Cycloastragenol Regulates Mitochondrial Homeostasis–Mediated Renal Tubular Injury to Ameliorate Diabetic Kidney Disease by Directly Targeting ERK to Modulate TFEB

ABSTRACT Background and Aim Restoring mitochondrial homeostasis to inhibit apoptosis in renal tubular epithelial cells (RTECs) has emerged as a promising therapeutic strategy for diabetic kidney disease (DKD). This study focuses on the therapeutic effect and mechanism of the triterpenoid compound cycloastragenol (CAG) from Astragali Radix in the treatment of DKD. Experimental Procedure The DKD model was established in C57BL/6J db/db mice and AGEs‐induced HK‐2 cells. Various biological techniques such as WB and RT‐PCR revealed that CAG enhanced mitophagy via TFEB, reducing apoptosis in RTECs. Mechanistic studies combining CETSA, molecular docking, and molecular dynamics simulations confirmed the CAG‐ERK interaction. Key Results CAG improved renal function and reduced renal tubular injury in db/db mice. CAG effectively reduced the accumulation of mitoROS, enhanced mitochondrial membrane potential, promoted mitophagy and mitochondrial biogenesis, and restored mitochondrial homeostasis. Mechanistically, CAG enhanced mitophagy in db/db mice and AGEs‐induced HK‐2 cells by stimulating the autophagic flux via regulating TFEB. Moreover, CAG inhibited AGEs‐induced HK‐2 apoptosis, which was reversed by autophagy inhibitor chloroquine (CQ) and siRNA‐TFEB. Importantly, after mutating the valine (VAL) at position 39 of the ERK to alanine (ALA), the binding effect between CAG and ERK was significantly reduced, revealing that CAG directly bound ERK at 39VAL, inhibiting its phosphorylation, thus preventing the phosphorylation of the S142 site of TFEB and enabling TFEB to translocate into the nucleus. Conclusions and Implications CAG ameliorated renal tubule damage in DKD by regulating mitochondrial quality though targeting ERK to regulate TFEB. This research advances drug development and proposes lifestyle interventions (e.g., dietary supplements).