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Abstract: TH-OR45

Genome-Wide CRISPR Screen Reveals That Elamipretide (SS-31) Mediated PLSCR3 Activation Mitigates Mitochondrial Dysfunction During AKI

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Silvaroli, Josie A., The Ohio State University, Columbus, Ohio, United States
  • Bisunke, Bijay, The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
  • Kim, Ji Young, The Ohio State University, Columbus, Ohio, United States
  • Pabla, Navjot Singh P., The Ohio State University, Columbus, Ohio, United States
  • Bajwa, Amandeep, The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
Background

Mitochondrial dysfunction is a hallmark of several human disorders, including acute kidney injury (AKI). SS-31 a cell-permeable mitochondrial-targeted peptide restores healthy mitochondrial function and is currently undergoing clinical trials. SS-31 interacts with cardiolipin in the inner mitochondrial membrane; however, the pharmacological basis of its protective effects remains obscure. Importantly, the role of phospholipid scramblase 3 (PLSCR3), a mitochondrial cardiolipin binding protein, in SS-31 and renal biology is unknown.

Methods

We performed a genome-wide CRISPR screen in a nephrotoxic model of tubular epithelial cell-death with SS-31. The primary and secondary screens in BUMPT and HK-2 cells showed that PLSCR3 is essential for the protective effects of SS-31. Seahorse based analysis of mitochondrial function was also carried out in WT and PLSCR3 KO HK-2 cells. Conditional KO mice were generated by crossing PLSCR3-floxed with GGT1-Cre mice (Fig. 1). Next in vivo KO of PLSCR3 through hydrodynamic intravenous RNAi injections was done. The severity of renal injury (IRI and cisplatin) was monitored in control and KO littermates or in mice injected with control and PLSCR3 siRNA through measurement of BUN, serum creatinine, histological analysis, and biomarker analysis.

Results

In cell culture models of cisplatin nephrotoxicity, cell survival and mitochondrial protection provided by SS-31 is abrogated by PLSCR3 knockdown or knockout. In vivo, PLSCR3 gene ablation or knockdown in RTECs suppresses the protective effects of SS-31 in cisplatin and ischemia-reperfusion associated models of AKI (Fig.1). Using liposome-based assays, we also found that SS-31 activates PLSCR3 scramblase activity.

Conclusion

Our studies have discovered phospholipid scramblase 3 as the crucial mediator of the cell protective effects of SS-31. We propose that SS-31 activates PLSCR3 phospholipid scramblase activity resulting in mitochondrial protection under stress conditions associated with AKI.

Funding

  • NIDDK Support