1 Veterans Affairs Medical Center, Long Beach, 90822; and 2 University of California College of Medicine, Irvine, California 92697
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ABSTRACT |
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The kidneys play an important role in regulating vitamin B6 body homeostasis, but limited information exists regarding the mechanism of pyridoxine uptake by renal epithelial cells, and no study exists on its regulation. To address these issues, we used the renal opossum-derived tubular epithelial (opossum kidney; OK) cells and found pyridoxine uptake to 1) be temperature and energy dependent, 2) be pH dependent, with a higher uptake at alkaline or neutral buffer pH compared with acidic pH, 3) be Na+ independent, 4) involve a saturable component (apparent Michaelis- Menten constant of 2.40 ± 0.23 µM), 5) be inhibited by structural analogs, and 6) be amiloride sensitive. Maintaining OK cells in a vitamin B6-deficient growth medium (for 48 h) led to a significant upregulation of pyridoxine uptake. This upregulation was found to be specific for pyridoxine, inhibited by cyclohexamide and actinomycin D, reversible, and mediated via an increase in maximal velocity. Pretreating OK cells with modulates of a Ca2+/calmodulin-mediated pathway led to a significant downregulation in pyridoxine uptake via inhibition of maximal velocity. These results demonstrate that pyridoxine uptake by renal tubular epithelial OK cells is via a specialized pH-sensitive carrier-mediated mechanism. This mechanism appears to be regulated by extracellular vitamin B6 levels and an intracellular Ca2+/calmodulin-mediated pathway.
transport; calcium/calmodulin; opossum kidney cells
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INTRODUCTION |
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VITAMIN B6 is an essential micronutrient that exists in the diet as a mixture of three vitaminic forms in different proportions: pyridoxine, pyridoxal, and pyridoxamine and the corresponding phosphate derivatives. The vitamin is involved in a variety of critical metabolic reactions including carbohydrate metabolism, sphingolipid biosynthesis and degradation, amino acid metabolism (including that of homocysteine), and neurotransmitter metabolism. Therefore, deficiency of this essential micronutrient in humans leads to a variety of adverse conditions and to disturbances in normal cellular metabolism (for review, see Ref. 9).
Humans and other mammals lack the ability to synthesize vitamin B6 and thus must obtain it from exogenous sources. As a water-soluble compound with a relatively low molecular weight, vitamin B6 is readily filtered in the glomeruli and reabsorbed in the renal tubule. The kidney plays an important role in regulating normal vitamin B6 body homeostasis by salvaging the vitamin from luminal filtrate. Thus it is not surprising that diseases of this organ are associated with abnormally low plasma vitamin B6 levels in humans (reviewed in Ref. 9). The mechanism of renal uptake of vitamin B6 has been examined previously (3-5), but very little is known about regulation of the uptake process by extracellular and intracellular factors and conditions. Also unclear are the roles of Na+ and pH in the renal vitamin B6 uptake process. In this study, we used the opossum-derived renal tubule epithelial cell line, opossum kidney (OK), as an in vitro model system and pyridoxine as a substrate to address these issues. Our results show that pyridoxine uptake by these cells is via a pH-sensitive, Na+-independent, specialized carrier-mediated mechanism. This mechanism appears to be regulated by extracellular substrate levels and by an intracellular Ca2+/calmodulin-mediated pathway.
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MATERIALS AND METHODS |
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Custom-made [3H]pyridoxine (sp act 20 Ci/mmol, radiochemical purity > 97%) was obtained from American Radiolabeled Chemical (St. Louis, MO). All other chemicals and reagents used in this study were of analytical quality and were obtained from commercial sources.
Cell Culture
OK cells (American Type Culture Collection, passages 36-46) were grown in MEM in 75-cm2 plastic flasks (Costar) at 37°C in a 5% CO2-95% air atmosphere with media changes every 2-3 days. Cells were subcultured by trypsinization with 0.05% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free phosphate buffered solution and plated onto 12-well plates at a concentration of 5 × 105 cells/well. Uptake studies were performed on monolayers 2-3 days after confluency. Cell growth was observed by periodic monitoring with an inverted microscope. Viability of OK cells grown in control and vitamin B6-deficient media was determined using the trypan blue dye exclusion method and was found to be >99%.Uptake studies were performed at 37°C in Krebs-Ringer buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES at pH 7.4 (unless otherwise stated). [3H]pyridoxine was added to the incubation buffer at the onset of incubation, and uptake was terminated at the desired time by the addition of 1 ml of ice-cold buffer and subsequent immediate removal by aspiration. Monolayers were then rinsed twice with ice-cold buffer, digested with 1 ml of 1 N NaOH, neutralized by HCl, and counted for radioactivity in a liquid scintillation counter. Protein contents of cell digests were estimated on parallel wells using a Bio-Rad protein assay kit.
In the study to determine the effects of maintaining OK cells in vitamin B6-deficient growth medium on pyridoxine uptake, cells were grown for 48 h before uptake investigations in a vitamin B6-deficient growth medium (with no added pyridoxine). Control cells were maintained in parallel wells in control growth media (i.e., medium that contained 5 µM pyridoxine). Both media were supplemented with 10% dialyzed fetal bovine serum. When the effects of actinomycin D and cyclohexamide on the observed induction of pyridoxine by the vitamin B6-deficient condition was to be tested, these transcription/protein synthesis inhibitors were added at the onset of cell incubation with the vitamin B6-deficient medium.
The metabolic form of the transported substrate after incubation with 30 nM [3H]pyridoxine was determined using the thin-layer chromatography procedure (with silica-precoated thin-layer plates) and a mobile phase containing a 8:7:4:1 vol/vol ratio of t-butanol, acetone, water, and diethylamine (10).
Statistical Analysis
Data are means ± SE of multiple separate uptake determinations performed on different occasions and are expressed in femtomoles or picomoles per milligram of protein per unit of time. Some variations in the absolute amount of pyridoxine uptake were seen with different cell batches; for this reason, simultaneous controls were run with each set of experiments. P values were calculated using Student's t-test. Uptake kinetic parameters [i.e., maximal velocity (Vmax) and the apparent Michaelis constant (Km)] of the pyridoxine carrier-mediated process were calculated using a computerized nonlinear regression-analysis program of the Michaelis-Menten equation as described previously (18). Carrier-mediated uptake was determined by subtracting uptake by diffusion from total uptake; uptake by diffusion was calculated from the slope of a line drawn between the point of origin and uptake at high pyridoxine concentration (1 mM). ![]() |
RESULTS |
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General Characteristics of Pyridoxine Uptake by OK Cells
The effects of incubation temperature on the initial rate (i.e., 3 min) of pyridoxine (15 nM) uptake were examined. The results showed that the uptake was highly temperature dependent and significantly (P < 0.01) inhibited upon decreasing the incubation temperature (117.4 ± 3.2, 53.3 ± 1.5, and 12.4 ± 1.9 fmol · mg proteinIn another study, we examined the effect of incubation buffer pH on the
initial rate of pyridoxine (15 nM) uptake. The results showed that
lowering the incubation buffer pH from 8.0 to 7.0 had no effect on
pyridoxine uptake. However, further reduction in incubation buffer pH
led to a marked inhibition in pyridoxine uptake with uptake at pH 5.0 being <50% of that at pH 7.5 (Fig. 1).
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The role of Na+ in pyridoxine uptake by OK cells was
examined by testing the effect of replacing Na+ in the
incubation medium with an equimolar concentration of other monovalent
cations on the initial rate of the vitamin uptake. The results showed
no inhibition in pyridoxine uptake upon Na+ removal from
the incubation medium regardless of the monovalent cation used to
replace it (Fig. 2). This is in contrast
to the severe (P < 0.01) inhibition observed in uptake
of the unrelated vitamin biotin (10 nM) by these cells upon replacement
of Na+ in the incubation medium with K+
(87.40 ± 1.20 and 5.31 ± 0.39 fmol · mg
protein1 · 3 min
1, respectively).
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Uptake of pyridoxine (0.1 µM) by OK cells as a function of time was
also examined and found to be linear for up to 10 min of incubation and
occurred at a rate of 0.27 pmol · mg
protein1 · 3 min
1 (Fig.
3). We elected to use a 3-min incubation
(initial rate) in all of our studies [uptake of higher (10 µM) and
lower (15 nM) pyridoxine concentrations were also linear during this
time; data not shown].
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Evidence for Involvement of a Carrier-Mediated Mechanism for Pyridoxine Uptake and Its Specificity
In the first study, we examined the initial rate of pyridoxine uptake as a function of substrate concentration in the incubation medium (0.015-10 µM). The results showed that pyridoxine uptake includes a saturable component (Fig. 4). Kinetic parameters of this component were determined as described in MATERIALS AND METHODS and found to be 2.44 ± 0.23 µM and 5.99 ± 0.23 pmol · mg protein
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In another study, we examined the specificity of the pyridoxine uptake
process. This was performed by examining the effects of pyridoxine
structural analogs and unrelated compounds on the initial rate of
[3H]pyridoxine (15 nM) uptake. The results showed that
pyridoxal, 4-deoxypyridoxamine, and pyridoxamine cause
significant inhibition in pyridoxine uptake. On the other hand,
pyridoxic acid (the principle urinary metabolite of vitamin
B6; see Ref. 3) and pyridoxal-5-phosphate as
well as the unrelated compounds tetraethylammonium chloride (TEA) and
N'-methylnicotinamide (NMN) were without effect (Table 1).
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Effects of Membrane Transport Inhibitors and Metabolic Poisons
In these studies, we examined the effects of the membrane transport inhibitors DIDS, probenecid, furosemide, and amiloride (all at 1 mM) on the initial rate of uptake of pyridoxine (15 nM) by OK cells. The results showed that with the exception of amiloride, which caused a modest but significant (P < 0.05) inhibition in pyridoxine uptake, none of the other compounds significantly affected vitamin uptake (Table 2). It is worth stating here that amiloride at the concentration used (1 mM) inhibits all isoforms of the Na+/H+ exchanger.
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We also examined the effects of the metabolic inhibitors dinitrophenol (10 mM), iodoacetate (1 mM), and azide (1 mM) on the initial rate of pyridoxine (15 nM) uptake. Significant (P < 0.01 for all) inhibition was observed with all compounds (Table 2).
Regulation of Pyridoxine Uptake Process in OK Cells
Effect of extracellular substrate level.
In this study, we examined the effect of growing OK cells in a vitamin
B6-deficient growth medium on the ability of the cells to
take up [3H]pyridoxine (see MATERIALS AND
METHODS). Significant (P < 0.01) induction in
pyridoxine (15 nM) uptake was observed in cells grown under vitamin
B6-deficient conditions compared with those grown in
control growth medium, with the induction being significantly (P < 0.01) inhibited when actinomycin D (0.23 µM)
and cyclohexamide (40 µM) were present in the deficient growth medium
(Fig. 5). In addition, this upregulation
in [3H]pyridoxine (15 nM) uptake was found to be
reversible after reintroduction of pyridoxine (5 µM) to the vitamin
B6-deficient growth medium (uptake of
[3H]pyridoxine of 119.99 ± 3.66, 223.52 ± 2.05, and 123.23 ± 2.05 fmol · mg
protein1 · 3 min
1 for cells grown
in control growth medium, vitamin B6-deficient growth
medium, and vitamin B6-deficient medium followed by
incubation for 24 h in the presence of pyridoxine, respectively).
In contrast to the effects of vitamin B6-deficient
conditions on pyridoxine uptake, uptake of the unrelated vitamin,
thiamin (30 nM), was found to be similar in cells grown in control and
vitamin B6-deficient growth medium (98.1 ± 0.7 and
93.8 ± 4.0 fmol · mg protein
1 · 3 min
1, respectively). In other studies, we determined
whether the effects of the vitamin B6-deficient
condition are mediated through effects on the
Vmax and/or the apparent
Km of the pyridoxine-uptake process. This was
performed by examining pyridoxine uptake as a function of concentration
in the two cell subtypes. The results (Fig.
6) showed a marked induction
(P < 0.01) in the Vmax of the
pyridoxine uptake process in cells grown under vitamin
B6-deficient conditions compared with those grown in
control growth medium (Vmax values of 7.25 ± 0.64 and 22.45 ± 1.03 pmol · mg
protein
1 · 3 min
1 for cells grown
in vitamin B6-deficient and control media, respectively) with a slight decrease in the apparent Km
(4.05 ± 1.07 and 2.22 ± 0.34 µM, respectively).
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Role of intracellular regulatory pathways.
In these studies, we examined the possible regulation of pyridoxine
uptake by OK cells via specific intracellular regulatory mechanisms. We
focused on pathways that have been shown to be involved in the
regulation of other substrates (including other water-soluble vitamins)
in renal and other epithelial cell membrane transport events including
Ca2+/calmodulin, nitric oxide (NO), and protein kinase A
(PKA)-mediated pathways (6, 8, 12-15). The results
showed that pretreating OK cells (for 1 h) with calmidazolium (25 µM; an inhibitor of the Ca2+/calmodulin-mediated pathway)
led to significant inhibition in pyridoxine uptake (122.6 ± 5.2 and 66.4 ± 4.9 fmol · mg
protein1 · 3 min
1 for control and
the presence of calmidazolium, respectively). Similarly, pretreatment
with two other inhibitors of the Ca2+/calmodulin-mediated
pathway inhibitors, trifluoperazine (TFP) and KN62, also caused
significant (P < 0.01 for all) inhibition in
pyridoxine (15 nM) uptake (132.3 ± 4.7, 94.0 ± 7.3, 76.4 ± 4.9, and 107.0 ± 2.6 fmol · mg
protein
1 · 3 min
1 for control and
in the presence of 50 and 100 µM TFP and 25 µM KN62, respectively).
We also examined the effects of pretreatment with calmidazolium (50 µM) on kinetic parameters (Vmax and apparent Km) of the pyridoxine-uptake process. The
results (Fig. 7) showed that
calmidazolium causes a significant (P < 0.01)
inhibition in Vmax but not in apparent
Km of the pyridoxine-uptake process (Vmax of 8.06 ± 0.49 and 2.44 ± 0.09 pmol · mg protein
1 · 3 min
1; apparent Km of 2.74 ± 0.58 and 2.96 ± 0.38 µM for control and calmidazolium-pretreated cells, respectively).
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DISCUSSION |
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Renal tubules play a critical role in salvaging filtered water-soluble nutrients such as vitamin B6. Any defect or interference with the normal vitamin B6 renal uptake process may lead to disturbance in normal vitamin B6 body homeostasis. Thus a detailed understanding of the renal uptake process of vitamin B6 is of significant importance. The present study examined the mechanism and regulation of uptake of pyridoxine, a member of the vitamin B6 family, by renal epithelial cells using the renal tubular epithelial OK cells as a model system. This well-established renal epithelial system has been widely used in similar investigations on cell physiology of renal transport (1, 7, 16, 17). The results showed that uptake of pyridoxine during the initial 3 min of incubation proceeded with no metabolic alterations in the transported substrate. The uptake process of pyridoxine was found to be both temperature and pH sensitive in nature. Lowering incubation temperature from 37 to 21 and 4°C was associated with a marked inhibition in pyridoxine uptake. With regard to pH sensitivity, although decreasing the incubation buffer pH from 8.0 to 7.0 did not affect the initial rate of pyridoxine uptake, further reduction in the buffer pH to 5.0 led to a gradual decrease in substrate uptake. Further investigations are required to establish the exact mechanism mediating the pH effect; i.e., whether it is mediated via an effect on the involved carrier system itself and/or through the pH gradient imposed across the membrane. In contrast to the effect of buffer pH, pyridoxine uptake by OK cells was found to be Na+ independent, because total replacement of Na+ in the incubation medium with equimolar concentrations of other monovalent cations did not affect pyridoxine uptake. This is in contrast to the severe inhibition that was observed in the uptake of the unrelated vitamin biotin by the same cells after Na+ replacement in the incubation medium (biotin uptake by renal epithelial cells is known to be Na+ dependent in nature; see Refs. 2 and 11). Our observation of a lack of a role for Na+ in pyridoxine uptake is similar to the previous observations by Bowman and McCormick (3) with isolated rat renal epithelial cells and by Middleton (10) with rat intestine. It is, however, different from a subsequent study by Bowman and colleagues (4) with renal brush-border membrane vesicles, which reported the process to be Na+ driven. The cause of this discrepancy in the previous reports (3, 4) is not clear but cannot be attributed to species variations because rats were used in both of the referenced studies.
Uptake of pyridoxine by OK cells was found to be carrier mediated in nature. This conclusion is based on the observation that uptake of the substrate as a function of concentration includes a saturable component and by the observation of significant inhibition in [3H]pyridoxine uptake by close structural analogs (pyridoxal and 4-deoxypyridoxamine). This pyridoxine carrier system displayed clear specificity, as indicated by the lack of effect of certain other analogs of the substrate (pyridoxic acid and pyridoxal-5-phosphate) and unrelated compounds (TEA and NMN) on its uptake. Uptake of pyridoxine by OK cells was found to be insensitive to the presence of membrane transport inhibitors DIDS, furosemide, and probenecid. However, a slight but significant inhibition in pyridoxine uptake was observed with the diuretic agent amiloride. The latter finding is similar to that reported previously for the vitamin uptake in isolated rat renal epithelial cells (3) and by certain strains of yeast (19).
After the characterization of the uptake mechanism of pyridoxine in OK cells, we used this cellular system to study regulation of the pyridoxine uptake by extracellular substrate-level and by intracellular regulatory mechanisms. Our results showed that growing OK cells in a vitamin B6-deficient growth medium leads to a significant upregulation in pyridoxine uptake. This upregulation was significantly inhibited when the transcription or translation inhibitors actinomycin D and cyclohexamide, respectively, were present in the growth medium. The latter observations suggest possible involvement of transcription/translation mechanisms in the upregulation process of pyridoxine uptake under vitamin B6-deficient conditions. Further studies at the molecular level are required to confirm this suggestion. The upregulation in pyridoxine uptake by vitamin B6-deficient conditions was also found to be specific for pyridoxine, because uptake of the unrelated vitamin thiamine was not affected by such a condition. Furthermore, the induction was reversible after reintroduction of pyridoxine into the vitamin B6-deficient growth medium. The induction in pyridoxine uptake by vitamin B6-deficient conditions was found to be mainly mediated via a significant increase in the Vmax of pyridoxine uptake with a slight change in the apparent Km. These findings suggest that the induction in pyridoxine is most likely the result of an increase in the number (and/or activity) of the pyridoxine-uptake carriers with slight changes in the corresponding affinities.
The possible role of intracellular regulatory pathways in the regulation of vitamin B6 uptake by renal epithelial cells was also investigated using this renal epithelial cell line as a model. In these investigations, we focused our effort on pathways that have been shown to play a role in regulating transport of other substrates, including other water-soluble vitamins (6, 8, 12-15). Our results showed that inhibition of the Ca2+/calmodulin-mediated pathway with the use of different inhibitors leads to a significant inhibition (downregulation) of pyridoxine uptake. This inhibition was found to be mediated via an inhibition in the Vmax of the pyridoxine-uptake process with no significant effect on the apparent Km. These findings suggest that the inhibition in pyridoxine uptake by the Ca2+/calmodulin inhibitor calmidazolium is mediated via a decrease in the activity (and/or number) of the pyridoxine-uptake carriers with no effect on carrier affinity. Further studies are required to determine the precise mechanisms through which this intracellular regulatory pathway exerts its effect on pyridoxine uptake in renal epithelia. In contrast to the apparent role of the Ca2+/calmodulin-mediated pathway in regulating pyridoxine uptake in OK cells, no role for NO- or PKA-mediated pathways was found. This conclusion is based on the observation that modulators of these pathways were without effect on pyridoxine uptake.
In summary, our study demonstrates that pyridoxine uptake by cultured renal tubular epithelial OK cells is via a pH-sensitive, Na+-independent, specialized carrier-mediated mechanism. This uptake mechanism appears to be regulated by extracellular substrate levels and by an intracellular Ca2+/calmodulin-mediated pathway. These findings should serve as a base for future investigations into the molecular mechanisms involved in the transport of vitamin B6 in renal epithelial cells and its regulation.
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ACKNOWLEDGEMENTS |
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This study was supported by the Department of Veterans Affairs and the National Institutes of Health (Grants DK-56061 and DK-58057).
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. M. Said, VA Medical Center, 151, Long Beach, CA 90822 (E-mail: hmsaid{at}uci.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00267.2001
Received 27 August 2001; accepted in final form 24 October 2001.
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