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Abstract：
The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman\'s syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na(+)-activated 80-150pS K(+) channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10(-/-) mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K(+) conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10(-/-) mice. Application of AVP significantly stimulated the 80-150pS K(+) channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80-150pS K(+) channel and the K(+) channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K(+) channel, suggesting that cAMP-PKA pathway stimulates the 80-150pS K(+) channels. The role of the basolateral K(+) conductance in maintaining transcellular Cl(-) transport is further suggested by the finding that the inhibition of basolateral K(+) channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl(-) channels. We conclude that vasopressin stimulates the 80-150pS K(+) channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K(+) channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K(+) conductance which is essential for the transport function in the TAL.
摘要：
EAST综合征的肾脏表型,由Kcnj10（Kir4.1）的功能丧失突变引起的疾病，是对Gitelman综合征的回忆，其特征是远曲小管（DCT）的功能缺陷。本研究的目的是测试抗利尿激素（加压素）诱导的Na（）激活的80-150pSK（）通道刺激是否负责补偿Kcnj10在粗大的上肢（TAL）中的功能丧失。患有EAST综合征的受试者。免疫染色和蛋白质印迹显示，水通道蛋白2（AQP2）在Kcnj10（-/-）小鼠中的表达明显高于WT同窝小鼠，表明Kcnj10的破坏刺激了肾脏中的加压素反应。发现精氨酸加压素（AVP）的应用使Kcnj10（-/-）小鼠的TAL中的膜超极化，这强烈表明了加压素在刺激TAL的基底外侧K（）电导中的作用。AVP的应用显着刺激了TAL中的80-150pSK（）通道，并且该作用被托伐普坦（V2受体拮抗剂）或通过抑制PKA阻断。此外，24h限水显著增加了在TAL中找到80-150pSK(+)通道和K(+)通道开放概率的概率。膜通透性cAMP类似物的应用也模拟了AVP的作用并激活了该K（）通道，表明cAMP-PKA途径刺激80-150pSK(+)通道。发现基底外侧K（）电导在维持跨细胞Cl（-）转运中的作用进一步表明，基底外侧K（）通道的抑制显着减少了AVP诱导的基底外侧10pSCl（-）通道的刺激。我们得出的结论是，加压素通过cAMP依赖性机制刺激TAL中的80-150pSK（）通道。加压素诱导的K（）通道刺激负责补偿Kcnj10的功能丧失，从而挽救基底外侧K（）电导，这对于TAL中的运输功能至关重要。
