Wang Shenghan, Lei Zhentao, Liu Wei, Shi Yuqiang, Sherryn Sherryn, Gao Qiang, Zhang Bao
Kidney Blood Press Res. 2025 Jun 22:1-16. doi: 10.1159/000546897.
Kidney stones caused by calcium oxalate (CaOx) is a chronic kidney disease. Acyl coenzyme A thioesterases (ACOTs) serve as the key regulators of fatty acids metabolism. However, ACOTs' effect on CaOx kidney stone formation remains to be explored. Here, we aimed to investigate the effect of ACOTs on CaOx kidney stone formation.
HK-2 and M-1 cells were cultured in DMEM/F12 medium supplemented with 10% FBS. Cells were treated with varying concentrations of calcium oxalate (CaC2O4) and transfected with siRNA or plasmid vectors targeting ACOT4 and ACOT6 using Lipofectamine RNAiMAX or Lipofectamine 3000. RT-qPCR and Western blotting were used to analyze gene and protein expression. Cell viability was assessed with CCK-8, and cell apoptosis was measured by flow cytometry. Crystal adhesion was visualized under a microscope. Lactate dehydrogenase (LDH) release was measured using a cytotoxicity assay kit. A kidney stone mouse model was established by injecting glyoxylic acid to induce kidney stones, and tissues were analyzed by Western blotting.
The mRNA and protein levels of several ACOT family members were upregulated in HK-2 cells treated with CaOx (inducing cell injury). Knockdown of ACOT4 and ACOT6 significantly suppressed the activity of CaOx-pretreated HK-2 and M-1 cells, and promoted the crystal formation and LDH release, whereas overexpression of ACOT4 and ACOT6 reduced CaOx crystal-induced kidney cell injury. Furthermore, the levels of p-AKT and p-S6 decreased after ACOT4 and ACOT6 knockdown and increased following ACOT4 and ACOT6 overexpression, suggesting that both ACOT4 and ACOT6 activated Akt/mTOR signaling pathway in HK-2 cells. We also observed that knockdown of ACOT4 and ACOT6 induced the apoptosis of HK-2 cells after CaOx treatment. Inhibition of apoptosis using Z-VAD-FMK reversed the enhanced cell injury caused by CaOx treatment and ACOT4/6 knockdown, suggesting that knockdown of ACOT4 and ACOT6 promoted cell injury via inducing cell apoptosis.
ACOT4 and ACOT6 could be protecting factors for kidney cell injury induced by CaOx via reducing apoptosis and activating Akt/mTOR signaling pathway. The study of the role of ACOT4 and ACOT6 in kidney cell injury provides a new insight into the cause of CaOx kidney stone formation. Its in-depth study may provide new targets for stone treatment.
草酸钙（CaOx）所致肾结石是一种慢性肾病。酰基辅酶A硫酯酶（ACOTs）是脂肪酸代谢的关键调节因子。然而，ACOTs对草酸钙肾结石形成的影响尚待探索。在此，我们旨在研究ACOTs对草酸钙肾结石形成的影响。
HK-2和M-1细胞在补充有10%胎牛血清的DMEM/F12培养基中培养。细胞用不同浓度的草酸钙（CaC2O4）处理，并使用Lipofectamine RNAiMAX或Lipofectamine 3000用靶向ACOT4和ACOT6的小干扰RNA（siRNA）或质粒载体转染。采用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法分析基因和蛋白质表达。用细胞计数试剂盒-8（CCK-8）评估细胞活力，通过流式细胞术检测细胞凋亡。在显微镜下观察晶体黏附情况。使用细胞毒性检测试剂盒测量乳酸脱氢酶（LDH）释放。通过注射乙醛酸诱导肾结石建立肾结石小鼠模型，并通过蛋白质免疫印迹法分析组织。
在用草酸钙处理的HK-2细胞（诱导细胞损伤）中，几个ACOT家族成员的信使核糖核酸（mRNA）和蛋白质水平上调。敲低ACOT4和ACOT6显著抑制了草酸钙预处理的HK-2和M-1细胞的活性，并促进了晶体形成和LDH释放，而ACOT4和ACOT6的过表达减轻了草酸钙晶体诱导的肾细胞损伤。此外，敲低ACOT4和ACOT6后，p-AKT和p-S6水平降低，而ACOT4和ACOT6过表达后升高，这表明ACOT4和ACOT6均激活了HK-2细胞中的Akt/mTOR信号通路。我们还观察到，敲低ACOT4和ACOT6可诱导草酸钙处理后HK-2细胞的凋亡。使用Z-VAD-FMK抑制凋亡可逆转草酸钙处理和ACOT4/6敲低所导致的增强的细胞损伤，这表明敲低ACOT4和ACOT6通过诱导细胞凋亡促进细胞损伤。
ACOT4和ACOT6可能是通过减少凋亡和激活Akt/mTOR信号通路来保护草酸钙诱导的肾细胞损伤的因子。对ACOT4和ACOT6在肾细胞损伤中作用的研究为草酸钙肾结石形成的原因提供了新的见解。对其深入研究可能为结石治疗提供新的靶点。
