La interación de HSP70/CHIP promueve la ubiquitinación y degradación de NOX4 como parte del efecto antioxidante del Losartan en células proximales tubulares

GIL LORENZO ANDREA; COSTANTINO VALERIA; LOPEZ APPIOLAZZA MARTÍN; BOCANEGRA VICTORIA; BENARDON MARÍA EUGENIA; CACCIAMANI VALERIA; GARRAMUÑO VALLÉS, PATRICIA

Tucumán

Congreso; XXII Congreso Argentino de Hipertensión Arterial. Sociedad Argentina de Hipertensión Arterial; 2015

We tested whether Heat shock protein 70 (Hsp70) and C-terminus of Hsp70-Interacting Protein (CHIP) interaction contributes to the negative regulation of Nox4 through its ubiquitinizaton and proteasomal degradation after AT1R receptor blockage in primary culture of proximal tubule epithelial cells (PTCs). SHR and WKY PTCs were stimulated with Angiotensin II (100 nM) (AII), treated with Losartan (10 µM) (L) or with Losartan plus Angiotensin II (L+AII). Whereas SHR PTCs stimulated with Angiotensin II upregulated AT1R, Nox4 NADPH oxidase and slightly decreased the caveolin-1 protein levels, Losartan decreased AT1R and Nox4 protein levels while increasing caveolin-1 and Hsp70 expression in SHR PTC membranes. Nox4 gene expression showed no differences among groups. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP that contrasts with decreased Nox4 in SHR PTC membranes (L) vs PTCs (AII). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate Angiotensin II effects on Nox4 expression and oxidative activity. Moreover, PTCs (L) exposed to the proteasome inhibitor MG132 blocked the degradation of ubiquitinated Nox4. Immunofluorescence analysis demonstrated Nox4 ubiquitination by increased colocalization of Nox4/Ubiquitin in PTCs (L) exposed to MG132. Conversely, Hsp72 knockdown PTCs (L) reduced Nox4/Ubiquitin colocalization, resulting in Nox4 upregulation due to the proteasomal degradation inhibition, even Losartan treatment. Our data suggest that Hsp70 and CHIP interaction mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR PTCs