Cold Storage Exacerbates Complement Pathway Activation in Kidneys following Transplantation

Background: Cold storage (CS) of kidneys is a vital process that enables the transport of kidneys sourced from deceased organ donors to the desired recipient. Our lab previously reported that the complement system (C3 and C5b-9) is activated in kidneys after CS combined with kidney transplantation (CS+Tx). Here, we have chosen to focus on the C5 component upstream of C5b-9, as it is a clinically relevant therapeutic target in organ transplants. It is published that reactive oxygen species (ROS) increases localization of C5aR1, a receptor for the cleaved C5 protein C5a, to mitochondrial membranes contributing to the dysfunction of oxidative phosphorylation. Interrogating oxidative stress caused by Tx and the involvement of the C5a/C5aR1 axis is vital. Methods: Lewis rat kidneys were removed from the donor rat, flushed, and placed in University of Wisconsin (UW) or CS solution at 4°C. After 18 hours of CS exposure, the left kidney was transplanted into a recipient rat for 1 post-operative day of reperfusion. Autotransplant (transplant with no CS) and Sham surgeries were used as controls. The proteome of rat kidney homogenates was analyzed using tandem mass-tag (TMT) mass spectrometry. C5 and C5aR1 were further characterized via SDS-PAGE western blotting and immunohistochemistry (IHC). Results: In the TMT dataset, 5,951 individual proteins were identified in all animal groups. Pathway analysis revealed significant complement dysregulation, and 34 complement proteins were differentially abundant in CS+Tx compared to ATx, suggesting that CS intensifies complement activation. The level of C5 protein was increased after CS+Tx in rat kidneys. Intriguingly, C5 was increased in isolated mitochondria fractions, and this increase correlated with mitochondrial injury. C5aR1 predominantly localizes in the renal medulla and distal tubules in Sham and ATx, but displays diffusely positive staining in the cortex after CS+Tx. Conclusion: We have identified significant dysregulation of the complement pathway during CS+Tx. Increased C5 in the mitochondrial fraction after CS+Tx suggests the involvement of the C5a/C5aR1 axis. Future studies will clarify the role of C5a/C5aR1 in the mitochondria during CS+Tx. Funding: NIDDK Support, Private Foundation Support