BRIEF REVIEW www.jasn.org The Uremic Toxicity of Indoxyl Sulfate and p-Cresyl Sulfate: A Systematic Review Raymond Vanholder, Eva Schepers, Anneleen Pletinck, Evi V. Nagler, and Griet Glorieux Nephrology Section, Ghent University Hospital, Ghent, Belgium ABSTRACT A growing number of publications supports a biologic effect of the protein-bound overestimated toxic impact. This bias has uremic retention solutes indoxyl sulfate and p-cresyl sulfate. However, the use of provoked justified objections against unrealistically high free concentrations of these compounds and/or inappropriately low recognizing protein-bound molecules albumin concentrations may blur the interpretation of these results. Here, we as real uremic toxins. In a recent edito- performed a systematic review, selecting only studies in which, depending on the rial,25 indoxyl sulfate, one of the main albumin concentration, real or extrapolated free concentrations of indoxyl sulfate and protein-bound solutes, was for that rea- p-cresyl sulfate remained in the uremic range. The 27 studies retrieved comprised in son called “long suspected but not yet vitro and animal studies. A quality score was developed, giving 1 point for each of the guilty.” following criteria: six or more experiments, confirmation by more than one experimen- Nevertheless, a careful check of the tal approach, neutralization of the biologic effect by counteractive reagents or anti- literature reveals that investigators in a bodies, use of a real-life model, and use of dose–response analyses in vitro and/or number of studies have applied accept- animal studies. The overall average score was 3 of 5 points, with five studies scoring 5 of able free concentrations by administering 5 points and six studies scoring 4 of 5 points, highlighting the superior quality of a those toxins to animals up to acceptable substantial number of the retrieved studies. In the 11 highest scoring studies, most total concentrations, adding relevant functional deteriorations were related to uremic cardiovascular disease and kidney quantities of albumin to in vitro media damage. We conclude that our systematic approach allowed the retrieval of method- by using whole blood, plasma, or serum, ologically correct studies unbiased by erroneous conditions related to albumin binding. or in the absence of albumin, applying Our data seem to confirm the toxicity of indoxyl sulfate and p-cresyl sulfate and support concentrations corresponding to the their roles in vascular and renal disease progression. free uremic fraction. At this moment, it is unclear, however, how many method- J Am Soc Nephrol 25: 1897–1907, 2014. doi: 10.1681/ASN.2013101062 ologically suitable publications are avail- able and what they teach us about the contribution of the compounds to the As CKD evolves, uremic retention observed in human CKD in in vitro testing uremic syndrome. 4,20 becomes a progressively more important or in vivo animal experiments. More- In the present analysis, we applied a contributor to overall organ dysfunc- over, for protein-bound toxins, even with systematic search to filter out those tion.1–3 Among the three physicochemical seemingly acceptable total concentrations, studies in which unacceptably high free types of uremic solutes,4 the pathophysi- the quantities of albumin or protein pres- concentration of protein-bound solutes ologic importance of protein-bound tox- ent are often too low, resulting in unac- had been applied. We aimed to analyze ins has been neglected for a long time,5–7 ceptably high and thus, irrelevant free the effect of indoxyl sulfate and p-cresyl leading to a shortage of specificremoval concentrations. sulfate in vitro and in animals. Based on strategies.8,9 In the past decade, however, a In analogy to drugs, albumin is likely to growing number of publications docu- act as a buffer, attenuating potential bio- mented the impact of protein-bound uremic chemicaleffectsofitsligands,21–23 whereas Published online ahead of print. Publication date toxins on vital processes and an associa- recent data clearly showed that, for both available at www.jasn.org. tion of their concentration with clinical indoxyl sulfate and p-cresyl sulfate, albu- outcome parameters.6,10–19 min is the main binding protein.24 Correspondence: Dr. Raymond Vanholder, Ne- phrology Section, 0K12, Ghent University Hospital, One factor blurring the interpretation Absence of appropriate albumin De Pintelaan 185, B9000 Ghent, Belgium. Email: of the biochemical effects of uremic toxins quantities results in too high free solute [email protected] is the application of unrealistic concen- concentrations, inducing exaggerated bi- Copyright © 2014 by the American Society of trationscomparedwiththeconcentrations ologic responses with a potential for an Nephrology J Am Soc Nephrol 25: 1897–1907, 2014 ISSN : 1046-6673/2509-1897 1897 BRIEF REVIEW www.jasn.org preset standards, 27 articles of adequate studies in total were performed in Eu- Dahl salt-sensitive hypertensive rat quality were retrieved and are reported rope, the United States, or Australia. strain,37,38,40–43,47,49,50 although in some here together with the shown effects. The following cell and organ systems of these studies, an effect in the wild were investigated (n of each study is in type was also observed.37,38,43,47,50 One parentheses) (Table 2): renal tubules study only evaluated Dahl wild-type rats RESULTS (9),34,36–39,43,45,47,50 endothelium and found a significant effect of indoxyl (6),26–30,33 leukocytes (2),30,31 whole sulfate in that strain.42 The literature was searched according to vessels (2),40,41 whole kidney (2),40,42 Of the in vitro studies, eight studies preset methods (see below) to retrieve cardiac cells (2),35,49 smooth muscle applied an appropriate concentration of studies on biologically relevant toxic cells (2),46,51 hepatocytes (2),32,51 intes- albumin, resulting in relevant free toxin effects of indoxyl sulfate and p-cresyl tinal cells (1),48 and adipocytes (1).44 A concentrations,26–29,31,46,51,52 whereas in sulfate following well defined quality few studies concentrated on more than the remaining seven studies, a concen- standards. One of the conditions was one target system: one study was on tration compatible with the uremic free that the concentrations used in those both the whole vessel and kidney40 and fraction was pursued.32–36,39,48 studies conformed with the concentra- one study was on leukocyte–endothelial A large array of mediators, pathways, tions observed in uremia, which is illus- interaction.30 and messenger molecules contributed to trated in Table 1. The concentrations Twenty-four studies concentrated on the observed system effects (Table 4). considered appropriate were in the indoxyl sulfate,26–30,32–43,46–52 and six Several were confirmed in more than one highuremicrangetocompensatefor studies concentrated on p-cresyl sul- study of this series.27,30,34–36,38,40,43,45,48–50 the often short exposure time in the ex- fate.31,34,36,39,44,45 Three studies evalu- Several of the observed effects were re- perimental studies, especially in vitro.To ated both compounds.34,36,39 Some lated to inflammation/free radical pro- make a comparison with the values cur- studies also assessed alterations induced duction27,30,34–36,44,45,48–50 and fibro- rentlyobservedinanaveragedialysis by other uremic toxins, but only two of sis.36,38–40,43,49 The mutual connections population, we give in Table 1, also as those studies showed a significant effect of these factors into global pathways for reference, data retrieved from a recent for indole acetic acid,46,48 hippuric acid,48 endothelial and proximal tubular cells study from the European Uremic Toxin and carboxy-methyl-propyl-furanpropionic are shown in Figure 2. Work Group as a real-life orientation for acid.48 With regards to quality, all retained the reader.20 We identified 336 citations All studies evaluating indoxyl sulfate studies together had a median score of 3.0 through electronic searching and added showed a negative (toxic) effect (Table 3), from a possible total of 5 points, indi- 61 citations from reference lists of re- except the study by Odamaki et al.,52 cating that approximately one half of the trieved publications (Figure 1). Based which showed an increase of hepatocyte retained studies had a score of 3 points or on the selection criteria, we included albumin production in the presence of above (Supplemental Material 1). Five stud- 27 studies in the review (Table 2).26–52 indoxyl sulfate. Four studies on p-cresyl ies reached a score of 5 of 5 points,35,36,39,44,45 The number of retrievable studies sulfate53–56 and one study on indoxyl and six studies obtained a score of 4 of 5 increased considerably over the past sulfate55 showed no significant changes points.27,30,38,46,49,50 These 11 studies years, suggesting a risingawarenessabout (data not shown). with a high quality score covered a broad the topic. The distribution among coun- Of 14 animal studies, 10 studies were array of pathophysiologic mechanisms tries points to a broad worldwide inter- in rats,37,38,40–43,45,47,49,50 and four mainly related to cardiovascular damage est. The majority (n=20) was generated studies were in mice.30,36,39,44 Several and progression of kidney disease: reac- in Asia, especially Japan; however, seven of the rat studies were in the specific tive oxygen species generation (3),27,45,50 endothelial dysfunction (2),27,30 epithelial- to-mesenchymal transition (2),36,38 Table 1. Threshold values of concentration leukocyte–endothelial interaction (1),30 Normal (mg/L) Uremia (mg/L) Comments deterioration of cardiac cell functional 35 39 IS (molecular weight 212) capacity (1), Klotho expression (1), 4 expression of tissue factor in vascular IStotal maximum — 236.0 Highest individual value 20 20 46 IStotal high 0.5 44.5 Highest mean smooth muscle cells (1), and insulin re- 20 44 ISfree high Less than LOD 4.5 Highest mean sistance (1).