Endothelial cell-specific knockout of ATG5 ameliorates inflammation and renal fibrosis by regulating pyroptosis

Objectives: ATG5, a key regulator of autophagy-associated inflammation, is markedly upregulated under inflammatory conditions. In this study, we observed significantly elevated ATG5 expression in the kidneys of patients with renal fibrosis and in mouse kidneys after unilateral ureteral obstruction (UUO), particularly in renal tubules and glomeruli. Recent studies suggest that enhanced ATG5-mediated autophagy in tubular epithelial cells targets the NF-κB pathway to alleviate renal injury. The glomerulus is a dense capillary network primarily composed of endothelial cells, which represent a key immune cell population in the kidney. However, whether endothelial ATG5 exerts a function similar to that of tubular ATG5 during the progression of renal fibrosis remains unclear. Methods: First, Sirius red staining, immunohistochemistry staining and other and pathological methods were used to analyze the expression of ATG5 in relation to the progression of renal fibrosis in patients and UUO model mice. We next observed the effects of endothelial-specific knockout of ATG5 in mice with renal fibrosis induced by the UUO model by ultrasound image assessment, HE staining, and Masson staining. Human umbilical vein endothelial cells (HUVECs) were isolated, and their regulation of inflammation and promotion of renal fibrosis were analyzed by overexpression and knockdown of ATG5 using in vitro synthetic mRNA and siRNA interference techniques. Transmission electron microscopy (TEM), qPCR and Western blotting were used to determine the effect of endothelial ATG5 on promoting inflammation-dependent pyroptosis in vitro and in vivo. Finally, the expression of GSDMD, a key pyroptosis gene, was examined in the kidneys of clinical renal fiber patients by immunohistochemistry staining. Results: We successfully generated endothelial-specific ATG5 knockout (Tek-Cre ATG5 KO) mice and found that endothelial cell deletion of ATG5 attenuated UUO-induced renal fibrosis. Mechanistically, endothelial-specific ATG5 knockout suppressed pyroptosis and the release of inflammatory factors in the UUO model, primarily by inhibiting GSDMD expression and its cleavage into the N-GSDMD fragment during canonical NLRP3 inflammasome activation. Electron microscopy results showed that ATG5 mainly caused pyroptosis independent of plasma membrane pores in endothelial cells. Conditioned medium from human umbilical vein endothelial cells with ATG5 knockdown inhibited the expression of the key fibrotic marker TGF-β1 in renal tubular epithelial HK-2 cells. Consistent results were obtained when HK-2 cells were cocultured with serum from UUO mice with endothelial-specific ATG5 knockout. Furthermore, GSDMD expression was positively correlated with the degree of fibrosis in patients with renal fibrosis. Conclusions: Collectively, our findings demonstrate that ATG5 modulates the inflammatory response by mediating pyroptosis and exacerbates UUO-induced renal fibrosis in endothelial cells, which is in striking contrast to its role in renal tubular epithelial cells. These results highlight the cell-type-specific functions of ATG5 during renal fibrogenesis. Furthermore, our study provides a novel theoretical basis for the development of targeted therapeutic strategies against renal fibrosis in clinical practice.