Saikosaponin D Regulates <scp>HK2</scp> ‐Mediated Glycolytic Lactate Signaling to Alleviate <scp>CKD</scp> ‐Induced Renal Fibrosis

Chronic kidney disease (CKD) is characterized by tubulointerstitial fibrosis and has a high prevalence, with limited clinical treatment options available. Saikosaponin D (SSD) is a major component of the traditional Chinese medicine compound Chaihuang Yishen Granules (CHYS) and exhibits favorable anti-fibrotic effects. However, its role and underlying mechanisms in renal fibrosis remain unclear. To elucidate the protective effects of SSD on CKD-induced renal fibrosis and investigate the underlying mechanism by which SSD alleviates renal fibrosis through regulating hexokinase-2 (HK2)-mediated Smad3 activation, CKD models were established using unilateral ureteral obstruction (UUO) and adenine (ADE) induction. Subsequently, SSD was administered via oral gavage as a therapeutic intervention to observe its protective effects against CKD-induced renal fibrosis. Mechanistically, in vitro experiments involving HK2 overexpression and knockdown, as well as the use of SIS3 to inhibit Smad3 activation, evaluated the regulatory role of HK2 on glycolysis and Smad3. The results demonstrated that SSD treatment significantly improved the abnormal serum creatinine (CRE) and blood urea nitrogen (BUN) levels in CKD mice, alleviated renal pathological damage, and reduced the expression of fibrosis-related proteins (Col-I, FN, α-SMA). HK2 was found to promote glycolysis-related enzymes and Smad3 activation. Inhibition of Smad3 activation with 4 μM SIS3 significantly attenuated TGF-β-induced fibrosis in tubular cells but had no effect on HK2 expression or glycolysis. Direct suppression of LDHA-mediated lactate production using 25 mM oxamic acid sodium (OX) markedly reduced HK2-induced Smad3 activation and tubular cell fibrosis. This study reveals that SSD significantly alleviates CKD-induced renal fibrosis by inhibiting HK2-mediated Smad3 activation. Lactate, not only as the end product of HK2-driven glycolysis, but also acts as a signaling mediator in HK2-regulated Smad3 activation, facilitating its activation.