Dietary supplementation with integral chia and flax flours ameliorates systemic inflammation.

INTRODUCTION: Chia and flax seeds are rich in alphalinolenic acid (ALA), which is bioconverted into the active derivatives eicosapentaenoic (EPA) and docosahexaenoic (DHA) having multiple beneficial effects. However, there is limited knowledge about the antiinflammatory effects of chia and flax integral flours diets rich in ALA. OBJECTIVE: The study aimed to evaluate the antiinflammatory effect of dietary supplementation with integral chia and flax flours in a murine model of LPSinduced systemic inflammation. METHODS: Balb/c mice were distributed into three groups: diet A (control), diet B (supplemented with integral chia flour), and diet C (supplemented with integral flax flour). Nutritional, hematological, and biochemical determinations were performed. ALA, EPA, and DHA were assessed by GC-MS in the liver, brain, cardiac and skeletal muscles. NF-kB immunoassays were performed in kidney, liver, and peritoneal macrophages, respectively. The phagocytic capacity was determined in peritoneal macrophages and the expression of the pro- and anti-inflammatory cytokines was assessed by RT-qPCR in the kidney, liver, and spleen. RESULTS: Diets B and C exhibited optimal nutritional adequacy and caused increased levels of ALA, EPA, and DHA in critical tissues compared to the control. The phagocytic capacity of murine peritoneal macrophages (p< 0.01) and IL-10 transcription increased, whereas the expression of NF-κB, IL-1Β, IL-6, and TNF-α decreased in animals fed both experimental diets. CONCLUSIONS: This work contributes to the current knowledge of the anti-inflammatory effects of chia and flax integral flours rich in ALA and reinforces the health advantages of their consumption.

Introducción: Las semillas de chía y lino son ricas en ácido alfa-linolénico (ALA), sus derivados activos eicosapentaenoico (EPA) y docosahexaenoico (DHA) ejercen probados efectos beneficiosos. Existe un conocimiento limitado sobre los efectos protectores de ambas semillas bajo la forma de harinas integrales, siendo de particular interés el efecto antiinflamatorio. OBJETIVO: El objetivo de este trabajo fue evaluar el efecto antiinflamatorio de la suplementación dietaria con harinas integrales de semillas de chía y lino en un modelo murino de inflamación sistémica inducido por LPS. Métodos: Ratones de la cepa Balb/c fueron distribuidos en tres grupos: dieta A (control), dieta B (suplementada con harina integral de chía) y dieta C (suplementada con harina integral de lino). Se efecturaron determinaciones nutricionales, hematológicas y bioquímicas. El contenido de ALA, EPA y DHA en hígado, cerebro, corazón y músculo esquelético se determinó por cromatografía GC-MS. Se realizó la inmunodetección de NF-kB en macrófagos peritoneales, riñón e hígado. Se determinó la capacidad fagocítica de macrófagos peritoneales y se evaluó la expresión de citoquinas pro y antiinflamatorias por RT-qPCR en riñón, hígado y bazo. RESULTADOS: Las dietas B y C mostraron una adecuación nutricional óptima y generaron niveles elevados de ALA, EPA y DHA en tejidos críticos. La capacidad fagocítica de los macrófagos peritoneales (p< 0.01) y la transcripción de IL-10 aumentó, mientras que la expresión de NF-κB, IL-1Β, IL-6 y TNF-α disminuyó en animales de los grupos B y C. CONCLUSIONES: Este trabajo contribuye al conocimiento actual de los efectos antiinflamatorios de ambas harinas integrales y refuerza los beneficios de su consumo.

The Hypoxic Microenvironment Induces Stearoyl-CoA Desaturase-1 Overexpression and Lipidomic Profile Changes in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cell carcinoma (RCC). It is characterized by a high cell proliferation and the ability to store lipids. Previous studies have demonstrated the overexpression of enzymes associated with lipid metabolism, including stearoyl-CoA desaturase-1 (SCD-1), which increases the concentration of unsaturated fatty acids in tumor cells. In this work, we studied the expression of SCD-1 in primary ccRCC tumors, as well as in cell lines, to determine its influence on the tumor lipid composition and its role in cell proliferation. The lipidomic analyses of patient tumors showed that oleic acid (18:1n-9) is one of the major fatty acids, and it is particularly abundant in the neutral lipid fraction of the tumor core. Using a ccRCC cell line model and in vitro-generated chemical hypoxia, we show that SCD-1 is highly upregulated (up to 200-fold), and this causes an increase in the cellular level of 18:1n-9, which, in turn, accumulates in the neutral lipid fraction. The pharmacological inhibition of SCD-1 blocks 18:1n-9 synthesis and compromises the proliferation. The addition of exogenous 18:1n-9 to the cells reverses the effects of SCD-1 inhibition on cell proliferation. These data reinforce the role of SCD-1 as a possible therapeutic target.

Erythropoietin attenuates LPS-induced microvascular damage in a murine model of septic acute kidney injury.

Acute kidney injury (AKI) is a frequent complication of sepsis, with a high mortality. Hallmarks of septic-AKI include inflammation, endothelial injury, and tissue hypoxia. Therefore, it would be of interest to develop therapeutic approaches for improving the microvascular damage in septic-AKI. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone. Thus, the aim of this study was to evaluate the protective effects of EPO on microvascular injury in a murine model of endotoxemic AKI. Male Balb/c mice were divided into four groups: control, LPS (8 mg/kg, ip.), EPO (3000 IU / kg, sc.) and LPS + EPO. A time course study (0-48 h) was designed. Experiments include, among others, immunohistochemistry and Western blottings of hypoxia-inducible transcription factor (HIF-1α), erythropoietin receptor (EPO-R), vascular endothelial growth factor system (VEGF/VEGFR-2), platelet and endothelial adhesion molecule-1 (PeCAM-1), inducible nitric oxide synthase (iNOS) and phosphorylated nuclear factor kappa B p65 (NF-κB). Data showed that EPO attenuates renal microvascular damage during septic-AKI progression through a) the decrease of HIF-1 alpha, iNOS, and NF-κB and b) the enhancement of EPO-R, PeCAM-1, VEGF, and VEGFR-2 expression. In summary, EPO renoprotection involves the attenuation of septic-induced renal hypoxia and inflammation as well as ameliorates the endotoxemic microvascular injury.

Tumor biology of non-metastatic stages of clear cell renal cell carcinoma; overexpression of stearoyl desaturase-1, EPO/EPO-R system and hypoxia-related proteins.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinomas. There is great interest to know the molecular basis of the tumor biology of ccRCC that might contribute to a better understanding of the aggressive biological behavior of this cancer and to identify early biomarkers of disease. This study describes the relationship among proliferation, survival, and apoptosis with the expression of key molecules related to tumoral hypoxia (hypoxia-inducible factor (HIF)-1α, erythropoietin (EPO), vascular endothelial growth factor (VEGF)), their receptors (EPO-R, VEGFR-2), and stearoyl desaturase-1 (SCD-1) in early stages of ccRCC. Tissue samples were obtained at the Urology Unit of the J.R. Vidal Hospital (Corrientes, Argentina), from patients who underwent radical nephrectomy for renal cancer between 2011 and 2014. Four experimental groups according to pathological stage and nuclear grade were organized: T1G1 (n = 6), T2G1 (n = 4), T1G2 (n = 7), and T2G2 (n = 7). The expression of HIF-1α, EPO, EPO-R, VEGF, VEGFR-2, Bcl-xL, and SCD-1 were evaluated by immunohistochemistry, Western blotting, and/or RT-PCR. Apoptosis was assessed by the TUNEL in situ assay, and tumor proliferation was determined by Ki-67 immunohistochemistry. Data revealed that HIF-1α, EPO, EPO-R, VEGF, and VEGF-R2 were overexpressed in most samples. The T1G1 group showed the highest EPO levels, approximately 200 % compared with distal renal tissue. Bcl-xL overexpression was concomitant with the enhancement of proliferative indexes. SCD-1 expression increased with the tumor size and nuclear grade. Moreover, the direct correlations observed between SCD-1/HIF-1α and SCD-1/Ki-67 increments suggest a link among these molecules, which would determine tumor progression in early stages of ccRCC. Our results demonstrate the relationship among proliferation, survival, and apoptosis with the expression of key molecules related to tumoral hypoxia (HIF-1α, EPO, VEGF), their receptors (EPO-R, VEGFR-2), and SCD-1 in early stages of ccRCC.

Erythropoietin attenuates renal and pulmonary injury in polymicrobial induced-sepsis through EPO-R, VEGF and VEGF-R2 modulation.

Sepsis remains the most important cause of acute kidney injury (AKI) and acute lung injury (ALI) in critically ill patients. The cecal ligation and puncture (CLP) model in experimental mice reproduces most of the clinical features of sepsis. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and pro-angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO protection through the expression of the EPO/EPO receptor (EPO-R) and VEGF/VEF-R2 systems in kidneys and lungs of mice undergoing CLP-induced sepsis. Male inbred Balb/c mice were divided in three experimental groups: Sham, CLP, and CLP+EPO (3000IU/kg sc). Assessment of renal functional parameters, survival, histological examination, immunohistochemistry and/or Western blottings of EPO-R, VEGF and VEGF-R2 were performed at 18h post-surgery. Mice demonstrated AKI by elevation of serum creatinine and renal histologic damage. EPO treatment attenuates renal dysfunction and ameliorates kidney histopathologic changes. Additionally, EPO administration attenuates deleterious septic damage in renal cortex through the overexpression of EPO-R in tubular interstitial cells and the overexpression of the pair VEGF/VEGF-R2. Similarly CLP- induced ALI, as evidenced by parenchymal lung histopathologic alterations, was ameliorated through pulmonary EPO-R, VEGF and VEGF-R2 over expression suggesting and improvement in endothelial survival and functionality. This study demonstrates that EPO exerts protective effects in kidneys and lungs in mice with CLP-induced sepsis through the expression of EPO-R and the regulation of the VEGF/VEGF-R2 pair.

Expresión diferencial de isoformas del factor inducible por hipoxia (hif-1αy hif-2 α) en bazo y médula ósea en un modelo murino / Differential expression of hypoxia inducible factor isoforms (hif-1α and hif-2 α) in spleen and bone marrow in a murine model

Los factores inducibles por hipoxia (HIFs) regulan la adaptación a la hipoxia (H) y protegen a las células induciendo la transcripción de múltiples genes. Se propone estudiar la expresión de las isoformas HIF1αy HIF2αen tejidos hematopoyéticos MO y Bz durante 15 días de Hipoxia Hipobárica (HH) relacionarlos con la cinética de expresión de EPO-R y factores determinantes de la eritropoyesis GATA-1 y NFE2. Se utilizaron ratones CF1, sometidos a HH 0,4 atm de 0 a 15 días. A cada tiempo, se extrajeron fémures y Bz para la obtención de extractos, fraccionamiento proteico e inmunoblotting. Se determinaron parámetros hematológicos standard. La apoptosis fue cuantificada por TUNEL. HIFαfue evaluado en sus dos isoformas. En MO y Bz el factor de transcripción aumenta y es mayor desde el día 1 de HH en Bz. Niveles máximos de HIF1αse verifican al día 3 en MO y a partir del día 5 en Bz. HIF2αen MO presenta expresión máxima al día 2 con posterior descenso, donde la MO retoma el control de la eritropoyesis. En Bz, HIF2 exhibe patrón irregular, conservando aumentos de su inmunodetección en función de la H. Epo-R se expresa desde día 1 en MO y Bz en un patrón similar de comportamiento a GATA-1. NFE2 tiene una cinética diferencial con progresión de ascenso en MO conforme aumentan los días de H observándose un máximo al día 15 mientras que el Bz muestra paulatino descenso en función de la adaptación medular a la H. Esto sugiere que en el Bz y MO se verifican procesos adaptativos coexistentes de expansión/sobrevivencia y apoptosis de progenitores eritroides. En Bz predomina una eritropoyesis compensatoria. En MO por el contrario predomina apoptosis temprana con posterior recomposición y control de la expansión del compartimiento eritroideo. HIF1 y HIF2 se sobreexpresan en ambos tejidos, sin embargo la eritropoyesis esplénica está ligada al control de HIF1αya que aparentemente HIF2αestaría asociada a la supervivencia de otros tipos celulares esplénicos durante el estrés hipóxicoPalabras clave: Hipoxia hipobárica; Factores Inducibles por Hipoxia; Eritropoyesis

Amelioration of lipopolysaccharide-induced acute kidney injury by erythropoietin: involvement of mitochondria-regulated apoptosis.

Sepsis remains the most important cause of acute kidney injury (AKI) in critically ill patients and is an independent predictor of poor outcome. The administration of lipopolysaccharide (LPS) to animals reproduces most of the clinical features of sepsis, including AKI, a condition associated with renal cellular dysfunction and apoptosis. Erythropoietin (EPO) is a well known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO renoprotection through the expression of the EPO receptor (EPO-R) and the modulation of the intrinsic apoptotic pathway in LPS-induced AKI. Male inbred Balb/c mice were divided in four experimental groups: Control, LPS (8 mg/kg i.p.), EPO (3000 IU sc) and LPS+EPO. Assessment of renal function, histological examination, TUNEL in situ assay, immunohistochemistry and Western blottings of caspase-3, Bax, Bcl-xL, EPO-R and Cytochrome c were performed at 24h post treatment. LPS+EPO treatment significantly improved renal function and ameliorated histopathological injury when compared to the LPS treated group. Results showed that EPO treatment attenuates renal tubular apoptosis through: (a) the overexpression of EPO-R in tubular interstitial cells, (b) the reduction of Bax/Bcl-xL ratio, (c) the inhibition Cytochrome c release into the cytosol and (d) the decrease of the active caspase-3 expression. This study suggests that EPO exerts renoprotection on an experimental model of LPS-induced AKI. EPO induced renoprotection involves an anti-apoptotic effect through the expression of EPO-R and the regulation of the mitochondrial apoptotic pathway.