*Purpose: Mouse kidney transplantation has been widely used to study the immune response to allogeneic grafts. This response includes a circulating systemic compartment and a resident non circulating one. A distinction between these compartments remains an important caveat to the interpretation of the resident or local immune response’s importance and function. This study aimed at developing a mouse kidney allograft re-transplantation model as a tool to investigate the local immunity in the grafts.

*Methods: In primary kidney transplantation, the donor aorta (AO) was anastomosed to the recipient AO using “interrupted 6-stitch end-to-side” method. The donor renal vein was anastomosed to the recipient inferior vena cava (IVC) using end-to-side continuous suturing method. The donor ureter was implanted into the recipient bladder and secured to the posterior wall of the recipient bladder. In the re-transplantation, the graft renal artery with a segment of the primary donor mouse’s AO and a segment of the primary recipient mouse’s AO was end-to-side anastomosed to the secondary recipient mouse’s AO. The graft renal vein with a segment of the primary recipient’s IVC was end-to-side anastomosed to the IVC of the secondary recipient mouse. A total of 3-4 well-proportioned stitches are applied to fix the ureter graft onto the posterior wall of the secondary recipient mouse’s bladder.

*Results: In syngeneic re-transplantation, the graft tissue architectures were normal and well maintained comparable with the primarily transplanted syngeneic graft. In allogenic re-transplantation, we firstly confirmed resident memory T cells formed and functioned in the kidney allografts by using this model. Our detailed, stepwise protocol can be stably replicated for both the primary and secondary, donor and recipient operations. The techniques in this protocol can be efficiently completed by an individual proficient in mouse kidney transplantation surgical procedures.

*Conclusions: We successfully developed a mouse kidney re-transplantation model, which had not been previously described and can be harnessed in studying local immunity in the grafts.

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