Aryl hydrocarbon receptor is activated in patients and mice with chronic kidney disease

Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR–/– CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR–/– mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD. Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR–/– CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR–/– mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD. Chronic kidney disease (CKD) is an emerging epidemic. CKD increased by 73% from 1990 to 2013 as a cause of deaths worldwide.1GBD 2013 Mortality and Causes of Death CollaboratorsGlobal, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013.Lancet. 2015; 385: 117-171Abstract Full Text Full Text PDF PubMed Scopus (5402) Google Scholar CKD is associated with an increased risk of death, especially from cardiovascular disease (CVD).2Gansevoort R.T. Correa-Rotter R. Hemmelgarn B.R. et al.Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention.Lancet. 2013; 382: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar, 3Tonelli M. Muntner P. 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De Smet R. et al.Normal and pathologic concentrations of uremic toxins.J Am Soc Nephrol. 2012; 23: 1258-1270Crossref PubMed Scopus (634) Google Scholar Among this last group, increased levels of the indolic toxins indoxyl sulfate (IS) and indole-3 acetic acid (IAA) are associated with increased risk of death and cardiovascular events.7Barreto F.C. Barreto D.V. Liabeuf S. et al.Serum indoxyl sulfate is associated with vascular disease and mortality in chronic kidney disease patients.Clin J Am Soc Nephrol. 2009; 4: 1551-1558Crossref PubMed Scopus (662) Google Scholar, 8Dou L. Sallée M. Cerini C. et al.The cardiovascular effect of the uremic solute indole-3 acetic Acid.J Am Soc Nephrol. 2015; 26: 876-887Crossref PubMed Scopus (196) Google Scholar In addition, numerous studies have demonstrated the deleterious effect of indolic toxins on renal and vascular cells.9Vanholder R. Schepers E. Pletinck A. et al.The uremic toxicity of indoxyl sulfate and p-cresyl sulfate: a systematic review.J Am Soc Nephrol. 2014; 25: 1897-1907Crossref PubMed Scopus (437) Google Scholar The cellular receptor of indolic solutes was identified as aryl hydrocarbon receptor (AHR).10Heath-Pagliuso S. Rogers W.J. Tullis K. et al.Activation of the Ah receptor by tryptophan and tryptophan metabolites.Biochemistry. 1998; 37: 11508-11515Crossref PubMed Scopus (240) Google Scholar, 11Schroeder J.C. Dinatale B.C. Murray I.A. et al.The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor.Biochemistry. 2010; 49: 393-400Crossref PubMed Scopus (218) Google Scholar AHR is an intracellular receptor for xenobiotics such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), and benzo[a]pyrene, a chemical found in tobacco smoke.12Hankinson O. The aryl hydrocarbon receptor complex.Annu Rev Pharmacol Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1431) Google Scholar AHR also binds endogenous ligands, including metabolites of arachidonic acid and tryptophan, such as kynurenine.13Denison M.S. Nagy S.R. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals.Annu Rev Pharmacol Toxicol. 2003; 43: 309-334Crossref PubMed Scopus (1437) Google Scholar, 14Stockinger B. Hirota K. Duarte J. et al.External influences on the immune system via activation of the aryl hydrocarbon receptor.Semin Immunol. 2011; 23: 99-105Crossref PubMed Scopus (142) Google Scholar AHR resides in the cytoplasm of mammalian cells in a multiprotein complex that includes HSP90 and AHR-interacting protein.12Hankinson O. The aryl hydrocarbon receptor complex.Annu Rev Pharmacol Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1431) Google Scholar, 15Denison M.S. Soshilov A.A. He G. et al.Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor.Toxicol Sci. 2011; 124: 1-22Crossref PubMed Scopus (532) Google Scholar, 16Ma Q. Whitlock J.P. A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.J Biol Chem. 1997; 272: 8878-8884Crossref PubMed Scopus (362) Google Scholar AHR-ligand complex translocates to the nucleus, where it forms a heterodimeric complex with the aryl hydrocarbon nuclear translocator.12Hankinson O. The aryl hydrocarbon receptor complex.Annu Rev Pharmacol Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1431) Google Scholar, 15Denison M.S. Soshilov A.A. He G. et al.Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor.Toxicol Sci. 2011; 124: 1-22Crossref PubMed Scopus (532) Google Scholar The complex binds to a DNA consensus sequence12Hankinson O. The aryl hydrocarbon receptor complex.Annu Rev Pharmacol Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1431) Google Scholar, 15Denison M.S. Soshilov A.A. He G. et al.Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor.Toxicol Sci. 2011; 124: 1-22Crossref PubMed Scopus (532) Google Scholar present in the promoters of a wide variety of genes, including those coding for enzymes involved in xenobiotic detoxification (CYP1A1, CYP1A2, and CYP1B1) and the AHR repressor, AHRR.12Hankinson O. The aryl hydrocarbon receptor complex.Annu Rev Pharmacol Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1431) Google Scholar, 15Denison M.S. Soshilov A.A. He G. et al.Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor.Toxicol Sci. 2011; 124: 1-22Crossref PubMed Scopus (532) Google Scholar A genomic, aryl hydrocarbon nuclear translocator–independent pathway for AHR-mediated gene expression has recently been reported,17Murray I.A. Patterson A.D. Perdew G.H. Aryl hydrocarbon receptor ligands in cancer: friend and foe.Nat Rev Cancer. 2014; 14: 801-814Crossref PubMed Scopus (532) Google Scholar as well as a role of AHR as a component of numerous signaling pathways, independent of its ability to bind to DNA.17Murray I.A. Patterson A.D. Perdew G.H. Aryl hydrocarbon receptor ligands in cancer: friend and foe.Nat Rev Cancer. 2014; 14: 801-814Crossref PubMed Scopus (532) Google Scholar In humans exposed to AHR agonists such as TCDD or PCB 126, the risk for cardiovascular events is increased.18Sallée M. Dou L. Cerini C. et al.The aryl hydrocarbon receptor-activating effect of uremic toxins from tryptophan metabolism: a new concept to understand cardiovascular complications of chronic kidney disease.Toxins. 2014; 6: 934-949Crossref PubMed Scopus (159) Google Scholar This association was recognized by the US government after exposure of veterans to Agent Orange.19Committee to Review the Health Effects in Vietnam Veterans of Exposure to Herbicides (Tenth Biennial Update), Board on the Health of Select Populations, Institute of Medicine et al. Veterans and Agent Orange: Update 2014. Washington, DC: National Academies Press; 2016.Google Scholar In ApoE–/– mice, AHR activation by TCDD is associated with acceleration of atherosclerosis.20Shan Q. Wang J. Huang F. et al.Augmented atherogenesis in ApoE-null mice co-exposed to polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin.Toxicol Appl Pharmacol. 2014; 276: 136-146Crossref PubMed Scopus (15) Google Scholar In rats, PCB 126 exposure increases CVD risk factors: serum cholesterol, blood pressure, and heart weight.21Lind P.M. Orberg J. Edlund U.-B. et al.The dioxin-like pollutant PCB 126 (3,3’,4,4’,5-pentachlorobiphenyl) affects risk factors for cardiovascular disease in female rats.Toxicol Lett. 2004; 150: 293-299Crossref PubMed Scopus (97) Google Scholar The activation of AHR by IS and IAA has been demonstrated to contribute to vascular dysfunction.8Dou L. Sallée M. Cerini C. et al.The cardiovascular effect of the uremic solute indole-3 acetic Acid.J Am Soc Nephrol. 2015; 26: 876-887Crossref PubMed Scopus (196) Google Scholar, 22Gondouin B. Cerini C. Dou L. et al.Indolic uremic solutes increase tissue factor production in endothelial cells by the aryl hydrocarbon receptor pathway.Kidney Int. 2013; 84: 733-744Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar In endothelial cells and in vascular smooth muscle cells, AHR activation increases the expression and activity of tissue factor, leading to a procoagulant state.22Gondouin B. Cerini C. Dou L. et al.Indolic uremic solutes increase tissue factor production in endothelial cells by the aryl hydrocarbon receptor pathway.Kidney Int. 2013; 84: 733-744Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 23Shivanna S. Kolandaivelu K. Shashar M. et al.The aryl hydrocarbon receptor is a critical regulator of tissue factor stability and an antithrombotic target in uremia.J Am Soc Nephrol. 2016; 27: 189-201Crossref PubMed Scopus (77) Google Scholar It also induces an increased expression and activity of the pro-inflammatory enzyme cyclooxygenase-2 in endothelial cells.8Dou L. Sallée M. Cerini C. et al.The cardiovascular effect of the uremic solute indole-3 acetic Acid.J Am Soc Nephrol. 2015; 26: 876-887Crossref PubMed Scopus (196) Google Scholar The vascular dysfunction induced by AHR activation could lead to atherothrombosis and plays a role in the increased risk of myocardial infarction, peripheral artery disease, and stroke observed in CKD.24Vanholder R. Fouque D. Glorieux G. et al.Clinical management of the uraemic syndrome in chronic kidney disease.Lancet Diabetes Endocrinol. 2016; 4: 360-373Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Despite strong evidence of AHR activation by indolic uremic toxins in vitro, studies of AHR activation in CKD are scarce. The present study aimed to demonstrate that AHR is activated in patients with various stages of CKD. In CKD mice, we also studied AHR activation in the vascular wall and the role of IS as a representative uremic AHR agonist. We studied a cohort of 116 patients with CKD (51 with stage 3–5 CKD and 65 with stage 5D CKD) (Table 1) and compared these patients with 52 healthy controls. We analyzed the activation of AHR by serum samples using the AHR-responsive chemically activated luciferase expression cell bioassay, a method commonly used for the screening of samples for the presence of TCDD, dioxin-like compounds, and AHR agonists and/or antagonists.25Denison M.S. Zhao B. Baston D.S. et al.Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals.Talanta. 2004; 63: 1123-1133Crossref PubMed Scopus (94) Google Scholar, 26Brennan J.C. He G. Tsutsumi T. et al.Development of species-specific Ah receptor-responsive third generation CALUX cell lines with enhanced responsiveness and improved detection limits.Environ Sci Technol. 2015; 49: 11903-11912Crossref PubMed Scopus (57) Google Scholar The serum AHR-activating potential (AHR-AP) was significantly higher in patients with stage 3 to 5 CKD (P < 0.05) and stage 5D CKD (P < 0.0001) than in controls (Figure 1a). Mean ± SD values of AHR-AP were 22 ± 9 arbitrary units (AU) (range: 5–44 AU), 37 ± 24 AU (range: 6–121 AU), and 79 ± 56 AU (range: 8–259 AU), respectively, in controls, in patients with stage 3 to 5 CKD, and in patients with stage 5D CKD (Figure 1a). We then examined whether AHR-AP of uremic serum could be counteracted by an AHR antagonist. The addition of the AHR antagonist CH223191 reduced by 46% the AHR-AP of serum from patients with stage 5D CKD (P < 0.01) (Figure 1b). CH223191 alone had no effect, and induced a slight, not significant decrease in the AHR-AP of control serum (Figure 1b).Table 1Baseline characteristics of the CKD populationCharacteristicsAll patients (n = 116)AHR-AP < 44AUAHR-AP ≥ 44AUP valueAge (yr)68 (23; 89)63 (31; 89)72 (23; 89)<0.05Gender ratio (W:M)40:7619:3621:401Body mass index (kg/m2)24.6 (15.8; 47)24.9 (16.8; 37.9)24.5 (15.8; 47)0.7Dialyzed patients (%)65 (56%)19 (34%)46 (75%)<0.0001eGFRaCalculated by Modification of Diet in Renal Disease (MDRD) study formula for nondialyzed CKD patients (n = 51). (ml/min per 1.73 m2)25 (8; 59)30 (11-59)14 (8; 53)<0.01Kidney disease Glomerulonephritis22 (19%)8 (15%)14 (23%)0.3 ADPKD11 (9%)6 (11%)5 (8%)0.7 Vascular32 (28%)13 (24%)19 (31%)0.4 Interstitial23 (20%)15 (27%)8 (13%)0.06 Other hereditary7 (6%)5 (9%)2 (4%)0.2 Unknown21 (18%)8 (14%)13 (21%)0.4Hypertension101 (87%)47 (85%)54 (89%)0.8Systolic blood pressure (mm Hg)141± 24143 ± 28139 ± 190.5Diastolic blood pressure (mm Hg)77 ± 1479 ± 1574 ± 12<0.05Current smokers47 (41%)20 (36%)27 (44%)0.4History of cardiovascular diseases41 (35%)17 (31%)24 (39%)0.4Phosphate binders60 (52%)19 (34%)41 (67%)<0.001Antihypertensive drugs86 (74%)43 (78%)43 (70%)0.4Statins37 (32%)13 (24%)24 (39%)0.07Antiplatelet drugs47 (40%)17 (31%)30 (49%)0.058Anticoagulant drugs26 (22%)11 (20%)15 (24%)0.6Erythropoietin therapy58 (50%)17 (31%)41 (67%)<0.001Serum CRP level (mg/l)4 (0; 78)4.6 (0.1; 54)4 (0; 78)0.5Hemoglobin (g/dl)12.0 (8.8; 16.3)12.4 (9.5; 16.3)11.3 (8.8; 14.4)<0.001Serum bicarbonate level (mmol/l)22.7 ± 3.023.7 ± 3.121.8 ± 2.6<0.01Serum albumin level (g/l)36 (26; 44)36 (26; 44)36 (28; 43)0.7Serum calcium level (mmol/l)2.34 ± 0.122.34 ± 0.102.34 ± 0.140.9Serum phosphate level (mmol/l)1.33 (0.65; 3.17)1.23 (0.65; 3.17)1.5 (0.7; 2.9)<0.05Serum cholesterol level (mmol/l)4.6 (1.9; 9.1)5.5 (2.7; 9.1)4.3 (1.9; 7.3)<0.001Serum LDL cholesterol level (mmol/l)2.9 ± 1.13.2 ± 1.12.6 ± 0.9<0.01Serum triglyceride level (mmol/l)1.5 (0.4; 5.9)1.4 (0.4; 5.9)1.6 (0.5; 3.6)0.7Serum β2 microglobulin level (mg/l)22.6 (2.8; 66.9)7.9 (2.8; 66.9)27.6 (3.8; 56.4)<0.0001Serum Indole-3 acetic acid level (μM)2.9 (0.6; 19.1)2.2 (0.6; 16.3)3.5 (0.7; 19.1)<0.05Serum indoxyl sulfate level (μM)43.7 (0.2; 256.2)13.4 (0.2; 157.8)78.9 (1.2; 256.2)<0.0001AHR, aryl hydrocarbon receptor; AP, activating potential; ADPKD, autosomal dominant polycystic kidney disease; AU, arbitrary units; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein; M, men; W, women.Results are given as mean ± SD if distribution is Gaussian, or in median (min; max) if not.a Calculated by Modification of Diet in Renal Disease (MDRD) study formula for nondialyzed CKD patients (n = 51). Open table in a new tab AHR, aryl hydrocarbon receptor; AP, activating potential; ADPKD, autosomal dominant polycystic kidney disease; AU, arbitrary units; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein; M, men; W, women. Results are given as mean ± SD if distribution is Gaussian, or in median (min; max) if not. At baseline, AHR-AP from all patients with stage 3 to 5D CKD negatively correlated with hemoglobin (r = –0.31, P < 0.01), serum bicarbonate (r = –0.39, P < 0.001), serum cholesterol (r = –0.37, P < 0.0001), low-density lipoprotein cholesterol (r = –0.37, P < 0.0001), and diastolic blood pressure (r = –0.27, P < 0.01) (Table 2). AHR-AP positively correlated with age (r = 0.26, P < 0.01), CKD stage (r = 0.52, P < 0.0001), phosphate binder therapy (r = 0.37, P < 0.0001), erythropoietin therapy (r = 0.39, P < 0.0001), serum phosphate (r = 0.25, P < 0.01), and the uremic toxins urea (r = 0.33, P < 0.001), creatinine (r = 0.46, P < 0.0001), β2-microglobulin (r = 0.41, P < 0.0001), IS (r = 0.62, P < 0.0001), and IAA (r = 0.26, P < 0.01) (Table 2). In multivariate linear regression analysis in stage 3 to 5D CKD patients, age (estimate = 0.68, P < 0.05), serum bicarbonate (estimate = –4.12, P < 0.05), and serum IS (estimate = 0.62, P < 0.001) were significantly associated with AHR-AP (Table 3).Table 2Spearman correlations of baseline characteristics with AHR-AP of serum in the stage 3 to 5D CKD cohort (n = 116)VariablesAHR-APrP valueAge0.26<0.01Gender–0.030.7Body mass index0.020.8Systolic blood pressure–0.130.2Diastolic blood pressure–0.27<0.01Current smoking–0.010.9eGFRaCalculated by Modification of Diet in Renal Disease (MDRD) study formula for nondialyzed CKD patients (n = 51).–0.56<0.0001CKD stage0.52<0.0001Kidney disease0.040.6Phosphate binders0.37<0.0001Antihypertensive drugs–0.150.1Statins0.140.1Antiplatelet drugs0.180.06Anticoagulant drugs0.030.7Erythropoietin therapy0.39<0.0001Serum CRP level0.070.4Hemoglobin–0.31<0.01Serum bicarbonate level–0.39<0.001Serum albumin level–0.050.6Serum calcium level0.070.4Serum phosphate level0.25<0.01Serum cholesterol level–0.37<0.0001Serum LDL cholesterol level–0.37<0.0001Serum triglyceride level0.080.3Serum urea level0.33<0.001Serum creatinine level0.46<0.0001Serum β2 microglobulin level0.41<0.0001Serum Indole-3 acetic acid level0.26<0.01Serum indoxyl sulfate level0.62<0.0001AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein.a Calculated by Modification of Diet in Renal Disease (MDRD) study formula for nondialyzed CKD patients (n = 51). Open table in a new tab Table 3Multivariate linear regression analysis for evaluating the relation between independent variables and AHR-AP in stage 3 to 5D CKD patients (n = 116)VariablesEstimateAHR-AP95% CIP valueAge0.68[0.04 to 1.33]<0.05Diastolic blood pressure0.12[−0.49 to 0.73]0.7CKD stage Stage 4–6.15[–38.59 to 26.29]0.7 Stage 523.7[–24.27 to 71.68]0.3 Stage 5D22.03[–30.87 to 74.92]0.4Phosphate binders3.56[−19.44 to 26.57]0.7Erythropoietin therapy12.68[−6.64 to 32]0.2Hemoglobin5.06[−1.57 to 11.69]0.13Serum bicarbonate level–4.12[−7.27 to -0.98]<0.05Serum phosphate level–10.96[−32.29 to 10.37]0.3Serum cholesterol level–0.67[−7.64 to 6.3]0.8Serum urea level–0.15[−1.94 to 1.64]0.8Serum creatinine level–0.06[−0.12 to 0.01]0.11Serum β2 microglobulin level0.15[−1.18 to 1.49]0.8Serum Indole-3 acetic acid level–0.29[−2.22 to 1.63]0.7Serum indoxyl sulfate level0.62[0.41 to 0.83]<0.001AHR, aryl hydrocarbon receptor; AP, activating potential; CI, confidence interval; CKD, chronic kidney disease. Open table in a new tab AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein. AHR, aryl hydrocarbon receptor; AP, activating potential; CI, confidence interval; CKD, chronic kidney disease. We analyzed the correlations of baseline characteristics and serum AHR-AP in the stage 3 to 5 CKD subcohort, and in the hemodialyzed stage 5D CKD subcohort. In the stage 3 to 5 CKD subcohort (Table 4), AHR-AP negatively correlated with estimated glomerular filtration rate (eGFR; r = –0.56, P < 0.0001) estimated by the MDRD simplified formula (Table 4 and Figure 1c), hemoglobin (r = –0.41, P < 0.001), serum bicarbonate (r = –0.39, P < 0.01), serum calcium (r = –0.46, P < 0.001), and serum cholesterol (r = –0.37, P < 0.01) (Table 4). AHR-AP positively correlated with CKD stage (r = 0.56, P < 0.0001), phosphate binder therapy (r = 0.46, P < 0.01), erythropoietin therapy (r = 0.29, P < 0.05), serum phosphate (r = 0.35, P < 0.01), urea (r = 0.53, P < 0.0001), creatinine (r = 0.64, P < 0.0001), β2-microglobulin (r = 0.69, P < 0.0001), and IS (r = 0.60, P < 0.0001) (Table 4). In multivariate linear regression analysis in stage 3 to 5 CKD patients, serum β2-microglobulin (estimate = 3.7, P < 0.05), and serum IS (estimate = 0.81, P < 0.05) were significantly associated with AHR-AP (Table 5).Table 4Spearman correlations of baseline characteristics with AHR-AP of serum in the CKD stage 3 to 5 subcohort (n = 51)VariablesAHR-APrP valueAge0.120.4Gender0.040.7Body mass index–0.190.2Systolic blood pressure–0.140.3Diastolic blood pressure–0.190.2Current smoking0.250.08eGFRaCalculated by Modification of Diet in Renal Disease (MDRD) study formula.–0.56<0.0001CKD stage0.56<0.0001Kidney disease0.100.5Phosphate binders0.46<0.01Antihypertensive drugs–0.160.2Statins0.240.08Antiplatelet drugs0.130.4Anticoagulant drugs–0.010.9Erythropoietin therapy0.29<0.05Serum CRP level0.070.6Hemoglobin–0.41<0.001Serum bicarbonate level–0.39<0.01Serum albumin level–0.180.2Serum calcium level–0.46<0.001Serum phosphate level0.35<0.01Serum cholesterol level–0.37<0.01Serum LDL cholesterol level–0.270.06Serum triglyceride level0.060.7Serum urea level0.53<0.0001Serum creatinine level0.64<0.0001Serum β2 microglobulin level0.69<0.0001Serum Indole-3 acetic acid level0.240.1Serum indoxyl sulfate level0.60<0.0001AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein.a Calculated by Modification of Diet in Renal Disease (MDRD) study formula. Open table in a new tab Table 5Multivariate linear regression analysis for evaluating the relation between independent variables and AHR-AP in CKD stage 3 to 5 subcohort (n = 51)VariablesEstimateAHR-AP95% CIP valueAge0.09[–0.49 to 0.68]0.7Diastolic blood pressure0.27[–0.4 to 0.94]0.4CKD stage Stage 4–4.53[–23.67 to 14.61]0.6 Stage 520.37[–17.4 to 58.13]0.3Phosphate binders2.67[−25.31 to 30.66]0.8Erythropoietin therapy12.7[−6.31 to 31.71]0.2Hemoglobin–0.48[−5.78 to 4.82]0.9Serum bicarbonate level–1.07[−3.39 to 1.24]0.3Serum calcium level–72.87[−160.74 to 15]0.1Serum phosphate level–28.11[–61.51 to 5.28]0.1Serum cholesterol level–3.86[9.98 to 2.25]0.2Serum urea level0.83[–1.08 to 2.75]0.4Serum creatinine level–0.2[−0.44 to 0.03]0.1Serum β2 microglobulin level3.7[0.07 to 7.32]<0.05Serum Indole-3 acetic acid level–1.3[−3.63 to 1.03]0.3Serum indoxyl sulfate level0.81[0.21 to 1.41]<0.05AHR, aryl hydrocarbon receptor; AP, activating potential; CI, confidence interval; CKD, chronic kidney disease. Open table in a new tab AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; LDL, low-density lipoprotein. AHR, aryl hydrocarbon receptor; AP, activating potential; CI, confidence interval; CKD, chronic kidney disease. In the hemodialyzed stage 5D CKD subcohort (Table 6), AHR-AP negatively correlated with serum bicarbonate (r = –0.33, P < 0.01) and positively correlated with IS (r = 0.51, P < 0.0001) (Table 6 and Figure 1d). In multivariate linear regression analysis in hemodialyzed stage 5D CKD patients, serum IS (estimate = 0.62, P < 0.001) was significantly associated with AHR-AP (Table 7).Table 6Spearman correlations of baseline characteristics with AHR-AP of serum in the hemodialyzed CKD stage 5D subcohort (n = 65)VariablesAHR-APrP valueAge0.180.1Gender–0.020.9Body mass index0.10.5Systolic blood pressure–0.090.5Diastolic blood pressure–0.040.8Current smoking–0.050.7Kidney disease–0.120.3Phosphate binders–0.060.6Antihypertensive drugs–0.040.7Statins0.020.9Antiplatelet drugs–0.120.3Anticoagulant drugs–0.080.5Erythropoietin therapy0.190.1Serum CRP level–0.140.3Hemoglobin–0.040.8Serum bicarbonate level–0.33<0.01Serum albumin level–0.20.1Serum calcium level0.20.1Serum phosphate level–0.090.5Serum cholesterol level–0.010.9Serum LDL cholesterol level–0.070.6Serum triglyceride level0.150.3Serum urea level0.040.7Serum creatinine level0.001Serum β2 microglobulin level–0.190.1Serum Indole-3 acetic acid level–0.170.2Serum indoxyl sulfate level0.51<0.0001AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; LDL, low-density lipoprotein. Open table in a new tab Table 7Multivariate linear regression analysis for evaluating the relation between independent variables and AHR-AP in hemodialyzed CKD stage 5D subcohort (n = 65)VariablesEstimateAHR-AP95% CIP valueAge0.71[–0.42 to 1.83]0.2Diastolic blood pressure0.28[–0.66 to 1.23]0.5Phosphate binders1.02[−33.84 to 35.88]0.9Erythropoietin therapy15.66[−13.86 to 45.18]0.3Hemoglobin12.11[−0.41 to 24.63]0.06Serum bicarbonate level–6.71[−13.72 to 0.3]0.06Serum phosphate level–2.54[–32.64 to 27.56]0.9Serum cholesterol level2.54[9.81 to 14.88]0.7Serum urea level–0.99[–4.06 to 2.08]0.5Serum creatinine level–0.07[−0.17 to 0.03]0.1Serum β2 microglobulin level0.23[–1.6 to 2.05]0.8Serum Indole-3 acetic acid level–0.49[−3.32 to 2.34]0.7Serum indoxyl sulfate level0.62[0.33 to 0.9]<0.001AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CI, confidence interval. Open table in a new tab AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CRP, C-reactive protein; LDL, low-density lipoprotein. AHR, aryl hydrocarbon receptor; AP, activating potential; CKD, chronic kidney disease; CI, confidence interval. We then analyzed whether the level of AHR agonists in serum could be decreased after a dialysis session. As shown in Figure 1e, the AHR-AP of sera drawn after a dialysis session was decreased compared with AHR-AP of sera drawn before dialysis (P < 0.01). This decrease corresponded to an AHR-AP reduction ratio of 27% (Supplementary Table S1). However, the values of AHR-AP after hemodialysis sessions remained higher than the values of AHR-AP of control serum. We measured the reduction of AHR agonists, IS and IAA, after a dialysis session. We observed an IS reduction ratio of 62% and an IAA reduction ratio of 60% (Supplementary Figure S1A and S1B, and Supplementary Table S1). The reduction of serum AHR-AP after dialysis was related to the reduction of serum IS (r = 0.47, P < 0.05), but not to the reduction of IAA (Table 8), small soluble uremic toxin urea, or middle molecule β2microglobulin (Table 8).Table 8Spearman correlations between the decrease in serum AHR-AP and the decrease in uremic toxin levels during hemodialysis sessionVar