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Int. J. Mol. Sci. 2018, 19(9), 2685; https://doi.org/10.3390/ijms19092685

Uremic Toxins Activates Na/K-ATPase Oxidant Amplification Loop Causing Phenotypic Changes in Adipocytes in In Vitro Models

1
Department of Internal Medicine, Marshall University Joan C Edwards School of Medicine, Huntington, WV 25755, USA
2
Department of Surgery and Biomedical Sciences, Marshall University Joan C Edwards School of Medicine, Huntington, WV 25701, USA
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 1 August 2018 / Revised: 31 August 2018 / Accepted: 6 September 2018 / Published: 10 September 2018
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Abstract

Background: Oxidant stress plays a key role in the development of chronic kidney disease (CKD). Experimental CKD leads to accumulation of uremic toxins (UT) in the circulation resulting in increased ROS production, which in turn, is known to activate the Na/K-ATPase/ROS amplification loop. Studies in a murine model of obesity have shown that increased oxidative stress in plasma is due to increased ROS and cytokine production from dysfunctional adipocytes. Therefore, we hypothesized that adipocytes exposed to UTs will activate the Na/K-ATPase oxidant amplification loop causing redox imbalance and phenotypic alterations in adipocytes. We also aimed to demonstrate that the Na/K-ATPase signaling antagonist, pNaKtide, attenuates these pathophysiological consequences. Methods: In the first set of experiments, 3T3-L1 murine pre-adipocytes were treated with varying concentrations of UTs, indoxyl sulfate (IS) (50, 100 and 250 µM) and p-cresol (50, 100 and 200 µM), with or without pNaKtide (0.7 µM) for five days in adipogenic media, followed by Oil Red O staining to study adipogenesis. RT-PCR analysis was performed to study expression of adipogenic, apoptotic and inflammatory markers, while DHE staining evaluated the superoxide levels in UT treated cells. In a second set of experiments, visceral fat was obtained from the West Virginian population. MSCs were isolated and cultured in adipogenic media for 14 days, which was treated with indoxyl sulfate (0, 25, 50 and 100 µM) with or without pNaKtide (1 µM). MSC-derived adipocytes were evaluated for morphological and molecular analysis of the above markers. Results: Our results demonstrated that 3T3-L1 cells and MSCs-derived adipocytes, treated with UTs, exhibited a significant decrease in adipogenesis and apoptosis through activation of the Na/K-ATPase/ROS amplification loop. The treatment with pNaKtide in 3T3-L1 cells and MSC-derived adipocytes negated the effects of UTs and restored cellular redox in adipocytes. We noted a varying effect of pNaKtide, in adipocytes treated with UTs, on inflammatory markers, adipogenic marker and superoxide levels in 3T3-L1 cells and MSC-derived adipocytes. Conclusions: This study demonstrates for the first time that the Na/K-ATPase/ROS amplification loop activated by elevated levels of UTs has varying effect on phenotypic alterations in adipocytes in various in vitro models. Thus, we propose that, if proven in humans, inhibition of Na/K-ATPase amplification of oxidant stress in CKD patients may ultimately be a novel way to combat adipocyte dysfunction and metabolic imbalance in these patients. View Full-Text
Keywords: adipocytes; Na/K-ATPase signaling; ROS; uremic toxins; CKD; oxidative stress adipocytes; Na/K-ATPase signaling; ROS; uremic toxins; CKD; oxidative stress
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Bartlett, D.E.; Miller, R.B.; Thiesfeldt, S.; Lakhani, H.V.; Khanal, T.; D. Pratt, R.; Cottrill, C.L.; Klug, R.L.; Adkins, N.S.; Bown, P.C.; Nease, D.B.; Shapiro, J.I.; Sodhi, K. Uremic Toxins Activates Na/K-ATPase Oxidant Amplification Loop Causing Phenotypic Changes in Adipocytes in In Vitro Models. Int. J. Mol. Sci. 2018, 19, 2685.

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