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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Roles of IL-6-gp130 Signaling in Vascular Inflammation

Interleukin-6 (IL-6) is a well-established, independent indicator of multiple distinct types of cardiovascular disease and all-cause mortality. In this review, we present current understanding of the multiple roles that IL-6 and its signaling pathways through glycoprotein 130 (gp130) play in cardiovascular homeostasis. IL-6 is highly inducible in vascular tissues through the actions of the angiotensin II (Ang II) peptide, where it acts in a paracrine manner to signal through two distinct mechanisms, the first being a classic membrane receptor initiated pathway and the second, a trans-signaling pathway, being able to induce responses even in tissues lacking the IL-6 receptor. Recent advances and new concepts in how its intracellular signaling pathways operate via the Janus kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) are described. IL-6 has diverse actions in multiple cell types of cardiovascular importance, including endothelial cells, monocytes, platelets, hepatocytes and adipocytes. We discuss central roles of IL-6 in endothelial dysfunction, cellular inflammation by affecting monocyte activation/differentiation, cellular cytoprotective functions from reactive oxygen species (ROS) stress, modulation of pro-coagulant state, myocardial growth control, and its implications in metabolic control and insulin resistance. These multiple actions indicate that IL-6 is not merely a passive biomarker, but actively modulates adaptive and pathological responses to cardiovascular stress

Angiogenesis induced by tumor necrosis factor-agr; is mediated by α4 integrins

Tumor necrosis factor-α (TNF-α) and fibroblast growth factor-2 (FGF-2 or bFGF) are potent stimulators of angiogenesis. TNF-α, but not FGF-2, can induce the expression of vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells. The soluble form of VCAM-1 has recently been demonstrated to function as an angiogenic mediator. Here we demonstrate that monoclonal antibodies directed against VCAM-1 or its α4 integrin counter-receptor inhibited TNF-α-induced endothelial cell migration in vitro. Angiogenesis induced in vivo in rat corneas by TNF-α was inhibited by a neutralizing antibody directed against the rat α4 integrin subunit. A peptide antagonist of the a4 integrins blocked TNF-α-induced endothelial cell migration in vitro and angiogenesis in rat corneas in vivo. No inhibition by the antibodies or peptide antagonist was observed either in vitro or in vivo when FGF-2 was used as the stimulus. The peptide antagonist did not inhibit TNF-a binding to its receptor nor did it block the function of αvβ3, an integrin previously implicated in TNF-a and FGF-2 mediated angiogenesis. These results demonstrate that angiogenic processes induced by TNF-α are mediated in part by agr;4 integrins possibly by a mechanism involving the induction of soluble VCAM-1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41761/1/10456_2004_Article_188219.pd

Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

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.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<or=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 PPARbeta/delta mRNA.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

SHIV-162P3 Infection of Rhesus Macaques Given Maraviroc Gel Vaginally Does Not Involve Resistant Viruses

Maraviroc (MVC) gels are effective at protecting rhesus macaques from vaginal SHIV transmission, but breakthrough infections can occur. To determine the effects of a vaginal MVC gel on infecting SHIV populations in a macaque model, we analyzed plasma samples from three rhesus macaques that received a MVC vaginal gel (day 0) but became infected after high-dose SHIV-162P3 vaginal challenge. Two infected macaques that received a placebo gel served as controls. The infecting SHIV-162P3 stock had an overall mean genetic distance of 0.294±0.027%; limited entropy changes were noted across the envelope (gp160). No envelope mutations were observed consistently in viruses isolated from infected macaques at days 14–21, the time of first detectable viremia, nor selected at later time points, days 42–70. No statistically significant differences in MVC susceptibilities were observed between the SHIV inoculum (50% inhibitory concentration [IC50] 1.87 nM) and virus isolated from the three MVC-treated macaques (MVC IC50 1.18 nM, 1.69 nM, and 1.53 nM, respectively). Highlighter plot analyses suggested that infection was established in each MVC-treated animal by one founder virus genotype. The expected Poisson distribution of pairwise Hamming Distance frequency counts was observed and a phylogenetic analysis did not identify infections with distinct lineages from the challenge stock. These data suggest that breakthrough infections most likely result from incomplete viral inhibition and not the selection of MVC-resistant variants

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Relationship between serum 25hydroxyvitamin D and lower extremity arterial disease in type 2 diabetes mellitus patients and the analysis of the intervention of vitamin D

The aim of this study was to explore the relationship between serum 25-hydroxyvitamin D [25(OH)D] concentrations and lower extremity arterial disease (LEAD) in type 2 diabetes mellitus (T2DM) patients and to investigate the intervention effect of vitamin D. 145 subjects were assigned to a control group (Group NC), T2DM group (Group DM1), and T2DM complicated with LEAD group (Group DM2); then Group DM2 were randomly divided into Group DM3 who received oral hypoglycemic agents and Group DM4 who received oral hypoglycemic drugs and vitamin D3 therapy. Compared to Group NC, 25(OH)D was significantly lower in Group DM2 and marginally lower in Group DM1. In contrast to baseline and Group DM3, 25(OH)D rose while low density lipoprotein (LDL), retinol binding protein 4 (RBP4), and HbA1c significantly lowered in Group DM4. Statistical analysis revealed that 25(OH)D had a negative correlation with RBP4, duration, HbA1c, homeostasis model assessment for insulin resistance (HOMA-IR), and fasting plasma glucose (FPG). LDL, systolic blood pressure (SBP), FPG, and smoking were risk factors of LEAD while high density lipoprotein (HDL) and 25(OH)D were protective ones. Therefore, we deduced that low level of 25(OH)D is significantly associated with the occurrence of T2DM complicated with LEAD