Abstract: TH-PO346

Highly Specific RIPK1 Inhibition Significantly Improves Renal Ischemia Reperfusion Injury in Mice

Session Information

Category: Acute Kidney Injury

  • 002 AKI: Repair and Regeneration

Authors

  • Gallagher, Kevin Michael, University of Edinburgh, Edinburgh, United Kingdom
  • Harrison, Ewen M, University of Edinburgh, Edinburgh, United Kingdom
  • Hughes, Jeremy, The Queens Medical Research Institute, Edinburgh, United Kingdom
  • Ross, James A, University of Edinburgh, Edinburgh, United Kingdom
  • Marson, Lorna, Queen's Medical Research Institute, Edinburgh, United Kingdom
  • Beh, Sheryl, University of Edinburgh, Edinburgh, United Kingdom
  • Beal, Allison M, GlaxoSmithKline, Collegeville, Pennsylvania, United States
  • Bertin, John, GlaxoSmithKline, Collegeville, Pennsylvania, United States
  • Wigmore, Stephen J, University of Edinburgh, Edinburgh, United Kingdom
Background

Non-specific RIPK1 inhibition with Nec-1, is beneficial in murine ischemia reperfusion injury (IRI). It is not known if tubular epithelial cells (TECs) undergo necroptosis nor how RIPK1 inhibition is beneficial. We aimed to determine if a novel, highly specific RIPK1 inhibitor (GSK963a) is beneficial in murine IRI and determine if TECs undergo necroptosis during IRI.

Methods

Mice were subjected to 23m (severe injury) bilateral renal vascular clamp then 24h reperfusion. Outputs included: Serum urea and creatinine; acute tubular necrosis(ATN) scoring and immunofluorescent(IF) staining of phosphorylated mixed lineage kinase like domain protein (pMLKL) (end effector of necroptosis) using MLKL phospho-S345 specific antibody. Dephosphorylation controls confirmed phospho-specific staining. The effect of GSK963a on TEC (HK2 cell line) viability, cytotoxicity and mitochondrial health (mito-tracker red) was also assessed in in-vitro models of TEC ischemic injury.

Results

GSK963a significantly reduced creatinine (131 umol/L (95%CI 134-175) vs 174 umol/L (154-194) p=0.01 N=7) and ATN score (Mean 3.29/4 (95% CI 2.83-3.74) vs 1.75/4 (1.16-2.34)p=0.008) compared to vehicle. Results with Nec-1s were similar. pMLKL was detected extensively with IF in injured moderately injured tubules (15m ischemia) but not glomeruli. With severe IRI (23 minutes), pMLKL was also detected within glomeruli. In-vitro, GSK963a significantly decreased TEC death in an ATP depletion and glucose deprivation model (12h injury % cell death (CellToxGreen) was: Vehicle: 67.2% (95%CI 62.8-71.5) vs GSK963a: 40.4% (34.3-46.5) N=6 p=<0.001). GSK963a also reduced mitochondrial perinuclear condensation and loss of membrane potential after 1.5 hours of physical hypoxia and glucose deprivation in HK2 cells (N=3).

Conclusion

Highly specific RIPK1 inhibition significantly improves biochemical and histological injury in severe murine IRI. We provide preliminary evidence that renal tubular cells undergo necroptosis in IRI.

Funding

  • Commercial Support