Na+/K+-ATPase stabilization by HSP70 in outer stripe of outer medulla (OSOM) during recovery from low protein diet

RUETE MC; CARRIZO L; BOCANEGRA V; MANUCHA W; VALLES P

Rio de Janeiro, Brasil

Congreso; World Congress of Nephrology 2007; 2007

Introduction: Renal ischemia induces loss of tubular cell polarity, which is associated with a time-dependent translocation of Na+K+ATPase from the basolateral into the apical membrane domain of proximal tubular cells. Overexpression of heat shock proteins (HSPs) has been implicated in the restoration of the cytoeskeletal anchorage of Na+K+ATPase. Low protein (LP) diet induces ischemic renal injury involving epithelial cells from outer medulla. In the present study we tested whether HSP70 would stabilize renal Na+K+ATPase attachment to the cytoeskeleton during recovery from LP feeding. Methodology: After weaning, Wistar rats (n=8), were fed for 14 days with a LP diet (8%), then the animals were recovered by means of a normal protein diet (24%,RP), each group had an age-matched control group(24%, NP). Tissue samples from cortex and outer stripe of outer medulla (OSOM) were homogeneized in extraction buffer plus 0.1% Triton X-100. Protein levels of Na+K+ATPase and HSP70 were measured by western blot. In vitro coincubation of soluble non cytoeskeletal and insoluble isolated cytoeskeletal-associated fractions in the presence or absence of anti HSP 70 antibody was performed. Interaction between proteins was determined by coimmunoprecipitation. Results: Increased Na+K+ATPase dissociation was demonstrated in soluble fraction from OSOM as a result of LP diet vs. NP (196.5±1.1 vs. 151±1.3, p< 0.05). Meanwhile, decreased HSP70 levels in the same fraction was shown (LP:179.3±10.5 vs. NP 224.7±1.85, p< 0.05). Translocation of HSP70 to the cytoeskeletal injured fraction associated with stabilization of Na+K+ATPase was shown in OSOM from LP, after in vitro coincubation of the cytoeskeletal fraction of LP and non cytoeskeletal fraction of RP (showing high HSP70 expression). These effects were abolished by the addition of anti-HSP70 antibody. Coimmunoprecipitation showed that the amount of Na+K+ATPase coprecipitating with HSP70 increased in OSOM from LP, the level of Na+K+ATPase that coprecipitated with HSP70 in RP was similar to that seen in control. In renal cortex, absence of significant differences was shown in the Na+K+ATPase and HSP70 expression at in vivo and in vitro experiments among groups. In LP and RP cortex tissues, interaction of both, Na+K+ATPase and HSP70, proteins by coimmunoprecipitation was similar to control. Conclusion: Our results allow us to suggest that HSP70 has a critical protective role in the integrity of the cytoskeletal anchorage of Na+K+ATPase during recovery from ischemic LP injury in outer stripe of the outer medulla.