Angiotensina II y Gremlin activan la vía Smad durante la transición epitelio-mesenquinal, un mecanismo báisco de progresión de daño renal

Tesis doctoral inédita realizada en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21 de Junio de 2010

Gremlin activates the smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells

Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-(TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation.These data showthatGremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlininduced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-βThis work was supported by grants from the Instituto de Salud Carlos III (PI11/01854 and REDINREN ISCIIIRETIC RD12/0021/0002 and 0001), Sociedad Española de Nefrología, PCI Iberoamerica (A/9571/07), CYTED IBERERC, FONDECYT Chile 1080083 and 1120480, Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD- 2378), Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín Entralgo/CM) to A.O. Fundación para el fomento en Asturias de la investigaciónn cientíica aplicada y la tecnología (FICYT)

Desarrollo de normas técnicas de seguridad industrial para mantenimiento y seguridad vial S.A.S.

El presente proyecto consiste en el desarrollo de las normas técnicas de seguridad industrial en Mantenimiento y Seguridad Vial S A S, compañía fabricante de pinturas para tráfico, resinas termoplásticas e imprimantes utilizadas en labores de señalización horizontal. Contemplado a tratar los distintos factores que intervienen en la seguridad tanto humana como material, la finalidad del proyecto consiste en mitigar o eliminar comportamientos inadecuados logrando hábitos seguros de trabajo, formando al personal para proteger su propia salud, previniendo accidentes de trabajo, identificando variables que originan dichos riesgos. La investigación de campo se realizó visitando en varias ocasiones obras civiles e instalaciones de la organización donde a través de distintas herramientas y técnicas se logra detectar actos inadecuados y deficiencias que poseen en materia de seguridad industrial es así como se obtienen datos cualitativos y cuantitativos que determinan acciones reguladas dentro del marco legal aplicable. Por medio del establecimiento de medidas preventivas, protocolos, programas y procedimientos donde se contemplan las actividades de prevención y seguimiento se pretende demostrar un mejoramiento continuo del sistema de seguridad y salud ocupacional aportándole a la organización una ventaja competitiva en el mercado de señalización vial en cuanto al cumplimiento de requerimientos solicitados para participar en grandes contrataciones.This project consists of the development of industrial safety standards in Maintenance and Road Safety SAS, manufacturer of traffic paints, thermoplastic resins and primers used in road marking work. Referred to try the different factors involved in the safety of both human and material, the purpose of the project is to mitigate or eliminate inappropriate behavior achieving safe work habits, training staff to protect their own health, preventing accidents, identifying variables that cause such risks. The field research was conducted repeatedly visiting civil works and facilities of the organization where through various tools and techniques are able to detect improper and weaknesses they possess in industrial safety so as obtained qualitative and quantitative data that determine regulated activities within the applicable legal framework. Through the establishment of preventive measures, protocols, programs and procedures which provide for the prevention and monitoring activities intended to demonstrate continual improvement of system safety and occupational health of the organization giving it a competitive advantage in the market as Road Signs compliance with requirements requested to participate in large contracts

Angiotensin II activates the Smad pathway during epithelial mesenchymal transdifferentiation

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor β, TGFβ) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-β mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-β upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-β however, Smad-dependent transcription after 1 day was TGF-β mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-β-mediated processes; however, early changes were independent of endogenous TGF-β. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-β-independent processes, in vivo and in vitro, causing renal fibrosis

CTGF Promotes inflammatory cell infiltration of the renal interstitium by activating

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-κB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-κB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-γ, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-κB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-κB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-κB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-κB pathwayThis work has been supported by grants from Ministerio de Educación y Ciencia (SAF 2005–03378), Sociedad Española de Nefrología, FIS (PI020822 and PI081564), Red tema´ tica de Investigación Renal REDINREN (ISCIII-RETIC RD06/0016) from the Instituto de Salud Carlos III from Ministerio de Sanidad y Consumo, the EU project DIALOK: LSHB-CT-2007–036644, PCI Iberoamerica (A/9571/07), and FONDECYT, Chile (1080083). Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to A.O. We want to thank Ma Mar Gonzalez Garcia-Parreñoo for her technical hel