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INTRODUCTION: Tumors can be targeted by modulating the immune response of the patient. Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are critical immune checkpoints in cancer biology. The efficacy of certain cancer immunotherapies has been achieved by targeting these molecules using monoclonal antibodies. METHOD: Small-molecule drugs have also been developed as inhibitors of the PD-1/PD-L1 axis, with a mechanism of action that is distinct from that of antibodies: they induce the formation of PD-L1 homodimers, causing their stabilization, internalization, and subsequent degradation. Drug repurposing is a strategy in which new uses are sought after for approved drugs, expediting their clinical translation based on updated findings. In this study, we generated a pharmacophore model that was based on reported small molecules that targeted PD-L1 and used it to identify potential PD-L1 inhibitors among FDA-approved drugs. RESULTS: We identified 12 pharmacophore-matching compounds, but only 4 reproduced the binding mode of the reference inhibitors in docking experiments. Further characterization by molecular dynamics showed that pranlukast, an antagonist of leukotriene receptors that is used to treat asthma, generated stable and energyfavorable interactions with PD-L1 homodimers and induced homodimerization of recombinant PD-L1. CONCLUSION: Our results suggest that pranlukast inhibits the PD-1/PD-L1 axis, meriting its repurposing as an antitumor drug.

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Proper kidney function depends highly on mitochondria homeostasis. This organelle is the primary source of ATP production in the kidney and regulates other cellular processes such as redox and calcium homeostasis. Although the mitochondria's primary recognized function is cellular energy production, through the function of the Krebs cycle, electron transport system (ETS), as well as oxygen and electrochemical gradient consumption, this function is interconnected with multiple signaling and metabolic pathways, making bioenergetics a central hub in renal metabolism. Furthermore, mitochondrial biogenesis, dynamics, and mass are also strongly related to bioenergetics. This central role is not surprising given that mitochondrial impairment, including functional and structural alterations, has been recently reported in several kidney diseases. Here, we describe assessment of mitochondrial mass, structure, and bioenergetics in kidney tissue and renal-derived cell lines. These methods allow investigation of mitochondrial alterations in kidney tissue and renal cells under different experimental conditions.

Asunto(s)

Metabolismo Energético , Mitocondrias , Mitocondrias/metabolismo , Riñón/metabolismo , Técnicas Histológicas , Microscopía Electrónica de Transmisión

RESUMEN

The endoplasmic reticulum (ER) is a complex multifunctional organelle that maintains cell homeostasis. Intrinsic and extrinsic factors alter ER functions, including the rate of protein folding that triggers the accumulation of misfolded proteins and alters homeostasis, thus generating stress in the ER, which activates the unfolded protein response (UPR) pathway to promote cell survival and restore their homeostasis; however, if the damage is not corrected, it could also trigger cell death. In addition, ER stress and oxidative stress are closely related because excessive production of reactive oxygen species (ROS), a well-known inducer of ER stress, promotes the accumulation of misfolded proteins; at the same time, the ER stress enhances ROS production, generating a pathological cycle. Furthermore, it has been described that the dysregulation of the UPR contributes to the progression of various diseases, so the use of compounds capable of regulating ER stress, such as antioxidants, has been used in several experimental models of diseases to alleviate the damage induced by the maladaptive signaling of the UPR, the mechanism of action of antioxidants generally is dose-dependent, and it is specific in each tissue and pathology, could decrease or enhance specific proteins of the UPR to have beneficial or detrimental effects.

Asunto(s)

Antioxidantes , Estrés del Retículo Endoplásmico , Humanos , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Respuesta de Proteína Desplegada , Retículo Endoplásmico/metabolismo

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Kidney diseases encompass many pathologies, including obstructive nephropathy (ON), a common clinical condition caused by different etiologies such as urolithiasis, prostatic hyperplasia in males, tumors, congenital stenosis, and others. Unilateral ureteral obstruction (UUO) in rodents is an experimental model widely used to explore the pathophysiology of ON, replicating vascular alterations, tubular atrophy, inflammation, and fibrosis development. In addition, due to the kidney's high energetic demand, mitochondrial function has gained great attention, as morphological and functional alterations have been demonstrated in kidney diseases. Here we explore the kidney mitochondrial proteome differences during a time course of 7, 14, and 21 days after the UUO in rats, revealing changes in proteins involved in three main metabolic pathways, oxidative phosphorylation (OXPHOS), the tricarboxylic acid cycle (TCA), and the fatty acid (FA) metabolism, all of them related to bioenergetics. Our results provide new insight into the mechanisms involved in metabolic adaptations triggered by the alterations in kidney mitochondrial proteome during the ON.

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The transcription factor Nrf2 is a master regulator of multiple cytoprotective genes that maintain redox homeostasis and exert anti-inflammatory functions. The Nrf2-Keap1 signaling pathway is a paramount target of many cardioprotective strategies, because redox homeostasis is essential in cardiovascular health. Nrf2 gene variations, including single nucleotide polymorphisms (SNPs), are correlated with cardiometabolic diseases and drug responses. SNPs of Nrf2, KEAP1, and other related genes can impair the transcriptional activation or the activity of the resulting protein, exerting differential susceptibility to cardiometabolic disease progression and prevalence. Further understanding of the implications of Nrf2 polymorphisms on basic cellular processes involved in cardiometabolic diseases progression and prevalence will be helpful to establish more accurate protective strategies. This review provides insight into the association between the polymorphisms of Nrf2-related genes with cardiometabolic diseases. We also briefly describe that SNPs of Nrf2-related genes are potential modifiers of the pharmacokinetics that contribute to the inter-individual variability.

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Mitochondria are complex organelles that orchestrate several functions in the cell. The primary function recognized is energy production; however, other functions involve the communication with the rest of the cell through reactive oxygen species (ROS), calcium influx, mitochondrial DNA (mtDNA), adenosine triphosphate (ATP) levels, cytochrome c release, and also through tricarboxylic acid (TCA) metabolites. Kidney function highly depends on mitochondria; hence mitochondrial dysfunction is associated with kidney diseases. In addition to oxidative phosphorylation impairment, other mitochondrial abnormalities have been described in kidney diseases, such as induction of mitophagy, intrinsic pathway of apoptosis, and releasing molecules to communicate to the rest of the cell. The TCA cycle is a metabolic pathway whose primary function is to generate electrons to feed the electron transport system (ETS) to drives energy production. However, TCA cycle metabolites can also release from mitochondria or produced in the cytosol to exert different functions and modify cell behavior. Here we review the involvement of some of the functions of TCA metabolites in kidney diseases.

Asunto(s)

Ciclo del Ácido Cítrico , Enfermedades Renales/metabolismo , Metaboloma , Animales , Biomarcadores/metabolismo , Humanos , Modelos Biológicos

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The fibrotic process could be easily defined as a pathological excess of extracellular matrix deposition, leading to disruption of tissue architecture and eventually loss of function; however, this process involves a complex network of several signal transduction pathways. Virtually almost all organs could be affected by fibrosis, the most affected are the liver, lung, skin, kidney, heart, and eyes; in all of them, the transforming growth factor-beta (TGF-ß) has a central role. The canonical and non-canonical signal pathways of TGF-ß impact the fibrotic process at the cellular and molecular levels, inducing the epithelial-mesenchymal transition (EMT) and the induction of profibrotic gene expression with the consequent increase in proteins such as alpha-smooth actin (α-SMA), fibronectin, collagen, and other extracellular matrix proteins. Recently, it has been reported that some molecules that have not been typically associated with the fibrotic process, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4), mammalian target of rapamycin (mTOR), histone deacetylases (HDAC), and sphingosine-1 phosphate (S1P); are critical in its development. In this review, we describe and discuss the role of these new players of fibrosis and the convergence with TGF-ß signaling pathways, unveiling new insights into the panorama of fibrosis that could be useful for future therapeutic targets.

Asunto(s)

Esfingosina , Factor de Crecimiento Transformador beta , Fibrosis , Amigos , Histona Desacetilasas , Humanos , NADPH Oxidasa 4 , Esfingosina/metabolismo , Serina-Treonina Quinasas TOR , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo

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Renal fibrosis is a well-known mechanism that favors chronic kidney disease (CKD) development in obstructive nephropathy, a significant pathology worldwide. Fibrosis induction involves several pathways, and although mitochondrial alterations have recently emerged as a critical factor that triggers renal damage in the obstructed kidney, the temporal mitochondrial alterations during the fibrotic induction remain unexplored. Therefore, in this work, we evaluated the time course of mitochondrial mass and bioenergetics alterations induced by a unilateral ureteral obstruction (UUO), a widely used model to study the mechanism involved in kidney fibrosis induction and progression. Our results show a marked reduction in mitochondrial oxidative phosphorylation (OXPHOS) in the obstructed kidney on days 7 to 28 of obstruction without significant mitochondrial coupling changes. Besides, we observed that mitochondrial mass was reduced, probably due to decreased biogenesis and mitophagy induction. OXPHOS impairment was associated with decreased mitochondrial biogenesis markers, the peroxisome proliferator-activated receptor γ co-activator-1alpha (PGC-1α), and nuclear respiratory factor 1 (NRF1); and also, with the induction of mitophagy in a PTEN-induced kinase 1 (PINK1) and Parkin independent way. It is concluded that the impairment of OXPHOS capacity may be explained by the reduction in mitochondrial biogenesis and the induction of mitophagy during fibrotic progression.

Asunto(s)

Obstrucción Ureteral , Animales , Fibrosis , Mitocondrias , Mitofagia , Biogénesis de Organelos , Ratas

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Five-sixths nephrectomy (5/6Nx) model is widely used for studying the mechanisms involved in chronic kidney disease (CKD) progression, a kidney pathology that has increased dramatically in recent years. Mitochondrial impairment is a key mechanism that aggravates CKD progression; however, the information on mitochondrial bioenergetics and redox alterations along a time course in a 5/6Nx model is still limited and in some cases contradictory. Therefore, we performed for the first time a time-course study of mitochondrial alterations by high-resolution respirometry in the 5/6Nx model. Our results show a decrease in mitochondrial ß-oxidation at early times, as well as a permanent impairment in adenosine triphosphate (ATP) production in CI-linked respiration, a permanent oxidative state in mitochondria and decoupling of these organelles. These pathological alterations are linked to the early decrease in complex I and ATP synthase activities and to the further decrease in complex III activity. Therefore, our results may suggest that mitochondrial bioenergetics impairment is an early event in renal damage, whose persistence in time aggravates CKD development in the 5/6Nx model.

Asunto(s)

Mitocondrias/metabolismo , Nefrectomía/efectos adversos , Estrés Oxidativo/fisiología , Insuficiencia Renal Crónica , Animales , Progresión de la Enfermedad , Metabolismo Energético , Hemodinámica/fisiología , Riñón/irrigación sanguínea , Riñón/metabolismo , Riñón/patología , Riñón/cirugía , Masculino , Mitocondrias/patología , Nefrectomía/métodos , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Complicaciones Posoperatorias/metabolismo , Complicaciones Posoperatorias/patología , Ratas , Ratas Wistar , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Factores de Tiempo

RESUMEN

Obstructive nephropathy favors the progression to chronic kidney disease (CKD), a severe health problem worldwide. The unilateral ureteral obstruction (UUO) model is used to study the development of fibrosis. Impairment of renal mitochondria plays a crucial role in several types of CKD and has been strongly related to fibrosis onset. Nevertheless, in the UUO model, the impairment of mitochondria, their relationship with endoplasmic reticulum (ER) stress induction and the participation of both to induce the fibrotic process remain unclear. In this review, we summarize the current information about mitochondrial bioenergetics, redox dynamics, mitochondrial mass, and biogenesis alterations, as well as the relationship of these mitochondrial alterations with ER stress and their participation in fibrotic processes in UUO models. Early after obstruction, there is metabolic reprogramming related to mitochondrial fatty acid ß-oxidation impairment, triggering lipid deposition, oxidative stress, (calcium) Ca2+ dysregulation, and a reduction in mitochondrial mass and biogenesis. Mitochondria and the ER establish a pathological feedback loop that promotes the impairment of both organelles by ER stress pathways and Ca2+ levels dysregulation. Preserving mitochondrial and ER function can prevent or at least delay the fibrotic process and loss of renal function. However, deeper understanding is still necessary for future clinically-useful therapies.

Asunto(s)

Fibrosis/genética , Mitocondrias/genética , Insuficiencia Renal Crónica/genética , Obstrucción Ureteral/genética , Señalización del Calcio/genética , Reprogramación Celular/genética , Estrés del Retículo Endoplásmico/genética , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Mitocondrias/patología , Biogénesis de Organelos , Oxidación-Reducción , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología

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Technological advances allowed the development of high-throughput instruments such as IntelliCyt iQue Screener PLUS®. Here, we took advantage of this technology to transfer a previously validated cytotoxicity assay. The evaluated parameters were cell permeability, caspase activation and phosphatidyl serine exposure. The assay was accurate (r2 = 0.90), precise (%CV ≤ 18.90) and specific. These results showed that this technology is suitable to be used in control quality environments. In addition, the automation provided a faster acquisition and analysis of data with precise and accurate results. This application could be implemented to evaluate another in vitro mechanism of action of different biotherapeutics.

RESUMEN

Transferon® is a complex drug based on a mixture of low molecular weight peptides. This biotherapeutic is employed as a coadjuvant in clinical trials of several diseases, including viral infections and allergies. Given that macrophages play key roles in pathogen recognition, phagocytosis, processing, and antigen presentation, we evaluated the effect of Transferon® on phenotype and function of macrophage-like cells derived from THP-1 monocytes. We determined the surface expression of CD80 and CD86 by flow cytometry and IL-1ß, TNF-α, and IL-6 levels by ELISA. Transferon® alone did not alter the steady state of PMA-differentiated macrophage-like THP-1 cells. On the contrary, simultaneous stimulation of cells with Transferon® and LPS elicited a significant increase in CD80 (P ≤ 0.001) and CD86 (P ≤ 0.001) expression, as well as in IL-6 production (P ≤ 0.05) compared to the LPS control. CD80 expression and IL-6 production exhibited a positive correlation (r = 0.6, P ≤ 0.05) in cells exposed to Transferon® and LPS. Our results suggest that the administration of Transferon® induces the expression of costimulatory molecules and the secretion of cytokines in LPS-activated macrophages. Further studies are necessary to determine the implication of these findings in the therapeutic properties of Transferon®.

Asunto(s)

Antígeno B7-1/genética , Interleucina-6/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Factor de Transferencia/farmacología , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Diferenciación Celular/efectos de los fármacos , Citocinas/inmunología , Citometría de Flujo , Humanos , Recuento de Leucocitos , Monocitos/efectos de los fármacos , Células THP-1

RESUMEN

The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy. Induction of ADCC can be evaluated by employing effector cells from different sources, such as peripheral blood mononuclear cells (PBMC) and genetically modified cell lines (e.g., transfected NKs or Jurkat cells), and different approaches can be used for detection and results interpretation depending on the type of effector cells used. In this regard, validation of the assays is relevant to ensure the reliability of the results according to the intended purpose. Herein, we show the standardization and validation of ADCC assays to test the potency of three biotherapeutic proteins using primary NK cells obtained from fresh blood as effector cells and detecting cell death by flow cytometry. The advantage of using primary NKs instead of modified cells is that the response is closer to that occurring in vivo since cytotoxicity is evaluated in a direct manner. Our results indicate that in all cases, the assays exhibited a characteristic sigmoidal dose/response curve complying with accurate, precise and specific parameters. Thereby, the validated ADCC assay is an appropriate alternative to evaluate the biological activities of these type of biotherapeutics.

Asunto(s)

Adalimumab/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antineoplásicos Inmunológicos/farmacología , Linfoma de Burkitt/tratamiento farmacológico , Separación Celular/métodos , Etanercept/farmacología , Citometría de Flujo , Células Asesinas Naturales/efectos de los fármacos , Rituximab/farmacología , Animales , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Células CHO , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Células Asesinas Naturales/inmunología , Cultivo Primario de Células , Reproducibilidad de los Resultados

RESUMEN

BACKGROUND: Entamoeba histolytica is a protozoan parasite that infects humans and causes amebiasis affecting developing countries. Phagocytosis of epithelial cells, erythrocytes, leucocytes, and commensal microbiota bacteria is a major pathogenic mechanism used by this parasite. A Toll/IL-1R/Resistance (TIR) domain-containing protein is required in phagocytosis in the social ameba Dictyostelium discoideum, an ameba closely related to Entamoeba histolytica in phylogeny. In insects and vertebrates, TIR domain-containing proteins regulate phagocytic and cell activation. Therefore, we investigated whether E. histolytica expresses TIR domain-containing molecules that may be involved in the phagocytosis of erythrocytes and bacteria. METHODS: Using in silico analysis we explored in Entamoeba histolytica databases for TIR domain containing sequences. After silencing TIR domain containing sequences in trophozoites by siRNA we evaluated phagocytosis of erythrocytes and bacteria. RESULTS: We identified an E. histolytica thioredoxin containing a TIR-like domain. The secondary and tertiary structure of this sequence exhibited structural similarity to TIR domain family. Thioredoxin transcripts silenced in E. histolytica trophozoites decreased erythrocytes and E. coli phagocytosis. CONCLUSION: TIR domain-containing thioredoxin of E. histolytica could be an important element in erythrocytes and bacteria phagocytosis.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Entamoeba histolytica/fisiología , Fagocitosis , Tiorredoxinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Biología Computacional , Entamoeba histolytica/genética , Eritrocitos , Escherichia coli , Silenciador del Gen , Conformación Proteica , Tiorredoxinas/química , Tiorredoxinas/genética