HEAT SHOCK PROTEIN 70 / CHIP / NOX4 NAD(P)H OXIDASE INTERACTION IN THE ANTIOXIDATIVE EFFECT OF LOSARTAN ON PROXIMAL TUBULE CELLS FROM SPONTANEOUSLY HYPERTENSIVE RATS (SHR)

VALLES PATRICIA; GIL LORENZO ANDREA; . BOCANEGRA VICTORIA

San Diego

Congreso; ASN Kidney Week 2012; 2012

Full expression of Angiotensin II signaling is dependent on the reactive oxygen species derived from NAD(P)H oxidase and the dynamic association of the Angiotensin II Type I receptor (AT1R) with caveolae/lipid rafts.The chaperone Hsp70 regulates a diverse set of signaling pathways.CHIP (Carboxyl terminus of the Hsc70-Interacting Protein) is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation.We investigated Hsp70/CHIP contribution to the regulation of Nox4 after AT1R receptor blockade,in primary culture of proximal tubule epithelial cells (PTCs) from SHR. PTCs from 8 week SHR and WKY rats were stimulated with Angiotensin II (100 nmol/L) for15 min (AII), pretreated with Losartan (100 μmol/L) 90 min (L) and with Losartan75 min plus Angiotensin15 min(L+AII).Whereas SHR PTCs exposure to Angiotensin II downregulated membrane Caveolin-1 expression and overexpressed Nox4 NAD(P)H-oxidase,Losartan increased Caveolin-1expression, increased Hsp70 and decreased Nox4 protein levels in SHR (L)PTC membranes.Decreased Hsp70 in SHR(L) vs SHR(AII) in cytosolic fraction confirm Hsp70 translocation to PTC membranes.No differences were shown in Nox4 gene expression among groups.Interaction and localization of Nox4, Hsp70 and CHIP were determined by immunoprecipitation and immunofluorescence confocal microscopy. Increased levels of Hsp7/CHIP contrasts with the decreased immunoprecipitation of Nox4 in membrane from PTCs SHR (L) vs SHR(AII).To validate these results, we transfected PTCs with p-SIREN RetroQ shHsp72 plasmid vector to knockdown Hsp72 or control empty vector.Hsp72 depletion was associated with higher Nox4 expression and increased NAD(P)H oxidase activity in SHR (L+AII) related to SHR (L+AII) without transfection.After Hsp72 silencing of PTCs from SHR (AII),Losartan could not prevent Angiotensin II-enhanced Nox 4 expression and NAD(P)H oxidase activity. Membrane interaction of Hsp70/CHIP may induce Nox4 protein degradation, which could be involved in the cytoprotective effect of Losartan in PCTs from SHR.