Tetrandrine attenuates diabetic kidney disease by suppressing the TLR4-NF-κB-NLRP3 inflammatory signaling pathway in proximal tubular cells.
Abstract
Background
Diabetic kidney disease (DKD) involves persistent tubular inflammation. Tetrandrine (Tet), a bioactive alkaloid from Stephania tetrandra S. Moore, has renoprotective potential, but its effects on TLR4-associated inflammatory signaling remain incompletely understood.
Methods
Male KK-Ay mice received Tet (10, 20, or 40 mg/kg/day) for 12 weeks. Biochemical parameters, urinary albumin-to-creatinine ratio, renal histopathology, and single-nucleus transcriptomes were assessed. Pathway-associated changes were examined in kidney tissues and palmitic acid (PA)-stimulated HK-2 cells. Molecular docking, molecular dynamics simulation, cellular thermal shift assay (CETSA), reciprocal co-immunoprecipitation, and siRNA-mediated TLR4 silencing were used to investigate the mechanism.
Results
Tet improved metabolic and renal injury indices and alleviated renal histopathological damage in KK-Ay mice. Single-nucleus RNA sequencing revealed bidirectional transcriptional remodeling in proximal tubular cells, including reduced expression of selected inflammation- and injury-associated genes. Structural analyses supported a plausible Tet-TLR4 interaction, while CETSA showed increased TLR4 thermal stability. Reciprocal co-immunoprecipitation supported reduced TLR4-MyD88 association after normalization to the respective immunoprecipitated proteins. Tet reduced TLR4 expression, NF-κB p65 activation, and NLRP3-associated inflammatory signaling, with lower mature IL-1β levels in PA-stimulated HK-2 cells. TLR4 silencing reduced MyD88 abundance, the p-p65/total p65 ratio, and mature IL-1β and IL-6 levels compared with negative-control siRNA.
Conclusion
Tet ameliorates experimental diabetic kidney injury, improves metabolic parameters, and attenuates proximal tubular inflammatory responses. The findings support TLR4 as a candidate cellular target and link Tet treatment to reduced TLR4-MyD88 association and attenuation of NF-κB/NLRP3-associated inflammatory signaling.