Indoleamine 2,3-dioxygenase-1, a Novel Therapeutic Target for Post-Vascular Injury Thrombosis in CKD.

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia.

Patients with CKD suffer high rates of thrombosis, particularly after endovascular interventions, yet few options are available to improve management and reduce thrombotic risk. We recently demonstrated that indoxyl sulfate (IS) is a potent CKD-specific prothrombotic metabolite that induces tissue factor (TF) in vascular smooth muscle cells (vSMCs), although the precise mechanism and treatment implications remain unclear. Because IS is an agonist of the aryl hydrocarbon receptor (AHR), we first examined the relationship between IS levels and AHR-inducing activity in sera of patients with ESRD. IS levels correlated significantly with both vSMC AHR activity and TF activity. Mechanistically, we demonstrated that IS activates the AHR pathway in primary human aortic vSMCs, and further, that AHR interacts directly with and stabilizes functional TF. Antagonists directly targeting AHR enhanced TF ubiquitination and degradation and suppressed thrombosis in a postinterventional model of CKD and endovascular injury. Furthermore, AHR antagonists inhibited TF in a manner dependent on circulating IS levels. In conclusion, we demonstrated that IS regulates TF stability through AHR signaling and uncovered AHR as an antithrombotic target and AHR antagonists as a novel class of antithrombotics. Together, IS and AHR have potential as uremia-specific biomarkers and targets that may be leveraged as a promising theranostic platform to better manage the elevated thrombosis rates in patients with CKD.

Klotho Protects Against Indoxyl Sulphate-Induced Myocardial Hypertrophy.

Left ventricular hypertrophy (LVH) is a common complication in patients with CKD and an independent risk factor for death. Changes in the levels of uremic solutes or Klotho have been reported to be related to CKD, whereas the relationships between these factors and CKD-associated LVH remain unclear. Here, we investigated the interaction between Klotho and indoxyl sulfate (IS), a typical uremic solute, in CKD-associated LVH. In a survey of 86 patients with CKD, a negative relationship was found between serum levels of IS and Klotho (r=-0.59, P<0.001). Furthermore, serum levels of IS and Klotho were independently associated with LVH (for IS: r=0.69, P<0.001; for Klotho: r=-0.49, P<0.001). In normal mice, intraperitoneal injection of IS for 8 weeks induced LVH accompanied by substantial downregulation of renal Klotho. Notably, IS-induced LVH was more severe in heterozygous Klotho-deficient (kl/+) mice. In vitro, treatment with Klotho strongly inhibited IS-induced cardiomyocyte hypertrophy by blocking oxidative stress and inhibiting p38 and extracellular signal-regulated protein kinase 1/2 signaling pathways. In a mouse model of CKD-associated LVH, the renal expression of Klotho was lower and the level of serum IS was higher than in healthy controls. Moreover, treatment of CKD mice with Klotho protein significantly restrained the development of LVH. Taken together, these results suggest that Klotho is an endogenous protector against IS-induced LVH, and the imbalance between Klotho and IS may contribute to the development of LVH in CKD.

Indoxyl sulfate promotes vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A through oxidative stress.

We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated ß-galactosidase (SA-ß-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-ß-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-ß-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.

Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells.

Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.

Counter-regulatory effects of procalcitonin and indoxyl sulphate on net albumin secretion by cultured rat hepatocytes.

BACKGROUND: Although hypoalbuminaemia is a significant predictor of mortality in haemodialysis (HD) patients, the pathophysiological mechanisms involved remain to be determined. Albumin is a negative acute-phase reactant and many proinflammatory substances are elevated in HD patients. We investigated factors that may affect liver albumin synthesis. METHODS: Hepatocytes were isolated from rat livers and were cultured with interleukin (IL)-4, IL-6, IL-12, tumor necrosis factor (TNF)-alpha, procalcitonin (PCT), a sensitive marker of infection, and indoxyl sulphate (IS), a uraemic toxin. Albumin levels in the supernatant were measured by enzyme-linked immunosorbent assay. Albumin mRNA expression was determined by reverse transcriptase polymerase chain reaction. RESULTS: IL-6 and TNF-alpha significantly decreased albumin levels in a dose-dependent manner (P<0.01 and P<0.05, respectively). In contrast, IL-4 and IL-12 did not modulate albumin production. PCT and IS significantly and dose-dependently increased albumin levels (both P<0.01). PCT increased albumin mRNA expression in the hepatocytes (P = 0.05) and dose-dependently abrogated IL-6-induced suppression of albumin synthesis (P<0.01). IS also blocked the IL-6-induced decrease in net albumin secretion (P<0.01). CONCLUSION: Our findings indicate that PCT and IS protect against suppression of hepatic albumin synthesis caused by proinflammatory cytokines, suggesting their potential role in preventing hypoalbuminaemia in HD patients.