Effect of angiotensin II on the cellular mechanisms involved in the podocytes injury

Abstract

It has been observed that high plasma, or kidney tissue concentrations of angiotensin II (Ang II) leads to changes in renal hemodynamics, severe glomerular injury, increased synthesis of glomerular extracellular matrix components, oxidative stress and apoptosis in glomerular cells, including podocyte and mesangial cells, among other things. The increase in reactive oxygen species (ROS) induces increased caspase 3 activity, a protein that mediates apoptosis. During apoptosis the activation of the isoform 1 of the Na+/H+ (NHE1) is important, since this protein is associated with cellular events such as proliferation control, migration, apoptosis resistance, and cell phenotype maintenance. In several cellular models Ang II regulates the activity of NHE1 through activation of signaling proteins such as ezrin and mitogen-activated protein kinase (p38MAP kinase). Podocytes has its own Renin-Angiotensin System (RAS), and expresses the AT1 and AT2 receptors for Ang II and NHE1. In addition, NHE1 makes interactions with podocyte integrins, that are present in the basement membrane of the foot processe to ensure stability for this cells in the glomerular basement membrane. Our hypothesis is that Ang II increases reactive oxygen species, and this increase can activate apoptosis in these cells. In this condition the NHE1, which is also activated by Ang II via p38 MAPK, may contribute to cellular resistance to apoptotic stimuli. Thus, the main goal of this study is to investigate in podocytes the role of Ang II through AT1 receptors in the induction of oxidative stress, and its relation with apoptosis, protein synthesis, factors that contribute to the adhesion of foot processes in the glomerular basement membrane and intracellular events linked to the NHE1 activity. Methods: It will be used mice podocytes in culture and the experimental groups are: control, treated with Ang II (10-12, 10-9 and 10-8 M) and/or losartan (10-5 M 1 receptor antagonist). Tests will be performed to evaluate apoptosis and superoxide anion production; polymerase chain reaction in real time (qPCR) to evaluate the expression of mRNA for the genes ²1 integrin, nephrin, podocina, laminin 521, tumor necrosis factor alpha (TNF-±), interleukin 6 and GAPDH (endogenous gene); evaluation of intracellular pH by fluorescence method (to investigate the activity of NHE1) and protein expression will be assessed by Western blot. Challenge: With this study we hope to contribute to the clarification of the intracellular signaling pathways that involves the participation of podocytes in glomerular injury induced by Ang II in diseases such as hypertension. (AU)