Background: 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.
Method: Here we examined the AT1 receptor antagonist Losartan effect on caveolin-1 and Hsp70 protein association in spontaneously hypertensive rat (SHR) proximal tubules. Hsp70 involvement in Losartan oxidative stress regulation was also studied. Five-week-old SHRs were randomized for receiving Losartan (40 mg/kg per day) (SHRLos) or no treatment (SHRH2O) during 6 weeks. Wistar–Kyoto rats (WKY) were normotensive controls.
Results: By western blotting, the relative abundance of caveolin-1 was two-fold higher in microdissected proximal tubule membrane fractions from treated SHRs vs. WKYH2O. Hsp70 membrane translocation was demonstrated in SHRLos through out the up-regulation of Hsp70 expression in microdissected proximal tubule membrane fractions when compared with WKYH2O (P < 0.001). Conversely, decreased Hsp70 protein levels were shown in microdissected proximal tubule cytosol fraction from SHRLos (P < 0.01). Interaction between caveolin-1 and Hsp70 was further determined by coimmunoprecipitation and by immunofluorescence co-localization in SHRLos proximal tubule membranes. After membrane translocation of Hsp70, the decreased NADPH oxidase activity (RFU/μprot per min incubation) near controls demonstrated on microdissected proximal tubule membranes from SHRLos vs. SHRH2O (P < 0.01) was reversed by the preincubation 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 SHRs.
Conclusion: After Losartan administration interaction of caveolin-1 and Hsp70 was shown in SHR proximal tubules. Translocation of Hsp70 to proximal tubule membranes in SHRLos might exert a cytoprotective effect by down-regulation of NADPH subunits Nox4.