Indoxyl Sulfate Promotes Uremic Arterial and Venous Thrombosis by Stabilizing IDO1 via the E3 Ligase SHFM3

BACKGROUND: The uremic milieu of chronic kidney disease (CKD) is characterized by the accumulation of protein-bound uremic toxins, which contribute to thrombosis. Indoxyl sulfate, a bona fide uremic toxin, upregulates indoleamine 2,3-dioxygenase 1 (IDO1), thereby augmenting kynurenine biogenesis. Both toxins activate tissue factor and thrombosis. Despite this pathogenic synergy, the mechanisms of indoxyl sulfate-mediated IDO1 stabilization remain undefined. METHODS: We employed targeted metabolomics, super-resolution microscopy, and gain-and loss-of-function experiments using nanoparticle-mediated gene delivery in mice to modulate SHFM3 expression. This was followed by carotid artery or inferior vena cava thrombosis assays in indoxyl sulfate-fed and adenine-induced CKD models. RESULTS: Among several F-box-containing E3 ligases, split-hand/foot malformation 3 (SHFM3) downregulates IDO1 in endothelial cells. SHFM3 constitutively interacts with IDO1, which is partially disrupted by indoxyl sulfate-induced nuclear sequestration of SHFM3 at concentrations corresponding to early to advanced CKD. SHFM3 interacts with the N-terminus of IDO1, ubiquitinates and destabilizes it. Indoxyl sulfate upregulates IDO1 activity, thereby increasing kynurenine biogenesis in N-terminus- and SHFM3-dependent manners. In an indoxyl sulfate-fed mouse model, endothelial cell-specific SHFM3 knockdown increased IDO1 and tissue factor and accelerated arterial thrombosis, and these effects were reversed by endothelial cell-specific SHFM3 overexpression. CKD mice exhibited 55% higher venous thrombogenicity than controls. Endothelial cell-specific SHFM3 overexpression significantly reversed these effects in control mice and to a lesser extent in CKD mice. Serum indoxyl sulfate levels significantly correlated with the global IDO1 activity and venous clot weights. CONCLUSIONS: SHFM3 functions as an indoxyl sulfate-sensitive potent E3 ligase of IDO1, targeting it for degradation, even with partial interaction with IDO1 in the uremic milieu. This work also described CKD-induced venous thrombosis model and supports the selective rewiring of uremic toxins in post-translational proteostasis, amplifying indoxyl sulfate-mediated arterial and venous thrombosis.