Impact of cholesterol lowering treatment on plasma kynurenine and tryptophan concentrations in chronic kidney disease: Relationship with oxidative stress improvement

Accepted Manuscript Impact of cholesterol lowering treatment on plasma kynurenine and tryptophan concentrations in chronic kidney disease: relationship with oxidative stress improvement Angelo Zinellu , Dr., Salvatore Sotgia , Arduino A. Mangoni , Manuela Sanna , Andrea E. Satta , Ciriaco Carru , Dr. PII:

S0939-4753(14)00343-3

DOI:

10.1016/j.numecd.2014.11.004

Reference:

NUMECD 1377

To appear in:

Nutrition, Metabolism and Cardiovascular Diseases

Received Date: 18 July 2014 Revised Date:

2 October 2014

Accepted Date: 16 November 2014

Please cite this article as: Zinellu A, Sotgia S, Mangoni AA, Sanna M, Satta AE, Carru C, Impact of cholesterol lowering treatment on plasma kynurenine and tryptophan concentrations in chronic kidney disease: relationship with oxidative stress improvement, Nutrition, Metabolism and Cardiovascular Diseases (2014), doi: 10.1016/j.numecd.2014.11.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Impact of cholesterol lowering treatment on plasma kynurenine and tryptophan concentrations in chronic kidney disease: relationship with oxidative stress

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improvement

Angelo Zinellu1, *, Salvatore Sotgia1, Arduino A. Mangoni2, Manuela Sanna1, Andrea E. Satta3,

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Ciriaco Carru1,4,*

of Biomedical Sciences - University of Sassari, Sassari, Italy

2Department

of Clinical Pharmacology, School of Medicine, Flinders University, Adelaide,

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1 Department

Australia 3Department

of Surgical, Microsurgical and Medical Sciences - University of Sassari, Sassari,

Italy

Control Unit, Hospital University of Sassari (AOU), Sassari, Italy

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Running title: Cholesterol lowering therapy and Kynurenine concentrations in renal disease

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*Correspondence:

Dr. Angelo Zinellu, e-mail: [email protected]

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Dr. Ciriaco Carru, e-mail: [email protected] Dept. Biomedical Sciences - University of Sassari, Viale San Pietro 43/B – 07100 SASSARI (Italy) - fax: +39-079228275

Abbreviations used: CKD, chronic kidney disease; IDO, indoleamine (2,3)-dioxygenase; Kyn, Kynurenine; OS, Oxidative stress; Trp, Tryptophan; Keywords: Chronic kidney disease; Inflammation; Kynurenine; Oxidative stress; Tryptophan

ACCEPTED MANUSCRIPT Abstract Background: Tryptophan (Trp) degradation via indoleamine (2,3)-dioxygenase (IDO), with

consequent increased of Kynurenine (Kyn) concentrations, has been proposed as marker of immune system activation. Oxidative stress (OS) might contribute to the pro-inflammatory state in chronic

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kidney disease (CKD) through the activation of NF-kB, with consequent activation and recruitment of immune cells.

Methods and results: Serum concentrations of Trp and Kyn, oxidative stress indices

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malondialdehyde (MDA) and allantoin/uric acid (All/UA) ratio and anti-oxidant amino acid taurine were measured in 30 CKD patients randomized to 40 mg/day simvastatin (group 1),

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ezetimibe/simvastatin 10/20 mg/day (group 2) or ezetimibe/simvastatin 10/40 mg/day (group 3) and treated for 12 months. Baseline Kyn and Kyn/Trp ratio were higher in CKD patients vs. healthy controls (1.67±0.62 µmol/L vs 1.25±0.40 µmol/L, p<0.01 and 0.036±0.016 vs 0.023 ± 0.010, p<0.001 respectively). Both Kyn and Kyn/Trp ratio significantly decreased after cholesterol

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lowering treatment, to values comparable with healthy controls after one year treatment (1.67±0.11 µmol/L vs 1.31±0.09 µmol/L, p<0.0001 and 0.036±0.003 vs 0.028±0.002, p<0.0001, respectively). This was paralleled by a significant decrease of MDA (218±143 nmol/L vs 176±123 nmol/L,

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p<0.01) and All/UA ratio (1.47±0.72 vs 1.19±0.51, p<0.01) in CKD patients. Conclusions: Amelioration of both oxidative and inflammation status after cholesterol lowering

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treatment in CKD might be mediated by restoration of antioxidant taurine concentrations during therapy (from 51.1±13.3 µmol/L at baseline to 63.1 ± 16.4 µmol/L, p<0.001 by ANOVA), suggesting that improvement of both oxidative and inflammation status in CKD patients could be explained, at least partly, by the cholesterol lowering effects.

ACCEPTED MANUSCRIPT Introduction The essential amino acid tryptophan acts as a precursor of several metabolic pathways involving different end products, such as proteins, serotonin, melatonin and kynurenines [1]. About 95% of Trp is metabolized via the kynurenines pathway [2]. Trp is oxidized by cleavage of the indole-ring,

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initiated either by tryptophan 2,3-dioxygenase (TDO) and/or indoleamine 2,3-dioxygenase (IDO) [3]. TDO is expressed primarily in the liver and is induced by Trp or corticosteroids [3]. IDO, on the other hand, is predominant in extra-hepatic cells, including macrophages, microglia, neurons

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and astrocytes [4-5]. Under physiological conditions, about 99 % of Trp is metabolized to Kyn in the liver by TDO. In inflammatory conditions, infections or oxidative stress, the first rate limiting

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enzymatic step involving IDO, is activated. IDO activity is enhanced by pro-inflammatory cytokines such as IFNγ [6], IL-1, IL-2, IL-6 and TNFα, and inhibited by the anti-inflammatory cytokine IL-4 [7]. Thus, the Kyn pathway is systemically up-regulated when the immune response is activated. Thereby the ratio of Kyn to Trp concentrations reflects the Trp breakdown linked with

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conditions of inflammation [8]. Inflammation is a significant burden to patients with chronic kidney disease. Chronic elevation of inflammatory markers appears to be exacerbated by disease progression [9]. Inflammation might favor a decline of renal function by promoting endothelial

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dysfunction, atherosclerosis, and glomerular damage. Although the mechanisms responsible for inflammation in CKD are not fully clear, oxidative stress has been proposed as potential contributor

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to inflammation in renal disease. Oxidative stress and inflammation are inseparably linked as they form a vicious cycle in which oxidative stress provokes inflammation by several mechanisms including activation of NF-κB, with consequent activation and recruitment of immune cells. Inflammation, in turn, provokes oxidative stress via production of reactive oxygen, nitrogen and halogen species by activated leukocytes and resident cells [10]. In recent years, it has become evident that inflammation is among the strongest predictors of poor clinical outcome in CKD patients. Elevated plasma levels of pro-inflammatory cytokines, IL-1, IL-6, and tumor necrosis factor (TNF)-α are also associated with increased mortality [11]. Cardiovascular disease is the

ACCEPTED MANUSCRIPT leading cause of morbidity and mortality in CKD [12]. Among patients with stages III-IV CKD, the prevalence of CVD is 4- to 5-fold higher than that observed for the general population. CKD patients are often affected by diabetes, hypertension, and obesity, which are known traditional CVD risk factors in general population [13]. Moreover, in proteinuric patients, dyslipidemia has a highly

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atherogenic profile, with increased total and low-density lipoprotein (LDL) cholesterol, triglycerides, and decreased high-density lipoprotein (HDL) cholesterol [14]. Since it is well known that cholesterol lowering with statins in hypercholesterolaemic patients decreases cardiovascular

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morbidity and mortality, management of hypercholesterolaemia is an important target in CKD [15]. Experimental and clinical evidences show that statins, in addition to lowering plasma cholesterol

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concentrations, may have specific renoprotective properties and, when combined with reninangiotensin system (RAS) inhibitor therapy, may have additive antiproteinuric effects [16]. Preliminary data suggest that the combination of statin with ezetimibe (EZE), a recently marketed cholesterol absorption inhibitor, provide complementary effects on lipids over that achieved with

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statin monotherapy [17]. Recent data also suggest as atorvastatin treatment, in addition to its beneficial effect on cholesterol levels, improved the inflammatory state of CKD patients by reducing plasma levels of CRP, TNF-α and IL-1 [18]. We hypothesized that cholesterol lowering

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treatment, by exerting anti-inflammatory effects, might directly affect IDO activity in CKD, with consequent changes in the product/substrate (Kyn/Trp) ratio. Concomitantly we sought to address

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whether these effects were associated with changes in markers of oxidative stress (malondialdehyde and allantoin/uric acid ratio) and taurine, a sulfur amino acid with a marked antioxidant effect.

Methods Subjects Thirty CKD patients (age 60.2±10.5 years, 19 males) were selected at the Istituto di Patologia Medica - Azienda Ospedaliero Universitaria, with the following inclusion criteria: age >18 years, plasma LDL-cholesterol concentrations > 100 mg/dl (without concomitant

ACCEPTED MANUSCRIPT hypolipidemic drugs), presence of proteinuric CKD defined as creatinine clearance >20 ml/min/1.73 m2 combined with urinary protein excretion rate > 0.3 g/24h, without evidence of urinary tract infection or overt heart failure (New York Heart Association class III or more). Patients were CKD stage 3 or 4, not receiving dialysis. Exclusion criteria were: previous or

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concomitant treatment with steroids, anti-inflammatory and immunosuppressive agents, vitamin B6, B12, folate or statin; evidence or clinical suspicion of renovascular disease, obstructive uropathy, type 1 diabetes and vasculitis. All patients were on stable treatment with RAS inhibitor

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therapy (ACE inhibition by benazepril plus angiotensin II antagonism by valsartan) for at least six months. Enrolled patients were randomized to 40 mg/day simvastatin (group 1, n=10),

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ezetimibe/simvastatin 10/20 mg/day (group 2, n=10) or ezetimibe/simvastatin 10/40 mg/day (group 3, n=10). Patients were treated for 12 months and were evaluated at baseline and at 4, 8 and 12 months of therapy. A control group including 30 age- and sex-matched subjects (age 58.1±13.8 years, 18 males) was also recruited. Exclusion criteria for control subjects were a history of

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diabetes, hypertension, cardiovascular or cerebrovascular disease, renal failure, blood dyscrasias, cancer, retinal vascular disorders, age <18 years, and current medication with vitamin B6, B12, or folic acid. Informed consent was obtained from each patient and control, and the study was

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approved by our Institution’s Ethics Committee. The study complied with the principles of the

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Helsinki Declaration and was registered with clinicaltrials.gov (NCT00861731).

Biochemical analysis

Kyn, Trp, MDA, All/UA ratio and taurine were determined by capillary electrophoresis UV detection as previously described [19-22]. Total plasma cholesterol, LDL, HDL and tryglicerides were measured by enzymatic methods using commercial kits (Boehringer-Mannheim, Mannheim, Germany). Serum CPK levels were measured by using a fully automated biochemical analyzer (Abbot).

ACCEPTED MANUSCRIPT Statistical analysis All results are expressed as mean values (mean ± SD) or median values (median and range). The variables distribution in the study group was assessed by the Kolmogorov-Simirnov test. The statistical differences among controls and patients were compared using unpaired Student’s t-test or

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Mann-Whitney rank sum test, as appropriate. The effect of the drug treatments was evaluated by one way repeated measures ANOVA. Correlation analysis between variables was performed by Pearson's correlation or Spearman’s correlation as appropriate. Multiple linear regression analysis

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was used to assess the contribution of different variables to serum Kyn concentration and Kyn/Trp ratio. Statistical analyses were performed using MedCalc for Windows, version 12.5 64 bit

Corporation; Armonk, NY, USA).

Results

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(MedCalc Software, Ostend, Belgium) and SPSS for Windows, version 14.0 32 bit (IBM

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Clinical characteristics of controls and all CKD patients, at baseline, are reported in Table 1. As reported in our previous work [22] CKD patients showed higher concentrations of plasma

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triglycerides, total cholesterol, LDL cholesterol and higher LDL/HDL ratio vs controls. CKD patients also showed higher concentrations of MDA and All/UA ratio and lower levels of taurine.

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We also found higher plasma Kyn levels and lower concentrations of Trp with an increased Kyn/Trp ratio in nephropatic patients (Table 1) . Baseline Kyn concentrations in CKD were positively correlated with All/AU ratio (r=0.52, p=0.003), serum creatinine (r=0.55, p=0.002) and age (r=0.52, p=0.003). Kyn/Trp ratio was positively correlated to All/AU ratio (r=0.36, p=0.049), MDA (r=0.60, p<0.001), serum creatinine (r=0.55, p=0.002) and age (r=0.58, p<0.001). Multiple linear regression with Kyn concentrations or Kyn/Trp ratio as dependent variable and sex, LDL, age, All/AU, MDA, and creatinine as independent variables showed that only All/AU (t-test, 2.67; P <0.014) were independently associated with baseline Kyn/Trp ratio in CKD. An association trend was also evident with LDL cholesterol levels even if data was not statistically significant (t-test 1.76

ACCEPTED MANUSCRIPT p=0.091). Treatment with statins and ezetimibe was well tolerated. No patient reported adverse drug reactions. After randomization, no significant differences were found among the three treatment groups except for All/UA ratios (Table 2). Drug treatment did not significantly affect GFR (baseline 55±30 ml/min x 1.73 m2 at baseline vs. 59±40 ml/min x 1.73 m2 after 12 months treatment for all

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patients, p=0.18 by ANOVA) or proteinuria (0.99 ± 1.27 g/24h at baseline vs 0.85 ± 0.85 after 12 months treatment for all patients, p=0.75 by ANOVA). As previously described [23], a significant improvement in lipid profile was observed in all groups after 4 months of therapy. A decrease of

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40% in total cholesterol, 62% in LDL, 21% in triglycerides, and 66% in LDL/HDL ratio was observed after 12 months. A significant decrease in MDA and All/UA ratios was also observed (-

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19% for both MDA concentration and All/UA ratio after 12 months) with a trend for a greater effect in patients of group 3 (-26% for MDA concentrations and -28% for All/UA ratio after 12 months). Moreover as previously reported [24] taurine concentration significantly increased all over drug treatment (from 51.1±13.3 µmol/L at baseline to 63.1 ± 16.4 µmol/L, p<0.001 by ANOVA). The

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rise of taurine was more striking for the group with the concomitant administration of ezetimibe/simvastatin 10/40 mg/day (+31.6 % after one year of therapy). As reported in Figure 1 drug treatment significantly reduced Kyn concentrations in all patients (-21%) as well as in

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individual treatment groups, with a greater effect in patients of group 3 (-23%). By contrast, Trp concentrations increased in all patients (+8%) as well as in each treatment group, although the

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increment was not statistically significant (data not shown). A significant decrease in Kyn/Trp ratios was observed during therapy for all patients and when assessing individual treatment groups (Figure 2). Treatment-induced decrement trends in Kyn concentrations and Kyn/Trp ratios paralleled those in MDA and All/UA in all patients (Figure 3). The reduction of Kyn concentrations and Kyn/Trp ratio during therapy was significantly associated to the reduction in MDA concentrations (r=0.965 p=0.037 and r=0.997 p=0.003, respectively) and All/AU ratios (r=0.964 p=0.036 and r=0.998 p=0.002 respectively). Moreover we found a correlation between the drop of LDL cholesterol and the decrease of All/AU ratio during treatment (rho=0.39, p=0.03) and a trend between the decrease

ACCEPTED MANUSCRIPT of LDL cholesterol and the reduction of Kyn/Trp ratio (rho=0.36, p=0.06). CPK concentrations, measured as a marker of drug-induced toxicity, significantly increase during treatment (93±8 U/L at baseline, 111± 12 U/L after 4 months, 106 ± 14 after 8 months and 148±23 U/L after 12 months, p=0.012 by ANOVA). In particular the differences between the baseline values vs CPK levels after

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one year of treatment were significantly different (p=0.032 by ANOVA with Bonferroni correction). No significant differences were observed among the three treatment groups. Only one

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patient showed CPK values above the reference range at the end of the study.

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Discussion

CKD is a well-known risk factor for cardiovascular disease [12]. It has been reported that the increased cardiovascular morbidity and mortality is present across the whole renal dysfunction spectrum, even in patients with moderate CKD. This high risk for cardiovascular disease seems to be the consequence of a higher prevalence of risk factors in patients with CKD than in the general

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population. CKD patients are often affected by diabetes, hypertension, and obesity [13]. Increased OS markers have been also found in patients with CKD. These include products of lipid oxidation and oxidized LDL advanced oxidation protein products, F2 isoprostanes, and the marker of

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oxidative DNA damage 8-hydroxyl 2-deoxyguanosine[10]. The mechanisms responsible for OS in

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CKD remain unclear. Impaired oxidative balance may derive from a combination of increased reactive oxygen species (ROS) production and reduced clearance as well as an unsuccessful antioxidant defense. CKD is also characterized by a marked pro-inflammatory state. The latter is an important indicator of patient health and outcome. Although precise mechanisms that contribute to the high prevalence of inflammation in CKD are not well established, ROS might have an important role particularly when renal function declines. Inflammation is a redox-sensitive mechanism, as oxidative stress is able to activate transcriptor factors such as NF-kB, which regulates inflammatory mediator gene expression [10]. In addition, to oxidative stress and inflammation, increasing cardiovascular risk per se, CKD patients often have a dyslipidemic profile with increased total and

ACCEPTED MANUSCRIPT LDL cholesterol, triglyceride, and a decreased HDL cholesterol. Lowering LDL cholesterol is an important target for cardiovascular disease prevention in CKD. Statins are considered the most effective treatment strategy in this context as they reduce cholesterol synthesis by inhibiting 3hydroxy-3-methyl-glutaryl-CoA reductase. The treatment with statins has been associated with

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improved outcomes both in primary and in secondary prevention of cardiovascular disease. This benefit has been attributed not only to cholesterol lowering but also to various pleiotropic antiatherosclerotic properties of these drugs (namely anti-inflammatory, anti-oxidative and anti-

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thrombotic) [25]. Nevertheless, not all patients tolerate high-dose statins and side effects in liver function or myopathy may increase in a dose-dependent manner with this class of drugs [26]. On

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the other hand, some patients do not respond satisfactorily to treatment. The combination of statins with cholesterol-lowering drugs having a different mechanism of action often provides additional effects on cholesterol concentrations [27]. Through the block of Niemann–Pick C1-like 1 protein for cholesterol transport, ezetimibe strongly inhibits the intestinal absorption of cholesterol from

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dietary and biliary sources [13]. By this work we aimed to investigate the effect of lipid lowering therapy on plasma kynurenine and tryptophan levels in nephropatic proteinuric patients. Dietary tryptophan is mostly metabolized by the kynurenine pathway, where Trp is catabolized by either

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tryptophan 2,3-dioxygenase or indoleamine 2,3-dioxygenase to N-formylkynurenine, which is then converted to Kyn [3]. Under physiological circumstances the activity of IDO in Trp catabolism is

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low. The expression of IDO can be induced by pro-inflammatory cytokines interferon-γ which is up-regulated in response to infection and tissue inflammation [6]. The kynurenine-to-tryptophan ratio has been proposed as a sensitive tool to monitor the activation status of IDO and of cellular immunity both in vivo and in vitro [8]. A decrease in serum tryptophan and a parallel increase of kynurenine attributable to IDO activation is found in various diseases associated with T-cell activation, such as viral infections, autoimmune disorders, and malignant diseases [28]. In accordance with a recent observation [29] we found higher levels of plasma kynurenyne and Kyn/Trp ratio in CKD patients vs. controls. These data are also consistent with the general concept

ACCEPTED MANUSCRIPT that recurrent or chronic inflammatory processes are common in CKD [10]. We also found a correlation between Kyn/Trp ratio and OS indices MDA and All/AU ratio, which confirms previous observations on the association between inflammation and OS in

nephropathic pre-dialyzed

patients [10, 18]. As expected, drug treatment significantly improved the lipid profile in all patients

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with better results in subjects treated with ezetimibe/simvastatin 10/40 mg day. This was associated with a consistent reduction in Kyn and Kyn/Trp ratios regardless of treatment groups.

Our data are consistent with the findings of Goicochea et al. [18] on the effects of atorvastatin on

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inflammatory parameters of nephropatic patients. These authors reported that, in addition to the hypolipidemic effect, atorvastatin treatment significantly reduced plasma levels of CRP, IL-1, and

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TNF-α. As the main route of eliminating Kyn is renal excretion, it will be reasonable to hypothesize that the improvement in the excretory kidney activity due to lipid lowering therapy may be responsible for the restoring of Kyn levels to normal values. However the lack of a significant improvement in GFR or proteinuria during treatment in our study suggests that Kyn decrease was

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due to other factors. The significant association between the reduction of Kyn levels and Kyn/Trp ratios and the reduction in OS markers (MDA and All/AU ratio) further supports a close interplay between inflammation and oxidation. The observed reduction of plasma Kyn during cholesterol

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lowering therapy may be due to down-regulation of IDO as consequence of OS decrease. In addition an indicative even if not significant increase in

Trp levels (+8%) suggest a larger

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preservation of this amino acid during cholesterol lowering treatment. The mechanisms involved in oxidative stress improvement during cholesterol lowering treatment have been extensively discussed in our previous work [23]. Briefly, we propose that the reduction of cholesterol following drug administration restores normal taurine concentrations. It is well known that in hypercholesterolemia there is a significant decrease in the concentration of taurine in serum, liver and heart due to excessive consumption for bile salts synthesis [30]. Taurine restoring during cholesterol lowering therapy might yield OS lowering through the reduction of superoxide anion production at the respiratory chain activity level [30].

ACCEPTED MANUSCRIPT Interestingly, the more beneficial effect of drugs on Kynurenine levels has been found in group 3, which also showed not significant trends towards greater efficacy on lipid lowering, increased taurine plasma leves and reduction of OS indices if compared with the other two groups. In summary, treatment with simvastatin alone or in combination with ezetimibe for one year in

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nondialysis CKD patients induced a significant decrease in Kyn levels and Kyn/Trp ratio confirming the important effects of these molecules in the reduction of inflammation status. It is well known that the protective effect of statins in CKD patients might be explained by a

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combination of cholesterol-lowering and pleiotropic effects on endothelial function, inflammation and oxidation. By this work we hypothesized a possible relationship between cholesterol-lowering

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and reduction of inflammation. In particular we suggest that the improvement in inflammatory status could be mediated, at least in part, by a reduction of OS due to the restoring of normal taurine

Acknowledgements

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concentrations. However, this hypothesis requires further larger, appropriately powered, studies.

This study was supported by the “Fondazione Banco di Sardegna – Sassari – Italy ” and by the

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“Ministero dell’Università e della Ricerca” Italy. The manuscript language revision by Mrs Maria

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Antonietta Meloni is greatly appreciated.

ACCEPTED MANUSCRIPT REFERENCES

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1. Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA. Tryptophan metabolism in the central nervous system: medical implications. Expert Rev Mol Med. 2006;8:1–27 2. Gál EM, Sherman AD. L-kynurenine: Its synthesis and possible regulatory function in brain. Neurochem Res 1980; 5:223-9 3. Salter M, Pogson CI. The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes. Biochem J 1985;229:499–504 4. Takikawa O. Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated L-tryptophan metabolism. Biochem Biophys Res Commun 2005;338:12–19. 5. Guillemin GJ, Smythe G, Takikawa O, Brew BJ. Expression of indoleamine 2,3dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia 2005;49:15–23. 6. Maes M, der Planken V, Van Gastel A, Desnyder R. Blood coagulation and platelet aggression in major depression. J Affect Disord 1996;40:35–40. 7. Musselman DL, Miller AH, Porter MR, Manatunga A, Gao F, Penna S. Higher than normal plasma interleukin-6 concentrations in cancer patients with depression: preliminary findings. Am J Psychiatry 2001;158:1252–7. 8. Capuron L, Schroecksnadel S, Feart C, Aubert A, Higueret D, Barberger-Gateau P, et al. Chronic low-grade inflammation in elderly persons is associated with altered tryptophan and tyrosine metabolism: role in neuropsychiatric symptoms. Biol Psychiatry 2011;70:175–82 9. Nakanishi I, Moutabarrik A, Okada N, Kitamura E, Hayashi A, Syouji T, et al. Interleukin-8 in chronic renal failure and dialysis patients. Nephrol Dial Transplant 1994;9:1435-42 10. Cachofeiro V, Goicochea M, de Vinuesa SG, Oubiña P, Lahera V, Luño J. Oxidative stress and inflammation, a link between chronic kidney disease and cardiovascular disease. Kidney Int Suppl 2008;111:S4-S9 11. Kimmel PL, Phillips TM, Simmens SJ, Peterson RA, Weihs KL, Alleyne S, et al. Immunologic function and survival in hemodialysis patients. Kidney Int 1998;54:236-44. 12. Manjunath G, Tighiouart H, Coresh J, Macleod B, Salem DN, Griffith JL, et al. Level of kidney function as a risk factor for cardiovascular outcomes in the elderly. Kidney Int 2003;63:1121–29. 13. Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B, Hamm LL, et al. American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention: 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. Circulation 2003;108:2154–69. 14. Vaziri ND. Dyslipidemia of chronic renal failure: the nature, mechanisms, and potential consequences. Am J Physiol Renal Physiol 2006;290:F262–F272. 15. Sever PS, Dahlöf B, Poulter NR, Wedel H, Beevers G, Caulfield M, et al. ASCOT investigators: Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations, in the Anglo-

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA): a multicentre randomised controlled trial. Lancet 2003;361:1149 – 58. 16. Zoja C, Corna D, Gagliardini E, Conti S, Arnaboldi L, Benigni A, Remuzzi G. Adding a statin to a combination of ACE inhibitor and ARB normalizes proteinuria in experimental diabetes, which translates into full renoprotection. Am J Physiol Renal Physiol 2010;299: F1203-F1211. 17. Dembowski E, Davidson MH. Statin and ezetimibe combination therapy in cardiovascular disease. Curr Opin Endocrinol Diabetes Obes 2009;16:183–88. 18. Goicoechea M, de Vinuesa SG, Lahera V, Cachofeiro V, Gómez-Campderá F, Vega A,Abad S, Luño J. Effects of atorvastatin on inflammatory and fibrinolyticparameters in patients with chronic kidney disease. J Am Soc Nephrol 2006;17:S231-5. 19. Zinellu A, Sotgia S, Deiana L, Talanas G, Terrosu P, Carru C. Simultaneous analysis of kynurenine and tryptophan in human plasma by capillary electrophoresis with UV detection. J Sep Sci 2012;35:1146-51 20. Zinellu A, Sotgia S, Deiana L, Carru C. Field-amplified online sample stacking capillary electrophoresis UV detection for plasma malondialdehyde measurement. Electrophoresis 2011; 32:1893-97 21. Zinellu A, Sotgia S, Deiana L, Carru C.Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma. Anal Bioanal Chem 2011;399: 2855-61 22. Zinellu A, Sotgia S, Scanu B, Chessa R, Gaspa L, Franconi F, et al. Taurine determination by capillary electrophoresis with laser-inducedfluorescence detection: from clinical field to quality food applications. Amino Acids 2009;36:35-41. 23. Zinellu A, Loriga G, Scanu B, Pisanu E, Sanna M, Deiana L, et al. Increased Low-Density Lipoprotein S-Homocysteinylation in Chronic Kidney Disease. Am J Nephrol 2010;32: 2428 24. Zinellu A, Sotgia S, Loriga G, Deiana L, Satta AE, Carru C. Oxidative stress improvement is associated with increased levels of taurine in CKD patients undergoing lipid-lowering therapy. Amino Acids 2012;43:1499-507 25. Kostapanos MS, Milionis HJ, Elisaf MS. An overview of the extra-lipid effects of rosuvastatin. J Cardiovasc Pharmacol Ther 2008;13:157–74 26. Conard S, Bays H, Leiter LA, Bird S, Lin J, Hanson ME, et al. Ezetimibe added to atorvastatin compared with doubling the atorvastatin dose in patients at high risk for coronary heart disease with diabetes mellitus, metabolic syndrome or neither. Diabetes Obes Metab 2010;12:210-8 27. Stein EA, Ballantyne CM, Windler E, Sirnes PA, Sussekov A, Yigit Z, et al. Efficacy and tolerability of fluvastatin XL 80 mg alone, ezetimibe alone, and the combination of fluvastatin XL 80 mg with ezetimibe in patients with a history of muscle-related side effects with other statins. Am J Cardiol 2008;101:490–96 28. Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Wachter H. Characteristics of interferon-induced tryptophan metabolism in human cells in vitro. Biochem Biophys Acta 1989;1012:140–7 29. Zhao J. Plasma kynurenic acid/tryptophan ratio: a sensitive and reliable biomarker for the assessment of renal function. Ren Fail. 2013;35:648-53

ACCEPTED MANUSCRIPT 30. Yokogoshi H, Oda H. Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Amino Acids 2002;23:433-9

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Figure Legends

Figure 1. Effect of drug treatment on Kynurenine plasma levels in all patients (A) and after categorization for therapy type: B = Group 1 (n=10); C = Group 2 (n=10); D = Group 3 (n=10).

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*= p<0.05; ** p< 0.01 vs baseline. P-values as been evaluated by one way repeated measures

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ANOVA with Bonferroni correction.

Figure 2. Effect of drug treatment on Kyn/Trp ratio values in all patients (A) and after categorization for therapy type: B = Group 1 (n=10); C = Group 2 (n=10); D = Group 3 (n=10). *= p<0.05; ** p< 0.01; *** p<0.001 vs baseline. P-values as been evaluated by one way repeated

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measures ANOVA with Bonferroni correction.

Figure 3. Decrement trends for all patients of Kyn and MDA plasma levels (A) and Kyn/Trp ratio

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11/19 (37%) 11/19 (37%) Sex, F/M(%F) 59±10 60±11 Age, Years -27.9±4.4 BMI Blood pressure Systolic BP, mmHg 126±8 130 ± 9 Diastolic BP, mmHg 80 (70-90) 80 (60-95) Kidney profile Creatinine, mg/dL 0.87±0.24 1.75 ± 0.77*** 2 GFR, ml/min per 1.73 m -55 ± 30 Proteinuria, g/24h -0.99 ± 1.27 Lipid profile Total cholesterol, mg/dL 208±42 239 ± 43** LDL-C, mg/dL 131±39 160 ± 37** HDL-C, mg/dL 56±18 49 ±15 2.5±1.1 3.5 ±1.3** LDL/HDL ratio Triglycerides, mg/dL 108 ± 54 143 ± 69* Oxidative stress indices MDA, nmol/L 140±81 218±147* All/UA ratio, (%) 0.90±0.10 1.5±0.7*** Taurine, µmol/L 62.3 ± 16.1 51.1±13.3** Trp degradation indices Kyn, µmol/L 1.25±0.40 1.67±0.62** Trp, µmol/L 57.1 ± 9.6 48.9±10.9** 0.023 ± 0.010 0.036±0.016*** Kyn/Trp ratio *= p<0.05, ** p< 0.01, *** p<0.001 vs Controls;

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Table 1. Demographic and clinical characteristics of patients and controls

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2/8 (20%) 4/6 (40%) 5/5 (50%) Sex, F/M(%F) 63±11 58±12 59±9 Age, Years 27.5±4.0 25.7±3.90 30±4.4 BMI Blood pressure Systolic BP, mmHg 131 ± 9 127 ± 11 132 ± 8 Diastolic BP, mmHg 80 (70-85) 80 (60-95) 80 (70-90) Kidney profile Creatinine, mg/dL 1.92 ± 0.98 1.63 ± 0.62 1.70 ± 0.71 2 GFR, ml/min per 1.73 m 61 ± 48 52 ± 19 53 ± 8 Proteinuria, g/24h 0.91 ± 0.63 0.81 ± 0.81 1.25 ± 2.00 Lipid profile Total cholesterol, mg/dL 232 ± 34 230 ± 41 254 ± 53 LDL-C, mg/dL 164 ± 34 156 ± 32 165 ± 47 HDL-C, mg/dL 44 ± 8 47 ± 12 57 ± 19 3.8 ± 1.0 3.5 ± 1.1 3.3 ± 1.7 LDL/HDL ratio Triglycerides, mg/dL 141 ± 70 136 ± 62 151 ± 80 Oxidative stress indices MDA, nmol/L 248±95 174±163 230±163 All/UA ratio, (%) 1.7±0.10 1.6±0.6 1.1±0.4° Taurine, µmol/L 53.3±13.0 51.0±10.5 48.9±16.7 Trp degradation indices Kyn, µmol/L 1.68±0.86 1.70±0.55 1.73±0.45 Trp, µmol/L 49.0±14.0 49.0±11.1 48.7±7.9 0.038±0.023 0.033±0.011 0.036±0.011 Kyn/Trp ratio After randomization baseline characteristics of the three sub-groups were quite similar except for ° p<0.05 vs Group 1 and Group 3

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Table 2. Demographic and clinical characteristics of CKD randomized groups. Group 1 (n=10) Group 2 (n=10) Group 3 (n=10) Simvastatin Eze/Simva Eze/Simva 40 10/20 mg/day 10/40 mg/day mg/day

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