We previously reported that radicicol (Hsp90 inhibitor) induced a reduction in the renal blood flow and glomerular filtration rate, in part due to a reduction in urinary NO2/NO3 excretion, suggesting that Hsp90 regulates renal vascular tone in physiological conditions. However, there is a lack of information concerning Hsp90α or Hsp90β role on eNOS activity and their association with acute kidney injury (AKI) characterized by an inadequate NO production. This study evaluated the effects of Hsp90α or Hsp90β intra-renal transfection under ischemia/reperfusion (IR) injury.Uninephrectomized (Nx) rats were intra-renally transfected through injections with Hsp90α or Hsp90β cloned into pcDNA3.1(+) or empty vector (EV) at 48 h before inducing IR, as indicated in the following groups: (i) Nx+sham, (ii) Nx+IR, (iii) Nx+IR+EV, (iv) Nx+IR+Hsp90α and (v) Nx+IR+Hsp90β. After 24 h, physiological, histopathological, biochemical and molecular studies were performed.IR-induced renal dysfunction, structural injury, tubular proliferation, the elevation of urinary Hsp72 and the reduction of urinary NO2/NO3 excretion. These alterations were associated with reduced eNOS-Hsp90 coupling and changes in the eNOS phosphorylation state mediated through a reduction in PKCα and increased Rho kinase expression. In contrast, intra-renal transfection of Hsp90α or Hsp90β prevented IR injury that was associated with the restoration of eNOS-Hsp90 coupling, eNOS activating phosphorylation and PKCα and Rho kinase levels.Here we showed that eNOS-Hsp90 uncoupling plays a critical role in promoting NO reduction during IR. This effect was effectively reversed through Hsp90α or Hsp90β intra-renal transfection, suggesting their implication in regulating NO/eNOS pathway and the renal vascular tone.