Canagliflozin Attenuates PromarkerD Diabetic Kidney Disease Risk Prediction Scores.

PromarkerD is a biomarker-based blood test that predicts kidney function decline in people with type 2 diabetes (T2D) who may otherwise be missed by current standard of care tests. This study examined the association between canagliflozin and change in PromarkerD score (Δ score) over a three-year period in T2D participants in the CANagliflozin cardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline and Year 3 in 2008 participants with preserved kidney function (baseline eGFR ≥60 mL/min/1.73 m2). Generalized estimating equations were used to assess the effect of canagliflozin versus placebo on PromarkerD scores. At baseline, the participants (mean age 62 years, 32% females) had a median PromarkerD score of 3.9%, with 67% of participants categorized as low risk, 14% as moderate risk, and 19% as high risk for kidney function decline. After accounting for the known acute drop in eGFR following canagliflozin initiation, there was a significant treatment-by-time interaction (p < 0.001), whereby participants on canagliflozin had decreased mean PromarkerD scores from baseline to Year 3 (Δ score: -1.0% [95% CI: -1.9%, -0.1%]; p = 0.039), while the scores of those on placebo increased over the three-year period (Δ score: 6.4% [4.9%, 7.8%]; p < 0.001). When stratified into PromarkerD risk categories, participants with high risk scores at baseline who were randomized to canagliflozin had significantly lower scores at Year 3 (Δ score: -5.6% [-8.6%, -2.5%]; p < 0.001), while those on placebo retained high scores (Δ score: 4.5% [0.3%, 8.8%]; p = 0.035). This post hoc analysis of data from CANVAS showed that canagliflozin significantly lowered PromarkerD risk scores, with the effect greatest in those T2D participants who were classified at study entry as at high risk of a subsequent decline in kidney function.

Assessment of biomarkers associated with rapid renal decline in the detection of retinopathy and its progression in type 2 diabetes: The Fremantle Diabetes Study Phase II.

AIMS: To determine whether biomarkers for diabetic kidney disease (DKD) can be used to determine the prevalence, progression and/or incidence of diabetic retinopathy (DR) complicating type 2 diabetes. METHODS: Proteomic biomarkers were measured in baseline fasting plasma from 958 Fremantle Diabetes Study Phase II participants whose baseline and, in those returning for follow-up (n = 764), Year 4 fundus photographs were graded for DR presence/severity. The performance of PromarkerD (three biomarkers and readily available clinical variables which identify prevalent DKD and predict incident DKD and estimated glomerular filtration rate decline ≥30% over four years) for detecting DR prevalence, progression and incidence was assessed using the area under the receiver operating curve (AUC). Logistic regression determined whether individual proteins were associated with DR outcomes after adjusting for the most parsimonious model. RESULTS: Plasma apolipoprotein A-IV (APOA4) was independently associated with moderate non-proliferative DR at baseline (OR (95% CI): 1.64 (1.01, 2.67), P = 0.047). Model discrimination was poor for all PromarkerD predicted probabilities against all DR outcomes (AUC ≤0.681). CONCLUSIONS: PromarkerD and its constituent biomarkers were not consistently associated with DR prevalence or temporal change. APOA4 was associated with prevalent DR, but not DR incidence or progression. Distinct pathophysiological mechanisms may underlie DKD and DR.

PromarkerD Predicts Renal Function Decline in Type 2 Diabetes in the Canagliflozin Cardiovascular Assessment Study (CANVAS).

The ability of current tests to predict chronic kidney disease (CKD) complicating diabetes is limited. This study investigated the prognostic utility of a novel blood test, PromarkerD, for predicting future renal function decline in individuals with type 2 diabetes from the CANagliflozin CardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline in 3568 CANVAS participants (n = 1195 placebo arm, n = 2373 canagliflozin arm) and used to predict incident CKD (estimated glomerular filtration rate (eGFR) <60 mL/min/1.73m2 during follow-up in those above this threshold at baseline) and eGFR decline ≥30% during the 4 years from randomization. Biomarker concentrations (apolipoprotein A-IV (apoA4), CD5 antigen-like (CD5L/AIM) and insulin-like growth factor-binding protein 3 (IGFBP3) measured by mass spectrometry were combined with clinical data (age, serum high-density lipoprotein (HDL)-cholesterol, eGFR) using a previously defined algorithm to provide PromarkerD scores categorized as low-, moderate- or high-risk. The participants (mean age 63 years, 33% females) had a median PromarkerD score of 2.9%, with 70.5% categorized as low-risk, 13.6% as moderate-risk and 15.9% as high-risk for developing incident CKD. After adjusting for treatment, baseline PromarkerD moderate-risk and high-risk scores were increasingly prognostic for incident CKD (odds ratio 5.29 and 13.52 versus low-risk, respectively; both p < 0.001). Analysis of the PromarkerD test system in CANVAS shows the test can predict clinically significant incident CKD in this multi-center clinical study but had limited utility for predicting eGFR decline ≥30%.

Proteomic profiling of developing wheat heads under water-stress.

A replicated iTRAQ (isobaric tags for relative and absolute quantification) study on developing wheat heads from two doubled haploid (DH) lines identified from a cross between cv Westonia x cv Kauz characterized the proteome changes influenced by reproductive stage water-stress. All lines were exposed to 10 days of water-stress from early booting (Zadok 40), with sample sets taken from five head developmental stages. Two sample groups (water-stressed and control) account for 120 samples that required 18 eight-plex iTRAQ runs. Based on the IWGSC RefSeq v1 wheat assembly, among the 4592 identified proteins, a total of 132 proteins showed a significant response to water-stress, including the down-regulation of a mitochondrial Rho GTPase, a regulator of intercellular fundamental biological processes (7.5 fold) and cell division protein FtsZ at anthesis (6.0 fold). Up-regulated proteins included inosine-5'-monophosphate dehydrogenase (3.83 fold) and glycerophosphodiester phosphodiesterase (4.05 fold). The Pre-FHE and FHE stages (full head emerged) of head development were differentiated by 391 proteins and 270 proteins differentiated the FHE and Post-FHE stages. Water-stress during meiosis affected seed setting with 27% and 6% reduction in the progeny DH105 and DH299 respectively. Among the 77 proteins that differentiated between the two DH lines, 7 proteins were significantly influenced by water-stress and correlated with the seed set phenotype response of the DH lines to water-stress (e.g. the up-regulation of a subtilisin-like protease in DH 299 relative to DH 105). This study provided unique insights into the biological changes in developing wheat head that occur during water-stress.

Protein Network Analysis Identifies Changes in the Level of Proteins Involved in Platelet Degranulation, Proteolysis and Cholesterol Metabolism Pathways in AECOPD Patients.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infection, increase in the rates of morbidity and mortality. Previous proteomic studies have focussed on identifying proteins involved in COPD pathogenesis in samples collected from the lung (e.g. lung tissue biopsies, bronchoalveolar lavage and sputum) but not from blood of patients who experienced AECOPD. In this study, plasma was analysed by two independent quantitative proteomics techniques; isobaric tag for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) to identify differential expression of circulating proteins in patients with stable COPD (sCOPD) and AECOPD. Firstly, iTRAQ performed on pooled plasma samples from AECOPD, sCOPD, and healthy non-smoking controls (HC) revealed 15 differentially expressed proteins between the 3 groups. MRM subsequently performed on a separate cohort of AECOPD, sCOPD, and HC patients confirmed 9 proteins to be differentially expressed by AECOPD compared to HC (Afamin, alpha-1-antichymotrypsin, Apolipoprotein E, Beta-2-glycoprotein 1, Complement component C9, Fibronectin, Immunoglobulin lambda like polypeptide 5, Inter-alpha-trypsin inhibitor heavy chain H3, Leucine rich alpha-2-glycoprotein 1). Network analysis demonstrates that most of these proteins are involved in proteolysis regulation, platelet degranulation and cholesterol metabolism. In conclusion, several potential plasma biomarkers for AECOPD were identified in this study. Further validation studies of these proteins may elucidate their roles in the development of AECOPD.

Validation of a protein biomarker test for predicting renal decline in type 2 diabetes: The Fremantle Diabetes Study Phase II.

AIMS: To validate the prognostic utility of a novel plasma biomarker panel, PromarkerD, for predicting renal decline in an independent cohort of people with type 2 diabetes. METHODS: Models for predicting rapid estimated glomerular filtration rate (eGFR) decline defined as i) incident diabetic kidney disease (DKD), ii) eGFR decline ≥30% over four years, and iii) annual eGFR decline ≥5 mL/min/1.73 m2 were applied to 447 participants from the longitudinal observational Fremantle Diabetes Study Phase II. Model performance was assessed using discrimination and calibration. RESULTS: During 4.2 ±â€¯0.3 years of follow-up, 5-10% of participants experienced a rapid decline in eGFR. A consensus model comprising apolipoprotein A-IV (apoA4), CD5 antigen-like (CD5L), insulin-like growth factor-binding protein 3 (IGFBP3), age, serum HDL-cholesterol and eGFR showed the best performance for predicting incident DKD (AUC = 0.88 (95% CI 0.84-0.93)); calibration Chi-squared = 5.6, P = 0.78). At the optimal score cut-off, this model provided 86% sensitivity, 78% specificity, 30% positive predictive value and 98% negative predictive value for four-year risk of developing DKD. CONCLUSIONS: The combination of readily available clinical and laboratory features and the PromarkerD biomarkers (apoA4, CD5L, IGFBP3) proved an accurate prognostic test for future renal decline in an independent validation cohort of people with type 2 diabetes.

Identification of Novel Circulating Biomarkers Predicting Rapid Decline in Renal Function in Type 2 Diabetes: The Fremantle Diabetes Study Phase II.

OBJECTIVE: To assess the ability of plasma apolipoprotein (apo) A-IV (apoA4), apo C-III, CD5 antigen-like (CD5L), complement C1q subcomponent subunit B (C1QB), complement factor H-related protein 2, and insulin-like growth factor binding protein 3 (IBP3) to predict rapid decline in estimated glomerular filtration rate (eGFR) in type 2 diabetes. RESEARCH DESIGN AND METHODS: Mass spectrometry was used to measure baseline biomarkers in 345 community-based patients (mean age 67.0 years, 51.9% males) from the Fremantle Diabetes Study Phase II (FDS2). Multiple logistic regression was used to determine clinical predictors of rapid eGFR decline trajectory defined by semiparametric group-based modeling over a 4-year follow-up period. The incremental benefit of each biomarker was then assessed. Similar analyses were performed for a ≥30% eGFR fall, incident chronic kidney disease (eGFR <60 mL/min/1.73 m2), and eGFR decline of ≥5 mL/min/1.73 m2/year. RESULTS: Based on eGFR trajectory analysis, 35 participants (10.1%) were defined as "rapid decliners" (mean decrease 2.9 mL/min/1.73 m2/year). After adjustment for clinical predictors, apoA4, CD5L, and C1QB independently predicted rapid decline (odds ratio 2.40 [95% CI 1.24-4.61], 0.52 [0.29-0.93], and 2.41 [1.14-5.11], respectively) and improved model performance and fit (P < 0.001), discrimination (area under the curve 0.75-0.82, P = 0.039), and reclassification (net reclassification index 0.76 [0.63-0.89]; integrated discrimination improvement 6.3% [2.1-10.4%]). These biomarkers and IBP3 contributed to improved model performance in predicting other indices of rapid eGFR decline. CONCLUSIONS: The current study has identified novel plasma biomarkers (apoA4, CD5L, C1QB, and IBP3) that may improve the prediction of rapid decline in renal function independently of recognized clinical risk factors in type 2 diabetes.

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.

Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.

Comprehensive mass spectrometry based biomarker discovery and validation platform as applied to diabetic kidney disease.

A protein biomarker discovery workflow was applied to plasma samples from patients at different stages of diabetic kidney disease. The proteomics platform produced a panel of significant plasma biomarkers that were statistically scrutinised against the current gold standard tests on an analysis of 572 patients. Five proteins were significantly associated with diabetic kidney disease defined by albuminuria, renal impairment (eGFR) and chronic kidney disease staging (CKD Stage ≥1, ROC curve of 0.77). The results prove the suitability and efficacy of the process used, and introduce a biomarker panel with the potential to improve diagnosis of diabetic kidney disease.

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat.

In this study, proteomics and metabolomics were used to study the wheat response to exposure to the SnToxA effector protein secreted by the fungal pathogen Stagonospora nodorum during infection. Ninety-one different acidic and basic proteins and 101 metabolites were differentially abundant when comparing SnToxA- and control-treated wheat leaves during a 72-h time course. Proteins involved in photosynthesis were observed to increase marginally initially after exposure, before decreasing rapidly and significantly. Proteins and metabolites associated with the detoxification of reactive oxygen species in the chloroplast were also differentially abundant during SnToxA exposure, implying that the disruption of photosynthesis causes the rapid accumulation of chloroplastic reactive oxygen species. Metabolite profiling revealed major metabolic perturbations in central carbon metabolism, evidenced by significant increases in tricarboxylic acid (TCA) cycle intermediates, suggestive of an attempt by the plant to generate ATP and reducing equivalents in response to the collapse of photosynthesis caused by SnToxA. This was supported by the observation that the TCA cycle enzyme malate dehydrogenase was up-regulated in response to SnToxA. The infiltration of SnToxA also resulted in a significant increase in abundance of many pathogenicity-related proteins, even in the absence of the pathogen or other pathogen-associated molecular patterns. This approach highlights the complementary nature of proteomics and metabolomics in studying effector-host interactions, and provides further support for the hypothesis that necrotrophic pathogens, such as S. nodorum, appear to exploit existing host cell death mechanisms to promote pathogen growth and cause disease.

Evidence of altered guinea pig ventricular cardiomyocyte protein expression and growth in response to a 5 min in vitro exposure to H(2)O(2).

Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 microM H(2)O(2) for 5 min followed by 10 units/mL catalase to degrade the H(2)O(2), and effects on protein expression were examined 48 h later. Transient exposure to H(2)O(2) increased the level of protein synthesis more than 2-fold, assessed as incorporation of [(3)H]leucine (n = 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H(2)O(2) altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H(2)O(2) increased the magnitude of the mitochondrial NADH signal 10.5 +/- 2.3% compared to cells exposed to 0 microM H(2)O(2) for 5 min followed by 10 units/mL catalase (n = 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H(2)O(2), assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 microM H(2)O(2) is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy.

Contribution of the membrane-distal tyrosine in intracellular signaling by the granulocyte colony-stimulating factor receptor.

We have evaluated the contribution of intracellular tyrosine residues of the granulocyte colony-stimulating factor receptor (GCSF-R) to its signaling and cellular outcomes. We began with stable BaF3 cell lines overexpressing wild-type or mutant GCSF-Rs. When all four intracellular tyrosines of the GCSF-R were replaced with phenylalanine (FFFF GCSF-R), cell proliferation and survival were compromised. Replacement of only the membrane-distal tyrosine (YYYF GCSF-R) also showed reduced survival following a GCSF withdrawal/replacement protocol, suggesting a role for this tyrosine. Proliferation by FFFY GCSF-R cells was attenuated by approximately 70%. In evaluating the biochemical steps involved in signaling, we then showed that the membrane-distal tyrosine was necessary and sufficient for c-Jun N-terminal kinase (JNK) activation. With the use of a cell-permeable JNK-inhibitory peptide, JNK was implicated in the proliferation of the FFFY GCSF-R mutant. To further define the events linking the membrane-distal tyrosine and JNK activation, the Src homology 2 domains of Shc, Grb2, and 3BP2 were shown to bind the full-length GCSF-R and a phosphopeptide encompassing the membrane-distal tyrosine. When binding to variant phosphopeptides based on this membrane-distal tyrosine was tested, altering the amino acids immediately following the phosphotyrosine could selectively abolish the interaction with Shc or Grb2, or the binding to both Grb2 and 3BP2. When these changes were introduced into the full-length GCSF-R and new cell lines created, only the mutant that did not interact with Grb2 and 3BP2 did not activate JNK. Our results suggest that direct binding of Shc by the GCSF-R is not essential for JNK activation.