Abstract: TH-PO119
PPP1R3G Promotes Necroptosis in Ischemia Reperfusion-Induced AKI by RIPK1 Dephosphorylation
Session Information
- AKI: Mechanisms - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Tran, Minh Hoang, University of South Florida, Tampa, Florida, United States
- Wang, Lei, University of South Florida, Tampa, Florida, United States
Background
Recent discoveries have revealed necroptosis as a major contributor to the pathogenesis of acute kidney injury (AKI). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been linked to the essentiality of RIPK1-dependent apoptosis and type I necroptosis. Nonetheless, the involvement of PPP1R3G in the regulation of necroptosis during ischemia reperfusion-induced acute kidney injury (IR-AKI) has yet to be elucidated.
Methods
Here, we investigated the role of PPP1R3G in the regulation of necroptosis in IR using an in vitro cellular I/R model and in vivo IRI-AKI mouse model. Primary proximal tubular cells (PTCs) from C57BL/6 and Ppp1r3g−/− mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and necroptosis were analyzed using a LUNA-II cell counter and flow cytometry, respectively. The expression levels of necroptosis, oxidative stress, and inflammation factors were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. In the in vivo IRI-AKI model, the kidney injury and function were evaluated by plasma creatine, BUN, and KIM-1 levels and GFR values, respectively. Morphological evidence of kidney injury was assessed by PAS staining and cell death by immunofluorescent staining.
Results
We found that knockout of PPP1R3G significantly decreased (by 35-50%) hypoxia-inducible factor-1α (HIF-1α), as well as the levels of necroptosis factors (RIP1, RIP3, and Sirtuin-2), and inflammatory factors (IL-6, IL-10 and TNF-α) in I/R injury cells and IR-AKI mice. Deletion of PPP1R3G attenuated the I/R induced kidney injury indicated by 54% decrease in the plasma creatine (2.28±0.12mg/dL vs. 1.09±0.07mg/dL, p<0.0001) and 45% higher GFR values than WT mice at day 3 after reperfusion. Histology showed less diffuse renal tubular necrosis, casts, and debris in Ppp1r3g−/− mice, as well as 60% less cell death compared to WT mice. Moreover, the application of chemical compounds to prevent inhibitory phosphorylations of RIPK1 significantly reinstated cell death in Ppp1r3g−/− cells.
Conclusion
In conclusion, our findings demonstrate, for the first time, that Ppp1r3g-mediated necroptosis significantly promotes IRI-AKI. Therefore, targeting Ppp1r3g to reduce necroptosis in the kidney holds promising potential for significant clinical benefits.
Funding
- NIDDK Support