With no lysine kinase 1 in hepatic stellate cells promotes mouse and human liver fibrosis through glycolytic triosephosphate isomerase 1

BACKGROUND AND AIMS: Liver fibrosis and cirrhosis are major global health burdens with limited treatments. Activated hepatic stellate cells (HSCs) drive fibrosis through increased proliferation, migration, collagen deposition and glycolysis. The aim of this study is to elucidate the signaling events that lead to enhanced glycolysis, subsequent HSC activation, and liver fibrosis. APPROACH AND RESULTS: Utilizing a phospho-kinase array, we identified with no lysine kinase 1 (WNK1) as a new kinase in primary human HSCs activated by the pro-fibrotic platelet-derived growth factor-B (PDGF). PDGF-mediated glycolysis in HSCs was attenuated by WNK1 selective inhibitor, WNK-IN-11, as measured by live-cell metabolic assay, Glifon-300 fluorescence, Glucose-Glo assay and glucose transporter 1 immunofluorescence. To understand how WNK1 promotes glycolysis, we employed chemical genetics where an engineered WNK1T301G mutant with a modified ATP pocket accommodated a specific ATP-γ-S analogue to thio-phosphorylate its substrates. Thio-phosphorylated proteins were analyzed by mass spectrometry which identified the glycolytic protein, triosephosphate isomerase 1 (TPI1), as a novel WNK1 direct substrate. TPI1 interacted with WNK1 in condensates, membrane-less phase separation structures. Condensate disruption and knockdown of TPI1 or WNK1 reduced HSC proliferation and migration in vitro. In vivo, pharmacological or genetic WNK1 inhibition significantly attenuated liver fibrosis in carbon tetrachloride and high-fat, choline-deficient, L-amino-acid diet mouse models. Finally, WNK1 inhibition reduced fibrogenesis ex vivo in human precision-cut liver slices. CONCLUSIONS: These findings identify WNK1 as a previously unrecognized kinase that promotes HSC activation and liver fibrosis via TPI1-mediated glycolysis, highlighting the WNK1-TPI1 axis as a potential therapeutic target.