Gasdermin D is involved in switching from apoptosis to pyroptosis in TLR4-mediated renal tubular epithelial cells injury in diabetic kidney disease

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.