Ischemia reperfusion injury (IRI) is associated with poor transplant outcome. IRI is often a sum of cold ischemic graft storage (CIS) plus unavoidable warm ischemia (WI) during surgery. Using a mouse model of life-supporting renal allograft transplantation, we previously reported that prolonged CIS leads to enhanced generation of class II donor specific alloantibodies (DSA) and the development of late transplant glomerular injury. The goal of the current study was to dissect the effect of prolonged WI on the induction of pathogenic anti-donor immune responses.

BALB/c renal allografts were stored on ice for 30 min prior to transplantation into bilaterally nephrectomized C57BL/6 (B6) recipients. The microvascular clamps were removed from recipient infrarenal aorta and IVC either immediately (minWI group) or after 30 min after completing anastomoses (30minWI). Except for additional WI time, both groups received identical pre- and post-operative care. Anti-donor immune responses were evaluated on d. 7 posttransplant. The frequencies of donor-reactive IFNγ producing spleen T cells were determined by ELISPOT assay, and the serum levels of class I and class II reactive DSA and autoantibodies against previously identified antigen DNA Topoisomerase I (TI) by ELISA.  Urine NGAL and KIM-1 and serum BUN levels were measured on d. 2 and 7 posttransplant, respectively.

At d. 2 posttransplant, recipients of 30minWI allografts had elevated urine levels of acute kidney injury markers NGAL and KIM-1 compared to minWI group. Prolonged WI led to significantly increased activation of donor-reactive T cells  (2,130 vs 934 IFNγ producing T cells per million splenocytes in minWI group). In contrast, the serum levels of class I, class II, and anti-TI antibodies were similar in both groups. Recipients of 30minWI grafts had inferior transplanted kidney function on d. 7 posttransplant as evidenced by serum BUN. Consistently with enhanced posttransplant injury and anti-donor immune responses, 8/14 30minWI recipients rejected kidney allografts by d. 25 and 6/14 survived for > 60 days. The majority of minWI grafts were spontaneously accepted for > 60 days. 

Taken together, our results show that in contrast to prolonged CIS, extended WI time exacerbates anti-donor cellular immune responses but has no significant influence on DSA or autoantibody development. These findings may guide the management of transplant recipients with marginal donor grafts or surgery complications