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

肾脏上皮细胞的Na~+转运对血压和细胞外液容量的控制非常重要，并受血浆渗透压和激素的调节。我们已经证明，肾上皮细胞A6细胞的细胞外低渗性刺激了Na~+的重吸收，是研究Na~+重吸收调控机制的模型细胞系。然而，这背后的监管机制并没有得到很好的理解。低渗休克引起细胞初始肿胀，随后调节性体积减小(RVD)，这与细胞内氯离子浓度([Cl^-]c)的降低有关。本研究旨在阐明RVD在低渗调节肾上皮细胞Na~+重吸收中的重要作用。我们发现：1)低渗休克通过用氯离子荧光染料(N-(6-甲氧基喹啉)乙酰氧基乙酸酯，MQAE)测定[Cl-]c来降低RVD过程中的[Cl2-]c；2)低渗休克引起胎盘黏附激酶(FAK)的去磷酸化，而FAK可被氯离子通道阻滞剂NPPB阻断RVD而取消；3)阻断RVD导致FAK持续酪氨酸磷酸化；4)低渗诱导的Na+重吸收和上皮细胞Na+通道基因的表达被NPPB阻断RVD所抑制。综上所述，我们认为低张休克可刺激肾上皮细胞A6细胞钠离子重吸收，RVD依赖的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.