HDAC2 Partially Contributes to the Differential Nephrotoxicity Induced by AA-I, AA-IVa, or AL-I

Background: ASARI RADIX ET RHIZOMA (Xixin), extensively utilized in clinical settings, contains aristolochic acid analogs (AAAs), primarily AA-IVa, with minor quantities of AL-I and negligible levels of AA-I. AA-I mediates aristolochic acid-induced nephropathy; therefore, the safety of Xixin is controversial. The toxicity of AAAs is varied, and the mechanisms are ambiguous. Objective: To compare the toxicity of AA-I, AA-IVa, and AL-I toward renal tubular epithelial cells and explain the molecular causes of such. Materials and Methods: C57BL/6 mice were administered AA-I, AA-IVa, or AL-I for 24 weeks; renal toxicity was assessed via survival and histopathology. In vitro, AAAs toxicity was evaluated in human renal tubular epithelial cells (HK-2) by high-content Hoechst 33342/PI apoptotic tests and fibrosis marker quantification. Transcriptomics revealed alterations in genes and pathways induced by AAAs. Click chemistry-based activity-based protein profiling, molecular docking, surface plasmon resonance, enzyme activity assays, and gene knockdown identified the AAAs' direct targets. Results: AA-I induced dose-dependent mortality, severe kidney injury, and fibrosis; AL-I showed less toxicity, and AA-IVa was not discernibly toxic in mice. In vitro, AA-I prompted apoptosis and fibrosis in HK-2 via transcriptional modification; other AAAs exhibited diminished effects. AA-I, AL-I and AA-IVa showed stable binding ability to HDAC2; AA-I and AL-I enhanced HDAC2 expression; and AA-I facilitated HDAC2 nuclear translocation and enzymatic activity. AA-I, unlike AL-I and AA-IVa, augmented MYC expression, disturbed the BCL2/BAX equilibrium, activated Caspase-7, and triggered apoptosis and pro-fibrotic transformation. Conclusions: AA-I induces apoptosis and fibrosis through HDAC2, and HDAC2 partially contributes to the various toxicities of AA-I, AA-IVa, and AL-I. This study provides fundamental evidence for the safety evaluation of Xixin.