Department of Nephrology, The Second Xiangya Hospital of Central South University, Hunan Key Laboratory of Kidney Disease and Blood Purification, Changsha, Hunan, China.
Department of Nephrology, The Second Xiangya Hospital of Central South University, Hunan Key Laboratory of Kidney Disease and Blood Purification, Changsha, Hunan, China; State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Arch Biochem Biophys. 2022 Sep 30;727:109347. doi: 10.1016/j.abb.2022.109347. Epub 2022 Jul 7.
Toll-like receptor 4 (TLR4) mediated pathway plays a pivotal role in promoting tubulointerstitial inflammation and contributes to the progression in type 2 diabetic kidney disease (T2DKD). As the first identified key pyroptosis executor, gasdermin D (GSDMD) is activated by caspases and might be the key protein to switch apoptosis to pyroptosis. It remains unclear that role of TLR4 on canonical pyroptosis pathway, and whether GSDMD is involved in switching from apoptosis to pyroptosis in the TLR4-related tubular injury in T2DKD.
Immunohistochemistry staining was used to detect the expression of pyroptosis-related proteins in renal tissues of T2DKD patients. T2DKD models was induced in TLR4 knockout (TLR4) mice through a high-fat diet combined with streptozotocin. Pyroptosis (caspase-1, GSDMD, interleukin 18(IL-18), interleukin 1β(IL-1β)) and apoptosis levels (caspase-3, Bax and Bcl-2) were detected by Western blot. HK-2 cells were cultured under high-glucose (HG) conditions as an in vitro model and then challenged with a TLR4-specific antagonist (TAK-242). GSDMD small interfering RNA (siRNA) and overexpression plasmid were transfected into HK-2 cells to down- or up-regulate GSDMD. The pyroptosis and apoptosis rates were determined by flow cytometry.
The expression levels of caspase-1, GSDMD, IL-18 and IL-1β were increased in renal biopsy tissues of T2DKD patients and GSDMD expression was positively correlated with tubular injury. Silencing GSDMD attenuated HG-induced IL-18, IL-1β, FN and α-SMA, and reduced pyroptotic cells rate in HK-2 cells. Up-regulation of GSDMD inhibited HG-induced expression of Bax and cleaved caspase-3 and reduced apoptosis rate. TLR4 knockout alleviated tubular injury and interstitial macrophages infiltration, improved impaired renal dysfunction, and decreased the expressions of active N-terminal of GSDMD(GSDMD-N), cleaved caspase-1(cl-caspase-1) and cleaved caspase-3(cl-caspase-3) in T2DKD mice. TLR4 inhibition reduced HG-induced pyroptosis and apoptosis level in HK-2 cells, while GSDMD up-regulation increased pyroptosis rate and decreased apoptosis rate.
TLR4 could exacerbate tubular injury and fibrosis via GSDMD-mediated canonical pyroptosis pathway in T2DKD. Activation of GSDMD could inhibit apoptosis and activate pyroptosis, which may involve the potential switch mechanism between TLR4-mediated pyroptosis and apoptosis in T2DKD.
Toll 样受体 4（TLR4）介导的途径在促进肾小管间质性炎症中起着关键作用，并有助于 2 型糖尿病肾病（T2DKD）的进展。Gasdermin D（GSDMD）作为第一个被鉴定的关键细胞焦亡执行者，被半胱天冬酶激活，可能是将细胞凋亡切换为细胞焦亡的关键蛋白。TLR4 对经典细胞焦亡途径的作用以及 GSDMD 是否参与 T2DKD 中 TLR4 相关肾小管损伤中的细胞凋亡向细胞焦亡的转换尚不清楚。
免疫组织化学染色检测 T2DKD 患者肾组织中细胞焦亡相关蛋白的表达。通过高脂肪饮食联合链脲佐菌素诱导 TLR4 敲除（TLR4）小鼠建立 T2DKD 模型。Western blot 检测细胞焦亡（半胱天冬酶-1、GSDMD、白细胞介素 18（IL-18）、白细胞介素 1β（IL-1β））和细胞凋亡水平（半胱天冬酶-3、Bax 和 Bcl-2）。将 HK-2 细胞在高糖（HG）条件下培养作为体外模型，然后用 TLR4 特异性拮抗剂（TAK-242）处理。转染 GSDMD 小干扰 RNA（siRNA）和过表达质粒下调或上调 HK-2 细胞中的 GSDMD。通过流式细胞术测定细胞焦亡和细胞凋亡率。
T2DKD 患者肾活检组织中 caspase-1、GSDMD、IL-18 和 IL-1β 的表达水平升高，GSDMD 表达与肾小管损伤呈正相关。沉默 GSDMD 可减轻 HG 诱导的 IL-18、IL-1β、FN 和α-SMA，并降低 HK-2 细胞中的细胞焦亡细胞率。GSDMD 的上调抑制 HG 诱导的 Bax 和 cleaved caspase-3 的表达，并降低细胞凋亡率。TLR4 敲除减轻了肾小管损伤和间质巨噬细胞浸润，改善了受损的肾功能，并降低了 T2DKD 小鼠中 GSDMD-N、cleaved caspase-1（cl-caspase-1）和 cleaved caspase-3（cl-caspase-3）的活性。TLR4 抑制减少了 HG 诱导的 HK-2 细胞中的细胞焦亡和细胞凋亡水平，而 GSDMD 的上调增加了细胞焦亡率并降低了细胞凋亡率。
TLR4 可通过 GSDMD 介导的经典细胞焦亡途径加重 T2DKD 中的肾小管损伤和纤维化。GSDMD 的激活可抑制细胞凋亡并激活细胞焦亡，这可能涉及 T2DKD 中 TLR4 介导的细胞焦亡和细胞凋亡之间的潜在转换机制。
