Department of Internal Medicine, Marshall University Joan C Edwards School of Medicine, Huntington, WV 25755, USA.
Department of Surgery and Biomedical Sciences, Marshall University Joan C Edwards School of Medicine, Huntington, WV 25701, USA.
Int J Mol Sci. 2018 Sep 10;19(9):2685. doi: 10.3390/ijms19092685.
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.
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.
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.
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.
氧化应激在慢性肾脏病（CKD）的发展中起着关键作用。实验性 CKD 导致尿毒症毒素（UT）在循环中积累，导致 ROS 产生增加，这反过来又已知会激活 Na/K-ATPase/ROS 扩增环。肥胖症小鼠模型的研究表明，血浆中氧化应激的增加是由于功能失调的脂肪细胞中 ROS 和细胞因子产生增加所致。因此，我们假设暴露于 UT 的脂肪细胞将激活 Na/K-ATPase 氧化剂扩增环，导致脂肪细胞中的氧化还原失衡和表型改变。我们还旨在证明 Na/K-ATPase 信号转导拮抗剂 pNaKtide 可减轻这些病理生理后果。
在第一组实验中，将 3T3-L1 鼠前脂肪细胞用不同浓度的 UT、吲哚硫酸酯（IS）（50、100 和 250 μM）和对羟苯甲酸钠（50、100 和 200 μM）处理，在成脂培养基中处理五天，然后用油红 O 染色研究脂肪生成。进行 RT-PCR 分析以研究脂肪生成、凋亡和炎症标志物的表达，而 DHE 染色评估 UT 处理细胞中的超氧化物水平。在第二组实验中，从西弗吉尼亚州人群中获得内脏脂肪。将 MSCs 分离并在成脂培养基中培养 14 天，用吲哚硫酸酯（0、25、50 和 100 μM）处理，并用 pNaKtide（1 μM）处理。评估 MSC 衍生的脂肪细胞的上述标记物的形态和分子分析。
我们的结果表明，用 UT 处理的 3T3-L1 细胞和 MSC 衍生的脂肪细胞表现出脂肪生成和凋亡的显著减少，这是通过激活 Na/K-ATPase/ROS 扩增环实现的。在 3T3-L1 细胞和 MSC 衍生的脂肪细胞中用 pNaKtide 处理可消除 UT 的作用并恢复脂肪细胞中的细胞内氧化还原。我们注意到，在用 UT 处理的脂肪细胞中，pNaKtide 对炎症标志物、脂肪生成标志物和 3T3-L1 细胞和 MSC 衍生的脂肪细胞中超氧化物水平的影响各不相同。
这项研究首次表明，由升高的 UT 水平激活的 Na/K-ATPase/ROS 扩增环对各种体外模型中脂肪细胞的表型改变具有不同的影响。因此，我们假设，如果在人类中得到证实，抑制 CKD 患者氧化应激的 Na/K-ATPase 扩增可能最终成为一种治疗这些患者脂肪细胞功能障碍和代谢失衡的新方法。
