NF-κB-inducing kinase increases renal tubule epithelial inflammation associated with diabetes.

The impact of increased NF-κB-inducing kinase (NIK), a key component of the NF-κB activation pathways, on diabetes-induced renal inflammation remains unknown. We overexpressed NIK wild type (NIKwt) or kinase-dead dominant negative mutants (NIKdn) in HK-2 cells and demonstrated that RelB and p52, but not RelA, abundance and DNA binding increased in nuclei of NIKwt but not NIKdn overexpressed cells, and this corresponded with increases in multiple proinflammatory cytokines. Since TRAF3 negatively regulates NIK expression, we silenced TRAF3 by >50%; this increased nuclear levels of p52 and RelB, and transcript levels of proinflammatory cytokines and transcription factors. In HK-2 cells and mouse primary proximal tubule epithelial cells treated with methylglyoxal-modified albumin, multiple proinflammatory cytokines and NIK were increased in association with increased nuclear RelB and p52. These observations indicate that NIK regulates proinflammatory responses of renal proximal tubular epithelial cells via mechanisms involving TRAF3 and suggest a role for NF-κB noncanonical pathway activation in modulating diabetes-induced inflammation in renal tubular epithelium.

Klotho depletion contributes to increased inflammation in kidney of the db/db mouse model of diabetes via RelA (serine)536 phosphorylation.

OBJECTIVE: Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys. RESEARCH DESIGN AND METHODS: Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)(536). RESULTS: Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser(536) phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding. CONCLUSIONS: These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB-linked inflammatory proteins via a mechanism that involves phosphorylation of Ser(536) in the transactivation domain of RelA.

Potent in vivo suppression of inflammation by selectively targeting the high affinity conformation of integrin α4ß1.

The development of antagonists to the α4 integrin family of cell adhesion molecules has been an active area of pharmaceutical research to treat inflammatory and autoimmune diseases. Presently being tested in human clinical trials are compounds selective for α4ß1 (VLA-4) as well as several dual antagonists that inhibit both α4ß1 and α4ß7. The value of a dual versus a selective small molecule antagonist as well as the consequences of inhibiting different affinity states of the α4 integrins have been debated in the literature. Here, we characterize TBC3486, a N,N-disubstituted amide, which represents a unique structural class of non-peptidic, small molecule VLA-4 antagonists. Using a variety of adhesion assay formats as well as flow cytometry experiments using mAbs specific for certain activation-dependent integrin epitopes we demonstrate that TBC3486 preferentially targets the high affinity conformation of α4ß1 and behaves as a ligand mimetic. The antagonist is capable of blocking integrin-dependent T-cell co-activation in vitro as well as proves to be efficacious in vivo at low doses in two animal models of allergic inflammation. These data suggest that a small molecule α4 integrin antagonist selective for α4ß1 over α4ß7 and, specifically, selective for the high affinity conformation of α4ß1 may prove to be an effective therapy for multiple inflammatory diseases in humans.

Altered retinoic acid metabolism in diabetic mouse kidney identified by O isotopic labeling and 2D mass spectrometry.

BACKGROUND: Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, (18)O- and (16)O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change >or=1.5 and p

Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC - tandem mass spectrometry.

BACKGROUND: Proteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue. RESULTS: We report a large scale analysis of mouse renal cortical proteome using SCX prefractionation strategy combined with HPLC - tandem mass spectrometry. High-confidence identification of ~2,000 proteins, including cytoplasmic, nuclear, plasma membrane, extracellular and unknown/unclassified proteins, was obtained by separating tryptic peptides of renal cortical proteins into 60 fractions by SCX prior to LC-MS/MS. The identified proteins represented the renal cortical proteome with no discernible bias due to protein physicochemical properties, subcellular distribution, biological processes, or molecular function. The highest ranked molecular functions were characteristic of tubular epithelium, and included binding, catalytic activity, transporter activity, structural molecule activity, and carrier activity. Comparison of this renal cortical proteome with published human urinary proteomes demonstrated enrichment of renal extracellular, plasma membrane, and lysosomal proteins in the urine, with a lack of intracellular proteins. Comparison of the most abundant proteins based on normalized spectral abundance factor (NSAF) in this dataset versus a published glomerular proteome indicated enrichment of mitochondrial proteins in the former and cytoskeletal proteins in the latter. CONCLUSION: A whole tissue extract of the mouse kidney cortex was analyzed by an unbiased proteomic approach, yielding a dataset of ~2,000 unique proteins identified with strict criteria to ensure a high level of confidence in protein identification. As a result of extracting all proteins from the renal cortex, we identified an exceptionally wide range of renal proteins in terms of pI, MW, hydrophobicity, abundance, and subcellular location. Many of these proteins, such as low-abundance proteins, membrane proteins and proteins with extreme values in pI or MW are traditionally under-represented in 2-DE-based proteomic analysis.

Diabetes-induced changes in the renal cortical proteome assessed with two-dimensional gel electrophoresis and mass spectrometry.

To understand the spectrum of proteins affected by diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of db/db mice using 2-DE combined with MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >two-fold by diabetes, of which 170 mapped to gene identifiers representing 147 nonredundant proteins. Gene Ontology classification demonstrated that 80% of these proteins modulated physiological functions, 55% involved metabolism, approximately 25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11-15%). Twenty eight percent of the proteins identified as significantly altered by diabetes were mitochondrial proteins. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARalpha was the most common node of interaction for the numerous enzymes whose expression levels were influenced by diabetes. These differentially regulated proteins create a foundation for a systems biology exploration of molecular mechanisms underlying the pathophysiology of diabetic nephropathy.

Angiotensin II induces IL-6 expression and the Jak-STAT3 pathway in aortic adventitia of LDL receptor-deficient mice.

Angiotensin II (A-II), the major effector peptide of the renin angiotensin system potently accelerates progression of atherosclerosis. To investigate its effects on vascular inflammatory mechanisms, we elucidated vascular cytokine expression during early lesion development in A-II-infused atherosclerosis-prone LDLR-/- mice. Male LDLR-/- mice were placed on a "Western" high-fat diet for 4 weeks, followed by sham or A-II infusion for 7 weeks. Equal blood pressures and elevations in serum lipids were seen in both groups. Mice were sacrificed when significant A-II-induced plaque development was first detectable, aortae were explanted and culture media assayed for secreted cytokines. Nine cytokines were significantly induced with interleukin-6 (IL-6) being the most highly secreted. Local IL-6 production was confirmed by in situ mRNA hybridization and immunostaining, where the most abundant IL-6 was found in the aortic adventitia, with lesser production by the medial and intimal layers. Immunofluorescence colocalization showed IL-6 expression by fibroblasts and activated macrophages. Activation of downstream IL-6 signaling mediated by the Jak-STAT3 pathway was demonstrated by inducible phospho-Tyr705-STAT3 formation in the adventitia and endothelium (of IL-6+/+ mice only). These findings define cytokine profiles in the A-II infusion model and demonstrate that IL-6, produced by activated macrophages and fibroblasts in the adventitia, induces the Jak-STAT3 pathway during early A-II-induced atherosclerosis.

Diabetes-induced activation of canonical and noncanonical nuclear factor-kappaB pathways in renal cortex.

Evidence of diabetes-induced nuclear factor-kappaB (NF-kappaB) 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-kappaB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-kappaB-regulated 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-kappaB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-kappaB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of kappaB kinase-alpha and several fold increases in NF-kappaB-inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-kappaB 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-kappaB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-kappaB pathway proteins.