Tesis doctoral de Milica Božic Stanojevic
Abstract epithelial-mesenchymal transition (emt) is a widely recognized molecular event of vital significance in the progression of renal tubulointerstitial fibrosis (tif). Emt is characterized by the downregulation of e-cadherin, de novo expression of alpha-sma and vimentin, actin cytoskeleton reorganization and increased cell migration. A number of factors have been stated as potential inducers of tubular emt in diverse experimental models. Nevertheless, growing evidence establishes a crucial role for transforming growth factor-beta 1 (tgf-beta 1) signaling in mediating renal fibrosis. Tgf-beta 1 plays an important role in altering the phenotype of renal epithelial cells and is capable of initiating and completing the whole process of emt. n-methyl-d-aspartate receptor (nmdar) is an ionotropic glutamate receptor that acts as a calcium channel and its presence in proximal tubular epithelium is important in the maintenance of normal renal function. An important feature of nmdar is its high permeability to calcium, placing this receptor in a unique position to control numerous calcium-dependent processes. Taking into consideration knowledge of multiple characteristics of nmdar in a variety of tissues, including renal itself, we sought to examine the role of this receptor in the maintenance of the proximal tubular epithelial phenotype and its role in tubular emt in vitro. Furthermore, we examined the role of channel activation in tif induced by unilateral ureteral obstruction (uuo) in mice. to assess the function of the nmdar in hk-2 cells in basal conditions, we designed nr1 shrna vector for lentiviral infection and the expression of the nmdar1 subunit was disrupted by short hairpin rna. Knockdown of an essential nr1 subunit of the nmdar induced remarkable changes in epithelial phenotype of hk-2 cells, evident as a decrease of e-cadherin and an increase of alpha-sma, alongside with the changes in cell morphology. having established that the basal nmdar activation had a role in preserving the epithelial phenotype of hk-2 cells, we assessed if the activation of the channel could be a possible strategy in attenuating the phenotypic changes induced by tgf-beta 1. In vitro, hk-2 exposed to tgf-beta 1 demonstrated downregulation of e-cadherin and membrane-associated beta-catenin, f-actin reorganization, de novo expression of mesenchymal markers such as alpha-sma and vimentin, upregulation of snail1 and elevated cell migration. Co-treatment with nmda attenuated all described signs of emt induced by tgf-beta 1. Furthermore, tgf-beta 1 increased cell velocity on collagen and fibronectin matrices, which was inhibited by co-treatment with nmda. once determined that the activation of nmdar managed to attenuate emt induced by tgf-beta 1 in hk-2 cells, we investigated the signaling events that had led to nmdar-induced blockade of tgf-beta 1-initiated emt. There is a growing body of evidence linking the activation of extracellular signal-regulated kinase erk and serine/threonine kinase akt pathways to the induction of tgf-beta 1-mediated emt. Indeed, in our experimental set up, treatment of hk-2 cells with tgf-beta 1 resulted in a rapid increase in the phosphorylation of erk1/2 and akt within 30 min after stimulation, which was prevented by co-treatment with nmda in the case of both investigated kinases. In addition, nmdar activation remarkably blocked tgf-beta 1-mediated activation of ras pointing to the inhibition of the ras-mek pathway as the mechanism by which nmda antagonizes tgf-beta 1-induced emt. in vivo, administration of nmda significantly inhibited expression of alpha-sma in the obstructed mouse kidneys at 5 and 15 days after uuo. Collagen i expression was significantly diminished in obstructed kidneys of nmda-treated mice at day 15 after uuo. Furthermore, administration of nmda blunted the downregulation of e-cadherin and an increase of fsp1 induced by uuo. results obtained point to a paramount role of nmdar in the preservation of normal epithelial phenotype of proximal tubular cells and in the modulation of important steps of tubular emt.
Datos académicos de la tesis doctoral «Glutamatergic signaling in proximal tubular cells maintains the epithelial phenotype and decreases epithelial-mesenchymal transition«
- Título de la tesis: Glutamatergic signaling in proximal tubular cells maintains the epithelial phenotype and decreases epithelial-mesenchymal transition
- Autor: Milica Božic Stanojevic
- Universidad: Lleida
- Fecha de lectura de la tesis: 20/07/2011
Dirección y tribunal
- Director de la tesis
- Elvira Fernández Giráldez
- Tribunal
- Presidente del tribunal: José Miguel Lopez novoa
- anna Messeguer navarro (vocal)
- carles Cantí nicolás (vocal)
- Alberto Ortiz arduan (vocal)