Mechanisms of nucleophosmin (NPM)-mediated regulated cell death elucidated by Hsp70 during renal ischemia

Nucleophosmin (NPM), a nucleolar-based protein chaperone, promotes Bax-mediated mitochondrial injury and regulates cell death during acute kidney injury. However, the steps that transform NPM from an essential to a toxic protein during stress are unknown. To localize NPM-mediated events causing regulated cell death during ischemia, wild type (WT) and Hsp70 mutant proteins with characterized intracellular trafficking defects that restrict movement to either the nucleolar region (M45) or cytosol (985A) were expressed in primary murine proximal tubule epithelial cells (PTEC) harvested from Hsp70 null mice. After ischemia in vitro, PTEC survival was significantly improved and apoptosis reduced in rank order by selectively overexpressing WT > M45 > 985A Hsp70 proteins. Only Hsp70 with nuclear access (WT and M45) inhibited T95 NPM phosphorylation responsible for NPM translocation and also reduced cytosolic NPM accumulation. In contrast, WT or 985A > M45 significantly improved survival in Hsp70 null PTEC that expressed a cytosol-restricted NPM mutant, more effectively bound NPM, and also reduced NPM-Bax complex formation required for mitochondrial injury and cell death. Hsp70 knockout prevented the cytoprotective effect of suppressing NPM in ischemic PTEC and also increased cytosolic NPM accumulation after acute renal ischemia in vivo, emphasizing the inhibitory effect of Hsp70 on NPM-mediated toxicity. Distinct cytoprotective mechanisms by wild type and mutant Hsp70 proteins identify dual nuclear and cytosolic events that mediate NPM toxicity during stress-induced apoptosis and are rational targets for therapeutic AKI interventions. Antagonizing these early events in regulated cell death promotes renal cell survival during experimental AKI.