Mechanism of Impaired Protein Homeostasis in Kidney Grafts following Cold Storage and Transplant

Background: Cold ischemia is an unavoidable event during the cold storage (CS) process of donor kidneys before transplantation. We have previously reported that CS impaired protein homeostasis in transplanted rat kidneys and reduced graft function. However, the mechanisms of how CS contributes to graft damage are largely unknown. Methods: Isolated donor rat kidneys were stored in a University of Wisconsin (UW) solution at 4°C for 0 or 18 hours, followed by transplantation to recipient rats (CS+Tx). To simulate an in vitro model of CS+Tx, rat or human proximal tubular cells (PTCs) were incubated in UW solution at 4°C for CS, followed by rewarming (RW) with normal media at 37°C (CS+RW). Results: Two members of 70-KDa heat shock proteins (HSPs) were dysregulated—a robust increase of Hsp72 and a decrease of Hsc70—in kidney grafts after CS+Tx. CS+Tx decreased HSF1, a stress-activated transcription factor that induces HSPs, along with its band shift in western blot, suggesting that the CS injury contributes to a post-translational modification of HSF1 in rat kidneys. To further elucidate the CS-mediated changes of these proteins in PTCs, renal cells were exposed to CS+RW. Consistent with in vivo data, CS alone increased HSF1 in PTCs, and rewarming episode increased Hsp72 and decreased Hsc70 and HSF1 proteins in a time-dependent manner. To assess protein localization during CS+RW, we performed immunocytochemistry (ICC), which revealed a dense staining pattern of HSF1, Hsp72, and Hsc70 proteins after CS+RW. Unlike western blot data, Hsc70 or HSF1 levels remained unchanged after ICC, suggesting that CS+RW induces aggregation of Hsc70 or HSF1 in PTCs. Adding HS-72, a specific inhibitor of Hsp72, to the CS solution partially restored Hsc70 levels, reduced aggregation of Hsp72, increased HSF1 level, and improved cell viability after rewarming in PTCs. Finally, the addition of HS-72 during CS improved graft function after CS+Tx. Conclusion: These data indicate that the CS-mediated increase of Hsp72 protein is a negative regulator of kidney function. Inhibiting Hsp72 during CS improves PTCs health and graft function and therefore HS-72 could be a potential therapeutic during CS to improve outcomes after kidney transplantation. Funding: NIDDK Support