Increasing cold ischemic time in mouse kidneys primes renal proximal tubular cells to undergo mitochondrial-induced necroptosis following transplantation

We developed a C57Bl/6 mouse kidney model of cold storage in UW solution prior to transplantation to assess cellular injury during cold ischemic time (CIT). Compared to warm storage, cold storage reduced proximal tubule cell (PTC) death assessed by terminal deoxynucleotidase dUTP nick end labeling (TUNEL). Many PTCs showed increases in nuclear phospho-mixed lineage kinase domain-like protein (pMLKL) with concomitant nuclear swelling and increases in phospho-receptor interacting protein kinase (pRIPK)3, Z-DNA, and Z-DNA binding protein (ZBP)1 but not phospho-RIPK1, caspase 3 cleavage or TUNEL staining. Nuclear pMLKL was reduced by small molecule Bax inhibitor, BAI1 suggesting a link between mitochondrial damage and pMLKL activation. Kidneys subjected to 16-24h CIT before syngeneic transplant showed more extensive PTC death, sloughing of remaining PTCs, TUNEL staining, cytosolic pMLKL, kidney injury molecule 1 (KIM1) expression, and infiltration of neutrophils and monocytes at the cortical medullary boundary compared to kidneys transplanted without CIT. Kidneys with prolonged CIT transplanted into nephrectomized syngeneic recipients had elevated BUN and recipient death. Bax inhibition during CIT reduced PTC injury but not immune infiltration, BUN, or improve survival. Overall, these results indicate that CIT causes Z-DNA/ZBP1 activation which primes PTCs through pMLKL for necroptosis following reperfusion of transplanted kidneys.