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).