Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS
Endogenous cardiotonic steroids (CTSs), such as telocinobufagin (TCB) and marinobufagin (MBG) contain a lactone moiety critical to their binding and signaling through the Na<sup>+</sup>/K<sup>+</sup>-ATPase. Their concentrations elevate in response to sodium intake and under...
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2022-11-01
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author | Sabitri Lamichhane Chrysan J. Mohammed Steven T. Haller David J. Kennedy Dragan Isailovic |
author_facet | Sabitri Lamichhane Chrysan J. Mohammed Steven T. Haller David J. Kennedy Dragan Isailovic |
author_sort | Sabitri Lamichhane |
collection | DOAJ |
description | Endogenous cardiotonic steroids (CTSs), such as telocinobufagin (TCB) and marinobufagin (MBG) contain a lactone moiety critical to their binding and signaling through the Na<sup>+</sup>/K<sup>+</sup>-ATPase. Their concentrations elevate in response to sodium intake and under volume-expanded conditions. Paraoxonase 3 (PON3) is an enzyme that can hydrolyze lactone substrates. Here, we examine the role of PON3 in regulating CTS levels in a rat model of chronic kidney diseases (CKD). TCB and MBG were extracted from rat urine samples, and the analyses were carried out using ultra-high pressure liquid chromatography–Orbitrap-mass spectrometry (UHPLC-Orbitrap-MS). Ten-week-old Dahl salt-sensitive wild type (SS-WT) and Dahl salt-sensitive PON3 knockout (SS-PON3 KO) rats were maintained on a high-salt diet (8% NaCl) for 8 weeks to initiate salt-sensitive hypertensive renal disease characteristic of this model. CTS extraction recovery from urine >80% was achieved. For animals maintained on a normal chow diet, the baseline amount of TCB excreted in 24 h urine of SS-PON3 KO rats (6.08 ± 1.47 ng/24 h; or 15.09 ± 3.25 pmol) was significantly higher than for SS-WT rats (1.48 ± 0.69 ng/24 h; or 3.67 ± 1.54 pmol, <i>p</i> < 0.05). Similarly, for the same animals, the amount of excreted MBG was higher in the urine of SS-PON3 KO rats (4.74 ± 1.30 ng/24 h versus 1.03 ± 0.25 ng/24 h in SS-WT; or 11.83 ± 2.91 pmol versus 2.57 ± 0.56 pmol in SS-WT, <i>p</i> < 0.05). For animals on a high-salt diet, the SS-PON3 KO rats had significantly increased levels of TCB (714.52 ± 79.46 ng/24 h; or 1774.85 ± 175.55 pmol) compared to SS-WT control (343.84 ± 157.54 ng/24 h; or 854.09 ± 350.02 pmol, <i>p</i> < 0.05), and comparatively higher levels of MBG were measured for SS-PON3 KO (225.55 ± 82.61 ng/24 h; or 563.19 ± 184.5 pmol) versus SS-WT (157.56 ± 85.53 ng/24 h; or 393.43 ± 191.01 pmol, <i>p</i> > 0.05) rats. These findings suggest that the presence and absence of PON3 dramatically affect the level of endogenous CTSs, indicating its potential role in CTS regulation. |
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spelling | doaj.art-b78ab6db205d43e884e68b57de4f597b2023-11-24T05:10:04ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123211356510.3390/ijms232113565Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MSSabitri Lamichhane0Chrysan J. Mohammed1Steven T. Haller2David J. Kennedy3Dragan Isailovic4Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606, USADepartment of Medicine, University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614, USADepartment of Medicine, University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614, USADepartment of Medicine, University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614, USADepartment of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606, USAEndogenous cardiotonic steroids (CTSs), such as telocinobufagin (TCB) and marinobufagin (MBG) contain a lactone moiety critical to their binding and signaling through the Na<sup>+</sup>/K<sup>+</sup>-ATPase. Their concentrations elevate in response to sodium intake and under volume-expanded conditions. Paraoxonase 3 (PON3) is an enzyme that can hydrolyze lactone substrates. Here, we examine the role of PON3 in regulating CTS levels in a rat model of chronic kidney diseases (CKD). TCB and MBG were extracted from rat urine samples, and the analyses were carried out using ultra-high pressure liquid chromatography–Orbitrap-mass spectrometry (UHPLC-Orbitrap-MS). Ten-week-old Dahl salt-sensitive wild type (SS-WT) and Dahl salt-sensitive PON3 knockout (SS-PON3 KO) rats were maintained on a high-salt diet (8% NaCl) for 8 weeks to initiate salt-sensitive hypertensive renal disease characteristic of this model. CTS extraction recovery from urine >80% was achieved. For animals maintained on a normal chow diet, the baseline amount of TCB excreted in 24 h urine of SS-PON3 KO rats (6.08 ± 1.47 ng/24 h; or 15.09 ± 3.25 pmol) was significantly higher than for SS-WT rats (1.48 ± 0.69 ng/24 h; or 3.67 ± 1.54 pmol, <i>p</i> < 0.05). Similarly, for the same animals, the amount of excreted MBG was higher in the urine of SS-PON3 KO rats (4.74 ± 1.30 ng/24 h versus 1.03 ± 0.25 ng/24 h in SS-WT; or 11.83 ± 2.91 pmol versus 2.57 ± 0.56 pmol in SS-WT, <i>p</i> < 0.05). For animals on a high-salt diet, the SS-PON3 KO rats had significantly increased levels of TCB (714.52 ± 79.46 ng/24 h; or 1774.85 ± 175.55 pmol) compared to SS-WT control (343.84 ± 157.54 ng/24 h; or 854.09 ± 350.02 pmol, <i>p</i> < 0.05), and comparatively higher levels of MBG were measured for SS-PON3 KO (225.55 ± 82.61 ng/24 h; or 563.19 ± 184.5 pmol) versus SS-WT (157.56 ± 85.53 ng/24 h; or 393.43 ± 191.01 pmol, <i>p</i> > 0.05) rats. These findings suggest that the presence and absence of PON3 dramatically affect the level of endogenous CTSs, indicating its potential role in CTS regulation.https://www.mdpi.com/1422-0067/23/21/13565endogenous cardiotonic steroidstelocinobufaginmarinobufaginparaoxonaseurineSPE |
spellingShingle | Sabitri Lamichhane Chrysan J. Mohammed Steven T. Haller David J. Kennedy Dragan Isailovic Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS International Journal of Molecular Sciences endogenous cardiotonic steroids telocinobufagin marinobufagin paraoxonase urine SPE |
title | Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS |
title_full | Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS |
title_fullStr | Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS |
title_full_unstemmed | Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS |
title_short | Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS |
title_sort | quantification of cardiotonic steroids potentially regulated by paraoxonase 3 in a rat model of chronic kidney disease using uhplc orbitrap ms |
topic | endogenous cardiotonic steroids telocinobufagin marinobufagin paraoxonase urine SPE |
url | https://www.mdpi.com/1422-0067/23/21/13565 |
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