Huang, Chou‐Long

Background and Aims: Liver fibrosis and cirrhosis are major global health burdens with limited treatments. Activated 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 profibrotic PDGF-B. PDGF-mediated glycolysis in HSCs was attenuated by the 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 WNK1 carrying threonine 301 to glycine mutation (WNK1 T301G ) 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.

Key Points α Klotho as an antiaging protein can extend the lifespan of healthy animals; serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease. Transgenic α Klotho and recombinant α Klotho delayed cyst growth in Pkd1 mutant mouse kidneys. α Klotho exerted its proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1. Background α -Klotho ( α Klotho) is expressed mainly in the kidney, which is encoded by the KL gene, existing in a membrane-bound form and a soluble circulating form. Soluble serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease (ADPKD). However, the role of α Klotho in ADPKD remains unknown. Methods To investigate the roles of α Klotho in ADPKD, we generated transgenic α Klotho and Pkd1 mutant Pkd1-HOMO-KL Tg mice to evaluate cyst growth and investigated underlying mechanisms with single-cell RNA sequencing analysis. Results We showed that α Klotho was lower in diverse kidney cell types in Pkd1 mutant kidneys, which was restored by transgenic α Klotho. We identified differential expression genes and pathways mediated by transgenic α Klotho at single-cell resolution and determined the cytokines and their receptors mediated cell-to-cell communication networks in Pkd1 mutant and Pkd1-HOMO-KL Tg kidneys by CellChat analysis. We further showed that α Klotho exerted a proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1, which disturbed TNF and its receptor mediated prosurvival complex, leading to ( 1 ) the formation of predeath complex to increase cystic cell death and ( 2 ) the decrease of the activation of NF-κB, the expression of cytokines, and the recruitment of macrophages, resulting in a delay of cyst growth. Treatment with recombinant α Klotho also delayed cyst growth in Pkd1 mutant kidneys. Conclusions This study defined an unknown role and mechanism of α Klotho in the regulation of a TNF-dependent cystic cell apoptosis and inflammation in Pkd1 mutant kidneys.