Diabetes-Induced Activation of Canonical and Noncanonical Nuclear Factor-B Pathways in Renal Cortex
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糖尿病学杂志 2006年第5期
1 Division of Endocrinology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas
2 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas
3 Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas
4 Stark Diabetes Center, University of Texas Medical Branch, Galveston, Texas
DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; IB, inhibitor of B; IKK, inhibitor of B kinase; IL, interleukin; NF-kB, nuclear factor-B; NIK, NF-BeCinducing kinase; TNF, tumor necrosis factor
ABSTRACT
Evidence of diabetes-induced nuclear factor-B (NF-B) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-B canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-BeCregulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-B canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-B noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of B kinase- and several fold increases in NF-BeCinducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-B pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-B activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-B pathway proteins.
The concept that diabetes is a chronic inflammatory disease is supported by a growing body of clinical, epidemiological, and experimental data indicating that chronic, low-grade inflammation is a common denominator linking obesity, insulin resistance, atherosclerosis, dyslipidemia, and excessive glucose metabolism in diabetes (1eC5). Epidemiological studies (2,6eC8) have demonstrated that markers of inflammation predict development of both type 1 and 2 diabetes, and low-grade inflammation is now considered an important constituent of the metabolic syndrome leading to insulin resistance (9,10). The proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-, and IL-1, as well as plasminogen activator inhibitor are increased in diabetic patients and are independently correlated with duration of diabetes (11,12), suggesting that chronic hyperglycemia creates a proinflammatory milieu. Even acute hyperglycemia increases circulating levels of IL-6, TNF-, and IL-18 and exposure of human mononuclear cells obtained from healthy volunteers to elevated glucose increases TNF- and IL-6 production (13,14).
There is a growing consensus that glucose- and diabetes-induced activation of nuclear factor-B (NF-B) contributes to the proinflammatory diabetic milieu (15eC20). The NF-B family of transcription factors is controlled by two distinct activation mechanisms known as canonical (classical) and noncanonical pathways (21). The former plays an important role in the host innate immune response by inducing the expression of numerous proinflammatory cytokines, chemokines, adhesion molecules, inducible enzymes, and proangiogenic growth factors as well as inhibiting apoptosis (22,23). The latter, more recently identified noncanonical pathway plays an important role in the adaptive immune response, including secondary lymphoid organogenesis and lymphocyte maturation (24,25). Noncanonical also can refer to any NF-B gene activation pathway that does not follow the classical pathway, i.e., serine 536 phosphorylation of RelA.
The NF-B complex is a heterogeneous collection of protein dimers in different combinations of related proteins that share a conserved DNA-binding and dimerization domain called the Rel homology domain (22). Five mammalian Rel proteins have been identified, including p65/RelA, RelB, c-Rel, p50/NF-B1, and p52/NF-B2. Except for p52, these proteins are constitutively present in the cytoplasm bound to a number of inhibitory proteins (inhibitor of B [IB]-, IB-, p105, p100, and BclIII) that are characterized by the presence of six to seven ankyrin repeats that mediate binding to the Rel homology domain. Both p50 and p52 exist as precursor proteins (p105 and p100, respectively), contain the ankyrin repeat structure of the inhibitory proteins, are cleaved during proteolytic processing to p50 and p52, and lack transactivation domains characteristic of the other Rel proteins (22). Stimulation of NF-B is mediated by diverse signal transduction cascades that converge on a large enzyme complex, the IB kinase (IKK) (22,26). The IKK complex is composed of the catalytic subunits IKK-, IKK-, and the scaffold protein IKK- (NEMO), and like the Rel proteins, IKK- and IKK- undergo homo- and heterodimerization. The noncanonical NF-B activation pathway uses IKK- homodimers and is dependent on phosphorylation-dependent ubiquitination of NF-B2/p100, which allows degradation of the COOH-terminal portion of the protein, freeing p52 containing the Rel homology domain (24). Activation of this pathway is initiated by an upstream mitogen-activated 3 kinaseeClike kinase, NF-kBeCinducing kinase (NIK), and the predominant heterodimer is a p52:RelB complex (25,27).
In this study, we have quantified proteins comprising both canonical and noncanonical NF-B activation pathways in the renal cortex of male db/db mice and age-matched controls. We report significant activation of the canonical pathway based on the induction of numerous NF-BeCregulated cytokines and chemokines. The increased renal cytokine production occurred without changes in canonical pathway proteins and with significant alterations in noncanonical pathway proteins. Our observations suggest that the diabetes-induced chronic activation of innate immunity may involve noncanonical NF-B pathway proteins.
RESEARCH DESIGN AND METHODS
Male Leprdb (db/db) mice were purchased from The Jackson Laboratories (Bar Harbor, ME) at 4 weeks of age and were housed in the University of Texas Medical Branch Animal Resource Center in accordance with its Institutional Animal Care and Use Committee and the Public Health Service Guide for the Care and Use of Laboratory Animals. Mice with the same genetic background (C57BLKS/J) were used as controls. Mice were killed 1 and 3 months after hyperglycemia onset, defined as a group mean value >15 mmol/l, which occurred at 8 weeks of age. Control and db/db mice were housed two to three per cage in a room with a 12-h artificial light cycle and free access to standard diet and water. Body weight and nonfasting blood glucose were measured weekly.
Mice were anesthetized (ketamine/xylazine; 70/10 mg/kg i.p.) and anticoagulated (5 units heparin) then exsanguinated before rapid aortic perfusion with ice-cold PBS to quickly rinse kidneys free of blood and to deliver protease (10 e蘥/ml pepstatin A, 10 e蘥/ml leupeptin, 10 e蘥/ml soybean trypsin inhibitor, and 10 e蘥/ml aprotinin) and phosphatase (1 mmol/l orthovanadate and 30 mmol/l sodium fluoride) inhibitors, phenylmethylsulphonylfluoride (1 mmol/l), and dithiothreitol (DTT) (0.5 mmol/l). Plasma was frozen at eC80° C, and both kidneys were removed, decapsulated, weighed, and frozen at eC80° C until processed for protein extraction. Before freezing, a 2- to 3-mm midline transmural section was removed from each kidney for fixation in 10% formaldehyde for 24 h before processing for immunohistochemistry.
Metabolic and cytokine measurements.
Commercially available kits were used to measure plasma glucose (Sigma-Aldrich, St. Louis, MO), cholesterol, and triglycerides (Wako Chemicals, Richmond, VA). Insulin, leptin, and amylin (Linco, St. Louis, MO) and multiple cytokines and chemokines were measured on additional aliquots of plasma and on a sample of renal cortical extract collected after 3 months of hyperglycemia using the Bio-Plex system, which are multiplex bead-based assays used with the Luminex xMAP technology according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). Eight-point standard curves were performed for each cytokine using the same Luminex bead technology.
Preparation of cytosolic and nuclear protein extracts.
Renal cortex (50 mg) was homogenized on ice using Wheaton 1-ml Dounce homogenizers in a 20-fold excess (wt/vol) of low-salt solution A (50 mmol/l HEPES, pH 7.0, 10 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l EGTA, protease inhibitor cocktail [Sigma P8340, containing 240 e蘥/ml AEBSF, 5 e蘥/ml aprotinin, 10 e蘥/ml leupeptin, 14 e蘥/ml bestatin, 10 e蘥/ml pepstatin A, and 5 e蘥/ml E-64], phosphatase inhibitors [1 mmol/l orthovanadate and 30 mmol/l sodium fluoride], 1 mmol/l DTT, and 0.5 mmol/l phenylmethylsulphonylfluoride). The homogenate was spun for 30 s at 100g and 4°C to remove tissue debris, the supernatant was transferred to a fresh tube, 10% NP-40 was added for a final concentration of 0.05%, tubes were mixed by gentle inversion, and immediately spun for 7 min at 1,000g and 4°C. The supernatant was used as cytosolic extract, while the pellet was gently resuspended in solution A containing 1 mol/l sucrose, and spun for 5 min at 8,000g at 4°C. The supernatant was removed and discarded, and the pellet gently resuspended in 20 e蘬 solution A. Eighty microliters of a high-salt solution B (50 mmol/l HEPES, pH 7.0, 500 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 10% glycerol, protease and phosphatase inhibitors, 1 mmol/l DTT, and 0.5 mmol/l phenylmethylsulphonylfluoride) were added dropwise with gentle vortexing. The suspension was allowed to sit on ice for 30 min, vortexing for 15 s every 5 min, then spun at 16,100g for 20 min. The supernatant was used as nuclear extract. Protein concentrations of nuclear and cytosolic extracts were determined using the Bio-Rad (Hercules, CA) protein assay with bovine albumin standards, and both samples were assayed for cytosolic (-tubulin and glyceraldehyde-3-phosphate dehydrogenase) and nuclear (lamin B) proteins.
Western immunoblot analysis.
Western blots were quantified from three to five separate sets of cytosolic and nuclear protein extracts pooled from an equal mass of renal cortical extract of three control and three diabetic animals using a panel of antibodies for each mouse protein (Table 1). Tissue lysates were electrophoresed on 7.5 or 10% SDS-polyacrylamide gels and analyzed by immunoblotting after transfer to nitrocellulose membranes (Bio-Rad), using primary antibodies according to each manufacturer’s instructions, followed by species-specific secondary antibodies tagged with a fluorescent dye (IR Dye 800; Rockland) at a 1:5,000 dilution. Densitometric quantitation of each protein was performed using the LI-COR Bioscience Odyssey Imaging System with infared fluorescence detection (LI-COR Bioscience, Lincoln, NE). -Actin was measured on every blot using an IR Dye 700 for evaluation of protein loading.
Electrophoretic mobility shift assay.
Sense and antisense oligonucleotides containing an IL-6 NF-B consensus sequence (5'-CATGTGGGATTTTCCCATGAC-3') with a 5' IR Dye 700 label were purchased, and double-stranded DNA was prepared according to the manufacturer’s instructions (LI-COR Bioscience). An IR Dye 800eClabeled mutant oligonucleotide (5'-CATGTGAGATTTTCACATGAC-3') was used as a control. Unlabeled (cWT) IL-6 promoter was purchased from Sigma Genosys. A total of 17.5 e蘥 nuclear extract were diluted to a concentration of 4 e蘥/e蘬 by the addition of solution C (1:4 solutions A:B) then combined with 2 e蘬 ice-cold 10x binding buffer (100 mmol/l TRIS, 500 mmol/l NaCl, 10 mmol/l DTT, pH 7.5) and Milli-Q water to make a final volume of 20 e蘬. Where applicable, 80-fold excess unlabeled wild-type oligonucleotide or 80-fold excess mutant oligonucleotide were added to the mixture and allowed to equilibrate for 15 min. One microliter of fluorescent probe was added and allowed to equilibrate on ice for 30 min. Samples were mixed with 1 e蘬 loading dye, placed on a 4% native PAGE gel, and the gel was run at 160 V on ice for 1 h then imaged using the LI-COR Bioscience Odyssey Imaging System according to the manufacturer’s instructions. RelB (Santa Cruz 226x; 4 e蘥), p50 (Santa Cruz 114x; 4 e蘥), and p65 (600-401-271, 2 e蘥; Rockland) antibodies were separately added to the incubation mix for 30 min at room temperature before adding probe to perform supershift assays.
Immunohistochemistry.
Paraffin-embedded 5-e蘭 kidney sections were washed in xylene twice for 5 min each and rehydrated by successive rinsing in 100, 95, 75, and 50% ethanol followed by PBS, and antigen retrieval was performed using 10 mmol/l sodium citrate (pH 6.0) with 0.1% Triton X for 20 min at 95°C. Slides were blocked using nonimmune sera from the secondary antibody species diluted to 2% in PBS containing 0.2% cold fish skin gelatin, 0.1% saponin, and 0.05% Tween 20 for 2 h at room temperature. Primary antibodies directed against NIK (Cell Signaling 4994, 1:100 dilution), RelB (sc226, 1;100 dilution; Santa Cruz Biotechnology), p50 (Abcam 7971, 1:100 dilution), F4/80 (specific macrophage stain, 1:100 dilution; Abcam 16911), and myeloperoxidase (specific neutrophil stain,1:500 dilution; Spring Bioscience) were incubated overnight at 4°C in blocking buffer containing 2% BSA substituted for the nonimmune sera. Tissue sections were washed three times in PBS for 10 min each, and secondary antibody Texas RedeClabeled goat anti-rabbit IgG (1:100 dilution; Rockland) was added for 2 h at room temperature. Sections were again washed three times in PBS, and DAPI (1:10,000 dilution of a 100-e蘥/ml stock in PBS) was added for 15 min for nuclear counterstaining, the sections washed in PBS, mounted with coverslips, and viewed using a Nikon Eclipse 80i microscope with fluorescence illumination system and digital camera (DXM1200F). Negative controls included omitting the primary antibody.
RESULTS
Gravimetric, metabolic, and hormone data.
Body weights of db/db mice were increased significantly at the onset of hyperglycemia, peaked by 1 month of hyperglycemia (approximately twofold increase over age-matched controls), then remained constant over the next 2 months (Table 2). After 3 months of hyperglycemia, the db/db mice exhibited significant hypercholesterolemia, hyperinsulinemia, and hyperleptinemia, and plasma amylin levels were increased 10-fold versus control mice (Table 2).
Plasma and renal tissue cytokine levels.
Numerous cytokines and chemokines were significantly increased in plasma of diabetic versus control mice after 1 month of hyperglycemia, including RANTES (40% increase), IL-1 (45%), IL-1 (50%), IL-12(p70) (60%), TNF- (62%), IL-3 (84%), and IL12(p40) (100%) (Fig. 1). After 3 months of hyperglycemia, all of these cytokines were decreased 50eC80% below controls, while additional cytokines were increased approximately twofold, including KC, IL-6, G-CSF (P < 0.05 vs. controls), and MIP-1. Numerous proinflammatory cytokines were increased significantly in 3-month renal cortical extracts, ranging from a 14% increase for IL-2 to a threefold increase for KC (Fig. 2). In addition, anti-inflammatory cytokines, including IL-12(p40) and IL-10, were significantly decreased.
Western blot of NF-B pathway proteins.
Band intensities of NF-B pathway proteins were quantified from separate renal cortical cytosolic and nuclear extracts from mice diabetic for 3 months and their age-matched controls (Fig. 3). Results are expressed as a mean and SD of the ratio of diabetic to control band intensities after normalization to -actin from each lane. Quantitation of three separate sets of pooled extracts from three control and three diabetic mice revealed no significant change in band intensities for RelA/p65 (1.00 ± 0.19), p50 (1.03 ± 0.20), IB- (1.07 ± 0.12), and IB- (1.14 ± 0.09) in the canonical pathway, while NIK and RelB were increased 5.6-fold and 3-fold, and IKK- was decreased 38%. The nuclear content of canonical and noncanonical NF-B pathway proteins was decreased in diabetic renal cortex (Fig. 3). RelB increased threefold in the cytoplasm and decreased twofold in the nucleus of diabetic versus control mice (Fig. 4A). Under the experimental conditions utilized for Western immunoblotting, p100 was identifiable only in nuclear extracts (Fig. 4B) and in general was not visible on Western blots from total tissue extracts in which the nuclear proteins would be diluted into the much larger volume of cytosolic proteins (data not shown). p52 could not be reproducibly measured.
Analysis of phosphorylation sites on NF-B pathway proteins from whole-tissue extracts indicated that phosphorylation of serines 276, 529, and 536 on RelA/p65 was decreased in diabetic versus control tissue (Fig. 4C). Likewise, separate antibodies recognizing serine 32 or serines 32 and 36 of IB- indicated either no change or decreases in diabetic renal cortex. In contrast, antibodies recognizing phosphorylation sites of IKK- (serines 176 and 180) and IKK- (serines 177 and 181) indicated increased phosphorylation in diabetic renal cortex.
Electrophoretic mobility shift assay.
Increased binding of protein to DNA was observed in nuclear extracts of diabetic versus control renal cortex (Fig. 5, lanes 2 vs. 5). A single complex was identified that was specifically inhibited by an 80-fold excess of cold wild-type oligonucleotide (lanes 3 and 6) but not by the same excess of mutant oligonucleotide (lanes 4 and 7). Antibodies to RelB were capable of supershifting a portion of the protein binding to the DNA (lane 8), while antibodies to RelA and p50 did not, even though the latter antibodies were capable of gel shifting TNF-eCstimulated Hela cell complexes (data not shown).
Immunohistochemistry.
Immunostaining for NIK demonstrated renal tubular epithelial localization in kidney sections from both control and diabetic mice (Fig. 6B and C). In control mice, NIK staining was predominantly located near the basal aspect of proximal tubular epithelial cells (Fig. 6B). Distal tubules, glomeruli, and interstitial cells remained relatively unstained (Fig. 6B, arrowheads), as did nuclei. The cellular distribution of NIK was comparable in diabetic kidneys, although there was increased staining throughout proximal tubular epithelial cells (Fig. 6C). RelB staining was intense and predominantly located in proximal and distal tubular epithelial cells in both control and diabetic mice (Fig. 6E and F). Nuclear staining was present within controls (arrowheads) but much less evident within diabetic kidneys. RelB staining was present but much lighter within glomeruli (Fig. 6F). p50 was present within renal tubular epithelial cells but not glomeruli of both control and diabetic kidneys, although the distribution of staining was punctuate and not very common (Fig. 6H and I).
DISCUSSION
Diabetes-induced inflammation and renal cytokine production.
This study utilized the db/db mouse model of type 2 diabetes since it develops robust changes within 2 months of the onset of hyperglycemia that include increases in renal mass, glomerular volume, mesangial volume, glomerular filtration rate, basement membrane thickening, and urinary albumin excretion (28). Our observation that numerous cytokines and chemokines, previously shown to be regulated in part by canonical NF-B pathway activation (23,29), were increased in the renal cortex of diabetic mice indicates chronic activation of the innate immune response via this NF-B pathway. Several of the elevated cytokines and chemokines previously have been associated with diabetic nephropathy (TNF-, IL-6, MCP-1, and RANTES) (30), while many have not (IL-2, IL-3, IL-5, IL-9, IL-12, G-CSF, GM-CSF, and KC). Virtually all cytokines and chemokines listed in Figs. 1 and 2 contain B motifs within the promoter region of their DNA (23). Exceptions include IL-3, IL-4, and IL-5, which are regulated by nuclear factor of activated T-cells (29). These elevated cytokines represent renal production of cytokines and not plasma contamination since kidneys were rinsed free of blood and plasma levels of the tested cytokines were at or below control levels at the 3-month time point. The possibility that infiltrating neutrophils, monocytes, and macrophages are responsible for the cytokine production cannot be ruled out but appears unlikely given the low numbers of infiltrating macrophages previously reported at this time point (31), and our inability to demonstrate a significant diabetes-induced increase of either macrophages (using anti-F4/80 antibody) or neutrophils (using anti-myeloperoxidase antibody) in random kidney sections from db/db mice (data not shown). The major contribution of tubular epithelial cells to total NF-B protein content demonstrated with immunohistochemistry in this study, together with previous observations that tubular epithelial cells produce a variety of cytokines and chemokines in other renal inflammatory diseases (32), suggest that this cell is responsible for the diabetes-induced increased cytokine production. We are the first to report that NIK and RelB are predominantly localized to tubular epithelial cells, an observation strikingly similar to other renal inflammatory diseases (see below).
Diabetes-induced canonical NF-B activation.
Increased immunohistochemical localization of p50 and p65 in the nucleus has been used as evidence of diabetes-induced activation of NF-B (18eC20). Likewise, reports using electrophoretic mobility shift assays (EMSAs) on cell extracts from renal and vascular cells in tissue culture have provided evidence of glucose-induced activation of NF-B (15eC19). A major caveat of these approaches is that NF-B protein binding is not synonymous with transcriptional activation, and demonstrating nuclear localization and/or DNA binding via these two methods provides no insights into mechanisms of diabetes-induced NF-B transcriptional activation.
Within the kidney, most studies of NF-B activation have utilized mesangial cells exposed to a variety of stimuli in tissue culture (rev. in 17) and have evaluated RelA and p50 as the predominant NF-B proteins. Renal tubular epithelial cells have received less attention, although recent studies (19,33) have demonstrated active NF-B. Even fewer experimental animal studies (20,34,35) have evaluated renal NF-B activation, and results are more discordant than in vitro cell culture studies. Reasons for this may include more chronic exposure to hyperglycemia, diabetes models evaluated (type 1 vs. type 2), animal species, and more complex tissue extracts than single-cell culture systems. Despite the decreased amounts of all NF-B proteins in nuclear extracts of 3-month diabetic db/db mice, we also report an increased protein binding to DNA using EMSA. While paradoxical, these data are consistent with the observation that the pool of individual nuclear NF-B proteins is much larger than the total amount of NF-B proteins actually binding to DNA (36). In contrast to previous experiments, only RelB could be supershifted slightly. Several possibilities exist for the inability of RelA and p50 antibodies to supershift the DNA-binding complex. These possibilities include the following: 1) RelA is incapable of binding DNA, perhaps through formation of inactive heterodimers with RelB (37), and 2) antibody epitopes are inaccessible in the protein complex.
The absence of any significant detectable changes by Western blot in cytosolic NF-B pathway proteins comprising the canonical pathway, and the finding that nuclear levels of all NF-B pathway proteins were decreased by approximately one-half, indicate that diabetes-induced chronic activation of the innate immune response is neither dependent on significant movement of NF-B dimers into the nucleus, nor is it due to prolonged degradation of IB inhibitory proteins that function to keep RelA:p50 homo- and/or heterodimers in the cytoplasm. The latter observation is consistent with previous reports showing sustained NF-B activation in the absence of significant decreases in IB- expression in peripheral blood mononuclear cells exposed to chronic high glucose (18).
Noncanonical NF-kB activation pathway.
The major novel finding of this study is diabetes-induced dysregulation of the noncanonical NF-B activation pathway, including significant changes in NIK, RelB, and IKK-. Our finding in diabetic kidneys that cytosolic and nuclear IKK- was decreased 40% (Fig. 3) yet phosphorylated IKK- was increased 34% (Fig. 4C) indicates that the fraction of activated IKK- increased significantly and provides support for noncanonical NF-B pathway activation. While only a few noncanonical pathway target genes have been identified, the positive transcriptional role of this pathway is clearly manifested in mice lacking specific noncanonical NF-B proteins (38). Pathophysiological roles for NIK remain mostly unexplored and the significance of the several-fold increase in NIK in diabetic renal cortex remains unclear at this time. NIK activation has been reported in pig models of ischemia-reperfusion injury and in delayed graft function in patients receiving kidney transplants (39). In the latter, activation of NIK occurred within proximal tubular epithelial cells, in a pattern strikingly similar to diabetes-induced changes reported here. In addition, NIK activity was induced by thrombin in cultured proximal tubular epithelial cells (39).
RelB displays a number of unusual features compared with other Rel proteins; it is weakly controlled by IB proteins and can serve as both a promoter and repressor of NF-BeCdependent gene expression (22,23,40). RelB primarily forms complexes with p50 and p52, the only Rel proteins lacking transactivation domains, yet is a transcriptional activator in these dimer conformations. Previously, it has been shown to be constitutively present only in nuclei of immune cells (40), and we show here that this also is true for renal cells (Fig. 3). Like IB proteins, the expression of RelB is controlled by NF-B (41), and the threefold increase in cytosolic RelB reported in this study is consistent with NF-B canonical pathway activation.
Potential mechanisms responsible for prolonged activation of the NF-B canonical pathway in diabetic kidney.
At least three potential mechanisms may contribute to the prolonged activation of the canonical NF-B pathway observed in renal cortex of db/db mice. First, based on the phenotype of RelB-deficient mice, RelB has been implicated as an important repressor of the inflammatory process, modulating the transition from innate to adaptive immunity (42). These mice develop a severe, noninfectious, multiorgan inflammatory syndrome that significantly contributes to their premature death (43). Renal fibroblasts harvested from RelB-deficient mice exhibit dysregulated cytokine production following LPS stimulation, which can be corrected by transfection of RelB cDNA into these cells (42). At least two mechanisms may be responsible for the repressive function of RelB. First, it has been reported that RelB exerts transcriptional suppression via stabilization of IB- protein (44). A second mechanism is via inhibition of DNA binding by RelA through its complex formation with RelB (37). Despite the threefold increase in cytosolic RelB, the nuclear content was decreased twofold in diabetic kidneys, which may be sufficient to impact upon its ability to form dimers with IB- or RelA to terminate the innate immune response. A second potential mechanism for the diabetes-induced prolonged activation of the canonical NF-B pathway involves phosphorylation of RelA ser276, which is necessary for RelA/RelB dimerization (45). We report a decrease in ser276 phosphorylation in diabetic kidney (Fig. 4C). Third, it has recently been reported that IKK- plays a negative role in macrophage activation and inflammation by accelerating the turnover and removal of RelA from proinflammatory gene promoters (46). This was accomplished via phosphorylation of RelA ser536; impaired ser536 phosphorylation significantly prolonged the retention time of RelA on the promoter of NF-B genes. It is noteworthy that both IKK- protein levels (cytosolic and nuclear) and RelA ser536 phosphorylation were decreased in diabetic kidneys in this study.
In summary, we report sustained activation of innate immunity via the NF-B canonical pathway in the renal cortex of db/db mice after 3 months of hyperglycemia, manifested by significantly elevated tissue concentrations of numerous NF-BeCregulated cytokines and chemokines. This was associated with a twofold reduction in the nuclear content of all NF-kB pathway proteins, no changes in tissue cytosolic levels of all proteins comprising the canonical pathway, and with significant changes in proteins essential for noncanonical NF-B pathway activation, including NIK, IKK-, and RelB.
ACKNOWLEDGMENTS
This project was supported by an American Diabetes Association Research Grant (to R.G.T.) and a National Heart, Lung, and Blood Institute Grant HL055630 (to A.R.B.). B.C.T. is a recipient of a National Institute of Environmental Health Sciences Predoctoral Training Grant (M. Moslen, Director) and a McLaughlin Predoctoral Fellowship in Infection and Immunity (Stanley M. Lemon, Director).
We acknowledge the excellent technical assistance of Hung Sun, Hung Q. Doan, Negin N. Fouladi, and Chang Lee.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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2 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas
3 Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas
4 Stark Diabetes Center, University of Texas Medical Branch, Galveston, Texas
DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; IB, inhibitor of B; IKK, inhibitor of B kinase; IL, interleukin; NF-kB, nuclear factor-B; NIK, NF-BeCinducing kinase; TNF, tumor necrosis factor
ABSTRACT
Evidence of diabetes-induced nuclear factor-B (NF-B) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-B canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-BeCregulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-B canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-B noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of B kinase- and several fold increases in NF-BeCinducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-B pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-B activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-B pathway proteins.
The concept that diabetes is a chronic inflammatory disease is supported by a growing body of clinical, epidemiological, and experimental data indicating that chronic, low-grade inflammation is a common denominator linking obesity, insulin resistance, atherosclerosis, dyslipidemia, and excessive glucose metabolism in diabetes (1eC5). Epidemiological studies (2,6eC8) have demonstrated that markers of inflammation predict development of both type 1 and 2 diabetes, and low-grade inflammation is now considered an important constituent of the metabolic syndrome leading to insulin resistance (9,10). The proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-, and IL-1, as well as plasminogen activator inhibitor are increased in diabetic patients and are independently correlated with duration of diabetes (11,12), suggesting that chronic hyperglycemia creates a proinflammatory milieu. Even acute hyperglycemia increases circulating levels of IL-6, TNF-, and IL-18 and exposure of human mononuclear cells obtained from healthy volunteers to elevated glucose increases TNF- and IL-6 production (13,14).
There is a growing consensus that glucose- and diabetes-induced activation of nuclear factor-B (NF-B) contributes to the proinflammatory diabetic milieu (15eC20). The NF-B family of transcription factors is controlled by two distinct activation mechanisms known as canonical (classical) and noncanonical pathways (21). The former plays an important role in the host innate immune response by inducing the expression of numerous proinflammatory cytokines, chemokines, adhesion molecules, inducible enzymes, and proangiogenic growth factors as well as inhibiting apoptosis (22,23). The latter, more recently identified noncanonical pathway plays an important role in the adaptive immune response, including secondary lymphoid organogenesis and lymphocyte maturation (24,25). Noncanonical also can refer to any NF-B gene activation pathway that does not follow the classical pathway, i.e., serine 536 phosphorylation of RelA.
The NF-B complex is a heterogeneous collection of protein dimers in different combinations of related proteins that share a conserved DNA-binding and dimerization domain called the Rel homology domain (22). Five mammalian Rel proteins have been identified, including p65/RelA, RelB, c-Rel, p50/NF-B1, and p52/NF-B2. Except for p52, these proteins are constitutively present in the cytoplasm bound to a number of inhibitory proteins (inhibitor of B [IB]-, IB-, p105, p100, and BclIII) that are characterized by the presence of six to seven ankyrin repeats that mediate binding to the Rel homology domain. Both p50 and p52 exist as precursor proteins (p105 and p100, respectively), contain the ankyrin repeat structure of the inhibitory proteins, are cleaved during proteolytic processing to p50 and p52, and lack transactivation domains characteristic of the other Rel proteins (22). Stimulation of NF-B is mediated by diverse signal transduction cascades that converge on a large enzyme complex, the IB kinase (IKK) (22,26). The IKK complex is composed of the catalytic subunits IKK-, IKK-, and the scaffold protein IKK- (NEMO), and like the Rel proteins, IKK- and IKK- undergo homo- and heterodimerization. The noncanonical NF-B activation pathway uses IKK- homodimers and is dependent on phosphorylation-dependent ubiquitination of NF-B2/p100, which allows degradation of the COOH-terminal portion of the protein, freeing p52 containing the Rel homology domain (24). Activation of this pathway is initiated by an upstream mitogen-activated 3 kinaseeClike kinase, NF-kBeCinducing kinase (NIK), and the predominant heterodimer is a p52:RelB complex (25,27).
In this study, we have quantified proteins comprising both canonical and noncanonical NF-B activation pathways in the renal cortex of male db/db mice and age-matched controls. We report significant activation of the canonical pathway based on the induction of numerous NF-BeCregulated cytokines and chemokines. The increased renal cytokine production occurred without changes in canonical pathway proteins and with significant alterations in noncanonical pathway proteins. Our observations suggest that the diabetes-induced chronic activation of innate immunity may involve noncanonical NF-B pathway proteins.
RESEARCH DESIGN AND METHODS
Male Leprdb (db/db) mice were purchased from The Jackson Laboratories (Bar Harbor, ME) at 4 weeks of age and were housed in the University of Texas Medical Branch Animal Resource Center in accordance with its Institutional Animal Care and Use Committee and the Public Health Service Guide for the Care and Use of Laboratory Animals. Mice with the same genetic background (C57BLKS/J) were used as controls. Mice were killed 1 and 3 months after hyperglycemia onset, defined as a group mean value >15 mmol/l, which occurred at 8 weeks of age. Control and db/db mice were housed two to three per cage in a room with a 12-h artificial light cycle and free access to standard diet and water. Body weight and nonfasting blood glucose were measured weekly.
Mice were anesthetized (ketamine/xylazine; 70/10 mg/kg i.p.) and anticoagulated (5 units heparin) then exsanguinated before rapid aortic perfusion with ice-cold PBS to quickly rinse kidneys free of blood and to deliver protease (10 e蘥/ml pepstatin A, 10 e蘥/ml leupeptin, 10 e蘥/ml soybean trypsin inhibitor, and 10 e蘥/ml aprotinin) and phosphatase (1 mmol/l orthovanadate and 30 mmol/l sodium fluoride) inhibitors, phenylmethylsulphonylfluoride (1 mmol/l), and dithiothreitol (DTT) (0.5 mmol/l). Plasma was frozen at eC80° C, and both kidneys were removed, decapsulated, weighed, and frozen at eC80° C until processed for protein extraction. Before freezing, a 2- to 3-mm midline transmural section was removed from each kidney for fixation in 10% formaldehyde for 24 h before processing for immunohistochemistry.
Metabolic and cytokine measurements.
Commercially available kits were used to measure plasma glucose (Sigma-Aldrich, St. Louis, MO), cholesterol, and triglycerides (Wako Chemicals, Richmond, VA). Insulin, leptin, and amylin (Linco, St. Louis, MO) and multiple cytokines and chemokines were measured on additional aliquots of plasma and on a sample of renal cortical extract collected after 3 months of hyperglycemia using the Bio-Plex system, which are multiplex bead-based assays used with the Luminex xMAP technology according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). Eight-point standard curves were performed for each cytokine using the same Luminex bead technology.
Preparation of cytosolic and nuclear protein extracts.
Renal cortex (50 mg) was homogenized on ice using Wheaton 1-ml Dounce homogenizers in a 20-fold excess (wt/vol) of low-salt solution A (50 mmol/l HEPES, pH 7.0, 10 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l EGTA, protease inhibitor cocktail [Sigma P8340, containing 240 e蘥/ml AEBSF, 5 e蘥/ml aprotinin, 10 e蘥/ml leupeptin, 14 e蘥/ml bestatin, 10 e蘥/ml pepstatin A, and 5 e蘥/ml E-64], phosphatase inhibitors [1 mmol/l orthovanadate and 30 mmol/l sodium fluoride], 1 mmol/l DTT, and 0.5 mmol/l phenylmethylsulphonylfluoride). The homogenate was spun for 30 s at 100g and 4°C to remove tissue debris, the supernatant was transferred to a fresh tube, 10% NP-40 was added for a final concentration of 0.05%, tubes were mixed by gentle inversion, and immediately spun for 7 min at 1,000g and 4°C. The supernatant was used as cytosolic extract, while the pellet was gently resuspended in solution A containing 1 mol/l sucrose, and spun for 5 min at 8,000g at 4°C. The supernatant was removed and discarded, and the pellet gently resuspended in 20 e蘬 solution A. Eighty microliters of a high-salt solution B (50 mmol/l HEPES, pH 7.0, 500 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 10% glycerol, protease and phosphatase inhibitors, 1 mmol/l DTT, and 0.5 mmol/l phenylmethylsulphonylfluoride) were added dropwise with gentle vortexing. The suspension was allowed to sit on ice for 30 min, vortexing for 15 s every 5 min, then spun at 16,100g for 20 min. The supernatant was used as nuclear extract. Protein concentrations of nuclear and cytosolic extracts were determined using the Bio-Rad (Hercules, CA) protein assay with bovine albumin standards, and both samples were assayed for cytosolic (-tubulin and glyceraldehyde-3-phosphate dehydrogenase) and nuclear (lamin B) proteins.
Western immunoblot analysis.
Western blots were quantified from three to five separate sets of cytosolic and nuclear protein extracts pooled from an equal mass of renal cortical extract of three control and three diabetic animals using a panel of antibodies for each mouse protein (Table 1). Tissue lysates were electrophoresed on 7.5 or 10% SDS-polyacrylamide gels and analyzed by immunoblotting after transfer to nitrocellulose membranes (Bio-Rad), using primary antibodies according to each manufacturer’s instructions, followed by species-specific secondary antibodies tagged with a fluorescent dye (IR Dye 800; Rockland) at a 1:5,000 dilution. Densitometric quantitation of each protein was performed using the LI-COR Bioscience Odyssey Imaging System with infared fluorescence detection (LI-COR Bioscience, Lincoln, NE). -Actin was measured on every blot using an IR Dye 700 for evaluation of protein loading.
Electrophoretic mobility shift assay.
Sense and antisense oligonucleotides containing an IL-6 NF-B consensus sequence (5'-CATGTGGGATTTTCCCATGAC-3') with a 5' IR Dye 700 label were purchased, and double-stranded DNA was prepared according to the manufacturer’s instructions (LI-COR Bioscience). An IR Dye 800eClabeled mutant oligonucleotide (5'-CATGTGAGATTTTCACATGAC-3') was used as a control. Unlabeled (cWT) IL-6 promoter was purchased from Sigma Genosys. A total of 17.5 e蘥 nuclear extract were diluted to a concentration of 4 e蘥/e蘬 by the addition of solution C (1:4 solutions A:B) then combined with 2 e蘬 ice-cold 10x binding buffer (100 mmol/l TRIS, 500 mmol/l NaCl, 10 mmol/l DTT, pH 7.5) and Milli-Q water to make a final volume of 20 e蘬. Where applicable, 80-fold excess unlabeled wild-type oligonucleotide or 80-fold excess mutant oligonucleotide were added to the mixture and allowed to equilibrate for 15 min. One microliter of fluorescent probe was added and allowed to equilibrate on ice for 30 min. Samples were mixed with 1 e蘬 loading dye, placed on a 4% native PAGE gel, and the gel was run at 160 V on ice for 1 h then imaged using the LI-COR Bioscience Odyssey Imaging System according to the manufacturer’s instructions. RelB (Santa Cruz 226x; 4 e蘥), p50 (Santa Cruz 114x; 4 e蘥), and p65 (600-401-271, 2 e蘥; Rockland) antibodies were separately added to the incubation mix for 30 min at room temperature before adding probe to perform supershift assays.
Immunohistochemistry.
Paraffin-embedded 5-e蘭 kidney sections were washed in xylene twice for 5 min each and rehydrated by successive rinsing in 100, 95, 75, and 50% ethanol followed by PBS, and antigen retrieval was performed using 10 mmol/l sodium citrate (pH 6.0) with 0.1% Triton X for 20 min at 95°C. Slides were blocked using nonimmune sera from the secondary antibody species diluted to 2% in PBS containing 0.2% cold fish skin gelatin, 0.1% saponin, and 0.05% Tween 20 for 2 h at room temperature. Primary antibodies directed against NIK (Cell Signaling 4994, 1:100 dilution), RelB (sc226, 1;100 dilution; Santa Cruz Biotechnology), p50 (Abcam 7971, 1:100 dilution), F4/80 (specific macrophage stain, 1:100 dilution; Abcam 16911), and myeloperoxidase (specific neutrophil stain,1:500 dilution; Spring Bioscience) were incubated overnight at 4°C in blocking buffer containing 2% BSA substituted for the nonimmune sera. Tissue sections were washed three times in PBS for 10 min each, and secondary antibody Texas RedeClabeled goat anti-rabbit IgG (1:100 dilution; Rockland) was added for 2 h at room temperature. Sections were again washed three times in PBS, and DAPI (1:10,000 dilution of a 100-e蘥/ml stock in PBS) was added for 15 min for nuclear counterstaining, the sections washed in PBS, mounted with coverslips, and viewed using a Nikon Eclipse 80i microscope with fluorescence illumination system and digital camera (DXM1200F). Negative controls included omitting the primary antibody.
RESULTS
Gravimetric, metabolic, and hormone data.
Body weights of db/db mice were increased significantly at the onset of hyperglycemia, peaked by 1 month of hyperglycemia (approximately twofold increase over age-matched controls), then remained constant over the next 2 months (Table 2). After 3 months of hyperglycemia, the db/db mice exhibited significant hypercholesterolemia, hyperinsulinemia, and hyperleptinemia, and plasma amylin levels were increased 10-fold versus control mice (Table 2).
Plasma and renal tissue cytokine levels.
Numerous cytokines and chemokines were significantly increased in plasma of diabetic versus control mice after 1 month of hyperglycemia, including RANTES (40% increase), IL-1 (45%), IL-1 (50%), IL-12(p70) (60%), TNF- (62%), IL-3 (84%), and IL12(p40) (100%) (Fig. 1). After 3 months of hyperglycemia, all of these cytokines were decreased 50eC80% below controls, while additional cytokines were increased approximately twofold, including KC, IL-6, G-CSF (P < 0.05 vs. controls), and MIP-1. Numerous proinflammatory cytokines were increased significantly in 3-month renal cortical extracts, ranging from a 14% increase for IL-2 to a threefold increase for KC (Fig. 2). In addition, anti-inflammatory cytokines, including IL-12(p40) and IL-10, were significantly decreased.
Western blot of NF-B pathway proteins.
Band intensities of NF-B pathway proteins were quantified from separate renal cortical cytosolic and nuclear extracts from mice diabetic for 3 months and their age-matched controls (Fig. 3). Results are expressed as a mean and SD of the ratio of diabetic to control band intensities after normalization to -actin from each lane. Quantitation of three separate sets of pooled extracts from three control and three diabetic mice revealed no significant change in band intensities for RelA/p65 (1.00 ± 0.19), p50 (1.03 ± 0.20), IB- (1.07 ± 0.12), and IB- (1.14 ± 0.09) in the canonical pathway, while NIK and RelB were increased 5.6-fold and 3-fold, and IKK- was decreased 38%. The nuclear content of canonical and noncanonical NF-B pathway proteins was decreased in diabetic renal cortex (Fig. 3). RelB increased threefold in the cytoplasm and decreased twofold in the nucleus of diabetic versus control mice (Fig. 4A). Under the experimental conditions utilized for Western immunoblotting, p100 was identifiable only in nuclear extracts (Fig. 4B) and in general was not visible on Western blots from total tissue extracts in which the nuclear proteins would be diluted into the much larger volume of cytosolic proteins (data not shown). p52 could not be reproducibly measured.
Analysis of phosphorylation sites on NF-B pathway proteins from whole-tissue extracts indicated that phosphorylation of serines 276, 529, and 536 on RelA/p65 was decreased in diabetic versus control tissue (Fig. 4C). Likewise, separate antibodies recognizing serine 32 or serines 32 and 36 of IB- indicated either no change or decreases in diabetic renal cortex. In contrast, antibodies recognizing phosphorylation sites of IKK- (serines 176 and 180) and IKK- (serines 177 and 181) indicated increased phosphorylation in diabetic renal cortex.
Electrophoretic mobility shift assay.
Increased binding of protein to DNA was observed in nuclear extracts of diabetic versus control renal cortex (Fig. 5, lanes 2 vs. 5). A single complex was identified that was specifically inhibited by an 80-fold excess of cold wild-type oligonucleotide (lanes 3 and 6) but not by the same excess of mutant oligonucleotide (lanes 4 and 7). Antibodies to RelB were capable of supershifting a portion of the protein binding to the DNA (lane 8), while antibodies to RelA and p50 did not, even though the latter antibodies were capable of gel shifting TNF-eCstimulated Hela cell complexes (data not shown).
Immunohistochemistry.
Immunostaining for NIK demonstrated renal tubular epithelial localization in kidney sections from both control and diabetic mice (Fig. 6B and C). In control mice, NIK staining was predominantly located near the basal aspect of proximal tubular epithelial cells (Fig. 6B). Distal tubules, glomeruli, and interstitial cells remained relatively unstained (Fig. 6B, arrowheads), as did nuclei. The cellular distribution of NIK was comparable in diabetic kidneys, although there was increased staining throughout proximal tubular epithelial cells (Fig. 6C). RelB staining was intense and predominantly located in proximal and distal tubular epithelial cells in both control and diabetic mice (Fig. 6E and F). Nuclear staining was present within controls (arrowheads) but much less evident within diabetic kidneys. RelB staining was present but much lighter within glomeruli (Fig. 6F). p50 was present within renal tubular epithelial cells but not glomeruli of both control and diabetic kidneys, although the distribution of staining was punctuate and not very common (Fig. 6H and I).
DISCUSSION
Diabetes-induced inflammation and renal cytokine production.
This study utilized the db/db mouse model of type 2 diabetes since it develops robust changes within 2 months of the onset of hyperglycemia that include increases in renal mass, glomerular volume, mesangial volume, glomerular filtration rate, basement membrane thickening, and urinary albumin excretion (28). Our observation that numerous cytokines and chemokines, previously shown to be regulated in part by canonical NF-B pathway activation (23,29), were increased in the renal cortex of diabetic mice indicates chronic activation of the innate immune response via this NF-B pathway. Several of the elevated cytokines and chemokines previously have been associated with diabetic nephropathy (TNF-, IL-6, MCP-1, and RANTES) (30), while many have not (IL-2, IL-3, IL-5, IL-9, IL-12, G-CSF, GM-CSF, and KC). Virtually all cytokines and chemokines listed in Figs. 1 and 2 contain B motifs within the promoter region of their DNA (23). Exceptions include IL-3, IL-4, and IL-5, which are regulated by nuclear factor of activated T-cells (29). These elevated cytokines represent renal production of cytokines and not plasma contamination since kidneys were rinsed free of blood and plasma levels of the tested cytokines were at or below control levels at the 3-month time point. The possibility that infiltrating neutrophils, monocytes, and macrophages are responsible for the cytokine production cannot be ruled out but appears unlikely given the low numbers of infiltrating macrophages previously reported at this time point (31), and our inability to demonstrate a significant diabetes-induced increase of either macrophages (using anti-F4/80 antibody) or neutrophils (using anti-myeloperoxidase antibody) in random kidney sections from db/db mice (data not shown). The major contribution of tubular epithelial cells to total NF-B protein content demonstrated with immunohistochemistry in this study, together with previous observations that tubular epithelial cells produce a variety of cytokines and chemokines in other renal inflammatory diseases (32), suggest that this cell is responsible for the diabetes-induced increased cytokine production. We are the first to report that NIK and RelB are predominantly localized to tubular epithelial cells, an observation strikingly similar to other renal inflammatory diseases (see below).
Diabetes-induced canonical NF-B activation.
Increased immunohistochemical localization of p50 and p65 in the nucleus has been used as evidence of diabetes-induced activation of NF-B (18eC20). Likewise, reports using electrophoretic mobility shift assays (EMSAs) on cell extracts from renal and vascular cells in tissue culture have provided evidence of glucose-induced activation of NF-B (15eC19). A major caveat of these approaches is that NF-B protein binding is not synonymous with transcriptional activation, and demonstrating nuclear localization and/or DNA binding via these two methods provides no insights into mechanisms of diabetes-induced NF-B transcriptional activation.
Within the kidney, most studies of NF-B activation have utilized mesangial cells exposed to a variety of stimuli in tissue culture (rev. in 17) and have evaluated RelA and p50 as the predominant NF-B proteins. Renal tubular epithelial cells have received less attention, although recent studies (19,33) have demonstrated active NF-B. Even fewer experimental animal studies (20,34,35) have evaluated renal NF-B activation, and results are more discordant than in vitro cell culture studies. Reasons for this may include more chronic exposure to hyperglycemia, diabetes models evaluated (type 1 vs. type 2), animal species, and more complex tissue extracts than single-cell culture systems. Despite the decreased amounts of all NF-B proteins in nuclear extracts of 3-month diabetic db/db mice, we also report an increased protein binding to DNA using EMSA. While paradoxical, these data are consistent with the observation that the pool of individual nuclear NF-B proteins is much larger than the total amount of NF-B proteins actually binding to DNA (36). In contrast to previous experiments, only RelB could be supershifted slightly. Several possibilities exist for the inability of RelA and p50 antibodies to supershift the DNA-binding complex. These possibilities include the following: 1) RelA is incapable of binding DNA, perhaps through formation of inactive heterodimers with RelB (37), and 2) antibody epitopes are inaccessible in the protein complex.
The absence of any significant detectable changes by Western blot in cytosolic NF-B pathway proteins comprising the canonical pathway, and the finding that nuclear levels of all NF-B pathway proteins were decreased by approximately one-half, indicate that diabetes-induced chronic activation of the innate immune response is neither dependent on significant movement of NF-B dimers into the nucleus, nor is it due to prolonged degradation of IB inhibitory proteins that function to keep RelA:p50 homo- and/or heterodimers in the cytoplasm. The latter observation is consistent with previous reports showing sustained NF-B activation in the absence of significant decreases in IB- expression in peripheral blood mononuclear cells exposed to chronic high glucose (18).
Noncanonical NF-kB activation pathway.
The major novel finding of this study is diabetes-induced dysregulation of the noncanonical NF-B activation pathway, including significant changes in NIK, RelB, and IKK-. Our finding in diabetic kidneys that cytosolic and nuclear IKK- was decreased 40% (Fig. 3) yet phosphorylated IKK- was increased 34% (Fig. 4C) indicates that the fraction of activated IKK- increased significantly and provides support for noncanonical NF-B pathway activation. While only a few noncanonical pathway target genes have been identified, the positive transcriptional role of this pathway is clearly manifested in mice lacking specific noncanonical NF-B proteins (38). Pathophysiological roles for NIK remain mostly unexplored and the significance of the several-fold increase in NIK in diabetic renal cortex remains unclear at this time. NIK activation has been reported in pig models of ischemia-reperfusion injury and in delayed graft function in patients receiving kidney transplants (39). In the latter, activation of NIK occurred within proximal tubular epithelial cells, in a pattern strikingly similar to diabetes-induced changes reported here. In addition, NIK activity was induced by thrombin in cultured proximal tubular epithelial cells (39).
RelB displays a number of unusual features compared with other Rel proteins; it is weakly controlled by IB proteins and can serve as both a promoter and repressor of NF-BeCdependent gene expression (22,23,40). RelB primarily forms complexes with p50 and p52, the only Rel proteins lacking transactivation domains, yet is a transcriptional activator in these dimer conformations. Previously, it has been shown to be constitutively present only in nuclei of immune cells (40), and we show here that this also is true for renal cells (Fig. 3). Like IB proteins, the expression of RelB is controlled by NF-B (41), and the threefold increase in cytosolic RelB reported in this study is consistent with NF-B canonical pathway activation.
Potential mechanisms responsible for prolonged activation of the NF-B canonical pathway in diabetic kidney.
At least three potential mechanisms may contribute to the prolonged activation of the canonical NF-B pathway observed in renal cortex of db/db mice. First, based on the phenotype of RelB-deficient mice, RelB has been implicated as an important repressor of the inflammatory process, modulating the transition from innate to adaptive immunity (42). These mice develop a severe, noninfectious, multiorgan inflammatory syndrome that significantly contributes to their premature death (43). Renal fibroblasts harvested from RelB-deficient mice exhibit dysregulated cytokine production following LPS stimulation, which can be corrected by transfection of RelB cDNA into these cells (42). At least two mechanisms may be responsible for the repressive function of RelB. First, it has been reported that RelB exerts transcriptional suppression via stabilization of IB- protein (44). A second mechanism is via inhibition of DNA binding by RelA through its complex formation with RelB (37). Despite the threefold increase in cytosolic RelB, the nuclear content was decreased twofold in diabetic kidneys, which may be sufficient to impact upon its ability to form dimers with IB- or RelA to terminate the innate immune response. A second potential mechanism for the diabetes-induced prolonged activation of the canonical NF-B pathway involves phosphorylation of RelA ser276, which is necessary for RelA/RelB dimerization (45). We report a decrease in ser276 phosphorylation in diabetic kidney (Fig. 4C). Third, it has recently been reported that IKK- plays a negative role in macrophage activation and inflammation by accelerating the turnover and removal of RelA from proinflammatory gene promoters (46). This was accomplished via phosphorylation of RelA ser536; impaired ser536 phosphorylation significantly prolonged the retention time of RelA on the promoter of NF-B genes. It is noteworthy that both IKK- protein levels (cytosolic and nuclear) and RelA ser536 phosphorylation were decreased in diabetic kidneys in this study.
In summary, we report sustained activation of innate immunity via the NF-B canonical pathway in the renal cortex of db/db mice after 3 months of hyperglycemia, manifested by significantly elevated tissue concentrations of numerous NF-BeCregulated cytokines and chemokines. This was associated with a twofold reduction in the nuclear content of all NF-kB pathway proteins, no changes in tissue cytosolic levels of all proteins comprising the canonical pathway, and with significant changes in proteins essential for noncanonical NF-B pathway activation, including NIK, IKK-, and RelB.
ACKNOWLEDGMENTS
This project was supported by an American Diabetes Association Research Grant (to R.G.T.) and a National Heart, Lung, and Blood Institute Grant HL055630 (to A.R.B.). B.C.T. is a recipient of a National Institute of Environmental Health Sciences Predoctoral Training Grant (M. Moslen, Director) and a McLaughlin Predoctoral Fellowship in Infection and Immunity (Stanley M. Lemon, Director).
We acknowledge the excellent technical assistance of Hung Sun, Hung Q. Doan, Negin N. Fouladi, and Chang Lee.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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