3.1 | AAI-induced PANoptosis in vivo and in vitro
To determine the renal response to aristolochic acid treatment, kidney tissues were harvested 1, 2, and 3 days after AA administration (Figure 1A). AAI administration caused time-dependent kidney dysfunction, as evidenced by increased BUN and Cr levels over time (Figure 1B). The progression of the lesions in the kidney after AAI administration was observed by histopathological examination. Tubular dilation, necrosis, cast formation, and preservation of the brush border in the kidney were observed three days after the AAI injection (Figure 1C). We found that AAI triggers multiple forms of cell death (PANoptosis). For apoptosis, we found markedly increased levels of cleaved forms of executioner caspase-3 after the AAI injection. AAI treatment resulted in robust phosphorylation of the pseudokinase mixed lineage kinase-like domain (MLKL), an inducer of necroptosis. Next, we explored the effect of the treatments on pyroptosis markers. GSDMD is the primary executioner that forms the membrane pores under specific conditions. The results revealed significant activation and cleavage of GSDMD in the treatment group (Figure 1D, E). In vitro, mouse kidney tubular epithelial cells (mTECs) were treated with AAI (10 μM) for 24 h and harvested to explore the role of AAI on nephrotoxicity. This was consistent with the in vivo results that AAI treatment could cause increased activation of pyroptotic, apoptotic, and necroptotic molecules (Figure 1F–G).