Disruption of local circadian clocks in aristolochic acid-induced nephropathy in mice

Aristolochic acid I (AAI), an emerging biogenic contaminant widely present in Aristolochic plants, has been implicated in the progression of tubulointerstitial disease, known as aristolochic acid nephropathy (AAN). The circadian clock, a vital regulator of organ homeostasis, is susceptible to external chemical cues, including toxins. However, the reciprocal interactions between AAI and the circadian clock remain unexplored.

We initially assessed sex- and time-dependent nephropathy and behavioral responses in C57BL/6J mice exposed to AAI. Subsequently, we evaluated changes in the expression of circadian clock genes following treatment with AAI or its bioactive metabolite, aristolactam I, using real-time quantitative PCR and immunoblotting in renal tissues and cells. Additionally, real-time reporter assays were conducted on kidney explants from PER2::Luc knock-in reporter mice and Per2-dLuc/Bmal1-dLuc reporter cell lines. To further elucidate the regulatory role of circadian clocks in AAI-induced nephropathy, mice with global or kidney-specific knockout of Bmal1, as well as mice subjected to experimental jetlag, were utilized.

Our findings revealed a sex-dependent nephrotoxicity of AAI, with males exhibiting greater vulnerability. AAI-induced nephropathy was accompanied by impaired spatial cognitive function, disruptions in free-running locomotor activity, altered renal expression of multiple core clock genes, and disturbances in the circadian rhythm of renal PER2::Luc activity. Notably, kidney-specific ablation of the core clock gene Bmal1 significantly exacerbated renal injury and inflammation, whereas disruptions to the central clock, either genetically (through conventional knockout of Bmal1) or environmentally (mimicking jetlag), had minimal effects on AAI nephrotoxicity. Furthermore, both AAI and its bioactive metabolite aristolactam I demonstrated the ability to disrupt circadian clocks in human osteosarcoma cells (U2OS) and mouse renal tubular epithelial cells (mRTEC).

Collectively, these findings highlight the detrimental impact of aristolochic acids on local renal circadian clocks, ultimately exacerbating kidney damage. This study provides novel insights into the molecular mechanisms underlying AAI nephrotoxicity, potentially opening avenues for therapeutic interventions aimed at modulating the renal circadian clock to treat AAN.