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Bioinformatics analysis of biomarkers of aristolochic acid-induced early nephrotoxicity in embryonic stem cells.

ABSTRACT: The present study aimed to identify key genes as potential biomarkers for early nephrotoxicity induced by aristolochic acid (AA) in embryonic stem cells (ESCs). An MTT assay was performed to determine the cytotoxicity of AA in ESCs. Differentially expressed genes (DEGs) were identified using the DNA-Chip Analyzer following microarray analysis. Gene Ontology analysis was performed to determine functional terms enriched by the DEGs in the categories biological process, cellular component and molecular function. Furthermore, the DEGs were subjected to Kyoto Encyclopedia of Genes and Genomes analysis to determine pathways they were accumulated in. Furthermore, a protein-protein interaction network was constructed using Cytoscape 3.2 software. Tumor protein 53 apoptosis effector (Perp), cation transport regulator-like 1 (Chac1), adrenoceptor β2 and Wnt6 were selected for confirmation by reverse transcription-quantitative (RT-q) PCR analysis. A total of 72 DEGs (49 upregulated and 23 downregulated) were identified. The DEGs were enriched in functional terms and pathways associated with nephrotoxicity and participated in 92 pathways. A total of two hub genes, fructose-1,6-bisphosphatase (Fbp)1 and Fbp2, were filtered out from the interaction network. Perp and phorbol-12-myristate-13-acetate-induced protein 1 were demonstrated to have vital roles in the p53 signaling pathway which was indicated in the interaction network. The results of the RT-qPCR analysis were consistent with the microarray data. Taken together, the present study suggested that hub genes involved in the p53 pathway, including Fbp1, Fbp2 and Perp, may serve as potential biomarkers for early nephrotoxicity induced by AA.

SUBMITTER: Wang L

PROVIDER: S-EPMC8005694 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(-/-) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography-mass spectrometry (GC-MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(-/-) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(-/-) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.

Project description:With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.

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Bioinformatics analysis of biomarkers of aristolochic acid-induced early nephrotoxicity in embryonic stem cells.

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