Indoxyl sulfate enhances endothelin-1-induced contraction via impairment of NO/cGMP signaling in rat aorta

Takayuki Matsumoto 1 • Keisuke Takayanagi1 • Mihoka Kojima1 • Kumiko Taguchi1 • Tsuneo Kobayashi1

The microbiome-derived tryptophan metabolite, indoxyl sulfate, is considered a harmful vascular toxin. Here, we examined the effects of indoxyl sulfate on endothelin-1 (ET-1)-induced contraction in rat thoracic aortas. Indoxyl sulfate (10−3 M, 60 min) increased ET-1-induced contraction but did not affect isotonic high-K+-induced contraction. The ET-1-induced contraction was enhanced by endothelial denudation in both control and indoxyl sulfate-treated groups. BQ123 (10−6 M), an ETA receptor antagonist, reduced the ET-1-induced contraction in both control and indoxyl sulfate groups. BQ788 (10−6 M), an ETB receptor antagonist, increased the contraction in the control group but had no effect on the indoxyl sulfate group. Conversely, indoxyl sulfate inhibited relaxation induced by IRL1620, an ETB receptor agonist. L-NNA, an NO synthase (NOS) inhibitor, increased the ET-1-induced contractions in both the control and indoxyl sulfate groups, whereas L-NPA (10−6 M), a specific neuronal NOS inhibitor, did not affect the ET-1-induced contraction in both groups. However, ODQ, an inhibitor of soluble guanylyl cyclase, increased the ET-1-induced contraction in both groups. Organic anion transporter (OAT) inhibitor probenecid (10−3 M) and antioxidant N-acetyl-L-cysteine (NAC; 5 × 10−3 M) inhibited the effects of indoxyl sulfate. A cell-permeant superoxide scavenger reduced the ET-1-induced contraction in the indoxyl sulfate group. The aortic activity of SOD was reduced by indoxyl sulfate. The present study revealed that indoxyl sulfate augments ET-1-induced contraction in rat aortae. This enhancement may be due to the impairment of NO/cGMP signaling and may be attributed to impairment of the antioxidant systems via cellular uptake through OATs.

Keywords Aorta . Contraction . Endothelin-1 . Indoxyl sulfate . Nitric oxide


Endothelin-1 (ET-1) is one of major vasoactive substances with potent vasoconstrictor, proinflammatory, and proliferative effects [5, 12, 14, 16, 28, 42, 68]. The expression and functional effects of ET-1 and the signaling of its receptors, such as ETA and ETB receptors, are dysregulated in diseases such as diabetes, hypertension, pulmonary hypertension, hyperlipidemia, atherosclerosis, and chronic kidney disease (CKD) [5, 12, 14, 16, 29, 42, 50]. Since ET-1 is an obvious causative factor of vascular dysfunction, a comprehensive understanding of the regulation of the ET-1 system is urgently needed, including not only the signaling pathways in (patho)physiological states, but also the modulators for the system.
A growing body of evidence suggests that gut microbiota– derived substances play an important role in the regulation of host body functions including cardiovascular function [8, 67, 72, 73, 77, 79]. Among the gut-derived substances, indoxyl sulfate, a uremic toxin, accumulates in patients with CKD as a consequence of altered gut microbiota metabolism and a decline in renal excretion [22, 25, 67, 72]. Indoxyl sulfate has diverse adverse effects on vascular endothelial and smooth muscle cells, including proinflammatory and oxidative stress, prothrombotic events, endothelial cell senescence, reduction of nitric oxide (NO) bioavailability, promotion of vascular smooth muscle proliferation, migration, and calcification [22, 25, 49, 63, 67, 72]. Moreover, several reports suggest that indoxyl sulfate impairs endothelium-dependent relaxation in several blood vessels [51, 53, 55, 64]. However, few studies have investigated the relationship between indoxyl sulfate and endogenous vasoactive substances.
The cross-talk between indoxyl sulfate and ET-1 may play an important role in the regulation of vascular tone in the initiation and/or progression of vascular dysfunction. To date, however, no studies have demonstrated the relationship between indoxyl sulfate and vascular responses induced by ET-1.
In the present study, we hypothesized that the direct exposure of vasculature to indoxyl sulfate would alter vascular responsiveness to ET-1. We investigated whether changes in ET-1-induced responses of the thoracic aorta would occur as a result of acute direct exposure to indoxyl sulfate in rats. We used rat thoracic aorta because ET-1-mediated vascular dysfunction has been identified in chronic diseases such as diabetes [32, 56, 57] and CKD [36], as well as to complement our recent study that demonstrated the inhibitive effect of indoxyl sulfate on endothelium-dependent relaxation in the aorta of Wistar rats [52].

Materials and methods


1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and Nacetyl-L-cysteine (NAC) were purchased from Wako (Osaka, Japan), BQ123 and BQ788 were purchased from Adipogen Life Sciences (San Diego, CA USA), ET-1 and IRL1620 were purchased from PEPTIDE Institute Inc. (Osaka, Japan), and acetylcholine chloride (ACh) was purchased from DaiichiSankyo Pharmaceuticals (Tokyo, Japan). Phenylephrine (PE), NG-nitro-L-arginine (L-NNA), polyethylene glycolsuperoxide dismutase (PEG-SOD), and indoxyl sulfate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Probenecid was purchased from Tokyo Chemical Industry (Tokyo, Japan), Nω-propyl-L-arginine (L-NPA) was purchased from Tocris Bioscience (Bristol, UK), and SOD assay kit-WST was purchased from Dojindo Lab. (Kumamoto, Japan).


In this study, we used male Wistar rats aged 3–4 months (Sankyo Labo Service Corporation, Inc., Tokyo, Japan) as used in our previous report [49]. All procedures were conducted according to the Guiding Principles for the Care and Use of Laboratory Animals from the Committee for the Care and Use of Laboratory Animals of Hoshi University (Tokyo, Japan) (permission code: 20-010), where the studies were performed.

Vascular functional study

The vascular isometric forces of rat thoracic aorta were monitored as described in previous studies [2, 31, 45, 48, 52, 53]. In all experiments, rats were anesthetized with isoflurane via a nose cone and sacrificed via exsanguination from the abdominal aorta. Subsequently, the thoracic aorta was rapidly and carefully isolated and placed in an ice-cold, oxygenated, modified Krebs–Henseleit Solution (KHS). This solution consisted of 118.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO3, 1.8 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgSO4, and 11.0 mM glucose. Each aorta was separated from the surrounding fat and connective tissue, cut into rings (approximately 1.8 mm in diameter), and then mounted on the organ bath system. The isometric force was measured and recorded using the PowerLab system (AD Instruments) as reported previously for aortic rings [2, 31, 45, 48, 52, 53]. Endothelium-denuded rings were prepared by gently rubbing the intimal surface of the vessels using a pipette tip [2, 31, 45, 52, 53]. Arterial integrity was assessed by contracting the aortic rings with high-K+ (80 mM) and subsequently with PE (10–6 M), followed by relaxation with ACh: 10–6 for endothelium-intact rings and 10–5 M for endotheliumdenuded rings. After washing and re-stabilization, the rings were incubated with indoxyl sulfate (10−4, 3 × 10−4, or 10−3 M) [64] or without (control) for 60 min. Concentration– response curves for ET-1 (10−10–3 × 10−8 M), isotonic highK+ (10–80 mM), and IRL1620 (10−10–10−6 M) were obtained for the vascular contraction study. For the relaxation study, IRL1620 (10−10–10−6 M) was cumulatively applied after a plateau contraction was achieved with PE.
The effects of specific ET receptor antagonists and nitric oxide synthase (NOS) inhibitors on ET-1-induced contraction in the indoxyl sulfate-treated aorta were investigated The aortic ring was co-incubated for 60 min with indoxyl sulfate (10−3 M); BQ123 (10−6 M), a specific ETA receptor antagonist [32, 44]; BQ788 (10−6 M), a specific ETB receptor antagonist [32, 44]; L-NNA (10−4 M), a non-selective NOS inhibitor [44]; and L-NPA (10−6 M), selective neuronal NOS (nNOS) inhibitor [40]. A cumulative application of ET-1 (10−10–3 × 10−8 M) was performed immediately after.
To investigate the effect of soluble guanylyl cyclase (sGC) inhibitor on ET-1-induced aortic contraction, the aortic ring was co-incubated for 60 min with indoxyl sulfate (10−3 M); ODQ (10−5 M), a specific sGC inhibitor [46], or a vehicle, 0.1% dimethyl sulfoxide (DMSO), before ET-1 (10−10–3 × 10−8 M) cumulative application.
To investigate the effects of antioxidant NAC on ET-1induced contraction in the indoxyl sulfate-treated aorta, the aortic ring was preincubated with or without NAC (5 × 10−3 M) [59, 60] for 30 min. Thereafter, indoxyl sulfate (10−3 M) was applied for 60 min before ET-1 (10−10–3 × 10−8 M) cumulative application. To investigate the effects of a cell-permeant superoxide scavenger on ET-1-induced contraction in the indoxyl sulfate-treated aorta, the aortic ring was coincubated for 60 min with indoxyl sulfate (10−3 M) and PEG-SOD (41 U/mL) [47] before ET-1 (10−10–3 × 10−8 M) cumulative application.
To investigate the effects of an organic anion transporter (OAT) inhibitor on ET-1-induced contraction in the indoxyl sulfate-treated aorta, the aortic ring was preincubated with probenecid (10−3 M [17, 52] or a vehicle (1.0% ethanol) for 30 min. Thereafter, indoxyl sulfate (10−3 M) was applied for 60 min before ET-1 (10−10–3 × 10−8 M) cumulative application.

Measurement of aortic SOD activity

The aortic rings were incubated in KHS with or without indoxyl sulfate (10−3 M) for 60 min at 37 °C. After that, the rings were weighed and frozen in liquid N2 and store at −80°C. Aortic samples were homogenized in sucrose buffer (250 mM sucrose, 10 mM Tris-HCl, and 1 mM EDTA; pH 7.4) and then at 10,000×g for 60 min. The supernatant was used to measure SOD activity using SOD assay kits according to the manufacturer’s instructions. SOD activity was expressed as units per milligram of tissue.

Data analysis

The results are presented as means ± standard error (SE). Contractile responses are expressed as millinewton per milligram wet weight of the arterial ring. GraphPad Prism software (RRID: SCR_002798; GraphPad Prism 8 Program; GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. To determine a negative logarithm of EC50 (in ET-1 and IRL1620-induced responses) or EC50 (isotonic highK+induced contraction) values, individual concentration– response curves were fitted using a non-linear regression fitting program with a standard slope or variable slope with GraphPad Prism. Statistical evaluations were performed using Student’s t-test for comparisons between two groups (Fig. 7). Differences between values were evaluated using a two-way repeated measures analysis of variance (ANOVA) followed by Sidak’s (Figs. 1b and 4a, b) or Tukey’s (Figs. 1a, 2, 3a, b, 5a–c, 6a, b, and 8) multiple comparison test. P-value < 0.05 was considered significant. Results Indoxyl sulfate enhances contractile responses induced by ET-1 but not by high-K+ The cumulative administration of ET-1 (10−10–3 × 10−8 M) induced a concentration-dependent contraction in aortic rings. ET-1-induced contractions tended to increase in the indoxyl sulfate-treated groups (10−4 and 3 × 10−4 M) (vs. control group) (Fig. 1a; Table 1). Specifically, at intermediate concentrations (i.e., 3 × 10−9 M), ET-1-induced contractions were significantly stronger in rings exposed to indoxyl sulfate (10−3 M) than those in the control group (Fig. 1a). Exposure of aortic rings to isotonic high-K+ (10–80 mM) induced a concentration-dependent rise in tension. Indoxyl sulfate (10−3 M for 60 min; EC50 28.3 ± 1.3 mM [n = 5]; Emax 5.60 ± 0.29 mN/mg [n = 5]) did not change the isotonic high-K+-induced contraction compared with the control group (EC50 32.0 ± 2.2 mM [n = 5]; Emax 5.51 ± 0.34 mN/mg [n = 5]) (Fig. 1b). Effect of indoxyl sulfate on ET-1-induced aortic contraction in the presence and absence of endothelium ET-1-induced contraction in the absence and presence of endothelium was examined to determine whether the effect of indoxyl sulfate is through the endothelium or vascular smooth muscle. The ET-1-induced contractions were increased by endothelial denudation in both control and indoxyl sulfatetreated groups (Fig. 2; Table 1). In endothelium-denuded aorta, indoxyl sulfate slightly, but not significantly, increased ET1-induced contraction (Fig. 2; Table 1). ET-1 receptor subtype in the effect of indoxyl sulfate To determine whether the effect of indoxyl sulfate is through ETAor ETB receptor–mediated signaling, we examined ET1-induced contraction in the presence of ETAand ETB receptor antagonists (Fig. 3; Table 1). As illustrated in Fig. 3a, the ETA receptor antagonist BQ123 (10−6 M) reduced ET-1induced contractions in both the control and indoxyl sulfate groups. In BQ123-treated groups, ET-1-induced contraction was greater in the indoxyl sulfate group compared to the control group; however, the ETB receptor antagonist BQ788 (10−6 M) increased ET-1-induced contractions in the control group but had no effect in the indoxyl sulfate group (Fig. 3b). In BQ788-treated groups, ET-1-induced contraction was similar between the indoxyl sulfate and control groups. Indoxyl sulfate inhibits ETB receptor–mediated aortic relaxation The effect of indoxyl sulfate on ETB receptor–mediated responses in rat aortas was also evaluated. We examined the effect of IRL1620, an ETB receptor agonist, on relaxation in the presence and absence of indoxyl sulfate. The tension developed in response to PE was 2.23 ± 0.17 mN/mg in the control group (n = 7) and 2.80 ± 0.99 mN/mg in the indoxyl sulfate group (n = 7; no significant difference). As shown in Fig. 4a, IRL1620absence (control) and presence of indoxyl sulfate (10−3 M for 60 min). Contractions are presented as millinewton per milligram wet weight of the aortic rings. Data are expressed as mean ± SE; n = 9 or 10 (a), 5 (b). *P < 0.05, control vs. indoxyl sulfate (10−3 M) induced relaxation was significantly inhibited in the indoxyl sulfate-treated aortas (–log EC50 7.53 ± 0.91 M; n in the control aortas (–log EC50 8.44 ± 0.24 M; n = 7; Emax 50.2 ± 5.3%; n = 7). The functional contractile component of the ETB receptor in vascular smooth muscle unmasks arteries in some situations [35, 65, 70]. To determine whether indoxyl sulfate would induce ETB receptor function in vascular smooth muscle, we next examined the exposure of endothelium-denuded aortic rings to IRL1620 in both the control and indoxyl sulfate-treated groups (Fig. 4b). In such preparations, IRL1620 did not induce contraction in either group. Relationships among indoxyl sulfate, NO/cGMP signaling, and ET-1-induced contraction in aorta NO/cGMP signaling plays important roles in vasorelaxation and in the modulation of contractile responses in blood vessels [20, 23, 44]. To determine whether the augmented effect of indoxyl sulfate on ET-1-induced contraction would be attributable to NO/cGMP signaling, we applied a representative NOS inhibitor or an sGC inhibitor to the aortic rings. The incubation of rings with L-NNA (10−4 M), which blocks both basal and agonist-induced NOS activity, increased ET-1induced contraction in both control and indoxyl sulfate groups (Fig. 5a; Table 1). In the L-NNA-treated groups, indoxyl sulfate slightly but significantly increased ET-1-induced contraction. Together with eNOS, nNOS plays a role in the regulation of vascular function [7, 9, 10]. To investigate whether nNOS could modify ET-1-induced aortic contraction, we examined ET-1-induced contraction in the presence of L-NPA, a specific nNOS inhibitor (Fig. 5b; Table 1). L-NPA (10−6 M) did not affect ET-1-induced contractions in both control and indoxyl sulfate groups. In the L-NPA-treated groups, indoxyl sulfate slightly but significantly increased ET-1-induced contraction. Moreover, the incubation of rings with ODQ (10−5 M), an sGC inhibitor, increased ET-1-induced contractions in both groups (Fig. 5c; Table 1). In the ODQ-treated groups, indoxyl sulfate slightly, but not significantly increased ET-1-induced contraction. Meanwhile, ACh-induced endothelium-dependent and sodium nitroprusside (SNP)–induced endothelium-independent relaxations were weaker in the indoxyl sulfate group than in the control group (Supplementary Fig. S1). Effect of antioxidant and cell-permeant superoxide scavenger on ET-1-induced aortic contraction in the absence and presence of indoxyl sulfate Since indoxyl sulfate generates ROS and inhibits antioxidant systems [63, 72], we evaluated the effects of antioxidants and cell-permeant superoxide scavengers on ET1-induced contraction (Fig. 6; Table 1). When aortic rings were pretreatment with antioxidant NAC (5 × 10−3 M for 30 min) before indoxyl sulfate exposure (10−3 M for 60 min), treatment with NAC suppressed ET-1-induced contraction in both the control and indoxyl sulfate-exposed groups (Fig. 6a). As shown in Fig. 6b, PEG-SOD (41 U/mL) reduced ET-1induced contraction in the indoxyl sulfate group but not in the control group. Indoxyl sulfate reduces aortic SOD activity Since PEG-SOD suppressed the effect of indoxyl sulfate on ET-1induced contraction (Fig. 6b), we subsequently examined whether aortic SOD activity might be altered by indoxyl sulfate (Fig. 7). As shown in Fig. 7, the aortic SOD activity was significantly lower in the indoxyl sulfate group than in the control group. Effect of OAT inhibitor on ET-1-induced aortic contraction in the absence and presence of indoxyl sulfate Since OATs are involved in the uptake of indoxyl sulfate [17, 58], we examined the effect of probenecid, an OAT inhibitor, on ET-1-induced contraction. As shown in Fig. 8, probenecid (10−3 M) suppressed the effect of indoxyl sulfate on ET-1induced contraction, although it did not influence the contraction in the control group. Discussion The present investigation demonstrates that indoxyl sulfate is an amplifier of ET-1-mediated contraction in rat aortas (Fig. 9). ET-1 not only possesses many biological activities but plays important roles in the vascular system at various pathophysiological states, including vascular tone regulation, (10−3 M for 60 min) groups. (c) Concentration–response curves for ET-1 in aortas in the absence or presence of ODQ (10−5 M for 60 min) in both control (0.1% DMSO) and indoxyl sulfate (10−3 M plus 0.1% DMSO for 60 min) groups. Data are expressed as mean ± SE; n =8 (a, c) or 7 (b). *P < 0.05, control vs. indoxyl sulfate. #P < 0.05, control vs. control-inhibitor. inflammation, and proliferation/migration in vascular smooth muscle [5, 12, 14, 16, 28, 42, 50, 68]. On the other hand, indoxyl sulfate, a protein-bound uremic toxin, has adverse effects on the vascular system [22, 25, 49, 63, 67, 72]. In endothelial cells, indoxyl sulfate causes apoptosis, reduction of NO bioavailability, prothrombotic events, senescence, and release of extracellular vesicles [34, 39, 49, 55]. In vascular smooth muscle cells, indoxyl sulfate induces inflammation, proliferation/migration, calcification, and vascular tone modulation [11, 49, 54, 61, 75, 76]. Despite several common cellular events between both indoxyl sulfate and ET-1, little is known about the cross-talk between indoxyl sulfate and ET-1mediated responses in blood vessels. Thus, we examined ET1-induced contraction in normal rat aorta directly exposed indoxyl sulfate to investigate the relationship between ET-1 and indoxyl sulfate in vascular function. We observed that exposure of normal rat aorta to 10−3 M indoxyl sulfate for 60 min acutely enhanced ET-1-induced contraction (Fig. 1a); however, receptor-independent high-K+-induced contraction was not affected by indoxyl sulfate exposure for 60 min at 10−3 M, suggesting that indoxyl sulfate did not influence calcium-mediated contraction in rat aorta. Our recent studies have demonstrated that indoxyl sulfate impairs ACh-mediated relaxation in healthy rat aorta [52] and superior mesenteric artery [51] after exposure for 30 min at 10−4 M, which are milder conditions than the ones implemented in the present study. On the other hand, under such conditions, indoxyl sulfate did not change SNP-mediated relaxation in both the aorta and superior mesenteric artery [51, 52]. Indoxyl sulfate exposure for 60 min at 10−3 M could impair not only AChbut also SNP-induced relaxation in healthy rat aorta (Supplemental Fig. S1). These results and relevant evidence suggest that indoxyl sulfate to affect NO-mediated responsiveness is primarily targeted in endothelial cells at shorter exposures and lower concentrations and significantly impair both endothelial and vascular smooth muscle cell functions at longer exposure and higher concentrations. In support of this statement, indoxyl sulfate has been shown to directly inhibit ACh-induced relaxation of healthy mouse aorta in a concentrationand timedependent manner, an effect that was already apparent after 30 min exposure and became more marked after 4 days [64]. ET-1 is known to stimulate ETA receptors on vascular smooth muscle cells for vasocontraction and ETB receptors on endothelial cells for vasorelaxation [5, 12, 14, 16, 28, 42, 68]. We observed that treatment with BQ123, an ETA receptor antagonist, reduced ET-1-mediated contraction in both control and indoxyl sulfate-treated aortas (Fig. 3a). ETB receptors on endothelial cells are believed to confer protective effects by mediating vasorelaxation in response to ET-1 or ETB agonists, although vascular smooth muscle ETB receptors contribute to vasocontraction in some situations [35, 65, 70]. On the basis of our results, we suggest that the enhancement of ET-1mediated contraction induced by indoxyl sulfate may be partly attributed to the impairment of relaxation (viz. anti-contractile component) induced by endothelial ETB receptor activation. Furthermore, ETB receptor signaling in vascular smooth muscle cells may play a negligible role in ET-1-induced contraction in aorta. This speculation is supported by data showing that ET-1-induced contraction in aortae was increased by endothelial denudation in both control and indoxyl sulfate groups (Fig. 2), and that increased endothelial ETB receptor signaling reduced ET-1-inuced contraction [69]. Furthermore, evidences in the literature suggest that endothelium-derived relaxing factor (EDRF) regulates the vasoconstrictor property of ET, since its suppression augments ET-1-induced contraction [18, 37, 41]. NO/cGMP signaling plays a pivotal role in the regulation of vascular tone [3, 20, 23], specifically, inducing vasorelaxation and anti-contractile activity [20, 23]. Cross-talk between ET-1 and NO may play an important role in the vascular system [13, 30]. Indoxyl sulfate impairs NO signaling [51, 52, 55]. Indeed, we observed that (1) the representative NOS inhibitor L-NNA (Fig. 5a) and sGC inhibitor ODQ (Fig. 5c) both enhanced ET-1-mediated contraction in both control and indoxyl sulfate-treated groups, whereas indoxyl sulfate In addition to eNOS, nNOS is found not only in the endothelium but also in vascular smooth muscle cells and contributes to vascular function [7, 9, 10]. We observed that L-NPA, a specific nNOS inhibitor, did not affect the ET-1-induced contraction in both control and indoxyl sulfate groups. These results suggest that the contribution of nNOS-derived NO to ET-1-induced contraction might be cancelled out and might be thus not involved in the augmentation of the ET-1-induced contraction induced by indoxyl sulfate. However, there is a limitation in this present study regarding nNOS that should be mentioned. There is a growing body of research on the contribution of nNOS to vascular tone regulation in aortic smooth muscle [7, 9, 10]. As yet, we are unable to determine whether nNOS was present in the enhanced ET-1-induced contraction by indoxyl sulfate. Further investigation will be required on this issue, for example, by using different inhibitors of nNOS or loading conditions. Furthermore, ET-1-induced contraction in the indoxyl sulfate group was not affected by the ETB antagonist, but was further increased by L-NNA or ODQ, indicating that indoxyl sulfate could affect both ETB receptor-stimulated eNOS activity and basal eNOS activity. Another possibility is the partial impairment of the endothelial ETB receptor/eNOS/NO pathway by indoxyl sulfate. Indoxyl sulfate could modify various molecules related to vascular contraction, for example, Rho kinase, and mitogen-activated protein kinases [11, 49, 61]. Further investigations of the relationships among these vascular mechanisms and indoxyl sulfate will offer valuable insights. Oxidative stress, defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses, affects not only NO bioavailability but also vascular tone regulation [19, 26]. ET-1-induced contraction is augmented by ROS in some arteries [38, 43, 78]. On the other hand, indoxyl sulfate possibly induces oxidative stress by including ROS generation and inhibiting antioxidant activity [11, 54, 55]. We observed that the enhancement of ET-1-induced contraction by indoxyl sulfate was considerably suppressed by antioxidant NAC (Fig. 6a). NAC also exhibits antioxidant-independent abilities, including vasorelaxation [66] and vasorelaxation via the activation of voltage-gated K+ channels [24]. Although the suppression of ET-1induced contraction by NAC in the control group may be due to such antioxidant-independent properties, detailed mechanisms remain unclear. In addition to NAC, the effect of indoxyl sulfate on ET-1-induced contraction was greatly reduced by a cell-permeant superoxide scavenger PEGSOD. Moreover, our study showed that aortic SOD activity was reduced by treatment with indoxyl sulfate. In rat aorta, relaxation induced by ETB receptor activation is mediated by NO formation [21], superoxide impairs endotheliumdependent relaxation via NO inactivation [29], and indoxyl sulfate induces reduction of NO bioavailability via superoxide production [55]. The body of evidence in the literature and in the present study findings suggests that indoxyl sulfateinduced augmentation of ET-1-mediated aortic contraction is attributable to the inhibition of the NO/cGMP signaling pathway via the impairment of antioxidative systems. However, further studies are needed to elucidate how and to what extent indoxyl sulfate alters the balance of ROS generation and antioxidant systems in endothelial and vascular smooth muscle cells. As indoxyl sulfate affects cellular functions by transport into the cells through OATs [1, 17, 76], the effect of probenecid, an inhibitor of OATs [15, 52], on ET-1-induced contraction was investigated. We observed that probenecid prevents the enhancement of ET-1-mediated contraction induced by indoxyl sulfate (Fig. 8). These results suggest that indoxyl sulfate can enhance ET-1-mediated contraction via cellular uptake through OATs. Although probenecid may affect other components such as pannexin 1 channel [62] and pendrin inhibition, a Cl–/HCO – exchanger [4], the deleterious effects of indoxyl sulfate can be prevented by suppressing OATs [1, 17, 52]. Aryl hydrocarbon receptor (AhR) is a ligandactivated transcriptional factor and known as an intracellular receptor for indoxyl sulfate [6, 27, 34]. In vascular tissues and cells, indoxyl sulfate increases oxidative stress through AhR [55, 74] and possibly via AhR-independent signaling [27]. 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