Kidney Blood Press Res 2013;37:496-505 DOI: 10.1159/000355730 © 2013 S. Karger AG, Basel Published online: November 11, 2013 www.karger.com/kbr 496 Accepted:Feger et al.: September CORM-2-induced 17, 2013 inhibition of vitamin D31423-0143/13/0375-0496$38.00/0 activation This is an Open Access article licensed under the terms of the Creative Commons Attribution- NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Original Paper Effect of Carbon Monoxide Donor CORM-2 on Vitamin D3 Metabolism Martina Fegera,f Abul Fajola,f Aleksandra Lebedevaa,b Adrian Meissnera Diana Michaela Jakob Voelkla Ioana Alesutana Erwin Schleicherc Christoph Reichetzederd Berthold Hocherd Syed M. Qadria,e,g Florian Langa,g aDepartment of Physiology, University of Tübingen, Tübingen, Germany; bDepartment of Immunology, Institute of Experimental Medicine, St. Petersburg, Russia; cDepartment of Internal Medicine, University of Tübingen, Tübingen, Germany; dInstitute of Nutritional Science, University of Potsdam, Potsdam, Germany, eDepartment of Pharmacology, University of Saskatchewan, Saskatoon, Canada; fcontributed equally and thus share first authorship; gcontributed equally and thus share last authorship Key Words CORM-2 • 1,25-Dihydroxyvitamin D3 • Klotho • FGF23 • Phosphate • Calcium Abstract Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase 2+ (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca and phosphate transport as well as klotho expression. The present study explored, whether CORM- 2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis. Copyright © 2013 S. Karger AG, Basel Prof. Dr. Florian Lang Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tuebingen (Germany), Tel. +49 7071 29 72194, Fax +49 7071 29 5618 E-Mail [email protected] Kidney Blood Press Res 2013;37:496-505 DOI: 10.1159/000355730 © 2013 S. Karger AG, Basel Published online: November 11, 2013 www.karger.com/kbr 497 Feger et al.: CORM-2-induced inhibition of vitamin D3 activation Introduction Carbon monoxide (CO), a gas, which is highly toxic to humans and animals when encountered in higher concentrations, binds rapidly to hemoglobin (Hb), leading to the stable formation of carboxyhemoglobin (COHb) [1]. As a result of CO binding to Hb both O2 binding at high and O2 release at low O2 partial pressure are impaired [1] leading to a reduced oxygen carrying capacity of blood after CO inhalation [2]. Besides its toxic effects at high concentrations, CO is a gaseous transmitter molecule [3]. In mammalian cells CO is generated by the enzyme heme-oxygenase [4]. CO participates in the regulation of a wide variety of cellular functions [5-9] and contributes to the regulation of cell death [10-15]. CO may elicit both vasodilation and vasoconstriction [16, 17]. In addition, CO in vitro and in vivo [18, 19]. CO can be released in an organism by the tricarbonyl-dichlororuthenium (II) dimer (CORM-2) [6]. CO-releasing moleculesfosters coagulation have been and suggested attenuates as therapeuticfibrinolysis option in various pathologies, but the risks of CO interferes with the function of cytochromes [21-23]. The cytochrome P450 enzyme 25-hydroxyvitamintreatment with those D molecules3 1-alpha-hydroxylase have been insufficiently accomplishes addressed formation [6, 20]. of 1,25-dihydroxy- vitamin D3 (1,25(OH)2D3) from its inactive precursor (25-hydroxyvitamin D3) [24]. The enzyme 25-hydroxyvitamin D3 1-alpha-hydroxylase is encoded by the Cyp27b1 gene [24]. 1,25(OH)2D3 is inactivated by 1,25-dihydroxyvitamin D3 24-hydroxylase, an enzyme encoded by the Cyp24a1 gene [25]. 1,25(OH)2D3 is a powerful regulator of CaHPO4 metabolism and lack of 1,25(OH)2D3 leads to rickets and osteomalacia [26]. Reduced levels of 1,25(OH)2D3 are further associated with the onset and progression of various diseases such as respiratory infections, cardiovascular disease and cancer [27-30]. 1,25(OH)2D3 formation is inhibited by joint action of klotho and FGF23, proteins regulating phosphate homeostasis and implicated in various disorders including arteriosclerosis, cancer metastasis, and chronic kidney disease [31-33]. The present study explored, whether the CO donor tricarbonyl-dichlororuthenium (II) Furthermore, heme-oxygenase-1 is involved3 metabolism in bone [6, metabolism 14]. To this andend, inflammation vitamin D metabolism [34, 35]. was investigated after CORM-2 injection into mice. dimer (CORM-2) influences vitamin D Materials and Methods All animal experiments were conducted according to the German law for the welfare of animals and were approved by local authorities. Male and female BALB/c mice had free access to food (ssniff Spezialdiäten, Soest, Germany) containing 7000 mg/kg phosphorus and to tap drinking water ad libitum. Where indicated, Germany), solubilized in dimethyl sulfoxide (DMSO, Sigma Aldrich), was injected intravenously (20 mg/kg bodythe CO-releasing weight). Control molecule mice tricarbonyldichlororutheniumwere injected with vehicle (10% (II) DMSO dimer in (CORM-2, saline solution). Sigma Aldrich, Taufkirchen, To determine excretion of phosphate, the mice were placed individually in metabolic cages (Techniplast, Hohenpeissenberg, Germany) for 24-hour urine collection [36, 37]. They were allowed a 2-day habituation period. Subsequently, 24-hour collection of urine was performed. To assure quantitative urine collection, metabolic cages were siliconized, and urine was collected under water-saturated oil [38]. The urinary phosphate concentration was determined colorimetrically utilizing a commercial diagnostic kit (Roche Diagnostics, Mannheim, Germany) [39]. Wiesbaden- Delkenheim, Germany) and about 50 - 200 µl of blood was withdrawn into heparinized capillaries by puncturingTo obtain the retro-orbital blood specimens, plexus mice[40]. Thewere plasma lightly concentrations anaesthetized of with phosphate isoflurane and Ca(Abott,2+ were measured by a photometric method (FUJI FDC 3500i, Sysmex, Norsted, Germany). The creatinine concentrations in plasma were measured using a commercial enzymatic kit (creatinine PAP, Labor & Technik, Berlin, Germany, based on the creatininase method). An EIA kit was employed to determine plasma concentrations of 1,25(OH)2D3 (IDS, Boldon, UK). Plasma FGF23 (C-Term) concentrations were determined utilizing a commercial ELISA Kit (Immutopics, San Clemente, USA) [41]. Kidney Blood Press Res 2013;37:496-505 DOI: 10.1159/000355730 © 2013 S. Karger AG, Basel Published online: November 11, 2013 www.karger.com/kbr 498 Feger et al.: CORM-2-induced inhibition of vitamin D3 activation cervical dislocation. The kidneys were removed and immediately snap frozen in liquid nitrogen [42]. To determineAfter protein24 hours expression of CORM-2 by treatment,western blotting, mice were kidneys anesthetized were lysed with with isoflurane ice-cold andlysis sacrificedbuffer (54.6 by mM HEPES; 2.69 mM Na4P2O7; 360 mM NaCl; 10% [vol/vol] Glycerol; 1% [vol/vol] NP40) containing phosphatase and protease inhibitors (Complete mini, Roche, Mannheim, Germany). Homogenates were Tris-Glycine), transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), blocked clarifiedfor 1 h in by blocking centrifugation buffer (5%at 12000 fat-free rpm milk for in20 Tris-bufferedmin [43]. Total saline protein (TBS) was containing separated 0.1% by SDS-PAGE Tween (TBS- (8% T)),and incubated overnight with an anti-klotho antibody (1:1000 in 5% fat-free milk in TBS-T, KM2076 (Rat), kindly provided by Kyowa Hakko Kirin Co. Ltd, Japan). After incubation with an anti-rat secondary antibody (1:2000 in 5% fat-free milk in TBS-T, Cell Signaling), the bands were visualized with enhanced chemiluminescence according to the manufacturer's instructions. Homogenates were also probed with a primary Gapdh antibody (1:2000 in 5% BSA in TBS-T, Cell Signaling) as a loading control. Densitometric analysis of klotho and Gapdh was performed using Quantity One software (Bio-Rad Laboratories, München, Germany). containing 4,5 g/l glucose (PAA Laboratories,