Hunan Key Laboratory of Kidney Disease and Blood Purification, Department of Nephrology, The Second Xiangya Hospital of Central South University, Changsha, 410011, Hunan, China.
Center for Medical Research, The Second Xiangya Hospital of Central South University, Changsha, China.
Biol Res. 2023 Feb 2;56(1):5. doi: 10.1186/s40659-023-00416-7.
Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1β in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1β and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined.
Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1β, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1β and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD.
The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68 macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1β in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1β, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA.
Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.
α-激酶 1（ALPK1）是炎症的主要调节因子，已被证明通过促进糖尿病肾病（DN）小鼠中 IL-1β的产生来促进肾纤维化。焦亡参与高糖（HG）诱导的管状细胞损伤，其特征为 Gasdermin D（GSDMD）的激活和 IL-1β和 IL-18 的释放，导致 DN 中的炎症损伤。可以合理地假设 ALPK1 参与 DN 中与焦亡相关的管状损伤。然而，其机制仍未得到明确。
通过免疫组织化学（IHC）染色检测 DN 患者和 DN 小鼠肾组织中焦亡和纤维化相关蛋白的表达。通过链脲佐菌素联合高脂肪饮食诱导建立 DN 模型。通过 Western blot 检测 ALPK1、NF-κB、Caspase-1、GSDMD、IL-1β、IL-18 和 α-SMA 的蛋白水平。将高糖（HG）处理的 HK-2 细胞用作体外模型。用 ALPK1 小干扰 RNA（siRNA）转染 HK-2 细胞以下调 ALPK1。通过流式细胞术测定焦亡率。通过 ELISA 试剂盒评估 IL-1β和 IL-18 的浓度。通过免疫荧光染色观察 NF-κB 和 GSDMD 的易位。
差异表达基因的热图显示，ALPK1、Caspase-1 和 GSDMD 在 DN 组中上调。IHC 显示 DN 患者肾活检组织中 ALPK1、Caspase-1、GSDMD 和 CD68 的表达水平增加。ALPK1 表达和 CD68 巨噬细胞与 DN 患者的管状损伤呈正相关。Western blot 分析显示，DN 小鼠和 HK-2 细胞中 ALPK1、磷酸化 NF-κB P65、GSDMD-NT 和 IL-1β 的表达增加，同时伴有肾纤维化相关蛋白（FN、α-SMA）和间质区巨噬细胞浸润增加。抑制 ALPK1 可减轻 HG 诱导的 HK-2 细胞中 NF-κB、焦亡相关蛋白 Caspase-1、GSDMD-NT、IL-1β、IL-18、α-SMA 和焦亡水平的上调。此外，在用 ALPK1 siRNA 处理后，HG 处理的 HK-2 细胞中 NF-κB 的强度和核易位以及 GSDMD 的膜易位得到改善。
我们的数据表明，ALPK1/NF-κB 途径启动了经典的 Caspase-1-GSDMD 焦亡途径，导致 DN 的管状损伤和间质炎症。
