a Medical Proteomics Unit, Office for Research and Development , Siriraj Hospital, Mahidol University , Bangkok , Thailand.
b Department of Immunology , Siriraj Hospital, Mahidol University , Bangkok , Thailand.
Cell Adh Migr. 2018 May 4;12(3):247-258. doi: 10.1080/19336918.2017.1356554. Epub 2017 Sep 14.
Hypokalemia is a common electrolyte disorder in hospitalized patients and those with chronic diseases and is associated with renal tubular injury. Our recent expression proteomics study revealed changes in levels of several proteins in renal tubular cells during K deficiency. However, functional significance and mechanisms underlying such changes remained unclear. The present study, thus, aimed to investigate functional changes of renal tubular cells induced by K deficiency. MDCK cells were maintained in normal-K (ANK; [K] = 5.0 mM), Low-K (ALK; [K] = 2.5 mM), or K-depleted (AKD; [K] = 0 mM) medium. Cell count and cell death assay showed that ALK and AKD groups had marked decrease in cell proliferation without significant change in cell death. Other functional investigations revealed that AKD cells had significantly increased levels of carbonylated proteins (by OxyBlot assay), impaired tissue repair (by scratch assay), defective tight junction (by Western blotting, immunofluorescence staining and measuring transepithelial electrical resistance), increased intracellular ATP level (by ATP measurement), decreased levels of ubiquitinated proteins (by Western blotting), and increased level of Na/K-ATPase (by Western blotting), which was consistent with the increased cellular K uptake after K repletion. Our findings have shown that AKD caused defects in cell proliferation, oxidative stress response, tissue repair and tight junction integrity, but on the other hand, enhanced energy production, proteasome function and cellular K uptake. These findings may shed light onto cellular response to K deficiency and better understanding of both pathogenic and compensatory mechanisms in hypokalemic nephropathy.
低血钾症是住院患者和慢性病患者中常见的电解质紊乱，与肾小管损伤有关。我们最近的蛋白质组学表达研究揭示了低钾血症时肾小管细胞中几种蛋白质水平的变化。然而，这些变化的功能意义和机制尚不清楚。本研究旨在探讨低钾血症引起的肾小管细胞的功能变化。MDCK 细胞在正常钾（ANK；[K] = 5.0 mM）、低钾（ALK；[K] = 2.5 mM）或低钾耗尽（AKD；[K] = 0 mM）培养基中维持。细胞计数和细胞死亡测定表明，ALK 和 AKD 组细胞增殖明显减少，而细胞死亡无明显变化。其他功能研究表明，AKD 细胞的羰基化蛋白水平显著升高（通过 OxyBlot 测定），组织修复受损（通过划痕测定），紧密连接缺陷（通过 Western 印迹、免疫荧光染色和测量跨上皮电阻），细胞内 ATP 水平升高（通过 ATP 测定），泛素化蛋白水平降低（通过 Western 印迹），Na/K-ATP 酶水平升高（通过 Western 印迹），这与钾补充后细胞内钾摄取增加一致。我们的研究结果表明，AKD 导致细胞增殖、氧化应激反应、组织修复和紧密连接完整性受损，但另一方面增强了能量产生、蛋白酶体功能和细胞内钾摄取。这些发现可能揭示了细胞对低钾血症的反应机制，并更好地理解低钾血症肾病的发病机制和代偿机制。
