Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms

【摘要】  Thiamin (vitamin B 1 ) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [ 3 H]thiamin uptake to be 1 ) temperature and energy dependent but Na + independent, 2 ) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3 ) saturable as a function of concentration over the nanomolar (apparent K m = 70.0 ± 18.4 nM) and micromolar (apparent K m = 2.66 ± 0.18 µM) ranges, 4 ) cis -inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5 ) trans -stimulated by unlabeled thiamin, and 6 ) competitively inhibited by amiloride with an apparent K i of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant ( P < 0.01) and specific upregulation in [ 3 H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca 2+ /calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca 2+ /CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

【关键词】  renal thiamin uptake transport mechanism transport regulation human thiamin transporter and human thiamin transporter

THIAMIN (vitamin B 1 ), a water-soluble micronutrient, is indispensable for life as its derivative thiamin pyrophosphate (TPP) is necessary for oxidative phosphorylation and the pentose phosphate pathway. TPP is a cofactor for transketolase and dehydrogenases of pyruvate, -ketoglutarate, and branched-chain -ketoacids ( 22 ). Thiamin is a quaternary amine consisting of a substituted pyrimidine nucleus linked to a thiazole ring that exists as a cation at physiological pH ( 11 ). Thiamin deficiency leads to poly-neuropathy ( 35 ), cardiomyopathy ( 31 ), and metabolic acidosis ( 23 ), while in severe conditions it leads to tissue damage and increased apoptotic cell death ( 14 ). Beriberi and the Wernicke-Korsakoff syndrome are well-known thiamin deficiency diseases ( 32 ). Thiamin deficiency occurs in a variety of conditions including chronic alcoholism, diabetes mellitus, and intestinal and renal diseases ( 13, 17, 30 ). In contrast to the negative consequences of thiamin deficiency, optimization of the body thiamin homeostasis may have positive consequences on health as it may prevent or delay the development of diabetic retinopathy ( 10 ). Thus studies that are aimed at furthering our understanding of the mechanisms and regulation of renal and intestinal thiamin uptake process are of significant physiological and nutritional importance. Such studies may assist in the development of effective strategies to optimize body thiamin homeostasis, especially in conditions of thiamin deficiency and suboptimal levels.

Humans and other mammals cannot synthesize thiamin de novo and must therefore obtain it from exogenous sources through intestinal absorption. Circulating thiamin is filtered in the renal glomeruli where it is subsequently salvaged via reabsorption by proximal renal tubular epithelial cells to prevent its loss in the urine. Thus the intestine and the kidneys play important roles in maintaining and regulating body thiamin homeostasis, hence understanding the mechanisms and regulation of the thiamin uptake processes by these tissues is of significant physiological and nutritional importance. With regards to thiamin uptake in the human intestine, studies from our laboratory ( 3, 15, 24, 26, 27 ) and others ( 12, 20 ) have characterized many of the cellular and molecular aspects of the uptake process. Results of these studies have shown, among other things, the involvement of both of the recently cloned human thiamin transporter-1 and -2 (hTHTR-1 and hTHTR-2; the products of the SLC19A2 and SLC19A3 genes, respectively) in the intestinal uptake process ( 24 ). With regards to the renal tubular thiamin reabsorption process, animal studies have examined the mechanism of thiamin uptake ( 9 ), but no study exists describing the mechanism of thiamin transport in human renal epithelial cells. In addition, nothing is known about regulation of the renal thiamin uptake process in any species or the potential contribution of the different transport system(s) toward total uptake of the vitamin. Our aims in this investigation were, therefore, to study the mechanism(s) and regulation of human renal thiamin uptake using the human-derived renal epithelial HEK-293 cells as an in vitro model system. We selected these well-differentiated human renal epithelial cells because previous studies have demonstrated their suitability in such type of physiological investigations and similar to findings with intact renal proximal tubular cells ( 7, 34 ). Our results showed thiamin uptake by these cells to be via a specialized and pH-dependent carrier-mediated process, which involves the activity of both the hTHTR-1 and hTHTR-2. Furthermore, thiamin uptake by these cells appears to be under the regulation of an intracellular Ca 2+ /CaM-mediated pathway, and it is also adaptively upregulated in thiamin deficiency through transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

MATERIALS AND METHODS

Radiolabeled [ 3 H]thiamin (specific activity 555 GBq/mmol; radiochemical 98%) was purchased from American Radiolabeled Chemicals (St. Louis, MO). TRIzol reagent and Lipofectamine were purchased from Life Technologies (Rockville, MD). DNA oligonucleotide primers were ordered from Sigma Genosys (The Woodlands, TX). Routine biochemicals, enzymes, dialyzed fetal bovine serum, and cell culture reagents were all of molecular biology quality and were purchased from either Fisher Scientific (Tustin, CA) or Sigma (St. Louis, MO).

Cell Growth and Transport Studies

The human-derived renal epithelial HEK-293 cells, obtained from ATCC (Manassas, VA), were grown and subcultured in DMEM supplemented with 2.5% fetal bovine serum (FBS), glutamine (0.29 g/l), sodium bicarbonate (2.2 g/l), penicillin (100,000 U/l), and streptomycin (10 mg/l) in an atmosphere of 5% CO 2 -95% air at 37°C. Thiamin uptake was performed using confluent monolayers (3-4 days after confluence) of HEK-293 cells, between passages 10 and 25. The physiological buffer used in these studies was Krebs-Ringer (K-R) buffer which contained (in mM) 133 NaCl, 4.93 KCl, 1.23 MgSO 4, 0.85 CaCl 2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4. Unless otherwise stated, all incubations were performed at 37°C for 7 min (initial rate; see RESULTS ). Labeled and unlabeled thiamin or other compounds were added to the incubation medium at the onset of uptake experiment and the reaction was terminated by the addition of 2 ml of ice-cold K-R buffer followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer, lysed with 1 ml of 1 N NaOH, neutralized with HCl, and then counted for radioactivity. The protein content of cell digests was measured from the experimental and control wells using a Bio-Rad kit (Bio-Rad, Richmond, VA).

In the studies to examine the effect of thiamin-deficient condition on [ 3 H]thiamin uptake by HEK-293 cells, the cells were maintained in custom-made thiamin-deficient DMEM medium containing 2.5% FBS (Hyclone). Control growth medium was the regular DMEM (i.e., containing 12 µM thiamin) to which 2.5% of FBS was added. Cells were maintained in the thiamin-deficient and the control DMEM medium for 96 h before their use in the uptake studies. For the studies of potential regulation of thiamin uptake by intracellular regulatory pathways, the HEK-293 cells were pretreated for 1 h with the modulators of the specific pathways followed by examination of [ 3 H]thiamin uptake.

In examining the metabolic form of 3 H radioactivity taken up by HEK-293 cells after incubation for 10 min with [ 3 H]thiamin (40 nM), the cells were homogenized in 100% ethanol and then centrifuged. The supernatant was then applied to a silica gel-precoated thin-layer chromatography (TLC) plate and run with a solvent system of isopropanol:acetate buffer (0.5 M, pH 4.5):water (65:15:20 vol/vol/vol) ( 26 ).

Pretreatment of HEK-293 Cells With Gene-Specific Small-Interference RNA

Custom-made gene-specific small-interference RNAs (siRNAs) against three targeted sequences of SLC19A2 (5'-CCTGACCGAGAGGGAGGTC-3', 5'-GTTACTGTCGAAGTGCCAC-3', and 5'-TGCTGGTTCTTGCCGAGGA-3') and three target sequences of SLC19A3 (5'-CAAATGAGATCTTCCCCGT-3', 5'-CTTCACTAAGCAGTTCCTG-3', and 5'-GGAGTGAAGACCATGCAGG-3') were chemically synthesized by a commercial vendor (Qiagen-Xeragon, Germantown, MD). Both strands of the siRNAs were modified by the addition of dTdT overhang at their 3' ends to increase their stability. HEK-293 cells at 50-60% confluency were transiently transfected with 1-2 µg siRNAs/well using the Lipofectamine reagents as per the manufacturer's instructions (Invitrogen, Carlsbad, CA). Control cells were transfected with lipofectamine alone as previously described ( 24 ). Cells were maintained until 3 to 4 days following confluence and then used in the different experiments.

Semiquantitative and Real-Time PCR Analysis

Total RNA was isolated from confluent HEK-293 cells using TRIzol (Life Technologies). Three micrograms of total RNA were reverse transcribed with oligo(dT) primers using Superscript II (Life Technologies) following the manufacturer's procedures. After the reverse transcription, all samples were diluted with sterile water and three different dilutions were used for each real-time PCR assay (QuantiTect SYBRgreen PCR Kit, Qiagen, Valencia, CA). Real-time PCR was carried out based on Light Cycler technology to accurately determine the level of expression of SLC19A2 and SLC19A3 in the HEK-293 cells. Gene-specific primers corresponding to the PCR targets were designed by using the specifications given by the vendors (Bio-Rad). For the SLC19A2, the primers were forward, 5'-AGCCAGACCGTCTCCTTGTA-3'; reverse, 5'-TAGAGAGGGCCCACCACAC-3'. For the SLC19A3, the primers were forward, 5'-TTCCTGGATTTACCCCACTG-3'; reverse, 5'-GTATGTCCAAACGGGGAAGA-3'. For -actin, the primers were forward, 5'-CATCCTGCGTCTGGACCT-3'; reverse, 5'-TAATGTCACGCACGATTTCC-3'. Each SYBRgreen reaction (20 µl total volume) contained 9 µl of diluted cDNA as template. The amplification program consisted of 1 cycle of 95°C with a 30-s hold ("hot start") followed by 40 cycles of 95°C for 1 min, specified annealing temperature with 15-s hold, 72°C with 30-s hold for extension, and data acquisition. A melting curve analysis program run for one cycle at 95°C with 0-s hold, 65°C with 10-s hold, and 95°C with 0-s hold at the step acquisition mode followed amplification. A negative control without cDNA template was run with every assay to assess specificity. For semiquantitative PCR analysis, the products were analyzed between cycles 19 and 26 for SLC19A2 and between cycles 26 and 32 for SLC19A3. Both the semiquantitative and real-time PCR data were normalized relative to the human -actin as described earlier ( 24 ).

Western Blot Analysis

Western blot analysis was performed using the membranous fractions of HEK-293 cells, isolated by homogenization of the cells in a buffer containing (in mM) 300 mannitol, 5 EGTA, and 12 Tris·HCl as well as a cocktail of protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, and 0.5 µg/ml leupeptin) ( 33 ). Protein (120 µg) samples were treated with Laemmli sample buffer and resolved on a 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were electroblotted onto an immunoblot polyvinylidene difluoride membrane (Bio-Rad) overnight, washed twice with PBS-Tween 20 for 10 min, and blocked with 5% dried milk in PBS-Tween 20. The membrane samples were then probed with either anti-human hTHTR-1-specific polyclonal antibodies (1:5,000 diluted in PBS-Tween 20) or anti-human hTHTR-2-specific polyclonal antibodies (1:5,000 diluted in PBS-Tween 20) as described previously ( 24, 27 ). Blots were then washed twice with PBS-Tween 20 buffer (Sigma) and reacted with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,500 diluted in PBS-Tween 20) for 1 h at room temperature. The blots were finally washed twice with PBS-Tween 20 for 10 min each time, and the luminescence was developed using an enhanced chemiluminescence kit (Amersham). -Actin Western blotting was performed as a loading control. Blots were incubated with a 1:500 dilution of a goat anti- -actin antibody (Santa Cruz Biotechnology) and developed as described above.

Promoter Activity-Transfection and Reporter Gene Assay

The full-length SLC19A2 and SLC19A3 promoter-luciferase reporter constructs, generated previously in our laboratory ( 16, 19 ), were used in the present investigations. The human renal epithelial HEK-293 cells were cotransfected in 12-well plates at less than 50% confluency with 2 µg of each test construct and 100 ng of the Renilla transfection control plasmid Renilla luciferase-thymidine kinase (pRL-TK; Promega, Madison, WI). Transfection was performed with Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Cells were then harvested at 3-4 days after transfection (confluence), and Renilla -normalized firefly luciferase activity was determined by using the Dual Luciferase Assay system (Promega). Data are presented as means ± SE of at least three independent experiments and given as folds over pGL3-Basic expression that was set arbitrarily at 1.

Statistical Analysis

Experimental points of transport studies are means ± SE of multiple separate uptake determinations and are expressed in terms of either femtomoles or picomoles per milligram of protein per 7 min (unless otherwise stated). Some variability in the absolute amount of thiamin uptake was observed in different patches of cells, and for this reason appropriate controls were run simultaneously with each set of experiments. Kinetic parameters of the saturable thiamin uptake process [determined by subtracting the diffusing component (determined from the slope of the uptake line between a high pharmacological concentration of thiamin of 1 mM and the point of origin) from total uptake at each concentration] were calculated using a computerized model of the Michaelis-Menten equation as described previously by Wilkinson ( 36 ). Statistical analysis was performed using Student's t -test or one-way ANOVA with statistical significance being 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 presented are from a representative set of experiments.

RESULTS

Thiamin Uptake By Human-Derived Renal Epithelial HEK-293 Cells

General characteristics. Uptake of thiamin (15 nM and 10 µM) by HEK-293 cells as a function of time was linear ( r = 0.99 for both) for up to 10 min of incubation and occurred at a rate of 21.2 fmol/mg protein and 8.4 pmol/mg protein, respectively ( Fig. 1 ). A 7-min incubation time was selected to represent the initial rate of uptake and used as the standard incubation time in all subsequent studies. Uptake during this period occurred without metabolic alterations as 97% of the transported radioactivity was found, by thin-layer chromatography (see MATERIALS AND METHODS ), to be in the form of intact thiamin.

Fig. 1. Uptake of thiamin by HEK-293 cells as a function of time. Confluent monolayers of HEK-293 cells were incubated at 37°C in Krebs-Ringer (K-R) buffer (pH 7.4) for different periods of time with low (15 nM; A ) or high (10 µM; B ) concentrations of thiamin. Data are means ± SE of 3-6 separate uptake determinations performed on 2 separate occasions. When not shown, error bars are smaller than symbols.

Effect of incubation buffer pH on thiamin uptake by HEK-293 cells was examined to determine whether the process is influenced by the prevailing H + concentration. The results showed the initial rate of thiamin (15 nM) uptake to progressively decrease as a function of decreasing the incubation buffer pH from 7.4 to 5.0 ( Fig. 2 ); uptake at pH 7.4 was 3.5-fold higher than that at pH 5.0. Hence, we used buffer pH 7.4 in all experiments. The effect of incubation temperature on thiamin (15 nM) uptake was also examined. A significant ( P < 0.01) and progressive decrease in thiamin uptake was observed with decreasing temperature from 37 to 4°C (234.2 ± 2.6, 151.0 ± 2.2, and 63.9 ± 3.6 fmol·mg protein -1 ·7 min -1 at 37, 22, and 4°C, respectively).

Fig. 2. Effect of incubation buffer pH on the initial rate of thiamin uptake by HEK-293 cells. Confluent monolayers of HEK-293 were incubated at 37°C in K-R buffer of varying pH (5.0-8.0) for 7 min in the presence of 15 nM [ 3 H]thiamin. Values are mean ± SE of 3-4 separate uptake determinations.

Involvement of Na + in thiamin uptake by HEK-293 cells was investigated by measuring thiamin uptake in the presence and absence of Na + (in the latter condition, NaCl was iso-osmotically replaced with KCl, LiCl, choline chloride, or mannitol). The results showed no substantial effect on the initial rate of thiamin (15 nM) uptake on Na + replacement [266.6 ± 11.7, 260.4 ± 4.9, 262.0 ± 4.9, 246.7 ± 10.6, and 265.5 ± 13.3 fmol·mg protein -1 ·7 min -1 in the presence of Na + (control), K +, Li +, choline, and mannitol, respectively]. Meanwhile, pretreatment of HEK-293 cells (for 30 min) with the Na-K-ATPase inhibitor ouabain (1 mM) had no effect on thiamin (15 nM) uptake process (215.5 ± 5.4 and 214.6 ± 9.5 fmol·mg protein -1 ·7 min -1, in the absence and presence of ouabain, respectively).

To determine whether thiamin uptake is energy dependent, HEK-293 cells were pretreated with the metabolic inhibitors 2,4-dinitrophenol (DNP), azide, p -chloromercuriphenyl sulfonate ( p -CMPS), and iodoacetate (all at 1 mM) for 30 min and uptake was then examined. The results showed significant ( P < 0.01 for all) inhibition in thiamin uptake by all inhibitors tested (215.5 ± 5.4, 126.9 ± 6.7, 159.8 ± 8.4, 170.3 ± 8.6, and 115.5 ± 6.4 fmol·mg protein -1 ·7 min -1 for control and in cells pretreated with DNP, iodoacetate, azide, and p -CMPS, respectively).

Existence For Involvement of Carrier-Mediated Process For Thiamin Uptake in Human Renal Epithelial HEK-293 Cells

Evidence for existence of two saturable thiamin uptake systems. Concentration dependency of 3 H-thiamin uptake was studied over a wide range of substrate concentrations that span the nanomolar (5-100 nM) and the micromolar (0.1-20 µM) ranges. The results showed the involvement of two saturable thiamin uptake systems, one being at the nanomolar range ( Fig. 3 A ) and the other at the micromolar range ( Fig. 3 B ). For both processes, uptake by the saturable components was determined as described in MATERIALS AND METHODS. Kinetic parameters of the saturable nanomolar component were 70.0 ± 18.4 nM and 1,370 ± 185.0 fmol·mg protein -1 ·7 min -1 for the apparent K m and V max, respectively. For the saturable micromolar components, the apparent K m and V max were 2.66 ± 0.18 µM and 25.4 ± 0.5 fmol·mg protein -1 ·7 min -1, respectively. We also applied the entire concentration-dependent uptake data to the Eadie-Hofstee plot, which produced a nonlinear relationship that fit well for a model describing the involvement of two saturable transport systems for thiamin uptake in HEK-293 cells ( Fig. 3 C ).

Fig. 3. Uptake of thiamin by HEK-293 cells as a function of nanomolar ( A ), micromolar ( B ) concentrations, and Eadie-Hofstee plot ( C ) of the uptake data. Confluent monolayers of HEK-293 cells were incubated at 37°C in K-R buffer (pH 7.4) for 7 min (i.e., initial rate) in the presence of different nanomolar (5-100 nM; A ) and micromolar (1-20 µM; B ) concentrations of thiamin. Uptake plots shown are those of the saturable components calculated as described in text. Eadie-Hofstee plot (V/S Vs V) where V is the uptake rate in fmol and pmol·mg protein -1 ·7 min -1, and S is the thiamin concentration in µM. Data are means ± SE of 3 to 5 separate uptake determinations performed on 2 separate occasions. When not shown in A and B, error bars are smaller than symbols.

To confirm the involvement of a specialized uptake process in thiamin uptake by HEK-293 cells, we examined the effect of different concentrations of the thiamin structural analogs amprolium, oxythiamine, and benfotiamine on the initial rate of [ 3 H]thiamin (15 nM) uptake. The results showed that these structural analogs cause a significant ( P < 0.01 for all) and concentration-dependent inhibition in [ 3 H]thiamin (15 nM) uptake ( Table 1 ). We also examined possible trans -stimulation in [ 3 H]thiamin efflux by unlabeled thiamin. In this experiment, we first preloaded the HEK-293 cells with 3 H-thiamin (by incubation with 30 nM [ 3 H]thiamin for 10 min at 37°C), then incubated the preloaded cells (for 10 min) in K-R buffer in the presence and absence of 100 µM unlabeled thiamin. The results showed the cell content of the 3 H radioactivity to be significantly ( P < 0.01) lower in HEK-293 cells incubated in the presence of unlabeled thiamin compared with those incubated in buffer alone (cell content of 3 H radioactivity was 298.4 ± 11.2 and 518.7 ± 5.9 fmol·mg protein -1 ·7 min -1, respectively). In another investigation, we examined the influence of unrelated organic cations on the uptake of the cationic thiamin (15 nM) and found that neither tetraethylammonium (TEA) nor N -methylnicotin amide (NMN; 100 µM) to significantly influence the substrate uptake (244.4 ± 7.7, 249.7 ± 6.7, and 249.8 ± 9.1 fmol·mg protein -1 ·7 min -1 for control and in the presence of TEA and NMN, respectively).

Table 1. Effect of thiamin structural analogs on initial rate of [ 3 H]thiamin uptake by HEK-293 cells

Effect of membrane transport inhibitors on [ 3 H]thiamin uptake by HEK-293 cells. The effects of the membrane transport inhibitors DIDS, amiloride, probenecid, and furosemide (all at 1 mM) on the initial rate of thiamin (15 nM) uptake were examined. With the exception of amiloride, which caused significant ( P < 0.01) inhibition in thiamin uptake, none of the other tested membrane transport inhibitors affected thiamin uptake (249.7 ± 6.3, 239.8 ± 4.3, 251.5 ± 14.9, 244.7 ± 6.9, and 91.2 ± 6.2 fmol·mg protein -1 ·7 min -1 for control and in the presence of DIDS, probenecid, furosemide, and amiloride, respectively). Furthermore, analysis of the inhibitory effect of amiloride (0.1-1.0 mM) was then performed and the inhibition was found, by the Dixon method, to be competitive in nature with an apparent K i value of 0.6 mM ( Fig. 4 ).

Fig. 4. Dixon plot for the effect of amiloride on thiamin uptake by HEK-293 cells. Confluent monolayers of HEK-293 cells were incubated at 37°C in K-R buffer (pH 7.4) in the presence of different concentrations of amiloride (0.1-1.0 mM). Thiamin uptake was measured during initial rate of uptake (7 min), and results were applied to Dixon plot as shown. Results are means ± SE of 3-5 separate uptake determinations performed on 2 separate occasions.

Molecular Aspects of Thiamin Uptake Process of the Human Renal Epithelial HEK-293 Cells

Expression of hTHTR-1 and hTHTR-2 in HEK-293 cells. In these investigations, we examined whether the hTHTR-1 and hTHTR-2 are expressed in HEK-293 cells at the mRNA and protein levels by real-time PCR and Western blot analysis. We also tested the relative activity of the promoters for SLC19A2 and SLC19A3, the genes that are responsible for coding of the hTHTR-1 and hTHTR-2, respectively, in these cells. We performed semiquantitative PCR with the use of gene-specific primers corresponding to the sequences of SLC19A2 and SLC19A3, and the results showed both of SLC19A2 and SLC19A3 to be expressed in HEK-293 cells ( Fig. 5 A, inset ). Furthermore, to quantitate the relative level of expression of SLC19A2 and SLC19A3 in HEK-293 cells, we used real-time PCR with specific primers (see MATERIALS AND METHODS ), and our results showed the expression of SLC19A2 to be significantly ( P < 0.01) higher than the level of expression of SLC19A3 ( Fig. 5 A ). We also examined expression of the hTHTR-1 and hTHTR-2 in these cells at the protein level by means of Western blot analysis using specific anti-hTHTR-1 and anti-hTHTR-2 polyclonal antibodies ( 24, 27 ). The results ( Fig. 5 B ) showed both the hTHTR-1 and hTHTR-2 proteins are expressed, with the expression of hTHTR-1 protein being higher than that of hTHTR-2. Relative activity of the full-length SLC19A2 and SLC19A3 promoters in HEK-293 cells was also examined and the results showed a significantly ( P < 0.01) higher activity for the former compared with the latter promoter ( Fig. 5 C ).

Fig. 5. Expression of the hTHTR-1 and hTHTR-2 in HEK-293 cells at the mRNA and protein levels and on activity of SLC19A2 and SLC19A3 promoters. A : real-time PCR analysis of SLC19A2 and SLC19A3 mRNA levels of HEK-293 cells. Inset : semiquantitative PCR product obtained using the same SLC19A2 - and SLC19A3 -specific primers described in MATERIALS AND METHODS. All experiments were run on at least 3 separate occasions. B : expression of hTHTR-1 and hTHTR2 proteins in HEK-293 cells. Western blot analysis was performed using membranous fractions of HEK-293 cells grown in DMEM growth medium. Data shown are representative of 3 separate sets of experiments. C : cells maintained in DMEM growth medium were cotransfected with promoter-luciferase reporter plasmids and a control pGL3-basic vector as described in MATERIALS AND METHODS. Values represent means ± SE of a representative experiment performed in triplicate. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase.

Relative contribution of hTHTR-1 and hTHTR-2 toward total carrier-mediated thiamin uptake by HEK-293 cells. We used a well-established approach of siRNA to selectively and specifically silence the SLC19A2 and SLC19A3 genes of the HEK-293 cells, then examined the effect of such silencing on carrier-mediated thiamin uptake ( 24 ). First, we established that such a pretreatment does indeed lead to a knockdown of the hTHTR-1 and hTHTR-2 mRNA and protein levels. Results with real-time PCR showed that pretreating HEK-293 cells with gene-specific siRNAs lead to a marked reduction in the endogenous level of the SLC19A2 and SLC19A3 transcripts compared with their respective controls ( Fig. 6 A ). Similarly, results of Western blot analysis showed the level of the hTHTR-1 and hTHTR-2 proteins to be substantially reduced in the siRNA-pretreated cells compared with controls ( Fig. 6 B ). However, there was no reduction in levels of the -actin. These findings confirm the effectiveness and specificity of the siRNA approach. Subsequently, we examined the effect of the siRNA treatment on the initial rate of carrier-mediated [ 3 H]thiamin (15 nM) uptake. Treating cells with hTHTR-1-specific siRNAs led to an 50% inhibition ( P < 0.01) in carrier-mediated thiamin uptake compared with control (356.9 ± 20.3 and 706.9 ± 32.6 fmol·mg protein -1 ·7 min -1, respectively). Similarly, treating the cells with siRNA specific to hTHTR-2 led to an 33% inhibition ( P < 0.01) in carrier-mediated thiamin uptake compared with control (477.2 ± 26.4 and 706.9 ± 32.6 fmol·mg protein -1 ·7 min -1, respectively). In contrast, uptake of unrelated [ 3 H]biotin (15 nM) was not affected by the treatment with either gene-specific siRNAs (data not shown).

Fig. 6. Effect of pretreatment of HEK-293 cells with gene-specific siRNAs on mRNA and protein levels of the hTHTR-1 and hTHTR-2. A : real-time PCR analysis of SLC19A2 and SLC19A3 mRNA levels. The analysis was performed after pretreating the cells with SLC19A2 and SLC19A3 gene-specific siRNAs as described in MATERIALS AND METHODS and compared with control. B : Western blot analysis of hTHTR-1 and hTHTR-2 proteins in membranous fractions of HEK-293 cells. 1 : Levels of expression of hTHTR-1 protein in control and SLC19A2 gene-specific siRNAs pretreated HEK-293 cells. 2 : Levels of hTHTR-2 protein in control and SLC19A3 gene-specific siRNAs pretreated HEK-293 cells. Analysis was performed as described in MATERIALS AND METHODS. Data shown are representative of 3 separate sets of experiments.

Regulatory Aspects of Thiamin Uptake Process of the Human Renal Epithelial HEK-293 Cells

Effect of thiamin deficiency. The effect of a thiamin-deficient condition on thiamin uptake by the renal epithelial HEK-293 cells was examined by maintaining the cells in thiamin-deficient growth medium for 96 h (see MATERIALS AND METHODS ). The results showed a significant ( P < 0.01) induction in thiamin (15 nM) uptake in cells maintained under a thiamin-deficient condition compared with control (526.3 ± 17.4 and 320.7 ± 8.3 fmol·mg protein -1 ·7 min -1, respectively). We also examined the effect of a thiamin-deficient condition on the level of expression of hTHTR-1 and hTHTR-2 mRNA and its protein by means of real-time PCR and Western blotting, respectively. Results of the real-time PCR showed a significant ( P < 0.01 for both) increase in the levels of both SLC19A2 and SLC19A3 mRNA in HEK-293 cells maintained under a thiamin-deficient condition compared with those maintained in control growth medium ( Fig. 7 A ). Similarly, results of the Western blotting showed that the hTHTR-1 and hTHTR-2 proteins are substantially upregulated in the cells maintained under a thiamin-deficient condition compared with those maintained in regular growth medium ( Fig. 7 B ). We also examined the effect of maintaining HEK-293 cells in thiamin-deficient growth medium on activity of the SLC19A2 and SLC19A3 promoters. The full-length promoter constructs fused to the Firefly luciferase reporter gene were transfected into the HEK-293 cells maintained in thiamin-deficient and control growth media, and promoter activity was assayed after 72 h following transfection. The results showed that activity of both the SLC19A2 and the SLC19A3 promoters to be significantly ( P < 0.01) elevated in HEK-293 cells maintained under a thiamin-deficient condition compared with those maintained under a control condition ( Fig. 7 C ).

Fig. 7. Effect of maintaining HEK-293 cells in thiamin-deficient growth medium on mRNA and protein levels of hTHTR-1 and hTHTR-2, and on activity of SLC19A2 and SLC19A3 promoters. A : real-time PCR analysis of SLC19A2 and SLC19A3 mRNA of HEK-293 cells grown under thiamin-deficient and control conditions. Data are normalized relative to the housekeeping gene, -actin. B : Western blot analysis of the hTHTR-1 ( A ) and hTHTR-2 ( B ) proteins in membranous fractions of HEK-293 cells. Level of expression of the transport proteins in control cells and those grown under thiamin-deficient conditions is shown. C : cells maintained in thiamin-deficient and control conditions were cotransfected with promoter-luciferase reporter plasmids and a control pGL3-basic vector as described in MATERIALS AND METHODS. Values represent means ± SE of a representative experiment performed in triplicates. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase.

Role of intracellular regulatory pathways. Involvement of specific intracellular regulatory pathways mediated by protein kinase C (PKC), protein kinase A (PKA), protein tyrosine kinase (PTK), Ca 2+ /calmodulin (Ca 2+ /CaM), and nitric oxide (NO) in the regulation of the renal thiamin uptake process was studied. We focused here on these pathways because previous studies documented their role in the regulation of transport of other nutrients/substrates in a variety of epithelial cells ( 1, 8, 25, 29 ).

The effect of a Ca 2+ /CaM-mediated pathway in the regulation of thiamin uptake by HEK-293 cells was investigated by examining the effect of pretreating the cells (for 1 h) with a number of specific modulators of this pathway, namely, calmidazolium (10 and 50 µM), trifluroperazine (TFP; 25 µM), N -(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13; 100 µM), and KN 62 (50 µM) on the initial rate of 3 H-thiamin uptake (15 nM). The findings showed that pretreatment with calmidazolium leads to a significant ( P < 0.01) and concentration-dependent inhibition of thiamin uptake (234.4 ± 5.7, 85.6 ± 3.3, and 59.6 ± 4.1 fmol·mg protein -1 ·7 min -1 for control and in the presence of 10 and 50 µM calmidazolium, respectively). Similarly, pretreating the cells with other modulators also led to a significant ( P < 0.01 for all) inhibition in thiamin uptake (234.4 ± 5.7, 91.5 ± 4.8, 151.8 ± 4.4, and 75.9 ± 1.0 fmol·mg protein -1 ·7 min -1 for control and the presence of TFP, W13, and KN 62, respectively).

The possible role of a PKA-mediated pathway in the regulation of thiamin uptake by HEK-293 cells was tested by examining the effect of pretreating the cells (for 1 h) with the modulators of this pathway, namely forskolin (100 µM) and dibutyryl cAMP (1 mM). The results showed that neither of these modulators has any significant effect on thiamin uptake (256.8 ± 4.8, 261.0 ± 5.9, and 246.1 ± 13.5 fmol·mg protein -1 ·7 min -1 for control, and following pretreatment with forskolin and dibutyryl cAMP, respectively). The effect of modulators of the PTK-mediated pathway was also investigated by testing the effect of pretreatment (for 1 h) of cells with genisteine (25 µM) and tyrphostin A25 (10 µM). Neither of these compounds had a significant effect on thiamin uptake (255.7 ± 3.9, 247.2 ± 3.4, and 247.6 ± 3.4 fmol·mg protein -1 ·7 min -1 for control, and following pretreatment with genisteine and tyrphostin A25, respectively). Similarly, no role for the PKC-mediated pathway was apparent as pretreatment with modulators of this pathway failed to affect thiamin uptake by HEK-293 cells (216.6 ± 3.2, 218.7 ± 4.6, and 213.5 ± 8.3 fmol·mg protein -1 ·7 min -1 for control and following pretreatment with 10 µM staurosporine and 10 µM chelerythrine, respectively). Finally, we determined the potential role of the NO-mediated pathway in the regulation of thiamin uptake by HEK-293 cells by examining the effect of pretreating the cells (for 1 h) with inhibitors of this pathway, namely S -nitrose- N -acetylpenicillamine (SNAP; 0.1 mM) and 8-bromo cGMP (8-BrcGMP; 0.5 mM). Neither compound had significant effect on thiamin uptake (234.6 ± 5.6, 226.6 ± 9.8, and 230.6 ± 4.2 fmol·mg protein -1 ·7 min -1 for control, and following pretreatment with SNAP and 8-BrcGMP, respectively).

DISCUSSION

The kidneys play an important role in regulating and maintaining normal thiamin homeostasis due to their ability to reabsorb the vitamin after its filtration in the renal glomeruli. This reabsorption process, which is designed to prevent losses of thiamin in the urine, is the function of the renal epithelial cells. Impairment or interference with this reabsorptive function could lead to disturbances in normal thiamin body homeostasis. Thus studies on the mechanism(s) and regulation of the human renal thiamin reabsorption process are of significant physiological and nutritional importance. The mechanism and regulation of thiamin uptake by human renal epithelial cells have not been investigated before and were therefore delineated in the present investigation. We used the human-derived renal epithelial HEK-293 cell line as an in vitro model system in these investigations because it represents a good model for human proximal tubular epithelial cells and has been used in similar physiological investigations with data that are similar to those found in the native renal epithelial cells ( 7, 34 ).

Our studies showed that the initial rate of thiamin uptake by HEK-293 (which occurs without metabolic alterations in the transported substrate) was temperature and energy dependent and was Na independent in nature. The latter conclusion is based on the observations that thiamin uptake was not affected by the isosmotic replacement of Na + in the incubation medium with other monovalent cations (or with mannitol) and by the inability of the Na + -K + -ATPase inhibitor, ouabain, to inhibit thiamin uptake. Uptake of thiamin by HEK-293 cells was pH dependent and substantially higher at alkaline/neutral buffer pH (a situation similar to that of the lumen of the renal proximal tubules) compared with acidic pH. The above-described observations are similar to those reported for thiamin uptake in human intestinal and hepatic epithelial cells where the process was suggested to involve a thiamin/H + exchange mechanism ( 21, 27, 28 ). Also similar to the thiamin uptake process in other human epithelial cells, the thiamin uptake process of the human renal epithelial HEK-293 cells was sensitive to amiloride (a known inhibitor of the Na + /H + exchange mechanism), with a reported apparent K i of 0.6 mM ( 2, 4, 26 ). The above-described findings on thiamin uptake by the human renal epithelial cells are similar to those seen previously with animal renal preparations ( 9 ).

The results on thiamin uptake by the HEK-293 renal epithelial cells as a function of concentration indicated the involvement of two saturable uptake processes; one being functional at the nanomolar range with high affinity but low capacity (apparent K m of 70.0 ± 18.4 nM and V max of 1,370 ± 185.0 fmol·mg protein -1 ·7 min -1, respectively) and the other being functional at the micromolar range with low affinity but higher capacity (apparent K m of 2.66 ± 0.18 µM and V max of 25.4 ± 0.5 pmol·mg protein -1 ·7 min -1, respectively). When the concentration-dependent uptake data were applied to the Eadie-Hofstee plot, clear confirmation for the involvement of two separate uptake systems was obtained. The ability of the thiamin structural analogs amprolium, oxythiamine, and benfotiamine, but not the unrelated organic cations TEA and NMN, to inhibit the uptake of cationic [ 3 H]thiamin by HEK-293 cells confirms the carrier-mediated nature of the renal thiamin uptake process and also demonstrates specificity of the uptake process. Finally, the observation of trans -stimulation in [ 3 H]thiamin efflux from HEK-293 cells by unlabeled thiamin adds further support for the carrier-mediated nature of the renal uptake of this vitamin.

We also elucidated the molecular identity of the systems involved in thiamin uptake by HEK-293 cells and found that both of the recently cloned human thiamin transporters hTHTR-1 and hTHTR-2 are expressed in these cells at the mRNA and protein levels, i.e., similar to what has been reported with native human kidney ( 5, 6 ). Expression of hTHTR-1, however, was greater than that of hTHTR-2. We also found the activity of the SLC19A2 promoter to be higher compared with the activity of the SLC19A3 promoter in HEK-293 cells, a finding that corresponds to the higher mRNA and protein levels of hTHTR-1 compared with hTHTR-2. To determine the relative contribution of the hTHTR-1 and the hTHTR-2 toward total carrier-mediated thiamin uptake by the human renal epithelial HEK-293 cells, we used the approach of gene silencing of the individual thiamin transporters (using gene-specific siRNA) and examined the effect of that silencing on thiamin uptake. We first verified that pretreating the cells with gene-specific siRNA leads to a marked knockdown in the mRNA and protein levels of hTHTR-1 and hTHTR-2. These effects were specific as no changes in the levels of expression of mRNA of the human -actin were observed in the siRNA-pretreated cells compared with control cells. When the effect of siRNA pretreatment was subsequently examined on thiamin uptake, the results showed that treating the cells with the hTHTR-1 and hTHTR-2 gene-specific siRNAs led to a significant (50 and 33%, respectively) inhibition in thiamin uptake. These findings suggest that both the hTHTR-1 and hTHTR-2 are involved in thiamin uptake by HEK-293 cells with the former transporter being somewhat more active.

Following establishment of the thiamin uptake mechanism in the human renal epithelial HEK-293 cells and elucidation of the systems involved, we examined potential regulation of the vitamin uptake process by extracellular and intracellular factors/conditions. The thiamin uptake by the renal epithelial HEK-293 cells appeared to be under the regulation of the extracellular substrate level. Maintaining the HEK-293 cells in a thiamin-deficient growth medium was found to lead to a specific and significant upregulation in initial rate of [ 3 H]thiamin uptake. This increase in uptake was associated with a substantial increase in endogenous hTHTR-1 and hTHTR-2 mRNA and protein levels. These findings suggest that the increase in thiamin uptake by renal epithelial HEK-293 cells maintained in a thiamin-deficient condition is transcriptionally mediated. To directly test the latter possibility, we examined the effect of maintaining the HEK-293 cells in a thiamin-deficient growth medium on activity of the SLC19A2 and SLC19A3 promoters. The results showed the activity of both promoters to be significantly induced in cells grown under a thiamin-deficient condition compared with those grown under a regular (control) condition. These results are similar to those found in our laboratory in mice where the renal thiamin uptake process was adaptively upregulated (and in a specific manner) in thiamin deficiency with the effect being mediated via a transcriptional regulatory mechanism(s) that involves both thiamin transporters ( 18 ).

Our results also showed that the intracellular PKA-, PKC- PTK-, and the NO-mediated regulatory pathways have no role in regulating the thiamin uptake process of HEK-293 cells. However, an intracellular Ca 2+ /CaM-mediated pathway appears to play a role in this process. Modulators of the Ca 2+ /CaM-mediated pathway had a significant inhibition in thiamin uptake, a finding that is similar to what has been seen for thiamin uptake in other cellular systems, e.g., the intestine ( 26, 27 ). Such findings suggest the possible involvement of a common intracellular pathway for the regulation of thiamin uptake in different human tissues. The cellular mechanism(s) through which the Ca 2+ /CaM-mediated pathway exerts its effect on thiamin uptake is (are) not clear and further investigations are required to address the issue.

In summary, results of the current investigations demonstrate that thiamin uptake by the human-derived renal proximal tubular epithelial HEK-293 cells is via a specialized carrier-mediated mechanism, which is pH dependent in nature. The results also show that both the hTHTR-1 and hTHTR-2 are involved in the thiamin uptake process. In addition, the renal thiamin uptake process appears to be under the regulation of an intracellular Ca 2+ /CaM-mediated pathway. Finally, this process is also adaptively regulated in thiamin deficiency via a transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

GRANTS

This study was supported by National Institutes of Health Grants DK-58057 and DK-56061 and the Department of Veterans Affairs.

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作者单位：1 Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and 2 Department of Veterans Affairs Medical Center, Long Beach, California