Indoxyl Sulfate and P-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance

Home , Destrin, ENAH (gene), Fibrinogen beta chain, GM2A, Indoxyl sulfate, IRGM

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Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calciﬁcation and Associate with Glucose Intolerance

Britt Opdebeeck ,1 Stuart Maudsley,2,3 Abdelkrim Azmi,3 Annelies De Maré,1 Wout De Leger,4 Bjorn Meijers,5,6 Anja Verhulst,1 Pieter Evenepoel,5,6 Patrick C. D’Haese,1 and Ellen Neven1

1Laboratory of Pathophysiology, Department of Biomedical Sciences, 2Receptor Biology Lab, Department of Biomedical Sciences, and 3Translational Neurobiology Group, Flanders Institute of Biotechnology Center for Molecular Neurology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium; 4Division of Molecular Design and Synthesis, Department of Chemistry and 6Laboratory of Nephrology, Department of Immunology and Microbiology, Catholic University of Leuven, Leuven, Belgium; and 5Division of Internal Medicine, Nephrology, University Hospitals Leuven, Leuven, Belgium

ABSTRACT Background Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. Methods To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. Results Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucome- tabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. Conclusions In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis.

J Am Soc Nephrol 30: 751–766, 2019. doi: https://doi.org/10.1681/ASN.2018060609

Vascular calcification refers to the pathologic deposi- tion of calcium-phosphate crystals in the arterial wall. This pathologic event represents a major clinical is- Received June 12, 2018. Accepted February 13, 2019. fi sue, because it signi cantly contributes to the high Published online ahead of print. Publication date available at cardiovascular morbidity and mortality observed www.jasn.org. 1–3 in patients with CKD. Except for phosphate and Correspondence: Dr. Patrick C. D’Haese, Laboratory of Pathophys- inflammation, other uremia-related factors may con- iology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, tribute to vascular calcification in patients with CKD. B-2610 Wilrijk, Belgium. Email: [email protected] Among uremic retention solutes, indoxyl sulfate (IS) Copyright © 2019 by the American Society of Nephrology

J Am Soc Nephrol 30: 751–766, 2019 ISSN : 1046-6673/3005-751 751 BASIC RESEARCH www.jasn.org and p-cresyl sulfate (PCS) have been associated with Significance Statement cardiovascular disease and mortality.4–6 Both uremic toxins originate from protein fermentation in the intestine and ac- Vascular calcification contributes to high cardiovascular mortality in cumulate in patients with CKD due to impaired renal clear- patients with CKD. Although research findings have suggested an ance and increased intestinal production. p-Cresol and indole association between the uremic toxins indoxyl sulfate and p-cresyl sulfate and cardiovascular disease, direct evidence has been lacking. are generated by the intestinal microbial breakdown of tyro- In this study, the authors demonstrate in a rat model of CKD that sine/phenylalanine and tryptophan, respectively. After intes- continuous exposure to indoxyl sulfate or p-cresyl sulfate promotes tinal absorption, these solutes undergo phase 2 reactions, moderate to severe calcification in the aorta and peripheral vessels. predominantly by conjugation with sulfate.7,8 In vitro exper- Activation of inflammation and coagulation pathways in the arterial fi iments have demonstrated that both uremic toxins can induce wall plays a pivotal role in toxin-induced calci cation and strongly associates with hyperglycemia and insulin resistance. These findings 9–11 endothelial dysfunction. IS also induces vascular smooth provide etiologic evidence for indoxyl sulfate and p-cresyl sulfate as muscle cell proliferation and upregulation of bone-specific major contributors to vascular calcification and suggest new avenues proteins in these cells,12,13 which is a hallmark of the calcifi- for identifying novel therapeutic targets to prevent or treat calcification process in the vessel wall.14 These results were further cation in the vessel wall of patients with CKD. supported by the work by Adijiang et al.,15 which reported that exposure of hypertensive rats to IS promotes aortic cal- Animal Study #1 cification with the expression of bone-specific markers, such To induce CKD, 42 male Wistar rats (225–250 g; Iffa Credo, as runx2, alkaline phosphatase, and osteopontin.15 Addition- Brussels, Belgium) were exposed to a 10-day adenine sulfate ally, treatment with AST-120, an oral spherical activated (Acros, Geel, Belgium) treatment via daily oral gavage carbon adsorbent that reduces the serum concentrations of (600 mg/kg per day). CKD rats fed a phosphate-enriched uremic toxins by limiting the intestinal absorption of gener- diet (1.2% Pi and 1.06% Ca; SSNIFF Spezialdiäten, Soest, ated microbial metabolites, went along with beneficial effects Germany) were randomly assigned to three treatment groups: on the cardiovascular system, including attenuation of cardiac (1) vehicle (n=14), (2) 150 mg/kg IS (IS potassium salt; Sigma- hypertrophy and fibrosis11,16 and a decline in vascular stiff- Aldrich, St. Louis, MO; n=14), or (3) 150 mg/kg PCS (De- ness17 as well as reduced aortic calcification in predialysis partment of Chemistry, KU Leuven, Leuven, Belgium; n=14). patients with CKD.18 These findings suggest that protein- From the start of CKD induction onward until week 2, either bound uremic retention solutes, which are only poorly IS or PCS was administered via the drinking water, which cleared by conventional dialysis, may play an important role contained either of these toxins at a concentration corre- in vascular calcification, and thus, they may contribute to the sponding to a daily intake of 150 mg/kg, followed by daily high cardiovascular disease burden in patients with end stage oral gavage of vehicle, IS (150 mg/kg), or PCS (150 mg/kg) renal failure. However, a recent clinical study found no associa- from week 3 onward until euthanasia at week 7. Before the tion of predialysis total PCS and IS with cardiac death, sudden start of the study, at week 5, and at euthanasia, animals were cardiac death, and first cardiovascular event.19 As commented by placed in metabolic cages to collect 24-hour urine samples Evenepoel et al.,20 these conflicting results point out that it is still followed by blood sampling. At week 3, blood sampling was unclear whether these uremic toxins are harmful to the vascu- also performed. To enable measurement of dynamic bone lature. As such, experimental studies are required to explore the parameters, all animals were labeled with tetracycline role of these uremic retention solutes in CKD-related vascular (30 mg/kg intravenously) 7 days before euthanasia and deme- complications. Here, we aim to provide evidence for an etiologic clocycline (25 mg/kg intravenously) 3 days before euthanasia. role for IS and PCS as major contributors to the onset and de- Mortality was noted in the three study groups due to CKD; velopment of calcification in the vessel wall of patients with CKD however, exposure to IS and particularly, PCS led to substan- by use of a CKD rat model. Using a high-dimensionality tially higher mortality rates during the course of the study. unbiased proteomic approach, we have also investigated the po- Before the planned euthanasia, four vehicle-, six IS-, and eight tential complex molecular signaling events associated with PCS-exposed CKD rats died. Despite this increased mortality toxin-mediated arterial calcification. rate, our experimental animal cohorts remained sufficiently large to conclude our protocols effectively.

METHODS Animal Study #2 To explore early molecular changes in the arterial wall caused Animal Experiments by the toxins IS and PCS before effective induction of vascular All animal experiments were performed in accordance with injury and calciﬁcation, an additional study was performed the National Institutes of Health Guide for the Care and Use of including animals that were exposed to these toxins for Laboratory Animals 85–23 (1996) and approved by the Univer- 4 days. To induce CKD, 12 male Wistar rats were exposed to sity of Antwerp Ethics Committee (permit number 2012–13). adenine sulfate via daily oral gavage (600 mg/kg per day) for Animals were housed two per cage, exposed to 12-hour light/ 4 days. CKD rats were fed a phosphate-enriched diet (1.2% dark cycles, and had ad libitum access to food and water. Pi and 1.06% Ca), and they were randomly assigned to three

752 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH treatment groups: (1)vehicle(n=4), (2)150mg/kgIS(n=4), of each artery was measured in the digestion liquid with flame or (3)150mg/kgPCS(n=4) administered via the drinking atomic absorption spectrometry. water until euthanasia at day 4. At euthanasia, rats of both animal studies were exsanguinated Analyses of Bone Parameters through the retro-orbital plexus after anesthesia with 60 mg/kg At euthanasia, the left tibia was fixed in 70% ethanol over- sodium pentobarbital (Nembutal; Ceva Santé Animale) via in- night at 4°C, dehydrated, and embedded in 100% methyl- traperitoneal injection. methacrylate (Merck, Hohenbrunn, Germany) for bone histomorphometry. For analysis of the static bone parame- Analyses of Biochemical Parameters ters, 5-mm sections of the left tibia were Goldner stained for Toassess renal function, creatinine was measured in serum and histomorphometric analysis of the proximal metaphysis urine samples according to the Jaffé method. Total serum and with AxioVision Release 4.5 software. By using the formulas urine phosphorus levels were analyzed with the Ecoline of Parfitt,21 the following static bone parameters were ob- S Phosphate kit (Diasys, Holzheim, Germany), and serum tained: bone area and mineralized area on total tissue area, and urinary calcium levels were determined with flame atomic osteoid width, osteoid area on total bone area, osteoid pe- absorption spectrometry (Perkin-Elmer, Wellesley, MA) after rimeter, eroded perimeter, osteoblast perimeter, and osteo- appropriate dilution in 0.1% La(NO3)3 to eliminate chemical clast perimeter on total bone perimeter. For the analysis of interference. Serum immunoreactive parathyroid hormone dynamic bone parameters, tetracycline labels were visual- (PTH) levels were measured using ELISA (Immutopics, San ized on 10-mm unstained tibia sections with fluorescence Clemente, CA). Serum IS and PCS levels were determined by microscopy, and the length and distance between both labels liquid chromatography-tandem mass spectrometry (MS). Be- were measured, enabling us to calculate additional dynamic fore the start of the study and at euthanasia, blood glucose bone parameters according to Dempster and colleagues.21 levels were measured using the GlucoMen Lx Plus+ automated whole-blood glucometer. MS and Quantitative Proteomics of Aortic Samples Toelucidatethemolecularsignaling pathwaysresponsiblefor Quantification of Adenine Crystals in the Kidney the toxin-induced calcification in the aorta, an unbiased At euthanasia, one slice of the left kidney was fixed in neutral quantitative proteomic approach using iTRAQ (isobaric buffered formalin for 4 hours and embedded in a paraffin mass tag labeling for relative and absolute quantitation) la- block. Four-micrometer sections were stained with periodic beling and MS was applied to simultaneously identify and acid–Schiff to quantify renal adenine crystals. The percentage quantify the proteins that are differentially up- or downre- of the area covered by adenine crystals was determined using gulated in aortic samples of CKD animals exposed to IS (n=5 Axiovision image analysis software (Release 4.5; Carl Zeiss, for animal study #1; n=3 for animal study #2) or PCS (n=5 Oberkochen, Germany) by calculating the ratio of the number for animal study #1; n=3 for animal study #2) versus vehicle of pixels taken by the crystals to the number of pixels taken by (n=4 for animal study #1; n=3 for animal study #2). The the total renal tissue. aorta samples were ground completely in the protein extrac- tion buffer (8 M urea, 2 M thiourea, and 0.1% SDS in 50 mM Analyses of Vascular Calcification triethylammonium bicarbonate solution). The concentra- Vascular calcification was analyzed histomorphometrically on tions of the proteins extracted from the aorta were quanti- Von Kossa–stained sections. At euthanasia, the thoracic aorta fied using the RCDC kit (Bio-Rad, Hercules, CA). Equal was fixed in neutral buffered formalin for 90 minutes and cut amounts of proteins from each sample were reduced and into sections of 2–3 mm. These sections were embedded up- alkylated by tris-2-carboxyethyl phosphine and 5-methyl- right in a paraffin block, and 4-mm sections were stained for methanethiosulfate, respectively, before trypsin digestion. calcification with the Von Kossa method and counterstained The resulting peptides from each sample were labeled using with hematoxylin and eosin. The percentage calcified area was iTRAQ reagents (Sciex, Framingham, MA) following calculated using Axiovision image analysis software (Release the manufacturer’s instructions. To improve LC-MS/MS 4.5), in which two color separation thresholds measure the proteome coverage, samples were subjected to a 2D-LC frac- total tissue area and the Von Kossa–positive area. After sum- tionation system (Dionex ULTIMATE 3000; ThermoScien- ming both absolute areas, the percentage of calcified area was tific, Waltham, MA). The mixed peptides were first calculated as the ratio of the Von Kossa–positive area to the fractionated on a strong cationic exchange chromatography total tissue area. Calcification in the aorta and smaller arteries polysulfoethyl aspartamide column (13150 mm; Dionex) was also quantified by calcium bulk analysis. For the purpose and then, separated on a nano-LC C18 column (200 Å, of this, the proximal section of the abdominal aorta and the 2 mm, 75 mm325cm;Dionex).Thenano-LCiscoupled left carotid and femoral arteries were isolated and directly online to a QExactive-Plus Orbitrap (ThermoScientific) weighed on a precision balance. The arterial samples were mass spectrometer. The nano-LC eluents were infused digested in 65% HNO3 at 60°C overnight, after which the to the Orbitrap mass spectrometer with a capillary at calcium content (milligrams of calcium per gram wet tissue) 1.7 kV on a nano-ESI source at a flow rate of 300 nl/min.

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Data-dependent acquisition in positive ion mode was per- (Rn00801649_g1), klotho (Rn00580123_m1), TGF-b formed for a selected mass range of 350–1800 m/z at MS1 (Rn00572010_m1), TNF-a (Rn00562055_m1), and vascular level (140,000 resolution) and MS2 level (17,500 resolution). cell adhesion molecule 1 (Rn00563627_m1) in the left kidney. The raw data were analyzed by Proteome Discoverer 2.1 Software (ThermoScientific) using Sequest HT as the search Statistical Analyses engine against the Rattus norvegicus (UP000002494) Uni- Statistical comparisons were made by nonparametric testing Prot/SwissProt database (2017) with threshold of confidence (SPSS 23.0 software; IBM Corp., Armonk, NY). To investigate above 99% (false discovery rate ,1%). The list of identified the statistical difference between groups at one timepoint, a proteins containing iTRAQ ratios of expression levels over Kruskal–Wallis test followed by Mann–Whitney U test was control samples was generated. The MS proteomics data applied. Comparisons between timepoints within each group have been deposited in the ProteomeXchange Consortium were performed with a Friedman related samples test fol- via the PRIDE22 partner repository with the dataset identi- lowed by a Wilcoxon signed rank test. Bonferroni correction fier PXD012582. was assessed when more than two groups were compared. To investigate the relationship between aortic calcification and Bioinformatic Analyses biochemical parameters, a Spearman rho univariate correla- Proteins identified according to the statistical MS cutoffs de- tion analysis was performed. Representative data are presen- scribed previously were then subsequently used for additional ted as mean6SEM and considered significant with a P value bioinformatics analyses. To identify the significantly altered #0.05. proteins (i.e., proteins differentially expressed due to IS and PCS exposure compared with vehicle exposure), raw iTRAQ ratios (IS to vehicle or PCS to vehicle) were first log2 trans- RESULTS formed. After log2 ratio transformation, differentially expressed protein lists were created by identifying only proteins Animal Study #1 , that possessed log2-transformed iTRAQ ratios 2 SD (P 0.05) IS or PCS Exposure Does Not Aggravate Chronic Renal Failure from the calculated mean background expression variation Chronic renal failure, mimicking CKD, was effectively induced level. Significant differentially expressed protein lists (compris- in our experimental cohorts via adenine dosing as evidenced ing proteins elevated or reduced in their expression in response by a significant increase in serum creatinine levels (Figure 1A) to IS or PCS) were then used for additional bioinformatics and a decline in creatinine clearance (Figure 1B). Vehicle- deconvolution using diverse informatics platforms, including exposed CKD rats demonstrated an increase in serum phospho- Ingenuity Pathway Analysis (IPA; Canonical Signaling Pathway rus levels, reflecting a moderate state of hyperphosphatemia and Upstream Regulator applications; https://www.qiagen- (Figure 1C) and a nonsignificant trend toward hypocalcemia bioinformatics.com/products/ingenuity-pathway-analysis/), (Figure 1D)—both characteristic facets of CKD. Comparable GeneIndexer23,24 (https://geneindexer.com/), WebGestalt,25 creatinine, phosphorus, and calcium values were also observed and VennPlex26 (https://www.irp.nia.nih.gov/bioinformatics/ in CKD rats exposed to PCS. IS-exposed CKD rats exhibited vennplex.html). significantly lower serum creatinine and phosphorous levels at weeks 5 and 7 as well as significantly higher serum calcium Analyses of Molecular Gene Expression in the Aorta concentrations at weeks 3 and 7 compared with vehicle- and Kidney exposedCKDrats(Figure1,A,C,andD).Furthermore,al- The mRNA transcript expression of glyceraldehyde-3- terations in calcium and phosphorus homeostasis were in line phosphate dehydrogenase (GAPDH; Rn99999916_s1) and with a significant increase of serum PTH levels in CKD rats GLUT1 (Rn01417099_m1) was determined in the distal part exposed to vehicle (14,40762283 pg/ml; P=0.02), IS (79326 of the abdominal aorta. Total mRNA was extracted using the 2432 pg/ml), and PCS (10,92363415 pg/ml; P=0.03) com- RNeasy Fibrous Tissue mini kit (Qiagen, Hilden, Germany) pared with historical control values of rats with normal renal and reverse transcribed to cDNA by the High Capacity cDNA function (938678 pg/ml). However, no differences in serum PTH archive kit (Applied Biosystems, Foster City, CA). Real-time levels between CKD rats exposed to vehicle, IS, or PCS were ob- PCR with an ABI Prism 7000 Sequence Detection System served at the end of the study. (Applied Biosystems) on the basis of TaqMan fluorescence To rule out the possibility of the lower serum creatinine methodology was used for mRNA quantification. TaqMan levels in the IS group being due to interference of the toxin probe and primers were purchased as TaqMan gene expres- with the adenine metabolism and thus, with the renal 2,8- sion assays on demand from Applied Biosystems. Each gene dihydroxyadenine crystal formation, we also quantified the was tested in triplicate and normalized to the expression of renal crystal content in our rats. Our results demonstrate no the housekeeping transcript GAPDH. differences in the percentages of renal tissue area covered by A similar experimental protocol was performed to inves- 2,8-dihydroxyadenine crystals in CKD rats exposed to IS tigate the mRNA transcript expression of GAPDH (3.41%60.31%), PCS (2.97%60.24%), or vehicle (3.10%6 (Rn99999916_s1), IL-1b (Rn00580432_m1), collagen I 0.15%).

754 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH

A B ** * * * * # ### #### ## 5 2.5 p=0.056 p=0.056 Vehicle IS PCS 4 2.0

3 1.5

2 1.0 Serum creatinine (mg/dl)

1 Creatinine clearance (ml/min) 0.5

0 0.0 0357 0357 Week Week C * ***D # ## 22 18

20 16 18 14 16 12 14 12 10

10 8 8 6

6 Serum calcium (mg/dl) Serum phosphorus (mg/dl) 4 4 2 2 0 0 0357 0357 Week Week

Figure 1. Indoxyl sulfate (IS) or p-cresyl sulfate (PCS) exposure does not aggravate chronic renal failure. (A) Serum creatinine, (B) creatinine clearance, (C) serum phosphorus, and (D) serum calcium levels. Data are presented as mean6SEM. *P,0.05 versus vehicle; #P,0.05 versus week 0.

IS and PCS Exposure Does Not Deteriorate Renal Fibrosis and significantly decreased mRNA expression of the fibrosis mark- Inflammation ers collagen I and TGF-b compared with vehicle-exposed To investigate whether exposure to uremic toxins modulates CKD rats (Figure 2, D and E). In addition, compared with renal fibrosis and inflammation, a characteristic feature of vehicle-exposed CKD rats, no difference in klotho mRNA ex- adenine-induced CKD, mRNA expression profiles of inflam- pression was observed for either IS- or PCS-exposed CKD rats matory and fibrotic markers were studied in the kidney. (Figure 2F). Although a trend toward lower mRNA expression of the inflammatory genes IL-1b,TNF-a, and vascular cell adhesion Time-Averaged Serum Concentrations of IS or PCS molecule 1 was detected in CKD rats exposed to IS or PCS On the basis of the IS and PCS serum levels at weeks 0, 3, 5, and compared with vehicle-exposed CKD rats, no significant dif- 7, time-averaged concentrations of IS and PCS were calculated. ferences between the study groups were found (Figure 2, CKD rats exposed to IS showed a significant increase in time- A–C). Nevertheless, CKD rats exposed to IS demonstrated a averaged concentration of IS (69.465.7 mM; P,0.001)

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ABC IL1-beta TNF-alpha VCAM-1 1.8 1.8 1.8

1.6 1.6 1.6

1.4 1.4 1.4

1.2 1.2 1.2

1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0 Vehicle IS PCS Vehicle IS PCS Vehicle IS PCS

D E F Collagen I TGF-beta Klotho 1.8 1.8 1.8 ** 1.6 1.6 1.6

1.4 1.4 1.4 Kidney gene expression (mRNA/GAPDH)

1.2 1.2 1.2

1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0 Vehicle IS PCS Vehicle IS PCS Vehicle IS PCS

Figure 2. Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) exposure does not deteriorate renal inflammation and fibrosis. Total mRNA expression of (A–C) inflammatory genes, (D and E) fibrosis-related genes, and (F) klotho gene in the kidney of IS- or PCS-exposed CKD rats versus vehicle-exposed CKD rats. Data are presented as mean6SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VCAM-1, vascular cell adhesion molecule 1. *P,0.05 versus vehicle. compared with vehicle-exposed CKD rats (35.363.6 mM). calcification, the effect of both uremic toxins on the glucose Additionally, the time-averaged concentration of PCS metabolism was investigated. CKD rats demonstrated sig- was also significantly elevated in PCS-exposed CKD rats nificantly increased serum glucose concentrations after (151.0640.7 mM; P=0.002) compared with vehicle-exposed 7 weeks of IS or PCS exposure, which was not the case in CKD rats (4.860.8 mM). vehicle-exposed CKD rats (Figure 3A). Furthermore, CKD rats exposed to IS and PCS had a lower expression of IS and PCS Exposure Influences Glucose Metabolism GLUT1, a major glucose uptake transporter, in the aorta— Because circulating glucose and altered insulin recep- this attenuation achieved significance in the PCS group tor (INSR) activities have been associated with vascular (Figure 3B).

756 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH

A B #* #** 250 3.0 Vehicle IS

PCS 2.5 200

2.0

150

1.5

100

Serum glucose (mg/dl) 1.0

50 Glut1 gene expression (mRNA/GAPDH) 0.5

0 0.0 07 Vehicle IS PCS Week

Figure 3. Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) exposure elevate resting glucose and decrease glucose transporter 1 (GLUT1) transcript expression in the aorta. (A) Serum glucose levels and (B) mRNA expression of GLUT1 in vehicle-, IS-, and PCS-exposed rats. Data are presented as mean6SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P,0.05 versus vehicle; #P,0.05 versus week 0.

IS and PCS Promote Calcification in the Aorta and Peripheral PCS also affected bone histology. Table 1 represents the mea- Arteries sured values of the static and dynamic bone parameters of The effect of IS and PCS on vascular calcification in CKD rats was CKD rats exposed to vehicle, IS, or PCS. As evidenced from investigated by determining the calcium content in several arter- various previous reports,28–30 adenine-induced CKD in the rat ies. Both IS and PCS exposure significantly augmented aortic goes along with the development of high-bone turnover dis- calcification. Aortic calcium content in both IS- and PCS-exposed ease as indicated by increased osteoid, eroded zones, and ele- CKD rats was tenfold higher compared with vehicle-exposed ones vated osteoblast as well as osteoclast numbers. In this study, (Figure 4A). Significant increases in calcium content were also CKD rats exposed to vehicle and PCS displayed similar values observed in the carotid and femoral arteries of CKD rats exposed of all static bone parameters under study. Compared with the to IS and PCS (Figure 4, B and C). These results were reinforced vehicle CKD group, IS-exposed CKD rats showed significantly by a distinct Von Kossa positivity on aortic cross-sections of IS- lower levels of the osteoid perimeter, osteoid width, and oste- and PCS-exposed CKD rats. In line with the total calcium oblast perimeter, which were similar to those observed in content, a significantly increased calcified tissue surface was found bone of rats with normal kidney function (historical data). in CKD rats exposed to the uremic toxins (Figure 4D). Von Kossa Dynamic bone parameters, (i.e., bone formation rate and staining also revealed that calcifications were uniquely located in mineralization apposition rate) did not differ significantly themediallayeroftheaorticwall(Figure4E).Aorticcalcification between the study groups. was significantly correlated with serum calcium (week 5: r=0.48; P,0.05 and week 7: r=0.65; P,0.01), serum IS (week 3: r=0.61; IS- and PCS-Induced Calcification Associates with Activation P,0.01), serum PCS (week 3: r=0.52; P,0.05 and week 5: r=0.64; of Inflammation and Coagulation Pathways in the Aorta P,0.01), and serum glucose (week 7: r=0.52; P,0.01). To comprehensively study the molecular signaling behavior associated with toxin-mediated arterial calcification in the set- IS and PCS Do Not Disturb Static and Dynamic Bone ting of CKD, we decided to use relative quantitative proteomic Parameters analysis of aortas extracted from IS- and PCS-exposed CKD As in CKD, the development of vascular calcification has been rats versus vehicle-exposed ones. Using stringent and signifi- associated with a disturbed bone metabolism (i.e., the bone- cant cutoff criteria for protein identification (99% percentile vascular axis),27 and we investigated whether exposure to IS or peptide identification confidence) and significant deviation

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ABE 100 **100 * Vehicle

10 10

1 1 200 μm Calcium aorta (mg/g wet tissue)

Calcium a. femoralis (mg/g wet tissue) IS

0.1 0.1 Vehicle IS PCS Vehicle IS PCS

C D 100 * **100 *

10 PCS

1 0.1 Calcium a. carotis (mg/g wet tissue) % Calcified surface in aorta (von Kossa)

0.1 0.01 Vehicle IS PCS Vehicle IS PCS

Figure 4. Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) promote calcification in the aorta and peripheral arteries. Calcium contents of the (A) aorta, (B) femoral artery, and (C) carotid artery and (D) the percentage of calcified aortic area of CKD rats exposed to vehicle, IS, or PCS. Black dots with a white stripe represent rats that died before the planned euthanasia. Black bars represent the median. *P#0.05 versus vehicle. (E) Visualization of calcification in aortic sections. Representative Von Kossa–stained aortic sections of a vehicle-, IS-, and PCS-exposed CKD rat. Magnification, 362.5. from the expression background (95% percentile deviation 203 of 530 proteins were common between IS and PCS), from control levels in control animals), we found that IS and we further investigated the global similarities at the pro- and PCS exposure resulted in a significant alteration of 268 teomic level in IS- and PCS-exposed aortic tissues with quan- (120 upregulated and 148 downregulated) (Supplemental titative Venn diagram analyses. The ten most upregulated or Table 1) and 262 (115 upregulated and 147 downregulated) downregulated proteins that were common between IS- or (Supplemental Table 2) proteins, respectively (Figure 5A). PCS-exposed rat aortic samples are presented in Supplemen- At a simple protein identity level, the proteomic expression tal Table 3. responses in aortic tissue were highly similar between IS and WebGestalt-based gene ontology (GO) biologic process PCS (Figure 5A) (38% of the proteins were common; i.e., analysis of the common IS/PCS proteomic responses was

758 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH

Table 1. Static and dynamic bone parameters of CKD rats exposed to vehicle, indoxyl sulfate, or p-cresyl sulfate Reference Values of Rats with Bone Parameters Vehicle IS PCS Normal Renal Function Static bone parameters Bone area (% of tissue area) 31.462.4 24.963.8 36.166.1 24.4162.0 Mineralized area (% of tissue area) 29.862.4 24.163.6 34.465.9 23.2861.88 Osteoid perimeter (% of total perimeter) 34.863.1 23.162.4a 30.263.9 27.4668.24 Eroded perimeter (% of total perimeter) 10.861.0 9.361.4 14.062.4 10.2660.75 Osteoid width (mm) 6.2660.4 4.7260.28a 6.7160.6 5.3860.74 Osteoblast perimeter (% of total perimeter) 25.663.0 13.862.4a 21.463.1 11.363.7 Osteoclast perimeter (% of total perimeter) 3.760.5 3.060.5 4.260.6 3.5660.51 Dynamic bone parameters Bone formation rate (mm2/mm2 per day) 56026528 45586971 34456997 19946213 Mineralization apposition rate (mm/d) 4.7360.19 4.5960.42 4.5960.66 2.5560.19 Data are presented as mean6SEM. IS, indoxyl sulfate; PCS, p-cresyl sulfate. aP,0.05 versus vehicle. performed to link and coherently cluster these proteins to overlap (at both the protein identity and expression polarity simple biologic processes. The 203 proteins common to levels) between our input IS/PCS common dataset and mul- both significantly regulated IS and PCS datasets (Figure 5A) tiple curated perturbagen datasets. Both upstream chemical populated (at a P,0.05 significance level) the following bi- factors and protein/genetic factors linked to our common ologic processes: “Acute Phase Response,”“Iso-Citrate Meta- IS/PCS proteome dataset revealed a consistent and strong bolic Process,” and “Complement Activation” (Figure 5B). In role for altered energy metabolism coincident with the addition to GO-based biologic process clustering, we also used IS/PCS-induced calcification process, including elevated glu- signaling pathway analysis (IPA-based canonical signaling cose levels and impaired insulin signaling/sensitivity as indicated pathway analysis) to generate an orthogonal and more mech- by reduced INSR as well as peroxisome proliferation–activated anistic signaling appreciation of our IS/PCS common dataset receptor-a (PPAR a) and PPARg expression (Figure 6A). (Supplemental Table 4). In accordance with our GO term Almost all IS- and PCS-exposed CKD animals developed clustering, we found that “Acute Phase Signaling” was prom- significant aortic calcifications. Each group, however, con- inent in the signaling pathways, which were significantly pop- tained one or two animals that failed to demonstrate calcifi- ulated by proteins from the common IS/PCS dataset cations (i.e., “escapees” from the procalcifying effects of the (Figure 5C). The “Acute Phase Signaling” pathway was popu- uremic toxins). To investigate this potential pathologic escape lated predominantly by upregulated proteins from the com- mechanism from vascular calcification, we compared the mul- mon IS/PCS dataset, suggesting that uremic toxin exposure tiple protein expression pattern differences between vehicle- induces a potent stimulation of this signaling pathway. Several exposed CKD rats and noncalcified IS/PCS-exposed CKD of the other common IS/PCS dataset significantly populated rats using our iTRAQ proteomic platform. In “noncalcified” and positively stimulated pathways were associated with pro- IS- and PCS-exposed aortic samples, 183 proteins (109 upre- coagulative activity (“Intrinsic/Extrinsic Prothrombin Activa- gulated and 74 downregulated) (Supplemental Table 5) and tion Pathways” and “Coagulation System”) (Figure 5C). The 232 proteins (132 upregulated and 100 downregulated) (Sup- common signaling pathways strongly populated in an inhibitory plemental Table 6), respectively, were significantly altered manner (i.e., largely populated by downregulated proteins) by compared with vehicle. Interestingly, in contrast to the the IS/PCS-exposed datasets were consistently associated with “calcified” IS/PCS proteome (Supplemental Tables 1 and 2), stress responsiveness (“Glutathione Mediated Detoxification,” IS- and PCS-exposed CKD rats that did not develop calcifica- “Glutathione Redox Reactions I,” and “Glucocorticoid Receptor tions in the aortic wall showed no significant activation in the Signaling”), metabolic activity (“Fatty Acid b-Oxidation I” and previously mentioned “procalcifying” pathways, including “TCA Cycle II”), and calcium-associated processes (“Calcium “Acute Phase Response,”“Intrinsic Prothrombin Activation,” Signaling”)(Figure5C). and “Coagulation System.” In contrast, however, a significant Tosupplement both our GO biologic process clustering and activation of the farnesoid/liver X-retinoid X receptor signaling pathway analysis, we used perturbagen response pat- functionality pathway (“LXR/FXR–RXR Activation” [lipid tern matching (using the IPA-based “Upstream Analysis” metabolism]) was predicted (Figure 6B). Differential pathway suite) between our IS/PCS differential expression dataset analysis thus demonstrated that, with respect to the strength of (203 proteins) and IPA-curated perturbagen (chemical or genet- pathway enrichment, the “Acute Phase Response, “Intrinsic ic/proteomic) response patterns. This analysis aims to identify Prothrombin Activation,” and “Coagulation System” path- upstream regulators that are responsible for the up- or down- ways promoted calcification, whereas LXR/RXR antagonized regulation of common IS/PCS proteins by assessing the degree of vascular calcification. Likewise, we found that the predicted

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A B glutathione metabolic NADH process metabolic actute-phase response 82 process 36 33 2 regulation of 29 26 119 ATPase activity

response to isocitrate IS PCS antibiotic metabolic process positive regulation of tumor necrosis factor biosynthetic process regulation of lipid digestion mRNA 3’-end complement activation, processing C alternative pathway

Upregulated 10 Downregulated # of upregulated

proteins in pathway 5

-5 # of downregulated proteins in pathway -10 ILK Signaling IL-6 Signaling iNOS Signaling Calcium Signaling TR/RXR Activation LXR/RXR Activation Coagulation System FXR/RXR Activation Fatty Acid β -oxidation I TCA Cycle II (Eukaryotic) Actin Cytoskeleton Signaling Cardiac Hypertrophy Signaling Glutathione Redox Reactions I Role of Tissue Factor in Cancer Acute Phase Response Signaling Glucocorticoid Receptor Signaling Glutathione-mediated Detoxification Aryl Hydrocarbon Receptor Signaling Intrinsic Prothrombin Activation Pathway Extrinsic Prothrombin Activation Pathway Superpathway of Methionine Degradation LPS/IL-1 Mediated Inhibition of RXR Function IL-12 Signaling and Production in Macrophages Hepatic Fibrosis / Stellate Cell Activation Neuroprotective Role of THOP1 in Alzheimer’s Disease

Figure 5. Indoxyl sulfate (IS)/p-cresyl sulfate (PCS) induced aortic calciﬁcation associates with activation of inﬂammation and coagulation pathways in the aorta. (A) Quantitative Venn diagram analysis of the global similarities at the proteomic level in IS- and PCS- exposed aortic tissues versus vehicles. The intersection between both circles shows the number of proteins that are common between IS- and PCS-exposed CKD rats. Bold and underlined numbers represent upregulated and downregulated proteins, respectively. The number indicated in italics represents the number of proteins that were contraregulated between IS- and PCS-exposed aortic samples. (B) Gene ontology and (C) ingenuity pathway analysis of the common IS/PCS proteome with the number of upregulated (black bars) or downregulated (gray bars) proteins in each pathway.

760 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH

A Upstream chemical factors B lipopolysaccharide D-glucose IS CalcifiedIS Non-calcifiedPCS CalcifiedPCS Non-calcified dinoprost calcitriol Acute Phase Response Signaling tamoxifen Intrinsic Prothrombin Activation actinomycin D hydrocortisone D-glucose Coagulation System mono-(2-ethylhexyl)phthalate Pro-calcifying troglitazone pirinixic acid LXR/RXR Activation fenofibrate rosiglitazone PPARα/RXRα Activation -40 -30 -20 -10 0 10 20 Anti-calcifying

Upstream protein/gene factors C HRAS TNF OSM IL6 IS CalcifiedIS Non-calcifiedPCS CalcifiedPCS Non-calcified IL22 INSR PPARA STAT3 PDLIM2 PPARG PPARG MAP4K4 RAF1 PPARA INSR RXRA INSR D-glucose PPARG PPARA SRF Activation z-score TP53

-50 -40 -30 -20 -10 0 10 20 30 -2.449 3.464

Figure 6. Inflammation, coagulation and glucometabolism pathways strongly associate with indoxyl sulfate (IS)/p-cresyl sulfate (PCS) induced calcification while escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. (A) Identification of the upstream chemical and protein/gene factors that are responsible for the up- or downregulation of genes in the common IS/PCS proteome was performed. Ingenuity Pathway Analysis ascribes a quantified z-score number (ordinate axis) that determines whether an upstream regulator has significantly more “activated” predictions (z.0) than “inhibited” predictions (z,0). A consistent and strong role for altered energy metabolism was observed, including elevated glucose levels (gray bar in upper panel) as well as decreased insulin receptor (INSR) function and peroxisome proliferation–activated receptor (PPAR) activity (gray bars in lower panel). The upregulation (orange) and downregulation (blue) of (B) the signaling pathways “Acute Phase Response Signaling,”“Intrinsic Prothrombin Activation,”“Coagulation System,” and “LXR/RXR Activation (Lipid Metabolism)” and (C) upstream proteomic perturbagens “PPARa,”“PPARg,”“Insulin Receptor (INSR),” and “D-Glucose” in IS- and PCS-exposed CKD rats with or without aortic calcification. effects on upstream proteome regulation (using the IPA-based CKD rats exposed to IS had significantly elevated serum IS “Upstream Analysis” suite) were highly divergent between levels, whereas those exposed to PCS showed significantly calcified and noncalcified IS- and PCS-exposed CKD rats. Al- higher serum PCS values (Supplemental Table 7). No calcifi- though aortic calcification was strongly linked with downregu- cation in the aorta was observed after 4 days of vehicle or toxin lation of INSR and PPAR activity and elevated glucose levels, exposure in CKD rats exposed to IS (0.1160.01 mg Ca/g tis- noncalcified IS/PCS vessels were associated with an opposing sue), PCS (0.1460.08 mg Ca/g tissue), or vehicle (0.0860.01 degree of INSR and glucose modulation (Figure 6C). mg Ca/g tissue) as evidenced by the very low aortic calcium levels, which are in the range of values from control rats with Animal Study #2 normal renal function.27 Relative quantitative proteomic anal- To further investigate whether the induction of inflammation ysis of aortas extracted from IS- and PCS-exposed CKD rats and coagulation pathways in the aorta is directly caused by versus vehicle-exposed ones followed by canonical signaling these uremic toxins and is not just the consequence of the pathway analysis revealed that the “Acute Phase Response Sig- calcification process in the vessel wall itself, we additionally naling Pathway,”“Intrinsic/Extrinsic Prothrombin Activation studied the changes in molecular signaling pathways during the Pathways,” and “Coagulation System” are predominantly ac- induction phase (i.e., before arterial injury and calcification is tivated in the aorta of CKD rats after short-term exposure of IS well established). CKD rats exposed to vehicle, IS, or PCS for or PCS (Figure 7), which corresponds very well with the out- 4 days showed an equally fourfold increase in serum creatinine come of the proteomic analysis after long-term exposure to values after 4 days of CKD induction (Supplemental Table 7). these toxins as described in animal study #1. Remarkably,

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AB 10 6

8 4 6

4 2 # of downregulated # of upregulated proteins in pathway 2 proteins in pathway 0 0

2 2

4 # of downregulated proteins in pathway 4 # of downregulated proteins in pathway

6 EGF Signaling RhoA Signaling RhoGDI Signaling LXR/RXR Activation Coagulation System FXR/RXR Activation Sumoylation Pathway GP6 Signaling Pathway Atherosclerosis Signaling Death Receptor Signaling TR/RXR Activation Role of Tissue Factor in Cancer LXR/RXR Activation Coagulation System FXR/RXR Activation Acute Phase Response Signaling Signaling by Rho Family GTPases Fatty Acid β -oxidation I GP6 Signaling Pathway Iron homeostasis signaling pathway SPINK1 Pancreatic Cancer Pathway Atherosclerosis Signaling Epithelial Adherens Juction Signaling Intrinsic Prothrombin Activation Pathway Clathrin-mediated Endocytosis Signaling Extrinsic Prothrombin Activation Pathway Regulation of Actin-based Motility by Rho Caveolar-mediated Endocytosis Signaling Role of Tissue Factor in Cancer Stearate Biosynthesis I (Animals) Agrin Interactions at Neuromuscular Junction Acute Phase Response Signaling IL-12 Signaling and Production in Macrophages Apelin Adipocyte Signaling Pathway Hepatic Fibrosis / Stellate Cell Activation Epithelial Adherens Junction Signaling Agranulocyte Adhesion and Diapedesis Intrinsic Prothrombin Activation Pathway Clathrin-mediated Endocytosis Signaling Production of Nitric Oxide and ROS in Macrophages Extrinsic Prothrombin Activation Pathway Mitochondrial L-carnitine Shuttle Pathway Caveolar-mediated Endocytosis Signaling Agrin Interactions at Neuromuscular Junction LPS/IL-1 Mediated Inhibition of RXR Function IL-12 Signaling and Production in Macrophages Hepatic Fibrosis / Stellate Cell Activation Production of Nitric Oxide and ROS in Macrophages Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency

Figure 7. Short-term indoxyl sulfate (IS) and p-cresyl sulfate (PCS) exposure generates a functional signature reminiscent of long-term uremic toxin exposure. Ingenuity canonical signaling pathway analysis (top 25 most signiﬁcantly populated pathways) with the number of upregulated (black bars) or downregulated (gray bars) proteins in each pathway after short-term exposure of (A) IS and (B) PCS in CKD rats.

glycoprotein 6 (GP6) signaling popped up after short-term IS could deliberately be restricted to only 2 of 14 vehicle-exposed and PCS exposure (Figure 7). GP6 is a platelet membrane re- CKD rats. CKD rats exposed to either IS or PCS, however, ceptor involved in platelet activation. distinctly and significantly promoted the development of moderate to severe arterial calcifications, which implicates a direct role for both toxins as inducers of vascular calcification in the DISCUSSION setting of CKD. Both IS and PCS induced distinct calcifications in the arteries of CKD rats. These effects were seen at serum IS There is substantial evidence supporting the hypothesis that and PCS levels comparable with the reported serum IS (104.7 the uremic toxins IS and PCS are related to cardiovascular [67.2–134.6] mM) and PCS (183.6 [114.4–305.3] mM) values mortality.5,31,32 However, no direct in vivo evidence for a in patients on hemodialysis.33 causal role of these toxins in the pathology of CKD-related Because the development and progress of vascular calcifi- vascular calcification has been reported so far. In this study, we cation greatly depend on the severity of renal failure2 and/or a demonstrate that IS and PCS promote calcification in the disordered bone metabolism either going or not going along aorta and peripheral vessels in rats with adenine-induced herewith,27 we investigated the possibility for the effect of IS or CKD. PCS on vascular calcification to be due to a direct effect of Adenine dosing of rats led to the typical clinical picture of these compounds on renal function and bone metabolism. CKD evidenced by decreased creatinine clearance as well as The literature has demonstrated that IS and PCS stimulate altered mineral homeostasis characterized by abnormal levels the progression of renal failure by increasing the inflamma- of phosphorus, calcium, and PTH and a disturbed bone me- tion in the proximal tubular cells and enhancing kidney tabolism. However, with the adenine dosing strategy applied in fibrosis.34,35 Moreover, Sun et al.36 suggested that IS and this study, development of significant vascular calcification PCS suppress renal klotho expression through epigenetic

762 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 www.jasn.org BASIC RESEARCH modifications. Several studies have considered klotho as a reno- signaling play a crucial causal role in the onset and develop- protective factor, because reduced expression of this protein has ment of toxin-induced vascular calcification.Thisiseven been associated with increased kidney damage.37,38 Conversely, more emphasized by the activation of GP6 signaling in aortas results of this study demonstrate that exposure to IS and PCS did after short-term exposure to IS and PCS. GP6 indeed is the not aggravate renal impairment, inflammation, and fibrosis in primary receptor for platelet/collagen interactions and subse- CKD rats, and it did not worsen serum biochemistry in terms of quent platelet activation. Activation of this GP6 pathway in creatinine, phosphorus, and calcium levels in comparison with short-term toxin-exposed aortas that did not yet develop calci- CKD rats exposed to vehicle. In addition, no significant differ- fications strongly suggests that platelet activation is initially in- ences in klotho expression for either IS- or PCS-exposed CKD volved in this toxin-mediated calcification process. rats versus those exposed to vehicle were observed. Interestingly, our experimental findings are in accordance Furthermore, other research groups have suggested that IS with those observed in patients suffering from calcific aortic and PCS may also contribute to bone impairment by increasing stenosis.41 In these patients, an integrated proteomic ap- reactive oxygen species in osteoblasts.39,40 However, we found proach of serum samples indeed showed significant alter- that exposure to PCS did not alter static and dynamic bone ations in coagulation, inflammation, oxidative stress, and lipid parameters compared with vehicle exposure in CKD animals. metabolism. Activation of these signaling pathways plays a key Exposure to IS showed that all static bone parameters evolve role in the process of arterial calcification. toward values observed in the bone of rats with normal func- Subsequently, curated chemical and genomic/proteomic tion, which was most probably due to the more preserved perturbagen matching analysis—predicting upstream regula- kidney function and smaller alterations in calcium and phos- tors that could be responsible for the observed changes in the phorus homeostasis in this study group. Taken together, both arterial proteins linked to inflammation and coagulation sig- IS and PCS exposure did not aggravate CKD, and it did not naling pathways—revealed a major role for altered energy and further disturb bone metabolism, which strongly suggests that glucose metabolism, including elevated glucose levels, INSR both uremic toxins trigger the development/progression of dysfunction, and impaired PPARa and PPARg function. moderate to severe vascular calcifications by direct deleterious PPAR, particularly the PPARg isoform, is an important regu- effects on the vessel wall. lator of glucose metabolism, because activation of this nuclear Toinvestigate whether IS and PCS directly affect the arterial receptor in adipocytes modulates the insulin signaling wall and to identify the molecular mechanisms and cellular cascade, which improves insulin sensitivity.42 Interestingly, signaling pathways by which these toxins exert their harmful IS- and PCS-exposed CKD rats showed significantly altered effect on the vasculature, an unbiased quantitative proteomic vascular GLUT1 expression and significantly increased serum approach using iTRAQ protein labeling and MS was applied. glucose levels, which were also significantly correlated with the This methodology allowed us to simultaneously identify and calcium content in the aorta. These results strongly support quantify the proteins (in a high-dimensionality manner) that our hypothesis that activation of inflammation and coagula- are differentially up- or downregulated in aortic samples of tion pathways by both uremic toxins is highly linked with in- CKD rats exposed to IS and PCS versus those exposed to ve- creased circulating glucose levels and insulin resistance, which hicle. Because the proteomic expression responses in arterial ultimately contribute to calcification of the arteries. Similar tissue were highly similar between IS and PCS versus vehicle findings were reported by Koppe et al.,43 who found that condition, we focused on the common IS and PCS functional chronic PCS administration promoted insulin resistance and signature. By using functional annotation analysis of the com- hyperglycemia in CKD mice. Systemic inflammation has been mon IS/PCS proteome, the following pathways were identified reported to enhance vascular calcification,44,45 whereas proin- as being significantly activated by both toxins in aortic tissue flammatory cytokines were found to induce a phenotypic samples: (1)the“Acute Phase Response Signaling” pathway switch of vascular cells into bone-like cells by which matrix (inflammation) and (2) the “Intrinsic and Extrinsic Pro- vesicles containing hydroxyapatite are released into the vessel thrombin Activation” pathway (coagulation). Activation of wall.46,47 Coagulation and inflammation are closely linked inflammation and coagulation pathways coincided with calci- processes, because coagulation factors (fibrinogens and pro- fication in the aorta after long-term exposure of IS and PCS, thrombin) are also members of the acute-phase proteins.48 which makes it difficult to conclude whether these molecular Both “Intrinsic and Extrinsic Prothrombin Activation” path- changes are causally implicated in toxin-induced vascular cal- ways were significantly activated in calcified aortas of IS- and cification. An additional study showed that the “Acute Phase PCS-exposed CKD animals. Interestingly, vascular smooth Response Signaling Pathway,”“Prothrombin Activation Path- muscle cells exposed to uremic serum have been reported to ways,” and “Coagulation System” are also predominantly ac- induce a significantly increased clot formation.49 Other stud- tivated after short-term exposure to IS or PCS and before ies have also associated high-serum IS levels with thrombotic calcification had developed. These findings thus correspond complications in patients with CKD.50,51 Furthermore, well with the molecular signaling pathways that are involved Kapustin et al.52 found that vascular smooth muscle cell exo- after long-term exposure to these toxins. They further illus- somes, recently recognized as important mediators of calcifi- trate that acute-phase response signaling and coagulation cation and coagulation,53 are loaded with coagulation factors,

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In conclusion, both protein-bound ure- Circulation Calcified aorta mic toxins, IS and PCS, must be considered IS as important harmful vascular toxins and CKD indoxyl sulfate promoters of insulin resistance in CKD. Di- glucose etary interventions, such as probiotics to inflammatory lower gut-derived uremic toxins, should insulin & coagulation pathways be considered as a promising treatment PCS resistance for CKD-related vascular calcification. p-cresyl sulfate Identification of specifictherapeutictar- gets in the molecular signaling pathways Figure 8. Mechanisms and pathways central to vascular calcification induced by in- underlying the calcification process (e.g., doxyl sulfate (IS) and p-cresyl sulfate (PCS). antidiabetic interventions, such as PPAR modulators, or insulin sensitizers, such as such as prothrombin. Prothrombin accumulates in calcified metformin) is an important step to establishing novel therapies human vessels, and a negative correlation between plasma aimed at preventing or slowing down the progression of this prothrombin levels and vascular calcification has been report- vascular pathology. ed.52 In vitro studies have also revealed that prothrombin in- hibits vascular smooth muscle calcification,52 suggesting that, in our study, increased prothrombin acted as an inadequate defense ACKNOWLEDGMENTS mechanism. In addition, several clinical studies have associated coagulation with elevated coronary artery calcification We thank Geert Dams, Hilde Geryl, Ludwig Lamberts, Rita Marynissen, scores.54,55 It is clear, therefore, that a close association between and Simonne Dauwe for their excellent technical assistance and Dirk De coagulation and vascular calcification exists, which opens a new Weerdt for his help with the graphics. research avenue for the identification of novel druggable targets. E. Neven is a postdoctoral fellow of the Fund for ScientificRe- Although IS and PCS exposure to CKD rats resulted in search–Flanders (FWO). B. Opdebeeck and A. De Maré are fellows of moderate to severe vascular calcification in the majority of the FWO. the animals, some IS- and PCS-exposed rats failed to demonstrate significant arterial calcifications. Toinvestigate a possible escape mechanism from calcifications in the arterial wall, a DISCLOSURES comparison between the aortic proteome profiles of noncalci- None. fied and calcified IS/PCS animals was performed. Interestingly, it was clear that, with respect to the strength (degree of enrichment probability and IPA-generated pathway z score) of pathway SUPPLEMENTAL MATERIAL enrichment, the “Acute Phase Response,”“Intrinsic Prothrom- bin Activation,” and “Coagulation System” pathways promoted This article contains the following supplemental material online at calcification, whereas “LXR/RXR (Lipid Metabolism)” pathways http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018060609/-/ antagonized arterial calcification. The latter finding is supported DCSupplemental. by a study by Miyazaki-Anzai et al.56 that reported that activation Supplemental Table 1. Differentially up- or downregulated pro- of FXR by a synthetic FXR agonist prevented the mineralization teins in IS-exposed calcified versus vehicle-exposed noncalcified of bovine-calcifying vascular cells and inhibited vascular calcifi- aortic samples. 2 2 cation in ApoE / mice with CKD. Hence, it seems that Supplemental Table 2. Differentially up- or downregulated pro- procoagulation and inflammatory processes seem to be teins in PCS-exposed calcified versus vehicle-exposed noncalcified strongly linked with calcification events, whereas escape from aortic samples. these is rather associated with lipid metabolic functions. More- Supplemental Table 3. The top ten upregulated or downregulated over, with respect to predicted upstream proteome perturba- proteins common to both IS- and PCS-exposed rat aortic samples. gens responsible for the vascular proteomes, we found that, Supplemental Table 4.The top 25most significantlyaltered canonical although calcification was associated with high glucose levels signaling pathways in the common IS and PCS aortic proteome. and downregulation of INSR activity, escape from vascular cal- Supplemental Table 5. Differentially up- or downregulated pro- cification was linked with an opposing degree of glucose and teins in IS-exposed noncalcified versus vehicle-exposed noncalcified INSR modulation. Taken together, this further supports our aortic samples. hypothesis that IS and PCS promote calcification in the aorta SupplementalTable6.Differentiallyup-ordownregulatedproteinsin and peripheral arteries of CKD rats via the induction of hyper- PCS-exposed noncalcified versus vehicle-exposed noncalcified aortic glycemia and insulin resistance, which contribute to activation samples. of the acute-phase response signaling and coagulation path- Supplemental Table 7. Biochemical parameters of CKD rats ex- ways in the arterial wall (Figure 8). posed to vehicle, indoxyl sulfate, or p-cresyl sulfate for 4 days.

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766 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 751–766, 2019 Supplemental material

Table of Contents

Table S1 : Differentially up- or downregulated proteins in IS exposed calcified versus vehicle exposed non-calcified aortic samples...... 2

Table S2 : Differentially up- or downregulated proteins in PCS exposed calcified versus vehicle exposed non-calcified aortic samples...... 9

Table S3: The top 10 upregulated or downregulated proteins common to both IS and PCS exposed rat aortic samples...... 15

Table S4: The top 25 of most significantly altered canonical signaling pathways in the common IS and PCS aortic proteome...... 16

Table S5: Differentially up- or downregulated proteins in IS exposed non-calcified versus vehicle exposed non-calcified aortic samples...... 17

Table S6: Differentially up- or downregulated proteins in PCS exposed non-calcified versus vehicle exposed non-calcified aortic samples...... 21

Table S7: Biochemical parameters of CKD rats exposed to vehicle, indoxyl sulfate or p-cresyl sulfate for 4 days...... 26

Table S1 : Differentially up- or downregulated proteins in IS exposed calcified versus vehicle exposed non-calcified aortic samples.

Protein Description Gene Symbol Log2 iTRAQ Ratio Globin a1 LOC103694855 1.537854946 Sideroflexin-3 Sfxn3 1.532657025 Epsilon 1 globin Hbe1 1.510884441 WD repeat-containing protein 5B Wdr5b 1.489274026 Unc-93 homolog B1 (C. elegans) Unc93b1 1.451848419 TGF-beta activated kinase 1 (MAP3K7) binding protein 1 Tab1 1.372491732 FAT atypical cadherin 4 Fat4 1.32132827 Globin c2 Hba-a2 1.291530096 Globin a4 Hbb 1.277356791 Myelin protein P0 Mpz 1.212255504 WD repeat and HMG-box DNA binding protein 1 (Predicted) Wdhd1 1.073430575 Anion exchange protein Slc4a1 1.030158921 Haptoglobin Hp 1.005444277 Neurofilament medium polypeptide Nefm 0.994342922 Alpha globin Hba-a3 0.978506139 Actinin alpha 3 Actn3 0.973132227 Neurofilament light polypeptide Nefl 0.972158491 Annexin A8 Anxa8 0.965831739 Solute carrier family 43 member 1 Slc43a1 0.947746593 Ring finger protein 219 Rnf219 0.942127968 Serine protease HTRA1 Htra1 0.90886013 Platelet factor 4 Pf4 0.905152246 Ig gamma-2A chain C region Igg-2a 0.898287479 RCG59107, isoform CRA_a Wdr17 0.893374883 40S ribosomal protein S15 Rps15 0.892775815 Carbonic anhydrase 1 Ca1 0.892030196 Fibrinogen gamma chain Fgg 0.891299291 RNA pseudouridylate synthase domain containing 2 (Predicted) LOC100911166 0.891252577 Centrosomal protein 112 Cep112 0.888177744 Ubiquitin-conjugating enzyme E2, J1 Ube2j1 0.864271425 RNA terminal phosphate cyclase domain 1 Rtcd1 0.86054284 UDP-N-acetylglucosamine pyrophosphorylase 1 Uap1 0.860388383 ADP-ribosylation factor-like protein 8B Arl8b 0.85939809 Exdl2 protein Exd2 0.856582852 Solute carrier family 2, facilitated glucose transporter member 4 Slc2a4 0.849902548 Lysyl oxidase-like 3 Loxl3 0.835129742 Heme oxygenase 2 Hmox2 0.831619366 C-reactive protein Crp 0.823525007 Carboxylesterase 1C Ces1c 0.798655384

Vitronectin Vtn 0.796801623 Cartilage oligomeric matrix protein Comp 0.793706665 Inositol-trisphosphate 3-kinase B Itpkb 0.791242098 Alpha-2-HS-glycoprotein Ahsg 0.781002744 Integral membrane protein 2B Itm2b 0.780612605 Hyaluronan and proteoglycan link protein 4 Hapln4 0.77443381 60S ribosomal protein L32 Rpl32 0.768843487 Tubulin beta-3 chain Tubb3 0.758806118 Galectin-5 Lgals5 0.753996614 Serotransferrin Tf 0.751218273 cAMP-dependent protein kinase catalytic subunit beta Prkacb 0.749542931 Protein S100-B S100b 0.737079153 Carboxylic ester hydrolase Bche 0.734896155 Alpha-2-HS-glycoprotein Ahsg 0.7321129 Sodium channel protein Scn7a 0.7321129 Apolipoprotein A-I Apoa1 0.730685808 Peripherin Prph 0.729896164 Ac1873 Fga 0.725024593 Tenascin C Tnc 0.721861402 40S ribosomal protein S24 LOC100363469 0.720099153 Fibronectin Fn1 0.717136996 Collagen alpha-1(XII) chain Col12a1 0.711071749 Collagen type XVIII alpha 1 chain Col18a1 0.705171148 Actin-related protein 2/3 complex subunit 1B Arpc1b 0.705148812 RCG27247 Zc3h8 0.704970316 Gamma-aminobutyric acid type B receptor subunit 1 Gabbr1 0.694597078 Serine protease inhibitor A3L Serpina3l 0.69395455 Collagen alpha-1(II) chain Col2a1 0.693297047 Osteopontin Spp1 0.693297047 Similar to RIKEN cDNA 9030624J02 LOC361635 0.687987827 Calcium/calmodulin-dependent protein kinase type II subunit delta Camk2d 0.68663013 Embigin Emb 0.68458094 Complement C1s subcomponent C1s 0.681486827 Complement C1q tumor necrosis factor-related protein 5 C1qtnf5 0.681324406 Matrix Gla protein Mgp 0.677271809 Ig gamma-2B chain C region Igh-1a 0.677157058 Sad1 and UNC84 domain-containing 1 Sun1 0.676993688 Fibrinogen beta chain Fgb 0.675575489 Nuclear cap-binding protein subunit 1 Ncbp1 0.66965013 MON2 homolog, regulator of endosome-to-Golgi-trafficking Mon2 0.664506827 C-type lectin domain family 3, member B Clec3b 0.663177059 Tenascin N Tnn 0.663177059 Fetub protein Fetub 0.661362894 Aquaporin-1 Aqp1 0.660681224 Ankyrin 1 Ank1 0.659942635

Collagen type IV alpha 2 chain Col4a2 0.65707686 Prostacyclin synthase Ptgis 0.650950757 RCG20461 Stum 0.650950757 Carbonic anhydrase 2 Ca2 0.650329931 Progressive ankylosis protein homolog Ankh 0.650235893 Family with sequence similarity 49, member A Fam49a 0.650235893 Serine protease inhibitor A3K Serpina3k 0.647910783 Alpha-1-macroglobulin A1m 0.640697559 Aggrecan core protein Acan 0.637027501 Neuroplastin Nptn 0.636095657 Lipopolysaccharide binding protein Lbp 0.627980951 EGF-containing fibulin-like extracellular matrix protein 2 Efemp2 0.626182852 Laminin subunit beta 1 Lamb1 0.626182852 Schlafen family member 5 Slfn5 0.618293001 Prothrombin F2 0.612863351 Retinol-binding protein 4 Rbp4 0.612126788 RNA-binding protein with multiple-splicing Rbpms 0.607486246 Alpha-1-antiproteinase Serpina1 0.607486246 Collagen alpha-1(I) chain Col1a1 0.607323824 Transporter Slc6a2 0.607323824 Protein VAC14 homolog Vac14 0.60205236 Amine oxidase Aoc3 0.598742243 Laminin subunit gamma 1 Lamc1 0.594612773 Pre-mRNA processing factor 8, isoform CRA_a Prpf8 0.594612773 Complement C3 C3 0.591371639 Elongator complex protein 5 Elp5 0.5911228 Small nuclear ribonucleoprotein U1 subunit 70 Snrnp70 0.5911228 Beta-hexosaminidase subunit alpha Hexa 0.585626812 Histone H1.4 Hist1h1e 0.57961793 ATP synthase mitochondrial F1 complex assembly factor 2 Atpaf2 0.574571623 HEAT repeat-containing 5B Heatr5b 0.574571623 V-type proton ATPase 16 kDa proteolipid subunit Atp6v0c 0.568646812 Translocon-associated protein subunit delta Ssr4 0.568646812 Collagen alpha-1(XXIII) chain Col23a1 0.563129807 Eukaryotic translation initiation factor 3 subunit M Eif3m 0.563129807 Wolfram syndrome 1 homolog (Human) Wfs1 0.557591623 Mast cell carboxypeptidase A (Fragment) Cpa3 0.552032098 Uncharacterized protein Pkn3 -0.557885594 SLAIN motif family, member 2 Slain2 -0.557885594 ATP synthase mitochondrial F1 complex assembly factor 2 Atpaf2 -0.557885594 Trafficking protein particle complex 6B Trappc6b -0.569958426 Myl9 protein Myl9 -0.57743147 PDZ and LIM domain protein 5 Pdlim5 -0.57743147 Histone H2A H2afx -0.58213314 Neurabin-2 Ppp1r9b -0.58213314

Tropomyosin 1, alpha, isoform CRA_p Tpm1 -0.589815195 Dimethylaniline monooxygenase [N-oxide-forming] 1 Fmo1 -0.59441147 Adenylate kinase 2, mitochondrial Ak2 -0.59620957 10 kDa heat shock protein, mitochondrial Hspe1 -0.59620957 RCG34610, isoform CRA_c Srsf1 -0.59620957 Serine/arginine-rich splicing factor 2 Srsf2 -0.598640754 Adenosylhomocysteinase 2 Ahcyl1 -0.602306139 Coiled-coil domain-containing 88C Ccdc88c -0.606795195 Syntaxin 16 Stx16 -0.606795195 Propionyl coenzyme A carboxylase, beta polypeptide Pccb -0.60952907 RNA-binding protein 3 Rbm3 -0.60952907 SP100 nuclear antigen Sp100 -0.60952907 Alpha-1B-glycoprotein A1bg -0.610856346 Uncharacterized protein Cep192 -0.610856346 60S ribosomal protein L3 Rpl3 -0.610856346 40S ribosomal protein S9 Rps9 -0.610856346 Alpha-crystallin B chain Cryab -0.614906176 Glutathione S-transferase Mu 2 Gstm2 -0.614906176 PDZ and LIM domain 3, isoform CRA_a Pdlim3 -0.614906176 Xin actin-binding repeat-containing 1 Xirp1 -0.616255564 Four and a half LIM domains protein 2 Fhl2 -0.619286139 Ectonucleotide pyrophosphatase/phosphodiesterase family member 3 Enpp3 -0.620500188 Protein tyrosine kinase 9-like (A6-related protein) (Predicted), isoform CRA_b Twf2 -0.620500188 Charged multivesicular body protein 1A Chmp1a -0.624985169 Eukaryotic translation initiation factor 5 Eif5 -0.62506638 Long-chain-fatty-acid--CoA ligase 1 Acsl1 -0.625653348 2,4-dienoyl CoA reductase 1, mitochondrial, isoform CRA_a Decr1 -0.625653348 Golgi associated, gamma adaptin ear containing, ARF binding protein 1 Gga1 -0.625653348 Perilipin Plin1 -0.625653348 Peptidyl-prolyl cis-trans isomerase Fkbp4 -0.627617227 Methionine sulfoxide reductase B3 Msrb3 -0.627617227 Synaptopodin 2 Synpo2 -0.627617227 Transforming growth factor beta-1-induced transcript 1 protein Tgfb1i1 -0.627617227 Transformer-2 protein homolog beta Tra2b -0.631555377 Thymosin beta-4 Tmsb4x -0.635308379 Sushi domain containing 5 (Predicted) Susd5 -0.638579175 Aminoacylase-1A Acy1a -0.640441268 Purkinje cell protein 4-like 1 Pcp4l1 -0.640441268 RCG35999, isoform CRA_a Smtn -0.640441268 Carnitine O-palmitoyltransferase 2, mitochondrial Cpt2 -0.640603689 AIP1, isoform CRA_a Dpy30 -0.640603689 Cytochrome b-c1 complex subunit 6, mitochondrial Uqcrh -0.640603689 Histone cluster 1 H1 family member c Hist1h1c -0.641663904 Serine and arginine-rich-splicing factor 4 Srsf4 -0.642695934 Synaptopodin 2-like Synpo2l -0.65392319

Methylthioribose-1-phosphate isomerase Mri1 -0.655583621 Histone H3 Hist2h3c2 -0.655710581 Creatine kinase S-type, mitochondrial Ckmt2 -0.657421268 Glutathione S-transferase alpha-3 Gsta3 -0.657421268 High mobility group nucleosomal binding domain 2 LOC100360316 -0.657421268 40S ribosomal protein S5 Rps5 -0.657421268 Calreticulin Calr -0.664371297 Zyxin Zyx -0.667802311 Asporin Aspn -0.670360323 Complement factor D Cfd -0.670977338 Protein phosphatase 1, regulatory subunit 12C Ppp1r12c -0.671539871 Myosin regulatory light chain RLC-A Rlc-a -0.674355805 Heme oxygenase 1 Hmox1 -0.676643266 Protein S100-A6 S100a6 -0.676643266 RCSD domain containing 1 (Predicted), isoform CRA_a Rcsd1 -0.679073595 Crk-like protein Crkl -0.679611865 Putative uncharacterized protein Srsf3 -0.684099269 LIM and cysteine-rich domains 1 Lmcd1 -0.688138905 Plectin Plec -0.696591865 Heterogeneous nuclear ribonucleoprotein U-like 1 Hnrnpul1 -0.699726879 Calumenin Calu -0.699726879 ABRA C-terminal-like Abracl -0.702004234 Protein AMBP Ambp -0.702004234 CD163 antigen (Predicted) Cd163 -0.702004234 Isocitrate dehydrogenase [NAD] subunit, mitochondrial Idh3a -0.702004234 Tropomyosin beta chain Tpm2 -0.705096969 Cysteine-rich protein 1 Crip1 -0.709888688 Myelin protein P0 Mpz -0.709888688 Protocadherin 7 Pcdh7 -0.711408684 Calponin-3 Cnn3 -0.712330232 60S acidic ribosomal protein P1 Rplp1 -0.712595043 60S ribosomal protein L7 Rpl7 -0.71777155 Filamin binding LIM protein 1, isoform CRA_a Fblim1 -0.719875735 Maleylacetoacetate isomerase Gstz1 -0.719875735 LIM and senescent cell antigen-like-containing domain protein Lims2 -0.719875735 Serine/arginine-rich splicing factor 5 Srsf5 -0.724661259 GM2 ganglioside activator Gm2a -0.733713093 Heat shock 27kDa protein 1 Hspb1 -0.734191768 Calponin-1 Cnn1 -0.743504768 SH3 domain-containing 19 Sh3d19 -0.747032594 Atypical kinase COQ8A, mitochondrial Coq8a -0.749832759 Cytoglobin Cygb -0.749832759 D-dopachrome decarboxylase Ddt -0.749832759 Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 Nucks1 -0.750024484 Cysteine and glycine-rich protein 2 Csrp2 -0.759318306

Long-chain specific acyl-CoA dehydrogenase, mitochondrial Acadl -0.766134571 Glycerol-3-phosphate dehydrogenase [NAD(+)] Gpd1l -0.766134571 Uncharacterized protein LOC679794 -0.766134571 Serum deprivation-response protein Sdpr -0.766134571 Eukaryotic translation initiation factor 4E binding protein 2 Eif4ebp2 -0.77128014 Destrin Dstn -0.771589542 Ring finger protein 219 Rnf219 -0.778275663 Death-associated protein 1 Dap -0.78355847 Pleckstrin homology domain containing, family B (Evectins) member 2 (Predicted), isoform CRA_b Plekhb2 -0.78355847 Ab2-292 Sec62 -0.78355847 Nucleolin Ncl -0.785748098 Hypothetical LOC289568 RGD1311575 -0.795942195 Mast cell protease 1 Mcpt1 -0.799301435 Alkaline phosphatase, tissue-nonspecific isozyme Alpl -0.807190882 SH3 domain-binding glutamic acid-rich-like protein Sh3bgrl -0.808727661 Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial Hadh -0.816175254 Superoxide dismutase [Cu-Zn] Sod1 -0.816175254 Polymerase I and transcript release factor Ptrf -0.82092 Prothymosin alpha Ptma -0.82313189 Glutamine synthetase Glul -0.833248767 Cysteine desulfurase, mitochondrial Nfs1 -0.833744227 Non-muscle caldesmon Cald1 -0.847883348 Transforming growth factor beta-3 Tgfb3 -0.859507323 Vacuolar protein sorting-associated protein 29 Vps29 -0.859507323 Electron transfer flavoprotein subunit alpha, mitochondrial Etfa -0.868014185 Legumain Lgmn -0.868014185 3-ketoacyl-CoA thiolase, mitochondrial Acaa2 -0.885716187 Spliceosome-associated protein CWC15 homolog Cwc15 -0.889202222 Transgelin Tagln -0.89484366 Electron transfer flavoprotein subunit beta Etfb -0.921785442 Microsomal glutathione S-transferase 1 Mgst1 -0.921785442 Insulin-like growth factor binding protein 7, isoform CRA_b Igfbp7 -0.94000155 Serotransferrin Tf -0.941214234 Cysteine and glycine-rich protein 1 Csrp1 -0.942486761 Serum albumin Alb -0.95698155 Mitochondrial brown fat uncoupling protein 1 Ucp1 -0.958779649 RCG61762, isoform CRA_d Srsf7 -0.959419288 Deoxyribonuclease Dnase1l1 -0.967422663 Four and a half LIM domains 1 Fhl1 -0.973767085 IQ motif-containing with AAA domain 1 Iqca1 -0.993852838 Thyroid hormone receptor-associated protein 3 Thrap3 -0.994629097 Aconitate hydratase, mitochondrial Aco2 -0.996747499 Carbonic anhydrase 3 Ca3 -1.021832694 Solute carrier family 43, member 3 Slc43a3 -1.035741631

Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Gpd1 -1.07581907 Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 Nucks1 -1.089736758 Fatty acid-binding protein, adipocyte Fabp4 -1.096283173 Selenoprotein P Selenop -1.096283173 Parathymosin Ptms -1.103691924 Prostaglandin E synthase 3 Ptges3 -1.177369449 Disks large homolog 1 Dlg1 -1.181009649 LRRN4 C-terminal-like Lrrn4cl -1.278883758 T-kininogen 1 Map1 -1.392676176 Zinc finger, DBF-type-containing 2 Zdbf2 -1.657421268 Family with sequence similarity 175, member B Fam175b -2.016932542

Table S2 : Differentially up- or downregulated proteins in PCS exposed calcified versus vehicle exposed non-calcified aortic samples.

Proein Description Gene Symbol Log2 iTRAQ Ratio Epsilon 1 globin Hbe1 1.826868402 Globin a1 LOC103694855 1.770184922 WD repeat-containing protein 5B Wdr5b 1.754727328 WD repeat and HMG-box DNA binding protein 1 (Predicted) Wdhd1 1.482202653 Unc-93 homolog B1 (C. elegans) Unc93b1 1.472397598 TGF-beta activated kinase 1 (MAP3K7) binding protein 1 Tab1 1.396056038 Globin c2 Hba-a2 1.354759037 FAT atypical cadherin 4 Fat4 1.344333844 Sideroflexin-3 Sfxn3 1.328169025 Globin a4 Hbb 1.323959262 Hyaluronan and proteoglycan link protein 4 Hapln4 1.187410688 Fibronectin Fn1 1.114977322 RT1 class Ia, locus A1 RT1-A1 1.113881653 Uncharacterized protein Ighm 1.103722815 C-reactive protein Crp 1.102959719 Anion exchange protein Slc4a1 1.096718183 C-reactive protein Crp 1.087760847 Alpha-2-HS-glycoprotein Ahsg 1.083981341 Ring finger protein 219 Rnf219 1.080277036 Protein FAM65B Fam65b 1.076305577 Gamma-aminobutyric acid type B receptor subunit 1 Gabbr1 1.071985663 Haptoglobin Hp 1.069142996 Cartilage oligomeric matrix protein Comp 1.054320087 ADP-ribosylation factor-like protein 8B Arl8b 1.042584746 Dipeptidyl peptidase 1 Ctsc 1.038335273 Alpha globin Hba-a3 1.032695832 Fibrinogen gamma chain Fgg 1.026418812 Platelet factor 4 Pf4 1.010303619 Solute carrier family 43 member 1 Slc43a1 0.995137584 Collagen alpha-1(XII) chain Col12a1 0.985776827 Tenascin C Tnc 0.979793046 Ferritin heavy chain Fth1 0.973090159 Ig gamma-2A chain C region Igg-2a 0.973043813 Collagen type VIII alpha 1 chain Col8a1 0.954735429 Carbonic anhydrase 1 Ca1 0.946076766 Centrosomal protein 112 Cep112 0.940714414 Ubiquitin-conjugating enzyme E2, J1 Ube2j1 0.938520799 UDP-N-acetylglucosamine pyrophosphorylase 1 Uap1 0.936974558 Transthyretin Ttr 0.923556312 Lysyl oxidase-like 3 Loxl3 0.90634236 Apolipoprotein C-I Apoc1 0.899980226

Serum amyloid P-component Apcs 0.897627847 RNA pseudouridylate synthase domain containing 2 (Predicted) LOC100911166 0.896026746 Annexin A8 Anxa8 0.895288656 Carboxylesterase 1C Ces1c 0.883746358 C4b-binding protein alpha chain C4bpa 0.878817998 Aggrecan core protein Acan 0.874073054 Vitronectin Vtn 0.873302098 Serine protease HTRA1 Htra1 0.867721066 Platelet factor 4 Pf4 0.866813487 GMP synthase [glutamine-hydrolyzing] Gmps 0.855917596 CDP-diacylglycerol--inositol 3-phosphatidyltransferase Cdipt 0.84820263 Immunity-related GTPase family M protein Irgm 0.845178497 Ac1873 Fga 0.833773734 Alpha-1-inhibitor 3 A1i3 0.832647816 Fibrinogen beta chain Fgb 0.822278095 Inositol-trisphosphate 3-kinase B Itpkb 0.816495742 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 Ndufa11 0.81296545 40S ribosomal protein S15 Rps15 0.812716424 Exdl2 protein Exd2 0.810690121 Tenascin N Tnn 0.809746555 Serine protease inhibitor A3K Serpina3k 0.805016697 Embigin Emb 0.803118812 V-type proton ATPase 16 kDa proteolipid subunit Atp6v0c 0.799084558 Transporter Slc6a2 0.799008316 RNA terminal phosphate cyclase domain 1 Rtcd1 0.798100737 Fatty acid synthase Fasn 0.797023907 Alpha-1-macroglobulin A1m 0.793010844 Apolipoprotein A-II Apoa2 0.793010844 Apolipoprotein A-I Apoa1 0.784951074 C-type lectin domain family 3, member B Clec3b 0.781306314 Complement component C9 C9 0.777852053 Dehydrogenase/reductase SDR family member 4 Dhrs4 0.777852053 Complement C3 C3 0.776846025 Transportin 3 Tnpo3 0.77607458 Matrix Gla protein Mgp 0.775357047 RCG27247 Zc3h8 0.773735599 Heme oxygenase 2 Hmox2 0.771049505 Cyclin-dependent-like kinase 5 Cdk5 0.762090351 Aquaporin-1 Aqp1 0.761941218 Osteopontin Spp1 0.757398656 Calcium/calmodulin-dependent protein kinase type II subunit delta Camk2d 0.757315122 Complement C1q tumor necrosis factor-related protein 5 C1qtnf5 0.751311574 Thrombospondin 1 Thbs1 0.747969243 Sodium channel protein Scn7a 0.746935976 Protein S100-B S100b 0.736481619

Alanyl (Membrane) aminopeptidase Anpep 0.735623362 Retinol-binding protein 4 Rbp4 0.728693258 Actinin alpha 3 Actn3 0.723023325 Family with sequence similarity 49, member A Fam49a 0.720656651 Ig gamma-2B chain C region Igh-1a 0.720656651 Progressive ankylosis protein homolog Ankh 0.718839134 Serine protease inhibitor A3L Serpina3l 0.71684439 Galectin-5 Lgals5 0.715342852 E3 ubiquitin-protein ligase Itch 0.714474805 Lipopolysaccharide binding protein Lbp 0.714247361 Prothrombin F2 0.710312273 Integrin subunit alpha 2b Itga2b 0.710009407 Uridine kinase Uck1 0.709622931 Integral membrane protein 2B Itm2b 0.708570766 ATP synthase mitochondrial F1 complex assembly factor 2 Atpaf2 0.706344241 Ankyrin 1 Ank1 0.704656173 Alpha-1-antiproteinase Serpina1 0.702101008 Translocon-associated protein subunit delta Ssr4 0.699874954 Small nuclear ribonucleoprotein U1 subunit 70 Snrnp70 0.693889742 cAMP-dependent protein kinase catalytic subunit beta Prkacb 0.690060665 Nuclear cap-binding protein subunit 1 Ncbp1 0.685128369 Acetyl-coenzyme A transporter 1 Slc33a1 0.685128369 Guanine nucleotide-binding protein subunit gamma Gng2 0.68304236 Myosin-9 Myh9 0.680179153 Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 Enpp1 0.670275001 Fetub protein Fetub 0.661099807 Elongator complex protein 5 Elp5 0.661099807 RCG43880 Tktl1 0.637853524 Collagen type IV alpha 2 chain Col4a2 0.63128087 Thioredoxin domain containing 13 Tmx4 0.63128087 Thrombospondin 2 Thbs2 0.63128087 GM2 ganglioside activator Gm2a -0.63571155 Glutathione S-transferase alpha-3 Gsta3 -0.63571155 Mitochondrial pyruvate carrier 2 Mpc2 -0.63571155 Complement factor D Cfd -0.651653093 Carnitine O-palmitoyltransferase 2, mitochondrial Cpt2 -0.651653093 2,4-dienoyl CoA reductase 1, mitochondrial, isoform CRA_a Decr1 -0.651653093 AIP1, isoform CRA_a Dpy30 -0.651653093 10 kDa heat shock protein, mitochondrial Hspe1 -0.651653093 Transketolase Tkt -0.651653093 Glutathione S-transferase Mu 2 Gstm2 -0.651653093 60S ribosomal protein L3 Rpl3 -0.651653093 Adenosylhomocysteinase 2 Ahcyl1 -0.652560672 Vinculin Vcl -0.652560672 ATP synthase subunit epsilon, mitochondrial Atp5e -0.666366472

Cysteine-rich protein 2 Crip2 -0.666366472 Crk-like protein Crkl -0.666366472 ABRA C-terminal-like Abracl -0.667772759 Histone H1.4 Hist1h1e -0.667772759 Phosphoglucomutase 5 Pgm5 -0.680305663 Alpha-1B-glycoprotein A1bg -0.684074571 Atypical kinase COQ8A, mitochondrial Coq8a -0.684074571 Thymosin beta-4 Tmsb4x -0.684074571 Myosin light polypeptide 6 Myl6 -0.691957869 Peptidyl-prolyl cis-trans isomerase Fkbp4 -0.694380848 Purkinje cell protein 4-like 1 Pcp4l1 -0.694380848 cGMP-inhibited 3',5'-cyclic phosphodiesterase A Pde3a -0.694380848 PDZ and LIM domain 3, isoform CRA_a Pdlim3 -0.694380848 Profilin-1 Pfn1 -0.694380848 Testin Tes -0.694380848 Plectin Plec -0.697341268 Protein AMBP Ambp -0.700562694 Perilipin Plin1 -0.700562694 Aminoacylase-1A Acy1a -0.70187472 Uncharacterized protein C11orf96 homolog Ag2 -0.709340578 Decorin Dcn -0.710280323 cAMP-dependent protein kinase type II-alpha regulatory subunit Prkar2a -0.710280323 ATP-binding cassette sub-family B member 7, mitochondrial Abcb7 -0.717549266 Alpha-crystallin B chain Cryab -0.72295 Methionine sulfoxide reductase B3 Msrb3 -0.72295 PDZ and LIM domain protein 7 Pdlim7 -0.72295 Polymerase I and transcript release factor Ptrf -0.72295 Calnexin Canx -0.729044905 Charged multivesicular body protein 1A Chmp1a -0.729044905 Protocadherin 7 Pcdh7 -0.729044905 Zyxin Zyx -0.73179144 CD163 antigen (Predicted) Cd163 -0.734115254 Mast cell carboxypeptidase A (Fragment) Cpa3 -0.734115254 D-dopachrome decarboxylase Ddt -0.734115254 Eukaryotic translation initiation factor 5 Eif5 -0.734115254 Glycerol-3-phosphate dehydrogenase [NAD(+)] Gpd1l -0.734115254 Serum albumin Alb -0.736511865 Transforming growth factor beta-1-induced transcript 1 protein Tgfb1i1 -0.73744957 Actin-related protein 2/3 complex subunit 5-like protein Arpc5l -0.740632879 Clathrin light chain B Cltb -0.740632879 Creatine kinase S-type, mitochondrial Ckmt2 -0.749808688 Death-associated protein 1 Dap -0.749808688 Cysteine-rich protein 1 Crip1 -0.751188767 Synaptopodin 2 Synpo2 -0.752096346 Putative uncharacterized protein Srsf3 -0.760390424

Carbonic anhydrase 3 Ca3 -0.763229203 Ab2-292 Sec62 -0.764091852 Isocitrate dehydrogenase [NAD] subunit, mitochondrial Idh3a -0.768466758 Myosin light chain kinase Mylk -0.781843689 Filamin binding LIM protein 1, isoform CRA_a Fblim1 -0.781843689 Profilin Pfn2 -0.784515538 Glutamine synthetase Glul -0.785954185 Uncharacterized protein LOC679794 -0.785954185 Eukaryotic translation initiation factor 1A Eif1a -0.787938594 Myosin regulatory light chain RLC-A Rlc-a -0.790359493 Histone H2A H2afx -0.790450672 Propionyl coenzyme A carboxylase, beta polypeptide Pccb -0.792243947 Tropomyosin 1, alpha, isoform CRA_p Tpm1 -0.796950581 Nucleosome assembly protein 1-like 1 Nap1l1 -0.800011426 Cytochrome b-c1 complex subunit 6, mitochondrial Uqcrh -0.803656187 Coiled-coil domain-containing 88C Ccdc88c -0.804256472 Similar to RPE-spondin (Predicted) Sbspon -0.804256472 High mobility group nucleosomal binding domain 2 LOC100360316 -0.81218614 Transforming growth factor beta-3 Tgfb3 -0.81218614 Spliceosome-associated protein CWC15 homolog Cwc15 -0.812217338 Myl9 protein Myl9 -0.812217338 Protein phosphatase 1, regulatory subunit 12C Ppp1r12c -0.812217338 RCG35999, isoform CRA_a Smtn -0.815591414 Calreticulin Calr -0.821578095 Vacuolar protein sorting-associated protein 29 Vps29 -0.82446447 Serine/arginine-rich splicing factor 5 Srsf5 -0.832782911 Calumenin Calu -0.836848195 Electron transfer flavoprotein subunit alpha, mitochondrial Etfa -0.839725442 Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial Hadh -0.839725442 IQ motif-containing with AAA domain 1 Iqca1 -0.839725442 Legumain Lgmn -0.839725442 Serum deprivation-response protein Sdpr -0.839725442 Superoxide dismutase [Cu-Zn] Sod1 -0.839725442 LIM and senescent cell antigen-like-containing domain protein Lims2 -0.843244234 RCSD domain containing 1 (Predicted), isoform CRA_a Rcsd1 -0.857768614 Pleckstrin homology domain containing, family B (Evectins) member 2 (Predicted), isoform CRA_b Plekhb2 -0.861939176 SP100 nuclear antigen Sp100 -0.861939176 Calponin-3 Cnn3 -0.87373851 Long-chain specific acyl-CoA dehydrogenase, mitochondrial Acadl -0.876719649 Microsomal glutathione S-transferase 1 Mgst1 -0.876719649 60S ribosomal protein L7 Rpl7 -0.876719649 LIM and cysteine-rich domains 1 Lmcd1 -0.887474268 Alcohol dehydrogenase 1 Adh1 -0.889986346 RCSD domain containing 1 (Predicted), isoform CRA_a Rcsd1 -0.891072759 3-ketoacyl-CoA thiolase, mitochondrial Acaa2 -0.895578676

Electron transfer flavoprotein subunit beta Etfb -0.895578676 Mast cell protease 1 Mcpt1 -0.895578676 Protein S100-A6 S100a6 -0.900413323 Cysteine desulfurase, mitochondrial Nfs1 -0.913469476 Serine and arginine-rich-splicing factor 4 Srsf4 -0.913469476 Histone H3 Hist2h3c2 -0.914687499 Nucleolin Ncl -0.920666182 Trafficking protein particle complex 6B Trappc6b -0.934840581 60S acidic ribosomal protein P1 Rplp1 -0.947360839 LRRN4 C-terminal-like Lrrn4cl -0.953362203 Hypothetical LOC289568 RGD1311575 -0.956738303 Insulin-like growth factor binding protein 7, isoform CRA_b Igfbp7 -0.957415254 SH3 domain-binding glutamic acid-rich-like protein Sh3bgrl -0.967691299 Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 Nucks1 -0.969205415 Calponin-1 Cnn1 -0.974488767 Prothymosin alpha Ptma -0.974488767 Fatty acid-binding protein, adipocyte Fabp4 -0.99375907 Asporin Aspn -1.012843093 Heat shock 27kDa protein 1 Hspb1 -1.026956187 Aconitate hydratase, mitochondrial Aco2 -1.034981733 Cysteine and glycine-rich protein 2 Csrp2 -1.050816724 Tropomyosin beta chain Tpm2 -1.065079573 Histone cluster 1 H1 family member c Hist1h1c -1.09911207 Cysteine and glycine-rich protein 1 Csrp1 -1.10126382 Transgelin Tagln -1.107348295 RCG61762, isoform CRA_d Srsf7 -1.118878676 Mitochondrial brown fat uncoupling protein 1 Ucp1 -1.121138377 Non-muscle caldesmon Cald1 -1.137987499 Alkaline phosphatase, tissue-nonspecific isozyme Alpl -1.140240338 Carnitine O-palmitoyltransferase 1, muscle isoform Cpt1b -1.166226266 Thyroid hormone receptor-associated protein 3 Thrap3 -1.166226266 Destrin Dstn -1.176275783 Maleylacetoacetate isomerase Gstz1 -1.176981631 Solute carrier family 43, member 3 Slc43a3 -1.189309879 Disks large homolog 1 Dlg1 -1.196881188 Selenoprotein P Selenop -1.212768852 Four and a half LIM domains 1 Fhl1 -1.32241207 Prostaglandin E synthase 3 Ptges3 -1.32241207 Parathymosin Ptms -1.344438377 Deoxyribonuclease Dnase1l1 -1.346019017 T-kininogen 1 Map1 -1.362146476 Zinc finger, DBF-type-containing 2 Zdbf2 -1.438606999 Myelin basic protein Mbp -1.776775735 Family with sequence similarity 175, member B Fam175b -2.258687266

Table S3: The top 10 upregulated or downregulated proteins common to both IS and PCS exposed rat aortic samples. Protein Description Gene Symbol IS PCS MEAN

Upregulated proteins hemoglobin subunit epsilon 1 Hbe1 1.510884 1.826868 1.668876 hemoglobin subunit beta-2-like LOC103694855 1.537855 1.770185 1.65402 WD repeat domain 5B Wdr5b 1.489274 1.754727 1.622001 unc-93 homolog B1 (C. elegans) Unc93b1 1.451848 1.472398 1.462123 sideroflexin 3 Sfxn3 1.532657 1.328169 1.430413 TGF-beta activated kinase 1 (MAP3K7) binding protein 1 Tab1 1.372492 1.396056 1.384274 FAT atypical cadherin 4 Fat4 1.321328 1.344334 1.332831 hemoglobin subunit alpha 2 Hba-a2 1.29153 1.354759 1.323145 hemoglobin subunit beta Hbb 1.277357 1.323959 1.300658 WD repeat and HMG-box DNA binding protein 1 Wdhd1 1.073431 1.482203 1.277817

Downregulated proteins abraxas 2, BRISC complex subunit Fam175b -2.01693 -2.25869 -2.13781 zinc finger DBF-type containing 2 Zdbf2 -1.65742 -1.43861 -1.54801 mannosidase processing 1 Map1 -1.39268 -1.36215 -1.37741 prostaglandin E synthase 3 Ptges3 -1.17737 -1.32241 -1.24989 parathymosin Ptms -1.10369 -1.34444 -1.22407 discs large MAGUK scaffold protein 1 Dlg1 -1.18101 -1.19688 -1.18895 deoxyribonuclease 1 like 1 Dnase1l1 -0.96742 -1.34602 -1.15672 selenoprotein P Selenop -1.09628 -1.21277 -1.15453 four and a half LIM domains 1 Fhl1 -0.97377 -1.32241 -1.14809 LRRN4 C-terminal like Lrrn4cl -1.27888 -0.95336 -1.11612

For each protein significantly (p<0.05) and differentially expressed in aortic samples of IS/PCS versus vehicle exposed CKD rats, the protein description, official gene symbol and the Log2 transformed iTRAQ expression ratio is depicted. The mean Log2 iTRAQ expression ratio value for IS and PCS exposure is also indicated.

Table S4: The top 25 of most significantly altered canonical signaling pathways in the common IS and PCS aortic proteome.

Canonical Pathways -log(p-value) Upregulated Downregulated #Modulated*P Proteins Acute Phase Response Signaling 12.5 14 2 200 FN1,AMBP,AHSG,SERPINA3,F2,FGG,HMOX2,ALB,HP,APOA1,CRP,FGB,LBP,FGA,TAB1,RBP4 LXR/RXR Activation 7.26 6 4 72.6 ALB,APOA1,VTN,AMBP,AHSG,LBP,FGA,A1BG,HADH,RBP4 FXR/RXR Activation 5.95 6 3 53.55 ALB,APOA1,VTN,AMBP,AHSG,FETUB,FGA,A1BG,RBP4 Extrinsic Prothrombin Activation Pathway 5.03 4 0 20.12 FGB,FGA,FGG,F2 Aryl Hydrocarbon Receptor Signaling 3.04 0 6 18.24 GSTA3,GSTM1,MGST1,TGFB3,PTGES3,HSPB1 Intrinsic Prothrombin Activation Pathway 4.11 4 0 16.44 FGB,FGA,FGG,F2 Coagulation System 3.84 4 0 15.36 FGB,FGA,FGG,F2 Calcium Signaling 2.5 2 4 15 PRKACB,MYL9,CALR,CAMK2D,Tpm1,Tpm2 Hepatic Fibrosis / Hepatic Stellate Cell Activation 2.49 4 2 14.94 MYL9,FN1,COL12A1,TGFB3,LBP,COL4A2 Glucocorticoid Receptor Signaling 2.12 3 4 14.84 PRKACB,FKBP4,TGFB3,CD163,FGG,TAB1,PTGES3 Glutathione-mediated Detoxification 3.57 0 4 14.28 GSTA3,GSTZ1,GSTM1,MGST1 ILK Signaling 2.37 2 4 14.22 MYL9,TGFB1I1,FBLIM1,FN1,LIMS2,Actn3 LPS/IL-1 Mediated Inhibition of RXR Function 2.09 1 5 12.54 GSTA3,GSTM1,MGST1,CPT2,FABP4,LBP Actin Cytoskeleton Signaling 2.04 4 2 12.24 MYL9,FN1,CRKL,Actn3,LBP,F2 Glutathione Redox Reactions I 2.77 0 3 8.31 GSTZ1,GSTM1,MGST1 Role of Tissue Factor in Cancer 1.82 4 0 7.28 FGB,FGA,FGG,F2 TCA Cycle II (Eukaryotic) 2.42 1 2 7.26 ACO2,IDH3A,NAD+ IL-6 Signaling 1.78 3 1 7.12 CRP,LBP,TAB1,HSPB1 Cardiac Hypertrophy Signaling 1.4 2 3 7 PRKACB,MYL9,TGFB3,TAB1,HSPB1 Fatty Acid β-oxidation I 2.31 1 2 6.93 HADH,ACAA2,NAD+ IL-12 Signaling and Production in Macrophages 1.55 2 2 6.2 ALB,APOA1,TGFB3,RBP4 Superpathway of Methionine Degradation 1.85 1 2 5.55 AHCYL1,PCCB,NAD+ TR/RXR Activation 1.33 2 1 3.99 HP,UCP1,FGA Neuroprotective Role of THOP1 in Alzheimer's Disease 1.32 2 0 2.64 PRKACB,SERPINA3 iNOS Signaling 1.3 2 0 2.6 LBP,TAB1 For each signaling pathway the significance level (-log(p-value) – generated using Fisher’s Exact T-test), the amount of significantly upregulated or downregulated proteins, the amount of modulated proteins (#Modulated*P) and the protein IDs that populate each pathway is depicted.

Table S5: Differentially up- or downregulated proteins in IS exposed non-calcified versus vehicle exposed non-calcified aortic samples.

Protein Description Gene Symbol Log2 iTRAQ ratio seminal vesicle secretory protein 6 Svs6 2.081 seminal vesicle secretory protein 3A Svs3b 1.843 ring finger protein 219 Rnf219 1.803 ubiquinol-cytochrome c reductase, complex III subunit X Uqcr10 1.781 seminal vesicle secretory protein 4 Svs4 1.747 seminal vesicle secretory protein 5 Svs5 1.642 potassium channel tetramerisation domain containing 12b LOC681355 1.594 creatine kinase, M-type Ckm 1.568 albumin Alb 1.453 creatine kinase, M-type Ckm 1.444 ubiquinol-cytochrome c reductase, complex III subunit X Uqcr10 1.413 haptoglobin Hp 1.354 ARP1 actin-related protein 1 homolog A, centractin alpha Actr1a 1.26 F-box protein 6 Fbxo6 1.212 taste receptor, type 2, member 110 Tas2r110 1.168 fetuin B Fetub 1.166 myelin protein zero Mpz 1.113 beta-2-microglobulin B2m 1.11 phosphoenolpyruvate carboxykinase 1 Pck1 1.109 bridging integrator 1 Bin1 1.102 ribosomal protein L27a Rpl27a 1.093 ethylmalonyl-CoA decarboxylase 1 Echdc1 1.081 toll interacting protein Tollip 1.067 uncoupling protein 1 Ucp1 1.059 3-hydroxyisobutyrate dehydrogenase Hibadh 1.057 fatty acid binding protein 4 Fabp4 1.055 cysteine rich protein 2 Crip2 1.038 transferrin Tf 1.035 tubulin polymerization promoting protein family member 3 Tppp3 1.034 apolipoprotein N Apon 1.033 PBX homeobox interacting protein 1 Pbxip1 1.018 serpin family A member 1 Serpina1 1.018 glyceraldehyde-3-phosphate dehydrogenase Gapdh 1.013 C-reactive protein Crp 0.991 diazepam binding inhibitor, acyl-CoA binding protein Dbi 0.991 orosomucoid 1 Orm1 0.988 myosin heavy chain 4 Myh4 0.982 RAB8A, member RAS oncogene family Rab8a 0.971 quiescin sulfhydryl oxidase 1 Qsox1 0.97 alcohol dehydrogenase 1C (class I), gamma polypeptide Adh1 0.957 myelin basic protein Mbp 0.937 eukaryotic translation initiation factor 3 subunit M Eif3m 0.935 glycerol-3-phosphate dehydrogenase 1 Gpd1 0.933 myosin light chain, phosphorylatable, fast skeletal muscle Mylpf 0.921 germ cell-less homolog 1 (Drosophila)-like LOC302576 0.92

17 phosphofructokinase, liver type Pfkl 0.918 Sjogren syndrome antigen B Ssb 0.913 hemoglobin subunit beta Hbb 0.904 protein kinase cAMP-dependent type II regulatory subunit alpha Prkar2a 0.903 hemopexin Hpx 0.888 MAP7 domain containing 1 Map7d1 0.887 mast cell protease 1 Mcpt1 0.884 hydroxyacyl-CoA dehydrogenase Hadh 0.876 RIKEN cDNA 9530003J23 gene RGD1306474 0.874 malic enzyme 1 Me1 0.874 guanylate cyclase 1 soluble subunit beta Gucy1b3 0.872 alpha 2-HS glycoprotein Ahsg 0.87 selenoprotein P Selenop 0.865 enolase 3 Eno3 0.857 synuclein gamma Sncg 0.855 kininogen 2 Kng1 0.849 acetyl-CoA carboxylase alpha Acaca 0.84 peripherin Prph 0.837 arrestin domain containing 2 Arrdc2 0.836 exocyst complex component 1 Exoc1 0.836 transthyretin Ttr 0.836 nicalin Ncln 0.825 carnitine palmitoyltransferase 1B Cpt1b 0.816 glutamic--pyruvic transaminase Gpt 0.816 ADP ribosylation factor 3 Arf3 0.807 myosin light chain 1 Myl1 0.807 EWS RNA-binding protein 1 Ewsr1 0.806 alpha-2-glycoprotein 1, zinc-binding Azgp1 0.803 fatty acid synthase Fasn 0.795 carboxylesterase 1C Ces1c 0.789 enolase 3 Eno3 0.779 ATP citrate lyase Acly 0.773 RIKEN cDNA 1300017J02 gene RGD1310507 0.772 exonuclease 3'-5' domain containing 2 Exd2 0.771 2',3'-cyclic nucleotide 3' phosphodiesterase Cnp 0.768 acylphosphatase 1 Acyp1 0.766 myoglobin Mb 0.766 family with sequence similarity 213 member A Fam213a 0.761 complement C3 C3 0.757 fascin actin-bundling protein 1 Fscn1 0.756 septin 5 Sept5 0.756 hemoglobin subunit beta-2-like LOC103694855 0.752 neurofilament light Nefl 0.752 C-type lectin domain family 3 member B Clec3b 0.748 hydroxysteroid dehydrogenase like 2 Hsdl2 0.727 TGF-beta activated kinase 1 (MAP3K7) binding protein 1 Tab1 0.701 phosphodiesterase 3A Pde3a 0.691 collagen type II alpha 1 chain Col2a1 0.682 matrix metallopeptidase 2 Mmp2 0.681 ADP ribosylation factor like GTPase 1 Arl1 0.661 histone cluster 1 H2B family member o Hist1h2bo 0.656 huntingtin Htt 0.656

NADH:ubiquinone oxidoreductase subunit B6 Ndufb6 0.651 tubulin beta 2B class IIb Tubb2b 0.651 aminopeptidase-like 1 Npepl1 0.646 neurofilament, medium polypeptide Nefm 0.644 arrestin beta 1 Arrb1 0.635 coiled-coil domain containing 6 Ccdc6 0.635 exonuclease 3'-5' domain containing 2 Exd2 0.635 mitochondrial pyruvate carrier 2 Mpc2 0.635 integrin subunit beta 3 Itgb3 0.634 tubulin, alpha 1B Tuba1b 0.634 seminal vesicle secretory protein 1 Svs1 0.614 protein kinase AMP-activated catalytic subunit alpha 1 Prkaa1 0.609 alpha-1-microglobulin/bikunin precursor Ambp -0.602 olfactory receptor family 1 subfamily M member 1 Olr1121 -0.608 ribosomal protein S5 Rps5 -0.608 elongin B Elob -0.614 ATPase H+ transporting V1 subunit B2 Atp6v1b2 -0.625 structure specific recognition protein 1 Ssrp1 -0.625 mannose receptor C-type 1 Mrc1 -0.637 UDP glucuronosyltransferase 1 family, polypeptide A7C Ugt1a7c -0.637 ADP ribosylation factor interacting protein 1 Arfip1 -0.646 tumor protein D52 Tpd52 -0.658 ATPase inhibitory factor 1 Atpif1 -0.681 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2 Smarcd2 -0.686 lysozyme Lyz2 -0.723 similar to 60S ribosomal protein L29 (P23) RGD1563300 -0.724 keratin 1 Krt1 -0.728 myosin light chain 6 Myl6 -0.729 fibrinogen like 2 Fgl2 -0.733 tumor protein D52 like 2 Tpd52l2 -0.736 chromosome 14 open reading frame 93 RGD1565222 -0.746 G protein-coupled receptor 4 Gpr4 -0.751 eukaryotic translation elongation factor 1 delta Eef1d -0.755 matrix Gla protein Mgp -0.755 S100 calcium binding protein A6 S100a6 -0.755 serine/arginine-rich splicing factor 5 Srsf5 -0.755 microtubule associated protein 1A Map1a -0.762 kelch repeat and BTB domain containing 3 Kbtbd3 -0.764 tropomyosin 4 Tpm4 -0.765 cytidine monophosphate N-acetylneuraminic acid synthetase Cmas -0.767 mitochondrial carrier 1 Mtch1 -0.775 caldesmon 1 Cald1 -0.778 kininogen 2 Kng2 -0.779 FK506 binding protein 9 Fkbp9 -0.78 desmin Des -0.787 acyl-CoA thioesterase 13 Acot13 -0.788 ezrin Ezr -0.793 heat shock protein family B (small) member 1 Hspb1 -0.801 xin actin binding repeat containing 1 Xirp1 -0.801 caldesmon 1 Cald1 -0.804 tropomyosin 2, beta Tpm2 -0.814

19 diaphanous related formin 1 Diaph1 -0.815 microfibril-associated glycoprotein 4-like LOC102553715 -0.829 scavenger receptor class A member 5 Scara5 -0.832 histone cluster 2 H2A family member c Hist2h2ac -0.835 secreted phosphoprotein 2 Spp2 -0.836 tropomyosin 1, alpha Tpm1 -0.837 fibronectin 1 Fn1 -0.843 small nuclear ribonucleoprotein D3 polypeptide Snrpd3 -0.843 serine/threonine kinase 24 Stk24 -0.843 procollagen C-endopeptidase enhancer Pcolce -0.845 ferritin heavy chain 1 Fth1 -0.846 milk fat globule-EGF factor 8 protein Mfge8 -0.857 translocase of outer mitochondrial membrane 22 Tomm22 -0.872 ARP1 actin-related protein 1 homolog B, centractin beta Actr1b -0.884 lysyl oxidase Lox -0.887 ribosomal protein lateral stalk subunit P1 Rplp1 -0.893 PCI domain containing 2 Pcid2 -0.898 CD38 molecule Cd38 -0.926 EGF containing fibulin like extracellular matrix protein 1 Efemp1 -0.934 charged multivesicular body protein 5 Chmp5 -0.935 CD163 molecule Cd163 -0.941 alkaline phosphatase, liver/bone/kidney Alpl -0.962 transmembrane p24 trafficking protein 5 Tmed5 -1 CDC42 effector protein 1 Cdc42ep1 -1.018 periostin Postn -1.02 signal sequence receptor subunit 2 Ssr2 -1.03 cytochrome P450 family 4 subfamily B member 1 Cyp4b1 -1.113 platelet and endothelial cell adhesion molecule 1 Pecam1 -1.113 keratin 5 Krt5 -1.174 keratin 10 Krt10 -1.32 glial fibrillary acidic protein Gfap -1.35 alpha-N-acetylgalactosaminidase Naga -1.363 charged multivesicular body protein 3 Chmp3 -1.473 LUC7 like 2, pre-mRNA splicing factor Luc7l2 -1.543

Table S6: Differentially up- or downregulated proteins in PCS exposed non-calcified versus vehicle exposed non-calcified aortic samples.

Protein Description Gene Symbol Log2 iTRAQ ratio taste receptor, type 2, member 110 Tas2r110 1.871 ribosomal protein L27a Rpl27a 1.723 ubiquinol-cytochrome c reductase, complex III subunit X Uqcr10 1.628 propionyl-CoA carboxylase beta subunit Pccb 1.612 F-box protein 6 Fbxo6 1.465 alpha-N-acetylgalactosaminidase Naga 1.329 3-hydroxyisobutyrate dehydrogenase Hibadh 1.325 potassium channel tetramerisation domain containing 12b LOC681355 1.255 cytochrome P450 family 4 subfamily B member 1 Cyp4b1 1.177 phosphoenolpyruvate carboxykinase 1 Pck1 1.173 KH-type splicing regulatory protein Khsrp 1.066 bridging integrator 1 Bin1 1.053 apolipoprotein N Apon 1.045 potassium channel tetramerisation domain containing 12b LOC681355 1.036 fibrinogen gamma chain Fgg 0.991 fetuin B Fetub 0.978 ethylmalonyl-CoA decarboxylase 1 Echdc1 0.969 fatty acid binding protein 4 Fabp4 0.968 hemoglobin subunit beta Hbb 0.96 carnitine palmitoyltransferase 1B Cpt1b 0.938 dipeptidase 1 (renal) Dpep1 0.919 fibrinogen alpha chain Fga 0.911 alpha-2-macroglobulin A2m 0.908 fascin actin-bundling protein 1 Fscn1 0.907 albumin Alb 0.904 ubiquinol-cytochrome c reductase, complex III subunit X Uqcr10 0.893 ethylmalonyl-CoA decarboxylase 1 Echdc1 0.891 MAP7 domain containing 1 Map7d1 0.88 RAB8A, member RAS oncogene family Rab8a 0.869 carbonyl reductase 1 LOC102556347 0.867 dipeptidase 1 (renal) Dpep1 0.86 histone cluster 1 H3 family member g Hist2h3c2 0.86 C-reactive protein Crp 0.844 hydroxyacyl-CoA dehydrogenase Hadh 0.839 histone cluster 1 H1 family member c Hist1h1c 0.837 cathepsin S Ctss 0.836 ADP ribosylation factor 3 Arf3 0.824 alpha-1-microglobulin/bikunin precursor Ambp 0.823 retinol binding protein 4 Rbp4 0.823 diazepam binding inhibitor, acyl-CoA binding protein Dbi 0.82 aldehyde dehydrogenase 3 family member A2 Aldh3a2 0.819 glycerol-3-phosphate dehydrogenase 1 Gpd1 0.815 family with sequence similarity 213 member A Fam213a 0.812 hydroxysteroid dehydrogenase like 2 Hsdl2 0.81 chromosome 14 open reading frame 93 RGD1565222 0.797 hemoglobin subunit beta Hbb 0.789

21 hemoglobin subunit beta-2-like LOC103694855 0.789 glutamic--pyruvic transaminase Gpt 0.786 glyceraldehyde-3-phosphate dehydrogenase Gapdh 0.782 RNA exonuclease 2 Rexo2 0.771 ribosomal protein L34 Rpl34 0.771 perilipin 1 Plin1 0.767 periostin Postn 0.762 eukaryotic translation initiation factor 3 subunit M Eif3m 0.759 electron transfer flavoprotein beta subunit Etfb 0.759 malate dehydrogenase 1 Mdh1 0.749 similar to 60S ribosomal protein L29 (P23) RGD1563300 0.749 serpin family A member 1 Serpina1 0.749 ATP binding cassette subfamily E member 1 Abce1 0.748 acyl-CoA synthetase long-chain family member 1 Acsl1 0.742 microtubule associated protein 6 Map6 0.74 fibrinogen beta chain Fgb 0.739 germ cell-less homolog 1 (Drosophila)-like LOC302576 0.739 SH3 domain containing GRB2 like, endophilin B1 Sh3glb1 0.733 phosphodiesterase 3A Pde3a 0.731 apolipoprotein A1 Apoa1 0.729 apolipoprotein A2 Apoa2 0.725 2,4-dienoyl-CoA reductase 1 Decr1 0.725 carboxylesterase 1 Ces1d 0.72 NADH:ubiquinone oxidoreductase subunit B6 Ndufb6 0.72 platelet and endothelial cell adhesion molecule 1 Pecam1 0.72 acetyl-CoA acyltransferase 2 Acaa2 0.716 microsomal glutathione S-transferase 1 Mgst1 0.712 adenylate kinase 2 Ak2 0.71 TGF-beta activated kinase 1 (MAP3K7) binding protein 1 Tab1 0.71 D-dopachrome tautomerase Ddt 0.708 histone cluster 1 H2B family member o Hist1h2bo 0.705 ribosomal protein L19 Rpl19 0.705 ribosomal protein L37a Rpl37a 0.705 ubiquinol-cytochrome c reductase complex assembly factor 1 Uqcc1 0.703 von Willebrand factor A domain containing 5A Vwa5a 0.695 phosphofructokinase, liver type Pfkl 0.694 aconitase 2 Aco2 0.692 dual specificity tyrosine phosphorylation regulated kinase 4 Dyrk4 0.692 succinate dehydrogenase complex subunit C Sdhc 0.692 enoyl-CoA delta isomerase 1 Eci1 0.689 high mobility group nucleosome binding domain 1 LOC100911295 0.678 similar to Histone H1.2 (H1 VAR.1) (H1c) LOC684828 0.678 protein kinase cAMP-dependent type II regulatory subunit alpha Prkar2a 0.675 neurolysin Nln 0.674 aldo-keto reductase family 1, member C-like Akr1cl 0.671 cysteine rich protein 2 Crip2 0.671 ribosomal protein S6 Rps6 0.664 calcium/calmodulin dependent protein kinase II gamma Camk2g 0.66 electron transfer flavoprotein alpha subunit Etfa 0.66 oxoglutarate dehydrogenase Ogdh 0.66 apolipoprotein M Apom 0.657 carnitine palmitoyltransferase 2 Cpt2 0.657

22 orosomucoid 1 Orm1 0.657 succinate-CoA ligase ADP-forming beta subunit Sucla2 0.657 NADH:ubiquinone oxidoreductase subunit B10 Ndufb10 0.653 6-phosphogluconolactonase Pgls 0.65 Parkinsonism associated deglycase Park7 0.647 ABRA C-terminal like Abracl 0.645 Sjogren syndrome antigen B Ssb 0.643 NADH:ubiquinone oxidoreductase core subunit S8 Ndufs8 0.641 charged multivesicular body protein 5 Chmp5 0.639 enoyl-CoA hydratase 1 Ech1 0.638 histone cluster 1 H4 family member j Hist1h4b 0.638 inositol polyphosphate-1-phosphatase Inpp1 0.638 fat storage inducing transmembrane protein 2 Fitm2 0.63 glutathione S-transferase alpha 3 Gsta3 0.626 ribosomal protein S24-like LOC100363469 0.614 FAU, ubiquitin like and ribosomal protein S30 fusion LOC100360647 0.606 coenzyme Q8A Coq8a 0.602 fibronectin 1 Fn1 0.589 collagen type XVIII alpha 1 chain Col18a1 0.583 signal sequence receptor subunit 4 Ssr4 0.583 actinin alpha 3 Actn3 0.579 mitochondrial pyruvate carrier 2 Mpc2 0.579 G protein subunit alpha 13 Gna13 0.575 microtubule associated protein RP/EB family member 1 Mapre1 0.572 collagen type IV alpha 2 chain Col4a2 0.564 HtrA serine peptidase 1 Htra1 0.564 ribosomal protein L39 Rpl39 0.563 milk fat globule-EGF factor 8 protein Mfge8 0.545 collagen type XV alpha 1 chain Col15a1 0.537 phosphotriesterase related Pter 0.537 D-dopachrome tautomerase Ddt 0.518 collagen type XII alpha 1 chain Col12a1 0.506 ryanodine receptor 2 Ryr2 0.506 farnesyl diphosphate synthase Fdps 0.494 carboxypeptidase A3 Cpa3 -0.494 ezrin Ezr -0.494 myosin VI Myo6 -0.502 UDP glucuronosyltransferase 1 family, polypeptide A7C Ugt1a7c -0.502 drebrin 1 Dbn1 -0.51 serine and arginine rich splicing factor 3 Srsf3 -0.519 ubiquitin specific peptidase 9, X-linked Usp9x -0.519 DnaJ heat shock protein family (Hsp40) member B11 Dnajb11 -0.535 BCL2 associated athanogene 3 Bag3 -0.543 cell growth regulator with EF-hand domain 1 Cgref1 -0.543 ectonucleoside triphosphate diphosphohydrolase 2 Entpd2 -0.543 histidine-rich glycoprotein Hrg -0.543 sphingomyelin phosphodiesterase acid like 3B Smpdl3b -0.543 CSK, non-receptor tyrosine kinase Csk -0.56 protein O-glucosyltransferase 1 Poglut1 -0.56 collagen, type I, alpha 2 NEWGENE_621351 -0.564 fibrillin 1 Fbn1 -0.573 mannose receptor C type 2 Mrc2 -0.576

23 cell adhesion molecule 3 Cadm3 -0.582 galactokinase 1 Galk1 -0.593 enabled homolog (Drosophila) Enah -0.596 tubulin beta 3 class III Tubb3 -0.602 kelch repeat and BTB domain containing 3 Kbtbd3 -0.609 proline and arginine rich end leucine rich repeat protein Prelp -0.609 galectin 1 Lgals1 -0.619 ezrin Ezr -0.62 fibromodulin Fmod -0.629 oxysterol binding protein Osbp -0.637 cysteine and glycine rich protein 2 Csrp2 -0.638 fibulin 5 Fbln5 -0.638 translocase of outer mitochondrial membrane 22 Tomm22 -0.638 ATP synthase, H+ transporting, mitochondrial Fo complex subunit F6 Atp5j -0.646 histone cluster 1 H2A family member b Hist1h2ak -0.646 myelin basic protein Mbp -0.646 hippocalcin like 4 Hpcal4 -0.654 cutA divalent cation tolerance homolog Cuta -0.656 nephroblastoma overexpressed Nov -0.656 sulfatase 1 Sulf1 -0.656 olfactory receptor family 1 subfamily M member 1 Olr1121 -0.665 solute carrier family 20 member 2 Slc20a2 -0.666 GATA binding protein 3 Gata3 -0.674 EWS RNA-binding protein 1 Ewsr1 -0.683 signal recognition particle 68 Srp68 -0.688 gamma-butyrobetaine hydroxylase 1 Bbox1 -0.692 fibrinogen like 2 Fgl2 -0.692 ATPase inhibitory factor 1 Atpif1 -0.696 myosin light chain 9 Myl9 -0.699 RuvB like AAA ATPase 1 Ruvbl1 -0.702 keratin 1 Krt1 -0.703 G protein-coupled receptor 4 Gpr4 -0.707 brain abundant, membrane attached signal protein 1 Basp1 -0.709 myosin light chain 6 Myl6 -0.71 pro-apoptotic WT1 regulator Pawr -0.712 glutathione peroxidase 3 Gpx3 -0.714 PYD and CARD domain containing Pycard -0.718 CD34 molecule Cd34 -0.723 dermatopontin Dpt -0.725 CD163 molecule Cd163 -0.727 CD38 molecule Cd38 -0.727 hydroxysteroid 17-beta dehydrogenase 11 Hsd17b11 -0.728 matrix Gla protein Mgp -0.728 desmin Des -0.742 caldesmon 1 Cald1 -0.743 eukaryotic translation initiation factor 2 subunit beta Eif2s2 -0.746 elongin B Elob -0.76 osteoglycin Ogn -0.766 lumican Lum -0.77 signal sequence receptor subunit 2 Ssr2 -0.778 heat shock protein family B (small) member 1 Hspb1 -0.783

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2 Smarcd2 -0.804 keratin 5 Krt5 -0.805 tropomyosin 2, beta Tpm2 -0.808 CDC42 effector protein 1 Cdc42ep1 -0.809 collagen type I alpha 1 chain Col1a1 -0.813 hyaluronan and proteoglycan link protein 1 Hapln1 -0.813 mitochondrial carrier 1 Mtch1 -0.814 EGF containing fibulin like extracellular matrix protein 1 Efemp1 -0.821 ARP1 actin-related protein 1 homolog B, centractin beta Actr1b -0.834 asporin Aspn -0.836 lysyl oxidase Lox -0.853 tropomyosin 1, alpha Tpm1 -0.856 purine rich element binding protein B Purb -0.857 G protein pathway suppressor 1 Gps1 -0.862 ferritin heavy chain 1 Fth1 -0.873 decorin Dcn -0.88 microtubule associated protein 1A Map1a -0.886 microfibril-associated glycoprotein 4-like LOC102553715 -0.888 procollagen C-endopeptidase enhancer Pcolce -0.923 diaphanous related formin 1 Diaph1 -0.937 glial fibrillary acidic protein Gfap -0.939 alkaline phosphatase, liver/bone/kidney Alpl -0.946 interleukin enhancer binding factor 2 Ilf2 -0.994 charged multivesicular body protein 3 Chmp3 -1.026 mast cell protease 1 Mcpt1 -1.044 PCI domain containing 2 Pcid2 -1.052 keratin 10 Krt10 -1.077 secreted phosphoprotein 2 Spp2 -1.078 tumor protein D52 like 2 Tpd52l2 -1.143 myelin protein zero Mpz -1.47 LUC7 like 2, pre-mRNA splicing factor Luc7l2 -1.613

Table S7: Biochemical parameters of CKD rats exposed to vehicle, indoxyl sulfate or p-cresyl sulfate for 4 days.

Biochemical parameter Day 0 Day 4

Serum creatinine (mg/dl) Vehicle 0.32 ± 0.02 1.46 ± 0.24

IS 0.31 ± 0.03 1.51 ± 0.21

PCS 0.31 ± 0.01 1.56 ± 0.01

Serum IS (µM) Vehicle 4.67 ± 0.54 35.98 ± 9.32

IS 2.59 ± 0.72 316.84 ± 132.80 a

PCS 2.84 ± 0.64 65.16 ± 31.99

Serum PCS (µM) Vehicle 0.09 ± 0.04 12.66 ± 6.61

IS 0.10 ± 0.04 15.44 ± 9.34

PCS 0.06 ± 0.02 205.12 ± 74.75 a

Data are presented as mean ± SEM. a P<0.05 versus vehicle.