Inhibition of the glycogen synthase kinase 3β–hypoxia‐inducible factor 1α pathway alleviates NLRP3‐mediated pyroptosis induced by high glucose in renal tubular epithelial cells

What is the central question of this study? Activation of the glycogen synthase kinase 3 β (GSK-3β)-hypoxia-inducible factor 1 α (HIF-1α) pathway results in stimulation of pyroptosis under high glucose, and exerts actions in a number renal diseases: does this pathway have a role in renal tubular epithelial cells? What is the main finding and its importance? Down-regulation of GSK-3β can inhibit pyroptosis of renal tubular epithelial cells induced by high glucose and this may be related to down-regulation of HIF-1α. This role of the GSK-3β-HIF-1α pathway has not previously been reported and identifies a potential new therapeutic target in diabetic nephropathy.Diabetic nephropathy (DN) is not only one of the main complications of diabetes, but also has a high incidence rate and a high mortality rate. Glycogen synthase kinase 3 β (GSK-3β) and hypoxia-inducible factor 1 α (HIF-1α) have been demonstrated to influence DN by regulating pyroptosis. This study aimed to investigate the effect of the GSK-3β-HIF-1α pathway on pyroptosis of high-glucose (HG)-induced renal tubular cells. Mouse renal proximal tubular epithelial cells (TKPT cells) were induced by HG to simulate DN cell and we transfected TKPT cells with GSK-3β knockdown lentivirus. Western blot analysis confirmed the transfection effects and detected the expression of GSK-3β, HIF-1α, Nod-like receptor protein 3 (NLRP3), cleaved-caspase-1, pro-caspase-1, gasdermin D (GSDMD) and GSDMD-N. The expression of GSDMD-N and HIF-1α were also verified by immunofluorescence. The levels of interleukin (IL)-1β and IL-18 were measured by enzyme linked immunosorbent assay. Flow cytometric analysis determined the apoptosis rate. Results showed that HIF-1α expression was increased in HG-induced TKPT cells, and GSK-3β knockdown could decrease the levels of NLRP3, cleaved-caspase-1, GSDMD-N and HIF-1α, verified by immunofluorescence. Moreover, GSK-3β knockdown suppressed the expression of IL-1β and IL-18, and reduced the apoptosis rate. Lithium chloride (LiCl) interference could cause the same changes as GSK-3β knockdown for HG-induced TKPT cells, and dimethyloxallyl glycine could reverse the effect of GSK-3β-knockdown interference. Our studies definitively demonstrate that the GSK-3β-HIF-1α signalling pathway mediates HG-stimulated pyroptosis in renal tubular epithelial cells and that down-regulation of GSK-3β inhibited HG-induced pyroptosis by inhibiting the expression of HIF-1α. These findings suggest a new potential target for the treatment of DN.