Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

Phosphorylated tau as a toxic agent in synaptic mitochondria: implications in aging and Alzheimer's disease.

During normal aging, there is a decline in all physiological functions in the organism. One of the most affected organs is the brain, where neurons lose their proper synaptic function leading to cognitive impairment. Aging is one of the main risk factors for the development of neurodegenerative diseases, such as Alzheimer's disease. One of the main responsible factors for synaptic dysfunction in aging and neurodegenerative diseases is the accumulation of abnormal proteins forming aggregates. The most studied brain aggregates are the senile plaques, formed by Aß peptide; however, the aggregates formed by phosphorylated tau protein have gained relevance in the last years by their toxicity. It is reported that neurons undergo severe mitochondrial dysfunction with age, with a decrease in adenosine 5'-triphosphate production, loss of the mitochondrial membrane potential, redox imbalance, impaired mitophagy, and loss of calcium buffer capacity. Interestingly, abnormal tau protein interacts with several mitochondrial proteins, suggesting that it could induce mitochondrial dysfunction. Nevertheless, whether tau-mediated mitochondrial dysfunction occurs indirectly or directly is still unknown. A recent study of our laboratory shows that phosphorylated tau at Ser396/404 (known as PHF-1), an epitope commonly related to pathology, accumulates inside mitochondria during normal aging. This accumulation occurs preferentially in synaptic mitochondria, which suggests that it may contribute to the synaptic failure and cognitive impairment seen in aged individuals. Here, we review the main tau modifications promoting mitochondrial dysfunction, and the possible mechanism involved. Also, we discuss the evidence that supports the possibility that phosphorylated tau accumulation in synaptic mitochondria promotes synaptic and cognitive impairment in aging. Finally, we show evidence and argue about the presence of phosphorylated tau PHF-1 inside mitochondria in Alzheimer's disease, which could be considered as an early event in the neurodegenerative process. Thus, phosphorylated tau PHF-1 inside the mitochondria could be considered such a potential therapeutic target to prevent or attenuate age-related cognitive impairment.

Palmitic acid reduces the autophagic flux in hypothalamic neurons by impairing autophagosome-lysosome fusion and endolysosomal dynamics.

High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.

The TGF-ß profibrotic cascade targets ecto-5'-nucleotidase gene in proximal tubule epithelial cells and is a traceable marker of progressive diabetic kidney disease.

Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-ß and adenosinergic signaling contributes to the phenotypic transition of cells and to renal fibrosis in DN models. We evaluated the role of TGF-ß on NT5E gene expression coding for the ecto-5`-nucleotidase CD73, the limiting enzyme in extracellular adenosine production. We showed that high d-glucose may predispose HK-2 cells towards active transcription of the proximal promoter region of the NT5E gene while additional TGF-ß results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-ß with occupancy by the p300 co-activator and the phosphorylated forms of the Smad2/3 complex and RNA Pol II. Transcriptional induction at NT5E in response to TGF-ß was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-ß in HK-2 cells. Interestingly, we found increased CD73 content in urinary extracellular vesicles only in diabetic patients with renal repercussions. Further, CD73-mediated AMPase activity was increased in the urinary sediment of DN patients. We conclude that the NT5E gene is a target of the profibrotic TGF-ß cascade and is a traceable marker of progressive DN.

Intraglomerular Monocyte/Macrophage Infiltration and Macrophage-Myofibroblast Transition during Diabetic Nephropathy Is Regulated by the A2B Adenosine Receptor.

Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage-myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-ß induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Blockade of the Adenosine A3 Receptor Attenuates Caspase 1 Activation in Renal Tubule Epithelial Cells and Decreases Interleukins IL-1ß and IL-18 in Diabetic Rats.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1ß and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1ß, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1ß and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

Deficient mitochondrial biogenesis in IL-2 activated NK cells correlates with impaired PGC1-α upregulation in elderly humans.

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence.

Rhodolirium andicola: a new renewable source of alkaloids with acetylcholinesterase inhibitory activity, a study from nature to molecular docking

ABSTRACT Acetylcholinesterase is an important target for control of neurodegenerative diseases causing cholinergic signaling deficit. Traditionally, galanthamine has been used as an Amaryllidaceae-derived acetylcholinesterase inhibitor, although new Amaryllidaceae plants could serve as source for better acetylcholinesterase inhibitors. Therefore, the objective of this study was to characterize the alkaloid composition from bulbs of Rhodolirium andicola (Poepp.) Traub, a native Chilean Amaryllidaceae specie, and assess their inhibitory activity on acetylcholinesterase by in vitro and in silico methodologies. Alkaloidal extracts from R. andicola exhibited an inhibitory activity with IC50 values between 11.25 ± 0.04 and 57.78 ± 1.92 µg/ml that included isolated alkaloid, galanthamine (2.3 ± 0.18 µg/ml), Additionally, 12 alkaloids were detected using gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0. To better understand the bioactivity of isolated compounds and alkaloidal extracts against acetylcholinesterase, a molecular docking approach was performed. Results suggested that alkaloids such as lycoramine, norpluvine diacetate and 6α-deoxy-tazettine expand the list of potential acetylcholinesterase inhibitors to not only galanthamine. The role of R. andicola as a source for acetylcholinesterase inhibitors is further discussed in this study.

Marsupialización modificada de ránula gigante intraoral: reporte de caso / Modified marsupialisation of a giant intraoral ranula: case report

La ránula intraoral es una enfermedad benigna seudoquística de extravasación que tiene su origen en las glándulas salivales sublinguales. Es una entidad poco prevalente. Es más frecuente en niños y adolescentes, con características clínicas propias que la diferencian de otras entidades de la boca. Su tratamiento es quirúrgico y varias técnicas han sido descritas en la literatura: marsupialización simple y modificada, enucleación de la lesión, extirpación de la glándula junto con enucleación de la ránula. Si bien la extirpación de la glándula junto con la enucleación de la lesión es el tratamiento definitivo radical y más aceptado, la marsupialización modificada puede ser útil como alternativa de tratamiento quirúrgico conservador en casos de ránulas intraorales gigantes, disminuyendo los riesgos de morbilidad quirúrgica y presentando bajos índices de recurrencia, mientras su aplicación sea cuidadosa. El siguiente reporte de caso describe el manejo clínico de forma quirúrgica conservadora mediante marsupialización modificada, con un seguimiento de 12 meses sin signos de recidiva.

The intraoral ranula is a benign pseudo-cystic extravasation with its origin in the sublingual salivary glands. It is an infrequent disease. It is more common in children and adolescents, with its own clinical characteristics that differentiate it from other entities in the mouth. The treatment for this condition is surgical, and several techniques have been described in the literature: simple and modified marsupialisation, enucleation of the lesion, and removal of the gland with enucleation of the ranula. While the removal of the gland with enucleation of the lesion is the widely accepted radical and definitive treatment, marsupialisation may be useful as an alternative to conservative surgical treatment in cases of giant intraoral ranula, reducing the risk of surgical morbidity, and presenting with low rates of recurrence, while its application should be performed with care. The case is presented that describes the conservative surgical treatment with marsupialisation in its clinical management, with a 12 months follow up without signs of recurrence.

Perfil bioquímico en sangre de cordón en prematuros extremos y crecimiento fetal / Intrauterine growth and biochemical levels in cord blood samples in infants less than 31 weeks gestation

Introducción: Con mayor aporte de proteínas y energía en la primera semana se ha observado hipofosfemia en prematuros extremos. Los menores niveles de fósforo se han presentado en prematuros con antecedentes de restricción de crecimiento intrauterino. Objetivos: Caracterizar los niveles plasmáticos bioquímicos en el cordón de prematuros extremos, nacidos adecuados (AEG) y pequeños para edad gestacional (PEG) y la relación con calcemia y fosfemia en la primera semana de vida. Pacientes y método: Estudio clínico realizado en Neonatología del Hospital Doctor Sótero del Río, en los años 2013 y 2014. Se analiza el perfil bioquímico en el cordón y la calcemia y fosfemia en los primeros 7 días de vida, registrados en la ficha clínica según fueran AEG o PEG, según las curvas de Alarcón-Pittaluga. Análisis con significación de p < 0,05. Resultados: Los niveles de colesterol, transaminasas, albúmina y creatinina fueron similares para los PEG y AEG. Los niveles de pH, fósforo, calcio, y fosfatasas alcalinas fueron menores en los PEG. El nitrógeno ureico, el ácido úrico y los triglicéridos fueron mayores en los PEG. Los PEG muestran marcada reducción de fosfemia en la primera semana, la calcemia tiende a subir proporcionalmente al descenso de la fosfemia. Conclusiones: En prematuros extremos la desnutrición intrauterina se expresa en modificación de los niveles plasmáticos de calcio, fósforo, fosfatasas alcalinas, nitrógeno ureico, ácido úrico y triglicéridos. Posnatalmente, al recibir aporte nutricional, se manifiesta una disminución de la fosfemia y un aumento de calcemia, concordante con aportes insuficientes de fósforo durante el período.

Introduction: The use of greater amounts of protein and energy during the first week of life is associated with hypophosphataemia in extreme preterm babies. The lowest phosphorus levels are described in intrauterine growth restricted (IUGR) babies. Objectives: To describe biochemistry levels in cord blood plasma in extreme premature, adequate and small for gestational age babies (AGA/SGA) and their relationship with plasma calcium and phosphorus levels during the first week of life. Patients and method: A descriptive clinical study was performed in the Neonatology Service at Hospital Dr. Sótero del Río during 2013 and 2014. A biochemical analysis of cord blood was performed on 43 premature babies, as well as plasma calcium and phosphorus levels during the first week. The adequacy for gestational age was obtained using Alarcón- Pittaluga growth curves. Statistical significance was P < .05. Results: Cholesterol, transaminases, albumin and creatinine levels were similar for both AGA and SGA babies. Levels of pH, phosphorus, calcium and alkaline phosphatase were significantly lower in SGA babies. Urea nitrogen, uric acid and triglycerides levels were higher in SGA. The analysis during the first week showed a strong reduction in phosphorus levels, as well as an increase in calcium levels in proportion to the decrease in phosphorus in the SGA sub- group. Conclusions: Intrauterine malnutrition in preterm babies is expressed in the modulation of plasma levels of calcium, phosphorus, alkaline phosphatase, urea nitrogen, uric acid and triglycerides. During post-natal life, when nutritional intake begins, a decrease in phosphorus and an increase in calcium levels appear, consistent with insufficient phosphorus intake during this period.

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

Actividad antioxidante y antimicrobiana de mieles monoflorales de plantas nativas chilenas / Antioxidant and antimicrobial activity of unifloral honeys of plants native to Chile

Chile has a great diversity of endemic and native species which gives floral origin to honey. Due the diversity of unifloral and multifloral honey previously identified it would be necessary to have information about the antioxidant content and biological activity. The objective of this study was to determine total phenols, antioxidant activity (DPPH and FRAP methods) and biological activity of unifloral honeys of native plants from Chile. For this purpose 59 beehoneys of different geographical origin were analyzed by melisopallinological method to determine the pollen present. Antimicrobial activity was tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes determining minimal bactericidal concentration (MBC). Results indicate that Chilean native honeys showed significant differences in their antioxidant as well as biological activity, which depends on the botanical and geographical origin, and it can be associated with polyphenol content.

Chile presenta una gran diversidad de especies vegetales endémicas y nativas que pueden dar origen a mieles producidas por Apis mellifera. En base a la diversidad de mieles poliflorales y monoflorales que han sido identificadas anteriormente en Chile, se propuso estudiar la actividad antioxidante y biológica para controlar el crecimiento de microorganismos patógenos. El objetivo de este estudio fue determinar el contenido de fenoles totales, actividad antioxidante (Métodos de FRAP y DPPH) y la actividad antibacteriana de mieles monoflorales de plantas nativas chilenas. Se utilizaron 59 mieles de diferente origen geográfico para determinar su origen botánico, mediante análisis melisopalinológico. La actividad antibactariana se evaluó contra Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus y Streptococcus pyogenes, determinando la concentración mínima bactericida (CMB). Los resultados indicaron que las mieles nativas de Chile muestran diferencias significativas tanto en la actividad antioxidante como en la actividad contra patógenos, la que depende del origen botánico y geográfico, pudiendo estar asociada al contenido de polifenoles.