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首页> 外文期刊>American Journal of Physiology >Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK1 cells.
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Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK1 cells.

机译：心脏糖苷下调LLC-PK1细胞中的NHE3活性和表达。

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摘要

Ouabain, a cardiotonic steroid and a specific inhibitor of the Na(+)-K(+)-ATPase, has been shown to significantly inhibit transcellular Na(+) transport without altering the intracellular Na(+) concentration ([Na(+)](i)) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na(+)/H(+) exchanger isoform 3 (NHE3) representing the major route of apical Na(+) reabsorption in LLC-PK(1) cells. Chronic basolateral, but not apical, exposure to low-concentration ouabain (50 and 100 nM) did not change [Na(+)](i) but significantly reduced NHE3 activity, NHE3 protein, and mRNA expression. Inhibition of c-Src or phosphoinositide 3-kinase (PI3K) with PP2 or wortmannin, respectively, abolished ouabain-induced downregulation of NHE3 activity and mRNA expression. In caveolin-1 knockdown LLC-PK(1) cells, ouabain failed to downregulate NHE3 mRNA expression and NHE3 promoter activity. Ouabain response elements were mapped to a region between -450 and -1,194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements was documented in vitro and in vivo by protein/DNA array analysis, EMSA, supershift, and chromatin immunoprecipitation. These data suggest that, in LLC-PK(1) cells, ouabain-induced signaling through the Na(+)-K(+)-ATPase-Src pathway results in decreased Sp1 and TR DNA binding activity and consequently in decreased expression and activity of NHE3. These novel findings may represent the underlying mechanism of cardiotonic steroid-mediated renal compensatory response to volume expansion and/or hypertension.
机译：哇巴因，一种强心性类固醇和Na（+）-K（+）-ATPase的特异性抑制剂，已显示在不改变细胞内Na（+）浓度（[Na（+） ]（i））来自肾近端小管的上皮细胞。因此，我们研究了哇巴因是否影响LLC-PK（1）细胞中Na（+）/ H（+）交换异构体3（NHE3）的活性和表达，它们代表了根尖Na（+）重吸收的主要途径。慢性基底外侧暴露于低浓度的哇巴因（50和100 nM），但没有根尖[Na（+）]（i）不变，但显着降低了NHE3活性，NHE3蛋白和mRNA表达。 PP2或渥曼青霉素分别抑制c-Src或磷酸肌醇3激酶（PI3K），消除了哇巴因诱导的NHE3活性和mRNA表达下调。在caveolin-1敲低LLC-PK（1）细胞中，哇巴因未能下调NHE3 mRNA表达和NHE3启动子活性。哇巴因反应元件定位在-450和-1,194 nt之间的区域，通过蛋白质/ DNA阵列分析，EMSA和体外方法，在体内和体外都记录了甲状腺激素受体（TR）和Sp1与其同源顺式元件的结合减少。超移位和染色质免疫沉淀。这些数据表明，在LLC-PK（1）细胞中，哇巴因通过Na（+）-K（+）-ATPase-Src途径引起的信号传导导致Sp1和TR DNA结合活性降低，从而导致表达和活性降低NHE3。这些新发现可能代表了强心类固醇介导的肾脏对体积膨胀和/或高血压的代偿反应的潜在机制。

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