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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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

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.

Purpose: To explore the effects of brief intravenous (IV) infusion of vascular endothelial growth factor (VEGF) on vascular albumin permeability, blood flow, and vascular conductance (blood flow normalized to arterial blood pressure) in ocular tissues and brain and to assess the role of nitric oxide in mediating these changes., Methods: A quantitative, double-tracer, radiolabeled albumin permeation method was combined with radiolabeled microspheres for assessment of changes in vascular permeability and blood flow, respectively, induced in ocular tissues by IV infusion of recombinant human VEGF165 for 20 minutes (80-450 picomoles/kg body weight). An inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (L-NMMA; 50 micromoles/kg body weight infused simultaneously with VEGF), was used to explore the role of nitric oxide in mediating the vascular changes induced by VEGF., Results: Infusion of VEGF165 in thiopental-anesthetized rats dose-dependently increased 125I albumin permeation in the retina, anterior uvea, and choroid/sclera and in brain, aorta, lung, kidney, small intestine, and peripheral nerve. Mean arterial blood pressure, cardiac output, and stroke volume were decreased only at the highest dose of VEGF, whereas heart rate remained unchanged. Blood flow was increased in the anterior uvea, and vascular conductance was increased in retina, anterior uvea, choroid/sclera, and brain at the highest dose of VEGF. The NOS inhibitor, L-NMMA, blocked VEGF-induced vascular hyperpermeability in all ocular and nonocular tissues, prevented the increase in vascular conductance in all ocular tissues, and blocked the decrease in mean arterial blood pressure, cardiac output, and stroke volume. Infusion of L-NMMA alone decreased vascular conductance in choroid/sclera and kidney, slightly increased mean arterial blood pressure, and in general, did not affect 125I-albumin permeation. (L-NMMA slightly decreased albumin permeation in the retina and increased it in the brain.), Conclusions: Intravenous infusion of VEGF can acutely impair endothelial cell barrier functional integrity and relax resistance arterioles in ocular tissues and brain through a mechanism involving activation of NOS.