Department of Radiology, Affiliated Hospital of North Sichuan Medical College, Medical Imaging Key Laboratory of Sichuan Province, Nanchong, Sichuan Province, China.
Department of Clinical Laboratory, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan Province, China.
Ren Fail. 2024 Dec;46(1):2322037. doi: 10.1080/0886022X.2024.2322037. Epub 2024 Mar 6.
Acute kidney injury (AKI) is a frequent complication of severe acute pancreatitis (SAP). Previous investigations have revealed the involvement of FTO alpha-ketoglutarate-dependent dioxygenase (FTO) and aquaporin 3 (AQP3) in AKI. Therefore, the aim of this study is to explore the association of FTO and AQP3 on proximal tubular epithelial cell damage in SAP-induced AKI.
An AKI model was established in human proximal tubular epithelial cells (PTECs) HK-2 tumor necrosis factor-α (TNF-α) induction (20 ng/mL), after which FTO and AQP3 expression was manipulated and quantified by quantitative real-time PCR and Western blotting. The viability and apoptosis of PTECs under various conditions, and reactive oxygen species (ROS), superoxide dismutase (SOD), and malonaldehyde (MDA) levels within these cells were measured using commercial assay kits and flow cytometry. Methylated RNA immunoprecipitation and mRNA stability assays were performed to elucidate the mechanism of FTO-mediated N6-methyladenosine (mA) modification. Western blotting was performed to quantify β-catenin protein levels in the PTECs.
FTO overexpression attenuated the TNF-α-induced decrease in viability and SOD levels, elevated apoptosis, increased levels of ROS and MDA, and diminished TNF-α-induced AQP3 expression and reduced β-catenin expression, but its silencing led to contradictory results. FTO negatively modulates AQP3 levels in RTECs in an mA-depednent manner and compromises AQP3 stability. In addition, all FTO overexpression-induced effects in TNF-α-induced PTECs were neutralized following AQP3 upregulation.
FTO alleviates TNF-α-induced damage to PTECs by targeting AQP3 in an mA-dependent manner.
急性肾损伤（AKI）是重症急性胰腺炎（SAP）的常见并发症。先前的研究表明，FTO α-酮戊二酸依赖性双加氧酶（FTO）和水通道蛋白 3（AQP3）参与了 AKI。因此，本研究旨在探讨 FTO 和 AQP3 在 SAP 诱导的 AKI 中对近端肾小管上皮细胞损伤的相关性。
采用肿瘤坏死因子-α（TNF-α，20ng/ml）诱导人近端肾小管上皮细胞（PTECs）HK-2 建立 AKI 模型，然后通过定量实时 PCR 和 Western blot 检测 FTO 和 AQP3 的表达情况。采用商业试剂盒和流式细胞术检测不同条件下 PTECs 的活力和凋亡情况，以及细胞内活性氧（ROS）、超氧化物歧化酶（SOD）和丙二醛（MDA）水平。采用甲基化 RNA 免疫沉淀和 mRNA 稳定性测定法阐明 FTO 介导的 N6-甲基腺苷（mA）修饰的机制。采用 Western blot 检测 PTECs 中β-连环蛋白蛋白水平。
FTO 过表达可减弱 TNF-α诱导的活力下降和 SOD 水平降低，增加凋亡，升高 ROS 和 MDA 水平，并降低 TNF-α诱导的 AQP3 表达和减少β-连环蛋白表达，但沉默则导致相反的结果。FTO 以 mA 依赖的方式负调控 RTECs 中的 AQP3 水平，并损害 AQP3 的稳定性。此外，在 TNF-α诱导的 PTECs 中，AQP3 上调可中和 FTO 过表达诱导的所有作用。
FTO 通过以 mA 依赖的方式靶向 AQP3 减轻 TNF-α诱导的 PTECs 损伤。
