Caveolin-1 and Hsp70 expression in spontaneously hypertensive rats (SHR) proximal tubule cells. Effect of Angiotensin receptor type 1 inhibition

MANUCHA WALTER; BOCANEGRA VICTORIA; RODRIGUEZ PEÑA MARCELO; RUETE MARIA CELESTE; CARRIZO LILIANA; VALLES PATRICIA

Congreso; 14th Congress of the International Pediatric Nephrology Association.; 2007

Caveolin proteins functions as to recruits proteins and lipids to caveolae for participation in intracellular trafficking of cellular components and operation in dignal transduction.  A direct interaction with caveolin is required to traffic the AT1-R trough the exocityc pathway. The chaperone Hsp70 participates in the translocation and assembly of proteins. In this study, we examined the effect of the Angiotensin II receptor type I inhibition on Cav-1 and Hsp70 protein association in SHR proximal tubules (PT). Hsp70 involvement on the cytoprotective effect of Lsartan was also studied. Methods: Four week old SHR were randomized for receiving the Angiotensin II type I antagonist Losartan (40 mg/kg/day) (SHRLos) or no treatment (SHRH2O) during 8 weeks. Wistar-Kyoto (WKY) were controls. Western blot analysis of Caveolin 1 (Cav-1)and Hsp70 was performed in membrane fractions from microdissected PT and from cortex tissue. Interaction between both proteins were determined by coimmunoprecipitation and by immunocytochemical colocalization (confocal microscopy). For apoptosis study, TUNEL, Caspase 3 activity and RNA expression of Bax/bcl2 was determined. NADPH oxidase was evaluated by luminal enhanced chemiluminiscence. The results were assessed by ons-way ANOVA and Bonferroni post test. Results: Losartan reduced systolic blood pressure in SHRLos vs SHRH2O: 135 ± 6 vs 170 ±4 mmHg, p<o.01. Blood pressure in SHRLos was similar tTo controls; WKY Los and WKYH2O. In SHRLos increased Cav-1 expression was demonstrated in PT membrane fractions related to WKYH2O: 1.5±0.02, p<0.05, n=4 and in cortex membrane fractions, 1.3 ±0.01, p<0.05, n=6, and 1.40±0.001, p<0.05, n=6 compared to SHRH2O and WKYH2O respectively. Membrane translocation of Hsp70 was demonstrated only in SRLos treatment trough out the Hsp70 expression in membrane fractions from PT and cortex. In addition, interaction between Cav-1 and Hsp70 in cortex membrane by coimmuoprecipitation and by immunofluorescence colocalization in PT cell membranes were shown only in SHRLos. The decreades NADPH oxidase activity (RFU/ug prot/min. incubation) near controls demonstrated in PT from SHRLos vs SHRH2O 41±6.1 vs 142±5.7, p<0.01, was reversed by the preincubation with anti-Hsp70 antibody. No differences in number of PT cells, caspase 3 activity and in proapoptotic rate Bax/bcl2 in cortex membrane fractions were demonstrated among groups. Conclussion: due to the interaction between Cav-1 and Hsp70 after AT1 Angiotensin II receptor inhibition, a role of both proteins in AT1 regulation could be suggested in SHR proximal tubules. Translocation of Hsp70 to proximal tubule membranes in SHRLos might exert a cytoprotective effect by decreasing oxidative stress.