Quantitative analysis of aquaporin mRNA expression in rat tissues by RNase protection assay
Quantitative analysis of aquaporin mRNA expression in rat tissues by RNase protection assay
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DOI：
10.1089/dna.1996.15.475
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发表时间：
1996-06-01
影响因子：
3.1
通讯作者：
Gropper, MA
中科院分区：
文献类型：
--
作者：
Umenishi, F;Verkman, AS;Gropper, MA
应用核糖核酸酶保护实验，定量检测了18种不同组织中5种主要哺乳动物水通道基因的表达，并探讨了脱水对肾脏水通道表达的影响。从大鼠CHIP28(AQP-1，编码序列238-575)、AQP-CD(AQP2，BP 53-606)、MIWC(AQP4，BP 235-572)、CLIP(AQP3，BP 219-604)和AQP5(BP 56-612)转录的标记CRNAs构成了探针。结果通过放射自显影定量密度法归一化为大鼠β-肌动蛋白的表达。CHIP28在心、肾、胎盘、骨骼肌和膀胱中表达较强，在眼、肺、气管、脾、肝、结肠、前列腺和皮肤中表达较弱。AQP-CD仅在肾脏中检测到。MIWC基因在脑中的表达最高，其次是眼、气管、肺、胃、肾和骨骼肌。GLIP存在于眼、气管、肾脏、膀胱、皮肤、前列腺、胎盘和骨骼肌中。在唾液腺、眼、肺和气管中检测到AQP5。通过核糖核酸酶保护实验，在肺和肾脏中也发现了MIWC的选择性剪接形式(SMIWC)，对应于MIWC外显子2的缺失。在脱水(3天，体重-15%)时，肾脏CHIP28和MIWC的表达没有变化，而AQP-CD和CLIP的表达分别显著增加2.18+/-0.04和1.36+/-0.11倍(SE，n=5)。这些结果为水通道蛋白在多种哺乳动物组织中的转录表达建立了定量的数值。敏感的核糖核酸酶保护实验显示，水通道在几个以前没有研究过的组织中表达，或者在这些组织中的mRNA水平太低，无法用Northern印迹分析来检测到。观察到脱水对肾脏的上调作用，提示其在尿液浓缩机制中起一定作用。
The RNase protection assay was applied to quantify mRNA expression of five principal mammalian water channels in 18 different rat tissues, and to determine the influence of dehydration on renal water channel expression. Probes consisted of labeled cRNAs transcribed from cDNA fragments of rat CHIP28 (AQP-1, bp 238-575 of coding sequence), AQP-CD (AQP2, bp 53-606), MIWC (AQP4, bp 235-572), CLIP (AQP3, bp 219-604), and AQP5 (bp 56-612). Results were normalized to expression of rat beta-actin by Quantitative densitometry of autoradiograms. CHIP28 mRNA was expressed strongly in heart, kidney > placenta, skeletal muscle, and urinary bladder and detected weakly in eye, lung, trachea, spleen, liver, colon, prostate, and skin. AQP-CD was detected only in kidney. MIWC mRNA expression was highest in brain, followed by eye, trachea, lung, stomach, kidney, and skeletal muscle. GLIP was found in eye, trachea, kidney, urinary bladder, skin, prostate, placenta, and skeletal muscle. AQP5 was detected in salivary gland, eye, lung, and trachea. An alternatively spliced form of MIWC (sMIWC) was also identified in lung and kidney by RNase protection assay, corresponding to deletion of exon 2 of MIWC. In response to dehydration (3 days, -15% body weight), renal expression of CHIP28 and MIWC were unchanged, whereas expression of AQP-CD and CLIP were increased significantly by 2.18 +/- 0.04 and 1.36 +/- 0.11 fold (SE, n = 5), respectively. These results establish quantitative values for aquaporin transcript expression in multiple mammalian tissues. The sensitive RNase protection assay revealed the expression of water channels in several tissues not studied previously or in which mRNA levels were too low to detect by Northern blot analysis. The observation of CLIP up-regulation in kidney by dehydration suggests a role in the urinary concentrating mechanism.