Vol. 274, Issue 1, F104-F110, January 1998
Mechanism and regulation of riboflavin uptake by human renal
proximal tubule epithelial cell line HK-2
Chandira K.
Kumar1,
Norimoto
Yanagawa2,
Alvaro
Ortiz1, and
Hamid M.
Said1,3
3 Medical Research Service,
Veterans Affairs Medical Center, Long Beach
90822; 1 Departments of
Medicine, Pediatrics and Physiology/Biophysics, University
of California School of Medicine, Irvine 92697; and
2 Nephrology Division,
Sepulveda Veterans Affairs Medical Center, Sepulveda, California
91343
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ABSTRACT |
Riboflavin (RF), a water-soluble vitamin, is essential for
normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the
human situation, are poorly understood. The aim of this study was
therefore to address these issues, using a recently established human
normal renal epithelial cell line, HK-2, as a model. Uptake of RF by
HK-2 cells was found to be 1) linear
with time for 5 min of incubation and occurring with minimal metabolic
alterations, 2) temperature
dependent, 3)
Na+ independent,
4) saturable as a function of
concentration [apparent Michaelis constant
(Km) of 0.67 ± 0.21 µM and maximal velocity (Vmax) of 10.05 ± 0.87 pmol · mg
protein
1 · 3 min1],
5) inhibited by structural analogs
and anion transport inhibitors, and
6) energy dependent. Protein kinase
C-, protein kinase A-, and protein tyrosine kinase-mediated pathways
were found to have no role in regulating RF uptake. On the other hand,
a Ca2+/calmodulin-mediated pathway
appeared to play a role in the regulation of RF uptake by HK-2 cells
via an effect on the
Vmax, as well as
on the apparent
Km of the RF
uptake process. The uptake process of RF was also found to be
adaptively regulated by the level of the substrate in the growth
medium, with the effect being mediated through changes in the apparent
Km and the
Vmax of the
uptake process. These results demonstrate that RF uptake by the
human-derived renal epithelial cell line HK-2 is via a carrier-mediated
system that is temperature and energy dependent and appears to be under the regulation of a
Ca2+/calmodulin-mediated pathways
and substrate level in the growth medium.
uptake regulation
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
[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 CaCl2, 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
(Vmax) and the
apparent Michaelis constant
(Km), were
calculated using a computerized model of the Michaelis-Menten equation
as described by Wilkinson (26).
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RESULTS |
Mechanism of RF Uptake by HK-2 Cells
We first confirmed the functional integrity of HK-2 cells by examining
the uptake of AMG (0.685 µM) in the presence and absence of
Na+ in the incubation medium as
well as the effect of phlorizin on the uptake process. The results
showed that the uptake of AMG is similar to that of the native renal
cells in that it is Na+ dependent
in nature [1.58 ± 0.03 (n = 6), 0.51 ± 0.04 (n = 6), 0.64 ± 0.07 (n = 6), and 0.64 ± 0.07 (n = 6)
pmol · mg
protein1 · 3 min1 for control (i.e.,
Na+) and in the absence of
Na+ but the presence of
Li+, choline, and mannitol,
respectively]. It was also similar in that it was inhibited by
phlorizin (1 mM) [1.46 ± 0.14 (n = 6) and 0.23 ± 0.08 (n = 6) pmol · mg
protein1 · 3 min1 for control and in the
presence of phlorizin, respectively]. Furthermore, these cells
were found to take up phosphate by a process that is similar to that of
the native renal epithelia in being sensitive to the inhibitory effect
of parathyroid hormone (PTH; 200 nM) [44 ± 3 (n = 4) and 33.7 ± 2 (n = 4) pmol · mg
protein1 · 3 min1
(P < 0.05) for control and the
PTH-treated cells, respectively]. These findings further confirm
the suitability of this cell line as a model for renal proximal tubule
epithelial cells, and thus it was used in this study to examine the
mechanism and cellular regulation of RF uptake.
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).
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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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In another experiment, we studied the effect of incubation temperature
on the uptake of RF (4.2 nM). Uptake was significantly (P < 0.01) higher at 37°C
compared with uptake at 4°C [87 ± 6 (n = 6) and 31 ± 2 (n = 6) fmol · mg
protein1 · 3 min1, respectively].
Metabolic form of the radioactivity taken up by HK-2 cells after 3- and
15-min incubations with
[3H]RF (21.6 nM) was
studied. Cells were washed immediately at the end of incubation with
ice-cold buffer and suspended in 50% aqueous methanol solution as
previously described (9, 23). The cells were then homogenized and
centrifuged. The supernatant was applied onto silica-precoated
thin-layer chromatography plates. The plates were run, using a solvent
system of 0.1 M anhydrous
Na2HPO4
solution. A percentage (94 and 89.8%, respectively) of the
3H radioactivity taken up by HK-2
cells was found to be in the form of intact
[3H]RF after 3- and
15-min incubation.
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
Km and 10.05 ± 0.87 pmol · mg
protein1 · 3 min1 for the
Vmax of the
uptake process.
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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.
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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].
In a separate experiment, we examined the effect of anion transport
inhibitors DIDS, SITS, and probenecid (all at 1 mM) on the uptake of RF
(4.2 nM) by HK-2 cells. The results showed significant inhibition
(P < 0.01 for all) in RF uptake by
all compounds tested [73 ± 5 (n = 7), 29 ± 4 (n = 7), 29 ± 4 (n = 7), and 30 ± 1 (n = 7) fmol · mg
protein1 · 3 min1 for control and in the
presence of DIDS, SITS, and probenecid, respectively].
We also examined the effect of organic anions such as
p-aminohippurate, salicylate, and
penicillin G (10 mM each) on the uptake of RF (4.2 nM).
The results showed a significant (P < 0.01) inhibition by the compounds tested [82 ± 0.7 (n = 4), 12 ± 1.6 (n = 4), 17 ± 1.7 (n = 4), and 9.4 ± 1.6 (n = 4) fmol · mg
protein1 · 3 min1 for control and in the
presence of p-aminohippurate,
salicylate, and penicillin G, respectively].
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.
The possible role of PKC in the regulation of RF uptake by HK-2 cells
was tested by examining the effect of pretreating HK-2 cells for 1 h
with either the PKC activator phorbol 12-myristate 13-acetate (PMA) or
with the PKC inhibitors bisindolylmaleimide I and chelerythrine on the
uptake of 4.2 nM RF. The results showed (Table
2) that PMA and chelerythrine did not
significantly affect RF uptake. Although bisindolylmaleimide I
inhibited the uptake, its inactive analog bisindolylmaleimide V also
caused inhibition to the same degree, indicating that the effect is
nonspecific in nature.
Involvement of a PKA-mediated pathway in the regulation of RF uptake
was tested by examining the effect of pretreating HK-2 cells for 1 h
with compounds that are known to increase intracellular cAMP levels
(dibutyryladenosine 3',5'-cyclic monophosphate,
3-isobutyl-1-methylxanthine, cholera toxin, and forskolin) and thus
activate PKA on RF (4.2 nM) uptake. We also examined the
effect of the specific PKA inhibitor H-89 on RF uptake. The results
showed that none of these compounds significantly affected RF
uptake (Table 3).
In another study, we tested for the involvement of PTK in the
regulation of RF uptake by HK-2 cells. This was done by examining the
effect of pretreating the HK-2 cells for 1 h with the PTK inhibitors
genistein and tyrphostin 25 on the uptake of 4.2 nM RF.
Genistin and tyrphostin 1, respectively, were used as negative controls
for these inhibitors. The results (Table 4)
showed that genistein and tyrphostin 25 did not have any effect on RF
uptake.
The role of
Ca2+/calmodulin-mediated pathways
in the regulation of RF uptake by HK-2 cells was tested by examining
the effect of pretreating (for 1 h) these cells with the calmodulin
inhibitors calmidazolium and trifluoperazine and with the inhibitor of
Ca2+/calmodulin-dependent protein
kinase II, KN-62, on the uptake of RF (4.2 nM). The
results showed that these compounds caused a significant
(P < 0.01) and
concentration-dependent inhibition in RF uptake (Table
5). We also examined the effect of
calmidazolium on the kinetic parameters of RF uptake by HK-2 cells.
This was done by examining the effect of calmidazolium (50 µM) on the
uptake of RF as a function of concentration. The results showed RF
uptake to be saturable both in the absence and presence of
calmidazolium; however, uptake in the presence of calmidazolium was
lower than that of control. Kinetic parameters were then calculated as
described in MATERIALS AND METHODS.
There was a decrease (20%) in the
Vmax of RF uptake
in calmidazolium-pretreated cells compared with control cells (8.03 ± 0.47 vs. 10.05 ± 0.87 fmol · mg
protein1 · 3 min1, respectively)
and an increase (76%) in the apparent
Km (1.18 ± 0.18 vs. 0.67 ± 0.21 µM, respectively).
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Table 5.
Effect of antagonists of Ca2+/ calmodulin-mediated
pathways on uptake of RF by confluent HK-2 monolayers
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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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Table 6.
Effect of growing confluent HK-2 cells in RF-deficient and
RF-oversupplemented growth medium on uptake of [3H]RF
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In another study, we examined the effect of RF levels in the growth
media on the kinetic parameters of the RF uptake process in HK-2 cells.
This was done by examining the initial rate of RF uptake as a function
of increasing the substrate concentration in the incubation buffer in
HK-2 monolayers grown in RF-sufficient, RF-oversupplemented, and
RF-deficient growth medium. Kinetic parameters of the saturable uptake
process were then determined as described in MATERIALS AND
METHODS. As shown in Table
7, there were changes in both apparent
Km and
Vmax of the
uptake process under conditions studied:
Vmax was found to
be higher (66%) in cells grown in RF-deficient medium than
Vmax in cells
grown in control (RF sufficient) medium, which in turn was higher
(74%) than in cells grown in RF-oversupplemented medium. The apparent
Km followed an
opposite trend and was higher (27%) in cells grown in
RF-oversupplemented medium compared with Km of those grown
in control medium, which was in turn higher (69%) than in cells grown
in RF-deficient medium.
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Table 7.
Effect of growing confluent HK-2 cells in RF-deficient and
RF-oversupplemented growth medium on kinetic parameters of
RF uptake by HK-2 cells
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In a separate experiment, we studied the effect of growing cells (for
48 h) in medium in which RF was replaced by different levels of LF
(i.e., 0, 1, and 50×) on the initial rate of
[3H]RF (4.2 nM)
uptake. Similar to the effect of growing cells in the presence of
different RF levels, uptake of
[3H]RF by cells grown
in "LF-deficient" medium was significantly (P < 0.01) higher than the uptake by
cells grown in "control" medium, which was in turn higher than
uptake by cells grown in "LF-oversupplemented" medium (91 ± 2.4, 82 ± 1.2, and 41 ± 1.8 fmol · mg
protein1 · 3 min1 for LF-deficient, LF
control, and LF-oversupplemented conditions, respectively).
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DISCUSSION |
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 Km and
Vmax 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 Vmax of the
RF uptake process and an increase in the apparent
Km 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 Km
and Vmax of the
RF uptake process.
Vmax of the RF
uptake process increased, whereas the apparent
Km 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.
| |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Department of Veterans
Affairs and the National Institute of Diabetes and Digestive and Kidney
Diseases (DK-47203).
| |
FOOTNOTES |
Address for reprint requests: H. M. Said, Univ. of California Irvine
and Long Beach Veterans Affairs Medical Program, Veterans Affairs
Medical Center (151), Long Beach, CA 90822.
Received 12 May 1997; accepted in final form 11 September 1997.
| |
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Abstract
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