Disruption of PPP1R3G-PP1γ Interaction by Deletion or Pharmacologic Inhibition Mitigates Kidney Fibrosis Progression

Background: Crosstalk between dying tubular cells and inflammation is a key driver of kidney fibrosis. Necroptosis, a type of programmed cell death involving receptor-interacting protein kinase 1 (RIPK1), plays a major role in this process. Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) collaborates with protein phosphatase 1γ (PP1γ) to regulate kinase activity, but its role in RIPK1 activation and tubular cell death during fibrosis development remains unknown. Methods: Fibrosis was induced both in primary proximal tubular cells (PTCs) using TGF-β and in vivo using the reversible unilateral ureteral obstruction (RUUO) model in wild-type and Ppp1r3g-/- mice. Cell viability, cell death, kidney injury, fibrosis, and renal function were assessed. The phosphorylation status of RIPK1 and NLRP3 inflammasome formation were analyzed using RT-PCR and Western blotting, and these findings were correlated with the extent of kidney fibrosis and functional decline. Additionally, compounds designed to disrupt the PPP1R3G/PP1γ interaction were synthesized and tested in kidney fibrosis models. Results: Renal cellular injury increases PPP1R3G expression and activates the RIPK1-mediated necroptosis pathway. Deletion of PPP1R3G significantly attenuated tubular cell death, reduced necroptosis markers (phospho-RIPK1, RIPK3, and MLKL), suppressed inflammatory responses, and led to marked reductions in kidney injury and fibrosis, evidenced by approximately 85% of KO cells survival from fibrosis, while only 36% of WT cells exhibited survival under the same conditions and a 62% reduction in plasma creatinine (0.74±0.16 mg/dl vs. 0.87 ± 0.18 mg/dL, p<0.001, n=5) and a 46% increase in GFR (n=5). Histological analysis confirmed decreased necroptosis and interstitial fibrosis in the Ppp1r3g-/- mice. Peptide inhibitors, PGB-1, effectively blocked RIPK1-dependent tubular cell death by disrupting the PPP1R3G–PP1γ interaction. PGB-1 treatment reduced fibrosis signals and improved kidney function, lowering plasma creatinine by 74% compared to the untreated group. Conclusion: This study reveals that PPP1R3G-mediated necroptosis significantly contributes to kidney fibrosis progression and that disrupting the PPP1R3G/PP1γ interaction with PGB-1 is a promising therapeutic intervention for human chronic kidney disease. Funding: NIDDK Support