title: Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent manner.
abstract: Advanced chronic kidney disease (CKD) patients encounter anemia through insufficient erythropoietin (EPO) production by peritubular fibroblasts. Recent studies showed an increase in EPO production by pharmacological activation of hypoxia-inducible transcription factors (HIFs) in dialysis patients, suggesting that desensitization of the oxygen-sensing mechanism is responsible for the development of renal anemia. Our recent work demonstrated that indoxyl sulfate (IS), a uremic toxin, dysregulates oxygen metabolism in tubular cells. Here we provide evidence of an additional property that IS impairs oxygen sensing in EPO-producing cells. HepG2 cells were stimulated with cobalt chloride (CoCl(2)) or hypoxia under varying concentrations of IS. EPO mRNA was evaluated by quantitative PCR. Nuclear accumulation of HIF-α was evaluated by western blotting. Transcriptional activity of HIF was checked by hypoxia-responsive element (HRE)-luciferase reporter assay. The impact of IS was further evaluated in vivo by administering rats with indole, a metabolic precursor of IS, and subjecting them to CoCl(2) stimulation, in which renal EPO mRNA as well as plasma EPO levels were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Although IS induced cellular toxicity at relatively high concentrations (2.5 mM), EPO mRNA expression was significantly suppressed by IS at concentrations below cytotoxic ranges. In HepG2 cells, IS treatment decreased nuclear accumulation of HIF-α proteins and suppressed HRE-luciferase activity following hypoxia. Furthermore, administration of rats with indole suppressed renal EPO mRNA expression and plasma EPO levels, corroborating in vitro findings. Results of the present study provide a possible connection between a uremic toxin and the desensitization of the oxygen-sensing mechanism in EPO-producing cells, which may partly explain inadequate EPO production in hypoxic kidneys of CKD patients.
authors.affiliation: Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Japan.
authors.forename: Chih-Kang
authors.initials: CK
authors.lastname: Chiang
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Tanaka
authors.forename: Reiko
authors.initials: R
authors.lastname: Inagi
authors.forename: Toshiro
authors.initials: T
authors.lastname: Fujita
authors.forename: Masaomi
authors.initials: M
authors.lastname: Nangaku
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003035:Cobalt; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D007200:Indican / Q000633:toxicity*; D007211:Indoles / Q000008:administration & dosage / Q000494:pharmacology; D007676:Kidney Failure, Chronic / Q000503:physiopathology*; D008156:Luciferases; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D014511:Uremia / Q000097:blood*
chemical_list: D051793:Hypoxia-Inducible Factor 1; D007211:Indoles; D004921:Erythropoietin; D003035:Cobalt; C030374:indole; D008156:Luciferases; C018021:cobaltous chloride; D007200:Indican; D010100:Oxygen