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Using Genome-wide CRISPR Screen to Identify Cannabidiol as an E2F1 Inhibitor against Aristolochic Acid Nephrotoxicity

ABSTRACT: Aristolochic Acid I (AAI), one of the most abundant aristolochic acids (AAs), is nearly ubiquitous in herbal and traditional Chinese medicines within the Aristolochia genus. Renal tubular epithelial cell damage has been identified as a primary cause of AAI-induced nephrotoxicity. Genome-wide CRISPR library screening has proven valuable in identifying key genes associated with the toxicity of exogenous substances. To screen for genes related to AAI-induced renal toxicity, we conducted a genome-wide CRISPR library screening in HK-2 cell line. Among the altered sgRNAs, we observed significant enrichment of sgRNAs targeting E2F1 gene in survival HK-2 cells of the AAI-treated group, correlating with the promotion of AAI-induced cell apoptosis. Subsequent investigations revealed that E2F1 primarily promotes cellular apoptosis by activating the p53 signaling pathway, thereby enhancing the expression of pro-apoptotic genes, BAK and BAX. Additionally, we screened cannabidiol (CBD) as an inhibitor of E2F1 by utilizing the HERB database (http://herb.ac.cn/). Validation in both in vitro cell models and in vivo mouse models confirmed that CBD inhibits the expression of E2F1, consequently suppressing p53 signaling pathway-associated cell apoptosis. In conclusion, our results underscore the crucial role of E2F1 in AAI-induced renal cell apoptosis. Furthermore, the identification of CBD as a potential therapeutic candidate for AAI-induced renal toxicity provides new insights for understanding and treating AAI-induced nephrotoxicity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE267986 | GEO | 2025/07/01

REPOSITORIES: GEO

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Project description:Exposure to aristolochic acid (AA) is linked to kidney disease and urothelial cancer in humans. The major carcinogenic component of the AA plant extract is aristolochic acid I (AAI). The transcription factor p53 acts as a tumour suppressor and is frequently mutated in AA-induced tumours. Using a mouse model, we previously showed that Trp53 genotype impacts on AAI-induced nephrotoxicity in vivo (i.e. p53 protects from AAI-induced renal proximal tubular injury), but the underlying mechanism(s) involved remain to be further explored. In the present study, we investigated the impact of p53 on AAI-induced gene expression in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 6 days. The Clariom™ S Assay microarray was used to elucidate gene expression profiles in mouse kidneys after AAI treatment in order to identify potential mechanisms by which AAI drives renal injury in Trp53(-/-) kidneys. Principle component analysis and hierarchical clustering in Qlucore Omics Explorer showed that gene expression in AAI-exposed Trp53(+/+), Trp53(+/-) and Trp53(-/-) kidneys is treatment-dependent. However, gene expression profiles did not segregate in a clear-cut manner according to Trp53 genotype, hence further investigations were performed by pathway analysis with MetaCore™. Several pathways, such as those related to epithelial-to-mesenchymal transition, transcription of hypoxia-inducible factor 1 targets, renal injury and secretion of xenobiotics were significantly altered to varying degrees for AAI-exposed kidneys. The top ten up-regulated genes included cyclin-dependent kinase inhibitor 1a (Cdkn1a), a mediator of cell cycle arrest; and neutrophil gelatinase-associated lipocalin (Ngal), which has been shown to play a role in nephritis by promoting inflammation and apoptosis. Members of the solute carrier (Slc) family (i.e. Slc22a2, Slc22a6, Slc22a7, Slc22a8) were amongst the top ten down-regulated genes. Pathway analysis also identified genes that are uniquely affected by AAI treatment in Trp53(+/+), Trp53(+/-) and Trp53(-/-) kidneys. Apoptotic pathways were modulated in Trp53(+/+) kidneys; whereas oncogenic and pro-survival pathways were significantly altered for Trp53(+/-) and Trp53(-/-) kidneys, respectively. Microarray gene expression analysis identified significant toxicogenomic responses to AAI that give novel insights into its mechanism of nephrotoxicity. Alterations of biological processes by AAI in Trp53(+/+), Trp53(+/-) and Trp53(-/-) kidneys could explain the mechanisms by which p53 protects from or p53 loss drives AAI-induced renal injury in vivo.

Project description:Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumours. These tumours contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and non-target (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfkb, aryl hydrocarbon receptor, Tp53 and cell cycle signalling as the most important pathways modulated in kidney. Expression of Nfkb1 and other Nfkb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfkb1 protein. Myc oncogene, frequently over-expressed in urothelial tumours, was up-regulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies. Keywords: Carcinogen treatment Two-color Agilent array. A reference design was chosen that all samples were hybridised to universal mouse reference RNA (UMRR). 12-condition experiment (2 mouse organs: kidney and liver; 2 treatments: AAI and water; 3 time points: 3, 12 and 21 days). Three biological replicates for each condition. One replicate per array.

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Using Genome-wide CRISPR Screen to Identify Cannabidiol as an E2F1 Inhibitor against Aristolochic Acid Nephrotoxicity

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