Role of High Glucose-Induced Nuclear Factor-κB Activation in Monocyte Chemoattractant Protein-1 Expression by Mesangial Cells

Mesangial Cell Culture

Dulbecco’s modified Eagle’s medium (DMEM) containing 5.6 mM glucose was used to culture MC, unless otherwise stated. A murine MC line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD); these transformed cells have been shown to exhibit similar characteristics to those of primary cultures of murine MC (27). Primary mouse and rat MC were also used to confirm the responses obtained with MES-13. Primary MC were obtained by culturing glomeruli isolated from kidneys of 100- to 150-g male Sprague-Dawley rats or 15- to 20-g male BALB/C mice by conventional sieving methods as described previously and characterized (28). Cells were cultured in DMEM containing 20% fetal bovine serum (FBS; Life Technologies BRL, Gaitherburg, MD), 100 U/ml penicillin, 100 μg/ml streptomycin, 44 mM NaHCO₃, and 14 mM N-hydroxy-ethylpiperazine-N′-2-ethane sulfonic acid (HEPES). For MES-13, DMEM containing 5% FBS was used. Cells were cultured in 100-mm culture dish for electrophoretic mobility shift assay (EMSA) and PKC measurement, on cover glass coated with polylysine for intracellular ROS measurement, and 6-well culture plate for enzyme-linked immunosorbent assay (ELISA). Near-confluent MC were incubated with serum-free media for 48 h to arrest and synchronize the cell growth. After this time period, the media were changed to fresh serum-free DMEM containing 5.6 (control) or 30 mM (high) glucose for up to 48 h. Incubation of cells in control as well as HG in serum-free condition for up to 96 h did not have significant effect on cell viability as determined by lactate dehydrogenase (LDH) release. In some experiments, cells were pretreated with 0.2 μM cytochalasin B, 100 nM calphostin C, 24 nM 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GFX 109203X, a bisindolylmaleimide PKC inhibitor; Calbiochem-Novachem Corporation, San Diego, CA), 100 μM pyrrolidine dithiocarbamate (PDTC), 5 mM n-acetyl-l-cysteine (NAC), 500 μM trolox (Aldrich Chemical Co., Milwaukee, WI) for 1 h and incubated with control and HG media for a given period. Cytochalasin B was used to inhibit cellular uptake of glucose. Preliminary study demonstrated that cytochalasin B up to 2 μM did not cause cytotoxicity as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The effective concentrations were decided on the basis of published data and our own preliminary study. In the study using depletion of cellular PKC, MC were incubated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h. Cells were processed for nuclear protein extraction, measurement of ROS and PKC activity, and MCP-1 mRNA and protein expression at the end of the incubation period as described below. Experiments using primary rat and mouse MC were performed using cells between the 6th and 8th passages.

Establishment of Mesangial Cells Stably Transfected with IκBαM

Establishment of MC stably transfected with ΙκBαM has been previously described and well characterized (26). In brief, overexpression of ΙκBαM were created in MC obtained from a male Harlan Sprague-Dawley rat. pLΙκBαMSN (a gift from Dr. Inder Verma, Salk Institute, La Jolla, CA) comprising IκBαM cDNA and a neomycin phosphotransferase gene (neo) were transfected into a helper-free ecotopic packaging line ΩE. Stable transfectants were selected in the presence of the neomycin analogue G418 (500 μg/ml). Conditioned media of the transfectants were used as sources of the IκBαM retrovirus. In the presence of 10 μg/ml of polybrene, MC were exposed to a diluted retrovirus, stable infectants were selected in the presence of G418 (750 μg/ml), and the IκBαM transfectants were established. As control cells, mock-transfected MC that express neo alone were created. Both IκBαM- and mock-transfected cells have been shown to retain most phenotypic characteristics of MC and IκBαM transfectants are resistant to both basal and interleukin-1β (IL-1β) and HG-induced NF-κB activation as shown in Figure 9A.

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Figure 9. Role of NF-κB in HG-induced MCP-1 expression in mesangial cells. Synchronized quiescent rat MC transfected with control vector or IκBαM were incubated under control (CG), HG, or 0.1 ng/ml interleukin-1β (IL-1β). (A) After 1 h, nuclear proteins were prepared and NF-κB activity was assessed by EMSA. A representative data from five experiments. (B) After 48 h, MCP-1 secreted were measured by enzyme-linked immunosorbent assay (ELISA). Values are expressed as mean ± SE of five experiments. (C) After 24 h, total RNA was isolated and subjected to Northern blot analyses for MCP-1 and GAPDH mRNA. Values are expressed as mean ± SE of four experiments.

Preparation of Nuclear Extracts

Nuclear extracts were prepared according to the method described by Lee et al. (29) with modifications (30). In brief, cells were washed twice with ice-cold phosphate-buffered saline after incubations and suspended in one packed cell volume (PCV) of buffer A containing 10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 1 μg/ml leupeptin and aprotinin, pH 7.9, on ice for 10 min and then lysed by passing the cell suspension through a 27-gauge needle 5 times. Crude nuclei were washed twice with buffer A to prevent cytosolic contamination, and the nuclear proteins were extracted with two-thirds PCV of ice-cold buffer B containing 20 mM HEPES, 420 mM KCl, 1.5 mM MgCl₂, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin and aprotinin, and 25% glycerol, pH 7.9. A two-thirds PCV of ice-cold buffer C (20 mM HEPES, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin and aprotinin, 20% glycerol, pH 7.9) was added. The mixture was then centrifuged at 14,000 rpm at 4°C for 15 min. The nuclear proteins were transferred to new tubes as aliquots and stored at −70°C until use. The protein concentration was determined by a Bio-Rad assay (Rio-Rad, Richmond, CA).

Electrophoretic Mobility Shift Assay

For EMSA, the following oligonucleotide with the NF-κB consensus binding sequence was used: 5′-AGTTGAGGGACTTTCCCAGGC-3′ (Santa Cruz Biotechnology, Santa Cruz, CA). A mutant motif with G to C substitution (5′-AGTTGAGCGACTTTCCCAGGC-3′, Santa Cruz Biotechnology) served as a control. The consensus oligonucleotide was labeled with [γ-³²P]ATP (DuPont NEN, Boston, MA) according to the manufacturer’s description. The binding reaction was performed in a final volume of 20 μl containing binding buffer (10 mM HEPES, 60 mM KCl, 1 mM DTT, 1 mM EDTA, 7% glycerol, pH 7.6), 0.0175 pmol of labeled probe (>10,000 cpm), 20 μg of nuclear protein, and 2 μg of poly dIdC (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Reactions were started by addition of nuclear extracts and allowed for 30 min at room temperature. Samples were loaded on 6% polyacrylamide nondenaturing gel and electrophoresed for 2 h at 180 V. The dried gel was exposed to Kodak XR5 film (Eastman Kodak, Rochester, NY) on intensifying screen for 10 to 20 h at −70°C. For competition assay, an unlabeled NF-κB oligonucleotide was added in 100-fold excess. For supershift assay, 1 μl of anti-p65 or anti-p50 antibody (Santa Cruz Biotechnology) was incubated with nuclear extract for 30 min at room temperature before binding reaction.

Assay of Intracellular ROS

Intracellular ROS production was measured by the method of Bass et al. (31), as modified for confocal microscopy (32). In brief, coverglasses of confluent cells at various times after stimulation with HG were washed with Dulbecco’s phosphate-buffered saline and incubated in the dark for 5 min in Krebs-Ringer solution containing 5 mM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes Inc., Eugene, OR). CM-H₂DCFDA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative 2′,7′-dichlorofluorescin (DCFH) and thereby trapped within the cells (31). In the presence of a proper oxidant, DCFH is oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Culture dishes were transferred to a Leica DM IRB/E inverted microscope (Leica, Wetzlar, Germany) equipped with a ×20 Fluotar objective and Leica TCS NT confocal attachment, and the ROS generation was detected as a result of the oxidation of DCFH (excitation, 488 nm; emission, 515 to 540 nm). The effect of DCFH photooxidation was minimized by collecting the fluorescence image with a single rapid scan (line average, 4 s; total scan time, 5.2 s) under identical conditions, such as contrast and brightness, for all samples. The cells were then imaged by differential interface contrast microscopy. Preliminary study demonstrated that HG induced intracellular ROS in MC as early as 15 min and gradually increased up to 4 h.

Assay of PKC Activity

Membrane and cytosol fractions were obtained according to the method previously described (33), and PKC activity was measured by a PepTag nonradioactive detection kit (Promega Corporation, Madison, WI) using fluorescence peptide (PLSRTLSVAAK), which is a highly specific substrate for PKC, according to the manufacturer’s descriptions. According to manufacturer, <10 ng of kinase can be detected by this assay. Phosphorylation of the substrate by PKC alters the peptide’s net charge from +1 to −1. The phosphorylated substrate was separated by an agarose gel at pH 8.0. Negatively charged bands from the gel were removed and heated at 95°C until the gel slice melted. Fluorescence of the solubilized solution was measured by a microplate spectrofluorometer (Luminescence spectrometer LS50B; Perkin Elmer, Norwalk, CT). Specific PKC activity was reported as relative PKC activity per mg of total protein.

Isolation of Total RNA and Northern Blot Analyses

Standard Northern blot analyses were performed as described previously (10,28) after isolation of total RNA using the method of Chomczynski and Sacchi (34). Partial cDNAs for rat MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cloned after reverse transcription-PCR and confirmed by DNA sequencing were used for the respective DNA probe. In brief, 20 μg of total RNA was denatured, separated by electrophoresis through a 1.2% agarose gel containing 2.2 M formaldehyde, transferred onto nylon membranes using a capillary transfer, and covalently crosslinked to the membrane with ultraviolet light using a gene-linker (Bio-Rad). The membranes were hybridized with ³²P-labeled cDNA probes for MCP-1 and then rehybridized with a probe for GAPDH as an internal control to assess RNA quantity and integrity. Autoradiography was performed by exposing the blots to Kodak x-ray film with intensifying screens at −70°C for 1 to 5 d. Quantitation of mRNA signals was performed by densitometry using MCID (Imaging Research Inc., St. Catharines, Ontario, Canada) and normalized with GAPDH mRNA signals.

Measurements of MCP-1 by ELISA

To quantify the level of MCP-1 protein under different experimental conditions, MCP-1 was measured in MC culture supernatant using a commercial solid phase quantitative sandwich ELISA kit for MCP-1 (Biosource International, Inc, Camarillo, CA) according to the manufacturer’s descriptions. According to the manufacturer, the ELISA kit for MCP-1 is specific for rat MCP-1 and sensitive to 8 pg MCP-1/ml.

Statistical Analyses

All results are expressed as mean ± SE. ANOVA was used to assess the differences between multiple groups. If the F statistic was significant, the mean values obtained from each group were then compared by Fisher’s least significant difference method. P < 0.05 was used as the criterion for a statistically significant difference.

HG-Induced NF-κB Activation in Mesangial Cells

As presented in Figure 1A, basal NF-κB activation was observed in MC maintained in serum-free DMEM for 48 h. NF-κB activation increased significantly at 30 mM glucose and remained elevated at 50 and 100 mM glucose. Thus 30 mM d-glucose was used as HG for this study. The density of the upper two bands increased in parallel under HG, and the difference in optical density between control and different glucose concentrations remained unchanged whether each band alone or both were analyzed. Thus, the sum of optical density of two bands is shown in Figure 1B. Unlike d-glucose, the addition of 25 mM l-glucose to the media containing 5.6 mM d-glucose did not activate NF-κB activation in MC compared with control (Figure 1C), suggesting that HG-induced NF-κB activation is not the result of high osmolality per se.

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Figure 1. Effects of glucose on nuclear factor-κB (NF-κB) DNA binding activity in mesangial cells. Synchronized quiescent MES-13 were stimulated under experimental condition for 1 h. Nuclear proteins were prepared and subjected to electrophoretic mobility shift assay (EMSA) as described in Materials and Methods. (A) A representative EMSA under different concentrations of glucose. (B) Bar graph showing the mean ± SE from five experiments. The density of upper two bands increased in parallel under different concentrations of glucose and the difference in optical density among different concentrations of glucose remained unchanged whether each band alone or both were analyzed. Thus, the sum of optical density of two bands is shown. *P < 0.05 compared with 5.6 mM (control) glucose. (C) A representative EMSA under d- or l-glucose.

To ensure that the mobility shift bands under the present experimental condition were the NF-κB/oligonucleotide complex, we used an NF-κB mutant oligonucleotide with ‘G’ to ‘C’ substitution in the NF-κB binding motif as a control. Addition of this cold NF-κB mutant oligonucleotide (100-fold in excess) did not affect the bands (p50/p65 and p50/p50) that were associated with the labeled NF-κB oligonucleotide probe, whereas the cold consensus NF-κB oligonucleotide (100-fold in excess) completely abolished these bands (Figure 2A). AP-1 or Oct-1 consensus oligonucleotide probe also failed to displace the labeled NF-κB oligonucleotide probe (Figure 2A). These results provided the specificity of the NF-κB binding activity. Incubation of nuclear extracts with antisera against NF-κB p50 or p65 subunit caused a supershift of the corresponding NF-κB/oligonucleotide band (Figure 2B). Antibody to p50 interacted and retarded both of upper two bands. Antibody to p65 retarded the slowest migrating band, leaving most of the lower part of upper two bands unaffected. These results suggest that most of the binding activity arose from p50/p50 and p50/p65 dimers. The molecular weight of other members of NF-κB family are more than 50 kD; therefore, the band between p50/p50 homodimer and unbound probe was considered to be nonspecific. Both primary rat and primary mouse MC exhibited similar pattern of NF-κB activation in response to HG (data not shown).

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Figure 2. Specificity of high glucose-induced NF-κB activation in mesangial cells. Synchronized quiescent MES-13 were cultured under 30 mM d-glucose for 1 h. (A) Competition assay verifying upper two bands are specific NF-κB DNA complex. Nuclear extracts (20 μg each) were pretreated with 100-fold excess cold wild type or mutant NF-κB, AP-1, or Oct-1 oligonucleotide. (B) Supershift assay showing the slowest migrating band is p50/p65 heterodimer and the lower part of upper two bands is p50/p50 homodimer. Nuclear extracts were incubated with 1 μl of either p50 or p65 antibodies for 30 min at room temperature prior to binding reaction.

In a time-response study, HG significantly activated NF-κB activity at 30 min (Figure 3, A and B). HG-induced NF-κB activation was maximal at 1 h and gradually decreased but remained increased compared with control for up to 48 h. In subsequent experiments, we determined NF-κB binding activity at 1 h after HG treatment.

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Figure 3. Time response of high glucose-induced NF-κB activation in mesangial cells. (A) Synchronized quiescent MES-13 were incubated with 30 mM d-glucose for the indicated period. NF-κB activity was assessed by EMSA. (B) Bar graph showing the mean ± SE from six experiments. *P < 0.05 compared with basal NF-κB activity; ⁺P < 0.05 compared with 5.6 mM glucose at the corresponding incubation period.

Role of ROS in HG-Induced NF-κB Activation in Mesangial Cells

Exogenous H₂O₂ induced mesangial NF-κB activation in a dose-dependent manner and was significant at 100 μM (Figure 4A). Structurally different cell-permeable antioxidants, PDTC, NAC, and trolox, abolished HG-induced, but not basal, NF-κB activation in MC cultured under HG (Figure 4, B and C), suggesting that the effect of HG on NF-κB activation in MC is mediated by ROS. In addition, as summarized in Figure 5, HG induced intracellular ROS in MC at 1 h. Unlike d-glucose, the addition of 25 mM of either l-glucose or 3-O-methyl-d-glucose to the media containing 5.6 mM d-glucose failed to induce intracellular ROS in MC (Figure 5). A glucose transporter inhibitor, cytochalasin B, effectively inhibited HG-induced intracellular ROS (Figure 5, inset picture a). These results suggest that glucose metabolism, but not glucose autooxidation, is necessary for HG-induced ROS generation in MC, because 3-O-methyl-d-glucose is an autooxidizable glucose analog that enters into the cell but is not metabolized. ROS visualized by confocal microscopy is often restricted in the cytoplasm as presented in Figure 5 (inset picture b). Our preliminary study (35) demonstrated that structurally different antioxidants and ROS scavenger PDTC, trolox, dimethylthiourea, and catalase abolished HG-induced ROS generation in MC.

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Figure 4. Role of reactive oxygen species (ROS) in high glucose (HG)-induced NF-κB activation in mesangial cells. (A) Synchronized quiescent MES-13 were incubated with H₂O₂ for 1 h. NF-κB activity was assessed by EMSA. (B) Synchronized quiescent MES-13 were pretreated with 100 μM pyrrolidine dithiocarbamate (PDTC), 5 mM n-acetyl-l-cysteine (NAC), and 500 μM trolox for 1 h and incubated under 5.6 mM control or 30 mM HG for 1 h. (C) Bar graph showing the mean ± SE from five to seven experiments. *P < 0.05 compared with control glucose without antioxidant. ⁺P < 0.05 compared with HG without antioxidant.

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Figure 5. Specificity of 2′,7′-dichlorofluorescein (DCF)-sensitive ROS in mesangial cells cultured under HG. Synchronized quiescent MES-13, primary mouse, or rat MC were incubated under control glucose and after the addition of 25 mM (for total of 30 mM) of d-glucose, 3-O-methyl-d-glucose, or l-glucose for 1 h, and intracellular DCF-sensitive ROS was visualized by a confocal microscopy. (Inset image a) To confirm the role of glucose uptake in HG-induced ROS generation, MC were pretreated with 0.2 μM cytochalasin B for 1 h before the addition of 25 mM d-glucose. (Inset image b) Intracellular ROS is distributed in cytoplasm but not in the nuclei (arrowhead).

Role of PKC in HG-Induced NF-κB Activation and Intracellular ROS Generation in Mesangial Cells

Blockade of PKC either by a PKC inhibitor calphostin C 100 nM or by depletion of PKC by preincubating MC with 100 nM PMA for 24 h effectively inhibited HG-induced, but not basal, NF-κB binding activity (Figure 6, A and B) and ROS generation (Figure 7) in MC. PMA at 100 nM increased ROS generation fourfold and NF-κB activity threefold (data not shown). Both pretreatment with calphostin C and PKC depletion effectively inhibited PMA-induced ROS generation and NF-κB activation in MC. Structurally different PKC inhibitor GFX 109203X at 24 nM also effectively blocked PMA- and HG-induced intracellular ROS generation in MC without significant effect on basal ROS generation (data not shown). In addition, as summarized in Figure 8, HG induced PKC translocation from cytosol to membrane in MC as early as 15 min and continuously up to 48 h. PKC translocation at 48 h was significantly higher than that at 24 h. Total PKC activity was also increased after 24 h (data not shown).

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Figure 6. Role of protein kinase C (PKC) in HG-induced NF-κB activation in mesangial cells. (A) Synchronized quiescent MES-13 were incubated under control or HG in the presence and absence of PKC inhibition. NF-κB activity was assessed by EMSA. (B) Bar graph showing the mean ± SE from five experiments.

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Figure 7. Role of PKC in HG-induced ROS generation in mesangial cells. Synchronized quiescent MES-13 were incubated under control or HG in the presence and absence of PKC inhibition. 100 nM calphostin C was added 1 h before the addition of HG.

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Figure 8. Effects of HG on PKC activity in mesangial cells. Synchronized quiescent MES-13 were incubated under control or high d-glucose for the indicated period. Membrane and cytosolic fractions were prepared and subjected to detection of PKC activity as described in Materials and Methods. *P < 0.05 compared with membrane fraction in control glucose (1.0); ⁺P < 0.05 compared with membrane fraction at 24 h.

Role of HG-Induced NF-κB Activation in MCP-1 mRNA and Protein Expression by Mesangial Cells

As summarized in Figure 9A, IκBαM-transfected MC did not exhibit NF-κB nuclear translocation at basal as well as in response to HG. IL-1β-induced NF-κB activation was impaired in IκBαM-transfected MC as previously demonstrated (26). HG increased MCP-1 protein secretion by 1.6-fold that of control in MC transfected with control vector at 48 h but not in MC transfected with dominant negative mutant of NF-κB (Figure 9B). MCP-1 mRNA expression was significantly upregulated (1.9-fold that of control) in mock-transfected, but not in IκBαM-transfected, MC cultured under HG for 24 h (Figure 9C), suggesting that HG-induced NF-κB activation is responsible for MCP-1 mRNA expression and protein secretion in MC.

Effects of PKC Inhibition and Antioxidants on HG-Induced MCP-1 Expression by Mesangial Cells

As summarized in Figure 10, HG increased MCP-1 protein secretion by 1.6-fold that of control glucose in MC transfected with control vector at 48 h. Blockade of PKC either by a PKC inhibitor GFX 109203X at 24 nM or by depletion of PKC by preincubating MC with 100 nM PMA for 24 h effectively inhibited HG-induced MCP-1 protein secretion. PDTC, NAC, and trolox at doses inhibiting NF-κB activation abolished HG-induced MCP-1 protein secretion in MC cultured under HG. PKC inhibition or antioxidants did not affect basal MCP-1 protein secretion (data not shown).

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Figure 10. Role of PKC and ROS in HG-induced MCP-1 secretion by mesangial cells. Synchronized quiescent MC transfected with control vector were incubated for 48 h under control or HG in the presence and absence of 24 nM GFX 109203X, PKC depletion, 100 μM PDTC, 5 mM NAC, and 500 μM trolox for 48 h. GFX 109203X, PDTC NAC, and trolox were added 1 h before the stimulation of HG. *P < 0.05 compared with control glucose, ⁺P < 0.05 compared with HG without PKC inhibition or antioxidants.