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Abstract：
Lithium (Li) administration causes deranged expression and function of renal aquaporins and sodium channels/transporters resulting in nephrogenic diabetes insipidus (NDI). Extracellular nucleotides (ATP/ADP/UTP), via P2 receptors, regulate these transport functions. We tested whether clopidogrel bisulfate (CLPD), an antagonist of ADP-activated P2Y(12) receptor, would affect Li-induced alterations in renal aquaporins and sodium channels/transporters. Adult mice were treated for 14 days with CLPD and/or Li and euthanized. Urine and kidneys were collected for analysis. When administered with Li, CLPD ameliorated polyuria, attenuated the rise in urine prostaglandin E2 (PGE2), and resulted in significantly higher urinary arginine vasopressin (AVP) and aldosterone levels as compared to Li treatment alone. However, urine sodium excretion remained elevated. Semi-quantitative immunoblotting revealed that CLPD alone increased renal aquaporin 2 (AQP2), Na-K-2Cl cotransporter (NKCC2), Na-Cl cotransporter (NCC), and the subunits of the epithelial Na channel (ENaC) in medulla by 25-130 %. When combined with Li, CLPD prevented downregulation of AQP2, Na-K-ATPase, and NKCC2 but was less effective against downregulation of cortical α- or γ-ENaC (70 kDa band). Thus, CLPD primarily attenuated Li-induced downregulation of proteins involved in water conservation (AVP-sensitive), with modest effects on aldosterone-sensitive proteins potentially explaining sustained natriuresis. Confocal immunofluorescence microscopy revealed strong labeling for P2Y(12)-R in proximal tubule brush border and blood vessels in the cortex and less intense labeling in medullary thick ascending limb and the collecting ducts. Therefore, there is the potential for CLPD to be directly acting at the tubule sites to mediate these effects. In conclusion, P2Y(12)-R may represent a novel therapeutic target for Li-induced NDI.
摘要：
锂（Li）给药导致肾水通道蛋白和钠通道/转运蛋白的表达和功能紊乱，导致肾性尿崩症（NDI）。细胞外核苷酸（ATP/ADP/UTP），通过P2受体，调节这些运输功能。我们测试了硫酸氢氯吡格雷(CLPD)ADP激活的P2Y（12）受体拮抗剂，会影响Li诱导的肾水通道蛋白和钠通道/转运蛋白的改变。成年小鼠用CLPD和/或Li处理14天并安乐死。收集尿液和肾脏用于分析。当使用Li时，CLPD改善多尿，减少尿前列腺素E2（PGE2）的升高，与单独使用Li治疗相比，并导致尿精氨酸加压素（AVP）和醛固酮水平显着升高。然而,尿钠排泄仍然升高。半定量免疫印迹显示，CLPD单独增加肾水通道蛋白2（AQP2），Na-K-2Cl协同转运蛋白(NKCC2),Na-Cl协同转运蛋白（NCC），和髓质上皮Na通道（ENaC）亚基的25-130％。当与Li结合时，CLPD阻止AQP2，Na-K-ATPase的下调，和NKCC2，但对皮质α-或γ-ENaC（70kDa条带）的下调效果较差。因此，CLPD主要减弱Li诱导的与节水有关的蛋白质的下调（AVP敏感），对醛固酮敏感的蛋白质有适度的影响，可能解释持续的利钠。共聚焦免疫荧光显微镜检查显示，在皮质的近端小管刷状边界和血管中，P2Y（12）-R的标记强烈，而在髓质粗大的上升肢体和收集管中的标记不那么强烈。因此,CLPD有可能直接作用于小管部位以介导这些效应。总之,P2Y(12)-R可以代表Li诱导的NDI的新治疗靶标。
