*Purpose: Renal transplant is an accepted therapy for end-stage renal failure. Chronic transplant dysfunction remains the leading cause of long-term graft loss and is, in part, caused by ischemia-reperfusion injury (IRI). Connexin proteins compromising cell-cell junctions, in particular Connexin43 (Cx43), govern many facets of endothelial cell functionality and play a pivotal role in the pathophysiology of IRI. We have developed and characterized a novel small molecule cell junction stabilizer, aCT1, which we have shown to improve endothelial integrity. Herein we explored the use of aCT1 as a pretreatment therapeutic to protect donor kidneys from the effects of IRI.

*Methods: Kidneys were harvested from 5 Yorkshire/Landrace pigs. Upon removal, kidneys were flushed with either HTK alone (n=6) or HTK supplemented with aCT1 (n=4). Kidneys were then cold stored for 36 hours prior to 6 hours of hypothermic pulsatile perfusion. On completion of perfusion 200 µm kidney sections were taken and organ cultures performed to simulate ex-vivo graft IRI. The effect of aCT1 therapy on machine perfusion dynamics, histopathology, and graft inflammation were analyzed in organ culture tissues.

*Results: aCT1 supplemention of HTK had no adverse effect on perfusion dynamics or kidney injury. Ex-vivo organ cultures demonstrated a significant increase in pro-inflammatory cytokines (IL-6, IL-8), adhesion molecules (P-selectin, ICAM, VCAM), and chemokine genes (MCP-1, IL-1β, TNF-α), key factors associated with IRI, which were all significantly reduced in aCT1 treated kidneys. Further, histological assessment of kidney sections demonstrated a significant reduction in tubular injury and the expression of KIM-1.

*Conclusions: Supplementation of standard of care HTK solution with cell junction modifying small molecule aCT1 significantly reduced ex-vivo kidney IRI and had no deleterious impact on graft performance during hypothermic pulsatile perfusion.

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