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Deleterious vascular effects of indoxyl sulfate and reversal by oral adsorbent AST-120
Atherosclerosis 243 (2015) 248e256
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Deleterious vascular effects of indoxyl sulfate and reversal by oral adsorbent AST-120 mond a, Jean Marc Chillon a, Sabrina Poirot a, Isabelle Six a, Priscilla Gross a, Mathieu C. Re Tilman B. Drueke a, Ziad A. Massy a, b, * a b
INSERM Unit 1088, Jules Verne University of Picardie, Amiens, France Division of Nephrology, Ambroise Par e University Hospital, AP-HP, Paris-Ile-de-France-Ouest University (UVSQ), Boulogne-Billancourt, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 10 September 2015 Accepted 14 September 2015 Available online 15 September 2015
Background: In chronic kidney disease (CKD), blood vessels are permanently exposed to uremic toxins such as indoxyl sulfate (IS). We hypothesized that IS could alter vascular tone and that reducing its serum concentration could be beneficial. Design: We studied acute and longer-term effects of IS and AST-120, an oral charcoal adsorbent, on vascular reactivity, endothelium integrity and expression of adhesion molecules VCAM-1 and ICAM-1 in aortic rings of normal and uremic wild type (WT) mice in vitro, and the cardiovascular effects of AST-120 in both WT and apoE/ mice with CKD in vivo. Results: In vitro, 1.0 mM IS acutely reduced vascular relaxation (64% for IS 1.0 mM vs. 80% for control, p < 0.05). The effect was more marked after 4 days exposure (39% for IS 1.0 mM 4 days; p < 0.001, prolonged vs. acute exposure), and was associated with endothelial cell loss and upregulation of ICAM-1/ VCAM-1 expression. In vitro, AST-120 restored normal vascular function and prevented IS induced endothelial cell loss and ICAM-1/VCAM-1 upregulation. In vivo, AST-120 treatment of CKD mice (1) improved vascular relaxation (72% vs. 48% maximal relaxation in treated vs. untreated mice, p < 0.001), (2) reduced aortic VCAM-1 and ICAM-1 expression, (3) decreased aorta systolic expansion rate (9 ± 3% CKD vs. 14 ± 3% CKD þ AST-120, p < 0.02), and (4) prevented the increase in pulse wave velocity (3.56 ± 0.17 m/s CKD vs. 3.10 ± 0.08 m/s CKD þ AST-120, p < 0.006). Similar changes were observed in apoE/ mice. Conclusion: IS appears to be an important contributor to the vascular dysfunction associated with CKD. AST-120 treatment ameliorates this dysfunction, possibly via a decrease in serum IS concentration. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Indoxyl sulfate Endothelial dysfunction CKD Mouse AST-120
1. Introduction Cardiovascular events are highly prevalent complications in patients with chronic kidney disease (CKD) [1]. CKD patients exhibit impaired endothelium dependent relaxation [2] and are thought to suffer from accelerated atherosclerosis [3,4]. Endothelial dysfunction is a non-traditional cardiovascular risk factor whose implication in CKD associated cardiovascular mortality has been repeatedly documented [5e7]. CKD is characterized by the accumulation of circulating uremic
Hospital, Paris Ile * Corresponding author. Division of Nephrology, Ambroise Pare de France Ouest (UVSQ) University, 09 Avenue Charles de Gaulle, 92100, Boulogne Billancourt, France. E-mail addresses: [email protected], [email protected] (Z.A. Massy). http://dx.doi.org/10.1016/j.atherosclerosis.2015.09.019 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
toxins [8]. Amongst them, indoxyl sulfate (IS) is a prototype of protein-bound uremic toxins. IS has been associated with overall mortality and cardiovascular disease and mortality in different stages of CKD [9e12]. Experimental and clinical evidence suggests that IS exerts deleterious effects on the vascular endothelium. It inhibits endothelial proliferation and wound repair [13], increases endothelium permeability [14], alters endothelial cell migration [15], increases cell senescence [16] and upregulates adhesion molecule expression [17e19]. These effects may result from a decrease of endothelial NO production and/or NO bioavailability due to induction of oxidative stress [15e17,20e22]. AST-120 is an oral charcoal adsorbent which is capable of limiting intestinal IS absorption. The administration of AST-120 has been shown to decrease oxidative stress [16,23] and improve flowmediated dilation in CKD patients [16] and endothelium dependent relaxation in uremic rat vessels [23].
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We have previously shown that the uremic milieu induces several cardiovascular abnormalities [24]. The specific contribution of IS to the uremic syndrome is still incompletely understood. Moreover, it remains to be determined whether excessive, ‘uremic’ IS concentrations alter vascular tone and reactivity only via endothelial effects or also via direct effects on vascular smooth muscle. The aims of the present study were to investigate (i) the precise role of IS in cardiovascular dysfunction in a more detailed way, in conditions of CKD as well as of intact renal function, and (ii) the potential of AST-120 to prevent altered vessel function and morphology induced by CKD in general and by IS specifically.
2. Materials and methods (see also Supplementary Materials and Methods) Human umbilical vein endothelial cells (HUVECs) were cultured on gelatin-coated flasks with Vascular Cell Basal Medium containing endothelial Cell Growth kit-BBE and supplemented with 2% FBS. Cell viability was evaluated by colorimetric MTT assay (catalog no.M5655-1G from Sigma Aldrich, Saint-Louis, MO, USA) and apoptosis was evaluated using annexin-V-phycoerythrin (PE) staining kit (catalog no.556421 from BD Biosciences San Jose, CA, USA).
2.1. Animals and diet Animal studies were performed in female C57BL/6J wild-type (WT) and apolipoprotein E knock-out (ApoE/) mice aged 8 weeks (Charles Rivers Laboratories, Lyon, France). Animals were handled in accordance with French legislation (Directive 2010/63/ EU of the European Parliament) and the protocol was approved by the local Institution's Animal Care and Use Committee (no 15031205). Mice were anesthetized with intraperitoneal injection of ketamine and xylazine (80 and 8 mg/kg, respectively) for CKD induction and measurements of intravascular blood pressure and pulse wave velocity (PWV).
2.2. Experimental procedure for CKD induction CKD was induced under general anesthesia using a previously described two-step procedure [24]. Mice were sacrificed at 2 or 10 weeks after induction of CKD.
2.3. Exposure to indoxyl sulfate in vitro Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (150 mg/kg) and vascular reactivity studies were carried out in aortic rings ex vivo as described previously [24]. Acute effects of IS were analysed by incubating aortic rings with IS (Sigma Aldrich, Saint-Louis, MO, USA) at concentrations recommended by EUTox, i.e 0.1, 0.5 and 1.0 mM [25], for 30 min before phenylephrine and then acetylcholine stimulations. To study longer-term effects of IS concentration, aortic rings were placed in medium in presence of 1.0 mM IS for 2 or 4 days, and vascular reactivity studies were realized. In some of the experiments, vessels were relaxed using the exogenous NO donor sodium nitroprussiate (SNP) to assess endothelium-independent vascular smooth muscle ability to relax. Aorta rings were included in OCT and the percentage of vessel lumen covered by endothelial cells and adhesion molecule expression were quantified as described previously [24].
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2.4. Exposure to AST-120 in vitro AST-120 was kindly provided by Kureha Industry Co. Tokyo, Japan. The effects on vascular reactivity, endothelium integrity and adhesion molecules expression were investigated in aorta rings maintained in culture medium for 4 days with IS in presence or absence of AST-120 (1.0 g/dL). An IS concentration of 1.0 mM was chosen to obtain a maximal effect on vascular reactivity. 2.5. AST-120 treatment of mice with CKD WT or ApoE/ mice were randomly assigned to one of the following four groups: non CKD mice treated with placebo or AST120 (Non-CKD, Non-CKD þ AST-120), CKD mice treated with placebo or AST-120 (CKD, CKD þ AST-120). AST-120 (4% w/w) or placebo was administered 2 weeks after induction of CKD and the treatment was continued for 8 weeks. Blood samples were drawn through retro-orbital sinus puncture before start of treatment (2 weeks of CKD) or via cardiac puncture at sacrifice. Before and at the end of the AST-120 treatment, serum urea, total cholesterol, phosphorus, total calcium and IS levels were measured. Two transthoracic echocardiography (TTE) scans were performed before induction of CKD and at the end of treatment as described previously [24]. Mitral pulsed Doppler was recorded in order to analyze the isovolumic relaxation time (IVRT) and calculate the Teï index. Left ventricular (LV) mass and aorta's systolic expansion rate (ESAo) was calculated [24]. After treatment with AST-120 or placebo for 8 weeks a subgroup of mice was used to measure blood pressure and PWV as described previously [24]. Another subgroup of mice was used to examine vascular reactivity and to quantify endothelial cell integrity and adhesion molecules expression. In ApoE/ mice, atherosclerotic lesion surface was quantified after Oil Red O staining, as described previously [24]. 2.6. Statistical analysis Results were expressed as means ± SEM. Comparisons between more than two means were performed by using analysis of variance (ANOVA) with or without repeat measurements. The changes over time of the various parameters in each experimental group were analyzed by one-way ANOVA (time). Logarithmic transformation was applied when the distribution was not normal. When a significant difference was found, Scheffe's test for multiple comparisons was used to identify inter-group differences. Wilcoxon test was used to compare echocardiographic parameters at baseline with those at final examination for each group. Inter-group differences were considered to be significant when p < 0.05. 3. Results 3.1. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with normal renal function Exposure of normal control vessels to IS for 30 min acutely altered vascular relaxation in a concentration-dependent manner (p < 0.05 for IS 1.0 mM vs control). (Fig. 1A) This effect was more marked after 4 days of exposure to 1.0 mM IS (p < 0.001 for 1.0 mM IS acutely vs. 1.0 mM IS at day 4) (Fig. 1B). When testing endothelium-independent relaxation in response to SNP, the response remained unchanged after exposure to IS for 4 days, as compared to vessels maintained in culture without addition of IS (maximal relaxation of 102 ± 16% for Control vs. 136 ± 13% for IS, NS).
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Fig. 1. Effects of indoxyl sulfate (IS) in vitro on aortic rings of wild type mice with normal renal function. A) Vascular reactivity of aortic rings incubated for 30 min with IS (0.1, 0.5 and 1.0 mM). *p < 0.05 Control vs. IS 1.0 mM. B) Vascular reactivity of aortic rings incubated for 30 min or 4 days with IS 1.0 mM *:p < 0.05 and ***:p < 0.001 vs. respective control group. C) Effects of IS (1.0 mM) treatment during 30 min or 4 days on CD31, ICAM-1 and VCAM-1 expression. ***:p < 0.001, xxx:p < 0.001 for ICAM-1 and VCAM-1 vs respective controls.
Culture of control vessels for 4 days did not lead to a change in the expression of CD31 or ICAM-1 and VCAM-1, when compared to freshly isolated vessels. IS exposure was associated with decreased CD31 expression both after 30 min and 4 days. The decrease in CD31 expression reflecting endothelial cell loss was found to be due to an increase in endothelial cell apoptosis after 30 min exposure to IS 1.0 mM (Annexin V positive cells of 100 ± 0% for Control vs. 290 ± 107% for IS, p < 0.05, n ¼ 3 experiments). Moreover, cell viability decreased after IS treatment: cell viability was 100 ± 0% for Control vs. 67 ± 10% for IS (p < 0.05, n ¼ 3 experiments).
Exposure to IS upregulated VCAM-1/ICAM-1 expressions with different profiles. ICAM-1 expression was maximal from 30 min at the difference to VCAM-1 with maximal expression after 4 days of exposure (Fig. 1C).
3.2. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with CKD of 2 or 10 weeks duration After 2 weeks, CKD and IS each induced endothelial dysfunction. However, the association of CKD and IS did not worsen endothelial
I. Six et al. / Atherosclerosis 243 (2015) 248e256
dysfunction further. After 10 weeks in Non-CKD vessels, IS induced endothelial dysfunction similar to endothelial dysfunction observed in 2 weeks Non-CKD vessels. Acute exposure to IS at 1.0 mM also did not aggravate this dysfunction further (Fig. 2A) but induced endothelial cell loss and amplified CKD induced ICAM-1 and VCAM-1 upregulation (Fig. 2B). After 2 weeks IS alone induced endothelial cell loss and an increase of adhesion molecule expression in Non-CKD group. The association of CKD and IS did not worsen these parameters further at 2 weeks. After 10 weeks IS alone induced endothelial cell loss and an increase of adhesion molecule expression. The association of CKD and IS worsened endothelial cell loss and the increase of
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ICAM-1 adhesion molecule expression. The effects of IS on endothelial cell integrity and on ICAM-1 expression was much stronger in 10 weeks CKD than in Non-CKD groups. 3.3. Effects of AST-120 in vitro on aortic rings of wild type with normal renal function Exposure of aortic rings to AST-120 for 4 days did not modify acetylcholine induced relaxation. Co-exposure of aortic rings to both IS and AST-120 prevented IS induced altered relaxation. (p < 0.005 for IS 1.0 mM vs AST-120 þ IS 1.0 mM). (Fig. 3A) Same results, i.e IS induced alteration of relaxation and prevention by
Fig. 2. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with CKD for 2 or 10 weeks. A) Effects of IS on vascular reactivity. *:p < 0.05 for Non-CKD vs. 3 other groups. B) Effects of IS on CD31, ICAM-1, and VCAM-1 expression. *:p < 0.05, **:p < 0.005, ***:p < 0.0001 vs Non-CKD and CKD groups at same time, £: p < 0.05, £££: p < 0.0001 vs NonCKD þ IS group at same time. x: p < 0.01, xx: p < 0.0001 vs. Non-CKD at same time.
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week CKD groups. (p < 0.05). In WT mice with CKD, AST-120 treatment restored IS levels to normal (p < 0.0005 for CKD vs CKD þ AST-120). A comparable AST120 treatment effect of borderline significance was found in ApoE/ mice (p ¼ 0.09 for CKD vs. CKD þ AST -120) (Tables 1 and 2). 3.4.2. Echocardiography (Table 3) There was no difference in baseline parameters between the different groups of mice (data not shown). In both ApoE/ and WT mice with CKD, a decrease in ESAo, diastolic dysfunction (increased IVRT and Tei index) and LV hypertrophy were observed on placebo treatment for 10 weeks. In both strains, ESAo was improved by AST-120 treatment. Moreover, AST-120 treatment led to a borderline significant (p ¼ 0.06 for the interaction) improvement of IVRT. In ApoE/ mice with CKD, AST-120 treatment prevented LV hypertrophy. 3.4.3. Arterial blood pressure and pulse wave velocity (Table 4) In both WT and ApoE/ mouse groups blood pressure values remained unchanged after 8 weeks of treatment. PWV was significantly higher in WT CKD than WT non-CKD groups. In ApoE/ mice, there was a similar trend of borderline significance. In WT mice, AST-120 treatment prevented the increase in PWV induced by CKD, with a similar trend in ApoE/ mice.
Fig. 3. Effects of AST-120 in vitro on aortic rings of wild type normal mice. A) Results of vascular reactivity of vessels incubated with 1 mM IS and/or 1 g/dL of AST-120 (AST120, IS 1 mM þ AST-120) for 4 days. ***:p < 0.001 for IS 1 mM vs. control, xx:p < 0.005 for IS 1 mM þ AST-120 vs. IS 1 mM. B) Results of 1 mM IS and/or 1 g/dL of AST-120 (AST-120, IS 1 mM þ AST-120) exposure on CD31, ICAM-1 and VCAM-1 expression. ***:p < 0.001 vs. control and AST-120 groups; x:p < 0.05, xxx:p < 0.001 vs. IS 1 mM.
AST-120 combined with 1 mM IS, were found after 2-day exposure (data not shown). AST-120 treatment prevented endothelial cell loss, at least partially, and the increased adhesion molecule expression induced by IS (Fig. 3B). 3.4. Effects of AST-120 treatment in wild type or ApoE/ mice with CKD
3.4.4. Vascular reactivity in vitro CKD induced an alteration of endothelial function characterized by a less marked relaxation in response to acetylcholine. AST-120 treatment, started 2 weeks after CKD induction, not only prevented the endothelial dysfunction induced by CKD, but restored relaxation to normal. (Fig. 4A) Ten weeks of CKD increased ICAM-1 expression compared to non-uremic controls and 2 weeks of CKD, respectively. VCAM-1 expression was upregulated after 2 weeks of CKD with no further aggravation after 10 weeks. (Fig. 2B) AST-120 treatment prevented ICAM-1 upregulation and restored normal VCAM-1 expression (Fig. 4B). Aortic rings from ApoE/ mice with normal kidney function displayed signs of endothelial dysfunction, characterized by a decrease of relaxation associated with a significant increase in ICAM-1 and VCAM-1 expression. Ten weeks of CKD aggravated the endothelial dysfunction in ApoE/ mice. Again, AST-120 treatment prevented the endothelial dysfunction induced by CKD. (Fig. 4A) AST-120 treatment also reduced the upregulation of VCAM-1 expression after 10 weeks of CKD (Fig. 4B). 3.4.5. Atherosclerotic lesions in aorta of ApoE/ mice In ApoE/ mice, CKD was associated with a higher degree of atherosclerosis (0.78 ± 0.33% of surface area covered by plaques in non CKD group and 1.90 ± 0.52% in CKD group, p < 0.05). AST-120 treatment did not improve the degree of atherosclerosis (1.90 ± 0.49% of surface area covered by plaques in CKD þ AST-120 group). 4. Discussion
3.4.1. Serum biochemistry After two weeks of CKD serum urea, total calcium, phosphorus and total cholesterol levels were increased and body weight decreased, compared with sham-op mice, with more marked differences after 8 weeks of CKD. In WT mice, AST-120 treatment increased serum total calcium as compared to placebo group. In CKD Apo E/ mice, AST-120 treatment improved serum urea (p ¼ 0.005 for the interaction). In both WT and ApoE/ mice, IS concentrations were increased at 10 weeks of CKD compared to non-CKD groups and 2-
Endothelial dysfunction, characterized by altered vascular reactivity, has been recognized as an important risk factor, contributing to the increase in cardiovascular mortality associated with CKD [5e7]. Patients with advanced stages of CKD are permanently exposed to uremic toxins as a result of progressive nephron loss. Particular interest has focused on protein bound uremic toxins since their extracorporeal removal by dialysis techniques is difficult, if not impossible, and several amongst them have been shown to play a role in cardiovascular disease and mortality in
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Table 1 Effects of chronic kidney disease (CKD) and AST-120 on body weight and serum biochemistry in wild type mice. IS: Indoxyl sulfate. Non-CKD þ AST-120
Non-CKD Before start of treatment (2 weeks of CKD) Body weight (g) 20.77 ± 0.94 Urea (mmol/L) 8.76 ± 0.33 Total calcium (mmol/L) 2.02 ± 0.06 Phosphorus (mmol/L) 1.84 ± 0.08 Total cholesterol (mmol/L) 1.89 ± 0.08 After 8 weeks of treatment (at sacrifice, 10 weeks of CKD) Body weight (g) 22.71 ± 0.19 Urea (mmol/L) 9.47 ± 0.24 Total calcium (mmol/L) 2.02 ± 0.01 Phosphorus (mmol/L) 1.61 ± 0.06 Total cholesterol (mmol/L) 1.77 ± 0.04 IS levels (mg/100 ml) 0.10 ± 0.06
CKD þ AST-120
CKD
CKD/AST-120/interaction p
20.69 9.16 2.04 1.99 1.87
± ± ± ± ±
0.16 0.28 0.01 0.06 0.03
19.81 32.52 2.40 2.09 2.52
± ± ± ± ±
0.13 0.90 0.02 0.04 0.04
19.87 35.92 2.49 1.94 2.50
± ± ± ± ±
0.3 2.57 0.04 0.07 0.09
<0.001/NS/NS <0.001/NS/NS <0.001/NS/NS <0.05/NS/NS <0.001/NS/NS
22.63 7.70 2.14 1.86 2.02 0.09
± ± ± ± ± ±
0.21 0.48 0.02 0.08 0.03 0.04
21.38 28.50 2.21 1.92 2.45 0.34
± ± ± ± ± ±
0.21 0.97 0.04 0.07 0.04 0.08
21.46 25.39 2.44 2.09 2.36 0.12
± ± ± ± ± ±
0.27 2.40 0.06 0.12 0.14 0.03
<0.001/NS/NS <0.001/NS/NS <0.001/<0.001/NS <0.005/NS/NS <0.001/0.003/NS <0.05/<0.05/0.06
Table 2 Effects of chronic kidney disease (CKD) and AST-120 on body weight and serum biochemistry in ApoE/ mice. IS: Indoxyl sulfate. Non-CKD þ AST-120
Non-CKD Before start of treatment (2 weeks of CKD) Body weight (g) 22.29 ± 0.32 Urea (mmol/L) 8.57 ± 0.18 Total calcium (mmol/L) 2.07 ± 0.01 Phosphorus (mmol/L) 1.72 ± 0.07 Total cholesterol (mmol/L) 6.22 ± 0.31 After 8 weeks of treatment (at sacrifice, 10 weeks of CKD) Body weight (g) 23.90 ± 0.49 Urea (mmol/L) 7.67 ± 0.32 Total calcium (mmol/L) 2.07 ± 0.02 Phosphorus (mmol/L) 2.07 ± 0.07 Total cholesterol (mmol/L) 5.84 ± 0.39 IS levels (mg/100 ml) 0.17 ± 0.02
CKD þ AST-120
CKD
CKD/AST-120/interaction p
22.63 9.91 2.02 1.87 5.86
± ± ± ± ±
0.29 0.47 0.02 0.07 0.23
20.46 33.99 2.42 2.04 11.53
± ± ± ± ±
0.27 1.16 0.02 0.08 0.37
20.92 31.13 2.50 2.03 10.71
± ± ± ± ±
0.19 1.02 0.03 0.06 0.40
<0.001/NS/NS <0.001/NS/NS <0.001/NS/NS <0.005/NS/NS <0.001/NS/NS
23.77 8.13 2.05 1.80 6.08 0.18
± ± ± ± ± ±
0.49 0.43 0.01 0.07 0.29 0.03
21.67 33.20 2.26 2.06 11.02 0.23
± ± ± ± ± ±
0.65 1.96 0.04 0.11 0.41 0.02
22.25 27.58 2.26 2.12 10.57 0.18
± ± ± ± ± ±
0.26 0.85 0.02 0.08 0.39 0.01
<0.001/NS/NS <0.001/0.02/0.005 <0.001/NS/NS NS/NS/NS <0.001/NS/NS NS/NS/NS
CKD [26e28]. Whether this association results mainly from the induction of endothelial dysfunction or is also due to other effects of uremic toxins is the subject of intensive research. In the present study we demonstrate the ability of IS to directly inhibit relaxation of healthy mouse aorta in a concentration and time dependent manner. This effect was already apparent after a 30 min exposure and was more marked after 4 days. The normal relaxation obtained in response to an exogenous NO donor in presence of IS demonstrates that endothelial cells are the main target of IS, leading to decreased NO production and/or bioavailability. In support of this conclusion, a decrease in NO production, via stimulated reactive oxygen production through NADPH oxidase activation and inhibition of antioxidant systems, has been reported after exposure of endothelial cells to IS [15e17,20e22]. In our in vitro experiments, both acute and longer-term exposure to IS resulted in increased ICAM-1 and VCAM-1 expression and
endothelial cell loss due to an increase of endothelial cell apoptosis. As described in another endothelial cell model using bEnd.3 cells, cell viability decreased after IS treatment [22]. The induction of ICAM-1/VCAM-1 expressions by IS has been reported by other groups [17e19]; however, it was surprising to observe a strong induction of adhesion molecule expression as early as 30 min after IS exposure. In line with this observation, Pletinck et al. recently demonstrated that same concentration of 1.0 mM IS led to a timedependent induction of leukocyte extravasation from rat peritoneal vascular bed, reaching the level of significance after exposure of 30 min [29]. The differing ICAM-1 and VCAM-1 expression profiles induced by IS may be explained by the fact that the basal expression of VCAM-1 is generally lower than that of ICAM-1 but increases more markedly after endothelial cell activation [30]. In CKD mouse vessels, the impairment of endothelium dependent relaxation was not worsened by an even high concentration of
Table 3 Effects of CKD and AST-120 on echocardiographic parameters in WT or ApoE/ mice. Non-CKD Wild type mice ESAo (%) IVRT (msec) Teï index LVM (mg) ApoE¡/¡ mice ESAo (%) IVRT (msec) Teï index LVM (mg)
Non-CKD þ AST-120
CKD þ AST-120
CKD
CKD/AST-120/interaction p
14 16 219 96
± ± ± ±
3 1 18 11
15 17 243 95
± ± ± ±
4 1 21 13
9 21 264 112
± ± ± ±
3 2$$ 51$ 28
14 19 292 107
± ± ± ±
3** 3 62 16
0.01/0.02/NS 0.001/NS/NS 0.001/NS/NS 0.01/NS/NS
13 15 234 99
± ± ± ±
2 2 39 9
12 15 245 99
± ± ± ±
3 2 23 12
9 19 278 116
± ± ± ±
2 2 31 21
12 17 279 99
± ± ± ±
3* 2 42 10**
0.004/NS/0.04 0.001/NS/NS 0.001/NS/NS 0.04/0.04/0.04
ESAo: the aorta's systolic expansion rate, IVRT: isovolumic relaxation time, LVM: the left ventricular mass. $: p < 0.05; $$: p < 0.005 vs. non-CKD placebo; *: p < 0.05, **: p < 0.005 vs. CKD placebo.
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Table 4 Effects of CKD and AST-120 on hemodynamic parameters in WT or ApoE/ mice. Non-CKD Wild type mice SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg PWV, m/sec ApoE¡/¡ mice SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg PWV, m/sec
Non-CKD þ AST-120
CKD þ AST-120
CKD
CKD/AST-120/interaction p
96.7 66.6 86.7 30.1 2.93
± ± ± ± ±
3.1 3.2 3.1 1.2 0.07
92.5 62.3 82.4 30.2 3.13
± ± ± ± ±
3.5 1.9 2.9 2.3 0.12
104.6 70.0 93.1 34.6 3.56
± ± ± ± ±
6.5 4.1 5.7 2.9 0.17$$
97.3 63.8 86.1 33.5 3.10
± ± ± ± ±
2.7 2.3 2.5 1.4 0.08*
NS/NS/NS NS/NS/NS NS/NS/NS 0.06/NS/NS 0.02/NS/0.006
99.7 70.4 89.9 29.2 3.20
± ± ± ± ±
4.7 4.1 4.5 1.6 0.23
105.6 71.3 94.2 34.3 3.40
± ± ± ± ±
7.1 6.0 6.7 1.8 0.28
105.5 71.1 94.1 34.5 3.96
± ± ± ± ±
3.4 3.5 3.4 1.3$$ 0.19$$
101.0 67.0 89.7 34.0 3.50
± ± ± ± ±
5.9 5.7 5.8 1.9 0.18*
NS/NS/NS NS/NS/NS NS/NS/NS 0.08/NS/NS 0.052/NS/NS
SAP, DAP, MAP: systolic, diastolic, mean arterial pressure respectively. PP: pulse pressure. PWV: pulse wave velocity. NS: non-significant. $$: p < 0.005 vs. non-CKD placebo, *p < 0.05 vs. CKD placebo.
Fig. 4. Effects of AST-120 treatment in wild type or ApoE/ mice with CKD 10 weeks. A) Vascular reactivity of aortic rings from WT or ApoE/ mice, Non-CKD or CKD, treated or not with AST-120. **:p < 0.01, ***:p < 0.001 vs. non-CKD; x:p < 0.05 for CKD vs CKD þ AST-120. B) ICAM-1 and VCAM-1 expression in aorta sections from WT or ApoE/ mice, NonCKD or CKD, treated or not with AST-120. ***:p < 0.001 vs. Non-CKD and Non-CKD þ AST-120 groups; x:p < 0.05, xxx:p < 0.001 for CKD vs CKD þ AST-120.
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IS but the toxin induced endothelial cell loss and amplified adhesion molecule upregulation. Since already early stages of CKD are associated with endothelial dysfunction [24] this could explain why IS did not impair endothelial function further. However, the increase in endothelial adhesion molecule expression and loss of CD31 expression in response to prolonged IS exposure in vitro would indicate that (i) the endothelium is effectively an important target for IS, and (ii) high IS concentrations can in addition exert toxic effects on endothelial structure. AST-120 treatment improved IS induced vascular dysfunction in vitro and prevented at least partially IS induced endothelial cell loss. In vivo, IS levels were measured before and at the end of the AST-120 treatment period. The CKD status of 10 weeks' duration induced an increase of IS concentrations. AST-120 treatment reduced the serum IS levels and concomitantly improved the reduced endothelium-dependent vascular response of the aorta of CKD mice. Thus, the beneficial effect of AST-120 on CKD induced vascular dysfunction could be related to the prevention of the deleterious role of IS in vivo. Namikoshi et al. [23] reported that AST-120 ameliorated CKD impaired arterial relaxation but did not demonstrate a major direct vascular effect of IS. Our study is the first to demonstrate a direct induction by IS of endothelial loss and a protective effect of AST-120 against endothelial damage. However, the protective effects of AST120 on the vasculature cannot be attributed to the preservation of endothelium integrity alone. In our two CKD mouse models we confirmed the beneficial AST120 effect on VCAM-1 expression as previously demonstrated by Inami et al. in the adenine rat model of CKD [31]. In our study, we found in addition a beneficial AST-120 effect on ICAM-1 expression in WT mice, although not in ApoE/ mice. The latter may be due to an already high expression of ICAM-1 in this mouse strain. Moreover, in ApoE/ mice AST-120 treatment did not prevent the CKD induced increase in aortic atheromatous plaques. This result is surprising in regard to the positive effects of the adsorbent on adhesion molecule expression and endothelial cell integrity. Adding further complexity to our findings, Yamamoto et al. [32] recently showed a reduction of CKD induced atherosclerosis in response to AST-120, using same ApoE/ mouse model. They evaluated the effect of AST-120, initiating treatment at time of CKD induction and pursuing treatment for 17 weeks. Moreover, they assessed atherosclerosis using en face preparations stained with Sudan IV. Thus, their study differed from ours in several aspects, including earlier treatment initiation, longer treatment time, and site of atherosclerosis measurement. Probably CKD severity was also lower (creatinine clearance approx. 50% of normal in their study vs. serum urea approx. 30 mM in ours) although a direct comparison is not possible. Collectively, these findings would be compatible with the suggestion that a prolonged reduction of serum IS starting in early CKD stages may be beneficial in slowing atherosclerosis progression. Several studies evaluated protective effects of AST120 treatment on CKD induced cardiovascular remodeling, both in animal models [33e35] and in patients [36,37], with mixed results. Some reports showed improvements in arterial stiffness [37], LV hypertrophy and myocardial fibrosis [35] while others only observed an improvement in concentric remodeling, but not hypertrophy [36] or only an improvement in myocardial fibrosis [33]. These highly variable results may be due in part to the different treatment modalities chosen to evaluate the effects of AST120 on the cardiovascular system and in part also due to different degrees of CKD severity and different lengths of treatment [33]. Our echocardiographic results are consistent with some of the earlier results as well as with the results obtained by other techniques in the current study. We observed that aortic compliance was preserved in AST-120-treated
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mice, consistent with our vascular reactivity and PWV findings and the observations of Nakamura et al. [37] We also observed a trend towards improved IVRT, suggesting an inhibitory effect on cardiac hypertrophy which was confirmed in the ApoE/ mice with CKD, in which the increase in LVM was prevented, consistent with the report by Fujii et al. [35]. One limitation of our study is the relatively short treatment time of only 8 weeks. This may explain our inability to detect an AST-120 effect on LVH, despite beneficial vascular actions. In a previous personal study testing the effect of the phosphate binder sevelamer in same experimental model, a treatment time of 8 weeks also was without effect on LVH, whereas treatment prolongation to 14 weeks led to LVH regression [38]. Of note, beneficial effects of sevelamer on vascular function were observed already after 8 weeks of treatment, similarly to the AST-120 effects in the present study. In conclusion, IS is a uremic toxin which exerts deleterious effects on vessel function and morphology via direct actions on vascular endothelium. AST-120 treatment is able to reverse these deleterious effects. Starting the administration of AST-120 in early stages of CKD could lead to an attenuation of the adverse effects of uremic toxins on vascular endothelium early on, prevent the negative consequences of vascular dysfunction for cardiac function and structure, and eventually reduce the occurrence of cardiovascular events and mortality. Funding and acknowledgment gional Priscilla Gross was supported by a grant from Conseil Re de Picardie, Amiens, France (Feder). The study was in part funded by a research grant from Kureha. We want to thank Cedric Boudot for valuable technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.09.019. References [1] A.S. Go, G.M. Chertow, D. Fan, C.E. McCulloch, C.Y. Hsu, Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization, N. Engl. J. Med. 351 (13) (2004 Sep 23), 1296-305. Erratum in: N Engl J Med. 2008;18(4): 4. €m, Oxidative stress and [2] M. Annuk, M. Zilmer, L. Lind, T. Linde, B. Fellstro endothelial function in chronic renal failure, J. Am. Soc. Nephrol. 12 (12) (2001 Dec) 2747e2752. [3] G.M. London, T.B. Drueke, Atherosclerosis and arteriosclerosis in chronic renal failure, Kidney Int. 51 (6) (1997 Jun) 1678e1695. [4] A. Lindner, B. Charra, D.J. Sherrard, B.H. Scribner, Accelerated atherosclerosis in prolonged maintenance hemodialysis, N. Engl. J. Med. 290 (13) (1974 Mar 28) 697e701. [5] R.N. Foley, P.S. Parfrey, M.J. Sarnak, Epidemiology of cardiovascular disease in chronic renal disease, J. Am. Soc. Nephrol. 9 (12 Suppl. l) (1998 Dec) S16eS23. €m, Endothelium-dependent vasodilation and [6] M. Annuk, M. Zilmer, B. Fellstro oxidative stress in chronic renal failure: impact on cardiovascular disease, Kidney Int. Suppl. 84 (2003 May) S50eS53. [7] F. Stam, C. van Guldener, A. Becker, J.M. Dekker, R.J. Heine, L.M. Bouter, C.D. Stehouwer, Endothelial dysfunction contributes to renal functionassociated cardiovascular mortality in a population with mild renal insufficiency: the Hoorn study, J. Am. Soc. Nephrol. 17 (2) (2006 Feb) 537e545. [8] N. Neirynck, R. Vanholder, E. Schepers, S. Eloot, A. Pletinck, G. Glorieux, An update on uremic toxins, Int. Urol. Nephrol. 45 (1) (2013 Feb) 139e150. [9] F.C. Barreto, D.V. Barreto, S. Liabeuf, N. Meert, G. Glorieux, M. Temmar, G. Choukroun, R. Vanholder, Z.A. Massy, European Uremic Toxin Work Group (EUTox). Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients, Clin. J. Am. Soc. Nephrol. 4 (10) (2009 Oct) 1551e1558. [10] M.L. Melamed, L. Plantinga, T. Shafi, R. Parekh, T.W. Meyer, T.H. Hostetter, J. Coresh, N.R. Powe, Retained organic solutes, patient characteristics and allcause and cardiovascular mortality in hemodialysis: results from the retained organic solutes and clinical outcomes (ROSCO) investigators, BMC Nephrol. 14
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[25] EUToX Uremic Solutes Database. Available online: http://eutoxdb.odeesoft. com/index.php. [26] S. Liabeuf, T.B. Drüeke, Z.A. Massy, Protein-bound uremic toxins: new insight from clinical studies, Toxins (Basel) 3 (7) (2011 Jul) 911e919. [27] S. Liabeuf, N. Neirynck, T.B. Drüeke, R. Vanholder, Z.A. Massy, Clinical studies and chronic kidney disease: what did we learn recently? Semin. Nephrol. 34 (2) (2014 Mar) 164e179. [28] S. Ito, M. Yoshida, Protein-bound uremic toxins: new culprits of cardiovascular events in chronic kidney disease patients, Toxins (Basel) 6 (2) (2014 Feb 20) 665e678. [29] A. Pletinck, G. Glorieux, E. Schepers, G. Cohen, B. Gondouin, M. Van Landschoot, S. Eloot, A. Rops, J. Van de Voorde, A. De Vriese, J. van der Vlag, P. Brunet, W. Van Biesen, R. Vanholder, Protein-bound uremic toxins stimulate crosstalk between leukocytes and vessel wall, J. Am. Soc. Nephrol. 24 (12) (2013 Dec) 1981e1994. s, M. Eppihimer, J. Russell, M. Gerritsen, D.C. Anderson, [30] D.D. Henninger, J. Pane D.N. Granger, Cytokine-induced VCAM-1 and ICAM-1 expression in different organs of the mouse, J. Immunol. 158 (4) (1997 Feb 15) 1825e1832. [31] Y. Inami, C. Hamada, T. Seto, Y. Hotta, S. Aruga, J. Inuma, K. Azuma, H. Io, K. Kaneko, H. Watada, Y. Tomino, Effect of AST-120 on endothelial dysfunction in adenine-induced uremic rats, Int. J. Nephrol. 2014 (2014) 164125. [32] S. Yamamoto, Y. Zuo, J. Ma, P.G. Yancey, T.E. Hunley, M. Motojima, A.B. Fogo, M.F. Linton, S. Fazio, I. Ichikawa, V. Kon, Oral activated charcoal adsorbent (AST-120) ameliorates extent and instability of atherosclerosis accelerated by kidney disease in apolipoprotein E-deficient mice, Nephrol. Dial. Transpl. 26 (8) (2011 Aug) 2491e2497. [33] S. Lekawanvijit, A.R. Kompa, M. Manabe, B.H. Wang, R.G. Langham, F. Nishijima, D.J. Kelly, Krum H.Chronic kidney disease-induced cardiac fibrosis is ameliorated by reducing circulating levels of a non-dialysable uremic toxin, indoxyl sulfate, PLoS One 7 (7) (2012) e41281. [34] M. Kuwahara, K. Bannai, H. Segawa, K. Miyamoto, H. Yamato, Cardiac remodeling associated with protein increase and lipid accumulation in earlystage chronic kidney disease in rats, Biochim. Biophys. Acta 1842 (9) (2014 Sep) 1433e1443. [35] H. Fujii, F. Nishijima, S. Goto, M. Sugano, H. Yamato, R. Kitazawa, S. Kitazawa, M. Fukagawa, Oral charcoal adsorbent (AST-120) prevents progression of cardiac damage in chronic kidney disease through suppression of oxidative stress, Nephrol. Dial. Transpl. 24 (7) (2009 Jul) 2089e2095. [36] K. Nakai, H. Fujii, K. Kono, S. Goto, M. Fukagawa, S. Nishi, Effects of AST-120 on left ventricular mass in predialysis patients, Am. J. Nephrol. 33 (3) (2011) 218e223. [37] T. Nakamura, Y. Kawagoe, T. Matsuda, Y. Ueda, N. Shimada, I. Ebihara, H. Koide, Oral ADSORBENT AST-120 decreases carotid intima-media thickness and arterial stiffness in patients with chronic renal failure, Kidney Blood Press Res. 27 (2) (2004) 121e126. [38] J. Maizel, I. Six, S. Dupont, E. Secq, B. Dehedin, F.C. Barreto, J. Benchitrit, re, T.B. Drueke, S. Poirot, M. Slama, C. Tribouilloy, G. Choukroun, J.C. Mazie Z.A. Massy, Effects of sevelamer treatment on cardiovascular abnormalities in mice with chronic renal failure, Kidney Int. 84 (3) (2013 Sep) 491e500.
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Deleterious vascular effects of indoxyl sulfate and reversal by oral adsorbent AST-120 mond a, Jean Marc Chillon a, Sabrina Poirot a, Isabelle Six a, Priscilla Gross a, Mathieu C. Re Tilman B. Drueke a, Ziad A. Massy a, b, * a b
INSERM Unit 1088, Jules Verne University of Picardie, Amiens, France Division of Nephrology, Ambroise Par e University Hospital, AP-HP, Paris-Ile-de-France-Ouest University (UVSQ), Boulogne-Billancourt, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 10 September 2015 Accepted 14 September 2015 Available online 15 September 2015
Background: In chronic kidney disease (CKD), blood vessels are permanently exposed to uremic toxins such as indoxyl sulfate (IS). We hypothesized that IS could alter vascular tone and that reducing its serum concentration could be beneficial. Design: We studied acute and longer-term effects of IS and AST-120, an oral charcoal adsorbent, on vascular reactivity, endothelium integrity and expression of adhesion molecules VCAM-1 and ICAM-1 in aortic rings of normal and uremic wild type (WT) mice in vitro, and the cardiovascular effects of AST-120 in both WT and apoE/ mice with CKD in vivo. Results: In vitro, 1.0 mM IS acutely reduced vascular relaxation (64% for IS 1.0 mM vs. 80% for control, p < 0.05). The effect was more marked after 4 days exposure (39% for IS 1.0 mM 4 days; p < 0.001, prolonged vs. acute exposure), and was associated with endothelial cell loss and upregulation of ICAM-1/ VCAM-1 expression. In vitro, AST-120 restored normal vascular function and prevented IS induced endothelial cell loss and ICAM-1/VCAM-1 upregulation. In vivo, AST-120 treatment of CKD mice (1) improved vascular relaxation (72% vs. 48% maximal relaxation in treated vs. untreated mice, p < 0.001), (2) reduced aortic VCAM-1 and ICAM-1 expression, (3) decreased aorta systolic expansion rate (9 ± 3% CKD vs. 14 ± 3% CKD þ AST-120, p < 0.02), and (4) prevented the increase in pulse wave velocity (3.56 ± 0.17 m/s CKD vs. 3.10 ± 0.08 m/s CKD þ AST-120, p < 0.006). Similar changes were observed in apoE/ mice. Conclusion: IS appears to be an important contributor to the vascular dysfunction associated with CKD. AST-120 treatment ameliorates this dysfunction, possibly via a decrease in serum IS concentration. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Indoxyl sulfate Endothelial dysfunction CKD Mouse AST-120
1. Introduction Cardiovascular events are highly prevalent complications in patients with chronic kidney disease (CKD) [1]. CKD patients exhibit impaired endothelium dependent relaxation [2] and are thought to suffer from accelerated atherosclerosis [3,4]. Endothelial dysfunction is a non-traditional cardiovascular risk factor whose implication in CKD associated cardiovascular mortality has been repeatedly documented [5e7]. CKD is characterized by the accumulation of circulating uremic
Hospital, Paris Ile * Corresponding author. Division of Nephrology, Ambroise Pare de France Ouest (UVSQ) University, 09 Avenue Charles de Gaulle, 92100, Boulogne Billancourt, France. E-mail addresses: [email protected], [email protected] (Z.A. Massy). http://dx.doi.org/10.1016/j.atherosclerosis.2015.09.019 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
toxins [8]. Amongst them, indoxyl sulfate (IS) is a prototype of protein-bound uremic toxins. IS has been associated with overall mortality and cardiovascular disease and mortality in different stages of CKD [9e12]. Experimental and clinical evidence suggests that IS exerts deleterious effects on the vascular endothelium. It inhibits endothelial proliferation and wound repair [13], increases endothelium permeability [14], alters endothelial cell migration [15], increases cell senescence [16] and upregulates adhesion molecule expression [17e19]. These effects may result from a decrease of endothelial NO production and/or NO bioavailability due to induction of oxidative stress [15e17,20e22]. AST-120 is an oral charcoal adsorbent which is capable of limiting intestinal IS absorption. The administration of AST-120 has been shown to decrease oxidative stress [16,23] and improve flowmediated dilation in CKD patients [16] and endothelium dependent relaxation in uremic rat vessels [23].
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We have previously shown that the uremic milieu induces several cardiovascular abnormalities [24]. The specific contribution of IS to the uremic syndrome is still incompletely understood. Moreover, it remains to be determined whether excessive, ‘uremic’ IS concentrations alter vascular tone and reactivity only via endothelial effects or also via direct effects on vascular smooth muscle. The aims of the present study were to investigate (i) the precise role of IS in cardiovascular dysfunction in a more detailed way, in conditions of CKD as well as of intact renal function, and (ii) the potential of AST-120 to prevent altered vessel function and morphology induced by CKD in general and by IS specifically.
2. Materials and methods (see also Supplementary Materials and Methods) Human umbilical vein endothelial cells (HUVECs) were cultured on gelatin-coated flasks with Vascular Cell Basal Medium containing endothelial Cell Growth kit-BBE and supplemented with 2% FBS. Cell viability was evaluated by colorimetric MTT assay (catalog no.M5655-1G from Sigma Aldrich, Saint-Louis, MO, USA) and apoptosis was evaluated using annexin-V-phycoerythrin (PE) staining kit (catalog no.556421 from BD Biosciences San Jose, CA, USA).
2.1. Animals and diet Animal studies were performed in female C57BL/6J wild-type (WT) and apolipoprotein E knock-out (ApoE/) mice aged 8 weeks (Charles Rivers Laboratories, Lyon, France). Animals were handled in accordance with French legislation (Directive 2010/63/ EU of the European Parliament) and the protocol was approved by the local Institution's Animal Care and Use Committee (no 15031205). Mice were anesthetized with intraperitoneal injection of ketamine and xylazine (80 and 8 mg/kg, respectively) for CKD induction and measurements of intravascular blood pressure and pulse wave velocity (PWV).
2.2. Experimental procedure for CKD induction CKD was induced under general anesthesia using a previously described two-step procedure [24]. Mice were sacrificed at 2 or 10 weeks after induction of CKD.
2.3. Exposure to indoxyl sulfate in vitro Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (150 mg/kg) and vascular reactivity studies were carried out in aortic rings ex vivo as described previously [24]. Acute effects of IS were analysed by incubating aortic rings with IS (Sigma Aldrich, Saint-Louis, MO, USA) at concentrations recommended by EUTox, i.e 0.1, 0.5 and 1.0 mM [25], for 30 min before phenylephrine and then acetylcholine stimulations. To study longer-term effects of IS concentration, aortic rings were placed in medium in presence of 1.0 mM IS for 2 or 4 days, and vascular reactivity studies were realized. In some of the experiments, vessels were relaxed using the exogenous NO donor sodium nitroprussiate (SNP) to assess endothelium-independent vascular smooth muscle ability to relax. Aorta rings were included in OCT and the percentage of vessel lumen covered by endothelial cells and adhesion molecule expression were quantified as described previously [24].
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2.4. Exposure to AST-120 in vitro AST-120 was kindly provided by Kureha Industry Co. Tokyo, Japan. The effects on vascular reactivity, endothelium integrity and adhesion molecules expression were investigated in aorta rings maintained in culture medium for 4 days with IS in presence or absence of AST-120 (1.0 g/dL). An IS concentration of 1.0 mM was chosen to obtain a maximal effect on vascular reactivity. 2.5. AST-120 treatment of mice with CKD WT or ApoE/ mice were randomly assigned to one of the following four groups: non CKD mice treated with placebo or AST120 (Non-CKD, Non-CKD þ AST-120), CKD mice treated with placebo or AST-120 (CKD, CKD þ AST-120). AST-120 (4% w/w) or placebo was administered 2 weeks after induction of CKD and the treatment was continued for 8 weeks. Blood samples were drawn through retro-orbital sinus puncture before start of treatment (2 weeks of CKD) or via cardiac puncture at sacrifice. Before and at the end of the AST-120 treatment, serum urea, total cholesterol, phosphorus, total calcium and IS levels were measured. Two transthoracic echocardiography (TTE) scans were performed before induction of CKD and at the end of treatment as described previously [24]. Mitral pulsed Doppler was recorded in order to analyze the isovolumic relaxation time (IVRT) and calculate the Teï index. Left ventricular (LV) mass and aorta's systolic expansion rate (ESAo) was calculated [24]. After treatment with AST-120 or placebo for 8 weeks a subgroup of mice was used to measure blood pressure and PWV as described previously [24]. Another subgroup of mice was used to examine vascular reactivity and to quantify endothelial cell integrity and adhesion molecules expression. In ApoE/ mice, atherosclerotic lesion surface was quantified after Oil Red O staining, as described previously [24]. 2.6. Statistical analysis Results were expressed as means ± SEM. Comparisons between more than two means were performed by using analysis of variance (ANOVA) with or without repeat measurements. The changes over time of the various parameters in each experimental group were analyzed by one-way ANOVA (time). Logarithmic transformation was applied when the distribution was not normal. When a significant difference was found, Scheffe's test for multiple comparisons was used to identify inter-group differences. Wilcoxon test was used to compare echocardiographic parameters at baseline with those at final examination for each group. Inter-group differences were considered to be significant when p < 0.05. 3. Results 3.1. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with normal renal function Exposure of normal control vessels to IS for 30 min acutely altered vascular relaxation in a concentration-dependent manner (p < 0.05 for IS 1.0 mM vs control). (Fig. 1A) This effect was more marked after 4 days of exposure to 1.0 mM IS (p < 0.001 for 1.0 mM IS acutely vs. 1.0 mM IS at day 4) (Fig. 1B). When testing endothelium-independent relaxation in response to SNP, the response remained unchanged after exposure to IS for 4 days, as compared to vessels maintained in culture without addition of IS (maximal relaxation of 102 ± 16% for Control vs. 136 ± 13% for IS, NS).
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Fig. 1. Effects of indoxyl sulfate (IS) in vitro on aortic rings of wild type mice with normal renal function. A) Vascular reactivity of aortic rings incubated for 30 min with IS (0.1, 0.5 and 1.0 mM). *p < 0.05 Control vs. IS 1.0 mM. B) Vascular reactivity of aortic rings incubated for 30 min or 4 days with IS 1.0 mM *:p < 0.05 and ***:p < 0.001 vs. respective control group. C) Effects of IS (1.0 mM) treatment during 30 min or 4 days on CD31, ICAM-1 and VCAM-1 expression. ***:p < 0.001, xxx:p < 0.001 for ICAM-1 and VCAM-1 vs respective controls.
Culture of control vessels for 4 days did not lead to a change in the expression of CD31 or ICAM-1 and VCAM-1, when compared to freshly isolated vessels. IS exposure was associated with decreased CD31 expression both after 30 min and 4 days. The decrease in CD31 expression reflecting endothelial cell loss was found to be due to an increase in endothelial cell apoptosis after 30 min exposure to IS 1.0 mM (Annexin V positive cells of 100 ± 0% for Control vs. 290 ± 107% for IS, p < 0.05, n ¼ 3 experiments). Moreover, cell viability decreased after IS treatment: cell viability was 100 ± 0% for Control vs. 67 ± 10% for IS (p < 0.05, n ¼ 3 experiments).
Exposure to IS upregulated VCAM-1/ICAM-1 expressions with different profiles. ICAM-1 expression was maximal from 30 min at the difference to VCAM-1 with maximal expression after 4 days of exposure (Fig. 1C).
3.2. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with CKD of 2 or 10 weeks duration After 2 weeks, CKD and IS each induced endothelial dysfunction. However, the association of CKD and IS did not worsen endothelial
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dysfunction further. After 10 weeks in Non-CKD vessels, IS induced endothelial dysfunction similar to endothelial dysfunction observed in 2 weeks Non-CKD vessels. Acute exposure to IS at 1.0 mM also did not aggravate this dysfunction further (Fig. 2A) but induced endothelial cell loss and amplified CKD induced ICAM-1 and VCAM-1 upregulation (Fig. 2B). After 2 weeks IS alone induced endothelial cell loss and an increase of adhesion molecule expression in Non-CKD group. The association of CKD and IS did not worsen these parameters further at 2 weeks. After 10 weeks IS alone induced endothelial cell loss and an increase of adhesion molecule expression. The association of CKD and IS worsened endothelial cell loss and the increase of
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ICAM-1 adhesion molecule expression. The effects of IS on endothelial cell integrity and on ICAM-1 expression was much stronger in 10 weeks CKD than in Non-CKD groups. 3.3. Effects of AST-120 in vitro on aortic rings of wild type with normal renal function Exposure of aortic rings to AST-120 for 4 days did not modify acetylcholine induced relaxation. Co-exposure of aortic rings to both IS and AST-120 prevented IS induced altered relaxation. (p < 0.005 for IS 1.0 mM vs AST-120 þ IS 1.0 mM). (Fig. 3A) Same results, i.e IS induced alteration of relaxation and prevention by
Fig. 2. Effects of indoxyl sulfate in vitro on aortic rings of wild type mice with CKD for 2 or 10 weeks. A) Effects of IS on vascular reactivity. *:p < 0.05 for Non-CKD vs. 3 other groups. B) Effects of IS on CD31, ICAM-1, and VCAM-1 expression. *:p < 0.05, **:p < 0.005, ***:p < 0.0001 vs Non-CKD and CKD groups at same time, £: p < 0.05, £££: p < 0.0001 vs NonCKD þ IS group at same time. x: p < 0.01, xx: p < 0.0001 vs. Non-CKD at same time.
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week CKD groups. (p < 0.05). In WT mice with CKD, AST-120 treatment restored IS levels to normal (p < 0.0005 for CKD vs CKD þ AST-120). A comparable AST120 treatment effect of borderline significance was found in ApoE/ mice (p ¼ 0.09 for CKD vs. CKD þ AST -120) (Tables 1 and 2). 3.4.2. Echocardiography (Table 3) There was no difference in baseline parameters between the different groups of mice (data not shown). In both ApoE/ and WT mice with CKD, a decrease in ESAo, diastolic dysfunction (increased IVRT and Tei index) and LV hypertrophy were observed on placebo treatment for 10 weeks. In both strains, ESAo was improved by AST-120 treatment. Moreover, AST-120 treatment led to a borderline significant (p ¼ 0.06 for the interaction) improvement of IVRT. In ApoE/ mice with CKD, AST-120 treatment prevented LV hypertrophy. 3.4.3. Arterial blood pressure and pulse wave velocity (Table 4) In both WT and ApoE/ mouse groups blood pressure values remained unchanged after 8 weeks of treatment. PWV was significantly higher in WT CKD than WT non-CKD groups. In ApoE/ mice, there was a similar trend of borderline significance. In WT mice, AST-120 treatment prevented the increase in PWV induced by CKD, with a similar trend in ApoE/ mice.
Fig. 3. Effects of AST-120 in vitro on aortic rings of wild type normal mice. A) Results of vascular reactivity of vessels incubated with 1 mM IS and/or 1 g/dL of AST-120 (AST120, IS 1 mM þ AST-120) for 4 days. ***:p < 0.001 for IS 1 mM vs. control, xx:p < 0.005 for IS 1 mM þ AST-120 vs. IS 1 mM. B) Results of 1 mM IS and/or 1 g/dL of AST-120 (AST-120, IS 1 mM þ AST-120) exposure on CD31, ICAM-1 and VCAM-1 expression. ***:p < 0.001 vs. control and AST-120 groups; x:p < 0.05, xxx:p < 0.001 vs. IS 1 mM.
AST-120 combined with 1 mM IS, were found after 2-day exposure (data not shown). AST-120 treatment prevented endothelial cell loss, at least partially, and the increased adhesion molecule expression induced by IS (Fig. 3B). 3.4. Effects of AST-120 treatment in wild type or ApoE/ mice with CKD
3.4.4. Vascular reactivity in vitro CKD induced an alteration of endothelial function characterized by a less marked relaxation in response to acetylcholine. AST-120 treatment, started 2 weeks after CKD induction, not only prevented the endothelial dysfunction induced by CKD, but restored relaxation to normal. (Fig. 4A) Ten weeks of CKD increased ICAM-1 expression compared to non-uremic controls and 2 weeks of CKD, respectively. VCAM-1 expression was upregulated after 2 weeks of CKD with no further aggravation after 10 weeks. (Fig. 2B) AST-120 treatment prevented ICAM-1 upregulation and restored normal VCAM-1 expression (Fig. 4B). Aortic rings from ApoE/ mice with normal kidney function displayed signs of endothelial dysfunction, characterized by a decrease of relaxation associated with a significant increase in ICAM-1 and VCAM-1 expression. Ten weeks of CKD aggravated the endothelial dysfunction in ApoE/ mice. Again, AST-120 treatment prevented the endothelial dysfunction induced by CKD. (Fig. 4A) AST-120 treatment also reduced the upregulation of VCAM-1 expression after 10 weeks of CKD (Fig. 4B). 3.4.5. Atherosclerotic lesions in aorta of ApoE/ mice In ApoE/ mice, CKD was associated with a higher degree of atherosclerosis (0.78 ± 0.33% of surface area covered by plaques in non CKD group and 1.90 ± 0.52% in CKD group, p < 0.05). AST-120 treatment did not improve the degree of atherosclerosis (1.90 ± 0.49% of surface area covered by plaques in CKD þ AST-120 group). 4. Discussion
3.4.1. Serum biochemistry After two weeks of CKD serum urea, total calcium, phosphorus and total cholesterol levels were increased and body weight decreased, compared with sham-op mice, with more marked differences after 8 weeks of CKD. In WT mice, AST-120 treatment increased serum total calcium as compared to placebo group. In CKD Apo E/ mice, AST-120 treatment improved serum urea (p ¼ 0.005 for the interaction). In both WT and ApoE/ mice, IS concentrations were increased at 10 weeks of CKD compared to non-CKD groups and 2-
Endothelial dysfunction, characterized by altered vascular reactivity, has been recognized as an important risk factor, contributing to the increase in cardiovascular mortality associated with CKD [5e7]. Patients with advanced stages of CKD are permanently exposed to uremic toxins as a result of progressive nephron loss. Particular interest has focused on protein bound uremic toxins since their extracorporeal removal by dialysis techniques is difficult, if not impossible, and several amongst them have been shown to play a role in cardiovascular disease and mortality in
I. Six et al. / Atherosclerosis 243 (2015) 248e256
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Table 1 Effects of chronic kidney disease (CKD) and AST-120 on body weight and serum biochemistry in wild type mice. IS: Indoxyl sulfate. Non-CKD þ AST-120
Non-CKD Before start of treatment (2 weeks of CKD) Body weight (g) 20.77 ± 0.94 Urea (mmol/L) 8.76 ± 0.33 Total calcium (mmol/L) 2.02 ± 0.06 Phosphorus (mmol/L) 1.84 ± 0.08 Total cholesterol (mmol/L) 1.89 ± 0.08 After 8 weeks of treatment (at sacrifice, 10 weeks of CKD) Body weight (g) 22.71 ± 0.19 Urea (mmol/L) 9.47 ± 0.24 Total calcium (mmol/L) 2.02 ± 0.01 Phosphorus (mmol/L) 1.61 ± 0.06 Total cholesterol (mmol/L) 1.77 ± 0.04 IS levels (mg/100 ml) 0.10 ± 0.06
CKD þ AST-120
CKD
CKD/AST-120/interaction p
20.69 9.16 2.04 1.99 1.87
± ± ± ± ±
0.16 0.28 0.01 0.06 0.03
19.81 32.52 2.40 2.09 2.52
± ± ± ± ±
0.13 0.90 0.02 0.04 0.04
19.87 35.92 2.49 1.94 2.50
± ± ± ± ±
0.3 2.57 0.04 0.07 0.09
<0.001/NS/NS <0.001/NS/NS <0.001/NS/NS <0.05/NS/NS <0.001/NS/NS
22.63 7.70 2.14 1.86 2.02 0.09
± ± ± ± ± ±
0.21 0.48 0.02 0.08 0.03 0.04
21.38 28.50 2.21 1.92 2.45 0.34
± ± ± ± ± ±
0.21 0.97 0.04 0.07 0.04 0.08
21.46 25.39 2.44 2.09 2.36 0.12
± ± ± ± ± ±
0.27 2.40 0.06 0.12 0.14 0.03
<0.001/NS/NS <0.001/NS/NS <0.001/<0.001/NS <0.005/NS/NS <0.001/0.003/NS <0.05/<0.05/0.06
Table 2 Effects of chronic kidney disease (CKD) and AST-120 on body weight and serum biochemistry in ApoE/ mice. IS: Indoxyl sulfate. Non-CKD þ AST-120
Non-CKD Before start of treatment (2 weeks of CKD) Body weight (g) 22.29 ± 0.32 Urea (mmol/L) 8.57 ± 0.18 Total calcium (mmol/L) 2.07 ± 0.01 Phosphorus (mmol/L) 1.72 ± 0.07 Total cholesterol (mmol/L) 6.22 ± 0.31 After 8 weeks of treatment (at sacrifice, 10 weeks of CKD) Body weight (g) 23.90 ± 0.49 Urea (mmol/L) 7.67 ± 0.32 Total calcium (mmol/L) 2.07 ± 0.02 Phosphorus (mmol/L) 2.07 ± 0.07 Total cholesterol (mmol/L) 5.84 ± 0.39 IS levels (mg/100 ml) 0.17 ± 0.02
CKD þ AST-120
CKD
CKD/AST-120/interaction p
22.63 9.91 2.02 1.87 5.86
± ± ± ± ±
0.29 0.47 0.02 0.07 0.23
20.46 33.99 2.42 2.04 11.53
± ± ± ± ±
0.27 1.16 0.02 0.08 0.37
20.92 31.13 2.50 2.03 10.71
± ± ± ± ±
0.19 1.02 0.03 0.06 0.40
<0.001/NS/NS <0.001/NS/NS <0.001/NS/NS <0.005/NS/NS <0.001/NS/NS
23.77 8.13 2.05 1.80 6.08 0.18
± ± ± ± ± ±
0.49 0.43 0.01 0.07 0.29 0.03
21.67 33.20 2.26 2.06 11.02 0.23
± ± ± ± ± ±
0.65 1.96 0.04 0.11 0.41 0.02
22.25 27.58 2.26 2.12 10.57 0.18
± ± ± ± ± ±
0.26 0.85 0.02 0.08 0.39 0.01
<0.001/NS/NS <0.001/0.02/0.005 <0.001/NS/NS NS/NS/NS <0.001/NS/NS NS/NS/NS
CKD [26e28]. Whether this association results mainly from the induction of endothelial dysfunction or is also due to other effects of uremic toxins is the subject of intensive research. In the present study we demonstrate the ability of IS to directly inhibit relaxation of healthy mouse aorta in a concentration and time dependent manner. This effect was already apparent after a 30 min exposure and was more marked after 4 days. The normal relaxation obtained in response to an exogenous NO donor in presence of IS demonstrates that endothelial cells are the main target of IS, leading to decreased NO production and/or bioavailability. In support of this conclusion, a decrease in NO production, via stimulated reactive oxygen production through NADPH oxidase activation and inhibition of antioxidant systems, has been reported after exposure of endothelial cells to IS [15e17,20e22]. In our in vitro experiments, both acute and longer-term exposure to IS resulted in increased ICAM-1 and VCAM-1 expression and
endothelial cell loss due to an increase of endothelial cell apoptosis. As described in another endothelial cell model using bEnd.3 cells, cell viability decreased after IS treatment [22]. The induction of ICAM-1/VCAM-1 expressions by IS has been reported by other groups [17e19]; however, it was surprising to observe a strong induction of adhesion molecule expression as early as 30 min after IS exposure. In line with this observation, Pletinck et al. recently demonstrated that same concentration of 1.0 mM IS led to a timedependent induction of leukocyte extravasation from rat peritoneal vascular bed, reaching the level of significance after exposure of 30 min [29]. The differing ICAM-1 and VCAM-1 expression profiles induced by IS may be explained by the fact that the basal expression of VCAM-1 is generally lower than that of ICAM-1 but increases more markedly after endothelial cell activation [30]. In CKD mouse vessels, the impairment of endothelium dependent relaxation was not worsened by an even high concentration of
Table 3 Effects of CKD and AST-120 on echocardiographic parameters in WT or ApoE/ mice. Non-CKD Wild type mice ESAo (%) IVRT (msec) Teï index LVM (mg) ApoE¡/¡ mice ESAo (%) IVRT (msec) Teï index LVM (mg)
Non-CKD þ AST-120
CKD þ AST-120
CKD
CKD/AST-120/interaction p
14 16 219 96
± ± ± ±
3 1 18 11
15 17 243 95
± ± ± ±
4 1 21 13
9 21 264 112
± ± ± ±
3 2$$ 51$ 28
14 19 292 107
± ± ± ±
3** 3 62 16
0.01/0.02/NS 0.001/NS/NS 0.001/NS/NS 0.01/NS/NS
13 15 234 99
± ± ± ±
2 2 39 9
12 15 245 99
± ± ± ±
3 2 23 12
9 19 278 116
± ± ± ±
2 2 31 21
12 17 279 99
± ± ± ±
3* 2 42 10**
0.004/NS/0.04 0.001/NS/NS 0.001/NS/NS 0.04/0.04/0.04
ESAo: the aorta's systolic expansion rate, IVRT: isovolumic relaxation time, LVM: the left ventricular mass. $: p < 0.05; $$: p < 0.005 vs. non-CKD placebo; *: p < 0.05, **: p < 0.005 vs. CKD placebo.
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Table 4 Effects of CKD and AST-120 on hemodynamic parameters in WT or ApoE/ mice. Non-CKD Wild type mice SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg PWV, m/sec ApoE¡/¡ mice SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg PWV, m/sec
Non-CKD þ AST-120
CKD þ AST-120
CKD
CKD/AST-120/interaction p
96.7 66.6 86.7 30.1 2.93
± ± ± ± ±
3.1 3.2 3.1 1.2 0.07
92.5 62.3 82.4 30.2 3.13
± ± ± ± ±
3.5 1.9 2.9 2.3 0.12
104.6 70.0 93.1 34.6 3.56
± ± ± ± ±
6.5 4.1 5.7 2.9 0.17$$
97.3 63.8 86.1 33.5 3.10
± ± ± ± ±
2.7 2.3 2.5 1.4 0.08*
NS/NS/NS NS/NS/NS NS/NS/NS 0.06/NS/NS 0.02/NS/0.006
99.7 70.4 89.9 29.2 3.20
± ± ± ± ±
4.7 4.1 4.5 1.6 0.23
105.6 71.3 94.2 34.3 3.40
± ± ± ± ±
7.1 6.0 6.7 1.8 0.28
105.5 71.1 94.1 34.5 3.96
± ± ± ± ±
3.4 3.5 3.4 1.3$$ 0.19$$
101.0 67.0 89.7 34.0 3.50
± ± ± ± ±
5.9 5.7 5.8 1.9 0.18*
NS/NS/NS NS/NS/NS NS/NS/NS 0.08/NS/NS 0.052/NS/NS
SAP, DAP, MAP: systolic, diastolic, mean arterial pressure respectively. PP: pulse pressure. PWV: pulse wave velocity. NS: non-significant. $$: p < 0.005 vs. non-CKD placebo, *p < 0.05 vs. CKD placebo.
Fig. 4. Effects of AST-120 treatment in wild type or ApoE/ mice with CKD 10 weeks. A) Vascular reactivity of aortic rings from WT or ApoE/ mice, Non-CKD or CKD, treated or not with AST-120. **:p < 0.01, ***:p < 0.001 vs. non-CKD; x:p < 0.05 for CKD vs CKD þ AST-120. B) ICAM-1 and VCAM-1 expression in aorta sections from WT or ApoE/ mice, NonCKD or CKD, treated or not with AST-120. ***:p < 0.001 vs. Non-CKD and Non-CKD þ AST-120 groups; x:p < 0.05, xxx:p < 0.001 for CKD vs CKD þ AST-120.
I. Six et al. / Atherosclerosis 243 (2015) 248e256
IS but the toxin induced endothelial cell loss and amplified adhesion molecule upregulation. Since already early stages of CKD are associated with endothelial dysfunction [24] this could explain why IS did not impair endothelial function further. However, the increase in endothelial adhesion molecule expression and loss of CD31 expression in response to prolonged IS exposure in vitro would indicate that (i) the endothelium is effectively an important target for IS, and (ii) high IS concentrations can in addition exert toxic effects on endothelial structure. AST-120 treatment improved IS induced vascular dysfunction in vitro and prevented at least partially IS induced endothelial cell loss. In vivo, IS levels were measured before and at the end of the AST-120 treatment period. The CKD status of 10 weeks' duration induced an increase of IS concentrations. AST-120 treatment reduced the serum IS levels and concomitantly improved the reduced endothelium-dependent vascular response of the aorta of CKD mice. Thus, the beneficial effect of AST-120 on CKD induced vascular dysfunction could be related to the prevention of the deleterious role of IS in vivo. Namikoshi et al. [23] reported that AST-120 ameliorated CKD impaired arterial relaxation but did not demonstrate a major direct vascular effect of IS. Our study is the first to demonstrate a direct induction by IS of endothelial loss and a protective effect of AST-120 against endothelial damage. However, the protective effects of AST120 on the vasculature cannot be attributed to the preservation of endothelium integrity alone. In our two CKD mouse models we confirmed the beneficial AST120 effect on VCAM-1 expression as previously demonstrated by Inami et al. in the adenine rat model of CKD [31]. In our study, we found in addition a beneficial AST-120 effect on ICAM-1 expression in WT mice, although not in ApoE/ mice. The latter may be due to an already high expression of ICAM-1 in this mouse strain. Moreover, in ApoE/ mice AST-120 treatment did not prevent the CKD induced increase in aortic atheromatous plaques. This result is surprising in regard to the positive effects of the adsorbent on adhesion molecule expression and endothelial cell integrity. Adding further complexity to our findings, Yamamoto et al. [32] recently showed a reduction of CKD induced atherosclerosis in response to AST-120, using same ApoE/ mouse model. They evaluated the effect of AST-120, initiating treatment at time of CKD induction and pursuing treatment for 17 weeks. Moreover, they assessed atherosclerosis using en face preparations stained with Sudan IV. Thus, their study differed from ours in several aspects, including earlier treatment initiation, longer treatment time, and site of atherosclerosis measurement. Probably CKD severity was also lower (creatinine clearance approx. 50% of normal in their study vs. serum urea approx. 30 mM in ours) although a direct comparison is not possible. Collectively, these findings would be compatible with the suggestion that a prolonged reduction of serum IS starting in early CKD stages may be beneficial in slowing atherosclerosis progression. Several studies evaluated protective effects of AST120 treatment on CKD induced cardiovascular remodeling, both in animal models [33e35] and in patients [36,37], with mixed results. Some reports showed improvements in arterial stiffness [37], LV hypertrophy and myocardial fibrosis [35] while others only observed an improvement in concentric remodeling, but not hypertrophy [36] or only an improvement in myocardial fibrosis [33]. These highly variable results may be due in part to the different treatment modalities chosen to evaluate the effects of AST120 on the cardiovascular system and in part also due to different degrees of CKD severity and different lengths of treatment [33]. Our echocardiographic results are consistent with some of the earlier results as well as with the results obtained by other techniques in the current study. We observed that aortic compliance was preserved in AST-120-treated
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mice, consistent with our vascular reactivity and PWV findings and the observations of Nakamura et al. [37] We also observed a trend towards improved IVRT, suggesting an inhibitory effect on cardiac hypertrophy which was confirmed in the ApoE/ mice with CKD, in which the increase in LVM was prevented, consistent with the report by Fujii et al. [35]. One limitation of our study is the relatively short treatment time of only 8 weeks. This may explain our inability to detect an AST-120 effect on LVH, despite beneficial vascular actions. In a previous personal study testing the effect of the phosphate binder sevelamer in same experimental model, a treatment time of 8 weeks also was without effect on LVH, whereas treatment prolongation to 14 weeks led to LVH regression [38]. Of note, beneficial effects of sevelamer on vascular function were observed already after 8 weeks of treatment, similarly to the AST-120 effects in the present study. In conclusion, IS is a uremic toxin which exerts deleterious effects on vessel function and morphology via direct actions on vascular endothelium. AST-120 treatment is able to reverse these deleterious effects. Starting the administration of AST-120 in early stages of CKD could lead to an attenuation of the adverse effects of uremic toxins on vascular endothelium early on, prevent the negative consequences of vascular dysfunction for cardiac function and structure, and eventually reduce the occurrence of cardiovascular events and mortality. Funding and acknowledgment gional Priscilla Gross was supported by a grant from Conseil Re de Picardie, Amiens, France (Feder). The study was in part funded by a research grant from Kureha. We want to thank Cedric Boudot for valuable technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.09.019. References [1] A.S. Go, G.M. Chertow, D. Fan, C.E. McCulloch, C.Y. Hsu, Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization, N. Engl. J. Med. 351 (13) (2004 Sep 23), 1296-305. Erratum in: N Engl J Med. 2008;18(4): 4. €m, Oxidative stress and [2] M. Annuk, M. Zilmer, L. Lind, T. Linde, B. Fellstro endothelial function in chronic renal failure, J. Am. Soc. Nephrol. 12 (12) (2001 Dec) 2747e2752. [3] G.M. London, T.B. Drueke, Atherosclerosis and arteriosclerosis in chronic renal failure, Kidney Int. 51 (6) (1997 Jun) 1678e1695. [4] A. Lindner, B. Charra, D.J. Sherrard, B.H. Scribner, Accelerated atherosclerosis in prolonged maintenance hemodialysis, N. Engl. J. Med. 290 (13) (1974 Mar 28) 697e701. [5] R.N. Foley, P.S. Parfrey, M.J. Sarnak, Epidemiology of cardiovascular disease in chronic renal disease, J. Am. Soc. Nephrol. 9 (12 Suppl. l) (1998 Dec) S16eS23. €m, Endothelium-dependent vasodilation and [6] M. Annuk, M. Zilmer, B. Fellstro oxidative stress in chronic renal failure: impact on cardiovascular disease, Kidney Int. Suppl. 84 (2003 May) S50eS53. [7] F. Stam, C. van Guldener, A. Becker, J.M. Dekker, R.J. Heine, L.M. Bouter, C.D. Stehouwer, Endothelial dysfunction contributes to renal functionassociated cardiovascular mortality in a population with mild renal insufficiency: the Hoorn study, J. Am. Soc. Nephrol. 17 (2) (2006 Feb) 537e545. [8] N. Neirynck, R. Vanholder, E. Schepers, S. Eloot, A. Pletinck, G. Glorieux, An update on uremic toxins, Int. Urol. Nephrol. 45 (1) (2013 Feb) 139e150. [9] F.C. Barreto, D.V. Barreto, S. Liabeuf, N. Meert, G. Glorieux, M. Temmar, G. Choukroun, R. Vanholder, Z.A. Massy, European Uremic Toxin Work Group (EUTox). Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients, Clin. J. Am. Soc. Nephrol. 4 (10) (2009 Oct) 1551e1558. [10] M.L. Melamed, L. Plantinga, T. Shafi, R. Parekh, T.W. Meyer, T.H. Hostetter, J. Coresh, N.R. Powe, Retained organic solutes, patient characteristics and allcause and cardiovascular mortality in hemodialysis: results from the retained organic solutes and clinical outcomes (ROSCO) investigators, BMC Nephrol. 14
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