The Uremic Toxicity of Indoxyl Sulfate and P-Cresyl Sulfate: a Systematic Review

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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 available 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). The reasons for scoring 4 PCS (molecular weight 188) of 5 points instead of 5 of 5 points were — 78 PCStotal maximum 105.0 Highest individual value lack of proof of concept by effect neutral- 77 78 PCStotal high 2.9 43.0 Highest mean ization two times, the number of experi- 20 20 PCSfree high 0.1 2.6 Highest mean ments was too low two times, lack of Highest individual value is the highest single value per patient ever reported; highest mean is the highest confirmation by an alternative approach mean for a patient group ever reported. Current average values observed in an average dialysis pop- one time, and absence of real-life condi- ulation as reported in the most recent review of the European Uremic Toxin Group are 23.1 mg/L for IStotal 20 fi and 20.9 mg/L for PCStotal. IS, indoxyl sulfate; LOD, limit of detection; PCS, p-cresyl sulfate. tions one time. There was no signi cant

1898 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1897–1907, 2014 www.jasn.org BRIEF REVIEW

damage40,42 are all are linked to the progression of CKD, another factor related to uremic morbidity and mortality.57 As a consequence, decrease in concentration of intestinally generated uremic retention solutes, like indole, by the intestinal sorbent AST-120 (Kremezin) has been accepted in mostly Asian countries as a strategy to restrain the progression of CKD. It is very likely that this sorbent affects the concentration of not only indoxyl sulfate but also, other protein- bound uremic toxins, one of which is p-cresyl sulfate.64 In at least two small- sized randomized controlled trials, AST- 120 had a positive effect on progression of CKD.65,66 In a recent large randomized controlled trial in the United States and Europe, however, this positive effect could not be confirmed.67 Until now, biologic data were per- ceived as not consistent or convincing enough to recognize protein-bound toxins as the real culprits.25,68,69 The results of the current analysis applying system- Figure 1. Flowchart of the review procedure and quality scoring. atic search methods linked to a quality assessment of the retrieved publications, correlation between year of publication disturbances,46 leukocyte activation,30,31 however, yielded several high-quality and quality score (data not shown). cross-talk of leukocytes and endothelium,30 papers applying, for the uremic setting, cardiac fibrosis and hypertrophy,35,49 and acceptable concentrations and state-of- insulin resistance44 as well as whole-vessel the-art test methods. Therefore, our DISCUSSION alterations per se40,41 are all linked to car- analysis offers, in our opinion, argu- diovascular damage and contribute to the ments that are solid enough to recon- This study reviews the literature evidence increased propensity for mortality and sider the reluctance to recognize the up until June 30, 2013, for the biologic cardiovascular events in CKD.57,58 Both protein-bound solutes indoxyl sulfate and/or toxic effects of two prototype indoxyl sulfate and p-cresyl sulfate per se and p-cresyl sulfate as toxic. protein-bound uremic toxins: indoxyl have repeatedly been related to cardiovas- This study is, to the best of our sulfate and p-cresyl sulfate. In total, 27 cular mortality.10–19 Although strategies knowledge, the first to apply to biologic studies from all over the world were that more efficiently remove protein- studies a stringent strategy that had been retained, with the principal topics being bound solutes, such as frequent dialysis59 defined in advance, in casu related to endothelial and proximal tubular cell and online hemodiafiltration,60,61 in con- uremic retention, resulting in a sys- dysfunction, although insulin resistance, trolled studies are associated with im- tematized summary of their toxicity. functional status of cardiac cells, leuko- proved outcomes,62,63 it remains difficult Although we tried to follow current cyte dysfunction, coagulation disturban- to interpret these data because of the standards for the conduct of systematic ces, and pharmacodynamics were also presence of confounding factors that af- reviews,70 there are methodological devi- covered. A quality assessment identified fect the outcomes on their own and a lack ations. In contrast to evidence-based sys- 11 of 27 (41%) studies with a score of of selectivity of removal, because dialysis tematic reviews, in our case, only a lim- at least 4 points from a possible maxi- strategies are usually not restricted to ited number of search engines was used. mum of 5 points. The majority of the protein-bound solutes alone. Of note, for biologic studies, especially changes found affects mechanisms that Next to the above cardiovascular in the area of uremia, there is, to the best are essential to the pathophysiology of the problems, the data linking these sol- of our knowledge, not a large repository uremic syndrome and its subsequent del- utes to tubular cell damage,39,45 tubular of data such as the one made available by eterious impact on prognosis. epithelial-to-mesenchymal transition,36,38 the Cochrane Library (Cochrane Cen- Endothelial dysfunction,26–30,33 smooth tubulointerstitial inflammation and fi- tral Register of Controlled Trials) for muscle cell lesions,46,51 coagulation brosis,34,36,37,39,47,50 or whole-kidney clinical studies. Our approach may serve

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Table 2. Retained articles and their main characteristics First Author, Year, Concentration Albumin/Type Cell/Organ System Toxin and Reference (mg/L)/Dose Model (1) In vitro, albumin: normal Dou, 200426 Endothelium IS 25–250 40 g/L Odamaki, 200452 Hepatocytes IS 50–100 40 g/L Faure, 200628 Endothelium IS 256 40 g/L Yamamoto, 200651 Smooth muscle cells IS 53–106 40 g/L Dou, 200727 Endothelium IS 125–250 40 g/L Schepers, 200731 Leukocytes PCS 121.0 Whole blood Itoh, 201229 Endothelium IS 29.9–57.2 40 g/L Chitalia, 201346 Smooth muscle cells IS 25 40 g/L Koppe, 201344 Adipocytes, myotubes PCS 40 35 g/L (2) In vitro, albumin: low, absent, or unspecified Tsujimoto, 201032 Liver microsomes IS 6.36 Absent Lekawanvijit, 201035 Cardiac fibroblasts/myocytes IS 0.02–63.6 5 g/L BSA/absent Yu, 201133 Endothelium IS 1.25–125 Unspecified Sun, 201334 Proximal tubular cells IS 1; 5 Absent PCS 1; 5 Sun, 201236 Proximal tubular cells IS 1–50 Absent PCS 1–50 Sun, 201239 Proximal tubular cells IS 1; 5 Unspecified PCS 1; 5 Tsujimoto, 201248 Intestinal cells (hepatic no effect) IS 4.24 0.8 g/L (10% FBS) (3) Animala Adijang, 200840 Aorta, kidney IS 23.1/200 mg/kg (30 wk) p.o. DH rat Ito, 201030b Endothelium/leukocyte interaction IS 15.7/200 mg/kg q.d. (10 d) p.o. Nx mouse Adijang, 201041 Aorta IS 15–20/200 mg/kg (32 wk) p.o. DN, DH rat Bolati, 201138b,c Tubular cells IS 9.4–18.9/200 mg/kg (32 wk) p.o. DH rat Sun, 201236 Tubular cells IS Unspecified/100 mg/kg q.d. (4 wk) i.p. 1/2 Nx mouse PCS Watanabe, 201345b Renal tubular cells PCS 32.6/50 mg/kg q.d. (4 wk) i.p. 5/6 Nx rat Sun, 201239 Proximal tubular cells IS 8.5 1/2 Nx mouse PCS 1.8 100 mg/kg q.d. (4 wk) i.p. Shimizu, 201237b,c Proximal tubular cells IS 9.4–18.9/200 mg/kg (32 wk) p.o. DN, DH rat Shimizu, 201342b Kidney (cortex) IS 9.4/200 mg/kg q.d. (32 wk) p.o. DN rat Shimizu, 201343b Tubular cells IS 9.4/200 mg/kg (32 wk) p.o. DN rat Koppe, 201344 Adipocytes, myocytes, various organs PCS 51/10 mg/kg b.i.d. (4 wk) i.p. Normal RF mouse Shimizu, 201347b,c Tubular cells IS 9.4–18.9/200 mg/kg (32 wk) p.o. DN, DH rat Yisireyili, 201349 Cardiac tissue IS 18.9/200 mg/kg q.d. (32 wk) p.o. DN, DH rat Bolati, 201350b Tubular cells IS 9.4/200 mg/kg q.d. (32 wk) p.o. DN, DH, rat IS, indoxyl sulfate; PCS, p-cresyl sulfate; BSA, bovine serum albumin; FBS, fetal bovine serum; wk, weeks; p.o., per os; q.d., once a day; i.p., intraperitoneal; b.i.d., twice per day; DH, Dahl salt-sensitive hypertensive; Nx, nephrectomized; DN, Dahl salt-resistance normotensive; RF, renal function. aFor animal experiments, we presumed that serum albumin was present at the same concentrations as usually in these animals; all solute concentrations were measured in serum, except for the study by Ito et al.30 (plasma), and steady state at the end of exposure on euthanasia of the animals, except for the study by Koppe et al.44 (peak after i.p. injection). Also, dose and administration route are mentioned; the Albumin/Type Model column contains both species and type of model. bIn vitro part not appropriate. cIf concentration is 9.4–18.9, it is 9.4 for Dahl wild-type rats and 18.9 for DH rats. as a basis for future analyses of the same ecules better and more consistently than in the nearby future. Anyway, the fact kind. at present. Alternatives to the standard that dietary components and residual re- If we consider a quality score of 4 or approaches that we now know to have a nal function seem to affect protein- 5 points of 5 points as the equivalent of potentially beneficial impact are extended bound solute concentration more than a high evidence label, the results reported dialysis,71 prebiotics,72,73 probiotics72,73 dialysis adequacy, as assessed by Kt/V,75 in the present article should provide a and extracorporeal sorbents.74 Because may suggest that alternative methods positive incentive to developing strate- our knowledge regarding this issue is focusing on preserving kidney function gies to remove these protein-bound mol- extending, alternative options may arise or affecting intestinal toxin generation

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Table 3. Main relevant pathophysiologic changes per study Cell/Organ System Toxin Main Observed Effect (1) Endothelium Dou, 200426 IS Inhibition proliferation and repair Faure, 200628 IS Increase endothelial microparticle release Dou, 200727 IS Induction ROS, inhibition glutathione Ito, 201030 IS Increase endothelial/leukocyte interaction (adhesion) Yu, 201133 IS Inhibition proliferation; increase senescence; increase ROS and decrease NO production Itoh, 201229 IS Induction ROS (2) Smooth muscle cells Yamamoto, 200651 IS Increase proliferation Chitalia, 201346 IS Generation tissue factor (linked to clot formation), tissue factor breakdown retarded (3) Leukocytes Schepers, 200731 PCS Activation oxidative burst Ito, 201030 IS Increase endothelial/leukocyte interaction (adhesion) (4) Whole vessels Adijang, 200840 IS Increase aortic calcification and stiffness, expression osteoblast markers, and OATs Adijang, 201041 IS Induction cell senescence (5) Cardiac cells Lekawanvijit, 201035 IS Cardiomyocyte proliferation, cardiac fibrosis, inflammation (6) Whole heart Yisireyili, 201349 IS Increase cardiomyocyte hypertrophy, cardiac fibrosis, oxidative stress (7) Renal tubular cells Bolati, 201138 IS Increase epithelial-to-mesenchymal transition Sun, 201334 IS, PCS Enhanced expression cytokine and inflammatory genes Sun, 201236 IS, PCS Activation RAAS and epithelial-to-mesenchymal transition, fibrosis, nephrosclerosis Shimizu, 201237 IS Increase expression MCP-1 Sun, 201239 IS, PCS Increase Klotho gene methylation, renal fibrosis Watanabe, 201245 PCS Increase renal tubular damage 43 Shimizu, 2013 IS Induction mediators for fibrosis (TGF-b1 and Smad3) Shimizu, 201347 IS Induction generation adhesion molecule (ICAM-1) Bolati, 201350 IS Increased oxidative stress (8) Whole kidneys Adijang, 200840 IS Increase fibrosis Shimizu, 201342 IS Increase angiotensinogen expression (9) Hepatocytes Odamaki, 200452 IS Increase albumin production Tsujimoto, 201032 IS Inhibition metabolic clearance losartan (10) Intestinal cells Tsujimoto, 201248 IS Inhibition pumps involved in clearance of pravastatin (11) Adipocytes Koppe, 201344 PCS Increase insulin resistance, decrease lipogenesis, increase lipolysis, ectopic lipid redistribution IS, indoxyl sulfate; ROS, reactive oxygen species; NO, nitric oxide; PCS, p-cresyl sulfate; OATs, organic anion transporters; RAAS, renin angiotensin aldosterone system; MCP-1, monocyte endothelial chemotactic protein-1; TGF-b1, transforming growth factor-b1; ICAM-1, intercellular adhesion molecule-1. may be as important as extracorporeal bound solutes, whereas several other whereas in the average uremic patient, removal. compounds in this group might have a the concentration will be lower. How- The question could be raised as to how biologic impact as well.3,5,6,76 We se- ever, a high concentration was applied far toxic mechanisms and pathways over- lected, however, the two molecules that essentially in vitro, where the concentra- lap for both toxins. It is only possible if have been most extensively evaluated. tion should be considered to compensate studies on the two compounds assess Some of the retained studies also show for short exposure to uremic toxins, in the same elements. To the best of our similar effects for other protein-bound contrast to real life, where it is continu- knowledge, for the time, such a parallel solutes,46,48 suggesting that the yielded ous, allowing solutes more time to reach approach has only been used in renal evidence could have been even more the intracellular compartments where tubular cells. Figure 2B confirms that, in- convincing if more compounds would biologic activity is exerted. It is also of deed, some mechanisms are overlapping. have been taken into account. Second, note that, in the in vivo animal studies, This study has a few drawbacks. First, we accepted concentrations up to the depending on the way of administration we focused only on two of the protein- upper uremic range (Table 1),4,20,77,78 (intraperitoneally versus per os), the

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Table 4. Most important affected pathophysiologic mechanisms Confirmed in Two Related to Related to Affected Mediators Effect Toxin or More Studies Inflammation Fibrosis a-Smooth muscle actin36,38,40,49 ↑ IS, PCS X X Cytokine generation34,35 ↑ IS, PCS X X E-cadherin36,38 ↓ IS, PCS X X Nuclear Factor (erythroid-derived 2) -like 249,50 ↓ IS X X Extracellular-regulated kinase 1/244 ↑ PCS X Fibronectin36 ↑ IS, PCS X Heme oxygenase-149,50 ↓ IS X X Intercellular Adhesion Molecule-147 ↑ IS X Inflammatory genes34 ↑ IS, PCS X Insulin receptor substrate-144 ↓ PCS X Klotho39 ↓ IS, PCS X Mitogen-activated protein kinase: MEK 1/2; p3835 ↑ IS X Multidrug resistance-associated protein-248 ↓ IS NADPH oxidase27,30,45 ↑ IS, PCS X X Nuclear Factor-kB35 ↑ IS X Organic Anion Transporters40,48 ↓ IS X Osteoblast-specific proteins40 ↑ IS X Phosphoinositide 3-kinase44 ↓ PCS X Protein kinase B/Akt44 ↓ PCS X Renin-angiotensin-aldosterone system36 ↑ IS, PCS X X E-selectin30 ↑ IS X Senescence proteins41a ↑ IS Smad2/3 and Smad436,43 ↑ IS, PCS X X Snail36 ↑ IS, PCS X Tissue factor46 ↑ IS Transforming Growth Factor-b36,40,43,49 ↑ IS, PCS X X Zonula occludens-138 ↓ IS X IS, indoxyl sulfate; PCS, p-cresyl sulfate. aSenescence proteins: senescence-associated b-galactosidase, 16INK4a, p21WAF1/CIP1, p53, and retinoblastoma protein. dose, and the type of model with regard such as endothelial microparticle release,56 sulfate and p-cresyl sulfate and taking to kidney function, fluctuating concen- tissue factor activation,79 or metabolic into account precise criteria for concentrations and different peak values might capacity for glucuronidation.80 trations that considered protein binding occur in the serum, which might, in Because studies on toxicity in tubular yielded 27 publications that conformed turn, impact the interpretation of the cells suggest a relation to progression of with the inclusion criteria. observed biologic effect. kidney failure that is especially important Eleven of these studies obtained a Third, our review data pointed over- in earlier stages of CKD, it can be argued high-quality score, indicating a substan- whelmingly to a toxic effect of indoxyl that lower concentrations should be tial scientific impact. Most frequently, sulfate and p-cresyl sulfate that may be pursued in these experiments. How- missing quality factors were insufficient skewed by submission bias. Investigators ever, even in dialysis patients, restrain- number of experiments, lack of a dose– may be tempted not to finalize or publish ing progression may have a substantial response analysis, and especially, ab- studies showing no ill effect. Neverthe- clinical impact, because residual renal sence of data showing the neutralization less, we retrieved five such studies by our function is definitely inversely related of the effect found by counteragents of analysis.52–56 Fourth, we should consider to mortality.81 In addition, in most of the presumed mechanism. Future stud- the possibility that we missed some stud- studies on tubular toxicity that were re- ies should comply with these conditions ies in our analysis because of the approach trieved,34,36–39,42,43,45,47,50 concentra- to be considered of high quality. The re- followed, but more data would only in- tions were in the same range as those trieved studies covered a broad array of crease the evidence delivered here. concentrations found in patients with effects, most of them related to clinically However, our approach, based on rigid CKD stages 3–5 not on dialysis13,82 (i.e., highly relevant aspects, such as cardio- threshold levels, must also have excluded lower than in dialysis patients). vascular disease or progression of CKD. a number of interesting studies based on In conclusion, a systematic retrieval These data, if matched to observational concentrations just above threshold77 approach searching for experimental outcome studies,6,10–19 are the best evi- and/or pathophysiologic key aspects studies evaluating the biologic (toxic) dence that we can obtain right now for highly relevant to the uremic syndrome, impact of the uremic solutes indoxyl the clinical impact of protein-bound

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Figure 2. Effects as seen in the (A) endothelial cell and (B) renal tubular cell. The effects of indoxyl sulfate and p-cresyl sulfate are represented by orange and purple arrows, respectively. The transcellular membrane transport of indoxyl sulfate and p-cresyl sulfate by the organic anion transporters is represented by the thick white arrow. a-SMA, a-smooth muscle actin; 5-Aza-2dc, 5-Aza-29-deoxycytidine; CpG, cytosine-phosphate-guanine (DNA sequence); DAG, diacylglycerol; DNMT, DNA methyltransferase; DPI, diphenylene iodonum chloride; EMT, epithelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; ER, endoplasmic reticulum; G, G protein- coupled receptor; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule-1; IKB, inhibitor of kB; IP3, inositol 1,4,5-triphosphate; IS, indoxyl sulfate; L-arg, L-arginine; LFA-1, lymphocyte function-associated antigen 1; MCP-1, monocyte chemoattractant protein-1; Me, Methylgroup; NAC, N-acetylcysteine; NADPH ox, NADPH oxidase; NO, nitric oxide; NQO1, NADPH guinone oxidoreductase 1; Nrf, NF

(erythroid-derived 2) -like; OAT, organic anion transporter; 8-OHdG, 8-hydroxydeoxyguanosine; p, protein; PCS, p-cresyl sulfate; PIP2, phosphatidylinositol 4,5-biphosphate; PLC, phospholipase C; PKC, protein kinase C; RAAS, renin angiotensin aldosterone system; ROS, reactive oxygen species; Vit C, vitamin C; Vit E, vitamin E; ZO-1, Zonula occludens protein 1. solutes. This knowledge may be support- evaluated until now. A literature search using or p-cresyl sulfate in vitro or in animals where ive of the development of strategies to Pubmed through Reference Manager was an acceptable free concentration was present. lower the concentration of these solutes undertaken with the following terms: p-cresyl For this reason, analytical studies, clinical in the body. sulfate or p-cresylsulfate, or indoxylsulfate or concentration or outcome studies, and stud- indoxyl sulfate. This search was completed ies on the origin and generation of the com- with additional references emanating from pounds of interest, medical interventions to CONCISE METHODS the originally retrieved publications. The decrease their concentration, and removal by search contained all obtained references dialysis or other strategies were all excluded. The study was limited to indoxyl sulfate and with June 30, 2013, as closing date (Figure 1). Also, studies not specifying the pursued solute p-cresyl sulfate, the two protein-bound ure- We only included primary studies assess- concentrations or unclear on the applied meth- mic toxins that have been most extensively ing the biologic effect of indoxyl sulfate and/ ods, making reproduction of the experiments

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Figure 2. Continued.

difficult, were rejected as well as reviews, ab- Based on this approach, two investigators of our search. Thus, we developed a quality stracts, and studies referring to conditions independently selected the studies and ex- score (Supplemental Material 2) that was other than CKD, including AKI. tracted all data, with discrepancies resolved given to each study based on the number of Only studies conforming to the preset by a third investigator (Figure 1). experiments (1 point if six or more experi- target concentrations, definedasvaluesnot The studies were then checked for their ments); confirmation of the obtained results exceeding the highest levels obtained in content using a data extraction list conceived by an alternative approach (same parameter, uremia (reported in Table 1), were in- as reported in Supplemental Material 1, with different methods, same pathway, different cluded.4,20,77,78 Additional conditions for specific attention to the experimental setting, parameters; 1 point); proof of neutralization accepting a study were as follows: in vitro the assessed problem, the molecule of inter- of the basic identified mechanism by the ap- studies with use of medium containing con- est, the applied concentrations, the presence propriate antibodies, counteractive agents, or centrations of albumin corresponding to (or absence) of appropriate concentrations of other interventions (1 point); use of a real- real-life in vivo conditions or animal studies albumin, the involved pathways, and the suc- life model (1 point; here, we excluded studies with injection or oral administration of the cessful application of neutralizing interven- in specific genetic animal strains instead of molecules of interest or their precursors and tions. wild type, especially because results between the appropriate concentration of toxin and From our analysis, it became clear that the these two groups in our search were not al- serum albumin. In the absence of albumin quality of the retrieved publications was not ways conformed20,40,42,49; also, specificcell in vitro, the use of solute concentrations always the same. However, to the best of our types that are not necessarily representative had to be in accordance with reported free knowledge, there are no checklists available of real-life conditions were excluded [e.g., toxin concentrations, which is also detailed for evaluating quality or reliability of primary human umbilical vein endothelial cells], be- in Table 2. studies of biologic effects, the primary target cause the phenotype of venous endothelium

1904 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1897–1907, 2014 www.jasn.org BRIEF REVIEW does not always match that of the arterial cell, 6. Vanholder R, Schepers E, Pletinck A, 19. Chiu CA, Lu LF, Yu TH, Hung WC, Chung FM, which is where uremic vascular damage and Neirynck N, Glorieux G: An update on pro- Tsai IT, Yang CY, Hsu CC, Lu YC, Wang CP, Lee tein-bound uremic retention solutes. JRen YJ: Increased levels of total P-Cresylsulphate atherosclerosis essentially take place83–85); Nutr 22: 90–94, 2012 and indoxyl sulphate are associated with coro- fi – and nally; performance of dose response 7. Glorieux G, Vanholder R: New uremic toxins - nary artery disease in patients with diabetic ne- experiments but only if the other conditions which solutes should be removed? Contrib phropathy. Rev Diabet Stud 7: 275–284, 2010 were appropriate (in the presence of albumin Nephrol 168: 117–128, 2011 20. Duranton F, Cohen G, De Smet R, Rodriguez or in the free concentration range; 1 point). 8. Neirynck N, Glorieux G, Schepers E, Pletinck M, Jankowski J, Vanholder R, Argiles A; Euro- A, Dhondt A, Vanholder R: Review of protein- pean Uremic Toxin Work Group: Normal and Thus, a maximum quality score of 5 points bound toxins, possibility for blood purifica- pathologic concentrations of uremic toxins. J could be obtained. tion therapy. Blood Purif 35[Suppl 1]: 45–50, Am Soc Nephrol 23: 1258–1270, 2012 These quality scores were attributed in- 2013 21. Hammarlund-Udenaes M: Active-site con- dependently by two of the authors and 9. Neirynck N, Vanholder R, Schepers E, Eloot S, centrations of chemicals - are they a better matched and double-checked by a third Pletinck A, Glorieux G: An update on uremic predictor of effect than plasma/organ/tissue toxins. Int Urol Nephrol 45: 139–150, 2013 concentrations? Basic Clin Pharmacol Tox- author in case of disagreement. 10. Meijers BK, Claes K, Bammens B, de Loor H, icol 106: 215–220, 2010 Because there are substantial ethical and Viaene L, Verbeke K, Kuypers D, Vanrenterghem 22. Joerger M, Huitema AD, Boogerd W, van der practicalobstaclestoperformingdose–response Y, Evenepoel P: p-Cresol and cardiovascular risk Sande JJ, Schellens JH, Beijnen JH: Inter- experiments for in vivo animal studies, 1 extra in mild-to-moderate kidney disease. Clin J Am actions of serum albumin, valproic acid and – point was added to all publications containing Soc Nephrol 5: 1182 1189, 2010 carbamazepine with the pharmacokinetics of 11. De Smet R, Van Kaer J, Van Vlem B, De phenytoin in cancer patients. Basic Clin animal studies as long as the same publication Cubber A, Brunet P, Lameire N, Vanholder R: Pharmacol Toxicol 99: 133–140, 2006 – did not include in vitro dose response studies Toxicity of free p-cresol: A prospective and 23. Smith BS, Yogaratnam D, Levasseur-Franklin as well. cross-sectional analysis. Clin Chem 49: 470– KE, Forni A, Fong J: Introduction to drug 478, 2003 pharmacokinetics in the critically ill patient. 12. Meijers BK, Bammens B, De Moor B, Verbeke Chest 141: 1327–1336, 2012 K, Vanrenterghem Y, Evenepoel P: Free 24. Viaene L, Annaert P, de Loor H, Poesen R, ACKNOWLEDGMENTS p-cresol is associated with cardiovascular dis- Evenepoel P, Meijers B: Albumin is the main ease in hemodialysis patients. Kidney Int 73: plasma binding protein for indoxyl sulfate 1174–1180, 2008 and p-cresyl sulfate. Biopharm Drug Dispos We thank Mrs. Caroline Vinck for linguistic 13. Liabeuf S, Barreto DV, Barreto FC, Meert N, 34: 165–175, 2013 correction of this publication. Glorieux G, Schepers E, Temmar M, 25. Sirich T, Meyer TW: Indoxyl sulfate: Long Choukroun G, Vanholder R, Massy ZA; Eu- suspected but not yet proven guilty. Clin J ropean Uraemic Toxin Work Group (EUTox): Am Soc Nephrol 6: 3–4, 2011 Free p-cresylsulphate is a predictor of mor- 26. Dou L, Bertrand E, Cerini C, Faure V, Sampol DISCLOSURES tality in patients at different stages of chronic J, Vanholder R, Berland Y, Brunet P: The None. kidney disease. Nephrol Dial Transplant 25: uremic solutes p-cresol and indoxyl sulfate 1183–1191, 2010 inhibit endothelial proliferation and wound 14. Lin CJ, Wu CJ, Pan CF, Chen YC, Sun FJ, repair. Kidney Int 65: 442–451, 2004 REFERENCES Chen HH: Serum protein-bound uraemic 27. Dou L, Jourde-Chiche N, Faure V, Cerini C, toxins and clinical outcomes in haemodial- Berland Y, Dignat-George F, Brunet P: The ysis patients. Nephrol Dial Transplant 25: uremic solute indoxyl sulfate induces oxida- 1. Meyer TW, Hostetter TH: Uremia. N Engl J 3693–3700, 2010 tive stress in endothelial cells. JThromb Med 357: 1316–1325, 2007 15. Wang CP, Lu LF, Yu TH, Hung WC, Chiu CA, Haemost 5: 1302–1308, 2007 2. Vanholder R, Baurmeister U, Brunet P, Cohen Chung FM, Yeh LR, Chen HJ, Lee YJ, Houng 28. Faure V, Dou L, Sabatier F, Cerini C, Sampol G, Glorieux G, Jankowski J; European Uremic JY: Serum levels of total p-cresylsulphate are J, Berland Y, Brunet P, Dignat-George F: El- Toxin Work Group: A bench to bedside view associated with angiographic coronary ath- evation of circulating endothelial micro- of uremic toxins. J Am Soc Nephrol 19: 863– erosclerosis severity in stable angina patients particles in patients with chronic renal failure. 870, 2008 with early stage of renal failure. Atheroscle- JThrombHaemost4: 566–573, 2006 3. Vanholder R, Van Laecke S, Glorieux G: What rosis 211: 579–583, 2010 29. Itoh Y, Ezawa A, Kikuchi K, Tsuruta Y, Niwa T: is new in uremic toxicity? Pediatr Nephrol 23: 16. Wang CP, Lu LF, Yu TH, Hung WC, Chiu CA, Protein-bound uremic toxins in hemodialysis 1211–1221, 2008 Chung FM, Hsu CC, Lu YC, Lee YJ, Houng JY: patients measured by liquid chromatogra- 4. Vanholder R, De Smet R, Glorieux G, Argilés Associations among chronic kidney disease, phy/tandem mass spectrometry and their A, Baurmeister U, Brunet P, Clark W, Cohen high total p-cresylsulfate and major adverse effects on endothelial ROS production. Anal G, De Deyn PP, Deppisch R, Descamps- cardiac events. JNephrol26: 111–118, 2013 Bioanal Chem 403: 1841–1850, 2012 Latscha B, Henle T, Jörres A, Lemke HD, 17. Wu IW, Hsu KH, Hsu HJ, Lee CC, Sun CY, Tsai 30. Ito S, Osaka M, Higuchi Y, Nishijima F, Ishii H, Massy ZA, Passlick-Deetjen J, Rodriguez M, CJ, Wu MS: Serum free p-cresyl sulfate levels Yoshida M: Indoxyl sulfate induces leukocyte- Stegmayr B, Stenvinkel P, Tetta C, Wanner C, predict cardiovascular and all-cause mortality endothelial interactions through up-regulation Zidek W; European Uremic Toxin Work in elderly hemodialysis patients—a prospective of E-selectin. JBiolChem285: 38869–38875, Group (EUTox): Review on uremic toxins: Clas- cohort study. NephrolDialTransplant27: 1169– 2010 sification, concentration, and interindividual 1175, 2012 31. Schepers E, Meert N, Glorieux G, Goeman J, variability. Kidney Int 63: 1934–1943, 2003 18. Wu IW, Hsu KH, Lee CC, Sun CY, Hsu HJ, Tsai Van der Eycken J, Vanholder R: P-cresylsul- 5. Vanholder R, De Smet R, Lameire N: Protein- CJ, Tzen CY, Wang YC, Lin CY, Wu MS: p- phate, the main in vivo metabolite of p-cresol, bound uremic solutes: The forgotten toxins. Cresyl sulphate and indoxyl sulphate predict activates leucocyte free radical produc- Kidney Int Suppl 78[Suppl 78]: S266–S270, progression of chronic kidney disease. tion. Nephrol Dial Transplant 22: 592–596, 2001 Nephrol Dial Transplant 26: 938–947, 2011 2007

J Am Soc Nephrol 25: 1897–1907, 2014 Uremic Toxicity of IS and pCS 1905 BRIEF REVIEW www.jasn.org

32. Tsujimoto M, Higuchi K, Shima D, Yokota H, Fouque D, Soulage CO: p-Cresyl sulfate 56. Meijers BK, Van Kerckhoven S, Verbeke K, Furukubo T, Izumi S, Yamakawa T, Otagiri M, promotes insulin resistance associated with Dehaen W, Vanrenterghem Y, Hoylaerts MF, Hirata S, Takara K, Nishiguchi K: Inhibitory CKD. JAmSocNephrol24: 88–99, 2013 Evenepoel P: The uremic retention solute effects of uraemic toxins 3-indoxyl sulfate 45. Watanabe H, Miyamoto Y, Honda D, Tanaka p-cresyl sulfate and markers of endothelial and p-cresol on losartan metabolism in vitro. H, Wu Q, Endo M, Noguchi T, Kadowaki D, damage. Am J Kidney Dis 54: 891–901, 2009 J Pharm Pharmacol 62: 133–138, 2010 Ishima Y, Kotani S, Nakajima M, Kataoka K, 57. Vanholder R, Massy Z, Argiles A, Spasovski 33. Yu M, Kim YJ, Kang DH: Indoxyl sulfate- Kim-Mitsuyama S, Tanaka M, Fukagawa M, G, Verbeke F, Lameire N; European Uremic induced endothelial dysfunction in patients Otagiri M, Maruyama T: p-Cresyl sulfate Toxin Work Group: Chronic kidney disease with chronic kidney disease via an induction causes renal tubular cell damage by inducing as cause of cardiovascular morbidity and of oxidative stress. Clin J Am Soc Nephrol 6: oxidative stress by activation of NADPH oxi- mortality. Nephrol Dial Transplant 20: 1048– 30–39, 2011 dase. Kidney Int 83: 582–592, 2013 1056, 2005 34. Sun CY, Hsu HH, Wu MS: p-Cresol sulfate 46. Chitalia VC, Shivanna S, Martorell J, Balcells 58. Matsushita K, van der Velde M, Astor BC, and indoxyl sulfate induce similar cellular M, Bosch I, Kolandaivelu K, Edelman ER: Woodward M, Levey AS, de Jong PE, Coresh inflammatory gene expressions in cultured Uremic serum and solutes increase post- J, Gansevoort RT; Chronic Kidney Disease proximal renal tubular cells. Nephrol Dial vascular interventional thrombotic risk through Prognosis Consortium: Association of esti- Transplant 28: 70–78, 2013 altered stability of smooth muscle cell tissue mated glomerular filtration rate and albu- 35. Lekawanvijit S, Adrahtas A, Kelly DJ, Kompa factor. Circulation 127: 365–376, 2013 minuria with all-cause and cardiovascular AR, Wang BH, Krum H: Does indoxyl 47. Shimizu H, Yisireyili M, Higashiyama Y, mortality in general population cohorts: A sulfate, a uraemic toxin, have direct effects Nishijima F, Niwa T: Indoxyl sulfate upregu- collaborative meta-analysis. Lancet 375: on cardiac fibroblasts and myocytes? Eur lates renal expression of ICAM-1 via pro- 2073–2081, 2010 Heart J 31: 1771–1779, 2010 duction of ROS and activation of NF-kB and 59. Fagugli RM, De Smet R, Buoncristiani U, 36. Sun CY, Chang SC, Wu MS: Uremic toxins p53 in proximal tubular cells. Life Sci 92: 143– Lameire N, Vanholder R: Behavior of non- induce kidney fibrosis by activating intrarenal 148, 2013 protein-bound and protein-bound uremic renin-angiotensin-aldosterone system asso- 48. Tsujimoto M, Hatozaki D, Shima D, Yokota H, solutes during daily hemodialysis. Am J ciated epithelial-to-mesenchymal transition. Furukubo T, Izumi S, Yamakawa T, Minegaki Kidney Dis 40: 339–347, 2002 PLoS ONE 7: e34026, 2012 T, Nishiguchi K: Influence of serum in he- 60. Meert N, Eloot S, Schepers E, Lemke HD, 37. Shimizu H, Bolati D, Higashiyama Y, modialysis patients on the expression of in- Dhondt A, Glorieux G, Van Landschoot M, Nishijima F, Shimizu K, Niwa T: Indoxyl sul- testinal and hepatic transporters for the Waterloos MA, Vanholder R: Comparison of fate upregulates renal expression of MCP-1 excretion of pravastatin. Ther Apher Dial 16: removal capacity of two consecutive gen- via production of ROS and activation of NF- 580–587, 2012 erations of high-flux dialysers during differ- kB, p53, ERK, and JNK in proximal tubular 49. Yisireyili M, Shimizu H, Saito S, Enomoto A, ent treatment modalities. Nephrol Dial cells. Life Sci 90: 525–530, 2012 Nishijima F, Niwa T: Indoxyl sulfate promotes Transplant 26: 2624–2630, 2011 38. Bolati D, Shimizu H, Higashiyama Y, cardiac fibrosis with enhanced oxidative 61. Meert N, Eloot S, Waterloos MA, Van Nishijima F, Niwa T: Indoxyl sulfate induces stress in hypertensive rats. Life Sci 92: 1180– Landschoot M, Dhondt A, Glorieux G, epithelial-to-mesenchymal transition in rat 1185, 2013 Ledebo I, Vanholder R: Effective removal of kidneys and human proximal tubular cells. 50. Bolati D, Shimizu H, Yisireyili M, Nishijima F, protein-bound uraemic solutes by different Am J Nephrol 34: 318–323, 2011 Niwa T: Indoxyl sulfate, a uremic toxin, convective strategies: A prospective trial. 39. Sun CY, Chang SC, Wu MS: Suppression of downregulates renal expression of Nrf2 Nephrol Dial Transplant 24: 562–570, 2009 Klotho expression by protein-bound uremic through activation of NF-kB. BMC Nephrol 62. Chertow GM, Levin NW, Beck GJ, Depner toxins is associated with increased DNA 14: 56, 2013 TA, Eggers PW, Gassman JJ, Gorodetskaya I, methyltransferase expression and DNA hy- 51. YamamotoH,TsuruokaS,IokaT,AndoH,Ito Greene T, James S, Larive B, Lindsay RM, permethylation. Kidney Int 81: 640–650, C, Akimoto T, Fujimura A, Asano Y, Kusano E: Mehta RL, Miller B, Ornt DB, Rajagopalan S, 2012 Indoxyl sulfate stimulates proliferation of rat Rastogi A, Rocco MV, Schiller B, Sergeyeva 40. Adijiang A, Goto S, Uramoto S, Nishijima F, vascular smooth muscle cells. Kidney Int 69: O, Schulman G, Ting GO, Unruh ML, Star RA, Niwa T: Indoxyl sulphate promotes aortic 1780–1785, 2006 Kliger AS; FHN Trial Group: In-center he- calcification with expression of osteoblast- 52. Odamaki M, Kato A, Kumagai H, Hishida A: modialysis six times per week versus three specific proteins in hypertensive rats. Neph- Counter-regulatory effects of procalcitonin times per week. NEnglJMed363: 2287– rol Dial Transplant 23: 1892–1901, 2008 and indoxyl sulphate on net albumin secre- 2300, 2010 41. Adijiang A, Higuchi Y, Nishijima F, Shimizu H, tion by cultured rat hepatocytes. Nephrol 63. Maduell F, Moreso F, Pons M, Ramos R, Niwa T: Indoxyl sulfate, a uremic toxin, pro- Dial Transplant 19: 797–804, 2004 Mora-Macià J, Carreras J, Soler J, Torres F, motes cell senescence in aorta of hypertensive 53. Vanholder R, De Smet R, Waterloos MA, Van Campistol JM, Martinez-Castelao A; ESHOL rats. Biochem Biophys Res Commun 399: 637– Landschoot N, Vogeleere P, Hoste E, Ringoir Study Group: High-efficiency postdilution 641, 2010 S: Mechanisms of uremic inhibition of online hemodiafiltration reduces all-cause 42. Shimizu H, Saito S, Higashiyama Y, Nishijima phagocyte reactive species production: mortality in hemodialysis patients. JAmSoc F, Niwa T: CREB, NF-kB, and NADPH oxi- Characterization of the role of p-cresol. Kid- Nephrol 24: 487–497, 2013 dase coordinately upregulate indoxyl sul- ney Int 47: 510–517, 1995 64. Kikuchi K, Itoh Y, Tateoka R, Ezawa A, fate-induced angiotensinogen expression in 54. Zhu JZ, Zhang J, Yang K, Du R, Jing YJ, Lu L, Murakami K, Niwa T: Metabolomic search for proximal tubular cells. Am J Physiol Cell Zhang RY: P-cresol, but not p-cresylsulphate, uremic toxins as indicators of the effect of an Physiol 304: C685–C692, 2013 disrupts endothelial progenitor cell function oral sorbent AST-120 by liquid chromatog- 43. Shimizu H, Yisireyili M, Nishijima F, Niwa T: in vitro. Nephrol Dial Transplant 27: 4323– raphy/tandem mass spectrometry. J Chro- Indoxyl sulfate enhances p53-TGF-b1- 4330, 2012 matogr B Analyt Technol Biomed Life Sci Smad3 pathway in proximal tubular cells. Am 55. Viaene L, Evenepoel P, Meijers B, 878: 2997–3002, 2010 JNephrol37: 97–103, 2013 Vanderschueren D, Overbergh L, Mathieu C: 65.AkizawaT,AsanoY,MoritaS,WakitaT, 44. Koppe L, Pillon NJ, Vella RE, Croze ML, Uremia suppresses immune signal-induced Onishi Y, Fukuhara S, Gejyo F, Matsuo S, Pelletier CC, Chambert S, Massy Z, Glorieux CYP27B1 expression in human monocytes. Yorioka N, Kurokawa K; CAP-KD Study G, Vanholder R, Dugenet Y, Soula HA, Am J Nephrol 36: 497–508, 2012 Group: Effect of a carbonaceous oral

1906 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1897–1907, 2014 www.jasn.org BRIEF REVIEW

adsorbent on the progression of CKD: A and adsorption. Artif Organs 32: 214–219, 81.vanderWalWM,NoordzijM,DekkerFW, multicenter, randomized, controlled trial. 2008 Boeschoten EW, Krediet RT, Korevaar JC, Am J Kidney Dis 54: 459–467, 2009 75. Eloot S, Van Biesen W, Glorieux G, Neirynck Geskus RB; Netherlands Cooperative Study 66. Konishi K, Nakano S, Tsuda S, Nakagawa A, N, Dhondt A, Vanholder R: Does the ade- on the Adequacy of Dialysis Study Group Kigoshi T, Koya D: AST-120 (Kremezin) initi- quacy parameter Kt/V(urea) reﬂect uremic (NECOSAD): Full loss of residual renal func- ated in early stage chronic kidney disease toxin concentrations in hemodialysis pa- tion causes higher mortality in dialysis pa- stunts the progression of renal dysfunction in tients? PLoS ONE 8: e76838, 2013 tients; ﬁndings from a marginal structural type 2 diabetic subjects. Diabetes Res Clin 76. Vanholder R, Glorieux G, De Smet R, Lameire model. Nephrol Dial Transplant 26: 2978– Pract 81: 310–315, 2008 N; European Uremic Toxin Work Group: New 2983, 2011 67. 2013. Available at: http://www.medpageto- insights in uremic toxins. Kidney Int Suppl 84: 82. Barreto FC, Barreto DV, Liabeuf S, Meert N, day.com/MeetingCoverage/ASN/35737. S6–S10, 2003 Glorieux G, Temmar M, Choukroun G, Accessed April 18, 2014 77. Meert N, Schepers E, Glorieux G, Van Vanholder R, Massy ZA; European Uremic 68. Winchester JF, Hostetter TH, Meyer TW: Landschoot M, Goeman JL, Waterloos MA, Toxin Work Group (EUTox): Serum indoxyl p-Cresol sulfate: Further understanding of its Dhondt A, Van der Eycken J, Vanholder R: sulfate is associated with vascular disease cardiovascular disease potential in CKD. Am Novel method for simultaneous determination and mortality in chronic kidney disease pa- JKidneyDis54: 792–794, 2009 of p-cresylsulphate and p-cresylglucuronide: tients. Clin J Am Soc Nephrol 4: 1551–1558, 69. Dobre M, Meyer TW, Hostetter TH: Search- Clinical data and pathophysiological impli- 2009 ing for uremic toxins. Clin J Am Soc Nephrol cations. Nephrol Dial Transplant 27: 2388– 83. Szasz T, Thakali K, Fink GD, Watts SW: A 8: 322–327, 2013 2396, 2012 comparison of arteries and veins in oxidative 70. Eden J, Levit L, Berg A, Morton S, editors: 78. Vanholder R, Bammens B, de Loor H, stress: Producers, destroyers, function, and Finding What Works in Health Care: Standards Glorieux G, Meijers B, Schepers E, Massy Z, disease. Exp Biol Med (Maywood) 232: 27– for Systematic Reviews. Washington, D.C., The Evenepoel P: Warning: The unfortunate end 37, 2007 National Academies Press, 2011. Available at: of p-cresol as a uraemic toxin. Nephrol Dial 84. Mateo T, Naim Abu Nabah Y, Losada M, http://www.nap.edu/openbook.php?re- Transplant 26: 1464–1467, 2011 Estellés R, Company C, Bedrina B, Cerdá- cord_id=13059. Accessed April 18, 2014 79. Gondouin B, Cerini C, Dou L, Sallee M, Nicolás JM, Poole S, Jose PJ, Cortijo J, 71. Sirich TL, Luo FJ, Plummer NS, Hostetter TH, Duval-Sabatier A, Pletinck A, Calaf R, Lacroix Morcillo EJ, Sanz MJ: A critical role for Meyer TW: Selectively increasing the clear- R, Jourde-Chiche N, Poitevin S, Arnaud L, TNFalpha in the selective attachment of ance of protein-bound uremic solutes. Neph- Vanholder R, Brunet P, Dignat-George F, mononuclear leukocytes to angiotensin- rol Dial Transplant 27: 1574–1579, 2012 Burtey S: Indolic uremic solutes increase tis- II-stimulated arterioles. Blood 110: 1895– 72. Evenepoel P, Meijers BK, Bammens BR, sue factor production in endothelial cells by 1902, 2007 Verbeke K: Uremic toxins originating from the aryl hydrocarbon receptor pathway. 85. Johnson AR: Human pulmonary endothelial colonic microbial metabolism. Kidney Int Kidney Int 84: 733–744, 2013 cells in culture. Activities of cells from arteries Suppl 114: S12–S19, 2009 80. Mutsaers HA, Wilmer MJ, Reijnders D, and cells from veins. J Clin Invest 65: 841– 73. Schepers E, Glorieux G, Vanholder R: The Jansen J, van den Broek PH, Forkink M, 850, 1980 gut: The forgotten organ in uremia? Blood Schepers E, Glorieux G, Vanholder R, van Purif 29: 130–136, 2010 den Heuvel LP, Hoenderop JG, Masereeuw 74. Meijers BK, Weber V, Bammens B, Dehaen R: Uremic toxins inhibit renal metabolic ca- W, Verbeke K, Falkenhagen D, Evenepoel pacity through interference with glucur- This article contains supplemental material online P: Removal of the uremic retention solute onidation and mitochondrial respiration. at http://jasn.asnjournals.org/lookup/suppl/doi:10. p-cresol using fractionated plasma separation Biochim Biophys Acta 1832: 142–150, 2013 1681/ASN.2013101062/-/DCSupplemental.

J Am Soc Nephrol 25: 1897–1907, 2014 Uremic Toxicity of IS and pCS 1907 SUPPLEMENTAL DATA 1

Adijang et al Nephrol Dial Transplant - 200840

Model: study of aortic calcification (morphologic and immunohistochemic characterization of osteoblast related proteins (osteopontin, core binding factor 1 (Cbfal), alkaline phosphatase (ALP), osteocalcin), indoxylsulfate (IS) and organic anion transporter 1 and 3 (OAT 1 and 3); morphology of kidneys (glomerular area, mesangial area, MT-positive tubulointerstitial area, TGF-ßl-positive glomerular area and TGF-ßl-positive tubulointerstitial area).

Setting: in vivo in Dahl salt resistant rats and in salt sensitive Dahl rats, treated or not by administration PO of indoxylsulfate for 30 weeks (no IS treated salt resistant group)

Toxin: indoxylsulfate; PO administration to rats (200 mg/kg)

Concentration: 23.1 +/- 3.6 mg/L at the end of the administration period (30 weeks – lower before).

Albumin concentration: Not measured, as can be expected in rats.

Pathophysiologic changes:

Increase in aortic thickness and calcification. Increase in osteoblast markers, IS and OATs. Increase of glomerular area, mesangial area, MT-positive tubulointerstitial area, TGF-ßl- positive glomerular area and TGF-ßl-positive tubulointerstitial in IS treated Dahl rats (n=10 – biochemical results in only 6)

Note: No salt resistant rats with IS administration

Adijang et al Biochem Biophys Res Commun - 201041

Model: Study of cell senescence evaluated by immunohistochemistry of senescence-associated β- galactosidase (SA-β-gal), and senescence-related proteins such as p16INK4a, p21WAF1/CIP1, p53 and retinoblastoma protein (Rb).

Setting: in vivo in Dahl salt resistant rats and in salt sensitive Dahl rats, treated or not by administration PO of indoxylsulfate for 32 weeks.

Toxin: indoxylsulfate; PO administration to rats (200 mg/kg)

Concentration: In indoxylsulfate treated Salt sensitive Dahl rats, indoxylsulfate is continuously fluctuating between 15 and 20 mg/L between weeks 8 and 32, with the highest values around 20 exactly at these two moments. Values are somewhat lower in non-sensitive rats, between 14 and 9.

Albumin concentration: Not measured, as can be expected in rats.

Pathophysiologic changes:

Increase in β-galactosidase (SA-β-gal), 16INK4a, p21WAF1/CIP1, p53 and retinoblastoma protein (Rb) in IS-treated Dahl sensitive rats as compared to Dahl-sensitive rats who were not treated by IS (n=8). Also aortic caclicification and wall thickness more important than in non- IS treated rats.

Note: Dahl sensitive rats not receiving IS showed the same lesions but significanty less pronounced than the IS treated Dahl sensitive rats. Salt-resistant rats receiving indoxylsulfate showed no significant changes.

Bolati et al Am J Nephrol - 201138

Model: Study of epithelial to mesanchymal transition in rat kidneys and cultured proximal tubular cells by immunohistochemistry, reverse transcription PCR and immunoblotting for epithelial markers E-cadherin and zonula occludens-1 and mesenchymal marker α-SMA. In vivo tests on normal and Dahl sensitive hypertensive rats.

Setting: in vitro and in vivo

Toxin: indoxylsulfate

Concentration: in vivo 9.4 +/-1.3 mg/L (normal rats) and 18.9 +/- 2.6 (Dahl rats). In vitro 250 µM (thus too high).

Albumin concentration: in vivo albumin as in living rats; in vitro 10% FBS

Pathophysiologic changes:

Reduction of expression of E-cadherin and ZO-1 in both normal and Dahl rats treated by indoxylsulfate and enhanced expression of α-SMA in vivo (n= 6). Similar results in vitro (n= 3 to 5).

Note: -

Bolati et al BMC Nephrol 201350

Model: In both wild type and salt sensitive hypertensive Dahl rats, IS-treated vs non-IS treated equivalent; study on proximal tubular cells; study of anti-oxidant regulators: erythroid-derived 2 like 2 (Nrf2); heme oxygenase-1 (HO-1; NAD(P)H quinine oxide reductase (NQO1); and of 8-hydroxydeoxyguanosine (8OHdG), marker of ROS activation.

Setting: Dahl wild type, IS vs. no IS

Toxin: indoxylsulfate

Concentration: 9.4 mg/dL.

Albumin concentration: as in rats.

Pathophysiologic changes:

Only in IS treated wild type: - Suppression of anti-oxidant Nrf2 and HO-1 and activation of 8OHdG vs. non-IS treated. - According to the data for IS-treated Dahl sensitive rats no significant difference from non treated. Thus only wild type data relevant.

Note: Study contains in vitro data but not appropriate. Also animal study in a second model, Sprague- Dawley with CKD (thus not IS treated) with neutralization by AST-120

Chitalia et al Circulation - 201346

Model: assessment of de-endothelialized vascular smooth muscle cells for tissue factor expression, activity, stability and posttranslational modification. Changes were associated to clot formation.

Setting: Uremic serum and IS induce tissue factor generation; this effect is related to clot formation. TF breakdown through ubiqination and this process is slowed down by indoxylsulfate. Experiments occurred in a flow loop mimicking coronary circulation.

Toxin: indoxylsulfate

Concentration: IS 25 mg/L.

Albumin concentration: Presumably 4 g/L, amail confirmation by author.

Pathophysiologic changes:

Activation of tissue factor generation (n=3). Effect seen from 4 hours on, continued over 24 hours. Also increasing effect with increasing dose of IS. SMC injury further enhances the effect.

Note: Same effect as with uremic toxins also seen with uremic serum; effect could be reduced by anti-TF neutralizing antibody. Also similar effect for uric acid and indole acetic acid.

Dou et al KI - 200426

Model: endothelium (HUVEC)

Setting: in vitro

Toxin: indoxylsulfate

Concentration: 25-250 mg/L

Albumin concentration: 4%

Pathophysiologic changes:

Inhibited endothelial proliferation (5-bromo-2-deoxyuridine incorporation) (BrdU) (25-250 mg/L) (dose response) (n=4)

Inhibited endothelial wound repair after injury (videomicroscopy) (125-250 mg/L – if with albumin) (dose response) (n=?)

Note: similar results reported with p-cresol, but p-cresol is not present as such in the body {Martinez, 2005 339 /id;de, 2005 322 /id;Vanholder, 2011 321 /id}; no induction of endothelial apoptosis (annexin-V testing by flow cytometry). Effect less pronounced here in presence than in absence of albumin. 3

Dou et al J Thromb Haemost - 200727

Model: endothelium (HUVEC)

Setting: in vitro

Toxin: indoxylsulfate

Concentration: 125-250 mg/L

Albumin concentration: 4% (g/dL)

Pathophysiologic changes:

Induction of reactive oxygen species (ROS) measured by cytofluorometry (125-250 mg/L) (dose response) (n=10)

Inhibition of NAD(P)H-oxidase neutralized this pro-inflammatory effect whereas inhibitors of xanthine oxidase, NO-synthase and mitochondrial RO production had no effect

Indoxylsulfate strongly inhibited glutathione, one of the most important antioxidant systems in the cell. This test was not performed in the presence of albumin, though

Note: inhibition of the pro-oxidant effects by the antioxidants Vit C, vit E and N-acetylcysteine (NAC)

Faure et al J Thromb Haemost - 200628

Model: endothelium (HUVEC)

Setting: in vitro

Toxin: indoxylsulfate

Concentration: 256 mg/L

Albumin concentration: 4% (g/dL)

Pathophysiologic changes:

Increase in number of released endothelial microparticles as detected by enhanced annexin- V labeling measured by flow cytometry (n=8)

Note: -

Ito et al J Biol Chem - 201030

Model: leukocyte endothelial interaction

Setting: in vivo studies after indoxylsulfate administration in drinking water in 5/6 nephrectomized mice; controls: 5/6 nephrectomized without indoxylsulfate

Toxin: indoxylsulfate

Concentration: 15.7 mg/L

Albumin concentration: no serum albumin mentioned but should be the usual ones for in vivo conditions

Pathophysiologic changes:

Interaction of leukocytes with endothelium of femoral arteries is enhanced in intravital microscopy studies (n=5)

Quantitative real-time PCR analysis also showed an increase of mRNA expression of E- selectin and the NAD(P)H oxidase subunits p47phox and p22phox vs normal rats and for E- selectin also vs nephrectomized rats not treated with indoxylsulfate (n=5). No (significant) changes however for ICAM-1 and VCAM-1

Note: the study contains an in vitro setting where no appropriate albumin is added and an in vivo setting in mice, where by definition appropriate albumin should be present. Injection of anti-selectin- E antibody neutralizes the interaction. Concentration IS low as compared to human uremia.

Itoh et al Anal Bioanal Chem - 201229

Model: endothelium (HUVEC)

Setting: in vitro

Toxin: indoxylsulfate

Concentration: 29.9 and 57.2 mg/L

Albumin concentration: 4% (g/dL)

Pathophysiologic changes:

Increase in endothelial ROS production compared to control (no toxin) (n=3)

Note: p-cresylsulfate no effect in absence of albumin; concentration 37.1 and 109.1 mg/L

Koppe et al J Am Soc Nephrol - 201344

Model: evaluation of several aspects of insulin resistance in vivo in mice and in vitro on cultured adipocytes (modified fibroblasts) and C2C12 myotubes (equivalent to myocytes after modification of C2C12 myoblasts).

Setting: in vivo assessment in PCS treated vs control mice of glucose response to insulin, insulin- induced phosphorylation of PKB/Akt and activation of phosphorylation of ERK1/2 in gastrocnemius, adipose tissue size, ectopic lipid distribution; in vitro assessment in presence of PCS compared to control in adipocytes of lipogenesis and lipolysis, in myotubes of glucose uptake in response to insulin as well as elements of the insulin pathway by Western blotting and ERK1/2 phosphorilation .

Toxin: p-cresylsulfate.

Concentration: In vivo 5.01 +/- 1.59 mg/dl total and 0.82 +/- 0.24 free (total OK with clinical reality, free 2.5 times above mean uremic concentration). In vitro 40 mg/l (OK).

Albumin concentration: In vivo not measured, as can be expected in healthy mice (but protein binding in the expected range: 90.5 +/- 2.0 %). In vitro albumin 35 g/l BSA.

Pathophysiologic changes:

In vivo with PCS: decreased glucose response to insulin (n= 5-8), inhibited insulin-induced phosphorylation of PKB/Akt and activation of phosphorylation of ERK1/2 in gastrocnemius (n= 4-6), decreased adipose tissue size (n = 8), increased ectopic lipid distribution (n=8); in vitro in presence of PCS: in adipocytes decreased lipogenesis and increased lipolysis (n=4), in myotubes inhibition of glucose uptake in response to insulin together with changes in the following elements of the insulin pathway (Western blotting) - serine 473 phosphorylation of PKB/Akt, tyrosine-phosphorylation of IRS-1, serine phosphorylation (Ser 636) of IRS-1, p85 subunit of PI3K coimmunoprecipitation: (n=4- 5), and ERK1/2 phosphorilation (n=6).

Note: Probenecid prevents PCS-induced disruption of insulin signaling pathways in C2C12 myotubes; the prebiotic AXOS decreased p-cresylsulfate levels and corrected metabolic changes.

Lekawanvijit et al Eur Heart J - 201035

Model: Cardiac fibroblast collagen synthesis, cardiomyocyte proliferation (3H-proline and 3H leucine incorporation). In THP1 cells (substitute for monocytes – leukemia celline) production of cytokine TNF- α, IL-6, IL-1β mRNA expression estimated by PT-PCR. Furthermore, estimation of activity of MAPKinase and NFκB.

Setting: in vitro

Toxin: indoxylsulfate

Concentration: fibroblast collagen synthesis from 3µM on; on myocyte hypertrophy from 0.01 µM on (i.e. below the normal free concentration); TNF-α and IL-1β 100 µM (too high); IL-6 1 µM; MAPKinase and NFκB: 10 µM.

Albumin concentration: myocytes and fibroblasts in 10% FB); TMP-1 in 0.5% BSA.

Pathophysiologic changes:

IS had pro-fibrotic (n=9 in triplicate), pro-hypertrophic (n=5 in triplicate)and pro- inflammatory effects (n=3). Decreasing IS might help in decreasing cardio hypertrophy in CKD. In THP1 cells increased production of TNF- α, IL-6, IL-1β mRNA expression estimated by PT-PCR. Furthermore activation of MAPKinase and NFκB (n= 3).

Note: neutralization by inhibition of RWJ-67657 and U0126, linked to p38 and MEK1/2 pathways

Odamaki et al Nephrol Dial Tranplant - 200452

Model: In vitro study of albumin production by isolated hepatocytes.

Setting: this is a study of several different compounds. The indoxylsulfate experimenyts were performed in the presence and absence of albumin

Toxin: indoxylsulfate

Concentration: 50 and 100 mg/L.

Albumin concentration: 4 g/dL.

Pathophysiologic changes:

Increase in albumin production.

Note: is in fact not a toxic but a positive effect . No difference with and without albumin .

Schepers et al Nephrol Dial Transplant - 200731

Model: leukocyte function (whole blood)

Setting: in vitro (healthy volunteers)

Toxin: P-cresylsulfate

Concentration: 121.0 mg/L

Albumin concentration: no serum albumin mentioned but should be the usual ones for in vivo conditions

Pathophysiologic changes:

Increase of oxidative burst activity of baseline leukocytes (monocytes and lymphocytes) (n=10); granulocytes trend but not significant

Note: Concentration P-CS slightly higher than accepted maximum

Shimizu et al Life Sci - 201237

Model: Study in proximal tubular cells on the effect on the expression of Monocyte Chemotactic Protein-1 (MCP-1), a pro-inflammatory agent responsible for recruiting leukocytes into tubulointerstitial tissue. This study was performed in salt sensitive hypertensive Dahl and wild type rats to whom indoxylsulfate was administered; the rats were not made renal insufficient. Furthermore also in vitro study in tubular cells of the effect on activation of ERK, p38, JNK, NF-κB and p53. In addition, study of the effect in absence and presence of an antioxidant and inhibitors of the above mediators.

Setting: in vitro and in vivo

Toxin: indoxylsulfate 7

Concentration: in vivo, in normal rats given indoxylsulfate 9.4 +/- 1.4 mg/L, in Dahl rats 18.9 +/- 2.8 (appropriate thus). In vitro 250 µMol (thus too high).

Albumin concentration: in vivo, serum albumin as can be expected in rats, in vitro 10% FBS

Pathophysiologic changes:

Induction of expression of MCP-1 at relevant concentrations in indoxylsulfate treated rats, both normal (n= 5) and Dahl (n= 5). All other experiments at inappropriate concentrations.

Note: indoxylsulfate administered per os (200 mg/kg in drinking water).

Shimizu et al Am J Physiol Cell Physiol - 201342

Model: In vitro and in vivo study on renal tubular cells: expression of angiotensinogen and cAMP response element-binding protein (CREB).

Setting: in vivo in Dahl salt sensitive rats for expression of angiotensinogen.

Toxin: indoxylsulfate: PO administration to rats (indoxylsulfate concentration: 9.4 mg/L +/- 1.3.

Concentration: In vivo: indoxylsulfate concentration: 9.4 mg/L +/- 1.3. In vitro experiments (all the rest) were at too high concentrations (250 µM) for protein-poor medium (10% FBS).

Albumin concentration: Not measured, as can be expected in rats.

Pathophysiologic changes:

Increase in agiotensinogen expression (n=8).

Note: -

Shimizu et Am J Nephrol - 201343

Model: evaluation in proximal tubular cells of the p53-TGF-β1-Smad3 pathway, involved in inducing fibrosis.

Setting: In vivo study on proximal tubular cells of salt resistant and salt sensitive Dahl hypertensive rats. Also in vitro studies but with only 10% FBS and for that protein concentration too high indoxylsulfate (250 µM).

Toxin: indoxylsulfate; PO administration to rats (indoxylsulfate concentration: 9.4 mg/L +/- 1.3.

Concentration: In vivo: indoxylsulfate concentration: 9.4 mg/L +/- 1.3. In vitro experiments (all the rest) were at too high concentrations (250 µM) for protein-poor medium (10% FBS).

Albumin concentration: Not measured, as in rats.

Pathophysiologic changes:

Only morphologic changes (increase TGF-β1 and Smad3 positive area). No statistical data. (n=8). 8

Note: -

Shimizu et al Life Sci - 201347

Model: assessment of renal regulation of ICAM-1, playing a role in attracting monocytes and macrophages.

Setting: Next to human tubular cells (HK-2) studied in vitro, also in vivo studies were performed with Dahl rats receiving IS. Two strains were receiving IS: Dahl salt sensitive rats and wild type. Rats received IS (200 mg/kg) for 32 weeks.

Toxin: indoxylsulfate

Concentration: in vivo assessment: 9.4 mg/L in wild strain and 18.9 mg/L in Dahl hypertensive rats; in vitro: 250 µmol but without albumin, only Dulbecco. Thus only in vivo experiments relevant.

Albumin concentration: As can be expected in normal rats.

Pathophysiologic changes:

Assessment of ICAM-1 mRNA expression in both wild strain and salt sensitive rats treated with IS (n= 5) (also Dahl hypertensive rats not receiving IS showed higher ICAM-1). Dahl hypertensive treated with IS had higher expression than Dahl hypertensive and no IS.

Note: Inhibitors of NADPH oxidase, NF-κB and p53 suppressed the increase of ICAM-1 mRNA expression (but only in vitro).

Sun et al Nephrol Dial Transplant - 201234

Model: Inflammatory gene expression in cultured renal proximal tubular cells

Setting: in vitro

Toxin: indoxylsulfate and p-cresylsulfate

Concentration: IS and PCS 1 and 5 mg/L (concentrations thus correspond to free fraction)

Albumin concentration: Serum starved cells

Pathophysiologic changes:

Stimulation of a whole series of pro-inflammatory genes (see below) (n= ?)

Note: The major genes from cytokines in the functional networks that were activated were Tgfb1, fasl, IL6/15, IL15, Csf1/3 and Cxcl10; those from the triggered intracellular signals were Stats, Smads, Nfkb2, Ikbkb, Bcl2 and Bax. Functional networks linked to Tgfb1: Col4a5, Cxc10, Fasl, Stat1 and Ikbkb.

Sun et al PLOS One - 201236

Model: Study of activation of the renal renin–angiotensin–aldosterone system (RAAS) in proximal tubular cells and of renal tubular epithelial-to-mesenchymal transition (EMT) and of TGF-β, Snail, fibronection, α-Smooth muscle actin (α-SMA) and E-cadherin production as mechanisms in the induction of kidney fibrosis. Direct study of fibrosis by assessment of nephrosclerosis scores.

Setting: in vitro and in vivo

Toxin: indoxylsulfate and p-cresylsulfate

Concentration: in vitro studies were showing a positive effect from 1 mg/L on for both IS and PCS with a further dose responsive increase for 5 and 50 mg/L. The in vivo data (TGFβ, Snail, fibronectin α-SMA and E-cadherin) were collected after peritoneal injection in mice but plasmatic level was not specified.

Albumin concentration: in vitro PBS, % not specified; in vivo as in animals.

Pathophysiologic changes:

Activation of RAAS , EMT , TGF-β , Snail , TGF-β , fibronectin , and α-SMA and decrease of E- cadherin production . Increase of nephrosclerosis , all with both IS and PCS (n=8).

Note: Neutralization effect with losartan.

Sun et al Kidney Int - 201239

Model: study of kidney fibrosis, methylation of Klotho gene, Klotho expression.

Setting: in vivo in B-6 mice; in vitro on renal tubular cells

Toxin: indoxylsulfate and p-cresylsulfate; in vivo injected to mice

Concentration: for indoxylsulfate in mice treated by indoxylsulfate 8.55±0.37,mg/L, treated by p- cresylsulfate 5.61±0.60 mg/l (corresponding to a very moderate increase in concentration); for p- cresylsulfate in mice treated by pCS 1.82 +/-0.33 and in those treated by IS 0.66 =/- 0.04 which are in fact numbers corresponding to normal. In vitro 1, 5 and 50 mg/L ( 1 (pCS) and 5 (IS) OK for free fraction).

Albumin concentration: in vivo albumin as in living mice; in vitro no protein content specified.

Pathophysiologic changes:

In mice (n= 8) induction of kidney fibrosis, hypermethylation of the Klotho gene and decreased Klotho expression; inhibition of DNA methyltransferase isoform 1 caused a decrease of methylation and increased Klotho expression in vitro.

Note: In mice treated by one toxin the other toxin rises as well, in absence of renal failure.

Tsujimoto et al J Pharm Pharmacol - 201032

Model: metabolic clearance of losartan – measurement of the metabolite EXP-3174 using pooled human liver microsomes

Setting: in vitro

Toxin: indoxylsulfate

Concentration: Indoxylsulfate is diluted in uremic serum at 3-300 µmol/L and then diluted into the medium with microsomes 10% vv. The concentration in normal serum is within the acceptable range. As the sample is diluted 1/10 the protein binding will certainly decrease, but the final concentration of 20 µmol remains well within the range also for free solute. An effect is seen at 300 µmol/L, thus 30 µmol/L diluted (as a matter of fact a bit too high for entirely free, but there is still some protein).

Albumin concentration: Final albumin concentration low but total concentration within the acceptable range of free concentration.

Pathophysiologic changes:

Inhibition of losartan metabolism (dose response but only starting at the uremic range, n=3 or 4) (uremic range is the highest concentration of the dose-response)

Note: Studies also on other toxins; for the cresols, however, only p-cresol was studied (of course, very likely, some cresol reaches the liver)

Tsujimoto et al Ther Apher Dial - 201248

Model: Investigation of pump systems involved in the non-renal clearance of pravastatin, in view of its accumulation in renal failure, in spite of it not being cleared by the kidneys: assessment of the role of uremic toxins and uremic milieu on the expression of OATP2B1 (at play in intestinal uptake of Pravastatin) and MRP-2 (at play in intestinal/bile secretion of Pravastatin) in Caco-2 cells (intestinal cell line) and of OATP1B1 and OATP2B1 (at play in intestinal and hepatic uptake of pravastatin) in HEP3B cells (hepatic cell line).

Setting: This is a pure in vitro study using HBSS (Hank’s balanced salt solution) as medium. The latter contains no protein or albumin but experiments were done at low enough concentrations to be representative for free fraction.

Toxin: indoxylsulfate

Concentration: 20 µmol, which corresponds to free concentration.

Albumin concentration: 10% FCS.

Pathophysiologic changes:

Clear inhibition only in Caco-2 for OATP2B1 and MRP-2 (n=3). No consistent changes in HEP3B (only slight decrease for OATP2B1.

Note: inhibitory effect not only seen for IS but also for IAA, hippuric acid and cmpf. No significance of effect given. Two effects taken together seem contradictory.

Watanabe et al Kidney Int - 201345

Model: evaluation of toxicity on renal tubular cells via activation of production of radical oxygen species (ROS).

Setting: in vitro assessment of activity of NADPH-oxidase; upregulation mRNA inflammatory mediators and TGF-β1 (associated with renal fibrosis) – cell cultures in K-SFM (keratinocyte serum free medium; no albumin); in vivo administration p-CS to 5/6 nephrectomized rats for 4 weeks with assessment of tubular damage and enhancement oxidative stress.

Toxin: p-cresylsulfate.

Concentration: in vitro: effect seen only at 100 and 500 µmol/L (thus too high in absence of protein in medium); no significant effect at 10 µmol/L (which is a correct concentration without protein). In vivo (5/6 nephrectomized rats): animals evolved with or without CKD till week 16, then one randomly selected CKD group received PCS up till 20 weeks, the other not. At week 16 (in all rats) PCS was between 2.0 and 2.99 µmol/L; at week 20 (non-PCS treated rats) PCS was 4.52 +/- 5.29 µmol/L; in the PCS treated rats this value was 32.57 +/- 19.94 µmol/L. 32 µmol/L is in the low human uremic range; 5 µmol/L is in the low normal range.

Albumin concentration: Not measured, as in rats.

Pathophysiologic changes:

Only in vivo changes count: in the presence of added PCS, an increase of tubule degeneration, TGFβ1 activity, free radical production, and NADPH-oxidase activity were observed. (N=9)

Note: Effect neutralized by Knock-down of p22phox and p22 neutralized the effect pointing to the importance of NADPH-oxidase. Also suppression effect by administration of probenecid and NAC. Mechanism similar as for indoxylsulfate. After 4 weeks of PCS not only PCS levels but also Screa and urea were slightly but significantly higher (pointing to a possible broader general uremic effect in this group, but then still PCS-induced – although the differences on PCS are much more dramatic than those in urea or creatinine; changes just point to an impact on kidney function.

Yisireyili et al Life Sci - 201349

Model: In Dahl salt sensitive hypertensive rats, investigation of heart weight, left ventricular weight, diameter of cardiomyocytes, parameters of cardiac fibrosis [Mason-trichrome, TGFβ1, α-SMA, type 1 collagen] and of oxidative stress [NADPH-oxidase (Nox4), malondialdehyde (MDA, 8-hydroxydeoxyguanosine (8OHdG)]; finally also expression of anti-oxidant regulators, constituting nuclear factor [(erythroid-derived 2) like 2 (Nrf2); heme oxygenase-1 (HO-1).

Setting: In vivo study on Dahl hypertensive rats administered IS compared to same rats not administered IS

Toxin: indoxylsulfate

Concentration: 18.9 mg/L.

Albumin concentration: as in rats.

Pathophysiologic changes:

In IS-treated rats: - Increase in diameter cardiomyocytes - Increased fibrotic area; increased staining for TGFβ1, SMA, type-1 collagen, - Increase of Nox4, 8-OHdG and MDA - Decreased staining for Nrf2 and HO-1

Note: no effect IS in wild type rat; study contains no in vitro arm

Yamamoto et al Kidney Int - 200651

Model: In vitro study of the effect of indoxylsulfate on smooth muscle cell proliferation.

Setting: although most experiments are in the absence of albumin, some have been performed in the presence of albumin

Toxin: indoxylsulfate

Concentration: 250 and 500 µM.

Albumin concentration: 4 g/dL.

Pathophysiologic changes:

Increased smooth muscle cell proliferation.

Note: data with and without albumin are expressed differently (fold-change vs. absolute values) making comparison difficult. Fold changes seem at least as prominent in the presence as in the absence of albumin.

Yu et al Clin J Am Soc Nephrol - 201133

Model: Proliferation, senescence and production of nitric oxide and oxygen free radicals by HUVEC as a model of endothelial damage; cell proliferation: incorporation of 3H-thymidine and by direct cell counting; cell senescence: in situ staining for SA-β-galactosidase

Setting: in vitro

Toxin: indoxylsulfate

Concentration: 1.25 to 125 mg/L; effect from 2.5 mg/L on

Albumin concentration: No mention of albumin

Pathophysiologic changes:

Inhibition of proliferation; enhancement of senescence; decrease NO production; increase ROS production (for 2.5 mg/L from the 3d hour on) (n= 4 in duplicate)

Note: This in vitro study is associated with an observational clinical study showing an in vivo improvement of endothelial function as demonstrated by flow-mediated endothelium-dependent vasodilatation (FMD) after 24 weeks of AST-120. This study arm was observational. There were no controls. All 40 patients received AST-120. Correction of in vitro effects by probenecid and the antioxidants NAC, rotenone and apocynin.

SUPPLEMENTAL DATA 2

Total quality score Dou 2004, KI26 2 Odamaki 2004, NDT52 2 Faure 2006, J Throm Haemost28 1 Yamamoto 2006, KI51 3 Dou 2007, J Throm Haemost27 4 Schepers 2007, NDT31 2 Adijang 2008, NDT40 3=2+1 Ito 2010, J Biol Chem30 4=3+1 Tsujimoto 2010, J Pharm Pharmacol32 1 Lekawanvijit 2010, Eur Heart J35 5 Adijang 2010, Biochem Biophys Res 3=2+1 Commun41 Yu 2011, cJASN33 3 Bolati 2011, Am J Nephrol38 4=3+1 Itoh 2012, Ann Bioanal Chem29 0 Sun 2013, NDT34 2 Sun 2012, Plos One36 5 Shimizu 2012, Life Sci37 2=1+1 Sun 2012, KI39 5 Watanabe 2013, KI45 5=4+1 Tsujimoto 2012, Ther Apher Dial48 1 Shimizu 2013, Am J Physiol Cell42 2=1+1 Shimizu 2013, Am J Nephrol43 3=2+1 Koppe 2013, JASN44 5=4+1 Chitalia 2013, Circulation46 4 Shimizu 2013, Life Sci47 3=2+1 Yisireyili 2013, Life Sci49 4=3+1 Bolati 2013, BMC Nephrol50 4=3+1