Abstract: TH-PO235

Nucleophosmin (NPM) Phosphorylation Mediates Renal Cell Death: A New Therapeutic Target for Ischemic AKI

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Wang, Zhiyong, BOSTON MEDICAL CENTER, BOSTON, Massachusetts, United States
  • Igwebuike, Chinaemere, Boston Univerisity School of Medicine , Boston, Massachusetts, United States
  • Salih, Erdjan, Boston University School of Medicine, Boston, Massachusetts, United States
  • Havasi, Andrea, Boston University Medical Center, Boston, Massachusetts, United States
  • Bonegio, Ramon G., Boston University School of Medicine, Boston, Massachusetts, United States
  • Schwartz, John H., Boston Medical Center-Evans Biomedical Center, Boston, Massachusetts, United States
  • Borkan, Steven C., BOSTON MEDICAL CENTER, BOSTON, Massachusetts, United States
Background

We hypothesize that renal ischemia alters site-specific NPM phosphorylation, converting NPM from an essential protein synthesis promoter to a killer Bax chaperone that causes renal cell death and AKI.

Methods

To detect site-specific phosphorylation and de-phosphorylation events, NPM was subjected to mass spectrometry before and after ischemic stress in both cell and cortical lysates harvested from: (1) normal vs. ATP deplete primary mouse and primary human proximal tubule epithelial cells (PTEC), (2) sham vs. ischemic mouse kidneys and (3) two pairs of human donor kidneys rejected for transplantation. One of the paired human kidneys was normally perfused, whereas the other was grossly ischemic due to perfusion pump malfunction. Site-specific NPM phosphorylation events were correlated with NPM alterations that mediate its cell death including: nuclear translocation, de-oligomerization, NPM-Bax complex formation, and mitochondrial complex accumulation. To confirm their biologic significance, NPM mutants with site-specific phospho-changes were generated in lentivirus and introduced into PTEC. Peptides that interfere with NPM phosphorylation were tested for their ability to prevent ischemic PTEC death.

Results

Mass spectrometry identified 92% of NPM residues that included all known serine, tyrosine and threonine residues capable of undergoing phosphorylation or de-phosphorylation. Five serine/threonine phosphorylation and de-phosphorylation events differed between NPM harvested from normal vs. ischemic conditions. NPM phosphorylation events were identical in both murine and human NPM harvested from either primary PTEC or intact renal cortex. Only the NPM phospho-mutant that mimicked phosphorylation events detected during renal ischemia caused cytosolic NPM translocation and de-oligomerization, NPM interaction with conformationally active Bax, mitochondrial complex accumulation, and increased PTEC death. In contrast, 2 distinct NPM peptides designed to interfere with NPM phosphorylation protected PTEC against ATP depletion-induced death.

Conclusion

Ischemia-induced NPM phosphorylation regulates Bax-induced cell death and contributes to human AKI. This study identifies a new, modifiable cell death pathway and reveals novel therapeutic approach for preventing and treating ischemic AKI.

Funding

  • NIDDK Support