Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients

Free Radical Biology and Medicine 89 (2015) 443–451

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Original Contribution

Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients Graziano Colombo a, Francesco Reggiani b, Manuel A. Podestà b, Maria Lisa Garavaglia a, Nicola M. Portinaro c, Aldo Milzani a, Salvatore Badalamenti b, Isabella Dalle-Donne a,n a

Department of Biosciences, Università degli Studi di Milano, Milan, Italy Humanitas Clinical and Research Center, Nephrology Unit, Rozzano, Milan, Italy c Humanitas Clinical and Research Center, Clinica ortopedica e traumatologica, Rozzano, Milan, Italy b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 June 2015 Received in revised form 7 August 2015 Accepted 25 August 2015 Available online 8 October 2015

The role of oxidative stress in patients with end stage renal disease (ESRD), which occurs at significantly higher levels than in the general population, is often underestimated in clinical practice. Emerging evidence highlights the strong correlation of oxidative stress with chronic inflammation and cardiovascular disease, which are highly prevalent in most patients on maintenance haemodialysis (HD) and are a major risk factor for mortality in this population. In this study, total plasma thiols and plasma S-thiolated proteins were measured in patients with ESRD, before and after a regular HD session, and compared to age-matched healthy subjects. We found a significant decrease in the level of total plasma thiols and, conversely, a significant increase in the level of S-thiolated proteins in these patients. In most patients, post-HD plasma level of total thiols did not differ from the one in healthy subjects, whereas plasma level of S-thiolated proteins was lower in HD patients than in age-matched healthy controls. This suggests that a single HD session restores plasma thiol redox status and re-establishes the antioxidant capacity of plasma thiols. Additionally, we determined protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to S-thiolated plasma proteins and protein free cysteinyl residues. Patients with ESRD had a significantly higher PTI compared to age-matched healthy subjects and HD was associated with a decrease in PTI to normal, or lower than normal, levels. Although this study is limited in size, our results suggest that PTI is a useful indicator of thiol-specific oxidative stress in patients with ESRD on maintenance HD. This study also emphasizes that PTI determination is a cheap and simple tool suitable for large-scale clinical studies that could be used for routine screening of thiol-specific oxidative stress. & 2015 Elsevier Inc. All rights reserved.

Keywords: Chronic kidney disease End stage renal disease Haemodialysis Oxidative stress Plasma thiols S-thiolated proteins Protein thiolation index Biomarker

1. Introduction Patients with chronic kidney disease (CKD) and, more markedly, those with end stage renal disease (ESRD, i.e. CKD stage V), show a significantly increased oxidative stress, which may results from uraemia per se and inflammation [1–5]. Indeed, the strong association between renal dysfunction and different mediators/ biomarkers of inflammation suggests that CKD is a low-grade inflammatory process in itself [6–10]. Other pro-oxidant conditions such as ageing, dyslipidemia, hypertension, diabetes mellitus,

obesity and infectious complications, which are commonly present in patients with CKD stages I-IV (i.e., patients with different degrees of CKD but with residual renal function) and are even more dominant in patients with ESRD, can further aggravate the oxidative stress status [11–15]. Particularly in patients with ESRD on haemodialysis (HD), both acute-phase inflammation and elevated levels of oxidative stress, besides accelerated atherogenesis, dyslipidemia, and endothelial dysfunction, are associated with a high rate of cardiovascular morbidity and hospitalization [16–18]. These risk factors are associated with a 10- to 100-fold increase in

Abbreviations: AOPP, advanced oxidation protein products; CKD, chronic kidney disease; CVD, cardiovascular disease; CysGly, cysteinylglycine; DMSO, dimethyl sulfoxide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; ECL, enhanced chemiluminescence; ESRD, end stage renal disease; GSH, glutathione; GSSG, glutathione disulphide; Hcy, homocysteine; HD, haemodialysis; HRP, horseradish peroxidase; IAA, iodoacetic acid; LMM-SH, low molecular mass thiols; NEM, N-ethylmaleimide; PBS, potassium phosphate buffer; PSH, protein thiols; PSSX, S-thiolated proteins; PTI, protein thiolation index; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis n Correspondence to: Department of Biosciences, University of Milan, via Celoria 26, I-20133 Milan, Italy. Fax: þ 39 02 50314781. E-mail address: [email protected] (I. Dalle-Donne). http://dx.doi.org/10.1016/j.freeradbiomed.2015.08.022 0891-5849/& 2015 Elsevier Inc. All rights reserved.

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cardiovascular and all-cause mortality when compared to agematched controls [10]. Furthermore, patients with CKD frequently present with lower levels of water-soluble antioxidant vitamins owing to their dietary restriction of fresh fruits and vegetables to avoid hyperkalaemia [2,19,20]. In particular, non-supplemented maintenance HD patients are at high risk for vitamin C deficiency [2,21,22] and a low plasma vitamin C level predicts fatal and major non-fatal adverse cardiovascular events among maintenance HD patients [21,22]. Moreover, the administration of intravenous iron to correct anaemia, a common finding in ESRD population, can further aggravate oxidative stress in these patients [23–25]. In addition, reactive oxygen species (ROS) released by peripheral polymorphonuclear leukocytes and monocytes during the recurrent contact of blood with dialysis membranes aggravates the already existing oxidative stress [2]. Taken together, these studies strongly suggest a prominent role of oxidative stress in haemodialyzed patients. Information on the occurrence of oxidative stress in humans derives, in most cases, from measurements carried out on blood or plasma with the assumption that any alteration of the hematic biomarkers should reflect the one occurring in other less accessible tissues. A series of biomarkers of oxidative stress were measured in HD patients, including the increased plasma level of carbonylated proteins [26] and advanced oxidation protein products (AOPP) [27,28]. Blood levels of both high molecular mass thiols (i.e., protein thiols, PSH) and low molecular mass thiols (LMM-SH), namely homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly), and glutathione (GSH), are frequently measured as biomarkers of oxidative stress [29]. PSH are also present as mixed disulphides with LMM-SH, as a whole referred to as S-thiolated proteins (PSSX) [30,31]. S-thiolation plays both a regulatory and an antioxidant role, because it protects PSH against irreversible oxidation [32,33]. Alteration in plasma levels of LMMSH [34,35], decreased plasma PSH [26,36], increased S-thiolated and homocysteinylated plasma proteins [37–39], and cysteinylated albumin [40,41] highlighted an oxidative shift in the plasma thiol redox status in patients with ESRD. The precise and accurate measurement of these thiol-specific oxidative stress biomarkers requires HPLC and/or mass spectrometry methods, as well as timeconsuming procedures and skilled personnel. Therefore, they are difficult to apply in large-scale clinical studies. A recently published spectrophotometric method suggests that it is possible to rapidly assess oxidative perturbations in the thiol redox status measuring simultaneously PSH and PSSX, obtaining the Protein Thiolation Index (PTI) [42]. Specifically, PTI¼[tLMM-SH]PSSX/[PSH], Table 1 Characteristics of study group. Data are expressed as mean 7SD. Haemodialyzed patients (n ¼20) Age (years) Sex Dialysis Vintage (years) CRPa (mg/dL) Albumin (g/dL) Fibrinogen (mg/dL) White Blood Cells (cells/mm3) Haemoglobin (g/dL) Urea (mg/dL) Creatinine (mg/dL) Sodium (mmol/L) Potassium (mmol/L) Calcium (mmol/L) Phosphorus (mmol/L) Ferritin (ng/mL) TIBCb (g/L) a b

CRP ¼C-reactive protein TIBC ¼total iron-binding capacity.

70.07 11.7 12M, 8F (1.5:1) 4.4 7 1.9 0.50 7 0.39 3.47 70.36 375.2 7 77.8 7286.7 7 1476.3 10.7 7 1.0 158.0 7 43.9 8.9 7 2.0 137.17 2.76 5.2 7 0.7 2.2 7 0.2 1.6 7 0.4 240.1 7 139.9 180.8 7 24.5

where [tLMM-SH]PSSX is the concentration of all LMM-SH (i.e., chiefly GSH, Cys, Hcy, and CysGly) released by all types of PSSX under reducing conditions and [PSH] is the concentration of protein free cysteinyl residues. The PTI was validated and applied to the plasma of both healthy humans and subjects affected by pathologies associated with increased oxidative stress. The PTI showed an age dependency with a near linear increase during ageing in healthy humans and was significantly higher in patients suffering from alkaptonuria (a genetically recessive metabolic disease associated with oxidative stress) [42]. The purpose of the present study was to determine the plasma thiol-specific oxidative stress in patients with ESRD, before and after HD, compared to age-matched healthy subjects. Thiol-specific oxidative stress was measured as total plasma thiols, S-thiolated plasma proteins, and PTI.

2. Materials and methods 2.1. Chemicals 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB; product code D8130), biotin-maleimide (N-biotinoyl-N′-(6-maleimidohexanoyl) hydrazide; product code B1267), dithiothreitol (DTT; product code D0632), N-ethylmaleimide (NEM; product code 04260), iodoacetic acid (IAA; product code I4386), ninhydrin (product code 33437) and hydrindantin (product code H17309) were purchased from Sigma-Aldrich (Milan, Italy). Horseradish peroxidase (HRP)-conjugated streptavidin (product code RPN1051) was purchased from GE Healthcare (Milan, Italy). All other reagents were of analytical grade (Sigma-Aldrich, Milan, Italy). 2.2. Study participants All the patients enrolled in the study belong to stage V of CKD and are referred to as ESRD patients. These patients don't have a residual renal function and thus require renal replacement therapy. Blood samples were collected after informed written consent was obtained from ESRD patients on maintenance HD at the Nephrology Unit of the Humanitas Clinical and Research Center (Rozzano, Milan, Italy). The presence of a clinically overt infectious process was the only exclusion criteria. For every patient an anamnestic record was collected. A de-identification of the samples was performed before any additional data processing. Twenty haemodialyzed patients were recruited in the study (Table 1). Control blood samples were collected from 20 age-matched voluntary healthy donors at the Analysis Laboratory of the University of Milan (Laboratorio Analisi Università di Milano), after obtaining informed verbal consent. Criteria included no known history of CKD or other diseases that could influence the analysis. In particular, healthy subjects were tested for serum creatinine in order to exclude CKD. 2.3. Sample collection From HD patients, venous blood samples of 10 ml were collected before HD and 5 ml were obtained after the same session. All samples were collected on the long inter-dialytic interval, i.e. two days apart from the previous HD session. Blood was withdrawn from the arteriovenous fistula or central venous catheter. K3EDTA was used as anticoagulant in all the blood samples. From healthy donors, 10 ml of venous blood was collected from the antecubital vein and treated with K3EDTA. All the samples were processed within the first hour from blood withdrawal through centrifugation for 10 min at 1000 g, obtaining pre- and post-dialysis plasma aliquots from haemodialyzed patients and plasma

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aliquots from healthy controls. Such aliquots were stored at 80 °C until the execution of the assays. 2.4. Total plasma thiol determination with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) The free thiol concentration of plasma samples was quantified by the Ellman assay measuring the increase in absorbance at 412 nm caused by the released TNB anion upon reaction of thiols with DTNB and using a molar absorption coefficient of 14.15 mM  1cm  1. In detail, 50 ml of plasma was diluted with 900 ml of 50 mM potassium phosphate buffer (PBS), pH 7.4, mixed with 50 μl of 3 mM DTNB prepared in PBS and incubated for 15 min at 25 °C. In order to subtract the intrinsic absorbance of plasma at 412 nm, a parallel sample was assembled mixing 950 ml of PBS with 50 μl of plasma for each sample. All measurements were performed in triplicate and the mean intrinsic absorbance was subtracted from the mean absorbance of TNB release. The molar concentration of thiols was calculated from the molar absorbance of the TNB anion. 2.5. Albumin thiol determination In order to label non-oxidized protein thiols, plasma samples were diluted to a final concentration of 1 mg/ml and mixed with 30 mM biotin–maleimide (stock solution 20 mM in dimethyl sulfoxide, DMSO). After mixing by gentle vortexing, the labelling reaction was carried out at room temperature in the dark for 60 min, with brief vortex-mixing every 15 min. The reaction was then stopped adding an equal volume of 2  reducing SDS-PAGE sample buffer. Samples (10 μg total protein) were resolved by SDSPAGE on 10% Tris–HCl resolving gels, electroblotted to Immobilon P polyvinylidene difluoride (PVDF) membrane and stored at 20 °C for later use. To detect biotin-labelled albumin Cys34, membranes were blocked for 1 h in 5% (w/v) non-fat dry milk in PBST [10 mM Na-phosphate, pH 7.2, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20] and probed with HRP-conjugated streptavidin (1:5000 dilution) for 2 h in 5% (w/v) non-fat dry milk in PBST. After washing in PBST, immunoreactive bands were detected by using enhanced chemiluminescence (ECL). 2.6. S-thiolated protein determination In order to measure LMM-SH [42] covalently bound to plasma proteins, a volume of plasma was mixed with an equal volume of 4 mM NEM and incubated at room temperature for 15 min. To precipitate plasma proteins, aliquots (100 μl) of NEM-derivatized samples were added with an equal volume of 10% trichloroacetic acid (TCA) and mixed by vortexing. Protein pellets were collected by centrifugation (14,000 g for 2 min), washed twice with 5% TCA and resuspended in 500 μl of 200 mM PBS, pH 7.4, added with 1 mM DTT. After incubation at room temperature for 20 min, 100 μl of 0.5 M IAA (dissolved in 0.2 M PBS, pH 7.4, fortified with 0.5 M NaOH to obtain an approximately final neutral pH value) was added followed by and additional incubation for 1 h. 5% TCA was then added to precipitate proteins that were collected by centrifugation at 14,000 g for 2 min. After centrifugation, 500 μl of the supernantant was collected for LMM-SH quantification. Ninhydrin reagent was freshly prepared as follows: 400 mg ninhydrin and 60 mg hydrindantin were dissolved in a solution consisting of 10 ml DMSO and 2.5 ml of 5 M sodium acetate buffer, pH 5.5. The reagent was kept protected from light and stored at room temperature during its use. Aliquots (500 μl) of the supernatants previously collected were then mixed with 100 μl of 5 M sodium acetate buffer, pH 5.5, and 400 μl of ninhydrin reagent, boiled for 10 min and left at room

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temperature for further 10 min. Absorbance spectra were then registered in the 700  500 nm wavelength range. To obtain blank, 500 μl of 200 mM PBS, pH 7.4, added with 1 mM DTT were processed as described above by the addition of 100 μl of 0.5 M IAA and further steps until boiling and cooling steps after ninhydrin addition. A calibration line was constructed from absorbance values of solutions containing a known concentration of LMM-SH. Stock solutions (200 mM) of Cys and GSH were freshly prepared in deionized water. Aliquots (500 μl) of LMM-SH in 200 mM PBS, pH 7.4, were incubated for 1 h with 100 μl of 500 mM IAA. Aliquots (600 μl) of IAA-conjugated standard thiols were then reacted with 100 μl of 5 M sodium acetate buffer, pH 5.5, and 400 μl of ninhydrin reagent, boiled for 10 min and left at room temperature for further 10 min. Final concentrations of standard thiols were 5, 10, 20, 30, and 40 mM. Blank samples were obtained by omitting LMM-SH. 2.7. Protein thiolation index (PTI) determination PTI was calculated as the molar ratio between the PSSX (where SX is usually Cys, CysGly, Hcy, or GSH) and the concentration of free, DTNB-titratable protein -SH groups [42]. 2.8. Statistical analysis The Student's independent t-test was used to test whether differences between the groups of patients and the healthy individuals, or patients with ESRD before and after HD, were significant.

3. Results There were significant differences in total plasma protein concentration in individual healthy subjects (Fig. 1A) as well as between the same ESRD patient before and after HD (Fig. 1B). This was particularly important to consider during the analysis of total plasma thiols and PSSX, since any increase/decrease in total plasma protein concentration can dramatically affect the measured values. Therefore, in this study total plasma thiols and PSSX are expressed, respectively, as μmol -SH/g protein or μmol -SX/g protein. However, the difference in total plasma protein concentration between healthy subjects and those with ESRD, both before and after HD, was not statistically different. Conversely, total plasma protein concentration measured in ESRD patients before and after an HD session differed significantly (p o 0.05). The spectrophotometric assay for evaluation of total plasma thiols with DTNB measures the reduced forms of PSH and LMM-SH available in the plasma. We found a 21% lower content of total plasma thiol in patients with ESRD compared with age-matched healthy subjects (3.72 μmol -SH/g protein70.66 SD vs. 4.73 μmol -SH/g protein 70.71 SD, respectively) (Fig. 2). Student's t-test proved that the difference in the mean value of total plasma thiols between the two groups was statistically significant. It is worth noting that the spectrophotometric analysis of total plasma thiols with DTNB mainly measures PSH, because they represent the most abundant thiol pool in human plasma, being in the 400–500 μM range, whereas concentration of LMM-SH is in the 0.1–20 μM range [30,31]. Therefore, the contribution of LMM-SH to the total plasma thiols measured by DTNB can be considered negligible. Albumin constitutes 50%–60% of total plasma proteins in humans (mean concentration ∼43 mg/ml, i.e., ∼600 μM) and, therefore, provides the bulk of total plasma PSH [43]. In particular, the exposed -SH group of Cys34 contributes to ∼80% of all PSH in human plasma [44], thus conferring a major role in the plasma

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Fig. 1. Plasma protein concentration in studied populations. (A) Total plasma protein concentration in individual age-matched healthy subjects (n¼ 20) ranged from 65.0 to 80.4 g/l (mean 76.1 g/l 73.86 SD). (B) Total plasma protein concentration in patients with ESRD (n ¼20) ranged from 61.2 to 81.9 g/l (mean 73.7 g/l 74.9 SD) and from 64.2 to 90.9 g/l (mean 78.9 g/l 7 7.2 SD) in samples taken before (grey circles) and after (black circles) HD, respectively.

Fig. 2. Total plasma thiols. Total plasma thiols (PSH þ LMM thiols) in patients with ESRD (n¼ 20) and age-matched healthy subjects (n¼ 20). Data are expressed as mean 7SD. Differences between the two groups were statistically evaluated using Student's t-test and resulted to be statistically significant (nnn ¼p o 0.00005).

antioxidant capacity to albumin [44,45]. In this regard, we measured the redox status of albumin Cys34 -SH group in patients with ESRD and age-matched healthy subjects by using a biotinbased tagging technique, which has been applied with success to monitor the oxidation of PSH by oxidative stress [46,47]. The biotin tag can be detected at a level of sensitivity in the picomole range using immunoblotting with HRP-conjugated streptavidin. The loss of the biotin signal is proportional to the degree of PSH modification. After PSH derivatization with biotin-maleimide, protein separation by SDS-PAGE, and Western-blotting with HRP-conjugated streptavidin, the thiol group of albumin Cys34 was detected in plasma samples of patients with ESRD compared to agematched healthy subjects (Fig. 3). In haemodialyzed patients, plasma albumin is substantially more oxidized than in healthy subjects [40,48,49]. Consistently, we found a 16% lower albumin thiol content in patients with ESRD compared to healthy subjects (940771462 densitometry units vs. 111977 13 densitometry units, respectively). Student's t-test proved that the difference in the mean value of plasma albumin Cys34 thiol group between haemodialyzed patients and healthy age-matched controls was statistically significant. We also analyzed the content/amount of total PSSX (where SX is usually Cys, CysGly, Hcy, or GSH) in the plasma of patients with

Fig. 3. Plasma albumin Cys34 thiol. Western blot analysis of albumin Cys34 thiol in patients with ESRD (n ¼20) and age-matched healthy subjects (n ¼20). Values are expressed in densitometry units and refer to densitometric analysis, performed by Image J 1.40d software (National Institutes of Health, Bethesda, MD, USA), of the ECL signal relative to the albumin Cys34 -SH group after Western blot. Data are expressed as mean 7 SD. Differences between means of the two groups were evaluated using Student's t-test (n ¼ p o 0.05).

ESRD as compared with that in age-matched healthy subjects by colorimetric reaction with ninhydrin, as described in Materials and Methods. Plasma levels of PSSX in patients with ESRD were 17% higher than those in age-matched healthy subjects (2.8 μmol -SX/ g protein 70.34 SD vs. 2.4 μmol -SX/g protein 70.3 SD, respectively), as shown in Fig. 4. The Student's t-test proved that the difference in the mean value of total plasma PSSX between the two groups was statistically significant. We calculated PTI, i.e., the molar ratio between the sum of all LMM-SH bound to plasma proteins (forming, as a whole, PSSX) and protein free cysteinyl residues [42], in plasma of patients with ESRD and age-matched healthy subjects. We found that PTI was 52% higher in patients with ESRD compared to age-matched healthy subjects (0.79 70.21 SD vs. 0.527 0.17 SD, respectively) (Fig. 5). Student's t-test proved that the difference in the mean value of PTI between haemodialyzed patients and related controls was statistically significant. HD is a procedure that alleviates symptoms of toxaemia associated with renal failure, prolonging the survival of patients with

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Fig. 4. S-thiolated plasma proteins. Plasma proteins of patients with ESRD (n¼ 20) and age-matched healthy subjects (n ¼20) were analyzed for total S-thiolated protein content by colorimetric reaction with ninhydrin as described in Materials and Methods. Data are expressed as mean7 SD. Differences between means of the two groups were evaluated using Student's t-test (nn ¼p o 0.001).

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ESRD for many years. To determine the effect of HD on the oxidative shift in the plasma thiol redox status, comparisons of total plasma thiols, total PSSX, and PTI were made in patients with ESRD before and immediately after the end of HD. We found a significant increase in the level of total plasma thiols after HD compared to the pre-dialysis baseline. Such an increase brought back total plasma thiols to normal levels or even to a value higher than normal. Scatter diagram of measured plasma value for total thiols in individual patients with ESRD is illustrated in Fig. 6A. As reported in Fig. 6B, the percentage of increase in total plasma thiols after HD in patients with ESRD ranged from 6.66 to 69.6% (mean 35.357 14.0 SD). Conversely, we found a significant decrease in the levels of plasma PSSX after HD compared with the pre-dialysis period: i.e., plasma PSSX dropped to normal, or lower than normal, levels. Scatter diagram of measured plasma value for total PSSX in individual patients with ESRD who received maintenance HD is illustrated in Fig. 7A. The percentage of decrease in PSSX levels after HD in haemodialyzed patients ranged from 17.9 to 43.0% (mean 30.9676.47 SD) (Fig. 7B). Finally, we found a significant decrease in PTI values in plasma of haemodialyzed patients after HD compared with the pre-dialysis period: i.e., values of PTI dropped to normal, or lower than normal, levels. Scatter diagram of determined plasma value for PTI in individual patients with ESRD who received maintenance HD is shown in Fig. 8A. The percentage of decrease in PTI after HD in haemodialyzed patients ranged from 23.0 to 61.5% (mean 48.17 79.17 SD) (Fig. 8B).

4. Discussion

Fig. 5. Protein thiolation index. Plasma of both haemodialyzed patients (n¼ 20) and age-matched healthy subjects (n¼ 20) was analyzed for PTI as described in Materials and Methods. Data are expressed as mean 7 SD. Differences between means of the two groups were evaluated using Student's t-test (nn ¼ p o0.001).

ESRD is a chronic condition, characterized by the irreversible loss of kidney function. HD is in most cases the renal replacement therapy of choice for ESRD patients. Mortality and morbidity rates for people with ESRD undergoing HD are high [50], largely due to a ∼15- to 30-fold increase in cardiovascular events [11,12,51]. Despite numerous advances in technology and patient care, only 50% of the US HD patients are still alive three years after start of HD. The risk of cardiovascular death of a 30-year-old patient with ESRD is similar to that of an 80-year-old in the general population [51,52]. As a whole, the leading cause of death in this population remains cardiovascular disease (CVD) [12,51,53]. The causes underlying this increase in cardiovascular morbidity and mortality

Fig. 6. Total plasma thiols before and after HD. (A) Effects of HD on total plasma thiol levels before (grey circles) and immediately after (black circles) HD were examined in patients with ESRD who received maintenance dialysis (n¼ 20). The horizontal solid line represents the mean of the total plasma thiol level in age-matched healthy subjects (see Fig. 2). The horizontal dashed lines represent the SD of the total plasma thiol level in age-matched healthy subjects (see Fig. 2). (B) Percentage of increase in total plasma thiols after HD in haemodialyzed patients.

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Fig. 7. S-thiolated plasma proteins before and after HD. (A) Effects of HD on total plasma PSSX levels before (grey circles) and immediately after (black circles) HD were examined in patients with ESRD who received maintenance dialysis (n¼ 20). The horizontal solid line represents the mean of plasma PSSX levels in age-matched healthy subjects (see Fig. 4). The horizontal dashed lines represent the SD of the plasma PSSX level in age-matched healthy subjects (see Fig. 4). (B) Percentage of decrease in plasma PSSX after HD in haemodialyzed patients.

Fig. 8. Values of PTI in plasma of patients with ESRD before and after HD. (A) Effects of HD on PTI before (grey circles) and immediately after (black circles) HD were examined in patients with ESRD who received maintenance HD (n ¼20). The horizontal solid line represents the mean value of PTI in age-matched healthy subjects (see Fig. 5). The horizontal dashed lines represent the SD of PTI value in age-matched healthy subjects (see Fig. 5). (B) Percentage of decrease in PTI after HD in plasma of haemodialyzed patients.

are still unclear. Increased levels of biomarkers of oxidative stress are present in patients with varying degrees of CKD, including patients with ESRD (CKD stage V) requiring chronic HD, which indicates that uraemia is a pro-oxidant state [54–56]. Increased biomarkers of oxidative stress have been demonstrated in patients with moderate to severe CKD (stages III-V) [6]. Moreover, the presence of carbonylated proteins, AOPP, nitrated plasma albumin, and dityrosine, which are also biomarkers of oxidative stress, has been documented in haemodialyzed patients [26–28,57–60]. Although CKD is strongly associated with risk factors for atherosclerosis, the excessive high cardiovascular risk seen in these patients is not fully accounted for by traditional cardiovascular risk factors [61]. Indeed, an additional risk factor that accelerates atherogenesis in CKD is oxidative stress [62]. The role of oxidative stress in CVD and CKD [63] suggests that antioxidant therapy may improve CVD morbidity and mortality of haemodialyzed patients [64]. However, a limited number of studies examined the relationship between biomarkers of oxidative stress and CVD in patients with CKD [62,65]. In addition, few prospective studies have examined the association between biomarkers of oxidative stress and clinical

outcomes in patients with ESRD on HD [62]. Although increasing evidence strongly supports oxidative stress as a plausible independent cardiovascular risk factor in patients with CKD and, even more, in those undergoing HD, the exact processes underlying such an increased oxidative stress remain to be elucidated. Furthermore, identification of biochemical and/or functional biomarkers that could be used to monitor oxidative imbalance in ESRD may allow development of optimized intervention strategies to reduce oxidative stress. Also, the use of biomarkers of oxidative stress in a panel of clinical biomarkers of processes known to impact on CKD and ESRD development could allow early detection and prevention of such processes. The collection of small blood volumes is mostly unproblematic. However, before clinical translation, biomarker assays must be rigorously standardized by a process involving the development, optimization, qualification, and validation of the assay, preferably done in different clinical and research laboratories [66]. Moreover, economic considerations by clinical and research laboratories for the choice of a bioassay will necessarily include such factors as cost of reagents and supporting equipment (such as spectrophotometer, HPLC with UV detection, or mass spectrometer), time-

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consuming procedures, skilled personnel training costs, etc. For example, many labs will tend to opt for spectrophotometric assays (e.g., for AOPP determination) rather than for other assays requiring substantial investments. In fact, increased plasma levels of AOPP have been used as an economic biomarker of oxidative stress in haemodialyzed patients [27,28,67] because of their ease of determination due to their absorbance at 340 nm under acidic conditions, even if the exact nature of AOPP remains not completely defined [59]. Unfortunately, several factors, chiefly sample turbidity following lipid precipitation in plasma samples, can markedly interfere with AOPP measurement [27,68]. Although a further improved method seems to detect AOPP with better reproducibility and accuracy compared to previously reported ones [58], another still unsolved problem, strictly related to poor reproducibility and accuracy of spectrophotometric AOPP detection, is the lack of reliable, validated AOPP reference values in healthy humans [69]. Other, more defined, biomarkers of oxidative stress, such as the decrease in plasma protein thiols [26,36] and the increase in S-thiolated proteins, have been assessed in patients with ESRD [37–41,70]. However, the measurement of these plasma biomarkers of thiol-specific oxidative stress requires HPLC with UV detection and/or mass spectrometry methods, as well as timeconsuming procedures and skilled personnel; therefore, their assessment may be difficult to carry out in large-scale clinical studies. In the present study, we determined the plasma redox status of patients with ESRD, before and after HD, compared to age-matched healthy subjects. We measured thiol-specific oxidative stress as total plasma thiols, S-thiolated plasma proteins and PTI, a new reliable biomarker of oxidative stress recently proposed [42]. Total plasma thiols were significantly lower in haemodialyzed patients compared to age-matched healthy subjects (Fig. 2). Actually, total plasma thiols are mainly constituted by PSH, whose concentration is mostly due to the single free thiol at Cys34 of albumin. As it has been proposed that reduced albumin represents a very abundant and important circulating antioxidant [43–45], it may also be an important defence against oxidative stress in HD patients. We found significantly lower content of albumin -SH groups in patient with ESRD compared to age-matched healthy subjects (Fig. 3). The pKa of the albumin Cys34 thiol is abnormally low (about 5), which is in contrast with pKa values of most PSH. Consequently, at physiological pH, the Cys34 –SH group of albumin exists primarily as the thiolate anion and is highly reactive [43]. This high reactivity, together with the paucity of thiol reducing systems in human plasma, may explain the fact that Cys34 binds LMM-SH in about one-half of the plasma albumin molecules, forming PSSX [30]. Protein S-thiolation is regarded as the adaptive response to protect PSH from losing their biological activity by irreversible oxidation [32]. Therefore, the increase in the level of PSSX in haemodialyzed patients compared to age-matched healthy subjects (Fig. 4) could be regarded as a protective effect of plasma LMM-SH towards plasma PSH. We also found that a single HD session transiently corrects the oxidative shift in the thiol redox status in patients with ESRD. The pre-dialysis concentration of total plasma thiols in patients with ESRD was significantly lower (with few exceptions) than that in healthy subjects, whereas post-dialysis concentration in most patients did not differ from that in controls (Fig. 6). HD was also associated with a decrease in PSSX levels below those in age-matched healthy controls (Fig. 7). Taken together, these results suggest that a generalized thiolspecific oxidative stress exists in patients with ESRD undergoing HD and that a single HD session is restorative of plasma thiol redox status, thus re-establishing the antioxidant capacity of plasma thiols. Furthermore, these data suggest the existence of unknown oxidising compounds that can be provisionally removed by a

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single HD session. However, in the period between consecutive HD sessions, pathological levels of total thiols and PSSX are restored. There is concordance between our results and those from previous studies examining oxidative perturbations in the thiol/disulphide homeostasis before and after HD in patients with ESRD [26,35,38,71,72]. A major finding in our present study is that PTI is a valid biomarker of thiol-specific oxidative stress in ESRD patients compared with age-matched healthy subjects as well as in ESRD patients before and after HD. Haemodialyzed patients had a significantly higher PTI compared to age-matched healthy subjects (Fig. 5) and HD was associated with a decrease in PTI (Fig. 8), whose post-dialysis value fell within or below the PTI range of age-matched healthy controls (Fig. 8). These results suggest that PTI is a useful indicator of thiol-specific oxidative stress in these patients. The low cost of reagents and supporting equipment, the easy implementation of the method, and the very short time of execution suggest that PTI determination is suitable for clinical analyses with a high daily throughput and can be used for routine measurements of thiol-specific oxidative stress in different types of patients, including those with ESRD and, more generally, CKD. Some limitations in our study should be mentioned. Firstly, this study was conducted on a small number of patients with ESRD receiving maintenance HD and in only one single centre (the Nephrology Unit at the Humanitas Clinical and Research Center). However, if on the one hand, the pilot nature of the study might justify the limited number of patients, on the other hand, the results of this study, the first one that uses PTI – a parameter where both PSH and PSSX are measured in the same plasma sample – to investigate the effect of HD on the oxidative perturbations in the plasma thiol redox status, could serve as a useful working hypothesis for further studies with a larger cohort from different dialysis facilities. Secondly, PTI does not reveal oxidative modifications other than protein mixed disulphides, since it relies on the detection of disulphides after reduction with DTT. Therefore, other types of thiol oxidations, such as sulphenic acid, S-nitrosothiols, intramolecular or intermolecular disulphides, cannot be detected. Thirdly, PTI does not reveal irreversible oxidative modifications of proteins such as carbonylated proteins, AOPP, and dityrosine. In this regard, it is important to note that, because of the complexity of oxidative stress in patients with ESRD, it is likely that a single biomarker of oxidative stress may not be sufficient and that a panel of biomarkers may be needed to encompass the different types of oxidative damage and perturbations to antioxidant defences [65]. Lastly, it should also be noted that, since PTI showed an age dependency with a nearly linear increase during ageing in healthy humans [42], one should always compare the results with a population of age-matched healthy subjects when determining the PTI in a population of patients, as we have done in this study. In conclusion, as discussed above, oxidative stress has an important pathophysiological role in patients with ESRD and earlier stages of CKD: indeed, oxidative stress is associated with the development and progression of CKD and, within the patient cohort on maintenance HD, higher markers of inflammation and oxidative stress are associated with greater adverse outcomes. Actually, the role of oxidative stress in patients with ESRD and CKD is often underestimated (or even ignored) in clinical practice, but emerging evidence continues to highlight its strong correlation with chronic inflammation, which is associated with complications in these patients, particularly CVD. Therefore, new biomarkers of oxidative stress, easy to measure and suitable for large-scale clinical studies, could be useful tools to monitor the development and progression of CKD and to check the efficiency of HD treatment in patients with ESRD. Moreover, such biomarkers of oxidative stress could be especially effective if used early in the course of renal disease before cardiovascular sequelae become

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irreversible or difficult to reverse. Although the present study suggests that PTI could make valuable contributions to clinical questions related to thiol-specific oxidative stress in patients with ESRD (and, likely, in all patients with earlier stages of CKD as well), the current challenge and long-term goal is to explore the most effective way in which PTI can be integrated with current clinical laboratory biomarkers to maximize their synergism for detection of CKD and ESRD complications, such as CVD. Therefore, it remains necessary to validate the clinical utility of PTI using a large, blinded set of samples, and to establish comparability and standards for quality control before a significant use of PTI can be advocated in patients undergoing maintenance HD therapy and, likely, in those with earlier stages of CKD as well.

Conflict of interest disclosure statement

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[15] [16]

[17]

[18] [19]

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We further confirm that the manuscript has been read and approved by all named authors and that the order of authors listed in the manuscript has been approved by all of us.

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Acknowledgments

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This research was supported by Fondazione Ariel, Rozzano (MI), Italy. The authors are grateful to Dr. Barbara Ponzini and all the personnel at the Analysis Laboratory, Department of Pathophysiology and Transplantation, University of Milan, for their invaluable support in providing blood samples from healthy subjects. Graphical abstract was prepared using and combining medical clip arts available within the Servier Medical Art section, by courtesy of Servier International.

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