■慢性肾脏病(CKD)缺乏有效的治疗方法,肾纤维化(RF)是CKD的结果之一。Dickkopf3(DKK3)已被鉴定为CKD的激动剂。然而,DKK3在CKD中的作用机制尚不完全清楚.
■H2O2处理的HK-2细胞和输尿管梗阻(UUO)小鼠用作RF模型。生物标志物,Masson染色,PAS染色,和TUNEL用于评估肾功能和细胞凋亡。还评估了氧化应激和线粒体功能。CCK-8和流式细胞术用于评估细胞活力和凋亡。西方印迹,IHC,和qRT-PCR检测分子表达水平。免疫荧光用于确定亚细胞定位。双荧光素酶测定,MeRIP,RIP,和ChIP用于验证m6A水平和分子相互作用。
■DKK3在UUO小鼠肾组织和H2O2处理的HK-2细胞中上调。敲除DKK3抑制氧化应激,维持线粒体稳态,减轻UUO小鼠的肾脏损伤和RF。此外,DKK3沉默抑制HK-2细胞凋亡,氧化应激,和线粒体裂变。机械上,DKK3上调与METTL3调控的高m6A水平有关。DKK3通过与其启动子结合而激活TCF4/β-连环蛋白并增强MFF转录表达。MFF的过表达逆转了DKK3敲低对细胞损伤的抑制作用。
■m6A修饰引起的DKK3上调激活了Wnt/β-catenin通路以增加MFF转录表达,导致线粒体功能障碍和氧化应激,从而促进RF进展。
UNASSIGNED: Chronic kidney disease (CKD) lacks effective treatments and renal fibrosis (RF) is one of CKD\'s outcomes. Dickkopf 3 (
DKK3) has been identified as an agonist in CKD. However, the underlying mechanisms of
DKK3 in CKD are not fully understood.
UNASSIGNED: H2O2-treated HK-2 cells and ureteric obstruction (UUO) mice were used as RF models. Biomarkers, Masson staining, PAS staining, and TUNEL were used to assess kidney function and apoptosis. Oxidative stress and mitochondria function were also evaluated. CCK-8 and flow cytometry were utilized to assess cell viability and apoptosis. Western blotting, IHC, and qRT-PCR were performed to detect molecular expression levels. Immunofluorescence was applied to determine the subcellular localization. Dual luciferase assay, MeRIP, RIP, and ChIP were used to validate the m6A level and the molecule interaction.
UNASSIGNED: DKK3 was upregulated in UUO mouse kidney tissue and H2O2-treated HK-2 cells. Knockdown of DKK3 inhibited oxidative stress, maintained mitochondrial homeostasis, and alleviated kidney damage and RF in UUO mice. Furthermore, DKK3 silencing suppressed HK-2 cell apoptosis, oxidative stress, and mitochondria fission. Mechanistically,
DKK3 upregulation was related to the high m6A level regulated by METTL3. DKK3 activated TCF4/β-catenin and enhanced MFF transcriptional expression by binding to its promoter. Overexpression of MFF reversed in the inhibitory effect of
DKK3 knockdown on cell damage.
UNASSIGNED: Upregulation of DKK3 caused by m6A modification activated the Wnt/β-catenin pathway to increase MFF transcriptional expression, leading to mitochondrial dysfunction and oxidative stress, thereby promoting RF progression.