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【关键词】 human
Veterans Affairs Medical Center, Long Beach
Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
ABSTRACT
Cellular and molecular regulation of renal biotin uptake in humans is not well defined. The contribution of the human Na+-dependent multivitamin transporter (hSMVT) to carrier-mediated biotin uptake by human proximal tubular epithelial cells is not clear. The aim of this study was to address these issues, with the human-derived proximal tubular epithelial HK-2 cells used as a model. First, we characterized the mechanism of biotin uptake by these cells and obtained evidence for involvement of an Na+-, temperature-, and energy-dependent carrier-mediated uptake system. This system was inhibited by the biotin structural analog desthiobiotin, pantothenic acid, and lipoate. These findings suggest involvement of the hSMVT system in the uptake process. This was confirmed by demonstrating that the hSMVT system is expressed in HK-2 cells at the protein and mRNA levels and by selective silencing of the hSMVT gene with the use of gene-specific small interfering RNAs, which led to specific and significant inhibition of carrier-mediated biotin uptake. Of the two recently cloned promoters of the hSMVT gene, promoter 1 was more active than promoter 2 in these cells. Pretreatment of HK-2 cells with modulators of PKC- and Ca2+/calmodulin-mediated pathways (but not those that modulate PKA-, protein tyrosine kinase-, or nitric oxide-mediated pathways) led to significant alterations in biotin uptake. Maintaining the HK-2 cells in a biotin-deficient growth medium led to a marked upregulation in biotin transport, which was associated with an increase in hSMVT protein and RNA levels and an increase in activity of the hSMVT promoters. These results demonstrate that biotin uptake by human renal epithelial cells occurs via the hSMVT system and that the process is regulated by intracellular PKC- and Ca2+/calmodulin-mediated pathways. The uptake process appears to be adaptively regulated by extracellular biotin level, which involves transcriptional regulatory mechanism(s).
transport regulation; renal biotin uptake; human sodium-dependent multivitamin transporter; human kidney epithelial cells
BIOTIN, A WATER-SOLUBLE MEMBER of the B-complex group of vitamins, is an essential micronutrient that is necessary for normal metabolism and growth in humans because of its involvement in a variety of metabolic reactions. The importance of biotin in normal human health is underscored by the serious clinical abnormalities that result from its deficiency, which include neurological and dermal disorders, growth retardation, fatigue, and depression (3, 24). Biotin deficiency occurs in a variety of conditions, including inborn errors of biotin metabolism, chronic use of anticonvulsant drugs, and prolonged use of parenteral nutrition (8, 12, 13, 24, 29, 30). Mammals have lost the ability to synthesize biotin endogenously and, therefore, must obtain the vitamin from exogenous sources via absorption in the gut. After absorption, biotin is circulated in the blood and is filtered in the renal glomeruli, where it is salvaged via reabsorption by renal proximal tubular epithelial cells. Thus intestinal and renal epithelial cells play critical roles in maintaining and regulating normal body biotin homeostasis. Extensive studies have focused on determining the mechanism of intestinal biotin absorption and its regulation (for review see Ref. 16). Much less, however, is known regarding the renal biotin reabsorption process, especially in humans. Previous studies have shown the involvement of an efficient Na+-dependent carrier-mediated mechanism for biotin uptake in the human kidneys (2). However, the contribution of the human Na+-dependent multivitamin transporter (hSMVT), a biotin transporter that is expressed in human kidneys (26), to overall carrier-mediated biotin uptake is not clear. Addressing this issue is important in light of recent reports of the possible existence of another low-affinity biotin uptake system in certain human cellular systems (keratinocytes and peripheral blood mononuclear cells) (7, 31). In addition, nothing is known about regulation of the renal biotin uptake process by intracellular and extracellular factors/conditions. To fill this gap in our knowledge of the human renal biotin reabsorption process, in the present study, we used human-derived proximal tubular epithelial HK-2 cells as a model (9, 15, 27). HK-2 cells are well-differentiated native renal epithelial cells and are well suited to study the mechanism and regulation of nutrient uptake processes (9, 15). The results showed that human renal biotin uptake occurs via the hSMVT system and that this system is under the regulation of intracellular PKC- and Ca2+/calmodulin (CaM)-mediated pathways. In addition, adaptive regulation was found to occur in human renal proximal tubular epithelial cell uptake of biotin in response to extracellular substrate levels via transcriptional regulatory mechanism(s).
MATERIALS AND METHODS
[3H]biotin (specific activity 58.2 Ci/mmol, radiochemical purity 97%) was obtained from DuPont NEN (Boston, MA). Cell culture medium and ingredients, as well as all other chemicals and reagents used in this study (which were of analytic/molecular biology grades), were obtained from Fisher Scientific (Tustin, CA) and Sigma (St. Louis, MO).
The human-derived renal proximal tubular epithelial HK-2 cells (American Type Culture Collection, Manassas, VA) were grown as monolayers, as described by us previously (9). Uptake studies were performed on cell monolayers 34 days after confluence. The physiological buffer used in these studies was Krebs-Ringer (KR) buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4). Unless otherwise stated, the cells were incubated at 37°C for 7 min (initial rate; see RESULTS). Labeled and unlabeled biotin or other compounds were added to the incubation medium at the beginning of the uptake experiment, and the reaction was terminated by the addition of 2 ml of ice-cold KR buffer followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer, digested with 1 ml of 1 N NaOH, neutralized with HCl, and then counted for radioactivity. A kit from Bio-Rad (Richmond, VA) was used to measure the protein content of cell digests from the experimental and control wells.
The metabolic form of the transported substrate after incubation of HK-2 cells with [3H]biotin (30 nM) for 7 min was tested by application of cell lysate to cellulose-precoated thin-layer chromatography plates. The plates were then run using a solvent system of butanol-acetic acid-water (4:1:1 vol/vol/vol). For the studies dealing with potential regulation of biotin uptake by intracellular regulatory pathways, HK-2 cells were pretreated for 1 h with modulators of these pathways and then examined for [3H]biotin uptake. For the studies related to potential regulation of biotin uptake by the extracellular level of biotin, HK-2 cells were maintained for 7296 h in biotin-deficient medium, and then [3H]biotin uptake was examined.
Pretreatment of HK-2 Cells With Small Interfering RNAs
Three custom-made hSMVT gene-specific small interfering RNAs (siRNAs), 21-nt double-stranded RNAs [5'-aagcgtgggcatgtctaccttdTdT-3' (siRNA-A1), and 5'-aaggccgtcatctggacagatdTdT-3' (siRNA-A2), and 5'-aaggctgctgtgctctcctgtdTdT-3' (siRNA-A3); GenBank accession no. AF081571], were chemically synthesized by a commercial vendor (Qiagen-Xeragon, Germantown, MD) and used as a pool for the present study. HK-2 cells at 5060% confluence were transiently transfected with 12 μg of siRNAs per well with use of oligofectamine reagents according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Control cells were transfected with oligofectamine alone or scrambled siRNAs (5'-aacctcactaaggttaacttcdTdT-3'). Cells were maintained until 34 days after confluence and then used in the specific experiments. Uptake studies with these cells were performed as described above.
Semiquantitative and Real-Time PCR Analysis
Total RNA was isolated from control and siRNA-pretreated HK-2 cells using TRIzol reagent according to the manufacturer's instructions (Life Technologies, Rockville, MD). Five micrograms of the total RNA were reverse transcribed with oligo(dT) primers using Superscript II (Life Technologies) enzyme. After reverse transcription, four different dilutions were made and used for PCR assays. The hSMVT primers and the PCR conditions are as follows: 5'-CGATTCAATAAAACTGTGCGAGT-3' (forward primer) and 5'-GGACAGCCA CAGATCAAAGC-3' (reverse primer) and 1 cycle at 95°C for 10 min and 2535 cycles at 95°C for 30 s, 57°C for 15 s, and 72°C for 30 s. The expression pattern of the housekeeping gene -actin was used as a control. For -actin, the primers and the PCR conditions were as follows: 5'-CATCCTGCGTCTGGACCT-3' (forward primer) and 5'-TAATGTCA CGCACGATTTCC-3' (reverse primer) and the conditions described above. We also measured the mRNA level of the unrelated human thiamine transporter-1 (hTHTR-1) in control and hSMVT siRNA-pretreated cells to confirm the specificity of the siRNAs used in the study. For semiquantitative PCR, the final products were analyzed on 3% agarose gels, data were normalized relative to human -actin, and the real-time PCR data were analyzed according to the manufacturer's instructions (Bio-Rad).
Western Blot Analysis
Membranous fractions of control and siRNA-pretreated HK-2 cells (150 μg/lane) were isolated (23) and resolved on a 10% SDS-PAGE and then electroblotted on a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then blocked with 5% dried milk in phosphate-buffered saline (pH 7.4) containing 0.1% Tween 20 and incubated for 2 h at 37°C or overnight at 4°C with specific rabbit polyclonal antipeptide antibodies raised against the LYHACRGWGRH TVGELLMADRK peptide of the human (and rat) SMVT sequence (Alpha Diagnostic, San Antonio, TX). The specificity of these polyclonal antibodies has been demonstrated in our laboratory (14). The hSMVT-immunoreactive bands were detected using a chemiluminescence kit (Amersham Pharmacia Biotech) and autoradiography (Kodak film). The hSMVT-specific bands were quantified using the Eagle Eye II System (Stratagene, La Jolla, CA).
Analysis of hSMVT Promoter Activity in HK-2 Cells
Activity of the hSMVT promoters P1 and P2, which have been recently cloned and characterized in our laboratory (4), was examined in HK-2 cells. Cells were seeded in a 12-well plate and grown in the presence of serum and antibiotics for 24 h. At 70% confluence, cells were transiently transfected with the hSMVT promoter constructs P1 and P2 cloned in pGL3-basic vector as previously described (4) in triplicates. After 4872 h of transfections, the cells were harvested and lysed in passive lysis buffer according to the instructions of the manufacturer (Promega, Madison, WI). Firefly luciferase (expressed by the hSMVT promoter-luciferase constructs) and Renilla luciferase were assayed using the dual-luciferase reporter assay (Promega) and a luminometer (model TD-20/20, Turner Designs, Sunnyvale, CA). The firefly luciferase activities were then divided by the Renilla luciferase activities to normalize for differences in transfection efficiencies, and data were analyzed as described by the manufacturer. The data are reported as expression relative to pGL3-basic, which was set arbitrarily at 1.
In the study on the effect of maintaining the HK-2 cells under biotin-deficient condition on activity of the hSMVT promoters, the cells were first synchronized for 24 h in regular growth medium and then released, first, by incubation in biotin-free growth medium containing 5% serum (which was pretreated with avidin to remove any biotin) or control growth medium for 7296 h. Transfections and measurement of promoter activity were then performed as described elsewhere (4).
Data Presentation and Statistical Analysis
Transport data are presented as means ± SE of multiple separate uptake determinations and expressed in terms of picomoles or femtomoles per milligram of protein per 7 min (unless otherwise stated). Kinetic parameters of the saturable biotin uptake process determined by subtracting the diffusing component (determined from the slope of the uptake line between a high pharmacological concentration of biotin of 1 mM and the point of origin, i.e., multiplication of the slope by individual concentration) were calculated by using a computerized model of the Michaelis-Menten equation as described previously by Wilkinson (28). Statistical analysis was performed using Student's t-test or one-way analysis of variance followed by Tukey's honestly significant difference test, with statistical significance set at 0.05 (P < 0.05). All transient transfection studies, semiquantitative and real-time PCR, Western blot analysis, and promoter analyses were performed on at least three separate occasions with comparable results. Data from a representative set of experiments are presented.
RESULTS
Mechanism of Biotin Uptake by Human-Derived Proximal Tubular Epithelial HK-2 Cells
Time course, effect of incubation temperature, and possible metabolism. In these experiments, we determined the linearity of uptake of biotin as a function of incubation time. Biotin uptake by HK-2 cells was proportional to incubation time in a linear manner up to 10 min of incubation: 18 and 1,010 nmol?mg protein1?min1 for 0.1 and 10 μM biotin, respectively (Fig. 1). Thus we chose a 7-min period as the standard incubation time in our investigations. The initial rate of uptake of 9.6 nM biotin was temperature dependent and decreased as a function of decreasing incubation temperature: 21.20 ± 1.06, 10.15 ± 1.48, and 4.82 ± 0.44 fmol?mg protein1?7 min1 for 37, 21, and 4°C, respectively. The metabolic form of the substrate transported into HK-2 cells after 7 min of incubation with 30 nM [3H]biotin was also tested using cellulose-precoated thin-layer chromatography plates and a solvent system of butanol-acetic acid-water (4:1:1) and found to be mostly (91%) in the form of intact biotin.
Effect of incubation buffer pH, role of Na+, and effect of metabolic and transport inhibitors on biotin uptake. The effect of incubation buffer pH on biotin uptake by HK-2 cells was examined to determine whether the process is influenced by pH (i.e., H+ concentration of the incubation medium). The results showed the initial rate of biotin (9.6 nM) uptake to decrease as a function of decreasing (or increasing) buffer pH from 7.4 (Fig. 2). Hence, we used buffer pH 7.4 in all experiments. The effect of Na+ in the incubation medium on the initial rate of biotin (9.6 nM) uptake was also examined by replacing the cation with an equimolar concentration of K+, Li+, NH4, Tris, choline, or mannitol and then examining substrate uptake. The results showed that replacing Na+ with any of these cations or with mannitol led to a significant inhibition of biotin uptake (Fig. 3). In related studies, we examined the effect of pretreating HK-2 cells (for 30 min) with the monovalent cation ionophore gramicidin (100 μM) or with the inhibitor of Na+-K+-ATPase ouabain (1 mM) on initial uptake of biotin. Significant inhibition (P < 0.01) of biotin uptake was caused by both treatments: 23.97 ± 1.88, 4.73 ± 0.81, and 9.35 ± 0.48 fmol?mg protein1?7 min1 for control and after pretreatment with gramicidin and ouabain, respectively.
In other experiments, we examined the effect of pretreating (for 30 min) the HK-2 cells with the metabolic inhibitors 2,4-dinitrophenol, azide, p-chloromercuriphenyl sulfonate, and iodoacetate (all at 1 mM) on the initial rate of biotin (9.6 nM) uptake. Our aim was to determine whether biotin uptake is energy dependent. The results showed significant (P < 0.01) inhibition of biotin uptake by all inhibitors tested: 25.36 ± 1.33, 12.57 ± 0.21, 18.08 ± 0.68, 4.74 ± 0.66, and 18.38 ± 0.39 fmol?mg protein1?7 min1 for control and cells pretreated with 2,4-dinitrophenol, azide, p-chloromercuriphenyl sulfonate, and iodoacetate, respectively. We also examined the effect of addition of the membrane transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, furosemide, and probenecid (all at 0.5 mM) to the incubation medium on the initial rate of uptake of biotin (9.6 nM), with the results showing significant (P < 0.01) inhibition by probenecid, but not by the other two compounds: 21.29 ± 3.60, 23.33 ± 2.74, 24.62 ± 4.13, and 9.76 ± 0.37 fmol?mg protein1?7 min1 for control and in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, furosemide, and probenecid, respectively.
Evidence for involvement of a carrier-mediated mechanism in biotin uptake by HK-2 cells. In these studies, we examined the initial rate of biotin uptake as a function of concentration over nanomolar (150 nM) and micromolar (150 μM) concentration ranges. The results showed biotin uptake to be linear as a function of concentration in the nanomolar range (Fig. 4A) but to contain a saturable component over the micromolar range (Fig. 4B). Kinetic parameters of the saturable component were determined (see MATERIALS AND METHODS) and found to be 12.16 ± 2.10 μM (Km) and 17.4 ± 3.1 pmol?mg protein1?7 min1 (Vmax). These results indicate the involvement of a carrier-mediated system for the biotin uptake system that saturates at the micromolar range.
In other studies, we examined the effect of nanomolar and micromolar concentrations of the biotin structural analogs on the initial rate of carrier-mediated [3H]biotin (2.6 nM) uptake. Our aim was to confirm the involvement of a carrier-mediated system for biotin uptake that saturates at the micromolar range. The results showed that 50 and 100 nM unlabeled biotin, desthiobiotin, and pantothenic acid had no effect on the initial rate of uptake of [3H]biotin (Fig. 5A). On the other hand, adding these compounds at 100 μM caused significant inhibition of carrier-mediated uptake of [3H]biotin (Fig. 5B). The biotin structural analogs biocytin and biotin methyl ester (100 μM) did not show any effect on the uptake of 2.6 nM [3H]biotin (Fig. 5B).
Molecular aspects of the biotin uptake process by HK-2 cells.
EXPRESSION OF HUMAN SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER IN HK-2 CELLS AT RNA AND PROTEIN LEVELS. To determine the expression of the hSMVT system at the RNA and protein levels in HK-2 cells, we isolated total RNA and protein from the confluent monolayers of these cells (see MATERIALS AND METHODS). Gene-specific primers corresponding to a sequence in the open reading frame of hSMVT were used to perform RT-PCR, and a fragment of the expected size was obtained (Fig. 6A). By Western blot analysis using specific anti-hSMVT polyclonal antibodies, expression of the hSMVT protein in membranous fractions of HK-2 cells was established (Fig. 6B).
DETERMINATION OF THE FUNCTIONAL CONTRIBUTION OF HUMAN SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER IN HK-2 CELLS. To determine the functional contribution of the hSMVT system to total carrier-mediated biotin uptake by the human renal proximal tubular epithelial HK-2 cells, we used gene-specific siRNAs to selectively silence the endogenous hSMVT in these cells and then examined carrier-mediated biotin uptake. First, we verified the ability of siRNA treatment to silence the hSMVT gene by performing semiquantitative RT-PCR. The results showed a substantial reduction in the level of endogenous hSMVT mRNA in siRNA-pretreated HK-2 cells compared with control and scrambled siRNA-pretreated cells (Fig. 7A). In contrast, mRNA levels of human -actin (Fig. 7A) and hTHTR-1 (data not shown) were not affected by the treatment. The level of the hSMVT protein was also substantially reduced in the siRNA-pretreated HK-2 cells compared with control cells (Fig. 7B), with no change in the protein level of the unrelated hTHTR-1 (data not shown). These findings demonstrate the effectiveness and selectivity of the siRNA approach in silencing the hSMVT gene in these cells. We then examined the initial rate of carrier-mediated biotin (9.6 nM) uptake in siRNA-pretreated and control HK-2 cells. The results showed a severe (>75%, P < 0.01) inhibition of biotin uptake in the hSMVT siRNA-pretreated cells compared with control cells (Fig. 7C).
IDENTIFICATION OF THE MORE ACTIVE HUMAN SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER PROMOTER IN HK-2 CELLS. We also determined which of the two recently cloned hSMVT promoters (4) is more active in the HK-2 cells. In this study, the full-length P1 (5846 to 5313, relative to translation initiation codon of hSMVT) and P2 (4417 to 4244) constructs were fused to the firefly luciferase reporter gene and then transfected into the HK-2 cells (see MATERIALS AND METHODS). The results showed that activity of the hSMVT P1 was significantly (P < 0.01) higher than that of P2 in these cells (Fig. 8).
Regulation of the Biotin Uptake Process in HK-2 Cells
Role of intracellular regulatory pathways. In these studies, we explored the possible regulation of the biotin uptake process of HK-2 cells by specific intracellular regulatory pathways. We focused on the intracellular regulatory pathways mediated by protein kinase C (PKC), Ca2+/CaM, protein tyrosine kinase (PTK), and nitric oxide (NO), because previous studies documented a role for these pathways in the regulation of transport of other substrates (including some other water-soluble vitamins) in epithelial cells (5, 6, 10, 11, 1720).
The possible role of a PKC-mediated pathway in the regulation of biotin uptake by HK-2 cells was examined by testing the effect of pretreatment of the cells for 1 h with modulators of this pathway on the initial rate of uptake of biotin (9.6 nM). The results showed that pretreatment of cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 μM (but not with its inactive derivative 4-PMA) led to a significant (P < 0.01) stimulation in carrier-mediated biotin uptake, whereas pretreatment with the PKC inhibitors staurosporine and chelerythrine led to significant (P < 0.01) inhibition of biotin uptake (Table 1). The effect of PMA on the kinetic parameters of the biotin uptake process by HK-2 cells was also examined. The results showed biotin uptake to be saturable in PMA-treated and control cells, with the uptake being higher in the former than in the latter (Fig. 9A). The kinetic parameters of the saturable process were as follows: Km = 10.38 ± 0.51 and 17.46 ± 0.28 μM and Vmax = 16.28 ± 1.96 and 28.27 ± 2.57 pmol?mg protein1?7 min1 for control and PMA-pretreated HK-2 cells, respectively.
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To assess the potential involvement of Ca2+/CaM-mediated pathways in biotin uptake by HK-2 cells, we examined the effect of pretreatment for 1 h with inhibitors of this pathway, trifluoperazine and calmidazolium, on the initial rate of biotin uptake. Both compounds caused significant (P < 0.01) inhibition of biotin uptake (Table 2). The effect of calmidazolium (50 μM) on kinetic parameters of carrier-mediated biotin uptake was also examined. The results (Fig. 9B) showed a decrease in Vmax of biotin uptake in calmidazolium-pretreated cells compared with control cells (10.67 ± 0.77 and 14.28 ± 1.46 pmol?mg protein1?7 min1, respectively), with no significant change in its apparent Km with such treatment (10.32 ± 0.60 and 11.02 ± 2.1 μM, respectively).
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The possible role of the PKA-mediated pathway was also investigated by examining the effect of pretreatment of HK-2 cells for 1 h with dibutyryl cAMP (1 mM) and 3-isobutyl-1-methylxanthine (0.5 mM) on the initial rate of biotin (9.6 nM) uptake. The results showed no significant effect on biotin uptake by either of these compounds: 21.98 ± 0.07, 21.86 ± 0.43, and 24.76 ± 2.16 fmol?mg protein1?7 min1 for control and dibutyryl cAMP- and 3-isobutyl-1-methylxanthine-pretreated cells, respectively. A role for a PTK-mediated pathway in the regulation of biotin uptake was also studied by examining the effect of pretreatment of the HK-2 cells for 1 h at 37°C with the PTK inhibitors genistein and tyrphostin A-25 on the initial rate of biotin (9.6 nM) uptake. Neither compound affected biotin uptake by these cells: 21.36 ± 3.08, 23.05 ± 1.48, and 19.30 ± 1.23 fmol?mg protein1?7 min1 for control and genistein- and tyrphostin A-25-pretreated cells, respectively. Similarly, the potential role of an NO-mediated pathway in the regulation of biotin uptake by HK-2 cells was examined by testing the effect on biotin (9.6 nM) uptake of pretreatment for 1 h with modulators of the NO-mediated pathway, 3-morpholinosydnonimine and S-nitroso-N-acetylpenicillamine, both at 1 mM. The results showed that neither of these compounds significantly affect biotin uptake: 23.07 ± 0.49, 22.15 ± 0.52, and 22.15 ± 0.89 fmol?mg protein1?7 min1 for control and S-nitroso-N-acetylpenicillamine- and 3-morpholinosydnonimine-pretreated cells, respectively.
Effect of extracellular biotin level. In this study, we examined the effect of maintaining (for 96 h) the HK-2 cells under biotin-deficient conditions on functionality and expression of hSMVT (see MATERIALS AND METHODS). Biotin uptake was significantly (P < 0.01) higher in cells maintained in biotin-deficient growth medium than in those maintained in control growth medium: 63.69 ± 3.04 vs. 21.23 ± 3.28 fmol?mg protein1?7 min1. We also examined the effect of the biotin-deficient condition on the level of expression of hSMVT mRNA and protein by real-time PCR and Western blot analysis, respectively. The real-time PCR showed a significant (P < 0.01, 2.5 fold) increase in the level of hSMVT mRNA in HK-2 cells maintained under biotin-deficient conditions compared with those maintained in control growth medium (Fig. 10A). On the other hand, no significant change was observed in the steady-state mRNA level of the unrelated vitamin transporter hTHTR-1 in cells maintained in biotin-deficient conditions compared with control (Fig. 10A). Similarly, results of the Western blot analysis showed a significantly (P < 0.01, 2.8-fold) higher level of expression of hSMVT protein in cells maintained under biotin-deficient conditions than in those maintained in control growth medium (Fig. 10B). We also examined the effect of maintaining HK-2 cells in biotin-deficient growth medium on activity of the hSMVT promoters P1 and P2. The results (Fig. 10C) showed a significantly (P < 0.01) higher activity for P1 and P2 in cells maintained in biotin-deficient medium than in cells maintained in control growth medium.
DISCUSSION
The aim of the present study was to delineate the mechanisms involved in regulation of the biotin uptake process in human renal epithelial cells, with HK-2 cells used as the model system. In addition, the functional contribution of the hSMVT system to the overall carrier-mediated renal biotin uptake process was investigated. We chose HK-2 cells, because they represent a good model for human proximal tubular epithelia and have been used in similar physiological investigations, with data that are relevant to those found in the native kidney (9, 15, 25, 27). First, we characterized the mechanism involved in the uptake of biotin by the HK-2 cells. The results showed that uptake occurred without metabolic alterations in the transported biotin and that the uptake process is temperature and energy dependent. The latter conclusions were based on the observations of a significant inhibition of the initial rate of biotin uptake as a result of lowering the incubation buffer temperature and after pretreatment of cells with metabolic inhibitors. The biotin uptake process of HK-2 cells was also highly dependent on the presence of Na+ in the incubation medium, in as much as replacement of this cation with other monovalent cations or with mannitol led to a significant inhibition in the initial rate of biotin uptake. This conclusion was further confirmed by the observed inhibition of biotin uptake after treatment of the HK-2 cells with the monovalent cation ionophore gramicidin and with the Na+-K+-ATPase inhibitor ouabain. The inhibition of biotin uptake by gramicidin suggests that it is not the presence of Na+ in the incubation medium that is sufficient for the function of the renal biotin uptake system; rather, it is the existence of an inwardly directed Na+ gradient that is important for the function. Similar findings have been reported for the role of Na+ in biotin uptake in brush border membrane vesicles isolated from native human kidneys as well as nonrenal tissues, such as the small intestine, liver, and Xenopus oocytes (2, 18, 21, 22).
The biotin uptake process of the HK-2 cells appeared to be mediated via a carrier-mediated system that operates in the micromolar range, with no evidence for the existence of a second system that operates in the nanomolar range. These conclusions are based on the observations (Fig. 4) that biotin uptake as a function of concentration is linear over the nanomolar range but is saturable over the micromolar range (apparent Km = 12.16 ± 2.10 μM). The ability of micromolar, but not nanomolar, concentrations of unlabeled biotin and its structural analogs, together with pantothenic acid, to inhibit the renal uptake of a physiological concentration of biotin (Fig. 5) further supports this conclusion. In addition, the ability of pantothenic acid and lipoate to inhibit the initial rate of renal biotin uptake suggests that the saturable uptake process is mediated via the hSMVT system. Indeed, the hSMVT system was expressed at the protein and RNA levels in HK-2 cells, as indicated by the results of the Western blot analysis and PCR, respectively. The role of the hSMVT system in biotin uptake by human HK-2 renal epithelial cells was further established by the study utilizing the gene-specific siRNA approach to silence the hSMVT gene. Such treatment led to a significant inhibition in RNA and protein levels of the hSMVT as well as in carrier-mediated uptake of a physiological concentration of biotin. These effects were specific, in as much as no changes in mRNA levels of the hTHTR-1 or in thiamin uptake were observed in siRNA-pretreated compared with control HK-2 cells. Thus the human proximal tubular epithelial HK-2 cells appear to utilize the hSMVT system as the main (if not the only) uptake system for transporting the vitamin and, therefore, join the human intestine and liver cells in this regard (1). Obviously, it is possible that the high-affinity biotin uptake system could have been lost during cell culturing; thus further studies with native human renal epithelial cells are required. Of the two recently cloned hSMVT promoters, P1 was significantly more active than P2 in the human HK-2 cells. This suggests that P1 may be more involved in transcriptional regulation of hSMVT expression than P2 under basal and regulated conditions. Further studies, however, are needed to address these issues.
After we established the mechanism of biotin uptake in the human proximal tubular HK-2 cells and demonstrated the involvement of hSMVT in the biotin uptake system, we examined the potential regulation of the renal biotin uptake process by intracellular and extracellular factors/conditions. The results showed that although the intracellular PKA-, PTK-, and NO-mediated regulatory pathways had no role in regulating the biotin uptake process in HK-2 cells, intracellular PKC- and Ca2+/CaM-mediated pathways appeared to have a role. Modulators of PKC activity altered biotin uptake by HK-2 cells, with a significant increase after pretreatment with the PKC activator PMA and a significant inhibition in vitamin uptake after pretreatment with the PKC inhibitors staurosporine and chelerythrine. The stimulatory effect of PMA was mediated via an increase in Vmax and apparent Km of the biotin uptake process, suggesting that the effect occurs via changes in the activity (and/or number) and affinity of the biotin uptake carriers. The apparent role of the PKC-mediated pathway in regulating renal biotin uptake appears to be different from its role in regulating biotin uptake by human intestinal epithelial cells (17). In the latter system, activation of the PKC-mediated pathway led to inhibition of biotin uptake. This difference adds to the already described differences in the way the intestinal and renal biotin uptake processes are regulated during ontogeny, where the former process appears to undergo ontogenic regulation, whereas the latter process does not respond to such regulation (14). The mechanisms through which the PKC-mediated regulatory pathway exerts its effect on the human renal biotin uptake process are not clear, but the hSMVT sequence appears to have two potential sites for PKC phosphorylation that could play a role in mediating the observed effect. Further studies are required to address this issue. Another intracellular regulatory pathway, the Ca2+/CaM-mediated pathway, also appeared to play a role in the regulation of biotin uptake by HK-2 cells. Inhibitors of this pathway, namely, calmidazolium and trifluoperazine, significantly inhibited the initial rate of biotin uptake by HK-2 cells. The effect of calmidazolium was mediated via changes in Vmax, suggesting that the effect is mediated via alterations in activity/numbers of the biotin uptake process. The mechanisms through which the Ca2+/CaM-mediated pathway exerts its effect on renal biotin uptake are not known, and further investigations are needed.
The biotin uptake process of the HK-2 cells also appeared to be under the regulation of extracellular substrate level. Maintaining the HK-2 cells in a biotin-deficient growth medium led to a specific and significant upregulation in the initial rate of biotin uptake. This increase in uptake was associated with an increase in hSMVT mRNA and protein levels. These findings suggest that a transcriptional mechanism(s) may be involved in the adaptive regulation of the human renal proximal tubular epithelial cell biotin uptake process in biotin deficiency. To directly test this possibility, we examined the effect of maintaining the HK-2 cells in biotin-deficient growth medium on activity of the hSMVT promoters P1 and P2. The activity of P1 and P2 was significantly higher in cells grown under biotin-deficient conditions than in those grown in control conditions (Fig. 10C). Further studies are needed to determine the exact molecular mechanism involved in this regulation.
In summary, results of these investigations indicate that biotin uptake by the human-derived renal proximal tubular epithelial HK-2 cells is via the hSMVT system. In addition, the results show that the uptake process is under the regulation of intracellular PKC- and Ca2+/CaM-mediated pathways and is also adaptively regulated by extracellular substrate levels. The latter mode of regulation appears to be mediated, at least in part, via transcriptional regulatory mechanism(s).
GRANTS
This study was supported by grants from the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-58057 and DK-56061.
ACKNOWLEDGMENTS
We thank Alvaro Ortiz for excellent technical help.
FOOTNOTES
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.
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