Nephropathy II Decoction Attenuates Renal Fibrosis <i>via</i> Regulating TLR4 and Gut Microbiota Along the Gut-Kidney Axis

Nephropathy II Decoction (NED) is a widely used Chinese medicinal formulation for managing chronic kidney disease (CKD). Despite its extensive application, the precise mechanisms underlying its therapeutic effects remain poorly understood. This study aims to elucidate the role of NED in attenuating renal fibrosis and to explore its impact on the gut-kidney axis. The principal constituents of NED were analyzed using ultra-performance LC-tandem mass spectrometry (UPLC-MS/MS). A bilateral renal ischemia-reperfusion injury (bIRI) model was employed to induce fibrosis. RT-qPCR was utilized to assess the expression of mRNA related to the toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) and nuclear factor-κB (NF-κB) signaling pathway. Western blotting analysis was performed to identify changes in renal fibrosis markers, TLR4/MyD88/NF-κB pathway proteins, and the colon proteins ZO-1 and Occludin-1. Serum levels of uremic toxins were quantified using enzyme-linked immunosorbent assay (ELISA), and 16S ribosomal RNA (rRNA) gene sequencing was conducted to explore changes in the gut microbiome of the mice. Our study demonstrated that mice in the NED group exhibited reduced serum creatinine, blood urea nitrogen, and urinary protein levels, alongside improvements in kidney damage and a decrease in renal fibrosis markers. In the bIRI group, TLR4/MyD88/NF-κB protein and mRNA levels, as well as intestinal tight junction proteins and enterogenic uremic toxins, were significantly reduced. NED treatment reversed these changes and modified the gut microbiota. Furthermore, fecal microbial transplantation (FMT) alleviated kidney damage and fibrosis in bIRI mice. In summary, NED ameliorates kidney injury and fibrosis by modulating the gut microbiota and may further attenuate fibrosis through the inhibition of TLR4 expression, thereby influencing the gut-kidney axis.