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.