Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression

Abstract

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr^96/101. We found that fructose (5 mM) increased transport-related O₂ consumption in TALs by 11.1 ± 3.2% (P < 0.05). The effect of fructose on O₂ consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% (P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5,  49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre^96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

INTRODUCTION

The thick ascending limb (TAL) of the loop of Henle plays an important role in the maintenance of salt homeostasis and blood pressure regulation (36). The TAL reabsorbs up to 25% of NaCl via the apical Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) (10, 35). Since the TAL reabsorbs NaCl, but not water, it dilutes the urine forming in the lumen (7, 9). NKCC2 is expressed in the apical membrane and subapical space of medullary and cortical TALs and also in macula densa cells (20, 25, 54, 59). The importance of NKCC2 in renal physiology is evident in humans, since mutations in NKCC2 cause Bartter’s syndrome, a disorder characterized by salt wasting, dehydration, and hypotension (62, 66, 68, 70). Opposite to Bartter’s syndrome, NKCC2 activity is enhanced in animal models of salt-sensitive hypertension (5, 42, 45–47).

Fructose consumption has increased dramatically in the last 40 years, reaching a maximum in 2010 (32, 71) because of its use as a sweetener. After ingestion, fructose is absorbed into the bloodstream; ~75% is metabolized by the liver, while the rest remains in circulation (71). Fructose is an isomer of glucose, which shares a similar molecular composition but not structure. Fructose is filtered, and a fraction is eliminated in urine (21, 34, 71). Excessive consumption of fructose has been linked to renal damage (28, 65) and hypertension (including salt-sensitive hypertension) (11, 37, 67). However, little is known about the effects of fructose on renal NaCl reabsorption, and its effect in TALs has not been studied. Under normal conditions, glucose filtered through the glomeruli is completely reabsorbed by the proximal tubule. However, fructose is not completely reabsorbed along the nephron, and a fraction (up to 50%) of filtered fructose is excreted in the urine (49). Therefore, fructose reaches all nephron segments. In the proximal tubule, fructose acutely stimulates Na⁺/H⁺ exchanger 3 activity and enhances the stimulatory effect of angiotensin II (11, 63). However, the effect of fructose in the loop of Henle or distal nephron has not been studied.

We previously found a direct relationship between apical surface NKCC2 expression and NKCC2 activity (1, 12, 56). NKCC2 activity may also be stimulated by phosphorylation at the amino-terminal Thr^96/101 (29, 30, 61). The effects of fructose on apical surface NKCC2 expression, phosphorylation at Thr^96/101, and, ultimately, NKCC2 activity have not been studied. We hypothesize that fructose, but not glucose, acutely enhances TAL-dependent NaCl absorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr^96/101.

METHODS

Suspensions of medullary TALs.

Suspensions of medullary (1, 12, 56) TALs were obtained from male Sprague-Dawley rats (200–250 g body wt; Charles River Breeding Laboratories, Wilmington, MA), which were maintained on a normal diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) with water ad libitum for ≥7 days. On the day of the experiment, the rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). The abdominal cavity was opened, and the kidneys were perfused retrogradely via the aorta with physiological solution (PS) containing 0.1% collagenase (Sigma, St. Louis, MO) and 100 U/ml heparin. The inner stripe of the outer medulla was cut from coronal slices, minced, incubated at 37°C for 30 min with 0.1% collagenase in PS, and gassed every 5 min with 100% O₂. The tissue was pelleted by gentle centrifugation at 120 g for 2 min, suspended in chilled PS, and stirred on ice for 30 min to release the tubules. The suspension was filtered through a 250-μm nylon mesh and centrifuged at 120 g for 2 min. The pellet was washed, centrifuged again, and finally suspended in 1 ml of chilled PS. The composition of the PS was (in mM) 130 NaCl, 2.5 NaH₂PO₄, 4.0 KCl, 1.2 MgSO₄, 6 l-alanine, 2 nitro-l-arginine methyl ester, 1.0 Na-citrate, 5.5 glucose, 2.0 Ca-lactate, and 10 HEPES, pH 7.40. All animal protocols were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

Transport-dependent O₂ consumption.

O₂ consumption is directly related to NaCl transport in the TAL. The loop diuretic furosemide completely inhibits NKCC2-dependent NaCl transport, which represents ~40% of total O₂ consumption by TALs (14, 22, 35, 52). An aliquot of freshly obtained TALs was suspended in 0.1 ml of bicarbonate-buffered PS (in mM: 114 NaCl, 2.5 NaH₂PO₄, 4.0 KCl, 1.2 MgSO₄, 6 l-alanine, 1.0 Na-citrate, 5.5 glucose, 2.0 Ca-lactate, and 25 NaHCO₃, pH 7.40) bubbled with 95% O₂-5% CO₂ at 37°C. Solutions were maintained at 37°C throughout the experiment to avoid air bubble formation caused by changes in O₂ solubility due to changes in temperature. O₂ consumption was measured continuously in a sealed chamber equilibrated at 37°C with a small injection port by a Clark-type electrode, as we described previously (26, 52, 58). After a constant O₂ consumption rate was established, 10 µl of freshly prepared fructose or glucose were added to reach 5 mM concentration in the chamber (in the case of glucose, the final concentration reaches 10.5 mM). Once the slope was constant, furosemide (100 μM) was added. O₂ consumption was measured as nanomoles of O₂ per minute per milligram of protein. On average, furosemide (100 μM) inhibited O₂ consumption by 39.2 ± 3.1% (n = 7, P < 0.05). At the end of the experiments, an aliquot of the suspension was used to determine protein content by the Bradford method. Data are expressed as percent change from baseline O₂ consumption. All experiments were completed within 15 min, as time controls showed no change in O₂ consumption rate during this time period (data not shown).

Measurement of NKCC activity by thallium influx in isolated perfused rat TALs.

This method was originally developed to measure K⁺ channel activity (6, 73) and was later adapted to measure NKCC1/NKCC2 activity in cell cultures (13, 27). It is very similar to a method we developed earlier using the Na⁺-sensitive dye Na⁺-binding benzofuran isophthalate (58). Briefly, after anesthesia, the rat abdominal cavity was opened and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated PS. Cortical TALs were dissected from the slices under a stereomicroscope at 4–10°C. TALs (0.5–1.0 mm long) were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C, as described previously (1, 57, 58). Isolated TALs were perfused through the lumen with PS containing 0 mM NaCl and 0 μM Tl⁺ and Tl⁺-sensitive fluorogenic indicator dye (FluxOR, Life Technologies, Carlsbad, CA) during the 30-min dye-loading period. Once inside the cell, the nonfluorescent acetoxymethyl ester form of the FluorOx dye is cleaved by endogenous esterases into a Tl⁺-sensitive indicator. To avoid extrusion of the dye through organic anion pumps, 125 µM probenecid was added to the bath solution and was present throughout the experiment, as performed by others (6, 13, 27, 73). Loading of the dye was followed by a 10-min wash period. After the dye was loaded, TALs were perfused through the lumen with PS containing 0 mM NaCl and 25 µM Tl⁺ and allowed to stabilize for 5 min at 37 ± 1°C. After the stabilization period, TALs were imaged on a Nikon TE2000 microscope (×100 magnification, 1.33 numerical aperture) and excited at 488 nm in a laser-scanning confocal microscope (VisiTech International, Sutherland, UK), and emission >500 nm was collected every 1 s. Experiments consisted of two periods. In the first period (baseline), we measured the initial rate of Tl⁺ entry at baseline conditions caused by switching the luminal solution from 0 to 130 mM NaCl while the concentration of Tl⁺ in the luminal solution was 25 µM. Fluorescence at multiple regions of interest encompassing individual TAL cells within a tubule was recorded at 1-s intervals for 2–3 min to determine the initial rate of Tl⁺ entry. After the initial rate of Tl⁺ entry at baseline conditions was obtained, the luminal solution was switched back to perfusion solution containing 0 mM NaCl and 0 µM Tl⁺ (to allow return to the baseline fluorescence) and fructose was added to the PS to reach a concentration of 5 mM. After 15 min of treatment, the luminal solution was changed to 0 mM NaCl and 25 µM Tl⁺ in the presence of 5 mM fructose. After a total of 20 min of treatment with 5 mM fructose, luminal PS was switched again from 0 to 130 mM NaCl containing 5 mM fructose, and fluorescence intensity [relative fluorescence units (RFU)] was measured over time. The initial rates of Tl⁺ influx (measured as RFU/s) were calculated for each individual region of a tubule and averaged. Time-control experiments showed no significant difference between measurements on the same tubule separated by 20-min periods (first measurement was 7.27 ± 1.4 RFU/s, and the second response after 20 min was  6.61 ± 0.9 RFU/s, n = 6), indicating no leakage of the dye, decreased sensitivity, or cell death.

Western blotting.

TALs were lysed with buffer containing 150 mM NaCl, 50 mM Tris·HCl, pH 7.4, 5 mM EDTA, 2% (vol/vol) Triton X-100, 0.2% (wt/vol) sodium dodecyl sulfate (SDS), protease inhibitors [10 µg/ml aprotinin, 5 µg/ml leupeptin, 4 mM benzamidine, 5 µg/ml chymostatin, and 5 µg/ml pepstatin A (Sigma, St. Louis, MO)], and phosphatase inhibitor cocktail (Roche, Branford, CT). Total protein content was measured in triplicate by colorimetric assay (Pierce Biotechnology) using the Bradford method. The lysates were boiled for 1 min and then spun at 4°C for 5 min at 10,000 g, loaded into each lane of a 6% SDS-polyacrylamide gel, separated by electrophoresis, and transferred to Immobilon-polyvinylidene difluoride membranes (Millipore, Bedford, MA). Primary antibody [rabbit anti-rat NKCC2 directed to amino acids 859–873 in rat NKCC2 COOH terminus (produced by GenScript, Piscataway, NJ)] was incubated for 120 min at room temperature in blocking buffer (containing 2% bovine serum albumin) at 1:20,000 dilution (1, 3, 4). For phosphorylated (Thr^96/101) NKCC2, we used a rabbit polyclonal antibody that reacts with NKCC2 only when it is phosphorylated at Thr^96/101, generated using the R5 antibody sequence optimized for rat (2, 29, 30). Monoclonal anti-GAPDH (Chemicon, Temecula, CA) was used at 1:10,000 dilution. Horseradish peroxidase-labeled secondary antibodies were detected by chemiluminescence and quantified by densitometry. Exposure times and amount of protein loaded were optimized so that each sample fell within the linear range of optical density.

Surface biotinylation of TAL suspensions.

Cell surface biotinylation of TAL suspensions was performed as we described previously (1, 3, 4, 56). Tubule suspensions from the same animal were divided into two, three, or four aliquots of equal volume. TALs were equilibrated for 20 min at 37°C and gassed every 5 min with 100% O₂. After equilibration, TALs were treated with vehicle or fructose/glucose (or other drugs of interest) for 20 min at 37°C, while they were gassed every 5 min. After treatments, suspensions were rapidly cooled to 4°C, washed twice with chilled PS, and centrifuged at 120 g for 2 min at 4°C. TALs were incubated with 0.75 ml of chilled biotinylation solution [HEPES-Ca²⁺-Mg²⁺ buffer (10 mM HEPES, 130 mM NaCl, 2 mM MgSO₄, 1 mM CaCl₂, and 5.5 mM glucose), pH 7.8– 8.0] containing 1.2 mg/ml NHS-SS-biotin (Pierce Biotechnology) in a rocker platform at 4°C for 15 min. Then 0.75 ml of freshly prepared NHS-SS-biotin (1.2 mg/ml) was added on top, and the samples were incubated for an additional 15 min. After biotinylation, tubules were washed three times at 4°C with PS containing 100 mM glycine to remove the excess NHS-SS-biotin. TALs were centrifuged (120 g) and lysed in buffer containing 150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 2% Triton X-100, 0.2% SDS, pH 7.5, and protease inhibitors [10 µg/ml aprotinin, 5 µg/ml leupeptin, 4 mM benzamidine, 5 µg/ml chymostatin, and 5 µg/ml pepstatin A (Sigma Aldrich)]. We previously found that this lysis buffer solubilizes most of the NKCC2 from TAL suspensions. Total protein content in each sample was measured in duplicate by colorimetric assay using the Bradford method (Pierce Biotechnology). Equal amounts of protein (50–75 μg) were incubated overnight at 4°C with streptavidin-coated agarose beads (10%) in lysis buffer. Beads were pulled down by centrifugation, and the supernatant was reincubated with streptavidin-coated agarose beads (10%) for 2 h at 4°C. The supernatant was saved for determination of intracellular NKCC2, whereas the beads were centrifuged and pooled with the beads from the first round. Beads were then washed twice in lysis buffer, twice in high-salt buffer (500 mM NaCl-50 mM HEPES, pH 7.4), and twice in no-salt buffer (50 mM HEPES, pH 7.4). Biotinylated proteins were extracted from the beads by boiling in 60 μl of SDS-loading buffer containing 50 μM dl-dithiothreitol and 5% β-mercaptoethanol. Proteins were resolved by SDS-PAGE (6% gels), and NKCC2 in the membrane was detected by Western blotting. In every experiment, 1/10th of the supernatant, containing intracellular nonbiotinylated proteins, was loaded in the same gels as NKCC2 samples recovered from the beads and resolved, and NKCC2 was measured by Western blotting. Optical densities from surface and intracellular NKCC2 bands were used to obtain the percent change from baseline (control) in the surface or total expression of NKCC2.

Statistical analysis.

Values are means ± SE. For paired protocols (O₂ consumption and Tl⁺ influx assay), Student’s t-test was used to determine differences in means between controls and treated groups. For surface biotinylation protocols, one-way analysis of variance was used to determine differences between means within different treatment groups. P < 0.05 was considered significant.

RESULTS

Effect of acute fructose on transport-related O₂ consumption.

Filtered fructose is excreted in urine and, therefore, reaches the distal nephron (49). The physiological effect of fructose on TAL-dependent NaCl reabsorption is not known. Part of our hypothesis is that fructose, but not glucose, acutely enhances TAL-dependent NaCl absorption. To begin studying the effect of fructose on TAL-dependent NaCl absorption in rats, we used transport-related O₂ consumption as an indirect measurement of Na⁺ transport in TALs. Suspensions of rat TALs were equilibrated for 20 min at 37°C. Each TAL suspension was then placed in a 600-µl air-sealed chamber, and baseline O₂ consumption was measured for 2–4 min. Then fructose was added to the chamber to achieve a final concentration of 5 mM, and O₂ consumption was continuously measured for a total of 15 min. Fructose treatment (5 mM) increased O₂ consumption from 207.6 ± 18.5 to 231.6 ± 22.0 nmol O₂·min⁻¹·mg protein⁻¹, an 11.5 ± 3.2% increase (P < 0.01; Fig. 1). To determine whether the stimulatory effect was selective for fructose, we studied the effect of adding the same concentration of glucose (5 mM) to the chamber. Glucose is an isomer of fructose that shares the same molecular composition, but glucose and fructose differ in their atomic arrangement; because glucose produces the same small increase in osmolality as fructose, it is a good control. Addition of glucose to the bath did not change O₂ consumption (from 184.6 ± 32.9 to 180.1 ± 29.9 nmol O₂·min⁻¹·mg protein⁻¹, −1.9 ± 1.2% change from baseline, n = 6, not significant), indicating that the effect of fructose is unique and not based on enhanced energy production from carbohydrates. To study whether the effect of fructose was caused by stimulation of NKCC2-mediated transport, in a separate group of TALs, we first inhibited NKCC2 with furosemide (100 µM), measured baseline O₂ consumption, and then added fructose (5 mM) to the chamber. Under these conditions, fructose did not increase O₂ consumption (from 100.4 ± 3.7 to 98.5 ± 3.6 nmol O₂·min⁻¹·mg protein⁻¹, n = 4, not significant). Together, our data suggest that fructose, but not glucose, increases transport-related O₂ consumption in TALs. This effect is likely mediated by stimulation of NKCC2, and not by enhanced metabolism, because it was not observed after inhibition of NKCC2 with furosemide.

Fig. 1.

Effect of fructose on transport-related O₂ consumption. Fructose stimulates O₂ consumption in thick ascending limbs. Cumulative data show the stimulatory effect of 5 mM fructose, but no effect of 5 mM glucose, on O₂ consumption. In thick ascending limbs pretreated with furosemide to inhibit Na⁺-K⁺-2Cl⁻ cotransporter activity, 5 mM fructose did not stimulate transport-related O₂ consumption: 11.5 ± 3.2%, −1.9 ± 1.2%, and −0.8 ± 0.6% change from baseline with fructose (n = 6), glucose (n = 6), and furosemide + fructose (n = 4), respectively. Values are means ± SE. *P < 0.05.

Effect of acute fructose on NKCC2 activity.

Since fructose increases transport-related O₂ consumption, we measured whether fructose directly increases NKCC2-mediated ion transport in TALs by measuring the initial rate of apical ion entry through NKCC2 in isolated and perfused rat TALs. We adapted an assay, previously developed by others to measure NKCC1 activity (14), that involves activation of a fluorescent dye by the cation Tl⁺, which is used as a K⁺ tracer (6, 73). We performed ion-substitution experiments similar to those we developed previously for isolated perfused TALs (58). NKCC2 provides the only known K⁺ entry pathway in the apical membrane of TALs. Thus we measured the initial rate of Tl⁺ entry caused by a switch in luminal NaCl solution from 0 to 130 mM in isolated and perfused rat TALs, similar to a method we developed previously (58). We found that incubation of rat TALs with 5 mM fructose in the lumen of the tubule increased the initial rate of Tl⁺ entry by 58.5 ± 16.9% (from 5.1 ± 1.0 to 7.8 ± 1.7 RFU/s, n = 6, P < 0.05; Fig. 2). In a different set of tubules, we tested whether Tl⁺ entry was dependent on NKCC2 activity. The protocol was repeated, except, after the first response to the luminal NaCl switch, we added furosemide (150 µM), instead of fructose. The increase in Tl⁺ entry caused by a switch in luminal NaCl was completely inhibited in the presence of furosemide in the luminal perfusion solution: 7.68 ± 1.6 RFU/s at baseline and  0.29 ± 0.2 RFU/s with furosemide (n = 4). These data indicate that fructose stimulates NKCC2-mediated ion entry in the TAL.

Fig. 2.

Effect of fructose on Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) activity. Fructose stimulates NKCC2-mediated ion transport in thick ascending limbs (TALs), as measured by Tl⁺ influx assay in isolated and perfused TALs. A: cumulative data showing a time-control effect on NKCC2 activity: 7.3 ± 1.4 and 6.6 ± 0.9 relative fluorescence units (RFU)/s at time 0 and 20 min, respectively (n = 6). B: cumulative data showing effect of 5 mM fructose on NKCC2 activity in TALs: 5.1 ± 1.0 and 7.8 ± 1.7 RFU/s at baseline and with fructose, respectively (n = 6). C: cumulative data showing effect of treatment with 300 µM furosemide for 20 min on NKCC2 activity: 7.7 ± 1.6 and 0.3 ± 0.2 RFU/s at baseline and with furosemide, respectively (n = 4). Values are means ± SE. *P < 0.05.

Effect of acute fructose on surface NKCC2 expression.

Surface NKCC2 expression is directly related to TAL-dependent NaCl reabsorption (1, 12, 56). The mechanism by which fructose increases NKCC2-mediated ion entry in the TAL is not known. To study whether the effect of fructose was caused by increased NKCC2 trafficking to the apical membrane, we used surface biotinylation of TAL suspensions and Western blotting to measure surface and intracellular NKCC2. Suspensions of TALs were divided into four samples, equilibrated for 20 min at 37°C, and then treated for 20 min with vehicle or increasing concentrations (1, 5, and 10 mM) of fructose. We found that fructose increased surface NKCC2 levels in a concentration-dependent manner: 122.4 ± 5.2% with 1 mM, 149.8 ± 10.2% with 5 mM, and 201.9 ± 59.3% with 10 mM fructose [P > 0.01 vs. basal (100%), n = 4; Fig. 3]. Total expression of NKCC2 was not changed after treatment with fructose, and the intracellular protein GAPDH was not detected in the surface protein fraction. These data indicate that fructose increases surface NKCC2 levels in TALs. Our data indicate that an increase in surface NKCC2 levels during the 20-min treatment is caused by a change in NKCC2 trafficking into the plasma membrane, rather than an increase in total NKCC2 expression.

Fig. 3.

Effect of fructose on surface Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) levels. Fructose acutely increases surface NKCC2 levels in thick ascending limbs. A: representative Western blots showing surface and intracellular NKCC2 levels in thick ascending limbs treated with vehicle (baseline, lane 1), 1 mM fructose (lane 2), 5 mM fructose (basal, lane 3), and 10 mM fructose (lane 4). B: concentration-response curve showing effect of fructose: 122.4 ± 5.2%, 149.8 ± 10.2%, and 201.9 ± 59.3% change from basal (100%) with 1, 5, and 10 mM fructose, respectively [n = 5, P > 0.01 vs. basal (100%)]. Values are means ± SE. *P < 0.05 vs. basal.

Effect of glucose or other hexoses on surface NKCC2 levels.

To test whether the increase in surface NKCC2 levels caused by fructose was unique to this monosaccharide, and not caused by changes in osmolality, metabolism, or merely addition of a sugar, we studied whether glucose or other fructose metabolites could increase surface NKCC2 levels. We repeated the previous protocol testing the effect of 5 mM fructose, 5 mM glucose, or 5 mM fructose 1,6-bisphosphate. Suspensions of TALs were equilibrated for 20 min at 37°C and then treated for 20 min with vehicle, fructose, glucose, or fructose 1,6-bisphosphate. We found that only fructose increased surface NKCC2 levels, whereas glucose or fructose 1,6-bisphosphate did not: 101.2 ± 7.0% with 5 mM glucose,  92 ± 5.6% with 5 mM fructose 1,6-bisphosphate, and 131.2 ± 8.8% with 5 mM fructose [n = 7, P < 0.05 vs. basal (100%); Fig. 4]. Our data indicate that fructose increases surface NKCC2 levels, but glucose does not.

Fig. 4.

Effect of fructose and glucose on surface Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) levels. Fructose acutely increases surface NKCC2 levels in thick ascending limbs. A: representative Western blots showing surface and intracellular NKCC2 levels in thick ascending limbs treated with vehicle (basal, lane 1), 5 mM glucose (lane 2), 5 mM fructose (lane 3), and 5 mM fructose 1,6-bisphosphate (fructose 1,6-biPO₄, lane 4). B: cumulative data showing effect of glucose, fructose, and fructose 1,6-bisphosphate on surface NKCC2 levels: 101.2 ± 7.0%,  131.2 ± 8.8%, and 92 ± 5.6% change from basal (100%) for 5 mM glucose, 5 mM fructose, and 5 mM fructose 1,6-biPO₄ (n = 7). Values are means ± SE. *P > 0.05 vs. basal (100%).

Effect of fructose on NKCC2 phosphorylation at Thr^96/101.

Phosphorylation of NKCC2 at Thr^96/101 has been associated with an increase in NKCC2 activity (29, 30, 61). Therefore, we studied the effect of acute fructose treatment on NKCC2 phosphorylation. Suspensions of TALs were equilibrated for 20 min at 37°C and then treated for 20 min with vehicle or increasing concentrations (1, 5, and 10 mM) of fructose. We found that fructose had no effect on NKCC2 phosphorylation at Thr^96/101: 114 ± 9% with 1 mM fructose, 108 ± 29% with 5 mM fructose, and 114 ± 43% with 10 mM fructose (n = 4, not significant; Fig. 5). Together, these data indicate that the increase in NKCC2 activity caused by acute fructose treatment is likely not related to enhanced NKCC2 phosphorylation at Thr^96/101, whereas enhanced NKCC2 trafficking is involved.

Fig. 5.

Effect of fructose on Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) phosphorylation at Thr^96/101. Acute (20 min) fructose treatment does not stimulate phosphorylation of NKCC2. Representative Western blot shows phosphorylation at Thr^96/101 and total NKCC2 expression in thick ascending limbs under baseline conditions or treated with fructose (1, 5, and 10 mM) for 20 min (n = 4).

DISCUSSION

Fructose consumption in humans has increased steadily from 1975, reaching a plateau in 2010. Excess fructose consumption has been implicated in the development of hypertension, diabetes, and obesity (28, 38–41, 53, 65). However, very little is known about the effect of fructose per se on renal function and tubular NaCl reabsorption. This is important, because the kidneys play a critical role in maintaining electrolyte and water balance and, thus, blood pressure. Fructose is metabolized very differently from glucose (51). Plasma fructose rises after oral ingestion; however, unlike glucose, after being filtered at the glomeruli, fructose is not completely reabsorbed by the proximal tubule. Therefore, a fraction of filtered fructose is excreted in urine after its oral ingestion (23, 49). Two reports indicate that fructose rapidly and directly stimulates proximal tubule Na⁺ transport (11, 63). However, the effects of fructose downstream from the proximal tubule have not been studied. We tested the hypothesis that fructose directly stimulates TAL-dependent NaCl reabsorption by enhancing NKCC2 activity. We provide evidence that fructose, but not glucose, stimulates O₂ consumption and NKCC2-mediated ion transport by TALs. The acute effect of fructose on NKCC2 activity is likely mediated by an effect on trafficking, because 20 min of treatment with fructose increased surface NKCC2 expression without affecting NKCC2 phosphorylation at Thr^96/101. To our knowledge, these are the first data showing that fructose acutely stimulates NKCC2-mediated NaCl reabsorption. The effect of fructose on NKCC2 trafficking and activity may in part explain the rise in blood pressure in rats fed both fructose and high salt (11), but this remains to be studied.

The overall effect of fructose intake on metabolism, bioavailability, and plasma levels was originally studied in rats, and this knowledge was later extrapolated to humans (53, 71). In humans and rats, baseline plasma fructose is maintained at 0.05–0.4 mM, depending on the fructose content of the diet (19, 44, 69). After an oral fructose load, plasma fructose levels rise to 0.6–2 mM (17, 55, 60, 72). Plasma fructose peaks within 20 min and decays within 180 min after consumption (17) as a result of hepatic and renal metabolism (primarily in the proximal tubule) and also urinary excretion (71). This is an important distinction between glucose and fructose, because a large fraction (up to 50%) of filtered fructose is excreted in urine (49). In humans, urinary fructose ranges from 0 at baseline to 7.8 mM after a fructose load (48). To our knowledge, the concentration of luminal fructose along the nephron after it is filtered has not been measured. However, fructose that escapes proximal tubule reabsorption may concentrate in the nephron lumen and could act directly on TALs to enhance NKCC2 activity. It is not known whether there are transport mechanisms that mediate fructose reabsorption beyond the proximal tubule.

Approximately 40% of O₂ consumed by TALs is required to maintain NKCC2-dependent NaCl transport (14, 22, 52). Fructose (5 mM) increased O₂ consumption by 11%, representing a 25–30% stimulation of NaCl transport. The effect of fructose was absent in furosemide-treated TALs, indicating that enhanced O₂ consumption was not due to an increased mitochondrial metabolic rate. Glucose (5 mM) did not affect O₂ consumption, ruling out an osmotic or metabolic effect of fructose on TALs. While transport-related O₂ consumption provides an estimate of NaCl transport in TALs, it does not specifically measure NKCC2-dependent NaCl reabsorption. Therefore, we studied the effect of fructose on NKCC2 activity by measuring the initial rate of NKCC2-dependent Tl⁺ influx. This method has been used by other investigators to measure NKCC1 and NKCC2 activity in different cells (13, 27). We adapted this method to isolated and perfused TALs. We found that this method has great sensitivity, because of the large increase in fluorescence caused by Tl⁺ entry, and specificity, because the Tl⁺ influx from the tubule lumen was completely absent in the presence of furosemide, making this a very robust protocol for measurement of NKCC2 activity. We treated isolated perfused TALs with 5 mM fructose for 20 min. Our data show that fructose stimulates NKCC2-mediated Tl⁺ entry in TALs (Fig. 2). Together, these data show, for the first time, that fructose stimulates NKCC2-mediated ion transport. Our data suggest that fructose could contribute to regulation of NaCl homeostasis and TAL function and, perhaps, could be involved in increases in blood pressure. Our data also show that glucose, at physiological concentrations (5–10 mM), does not directly affect TAL NaCl transport or metabolism, since no changes in O₂ consumption were observed.

We previously showed that one of the mechanisms by which NKCC2 activity is regulated is NKCC2 trafficking into and out of the apical membrane (1, 3, 12, 56). The mechanism by which fructose stimulates NKCC2 activity is not known. Therefore, we tested the effect of a concentration-response curve within the physiological range (1–10 mM) of fructose on surface NKCC2 levels. Our data show that fructose increases surface NKCC2 levels in a concentration-dependent manner. We believe that this stimulatory effect is very potent compared with the magnitude of the response to β-adrenergic receptor agonists or arginine vasopressin, which stimulate surface NKCC2 in TALs by 25–40% (12, 29, 33). To our knowledge, this is the first evidence that fructose has an acute effect on NKCC2 trafficking in the kidney.

To rule out the possibility of an osmotic or a metabolic effect of fructose, we first used glucose as a control, because it is an isomer of fructose that shares a similar molecular structure. Interestingly, 5 mM glucose did not increase surface NKCC2 levels (Fig. 4), indicating that the effect of fructose on surface NKCC2 levels is not due to an osmotic or a metabolic effect. Additional controls were performed by incubation of TALs with the glucose metabolite fructose 1,6-bisphosphate. The latter did not affect surface NKCC2 levels in TALs, ruling out an osmotic effect. Phosphorylated sugar metabolites are not transported into cells via fructose or glucose transporters (24). Thus it is expected that fructose metabolites would not directly affect NKCC2 trafficking. Together, these data indicate that fructose, but not glucose, increases surface NKCC2 levels and its transporter activity by a mechanism independent of an osmotic effect.

The signaling mechanism initiated by fructose that leads to increased surface NKCC2 and activity is unclear. In the proximal tubule, fructose is transported into the cell via the glucose transporter GLUT5 (16, 18). There have been no studies on fructose transporters in the TAL, while glucose transporters are known to be expressed in the TAL (15). GLUT2 and GLUT5 are the main hexose transporters with specificity for fructose, but GLUT5 seems to be restricted to proximal tubules, raising the possibility that GLUT2 may be expressed in TALs. In the liver, upon entering cells through GLUT2, fructose is partly metabolized by aldolase B to glyceraldehyde 3-phosphate (21, 34, 39); however, there is no evidence of this signaling pathway in the TAL. Previous studies showed that aldolase B is associated with the actin cytoskeleton and plays a role in protein trafficking (43, 50). Benziane et al. identified aldolase B as a binding partner of NKCC2 and showed that overexpression of aldolase B produced a downregulation of surface NKCC2 expression in cultured cells (8). The aldolase-dependent decrease in surface NKCC2 expression was accompanied by a decrease in NKCC2 activity. Aldolase B forms a complex with scaffold proteins involved in endocytosis, such as sorting nexin 9 and dynamin 2 (50). We previously showed that plasma membrane NKCC2 levels are maintained by a balance between exocytic delivery and endocytic retrieval and that dynamin 2 mediates NKCC2 endocytosis in TALs (1). Therefore, it could be speculated that fructose affects aldolase B activity, and this may inhibit NKCC2 endocytosis. However, it remains unknown whether the signaling pathway induced by fructose or the protein-protein interactions could be affected by fructose in the TAL. These possibilities, including the potential effect of fructose on other molecular mechanisms, such as exocytosis, are very interesting and should be studied in detail in the future.

In addition to NKCC2 trafficking, phosphorylation of NKCC2 at Thr^96/101 is associated with enhanced NKCC2 activity (29, 30, 61), and this phosphorylation is mediated by Ste20p-related proline/alanine-rich kinase (SPAK) or odd-skipped related transcription factor 1 (OSR1) kinases (15, 31, 64). We found that phosphorylation of NKCC2 at Thr^96/101 was not changed by acute fructose treatment. These data suggest that the acute (20–30 min) stimulation of NKCC2 by fructose is likely mediated by enhanced apical surface levels of NKCC2, rather than enhanced phosphorylation at Thr^96/101.

Cabral et al. showed that fructose acutely stimulates Na⁺/H⁺ exchanger 3 activity in the proximal tubule (11). We found that fructose acutely stimulates NKCC2 activity. However, it is unclear how these effects relate to blood pressure regulation. Cabral et al. found that chronic fructose intake (20% in drinking water) did not increase blood pressure in Sprague-Dawley rats fed a normal-salt diet for 2 wk. However, when combined with high salt intake, chronic fructose intake increased blood pressure. Thus it is possible that, despite increasing proximal tubule and TAL-dependent NaCl reabsorption, compensatory mechanisms exist to prevent an increase in blood pressure during a normal-salt diet. However, similar to genetic models of salt-sensitive hypertension, with consumption of a high-salt diet, fructose may prevent adequate salt handling by the kidney perhaps due to its actions in the TAL.

In summary, we found that, in normal Sprague-Dawley rats, fructose at concentrations found after ingestion acutely stimulates NKCC2 activity in TALs by increasing surface NKCC2 levels, but not its phosphorylation at Thr^96/101. The stimulatory effect of fructose was not mimicked by glucose or fructose 1,6-bisphosphate. Our data raise the interesting possibility that fructose may affect renal salt handling by increasing TAL-dependent NaCl reabsorption. We speculate that such an effect could contribute to the salt-sensitive hypertension caused by chronically elevated dietary fructose that we and others have reported.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-105818A1 (P. A. Ortiz).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

G.R.A. and P.A.O. conceived and designed research; G.R.A., K.M.K., and P.A.O. performed experiments; G.R.A., K.M.K., and P.A.O. analyzed data; G.R.A., K.M.K., and P.A.O. interpreted results of experiments; G.R.A., K.M.K., and P.A.O. prepared figures; G.R.A. and P.A.O. drafted manuscript; G.R.A. and P.A.O. edited and revised manuscript; G.R.A., K.M.K., and P.A.O. approved final version of manuscript.