Trimethylamine N-oxide mediated Y-box binding protein-1 nuclear translocation promotes cell cycle progression by directly downregulating Gadd45a expression in a cellular model of chronic kidney disease

AIMS:Cell cycle arrest plays critical roles in preventing renal tubular epithelial cell (RTEC) injury and maladaptation after the onset of chronic kidney disease (CKD), but the underlying mechanism governing this arrest has not been fully elucidated. This study was designed to determine the underlying role of YB-1 in promoting cell cycle progression and nuclear translocation in HK-2 cells induced by trimethylamine N-oxide (TMAO). MAIN METHODS:YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. Gene expression was analysed using RNA sequencing in HK-2 cells treated with TMAO. Cell cycle progression was analysed via flow cytometry. Luciferase assay and ChIP-PCR were performed to determine the relationship between transcription factor YB-1 and Gadd45a promoter region. Additionally, mice were fed with TMAO to test renal dysfunction and measure the expression of YB-1, GADD45a and CCNA2 in the kidney sections through immunohistochemistry. KEY FINDINGS:YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. RNA sequencing analysis showed that the cell cycle checkpoint genes growth arrest and DNA damage (Gadd)45a, Gadd45g, cyclin (Ccn)a2, Ccnb1, Ccne1 and Ccnf were differentially expressed in HK-2 cells after treated with 400 μM TMAO for 30 min. Flow cytometry results demonstrated that cell cycle progression was blocked at the G2/M checkpoint. In animal models, elevated dietary TMAO directly led to progressive renal tubulointerstitial dysfunction and inhibited the expression of YB-1 in kidney. Moreover, YB-1 was determined to regulate Gadd45a expression by directly binding to its promoter region. YB-1 expression was negatively correlated with the expression of Gadd45a and Gadd45g but positively correlated with Ccna2, Ccnb1, Ccne1 and Ccnf in CKD. SIGNIFICANCE:YB-1 may be a reliable molecular target and an effective prognostic biomarker for CKD.