Tangshenning formula alleviates tubular injury in diabetic kidney disease via the Sestrin2/AMPK/PGC-1α axis: Restoration of mitochondrial function and inhibition of ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE

Tangshenning (TSN) is a traditional Chinese medicinal formula developed on principles of kidney tonification and collateral unblocking. TSN, formulated from Astragalus mongholicus Bunge, Rheum palmatum L., Ligusticum chuanxiong Hort., and Rosa laevigata Michx., has demonstrated significant clinical efficacy in the treatment of diabetic kidney disease (DKD). Our previous studies have suggested that TSN mitigates tubular injury in DKD by inhibiting ferroptosis, however, the precise molecular targets and mechanistic pathways underlying these effects remain to be fully elucidated.

AIM OF THE STUDY

We investigated whether the Sestrin2/AMPK/PGC-1α axis serves as a key pathway mediating TSN's protective effects against tubular injury in DKD.

METHODS

In vivo, a spontaneous DKD mouse model was developed using KK-Ay mice. In vitro, human tubular epithelial cells (TECs) were used to establish high glucose and ferroptosis models, as well as a Sestrin2 knockdown model for further analysis. Molecular docking was utilized to examine the binding interactions between TSN's key active components and Sestrin2. Colocalization of Sestrin2 and GPX4 was assessed using dual fluorescence staining. Protein expression levels related to the Sestrin2/AMPK/PGC-1α pathway, ferroptosis markers (SLC7A11 and GPX4), and the tubular injury marker KIM-1 were quantified via Western blot analysis. In vivo, DHE staining, TUNEL staining, and ferrous ion content measurement were performed to evaluate ferroptosis levels in renal tissue. In vitro, the BODIPY 581/591 C11 probe and ferrous ion assay were used to assess ferroptosis levels in TECs. MitoSOX staining, JC-1 assay, and ATP level measurements were conducted to evaluate mitochondrial function in TECs.

RESULTS

In vivo, our results demonstrated that TSN improved renal function, alleviated tubular injury, and reduced pathological damage in DKD mice. Furthermore, TSN upregulated the protein expression of the Sestrin2/AMPK/PGC-1α axis and decreased ferroptosis-related markers in the DKD mouse model. Similarly, in vitro, TSN enhanced the expression of the Sestrin2/AMPK/PGC-1α pathway, restored mitochondrial function, and inhibited ferroptosis in TECs under high glucose and ferroptosis-inducing conditions. Additionally, downregulation of Sestrin2 impaired the therapeutic effects of TSN.

CONCLUSION

TSN alleviates tubular injury in DKD by activating the Sestrin2/AMPK/PGC-1α pathway, restoring mitochondrial function, and inhibiting ferroptosis in TECs.