Astragaloside Ⅳ negatively regulates Gpr97-TPL2 signaling to protect against hyperhomocysteine-exacerbated sepsis associated acute kidney injury.

• HHcy mice were susceptible to LPS-induced AKI. • Hcy caused tubular cell inflammation and cell death via Gpr97-TPL2 activation. • AS-Ⅳ negatively regulates Gpr97-TPL2 signaling to protect against HHcy exacerbated S-AKI. Hyperhomocysteine (HHcy) plays an important role in promoting inflammation and cell death of tubular epithelial cells. However, the role of HHcy and Astragaloside IV (AS-IV) in sepsis associated acute kidney injury (S-AKI) remain unclear. A significant aspect of this study aimed to elucidate the effect of AS-Ⅳ treatment on HHcy-exacerbated S-AKI and reveal its potential mechanism. Male C57BL/6 J mice fed with specific diet containing 2% methionine were established as in vivo models, and AS-Ⅳ was orally administrated continuously for 3 weeks, and then LPS (10 mg·kg⁻¹ bodyweight) was given by a single intraperitoneal injection. The renal morphological changes were evaluated by HE and PAS staining. RNA-sequencing analysis was applied to select key signaling. The NRK-52E cells exposed to Hcy or combined with LPS were used as in vitro models. The mRNA and protein expression levels of Gpr97-TPL2 signaling were examined by qRT-PCR and western blotting assays. In vivo , HHcy mice developed more severe renal injury and prevalent tubular inflammation after LPS injection. In vitro , the levels of NGAL, Gpr97 and TPL2 were significantly increased in NRK-52E cells induced by Hcy (1.6 mM) or in combination with LPS. Notably, the effects of Hcy on TPL2 signaling was abolished by transfecting TPL2 siRNA or treating TPL2 inhibitor, without alterations in Gpr97. However, the enhancement of Gpr97-TPL2 signaling induced by Hcy was counteracted by Gpr97 siRNA. Subsequently, our findings demonstrated that AS-Ⅳ treatment can improve renal function in HHcy-exacerbated S-AKI mice. Mechanistically, AS-Ⅳ alleviated renal tubular damage characterized by abnormal increases in KIM-1, NGAL, TPL2, Gpr97, Sema3A and TNF-α, and decreases in survivin in vivo and in vitro mainly through suppressing the activation of Gpr97-TPL2 signaling. The present study suggested that HHcy-exacerbated S-AKI was mediated mechanically by activation of Gpr97-TPL2 signaling for the first time. Furthermore, our research also illustrated that AS-Ⅳ protected against HHcy-exacerbated S-AKI by attenuating renal tubular epithelial cells damage through negatively regulating Gpr97-TPL2 signaling, proposing a natural product treatment strategy for HHcy-exacerbated S-AKI. The Gpr97 level was significantly increased in HHcy-exacerbated S-AKI in mice and renal tubular epithelial cell, and Gpr97 could regulate TPL2 and Sema3A. AS-Ⅳ inhibited HHcy-induced activation of renal inflammation and cell death by down-regulating the Gpr97-TPL2 pathway in renal tubular epithelial cells. [Display omitted] [ABSTRACT FROM AUTHOR]