Purpose: Renal cold storage (CS) is considered to be an alloantigen-independent risk factor for long-term transplant outcome. The precise molecular mechanisms responsible for CS-related renal damage are largely unknown; therefore, the overall goal of this study is to identify pathways responsible for inducing renal damage during CS and transplantation. Our recent study using porcine kidneys shows that CS induced oxidative stress and mitochondrial dysfunction, which were blunted by mitochondria-targeted antioxidant, Mitoquinone (MitoQ). In this study, we tested the hypothesis that CS triggers proteasome activation, which leads to mitochondrial dysfunction and renal damage following CS and transplantation.

Methods: Male rat kidneys were isolated and cold stored for 4 hr +/- Bortezomib /Velcade® (100 nM), a proteasome inhibitor, or MitoQ (100 ¯o;M) followed by transplantation. Mitochondrial function was assessed in the renal tissue extracts using a bioluminescent ATP assay. The proteasome activity was measured in the renal tissue extracts by hydrolysis of the fluorogenic based peptide substrates. Renal tubular injury was scored based on histologic evaluation after PAS staining. Generation of reactive oxygen species was assessed using nitrotyrosine immunohistochemistry. Finally, renal cell death was monitored by TUNEL assay.

Results: As expected, renal tubular damage, nitrotyrosine accumulation, and cell death were increased, and ATP levels were decreased following CS and transplantation. Interestingly, the proteasome activity was increased in the renal transplant, which was blunted by addition of MitoQ during CS. Excitingly, our preliminary data using Bortezomib addition during renal CS showed decrease of tubular cell death and increase of ATP levels after transplantation.

Summary: These results indicate that CS-induced oxidative stress triggers activation of the proteasome leading to mitochondrial dysfunction and renal damage, and our early results using Bortezomib suggest that the proteasome inhibition during CS could increase viability of renal transplants. Future studies using the rat model of renal transplantation will address whether Bortezomib inclusion during CS improves long-term transplant outcome.

Supported by NIH 5 T32 DK 61921-7 (NP) and NIH DK089659 (LAMC)

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