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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.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small molecule magnetic resonance (MR) probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis noninvasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, make them strong candidates for clinical translation.

Type 2 diabetes is a complex, multifactorial disease with varying presentation and underlying pathophysiology. Recent studies using data-driven cluster analysis have led to a stratification of type 2 diabetes into novel subgroups based on six clinical measurements. Whether these subgroups truly correspond to the underlying phenotypic differences is nevertheless unclear. Here, we apply an unsupervised, data-driven clustering method (Similarity Network Fusion) to characterize type 2 diabetes in two independent cohorts involving 1,134 subjects in total based on integrated plasma lipidomics and peptidomics data without pre-selection. Logistic regression was then used to explore clustering based on ≥ 180 circulating lipids and 1,195 protein biomarkers, alongside clinical signatures. Two subgroups were identified, one of which associated with elevated C-peptide levels, diabetic complications and more severe insulin resistance compared to the other. GWAS analysis against 403 type 2 diabetes risk variants revealed associations of several SNPs with clusters and altered molecular profiles. We thus demonstrate that heterogeneity in type 2 diabetes can be captured by circulating omics alone using an unsupervised bottom-up approach. Such multiomics signatures could reflect pathological mechanisms underlying type 2 diabetes and thus may help inform on precision medicine approaches to disease management.

OBJECTIVE To examine whether the circulating substrate mix may be related to the incidence of heart failure (HF) and cardiovascular (CV) mortality and how it is altered by canagliflozin treatment. RESEARCH DESIGN AND METHODS We measured fasting glucose, free fatty acids (FFA), glycerol, β-hydroxybutyrate, acetoacetate, lactate, and pyruvate concentrations in 3,581 samples from the CANagliflozin cardioVascular Assessment Study (CANVAS) trial at baseline and at 1 and 2 years after randomization. Results were analyzed by univariate and multivariate Cox proportional hazards models. RESULTS Patients in the lowest baseline FFA tertile were more often men with a longer duration of type 2 diabetes (T2D), higher urinary albumin excretion, lower HDL-cholesterol levels, higher history of CV disease (CVD), and higher use of statins and insulin. When all seven metabolites were used as predictors, FFA were inversely associated with incident hospitalized HF (hazard ratio [HR] 0.33 [95% CI 0.21–0.55]), while glycerol was a positive predictor (2.21 [1.45–3.35]). In a model further adjusted for 16 potential confounders, including prior HF and CVD and pharmacologic therapies, FFA remained a significant negative predictor. FFA and glycerol also predicted CV mortality (HR 0.53 [95% CI 0.35–0.81] and 1.81 [1.26–2.58], respectively) and all-cause death (0.50 [0.36–0.70] and 1.64 [1.22–2.18]). When added to these models, background insulin therapy was an independent positive predictor of risk of death. Canagliflozin treatment significantly increased plasma FFA and β-hydroxybutyrate regardless of background antihyperglycemic therapy. CONCLUSIONS A constitutive metabolic setup consisting of higher lipolysis may be beneficial in delaying or preventing hospitalized HF; a further stimulation of lipolysis by canagliflozin may reinforce this influence.

Individuals with diabetic kidney disease (DKD) are at risk for progression, heart failure, and death. We assessed the association of KidneyIntelX, a bioprognostictest validated for DKD progression, with a composite time-to-event endpoint of 57% eGFR decline, kidney failure, heart failure hospitalization (HFH) , or death in a posthoc analysis of a subgroup of CANVAS participants with DKD. sTNFR-1, sTNFR-2, and KIM-1 were measured via proprietary assays, and KidneyIntelX scores were calculated using the existing algorithm. Hazard ratios for high vs. low-risk strata for the composite outcome were adjusted for age, sex, race, treatment arm, baseline CVD, HbA1c, SBP, DBP, LDL, BMI) . During 5.6 years follow-up, 282 (22%) of the 1278 CANVAS participants with DKD experienced the outcome (57% decline in eGFR/ESKD 3%, HFH 6%, death 16%) . The adjusted HR for the composite outcome in KidneyIntelX high-risk group was 2.7 (95% CI 1.9 to 3.8) vs. the low-risk group. Canagliflozin reduced the risk for the composite event vs. placebo, with larger absolute risk reductions for the high-risk stratum (11%) , compared to intermediate (6%) or low-risk strata (4%) (p In conclusion, KidneyIntelX successfully risk-stratified adults with DKD for clinical outcomes. Absolute risk reductions with canagliflozin were greatest in the high-risk stratum, thereby potentially allowing its use to identify patients most likely to benefit from treatment. Disclosure G.N.Nadkarni: Advisory Panel; Renalytix, Consultant; AstraZeneca, Reata Pharmaceuticals, Inc., Renalytix, Siemens, Other Relationship; Renalytix, Stock/Shareholder; Renalytix. D.Takale: None. B.Neal: Other Relationship; Janssen Research & Development, LLC. K.W.Mahaffey: Consultant; Novo Nordisk. Y.Yavin: Employee; Janssen Research & Development, LLC. M.K.Hansen: Employee; Janssen Research & Development, LLC. F.Fleming: Employee; Renalytix. H.L.Heerspink: Consultant; AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Chinook Therapeutics Inc., CSL Behring, Gilead Sciences, Inc., Goldfinch Bio, Inc., Janssen Research & Development, LLC, Mitsubishi Tanabe Pharma Corporation, Mundipharma, Traveere Pharmaceuticals, Research Support; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk A/S. S.Coca: Advisory Panel; 3ive Labs, Nuwellis, Reprieve Cardiovascular, Consultant; Axon Therapies, Bayer AG, Boehringer Ingelheim International GmbH, Vifor Pharma Management Ltd., Stock/Shareholder; pulseData, Renalytix. Funding Renalytix

A third of people with type 2 diabetes (T2D) will develop chronic kidney disease (CKD) , the leading cause of end-stage renal disease (ESRD) . Current standard of care tests (urinary albumin:creatinine ratio (ACR) , estimated glomerular filtration rate (eGFR)) have limited ability to predict CKD progression. PromarkerD is a novel blood test that has prognostic value for the onset/progression of early-stage renal function decline in T2D. The ability of PromarkerD to predict later-stage renal decline in participants in the CANVAS trial was explored. Concentrations of PromarkerD biomarkers (CD5L, ApoA4, IGFBP3) were measured by mass spectrometry at baseline in 3,525 CANVAS participants (n=1,179 placebo arm, n=2,346 canagliflozin arm) and combined with clinical data (age, serum HDL-cholesterol, eGFR) to provide PromarkerD scores (0 to 100%) for adverse renal outcomes. Cox regression was used to assess the ability of PromarkerD to predict three composites: i) ≥40% eGFR decline, ESRD, or renal death ii) outcome 1 or cardiovascular disease death and iii) outcome 2 or progression to macroalbuminuria. At baseline, the participants (mean age 62.7 years, 67% males, median diabetes duration 12.5 years) had mean eGFR 77 mL/min/1.73m2, median ACR 11.6 mg/g and mean PromarkerD score 34.5%. During a mean 5.6 years of follow-up, 138 (3.9%) , 380 (10.8%) and 427 (12.1%) participants experienced composite outcomes 1 to 3, respectively. After adjusting for allocated treatment, each 10% increase in PromarkerD score was significantly associated with higher rates of adverse outcomes (outcome 1: HR 1.22 [1.to 1.38], outcome 2: HR 1.28 [1.to 1.37], outcome 3: HR 1.13 [1.to 1.21], all P≤0.001) with only modest attenuation after adjustment for both eGFR and ACR (all P≤0.013) . This post-hoc analysis of CANVAS data shows that PromarkerD can be used to predict late as well as early-stage renal decline in people with T2D. Disclosure K.E.Peters: None. P.Di prinzio: None. S.Bringans: None. W.A.Davis: None. T.Davis: Advisory Panel; Merck Sharp & Dohme Corp., Novo Nordisk, Roche Diagnostics, Research Support; Novo Nordisk, Speaker's Bureau; Novo Nordisk. R.J.Lipscombe: Employee; Proteomics International, Stock/Shareholder; Proteomics International. M.K.Hansen: Employee; Janssen Research & Development, LLC.

KidneyIntelX, a bioprognostic test for assessing risk of CKD progression, risk stratified individuals for kidney, heart failure, and death outcomes in the Canagliflozin Cardiovascular Assessment Study. Individuals scored as high risk seemed to derive more of benefit from treatment with canagliflozin versus placebo. These findings may serve to increase adoption of underutilized therapies for cardiorenal risk reduction in patients with diabetic kidney disease.

Background Canagliflozin reduces cardiovascular events including hospitalization for heart failure (HHF) in patients with type 2 diabetes and cardiovascular risk. Elevated amino-terminal pro–B-type natriuretic peptide (NT-proBNP) concentrations are associated with HF diagnosis and predict cardiovascular risk. Objectives The purpose of this study was to measure NT-proBNP in CANVAS (Canagliflozin Cardiovascular Assessment Study) participants. Methods Associations between baseline NT-proBNP and cardiovascular, renal, and mortality outcomes and intervention-associated changes were determined. Results Of the 4,330 participants in the CANVAS trial, NT-proBNP was measured in 3,587, 2,918, and 995 participants at baseline, 1 year, and 6 years, respectively. The median baseline NT-proBNP concentration was 91 pg/ml, and 39.3% had NT-proBNP ≥125 pg/ml. NT-proBNP was higher in those with investigator-reported HF (13% of participants at baseline) versus those without (187 pg/ml vs. 81 pg/ml), with substantial overlap between groups. By 1 year, NT-proBNP increased with placebo, whereas canagliflozin reduced NT-proBNP by 11% (geometric mean ratio for canagliflozin vs. placebo = 0.89 [95% confidence interval (CI): 0.84 to 0.94]; p Conclusions A substantial percentage of patients in the CANVAS trial had elevated NT-proBNP values. Canagliflozin reduced NT-proBNP concentrations versus placebo; however, reduction in NT-proBNP explained only a small proportion of the benefit of canagliflozin on HF events. (CANVAS [CANagliflozin cardioVascular Assessment Study]; NCT01032629 )

To test the hypothesis that the reduction in urinary kidney injury molecule-1 (KIM-1) observed with the sodium-glucose cotransporter-2 (SGLT2) inhibitor canagliflozin is mediated through its effects on urine albumin to creatinine ratio (UACR) and monocyte chemoattractant protein-1 (MCP-1) by assessing the proportion of the effect of canagliflozin on KIM-1 that is mediated through its effects on MCP-1 and UACR in patients with type 2 diabetes and albuminuric kidney disease.We measured KIM-1 and MCP-1 levels in urine samples from the CANVAS trial at baseline and Week 52 with the Mesoscale QuickPlex SQ 120 platform. KIM-1 and MCP-1 were standardized by urinary creatinine (Cr). The proportion of the effect of canagliflozin that is mediated through UACR and MCP-1/Cr on KIM-1/Cr was estimated with G-computation.In total, 763 patients with micro- or macroalbuminuria (17.6% of the total cohort) were included. Baseline characteristics were well balanced between the canagliflozin and placebo group. At Year 1, canagliflozin compared to placebo reduced UACR, MCP-1/Cr and KIM-1/Cr by 40.4% (95% CI 31.0, 48.4), 18.1% (95% CI 8.9, 26.4) and 30.9% (95% CI 23.0, 38.0), respectively. The proportion of the effect of canagliflozin on KIM-1/Cr mediated by its effect on UACR and in turn on MCP-1/Cr was 15.2% (95% CI 9.4, 24.5).Canagliflozin reduces urinary KIM-1, suggesting decreased tubular damage. This effect was partly mediated through a reduction in MCP-1, indicative of reduced tubular inflammation, which was in turn mediated by a reduction in UACR. This post hoc analysis suggests that urinary albumin leakage may lead to tubular inflammation and induction of injury, and provide mechanistic insight for how canagliflozin may ameliorate tubular damage, but further research is required to confirm these findings.

BACKGROUND AND OBJECTIVES: Clinical trials in nephrology are enriched for patients with micro- or macroalbuminuria to enroll patients at risk of kidney failure. However, patients with normoalbuminuria can also progress to kidney failure. TNF receptor-1, TNF receptor-2, and kidney injury marker-1 (KIM-1) are known to be associated with kidney disease progression in patients with micro- or macroalbuminuria. We assessed the value of TNF receptor-1, TNF receptor-2, and KIM-1 as prognostic biomarkers for CKD progression in patients with type 2 diabetes and normoalbuminuria. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: TNF receptor-1, TNF receptor-2, and KIM-1 were measured using immunoassays in plasma samples from patients with type 2 diabetes at high cardiovascular risk participating in the Canagliflozin Cardiovascular Assessment Study trial. We used multivariable adjusted Cox proportional hazards analyses to estimate hazard ratios per doubling of each biomarker for the kidney outcome, stratified the population by the fourth quartile of each biomarker distribution, and assessed the number of events and event rates. RESULTS: In patients with normoalbuminuria (n=2553), 51 kidney outcomes were recorded during a median follow-up of 6.1 (interquartile range, 5.8-6.4) years (event rate, 3.5; 95% confidence interval, 2.6 to 4.6 per 1000 patient-years). Each doubling of baseline TNF receptor-1 (hazard ratio, 4.2; 95% confidence interval, 1.8 to 9.6) and TNF receptor-2 (hazard ratio, 2.3; 95% confidence interval, 1.5 to 3.6) was associated with a higher risk for the kidney outcome. Baseline KIM-1, urinary albumin-creatinine ratio, and eGFR were not associated with kidney outcomes. The event rates in the highest quartile of TNF receptor-1 (≥2992 ng/ml) and TNF receptor-2 (≥11,394 ng/ml) were 5.6 and 7.0 events per 1000 patient-years, respectively, compared with 2.8 and 2.3, respectively, in the lower three quartiles. CONCLUSIONS: TNF receptor-1 and TNF receptor-2 are associated with kidney outcomes in patients with type 2 diabetes and normoalbuminuria.Clinical Trial registry name and registration number: CANagliflozin cardioVascular Assessment Study (CANVAS), NCT01032629.

Background Studies have suggested that sodium glucose co‐transporter 2 inhibitors exert anti‐inflammatory effects. We examined the association of baseline growth differentiation factor‐15 (GDF‐15), a marker of inflammation and cellular injury, with cardiovascular events, hospitalization for heart failure (HF), and kidney outcomes in patients with type 2 diabetes in the CANVAS (Canagliflozin Cardiovascular Assessment Study) and determined the effect of the sodium glucose co‐transporter 2 inhibitor canagliflozin on circulating GDF‐15. Methods and Results The CANVAS trial randomized 4330 people with type 2 diabetes at high cardiovascular risk to canagliflozin or placebo. The association between baseline GDF‐15 and cardiovascular (non‐fatal myocardial infarction, non‐fatal stroke, cardiovascular death), HF, and kidney (40% estimated glomerular filtration rate decline, end‐stage kidney disease, renal death) outcomes was assessed using multivariable adjusted Cox regression models. During median follow‐up of 6.1 years (N=3549 participants with available samples), 555 cardiovascular, 129 HF, and 137 kidney outcomes occurred. Each doubling in baseline GDF‐15 was significantly associated with a higher risk of cardiovascular (hazard ratio [HR], 1.2; 95% CI, 1.0‒1.3), HF (HR, 1.5; 95% CI, 1.2‒2.0) and kidney (HR, 1.5; 95% CI, 1.2‒2.0) outcomes. Baseline GDF‐15 did not modify canagliflozin’s effect on cardiovascular, HF, and kidney outcomes. Canaglifozin treatment modestly lowered GDF‐15 compared with placebo; however, GDF‐15 did not mediate the protective effect of canagliflozin on cardiovascular, HF, or kidney outcomes. Conclusions In patients with type 2 diabetes at high cardiovascular risk, higher GDF‐15 levels were associated with a higher risk of cardiovascular, HF, and kidney outcomes. Canagliflozin modestly lowered GDF‐15, but GDF‐15 reduction did not mediate the protective effect of canagliflozin.

Background Circulating biomarkers reflecting different mechanistic pathways may identify at-risk individuals with diabetes who may benefit from sodium-glucose cotransporter-2 (SGLT2) inhibitors. Objectives The purpose of this study was to determine if high-sensitivity cardiac troponin T (hs-cTnT), soluble suppression of tumorigenesis-2 (sST2), and insulin-like growth factor binding protein 7 (IGFBP7) levels, either alone or in combination, may modify the treatment benefits of canagliflozin. Methods In the CANVAS (CANagliflozin cardioVascular Assessment Study) biomarker substudy, we evaluated the prognostic significance of baseline biomarker measurements, the long-term trajectory of each, and response to canagliflozin on key cardiovascular and kidney outcomes. Results Among the 4,330 study participants, baseline hs-cTnT, sST2, and IGFBP7 were available in 3,503 (81%), 3,084 (71%), and 3,577 (83%). In total, 39% had elevated hs-cTnT ≥14 pg/mL, 6% had sST2 >35 ng/mL, and 49% had IGFBP7 >96.5 ng/mL. Canagliflozin significantly slowed increases of hs-cTnT (P = 0.027) and sST2 (P = 0.033) through 6 years. Each biomarker was significantly associated with cardiovascular and kidney outcomes, independent of clinical covariates. Canagliflozin reduced heart failure and kidney events regardless of baseline biomarker concentration. Patients with hs-cTnT ≥14 ng/L and those with sST2 >35 ng/mL derived greater relative benefit for major adverse cardiovascular events (MACE) (both Pinteraction ≤0.05). A panel of all 3 biomarkers predicted each cardiac and kidney outcome evaluated; participants with an increasing number of abnormal circulating biomarkers appeared to have greater relative reductions in MACE from canagliflozin treatment (Pinteraction trend = 0.005). Conclusions Canagliflozin delays longitudinal rise in hs-cTnT and sST2 compared with placebo out to 6 years. Canagliflozin reduced heart failure and kidney events regardless of baseline biomarker concentration. Elevated cardiovascular biomarkers, either alone or in combination, may identify individuals who may derive greater MACE benefit from SGLT2 inhibition. CANVAS (CANagliflozin cardioVascular Assessment Study; NCT01032629)

Introduction: KidneyIntelX is a composite risk score, incorporating biomarkers and clinical variables for predicting progression of diabetic kidney disease (DKD). The utility of this score in the context of sodium glucose co-transporter 2 inhibitors and how changes in the risk score associate with future kidney outcomes are unknown. Methods: We measured soluble tumor necrosis factor receptor (TNFR)-1, soluble TNFR-2, and kidney injury molecule 1 on banked samples from CANagliflozin cardioVascular Assessment Study (CANVAS) trial participants with baseline DKD (estimated glomerular filtration rate [eGFR] 30–59 mL/min/1.73 m2 or urine albumin-to-creatinine ratio [UACR] ≥30 mg/g) and generated KidneyIntelX risk scores at baseline and years 1, 3, and 6. We assessed the association of baseline and changes in KidneyIntelX with subsequent DKD progression (composite outcome of an eGFR decline of ≥5 mL/min/year [using the 6-week eGFR as the baseline in the canagliflozin group], ≥40% sustained decline in the eGFR, or kidney failure). Results: We included 1,325 CANVAS participants with concurrent DKD and available baseline plasma samples (mean eGFR 65 mL/min/1.73 m2 and median UACR 56 mg/g). During a mean follow-up of 5.6 years, 131 participants (9.9%) experienced the composite kidney outcome. Using risk cutoffs from prior validation studies, KidneyIntelX stratified patients to low- (42%), intermediate- (44%), and high-risk (15%) strata with cumulative incidence for the outcome of 3%, 11%, and 26% (risk ratio 8.4; 95% confidence interval [CI]: 5.0, 14.2) for the high-risk versus low-risk groups. The differences in eGFR slopes for canagliflozin versus placebo were 0.66, 1.52, and 2.16 mL/min/1.73 m2 in low, intermediate, and high KidneyIntelX risk strata, respectively. KidneyIntelX risk scores declined by 5.4% (95% CI: −6.9, −3.9) in the canagliflozin arm at year 1 versus an increase of 6.3% (95% CI: 3.8, 8.7) in the placebo arm (p < 0.001). Changes in the KidneyIntelX score at year 1 were associated with future risk of the composite outcome (odds ratio per 10 unit decrease 0.80; 95% CI: 0.77, 0.83; p < 0.001) after accounting for the treatment arm, without evidence of effect modification by the baseline KidneyIntelX risk stratum or by the treatment arm. Conclusions: KidneyIntelX successfully risk-stratified a large multinational external cohort for progression of DKD, and greater numerical differences in the eGFR slope for canagliflozin versus placebo were observed in those with higher baseline KidneyIntelX scores. Canagliflozin treatment reduced KidneyIntelX risk scores over time and changes in the KidneyIntelX score from baseline to 1 year associated with future risk of DKD progression, independent of the baseline risk score and treatment arm.

KidneyIntelX is a composite risk score incorporating biomarkers and clinical variables for predicting progression of prevalent diabetic kidney disease (DKD). The change of this score over time in response to SGLT2 inhibitors and how these changes associate with future kidney outcomes is unknown. We measured soluble Tumor Necrosis Factor Receptor 1 (sTNFR-1), soluble Tumor Necrosis Factor Receptor 2 (sTNFR-2), and Kidney Injury Molecule (KIM-1) on banked samples from CANVAS trial participants with baseline DKD (eGFR In conclusion, CANA treatment reduced KidneyIntelX risk scores. Changes in the KidneyIntelX score from baseline to 1 year predict future risk of DKD progression whether they resulted from kidney protection afforded by SGLT2i therapy, or decline in kidney function caused by disease progression. Disclosure D. W. Lam: None. G. N. Nadkarni: Advisory Panel; Self; Renalytix AI plc., Consultant; Self; AstraZeneca, Renalytix AI plc., Variant Bio, Research Support; Self; Renalytix AI plc., Stock/Shareholder; Self; Renalytix AI plc. B. Neal: Advisory Panel; Self; Abbott, Janssen Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Pfizer Inc., Roche Pharma, Servier Laboratories, Research Support; Self; Janssen Pharmaceuticals, Inc., Merck Schering Plough, Roche Pharma, Servier Laboratories. K. W. Mahaffey: Consultant; Self; Abbott, Amgen Inc., Anthos, AstraZeneca, Baim Institute for Clinical Research, Bayer Healthcare Pharmaceuticals Inc., Boehringer Ingelheim Pharmaceuticals, Inc., CSL Behring, Elsevier, Inova, Intermountain Health, Johnson & Johnson, Medscape, Mount Sinai, Mundipharma International, MyoKardia, National Institutes of Health, Novartis Pharmaceuticals Corporation, Novo Nordisk, Otsuka America Pharmaceutical, Inc., Portola Pharmaceuticals, Inc., Regeneron Pharmaceuticals Inc., SmartMedics, Theravance Biopharma, Research Support; Self; Afferent, AHA, Amgen Inc., Apple, AstraZeneca, Bayer Healthcare Pharmaceuticals Inc., Cardiva Medical, Inc, Eidos, Ferring Pharmaceuticals Inc., Gilead Sciences, Inc., Google, Johnson & Johnson, Luitpold Pharmaceuticals, Inc., Medtronic, Merck & Co., Inc., National Institutes of Health, Novartis Pharmaceuticals Corporation, Sanifit, Sanofi, St. Jude Medical. N. Rosenthal: Employee; Self; Janssen Research & Development, LLC. M. K. Hansen: Employee; Self; Janssen Pharmaceuticals, Inc. H. L. Heerspink: Consultant; Self; AbbVie Inc., Astellas Pharma Inc., AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Chinook, CSL Behring, Fresenius Medical Care, Gilead Sciences, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Mitsubishi Corporation Life Sciences Limited, Mundipharma International, Novo Nordisk, Retrophin, Inc., Research Support; Self; AbbVie Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Janssen Research & Development, LLC. S. Coca: Advisory Panel; Self; Akebia Therapeutics, Inc., Bayer AG, Boehringer Ingelheim International GmbH, Consultant; Self; CHF Solutions, Relypsa Inc., Renalytix AI plc., Takeda Pharmaceutical Co., Research Support; Self; inRegen, XORTX Therapeutics Inc., Stock/Shareholder; Self; Renalytix AI plc.

We have deployed a multi-omics approach in large cohorts of patients with existing type 2 diabetes to identify biomarkers for disease progression across three molecular classes, metabolites, lipids and proteins. A Cox regression analysis for association with time to insulin requirement in 2,973 patients in the DCS, ANDIS and GoDARTS cohorts identified homocitrulline, isoleucine and 2-aminoadipic acid, as well as the bile acids glycocholic and taurocholic acids, as predictive of more rapid deterioration. Increased levels of eight triacylglycerol species, and lowered levels of the sphingomyelin SM 42:2;2 were also predictive of disease progression. Of ∼1,300 proteins examined in two cohorts, levels of GDF-15/MIC1, IL-18RA, CRELD1, NogoR, FAS, and ENPP7 were associated with faster progression, whilst SMAC/DIABLO, COTL1, SPOCK1 and HEMK2 predicted lower progression rates. Strikingly, identified proteins and lipids were also associated with diabetes incidence and prevalence in external replication cohorts. Implicating roles in disease compensation, NogoR/RTN4R improved glucose tolerance in high fat-fed mice and tended to improved insulin signalling in liver cells whilst IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. Conversely, high NogoR levels led to islet cell apoptosis. This comprehensive, multi-disciplinary approach thus identifies novel biomarkers with potential prognostic utility, provides evidence for new disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Type 2 diabetes is a multifactorial disease with multiple underlying aetiologies. To address this heterogeneity, investigators of a previous study clustered people with diabetes according to five diabetes subtypes. The aim of the current study is to investigate the etiology of these clusters by comparing their molecular signatures. In three independent cohorts, in total 15,940 individuals were clustered based on five clinical characteristics. In a subset, genetic (N = 12,828), metabolomic (N = 2,945), lipidomic (N = 2,593), and proteomic (N = 1,170) data were obtained in plasma. For each data type, each cluster was compared with the other four clusters as the reference. The insulin-resistant cluster showed the most distinct molecular signature, with higher branched-chain amino acid, diacylglycerol, and triacylglycerol levels and aberrant protein levels in plasma were enriched for proteins in the intracellular PI3K/Akt pathway. The obese cluster showed higher levels of cytokines. The mild diabetes cluster with high HDL showed the most beneficial molecular profile with effects opposite of those seen in the insulin-resistant cluster. This study shows that clustering people with type 2 diabetes can identify underlying molecular mechanisms related to pancreatic islets, liver, and adipose tissue metabolism. This provides novel biological insights into the diverse aetiological processes that would not be evident when type 2 diabetes is viewed as a homogeneous disease.

Aims/hypothesisFive clusters based on clinical characteristics have been suggested as diabetes subtypes: one autoimmune and four subtypes of type 2 diabetes (T2D). In the current study we replicate and cross-validate these T2D clusters in three large cohorts using readily measured variables in the clinic.MethodsIn this cross-sectional study, 15,940 individuals were clustered based on age, BMI, HbA1c, random or fasting C-peptide and HDL in three independent cohorts. Clusters were cross-validated against the original clusters based on HOMA measures. In addition, between cohorts, clusters were cross-validated by re-assigning people based on each cohort’s cluster centres.ResultsFive distinct T2D clusters were identified and mapped back to the original four ANDIS clusters. Using C-peptide and HDL instead of HOMA-B and HOMA-S three of the clusters mapped with high sensitivity (80.6 – 90.7%) to the previously identified Severe Insulin Deficient (SIDD), Severe insulin resistant (SIRD) and Obese (MOD) clusters. The previously described ANDIS MARD cluster could be mapped to the two milder groups in our study – one characterised by high HDL, and the other having not any extreme characteristic (MDH cluster). When these two milder groups were combined they mapped well to the previously labelled MARD cluster (sensitivity 79.4%). In the cross-validation between cohorts, particularly the SIDD and MDH cluster cross-validated well with sensitivities ranging from 73.3% to 97.1%. SIRD and MD showed a lower sensitivity ranging from 36.1% to 92.3% where individuals shifted from SIRD to MD and vice versa.Conclusions/interpretationClusters based on C-peptide instead of HOMA measures result in clusters that resemble those based on HOMA measures, especially for SIDD, SIRD and MOD. By adding HDL, the MARD cluster based upon HOMA measures resulted in the current clustering in two clusters with one cluster having high HDL levels. Cross-validation between cohorts showed generally a good resemblance between cohorts. Together, our results show that the clustering based on clinical variables readily measured in the clinic (age, HbA1c, HDL, BMI and C-peptide) results in informative clusters that are representative of the original ANDIS clusters and stable across cohorts.

OBJECTIVE To analyze the association between concentrations of plasma insulin-like growth factor binding protein 7 (IGFBP7) with renal and cardiac outcomes among participants with type 2 diabetes and high cardiovascular risk. RESEARCH DESIGN AND METHODS Associations between IGFBP7 levels and clinical outcomes were assessed among participants in the Canagliflozin Cardiovascular Assessment Study (CANVAS) with type 2 diabetes and high cardiovascular risk. RESULTS Among CANVAS participants, 3,577 and 2,898 had IGFBP7 measured at baseline and 1 year, respectively. Per log-unit higher concentration, baseline IGFBP7 was significantly associated with the composite renal end point of sustained 40% reduction in estimated glomerular filtration rate, need for renal replacement therapy, or renal death (hazard ratio [HR] 3.51; P < 0.001) and the composite renal end point plus cardiovascular death (HR 4.90; P < 0.001). Other outcomes, including development or progression of albuminuria, were also predicted by baseline IGFBP7. Most outcomes were improved by canagliflozin regardless of baseline IGFBP7; however, those with baseline concentrations ≥96.5 ng/mL appeared to benefit more from canagliflozin relative to the first progression of albuminuria compared with those with lower baseline IGFBP7 (HR 0.64 vs. 0.95; Pinteraction = 0.003). Canagliflozin did not lower IGFBP7 concentrations by 1 year; however, at 1 year, higher IGFBP7 concentrations more strongly predicted the composite renal end point (HR 15.7; P < 0.001). Patients with rising IGFBP7 between baseline and 1 year had the highest number of composite renal events. CONCLUSIONS Plasma IGFBP7 concentrations predicted renal and cardiac events among participants with type 2 diabetes and high cardiovascular risk. More data are needed regarding circulating IGFBP7 and progression of diabetic kidney disease and its complications.

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) &lt, 60 mL/min/1.73m2 during follow-up in those above this threshold at baseline) and eGFR decline &ge, 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 &lt, 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 &ge, 30%.

Canagliflozin reduces cardiovascular events including hospitalization for heart failure (HHF) in patients with type 2 diabetes and cardiovascular risk. Elevated amino-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations are associated with HF diagnosis and predict cardiovascular risk.The purpose of this study was to measure NT-proBNP in CANVAS (Canagliflozin Cardiovascular Assessment Study) participants.Associations between baseline NT-proBNP and cardiovascular, renal, and mortality outcomes and intervention-associated changes were determined.Of the 4,330 participants in the CANVAS trial, NT-proBNP was measured in 3,587, 2,918, and 995 participants at baseline, 1 year, and 6 years, respectively. The median baseline NT-proBNP concentration was 91 pg/ml, and 39.3% had NT-proBNP ≥125 pg/ml. NT-proBNP was higher in those with investigator-reported HF (13% of participants at baseline) versus those without (187 pg/ml vs. 81 pg/ml), with substantial overlap between groups. By 1 year, NT-proBNP increased with placebo, whereas canagliflozin reduced NT-proBNP by 11% (geometric mean ratio for canagliflozin vs. placebo = 0.89 [95% confidence interval (CI): 0.84 to 0.94]; p 0.001). Lower NT-proBNP with canagliflozin was also observed at 6 years (p = 0.004). In adjusted models, baseline NT-proBNP ≥125 pg/ml was prognostic for incident HHF (hazard ratio [HR]: 5.40; 95% CI: 2.67 to 10.9), HHF/cardiovascular death (HR: 3.52; 95% CI: 2.38 to 5.20), and all-cause death (HR: 2.53; 95% CI: 1.78 to 3.61). Mediation analyses suggested that 10.4% of the effects of canagliflozin on HHF were reflected in NT-proBNP lowering.A substantial percentage of patients in the CANVAS trial had elevated NT-proBNP values. Canagliflozin reduced NT-proBNP concentrations versus placebo; however, reduction in NT-proBNP explained only a small proportion of the benefit of canagliflozin on HF events. (CANVAS [CANagliflozin cardioVascular Assessment Study]; NCT01032629).

We tested whether circulating metabolites reflecting cytosolic (lactate/pyruvate) or mitochondrial (β-hydroxybutyrate/acetoacetate [AcAc]) redox balance are associated with major clinical outcomes in patients with type 2 diabetes. The CANVAS study randomized participants to placebo (Plb) or canagliflozin (100mg or 300mg) (Tx) and followed them for up to 6 years. Metabolic markers were measured at baseline (2,373 in Tx [dose combined] and 1,185 in Plb), at year 1 (2,022 in Tx and 949 in Plb) and year 2 (1,793 in Tx and 834 in Plb). By linear mixed model analysis, plasma free fatty acids, AcAc, and β-hydroxybutyrate increased with Tx vs. Plb, whereas plasma pyruvate decreased with Tx while glycerol and lactate did not change significantly. Of note, these changes were independent of the background antihyperglycemic medication (metformin, sulfonylureas, insulin, or combinations thereof). The incidence of hospitalized heart failure or cardiovascular death (HHF/CVD) was 10.2%, the incidence of the renal composite endpoint (CombR; i.e., 40% reduction in eGFR or renal replacement/death) was 3.9%. In Cox models adjusting for sex, age, BMI, HbA1c, baseline systolic blood pressure, diabetes duration, smoking, baseline use of diuretics or statins, history of heart failure, prior CV disease, and Tx, baseline AcAc was a negative predictor of HHF/CVD (HR=0.83 [95%CI 0.75-0.94]), while baseline lactate was a positive predictor (HR=1.42 [95%CI 1.20-1.68]). Adding baseline ACR and eGFR to the same multivariate model for CombR as the outcome, AcAc was still a negative (HR=0.84 [95%CI 0.75-0.94]) and lactate still a positive predictor (HR=1.42 [95%CI 1.20-1.67]). For CV and renal outcomes, higher baseline plasma lactate is an independent positive risk marker potentially reflecting a reducing cytosolic redox shift, but higher baseline plasma AcAc is a protective factor, which may be related to efficient mitochondrial supply/usage of reducing equivalents. Further analysis is needed to test these hypotheses. Disclosure E. Ferrannini: Consultant; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Oramed Pharmaceuticals. Research Support; Self; AstraZeneca, Janssen Pharmaceuticals, Inc. S. Baldi: None. T. Scozzaro: None. F. Tesfaye: Employee; Self; Janssen Research & Development, LLC. W. Shaw: Employee; Self; Janssen Pharmaceuticals, Inc. N. Rosenthal: None. K.W. Mahaffey: Consultant; Self; Medscape, Mitsubishi, Myokardia, NIH, Novartis, Novo Nordisk, Portola, Radiometer, Regeneron, SmartMedics, Springer Publishing, UCSF. Research Support; Self; Afferent, Amgen, Apple, Inc, AstraZeneca, Cardiva Medical, Inc, Daiichi, Ferring, Google (Verily), Johnson & Johnson, Luitpold, Medtronic, Merck, NIH, Novartis, Sanofi, St. Jude, Tenax. B. Neal: Research Support; Self; Janssen Research & Development, LLC, Merck Schering Plough, Roche Pharma, Servier, Zydus Pharmaceuticals, Inc. Other Relationship; Self; Abbott, Janssen, Novartis, Pfizer, Roche, and Servier. V. Perkovic: Other Relationship; Self; See Other Relationship field. M.K. Hansen: Employee; Self; Janssen Research & Development, LLC. Funding Janssen Research & Development, LLC

Chronic kidney disease (CKD) develops in 1 in 3 people with type 2 diabetes (T2D) and is the leading cause of end-stage renal disease (ESRD). The ability of baseline urinary albumin: creatinine ratio or estimated glomerular filtration rate (eGFR) to predict onset and progression of CKD complicating diabetes is limited. PromarkerD is a novel blood test that can predict future renal function decline. This study sought to validate the prognostic utility of PromarkerD in individuals with T2D from CANVAS, a randomized controlled trial of INVOKANA® (canagliflozin). PromarkerD scores were measured at baseline in 3,570 CANVAS participants (n=1,196 placebo arm, n=2,375 canagliflozin arm). Biomarker concentrations (CD5L, ApoA4, IGFBP3) measured by mass spectrometry were combined with clinical data (age, serum HDL-cholesterol, eGFR) using previously defined algorithms to provide PromarkerD scores. The test score (predicted probability) ranges from 0 to 100% and is categorized as low-, moderate- or high-risk as determined by pre-specified cut-offs at 10% and 20%. Renal function decline was defined as incident DKD during the 4 years from randomization. At baseline, the participant sample (mean age 62.7 years, 67% males, median diabetes duration 12.4 years) had a median PromarkerD score of 2.88%, with 70.5% categorized as low-risk, 13.6% as moderate-risk and 15.9% as high-risk for renal function decline. There was no significant difference in baseline PromarkerD scores by allocated treatment (P=0.58). In a model adjusted for treatment, baseline PromarkerD moderate-risk and high-risk scores were increasingly prognostic for renal function decline (OR 5.29 [4.22, 6.64] and OR 13.52 [10.69, 17.11] versus low-risk, respectively; both P These data provide further validation of the role of PromarkerD in predicting clinically significant renal function decline and thus to facilitate preventive management strategies. Disclosure K. Peters: Other Relationship; Self; Proteomics International Laboratories Ltd. J. Xu: Employee; Self; Janssen Research & Development, LLC. S. Bringans: Other Relationship; Self; Proteomics International Laboratories Lt. W.A. Davis: Advisory Panel; Spouse/Partner; Lilly Diabetes, Merck Sharp & Dohme Corp., Novo Nordisk A/S. Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH. Speaker’s Bureau; Spouse/Partner; Lilly Diabetes, Merck Sharp & Dohme Corp., Mylan, Novo Nordisk A/S, Sanofi-Aventis. Other Relationship; Self; Proteomics International. Other Relationship; Spouse/Partner; Proteomics International. T. Davis: Advisory Panel; Self; Lilly Diabetes, Merck Sharp & Dohme Corp., Novo Nordisk A/S. Speaker’s Bureau; Spouse/Partner; Boehringer Ingelheim International GmbH. Speaker’s Bureau; Self; Lilly Diabetes, Merck Sharp & Dohme Corp., Mylan, Novo Nordisk A/S, Sanofi-Aventis. Other Relationship; Self; Protemics International. Other Relationship; Spouse/Partner; Protemics International. M.K. Hansen: Employee; Self; Janssen Research & Development, LLC. R.J. Lipscombe: Other Relationship; Self; Proteomics International Laboratories Ltd. Funding Janssen Research & Development, LLC

Introduction: Growth differentiation factor 15 (GDF-15) is a marker of inflammation and cellular injury. Small clinical studies have suggested that SGLT2 inhibitors exert anti-inflammatory effects. We examined the association of baseline GDF-15 with kidney outcomes in patients with type 2 diabetes mellitus (T2DM) participating in the CANagliflozin cardioVascular Assessment Study (CANVAS) study. We also assessed the effect of the SGLT2 inhibitor canagliflozin (CANA) on GDF-15. Methods: The CANVAS trial randomized 4330 people with T2DM at high cardiovascular risk to CANA or placebo. Plasma GDF-15 concentration was measured with GDF-15 Roche Elecsys assay at baseline and follow-up. The association between GDF-15 and the primary kidney outcome (40% eGFR decline, end-stage kidney disease, or renal death) was assessed using multivariable adjusted Cox regression. Linear mixed effects models were used to assess the effect of CANA versus placebo on GDF-15 levels. Results: We included 3557 CANVAS participants with available plasma samples (mean age 62.8 years, 33.1% female, mean eGFR 76.9 ml/min/1.73 m2, median uACR 11.6 mg/g, median GDF-15 1776.0 pg/ml). During a mean follow-up of 5.7 years, 137 kidney outcomes occurred. Higher GDF-15 levels were independently associated with higher risk of kidney events (HR 1.98 [95% CI 1.40 to 2.81] per log increment GDF-15). Treatment with CANA modestly lowered GDF-15 compared to placebo (-2% [95% CI -5 to 0; p=0.046]). The effect of CANA on the kidney outcome (HR 0.56, 95% CI 0.40-0.79) was consistent regardless of baseline GDF-15 levels (p-interaction=0.75). GDF-15 did however not mediate the renal protective effect of CANA (percent mediation 3.9% [95% CI -3.6 to 14.0]). Conclusion: CANA modestly lowers GDF-15, which is independently associated with a higher risk of kidney disease progression in patients with T2DM at high cardiovascular risk. The modest reduction in GDF-15 with CANA did not mediate the renal protective effect of CANA. Disclosure J. Li: Employee; Self; George Institute. T. Sen: None. B. Neal: Research Support; Self; Janssen Research & Development, LLC, Merck Schering Plough, Roche Pharma, Servier, Zydus Pharmaceuticals, Inc. Other Relationship; Self; Abbott, Janssen, Novartis, Pfizer, Roche, and Servier. B.L. Neuen: Research Support; Self; Australian National Health and Medical Research Council Postgraduate Scholarship, Oxford Australia Clarendon Scholarship from the University of Oxford, University Postgraduate Award from UNSW Sydney. Other Relationship; Self; Janssen Research & Development, LLC. V. Perkovic: Other Relationship; Self; See Other Relationship field. D. de Zeeuw: Advisory Panel; Self; AbbVie Inc., Bayer AG, Boehringer Ingelheim International GmbH, Fresenius Medical Care, Janssen Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation. K.W. Mahaffey: Consultant; Self; Medscape, Mitsubishi, Myokardia, NIH, Novartis, Novo Nordisk, Portola, Radiometer, Regeneron, SmartMedics, Springer Publishing, UCSF. Research Support; Self; Afferent, Amgen, Apple, Inc, AstraZeneca, Cardiva Medical, Inc, Daiichi, Ferring, Google (Verily), Johnson & Johnson, Luitpold, Medtronic, Merck, NIH, Novartis, Sanofi, St. Jude, Tenax. Y. Yavin: Employee; Self; Janssen Research & Development, LLC. N. Rosenthal: None. M.K. Hansen: Employee; Self; Janssen Research & Development, LLC. H.L. Heerspink: Consultant; Self; AbbVie Inc., AstraZeneca, Boehringer Ingelheim International GmbH, CSL Behring, Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Mundipharma International, Retrophin, Inc. Funding Janssen Research & Development, LLC

Introduction: Canagliflozin (CANA) slows progression of kidney disease, which may be at least partly mediated by improvements in tubular function. To test this hypothesis, we assessed effects of CANA on markers of tubular function and injury in the CANVAS trial. Methods: Urine biomarkers of tubule function (β2-microglobulin [B2M], cystatin C, and uromodulin), tubular injury (interleukin-18 [IL-18], kidney injury molecule-1 [KIM-1], neutrophil gelatinase associated lipocalin [NGAL], and osteopontin), and inflammation (monocyte chemotactic protein-1 [MCP-1]) were measured at baseline and after 12 months. Biomarker concentrations were standardized for urine creatinine concentration. Differences between CANA and placebo treatment were assessed using ANCOVA models. Results: We included 3723 CANVAS participants with available urine samples (eGFR 77.1 ml/min/1.73 m2 and median UACR 11.7 mg/g). Clinical characteristics and biomarker levels were well balanced between treatment groups at baseline. CANA compared to placebo significantly reduced urinary KIM-1, uromodulin, cystatin C, and osteopontin. It did not change NGAL or MCP-1 and increased IL-18 and B2M. Results were consistent in subgroups by baseline UACR except MCP-1, which decreased in patients with UACR ≥30 mg/g (Table). Conclusion: CANA had variable effects on markers of tubule cell injury and function, decreasing 4, while 2 remained stable and 2 increased. Disclosure T. Sen: None. J. Li: Employee; Self; George Institute. B. Neal: Research Support; Self; Janssen Research & Development, LLC, Merck Schering Plough, Roche Pharma, Servier, Zydus Pharmaceuticals, Inc. Other Relationship; Self; Abbott, Janssen, Novartis, Pfizer, Roche, and Servier. B.L. Neuen: Research Support; Self; Australian National Health and Medical Research Council Postgraduate Scholarship, Oxford Australia Clarendon Scholarship from the University of Oxford, University Postgraduate Award from UNSW Sydney. Other Relationship; Self; Janssen Research & Development, LLC. M.J. Jardine: Other Relationship; Self; See Other Relationship field. J. Ix: Research Support; Self; Baxter. M.G. Shlipak: Advisory Panel; Self; Cricket Health, Tai Diagnostics. V. Perkovic: Other Relationship; Self; See Other Relationshipfield. Y. Yavin: Employee; Self; Janssen Research & Development, LLC. N. Rosenthal: None. M.K. Hansen: Employee; Self; Janssen Research & Development, LLC. H.L. Heerspink: Consultant; Self; AbbVie Inc., AstraZeneca, Boehringer Ingelheim International GmbH, CSL Behring, Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Mundipharma International, Retrophin, Inc. Funding Janssen Research & Development, LLC

Background Diabetes-related kidney disease is associated with end-stage renal disease and mortality, but opportunities remain to quantify its association with cardiovascular and non-cardiovascular morbidity outcomes. Methods We used the Truven Health MarketScan Commercial Claims and Encounters Database, 2010–2014, which includes specific health services records for employees and their dependents from a selection of large employers, health plans, and government and public organizations. We used administrative claims data to quantify the association between diabetes-related kidney disease and end-stage renal disease, myocardial infarction, congestive heart failure, stroke, and infections. Cox proportional hazard regression models were used to estimate adjusted hazard ratios of developing complications. Results Among 2.2 million patients with diabetes, 7.1% had diabetes-related kidney disease: 13.5%, stage 1–2; 33.8%, stage 3; 13.2% stages 4–5; 39.5%, unknown stage. In multivariable Cox proportional hazard models adjusted for demographic characteristics, baseline comorbid conditions, and total hospital days during the baseline period, hazard ratios for each outcome increased with greater diabetes-related kidney disease severity (stage 1–2 vs. stage 4–5) compared with no diabetes-related kidney disease: myocardial infarction, 1.2 (95% confidence interval 1.1–1.4) and 3.1 (2.9–3.4); congestive heart failure, 1.7 (1.6–1.9) and 5.6 (5.3–5.8); stroke, 1.3 (1.2–1.5) and 2.3 (2.1–2.5); infection, 1.4 (1.3–1.5) and 2.9 (2.8–3.0). Among patients with stage 4–5 disease, 36-month cumulative incidence was nearly 22.8% for congestive heart failure, and 25.8% for infections. Conclusions Diabetes-related kidney disease appears to be formally diagnosed at a more advanced stage than might be expected, given clinical practice guidelines. Risks of cardiovascular and non-cardiovascular outcomes are high. Electronic supplementary material The online version of this article (10.1186/s12902-019-0417-9) contains supplementary material, which is available to authorized users.

Aims/hypothesis The sodium–glucose cotransporter 2 (SGLT2) inhibitor canagliflozin slows progression of kidney function decline in type 2 diabetes. The aim of this study was to assess the effect of the SGLT2 inhibitor canagliflozin on biomarkers for progression of diabetic kidney disease (DKD). Methods A canagliflozin mechanism of action (MoA) network model was constructed based on an in vitro transcriptomics experiment in human proximal tubular cells and molecular features linked to SGLT2 inhibitors from scientific literature. This model was mapped onto an established DKD network model that describes molecular processes associated with DKD. Overlapping areas in both networks were subsequently used to select candidate biomarkers that change with canagliflozin therapy. These biomarkers were measured in 296 stored plasma samples from a previously reported 2 year clinical trial comparing canagliflozin with glimepiride. Results Forty-four proteins present in the canagliflozin MoA molecular model overlapped with proteins in the DKD network model. These proteins were considered candidates for monitoring impact of canagliflozin on DKD pathophysiology. For ten of these proteins, scientific evidence was available suggesting that they are involved in DKD progression. Of these, compared with glimepiride, canagliflozin 300 mg/day decreased plasma levels of TNF receptor 1 (TNFR1; 9.2%; p

The SGLT2 inhibitor canagliflozin (CANA) slows progression of kidney function decline in type 2 diabetes. In this translational study, we determined in silico potential mechanisms of action (MoA) and identified representative candidate biomarkers to assess in vivo how CANA may delay progression of diabetic kidney disease (DKD). These biomarkers were then validated in a CANA clinical trial. A CANA MoA network model was constructed based on an in vitro transcriptomics experiment in human proximal tubular cells and molecular features linked to SGLT2 inhibitors from scientific literature. This model was mapped onto an established DKD network model that describes molecular processes of disease progression. Overlapping areas in both networks were subsequently used to select candidate biomarkers that change with CANA therapy and may be used to monitor CANA response. These biomarkers were measured in 296 plasma samples of the 2-year CANTATA-SU clinical trial comparing CANA with glimepiride. Forty-four proteins present in the CANA MoA molecular model overlapped with proteins in the DKD network model. These proteins were considered candidates serving as proxy for monitoring impact of CANA on DKD pathophysiology. For ten of these, scientific evidence was available suggesting that they are involved in DKD progression. Of these, CANA 300 mg decreased plasma levels of tumor necrosis factor receptor 1 (TNFR-1; 9.2%, p Disclosure H.L. Heerspink: Consultant; Self; AbbVie Inc., AstraZeneca. Advisory Panel; Self; Boehringer Ingelheim GmbH. Consultant; Self; Janssen Research & Development, Fresenius SE & Co. KGaA. Advisory Panel; Self; Merck & Co., Inc.. Consultant; Self; Mitsubishi Tanabe Pharma Corporation. P. Perco: None. J. Leierer: None. M.K. Hansen: Employee; Self; Janssen Research & Development. A. Heinzel: None. G. Mayer: None.

A decline in β-cell function is a prerequisite for the development of type 2 diabetes, yet the level of β-cell function in individuals at risk of the condition is rarely measured. This is due, in part, to the fact that current methods for assessing β-cell function are inaccurate, prone to error, labor-intensive, or affected by glucose-lowering therapy. The aim of the current study was to identify novel circulating biomarkers to monitor β-cell function and to identify individuals at high risk of developing β-cell dysfunction. In a nested case-control study from the Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) cohort (n = 1157), proteomics and miRNA profiling were performed on fasting plasma samples from 43 individuals who progressed to impaired glucose tolerance (IGT) and 43 controls who maintained normal glucose tolerance (NGT) over three years. Groups were matched at baseline for age, gender, body mass index (BMI), insulin sensitivity (euglycemic clamp) and β-cell glucose sensitivity (mathematical modeling). Proteomic profiling was performed using the SomaLogic platform (Colorado, USA); miRNA expression was performed using a modified RT-PCR protocol (Regulus Therapeutics, California, USA). Results showed differentially expressed proteins and miRNAs including some with known links to type 2 diabetes, such as adiponectin, but also novel biomarkers and pathways. In cross sectional analysis at year 3, the top differentially expressed biomarkers in people with IGT/ reduced β-cell glucose sensitivity were adiponectin, alpha1-antitrypsin (known to regulate adiponectin levels), endocan, miR-181a, miR-342, and miR-323. At baseline, adiponectin, cathepsin D and NCAM.L1 (proteins expressed by pancreatic β-cells) were significantly lower in those that progressed to IGT. Many of the novel prognostic biomarker candidates were within the epithelial-mesenchymal transition (EMT) pathway: for example, Noggin, DLL4 and miR-181a. Further validation studies are required in additional clinical cohorts and in patients with type 2 diabetes, but these results identify novel pathways and biomarkers that may have utility in monitoring β-cell function and/ or predicting future decline, allowing more targeted efforts to prevent and intercept type 2 diabetes.

Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus® and 0.59 g/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and U13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.

Insulin resistance is a characteristic of type 2 diabetes and is a major independent risk factor for progression to the disease. In particular, insulin resistance associates with increased body fat and almost certainly contributes to the dramatic increase in risk of type 2 diabetes associated with obesity. Therefore, in order to design truly effective insulin sensitising agents, targeted at the mechanism of disease development, we aimed to generate an obesity-related insulin resistant cell model. Rat hepatoma cells were grown in the presence of serum isolated from obese rodents or obese human volunteers, and the insulin sensitivity of the cells monitored over time by measuring a well-characterised insulin regulated gene promoter. Higher insulin concentrations were required to fully repress the gene in the cells grown in obese rodent serum compared with those grown in serum from lean rodents (almost a 10-fold shift in insulin sensitivity). This was reversed by restoration of normal growth medium, while the insulin resistance was prevented by pioglitazone or metformin. Meanwhile, growth of cells in serum collected from obese human volunteers with diabetes also reduced the insulin sensitivity of the rat cells. No clinical marker predicted the degree of insulin resistance that was generated by the human serum. We have developed a novel insulin resistant cell model for the study of the molecular development of obesity-linked insulin resistance, screen for compounds to overcome obesity-related insulin resistance and potentially search for novel serum biomarkers of insulin resistance.

Background/Aims: Patients treated with peroxisome proliferator-activated receptor analogs (PPAR) α or α/γ may develop a transient and reversible increase in serum creatinine, the mechanism of which remains obscure. This study evaluates whether treatment with either PPAR-α or -α/γ analogs, fenofibrate or tesaglitazar, may cause deterioration in renal hemodynamics or exert direct tubular or glomerular nephrotoxic effects in rats. Methods: Male Sprague-Dawley rats (300–320 g) were treated per os with fenofibrate (300 mg/kg/day), tesaglitazar (1.2 mg/kg/day) or vehicle, for 14 days. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by inulin clearance and ultrasonic flowmetry, and cumulative excretion of sodium and creatinine were assessed. Biomarkers of glomerular and tubular injury were measured, including urinary albumin excretion and renal mRNA levels of kidney injury molecule 1 (Kim-1), lipocalin 2 (Lcn2), and osteopontin (Spp1). Results: Fenofibrate and tesaglitazar improved the lipid profile, but caused no detectable decrease in GFR or RBF compared with vehicle-treated rats. Furthermore, the cumulative excretions of sodium and creatinine were not altered by the drugs. Finally, there was no significant difference between drug- and vehicle-treated groups in urinary albumin excretion or in the expression of renal injury biomarkers. Conclusions: In the rat, no direct nephrotoxic effect or deterioration in renal hemodynamics and function were observed following treatment with fenofibrate or tesaglitazar.

Context: Previous studies have identified a single-nucleotide polymorphism in the gene encoding peroxisome proliferator-activated receptor-δ (PPARD), rs2016520, that is associated with changes in metabolic disease in some but not all studies, which suggests that PPARD agonists may have therapeutic benefits for the treatment of metabolic disorders, including dyslipidemia, type 2 diabetes, and obesity. Objective: The objective of the study was to determine whether rs2016520 or other single-nucleotide polymorphism in the PPARD locus influenced the risk of developing various characteristics of metabolic disease. Design: Haplotype tagging analysis across PPARD was performed in 11,074 individuals from the Welcome Trust U.K. Type 2 Diabetes Case Control Collection. Results: In subjects with and without type 2 diabetes, rs2016520 was associated with body mass index, high-density lipoprotein cholesterol, leptin, and TNFα and was dependent on gender. Conclusion: The current results suggest differential effects of P...

Cholesteryl ester transfer protein (CETP) plays a key role in high-density lipoprotein (HDL) cholesterol metabolism, but normal mice are deficient in CETP. In this study, transgenic mice expressing both human apolipoprotein B 100 (ApoB-100) and human CETP (hApoB100/hCETP) were used to characterize the effects of CETP inhibition and peroxisome proliferator-activated receptor alpha (PPARalpha) agonism on lipid profiles. Torcetrapib (3, 10, and 30 mg/kg), a CETP inhibitor, fenofibrate (30 mg/kg), a weak PPARalpha agonist, and GW590735 (3 and 10 mg/kg), a potent and selective PPARalpha agonist were given orally for 14 days to hApoB100/hCETP mice and lipid profiles were assessed. The average percentages of HDL, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) cholesterol fractions in hApoB100/hCETP mice were 34.8%, 61.6%, and 3.6%, respectively, which is similar to those of normolipidemic humans. Both torcetrapib and fenofibrate significantly increased HDL cholesterol and reduced LDL cholesterol, and there was a tendency for torcetrapib to reduce VLDL cholesterol and triglycerides. GW590735 significantly increased HDL cholesterol, decreased LDL and VLDL cholesterol, and significantly reduced triglycerides. Maximal increases in HDL cholesterol were 37%, 53%, and 84% with fenofibrate, torcetrapib, and GW590735, respectively. These results, in mice that exhibit a more human-like lipid profile, demonstrate an improved lipid profile with torcetrapib, fenofibrate, and GW590735, and support the use of selective PPARalpha agonism for the treatment of lipid disorders. In addition, these data demonstrate the use of hApoB100/hCETP transgenic mice to identify, characterize, and screen compounds that increase HDL cholesterol.

Background and purpose: Peroxisome proliferator-activated receptor γ (PPARγ) agonists, such as rosiglitazone and pioglitazone, sensitize cells to insulin, and are therefore used to treat type 2 diabetes. However, in some patients, these drugs induce oedema, and the present study tests the hypothesis that this side effect reflects serum and glucocorticoid-inducible kinase 1 (SGK1)-dependent enhancement of epithelia Na+ absorption. Experimental approach: Na+ absorbing epithelial cells (H441 cells, mpkCCD cells) on permeable membranes were mounted in Ussing chambers, and the effects of rosiglitazone (2 µM) and pioglitazone (10 µM) on transepithelial Na+ absorption were quantified electrometrically. Changes in SGK1 activity were assessed by monitoring phosphorylation of residues within an endogenous protein. Key results: Both cell types absorbed Na+ via an electrogenic process that was enhanced by insulin. In mpkCCD cells, this stimulation of Na+ transport was associated with increased activity of SGK1, whereas insulin regulated Na+ transport in H441 cells through a mechanism that did not involve activation of this kinase. Rosiglitazone and pioglitazone had no discernible effect on transepithelial Na+ absorption in unstimulated or insulin-stimulated cells and failed to alter cellular SGK1 activity. Conclusions and implications: Our results do not support the view that PPARγ agonists stimulate epithelial Na+ absorption or alter the control of cellular SGK1 activity. It is therefore likely that other mechanisms are involved in PPARγ-mediated fluid retention, and a better understanding of these mechanisms may help with the identification of patients likely to develop oedema or heart failure when treated with these drugs.

Peroxisome proliferator-activated receptor (PPAR)-delta is a nuclear transcription factor that plays a key role in many metabolic processes, including energy metabolism, and lipid and glucose metabolism. Candidate gene studies have identified a putative functional variant, rs2016520, in the gene encoding PPARdelta (PPARD), which is associated in some studies with metabolic traits. In addition, this single-nucleotide polymorphism was associated with adult height in several whole-genome scans, but this association did not achieve whole genome significance.This study sought to determine whether PPARD variation influenced height.Haplotype tagging analysis across PPARD was performed in about 11,000 individuals from the Wellcome Trust U.K. Type 2 Diabetes Case Control Collection (Go-DARTS2).There was an association between rs2016520 and height in both patients with type 2 diabetes and controls without diabetes (combined P = 5 x 10(-5)). In a metaanalysis using published data from Caucasian cohorts totaling more than 38,000 participants, compelling evidence was found for this locus and its association with height (P = 10(-8)) with an overall effect size of about 0.5 cm per allele. A similar analysis in a group of 2700 prepubescent children also displayed a similar effect size to that seen in the adults.PPARD variation is clearly associated with a phenotype of reduced stature in both adults and children. Because height is an important indicator of metabolic and nutritional status, this provides additional support for a key role for PPARdelta in critical metabolic functions. PPARdelta may affect height through a variety of mechanisms including altered metabolic efficiency or effects on osteoclast function.

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than peripheral (subcutaneous) obesity, however the underlying differences in morphology and pathophysiology between subcutaneous and visceral adipose are largely unknown. To evaluate the effects of diabetes and rosiglitazone (RSG) treatment, the expression of mitochondrial Hsp60, UCP-1 and F4/80 in inguinal subcutaneous (SC) fat, composed of white and brown adipose tissues, and epididymal (EP) fat, mainly white adipose tissue, were evaluated. In diabetic db/db mice, there was significant increased number of aggregated macrophage foci compared to db/+ mice, especially in EP fat. On the other hand, the expression of mitochondrial Hsp60 protein was suppressed in both SC and EP fat of db/db mice compared to db/+ mice, and the expression level of mitochondrial Hsp60 in db/+ mice was lower in EP fat compared with SC. In db/db mice, RSG suppressed the number of aggregated macrophage foci in EP fat, but not in SC fat. RSG ameliorated the mitochondrial Hsp60 expression and induced the expression of UCP-1 in both SC and EP fat. Taken together, these data suggest that differences exist in mitochondrial and macrophage content, and in the response to RSG between visceral and subcutaneous adipose tissue, and adipose type and distribution may be important for obesity-linked insulin resistance.

Commercially available enzyme-linked immunosorbent assay (ELISA) kits are commonly used to assess levels of proinflammatory cytokines in biological samples. Most of these kits require sample volumes of at least 50 microl. Thus, in order to examine multiple cytokines, volumes greater than 100 microl must be collected. However, the volume of many biological samples, especially those collected from the central nervous system (i.e., microdialysates, push-pull perfusions, or cerebrospinal fluid samples), is much less than 100 microl. Therefore, we developed a method for analyzing multiple cytokines from a single, low-volume biological sample, which involves serially assaying the samples on multiple proinflammatory cytokine ELISA kits. In many cases, assaying for one cytokine does not interfere with subsequent assay for another cytokine in the same sample. Moreover, when interference is observed, the interfering factor can be identified and its effect minimized.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 × 107, 2.5 × 108, 2.5 × 109 colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and brain IL-1β levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.