Abstract 4139367: A major uremic toxin indoxyl sulfate impairs macrophage efferocytosis and accelerates atherogenesis: a potential mechanism for cardiovascular risk in chronic kidney disease

Background: Chronic kidney disease (CKD) is a global burden with high unmet medical needs. Despite the availability of potent drugs such as statins, CKD patients have accelerated atherogenesis and succumb to vascular complications. Underlying mechanisms, however, remain unclear. Elevated levels of a major uremic toxin, indoxyl sulfate (IS), is an independent cardiovascular risk factor in CKD patients. Using a multi-layer systems approach followed by in vitro and in vivo experiments, we investigated the mechanisms by which IS accelerates atherogenesis. Methods&Results: Gene expression meta-analysis of microarray datasets of patients with atherosclerosis or CKD (CKD n=102; atherosclerosis n=136) implicated efferocytosis as a shared mechanism between two diseases (Fig. A and B). Impaired efferocytosis contributes to the development of high-risk atherosclerotic plaques. In vitro validation using live cell imaging demonstrated that clinically relevant concentration of IS (1mmol/L) suppressed efferocytosis of human primary macrophages (n=6 PBMC donors) (Fig. C). In in vivo experiments, daily IS administration (100 mg/kg/day, i.p.) for 4 weeks in hyperlipidemic Ldlr-/- mice caused expansion of %-necrotic core area (n=10/group, Fig. D), typical features of high-risk plaques. Multi-omic integration of global proteomics and transcriptomics of IS-treated human primary macrophages and Ldlr-/- mice atherosclerotic plaque further enriched efferocytosis-related signals and identified reduced growth arrest specific protein 6 (GAS6), a tethering molecule between macrophages and dying cells, as a potential key mediator. IS treatment decreased the expression levels of GAS6 in both human primary macrophages (n=14, Fig. E) and Ldlr-/- mice atherosclerotic plaque (n=6, Fig. F). Moreover, in vivo gain-of-function studies in macrophage-selective Gas6 knock-in mice demonstrated that Gas6 restored IS-induced defective efferocytosis in peritoneal macrophages (Fig. G and H). Conclusions: IS suppresses GAS6, impairs human macrophage efferocytosis, and accelerates experimental atherogenesis. Our findings provide molecular bases for the future development of new therapies for cardiovascular complications in CKD patients.