PAX2 REACTIVATION PREVENTS NECROPTOSIS-MEDIATED MORTALITY BY CHANGING STAT1 PHOSPHORYLATION IN ACUTE TUBULAR NECROSIS

Acute kidney injury (AKI) is a critical clinical syndrome that adversely affects renal and overall patient prognosis, with acute tubular necrosis (ATN) representing a particularly severe form of kidney injury. Paired box 2 (Pax2), an essential transcription factor for kidney development, undergoes reactivation in injured proximal tubular epithelial cells (PTECs) during ATN. Our previous research demonstrated that Pax2 reactivation promotes PTEC proliferation by activating cyclin-dependent kinase 4 in ischemia-reperfusion injury models. However, the role of Pax2 in ATN cell death mechanisms remains unclear. This study aimed to investigate the role of Pax2 in regulating cell death pathways during ATN and its impact on kidney injury severity and survival outcomes.

We conducted experiments using 8-week-old male mice with proximal tubular epithelial cell-specific Pax2 knockout (Pax2 KO) mice under endogenous androgen control. We administered aristolochic acid (AA) at a dose of 5 mg/kg intraperitoneally for three consecutive days to induce severe toxic ATN. As a control group, we used C57BL/6 wild-type (WT) mice. Mice were sacrificed on days 1, 4, 7, and 42 after the end of AA administration. Kidney function was assessed by measuring serum urea nitrogen (BUN) and serum creatinine (Cr) levels at days 1, 4, 7, and 42. Gene expression of cell death-related markers, including apoptosis, necroptosis, and ferroptosis, was analyzed by qPCR. Histological examination included PAS staining, immunohistochemistry for cleaved caspase-3 and phosphorylated mixed lineage kinase domain-like protein (p-MLKL), and TUNEL staining. To evaluate the impact on mortality, we administered a single intraperitoneal dose of AA at 15 mg/kg and monitored survival for 42 days. In vitro experiments utilized human proximal tubular epithelial cells (HK-2) treated with AA (100 µM) and the PAX2 inhibitor EG1 (100 µM). Novel PAX2-necroptosis-associated factors were identified through STRING database analysis and validated by qPCR and Western blotting.

Survival analysis revealed significantly reduced survival in Pax2 KO mice compared to WT mice (20% vs 60%, p=0.013). In kidney function, on the first and fourth days, the two groups showed no significant differences. However, on the seventh day, serum creatinine levels increased in the Pax2 KO group (p=0.002). Histological examination showed clear tubular obstruction in the Pax2 KO group. TUNEL-positive cells were observed in both groups; however, the number of cleaved caspase 3-positive cells decreased in the Pax2 KO group (p=0.027). Instead, the number of p-Mlkl-positive cells increased (p<0.001). mRNA expression showed significant upregulation of necroptosis markers, including increased Mlkl and Ripk3. In HK-2 cells, PAX2 inhibition with EG1 following AA treatment led to increased MLKL and RIPK3 expression and significantly reduced cell viability. Network analysis identified STAT1 as a novel PAX2-necroptosis mediator, with qPCR confirming increased STAT1 mRNA expression, and Western blotting analysis revealed increased phosphorylation at Ser727 in AA+EG1-treated cells. These findings suggest that PAX2 plays a crucial role in determining the fate of injured PTECs, and its suppression can lead to necroptosis, a more severe form of cell death, and worsen kidney injury.

This study demonstrates that attenuation of Pax2 reactivation in ATN induces a critical shift in cell death modality from apoptosis to necroptosis, resulting in severe tubular obstruction, exacerbated kidney injury, and increased mortality. The identification of phosphorylated STAT1-Ser727 as a mediator linking PAX2 suppression to necroptosis activation reveals a novel regulatory mechanism in ATN pathogenesis. These findings establish PAX2 as a key regulator of cell death fate determination in injured PTECs and suggest that maintaining adequate PAX2 reactivation may be crucial for preventing maladaptive necroptotic cell death.