Abstract：
OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1).
METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2\'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB.
RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.
CONCLUSIONS: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.
METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2\'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB.
RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.
CONCLUSIONS: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.
摘要：
目的：肥胖引起的肾损伤参与了糖尿病肾病(DN)的发生发展。这里,我们在暴露于高糖(HG)和游离脂肪酸(FFA)的HK-2细胞中鉴定了泛素特异性肽酶19(USP19)的功能,并确定了其与TGF-β活化激酶1(TAK1)的相关性.
方法:将HK-2细胞暴露于HG和FFA的组合。通过定量RT-PCR(qRT-PCR)检测USP19mRNA表达,通过免疫印迹(IB)进行蛋白质分析。通过细胞计数试剂盒-8(CCK-8)活力和5-乙炔基-2'-脱氧尿苷(EdU)增殖测定来评估细胞生长。流式细胞术检测细胞周期分布和细胞凋亡。通过共免疫沉淀(Co-IP)测定和IB测定USP19/TAK1相互作用和泛素化TAK1水平。
结果：在HG+FFA攻击的HK-2细胞中，USP19被高度表达。在HK-2细胞中,USP19敲低减弱HG+FFA触发的生长抑制和凋亡促进。此外，USP19敲除减轻HG+FFA介导的PTEN诱导的推定激酶1(PINK1)/Parkin途径失活和增加的线粒体活性氧(ROS)在HK-2细胞中的产生。机械上,USP19通过去泛素化稳定TAK1蛋白。重要的是,TAK1表达的增加逆转了HG+FFA攻击的HK-2细胞中USP19敲低介导的表型改变和PINK1/Parkin通路激活。
结论：研究结果表明，USP19通过稳定TAK1在促进HG和FFA联合刺激诱导的HK-2细胞功能障碍中起关键作用，为对抗DN提供了潜在的治疗策略。
方法:将HK-2细胞暴露于HG和FFA的组合。通过定量RT-PCR(qRT-PCR)检测USP19mRNA表达,通过免疫印迹(IB)进行蛋白质分析。通过细胞计数试剂盒-8(CCK-8)活力和5-乙炔基-2'-脱氧尿苷(EdU)增殖测定来评估细胞生长。流式细胞术检测细胞周期分布和细胞凋亡。通过共免疫沉淀(Co-IP)测定和IB测定USP19/TAK1相互作用和泛素化TAK1水平。
结果：在HG+FFA攻击的HK-2细胞中，USP19被高度表达。在HK-2细胞中,USP19敲低减弱HG+FFA触发的生长抑制和凋亡促进。此外，USP19敲除减轻HG+FFA介导的PTEN诱导的推定激酶1(PINK1)/Parkin途径失活和增加的线粒体活性氧(ROS)在HK-2细胞中的产生。机械上,USP19通过去泛素化稳定TAK1蛋白。重要的是,TAK1表达的增加逆转了HG+FFA攻击的HK-2细胞中USP19敲低介导的表型改变和PINK1/Parkin通路激活。
结论：研究结果表明，USP19通过稳定TAK1在促进HG和FFA联合刺激诱导的HK-2细胞功能障碍中起关键作用，为对抗DN提供了潜在的治疗策略。
