Sergio de Frutos

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation.

The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.

Differential Regulation of Soluble Guanylyl Cyclase Expression and Signaling by Collagens: Involvement of Integrin-Linked Kinase

Glomerular diseases are characterized by an abnormal synthesis of extracellular matrix proteins, such as collagen type I. Evidence that growth on collagen type I downregulates soluble guanylyl cyclase (sGC) expression and the responsiveness of human mesangial cells to nitric oxide (NO) by activating specific integrin signals involving integrin-linked kinase (ILK) is presented. Human mesangial cells were grown on collagen type I or IV for 24 to 72 h. Compared with collagen IV, growth on collagen I reduced the protein expression and NO-stimulated enzyme activity of sGC. This downregulation was effected at the level of transcription, because steady-state sGC mRNA expression was reduced on collagen I, but inhibition of transcription with Actinomycin D revealed no differences in transcript stability between the two culture conditions. Collagen I also reduced the capacity of cells to relax in response to NO after H2O2 challenge and inhibited NO-induced phosphorylation of vasodilator-activated phosphoprotein, a target of cyclic guanine monophosphate-dependent protein kinase. Examination of the surface expression of integrins, the receptors for extracellular matrix components, revealed that alpha1 and alpha2 integrin subunits were more abundant on cells that were grown on collagen IV and that surface expression of beta1 integrin did not vary with collagen type. However, growth on collagen I induced beta1 integrin to adopt its active conformation, and this activation of beta1 integrin was accompanied by increased activity of its downstream effector ILK. Dominant-negative suppression of ILK signaling relieved the suppression of sGC expression and NO-induced vasodilator-activated phosphoprotein phosphorylation induced by collagen I.

Integrin-linked kinase regulates tubular aquaporin-2 content and intracellular location: a link between the extracellular matrix and water reabsorption

One of the clinical alterations observed in chronic renal disease (CRD) is the impaired urine concentration, known as diabetes insipidus (DI). Tubulointerstitial fibrosis of the kidney is also a pathological finding observed in CRD and involves composition of extracellular matrix (ECM). However, an association between these two events has not been elucidated. In this study, we showed that the extracellular‐to‐intracellular scaffold protein integrin‐linked kinase (ILK) regulates expression of tubular water channel aquaporin‐2 (AQP2) and its apical membrane presence in the renal tubule. Basally, polyuria and decreased urine osmolality were present in ILK conditional‐knockdown (cKD‐ILK) adult mice compared with nondepleted ILK littermates. No changes were observed in arginine‐vasopressin (AVP) blood levels, renal receptor (V2R), or AQP3 expression. However, tubular AQP2 was decreased in expression and apical membrane presence in cKD‐ILK mice, where the canonical V2R/cAMP axis activation is still functional, but independent of the absence of ILK. Thus, cKD‐ILK constitutes a nephrogenic diabetes insipidus (NDI) model. AQP2 and ILK colocalize in cultured inner medullary collecting duct (mIMCD3) cells. Specific ILK siRNAs and collagen I (Col) decrease ILK and AQP2 levels and AQP2 presence on the membrane of tubular mIMCD3 cells, which impairs the capacity of the cells to transport water under hypotonic stress. The present work points to ILK as a therapeutic target in NDI.—Cano‐Peñalver, J. L., Griera, M., Serrano, I., Rodríguez‐Puyol, D., Dedhar, S., de Frutos, S., Rodríguez‐Puyol, M. Integrin‐linked kinase regulates tubular aquaporin‐2 content and intracellular location: a link between the extracellular matrix and water reabsorption. FASEB J. 28, 3645–3659 (2014). www.fasebj.org

H-ras deletion protects against angiotensin II–induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H‐ras gene deletion produces mice hypotension via a soluble guanylate cyclase‐protein kinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H‐ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross‐sectional area were similar between AngII‐treated H‐Ras knockout (H‐ras−/−) and control wild‐type (H‐ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG‐Iβ protein expression in H‐ras−/− mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII‐induced cardiac hypertrophy and fibrosis markers in H‐ras−/− mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H‐Ras down‐regulates the B‐RAF/MEK/ERK pathway, which induces the glycogen synthase kinase‐3β‐dependent activation of the transcription factor, cAMP response element‐binding protein, which is responsible for PKG‐Iβ overexpression in H‐ras−/− mouse embryonic fibroblasts. This study demonstrates that H‐ras deletion protects against AngII‐induced cardiac remodeling, possibly via a mechanism in which PKG‐Iβ overexpression could play a partial role, and points to H‐Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.—Martín‐Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López‐Olañeta, M., Chiloeches, A., Lara‐Pezzi, E., de Frutos, S., Rodríguez‐Puyol, M., Calleros, L., Rodriíguez‐Puyol, D. H‐ras deletion protects against angiotensin II–induced arterial hypertension and cardiac remodeling through protein kinase G‐Iβ pathway activation. FASEB J. 32, 920–934 (2018). www.fasebj.org

Peripheral insulin resistance in ILK-depleted mice by reduction of GLUT4 expression

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blocked in vivo and in vitro, although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance.

Integrin linked kinase regulates the transcription of AQP2 by NFATC3

Two processes are associated with progressive loss of renal function: 1) decreased aquaporin-2 (AQP2) expression and urinary concentrating capacity (Nephrogenic Diabetes Insipidus, NDI); and 2) changes in extracellular matrix (ECM) composition, e.g. increased collagen I (Col I) deposition, characteristic of tubule-interstitial fibrosis. AQP2 expression is regulated by both the ECM-to-intracellular scaffold protein integrin-linked kinase (ILK) by NFATc/AP1 and other transcription factors. In the present work, we used in vivo and in vitro approaches to examine ILK participation in NFATc3/AP-1-mediated increases in AQP2 gene expression. Both NFATc3 knock-out mice and ILK conditional-knockdown mice (cKD-ILK) display symptoms of NDI (polyuria and reduced AQP2 expression). NFATc3 is upregulated in the renal medulla tubular cells of cKD-ILK mice but with reduced nuclear localization. Inner medullary collecting duct mIMCD3 cells were subjected to ILK depletion and transfected with reporter plasmids. Pharmacological activators or inhibitors determined the effect of ILK activity on NFATc/AP-1-dependent increases in transcription of AQP2. Finally, mIMCD3 cultured on Col I showed reduced activity of the ILK/GSK3β/NFATc/AQP2 axis, suggesting this pathway is a potential target for therapeutic treatment of NDI.

Elaboración y empleo de materiales didácticos para la mejora de la enseñanza práctica en la asignatura de Fisiología Humana en el grado de Ciencias de la Actividad Física y del Deporte. Evaluación de resultados

The need to advance towards a methodology that strengthens self-directed learning and competency development among students has become a key focus of university teaching in recent years. This focus has given rise to new methodological strategies that have been enhanced by the use of information and communication technologies (ICTs). Within the progress of these methodological strategies, the creation of educational resources that help to meet their objectives has become an important part of the work done by university lecturers. Along those lines, a group of lecturers in the Department of Physiology at the University of Alcalá (UAH), Spain, has created a series of educational resources using visual online tools for practical teaching in the Human Physiology subject. These resources were used for the first time in the 2011/12 academic year in the Human Physiology subject, taught in the first year of the bachelor’s degree course in Physical Activity and Sports Sciences. This article presents the resources created and the methodology used. The results obtained from their use are also assessed; these results refer to the students’ satisfaction with the resources, and the contribution that the resources and the methodology made to the students’ learning. A survey was conducted to ascertain the students’ opinions, and a comparative analysis was performed of the grades obtained by the students in the practical part of the subject and their impact on the overall grade, and those obtained in the previous academic year when the resources had not been used. The results show that the students were satisfied with both the educational resources and the methodology used, and that there was a significant improvement in the grades obtained in comparison to the previous academic year.ResumenLa necesidad de caminar hacia una metodología que potencie el aprendizaje autónomo y el desarrollo de las competencias en el alumno se ha convertido en estos últimos años en un elemento clave en la enseñanza universitaria. Esta constante ha impulsado el auge de nuevas estrategias metodológicas que se han visto dinamizadas por el uso de las nuevas tecnologías. Dentro del progreso de estas metodologías, la elaboración de materiales didácticos que favorezcan estos objetivos ha sido y sigue siendo una pieza importante de trabajo entre los docentes universitarios. En esta línea, un grupo de profesores del Departamento de Fisiología de la universidad hemos elaborado una serie de materiales didácticos empleando herramientas visuales y virtuales, para la enseñanza práctica de la asignatura Fisiología Humana. Estos materiales se utilizaron por primera vez durante el curso 2011–12 en la asignatura Fisiología Humana de primer curso del grado de Ciencias de la Actividad Física y del Deporte (CCAFyDE). En este trabajo presentamos los materiales elaborados y la metodología utilizada. También evaluamos el resultado obtenido tras su empleo en cuanto a la satisfacción del alumno y a la contribución que estos materiales y tal metodología han tenido para el aprendizaje de los estudiantes. Para ello se realizó una encuesta entre los alumnos y un análisis comparativo de las calificaciones obtenidas en la parte práctica de la asignatura y de su impacto en el conjunto de la nota total de esta misma, con respecto a las notas obtenidas por los estudiantes del curso anterior, en el que no se utilizaron estos materiales. Los resultados indican una aceptación satisfactoria por parte de los alumnos tanto de los materiales didácticos como de la metodología empleada, así como una mejora significativa en sus calificaciones con respecto al curso anterio

Creating and using educational resources to improve practical teaching in the Human Physiology subject on the bachelor’s degree course in Physical Activity and Sports Sciences. Assessment of results

The need to advance towards a methodology that strengthens self-directed learning and competency development among students has become a key focus of university teaching in recent years. This focus has given rise to new methodological strategies that have been enhanced by the use of information and communication technologies (ICTs). Within the progress of these methodological strategies, the creation of educational resources that help to meet their objectives has become an important part of the work done by university lecturers. Along those lines, a group of lecturers in the Department of Physiology at the University of Alcalá (UAH), Spain, has created a series of educational resources using visual online tools for practical teaching in the Human Physiology subject. These resources were used for the first time in the 2011/12 academic year in the Human Physiology subject, taught in the first year of the bachelor’s degree course in Physical Activity and Sports Sciences. This article presents the resources created and the methodology used. The results obtained from their use are also assessed; these results refer to the students’ satisfaction with the resources, and the contribution that the resources and the methodology made to the students’ learning. A survey was conducted to ascertain the students’ opinions, and a comparative analysis was performed of the grades obtained by the students in the practical part of the subject and their impact on the overall grade, and those obtained in the previous academic year when the resources had not been used. The results show that the students were satisfied with both the educational resources and the methodology used, and that there was a significant improvement in the grades obtained in comparison to the previous academic year.ResumenLa necesidad de caminar hacia una metodología que potencie el aprendizaje autónomo y el desarrollo de las competencias en el alumno se ha convertido en estos últimos años en un elemento clave en la enseñanza universitaria. Esta constante ha impulsado el auge de nuevas estrategias metodológicas que se han visto dinamizadas por el uso de las nuevas tecnologías. Dentro del progreso de estas metodologías, la elaboración de materiales didácticos que favorezcan estos objetivos ha sido y sigue siendo una pieza importante de trabajo entre los docentes universitarios. En esta línea, un grupo de profesores del Departamento de Fisiología de la universidad hemos elaborado una serie de materiales didácticos empleando herramientas visuales y virtuales, para la enseñanza práctica de la asignatura Fisiología Humana. Estos materiales se utilizaron por primera vez durante el curso 2011–12 en la asignatura Fisiología Humana de primer curso del grado de Ciencias de la Actividad Física y del Deporte (CCAFyDE). En este trabajo presentamos los materiales elaborados y la metodología utilizada. También evaluamos el resultado obtenido tras su empleo en cuanto a la satisfacción del alumno y a la contribución que estos materiales y tal metodología han tenido para el aprendizaje de los estudiantes. Para ello se realizó una encuesta entre los alumnos y un análisis comparativo de las calificaciones obtenidas en la parte práctica de la asignatura y de su impacto en el conjunto de la nota total de esta misma, con respecto a las notas obtenidas por los estudiantes del curso anterior, en el que no se utilizaron estos materiales. Los resultados indican una aceptación satisfactoria por parte de los alumnos tanto de los materiales didácticos como de la metodología empleada, así como una mejora significativa en sus calificaciones con respecto al curso anterio

Elaboració i ocupació de materials didàctics per a millorar l'ensenyament pràctic en l'assignatura de Fisiologia Humana en el grau de Ciències de l'Activitat Física i de l'Esport. Avaluació de resultats

The need to advance towards a methodology that strengthens self-directed learning and competency development among students has become a key focus of university teaching in recent years. This focus has given rise to new methodological strategies that have been enhanced by the use of information and communication technologies (ICTs). Within the progress of these methodological strategies, the creation of educational resources that help to meet their objectives has become an important part of the work done by university lecturers. Along those lines, a group of lecturers in the Department of Physiology at the University of Alcalá (UAH), Spain, has created a series of educational resources using visual online tools for practical teaching in the Human Physiology subject. These resources were used for the first time in the 2011/12 academic year in the Human Physiology subject, taught in the first year of the bachelor’s degree course in Physical Activity and Sports Sciences. This article presents the resources created and the methodology used. The results obtained from their use are also assessed; these results refer to the students’ satisfaction with the resources, and the contribution that the resources and the methodology made to the students’ learning. A survey was conducted to ascertain the students’ opinions, and a comparative analysis was performed of the grades obtained by the students in the practical part of the subject and their impact on the overall grade, and those obtained in the previous academic year when the resources had not been used. The results show that the students were satisfied with both the educational resources and the methodology used, and that there was a significant improvement in the grades obtained in comparison to the previous academic year.ResumenLa necesidad de caminar hacia una metodología que potencie el aprendizaje autónomo y el desarrollo de las competencias en el alumno se ha convertido en estos últimos años en un elemento clave en la enseñanza universitaria. Esta constante ha impulsado el auge de nuevas estrategias metodológicas que se han visto dinamizadas por el uso de las nuevas tecnologías. Dentro del progreso de estas metodologías, la elaboración de materiales didácticos que favorezcan estos objetivos ha sido y sigue siendo una pieza importante de trabajo entre los docentes universitarios. En esta línea, un grupo de profesores del Departamento de Fisiología de la universidad hemos elaborado una serie de materiales didácticos empleando herramientas visuales y virtuales, para la enseñanza práctica de la asignatura Fisiología Humana. Estos materiales se utilizaron por primera vez durante el curso 2011–12 en la asignatura Fisiología Humana de primer curso del grado de Ciencias de la Actividad Física y del Deporte (CCAFyDE). En este trabajo presentamos los materiales elaborados y la metodología utilizada. También evaluamos el resultado obtenido tras su empleo en cuanto a la satisfacción del alumno y a la contribución que estos materiales y tal metodología han tenido para el aprendizaje de los estudiantes. Para ello se realizó una encuesta entre los alumnos y un análisis comparativo de las calificaciones obtenidas en la parte práctica de la asignatura y de su impacto en el conjunto de la nota total de esta misma, con respecto a las notas obtenidas por los estudiantes del curso anterior, en el que no se utilizaron estos materiales. Los resultados indican una aceptación satisfactoria por parte de los alumnos tanto de los materiales didácticos como de la metodología empleada, así como una mejora significativa en sus calificaciones con respecto al curso anterio