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Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932
* To whom correspondence should be addressed. E-mail: kmcmar{at}lsuhsc.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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65 mmol/L) reduced the apically directed transport of folate by 20–25% without affecting the basolateral transport. A 5-d exposure of HPT cells to ethanol dose-dependently increased the content of both the FR and RFC proteins, with a greater effect on the RFC. Similarly, a 14-d exposure of rats to ethanol increased the in vivo expression of both the RFC and FR. These studies demonstrate that ethanol decreases the reabsorptive transport of folate by renal PT cells, which would increase urinary folate excretion. In contrast, subchronic exposure of PT cells, both in vivo and in vitro, to folate-depleting concentrations of ethanol leads to an upregulation of the 2 folate transport proteins. The increase in folate transporters partly counteracts the inhibitory effects of ethanol on folate transport activity, which explains the lower magnitude of ethanol's effect on transport with subchronic exposure compared with that with acute exposure.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Increased urinary folate excretion is a principal mechanism for the ethanol-induced folate deficiency. Acute or chronic administration of ethanol results in an increased urinary excretion of folate, which precedes a decrease in plasma folate levels (12,13). In humans as well as rats, acute ethanol ingestion markedly decreases plasma folate levels in 16–20 h, which then contributes to the development of folate deficiency (13–15). In rats, urinary folate excretion is increased 2-fold and 6-fold after chronic (12 wk) and acute (4 h) ethanol ingestion, respectively (16,17). Urinary folate excretion also contributes to the development of folate deficiency in monkeys or humans chronically consuming ethanol (12,18). The effects on urinary folate excretion occur only at high blood ethanol levels [33 mmol/L (150 mg/dL)] (17,19). These levels are relevant, as chronic alcoholic populations exhibit folate deficiencies at blood ethanol levels in the 43–65 mmol/L range (2).
The mechanisms by which ethanol increases urinary folate excretion are unknown. In the kidney, plasma folate is filtered at the glomerulus and then most of the folate in the tubular lumen is reabsorbed by the proximal tubule (PT)3 cells. Urinary folate can also result when folate is secreted from PT cells into the lumen. Experiments in vivo have demonstrated that ethanol does not increase the glomerular filtration rate or the amount of folate presented to the kidney (20). In contrast, ethanol has a direct effect on the isolated perfused rat kidney (IPRK), where ethanol alters the reabsorption of folate in the PT, resulting in increased excretion (21). Hence, ethanol's effects appear to result from a decreased reabsorptive transport of folate in the PT.
The roles of folate transporters in mediating the ethanol-induced inhibition of renal folate transport have not been defined. Two folate transporter systems are considered to be responsible for regulating folate homeostasis: the reduced folate carrier (RFC) (22,23) and the folate receptor (FR), also referred to as the folate-binding protein (22). The RFC has been identified in kidney epithelial cells, where it is involved in the transport of folates into the cytosol (24). The FR has also been located in renal PT cells, where it functions to concentrate folates at the apical surface of these cells for internalization, resulting in a net reabsorption of folates from the glomerular filtrate (25). Both the FR and RFC play important roles in renal folate transport (26).
In studying the mechanisms of folate excretion, a system is needed to distinguish between the apical and basolateral components of PT cells, because ethanol could increase excretion either by inhibiting reabsorption from the lumen (apical uptake) or by enhancing secretion into the lumen (regulated by basolateral uptake). In the present studies, culturing human PT (HPT) cells on microporous membrane inserts allowed for distinct measurements of apical and basolateral folate transport (27). The HPT cell culture model was thus used to determine the effects of acute and subchronic ethanol on the apical- and basolateral-directed uptake of 5-methyltetrahydrofolate. In addition, the effects of subchronic ethanol on the folate transporter proteins were investigated in both human (in vitro) and rat (in vivo) PT cells.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Preparation of primary cultures and general cell culture techniques. HPT cells were obtained as normal cortical renal tissue specimens as a result of nephrectomy due to tumor or trauma (Urology Department, Louisiana State University Health Sciences Center, Shreveport, LA). Kidney specimens were judged to be normal by the pathologist prior to use as a tissue source. Because the patient records were not retained and because the tissue obtained was regarded as waste for discarding, the Institutional Review Board for Human Research (Louisiana State University Health Sciences Center) exempted these studies. HPT cells were isolated from cortical tissue by a DNase-collagenase digestion method, described previously (28). To further limit the growth of contaminating cells and to enrich the population of HPT cells (29), isolated cells were cultured at 37°C in 5% CO2 in a serum-free media (DMEM:Ham's F-12 medium 50:50 with additional growth factors: insulin, transferrin, selenious acid, hydrocortisone, epidermal growth factor, triiodothyronine, and L-glutamine). At confluency, cells were subcultured by trypsinization into flasks or tissue culture inserts for experimentation. In inserts, confluency was determined by the development of transepithelial resistance (TER) (27), as measured using an EVOM Epithelial Voltohmmeter (World Precision Instruments).
Folate transport studies in HPT cells on inserts. Characterization of 5-methyltetrahydrofolate binding and transport by HPT cells was determined by methods described previously (30). The conditions used in these studies, including folate concentrations and incubation times, were based on kinetic studies of 5-methyltetrathydrofolate handing by HPT cells previously conducted (27,30). In brief, cells were subcultured on collagen-coated membrane inserts that isolate the apical and basolateral surfaces for transport studies (27). Once cells reached confluence, they were washed and then preincubated in incubation buffer (30) for 1 h to allow for recovery of TER (27). After the preincubation, [3H]-5-methyltetrahydrofolate (25 nmol/L, Moravek Biochemical) and [14C]-inulin (3.7 Bq/L, Amersham Pharmacia Biotech) were added to the insert to measure transport from the apical compartment or to the buffer in the well (outside the insert) for transport from the basolateral compartment. Incubations were conducted for 2 h at 37°C. Nonspecific controls included a 1000-fold excess of unlabeled 5-methyltetrahydrofolate. Specific binding and transport were calculated by subtracting the nonspecific binding and transport values from the total binding and transport values. Specific binding and transport values represented >90% of the total transport or binding value. These proportions were not affected by ethanol treatment.
After incubation, the buffers were removed to measure transmembrane transfer, i.e. the movement across the cell layer from the apical to basolateral side (or vice versa). Then, inserts were washed with buffer to remove nonspecifically bound 5-methyltetrahydrofolate. Binding to the apical FR was determined by removing ligand from the apical surface with a pH 3.0 buffer wash (30). Transport into the cells was then determined by solubilizing the cells using 0.1% Triton X-100 in PBS. The content of [3H]-5-methyltetrahydrofolate and [14C]-inulin in the various samples was determined using a dual-label-programmed liquid scintillation counter. The 5-methyltetrahydrofolate content was normalized as fmol/mg protein using the protein concentration of solubilized cells, determined by the Bio-Rad protein assay.
Effects of ethanol on folate binding and transport in HPT cells. For the acute treatment, ethanol [11–109 mmol/L (50–500 mg/dL)] was added to the incubation buffer during the normal 1-h preincubation needed to restore TER (as noted above). These concentrations of ethanol did not affect tight junctional status, because the TER measured after the 1-h recovery was the same in ethanol-containing and control inserts. After 1 h, [3H]-5-methyltetrahydrofolate was added for a further 2-h incubation, during which ethanol remained in the incubation buffer. Transport and binding of 5-methyltetrahydrofolate were determined as above.
For the 5-d treatment, we added ethanol (11–109 mmol/L) to the normal growth media to maintain cellular nutrition over the 5 d. The ethanol-containing media was replaced daily to minimize depletion of ethanol levels. After d 5, the cells were washed with buffer to remove the growth media and preincubated for 1 h in ethanol-containing incubation buffer to restore TER. [3H]-5-Methyltetrahydrofolate was added for the 2-h incubation, as above. In the 5-d studies, the protein content of insert-grown cells decreased by 20–30% at ethanol concentrations 65 mmol/L, but this was controlled for by normalizing the transport and binding data as fmol/milligram protein using the protein concentration of each insert.
To test the specificity of the ethanol effect on folate transport, the effect of acute ethanol on the transport of the glucose analogue, 
-methylglucopyranoside (-MG), was measured. Confluent HPT cells in 12-well plates were washed with buffer and incubated with methyl--D-[U- 14C] glucopyranoside (1 mmol/L, 18.5 MBq/L, Amersham Pharmacia), 3.7 MBq/L [3H]-inulin (to measure nonspecific uptake), and various concentrations of ethanol in incubation buffer for 1 h at 37°C. Then the cells were washed with buffer and solubilized with 0.2 mol/L NaOH. We determined the transport of [14C]--MG into the cells using a liquid scintillation counter. Values for [3H]-inulin uptake were subtracted from total [14C]--MG uptake to determine the specific transport of [14C]--MG.
Effect of ethanol on folate transport protein expression in HPT cells. To test the effects of 5-d ethanol exposure on the content of the folate transport proteins in kidney cells in vitro, confluent HPT cells were treated by adding ethanol [22, 65, and 109 mmol/L (100, 300, and 500 mg/dL)] to normal growth media in separate 75-cm2 flasks, with normal growth media in the control flasks (0 mmol/L ethanol). We placed each separate flask in a container that included a petri dish containing the same concentration of ethanol as in the flask. The media in the flask and petri dish were replaced daily with fresh media to maintain the ethanol content. We used an aliquot of the spent media to determine the ethanol concentration by GC using isopropanol as the internal standard (31). These concentrations were consistent with the target concentrations of 0, 21.7, 65.2, and 109 mmol/L (0.2 ± 0.2, 18.7 ± 1.3, 61.7 ± 3.3, and 102 ± 3.5 mmol/L, respectively, on d 1 and 0.4 ± 0.4, 19.6 ± 1.5, 64.3 ± 5.4, and 124 ± 9.8 mmol/L, respectively, on d 5), demonstrating that the cells were exposed to a constant level of ethanol during each 24-h period of the 5-d treatment. To minimize potential loss of cells during the 5-d ethanol exposure, we conducted these studies with minimal disturbance of the cell monolayer. Microscopic visualization on d 5 revealed a confluent monolayer with no apparent cell loss, even at 109 mmol/L. Also, the total cellular protein content did not differ among ethanol concentrations: 1.6 ± 0.2, 1.6 ± 0.2, 1.4 ± 0.1, and 1.2 ± 0.1 g/L for 0, 22, 65, and 109 mmol/L ethanol, respectively, indicating minimal cellular loss.
After the 5-d treatment, the media was removed and the cell monolayer was washed twice with PBS. A total of 5 mL of sucrose buffer (0.25 mol/L sucrose, 10 mmol/L MgCl2, 5 mmol/L Tris-HCl, pH 7.4) containing a protease inhibitor cocktail (1 mmol/L para-aminoethylbenzenesulfonyl fluoride, 100 µmol/L leupeptin, 10 µmol/L bestatin) were added before scraping cells from the flask. Scraped cells were homogenized using a glass homogenizer and plasma membranes were isolated by centrifugation (30,000 x g; 30 min at 4°C). Pellets were resuspended in wash buffer (10 mmol/L MgCl2, 50 mmol/L Tris-HCl, pH 7.4) containing protease inhibitor cocktail and stored at –80°C until their subsequent use in gel electrophoresis.
Effect of ethanol on folate transport protein expression in rat kidneys. To test the effects of subchronic ethanol exposure on the content of the folate transport proteins in kidney cells in vivo, male Sprague Dawley rats (250–300 g, Harlan) received either a nutritionally complete control diet or ethanol-containing (5% wt:v) liquid diet for 14 d (n = 8 per group) in a pair-fed manner (17). Diets, obtained from Dyets, were administered in a composition previously described (17). Both diets contained prescribed levels of folate (0.5 mg/L) but did not contain sulfa [used to induce tissue folate deficiency (17)], because ethanol can increase urinary folate excretion in the absence of sulfa (17). On d 13, the rats were placed in metabolic chambers and urine was collected every 8 h in tubes that contained ß-mercaptoethanol (50 µL/mL urine) to minimize oxidative breakdown of folates. The 3 samples were combined for a 24-h sample, which was stored at –20°C until analysis for ethanol concentration (31) and for folate content by Lactobacillus casei microtiter growth assay (32). The rats were returned to their home cages on d 14.
On d 15, the rats were anesthetized with sodium pentobarbital and their kidneys were rapidly excised and decapsulated. Preparation of the renal apical brush border membrane (BBM) fractions was adapted from previous studies (33). Renal cortices were homogenized in 25 volumes ice-cold sucrose buffer containing the protease inhibitor, phenylmethylsulfonyl fluoride (1 mmol/L), then centrifuged at 2000 x g; 10 min at 4°C. The supernatant was further centrifuged (30,000 x g; 30 min at 4°C). The pellet was washed 3 times in wash buffer, resuspended in wash buffer containing the protease inhibitor cocktail above (para-aminoethylbenzenesulfonyl fluoride, leupeptin, bestatin), and stored at –20°C until use in gel electrophoresis.
The animal protocols were approved by the Institutional Animal Care and Use Committee (LSUHSC-Shreveport) and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Western-blot analysis for FR and RFC protein content. Membrane protein samples from the HPT cells and rat kidneys were electrophoresed on precast 4–20% Tris-glycine gels (Invitrogen) in the presence of SDS (34) and were transferred to polyvinylidene difluoride membranes (Millipore). Nonspecific proteins were blocked using 5% bovine serum albumin (wt:v) in Tris-buffered saline with 0.05% Tween 20 and then membranes were probed with primary antibodies for the FR (1:500 dilution) or RFC (1:5000 dilution). The polyclonal antibody to the FR was raised in rabbits against the folate-binding protein purified from the rat placenta and was a gift from Dr. Sheldon Rothenberg (SUNY Downstate Medical Center, Brooklyn, NY). The polyclonal antibody to the human RFC was raised in rabbits using a purified glutathione S-transferase-RFC fusion protein as antigen. Anti-glutathione S-transferase-RFC antiserum was a gift from Dr. Larry Matherly (Karmanos Cancer Institute, Detroit, MI). Preparation of these antibodies for western blotting or immunoassay techniques and their validation have been described by these authors (35,36). Antibody titers for these studies were determined by dilutional analysis to minimize the background while maintaining signal bands. Immunoblot detection employed biotinylated rabbit IgG at 1:20,000 dilution and NeutrAvidin, horseradish peroxidase-conjugated at 1:10,000 dilution (Pierce), followed by enhanced chemiluminescence reagents (Amersham Pharmacia). Band intensities were analyzed using Bio-Rad Quantity One version 4.1 Quantitation software and the Bio-Rad Gel Doc imaging device.
Statistical analysis. Statistical analysis was performed using GraphPad Prism version 3.0 software. All folate transporter densitometry data were expressed as OD units and were normalized to control, with 0 mmol/L ethanol concentration (cells) or control diet (rats) as the control. The statistical methods used for the HPT cell studies were the 1-way ANOVA and Dunnett's or Tukey's multiple comparison test as the post test, with P < 0.05 being the level of significance. The statistical method used in the rat diet study was the Student's t test, with differences considered significant at P < 0.05. Values in the text are means ± SEM.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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1-H ethanol treatment.
The treatment of insert-grown HPT cells with ethanol for 1 h prior to the apical addition of [3H]-5-methyltetrahydrofolate had no effect on apical binding (Fig. 1). However, ethanol at 65 mmol/L (300 mg/dL) significantly reduced (by 15, 20, and 26%, respectively) the apically directed cellular transport of 5-methyltetrahydrofolate (Fig. 1). Similar ethanol treatment did not alter the basolaterally directed transport of 5-methyltetrahydrofolate (data not shown). In these studies, transmembrane transfer (apical to basolateral or vice versa) was negligible (<0.5% of total label), occurred by nonspecific pathways (30), and was not affected by ethanol treatment. Acute ethanol treatment, at concentrations 
87 mmol/L, did not alter the apical transport of -MG by HPT cells, although there was a significant decrease (29%) in transport at 109 mmol/L.
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Effect of 5-d ethanol on folate transport protein content in HPT cells
HPT cells were cultured in media containing ethanol for 5 d as an in vitro model of the effects of subchronic ethanol on expression of folate transporter proteins. This ethanol exposure resulted in an upregulation of the FR and RFC transporter proteins that was dependent on the ethanol concentration. Representative western blots for 2 different cell isolates are shown in Figure 2A,B; the duplex bands for the FR are typical for this protein with polyclonal antibodies, as has been reported by Villaneuva et al. (37). OD data from the blots from 4 different isolates of HPT cells were combined (Fig. 2C). Both the FR and RFC transporter proteins were upregulated with increasing ethanol concentrations (P < 0.001).
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As an in vivo model of the effects of subchronic ethanol on the regulation of the folate transporter proteins, a study was conducted in rats receiving either a nutritionally complete control diet or an ethanol liquid diet for 14 d. On d 13, the mean urine ethanol concentration was 39.3 ± 8.7 mmol/L. All rats had a urine ethanol level 21.7 mmol/L (range 21.7–81.7 mmol/L), except 1 rat that consumed only 2 g of diet overnight, thus resulting in an unusually low ethanol concentration (2.1 mmol/L). The urine folate excretion of the ethanol-treated rats was 9.0 ± 2.3 pmol/24 h, which was greater than that of the control rats (2.5 ± 0.5 pmol/24 h) (P < 0.05). After 14 d of ethanol exposure, rat renal apical membrane fractions were analyzed for folate transporter content. The FR and RFC transporter proteins were upregulated as a result of subchronic ethanol exposure (P < 0.01) (Fig. 3).
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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65 mmol/L) significantly decreased apically mediated cellular transport of 5-methyltetrahydrofolate without affecting the basolateral transport. Apical transport would represent the transport that is involved in the reabsorption of folate in the PT. The effective concentrations of ethanol were similar to those that increased folate excretion in the IPRK and in vivo (19). In contrast, these concentrations of ethanol did not inhibit apically mediated, sodium-dependent glucose transport, which is a unique characteristic of the PT cell (38). Acute ethanol exposure of HPT cells inhibited the glucose transport system, but only at very high concentrations (109 mmol/L), whereas ethanol inhibited folate transport at lower concentrations. These studies have therefore confirmed that acute ethanol directly reduced the transport of 5-methyltetrahydrofolate by the renal PT cell, which then leads to higher concentrations of folate in the lumen and eventually in the urine. Presumably, ethanol must affect the uptake of folate by either the FR or the RFC pathways, because these pathways have been shown to carry out the apically mediated transport in the PT cell (26). Our studies also showed that subchronic exposure of PT cells, both in vivo and in vitro, to folate-depleting concentrations of ethanol led to an upregulation of the folate transport proteins. For the FR transporter protein, the expression in the HPT cells was significantly increased to 1.7-fold and 2.2-fold of the control with 65 and 109 mmol/L ethanol, respectively, and the expression of the RFC transporter protein was significantly increased to 2.5-fold and 4.5-fold of the control, respectively. Ethanol therefore appears to have a modulating effect on the RFC transporter that is greater than its effect on the FR transporter. The various HPT cell isolates examined in these studies produced relatively consistent results. Because the isolates represent normal human kidney tissue from 4 individual patients, there is a vast potential for inter-individual variability. However, in all 4 isolates exposed to ethanol, the protein expression for the RFC was increased to a larger extent than that for the FR. The concentration effect was similar for all isolates, and when data from all isolates were combined, ethanol concentrations of 65 and 109 mmol/L significantly upregulated both folate transporters.
The larger increase in expression observed for the RFC transporter protein, as compared with the FR protein, was also demonstrated to occur for the in vivo model. The protein expression of the RFC transporter in rats administered subchronic ethanol for 2 wk increased 30% compared with controls, whereas the expression of the FR protein increased 20%. This upregulation in both folate transporters, although not as pronounced as in the HPT cells, was significant for both proteins. The urine ethanol concentration in the rats was 39 ± 9 mmol/L, which was below the ethanol level of 65 mmol/L in the HPT cells that was required to produce a significant upregulation in the folate transporter proteins. The lower apparent upregulation in the rats may have resulted from the rats' exposure to a lower concentration of ethanol compared with that in the media of the HPT cells. Alternatively, the transporters in humans may be more sensitive to ethanol than rat transporters. Even so, the increase in protein expression for both the FR and RFC transporters was consistent, with a larger effect observed for the RFC transporter and similar concentrations required for upregulation for both in vitro and in vivo models.
The effect of ethanol on folate excretion is related to the concentration of ethanol; it occurs only when blood ethanol levels increase to 43–54 mmol/L (19). In our studies, the urine ethanol concentrations of nearly all rats that received the ethanol diet were moderately intoxicating (22 mmol/L). This ethanol treatment resulted in a modest increase in urinary excretion of folate, similar to that in previous diet studies (17) but less than that observed after acute ethanol ingestion (13). At these ethanol levels (22–43 mmol/L), upregulation of the folate transport proteins was modest but significant. In the HPT cell studies, upregulation of the folate transporters at a concentration of 22 mmol/L was minimal, but was marked and significant at ethanol concentrations 65 mmol/L. Thus, the upregulation occurs only at very high ethanol levels, possibly as a response to the acute decrease in folate transport that occurs at these levels.
In contrast to the acute ethanol treatment, 5-d pretreatment with ethanol did not significantly affect 5-methyltetrahydrofolate transport by HPT cells. The absence of an effect of ethanol on folate transport after 5 d, compared with the significant decrease in transport after 1 h, could be explained by the significant increase in folate transport protein content (both FR and RFC) that also occurred after 5 d of ethanol exposure. The increase in folate transporter content should contribute to an increase in transport, thereby counteracting the inhibitory effects of acute ethanol exposure on folate transport activity and leading to the lesser overall effect on transport that occurs with subchronic exposure.
Urinary folate excretion has been shown to be markedly increased 5-fold after acute ethanol exposure but only 1-fold after chronic ethanol ingestion (12 wk) (16,17). In our studies, subchronic exposure to ethanol for 2 wk increased urinary folate excretion almost 3-fold, yet also produced an upregulation of folate transport proteins. As in the cellular studies, the increase in transport protein content should increase the uptake of folate, thereby counteracting the acute ethanol-induced decrease in folate transport activity. This upregulation of folate transporters could therefore explain the lesser effect on urine folate excretion in chronic ethanol studies, compared with the 5-fold increase after acute ethanol ingestion.
It is difficult to explain how ethanol increases folate excretion yet upregulates folate transporters. Previous studies have shown that the FR and RFC operate in the apical transport of folate independently (26). Which of these pathways is inhibited by ethanol cannot be determined from this data; studies with genetic or pharmacologic manipulation of the transporters will be needed. These studies suggest that ethanol does not appear to affect the interaction of 5-methyltetrahydrofolate with the FR protein per se, although it could inhibit flux through this pathway at a downstream event, such as the processing of endocytotic vesicles or the acidification of vesicles. Similarly, ethanol could indirectly inhibit the RFC transport activity at a downstream or regulatory step. In either case, the increased expression of the transporter could counteract the ethanol effect downstream by increasing the overall stream (increasing the number of folate-transport protein interactions by upregulating the proteins). This increased interaction at the membrane would reduce the downstream inhibition and result in a lower increase in urinary folate excretion, as noted above when comparing the magnitude of the acute and chronic ethanol exposures. An alternative explanation for how ethanol continues to increase urine folate excretion when apical folate transporters are upregulated may be that ethanol's effects are independent of the transporters, such as an alteration of glomerular filtration or renal blood flow. We cannot discount such effects, except that previous studies in rats did not indicate an effect of ethanol on glomerular filtration or on delivery of folate to the kidney (20).
Recent studies have shown that chronic ethanol exposure of rats for 12 wk decreases the binding and uptake of folic acid by isolated renal cortical BBM (39,40). The authors suggest that the reduced binding and uptake by PT cells represents 1 mechanism by which chronic ethanol exposure increases urinary folate excretion. The decrease in transport appears to result from a decrease in affinity of transporters for folic acid as well as an apparent decrease in the number of transport sites (40). Because most of the uptake of folate in their preparations was due to binding, the decrease in number of sites suggests an ethanol-induced decrease in the content of binding proteins. Our data do not support that decreased folate binding is a mechanism for the decrease in folate reabsorption; instead, our data support that ethanol inhibits reabsorption by an effect on an uptake mechanism downstream of the binding activity. In our studies, 5-d ethanol did not reduce 5-methyltetrahydrofolate binding activity by PT cell apical membranes and subchronic ethanol (2 wk) increased the content of the FR (folate-binding protein) in the rat kidney apical membranes, suggesting an increase in binding over longer exposures. However, this study measured the content of the FR by western blotting, not its binding activity per se, and also measured binding activity only after acute ethanol treatment. In contrast, the studies by Hamid and Kaur (39,40) used folic acid (not 5-methyltetrahydrofolate, the natural ligand) to measure binding and uptake by the BBM vesicles. Folic acid, because of its differences in affinity for the 2 transporters, would primarily measure effects on the FR, not the RFC. As noted here, subchronic ethanol had a greater effect on the RFC than on the FR. Hence, the methodological differences between these 2 studies do not allow a conclusive resolution at this time.
In summary, acute ethanol was shown to inhibit the apical transport of 5-methyltetrahydrofolate in cultured HPT cells, which confirms previous studies showing a decreased reabsorptive uptake from the tubular lumen and an increased amount of folate in the urine. In the subchronic ethanol studies in HPT cell isolates, increasing concentrations of ethanol resulted in an upregulation of the folate transporters. In male Sprague Dawley rats as an in vivo model, the FR and RFC transporter proteins were both upregulated in the rats receiving the ethanol diet. The subchronic ethanol studies, conducted in vitro and in vivo, have thus demonstrated that administration of intoxicating levels of ethanol results in a significant upregulation of both the FR and RFC transporter proteins. The ethanol-mediated protein upregulation is probably a compensatory response to counteract the effects of ethanol in inhibiting the reabsorption of folate. This effect also functions in the long term to diminish the excess loss of folate via the urine.
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FOOTNOTES |
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2 Author disclosures: R. L. Romanoff, no conficts of interest; D. M. Ross, no conficts of interest; and K. E. McMartin, no conficts of interest.
3 Abbreviations used: -MG, -methylglucopyranoside; BBM, apical or brush border membrane; FR, folate receptor; HPT, human proximal tubule (cells); IPRK, isolated perfused rat kidney; PT, proximal tubule; RFC, reduced folate carrier; TER, transepithelial resistance.
Manuscript received 12 July 2006. Initial review completed 18 August 2006. Revision accepted 25 January 2007.
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LITERATURE CITED |
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