Renal Division, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, PR China.
Renal Division, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, PR China.
Life Sci. 2021 Jan 1;264:118728. doi: 10.1016/j.lfs.2020.118728. Epub 2020 Nov 6.
Diabetic nephropathy (DN) is the most frequent complication of diabetes and causes millions of deaths each year. Finding novel therapy to DN is urgent, which requires a good understanding of the pathogenesis. Aims are to investigate the molecular mechanisms of DN by focusing on ANRIL/miR-497/TXNIP axis.
Kidney tissues were collected from diagnosed DN patients. High glucose (HG) treatment of human renal tubular epithelial cell cells (HK-2) was used as the cell model of DN. qRT-PCR and Western blotting were performed to measure levels of ANRIL, miR-497, TXNIP, IL-1β, IL-18, caspase-1, and NLRP3. LDH leakage and cell viability were determined with commercial LDH activity kit and MTT assay. ELISA was employed to examine secreted IL-1β and IL-18 levels. Flow cytometry was used to examine caspase-1 activity. Dual luciferase assay was performed to validate interactions of ANRIL/miR-497 and miR-497/TXNIP.
ANRIL and TXNIP were elevated in DN kidney tissues and HG-treated HK-2 cells while miR-497 was reduced. ANRIL bound miR-497 while miR-497 directly targeted TXNIP. Knockdown of ANRIL suppressed HG-induced LDH leakage, TXNIP/NLRP3/caspase-1 activation, and increases of IL-1β and IL-18 secreted levels. miR-497 knockdown or TXNIP overexpression reversed the effects of ANRIL knockdown on LDH leakage and pyroptosis-related signaling. miR-497 mimics inhibited caspase-1-dependent pyroptosis while co-overexpression of TXNIP blocked its effects in HG-treated HK-2 cells.
ANRIL promotes pyroptosis and kidney injury in DN via acting as miR-497 sponge to disinhibit TXNIP expression. These results shed light on the mechanisms of DN and provide targets for therapy development.
糖尿病肾病（DN）是糖尿病最常见的并发症，每年导致数百万人死亡。寻找治疗 DN 的新疗法迫在眉睫，这需要对发病机制有很好的了解。本研究旨在通过关注 ANRIL/miR-497/TXNIP 轴来研究 DN 的分子机制。
从确诊的 DN 患者中收集肾组织。高糖（HG）处理人肾小管上皮细胞（HK-2）作为 DN 的细胞模型。qRT-PCR 和 Western blot 用于测量 ANRIL、miR-497、TXNIP、IL-1β、IL-18、caspase-1 和 NLRP3 的水平。使用商用 LDH 活性试剂盒和 MTT 测定法测定 LDH 漏出和细胞活力。ELISA 用于检测分泌的 IL-1β 和 IL-18 水平。流式细胞术用于检测 caspase-1 活性。双荧光素酶报告基因实验验证 ANRIL/miR-497 和 miR-497/TXNIP 的相互作用。
在 DN 肾组织和 HG 处理的 HK-2 细胞中，ANRIL 和 TXNIP 升高，而 miR-497 降低。ANRIL 结合 miR-497，而 miR-497 直接靶向 TXNIP。敲低 ANRIL 抑制 HG 诱导的 LDH 漏出、TXNIP/NLRP3/caspase-1 激活以及增加的 IL-1β 和 IL-18 分泌水平。miR-497 敲低或 TXNIP 过表达逆转了 ANRIL 敲低对 LDH 漏出和焦亡相关信号的影响。miR-497 模拟物抑制 caspase-1 依赖性焦亡，而 TXNIP 的共过表达阻断其在 HG 处理的 HK-2 细胞中的作用。
ANRIL 通过作为 miR-497 海绵来抑制 TXNIP 的表达，从而促进 DN 中的细胞焦亡和肾损伤。这些结果揭示了 DN 的机制，并为治疗开发提供了靶点。
