Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

Fatty acids and inflammatory stimuli induce expression of long-chain acyl-CoA synthetase 1 to promote lipid remodeling in diabetic kidney disease.

Fatty acid handling and complex lipid synthesis are altered in the kidney cortex of diabetic patients. We recently showed that inhibition of the renin-angiotensin system without changes in glycemia can reverse diabetic kidney disease (DKD) and restore the lipid metabolic network in the kidney cortex of diabetic (db/db) mice, raising the possibility that lipid remodeling may play a central role in DKD. However, the roles of specific enzymes involved in lipid remodeling in DKD have not been elucidated. In the present study, we used this diabetic mouse model and a proximal tubule epithelial cell line (HK2) to investigate the potential relationship between long-chain acyl-CoA synthetase 1 (ACSL1) and lipid metabolism in response to fatty acid exposure and inflammatory signals. We found ACSL1 expression was significantly increased in the kidney cortex of db/db mice, and exposure to palmitate or tumor necrosis factor-α significantly increased Acsl1 mRNA expression in HK-2 cells. In addition, palmitate treatment significantly increased the levels of long-chain acylcarnitines and fatty acyl CoAs in HK2 cells, and these increases were abolished in HK2 cell lines with specific deletion of Acsl1(Acsl1KO cells), suggesting a key role for ACSL1 in fatty acid ß-oxidation. In contrast, tumor necrosis factor-α treatment significantly increased the levels of short-chain acylcarnitines and long-chain fatty acyl CoAs in HK2 cells but not in Acsl1KO cells, consistent with fatty acid channeling to complex lipids. Taken together, our data demonstrate a key role for ACSL1 in regulating lipid metabolism, fatty acid partitioning, and inflammation.

Tubular dysfunction impairs renal excretion of pseudouridine in diabetic kidney disease.

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.

Deletion of bone marrow myeloperoxidase attenuates chronic kidney disease accelerated atherosclerosis.

Increased myeloperoxidase (MPO) expression and activity are associated with atherosclerotic disease in patients with chronic kidney disease (CKD). However, the causal relationship between MPO and the development and progression of atherosclerosis in patients with CKD is unknown. Eight-week-old male low-density-lipoprotein-receptor-deficient mice were subjected to 5/6 nephrectomy, irradiated, and transplanted with bone marrow from MPO-deficient mice to induce bone marrow MPO deletion (CKD-bMPOKO) or bone marrow from WT mice as a control to maintain preserved bone marrow MPO(CKD-bMPOWT). The mice were maintained on a high-fat/high-cholesterol diet for 16 weeks. As anticipated, both groups of mice exhibited all features of moderate CKD, including elevated plasma creatinine, lower hematocrit, and increased intact parathyroid hormone but did not demonstrate any differences between the groups. Irradiation and bone marrow transplantation did not further affect body weight, blood pressure, creatinine, or hematocrit in either group. The absence of MPO expression in the bone marrow and atherosclerotic lesions of the aorta in the CKD-bMPOKO mice was confirmed by immunoblot and immunohistochemistry, respectively. Decreased MPO activity was substantiated by the absence of 3-chlorotyrosine, a specific by-product of MPO, in aortic atherosclerotic lesions as determined by both immunohistochemistry and highly sensitive LC-MS. Quantification of the aortic lesional area stained with oil red O revealed that CKD-bMPOKO mice had significantly decreased aortic plaque area as compared with CKD-bMPOWT mice. This study demonstrates the reduction of atherosclerosis in CKD mice with the deletion of MPO in bone marrow cells, strongly implicating bone-marrow-derived MPO in the pathogenesis of CKD atherosclerosis.

Elevated lipoxygenase and cytochrome P450 products predict progression of chronic kidney disease.

BACKGROUND: The clinical relevance of arachidonic acid (AA) metabolites in chronic kidney disease (CKD) progression is poorly understood. We aimed to compare the concentrations of 85 enzymatic pathway products of AA metabolism in patients with CKD who progressed to end-stage kidney disease (ESKD) versus patients who did not in a subcohort of Chronic Renal Insufficiency Cohort (CRIC) and to estimate the risk of CKD progression and major cardiovascular events by levels of AA metabolites and their link to enzymatic metabolic pathways. METHODS: A total 123 patients in the CRIC study who progressed to ESKD were frequency matched with 177 nonprogressors and serum eicosanoids were quantified by mass spectrometry. We applied serum collected at patients' Year 1 visit and outcome of progression to ESKD was ascertained over the next 10 years. We used logistic regression models for risk estimation. RESULTS: Baseline 15-hydroxyeicosatetraenoate (HETE) and 20-HETE levels were significantly elevated in progressors (false discovery rate Q ≤ 0.026). The median 20-HETE level was 7.6 pmol/mL [interquartile range (IQR) 4.2-14.5] in progressors and 5.4 pmol/mL (IQR 2.8-9.4) in nonprogressors (P < 0.001). In an adjusted model, only 20-HETE independently predicted CKD progression. Each 1 standard deviation increase in 20-HETE was independently associated with 1.45-fold higher odds of progression (95% confidence interval 1.07-1.95; P = 0.017). Principal components of lipoxygenase (LOX) and cytochrome P450 (CYP450) pathways were independently associated with CKD progression. CONCLUSIONS: We found higher odds of CKD progression associated with higher 20-HETE, LOX and CYP450 metabolic pathways. These alterations precede CKD progression and may serve as targets for interventions aimed at halting progression.

Myeloperoxidase-derived oxidants damage artery wall proteins in an animal model of chronic kidney disease-accelerated atherosclerosis.

Increased myeloperoxidase (MPO) levels and activity are associated with increased cardiovascular risk among individuals with chronic kidney disease (CKD). However, a lack of good animal models for examining the presence and catalytic activity of MPO in vascular lesions has impeded mechanistic studies into CKD-associated cardiovascular diseases. Here, we show for the first time that exaggerated atherosclerosis in a pathophysiologically relevant CKD mouse model is associated with increased macrophage-derived MPO activity. Male 7-week-old LDL receptor-deficient mice underwent sham (control mice) or 5/6 nephrectomy and were fed either a low-fat or high-fat, high-cholesterol diet for 24 weeks, and the extents of atherosclerosis and vascular reactivity were assessed. MPO expression and oxidation products-protein-bound oxidized tyrosine moieties 3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine-were examined with immunoassays and confirmed with mass spectrometry (MS). As anticipated, the CKD mice had significantly higher plasma creatinine, urea nitrogen, and intact parathyroid hormone along with lower hematocrit and body weight. On both the diet regimens, CKD mice did not have hypertension but had lower cholesterol and triglyceride levels than the control mice. Despite the lower cholesterol levels, CKD mice had increased aortic plaque areas, fibrosis, and luminal narrowing. They also exhibited increased MPO expression and activity (i.e. increased oxidized tyrosines) that co-localized with infiltrating lesional macrophages and diminished vascular reactivity. In summary, unlike non-CKD mouse models of atherosclerosis, CKD mice exhibit increased MPO expression and catalytic activity in atherosclerotic lesions, which co-localize with lesional macrophages. These results implicate macrophage-derived MPO in CKD-accelerated atherosclerosis.

Impaired ß-Oxidation and Altered Complex Lipid Fatty Acid Partitioning with Advancing CKD.

Studies of lipids in CKD, including ESRD, have been limited to measures of conventional lipid profiles. We aimed to systematically identify 17 different lipid classes and associate the abundance thereof with alterations in acylcarnitines, a metric of ß-oxidation, across stages of CKD. From the Clinical Phenotyping Resource and Biobank Core (CPROBE) cohort of 1235 adults, we selected a panel of 214 participants: 36 with stage 1 or 2 CKD, 99 with stage 3 CKD, 61 with stage 4 CKD, and 18 with stage 5 CKD. Among participants, 110 were men (51.4%), 64 were black (29.9%), and 150 were white (70.1%), and the mean (SD) age was 60 (16) years old. We measured plasma lipids and acylcarnitines using liquid chromatography-mass spectrometry. Overall, we identified 330 different lipids across 17 different classes. Compared with earlier stages, stage 5 CKD associated with a higher abundance of saturated C16-C20 free fatty acids (FFAs) and long polyunsaturated complex lipids. Long-chain-to-intermediate-chain acylcarnitine ratio, a marker of efficiency of ß-oxidation, exhibited a graded decrease from stage 2 to 5 CKD (P<0.001). Additionally, multiple linear regression revealed that the long-chain-to-intermediate-chain acylcarnitine ratio inversely associated with polyunsaturated long complex lipid subclasses and the C16-C20 FFAs but directly associated with short complex lipids with fewer double bonds. We conclude that increased abundance of saturated C16-C20 FFAs coupled with impaired ß-oxidation of FFAs and inverse partitioning into complex lipids may be mechanisms underpinning lipid metabolism changes that typify advancing CKD.

Gut Microbial Product Predicts Cardiovascular Risk in Chronic Kidney Disease Patients.

BACKGROUND: The gut microbiota is altered in patients with chronic kidney disease (CKD), and cardiovascular risk increases with progressive CKD. This study examined the potential link between short chain fatty acids (SCFAs), which are produced by the gut microbiota, and cardiovascular outcomes in patients with CKD. METHODS: SCFAs were measured using a targeted liquid chromatography-mass spectrometry platform in baseline plasma samples from 214 patients with CKD enrolled in the Clinical Phenotyping Resource and Biobank Core; 81 patients with coronary artery disease (CAD) and 133 without CAD were randomly assigned to training and validation subsets. The primary outcome was a history of CAD and the secondary outcome was a composite history of cardiovascular disease (CVD) at enrollment. RESULTS: We found significantly higher levels of the SCFA valerate among patients with CAD as compared with patients without CAD in the training set (p < 0.001). The valerate concentrations were also significantly higher among subjects with composite outcomes of CVD compared to those without CVD (p = 0.006). These results were subsequently replicated in the validation set. Logistic regression analysis revealed a strong independent association between plasma valerate levels and CVD in both training and validation sets. When valerate was added to the base clinical model comprising of diabetes, hypertension, urinary protein-creatinine ratio, and estimated glomerular filtration rate, it increased the c-statistics for predicting CVD from 0.68 to 0.79 (p = 0.02) in the training set, an observation which was confirmed in the validation set. -Conclusion: This study provides evidence for alterations in gut-microbiota-derived SCFAs with advancing CKD, demonstrates the association of higher plasma valerate levels with pre-existing CVD, and reveals areas for future exploration of cardiovascular risk in patients with CKD.

Myeloperoxidase Levels and Its Product 3-Chlorotyrosine Predict Chronic Kidney Disease Severity and Associated Coronary Artery Disease.

BACKGROUND: The role of myeloperoxidase in chronic kidney disease (CKD) and its association with coronary artery disease (CAD) is controversial. In this study, we compared myeloperoxidase and protein-bound 3-chlorotyrosine (ClY) levels in subjects with varying degrees of CKD and tested their associations with CAD. METHODS: From Clinical Phenotyping Resource and Biobank Core, 111 patients were selected from CKD stages 1 to 5. Plasma myeloperoxidase level was measured using enzyme-linked-immunosorbent assay. Plasma protein-bound 3-ClY, a specific product of hypochlorous acid generated by myeloperoxidase was measured by liquid chromatography mass spectrometry. RESULTS: We selected 29, 20, 24, 22, and 16 patients from stages 1 to 5 CKD, respectively. In a sex-adjusted general linear model, mean ± SD of myeloperoxidase levels decreased from 18.1 ± 12.3 pmol in stage 1 to 10.9 ± 4.7 pmol in stage 5 (p = 0.011). In patients with and without CAD, the levels were 19.1 ± 10.1 and 14.8 ± 8.7 pmol (p = 0.036). There was an increase in 3-ClY mean from 0.81 ± 0.36 mmol/mol-tyrosine in stage 1 to 1.42 ± 0.41 mmol/mol-tyrosine in stage 5 (p < 0.001). The mean 3-ClY levels in patients with and without CAD were 1.25 ± 0.44 and 1.04 ± 0.42 mmol/mol-tyrosine (p = 0.023), respectively. C-statistic of ClY when added to myeloperoxidase level to predict CKD stage 5 was 0.86, compared to 0.79 for the myeloperoxidase level alone (p = 0.0097). CONCLUSION: The myeloperoxidase levels decrease from stages 1 to 5, whereas activity increases. In contrast, both myeloperoxidase and ClY levels rise in the presence of CAD at various stages of CKD. Measuring both plasma myeloperoxidase and 3-CLY levels provide added value to determine the burden of myeloperoxidase-mediated oxidative stress.

Oxidative Modifications of Protein Tyrosyl Residues Are Increased in Plasma of Human Subjects with Interstitial Lung Disease.

RATIONALE: Interstitial lung diseases (ILDs) are associated with oxidative stress. Plasma biomarkers that are directly linked to oxidative stress responses in this disease have not been identified. Stable oxidation products of tyrosine residues in proteins may reflect the oxidative microenvironment in the lung or a systemic inflammatory state. OBJECTIVES: To determine if levels of protein tyrosine oxidation are elevated in plasma of patients with ILD compared with an age- and sex-matched healthy control cohort. METHODS: Three tyrosine oxidation products (3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine) were quantified by tandem mass spectrometry in cellular models, a mouse model of injury-induced fibrosis, and in plasma of healthy control subjects and patients with ILD (n = 42 in each group). MEASUREMENTS AND MAIN RESULTS: Plasma levels of 3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine were markedly elevated in patients with ILD compared with control subjects with receiver operating characteristic curves separating these groups of 0.872, 0.893, and 0.997, respectively. In a murine model of lung fibrosis, levels of all three oxidative tyrosine modifications were increased in plasma and lung tissue. Cellular models support a critical role for a heme peroxidase and enzymatic sources of reactive oxygen species in the generation of these oxidized products. CONCLUSIONS: We demonstrate an increase in oxidized tyrosine moieties within proteins in the circulating plasma of patients with ILD. These data support the potential for development of oxidative stress-related biomarkers in early diagnosis, prognostication, and/or in evaluating responsiveness to emerging therapies for ILD.

Evidence for a link between gut microbiota and hypertension in the Dahl rat.

The gut microbiota plays a critical role in maintaining physiological homeostasis. This study was designed to evaluate whether gut microbial composition affects hypertension. 16S rRNA genes obtained from cecal samples of Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats were sequenced. Bacteria of the phylum Bacteroidetes were higher in the S rats compared with the R rats. Furthermore, the family S24-7 of the phylum Bacteroidetes and the family Veillonellaceae of the phylum Firmicutes were higher in the S rats compared with the R rats. Analyses of the various phylogenetic groups of cecal microbiota revealed significant differences between S and R rats. Both strains were maintained on a high-salt diet, administered antibiotics for ablation of microbiota, transplanted with S or R rat cecal contents, and monitored for blood pressure (BP). Systolic BP of the R rats remained unaltered irrespective of S or R rat cecal transplantation. Surprisingly, compared with the S rats given S rat cecal content, systolic BP of the S rats given a single bolus of cecal content from R rats was consistently and significantly elevated during the rest of their life, and they had a shorter lifespan. A lower level of fecal bacteria of the family Veillonellaceae and increased plasma acetate and heptanoate were features associated with the increased BP observed in the S rats given R rat microbiota compared with the S rats given S rat microbiota. These data demonstrate a link between microbial content and BP regulation and, because the S and R rats differ in their genomic composition, provide the necessary basis to further examine the relationship between the host genome and microbiome in the context of BP regulation in the Dahl rats.

Altered Metabolic Profile With Sodium-Restricted Dietary Approaches to Stop Hypertension Diet in Hypertensive Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is increasingly recognized as a distinct entity with unique pathophysiology. In the Dietary Approaches to Stop Hypertension in Diastolic Heart Failure (DASH-DHF) study, the sodium-restricted Dietary Approaches to Stop Hypertension diet (DASH/SRD) was associated with improved blood pressure and cardiovascular function in 13 hypertensive patients with HFpEF. With the use of targeted metabolomics, we explored metabolite changes and their relationship with energy-dependent measures of cardiac function in DASH-DHF. METHODS AND RESULTS: With the use of chromatography and mass spectrometry, 152 metabolites including amino acids, free fatty acids, phospholipids, diglycerides, triglycerides, cholesterol esters, and acyl carnitines were measured. Comparison of baseline and post-DASH/SRD samples revealed increases in short-chain acetyl, butryl, and propionyl carnitines (P values .02, .03, .03, respectively). Increases in propionyl carnitine correlated with ventricular-arterial coupling ratio (Ees:Ea; r = 0.78; P = .005) and ventricular contractility (maximum rate of change of pressure-normalized stress [dσ*/dtmax]; r = 0.66; P = .03). Changes in L-carnitine also correlated with Ees:Ea (r = 0.62; P = .04) and dσ*/dtmax (r = 0.60; P = .05) and inversely with ventricular stiffness (r = -0.63; P = .03). CONCLUSIONS: Metabolite profile changes of patients with HFpEF during dietary modification with the use of DASH/SRD suggest improved energy substrate utilization. Additional studies are needed to clarify connections between diet, metabolic changes, and myocardial function in HFpEF.

Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression.

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.

Uremic solutes and risk of end-stage renal disease in type 2 diabetes: metabolomic study.

Here we studied plasma metabolomic profiles as determinants of progression to end-stage renal disease (ESRD) in patients with type 2 diabetes (T2D). This nested case-control study evaluated 40 cases who progressed to ESRD during 8-12 years of follow-up and 40 controls who remained alive without ESRD from the Joslin Kidney Study cohort. Controls were matched with cases for baseline clinical characteristics, although controls had slightly higher eGFR and lower levels of urinary albumin excretion than cases. Plasma metabolites at baseline were measured by mass spectrometry-based global metabolomic profiling. Of the named metabolites in the library, 262 were detected in at least 80% of the study patients. The metabolomic platform recognized 78 metabolites previously reported to be elevated in ESRD (uremic solutes). Sixteen were already elevated in the baseline plasma of our cases years before ESRD developed. Other uremic solutes were either not different or not commonly detectable. Essential amino acids and their derivatives were significantly depleted in the cases, whereas certain amino acid-derived acylcarnitines were increased. All findings remained statistically significant after adjustment for differences between study groups in albumin excretion rate, eGFR, or HbA1c. Uremic solute differences were confirmed by quantitative measurements. Thus, abnormal plasma concentrations of putative uremic solutes and essential amino acids either contribute to progression to ESRD or are a manifestation of an early stage(s) of the disease process that leads to ESRD in T2D.

High density lipoprotein is targeted for oxidation by myeloperoxidase in rheumatoid arthritis.

OBJECTIVE: Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD). METHODS: Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS. RESULTS: RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD. CONCLUSIONS: We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.

Kynurenine pathway metabolites predict subclinical atherosclerotic disease and new cardiovascular events in chronic kidney disease.

Introduction: Inflammation and oxidative stress contribute to the disproportionate burden of cardiovascular disease (CVD) in chronic kidney disease (CKD). Disordered catabolism of tryptophan via the kynurenine and indole pathways is linked to CVD in both CKD and dialysis patients. However, the association between specific kynurenine and indole metabolites with subclinical CVD and time to new cardiovascular (CV) events in CKD has not been studied. Methods: We measured kynurenine and indole pathway metabolites using targeted mass spectrometry in a cohort of 325 patients with moderate to severe CKD and a median follow-up of 2 years. Multiple linear regression and Cox regression analyses were used to assess the relationship between these tryptophan metabolites and subclinical CVD, including calcium scores, carotid intima-media thickness and time to new cardiovascular (CV) events. Results: Elevated quinolinic and anthranilic acids were independently associated with reduced time to new CVD [hazard ratio (HR) 1.28, P = .01 and HR 1.02, P = .02, respectively). Low tryptophan levels were associated with reduced time to new CV events when adjusting for demographics and CVD history (HR 0.30, P = .03). Low tryptophan levels were also associated with aortic calcification in a fully adjusted linear regression model (ß = -1983, P = .006). Similarly, high levels of several kynurenine pathway metabolites predicted increased coronary, aortic and composite calcification scores. Conclusions: We demonstrate the association of kynurenine pathway metabolites, and not indole derivatives, with subclinical and new CV events in an advanced CKD cohort. Our findings support a possible role for altered tryptophan immune metabolism in the pathogenesis of CKD-associated atherosclerosis.

Serum lipidomic determinants of human diabetic neuropathy in type 2 diabetes.

OBJECTIVE: The serum lipidomic profile associated with neuropathy in type 2 diabetes is not well understood. Obesity and dyslipidemia are known neuropathy risk factors, suggesting lipid profiles early during type 2 diabetes may identify individuals who develop neuropathy later in the disease course. This retrospective cohort study examined lipidomic profiles 10 years prior to type 2 diabetic neuropathy assessment. METHODS: Participants comprised members of the Gila River Indian community with type 2 diabetes (n = 69) with available stored serum samples and neuropathy assessment 10 years later using the combined Michigan Neuropathy Screening Instrument (MNSI) examination and questionnaire scores. A combined MNSI index was calculated from examination and questionnaire scores. Serum lipids (435 species from 18 classes) were quantified by mass spectrometry. RESULTS: The cohort included 17 males and 52 females with a mean age of 45 years (SD = 9 years). Participants were stratified as with (high MNSI index score > 2.5407) versus without neuropathy (low MNSI index score ≤ 2.5407). Significantly decreased medium-chain acylcarnitines and increased total free fatty acids, independent of chain length and saturation, in serum at baseline associated with incident peripheral neuropathy at follow-up, that is, participants had high MNSI index scores, independent of covariates. Participants with neuropathy also had decreased phosphatidylcholines and increased lysophosphatidylcholines at baseline, independent of chain length and saturation. The abundance of other lipid classes did not differ significantly by neuropathy status. INTERPRETATION: Abundance differences in circulating acylcarnitines, free fatty acids, phosphatidylcholines, and lysophosphatidylcholines 10 years prior to neuropathy assessment are associated with neuropathy status in type 2 diabetes.

Dose-dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor.

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.