DOI: 10.1111/eci.12297
ORIGINAL ARTICLE Modulation of NADPH oxidase activity by known uraemic retention solutes Anna Marta Schulz*, Cindy Terne*,†, Vera Jankowski‡, Gerald Cohen§, Mandy Schaefer*, Falko Boehringer*, Martin Tepel¶, Desiree Kunkel**, Walter Zidek* and Joachim Jankowski‡ on the behalf of European Uremic Toxin Work Group (EUTox) * -Universita €tsmedizin Berlin (CBF), Medizinische Klinik IV, Berlin, Germany, †Helmholtz Virtual Institute Charite “Multifunctional Biomaterials for Medicine”, Berlin, Germany, ‡University Hospital, RWTH Aachen, Institute for Molecular €t Wien, Univ. Klinik fu € r Innere Med III, Vienna, Cardiovascular Research (IMCAR), Aachen, Germany, §Medizinische Universita -Universita €tsmedizin Austria, ¶Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark, **Charite Berlin (CVK), Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
ABSTRACT Background Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Methods Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Results Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. Conclusions The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. Keywords NADPH oxidase, oxidative stress, uraemic retention solutes. Eur J Clin Invest 2014; 44 (8): 802–811
Introduction Reasons for the high prevalence of cardiovascular complications in patients suffering from chronic kidney disease (CKD) have been described in detail in large number of studies, for example [1–3]. Numerous recent as well as ongoing studies discussed the potential of accumulated uraemic retention solutes to affect the vascular damage in patients with CKD [4,5]. However, a clarification of the underlying mechanism of contribution to cardiovascular disease is yet to be found.
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Oxidative stress and increased production of reactive oxygen species (ROS) might be involved in the progression of cardiovascular diseases (CVD) [6,7]. There are several differentially localized and expressed enzyme systems contributing to ROS formation, including endothelial NO synthase enzymes of the respiratory chain, cytochrome P450 mono-oxygenases, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [8–10]. Although all of these enzymes contribute to oxidative burden, an initial generation of ROS by NADPH oxidases triggers their release by other enzymes [11].
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The NADPH oxidases are a group of enzymes which are located in the plasma membrane of mesodermal cells. Due to the reduction of oxygen, NADPH oxidases generate peroxides (O 2 compounds). The peroxides represent the origin of a variety of other reactive oxidants, including oxidized halogens, free radicals, hydrogen peroxide and singlet oxygen. ROS are used by phagocytes to defend against invading microorganisms, but in the case of overproduction, they may still lead to damage in the surrounding tissue. Thus, ROS generation is strictly regulated in healthy subjects [12,13]. Chronic kidney disease is strongly associated with cardiovascular disease (CVD) [14,15]. The NADPH oxidases seem to play a crucial role in both kidney and cardiovascular damage. Figure 1 illustrates the NADPH oxidase mechanism and representative physiologically and pathophysiologically relevant effects of NADPH oxidase [16]. The activity of NADPH oxidase has been reported to be increased in human heart failure [17]. In addition, increased NADPH oxidase expression was found in mice with diabetic nephropathy [18]. The vascular NADPH oxidases have also been suggested to have an impact on the development of hypertension, endothelial dysfunction, arteriosclerosis, restenosis and hypertrophy [19]. Finally, modified NADPH oxidase activity has been observed in haemodialysis patients [20]. Several studies indicate an overproduction of ROS in CKD5D (CKD stage 5 undergoing dialysis) patients treated by
Pathophysiological effects ,e. g. - Vasoconstriction - Smooth muscle proliferation - Endothelial damage - Inflammation
NADPH
NADPH oxidase
O2*
Intermediate products
NADP +
Mononuclear leukocyte
Figure 1 Scheme of the NADPH oxidase mechanism; NADPH, acting as an electron donor, is metabolized by leucocyte NADPH oxidase to NADP+. The electron is transferred to an oxygen molecule, and the oxygen radical is generated. This process is of central relevance in additional pathway reactions, due to resulting intermediates directly causing vasoconstriction, smooth muscle proliferation, endothelial damage and/or inflammation.
haemodialysis [20,21]. This may be caused by an accumulation of the oxidative stress stimulating factors or a removal of the antioxidants. Uraemic retention solutes have been implicated in the oxidative burden [22–24]. However, the interaction between accumulation of uraemic retention solutes and ROS production remains unclear. In addition, known uraemic retention solutes have, to our knowledge, not yet been analysed consistently with regard to their impact on the oxidative stress. Furthermore, an important feature of uraemia is the complexity of biochemical aspects and the wide variety of effects of the uraemic retention solutes on the intermediary metabolism. The accumulated uraemic retention solutes acting individually or in combination may modulate different enzymatic reactions and thus different metabolic pathways. The uraemic retention solutes represent a variety of substances, which have been classified regarding their physical properties and their origin. They are divided into three groups: (i) small water-soluble uraemic retention solutes, (ii) middle uraemic retention solutes and (iii) protein-bound uraemic retention solutes [25]. These groups are characterized by strongly different physical and chemical properties and wide concentration ranges affecting their physiological effects and dialytic removal. For this reason, the correct interpretation and comparison of their pathophysiological role appear to be strongly complex. Currently, in the context of the standardized handling of uraemic toxins in in vitro assays, the European Uremic Toxin Work Group (http://www. uraemic-toxins.org/home.html) investigates 91 known uraemic retention solutes systematically in different biological systems regarding their impact on cardiovascular-related control mechanisms. The present systematic study of the effects of known uraemic retention solutes on the mononuclear leucocyte NADPH oxidase activity may contribute to the physiological and pathophysiological classification of presently known uraemic retention solutes.
Materials and methods Chemicals Glyoxal, hyaluronic acid, malondialdehyde and methionineenkephaline were obtained from Fluka (Buchs, Switzerland) and taurocyamine from The Binding Site Ltd (Birmingham, UK). Phosphate-buffered saline containing calcium chloride and magnesium chloride was purchased from GIBCO Invitrogen (Auckland, NZ), LC-MS grade water from LAB SCAN (Gliwice, Poland), leptin from Rockland (Gilbertsville, USA), bovine serum albumin from Bio-Rad (Munich, Germany) and low-protein-bind tubes from Eppendorf (Wesseling-Berzdorf, Germany). All other substances were purchased from SigmaAldrich (Munich, Germany).
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Isolation and preparation of mononuclear leucocytes Mononuclear leucocytes were isolated from buffy coats obtained from the German Red Cross biobank (Berlin, Germany) in accordance with the protocol of van der Giet et al. [26]. Blood from healthy volunteers was centrifuged at 150 9 g for 10 min at 20 °C. Next, the buffy coats were separated from plasma and erythrocytes using Compomat G4 (Fresenius, Bad Homburg, Germany). The buffy coats were used within 18 h after the blood withdrawal. For the separation of the blood cells from plasma, the buffy coats were transferred into 11-mL tubes and centrifuged at 260 9 g for 30 min at room temperature. The blood cells were diluted with phosphate-buffered saline (PBS) containing calcium chloride and magnesium chloride (GIBCO Invitrogen, Auckland, NZ) at a ratio of 1:1. Ten millilitre of the cell suspension was applied to 4 ml of Lymphoprep and centrifuged at 750 9 g for 20 min at room temperature. The medial layer containing mononuclear leucocytes was aspirated and diluted with PBS (1:1). The mononuclear leucocytes suspension was washed by centrifugation for 10 min at 750 9 g at room temperature. The pellet of mononuclear leucocytes was resuspended in 5 mL of PBS and then centrifuged at 750 9 g for 10 min at room temperature. The pellet of mononuclear leucocytes was resuspended in 1 mL of PBS. The NADPH oxidase was obtained from the cell membranes of the mononuclear leucocytes after 3 freeze-thawing cycles. Peripheral blood mononuclear cells are established sources for determination of NADPH oxidase activity [27,28].
Flow cytometric analysis of the isolated leucocytes The flow cytometric analysis of the isolated fractions was carried out for three buffy coats from various healthy volunteers. The cells were washed once with PBS/BSA by centrifugation for 5 min at 300 9 g at room temperature. For surface staining, the pellet was resuspended and incubated for 10 min at 4 °C in the dark according to the manufacturer’s instructions with the following antibodies: anti-human CD14 Brilliant Violet421, anti-human CD45 FITC, anti-human CD3 PECy7, anti-human CD19 APCCy7 (each Biolegend) and anti-human CD15 APC (BD Biosciences, San Diego, CA, USA). After staining, the cells were washed with PBS/BSA, resuspended in PBS/BSA and acquired on a BD LSR II SORP with Diva Software (BD Biosciences). Analysis was carried out using FlowJo 9.6.2 (TreeStar, Ashland, OR, USA). Leucocytes were defined as CD45+ cells, and frequencies are given for the following subpopulations: lymphocytes (CD45 high, SSC low), monocytes (CD14+) and granulocytes (CD15+).
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tion at a ratio of 1:5 of mononuclear leucocytes from those healthy volunteers corresponded to a protein concentration of nearly 10 mg/mL.
Preparation of plasma Blood samples from healthy volunteers (n = 10) and patients with CKD-5D (n = 10) were collected using Vacutainer tubes (BD, Heidelberg, Germany) containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (18 mg EDTA per 1 mL blood). Plasma was separated from blood cells by centrifugation at 2500 9 g for 15 min at 4 °C and stored at 20 °C until further analysis.
Chronic renal failure patients The local ethics committees approved the study. We studied patients with CKD-5D by utilizing maintenance haemodialysis (n = 10). The inclusion criteria were the following: patients had to have chronic renal failure stage 5D and had to be treated with haemodialysis therapy at least for 2 months. All eligible patients in the dialysis unit were asked for participation. All patients had signed an informed consent before inclusion into the study. Patients younger than 18 years and pregnant patients were excluded. The clinical and biochemical characteristics are given in Table 1. Blood pressure was measured before and after haemodialysis. The mean dialysis vintage was 26 6 months (mean SEM). The patients were routinely dialysed for 4 h three times a week. Blood flow rates were seen to be between 250 and 300 mL/min, dialysate flow rates were 500 mL/min and dialysate conductivity was 135 mS. The dialysates used were bicarbonate-based. Kt/V values were measured according to the formula, Kt/V as ln (R003)+ (435 9 R) 9 UF/W; using R as post-/pre-plasma urea nitrogen ratio; UF as ultrafiltrate volume in litres was subtracted off; W as posthaemodialysis weight in kg, and the mean Kt/V value was 103 005.
Preparation of uraemic retention solutes All stock solutions were prepared in water and stored at 20°C prior to analysis. To ensure the stability of uraemic retention solutes, all stock solutions were prepared in low-protein-bind tubes. The uraemic retention solutes used in the assay were diluted immediately before used in PBS. The concentration of the uraemic retention solute used in the assay corresponds to cmax, the highest concentration of these ever reported for uraemia [25].
Bradford protein assay
Quantification of the effect of the uraemic retention solutes on nadph oxidase activity
The total protein concentrations of the lysed mononuclear leucocytes from various healthy volunteers were quantified using the Bradford protein assay [29]. The assay revealed that dilu-
The principle of the assay was based on a method published by van der Giet et al. [26]. In preliminary experiments, the optimal conditions for the quantification of NADPH oxidase activity
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Table 1 Clinical and biochemical characteristics of CKD-5D patients, (values are mean SEM) Parameters
Healthy control CKD-5D patients subjects (N = 10) (N = 10)
Significance
Age (years)
33 9
57 12
ns
8/2
ns
Gender (m/f) 7/3 Blood pressure (mm Hg) 123 10/70 6
119 25/69 14 ns
Body mass i ndex (kg/m2)
20 2
25 4
Creatinine (mg/dL)
096 013
1028 429
P < 00001***
Phosphate (mg/dL)
10 02
18 05
P < 00001***
Hb (g/dL)
128 203
104 13
P < 00001***
CRP (mg/dL)
16 31
616 72
P < 00001***
Systolic/ diastolic
ns
Statistics The data were analysed by GraphPad Prism 5.0 Software (GraphPad Software, San Diego, CA, USA). The results are presented as mean values SEM. Statistical significance was determined by Mann–Whitney t-test. Two-tailed values of P < 005 were considered as significant. Grubbs test was used to identify outlier values with a significance level of 95%.
Results
***Means high significance (P < 0.001).
were tested. The optimal NADH concentration was 100 mM, and it was located within the linear range of our system (Fig. 4). For the determination of NADPH oxidase activity, the lysed mononuclear leucocytes were diluted with PBS (pH 74) at a ratio of 1:3, and 50 lL of the lysed cells was transferred into a 96-well plate and incubated for 120 min at 37 °C with 150 lL of NADH (100 mM) in the absence or presence of uraemic retention solute or plasma sample (50 ll) and 50 ll of diphenyleneiodonium chloride (DPI) (48 mM), an inhibitor of NADPH oxidase [30]. The change in the absorbance of NADH at 340 nm within 120 minutes was quantified in sextuplicate (n = 6). A Multiskan Ascent photometer (Thermo Electron Corporation, Waltham, MA, USA) was used for these measurements. The NADH absorbance at time point 0 min was normalized to 100%. The enzyme activity of mononuclear leucocytes was calculated as the difference between time points 0 and 120 min. The specific effect of each uraemic retention solute and plasma on NADPH oxidase activity was calculated relatively to the effect of mononuclear leucocytes with DPI using Formula 1. se ¼
significance was determined by comparing the specific effect of the uraemic retention solutes to the effect of mononuclear leucocytes without DPI.
ðactivity of L +URSÞ ðactivity of L + DPIÞ ðactivity of LÞ ðactivity of L + DPIÞ
where se means specific effect, L leucocytes, URS uraemic retention solutes and DPI diphenyleneiodonium chloride. The resulting values were subjected a transformation as follow: (se 1)*(1)*100% to simplify the illustration of the results. The
To determine the percentage composition of the Lymphoprepisolated cell fractions from various buffy coats, we tested the isolated cells using flow cytometry. The percentages of cell contents of various buffy coats were comparable. The isolated cell fractions contained 710 22% lymphocytes, 197 02% monocytes and 13 05% granulocytes (Fig. 2). Because NADPH oxidase has been found in lymphocytes and monocytes [31,32], further cell sorting was not necessary. The Lymphoprep-isolated cell fractions from buffy coats were used to quantify the effect on the NADPH oxidase activity in the presence of plasma from healthy volunteers, patients with CKD-5D as well as uraemic retention solutes. A schematic illustration of the determination of NADPH oxidase activity is shown in Fig. 3. Furthermore, as shown in the Fig. 4, the NADH concentration used in the assay causes an UV absorption, which is located within the wide linear range of our assay system. The effect of plasma from healthy volunteers and patients with CKD-5D prior to haemodialysis on NADPH oxidase is shown in Fig. 5. The inhibitory effect of plasma from patients with CKD-5D before haemodialysis on NADPH oxidase activity is significantly increased compared with the effect in the presence of plasma from healthy controls (P < 001). The comparison of the effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D before and after haemodialysis shows a significantly decreased (P < 005) inhibitory effect on NADPH oxidase activity during haemodialysis (Fig. 6). Because the presence of endotoxin had no effect on the activity of leucocyte NADPH oxidase (Data S1), the effect of plasma on the activity is caused by endogenous plasma compounds. Next, we tested 48 commercial available uraemic retention solutes with unknown effect on NADPH oxidase [25]. The specific effect of each uraemic retention solute on NADPH oxidase activity was calculated relatively to the effect of DPI. Thirty-nine of the 48 tested uraemic retention solutes showed a significant effect on NADPH oxidase activity (P < 005).
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100
UV-Absorption λ 340 nm [%]
(a)
95 90 85 80 75 0
30
60
90
120
Time [min]
Figure 3 Schematic illustration of NADPH oxidase activity determination of mononuclear leucocytes (▲), mononuclear leucocytes and representative inhibitory uraemic retention solute (■), and mononuclear leucocytes and DPI (●). (b)
UV-Absorption λ 340 nm [AU]
3 R2 = 0·9994
2
1
0 0
200
400
600
800
1000
Concentration [mM]
Figure 4 NADH.
Determination of the linear concentration range of
(c) Inhibitory effect on the NADPH oxidase [%]
160
**
120 80 40
D CK
H ea lth y
Le uk oc y
te
s+ D PI
0
Figure 2 Flow cytometric analysis of Lymphoprep-isolated cell fraction from buffy coat. Frequencies are given for the following subpopulations: (a) lymphocytes (CD45 high, SSC low), (b) granulocytes (CD15+) and (c) monocytes (CD14+).
Figure 5 Comparison of the effect on NADPH oxidase activity in the presence of plasma from healthy volunteers and patients with CKD-5D; the values are relative to the effects of mononuclear leucocytes and DPI (bars represent mean SEM; **Means medium significance P < 001; n = 10).
Twenty of the small water-soluble uraemic retention solutes with a molecular weight (MW) of under 500 Da had a significant inhibitory effect (P < 005) on the enzyme activity: oxalate,
cytidine, malondialdehyde, myoinositol, a-N-acetylarginine, thymine, uridine, orotidine, threitol, sorbitol, taurocyamine, mannitol, xanthosine, 1-methylguanosine, benzylalcohol, N,
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Discussion
**
90 60
Inhibitory effect on the NADPH oxidase [%]
60
30
oc yt es + M O DP al on C xa I di yti lte αN M alde din -a yo h e ce in yd ty o e la sit rg o Th ini l ym ne U in O rid e ro in t e Th idin Ta r e ur So eito oc rb l ya ito l-m M m l et Xa an ine h N B yla nth nito ,N e d os l -d nz en in im yl os e et alc ine H hylg oho yp l le ox yci an ne th in Er Ura e O yth cil ro ri tic tol ac id
Figure 7 Effects of small water-soluble uraemic retention solutes (< 500 Da) on NADPH oxidase activity; the effects of arabitol, urea, uric acid and xanthine are not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
between accumulation of uraemic retention solutes and oxidative stress in uraemic patients still remains insufficiently clarified. Pilot experiments of the current study had shown that the inhibitory effect on NADPH oxidase activity of mononuclear leucocytes is significantly increased in the presence of plasma from patients with CKD-5D prior to haemodialysis compared with renal healthy volunteers. This increased effect on NADPH 120 90 60 30
30
sis rd D CK
be D CK
af te
fo re d
ia
ly
sis ia ly
PI D es + oc yt Le uk
Figure 6 NADPH oxidase effects in the presence of plasma from patients with CKD-5D before and after haemodialysis; the values are relative to the effect of mononuclear leucocytes and DPI (bars represent mean SEM; **Means medium significance P < 001; n = 10).
uk o H cyte om s K o +D yn cy P In uren stei I do ic ne xy a l s cid u Sp lfa er te H G min pyd l e hy dr H roc yox ox ip h al y- pu ino hi ri ne pp c ur aci i M c ac d el id at on K Ph in y M nu eno e l Q thy rnin ui lg ee no ly lin ox a Pu ic a l In tre cid do s l-3 p- cin -a cre e ce so tic l ac id
0 0
Le
Inhibitory effect on the NADPH oxidase [%]
120
90
0
Inhibitory effect on the NADPH oxidase [%]
Recent studies demonstrate that oxidative stress has a strong impact on the pathophysiology and especially on the progression of renal failure patients [35]. However, the relationship
120
Le uk
N-dimethylglycine, hypoxanthine, uracil, erythritol and orotic acid. Oxalate showed the strongest inhibitory effect. The four uraemic retention solutes arabitol, urea, uric acid and xanthine apparently had no significant effect on NADPH oxidase activity. The effect of small water-soluble uraemic retention solutes on NADPH oxidase is shown in Fig. 7. Then, the effects of seventeen substances in the group of protein-bound uraemic retention solutes on NADPH oxidase activity were tested (P < 005). Homocysteine, glyoxal, kynurenic acid, indoxyl sulphate, p-hydroxy-hippuric acid, hydrochinone, melatonin, spermine, hippuric acid, phenol, kynurenine, methylglyoxal, quinolinic acid, putrescine, p-cresol sulphate and indol-3-acetic acid were seen to decrease NADPH oxidase activity significantly. P-cresol sulphate was analysed within the study because recent studies demonstrate that the p-cresol sulphate is the major circulating conjugate of p-cresol [33,34]. Spermidine had no significant effect on the activity of NADPH oxidase. The quantitative effects of protein-bound uraemic retention solutes are shown in Fig. 8. Seven uraemic retention middle molecules were analysed. While b-endorphin, neuropeptide Y and endothelin inhibited NADPH oxidase activity significantly (P < 005), adrenomedullin, leptin, methionine-enkephaline and hyaluronic acid had no apparent effect on NADPH oxidase activity. The effects on NADPH oxidase activity in the group of middle uraemic retention solutes are shown in Fig. 9.
Figure 8 Effects of protein-bound uraemic retention solutes on NADPH oxidase activity; the effect of spermidine is not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
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Inhibitory effect on the NADPH oxidase [%]
120
90
60
30
Le
uk
oc
yt e β- s +D e N ndo PI eu ro rph i pe pt n i d En e do Y th el in
0
Figure 9 Effects of middle uraemic retention solutes (> 500 Da) on NADPH oxidase activity; the effects of adrenomedullin, leptin, methionine-enkephaline and hyaluronic acid are not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
oxidase activity in patients with CKD may be associated with the accumulation of uraemic retention solutes. Therefore, the objective of this study was to investigate the direct effect of known uraemic retention solutes on the mononuclear leucocyte NADPH oxidase activity. The inhibitory effect on the activity of NADPH oxidase was stronger in the presence of CKD-5D plasma compared with healthy plasma (Fig. 5). This may be associated with the accumulation of inhibitors of NADPH oxidase activity in plasma of patients with CKD. A comparison of the effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D before and after haemodialysis showed indeed a significant decrease in the inhibitory effect on activity of NADPH oxidase during haemodialysis. We hypothesized that the removal of NADPH oxidase inhibitors during haemodialysis may lead to an increased ROS generation. This, in turn, may be a link to the increased oxidative stress of patients with CKD affecting both accelerated progression of cardiovascular disease and increased immune reaction in dialysis patients. Therefore, the effect of 48 commercially available uraemic retention solutes without a known effect on NADPH oxidase activity was tested. Thirty-nine uraemic retention solutes have been shown to affect leucocyte NADPH oxidase activity and the formation of ROS significantly. The uraemic retention solutes caused an inhibition of NADPH oxidase activity. None of the uraemic retention solutes which were tested increased NADPH oxidase activity. These findings confirm the results of those pilot experiments and thus demonstrate that there is a
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decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after haemodialysis than before haemodialysis. The inhibitory effect on NADPH oxidase activity in the presence of plasma from healthy subjects was decreased, compared with the effects of plasma from patients with CKD-D5. This might be explained as a kind of compensatory mechanism of the organism on the increased oxidative burden. Although the uraemic retention solutes screened in this approach might be toxic in general, these uraemic retention solutes may have a specific protective effects concerning NADPH oxidase activity. However, these substances may cause opposed effects on other ROS-dependent processes like innate host defence. A decreased renal elimination and thus increased concentration of uraemic retention solutes in plasma of patients with CKD may modify the activity of NADPH oxidase. Because the concentration of uraemic retention solutes in the plasma is reduced by dialysis, the inhibitory effect on NADPH oxidase is reduced, resulting in increased enzymatic activity and increased oxidative burden. Increased oxidative stress may be somewhat induced by dialysis, at least from the point of view of the NADPH oxidase. These results are in accordance with former studies demonstrating an increasing oxidative stress with increasingly haemodialysis sessions [36]. Because there are only a few studies comparing the ROS production before and after dialysis, the increased oxidative stress is interpreted as a classical consequence of CKD not of the dialysis. However, as based on the screening approach of uraemic retention solutes, a general estimation of the haemodialysis effect on the oxidative status is limited because concentrations of each known and yet unknown inhibitors and yet unknown enhancers are most likely decreased during the haemodialysis session. Furthermore, dialysis efficiencies of different single uraemic retention solutes are strongly divergent most likely due to their different chemical properties like hydrophobicity and thus their solubility in water. This might be the reason for the clearly smaller differences in the inhibitory effect on the NADPH oxidase activity when comparing plasma from patients with CKD-5D after haemodialysis and before haemodialysis versus plasma from CKD-5D before haemodialysis and plasma from healthy subjects. In addition, the uraemic concentration of different uraemic retention solutes is spread over a broad range [25]. However, a high concentration is not always associated with a strong effect on the NADPH oxidase activity. For instance, the highest concentrated uraemic retention solute urea (46 g/L) did not cause any significant effects on the NADPH oxidase activity. Moreover, only a limited part of plasma compounds have been identified until now; therefore, the specific effect of the plasma compounds in general is not feasible; and we were able to analyse the overall effects of human plasma from renal healthy controls and
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patients with CKD-5D in dependence of the haemodialysis session. Furthermore, the uraemic milieu differs strongly from the healthy milieu not only in regard to the presence of uraemic retention solutes but also in regard to any exposure to such a medium. This may lead to secondary effects like post-translational modification of plasma components like plasma proteins as well as uraemic retention solutes affecting their native molecular structure and function as well as resulting binding properties [37]. Presently, the knowledge of the overall effects of uraemic retention solutes is insufficient because individual effects of the uraemic retention solutes do not affect only the oxidative pathways based on NADPH oxidase, but have an impact on other biological systems as well. For instance, it has been reported that an inhibitory effect of plasma ultrafiltrate on LDL oxidation was dramatically reduced as a consequence of the intradialytic removal of uraemic retention solutes like indoxyl sulphate, p-cresol and phenol, and in accordance with our findings, the plasma ultrafiltrate of patients with CKD had an enhanced antioxidant effect on oxidative modification of LDL compared with healthy controls [38]. The uraemic retention solute with the strongest inhibitory effect on NADPH oxidase, oxalate – which belongs to the group of small water-soluble compounds – is excreted by the kidney in healthy subjects and accumulated in patients with CKD. The maximum uraemic plasma concentration of oxalate in patients with CKD (76 mg/L) is 25 times higher than it is in healthy subjects (03 mg/L) [39]. Because oxalate has an inhibitory effect on NADPH oxidase of mononuclear leucocytes, a compensatory mechanism in the patient with CKD is conceivable. Because previous studies described an increasing ROS production by oxalate [40], oxalate acts besides its activity as an inhibitor of the NADPH oxidase on other NADPH oxidaseindependent ROS-producing enzymatic systems. While the results of the current study demonstrate inhibitory effect of indoxyl sulphate on NAPDH oxidase activity in leucocytes, this uraemic retention solute has an activating effect on ROS production in mesangial and tubular cells in general [41,42]. Because patients with CKD are suffering from an increased oxidative stress in general [6,7], the increased oxidative stress might result due to activity of other ROS-generating systems and/or inhibition of degrading systems like superoxide dismutase. Furthermore, indoxyl sulphate and p-cresol sulphate have strong affinities to plasma albumin using the identical binding site causing competitive effects of these substances to these binding sites [43]. The identity of other plasma proteins involved in the process of uraemic retention solutes adsorption is still unknown. Therefore, the determination of the single redox effect of these uraemic retention solutes under uraemic conditions is challenging.
The distinct effect of uraemic retention solutes – which is also exemplified by this study – emphasizes the necessity for further investigations of oxidative stress in uraemia because this might be of relevance for specific therapeutic targeting. The objective in the treatment of CKD should be the development of specific separation techniques, modulation of pathways for the generation of uraemic retention solutes and identification of biomarkers for indication of CKD at an early state and/or the progression of CKD. Furthermore, the development of a new, selective NADPH oxidase inhibitor could be effective for the decrease of oxidative stress and thus cardiovascular progression in patients with CKD. However, our knowledge about the pathogenic mechanisms and interactions of all uraemic retention solutes is currently just at the beginning. For the development of specific therapies, the pathobiochemical and pathophysical effects of uraemic retention solutes have to be clarified in more detail, in regard to their effect on different ROS-producing enzymatic systems. In addition, future investigations will have to focus on the identification of new, hitherto unknown uraemic retention solutes and their systematic and detailed pathophysiological characterization. Such uraemic retention solutes may have comparable effects on NADPH oxidase activity in patients with CKD. However, the removal of these substances by dialysis has an effect on the ROS production in patients with CKD-5D, and this may affect different physiological and pathophysiological processes in CKD, for example, cardiovascular status and immune defence status. In conclusion, this study demonstrates that uraemic retention solutes affect NADPH oxidase activity. Removal of these modulators of NADPH oxidase during dialysis may cause further oxidative stress in patients with CKD-5D. Further studies will aim at clarifying the pathophysiological impact of this modulation on, for example, immune defence, cardiovascular diseases and atherogenicity in patients with CKD. To assess the overall influence of uraemic retention solutes on the oxidative stress in patients with CKD, their effect on further ROS-generating systems has to be tested systematically, because uraemic retention solutes may have different effects on different enzyme systems. After detailed analyses of the effects of uraemic retention solutes in vitro, the overall effects of the most prominent uraemic retention solutes will then have to be evaluated in vivo, too.
Acknowledgements This study was supported by a grant from Federal Ministry of Education and Research (01GR0807) and by grant FP7HEALTH-2009-2.4.5-2 to “SYSKID” from the European Union.
Conflict of interest None declared.
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Address Charite-Universit€atsmedizin Berlin (CBF), Medizinische Klinik IV, Hindenburgdamm 30, D-12200 Berlin, Germany (A. M. Schulz, C. Terne, M. Schaefer, F. Boehringer, W. Zidek); Helmholtz Virtual Institute “Multifunctional Biomaterials for Medicine”, Berlin, Germany (C. Terne); RWTH University of Aachen, Institute of Molecular Cardiovascular Research, Pauwelsstrasse 30, D-52074 Aachen, Germany (V. Jankowski, J. Jankowski); Medizinische Universit€at Wien, Univ. Klinik f€ ur Innere Med III, W€ ahringer G€ urtel 18-20, A-1090 Vienna, Austria (G. Cohen); University of Southern Denmark, Institute of Molecular Medicine, Winsløwparken 21.3, DK-5000 Odense, Denmark (M. Tepel); Charite-Universit€atsmedizin Berlin (CVK), BerlinBrandenburg Center for Regenerative Therapies, Augustenburger Platz 1, D-13353 Berlin, Germany (D. Kunkel). Correspondence to: Prof. Dr. J. Jankowski, RWTH University of Aachen, Institute of Molecular Cardiovascular Research, Pauwelsstrasse 30, D-52074Aachen, Germany. Tel: +49 160 91438540; fax: +49 241 80 52716; e-mail: Joachim.Jankowski@ charite.de
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Received 23 November 2013; accepted 24 June 2014
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References
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endothelial cell nitric oxide synthase in hypertension. J Clin Invest 2003;111:1201–9. Babior BM. NADPH oxidase: an update.Blood 1999;93:1464–76. Lassegue B, Griendling KK. NADPH oxidases: functions and pathologies in the vasculature. Arterioscler Thromb Vasc Biol 2010;30:653–61. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med 2004;351:1296–305. Vanholder R, Massy Z, Argiles A, Spasovski G, Verbeke F, Lameire N. Chronic kidney disease as cause of cardiovascular morbidity and mortality. Nephrol Dial Transplant 2005;20:1048–56. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000;87:840–4. Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel JL, Hasenfuss G et al. Increased myocardial NADPH oxidase activity in human heart failure. J Am Coll Cardiol 2003;41:2164–71. Fujii H, Kono K, Nakai K, Goto S, Komaba H, Hamada Y et al. Oxidative and nitrosative stress and progression of diabetic nephropathy in type 2 diabetes. Am J Nephrol 2010;31:342–52. Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 2003;24:471–8. Galli F, Varga Z, Balla J, Ferraro B, Canestrari F, Floridi A et al. Vitamin E, lipid profile, and peroxidation in hemodialysis patients. Kidney Int Suppl 2001;78:S148–54. Chen MF, Chang CL, Liou SY. Increase in resting levels of superoxide anion in the whole blood of uremic patients on chronic hemodialysis. Blood Purif 1998;16:290–300. Dou L, Jourde-Chiche N, Faure V, Cerini C, Berland Y, DignatGeorge F et al. The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells. J Thromb Haemost 2007;5:1302–8. Palm F, Nangaku M, Fasching A, Tanaka T, Nordquist L, Hansell P et al. Uremia induces abnormal oxygen consumption in tubules and aggravates chronic hypoxia of the kidney via oxidative stress. Am J Physiol Renal Physiol 2010;299:F380–6. Rutkowski P, Slominska EM, Szolkiewicz M, Aleksandrowicz E, Smolenski RT, Wolyniec W et al. Relationship between uremic toxins and oxidative stress in patients with chronic renal failure. Scand J Urol Nephrol 2007;41:243–8. Vanholder R, De Smet R, Glorieux G, Argiles A, Baurmeister U, Brunet P et al. Review on uremic toxins: classification, concentration, and interindividual variability. Kidney Int 2003;63:1934–43. van der Giet M, Erinola M, Zidek W, Tepel M. Captopril and quinapril reduce reactive oxygen species. Eur J Clin Invest 2002;32:732–7. Dickerson R, Deshpande B, Gnyawali U, Lynch D, Gordillo GM, Schuster D et al. Correction of aberrant NADPH oxidase activity in blood-derived mononuclear cells from type II diabetes mellitus patients by a naturally fermented papaya preparation. Antioxid Redox Signal 2012;17:485–91. Kanegae MP, Condino-Neto A, Pedroza LA, de Almeida AC, Rehder J, da Fonseca LM et al. Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells. Biochem Biophys Res Commun 2010;393:551–4. Noble JE, Bailey MJ. Quantitation of protein. Methods Enzymol 2009;463:73–95. Osaki T, Uchida Y, Hirayama J, Nishina H. Diphenyleneiodonium chloride, an inhibitor of reduced nicotinamide adenine
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dinucleotide phosphate oxidase, suppresses light-dependent induction of clock and DNA repair genes in zebrafish. Biol Pharm Bull 2011;34:1343–7. Jackson SH, Devadas S, Kwon J, Pinto LA, Williams MS. T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nat Immunol 2004;5:818–27. Zhao X, Bey EA, Wientjes FB, Cathcart MK. Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67 (phox) and p47(phox). J Biol Chem 2002;277:25385–92. de Loor H, Bammens B, Evenepoel P, De Preter V, Verbeke K. Gas chromatographic-mass spectrometric analysis for measurement of p-cresol and its conjugated metabolites in uremic and normal serum. Clin Chem 2005;51:1535–8. Martinez AW, Recht NS, Hostetter TH, Meyer TW. Removal of P-cresol sulfate by hemodialysis. J Am Soc Nephrol 2005;16:3430–6. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655–73. Ferretti G, Bacchetti T, Masciangelo S, Pallotta G. Lipid peroxidation in hemodialysis patients: effect of vitamin C supplementation. Clin Biochem 2008;41:381–6. Vanholder R, Baurmeister U, Brunet P, Cohen G, Glorieux G, Jankowski J. A bench to bedside view of uremic toxins. J Am Soc Nephrol 2008;19:863–70. Ujhelyi L, Balla G, Jeney V, Varga Z, Nagy E, Vercellotti GM et al. Hemodialysis reduces inhibitory effect of plasma ultrafiltrate on LDL oxidation and subsequent endothelial reactions. Kidney Int 2006;69:144–51.
39 McConnell KN, Rolton HA, Modi KS, Macdougall AI. Plasma oxalate in patients with chronic renal failure receiving continuous ambulatory peritoneal dialysis or hemodialysis. Am J Kidney Dis 1991;18:441–5. 40 Scheid C, Koul H, Hill WA, Luber-Narod J, Kennington L, Honeyman T et al. Oxalate toxicity in LLC-PK1 cells: role of free radicals. Kidney Int 1996;49:413–9. 41 Gelasco AK, Raymond JR. Indoxyl sulfate induces complex redox alterations in mesangial cells. Am J Physiol Renal Physiol 2006;290: F1551–8. 42 Watanabe H, Miyamoto Y, Honda D, Tanaka H, Wu Q, Endo M et al. p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase. Kidney Int 2013;83:582–92. 43 Meijers BK, De Loor H, Bammens B, Verbeke K, Vanrenterghem Y, Evenepoel P. p-Cresyl sulfate and indoxyl sulfate in hemodialysis patients. Clin J Am Soc Nephrol 2009;4:1932–8.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Effect of endotoxin on NADPH oxidase activity and background oxidation of NADH.
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ORIGINAL ARTICLE Modulation of NADPH oxidase activity by known uraemic retention solutes Anna Marta Schulz*, Cindy Terne*,†, Vera Jankowski‡, Gerald Cohen§, Mandy Schaefer*, Falko Boehringer*, Martin Tepel¶, Desiree Kunkel**, Walter Zidek* and Joachim Jankowski‡ on the behalf of European Uremic Toxin Work Group (EUTox) * -Universita €tsmedizin Berlin (CBF), Medizinische Klinik IV, Berlin, Germany, †Helmholtz Virtual Institute Charite “Multifunctional Biomaterials for Medicine”, Berlin, Germany, ‡University Hospital, RWTH Aachen, Institute for Molecular €t Wien, Univ. Klinik fu € r Innere Med III, Vienna, Cardiovascular Research (IMCAR), Aachen, Germany, §Medizinische Universita -Universita €tsmedizin Austria, ¶Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark, **Charite Berlin (CVK), Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
ABSTRACT Background Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Methods Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Results Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. Conclusions The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. Keywords NADPH oxidase, oxidative stress, uraemic retention solutes. Eur J Clin Invest 2014; 44 (8): 802–811
Introduction Reasons for the high prevalence of cardiovascular complications in patients suffering from chronic kidney disease (CKD) have been described in detail in large number of studies, for example [1–3]. Numerous recent as well as ongoing studies discussed the potential of accumulated uraemic retention solutes to affect the vascular damage in patients with CKD [4,5]. However, a clarification of the underlying mechanism of contribution to cardiovascular disease is yet to be found.
802
Oxidative stress and increased production of reactive oxygen species (ROS) might be involved in the progression of cardiovascular diseases (CVD) [6,7]. There are several differentially localized and expressed enzyme systems contributing to ROS formation, including endothelial NO synthase enzymes of the respiratory chain, cytochrome P450 mono-oxygenases, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [8–10]. Although all of these enzymes contribute to oxidative burden, an initial generation of ROS by NADPH oxidases triggers their release by other enzymes [11].
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The NADPH oxidases are a group of enzymes which are located in the plasma membrane of mesodermal cells. Due to the reduction of oxygen, NADPH oxidases generate peroxides (O 2 compounds). The peroxides represent the origin of a variety of other reactive oxidants, including oxidized halogens, free radicals, hydrogen peroxide and singlet oxygen. ROS are used by phagocytes to defend against invading microorganisms, but in the case of overproduction, they may still lead to damage in the surrounding tissue. Thus, ROS generation is strictly regulated in healthy subjects [12,13]. Chronic kidney disease is strongly associated with cardiovascular disease (CVD) [14,15]. The NADPH oxidases seem to play a crucial role in both kidney and cardiovascular damage. Figure 1 illustrates the NADPH oxidase mechanism and representative physiologically and pathophysiologically relevant effects of NADPH oxidase [16]. The activity of NADPH oxidase has been reported to be increased in human heart failure [17]. In addition, increased NADPH oxidase expression was found in mice with diabetic nephropathy [18]. The vascular NADPH oxidases have also been suggested to have an impact on the development of hypertension, endothelial dysfunction, arteriosclerosis, restenosis and hypertrophy [19]. Finally, modified NADPH oxidase activity has been observed in haemodialysis patients [20]. Several studies indicate an overproduction of ROS in CKD5D (CKD stage 5 undergoing dialysis) patients treated by
Pathophysiological effects ,e. g. - Vasoconstriction - Smooth muscle proliferation - Endothelial damage - Inflammation
NADPH
NADPH oxidase
O2*
Intermediate products
NADP +
Mononuclear leukocyte
Figure 1 Scheme of the NADPH oxidase mechanism; NADPH, acting as an electron donor, is metabolized by leucocyte NADPH oxidase to NADP+. The electron is transferred to an oxygen molecule, and the oxygen radical is generated. This process is of central relevance in additional pathway reactions, due to resulting intermediates directly causing vasoconstriction, smooth muscle proliferation, endothelial damage and/or inflammation.
haemodialysis [20,21]. This may be caused by an accumulation of the oxidative stress stimulating factors or a removal of the antioxidants. Uraemic retention solutes have been implicated in the oxidative burden [22–24]. However, the interaction between accumulation of uraemic retention solutes and ROS production remains unclear. In addition, known uraemic retention solutes have, to our knowledge, not yet been analysed consistently with regard to their impact on the oxidative stress. Furthermore, an important feature of uraemia is the complexity of biochemical aspects and the wide variety of effects of the uraemic retention solutes on the intermediary metabolism. The accumulated uraemic retention solutes acting individually or in combination may modulate different enzymatic reactions and thus different metabolic pathways. The uraemic retention solutes represent a variety of substances, which have been classified regarding their physical properties and their origin. They are divided into three groups: (i) small water-soluble uraemic retention solutes, (ii) middle uraemic retention solutes and (iii) protein-bound uraemic retention solutes [25]. These groups are characterized by strongly different physical and chemical properties and wide concentration ranges affecting their physiological effects and dialytic removal. For this reason, the correct interpretation and comparison of their pathophysiological role appear to be strongly complex. Currently, in the context of the standardized handling of uraemic toxins in in vitro assays, the European Uremic Toxin Work Group (http://www. uraemic-toxins.org/home.html) investigates 91 known uraemic retention solutes systematically in different biological systems regarding their impact on cardiovascular-related control mechanisms. The present systematic study of the effects of known uraemic retention solutes on the mononuclear leucocyte NADPH oxidase activity may contribute to the physiological and pathophysiological classification of presently known uraemic retention solutes.
Materials and methods Chemicals Glyoxal, hyaluronic acid, malondialdehyde and methionineenkephaline were obtained from Fluka (Buchs, Switzerland) and taurocyamine from The Binding Site Ltd (Birmingham, UK). Phosphate-buffered saline containing calcium chloride and magnesium chloride was purchased from GIBCO Invitrogen (Auckland, NZ), LC-MS grade water from LAB SCAN (Gliwice, Poland), leptin from Rockland (Gilbertsville, USA), bovine serum albumin from Bio-Rad (Munich, Germany) and low-protein-bind tubes from Eppendorf (Wesseling-Berzdorf, Germany). All other substances were purchased from SigmaAldrich (Munich, Germany).
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Isolation and preparation of mononuclear leucocytes Mononuclear leucocytes were isolated from buffy coats obtained from the German Red Cross biobank (Berlin, Germany) in accordance with the protocol of van der Giet et al. [26]. Blood from healthy volunteers was centrifuged at 150 9 g for 10 min at 20 °C. Next, the buffy coats were separated from plasma and erythrocytes using Compomat G4 (Fresenius, Bad Homburg, Germany). The buffy coats were used within 18 h after the blood withdrawal. For the separation of the blood cells from plasma, the buffy coats were transferred into 11-mL tubes and centrifuged at 260 9 g for 30 min at room temperature. The blood cells were diluted with phosphate-buffered saline (PBS) containing calcium chloride and magnesium chloride (GIBCO Invitrogen, Auckland, NZ) at a ratio of 1:1. Ten millilitre of the cell suspension was applied to 4 ml of Lymphoprep and centrifuged at 750 9 g for 20 min at room temperature. The medial layer containing mononuclear leucocytes was aspirated and diluted with PBS (1:1). The mononuclear leucocytes suspension was washed by centrifugation for 10 min at 750 9 g at room temperature. The pellet of mononuclear leucocytes was resuspended in 5 mL of PBS and then centrifuged at 750 9 g for 10 min at room temperature. The pellet of mononuclear leucocytes was resuspended in 1 mL of PBS. The NADPH oxidase was obtained from the cell membranes of the mononuclear leucocytes after 3 freeze-thawing cycles. Peripheral blood mononuclear cells are established sources for determination of NADPH oxidase activity [27,28].
Flow cytometric analysis of the isolated leucocytes The flow cytometric analysis of the isolated fractions was carried out for three buffy coats from various healthy volunteers. The cells were washed once with PBS/BSA by centrifugation for 5 min at 300 9 g at room temperature. For surface staining, the pellet was resuspended and incubated for 10 min at 4 °C in the dark according to the manufacturer’s instructions with the following antibodies: anti-human CD14 Brilliant Violet421, anti-human CD45 FITC, anti-human CD3 PECy7, anti-human CD19 APCCy7 (each Biolegend) and anti-human CD15 APC (BD Biosciences, San Diego, CA, USA). After staining, the cells were washed with PBS/BSA, resuspended in PBS/BSA and acquired on a BD LSR II SORP with Diva Software (BD Biosciences). Analysis was carried out using FlowJo 9.6.2 (TreeStar, Ashland, OR, USA). Leucocytes were defined as CD45+ cells, and frequencies are given for the following subpopulations: lymphocytes (CD45 high, SSC low), monocytes (CD14+) and granulocytes (CD15+).
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tion at a ratio of 1:5 of mononuclear leucocytes from those healthy volunteers corresponded to a protein concentration of nearly 10 mg/mL.
Preparation of plasma Blood samples from healthy volunteers (n = 10) and patients with CKD-5D (n = 10) were collected using Vacutainer tubes (BD, Heidelberg, Germany) containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (18 mg EDTA per 1 mL blood). Plasma was separated from blood cells by centrifugation at 2500 9 g for 15 min at 4 °C and stored at 20 °C until further analysis.
Chronic renal failure patients The local ethics committees approved the study. We studied patients with CKD-5D by utilizing maintenance haemodialysis (n = 10). The inclusion criteria were the following: patients had to have chronic renal failure stage 5D and had to be treated with haemodialysis therapy at least for 2 months. All eligible patients in the dialysis unit were asked for participation. All patients had signed an informed consent before inclusion into the study. Patients younger than 18 years and pregnant patients were excluded. The clinical and biochemical characteristics are given in Table 1. Blood pressure was measured before and after haemodialysis. The mean dialysis vintage was 26 6 months (mean SEM). The patients were routinely dialysed for 4 h three times a week. Blood flow rates were seen to be between 250 and 300 mL/min, dialysate flow rates were 500 mL/min and dialysate conductivity was 135 mS. The dialysates used were bicarbonate-based. Kt/V values were measured according to the formula, Kt/V as ln (R003)+ (435 9 R) 9 UF/W; using R as post-/pre-plasma urea nitrogen ratio; UF as ultrafiltrate volume in litres was subtracted off; W as posthaemodialysis weight in kg, and the mean Kt/V value was 103 005.
Preparation of uraemic retention solutes All stock solutions were prepared in water and stored at 20°C prior to analysis. To ensure the stability of uraemic retention solutes, all stock solutions were prepared in low-protein-bind tubes. The uraemic retention solutes used in the assay were diluted immediately before used in PBS. The concentration of the uraemic retention solute used in the assay corresponds to cmax, the highest concentration of these ever reported for uraemia [25].
Bradford protein assay
Quantification of the effect of the uraemic retention solutes on nadph oxidase activity
The total protein concentrations of the lysed mononuclear leucocytes from various healthy volunteers were quantified using the Bradford protein assay [29]. The assay revealed that dilu-
The principle of the assay was based on a method published by van der Giet et al. [26]. In preliminary experiments, the optimal conditions for the quantification of NADPH oxidase activity
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Table 1 Clinical and biochemical characteristics of CKD-5D patients, (values are mean SEM) Parameters
Healthy control CKD-5D patients subjects (N = 10) (N = 10)
Significance
Age (years)
33 9
57 12
ns
8/2
ns
Gender (m/f) 7/3 Blood pressure (mm Hg) 123 10/70 6
119 25/69 14 ns
Body mass i ndex (kg/m2)
20 2
25 4
Creatinine (mg/dL)
096 013
1028 429
P < 00001***
Phosphate (mg/dL)
10 02
18 05
P < 00001***
Hb (g/dL)
128 203
104 13
P < 00001***
CRP (mg/dL)
16 31
616 72
P < 00001***
Systolic/ diastolic
ns
Statistics The data were analysed by GraphPad Prism 5.0 Software (GraphPad Software, San Diego, CA, USA). The results are presented as mean values SEM. Statistical significance was determined by Mann–Whitney t-test. Two-tailed values of P < 005 were considered as significant. Grubbs test was used to identify outlier values with a significance level of 95%.
Results
***Means high significance (P < 0.001).
were tested. The optimal NADH concentration was 100 mM, and it was located within the linear range of our system (Fig. 4). For the determination of NADPH oxidase activity, the lysed mononuclear leucocytes were diluted with PBS (pH 74) at a ratio of 1:3, and 50 lL of the lysed cells was transferred into a 96-well plate and incubated for 120 min at 37 °C with 150 lL of NADH (100 mM) in the absence or presence of uraemic retention solute or plasma sample (50 ll) and 50 ll of diphenyleneiodonium chloride (DPI) (48 mM), an inhibitor of NADPH oxidase [30]. The change in the absorbance of NADH at 340 nm within 120 minutes was quantified in sextuplicate (n = 6). A Multiskan Ascent photometer (Thermo Electron Corporation, Waltham, MA, USA) was used for these measurements. The NADH absorbance at time point 0 min was normalized to 100%. The enzyme activity of mononuclear leucocytes was calculated as the difference between time points 0 and 120 min. The specific effect of each uraemic retention solute and plasma on NADPH oxidase activity was calculated relatively to the effect of mononuclear leucocytes with DPI using Formula 1. se ¼
significance was determined by comparing the specific effect of the uraemic retention solutes to the effect of mononuclear leucocytes without DPI.
ðactivity of L +URSÞ ðactivity of L + DPIÞ ðactivity of LÞ ðactivity of L + DPIÞ
where se means specific effect, L leucocytes, URS uraemic retention solutes and DPI diphenyleneiodonium chloride. The resulting values were subjected a transformation as follow: (se 1)*(1)*100% to simplify the illustration of the results. The
To determine the percentage composition of the Lymphoprepisolated cell fractions from various buffy coats, we tested the isolated cells using flow cytometry. The percentages of cell contents of various buffy coats were comparable. The isolated cell fractions contained 710 22% lymphocytes, 197 02% monocytes and 13 05% granulocytes (Fig. 2). Because NADPH oxidase has been found in lymphocytes and monocytes [31,32], further cell sorting was not necessary. The Lymphoprep-isolated cell fractions from buffy coats were used to quantify the effect on the NADPH oxidase activity in the presence of plasma from healthy volunteers, patients with CKD-5D as well as uraemic retention solutes. A schematic illustration of the determination of NADPH oxidase activity is shown in Fig. 3. Furthermore, as shown in the Fig. 4, the NADH concentration used in the assay causes an UV absorption, which is located within the wide linear range of our assay system. The effect of plasma from healthy volunteers and patients with CKD-5D prior to haemodialysis on NADPH oxidase is shown in Fig. 5. The inhibitory effect of plasma from patients with CKD-5D before haemodialysis on NADPH oxidase activity is significantly increased compared with the effect in the presence of plasma from healthy controls (P < 001). The comparison of the effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D before and after haemodialysis shows a significantly decreased (P < 005) inhibitory effect on NADPH oxidase activity during haemodialysis (Fig. 6). Because the presence of endotoxin had no effect on the activity of leucocyte NADPH oxidase (Data S1), the effect of plasma on the activity is caused by endogenous plasma compounds. Next, we tested 48 commercial available uraemic retention solutes with unknown effect on NADPH oxidase [25]. The specific effect of each uraemic retention solute on NADPH oxidase activity was calculated relatively to the effect of DPI. Thirty-nine of the 48 tested uraemic retention solutes showed a significant effect on NADPH oxidase activity (P < 005).
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100
UV-Absorption λ 340 nm [%]
(a)
95 90 85 80 75 0
30
60
90
120
Time [min]
Figure 3 Schematic illustration of NADPH oxidase activity determination of mononuclear leucocytes (▲), mononuclear leucocytes and representative inhibitory uraemic retention solute (■), and mononuclear leucocytes and DPI (●). (b)
UV-Absorption λ 340 nm [AU]
3 R2 = 0·9994
2
1
0 0
200
400
600
800
1000
Concentration [mM]
Figure 4 NADH.
Determination of the linear concentration range of
(c) Inhibitory effect on the NADPH oxidase [%]
160
**
120 80 40
D CK
H ea lth y
Le uk oc y
te
s+ D PI
0
Figure 2 Flow cytometric analysis of Lymphoprep-isolated cell fraction from buffy coat. Frequencies are given for the following subpopulations: (a) lymphocytes (CD45 high, SSC low), (b) granulocytes (CD15+) and (c) monocytes (CD14+).
Figure 5 Comparison of the effect on NADPH oxidase activity in the presence of plasma from healthy volunteers and patients with CKD-5D; the values are relative to the effects of mononuclear leucocytes and DPI (bars represent mean SEM; **Means medium significance P < 001; n = 10).
Twenty of the small water-soluble uraemic retention solutes with a molecular weight (MW) of under 500 Da had a significant inhibitory effect (P < 005) on the enzyme activity: oxalate,
cytidine, malondialdehyde, myoinositol, a-N-acetylarginine, thymine, uridine, orotidine, threitol, sorbitol, taurocyamine, mannitol, xanthosine, 1-methylguanosine, benzylalcohol, N,
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Discussion
**
90 60
Inhibitory effect on the NADPH oxidase [%]
60
30
oc yt es + M O DP al on C xa I di yti lte αN M alde din -a yo h e ce in yd ty o e la sit rg o Th ini l ym ne U in O rid e ro in t e Th idin Ta r e ur So eito oc rb l ya ito l-m M m l et Xa an ine h N B yla nth nito ,N e d os l -d nz en in im yl os e et alc ine H hylg oho yp l le ox yci an ne th in Er Ura e O yth cil ro ri tic tol ac id
Figure 7 Effects of small water-soluble uraemic retention solutes (< 500 Da) on NADPH oxidase activity; the effects of arabitol, urea, uric acid and xanthine are not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
between accumulation of uraemic retention solutes and oxidative stress in uraemic patients still remains insufficiently clarified. Pilot experiments of the current study had shown that the inhibitory effect on NADPH oxidase activity of mononuclear leucocytes is significantly increased in the presence of plasma from patients with CKD-5D prior to haemodialysis compared with renal healthy volunteers. This increased effect on NADPH 120 90 60 30
30
sis rd D CK
be D CK
af te
fo re d
ia
ly
sis ia ly
PI D es + oc yt Le uk
Figure 6 NADPH oxidase effects in the presence of plasma from patients with CKD-5D before and after haemodialysis; the values are relative to the effect of mononuclear leucocytes and DPI (bars represent mean SEM; **Means medium significance P < 001; n = 10).
uk o H cyte om s K o +D yn cy P In uren stei I do ic ne xy a l s cid u Sp lfa er te H G min pyd l e hy dr H roc yox ox ip h al y- pu ino hi ri ne pp c ur aci i M c ac d el id at on K Ph in y M nu eno e l Q thy rnin ui lg ee no ly lin ox a Pu ic a l In tre cid do s l-3 p- cin -a cre e ce so tic l ac id
0 0
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Recent studies demonstrate that oxidative stress has a strong impact on the pathophysiology and especially on the progression of renal failure patients [35]. However, the relationship
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N-dimethylglycine, hypoxanthine, uracil, erythritol and orotic acid. Oxalate showed the strongest inhibitory effect. The four uraemic retention solutes arabitol, urea, uric acid and xanthine apparently had no significant effect on NADPH oxidase activity. The effect of small water-soluble uraemic retention solutes on NADPH oxidase is shown in Fig. 7. Then, the effects of seventeen substances in the group of protein-bound uraemic retention solutes on NADPH oxidase activity were tested (P < 005). Homocysteine, glyoxal, kynurenic acid, indoxyl sulphate, p-hydroxy-hippuric acid, hydrochinone, melatonin, spermine, hippuric acid, phenol, kynurenine, methylglyoxal, quinolinic acid, putrescine, p-cresol sulphate and indol-3-acetic acid were seen to decrease NADPH oxidase activity significantly. P-cresol sulphate was analysed within the study because recent studies demonstrate that the p-cresol sulphate is the major circulating conjugate of p-cresol [33,34]. Spermidine had no significant effect on the activity of NADPH oxidase. The quantitative effects of protein-bound uraemic retention solutes are shown in Fig. 8. Seven uraemic retention middle molecules were analysed. While b-endorphin, neuropeptide Y and endothelin inhibited NADPH oxidase activity significantly (P < 005), adrenomedullin, leptin, methionine-enkephaline and hyaluronic acid had no apparent effect on NADPH oxidase activity. The effects on NADPH oxidase activity in the group of middle uraemic retention solutes are shown in Fig. 9.
Figure 8 Effects of protein-bound uraemic retention solutes on NADPH oxidase activity; the effect of spermidine is not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
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Figure 9 Effects of middle uraemic retention solutes (> 500 Da) on NADPH oxidase activity; the effects of adrenomedullin, leptin, methionine-enkephaline and hyaluronic acid are not shown because these substances had no significant effect on NADPH oxidase activity (bars represent mean SEM; P < 005; n = 6).
oxidase activity in patients with CKD may be associated with the accumulation of uraemic retention solutes. Therefore, the objective of this study was to investigate the direct effect of known uraemic retention solutes on the mononuclear leucocyte NADPH oxidase activity. The inhibitory effect on the activity of NADPH oxidase was stronger in the presence of CKD-5D plasma compared with healthy plasma (Fig. 5). This may be associated with the accumulation of inhibitors of NADPH oxidase activity in plasma of patients with CKD. A comparison of the effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D before and after haemodialysis showed indeed a significant decrease in the inhibitory effect on activity of NADPH oxidase during haemodialysis. We hypothesized that the removal of NADPH oxidase inhibitors during haemodialysis may lead to an increased ROS generation. This, in turn, may be a link to the increased oxidative stress of patients with CKD affecting both accelerated progression of cardiovascular disease and increased immune reaction in dialysis patients. Therefore, the effect of 48 commercially available uraemic retention solutes without a known effect on NADPH oxidase activity was tested. Thirty-nine uraemic retention solutes have been shown to affect leucocyte NADPH oxidase activity and the formation of ROS significantly. The uraemic retention solutes caused an inhibition of NADPH oxidase activity. None of the uraemic retention solutes which were tested increased NADPH oxidase activity. These findings confirm the results of those pilot experiments and thus demonstrate that there is a
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decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after haemodialysis than before haemodialysis. The inhibitory effect on NADPH oxidase activity in the presence of plasma from healthy subjects was decreased, compared with the effects of plasma from patients with CKD-D5. This might be explained as a kind of compensatory mechanism of the organism on the increased oxidative burden. Although the uraemic retention solutes screened in this approach might be toxic in general, these uraemic retention solutes may have a specific protective effects concerning NADPH oxidase activity. However, these substances may cause opposed effects on other ROS-dependent processes like innate host defence. A decreased renal elimination and thus increased concentration of uraemic retention solutes in plasma of patients with CKD may modify the activity of NADPH oxidase. Because the concentration of uraemic retention solutes in the plasma is reduced by dialysis, the inhibitory effect on NADPH oxidase is reduced, resulting in increased enzymatic activity and increased oxidative burden. Increased oxidative stress may be somewhat induced by dialysis, at least from the point of view of the NADPH oxidase. These results are in accordance with former studies demonstrating an increasing oxidative stress with increasingly haemodialysis sessions [36]. Because there are only a few studies comparing the ROS production before and after dialysis, the increased oxidative stress is interpreted as a classical consequence of CKD not of the dialysis. However, as based on the screening approach of uraemic retention solutes, a general estimation of the haemodialysis effect on the oxidative status is limited because concentrations of each known and yet unknown inhibitors and yet unknown enhancers are most likely decreased during the haemodialysis session. Furthermore, dialysis efficiencies of different single uraemic retention solutes are strongly divergent most likely due to their different chemical properties like hydrophobicity and thus their solubility in water. This might be the reason for the clearly smaller differences in the inhibitory effect on the NADPH oxidase activity when comparing plasma from patients with CKD-5D after haemodialysis and before haemodialysis versus plasma from CKD-5D before haemodialysis and plasma from healthy subjects. In addition, the uraemic concentration of different uraemic retention solutes is spread over a broad range [25]. However, a high concentration is not always associated with a strong effect on the NADPH oxidase activity. For instance, the highest concentrated uraemic retention solute urea (46 g/L) did not cause any significant effects on the NADPH oxidase activity. Moreover, only a limited part of plasma compounds have been identified until now; therefore, the specific effect of the plasma compounds in general is not feasible; and we were able to analyse the overall effects of human plasma from renal healthy controls and
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patients with CKD-5D in dependence of the haemodialysis session. Furthermore, the uraemic milieu differs strongly from the healthy milieu not only in regard to the presence of uraemic retention solutes but also in regard to any exposure to such a medium. This may lead to secondary effects like post-translational modification of plasma components like plasma proteins as well as uraemic retention solutes affecting their native molecular structure and function as well as resulting binding properties [37]. Presently, the knowledge of the overall effects of uraemic retention solutes is insufficient because individual effects of the uraemic retention solutes do not affect only the oxidative pathways based on NADPH oxidase, but have an impact on other biological systems as well. For instance, it has been reported that an inhibitory effect of plasma ultrafiltrate on LDL oxidation was dramatically reduced as a consequence of the intradialytic removal of uraemic retention solutes like indoxyl sulphate, p-cresol and phenol, and in accordance with our findings, the plasma ultrafiltrate of patients with CKD had an enhanced antioxidant effect on oxidative modification of LDL compared with healthy controls [38]. The uraemic retention solute with the strongest inhibitory effect on NADPH oxidase, oxalate – which belongs to the group of small water-soluble compounds – is excreted by the kidney in healthy subjects and accumulated in patients with CKD. The maximum uraemic plasma concentration of oxalate in patients with CKD (76 mg/L) is 25 times higher than it is in healthy subjects (03 mg/L) [39]. Because oxalate has an inhibitory effect on NADPH oxidase of mononuclear leucocytes, a compensatory mechanism in the patient with CKD is conceivable. Because previous studies described an increasing ROS production by oxalate [40], oxalate acts besides its activity as an inhibitor of the NADPH oxidase on other NADPH oxidaseindependent ROS-producing enzymatic systems. While the results of the current study demonstrate inhibitory effect of indoxyl sulphate on NAPDH oxidase activity in leucocytes, this uraemic retention solute has an activating effect on ROS production in mesangial and tubular cells in general [41,42]. Because patients with CKD are suffering from an increased oxidative stress in general [6,7], the increased oxidative stress might result due to activity of other ROS-generating systems and/or inhibition of degrading systems like superoxide dismutase. Furthermore, indoxyl sulphate and p-cresol sulphate have strong affinities to plasma albumin using the identical binding site causing competitive effects of these substances to these binding sites [43]. The identity of other plasma proteins involved in the process of uraemic retention solutes adsorption is still unknown. Therefore, the determination of the single redox effect of these uraemic retention solutes under uraemic conditions is challenging.
The distinct effect of uraemic retention solutes – which is also exemplified by this study – emphasizes the necessity for further investigations of oxidative stress in uraemia because this might be of relevance for specific therapeutic targeting. The objective in the treatment of CKD should be the development of specific separation techniques, modulation of pathways for the generation of uraemic retention solutes and identification of biomarkers for indication of CKD at an early state and/or the progression of CKD. Furthermore, the development of a new, selective NADPH oxidase inhibitor could be effective for the decrease of oxidative stress and thus cardiovascular progression in patients with CKD. However, our knowledge about the pathogenic mechanisms and interactions of all uraemic retention solutes is currently just at the beginning. For the development of specific therapies, the pathobiochemical and pathophysical effects of uraemic retention solutes have to be clarified in more detail, in regard to their effect on different ROS-producing enzymatic systems. In addition, future investigations will have to focus on the identification of new, hitherto unknown uraemic retention solutes and their systematic and detailed pathophysiological characterization. Such uraemic retention solutes may have comparable effects on NADPH oxidase activity in patients with CKD. However, the removal of these substances by dialysis has an effect on the ROS production in patients with CKD-5D, and this may affect different physiological and pathophysiological processes in CKD, for example, cardiovascular status and immune defence status. In conclusion, this study demonstrates that uraemic retention solutes affect NADPH oxidase activity. Removal of these modulators of NADPH oxidase during dialysis may cause further oxidative stress in patients with CKD-5D. Further studies will aim at clarifying the pathophysiological impact of this modulation on, for example, immune defence, cardiovascular diseases and atherogenicity in patients with CKD. To assess the overall influence of uraemic retention solutes on the oxidative stress in patients with CKD, their effect on further ROS-generating systems has to be tested systematically, because uraemic retention solutes may have different effects on different enzyme systems. After detailed analyses of the effects of uraemic retention solutes in vitro, the overall effects of the most prominent uraemic retention solutes will then have to be evaluated in vivo, too.
Acknowledgements This study was supported by a grant from Federal Ministry of Education and Research (01GR0807) and by grant FP7HEALTH-2009-2.4.5-2 to “SYSKID” from the European Union.
Conflict of interest None declared.
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Address Charite-Universit€atsmedizin Berlin (CBF), Medizinische Klinik IV, Hindenburgdamm 30, D-12200 Berlin, Germany (A. M. Schulz, C. Terne, M. Schaefer, F. Boehringer, W. Zidek); Helmholtz Virtual Institute “Multifunctional Biomaterials for Medicine”, Berlin, Germany (C. Terne); RWTH University of Aachen, Institute of Molecular Cardiovascular Research, Pauwelsstrasse 30, D-52074 Aachen, Germany (V. Jankowski, J. Jankowski); Medizinische Universit€at Wien, Univ. Klinik f€ ur Innere Med III, W€ ahringer G€ urtel 18-20, A-1090 Vienna, Austria (G. Cohen); University of Southern Denmark, Institute of Molecular Medicine, Winsløwparken 21.3, DK-5000 Odense, Denmark (M. Tepel); Charite-Universit€atsmedizin Berlin (CVK), BerlinBrandenburg Center for Regenerative Therapies, Augustenburger Platz 1, D-13353 Berlin, Germany (D. Kunkel). Correspondence to: Prof. Dr. J. Jankowski, RWTH University of Aachen, Institute of Molecular Cardiovascular Research, Pauwelsstrasse 30, D-52074Aachen, Germany. Tel: +49 160 91438540; fax: +49 241 80 52716; e-mail: Joachim.Jankowski@ charite.de
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Received 23 November 2013; accepted 24 June 2014
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Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Effect of endotoxin on NADPH oxidase activity and background oxidation of NADH.
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