Date of Award

Spring 2020

Document Type

Thesis

First Reader

Dr. Timothy Knight

Second Reader

Dr. Blake Johnson

Third Reader

Dr. Allyson Phillips

Abstract

Renal transplantation is the preferred method of treatment for end stage kidney disease. The majority of donor kidneys come from deceased donors and have to be stored in cold storage solution (CS) until the recipient is identified. However, prolonged CS is associated with poor long-term outcome. Unfortunately, the mechanisms of CS-related damage are largely unknown. Our laboratory recently reported that the proteasome and renal function were significantly decreased in rat kidney transplants that involved CS combined with transplantation (CS/Tx), as opposed to those that did not undergo CS (auto-transplantation/ATx). The long-term goal is to improve the transplant outcome by identifying CS-mediated renal damage and by acquiring targeted therapies during CS. This study contributes to that objective by characterizing immunoproteasome (a proteasome variant) and complement (a group of serum proteins that participates in eliminating pathogens and debris) activation within the kidneys after CS/Tx. Our hypothesis is that CS/Tx will exacerbate the function of immunoproteasome and complement systems. Lewis rat kidneys exposed to 18 hours of cold storage were used for transplantation (CS/Tx). Kidneys with no CS exposure were transplanted (ATx) and used as a transplant control. The sham (Sh) kidneys with right nephrectomy were used as a control. Using paraffin embedded kidney sections and immunohistochemistry/immunofluorescence, immunoproteasome and complement levels/function were evaluated. Immunoproteasome function was significantly increased only after CS/Tx when compared to Sh and ATx. Immunohistochemistry of kidney sections revealed a modest increase of immunoproteasome catalytic subunits, LMP2 and LMP10, after ATx when compared to Sh; but a profound increase of these subunits was detected after CS/Tx. Similarly, complement proteins C3 (an upstream component) and C5b-9 (a cytolytic terminal activation product), were increased in kidneys after ATx (detected by immunofluorescence), but an excessive increase of these proteins was observed after CS/Tx. Furthermore, TUNEL assay revealed exacerbated cell death in kidney sections after CS/Tx, whereas ATx showed a slight increase of cell death. These results suggest that the prolonged CS worsens activation of the immunoproteasome and complement system leading to renal damage/dysfunction.

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