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Kidney International, Vol. 51 (1997), pp. 497-507 Effects of antioxidant vitamins on renal and hepatic erythropoietin production WoLrGANG JELKMANN, Horst PAGEL, THoMas HeLLwiG, and JOACHIM FANDREY Institute of Physiology, University of Lilbeck, Libeck, and Institute of Physiology, University of Bonn, Bonn, Germany Efects of antioxidant vitamins on hypoxia-induced erythropoictin production, An important role in O, sensing has been assigned to microsomal and membrane-bound b-type cyrochromes which generate regulatory reactive O, species (ROS). Recent!y, ROS have been shown to suppress the in vitro synthesis of erythropoietin (Epo). Wo investigated the potential of the antioxidant vitamins A, E and C to enhance renal and hopatic Epo production. Renal cffecis were studied in isolated serum-free perfused rat kidneys. In control experiments without antioxidant vitamins, Epo secretion amounted to 444 + 23 mU/g kidney (mean £ sem, A = 5) during the three hour period of hypoxic perfusion (arterial pO; 35 mm Hg). Epo secretion significantly increased to 674 + 92 mU/g kidney (N = 7) when vitamins A (0.5 pgml), E (0.5 ugimi) and C (10 ngiml) in combination were added to the perfusion medium. The effects of the single vitamins were studied in Fpo-producing hepatoma cell cultures (lines HepG2 and Hep3B). Vitamin A induced a dose-dependent increase (half-maximal stimulation at 0.2 pg/ml) in the production of immunore- active Epo during 24 hours of incubation (suck as 680 + 51 U Epofg cell protein in HepG2 cultures with 3 gg/m! retinol acetate compared to 261 + 15 U/g in untreated controls; N = 4). In contrast, vitamin E (tested from 0.05 to 500 ug/ml) and sitamin C (tested from 2 to 200 ugiml) did not increase Epo production in hepatoma cell cultures. Thus, while vitamins E. and C may have the potential to protect cells from oxidative damage, vitamin A cxerts a specific stimulation of Epo production. Preliminary evidence suggests that this effect of vitamin A invalves increased mRNA levels of hypoxia-inducible factor la (AIF-ta) Hemoproteins are involved in O, sensing in various tissues. An. important role has been assigned to microsomal and membrane- bound b-rype cytochromes because they generate reactive O, species such as HO, (1, 2], which may oxidize sulfhydryl-groups of regulatory proteins, With respect to the synthesis of erythro- poietin (Epo) we have previously shown that the ix vitro produe- tion of H,O, is dependent on O, tension (pO,) and that HO, suppresses Epo gene expression [3]. The present in vitro study investigated the potential of the antioxidant vitamins A, E and € to enhance renal and hepatic Epo synthesis. Renal effects were studicd in isolated pertuscd rat kidneys. Hepatic synthesis of Epo was studied in cultures of the human hepatoma cell lines HepG?2 and Hep3B. Hypoxie induc- tion of Epo gene expression is critically dependent on activation of an cnhancer clement located at the 3' flanking region of the Epo gene. This enhancer is responsive to hinding of a protein complex termed hypoxia-inducible factor 1 (HIF-1), which is composed of at least two subunits that have been designated HIF-la and HEF-18 [4, 5]. Theretore, im addition Northern blots O 1997 by the International Society of Nephrology for HIF-Ia mRNA were performed to detect a possible increase in the mRNA level for HIF-te. Methods Kidney perfusion To study renal Epo production, kidneys from adult male Sprague-Dawley rats were perfused at constant pressure (100 mm Hg) in a recirculation system for three hours as described in [6]. Where indicated, antioxidant vitamins were added to the perfu- sion medium (retinol acetate 0.5 gg/ml, a-tocophero! 0.5 ug/mi, vitamin C 10 ng/ml; al obtained from Sigma, Minchen, Germa- ny). The perfusion medium was a substrate and amino acids enriched Krebs-Henseleit solution supplemented with 60 giliter albumin and 5% human erythrocytes. It was dialyzed against a 25-fold volume of a protein- and cell-free medium. Epo was measured by enzyme-linked immunoassay in samples of the perfusion medium. The assay was carried out according to the manufacturer (EPO-ELISA Test; Medac GmbH, Hamburg, Ger- many), except that rat serum Epo previously calibrated by bioas- say was used as the standard instead ot human Epo. FHepatoma cell cultures The human hepatoma cell lines HepG2 (ATCC No. 8065) and Hep3B (ATCC No. 8064) were used to study effects of single antioxidant vitamins on Epo production. The cells were main- tained in RPMI 1640 medium supplemented with 10% fotal bovine serum (Sigma) and NaHCO, (2.2 g/liter) in a humidified atmosphere (5% CO, in air) at 37ºC (Heraeus Incubators, Hanau, Germany). Cel! monolayers were grown to confuence in 24-well polystyrene dishes (Falcon, Becton Dickinson, Heidelberg, Ger- many). The cultures had a density of 5 X 10º cells/cm? when used. The medium (0.5 mi/em?) was renewed 24 hours before the experiment. For study of Epo secretion, cells in 24-wcll polystyrene dishes were incubated wilh the respective vitamin in air-5% CO, for 24 hours. Under these conditions, 3 to 10 U Epo per hour and g cellular protein were usually produced because the cultures were hypoxic due to a diffusion-limiled O, supply [7]. The medium was collected after 24 hours of incubation and frozen at --20ºC. Epo was measured by radioimmunoassay as described earlier [7]. The ccll layer was washed with phosphate buffcred saline and Iysed with SDS/NaOH (5 g/liter sodium dodecyl sulfate in 0.1 M NaOH) Total cellular protein was determined by a micro-determination kit (Sigma Diagnosties, Taufkirchen, Germany). 497 498 Jelkmann ei al: Vitamins and enthropoietin produciion 10 075 ' » & ES] = É) o 3 5 050 5 ê & 0.25 o) 60 120 180 Perfusion period, minutes Fig. 1, Time-course ofthe production of tipo by isolated perfused rat kidneys and its dependence om arterial pO» (means * sem N = 5) 800 + 4 600 + £ B e > 8 400 + 8 g 8 uu L 200 O º T f f Za f 0.8 5 * V Epo, U/g kidney o > 1 0.2 5 7 0 Controls + Antioxidant vitamins 2. Effect of antioxidant vitamins (retinol acetate 0.5 Hgiml, a-tocoph- eroi 0.5 mgiral, vitamin C 10 pgiml) on the three hours rate of the production of Epo ir isolated hypoxically (arterial pO 35 mm Hg) perfused rat kidneys (means ! SEM, exp. no. at the bottom, *P <: 0.05 compared to controls). o 0.03 01 03 1 Vitamin A, pg/mi Cytotoxicity was assessed by means of the [3-(4,5-dimethylthia- zol-24]]2,5-diphenyiltetrazolium bromide assay (MTT) as re- cently described [8]. T + Fig. 3. Efect of vitamin A fretinol acetate) on 3 the 24 hours rate of the production of Epo in HepG2 cultures (means * SEM, N = 4 *P< 0.05 vs. control cultures). To study HIF-Lw gene expression, cells werc grown to contlu- ence in culture dishes with a gas-permeable bottom (3.5 em diameter; Petriperm, Heracus, Germany). After 10 hours of 500 Control 20% O, Control 20% O, Vitamin À 20% O, Vitamin À 20% O, Control 2% O, Control 2% O, Vitamin A 2% O, Vitamin A 2% O, AEERERNRER.S hypoxic incubation (2% 0,) at 37'C, RNA was isolated with the commercially available Trizol reagent (GIBCO BRL, Eggenstein, Germany) following the instructions of lhe manufacturer (this method is a modification of the single step protocol developed by Chomezynski and Sacchi [9]. RNA concentrations were determi- nated by measuring the optical density at 260 nm. RNA was separated in 1.4% agarose gel containing 0.7 M formaldehyde and visualized according to Gong [10]. After separation, the RNA was transferred onto Nylon membranes (Nytran plus; Schleicher & Schiill, Dassel, Germany) using a vacuam blotting apparatus (Pharmacia, Freiburg, Germany) and fixed to the membran by UV.-irradiation. Finally, the membranc was bakcd for 1.5 hours at 80ºC and stored dry in the dark until hybridization. Hybridization was performed in 45% formamide, 5 X sodium chloride sodium citrate (SSC), 5 x DenhardU's solution, 0.19% SDS and 100 ng/ml sonicated denatured salmon testis DNA at 42C for 36 to 48 hours. After hybridization filters were washed twice for 15 min- utes in (1.2 x S$C/0.1% SDS at SO“C, scaled in plastic bags and exposed to autoradiographie films at —80ºC for varying time intervals. HIF-le probe is a 542 bp PCR-fragment gencrated by primers: 5! GTCGGA CAG CCT CACCAA ACAG3' (upsiream primer) and 5º TAG GTA GTG AGC CAC CAG TGT CC 3' (down- stream primer). Primer sequences were derived from the HIF-Ja Jelkmann et al: Vitamins and erythropoietin production kb — 747 — 4.40 — 2.37 =—747 —4.40 —a.s7 Fig. 6. Nonhern-hybridization on RNA of Hep3B cell extracts with a HIF-lo probe (top) and corresponding gel (bottom). Cells were incubated with 1 ugiml vitamin À for 10 hours under normoxic and hypoxic conditions. nucleotide sequence published by Wang et al [4]. PCR-tempera- ture profiles were one minute at 93ºC, 1.30 minutes at 57ºC and two minutes at 72ºC for 35 cycles. Labeling was done using a random prime labeling kit (Boehringer, Mannheim, Germany) and a(2P) dCTP (3000 Cijmmol). Statistics Data are expressed as thc mean + standard error (SEM). Sludent's f-test was applied for estimation of significance (P < 0.05) of the differences between two group means. Dunnctt's test was applied to compare a control mean with several treatment means. Results Figure 1 shows Lhat isolated perfused rat kidneys produced Epo in à pO;-dependent manner. The rate of Epo production was significantly increased when vitamins A, E and € in combination were added to the pertusion medium (Fig. 2). Studies in HepG2 and Hep3B cultures showed that only vitamin A increased the rate of Epo production (Figs. 3 and 4) m contrast, vitamins E (dose range 0.05 to 500 pg/ml; data not shown) and C (Fig. 5) were without effect over a wide dosage range. Based on the MTT assay none of the vitamins was cytotoxic in the dose range under study. Jelkmann et al Vitamins and erythropoietin production Northem blots wilh total RNA from untreated and from vitamin A-treated Hep3B cells revealed a possible increase in HiF-ja mRNA levels by vitamin A treatment (Fig. 6). Discussion The antioxidant vitamins A, E and € increased the concentra- tion of immunoreactive Epo in the perfusate of isolated perfused rat kidncys when added in combination to the perfusion medium. This result indicated that at least one of the vitamins was capable of stimulating renal Epo production. When the single vitamins were tested in the human hepatoma cell lines HepG2 and Hep3B, only vitamin A proved to increase the concentration of immuno- reactive Epo in the culture medium. Okano et al [E] have proposed earlier that vitamin A activates Epo gene transcription, because Epo mRNA levels are elevated in vitamin A-treated HepG2 cultures. A renal cell culture model for the study of pO,-dependent Epo production has not yet been established. Thus, most of the knowledge of the molecular mechanisms of the control of Epo gene expression has been based on experiments with human hepatoma cells. The aceumulation of Epo mRNA following the induction of hypoxia results in these cells both from activated Epo gene expression and increased mRNA stability [12]. Cis-acting DNA-sequences neighboring the Epo gene mediate hypoxia in- ducibility both as promoter and enhancer elements [13-15], HIF-3 is presently considered the main transcription factor in the control of the Epo gene [4, 3). HIF-1 is composed of two subunits, HIF-La and HIF-1f, with the latter being identical to the aryl hydrocar- bon receptor nuclear translocator, ARNT [4]. The present Northern bloiting experiments resulted in in- creased HIP-la mRNA levels in vitamin A-treated Hep3B cells. This observation clearly needs to be inspected by a more quanti- tative assay of HIF-la mRNA. Clarification of this issue is crucial for the interpretation of the present results. If vitamin A increas HIF-1 mRNA levels, it is likely that this vitamin interferes with the O, sensing mechanism. An alternative explanation for the vitamin A effect might be that retinoic acid receptors bind to a steroid responsive clement (DR-2 sequence) in the 3'-enhancer region of the Epo gene [21]. However, lhe expression of a reporter gene linked to the Epo 3-enhancer region was not stimulated by retinoic acid in Hep3B celis [15]. Carotenoids arc considered antioxidants because of their ca- pacity to scavenge reactive O, species [16]. Therefore, the present findings arc in agreement with the hypothesis that reactivo O, species are inhibiting signals in Epo synthesis [2, 3]. It still necds to be clarified as to why severa! other antioxidant compounds — including vitamins E and C—do not stimulate Epo production. Because of its Hpophilic property vitamin E, similar to vitamin A, acis preliminary in lipid-rich membranes, where it converts O, radicals to less reactive forms [16]. With respect to the action of 501 vilamin €, ilhas to be noted that this it is hydrophilic and that jt may act both as an antioxidant and prooxidant. The present study shows that vitamin € did not stimulate Epo production. Neithcr does it induce HUE-1 DNA-binding activity [5]. Reprint requests io Wolfzang Jelkmann. MD, Institute of Physiology, Medical University of Luebeck, Ratzeburger Alle 160, 1)-23438 Luebeck, German: References FanpRey J. Sevori. FP, SikGrRS CP, JE KMANN W: Rolo of cyto- chrome Esso in the control of the production of erythropoietin. Life Sci 47:127-134, 1990 GORLACH A, HOLTERMANN G, FELKMANN W. 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