Regulatory mechanism of Na+ reabsorption by hypotonicity through regulatory volume decrease-dependent dephosphorylation of FAK.

肾脏中的上皮Na^+转运对于控制血压和细胞外液量很重要，并受血浆渗透压和激素的调节。我们已经在研究Na^+重吸收调节机制的模型细胞系肾上皮A6细胞中指出，细胞外低渗刺激Na^+重吸收。然而，这背后的监管机制并不清楚。低渗休克引起初始细胞肿胀，随后是调节性容积减少（RVD），这与胞质Cl^-浓度（[Cl^-] c）的降低有关。本研究的目的是阐明RVD在肾上皮Na^+重吸收的低渗调节中起关键作用的假说。用Cl^-荧光染料测定[Cl ^-]_c，发现低渗休克通过RVD过程降低[Cl ^-]_c（2）低渗休克引起黏着斑激酶（foacal adhesion kinase，FAK）的去磷酸化，而NPPB阻断RVD可使FAK的去磷酸化消失（一种Cl^-通道阻断剂）; 3）阻断RVD可引起FAK的持续酪氨酸磷酸化; 4）NPPB阻断RVD可消除低渗诱导的Na^+重吸收和上皮Na^+通道基因表达。综合以上结果，我们认为低渗休克刺激了肾上皮A6细胞Na^+重吸收RVD依赖性FAK酪氨酸去磷酸化。

Epithelial Na^+ transport in the kidney is important for control of blood pressure and extracellular fluid volume and is regulated by plasma osmolality and hormones. We have already indicated that Na^+ reabsorption is stimulated by extracellular hypotonicity in renal epithelial A6 cell that is a model cell line to study regulatory mechanism of Na^+ reabsorpption. However, the regulatory mechanism behind this is not well understood. Hypotonic shock causes the initial cell swelling followed by regulatory volume decrease(RVD) which is involved in the decrease in cytosolic Cl^- concentration ([Cl^-]_c). Our aim in this study is to clarify the hypothesis that RVD has a crucial role in hyposmotic regulation of Na^+ reabsorption in renal epithelium. We found that 1)hypotonic shock reduced [Cl^-]_c through RVD process by measuring [Cl^-]_c with Cl^- fluorescence dye (N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE), 2)hypotonic shock caused dephosphoryaltion of foacal adhesion kinase(FAK) which was abolished by blockade of RVD by NPPB (a Cl^- channel blocker), 3)blockade of RVD caused the sustained tyrosine phosphorylation of FAK, 4)hypotonicity-induced Na^+ reabsorption and epithelial Na^+ channel gene expression were abolished by blocking RVD by NPPB. Taken together these results, it is suggested that hypotonic shock stimulated Na^+ reabsorption RVD-dependent FAK tyrosine dephosphorylation in renal epithelial A6 cells.