The role of PPP1R3G in promoting kidney graft necroptosis during kidney transplantation

Death of renal cells is central to the pathophysiology of kidney graft injury and delayed graft function in kidney transplant (KTX). Necroptosis, a recently discovered form of regulated cell death, has emerged as a pivotal event in the development of various kidney disorders, mainly triggered by the activation of receptor-interacting protein kinase 1 (RIPK1). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been identified to work with protein phosphatase 1γ (PP1γ) in kinase activation. However, the role of PPP1R3G in RIPK1 activation and tubular cell death remains unexplored. This study aims to explore PPP1R3G’s role in tubular cell death during transplantation and develop peptidomimetics to block the PPP1R3G/PP1γ interaction to prevent kidney graft injury. In vitro cellular I/R and in vivo KTX mouse model were used in this study. Primary proximal tubular cells (PTCs) from Ppp1r3g+/+ (WT) and Ppp1r3g−/− (KO) mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and death were analyzed using CellTiter-Glo assay and Annexin V-Cy3/ SYTOX green staining, respectively. The activation of necroptosis pathway and inflammation factors were determined by real-time PCR and Western blotting. For the in vivo model, syngeneic KTX was performed using same-sex mice in WT and KO groups (10-12 weeks). Kidney graft injury and function were evaluated by measuring plasma creatine and BUN levels, PAS staining and cell death (immunofluorescent staining) and GFR values. In male C57BL/6 mice, a newly designed sulfono-γ-AApeptide foldamer, PGB-1 (PP1 gamma binding 1), was administrated to donor kidneys (5 µM in University of Wisconsin solution) during cold storage. Syngeneic KTX was conducted after 3 hours of cold storage, and graft injury and function were assessed at 1- and 3-days post-transplantation. We found that deleting PPP1R3G in PTCs significantly enhanced cell viability and reduced tubular cell death. This protective effect was associated with lower activation of necroptosis markers (p-RIP1, p-RIP3, and p-MLKL) and reduced expression of inflammatory factors. In the KTX, PPP1R3G deletion mitigated I/R-induced kidney graft injury, demonstrated by a 51% decrease in plasma creatinine (1.78±0.16 vs. 0.87±0.18 mg/dL, p<0.001, n=7) and a 55% improvement in GFR in KO mice. Histological analysis showed reduced activated MLKL (p-S345-MLKL) and lower TUNEL staining in KO mice than WT. Additionally, administering PGB-1 to donor kidneys significantly protected grafts from ischemic injury, reducing plasma creatinine by 65% compared to controls. This study demonstrates, for the first time, that Ppp1r3g-mediated necroptosis plays a significant role in kidney graft injury during transplantation. Blocking the PPP1R3G/PP1γ interaction with PGB-1 effectively halts cell death signaling, highlighting its potential as a therapeutic approach for human KTX. This research study is graciously supported by the NIH, NIDDK, and the home institute of the University of South Florida. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.