- Email: [email protected]
Amelioration of uremic toxin indoxyl sulfate-induced endothelial cell dysfunction by Klotho protein
Toxicology Letters 215 (2012) 77–83
Contents lists available at SciVerse ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Amelioration of uremic toxin indoxyl sulfate-induced endothelial cell dysfunction by Klotho protein Ke Yang, Ling Nie, Yunjian Huang, Jingbo Zhang, Tangli Xiao, Xu Guan, Jinghong Zhao ∗ Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China
h i g h l i g h t s IS can damage and disturb the functions of endothelial cells. Klotho protein has the ability to reverse IS-induced endothelial dysfunction. Klotho protein can inhibit ROS/p38MAPK and NF-B pathways triggered by IS.
a r t i c l e
i n f o
Article history: Received 18 June 2012 Received in revised form 4 October 2012 Accepted 8 October 2012 Available online 17 October 2012 Keywords: Uremic toxin Indoxyl sulfate Endothelial cells Dysfunction Klotho
a b s t r a c t Indoxyl sulfate (IS), a common kind of uremic toxin, is considered as a risk factor for aggravating endothelial function in CKD patients due to its oxidative activity. The anti-aging protein Klotho, which is produced by the kidneys and down-regulated in uremic conditions, has the ability to resist oxidative stress. Here, we carried out an in vitro study to investigate the deleterious effects of IS on endothelial cells and the protective role of Klotho protein. The cultured human umbilical vein endothelial cells (HUVECs) were incubated with IS in the presence or absence of Klotho protein. The release of reactive oxygen species (ROS) and the expression of monocyte chemoattractant protein-1 (MCP-1) were enhanced while the cell viability and production of nitric oxide (NO) were inhibited by IS in a concentration-dependent manner. Meanwhile, the phosphorylation of p38MAPK and the nuclear translocation of NF-B were increased in HUVECs treated with IS. Pretreatment with Klotho protein resulted in remarkable increase of cell viability and decrease of ROS production in IS-treated HUVECs. Like ROS scavenger, N-acetyl-l-cysteine (NAC), Klotho protein could inhibit the IS-induced activations of p38MAPK and NF-B. Moreover, Klotho protein could also attenuate IS-induced reduction of NO production and up-regulation of MCP-1 expression. These results suggest that IS can damage the functions of endothelial cells. Klotho protein has the ability to ameliorate the IS-induced endothelial dysfunction, which may be partly through inhibiting the ROS/p38MAPK and downstream NF-B signaling pathways. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is frequently associated with chronic kidney disease (CKD) and recognized as the major cause of death for CKD patients. Evidences suggest that atherosclerosis is an important pathological mechanism that leads to CVD (Sarnak et al., 2003; Tonelli et al., 2006). The progressive deterioration of renal function in CKD patients may induce accumulation of uremic toxins, which can enhance oxidative stress and inflammation, resulting in dysfunctions of vascular endothelial cells and consequently accelerating the development of atherosclerosis (Oberg et al., 2004).
∗ Corresponding author at: Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China. Tel.: +86 23 68774321; fax: +86 23 68774321. E-mail addresses: [email protected], [email protected] (J. Zhao). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.004
Indoxyl sulfate (IS) is a typical uremic toxin derived from dietary proteins and excreted into the urine. The reduction of renal clearance leads to elevated serum level of IS in CKD patients (Taki et al., 2007; Atoh et al., 2009). Recent studies reported that IS could cause vascular dysfunction, predominantly due to its capacities of inducing oxidative stress and subsequent inflammation (Dou et al., 2004; Namikoshi et al., 2009). Hence, inhibition of IS-induced oxidative stress is a potential avenue to abate endothelial injury and ameliorate atherosclerosis. Klotho gene, which is originally identified in mice and expressed in the kidneys, has strong anti-aging abilities. The mutation of this gene leads to a syndrome resembling aging phenotypes, such as atherosclerosis, osteoporosis, skin atrophy, short life span (Kuroo et al., 1997; Arking et al., 2002). Although Klotho is mainly expressed in the renal tubules, the extracellular domain of Klotho protein can act as a circulating hormone to exert physiological functions including protecting endothelial cells against oxidative injury, apoptosis and dysfunctions, upon multiple risk factors (Rakugi
78
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
et al., 2007; Saito et al., 1998, 2000). Whereas, a significant decline in Klotho gene and protein expression was reported in chronic renal failure patients (Koh et al., 2001). Similarly, our recent study also found that Klotho expression in the kidneys was decreased markedly in uremic atherosclerosis mice (Yu et al., 2010). Thus, the reduction of Klotho hormone is likely to aggravate endothelial dysfunction induced by uremic toxins in patients with uremia. To confirm this hypothesis, in this study we aimed to characterize the protective role of Klotho protein against IS-induced endothelial dysfunction and clarify the mechanisms involved.
Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, USA). Cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) (Invitrogen, USA) supplemented with 1800 mg/L NaHCO3 , 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere with 5% CO2 . Only cells between passages 2 and 5 were used for experiments.
10 mM KCl, 1.5 mM MgCl2 , 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 0.1% Nonidet P-40, 2.5 g/ml leupeptin, 5 g/ml antipain, and 5 g/ml aprotinin) and placed on ice for 10 min, followed by centrifugation for 30 s at the speed of 14,000 × g at 4 ◦ C. Proteins in the nuclei were resuspended in 50 1 ice-cold protein extraction buffer (420 mM NaCl, 20 mM HEPES, pH 7.9, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 2.5 g/ml leupeptin, 5 g/ml antipain, and 5 g/ml aprotinin). After being vigorous mixed and rocked at 4 ◦ C for 15 min on a shaking platform, the nuclear suspension was centrifuged for 10 min at the speed of 14,000 × g at 4 ◦ C. The supernatant extract was collected and stored at −80 ◦ C. The concentration of protein was determined using protein assay kits (Pierce Biotechnology, USA). Forty microgram protein of whole cell lysates and 30 g of nuclear isolated protein were loaded per well on a 10% or 12% SDS–PAGE and transferred to a nitrocellulose membrane. The blot was blocked with 5% nonfat dry milk and incubated with primary antibodies at 4 ◦ C for overnight. Then, secondary antibodies were applied and the signals developed with ECL plus Western blotting detection system (GE Healthcare, UK). Densitometry analysis was performed with an image analysis system (Bio-Rad, USA). As primary antibodies, anti-cleaved caspase3 antibody, anti-phospho-p38MAPKThr180/Tyr182 antibody, anti-p38MAPK antibody, anti-phospho-eNOSser1177 antibody, anti-eNOS antibody, anti-iNOS antibody (Cell Signaling Technology, USA) and anti-phospho-NF-B p65 antibody, anti--Actin antibody, anti-Histone H3 antibody (Santa Cruz, USA) were used. All experiments were repeated at least three times.
2.2. Cell viability assay
2.6. Measurement of NO production
The viability of cells was measured by using the 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl- 2H-tetrazolium bromide (MTT) uptake assay. The MTT assay was performed as previously described (Zhao et al., 2009). HUVECs were planted at a density of 8.0 × 103 cells/well in 96-well plate for 24 h, and then incubated with 500 M IS (Sigma–Aldrich, USA) for 24 h, 48 h and 72 h. To observe the dose-dependent effect of IS, HUVECs was incubated with various concentrations of IS (100 M, 250 M, 500 M, 1000 M) for 24 h at 37 ◦ C in humidified air supplemented with 5% CO2 . In another experiment, HUVECs were preincubated with 50, 100, 200, 400 pM of recombinant human Klotho protein (R&D Systems, USA) for 30 min and subsequently treated with 500 M IS for 24 h. After incubation with IS, 20 l of MTT working solution (5 mg MTT/ml of phosphate buffered saline) was added into each well and incubated for 5 h at 37 ◦ C. Then, the formazan was solubilized with 150 l of dimethylsulfoxide (DMSO). The absorbance was detected at an O.D. of 570 nm for test wavelength and 630 nm for reference wavelength with a microplate reader (Microplate Fluorescence Reader FL-600, BioTeK, USA). Each experiment was performed three times to validate the results.
HUVECs were cultured at a density of 1.0 × 105 cells/well in 24-well plate for 24 h. Then, the cells were incubated with different concentrations of IS (100 M, 250 M, 500 M, 1000 M) for 2 h. In an additional experiment, HUVECs were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 and 10 M PDTC for 30 min and whereafter treated with 500 M IS for 2 h. After 2 h, NO production was evaluated by measurement of nitrites, a stable oxidative product of NO, according to the Griess method (Green et al., 1982). Briefly, 100 l HUVECs culture supernatant incubated with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyldiamine dihydrochloride, and 2.5% orthophosphoric acid) for 15 min at room temperature. Absorption was measured at 550 nm with a microplate reader. The amount of nitrite was determined by comparison of unknowns using a NaNO2 standard curve. All experiments were repeated three times.
2. Materials and methods 2.1. Cell culture
2.3. Apoptosis assay To determine whether IS can induce cell apoptosis of HUVECs, 1.0 × 105 cells/well were seeded into 24-well plates and incubated with 500 M IS for 24 h or preincubated with 200 pM Klotho protein for 30 min. After being incubated with IS for 24 h, cells were harvested and washed twice with cold phosphate buffered saline. Afterwards, cells were incubated with 10 g/ml of Hoechst 33342 and 5 g/ml of propidium iodide (PI) for 5 min at room temperature. Dual fluorescence-stained cultures were washed and observed under an inverted fluorescence microscope (Olympus IX70-S1F2, Japan). Viable cells were identified by intact nuclei with Hoechst 33342+ stained. Necrotic cells were identified by intact nuclei with PI+ stained. Apoptotic cells were identified by detecting fragmented nuclei with either Hoechst 33342+ or Hoechst 33342+ /PI+ stained, and the quantitative analysis of cell apoptosis was determined by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA).
2.7. ELISA assay HUVECs were incubated with IS at various concentrations (100 M, 250 M, 500 M, 1000 M) for 24 h. In another experiment, HUVECs were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC for 30 min, and then treated with 500 M IS for 24 h. The released MCP-1 in the HUVECs culture supernatants was quantified by a human MCP-1 ELISA kit (R&D Systems, MN, USA), according to the manufacturer’s instructions. Briefly, diluted culture supernatant was added to 96-well microplates coated with polyclonal antibody specific for human MCP-1. After incubation at room temperature for 2 h followed by several times of washes, an enzyme-linked polyclonal antibody was added. After further incubation for 1 h at room temperature and repeated washes, color reagents were added. The intensity of the color was detected at 450 nm with a microplate reader. 2.8. Statistical analysis Data are expressed as the mean ± S.D. Multiple comparisons were evaluated using one-way ANOVA and significant differences between two groups were analyzed by Student–Newman–Keuls test. A value of P < 0.05 was considered statistical significance.
2.4. Measurement of ROS production The generation of intracellular ROS was detected using CM-H2 DCFDA, a ROS-sensitive fluorescent dye. HUVECs were incubated in 24-well plates (1.0 × 105 cells/well) for 24 h, and then incubated with 10 M CM-H2 DCFDA for 30 min in PBS. After removal of PBS, cells were incubated with IS at different concentrations (100 M, 250 M, 500 M, 1000 M) for 3 h. To observe the effect of Klotho protein on ROS production, cells were preincubated with 50, 100, 200, 400 pM of Klotho protein for 30 min, and then incubated with 500 M IS for 3 h. The fluorescence level was observed under inverted fluorescence microscope and fluorescence intensity was measured at excitation 480 nm and emission 525 nm with a microplate reader. The experiments were performed three times to validate the results. 2.5. Immunoblotting HUVECs were preincubated with 200 pM of recombinant human Klotho, 5 mM N-acetyl-l-cysteine (NAC), 10 M p38MAPK inhibitor SB203580, 10 M NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) (Sigma–Aldrich, USA) for 30 min respectively, and then treated with 500 M IS. Whole cell and nuclear protein were isolated from HUVECs. For nuclear protein isolation, 5 × 106 cells were collected, washed once with ice-cold PBS and centrifugated at the speed of 1500 × g for 5 min. The pellet were resuspended in 500 l of cold lysis buffer (10 mM HEPES pH 7.9,
3. Results 3.1. IS decreased the cell viability of HUVECs and Klotho protein could attenuate the effect of IS After incubation of HUVECs with IS for 24 h, 48 h and 72 h at a fixed concentration of 500 M, the cell viability gradually declined, and a significant decrease had already occurred at 24 h (Fig. 1A). When the cells were treated with IS at different concentrations from 100 to 1000 M for 24 h, the cell viability was decreased in a concentration-dependent manner. As shown in Fig. 1B, the cell viability of HUVECs treated with 500 M IS was markedly decreased to 69.26 ± 4.08% of control, while pretreatment with 200 pM and 400 pM recombinant human Klotho protein rescued the inhibitory effect of IS (Fig. 1C). Subsequently, Hoechst 33342/PI staining, capase-3 immunoblot and flow cytometry analysis were performed to explore whether IS could induce HUVECs apoptosis.
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
79
Fig. 1. The effects of IS and Klotho protein on HUVECs viability. (A) Cells were exposed to 500 M of IS for 24 h, 48 h and 72 h. Cell viability was detected by MTT assay. (B) Cells were exposed to various concentrations of IS (100, 250, 500, 1000 M) for 24 h. (C) Cells were pretreated with 50, 100, 200, 400 pM of Klotho protein for 30 min respectively, and then incubated with IS (500 M) for 24 h. Data were shown as mean ± S.D. (n = 10). Statistical differences are expressed as **P < 0.01 vs Control; ## P < 0.01 vs IS.
The protein level of cleaved caspase-3 and apoptotic cell counts were not significantly increased after incubation with IS for 24 h (Fig. 2). Therefore, these results indicate that incubation of HUVECs with IS can induce cell injury but not apoptosis, and the cell injury induced by IS can be reversed by Klotho protein. 3.2. Klotho protein abrogated ROS production in HUVECs induced by IS To determine the potential role of oxidative stress in IS-induced injury of HUVECs, ROS production was detected. As shown in Fig. 3A, after incubation with IS at different concentrations from
100 to 1000 M for 3 h, ROS production in HUVECs was increased in a concentration-dependent manner. Compared with control group, even 100 M of IS could significantly increase the production of ROS, and treatment with 500 M of IS led to a 1.6-fold increase in ROS production. Because Klotho protein was considered to have strong ability to resist oxidative stress and could act as a circulating hormone, the effect of Klotho protein on ROS production induced by IS in HUVECs was detected in the following experiment. The result displayed that pretreatment with 200 pM and 400 pM of recombinant human Klotho markedly suppressed the production of ROS induced by IS in HUVECs (Fig. 3B and C).
Fig. 2. The effects of IS and Klotho protein on HUVECs apoptosis. (A) The normal, apoptotic and necrotic cells were observed by Hoechst 33342/PI staining in HUVECs after being incubated with 500 M IS for 24 h in the presence or absence of 200 pM Klotho protein. The scale bar corresponds to 50 m. (B) Apoptotic cells were analyzed by flow cytometry after incubating with 500 M IS for 24 h. (C) Cleaved caspase-3 protein levels were examined by immunoblotting. -Actin was used as a loading control.
80
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
Fig. 3. ROS production in HUVECs. (A) Cells were exposed to various concentrations of IS (100, 250, 500, 1000 M) for 3 h. (B) Cells were pretreated with 50, 100, 200, 400 pM Klotho protein for 30 min, and then incubated with 500 M IS for 3 h. The generation of intracellular ROS was detected using CM-H2 DCFDA. (C) ROS production was observed under inverted fluorescence microscope. Data were shown as mean ± S.D. (n = 8). Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; P < 0.05 vs IS.
3.3. Klotho protein could inhibit IS-induced activations of p38MAPK and NF-ÄB in HUVECs
3.5. Klotho protein could reverse the increased release of MCP-1 from HUVECs induced by IS
To investigate the mechanisms of the effects of IS on HUVECs and the protective role of Klotho protein against IS-induced injury, the activations of ROS sensitive pathways including p38MAPK and NF-B were detected. As shown in Fig. 4, treatment of IS for 15 min markedly increased the phosphorylation of p38MAPK and nuclear translocation of NF-B, whereas the IS-stimulated p38 MAPK phosphorylation and NF-B nuclear translocation were significantly attenuated by Klotho protein, similar as the effects of ROS scavenger, NAC. These results suggest that p38MAPK pathway and its downstream NF-B can be activated by IS in HUVECs, while pretreatment of HUVECs with Klotho protein is able to inhibit ISstimulated activation of p38MAPK and NF-B, possibly through blocking the production of ROS.
To further study the protective role of Klotho protein against IS-induced dysfunction of HUVECs, the production of MCP-1, an inflammation-related factor regulated by NF-B, was analyzed subsequently. After incubation with IS at concentrations from 100 to 1000 M, a concentration-dependent increase of MCP-1 protein released into culture medium was observed. In contrast, pretreatment with Klotho protein suppressed the IS-induced release of MCP-1 in HUVECs. The same results were also found when the cells were pretreated with SB203580 and PDTC (Fig. 6). It indicated that Klotho protein as well as p38MAPK and NF-B inhibitors could rescue IS-induced dysfunctions of HUVECs.
4. Discussion
3.4. Klotho protein could abrogate the decrease of NO production and phosphorylation of eNOS in HUVECs induced by IS To observe the cell functions influenced by IS, the effects of IS on NO production, the expression levels of endothelial NOS (eNOS) and inducible NOS (iNOS) in HUVECs were detected. After 2 h incubation, IS decreased NO production in a concentrationdependent manner. As shown in Fig. 5A, incubation with 250 M, 500 M and 1000 M of IS significantly decreased NO production to 76.3%, 70.4% and 54.5% of control. Moreover, the phosphorylation of eNOS was significantly reduced in HUVECs treated with 500 M IS for 30 min. While Klotho protein, as well as p38MAPK inhibitor SB203580 and NF-B inhibitor PDTC, could significantly abrogate the decrease of NO production and phosphorylation of eNOS in HUVECs induced by IS (Fig. 5B and C). But the iNOS expression was not affected by IS and Klotho protein in cultured HUVECs (Fig. 5D).
Chronic kidney disease (CKD) is recognized as a serious public health problem which can dramatically increase the complications of atherosclerosis and cardiovascular events (Elsayed et al., 2007; Mallamaci et al., 2005). As known, the occurrence of cardiovascular disease (CVD) is frequently initiated by “traditional” and “nontraditional” risk factors. In the past, “traditional” risk factors such as dislipidemia, hypertension, diabetes mellitus and old age were taken as the main causes of CVD. However, the traditional risk factors could not explain the high prevalence and incidence of CVD in CKD patients. In recent years, more and more reports demonstrated that “non-traditional” risk factors, such as inflammation, oxidative stress and endothelial dysfunction, were significantly associated with accelerated atherosclerosis and CVD (Gosmanova and Le, 2011; Rodriguez-Iturbe and Correa-Rotter, 2010). Uremic toxins in CKD patients are regarded as one of the main sources of “non-traditional” risk factors (Shimoishi et al., 2007). IS is a representative uremic toxin with property to bind with serum proteins, which leads to difficult elimination by general
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
81
Fig. 4. Immunoblotting analysis of the expression of p38MAPK and NF-B in HUVECs. HUVECs were preincubated with 200 pM of recombinant human Klotho, 5 mM NAC, 10 M SB203580 or 10 M PDTC for 30 min respectively, and then treated with 500 M IS for another 15 min. Whole cell and nuclear protein were isolated to perform immunoblot. (A) and (B) The protein expression of phospho-p38 was standardized by p38. (C) and (D) The protein expression of NF-B p65 was standardized by Histone H3. Values are means ± S.D. from each group. *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
dialysis in CKD patients. Previous studies demonstrated that high level of IS could induce endothelial cell dysfunction through triggering oxidative stress, even though it might function as an antioxidant under physiological concentration (Miyamoto et al., 2010; Tumur and Niwa, 2009; Tumur et al., 2010). Thus, inhibition of IS-induced oxidative stress will play beneficial roles in maintaining endothelial function and preventing the development of atherosclerosis for uremia patients. As reported, Klotho protein, a novel humoral factor produced in the kidneys, confers resistance to oxidative stress in multifold pathological conditions (Saito et al., 2000). In this study, we examined the damage effects of IS on endothelial cells firstly, and then explore the protective role of Klotho protein against IS-induced endothelial dysfunction. It was reported that the serum levels of IS in uremic patients were increased approximately 50-fold compared to healthy individuals, and the mean and maximal serum level of IS were approximately 250 M and 500 M respectively (Vanholder et al., 2003). Accordingly, we first chose a fixed-concentration of 500 M of IS to treat the cultured HUVECs for different times and found that the cell viability was significantly inhibited after 24 h incubation. Then a wide range of IS (100 M to 1000 M) was selected to observe its concentration-effect on HUVECs. As shown in the results, after incubation with IS at different concentrations, the cell viability were inhibited by IS in a dose-dependent manner. Notably, pretreatment with 200 pM or 400 pM Klotho protein can alleviate the inhibitive effect of IS. We further found that IS could induce ROS production in HUVECs with a concentration-dependent manner. It is known that
high levels of ROS can regulate signaling pathways via stressactivated MAP kinases, such as p38MAPK, to affect cell functions. Nuclear transcription factor NF-B is normally a downstream target of p38MAPK signaling pathway (Ganju et al., 2005). The activation of NF-B is involved in many pathological events, especially in the promotion of endothelial dysfunction and the development of atherosclerosis (Kanters et al., 2003). Because the transgenic mice overexpressing Klotho have low level of endogenous ROS and can live longer, the relationship between Klotho and ROS sensitive pathway has caused extensive concerns. Hsieh et al. (2010) reported that the beneficial effects of Klotho on longevity were mediated, at least partially, by inactivation of p38MAPK signaling. Therefore, we next investigated the effects of IS and Klotho on the activations of p38MAPK and NF-B in HUVECs. It was found that p38MAPK phosphorylation and nuclear translocation of NF-B were markedly increased in HUVECs treated with IS, in line with the report by Masai et al. (2010). As expected, treatment with Klotho protein significantly suppressed IS-induced ROS production in a dose-dependent manner, and the activations of p38MAPK and NFB could be blocked by Klotho protein, similar as the effect of ROS scavenger, NAC. Therefore, it implies that Klotho protein has the ability to inhibit the IS-induced oxidative stress and activations of p38MAPK and NF-B, possibly through blocking ROS production. Endothelial dysfunction is normally characterized by the imbalanced release of vasodilator and vasoconstrictor factors. The imbalance is predominantly due to the loss of NO because of its inactivation by ROS (Münzel et al., 2008). In addition, endothelial function is also affected by vascular inflammation (Trepels
82
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
Fig. 5. NO production, eNOS and iNOS expression in HUVECs. (A) IS significantly decreased NO production in a concentration-dependent manner (n = 8). (B) Decrease of NO production induced by IS (500 M) in HUVECs was rescued by pretreatment with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC (n = 8). (C) Immunoblotting analysis of the protein expression of phospho-eNOS which was standardized by eNOS. (D) Immunoblotting analysis of the protein expression of iNOS which was standardized by -actin. Data were shown as mean ± S.D. Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
et al., 2006). Interestingly, the activated endothelial cells may also produce chemotactic factors such as MCP-1 and soluble vascular adhesion molecule-1 (sVCAM-1), which can recruit circulating monocytes and T lymphocytes into the arterial wall to join in the process of inflammation and atherosclerosis (Collins, 1993; Toborek et al., 2002). It was reported recently that IS could activate the MAPK/NF-B pathway and lead to induction of MCP-1 expression in HUVECs (Masai et al., 2010). Therefore, we further
investigate the effects of IS and Klotho protein on NO production and MCP-1 expression in HUVECs. Since the production of NO is regulated by NOS, which includes eNOS and iNOS (Rudic et al., 1998; Xie et al., 1992; Marczin et al., 1996), the expression levels of both eNOS and iNOS were also observed in this study. Our results demonstrated that the NO production and eNOS phosphorylation were reduced, while the MCP-1 production was significantly enhanced in HUVECs treated with IS. Interestingly,
Fig. 6. The release of MCP-1 protein from HUVECs. (A) Cells were exposed to various doses of IS for 24 h. (B) Cells were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC for 30 min and then treated with 500 M IS for 24 h. ELISA assay was applied to detect the released of MCP-1 in the culture supernatants. Data were shown as mean ± S.D. (n = 12). Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
the expression of iNOS was not affected by IS and Klotho protein in HUVECs, indicating that IS inhibits the production of NO in HUVECs mainly by suppressing the expression and phosphorylation of eNOS. Remarkably, the IS-induced reduction of NO production, the depression of eNOS phosphorylation and the up-regulation of MCP1 could be reversed by Klotho protein, similarly to the effects of p38MAPK and NF-B inhibitors. Because we had already found that Klotho protein was able to inhibit IS-stimulated activation of p38MAPK and NF-B, these results suggest that the protective role of Klotho protein against IS-induced dysfunction of HUVECs may be partly attributed to the attenuation of p38MAPK and NF-B signaling pathways. In conclusion, this study demonstrated that Klotho protein has the ability to reverse IS-induced endothelial dysfunction. The observations further confirmed that accelerated atherosclerosis in uremia is closely related to uremic toxins-induced endothelial dysfunction. An increase in the serum level of Klotho protein may be a potential measure for treatment of atherosclerosis in chronic kidney disease. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work is supported by the National Science Foundations of China (No. 81270290 and No. 30700316) and the Natural Science Foundation of Chongqing (No. 2007BB5024). References Arking, D.E., Krebsova, A., Macek Sr., M., Macek Jr., M., Arking, A., Mian, I.S., Fried, L., Hamosh, A., Dey, S., McIntosh, I., Dietz, H.C., 2002. Association of human aging with a functional variant of Klotho. Proceedings of the National Academy of Sciences of the United States of America 99, 856–861. Atoh, K., Itoh, H., Haneda, M., 2009. Serum indoxyl sulfate levels in patients with diabetic nephropathy: relation to renal function. Diabetes Research and Clinical Practice 83, 220–226. Collins, T., 1993. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Laboratory Investigation 68, 499–508. Dou, L., Bertrand, E., Cerini, C., Faure, V., Sampol, J., Vanholder, R., Berland, Y., Brunet, P., 2004. The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair. Kidney International 65, 442– 451. Elsayed, E.F., Tighiouart, H., Griffith, J., Kurth, T., Levey, A.S., Salem, D., Sarnak, M.J., Weiner, D.E., 2007. Cardiovascular disease and subsequent kidney disease. Archives of Internal Medicine 167, 1130–1136. Ganju, L., Padwad, Y., Singh, R., Karan, D., Chanda, S., Chopra, M.K., Bhatnagar, P., Kashyap, R., Sawhney, R.C., 2005. Anti-inflammatory activity of seabuckthorn (hippophae rhamnoides) leaves. International Immunopharmacology 5, 1675–1684. Gosmanova, E.O., Le, N.A., 2011. Cardiovascular complications in CKD patients: role of oxidative stress. Cardiology Research and Practice 2, 156326. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., 1982. Analysis of nitrate, nitrite, and [15 N] nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Hsieh, C.C., Kuro-o, M., Rosenblatt, K.P., Brobey, R., Papaconstantinou, J., 2010. The ASK1-signalosome regulates p38 MAPK activity in response to levels of endogenous oxidative stress in the Klotho mouse models of aging. Aging (Albany NY) 2, 597–611. Kanters, E., Pasparakis, M., Gijbels, M.J., Vergouwe, M.N., Partouns-Hendriks, I., Fijneman, R.J., Clausen, B.E., Förster, I., Kockx, M.M., Rajewsky, K., Kraal, G., Hofker, M.H., de Winther, M.P., 2003. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. Journal of Clinical Investigation 112, 1176–1185. Koh, N., Fujimori, T., Nishiguchi, S., Tamori, A., Shiomi, S., Nakatani, T., Sugimura, K., Kishimoto, T., Kinoshita, S., Kuroki, T., Nabeshima, Y., 2001. Severely reduced production of klotho in human chronic renal failure kidney. Biochemical and Biophysical Research Communications 280, 1015– 1020. Kuro-o, M., Matsumura, Y., Aizawa, H., Kawaguchi, H., Suga, T., Utsugi, T., Ohyama, Y., Kurabayashi, M., Kaname, T., Kume, E., Iwasaki, H., Iida, A., Shiraki-Iida, T., Nishikawa, S., Nagai, R., Nabeshima, Y.I., 1997. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390, 45–51.
83
Mallamaci, F., Tripepi, G., Cutrupi, S., Malatino, L.S., Zoccali, C., 2005. Prognostic value of combined use of biomarkers of inflammation, endothelial dysfunction, and myocardiopathy in patients with ESRD. Kidney International 67, 2330–2337. Marczin, N., Jilling, T., Papapetropoulos, A., Go, C., Catravas, J.D., 1996. Cytoskeletondependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells. British Journal of Pharmacology 118, 1085–1094. Masai, N., Tatebe, J., Yoshino, G., Morita, T., 2010. Indoxyl sulfate stimulates monocyte hemoattractant protein-1 expression in human umbilical vein. endothelial cells by inducing oxidative stress through activation of the NADPH oxidasenuclear factor-b pathway. Circulation Journal 74, 2216–2224. Miyamoto, Y., Iwao, Y., Tasaki, Y., Sato, K., Ishima, Y., Watanabe, H., Kadowaki, D., Maruyama, T., Otagiri, M., 2010. The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions. FEBS Letters 584, 2816–2820. Münzel, T., Sinning, C., Post, F., Warnholtz, A., Schulz, E., 2008. Pathophysiology, diagnosis and prognostic implications of endothelial dysfunction. Annals of Medicine 40, 180–196. Namikoshi, T., Tomita, N., Satoh, M., Sakuta, T., Kuwabara, A., Kobayashi, S., 2009. Oral adsorbent AST-120 ameliorates endothelial dysfunction independent of renal function in rats with subtotal nephrectomy. Hypertension Research 32, 194–200. Oberg, B.P., McMenamin, E., Lucas, F.L., McMonagle, E., Morrow, J., Ikizler, T.A., Himmelfarb, J., 2004. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney International 65, 1009–1016. Rakugi, H., Matsukawa, N., Ishikawa, K., Yang, J., Imai, M., Ikushima, M., Maekawa, Y., Kida, I., Miyazaki, J., Ogihara, T., 2007. Anti-oxidative effect of Klotho on endothelial cells through cAMP activation. Endocrine 31, 82–87. Rodriguez-Iturbe, B., Correa-Rotter, R., 2010. Cardiovascular risk factors and prevention of cardiovascular disease in patients with chronic renal disease. Expert Opinion on Pharmacotherapy 11, 2687–2698. Rudic, R.D., Shesely, E.G., Maeda, N., Smithies, O., Segal, S.S., Sessa, W.C., 1998. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. The Journal of Clinical Investigation 101, 731–736. Saito, Y., Yamagishi, T., Nakamura, T., Ohyama, Y., Aizawa, H., Suga, T., Matsumura, Y., Masuda, H., Kurabayashi, M., Kuro-o, M., Nabeshima, Y., Nagai, R., 1998. Klotho protein protects against endothelial dysfunction. Biochemical and Biophysical Research Communication 248, 324–329. Saito, Y., Nakamura, T., Ohyama, Y., Suzuki, T., Iida, A., Shiraki-Iida, T., Kuro-o, M., Nabeshima, Y., Kurabayashi, M., Nagai, R., 2000. In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochemical and Biophysical Research Communication 276, 767–772. Sarnak, M.J., Levey, A.S., Schoolwerth, A.C., Coresh, J., Culleton, B., Hamm, L.L., McCullough, P.A., Kasiske, B.L., Kelepouris, E., Klag, M.J., Parfrey, P., Pfeffer, M., Raij, L., Spinosa, D.J., Wilson, P.W., 2003. Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on kidney in cardiovascular disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Hypertension 42, 1050–1065. Shimoishi, K., Anraku, M., Kitamura, K., Tasaki, Y., Taguchi, K., Hashimoto, M., Fukunaga, E., Maruyama, T., Otagiri, M., 2007. An oral adsorbent, AST-120 protects against the progression of oxidative stress by reducing the accumulation of indoxyl sulfate in the systemic circulation in renal failure. Pharmaceutical Research 24, 1283–1289. Taki, K., Tsuruta, Y., Niwa, T., 2007. Indoxyl sulfate and atherosclerotic risk factors in hemodialysis patients. American Journal of Nephrology 27, 30–35. Toborek, M., Lee, Y.W., Garrido, R., Kaiser, S., Hennig, B., 2002. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. The American Journal of Clinical Nutrition 75, 119–125. Tonelli, M., Wiebe, N., Culleton, B., House, A., Rabbat, C., Fok, M., McAlister, F., Garg, A.X., 2006. Chronic kidney disease and mortality risk: a systematic review. Journal of the American Society of Nephrology 17, 2034–2047. Trepels, T., Zeiher, A.M., Fichtlscherer, S., 2006. The endothelium and inflammation. Endothelium 13, 423–429. Tumur, Z., Niwa, T., 2009. Indoxyl sulfate inhibits nitric oxide production and cell viability by inducing oxidative stress in vascular endothelial cells. American Journal of Nephrology 29, 551–557. Tumur, Z., Shimizu, H., Enomoto, A., Miyazaki, H., Niwa, T., 2010. Indoxyl sulfate upregulates expression of ICAM-1 and MCP-1 by oxidative stress-induced NF-B activation. American Journal of Nephrology 31, 435–441. Vanholder, R., De Smet, R., Glorieux, G., Argilés, A., Baurmeister, U., Brunet, P., Clark, W., Cohen, G., De Deyn, P.P., Deppisch, R., Descamps-Latscha, B., Henle, T., Jörres, A., Lemke, H.D., Massy, Z.A., Passlick-Deetjen, J., Rodriguez, M., Stegmayr, B., Stenvinkel, P., Tetta, C., Wanner, C., Zidek, W., 2003. European uremic toxin work group (eutox) review on uremic toxins: classification, concentration, and interindividual variability. Kidney International 63, 1934–1943. Xie, Q.W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Troso, T., Nathan, C., 1992. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256, 225–228. Yu, J., Deng, M., Zhao, J., Huang, L., 2010. Decreased expression of klotho gene in uremic atherosclerosis in apolipoprotein E-deficient mice. Biochemical and Biophysical Research Communications 391, 261–266. Zhao, J., Huang, Y., Song, Y., Zhao, X., Jin, J., Wang, J., Huang, L., 2009. Low osmolar contrast medium induces cellular injury and disruption of calcium homeostasis in rat glomerular endothelial cells in vitro. Toxicology Letters 185, 124–131.
Contents lists available at SciVerse ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Amelioration of uremic toxin indoxyl sulfate-induced endothelial cell dysfunction by Klotho protein Ke Yang, Ling Nie, Yunjian Huang, Jingbo Zhang, Tangli Xiao, Xu Guan, Jinghong Zhao ∗ Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China
h i g h l i g h t s IS can damage and disturb the functions of endothelial cells. Klotho protein has the ability to reverse IS-induced endothelial dysfunction. Klotho protein can inhibit ROS/p38MAPK and NF-B pathways triggered by IS.
a r t i c l e
i n f o
Article history: Received 18 June 2012 Received in revised form 4 October 2012 Accepted 8 October 2012 Available online 17 October 2012 Keywords: Uremic toxin Indoxyl sulfate Endothelial cells Dysfunction Klotho
a b s t r a c t Indoxyl sulfate (IS), a common kind of uremic toxin, is considered as a risk factor for aggravating endothelial function in CKD patients due to its oxidative activity. The anti-aging protein Klotho, which is produced by the kidneys and down-regulated in uremic conditions, has the ability to resist oxidative stress. Here, we carried out an in vitro study to investigate the deleterious effects of IS on endothelial cells and the protective role of Klotho protein. The cultured human umbilical vein endothelial cells (HUVECs) were incubated with IS in the presence or absence of Klotho protein. The release of reactive oxygen species (ROS) and the expression of monocyte chemoattractant protein-1 (MCP-1) were enhanced while the cell viability and production of nitric oxide (NO) were inhibited by IS in a concentration-dependent manner. Meanwhile, the phosphorylation of p38MAPK and the nuclear translocation of NF-B were increased in HUVECs treated with IS. Pretreatment with Klotho protein resulted in remarkable increase of cell viability and decrease of ROS production in IS-treated HUVECs. Like ROS scavenger, N-acetyl-l-cysteine (NAC), Klotho protein could inhibit the IS-induced activations of p38MAPK and NF-B. Moreover, Klotho protein could also attenuate IS-induced reduction of NO production and up-regulation of MCP-1 expression. These results suggest that IS can damage the functions of endothelial cells. Klotho protein has the ability to ameliorate the IS-induced endothelial dysfunction, which may be partly through inhibiting the ROS/p38MAPK and downstream NF-B signaling pathways. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is frequently associated with chronic kidney disease (CKD) and recognized as the major cause of death for CKD patients. Evidences suggest that atherosclerosis is an important pathological mechanism that leads to CVD (Sarnak et al., 2003; Tonelli et al., 2006). The progressive deterioration of renal function in CKD patients may induce accumulation of uremic toxins, which can enhance oxidative stress and inflammation, resulting in dysfunctions of vascular endothelial cells and consequently accelerating the development of atherosclerosis (Oberg et al., 2004).
∗ Corresponding author at: Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China. Tel.: +86 23 68774321; fax: +86 23 68774321. E-mail addresses: [email protected], [email protected] (J. Zhao). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.004
Indoxyl sulfate (IS) is a typical uremic toxin derived from dietary proteins and excreted into the urine. The reduction of renal clearance leads to elevated serum level of IS in CKD patients (Taki et al., 2007; Atoh et al., 2009). Recent studies reported that IS could cause vascular dysfunction, predominantly due to its capacities of inducing oxidative stress and subsequent inflammation (Dou et al., 2004; Namikoshi et al., 2009). Hence, inhibition of IS-induced oxidative stress is a potential avenue to abate endothelial injury and ameliorate atherosclerosis. Klotho gene, which is originally identified in mice and expressed in the kidneys, has strong anti-aging abilities. The mutation of this gene leads to a syndrome resembling aging phenotypes, such as atherosclerosis, osteoporosis, skin atrophy, short life span (Kuroo et al., 1997; Arking et al., 2002). Although Klotho is mainly expressed in the renal tubules, the extracellular domain of Klotho protein can act as a circulating hormone to exert physiological functions including protecting endothelial cells against oxidative injury, apoptosis and dysfunctions, upon multiple risk factors (Rakugi
78
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
et al., 2007; Saito et al., 1998, 2000). Whereas, a significant decline in Klotho gene and protein expression was reported in chronic renal failure patients (Koh et al., 2001). Similarly, our recent study also found that Klotho expression in the kidneys was decreased markedly in uremic atherosclerosis mice (Yu et al., 2010). Thus, the reduction of Klotho hormone is likely to aggravate endothelial dysfunction induced by uremic toxins in patients with uremia. To confirm this hypothesis, in this study we aimed to characterize the protective role of Klotho protein against IS-induced endothelial dysfunction and clarify the mechanisms involved.
Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, USA). Cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) (Invitrogen, USA) supplemented with 1800 mg/L NaHCO3 , 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a humidified atmosphere with 5% CO2 . Only cells between passages 2 and 5 were used for experiments.
10 mM KCl, 1.5 mM MgCl2 , 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 0.1% Nonidet P-40, 2.5 g/ml leupeptin, 5 g/ml antipain, and 5 g/ml aprotinin) and placed on ice for 10 min, followed by centrifugation for 30 s at the speed of 14,000 × g at 4 ◦ C. Proteins in the nuclei were resuspended in 50 1 ice-cold protein extraction buffer (420 mM NaCl, 20 mM HEPES, pH 7.9, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 2.5 g/ml leupeptin, 5 g/ml antipain, and 5 g/ml aprotinin). After being vigorous mixed and rocked at 4 ◦ C for 15 min on a shaking platform, the nuclear suspension was centrifuged for 10 min at the speed of 14,000 × g at 4 ◦ C. The supernatant extract was collected and stored at −80 ◦ C. The concentration of protein was determined using protein assay kits (Pierce Biotechnology, USA). Forty microgram protein of whole cell lysates and 30 g of nuclear isolated protein were loaded per well on a 10% or 12% SDS–PAGE and transferred to a nitrocellulose membrane. The blot was blocked with 5% nonfat dry milk and incubated with primary antibodies at 4 ◦ C for overnight. Then, secondary antibodies were applied and the signals developed with ECL plus Western blotting detection system (GE Healthcare, UK). Densitometry analysis was performed with an image analysis system (Bio-Rad, USA). As primary antibodies, anti-cleaved caspase3 antibody, anti-phospho-p38MAPKThr180/Tyr182 antibody, anti-p38MAPK antibody, anti-phospho-eNOSser1177 antibody, anti-eNOS antibody, anti-iNOS antibody (Cell Signaling Technology, USA) and anti-phospho-NF-B p65 antibody, anti--Actin antibody, anti-Histone H3 antibody (Santa Cruz, USA) were used. All experiments were repeated at least three times.
2.2. Cell viability assay
2.6. Measurement of NO production
The viability of cells was measured by using the 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl- 2H-tetrazolium bromide (MTT) uptake assay. The MTT assay was performed as previously described (Zhao et al., 2009). HUVECs were planted at a density of 8.0 × 103 cells/well in 96-well plate for 24 h, and then incubated with 500 M IS (Sigma–Aldrich, USA) for 24 h, 48 h and 72 h. To observe the dose-dependent effect of IS, HUVECs was incubated with various concentrations of IS (100 M, 250 M, 500 M, 1000 M) for 24 h at 37 ◦ C in humidified air supplemented with 5% CO2 . In another experiment, HUVECs were preincubated with 50, 100, 200, 400 pM of recombinant human Klotho protein (R&D Systems, USA) for 30 min and subsequently treated with 500 M IS for 24 h. After incubation with IS, 20 l of MTT working solution (5 mg MTT/ml of phosphate buffered saline) was added into each well and incubated for 5 h at 37 ◦ C. Then, the formazan was solubilized with 150 l of dimethylsulfoxide (DMSO). The absorbance was detected at an O.D. of 570 nm for test wavelength and 630 nm for reference wavelength with a microplate reader (Microplate Fluorescence Reader FL-600, BioTeK, USA). Each experiment was performed three times to validate the results.
HUVECs were cultured at a density of 1.0 × 105 cells/well in 24-well plate for 24 h. Then, the cells were incubated with different concentrations of IS (100 M, 250 M, 500 M, 1000 M) for 2 h. In an additional experiment, HUVECs were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 and 10 M PDTC for 30 min and whereafter treated with 500 M IS for 2 h. After 2 h, NO production was evaluated by measurement of nitrites, a stable oxidative product of NO, according to the Griess method (Green et al., 1982). Briefly, 100 l HUVECs culture supernatant incubated with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyldiamine dihydrochloride, and 2.5% orthophosphoric acid) for 15 min at room temperature. Absorption was measured at 550 nm with a microplate reader. The amount of nitrite was determined by comparison of unknowns using a NaNO2 standard curve. All experiments were repeated three times.
2. Materials and methods 2.1. Cell culture
2.3. Apoptosis assay To determine whether IS can induce cell apoptosis of HUVECs, 1.0 × 105 cells/well were seeded into 24-well plates and incubated with 500 M IS for 24 h or preincubated with 200 pM Klotho protein for 30 min. After being incubated with IS for 24 h, cells were harvested and washed twice with cold phosphate buffered saline. Afterwards, cells were incubated with 10 g/ml of Hoechst 33342 and 5 g/ml of propidium iodide (PI) for 5 min at room temperature. Dual fluorescence-stained cultures were washed and observed under an inverted fluorescence microscope (Olympus IX70-S1F2, Japan). Viable cells were identified by intact nuclei with Hoechst 33342+ stained. Necrotic cells were identified by intact nuclei with PI+ stained. Apoptotic cells were identified by detecting fragmented nuclei with either Hoechst 33342+ or Hoechst 33342+ /PI+ stained, and the quantitative analysis of cell apoptosis was determined by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA).
2.7. ELISA assay HUVECs were incubated with IS at various concentrations (100 M, 250 M, 500 M, 1000 M) for 24 h. In another experiment, HUVECs were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC for 30 min, and then treated with 500 M IS for 24 h. The released MCP-1 in the HUVECs culture supernatants was quantified by a human MCP-1 ELISA kit (R&D Systems, MN, USA), according to the manufacturer’s instructions. Briefly, diluted culture supernatant was added to 96-well microplates coated with polyclonal antibody specific for human MCP-1. After incubation at room temperature for 2 h followed by several times of washes, an enzyme-linked polyclonal antibody was added. After further incubation for 1 h at room temperature and repeated washes, color reagents were added. The intensity of the color was detected at 450 nm with a microplate reader. 2.8. Statistical analysis Data are expressed as the mean ± S.D. Multiple comparisons were evaluated using one-way ANOVA and significant differences between two groups were analyzed by Student–Newman–Keuls test. A value of P < 0.05 was considered statistical significance.
2.4. Measurement of ROS production The generation of intracellular ROS was detected using CM-H2 DCFDA, a ROS-sensitive fluorescent dye. HUVECs were incubated in 24-well plates (1.0 × 105 cells/well) for 24 h, and then incubated with 10 M CM-H2 DCFDA for 30 min in PBS. After removal of PBS, cells were incubated with IS at different concentrations (100 M, 250 M, 500 M, 1000 M) for 3 h. To observe the effect of Klotho protein on ROS production, cells were preincubated with 50, 100, 200, 400 pM of Klotho protein for 30 min, and then incubated with 500 M IS for 3 h. The fluorescence level was observed under inverted fluorescence microscope and fluorescence intensity was measured at excitation 480 nm and emission 525 nm with a microplate reader. The experiments were performed three times to validate the results. 2.5. Immunoblotting HUVECs were preincubated with 200 pM of recombinant human Klotho, 5 mM N-acetyl-l-cysteine (NAC), 10 M p38MAPK inhibitor SB203580, 10 M NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) (Sigma–Aldrich, USA) for 30 min respectively, and then treated with 500 M IS. Whole cell and nuclear protein were isolated from HUVECs. For nuclear protein isolation, 5 × 106 cells were collected, washed once with ice-cold PBS and centrifugated at the speed of 1500 × g for 5 min. The pellet were resuspended in 500 l of cold lysis buffer (10 mM HEPES pH 7.9,
3. Results 3.1. IS decreased the cell viability of HUVECs and Klotho protein could attenuate the effect of IS After incubation of HUVECs with IS for 24 h, 48 h and 72 h at a fixed concentration of 500 M, the cell viability gradually declined, and a significant decrease had already occurred at 24 h (Fig. 1A). When the cells were treated with IS at different concentrations from 100 to 1000 M for 24 h, the cell viability was decreased in a concentration-dependent manner. As shown in Fig. 1B, the cell viability of HUVECs treated with 500 M IS was markedly decreased to 69.26 ± 4.08% of control, while pretreatment with 200 pM and 400 pM recombinant human Klotho protein rescued the inhibitory effect of IS (Fig. 1C). Subsequently, Hoechst 33342/PI staining, capase-3 immunoblot and flow cytometry analysis were performed to explore whether IS could induce HUVECs apoptosis.
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
79
Fig. 1. The effects of IS and Klotho protein on HUVECs viability. (A) Cells were exposed to 500 M of IS for 24 h, 48 h and 72 h. Cell viability was detected by MTT assay. (B) Cells were exposed to various concentrations of IS (100, 250, 500, 1000 M) for 24 h. (C) Cells were pretreated with 50, 100, 200, 400 pM of Klotho protein for 30 min respectively, and then incubated with IS (500 M) for 24 h. Data were shown as mean ± S.D. (n = 10). Statistical differences are expressed as **P < 0.01 vs Control; ## P < 0.01 vs IS.
The protein level of cleaved caspase-3 and apoptotic cell counts were not significantly increased after incubation with IS for 24 h (Fig. 2). Therefore, these results indicate that incubation of HUVECs with IS can induce cell injury but not apoptosis, and the cell injury induced by IS can be reversed by Klotho protein. 3.2. Klotho protein abrogated ROS production in HUVECs induced by IS To determine the potential role of oxidative stress in IS-induced injury of HUVECs, ROS production was detected. As shown in Fig. 3A, after incubation with IS at different concentrations from
100 to 1000 M for 3 h, ROS production in HUVECs was increased in a concentration-dependent manner. Compared with control group, even 100 M of IS could significantly increase the production of ROS, and treatment with 500 M of IS led to a 1.6-fold increase in ROS production. Because Klotho protein was considered to have strong ability to resist oxidative stress and could act as a circulating hormone, the effect of Klotho protein on ROS production induced by IS in HUVECs was detected in the following experiment. The result displayed that pretreatment with 200 pM and 400 pM of recombinant human Klotho markedly suppressed the production of ROS induced by IS in HUVECs (Fig. 3B and C).
Fig. 2. The effects of IS and Klotho protein on HUVECs apoptosis. (A) The normal, apoptotic and necrotic cells were observed by Hoechst 33342/PI staining in HUVECs after being incubated with 500 M IS for 24 h in the presence or absence of 200 pM Klotho protein. The scale bar corresponds to 50 m. (B) Apoptotic cells were analyzed by flow cytometry after incubating with 500 M IS for 24 h. (C) Cleaved caspase-3 protein levels were examined by immunoblotting. -Actin was used as a loading control.
80
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
Fig. 3. ROS production in HUVECs. (A) Cells were exposed to various concentrations of IS (100, 250, 500, 1000 M) for 3 h. (B) Cells were pretreated with 50, 100, 200, 400 pM Klotho protein for 30 min, and then incubated with 500 M IS for 3 h. The generation of intracellular ROS was detected using CM-H2 DCFDA. (C) ROS production was observed under inverted fluorescence microscope. Data were shown as mean ± S.D. (n = 8). Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; P < 0.05 vs IS.
3.3. Klotho protein could inhibit IS-induced activations of p38MAPK and NF-ÄB in HUVECs
3.5. Klotho protein could reverse the increased release of MCP-1 from HUVECs induced by IS
To investigate the mechanisms of the effects of IS on HUVECs and the protective role of Klotho protein against IS-induced injury, the activations of ROS sensitive pathways including p38MAPK and NF-B were detected. As shown in Fig. 4, treatment of IS for 15 min markedly increased the phosphorylation of p38MAPK and nuclear translocation of NF-B, whereas the IS-stimulated p38 MAPK phosphorylation and NF-B nuclear translocation were significantly attenuated by Klotho protein, similar as the effects of ROS scavenger, NAC. These results suggest that p38MAPK pathway and its downstream NF-B can be activated by IS in HUVECs, while pretreatment of HUVECs with Klotho protein is able to inhibit ISstimulated activation of p38MAPK and NF-B, possibly through blocking the production of ROS.
To further study the protective role of Klotho protein against IS-induced dysfunction of HUVECs, the production of MCP-1, an inflammation-related factor regulated by NF-B, was analyzed subsequently. After incubation with IS at concentrations from 100 to 1000 M, a concentration-dependent increase of MCP-1 protein released into culture medium was observed. In contrast, pretreatment with Klotho protein suppressed the IS-induced release of MCP-1 in HUVECs. The same results were also found when the cells were pretreated with SB203580 and PDTC (Fig. 6). It indicated that Klotho protein as well as p38MAPK and NF-B inhibitors could rescue IS-induced dysfunctions of HUVECs.
4. Discussion
3.4. Klotho protein could abrogate the decrease of NO production and phosphorylation of eNOS in HUVECs induced by IS To observe the cell functions influenced by IS, the effects of IS on NO production, the expression levels of endothelial NOS (eNOS) and inducible NOS (iNOS) in HUVECs were detected. After 2 h incubation, IS decreased NO production in a concentrationdependent manner. As shown in Fig. 5A, incubation with 250 M, 500 M and 1000 M of IS significantly decreased NO production to 76.3%, 70.4% and 54.5% of control. Moreover, the phosphorylation of eNOS was significantly reduced in HUVECs treated with 500 M IS for 30 min. While Klotho protein, as well as p38MAPK inhibitor SB203580 and NF-B inhibitor PDTC, could significantly abrogate the decrease of NO production and phosphorylation of eNOS in HUVECs induced by IS (Fig. 5B and C). But the iNOS expression was not affected by IS and Klotho protein in cultured HUVECs (Fig. 5D).
Chronic kidney disease (CKD) is recognized as a serious public health problem which can dramatically increase the complications of atherosclerosis and cardiovascular events (Elsayed et al., 2007; Mallamaci et al., 2005). As known, the occurrence of cardiovascular disease (CVD) is frequently initiated by “traditional” and “nontraditional” risk factors. In the past, “traditional” risk factors such as dislipidemia, hypertension, diabetes mellitus and old age were taken as the main causes of CVD. However, the traditional risk factors could not explain the high prevalence and incidence of CVD in CKD patients. In recent years, more and more reports demonstrated that “non-traditional” risk factors, such as inflammation, oxidative stress and endothelial dysfunction, were significantly associated with accelerated atherosclerosis and CVD (Gosmanova and Le, 2011; Rodriguez-Iturbe and Correa-Rotter, 2010). Uremic toxins in CKD patients are regarded as one of the main sources of “non-traditional” risk factors (Shimoishi et al., 2007). IS is a representative uremic toxin with property to bind with serum proteins, which leads to difficult elimination by general
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
81
Fig. 4. Immunoblotting analysis of the expression of p38MAPK and NF-B in HUVECs. HUVECs were preincubated with 200 pM of recombinant human Klotho, 5 mM NAC, 10 M SB203580 or 10 M PDTC for 30 min respectively, and then treated with 500 M IS for another 15 min. Whole cell and nuclear protein were isolated to perform immunoblot. (A) and (B) The protein expression of phospho-p38 was standardized by p38. (C) and (D) The protein expression of NF-B p65 was standardized by Histone H3. Values are means ± S.D. from each group. *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
dialysis in CKD patients. Previous studies demonstrated that high level of IS could induce endothelial cell dysfunction through triggering oxidative stress, even though it might function as an antioxidant under physiological concentration (Miyamoto et al., 2010; Tumur and Niwa, 2009; Tumur et al., 2010). Thus, inhibition of IS-induced oxidative stress will play beneficial roles in maintaining endothelial function and preventing the development of atherosclerosis for uremia patients. As reported, Klotho protein, a novel humoral factor produced in the kidneys, confers resistance to oxidative stress in multifold pathological conditions (Saito et al., 2000). In this study, we examined the damage effects of IS on endothelial cells firstly, and then explore the protective role of Klotho protein against IS-induced endothelial dysfunction. It was reported that the serum levels of IS in uremic patients were increased approximately 50-fold compared to healthy individuals, and the mean and maximal serum level of IS were approximately 250 M and 500 M respectively (Vanholder et al., 2003). Accordingly, we first chose a fixed-concentration of 500 M of IS to treat the cultured HUVECs for different times and found that the cell viability was significantly inhibited after 24 h incubation. Then a wide range of IS (100 M to 1000 M) was selected to observe its concentration-effect on HUVECs. As shown in the results, after incubation with IS at different concentrations, the cell viability were inhibited by IS in a dose-dependent manner. Notably, pretreatment with 200 pM or 400 pM Klotho protein can alleviate the inhibitive effect of IS. We further found that IS could induce ROS production in HUVECs with a concentration-dependent manner. It is known that
high levels of ROS can regulate signaling pathways via stressactivated MAP kinases, such as p38MAPK, to affect cell functions. Nuclear transcription factor NF-B is normally a downstream target of p38MAPK signaling pathway (Ganju et al., 2005). The activation of NF-B is involved in many pathological events, especially in the promotion of endothelial dysfunction and the development of atherosclerosis (Kanters et al., 2003). Because the transgenic mice overexpressing Klotho have low level of endogenous ROS and can live longer, the relationship between Klotho and ROS sensitive pathway has caused extensive concerns. Hsieh et al. (2010) reported that the beneficial effects of Klotho on longevity were mediated, at least partially, by inactivation of p38MAPK signaling. Therefore, we next investigated the effects of IS and Klotho on the activations of p38MAPK and NF-B in HUVECs. It was found that p38MAPK phosphorylation and nuclear translocation of NF-B were markedly increased in HUVECs treated with IS, in line with the report by Masai et al. (2010). As expected, treatment with Klotho protein significantly suppressed IS-induced ROS production in a dose-dependent manner, and the activations of p38MAPK and NFB could be blocked by Klotho protein, similar as the effect of ROS scavenger, NAC. Therefore, it implies that Klotho protein has the ability to inhibit the IS-induced oxidative stress and activations of p38MAPK and NF-B, possibly through blocking ROS production. Endothelial dysfunction is normally characterized by the imbalanced release of vasodilator and vasoconstrictor factors. The imbalance is predominantly due to the loss of NO because of its inactivation by ROS (Münzel et al., 2008). In addition, endothelial function is also affected by vascular inflammation (Trepels
82
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
Fig. 5. NO production, eNOS and iNOS expression in HUVECs. (A) IS significantly decreased NO production in a concentration-dependent manner (n = 8). (B) Decrease of NO production induced by IS (500 M) in HUVECs was rescued by pretreatment with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC (n = 8). (C) Immunoblotting analysis of the protein expression of phospho-eNOS which was standardized by eNOS. (D) Immunoblotting analysis of the protein expression of iNOS which was standardized by -actin. Data were shown as mean ± S.D. Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
et al., 2006). Interestingly, the activated endothelial cells may also produce chemotactic factors such as MCP-1 and soluble vascular adhesion molecule-1 (sVCAM-1), which can recruit circulating monocytes and T lymphocytes into the arterial wall to join in the process of inflammation and atherosclerosis (Collins, 1993; Toborek et al., 2002). It was reported recently that IS could activate the MAPK/NF-B pathway and lead to induction of MCP-1 expression in HUVECs (Masai et al., 2010). Therefore, we further
investigate the effects of IS and Klotho protein on NO production and MCP-1 expression in HUVECs. Since the production of NO is regulated by NOS, which includes eNOS and iNOS (Rudic et al., 1998; Xie et al., 1992; Marczin et al., 1996), the expression levels of both eNOS and iNOS were also observed in this study. Our results demonstrated that the NO production and eNOS phosphorylation were reduced, while the MCP-1 production was significantly enhanced in HUVECs treated with IS. Interestingly,
Fig. 6. The release of MCP-1 protein from HUVECs. (A) Cells were exposed to various doses of IS for 24 h. (B) Cells were preincubated with 200 pM of recombinant human Klotho, 10 M SB203580 or 10 M PDTC for 30 min and then treated with 500 M IS for 24 h. ELISA assay was applied to detect the released of MCP-1 in the culture supernatants. Data were shown as mean ± S.D. (n = 12). Statistical differences are expressed as *P < 0.05, **P < 0.01 vs Control; # P < 0.05, ## P < 0.01 vs IS.
K. Yang et al. / Toxicology Letters 215 (2012) 77–83
the expression of iNOS was not affected by IS and Klotho protein in HUVECs, indicating that IS inhibits the production of NO in HUVECs mainly by suppressing the expression and phosphorylation of eNOS. Remarkably, the IS-induced reduction of NO production, the depression of eNOS phosphorylation and the up-regulation of MCP1 could be reversed by Klotho protein, similarly to the effects of p38MAPK and NF-B inhibitors. Because we had already found that Klotho protein was able to inhibit IS-stimulated activation of p38MAPK and NF-B, these results suggest that the protective role of Klotho protein against IS-induced dysfunction of HUVECs may be partly attributed to the attenuation of p38MAPK and NF-B signaling pathways. In conclusion, this study demonstrated that Klotho protein has the ability to reverse IS-induced endothelial dysfunction. The observations further confirmed that accelerated atherosclerosis in uremia is closely related to uremic toxins-induced endothelial dysfunction. An increase in the serum level of Klotho protein may be a potential measure for treatment of atherosclerosis in chronic kidney disease. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work is supported by the National Science Foundations of China (No. 81270290 and No. 30700316) and the Natural Science Foundation of Chongqing (No. 2007BB5024). References Arking, D.E., Krebsova, A., Macek Sr., M., Macek Jr., M., Arking, A., Mian, I.S., Fried, L., Hamosh, A., Dey, S., McIntosh, I., Dietz, H.C., 2002. Association of human aging with a functional variant of Klotho. Proceedings of the National Academy of Sciences of the United States of America 99, 856–861. Atoh, K., Itoh, H., Haneda, M., 2009. Serum indoxyl sulfate levels in patients with diabetic nephropathy: relation to renal function. Diabetes Research and Clinical Practice 83, 220–226. Collins, T., 1993. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Laboratory Investigation 68, 499–508. Dou, L., Bertrand, E., Cerini, C., Faure, V., Sampol, J., Vanholder, R., Berland, Y., Brunet, P., 2004. The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair. Kidney International 65, 442– 451. Elsayed, E.F., Tighiouart, H., Griffith, J., Kurth, T., Levey, A.S., Salem, D., Sarnak, M.J., Weiner, D.E., 2007. Cardiovascular disease and subsequent kidney disease. Archives of Internal Medicine 167, 1130–1136. Ganju, L., Padwad, Y., Singh, R., Karan, D., Chanda, S., Chopra, M.K., Bhatnagar, P., Kashyap, R., Sawhney, R.C., 2005. Anti-inflammatory activity of seabuckthorn (hippophae rhamnoides) leaves. International Immunopharmacology 5, 1675–1684. Gosmanova, E.O., Le, N.A., 2011. Cardiovascular complications in CKD patients: role of oxidative stress. Cardiology Research and Practice 2, 156326. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., 1982. Analysis of nitrate, nitrite, and [15 N] nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Hsieh, C.C., Kuro-o, M., Rosenblatt, K.P., Brobey, R., Papaconstantinou, J., 2010. The ASK1-signalosome regulates p38 MAPK activity in response to levels of endogenous oxidative stress in the Klotho mouse models of aging. Aging (Albany NY) 2, 597–611. Kanters, E., Pasparakis, M., Gijbels, M.J., Vergouwe, M.N., Partouns-Hendriks, I., Fijneman, R.J., Clausen, B.E., Förster, I., Kockx, M.M., Rajewsky, K., Kraal, G., Hofker, M.H., de Winther, M.P., 2003. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. Journal of Clinical Investigation 112, 1176–1185. Koh, N., Fujimori, T., Nishiguchi, S., Tamori, A., Shiomi, S., Nakatani, T., Sugimura, K., Kishimoto, T., Kinoshita, S., Kuroki, T., Nabeshima, Y., 2001. Severely reduced production of klotho in human chronic renal failure kidney. Biochemical and Biophysical Research Communications 280, 1015– 1020. Kuro-o, M., Matsumura, Y., Aizawa, H., Kawaguchi, H., Suga, T., Utsugi, T., Ohyama, Y., Kurabayashi, M., Kaname, T., Kume, E., Iwasaki, H., Iida, A., Shiraki-Iida, T., Nishikawa, S., Nagai, R., Nabeshima, Y.I., 1997. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390, 45–51.
83
Mallamaci, F., Tripepi, G., Cutrupi, S., Malatino, L.S., Zoccali, C., 2005. Prognostic value of combined use of biomarkers of inflammation, endothelial dysfunction, and myocardiopathy in patients with ESRD. Kidney International 67, 2330–2337. Marczin, N., Jilling, T., Papapetropoulos, A., Go, C., Catravas, J.D., 1996. Cytoskeletondependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells. British Journal of Pharmacology 118, 1085–1094. Masai, N., Tatebe, J., Yoshino, G., Morita, T., 2010. Indoxyl sulfate stimulates monocyte hemoattractant protein-1 expression in human umbilical vein. endothelial cells by inducing oxidative stress through activation of the NADPH oxidasenuclear factor-b pathway. Circulation Journal 74, 2216–2224. Miyamoto, Y., Iwao, Y., Tasaki, Y., Sato, K., Ishima, Y., Watanabe, H., Kadowaki, D., Maruyama, T., Otagiri, M., 2010. The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions. FEBS Letters 584, 2816–2820. Münzel, T., Sinning, C., Post, F., Warnholtz, A., Schulz, E., 2008. Pathophysiology, diagnosis and prognostic implications of endothelial dysfunction. Annals of Medicine 40, 180–196. Namikoshi, T., Tomita, N., Satoh, M., Sakuta, T., Kuwabara, A., Kobayashi, S., 2009. Oral adsorbent AST-120 ameliorates endothelial dysfunction independent of renal function in rats with subtotal nephrectomy. Hypertension Research 32, 194–200. Oberg, B.P., McMenamin, E., Lucas, F.L., McMonagle, E., Morrow, J., Ikizler, T.A., Himmelfarb, J., 2004. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney International 65, 1009–1016. Rakugi, H., Matsukawa, N., Ishikawa, K., Yang, J., Imai, M., Ikushima, M., Maekawa, Y., Kida, I., Miyazaki, J., Ogihara, T., 2007. Anti-oxidative effect of Klotho on endothelial cells through cAMP activation. Endocrine 31, 82–87. Rodriguez-Iturbe, B., Correa-Rotter, R., 2010. Cardiovascular risk factors and prevention of cardiovascular disease in patients with chronic renal disease. Expert Opinion on Pharmacotherapy 11, 2687–2698. Rudic, R.D., Shesely, E.G., Maeda, N., Smithies, O., Segal, S.S., Sessa, W.C., 1998. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. The Journal of Clinical Investigation 101, 731–736. Saito, Y., Yamagishi, T., Nakamura, T., Ohyama, Y., Aizawa, H., Suga, T., Matsumura, Y., Masuda, H., Kurabayashi, M., Kuro-o, M., Nabeshima, Y., Nagai, R., 1998. Klotho protein protects against endothelial dysfunction. Biochemical and Biophysical Research Communication 248, 324–329. Saito, Y., Nakamura, T., Ohyama, Y., Suzuki, T., Iida, A., Shiraki-Iida, T., Kuro-o, M., Nabeshima, Y., Kurabayashi, M., Nagai, R., 2000. In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochemical and Biophysical Research Communication 276, 767–772. Sarnak, M.J., Levey, A.S., Schoolwerth, A.C., Coresh, J., Culleton, B., Hamm, L.L., McCullough, P.A., Kasiske, B.L., Kelepouris, E., Klag, M.J., Parfrey, P., Pfeffer, M., Raij, L., Spinosa, D.J., Wilson, P.W., 2003. Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on kidney in cardiovascular disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Hypertension 42, 1050–1065. Shimoishi, K., Anraku, M., Kitamura, K., Tasaki, Y., Taguchi, K., Hashimoto, M., Fukunaga, E., Maruyama, T., Otagiri, M., 2007. An oral adsorbent, AST-120 protects against the progression of oxidative stress by reducing the accumulation of indoxyl sulfate in the systemic circulation in renal failure. Pharmaceutical Research 24, 1283–1289. Taki, K., Tsuruta, Y., Niwa, T., 2007. Indoxyl sulfate and atherosclerotic risk factors in hemodialysis patients. American Journal of Nephrology 27, 30–35. Toborek, M., Lee, Y.W., Garrido, R., Kaiser, S., Hennig, B., 2002. Unsaturated fatty acids selectively induce an inflammatory environment in human endothelial cells. The American Journal of Clinical Nutrition 75, 119–125. Tonelli, M., Wiebe, N., Culleton, B., House, A., Rabbat, C., Fok, M., McAlister, F., Garg, A.X., 2006. Chronic kidney disease and mortality risk: a systematic review. Journal of the American Society of Nephrology 17, 2034–2047. Trepels, T., Zeiher, A.M., Fichtlscherer, S., 2006. The endothelium and inflammation. Endothelium 13, 423–429. Tumur, Z., Niwa, T., 2009. Indoxyl sulfate inhibits nitric oxide production and cell viability by inducing oxidative stress in vascular endothelial cells. American Journal of Nephrology 29, 551–557. Tumur, Z., Shimizu, H., Enomoto, A., Miyazaki, H., Niwa, T., 2010. Indoxyl sulfate upregulates expression of ICAM-1 and MCP-1 by oxidative stress-induced NF-B activation. American Journal of Nephrology 31, 435–441. Vanholder, R., De Smet, R., Glorieux, G., Argilés, A., Baurmeister, U., Brunet, P., Clark, W., Cohen, G., De Deyn, P.P., Deppisch, R., Descamps-Latscha, B., Henle, T., Jörres, A., Lemke, H.D., Massy, Z.A., Passlick-Deetjen, J., Rodriguez, M., Stegmayr, B., Stenvinkel, P., Tetta, C., Wanner, C., Zidek, W., 2003. European uremic toxin work group (eutox) review on uremic toxins: classification, concentration, and interindividual variability. Kidney International 63, 1934–1943. Xie, Q.W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Troso, T., Nathan, C., 1992. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256, 225–228. Yu, J., Deng, M., Zhao, J., Huang, L., 2010. Decreased expression of klotho gene in uremic atherosclerosis in apolipoprotein E-deficient mice. Biochemical and Biophysical Research Communications 391, 261–266. Zhao, J., Huang, Y., Song, Y., Zhao, X., Jin, J., Wang, J., Huang, L., 2009. Low osmolar contrast medium induces cellular injury and disruption of calcium homeostasis in rat glomerular endothelial cells in vitro. Toxicology Letters 185, 124–131.