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Am J Physiol Renal Physiol 293: F1036-F1046, 2007. First published June 20, 2007; doi:10.1152/ajprenal.00034.2007
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Interleukin-6 stimulates {alpha}-MG uptake in renal proximal tubule cells: involvement of STAT3, PI3K/Akt, MAPKs, and NF-{kappa}B

Yu Jin Lee, Jung Sun Heo, Han Na Suh, Min Young Lee, and Ho Jae Han

Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Submitted 17 January 2007 ; accepted in final form 9 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have shown that interleukin 6 (IL-6) acts on the cellular proliferation-activating transduction signals during cellular regeneration. Therefore, this study examined the effect of IL-6 on the activation of Na+/glucose cotransporters (SGLTs) and its related signaling pathways in primary cultured renal proximal tubule cells (PTCs). IL-6 increased the level of {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) uptake in time- and dose-dependent manners. IL-6 also increased SGLT1 plus SGLT2 mRNA and protein expression level. The IL-6 receptors (IL-6R{alpha} and gp130) were expressed in PTCs. In addition, genistein and herbimycin A completely blocked the IL-6-induced increases in {alpha}-MG uptake and the protein expression level of SGLTs. On the other hand, IL-6 increased the level of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-sensitive cellular reactive oxygen species (ROS), and IL-6-induced increases in {alpha}-MG uptake and the protein expression level of SGLTs were blocked by ascorbic acid or taurine (antioxidants). IL-6 also increased the phosphorylation of signal transducer and activator of transcription-3 (STAT3), phosphoinositide-3 kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) in a time-dependent manner. A pretreatment with STAT3 inhibitor LY 294002, an Akt inhibitor, or MAPK inhibitors significantly blocked the IL-6-induced increase in {alpha}-MG uptake. In addition, IL-6 increased the level of nuclear factor-{kappa}B (NF-{kappa}B) phosphorylation. A pretreatment with SN50 or BAY 11-7082 also blocked the IL-6-induced increase in {alpha}-MG uptake. In conclusion, IL-6 increases the SGLT activity through ROS, and its action in renal PTCs is associated with the STAT3, PI3K/Akt, MAPKs, and NF-{kappa}B signaling pathways.

Na+/glucose cotransporters; kidney

INTERLEUKIN 6 (IL-6) has been demonstrated in the glomerular mesangium and in the urine of patients with proliferative glomerulonephritis or renal cell carcinoma, suggesting it has an important role in the pathogenesis of renal diseases (25). IL-6 is as a pleiotropic cytokine that may play a role in cellular proliferation, activating the transduction signals in cellular regeneration (24). It has been reported that renal epithelial cells in culture produce low levels of IL-6, which suggests that the IL-6 transcriptional machinery is constitutively activated in these cells (1). The expression of cytokines by human renal tubular epithelial cells has been observed in different studies (14, 44). In the kidney, IL-6 is implicated in the development of glomerular and tubular injury in various forms of immune-mediated renal diseases (4). Recent studies have shown that IL-6 stimulates tubular regeneration after glycerol-induced acute renal failure (24). In addition, previous reports demonstrated the constitutive expression of IL-6 in proximal tubule cells (PTCs) and distal tubule cells, as well as enhanced expression after stimulation with IL-1 (2). IL-6 interacts with its receptor complex consisting of the IL-6R{alpha} and two molecules of gp130, thereby initiating intracellular signaling (22). Signal transduction by the IL-6 family of cytokines involves the activation of two major pathways: the JAK/signal tranducer and activator of transcription (STAT) and MAPK pathways (21).

In the kidney, glucose is reabsorbed from the urinary filtrate through the action of several types of glucose transporters that are arranged in series along the PTCs (20). The Na+/glucose cotransporters (SGLTs) in the apical membrane catalyze active glucose transport intracellularly and are driven by a Na+ gradient. Glucose passively diffuses out of the cell through the basolateral glucose-facilitated transporters (GLUTs). Previous reports have demonstrated that IL-6-deficient mice showed markedly impaired glucose disposal during an intravenous glucose tolerance test compared with their littermate control mice (56). In addition, IL-6 enhances both the basal and insulin-stimulated glucose uptake in cultured 3T3-L1 adipocytes (46), while the level of glucose transport was increased in the jejunal tissue incubated with IL-6 compared with the controls (19). However, to date, there is little information regarding the effect of IL-6 on the Na+/glucose cotransport system in renal PTCs. In addition, the signal transduction pathways involved in eliciting these effects are not completely understood.

Primary cultured renal PTCs have been observed to retain a number of differentiated typical functions of the renal proximal tubules. When maintained in a hormonally defined serum-free medium, the cells show a polarized morphology that is distinctive of tubule epithelial cells. Differentiated functions including the apical Na+/glucose cotransport system (50), apical {gamma}-glutamyltranspeptidase (11), basolateral PAH transport system (58), and mitochondrial phosphoenolpyruvate carboxykinase (55), are indicative of the gluconeogenic capacity of cells (28). Moreover, the uptake studies with {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) indicate that the activity of the transport system is stably maintained in culture (15, 34). Therefore, membrane transport studies carried out with such PTCs have a particular advantage in that the results can be compared directly with the original renal tissue. In addition, PTCs in hormonally defined, serum-free culture conditions provide a valuable in vitro model for examining the effect of IL-6 on the renal SGLTs (16, 18). This study examined the effects of the IL-6 on {alpha}-MG uptake by the PTCs, along with the signal transduction pathways involved in eliciting these effects in PTCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. New Zealand White male rabbits (1.5–2.0 kg) were purchased from Dae Han Experimental Animal (Chungju, Korea). All procedures for animal management followed the standard operation protocols of Seoul National University. The Institutional Review Board of Chonnam University approved the research proposal and the relevant experimental procedures including animal care. Class IV collagenase and soybean trypsin inhibitor were purchased from Life Technologies (GIBCO BRL, Grand Island, NY). IL-6, actinomycin D, cycloheximide, genistein, herbimycin A, hydrogen peroxide (H2O2), LY 294002, PD 98059, SB 203580, SP 600125, ascorbic acid, FITC-conjugated anti-rabbit IgG, and monoclonal anti-beta-actin were obtained from Sigma (St. Louis, MO). STAT3 inhibitor, Akt inhibitor, and SN50 were acquired from Calbiochem (La Jolla, CA). BAY 11-7082 was supplied by Biomol (Plymouth Meeting, PA). {alpha}-MG was purchased from DuPont/NEN (Boston, MA). Phospho-STAT3 (Tyr705), IL-6R{alpha}, gp130, and phospho-Akt (Ser473) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-p44/42 MAPK (Thr202/Tyr204) antibody, p44/42 MAPK antibody, phospho-JNK (Thr183/Tyr185) antibody, JNK antibody, phospho-p38 MAPK (Thr180/Tyr182) antibody, p38 MAPK antibody, phospho-NF-{kappa}B, and NF-{kappa}B antibody were obtained from Cell Signaling Technology (Herts, UK). Rabbit anti-SGLT1 was supplied by Chemicon International (Temecula, CA), and rabbit anti-SGLT2 antibody from Alpha Diagnostic International (San Antonio, TX). Goat anti-rabbit IgG was acquired from Jackson ImmunoResearch (West Grove, PA). Goat anti-mouse IgG was purchased from BD Bioscience (Franklin Lakes, NJ). All other reagents were of the highest purity commercially available. Liquiscint was obtained from National Diagnostics (Parsippany, NJ).

Cell preparation and culture condition. The primary rabbit renal proximal tubule cell cultures were prepared using the method reported by Chung et al. (11). The PTCs were grown in a D-MEM/F-12 medium (GIBCO-BRL, Gaithersburg, MD) with 15 mM HEPES and 20 mM sodium bicarbonate (pH 7.4). Three growth supplements (5 µg/ml insulin, 5 µg/ml transferrin, and 5 x 10–8 M hydrocortisone) were added immediately before the medium was used. The kidneys of a rabbit were perfused through the renal artery, first with PBS and then with medium containing 0.5% iron oxide. Renal cortical slices were prepared and homogenized. The homogenate was poured first through a 253-µm mesh filter and then through an 83-µm mesh filter. Tubules and glomeruli on the top of the 83-µm filter were transferred into a sterile medium. The glomeruli (containing iron oxide) were removed using a magnetic stir bar. The remaining proximal tubules were incubated briefly in a medium containing collagenase (0.125 mg/ml) and 0.025% soybean trypsin inhibitor. The tubules were then washed by centrifugation, resuspended in a medium containing the three supplements, and transferred into tissue culture dishes. The medium was changed 1 day after plating and every 2 days thereafter. The primary cultured rabbit kidney PTCs were maintained at 37°C in a 5% CO2 humidified environment in a serum-free basal medium supplemented with the three growth supplements.

{alpha}-MG uptake studies. The {alpha}-MG uptake studies were carried out as described by the method reported by Sakhrani et al. (40). Briefly, the culture medium was removed by aspiration, and the monolayers were gently washed twice with the uptake buffer (in mM: 136 NaCl, 5.4 KC1, 0.41 MgSO4, 1.3 CaCl2, 0.44 Na2HPO4, 0.44 KH2PO4, 5 HEPES, and 2 glutamine, as well as 0.5 µg/ml BSA, pH 7.4). After washing, the monolayers were incubated for 30 min at 37°C in uptake buffer containing 0.5 mM {alpha}-MG and 14C-labeled {alpha}-MG (0.5 µCi/ml). At the end of the incubation period, the monolayers were again washed three times with ice-cold uptake buffer, and the cells were dissolved in 1 ml 0.1% SDS. To determine the {alpha}-[14C]MG incorporated intracellularly, 900 µl of each sample was removed and counted in a liquid scintillation counter (LS 6500, Beckmann Coulter, Fullerton, CA). The remainder of each sample was used to determine the level of protein using the Bradford method (5). The radioactive counts in each sample were then normalized to the protein level and corrected for the zero-time uptake per milligram protein. All the uptake measurements were made in triplicate.

Assay of cellular reactive oxygen species. The cellular production of reactive oxygen species (ROS) was measured using confocal microscopy according to the method reported by Lee et al. (33). To confirm involvement of ROS in IL-6-induced SGLT activation, the PTCs were treated with ascorbic acid (antioxidant, 10–3 M) before being treated for 30 min with either IL-6 or H2O2 (10–3 M). The cells were washed with Dulbecco's PBS and incubated for 15 min in Krebs-Ringer solution containing 5 µM 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate (CM-H2DCF-DA; Molecular Probes, Eugene, OR). The ROS generation was detected (excitation, 488 nm; emission, 515–540 nm) using a fluorescent microscope (Fluoview 300, Olympus, Tokyo, Japan).

Isolation of SGLT RNA and RT-PCR. Extraction of total RNA from PTCs was performed as described previously (9). PTCs were homogenated with STAT-60, a monophasic solution of phenol, and guanidine isothiocyanate obtained from Tel-Test (Friendswood, TX). Two micrograms of purified RNA were synthesized into cDNA using avian leukemia virus RT with oligo dT18 primers. PCR amplification was performed with 5 µl of RT product, 10 pmol of each primer, 1.25 U of Taq polymerase (Promega, Madison, WI), and 1 mM 2-deoxynucleotide 5'-triphosphate. Primers were 5'-TCCTCACCCTTTGGTACTGG-3' (forward) and 5'-ACAGGATACGGCTCACCATC-3' (reverse) for SGLT1 (7) and 5'-AGGATCCATCTGTTGGCA-3' (forward) and 5'-ACGGGGCACAAAGAGT-3' (reverse) for SGLT2 (49). After an initial incubation at 95°C for 5 min, 30 amplification cycles consisting of 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min were performed for SGLT1, and 30 amplification cycles consisting of 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 55 s were performed for SGLT2. As a control for the amount of cDNA, RT-PCR was performed using beta-actin primers. PCR products were visualized using ethidium bromide staining.

Cell membrane preparations for Western blotting. After confluent culture in each condition, the medium was then removed. The cells were then washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A (in mM: 137 NaCl, 8.1 Na2HPO4, 2.7 KCl, 1.5 KH2PO4, 2.5 EDTA, 1 dithiothreitol, and 0.1 PMSF, as well as 10 µg/ml leupeptin, pH 7.5). The resuspended cells were then lysed mechanically on ice by trituration with a 21.1-gauge needle. The lysates were first centrifuged at 1,000 g for 10 min at 4°C. The supernatants were centrifuged at 100,000 g for 1 h at 4°C to prepare the cytosolic and particulate fractions. The particulate fractions, which contained the membranes, were washed twice, and resuspended in buffer A containing 1% (vol/vol) Triton X-100. The protein in each fraction was quantified using the Bradford procedure (5).

Western blot analysis. The cell homogenates (30 µg of protein) were separated on 10% SDS-PAGE and transferred to nitrocellulose paper. The blots were then washed with H2O, blocked with 5% skimmed milk powder in TBST (10 mM Tris·HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 h and incubated with the primary polyclonal antibody at the dilutions recommended by the supplier. The specificity of the SGLT1 or SGLT2 antibody was confirmed using control peptides. A control peptide, because of its low molecular mass (<3 kDa), is not suitable for Western blot analysis. Thus we used it for ELISA or antibody blocking to confirm antibody specificity. The membrane was washed, and the primary antibodies were detected using goat anti-rabbit-IgG conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech).

Statistical analysis. The results are expressed as means ± SE. The difference between the two mean values was analyzed by ANOVA. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of IL-6 on {alpha}-MG uptake. To determine the effect of IL-6 on {alpha}-MG uptake, the PTCs were incubated with different concentrations of IL-6 (0–20 ng/ml) for 24 h or with 10 ng/ml for various times (0–48 h). As shown in Fig. 1, IL-6 increased the level of {alpha}-MG uptake in both a time- and dose-dependent manner. IL-6, at ≥1 ng/ml, significantly increased {alpha}-MG uptake after 24-h incubation (Fig. 1A). A statistically significant increase in {alpha}-MG uptake was found after 8-h incubation with 10 ng/ml IL-6, and the maximum effect was observed at 24 h (Fig. 1B). IL-6 significantly increased SGLT1 plus SGLT2 mRNA and protein expression level (Fig. 1, C and D). In addition, actinomycin D (transcription inhibitor, 10–7 M) and cycloheximide (translation inhibitor, 4 x 10–6 M) significantly blocked the IL-6-induced increase of SGLT1 and SGLT2 protein expression level (Fig. 1D). Actinomycin D or cycloheximide also completely blocked the IL-6-induced increase in {alpha}-MG uptake (Fig. 1E). Therefore, 24-h incubation with 10 ng/ml IL-6 was used in all further experiments.


Figure 1
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  		Fig. 1. Effect of interleukin-6 (IL-6) on {alpha}-methyl-D-[14C]glucopyranoside ({alpha}-MG) uptake. A: proximal tubule cells (PTCs) were treated with different doses (0–20 ng/ml) of IL-6 for 24 h. B: PTCs were treated with 10 ng/ml IL-6 for 0–48 h before measurement of {alpha}-MG uptake. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. C: expression of Na+/glucose cotransporter (SGLT1 and SGLT2) mRNA was determined by RT-PCR in PTCs treated with IL-6 (10 ng/ml) for 24 h. Bands represent 162-bp SGLT1, 707-bp SGLT2, and 350-bp beta-actin. Each example shown is a representative of 4 independent experiments. D: cells were incubated with actinomycin D (AD; 10–7 M) or cycloheximide (CHX; 4 x 10–6 M) for 30 min before treatment with IL-6 for 24 h. SGLT1 and SGLT2 protein expression level was determined by Western blotting with plasma membrane protein. The bands represent 70–77 kDa of SGLT1 and SGLT2 and 41 kDa of beta-actin, respectively. Each example shown is a representative of 3 independent experiments. Values are means ± SE of 3 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. E: cells were incubated with actinomycin D or cycloheximide for 30 min before treatment with IL-6 for 24 h before measurement of {alpha}-MG uptake. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone.

 
Involvement of IL-6 receptors (IL-6R{alpha} and gp 130) and tyrosine kinase in IL-6-induced {alpha}-MG uptake. Western blot analysis with membrane fraction of PTCs was performed to determine the involvement of the IL-6 receptor (IL-6R{alpha} and gp 130) activation in IL-6-induced {alpha}-MG uptake. IL-6 significantly enhanced the protein level of IL-6R{alpha} and gp 130 in a dose-dependent manner (Fig. 2). To determine whether the phosphorylation of tyrosine kinase is involved in the effect of IL-6 on {alpha}-MG uptake, the PTCs were treated with the tyrosine kinase inhibitors genistein or herbimycin A (10–6 M), before the IL-6 treatment. As shown in Fig. 3, genistein and herbimycin A completely blocked the IL-6-induced increase in {alpha}-MG uptake, as well as in SGLT1 and SGLT2 protein expression.


Figure 2
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  		Fig. 2. Effect of IL-6 on the expression of IL-6 receptors (IL-6R{alpha} and gp 130). IL-6R{alpha} (A) and gp130 (B) protein expression was determined by Western blotting with the membrane protein, which was treated with IL-6 (10 ng/ml) for 24 h. The bands represent 80 kDa of IL-6R{alpha}, 130 kDa of gp130, and 41 kDa of beta-actin. Each example shown is a representative of 3 independent experiments. Values are means ± SE of 3 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control (0 ng/ml).

 

Figure 3
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  		Fig. 3. Involvement of tyrosine kinase in the IL-6-induced {alpha}-MG uptake. A: PTCs were pretreated with the tyrosine kinase inhibitors genistein or herbimycin A (10–6 M) for 30 min before IL-6 incubation for 24 h. {alpha}-MG uptake was then measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were pretreated with genistein or herbimycin A for 30 min before IL-6 incubation for 24 h. SGLT1 and SGLT2 protein expression was determined by Western blotting with the membrane fraction. Each example shown is a representative of 3 independent experiments.

 
Effect of IL-6-induced ROS on {alpha}-MG uptake. Pretreatment with ascorbic acid (10–3 M) or taurine (2 x 10–3 M) abolished the IL-6-induced increase in {alpha}-MG uptake (Fig. 4A). This confirmed that IL-6 stimulates the production of ROS in PTCs. Moreover, Western blot analysis with the membrane fraction demonstrated an increase in the SGLT1 and SGLT2 protein level, which was blocked by ascorbic acid (Fig. 4B). In addition, the intracellular level of ROS stimulated by IL-6 using CM-H2DCF-DA was measured. Figure 4C shows that IL-6 increased the DCF-sensitive cellular ROS level, which was blocked by the pretreatment with ascorbic acid (10–3 M) for 30 min. This is in concordance with the hypothesis that IL-6 increases the SGLT activity via ROS, which may play a role in activating the intracellular signaling pathway.


Figure 4
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  		Fig. 4. Effect of IL-6 on the cellular level of reactive oxygen species (ROS). A: PTCs were incubated with the antioxidants ascorbic acid (10–3 M) or taurine (2 x 10–3 M) for 30 min before IL-6 treatment for 24 h. {alpha}-MG uptake was then measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were pretreated with ascorbic acid for 30 min before IL-6 treatment for 24 h. The cells were harvested, and the level of SGLT1 and SGLT2 protein expression was determined by Western blotting with the membrane fraction. Each example shown is a representative of 3 independent experiments. Values are means ± SE of 3 experiments for each condition determined from densitometry relative to beta-actin. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. C: dichlorofluorescein (DCF)-sensitive cellular ROS was measured by confocal microscopy. Shown are results from control (a), IL-6 (10 ng/ml) treatment for 30 min (b), H2O2 (10–3 M) treatment for 30 min (c), and pretreatment with ascorbic acid for 30 min before incubation with IL-6 for 30 min (d). Also shown is the relative fluorescent intensity compared with control. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone.

 
Involvement of the STAT3 and phosphoinositide-3 kinase/Akt pathway in the IL-6-induced increase in {alpha}-MG uptake. To examine the role of the STAT3 pathway in the IL-6-induced increase in {alpha}-MG uptake, the cells were pretreated with the STAT3 inhibitor (10–5 M) for 30 min before IL-6 treatment. The STAT3 inhibitor significantly blocked the IL-6-induced increase in {alpha}-MG uptake (Fig. 5A). In addition, Western blotting showed that the level of STAT3 phosphorylation was increased in a time-dependent manner (0–90 min) (Fig. 5B). The cells were pretreated with LY 294002 [a phosphoinositide-3 kinase (PI3K) inhibitor, 10–6 M] or an Akt inhibitor (10–5 M) for 30 min before the IL-6 treatment to determine the role of the PI3K/Akt pathways in IL-6-induced increase in {alpha}-MG uptake (Fig. 6A). In Western blotting, the level of Akt (Ser473) phosphorylation increased in a time-dependent manner (0–90 min) (Fig. 6B). Furthermore, as shown in Fig. 7A, pretreatment with ascorbic acid blocked the phosphorylation of STAT3. Moreover, the STAT3 inhibitor inhibited the level of Akt phosphorylation (Ser473) (Fig. 7B). In contrast, neither LY 294002 nor the Akt inhibitor blocked the level of STAT3 phosphorylation (Fig. 7C).


Figure 5
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  		Fig. 5. Involvement of signal transducer and activator of transcription-3 (STAT3) in the IL-6-induced {alpha}-MG uptake. A: PTCs were pretreated with the STAT3 inhibitor (10–5 M) for 30 min before incubation with IL-6 for 24 h, and then {alpha}-MG uptake was measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were incubated with IL-6 incubation for 0–90 min, and the total protein was then extracted and blotted with antibodies against phospho-STAT3 and total STAT3. Each example shown is a representative of 4 independent experiments. Values are means ± SE of 4 experiments for each condition determined from densitometry relative to the total STAT3. *P < 0.05 vs. control (0 min).

 

Figure 6
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  		Fig. 6. Involvement of phosphoinositide-3 kinase (PI3K)/Akt in IL-6-induced {alpha}-MG uptake. A: PTCs were pretreated with LY 294002 (10–6 M) and the Akt inhibitor (10–5 M) for 30 min before IL-6 incubation for 24 h, and then {alpha}-MG uptake was measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were incubated with IL-6 for 0–90 min, and total protein was then extracted and blotted with antibodies against phospho-Akt 473 and total Akt. Each example shown is a representative of 3 independent experiments. Values are means ± SE of 3 experiments for each condition determined from densitometry relative to total Akt. *P < 0.05 vs. control (0 min).

 

Figure 7
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  		Fig. 7. Interaction of STAT3 and PI3K/Akt in IL-6-induced signaling pathway. A: PTCs were pretreated with ascorbic acid for 30 min before the IL-6 treatment for 30 min. Total protein was extracted and blotted with the antibodies against phospho-STAT3 and total STAT3. B: cells were pretreated with STAT3 inhibitor for 30 min before the IL-6 treatment for 30 min. The total protein was extracted and blotted with antibodies against phospho-Akt 473 and total Akt. C: cells were pretreated with either LY 294002 or the Akt inhibitor for 30 min before the IL-6 treatment for 30 min. Total protein was extracted and blotted with antibodies against phospho-STAT3 or total STAT3. Each example shown is a representative of 4 independent experiments.

 
Involvement of the MAPKs in the IL-6-induced increase in {alpha}-MG uptake. The cells were pretreated with PD 98059 (p44/42 MAPK inhibitor, 10–5 M), SB 203580 (p38 MAPK inhibitor, 10–6 M), or SP 600125 (JNK inhibitor, 10–6 M) for 30 min before IL-6 treatment to determine whether the MAPKs are involved in the IL-6-induced increase in {alpha}-MG uptake. As shown in Fig. 8A, PD 98059, SB 203580, and SP 600125 reduced the IL-6-induced increase in {alpha}-MG uptake. In addition, IL-6 enhanced the level of p44/42 MAPKs, p38 MAPK, and JNK phosphorylation for 30–90 min after the IL-6 treatment (Fig. 8B). Moreover, the IL-6 induced phosphorylation of p44/42 MAPKs, p38 MAPK, and JNK were attenuated by pretreatment with ascorbic acid, LY 294002, or the Akt inhibitor (Fig. 9, A and B). The total expression of these MAPKs on the Western blot revealed a similar protein level. However, PD 98059, SB 203580, and SP 600125 did not block the IL-6-induced phosphorylation of Akt (Fig. 9C).


Figure 8
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  		Fig. 8. Involvement of MAPKs in IL-6-induced {alpha}-MG uptake. A: PTCs were pretreated with PD 98059 (p44/42 MAPK inhibitor, 10–5 M), SB 203580 (p38 MAPK inhibitor, 10–6 M), or SP 600125 (JNK inhibitor, 10–6 M) for 30 min before the IL-6 treatment. {alpha}-MG uptake was then measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were incubated with IL-6 for 0–90 min. Total protein was extracted and blotted with antibodies against phospho- or total p44/42 MAPKs, p38 MAPK, and JNK antibodies. Each example shown is a representative of 3 independent experiments.

 

Figure 9
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  		Fig. 9. Interaction of PI3K/Akt and MAPKs in IL-6-induced signaling pathway. PTCs were pretreated with ascorbic acid (A), LY 294002, and Akt inhibitor (B) for 30 min before incubation with IL-6 for 30 min. The level of p44/42 MAPKs, p38 MAPK, and JNK phosphorylation was then determined. Each example shown is a representative of 3 independent experiments. C: cells were pretreated with PD 98059, SB 203580, and SP 600125 for 30 min before incubation with IL-6 for 30 min. Total protein was extracted and blotted with antibodies against phospho-Akt 473 or total Akt. Each example shown is a representative of 4 independent experiments.

 
Involvement of NF-{kappa}B in the IL-6-induced increase in {alpha}-MG uptake. Cells were pretreated SN50 (nuclear translocation inhibitor, 500 ng/ml) and BAY 11-7082 (I-{kappa}B phosphorylation inhibitor, 2x10–5 M) for 30 min before the IL-6 treatment for 24 h to determine whether NF-{kappa}B activation is involved in the IL-6-induced increase in {alpha}-MG uptake. As shown in Fig. 10A, SN50 and BAY 11-7082 prevented the IL-6-induced increase in {alpha}-MG uptake. Western blotting showed that IL-6 enhanced the phosphorylation of NF-{kappa}B for 30–60 min after the IL-6 treatment (Fig. 10B). Moreover, a pretreatment with ascorbic acid, PD 98059, SB 203580, or SP 600125 attenuated the IL-6-induced phosphorylation of NF-{kappa}B (Fig. 11, A and B). The total expression of NF-{kappa}B on the Western blot revealed a similar protein level. However, SN 50 and BAY 11-7082 did not block the IL-6-induced phosphorylation of p44/42 MAPKs (Fig. 11C).


Figure 10
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  		Fig. 10. Involvement of NF-{kappa}B in the IL-6-induced {alpha}-MG uptake. A: PTCs were incubated with SN50 (500 ng/ml) or BAY 11-7082 (2 x 10–5 M) for 30 min before IL-6 treatment for 24 h. {alpha}-MG uptake was then measured. Values are means ± SE of 5 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. IL-6 alone. B: cells were incubated with IL-6 for 0–90 min. The total protein was extracted and blotted with antibodies against phospho- or total NF-{kappa}B. Each of the examples shown is a representative of 5 independent experiments. Values are means ± SE of 5 experiments for each condition determined from densitometry relative to total NF-{kappa}B. *P < 0.05 vs. control (0 min).

 

Figure 11
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  		Fig. 11. Interaction of MAPKs and NF-{kappa}B in IL-6-induced signaling pathways. PTCs were pretreated with ascorbic acid (A), PD 98059, SB 203580, and SP 600125 (B) for 30 min before incubation with IL-6 for 30 min. The level of NF-{kappa}B phosphorylation was then determined. C: cells were pretreated with SN50 and BAY 11-7082 for 30 min before incubation with IL-6 for 30 min. The level of p44/42 phosphorylation was then determined. Each of the examples shown is a representative of 4 independent experiments.

 

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These results show for the first time that IL-6 stimulates {alpha}-MG uptake in the primary PTCs, and that the STAT3, PI3K/Akt, MAPKs, and NF-{kappa}B signal pathways mediate IL-6-induced increases in {alpha}-MG uptake. The concentration of IL-6 used in this study has pathophysiological significance in the regulation of the renal proximal tubule functions for the following reasons: 1) normal tubular epithelial cells express low levels of IL-6 (10); 2) much higher levels of IL-6 are produced after IL-1 stimulation (29) as well as a during renal injury, caused by oxalate, renal cell carcinoma, and uric acid etc. (1, 17, 27); and 3) the concentration of IL-6 is increased after a renal transplant. Indeed, the IL-6 concentrations in the serum and in urine of humans are elevated during episodes of acute graft rejection within the first 2 mo after the transplant (54) (in serum, 17 ± 4.8 pg/ml; in urine, 114 ± 27 pg/ml). The differences in the effectiveness of various IL-6 concentrations might be due to uncertain quality of IL-6, differences in the cell types, marker indexes, or experimental conditions (in vitro vs. in vivo, serum-containing vs. serum-free media). Therefore, a range of IL-6 concentrations comparable to that the used in other studies was chosen.

We hypothesized that treatment with IL-6 would increase the SGLT activity in PTCs. This was based on previous studies showing that IL-6 acted by increasing the intrinsic glucose transporter activity (47), and the number of {alpha}-MG transporters induced by IL-1 increases in proportion to the metabolic need for glucose by the cells (19). To our knowledge, this is the first report of a direct effect of IL-6 on SGLTs. Therefore, the mechanism by which IL-6 increases the {alpha}-MG uptake into the PTCs was examined. In this study, IL-6 significantly increased SGLT1 and SGLT2 mRNA expression as well as protein expression. Both a transcription inhibitor (actinomycin D) and translation inhibitor (cycloheximide) were blocked IL-6-induced SGLT1 and SGLT2 protein expression. These findings suggest the possibility that the regulation of SGLT expression is involved in changes in transcription (43, 52) and amount of transporter within the plasma membrane (23). Recently, it was reported that a transporter-regulatory protein, RS1, inhibited hSGLT1 by inhibiting the dynamin-dependent release of SGLT1-containing vesicles from the trans-Golgi network (31, 32, 53). On the other hand, PKA and PKC regulate rabbit SGLT1 activity by modulating the number of cotransporters in the plasma membrane, which occurs through regulation of exocytosis and endocytosis (57).

In this study, IL-6 stimulated the protein expression of IL-6R{alpha}, a ligand binding subunit, and glycoprotein 130 (gp130, IL-6Rbeta), a signal-transducing component, in a dose-dependent manner. The binding of IL-6 to IL-6R{alpha} triggers the homodimerization of gp130 and activates the JAK (22). Subsequently, gp130 is phosphorylated by JAK, and the phosphotyrosines recruit STAT3 and protein tyrosine phosphatase 2 (13, 45). These results also suggest that the activation of a tyrosine kinase like JAK is essential for the action of IL-6. After tyrosine phosphorylation, STAT3 forms either a homodimer or heterodimer with STAT1 and enters the nucleus, where it regulates the expression of a specific set of genes (21).

ROS cause a wide spectrum of responses, ranging from proliferation to growth arrest or to cell death, depending on the type and level of ROS or cell type (3). Indeed, low levels of ROS (which is endogenous origin) have been demonstrated to cause an increase in cell cycle progression, while high levels of oxidative stress inhibited cell growth and function (15, 35). This study showed that the endogenous ROS production in PTCs exposed to IL-6 was higher than in the control, and an antioxidant blocked the activation of many signal molecules involving the IL-6-induced increase in {alpha}-MG uptake. This suggests that oxidative stress is a key factor in the increase in glucose uptake in renal PTCs. These results are consistent with a previous study, which suggests that IL-6 induces the generation of ROS in the synoviocytes of rheumatoid arthritis (48). Furthermore, previous studies have shown that elevated levels of ROS in BCR/ABL-transformed cells are secondary to a transformation-associated increase in glucose metabolism (41) and an overactive mitochondrial electron transport chain (30). Hence, it was suggested that the generation of ROS in response to IL-6 might contribute to various downstream signaling events as requisite second messengers, particularly those involving tyrosine phosphorylation as shown in these results.

IL-6 leads to the activation of STAT3 and then to the activation of PI3K and Akt, which was prevented by a STAT3 inhibitor. Previous reports have investigated the STAT3 SH2 domain-binding peptide to disrupt the IL-6-mediated activation of STAT3 in vitro (51). In addition, the IL-6-induced activation of the PI3K/Akt cascade in multiple myeloma cells is essential for tumor cell proliferation and viability (26). They also investigated these two potentially independent pathways of PI3K/Akt activation in multiple myeloma cells: one mediated by Ras signaling and the other mediated by the STAT3-containing complex. It was also observed that the PTCs treated with IL-6 lead to the activation of PI3K/Akt and LY 294002, and the Akt inhibitor blocked the IL-6-induced increase in {alpha}-MG uptake. These results are supported by the result of Western blot analysis of Akt. In addition, STAT3 protein expression was not blocked by LY 294002 and the Akt inhibitor, which suggests that the PI3K and Akt signal molecules are downstream of STAT3.

These processes subsequently lead to the activation of MAPKs, including p44/42 MAPKs, p38 MAPK, and JNK. This is consistent with previous results showing that IL-6 can activate the ERK1/2, p38 MAPKs (12, 36), and JNK pathways (38). In addition, previous studies have shown that the PI3K/Akt pathway has a regulatory function in JNK (39) and ERK (6) signaling. In this study, Akt protein expression was not blocked by the MAPK inhibitors, which suggested that MAPKs are downstream of STAT and PI3K/Akt. On the other hand, these results clearly demonstrated that IL-6 increases the NF-{kappa}B activity through the generation of ROS. Moreover, its activation may play a role in increasing the SGLT1 and SGLT2 levels in primary cultured renal PTCs. These findings strongly suggest a role of ROS in intracellular signaling processes, as supported by previous observations of H2O2-dependent NF-{kappa}B activation in HK-2 cells (37). In addition, Chen et al. (8) reported that the p38 MAPK and PI3K/Akt pathways, but not the JNK pathway, appear to regulate the activation of NF-{kappa}B. In this study, phospho-NF-{kappa}B was blocked by MAPK inhibitors, which suggests that NF-{kappa}B is downstream of the MAPKs. Furthermore, SN50 and BAY 11-7082 effectively blocked the {alpha}-MG uptake in PTCs exposed to IL-6, which indicates a role of NF-{kappa}B in the IL-6-induced increase in {alpha}-MG uptake. It appears that the stimulatory function of cross talk between the IL-6-activated multiple pathways may depend on the cell type. Figure 12 shows a hypothetical model of the signaling mechanisms involved in mediating the IL-6-induced increase in SGLT activity in renal PTCs.


Figure 12
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  		Fig. 12. Hypothesized model for the signal pathways involved in IL-6-induced {alpha}-MG uptake. IL-6 activates IL-6 receptor {alpha} (IL-6R{alpha}), gp130 (IL-6Rbeta), and tyrosine kinase (JAK), which generate intracellular ROS. In turn, ROS stimulate STAT3, which activates PI3K/Akt and MAPK. Subsequently, MAPK (p44/42, p38, and JNK) activation induces the phosphorylation of NF-{kappa}B. Finally, these molecules may induce an increase in SGLT expression involved in the increase in {alpha}-MG uptake.

 
Previous reports demonstrated that several proinflammatory cytokines markedly upregulated SGLT1 and GLUT1 expression (42) and SGLT1-mediated glucose uptake protected intestinal epithelial cells against LPS-induced apoptosis (59). Therefore, the enhanced uptake of glucose may play a role in the renal response to the hypermetabolic state or a cytoprotective mechanism. In summary, IL-6 increased SGLT activity through ROS, and its action is associated with the STAT3, PI3K/Akt, MAPKs, and NF-{kappa}B signaling pathways in renal PTCs.


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This research was supported by a grant (SC 2210) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea, and the authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (e-mail: hjhan{at}chonnam.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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