), were calculated using a computerized model of the Michaelis-Menten equation as described by Wilkinson (26).

	RESULTS

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Mechanism of RF Uptake by HK-2 Cells

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Uptake with time and effect of incubation temperature. Shown in Fig. 1, A and B, is the time-dependent uptake of low (A; 4.2 nM) and high (B; 1 µM) concentrations of RF by HK-2 cells. Uptake was linear with time for up to 5 min of incubation under both conditions and occurred at a rate of 0.05 and 0.75 pmol · mg protein¹ · min¹ for low and high concentrations, respectively. Based on these results, a 3-min incubation was used as the standard incubation time for all the experiments done in this study (unless otherwise specified).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Uptake of riboflavin (RF) by HK-2 cells as a function of time. HK-2 cells were incubated at 37°C in Krebs-Ringer-phosphate buffer, pH 6.5, in presence of low (4.2 nM; A) or high (1 µM; B) concentrations of RF. Each data point represents mean ± SE of 4-6 separate uptake determinations. A: y = 0.019x + 0.034, r = 0.977. B: y = 0.575x + 0.244, r = 0.968.

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Role of Na⁺ in the incubation buffer. The role of Na⁺ in RF uptake by HK-2 cells was investigated in this study. This was done by examining the effect of isosmotically replacing Na⁺ (123 mM) in the incubation buffer with chloride salts of other monovalent cations such as K⁺ or Li⁺ (123 mM) on the uptake of RF (4.2 nM). The results showed no significant change in RF uptake under all conditions tested [75 ± 1 (n = 4), 74 ± 3 (n = 4), and 77 ± 3 (n = 4) fmol · mg protein¹ · 3 min¹ for control (Na⁺), K⁺, and Li⁺, respectively]. In another study, we examined the effect of pretreating (for 30 min) HK-2 cells with the Na⁺-K⁺-adenosinetriphosphatase (Na⁺-K⁺-ATPase) inhibitor ouabain (10 mM) on the uptake of RF (4.2 nM). The result showed no significant effect of ouabain on RF uptake (68.5 ± 2 and 73.9 ± 4 fmol · mg protein¹ · 3 min¹ for control and in the presence of ouabain, respectively).

Uptake of RF as a function of concentration. Uptake of RF was examined as a function of increasing the substrate concentration in the incubation medium. It was found to be saturable at low concentrations and linear at high concentrations. Uptake by the saturable component was determined by subtracting the uptake by diffusion from the total uptake at each concentration (Fig. 2). Uptake by diffusion was determined from the slope of the linear uptake at high RF concentration. Kinetic parameters of the saturable uptake process were then calculated as described in MATERIALS AND METHODS and were found to be 0.67 ± 0.21 µM for the apparent K_m and 10.05 ± 0.87 pmol · mg protein¹ · 3 min¹ for the V_max of the uptake process.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Uptake of RF by HK-2 cells as a function of substrate concentration (conc) in incubation medium. Uptake was performed at 37°C in Krebs-Ringer buffer, pH 6.5, containing varying concentration of unlabeled RF and [3H]RF (4.2 nM). Each data point represents mean ± SE of 3 separate uptake determinations.

Effect of unlabeled RF and related compounds on the uptake of[³H]RF. The effect of unlabeled RF and its related compounds lumiflavine (LF), lumichrome, isoRF, 8-amino-RF, lumazine, and D-ribose as well as that of the unrelated compounds carnitine and biotin on the uptake of [³H]RF (4.2 nM) by HK-2 cells were examined in this study. Unlabeled RF, LF, lumichrome, isoRF, and 8-amino-RF (but not lumazine, D-ribose, biotin, or carnitine) caused a significant (P < 0.01) inhibition of [³H]RF uptake (Table 1).

Effect of metabolic and membrane transport inhibitors. The effect of the metabolic inhibitors sodium azide (10 mM), 2,4-dinitrophenol (1 mM), and iodoacetate (10 mM) and that of the sulfhydryl group inhibitor p-chloromercuriphenylsulfonate ( p-CMPS; 1 mM) on the uptake of RF (4.2 nM) were examined. HK-2 cells were preincubated with the aforementioned compounds for 30 min at 37°C before uptake measurements. [³H]RF was then added, and incubation was continued for 3 min. All the compounds tested were found to cause a significant (P < 0.01 for all) inhibition in RF uptake [65 ± 2 (n = 8), 34 ± 1 (n = 8), 11 ± 1 (n = 8), 7 ± 1 (n = 4), and 19 ± 5 (n = 8) fmol · mg protein¹ · 3 min¹ for control and after pretreatment with sodium azide, 2,4-dinitrophenol, iodoacetate, and p-CMPS, respectively].

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Regulation of RF Uptake in HK-2 Cells

Role of intracellular regulatory pathways. Several studies have demonstrated the involvement of intracellular regulatory pathways such as protein kinase C (PKC)-, protein kinase A (PKA)-, protein tyrosine kinase (PTK)-, and Ca²⁺/calmodulin-mediated pathways in the regulation of uptake of many substrates in epithelial cells (2, 3, 6-8, 10, 20, 21, 24). We have therefore examined the possible role played by these pathways in regulating the RF uptake process of the HK-2 cells identified.

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Effect of RF levels in the growth medium on RF uptake. The possible regulatory effect of RF levels in the growth medium (i.e., deficiency and oversupplementation) on RF uptake by HK-2 cells was studied. Experiments were carried out as described in MATERIALS AND METHODS. The initial rate (i.e., 3 min) of [³H]RF (4.2 nM) uptake was examined in HK-2 cells grown in control (RF sufficient), RF-deficient, and RF-oversupplemented media. Uptake of [³H]RF by cells grown in an RF-deficient medium was found to be significantly (P < 0.01) higher than the uptake by cells grown in control medium (i.e., RF sufficient), which was in turn higher than the uptake by cells grown in an RF-oversupplemented medium (Table 6). The changes in RF uptake appeared to be specific for RF, as uptakes of the unrelated biotin and carnitine were not affected by these conditions [5.7 ± 0.3, 6.2 ± 0.4, and 5.9 ± 0.3 (for biotin; 4.5 nM) and 3.6 ± 0.4, 3.2 ± 0.5, and 3.9 ± 0.4 (for carnitine; 4.7 nM) fmol · mg protein¹ · 3 min¹ for RF-sufficient, RF-deficient, and RF-oversupplemented conditions, respectively].

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	DISCUSSION

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The major aim of the present study was to investigate the mechanism and regulation of RF renal transport in a human cell line with the use of HK-2 cells as a model to system. We chose this normal human kidney epithelial cell line because HK-2 cells are well differentiated (on the basis of their histochemical, immunocytochemical, and functional characteristics) and because they have been shown to reproduce experimental results obtained with freshly isolated proximal tubule epithelial cells. This was confirmed in our laboratory. Uptake of AMG by these cells was found to be Na⁺ dependent and phlorizin inhibitable in nature. Furthermore, phosphate uptake by these cells was inhibited by PTH. These characteristics are similar to those seen in the native proximal tubule epithelial cells.

Using these cells, we found RF uptake to be temperature dependent and occurring with minimal metabolic alterations in the transported substrate. Na⁺ in the incubation medium appeared to play no role in RF uptake, as indicated by the lack of effect of Na⁺ removal on uptake as well as the lack of effect of the Na⁺-K⁺-ATPase inhibitor ouabain on the process. The uptake process of RF by HK-2 cells was saturable as a function of increasing the substrate concentration in the incubation medium with an apparent K_m and V_max of 0.67 ± 0.21 µM and 10.05 ± 0.87 pmol · mg protein¹ · 3 min¹, respectively. This finding indicates the involvement of a carrier-mediated system in the uptake process. This conclusion was further supported by the finding of a significant inhibition in [³H]RF uptake by unlabeled RF and by its structural analogs. The study on the effect of structural analogs on RF uptake also provided some information about the structural-functional relationship between RF and its uptake system. Whereas LF (4 µM) caused 83.6% inhibition in [³H]RF uptake, which was close to that caused by an equimolar concentration of unlabeled RF (89%), lumichrome (at 4 µM) caused only 55.5% inhibition. This suggests that replacement of the ribityl group of the riboflavin molecule by a smaller group such as methyl group, as in LF at position 10 of the RF molecule, does not affect the ability of the compound to interact with the uptake carrier and thereby inhibit the RF uptake. However, absence of any side chain or a group at this position (as in lumichrome) of the RF molecule seems to make the compound less effective as an inhibitor of the uptake process. Similarly, isoRF (4 µM) caused only 27.5% inhibition. The only difference between the structure of the RF molecule and that of isoRF is the shift in the position of the methyl group from position eight in the case of RF to position six in the case of isoRF. Thus it seems that the position of this methyl group is important for the interaction of the substrate with its renal uptake system.

The process of RF uptake by HK-2 cells was energy dependent, as indicated by the significant inhibition in the uptake process by different metabolic inhibitors. It was also sensitive to the effect of the anion transport inhibitors DIDS, SITS, and probenecid. Furthermore, RF uptake was inhibited by the presence of organic anions such as p-aminohippurate, salicylate, and antibiotic penicillin G in the incubation buffer. These results support the findings of Spector (25), who used rabbit kidney slices to study RF uptake and showed the inhibition by penicillin G. On the other hand, our results are in contrast to the findings of Bowers-Komro and McCormick (1), who found no inhibition of RF uptake by isolated rat kidney cells by these compounds. On the basis of these results, we suggest that the possible existence of a relationship between the RF uptake mechanism and the uptake mechanism of organic anions in the renal epithelia. Further studies are needed to clarify this issue. Nevertheless, the finding that organic anions may interfere with RF renal uptake could be of clinical importance, because a variety of commonly used drugs are organic anions and thus may have the potential of interacting with RF renal transport.

After the delineation of the mechanism of uptake of RF by this human renal epithelial cell model, we then examined the regulation of RF uptake by HK-2 cells. We concentrated on intracellular regulatory pathways that have been shown to play an important role in the regulation of uptake of other substrates by epithelial cells (PKC-, PKA-, PTK-, and Ca²⁺/calmodulin-mediated pathways) (2, 3, 6-8, 10, 20, 21, 24). Using specific modulators of these pathways, we found that PKC-, PKA-, and PTK-mediated pathways appear to have no role in regulating the RF uptake by HK-2 cells. In contrast, compounds that are antagonists to Ca²⁺/calmodulin-mediated pathways such as calmidazolium, trifluoperazine, and KN-62 caused a significant and concentration-dependent downregulation in RF uptake. The inhibitory effect of calmidazolium appeared to be mediated through a decrease in the V_max of the RF uptake process and an increase in the apparent K_m of the uptake process_. Based on these findings, we suggest that the inhibitory effect caused by calmidazolium is mediated through a decrease in the activity and/or the number as well as affinity of the RF uptake carriers.

RF levels in the growth medium were found to have a clear, adaptive regulatory effect on uptake of the vitamin by HK-2 cells. The growing of HK-2 cells in an RF-deficient medium was found to cause a significant upregulation in RF uptake, whereas growing them in a growth medium oversupplemented with RF caused a significant downregulation in RF uptake. These changes in RF uptake were specific for RF, because uptake of the unrelated biotin and carnitine under the same conditions was not affected. The adaptive regulatory effect of RF level in the growth medium on RF uptake was found to be mediated via changes in both the apparent K_m and V_max of the RF uptake process. V_max of the RF uptake process increased, whereas the apparent K_m decreased in the following order: RF-oversupplemented, RF-sufficient, and RF-deficient conditions. These findings suggest that the adaptive regulatory effect of substrate level on RF uptake is mediated via changes in the number and/or activity as well as the affinity of the RF uptake carrier. LF level in the growth medium also caused similar up- and downregulation in RF uptake by HK-2 cells. LF is a close structural analog to RF and seems to share the same uptake mechanism with the vitamin, as demonstrated in the present study (see RESULTS). However, LF cannot be phosphorylated or utilized as an RF-like vitamin by cells (18). Thus the observation that LF level in the growth medium could also regulate the RF uptake system in HK-2 cells raises the possibility that it is the presence of a transport substrate that is recognizable by the RF carrier system that is important for the induction of these adaptive changes in RF uptake. It is not known exactly how changes in RF/LF levels in the growth medium bring about the changes in RF carrier at this point. Further studies are needed to explore the mechanism.

In summary, our results demonstrate that uptake of RF by the human-derived kidney proximal tubule epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent but Na⁺ independent in nature. Furthermore, this system appears to be under the regulation of a Ca²⁺/calmodulin-mediated pathway and substrate level in the growth medium.