Caveolin-1 and Hsp70 interaction in microdissected proximal tubules from spontaneously hypertensive rats as an effect of Losartan

Abstract

Caveolin is required to traffic the AT1 receptor through the exocytic pathway. The chaperone Hsp70 regulates a diverse set of signaling pathways via their interactions with proteins. Here we examined the AT1 receptor antagonist Losartan effect on Caveolin-1 and Hsp70 protein association in SHR proximal tubules (PT). Hsp70 involvement on Losartan oxidative stress regulation was also studied. Four-week-old SHR were randomized for receiving Losartan (40 mg.kg-1day) (SHRLos) or no treatment (SHRH2O) during 8 weeks. Wistar-Kyoto rats (WKY) were normotensive controls. By western blotting, the relative abundance of Caveolin-1 was 2-fold higher in microdissected PT membrane fractions from treated SHR vs WKYH2O. Hsp70 membrane translocation was demonstrated in SHRLos through out the upregulation of Hsp70 expression in microdissected PT membrane fractions when compared with WKYH2O, p<0.001. Conversely, decreased Hsp70 protein levels were shown in microdissected PT cytosol fraction from SHRLos, p<0.01. Interaction between Cav-1 and Hsp70 was further determined by coimmunoprecipitation and by immunofluorescence colocalization in SHRLos PT membranes. After membrane translocation of Hsp70, the decreased NADPH oxidase activity (RFU/ìprot/min incubation) near controls demonstrated on microdissected PT membranes from SHRLos vs. SHRH2O, p<0.01, was reversed by the pre-incubation with anti-Hsp70 antibody. In addition, interaction between Hsp70 and Nox4 was determined by the coimmunoprecipitation strategy showing that membrane overexpression of Hsp70 was associated with decreased Nox4 after Losartan treatment in SHR. Conclusion: After Losartan administration interaction of Caveolin-1 and Hsp70 was shown in SHR proximal tubules. Translocation of Hsp70 to PT membranes in SHRLos might exert a cytoprotective effect by downregulation of NADPH isoform Nox4.