Cardiovascular Effects of Canagliflozin in Relation to Renal Function and Albuminuria

Background: People with type 2 diabetes mellitus (T2DM) have elevated cardiovascular (CV) risk, including for hospitalization for heart failure (HHF). Canagliflozin reduced CV and kidney events in patients with T2DM and high CV risk or nephropathy in the CANVAS (CANagliflozin cardioVascular Assessment Study) Program and the CREDENCE (Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation) trial. Objectives: The aim of this study was to assess the effects of canagliflozin on CV outcomes according to baseline estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (UACR) in pooled patient-level data from the CANVAS Program and CREDENCE trial. Methods: Canagliflozin effects on CV death or HHF were assessed by baseline eGFR (60 mL/min/1.73 m2) and UACR (300 mg/g). HRs and 95% CIs were estimated by using Cox regression models overall and according to subgroups. Results: A total of 14,543 participants from the CANVAS Program (N = 10,142) and the CREDENCE (N = 4,401) trial were included, with a mean age of 63 years, 35% female, 75% White, 13.2% with baseline eGFR 300 mg/g. Rates of CV death or HHF increased as eGFR declined and/or UACR increased. Canagliflozin significantly reduced CV death or HHF compared with placebo (19.4 vs 28.0 events per 1,000 patient-years; HR: 0.70; 95% CI: 0.62-0.79), with consistent results across eGFR and UACR categories (all P interaction >0.40). Conclusions: Risk of CV death or HHF was higher in those with lower baseline eGFR and/or higher UACR. Canagliflozin consistently reduced CV death or HHF in participants with T2DM and high CV risk or nephropathy regardless of baseline renal function or level of albuminuria. (Canagliflozin Cardiovascular Assessment Study [CANVAS], NCT01032629; A Study of the Effects of Canagliflozin [JNJ-24831754] on Renal Endpoints in Adult Participants With Type 2 Diabetes Mellitus [CANVAS-R], NCT01989754; and Evaluation of the Effects of Canagliflozin on Renal and Cardiovascular Outcomes in Participants With Diabetic Nephropathy [CREDENCE], NCT02065791

Effects of Canagliflozin on Cardiovascular, Renal, and Safety Outcomes in Participants With Type 2 Diabetes and Chronic Kidney Disease According to History of Heart Failure: Results From the CREDENCE Trial

We aimed to assess the efficacy and safety of canagliflozin in patients with type 2 diabetes and nephropathy according to prior history of heart failure in the Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation (CREDENCE) trial. We found that participants with a prior history of heart failure at baseline (15%) were more likely to be older, female, white, have a history of atherosclerotic cardiovascular disease, and use diuretics and beta blockers (all P0.150). These results support the efficacy and safety of canagliflozin in patients with type 2 diabetes and nephropathy regardless of prior history of heart failure

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

Cardiovascular Effects of Canagliflozin in Relation to Renal Function and Albuminuria

Background: People with type 2 diabetes mellitus (T2DM) have elevated cardiovascular (CV) risk, including for hospitalization for heart failure (HHF). Canagliflozin reduced CV and kidney events in patients with T2DM and high CV risk or nephropathy in the CANVAS (CANagliflozin cardioVascular Assessment Study) Program and the CREDENCE (Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation) trial. Objectives: The aim of this study was to assess the effects of canagliflozin on CV outcomes according to baseline estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (UACR) in pooled patient-level data from the CANVAS Program and CREDENCE trial. Methods: Canagliflozin effects on CV death or HHF were assessed by baseline eGFR (60 mL/min/1.73 m2) and UACR (300 mg/g). HRs and 95% CIs were estimated by using Cox regression models overall and according to subgroups. Results: A total of 14,543 participants from the CANVAS Program (N = 10,142) and the CREDENCE (N = 4,401) trial were included, with a mean age of 63 years, 35% female, 75% White, 13.2% with baseline eGFR 300 mg/g. Rates of CV death or HHF increased as eGFR declined and/or UACR increased. Canagliflozin significantly reduced CV death or HHF compared with placebo (19.4 vs 28.0 events per 1,000 patient-years; HR: 0.70; 95% CI: 0.62-0.79), with consistent results across eGFR and UACR categories (all P interaction >0.40). Conclusions: Risk of CV death or HHF was higher in those with lower baseline eGFR and/or higher UACR. Canagliflozin consistently reduced CV death or HHF in participants with T2DM and high CV risk or nephropathy regardless of baseline renal function or level of albuminuria. (Canagliflozin Cardiovascular Assessment Study [CANVAS], NCT01032629; A Study of the Effects of Canagliflozin [JNJ-24831754] on Renal Endpoints in Adult Participants With Type 2 Diabetes Mellitus [CANVAS-R], NCT01989754; and Evaluation of the Effects of Canagliflozin on Renal and Cardiovascular Outcomes in Participants With Diabetic Nephropathy [CREDENCE], NCT02065791

MBL interacts with HIV-EBOV GP via MBL carbohydrate recognition domains.

<p>We preincubated 5% serum containing native human MBL (3,621 ng/ml) with (A) 0, 1 and 10 mM of hexose monosaccharides or EDTA diluted in media, or (B) 0–100 µg/ml of mannan or polydisperse polyethylene glycol (PEG)(D) at room temperature for 30 minutes. Then we incubated the serum with HIV-EBOV GP (1200 pg p24/100 µl) at 37°C for 1 hour before infecting adherent HEK293F cells. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). We observed relatively more toxicity associated with 10 mM EDTA but this did not invalidate our results because of our adjustment for cell viability. (C) We repeated the previous experiments with 3F8, an anti-human MBL monoclonal antibody or an IgG1 isotype control (preincubation at 37°C for 30 minutes). Significant differences are shown. (D) We preincubated HIV-EBOV GP virion-like particles with cyanovirin (0–600 nM) at 37°C for 1 hour before incubating the particles with 5% serum in the presence or absence of rhMBL. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate.</p

MBL enhances HIV-EBOV GP infection of THP-1 cells and human monocyte-derived macrophages.

<p>(A) We stimulated 5×10⁴ THP-1 cells with PMA (10 ng/ml) and supplemented the cells with IL-4 (100 ng/ml) for 72 hours. We preincubated HIV-EBOV GP or HIV-<i>env</i> negative virion-like particles (1200 pg p24/100 µl) with or without rhMBL before infecting differentiated adherent THP-1 cells cultivated in 5% MBL-deficient serum. (B) We cultivated 2.5×10⁵ PBMC derived from human single-donor buffy coat samples in RPMI-1640 with 10% FBS and stimulated the cells with M-CSF (50 ng/ml) to induce differentiation of monocyte-derived macrophages. We infected cells with HIV-EBOV GP (WT), HIV-EBOV-ΔGP NTDL6 (NTDL6, mutated GP lacks 217 amino acids in the heavily glycosylated mucin-rich region) or HIV-<i>env</i> negative (env neg) in the presence or absence of rhMBL. The box plot represents outliers (dots), 10^th and 90^th percentiles (whiskers), 25^th and 75^th percentiles (box) and median values (line). Significant differences in infection rates are shown. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay) for all the above experiments, which were performed twice in quadruplicate.</p

RNA interference screen of candidate cellular receptors for EBOV and MBL.

<p>(A–D) We targeted 24 candidate lectin, scavenger and other putative receptors using pLKO.1 lentiviral vectors that expressed 4 or 5 unique short hairpin RNA (shRNA) constructs per gene. We transduced HEK293F cells in quadruplicate using 4.6×10⁸ viral particles (shRNA-expressing vectors or empty control vectors) with hexadimethrine bromide (6 µg/ml) at 37°C for 18 hours. We selected transduced cells with 5 µg/ml puromycin over 48 hours and determined cell viability with alamarBlue reagent (resazurin reduction assay). We then infected cells in quadruplicate with HIV-EBOV GP virion-like particles (1000 pg p24/100 µl) with or without rhMBL. After 48 hours we measured rates of single-round infection (luciferase assay) and adjusted results for cell viability. Percentage change in infection was normalized to the empty pLK0.1 control vector (CTRL). Shown are positive hits (A, C) which were defined as ≥66% reduction in infection by at least two shRNA constructs for any particular gene. Reductions in protein expression produced by shRNAs (western blots; B, D) relative to that produced by the empty pLK0.1 control vector are shown. Relative densitometry was performed with ImageJ (NIH) by adjusting for variations in the actin loading controls (adjusted relative densities for CLEC6A: lane 1, 0.60; lane 2, 0.56; lane 3, 0.51; lane 4, 0.35; control, 1.0. C1QBP: lane 1, 0.32; lane 2, 0.52; lane 3, 0.47; control 1.0).</p