Abstract
Background: Mitochondrial transcription factor A (TFAM) plays multiple pathophysiologic roles in mitochondrial DNA (mtDNA) maintenance. However, the role of TFAM in sepsis-induced acute kidney injury (AKI) remains largely unknown. Methods: Lipopolysaccharide (LPS) treatment of HK-2 cells mimics the in vitro model of AKI inflammation. pcDNA3.1 plasmid was used to construct pcDNA3.1-TFAM. sh-TFAM-543, sh-TFAM-717, sh-TFAM-765, sh-TFAM-904 and pcDNA3.1-TFAM were transfected into HK-2 cells using Lipofectamine 2000. MtDNA transcriptional levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was performed to assess the cell viability. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential in HK-2 cells were detected using the corresponding kits. Immunofluorescence experiment was used to investigate the displacement of TFAM. mRNA and protein expression levels of TFAM and its related genes were measured by qRT-PCR and western blot respectively. Mice in sepsis were administered cecal ligation and puncture surgery. Results: LPS treatment was a non-lethal influencing factor, leading to the upregulation of ROS levels and downregulation of mtDNA copy number and NADH dehydrogenase subunit-1 (ND1) expression, and caused damage to the mitochondria. As the LPS treatment time increased, TFAM was displaced from the periphery of the nucleus to cytoplasm. TFAM reduced ROS and P38MAPK levels by inhibiting toll-like receptor 4 (TLR4) expression, ultimately inhibiting inflammation and repairing mtDNA. Conclusions: Our results indicate that TFAM repairs mtDNA by blocking the TLR4/ROS/P38MAPK signaling pathway in inflammatory cells, thereby repairing septic tubular epithelial cells, and TFAM may serve as a new target for sepsis therapy.
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