POTENTIAL OF REPAIRING ISCHEMICALLY DAMAGED KIDNEYS EX VIVO (2004)

O258 Aims: Therapies accelerating recovery from ischemia could impact the number of kidneys procured from non-heartbeating donors. An acellular warm (32°C) perfusion was used to deliver growth factors to kidneys damaged by 2-hours of warm ischemia (WI). Fibroblast growth factors (FGF)-1 and 2 were selected for their receptor specific binding in the kidney, metabolic regulation and mitogenic effect. The impact of ex vivo FGFs treatment on pro- or anti-apoptotic pathways was assessed using a TUNEL assay. Two critical regenerative pathways requiring new synthesis for recovery following WI injury were evaluated. The junctional integrity protein, ZO-1 was used to determine recovery of cytoskeletal integrity. The up-regulation of proliferating cell nuclear antigen (PCNA) was used as a marker for recovery of synthetic functions. The ability to repair the injury occurring from 2-hours of WI was correlated with clinical outcomes Methods: A canine transplant model was used with one WI damaged kidney “repaired” ex vivo and reimplanted and the contralateral kidney similarly damaged used to obtain histochemical evidence of active cellular repair. Kidneys were divided into 4 groups: Group 1 (n=2)2-H WI Group 2 (n=2)2-H WI, 24 hours cold machine perfusion (4°C) and reimplantation Group 3 (n=2) 2-H WI, 24 hours warm perfusion without FGFs (32°C) and reimplantation Group 4 (n=4)2-H WI, 24 hours warm perfusion plus FGFs (32°C) and reimplantation Results: TUNEL assay – Kidneys damaged by 2-hours of WI were negative. WI damage followed by hypothermic perfusion or the absence of FGF(s) resulted in approximately 10% of the renal cells having fragmented DNA and following reimplantation, the number of TUNEL stained cells increased to 50 and 20% respectively. When WI damaged kidneys were warm perfused with FGF(s), the kidneys were TUNEL negative following both 24-hours of perfusion and reperfusion. ZO-1 - Two hours of WI insult resulted in >80% of tubules staining positive for ZO-1. WI damaged kidneys with subsequent cold perfusion at 4°C, demonstrated intense positive staining for ZO-1 accompanied by disintegration. The WI damaged kidneys that were warm perfused with growth factors were found to normalize ZO-1 expression in a time dependent fashion during the 24-hour period of ex vivo warm perfusion. Normalization was impaired in the absence of FGF(s). PCNA- WI damaged kidneys perfused at 4°C resulted in PCNA staining equivalent to 2 hours of WI (2%). In contrast, in kidneys damaged by 2-hours of WI that were warm perfused with growth factors an up-regulation of PCNA expression was observed with > 20% of cells staining positive at 6 hours and increasing to >80% by 24 hours. Absence of FGF(s) impaired PCNA expression. Function – FGFs induced up-regulation of PCNA, accompanied by down-regulation of ZO-1 during ex vivo perfusion was found to correlate with good outcomes. Reimplantation of these “repaired” kidneys resulted in life-sustaining function. Immediate reimplantation following WI, WI with 24 hours of cold perfusion or warm perfusion without FGF(s) resulted in non-viable kidneys. Conclusions: During 24-hours of ex vivo perfusion with FGF(s) metabolism was sufficiently restored to support up-regulation of cellular processes with the potential to modulate both injury and repair proteins in WI damaged kidneys. The results suggest the administration of growth factors FGF-1 and 2 during perfusion triggers pathways for new synthesis leading to cellular recovery rather than activation of the apoptotic pathway