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Reactive oxygen species induced by uric acid promote NRK‑52E cell apoptosis through the NEK7‑NLRP3 signaling pathway.

ABSTRACT: Increasing uric acid (UA) could induce renal tubular epithelial cell (NRK‑52E) injury. However, the specific mechanism by which UA induces renal tubular epithelial cell injury remains unknown. It was hypothesized that UA induces renal tubular epithelial cell injury through reactive oxygen species (ROS) and the Never in mitosis gene A (NIMA)‑related kinase 7 (NEK7)/NLR family pyrin domain containing 3 (NLRP3) signaling pathway. TUNEL assay and flow cytometry were applied to measure apoptosis, and the results of the present study showed that UA treatment induced apoptosis of NRK‑52E cells in a concentration‑dependent manner. Western blotting was performed to determine the expression levels of cleaved caspase‑3, Bax and Bcl‑xl, it was found that levels were significantly increased after UA treatment in NRK‑52E cells. ROS and apoptosis were predominantly induced in NRK‑52E cells and there was an association between ROS and apoptosis. Enhanced expression of NEK7, NLRP3, apoptosis‑associated speck‑like and caspase‑1 were observed in NRK‑52E cells treated with UA. The ROS inhibitor, N‑acetyl‑l‑cysteine, exerted a protective effect on the UA‑induced apoptosis of tubular epithelial cells by reducing excess ROS production, which significantly inhibited NEK7 and NLRP3 inflammasome activation. These results indicated that UA activates ROS and induces apoptosis of NRK‑52E cells. The mechanism might be related to the regulation of the NEK7/NLRP3 signaling pathway.

SUBMITTER: Li D

PROVIDER: S-EPMC8383041 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background/purposeHigh glucose (HG)-induced aberrant proliferation, apoptosis and odontoblastic differentiation of dental pulp cells (DPCs) have been implicated in the pathogenesis of impaired diabetic pulp healing; however, the underlying mechanism remains unclear. This study aimed to investigate the role of mitochondrial reactive oxygen species (mtROS) and mitochondria in HG-induced dysfunction and apoptosis of DPCs.Materials and methodsHuman DPCs (hDPCs) were cultured in a low-glucose, high-glucose, mannitol, and MitoTEMPO medium in vitro. Methylthiazol tetrazolium assay, Annexin V-FITC/PI staining and scratch-wound assay were used to analyze cell proliferation, apoptosis and migration, respectively. Alkaline phosphatase staining and alizarin red S staining were used to evaluate cell differentiation. DCF-DA staining, MitoSOX staining, MitoTracker Red staining, JC-1 staining, and adenosine triphosphate (ATP) kit assay were performed to investigate total ROS and mtROS generation, mitochondrial density, mitochondrial membrane potential (MMP), and ATP synthesis, respectively. Quantitative PCR assay was performed to detect the mRNA expression of mitochondrial biogenesis- and dynamics-related markers. Transmission electron microscopy was used to observe the mitochondrial ultrastructure.ResultsHG augmented the production of total ROS and mtROS, and triggered mitochondrial damage in hDPCs, as reflected by decreased mitochondrial density, depolarized MMP, reduced ATP synthesis, altered mRNA expression of mitochondrial biogenesis- and dynamics-related markers, and abnormal mitochondrial ultrastructure. Supplementation of MitoTEMPO alleviated the mitochondrial damage and reversed the aberrant proliferation, apoptosis, migration and odontoblastic differentiation of HG-stimulated hDPCs.ConclusionHG triggers mitochondrial damage via augmenting mtROS generation, resulting in the inhibited proliferation, migration, and odontoblastic differentiation of hDPCs and enhanced their apoptosis.

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Reactive oxygen species induced by uric acid promote NRK‑52E cell apoptosis through the NEK7‑NLRP3 signaling pathway.

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