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NDT Advance Access originally published online on August 27, 2007
Nephrology Dialysis Transplantation 2007 22(12):3381-3390; doi:10.1093/ndt/gfm210
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Review on uraemic toxins III: recommendations for handling uraemic retention solutes in vitro—towards a standardized approach for research on uraemia

Gerald Cohen1, Griet Glorieux2, Paul Thornalley3, Eva Schepers2, Natalie Meert2, Joachim Jankowski4, Vera Jankowski4, Angel Argiles5, Björn Anderstam6, Philippe Brunet7, Claire Cerini7, Laetitia Dou7, Reinhold Deppisch8, Bart Marescau9, Ziad Massy10, Alessandra Perna11, Jana Raupachova1, Mariano Rodriguez12, Bernd Stegmayr13, Raymond Vanholder2, Walter H. Hörl1 for the European Uremic Toxin Work Group (EUTox)

1Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Austria, 2Department of Internal Medicine, Nephrology Division, University Hospital Ghent, Gent, Belgium, 3Clinical Sciences Research Institute, University of Warwick, University Hospital, Coventry, UK, 4Charité (CBF); Medizinische Klinik IV, Berlin, Germany, 5SAS RD—Néphrologie and Institute of Functional Genomics, Montpellier, France, 6Department of Renal Medicine, Karolinska Institutet, Stockholm, Sweden, 7INSERM UMR 608, Aix-Marseille Université, Marseille, France, 8Gambro Corporate Research, Hechingen, Germany, 9Department of Biomedical Sciences, University of Antwerp, CDE, IBB, Wilrijk, Belgium, 10INSERM ERI-12, Amiens, France and Division(s) of Clinical Pharmacology and Nephrology, University of Picardie and CHU-Amiens, Amiens, France, 11First Division of Nephrology, Second University of Naples, Naples, Italy, 12University Hospital Reina Sofia, Unidad de Investigacion, Nephrology Service, Cordoba, Spain and 13Division of Nephrology,Department Internal Medicine, Norrlands Universitets Sjukhus, Medicin kliniken-University Hospital, Umea, Sweden

Correspondence and offprint requests to: Dr Gerald Cohen, Medizinische Universitätsklinik III, Währinger Gürtel 18-20, A-1090 Wien, Austria. Email: gerald.cohen{at}meduniwien.ac.at

Keywords: basic protocols; in vitro assays; renal failure; uraemic toxins



   Introduction
Top
 Introduction
General methodological aspects
 Recommendations for stock...
 Discussion
 Conclusions
 Acknowledgements
 References
 
The progression of chronic kidney disease (CKD) leads to the dysfunction of multiple organs with clinical features constituting the uraemic syndrome and is characterized by a variety of metabolic and enzymatic disturbances. Many different substances, which are normally secreted into the urine by the healthy kidneys, are retained in the body. Those uraemic retention solutes which are the main actors during the development and manifestation of the uraemic syndrome are called uraemic toxins [1]. The identification, characterization and analytical determination of toxins responsible for the adverse biological effects encountered in uraemia and the knowledge of their patho-physiological importance is crucial for future prevention and therapy in patients with CKD stage 5. The information obtained from these analyses will make it possible to evaluate existing therapeutic approaches and to define new prognostic markers for the removal of uraemic toxins and, more importantly, it will allow the design of new specific removal strategies or other interventions to decrease and even normalize the plasma levels of uraemic toxins.

Uraemic retention solutes represent a heterogeneous group of substances such as organic compounds and peptides in their ‘native’ form or altered with post-translational modifications. They exist as free substances or are bound to serum protein. Since the intact glomerular filter clears substances up to ~60 000 Da, molecules below this molecular weight (MW) reportedly are retained with impairment of kidney function. Compounds in a MW range of 500–60 000 Da are arbitrarily defined as middle molecules, smaller substances as low MW solutes. Uraemic retention solutes have also different degrees of hydrophobicity and protein-binding properties. In a previous report [2], we summarized the uraemic retention solutes known at that time and provided information about their serum concentrations in healthy (Cn) and uraemic persons (Cu) as well as the highest ever reported concentration in patients with kidney failure (Cmax). This publication offered a platform for more systematic future analytical approaches to define the relative bio-functional importance of all known uraemic retention solutes. So far, many studies focused only on one or a few potential uraemic toxins at a time and were performed at concentrations that did not conform to those encountered in uraemia. However, the choice of relevant concentrations for in vitro and in vivo studies is a prerequisite to obtain appropriate conclusions.

In vitro experiments evaluating the effect of individual solutes or groups of solutes on enzyme systems, cell types, co-cultures or organ systems representative for functional defects encountered in uraemia may be helpful to unravel the patho-physiology of uraemic retention solutes in CKD. In this way, these approaches represent a fast and straightforward way to select candidates for further in-depth investigation even if they are not investigated in their original biological environment, as compounds are usually tested individually or in groups of only a few substances. Until now, in the reports on uraemic toxicity, each research group, if not each study, has developed its own specific conditions, making it impossible to compare different studies and/or evaluations on different organ systems. The present work includes basic protocols that give information for those researchers who aim to screen the in vitro effect of uraemic retention solutes in any biological system.

The present report emanated out of the perception that there was a need for more consistent as well as coordinated research activities in the uraemic toxicity area and was developed by a group of European researchers, each with their own area of expertise, who work in a collaborative project in this area since more than 5 years (the European Uremic Toxin Work Group—EUTox: http://www.uremic-toxins.org). The present text is part of a series of statement publications [2–6] of this group. Information will be provided about the availability, solubility and, if not commercially available, methods to prepare uraemic retention solutes which have been identified in the previous encyclopaedic review by EUTox [2]. This type of research approach helps to upgrade the bio-analytical expertise and prepares the application of emerging bio-analytical methods, e.g. proteomics, mass spectroscopy, capillary electrophoresis, multiple label confocal microscopy or even high throughput discovery technologies.



   General methodological aspects
Top
 Introduction
 General methodological aspects
Recommendations for stock...
 Discussion
 Conclusions
 Acknowledgements
 References
 
Substances
The substances discussed in this report have been purchased from the companies listed in Tables 1 and 2.


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  		Table 1. Preparation of stock solutions of non-peptidic compounds

 

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  		Table 2. Peptidic factors

 
Model systems
We recommend the use of cell culture models which are representative of cellular dysfunction implicated in the major uraemic complications such as neutrophils (diminished immune defence, oxidant stress), endothelial cells (cardiovascular disease), smooth muscle cells (progression of atherosclerosis), epithelial cells (metabolic acidosis), hepatocytes (disturbed metabolism), fibroblasts (fibrosis), osteoblasts (renal osteodystrophy). The protocols as presented here, can be used for any model or (other) cell system, however. This recommendation implies the use of human cells, whenever possible. When experiments with potential uraemic toxins on isolated organs are performed, an animal model has to be chosen, preferentially using a species where the relevance of the effect under investigation for human subjects has already been proven.

Standard experimental approach
Concentrations
The standard experimental set-up proposed for evaluation of each compound is illustrated in Figure 1. We recommend the use of the highest reported concentration in uraemic plasma/serum (Cmax) as a starting point for testing the in vitro effect of the substances. To confirm that the desired level has been reached, one can measure the effective concentration of the substance in the incubation medium before testing its effects. This seems especially advisable for compounds, which tend to bind to serum proteins.


Figure 1
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  		Fig. 1. Standard experimental set-up. Recommended procedure for testing the in vitro effect of individual compounds.

 
Protein binding
A substantial fraction of the uraemic retention solutes is protein bound (indicated by PB in Tables 1 and 2 which show the total, i.e. bound + free concentration). However, only the free fraction is assumed to be responsible for biological effects. Therefore, it is advised to add human serum albumin at the average uraemic concentration of 35 g/l to any test system not containing protein and to the controls as well. The albumin preparations used should be shown to be inactive and free of contamination such as endotoxin. The recommendation to add albumin together with the highest total concentration of the substance (which is usually provided in the literature) ensures that the concentration of the free toxin reaches the desired value.

Experimental media and controls
Depending on the biological system under investigation, the test medium can be isolated cells in saline or buffered salt solution with or without serum, or anti-coagulated whole blood. The use of an appropriate control is crucial for the correct interpretation of the results and is discussed in detail subsequently.

Stock solutions
To obtain Cmax as the final concentration in the assay, a concentrated stock solution is to be added. We recommend the addition of 1/10 of the final volume. Therefore, a stock solution with a concentration 10-fold higher than the target concentration should be added, i.e. 10-fold (10x) Cmax. Information about the preparation of stock solutions is given in Table 1. Stock solutions can be stored at –20°C. In the case of a low target concentration of a substance in the assay, higher concentrated stock solutions should be prepared, since this improves the accuracy of weighing. Obviously, the smallest amount, which can be weighed, will depend on the sensitivity of the available balance. This procedure can then be followed by appropriate dilution so as to obtain the above-mentioned 1/10 range. When working with small volume/amounts of solutes great care should be taken with respect to unspecific adsorption to surfaces of laboratory flasks, tubes and pipette tips. In case of a significant biochemical/biological effect, the concentration dependence of the effect should be investigated by making serial dilutions covering the serum concentrations observed in healthy subjects (Cn) and the average concentration observed in uraemic persons (Cu) (dose–response curve) [2].

Solubility considerations
Most uraemic solutes are soluble in saline. Therefore, a sterile (pyrogen-free) NaCl solution (0.9% w/v) can be used as a solvent to prepare the 10x Cmax stock solution. However, for some compounds organic solvents such as methanol, ethanol or dimethyl sulphoxide (DMSO) are to be used in the preparation of concentrated stock solutions (Table 1). In these cases, the stock solution is prepared at least 100x Cmax and diluted accordingly to achieve a final solvent concentration of ≤1%. This is necessary to avoid solvent effect on cell function which should also be evaluated and validated in control experiments.

Use of pools of substances
It is recommended to test several substances together in a pool in the initial phase of screening. If a significant effect of the pool substances is found, the components need to be tested individually. The rationale to use pools of solutes is not only to reduce the number of experiments to screen retention solutes, but also to bring together compounds to mimic the uraemic condition where numerous solutes are present together. Our protocols show examples of pools (Table 3).


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  		Table 3. Examples of pools of solutes

 
Recently, Jacobs et al. [7] evaluated the effect of the phenolic protein-bound uraemic retention solutes hydroquinone, phenol and p-cresol on leucocyte oxidative burst activity. The pool strongly decreased the leucocyte response to stimulation by Escherichia coli. Each individual solute showed a similar trend. On the other hand, Schepers et al. [8] showed that a pool of seven guanidino compounds markedly enhanced the baseline and fMLP-stimulated oxidative burst activity in monocytes, but only two of the guanidino compounds were responsible for this effect.

How combinations of compounds are composed is left up to the individual researcher. The decision will be based on the solvent required, the chemical nature of the substances and the final concentrations. The disadvantage of such an approach is the risk that the effect of individual substances is overlooked if the effects of two components antagonize each other, but it can reasonably be accepted that such events then occur in the in vivo setting as well. On the other hand, testing of a pool of substances vis-à-vis single compounds gives hints on potential synergistic effects.

The standard approach for testing pools of substances is illustrated in Figure 2. If several substances are tested together in a pool, a 10th of the test volume of a stock solution containing these substances is to be added. As a consequence, the concentrations of the stock solutions of the individual substances are to be increased. For example, for a pool containing six solutes, each substance then should be 60-fold over-concentrated.


Figure 2
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  		Fig. 2. Stock solution for pools. Recommended procedure for the preparation of a stock solution (10x Cmax) for a pool consisting of n solutes.

 


   Recommendations for stock solutions
Top
 Introduction
 General methodological aspects
 Recommendations for stock...
Discussion
 Conclusions
 Acknowledgements
 References
 
Strategies to prepare stock solutions of the aforementioned compounds were developed and tested. Those, which were finally accepted as being most appropriate, are reported subsequently.

Non-peptidic compounds
Table 1 lists the information for preparing stock solutions of the non-peptidic solutes. Nx Cmax stands for the maximal solubility tested expressed in multiples of Cmax. A higher concentration than 10x Cmax is necessary when preparing a stock solution of a pool of substances and when a high dilution factor is needed. In Table 1, the column labelled ‘mg/10 ml Nx Cmax’ provides the corresponding amount to be weighed for a final volume of 10 ml. If it is not possible to weigh the requested amount with sufficient accuracy, a higher amount is to be weighed and the volume of the solvent has to be increased proportionally. If a compound cannot be dissolved in saline (NaCl), specific alternative recommendations are given in the text further. For those substances that were used as a salt or that contained crystal water and as a consequence had a higher MW than the genuine compound, a corrected Cmax value is given as indicated in Table 1 under ‘Comment’.

In what follows, we describe specific procedures per individual compound, if those deviate from the standard approach outlined earlier.

N2,N2-Dimethylguanosine (#30)
Since this compound cannot be dissolved in saline, 10 mg was dissolved in 1 ml DMSO. To obtain a 100-fold Cmax stock solution, 416 µl of this solution has to be diluted in saline up to a volume up to 100 ml. This approach avoids adding too much DMSO to the experimental set-up.

Hippuric acid (#17), hypoxanthine (#20), uracil (#48), uric acid (#50), and xanthine (#52), thymine (#47) and uridine (#51)
Hippuric acid, the purines hypoxanthine, uracil, uric acid and xanthine and the pyrimidines thymine and uridine cannot be dissolved in saline. Thymine, uracil, uridine and xanthine can be dissolved in 0.2 M NaOH in Tris-buffer pH = 7.7 (Tris 50 mM, NaCl 120 mM and KCl 5 mM), and hippuric acid, hypoxanthine and uric acid in 0.25 M NaOH in Tris-buffer pH = 7.7. The final pH of the solutions when diluted with Tris buffer to 1x Cmax is between 7.4 and 7.7.

Homocysteine (#18)
Homocysteine is, as such, not available on the market. One possibility is to apply a mixture of D- and L-homocysteine, but D-homocysteine is not present in nature. Since this D-homocysteine may be active per se or can compete with L-homocysteine for protein binding, it is recommended to use a preparation only containing L-homocysteine. In that case an HCl-bound thiolactone is available. Homocysteine is only active in its reduced form. A 0.3 mg/ml stock solution of reduced L-homocysteine can be obtained by incubating L-homocysteine thiolactone for 5 min in 200 µl NaOH (5 mol/l) at 40°C. In order to neutralize the solution, first 320 µl phosphate buffer (pH 7.4) can be added, followed by the addition of 400–450 µl 2.5 M HCl, dependent on what volume is necessary to reach a neutral pH. At the end, phosphate buffer (pH 7.4) is added until a final volume of 1 ml (adapted from van der Molen et al. [9]). The end product is not stable and reduction is lost within 16–24 h. On the other hand, only 1–2% of the homocysteine is reduced both in healthy and in uraemic plasma [9,10]. It is recommended to use the product immediately after the end of the reduction process. Furthermore, we suggest to construct a dose–response curve to assess the effect of reduced L-homocysteine at the concentrations observed in vivo. The homocysteine concentration range employed in the past in most in vitro experiments is far above (by at least one order of magnitude) that found in end-stage renal disease (ESRD) patients [11]. More recently, dose–response curves have been adjusted to values around the physiological and near pathological homocysteine concentration range [12].

Melatonin (#27), 2-methoxyresorcinol (#3) and indole-3-acetic acid (#21)
To dissolve melatonin, 2-methoxyresorcinol and indole-3-acetic acid in 10 ml 20% ethanol, it is recommended to dissolve these compounds first in 2 ml 100% ethanol to which 8 ml saline is added. Notably, in some countries a specific certificate to order and use melatonin is required stating that the compound will be used for in vitro experiments and not in vivo as a drug.

p-Cresol (#35)
p-Cresol has to be dissolved in methanol. We decided to use a p-cresol standard solution (in methanol) because dry p-cresol is a crystal-like compound that consists of relatively large particles, which are difficult to weigh. In addition, p-cresol is at one side hydrophilic and at the other lipophilic while it is also hygroscopic, meaning that it absorbs easily water from the air. This makes it even more difficult to weigh correctly the compound. Moreover, p-cresol has a very unpleasant smell and is toxic after inhalation and when contacting the skin. Since p-cresol solutions are available on the market as such, we purchased this compound already dissolved. The standard of p-cresol (5 mg/ml) that was used has a concentration of 122.85x Cmax (5000 µg/ml divided by 40.7 µg/ml). A 100x Cmax stock solution containing 81.3% MeOH is obtained by adding 228.5 µl saline to 1 ml of the p-cresol standard. It is of note that recent data show that p-cresol is not present as such in uraemia [13,14], but in conjugated forms (essentially p-cresylsulphate and p-cresylglucuronate). Both compounds are not commercially available and should be prepared chemically, taking care for the purity of the end product [15].

Peptides
Peptidic factors (Table 2) are provided in a diversity of buffers. When working with commercially available proteins, the exact constitution of the buffer in which the protein is dissolved as well as information about additives such as azide, stabilizers or protease inhibitors can be obtained from the data sheet provided by the manufacturer. If the purchased protein solution is to be diluted by a factor of 1000 or higher, the final concentrations of the buffer components can be neglected. Otherwise, it is recommended to use a control, which contains exactly the same components (except the peptide itself) at the same final concentrations as the peptide solution (see subsequently). If one or more of the components interfere with the biological assay, the peptide solution can be dialysed against saline or a buffer of choice e.g. by using dialysis membranes from Spectrum Laboratories, Inc. (Los Angeles, CA, USA) with the appropriate MW cut-off allowing the waste-out of additives without major loss of the compound of interest. Subsequently, we recommend determining the exact concentration of the peptide, because dialysis may lead to changes in sample volume and/or adsorption for the peptide to the membrane.

β2-microglobulin (#57)
Instead of being purchased β2-microglobulin can also be purified: It can be isolated from the ultrafiltrate obtained during a haemodialysis (HD) session with a high-flux membrane in one or more patients who are clinically free of dialysis-related amyloidosis as described by Nissen et al. [16]. The total volume of spent dialysate is then collected, concentrated and dialysed against ultrapure water (reversed osmosis), frozen and lyophilized. Then it should be re-suspended and passed through a gel chromatography column, Sephadex G75 (GE Healthcare—formerly Amersham Biosciences/Pharmacy Biotech—Uppsala, Sweden). The peaks are controlled by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The peak of interest corresponding to an MW of 11.8 kDa is then pooled and lyophilized, re-suspended and passed through a chromatofocusing column with a polybuffer exchanger (PBE) support and an elution buffer pH 7-4 (GE Healthcare). The unmodified β2-microglobulin elutes at pI of 5.7 as controlled by isoelectric focusing and is precipitated with ammonium sulphate, recovered and passed through a desalting column and lyophilized again. Endotoxins are to be removed at the end of the procedure as commented subsequently.

Retinol binding protein (RBP) (#75)
It is purified following the same procedure as the one for β2-microglobulin. The peak containing unmodified RBP elutes from the Sephadex G75 column at an MW of 21 kDa and at a pI of 4.9 in the chromatofocusing step.

Parathyroid hormone (PTH)
Beside intact PTH, a variety of PTH fragments with different actions on bone metabolism accumulate in sera of CKD patients [17]. Therefore, we recommend testing the intact hormone (1–84; #72), the amino-terminal fragment 1–34 (#73) which exerts the classical biological PTH effects on bone and kidney as well as fragment 7–84 (#74), a dominant carboxy-terminal structure which has been suggested to reduce the calcaemic effect of the intact molecule.

Post-translational protein modifications
Proteins exposed to the uraemic milieu tend to undergo post-translational modifications, which may alter their biological activities. We recommend investigating these two aspects separately: (i) the use of unmodified proteins, either purchased or isolated (e.g. β2-microglobulin, RBP) will reveal if the protein per se exerts a toxic effect. (ii) To investigate the effect of protein modifications, we suggest using human serum albumin as model protein. Albumin represents the most abundant serum protein and is no uraemic toxin in its unmodified form. Protein glycation adduct residues are listed in Table 4. It is conceivable that specific modifications of certain other proteins create a toxic effect. Whereas it seems not feasible to study all different possibilities, selected proteins such as β2-microglobulin, Ig light chains or RBP may be modified as well.


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  		Table 4. Protein glycation, oxidation and nitration-related uraemic toxins

 
AGE compounds
AGEs represent a specific group of uraemic retention solutes. Those related to protein glycation, oxidation and nitration are shown in Table 4. Data on AGEs which appeared after our previous work summarizing the known uraemic retention solutes [2] have significantly modified the classification and the perception about the concentrations of these compounds [18]. We decided to present the information related to AGEs taking into account these new data.

AGE-free adducts are the major form of AGE excreted in the urine. To assess effects on cells, experiments with AGE-free adducts and AGE-modified proteins (modified minimally with 1–2 equivalents of AGE residue per mole protein) at concentrations found in plasma can be used [18]. AGEs formed from amino acid residues within proteins are more appropriately called ‘AGE residues’. AGE modified proteins and free adducts can be stored at –20°C or –80°C. Working solutions during a working day can be stored on ice.



   Discussion
Top
 Introduction
 General methodological aspects
 Recommendations for stock...
 Discussion
Conclusions
 Acknowledgements
 References
 
Controls
The use of appropriate controls is crucial for the correct interpretation of the observed effects. Generally, all controls are to be performed at the same time and in exactly the same way as the analysis of the real sample.

If the uraemic compound under investigation is only available as a salt, we recommend the corresponding anion/cation is dissolved with a single inorganic counter-ion (e.g. KCl for K, etc.) at the same final molar concentration as the solute in the control medium.

For those substances, which have to be dissolved in organic solvents such as ethanol, methanol or DMSO, the same solvent at the same final concentration should be used as a control. Since organic solvents by themselves can exert an effect on the biological system under investigation, it is recommended to use the lowest possible final concentration. Furthermore, it is advisable to include a control with saline in the experiments. If the parameter tested shows a deviation in the presence of the organic solvent alone compared with saline, the obtained results should be interpreted accordingly. If the concentration dependency of uraemic solutes is tested, all the samples should contain the same concentration of organic solvent. A lower concentration of organic solvent does not necessarily lead to a smaller biological effect.

Some compounds only dissolve in acidic or alkaline milieu. Since biological systems are sensitive to changes in pH, it is recommended to use a final pH which is as close as possible to the physiological range. In case of a non-physiological final pH, we suggest to perform control experiments with the same final pH.

Since HCl-bound thiolactone homocysteine is used in the reduction procedure to obtain L-homocysteine, it is recommended to include an extra negative control when performing the tests for this compound. This will be done by performing the reduction procedure as described above in which L-homocysteine thiolactone. HCl is replaced by 71.3 µg/ml HCl [300 µg/ml x (36.5/153.6)].

Contaminations by lipopolysaccharide
Lipopolysaccharide (LPS; endotoxin) contamination which is a general problem frequently encountered in in vitro assays can be avoided or at least minimized by the use of pyrogen-free chemicals, solvents as well as preparation procedures. Of note, endotoxins cannot be removed by sterile filtration. If an effect that could be attributed to LPS- contamination is observed, it is recommended to measure the endotoxin content by commercially available test kits (e.g. E-TOXATE® reagent from Limulus polyphemus; Sigma). Additionally, the assay can be repeated in the presence of polymyxin B (Sigma–Aldrich), a cationic peptide which binds to the biologically active part of LPS and abolishes its activity [19]. It is recommended to include polymyxin B alone as a control to ensure that this compound per se does not interfere with the test. If polymyxin B partially or entirely abolishes the observed effect without having an effect by itself on the assay, the primary effect is attributable to LPS contamination rather than to the compound under evaluation. In this case, it is recommended to remove the contaminating endotoxin by affinity chromatography using immobilized polymyxin B (e.g. from Sigma–Aldrich or Bio-Rad, Hercules, CA, USA). Afterwards, it should be checked by concentration measurements whether the adsorptive system does not adsorb the test compound as well. Caution should be exercised where the uraemic toxin is polyanionic and may also bind to polymyxin B or displace LPS from polymyxin B. Albumin modified highly by AGEs is an acidic, polyanionic form of albumin and may produce this effect under some conditions.

Substances not commercially available
No specific information is given for substances, which are not commercially available [1-methylinosine, {alpha}-keto-{delta}-guanidinovaleric acid, argininic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), N6-methyladenosine, N6-threonylcarbamoyladenosine, phenylacetylglutamine, taurocyamine]. If there is a necessity to test them, they have to be synthesized.



   Conclusions
Top
 Introduction
 General methodological aspects
 Recommendations for stock...
 Discussion
 Conclusions
Acknowledgements
 References
 
Uraemic retention solutes represent a heterogeneous group of substances such as organic compounds, peptides and advanced glycation or oxidation end products. The present report includes basic protocols that give information for those researchers who aim to screen the in vitro effect of individual uraemic retention solutes or groups of solutes in any biological system. Information about the availability and solubility of uraemic retention solutes is given. Recommendations for the experimental set-up, preparation of stock solutions, the design of control experiments and the procedure to be followed when observing endotoxin-like effects are provided and should lead to more consistent research activities in the uraemic toxicity area and an easier comparison of different studies using uraemic retention solutes.



   Acknowledgements
Top
 Introduction
 General methodological aspects
 Recommendations for stock...
 Discussion
 Conclusions
 Acknowledgements
References
 
The European Uremic Toxin Work Group (EUTox) has been created within the European Society for Artificial Organs (ESAO) to discuss and analyse matters related to the identification, characterisation, analytic determination, and evaluation of biological activity of uraemic retention solutes. More information about the European Uremic Toxin Work Group, which is responsible for this publication, can be obtained at the web site: http://uremic-toxins.org. E-mail: raymond.vanholder{at}ugent.be

The current members of EUTox are: A Argiles, Montpellier, France; U Baurmeister, Berlin, Germany; J Beige, Leipzig, Germany; P Brouckaert, Gent, Belgium; P Brunet, Marseille, France; J Chudek, Katowice, Poland; G Cohen, Vienna, Austria; PP De Deyn, Antwerp, Belgium; T Drüeke, Paris, France; G Glorieux, Gent, Belgium; S Herget-Rosenthal, Essen, Germany; W Hörl, Vienna, Austria; J Jankowski, Berlin, Germany; A Jörres, Berlin, Germany; Z Massy, Amiens, France; H Mischak, Hannover, Germany; A Perna, Naples, Italy; M Rodriguez, Cordoba, Spain; G Spasovski, Skopje, Macedonia; B Stegmayr, Umea, Sweden; P Stenvinkel, Stockholm, Sweden; P Thornalley, Essex, United Kingdom; R Vanholder, Gent, Belgium; C Wanner, Würzburg, Germany; A Wiecek, Katowice, Poland, W Zidek, Berlin, Germany.

The activities of the EUTox group are supported by the following industries: Amgen; Baxter Healthcare Corporation; Fresenius Medical Care; Gambro; Genzyme; Hoffmann-La Roche; Membrana GMBH.

Conflict of interest statement. None declared.



   References
Top
 Introduction
 General methodological aspects
 Recommendations for stock...
 Discussion
 Conclusions
 Acknowledgements
 References
 

  1. Hörl WH. Genesis of the uraemic syndrome: role of uraemic toxins. Wien Klin Wochenschr (1998) 110:511–520.[Web of Science][Medline]
  2. Vanholder R, De Smet R, Glorieux G, et al. Review on uremic toxins: classification, concentration, and interindividual variability. Kidney Int (2003) 63:1934–1943.[CrossRef][Web of Science][Medline]
  3. Vanholder R, Argiles A, Baurmeister U, et al. Uremic toxicity: present state of the art. Int J Artif Organs (2001) 24:695–725.[Web of Science][Medline]
  4. Vanholder R, Glorieux G, De Smet R, Lameire N. New insights in uremic toxins. Kidney Int Suppl (2003) 84:S6–S10.[Medline]
  5. Vanholder R, Glorieux G, Lameire N. Uraemic toxins and cardiovascular disease. Nephrol Dial Transplant (2003) 18:463–466.[Free Full Text]
  6. Vanholder R, Massy Z, Argiles A, et al. Chronic kidney disease as cause of cardiovascular morbidity and mortality. Nephrol Dial Transplant (2005) 20:1048–1056.[Abstract/Free Full Text]
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Received for publication: 23.11.06
Accepted in revised form: 16. 3.07


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