Cold Storage-Mediated p38MAPK Activation: A Potential Contributor of Proteasome Dysfunction and Kidney Damage After Transplantation
- Mechanisms and Single-Cell Transcriptional Profiles in Transplant Rejection and Ischemia Reperfusion Injury
November 02, 2023 | Location: Room 115, Pennsylvania Convention Center
Abstract Time: 05:06 PM - 05:15 PM
- 2101 Transplantation: Basic
- Bhattarai, Dinesh, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
- Lee, Seongok, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
- Parajuli, Nirmala, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States
The majority of donor kidneys require cold storage (CS) prior to transplantation, but this process usually leads to suboptimal outcomes, due to CS activating cellular pathways that damage kidney tissue. Previously, we demonstrated that the CS followed by transplantation decreases proteasome function in rat renal grafts; however, the mechanism of this dysfunction is not known.
Established in vivo (CS+Tx) and in vitro (CS+RW) models of rat kidney cold storage followed by transplantation. The proteasome function was performed using rat kidney homogenates and fluorescent-based peptide substrates. The subunit level of the proteasome subunit was characterized using renal extracts and western blotting.
Western blots showed an unprecedented higher-molecular-weight band of Rpt6 subunit of the proteasome after CS+Tx, suggesting a post-translational modification (PTM) of the Rpt6 subunit. Non-denatured western blots for Rpt6 revealed aggregated proteasome levels after CS+Tx. Phosphatase treatment of renal extracts depleted the higher-molecular-weight band of the Rpt6 subunit in the CS+Tx, suggesting that the aggregation of Rpt6 was associated with phosphorylation. Renal (NRK) cells exposed to CS+RW showed a time-dependent increase p38MAPK activation. Treatment of NRK cells with p38MAPK inhibitor (SB202190 or VX-745) during CS followed by rewarming increased the cell viability and proteasome activity when compared to the CS+RW condition, suggesting p38 MAPK negatively regulates tubular cell viability during CS+Tx/RW. Finally, ex vivo treatment of the donor kidney with Bortezomib prior to transplantation (no CS) increased renal dysfunction after transplantation, suggesting normal proteasome function is needed to prevent renal injury in the transplants.
Together, our results suggest that CS-mediated activation of the p38MAPK may contribute to Rpt6 phosphorylation/aggregation, which then leads to decreased proteasome function and renal injury following CS+Tx. Therefore, p38MAPK could be a novel therapeutic target during CS to reduce CS+Tx-mediated graft failure.
- NIDDK Support