Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells
Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells
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DOI：
10.1046/j.1523-1755.2002.00672.x
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发表时间：
2002-12-01
影响因子：
19.6
通讯作者：
Shapiro, JI
中科院分区：
文献类型：
--
作者：
Liu, J;Periyasamy, SM;Shapiro, JI
背景慢性肾功能衰竭和肾容量扩张时近端小管钠重吸收的减少归因于循环洋地黄样物质（DLS）。然而，DLS的循环浓度并没有急性抑制钠泵的程度与观察到的近端小管钠重吸收的变化一致。我们检测了模拟近端小管（LLC-PK 1）和远端小管（MDCK）细胞的细胞系如何对DLS的急性（30 min）和长期（长达12 h）Na（+），K（+）-ATP酶抑制反应。在LLC-PK 1细胞中，低浓度的哇巴因以时间和剂量依赖性方式显著降低（86）Rb摄取，但MDCK细胞没有。在生长至汇合的LLC-PK 1细胞中，跨细胞（22）Na通量显著降低，与（86）Rb摄取的降低一致。用海蟾毒配基（MBG）和来自慢性肾衰竭患者的血清的脱蛋白提取物观察到类似的结果。然而，在低细胞外钾浓度下抑制Na（+），K（+）-ATP酶并不产生任何这些效应。Western和北方印迹法检测到哇巴因处理LLC-PK 1细胞12小时后，细胞内Na（+），K（+）-ATP酶蛋白和信使RNA均无变化。然而，这些细胞的Na（+），K（+）-ATP酶活性的降低与观察到的（86）Rb摄取的降低相当。差速离心和生物素化实验表明，随着哇巴因处理时间的延长，Na（+），K（+）-ATP酶从质膜上移。结果表明，强心苷类与近端（而非远端）肾小管细胞的结合导致Na（+），K（+）-ATP酶的内化，其净效应是放大Na（+），K（+）-ATP酶的抑制。由于DLS的循环浓度随着慢性肾功能衰竭和容量扩张而增加，我们认为这种现象解释了在这些条件下近端小管的钠重吸收减少。
Background. The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption.Methods. We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na(+),K(+)-ATPase inhibition with DLS.Results. In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased (86)Rb uptake profoundly in a time and dose dependent manner. In LLC-PK1 cells grown to confluence, transcellular (22)Na flux was markedly reduced in concert with the decreases in (86)Rb uptake. Similar findings were observed with marinobufagenin (MBG) and deproteinated extract of serum derived from patients with chronic renal failure. However, inhibition of the Na(+),K(+)-ATPase with low extracellular potassium concentrations did not produce any of these effects. Western and Northern blots detected no change in alphal Na(+),K(+)-ATPase protein and message RNA, respectively, in LLC-PK1 cells treated with ouabain for 12 hours. However, the decrease in enzymatic activity of Na(+),K(+)-ATPase of these cells was comparable to observed decreases in (86)Rb uptake. Differential centrifugation as well as biotinylation experiments demonstrated a shift of the Na(+),K(+)-ATPase from the plasmalemma with prolonged ouabain treatment.Conclusions. The results show that binding of cardiac glycosides by proximal (but not distal) tubular cells results in internalization of Na(+),K(+)-ATPase with the net effect to amplify inhibition of the Na(+),K(+)-ATPase. As the circulating concentrations of DLS increase with chronic renal failure and volume expansion, we suggest that this phenomenon explains some of the decreased sodium reabsorption by the proximal tubule seen in these conditions.