Qing-Re-Xiao-Zheng-yi-qi formula protects renal function by stabilizing lysosomal membranes and inhibiting NLRP3-inflammasome-mediated pyroptosis in diabetic kidney disease

Qing-Re-Xiao-Zheng-Yi-Qi formula (QRXZYQF), based on the "Shen-Luo-Zheng-Jia" principles of traditional Chinese medicine, has been reported to reduce 24-hour urinary total protein in diabetic kidney disease (DKD) patients, slow disease progression, and improve podocyte injury. This study aims to explore the mechanisms of QRXZYQF in improving podocyte injury. Diabetes was induced in male C57BL/6J mice by intraperitoneal injection of streptozotocin (STZ). After 12 weeks of QRXZYQF, blood glucose, blood urea nitrogen, serum creatinine, microalbumin, and the urinary albumin-to-creatinine ratio (UACR) were monitored. Renal pathological changes were evaluated using hematoxylin and eosin (H&E), Masson, and periodic acid–Schiff (PAS) staining. RNA sequencing (RNA-Seq) was performed to identify differences in renal mRNA expression and enrichment pathways. The involvement of autophagy–lysosomal and NOD-like receptor pathways was examined by western blotting and immunofluorescence in renal tissues and cultured podocytes. Following 12 weeks of QRXZYQF, renal function improved and ECM accumulation and glomerulosclerosis were markedly reduced. Subsequently, RNA-Seq analysis showed that the autophagy–lysosomal and NOD-like receptor signaling pathways were the potential pathways involved in the mechanism of QRXZYQF. Moreover, QRXZYQF reduced the levels of NLRP3, apoptosis-associated speck-like protein (ASC), as well as Caspase-1 in vitro. Furthermore, we performed interventions using the lysosomal membrane-permeabilizing agent (L-leucyl-L-leucine-O-methylester) and found that QRXZYQF inhibits NLRP3 overexpression by protecting lysosomal membranes and preventing the leakage of Cathepsin B (CB) into the cytoplasm. QRXZYQF inhibits NLRP3-mediated podocyte pyroptosis by stabilizing lysosomal membranes, providing insights into its protective mechanism and potential therapeutic targets for DKD.