Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Processing of Snake Venom Metalloproteinases: Generation of Toxin Diversity and Enzyme Inactivation

Submitted by Sandra Infurna ([email protected]) on 2016-12-27T14:45:10Z No. of bitstreams: 1 carolina_nicolau_etal_IOC_2016.pdf: 1379420 bytes, checksum: f93cf450154ddf56658d64ea57272944 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-27T14:51:42Z (GMT) No. of bitstreams: 1 carolina_nicolau_etal_IOC_2016.pdf: 1379420 bytes, checksum: f93cf450154ddf56658d64ea57272944 (MD5)Made available in DSpace on 2016-12-27T14:51:43Z (GMT). No. of bitstreams: 1 carolina_nicolau_etal_IOC_2016.pdf: 1379420 bytes, checksum: f93cf450154ddf56658d64ea57272944 (MD5) Previous issue date: 2016Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil.Snake venom metalloproteinases (SVMPs) are abundant in the venoms of vipers and rattlesnakes, playing important roles for the snake adaptation to different environments, and are related to most of the pathological effects of these venoms in human victims. The effectiveness of SVMPs is greatly due to their functional diversity, targeting important physiological proteins or receptors in different tissues and in the coagulation system. Functional diversity is often related to the genetic diversification of the snake venom. In this review, we discuss some published evidence that posit that processing and post-translational modifications are great contributors for the generation of functional diversity and for maintaining latency or inactivation of enzymes belonging to this relevant family of venom toxins

High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.Fil: Favoretto, Bruna C.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Portes Junior, José A.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Jacysyn, Jacqueline F.. Universidade de Sao Paulo; BrasilFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Faquim Mauro, Eliana L.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; Brasi

Assessing the binding of venoms from aquatic elapids to the nicotinic acetylcholine receptor orthosteric site of different prey models

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was ;, sea kraits representative was sea snakes representatives were two spp. and eight spp; and water cobras representative was . No prey-specific differences in crude venom binding were observed from any species tested, except for which showed slight evidence of prey-potency differences. For , , and , there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within <i>Micrurus</i> Venoms and Toxin Resistance in Prey Species

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey

Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

Made available in DSpace on 2015-09-21T17:25:46Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) carolina_nicolau_etal_IOC_2013.pdf: 1668584 bytes, checksum: 6ae4f3b359524876188b347a8f43cd15 (MD5) Previous issue date: 2013Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo CruzI. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.Universidade São Paulo. Instituto de Matemática e Estatística. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Instituto Butantan. Laboratório de Fisiopatologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Universidade Federal do Oeste do Pará. Santarém, Pará, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Instituto Butantan. Laboratório de Fisiopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Faculdades Integradas do Tapajó. Santarém, Pará, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo CruzI. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.nstituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotro´pico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes

Principal Component Analysis relative to toxin composition.

<p>Loading (top) and score (bottom) plots of the principal components 1 and 2 of the venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) according to their protein composition including as variables the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A), or the normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The Principal Component Analysis was based on the covariance matrix and all calculations were carried out in the software Minitab 16.</p

Comparison of the elution profiles of venoms from snakes classified in different genera.

<p>Samples containing 5 mg of crude lyophilized venom from <i>Bothrops atrox</i>, <i>Bothrops jararacussu</i>, <i>Bothropoides jararaca</i>, <i>Bothropoides neuwiedi</i>, <i>Rhinocerophis alternatus</i> and <i>Rhinocerophis cotiara</i>, species maintained at Instituto Butantan herpetarium, were applied to a Vydac C-18 column (4.6×250 mm, 10-µm particle size) coupled to an Agilent 1100 HPLC system. The fractions were eluted at 1 mL/min, with a gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, 45–70% B over 20 min and 70–100% B over 10 min). The separations were monitored at 214 nm.</p

Venom clustering according to toxin composition.

<p>The venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) were classified according to their protein composition by hierarchical clustering of the observations, including as a variable the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A) or normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The procedure used an agglomerative hierarchical method linked by the minimum Euclidean distance between an item in one cluster and an item in another cluster (nearest neighbor) using the Minitab 16 software.</p