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Abstract: PO0338

Mechanisms of Nucleophosmin-Mediated Regulated Cell Death During Renal Ischemia

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Wang, Zhiyong, Boston Medical Center, Boston, Massachusetts, United States
  • Havasi, Andrea, Boston Medical Center, Boston, Massachusetts, United States
  • Borkan, Steven C., Boston Medical Center, Boston, Massachusetts, United States
Background

Nucleophosmin (NPM) is a protein chaperone that potentiates Bax-mediated cell death during ischemic AKI by an unknown mechanism. In contrast, heat shock protein 70 (Hsp70) is a potent anti-apoptotic agent that promotes renal cell survival and preserves organ function. In this study, we characterize for the first time the intracellular events in which NPM and Hsp70 compete to regulate cell survival during ischemic stress.

Methods

Hsp70 wild type (WT) or Hsp70 mutants either restricted to the cytosol (Hsp70 985A) or unable to enter the nucleolar region (Hsp70 M45) were selectively over-expressed in primary murine proximal tubule epithelial cells (PTEC) harvested from Hsp70 null mice. Hsp70 expression, NPM nuclear translocation, NPM de-oligomerization, NPM-Bax complex formation, T95 phosphorylation responsible for NPM translocation, and cell survival were measured.

Results

Equivalent, selective over-expression of the hsp70 proteins significantly improved cell survival after ischemic stress in the following rank order: WT > M45 > 985A (each P < 0.05 vs. control). Only Hsp70 members with nuclear access (WT and M45) inhibited T95 NPM phosphorylation that mediates NPM translocation and reduced cytosolic NPM accumulation. Neither WT nor the Hsp70 mutants inhibited stress-induced NPM de-oligomerization. In contrast, WT > 985A > M45 Hsp70 significantly improved survival in Hsp70 null PTEC that expressed a cytosol-restricted NPM mutant, interacted with cytosolic NPM, and 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 protective effects of Hsp70 on NPM-mediated renal cell toxicity.

Conclusion

These observations identify key steps that mediate NPM toxicity during ischemia-induced cell death: (1) nuclear NPM de-oligomerization, (2) NPM translocation into the cytosol and (3) cytosolic NPM-Bax complex formation. Hsp70 promotes renal cell survival during ischemic acute kidney injury partly by inhibiting two of these toxic events in distinct cell compartments: nuclear NPM translocation and NPM-Bax interaction in the cytosol. Renal cell survival during ischemic AKI is substantially improved by interfering with events that render NPM toxic.

Funding

  • NIDDK Support