Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation

Abstract

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits β5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and β5. Peroxynitrite induced a dose-dependent decrease in PAC1 and β5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

Out of the Cold

“…Bhattarai et al had previously demonstrated that the peptidase activity of the constitutive proteasome was decreased in kidney cells after cold storage and that this led to reduced graft function, potentially by impairing mitochondrial function. 5 – 7 The immunoproteasome differs from the constitutive proteasome in that several of the constitutively expressed catalytic β subunits of the standard proteasome are replaced by immunoproteasome-specific inducible counterparts. 8 These include the immunoproteasome-specific subunits LMP2 for constitutive β 1, LMP10 for β 2, and LMP7 for β 5.…”