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Research shows that biochemical tumor necrosis factor-alpha interacts with nitric oxide (NO) to damage the blood-brain vascular barrier mechanism. Endothelial barrier integrity does not occur in the presence of an NO synthase enzyme inhibitor. Experiments to establish vascular barrier dysfunction in various tissues such as lung, liver and sciatic nerge are described.

Abstract 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.

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, Re1A 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 Ser536 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 Ser536 in the transactivation domain of RelA. [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. [ABSTRACT FROM AUTHOR]

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, 18O- and 16O-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 ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance: Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy. [ABSTRACT FROM AUTHOR]

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. [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]

Angiotensin-II (Ang-II), the major effector peptide of the renin angiotensin system, potently accelerates progression of atherosclemsis and vascular remodeling, and induces activation of NF-κB, a nuclear transcription factor regulating expression of numerous proinflammatory and proangiogenic cytokines, chemokines, growth factors, and adhesion molecules. To investigate Ang-II effects on diabetes-induced vascular inflammation, effects of chronic Ang-II infusion on NF-κB canonical and noncanonical proteins were studied in aorta of type 2, three month hyperglycemic db/db mice and congenic age-matched controls. Proteins were extracted from buffer (containing protease and phosphatase inhibitors)-perfused aortas obtained from four experimental groups, including untreated and Ang-II-infused controls, and untreated and Ang-II-infused db/db mice. Ang-II was infused subcutaneously via osmotic mini-pumps (0.5 mg/kg/day) for one month after two months of untreated diabetes. Protein levels of canonical (RelA/p65, IκBα, IκBβ, IKKβ, p50) and noncanonical (NIK, RelB, IKKα, p52, TRAF3) NF-κB proteins were quantified by Western blotting and normalized to tissue beta actin levels using infrared imaging. After 3 months of diabetes, most NF-κB canonical proteins were decreased in abundance, including IKKβ (40 % decrease), p50 (32 %), IκBα (26 %); RelA/p65 (20 %), and IκBβ (16%). Similar decreases were observed for noncaoonical NF-κB proteins, including IKKα (46 %), RelB (36%), and TRAF3 (32%), while p52 was unchanged and NIK was increased ∼60 %. In general, Ang-II infusion into diabetic mice produced further decreases in canonical NF-κB proteins as well as p52 and TRAF3 (both decreased ∼65 %), but increased RelB 30%. Ang-II increased the total amount of kinases, including IKKα (31%), IKKβ (61%), and NIK (45%). These results indicate that steady state levels of most NF-κB proteins are decreased in aorta of diabetic mice, and suggest that Ang-II has a divergent effect on canonical versus non-canonical NF-κB proteins. ADA-Funded Research [ABSTRACT FROM AUTHOR]

To understand the spectrum of proteins affected by type 2 diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of 3 month hyperglycemic db/db mice and age-matched congenic controls. Predominantly hydrophilic proteins were extracted from the cytosolic compartment of buffer (containing protease and phosphatase inhibitors)-perfused renal cortex, then equal amounts of extract pooled from 5 control and 5 diabetic mice were subjected to 2D gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. The staining intensity of approximately one third of all protein spots on 2D gels was affected by diabetes. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >2 fold by diabetes, of which 170 mapped to gene identifiers representing 147 non-redundant proteins. Based on Gene Ontology classification, 80% of these proteins modulated physiological functions, 55% involved metabolism, ∼25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Most of these proteins (∼85%) were increased in abundance. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11 - 15%). Mitochondrial proteins accounted for 28% of the proteins identified as significantly altered by diabetes. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARα 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. ADA-Funded Research [ABSTRACT FROM AUTHOR]

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