Frische Sebastian, Zolotarev Alexander S, Kim Young-Hee, Praetorius Jeppe, Alper Seth, Nielsen Søren, Wall Susan M
The Water and Salt Research Center, Bldg. 233/234, Univ. of Aarhus, DK-8000 Aarhus C, Denmark.
Am J Physiol Renal Physiol. 2004 Jun;286(6):F1163-70. doi: 10.1152/ajprenal.00409.2003. Epub 2004 Jan 28.
Three splice variants of anion exchanger (AE)2 (AE2a, b, and c) have been described in the rat, but their relative distribution in rat kidney is not known. The purpose of this study was to describe the segmental and cellular distribution of the AE2 isoforms in the rat kidney and to evaluate whether the expression levels of these AE2 isoforms are regulated independently in response to chronic NH(4)Cl loading. Two polyclonal antibodies were generated, respectively, recognizing a NH(2)-terminal peptide unique to AE2a and an amino acid sequence common to AE2a and AE2b. Antibody specificities were tested using cells transfected separately with the AE2a, AE2b, and AE2c isoforms. Immunohistochemistry on sections of paraffin-embedded rat kidneys showed a distribution of AE2a/AE2b labeling in the kidney similar to the distribution of AE2 in the rat kidney reported previously. AE2 is highly expressed in the medullary thick ascending limb, cortical thick ascending limb (cTAL), and macula densa. The pattern of AE2a-specific labeling differed from the pattern of AE2a/AE2b labeling in that relatively more of the total immunolabel was observed in the terminal inner medullary collecting duct. NH(4)Cl loading (0.033 mmol NH(4)Cl/g body wt for 7 days) did not change the labeling of AE2 isoforms in the medulla, whereas the labeling in the cortex was intensified and included more distal parts of the cTAL. Immunoblotting confirmed upregulation of AE2a/b expression in the cortex. These results indicate that AE2a and AE2b are differentially expressed and regulated in the rat kidney. The regulation following NH(4)Cl loading of AE2b in the cTAL suggests a role for AE2 in transepithelial bicarbonate reabsorption in this segment.
在大鼠中已发现阴离子交换蛋白（AE）2有三种剪接变体（AE2a、b和c），但它们在大鼠肾脏中的相对分布尚不清楚。本研究的目的是描述AE2亚型在大鼠肾脏中的节段性和细胞分布，并评估这些AE2亚型的表达水平是否会因慢性氯化铵负荷而独立调节。分别制备了两种多克隆抗体，一种识别AE2a特有的NH2末端肽，另一种识别AE2a和AE2b共有的氨基酸序列。使用分别转染AE2a、AE2b和AE2c亚型的细胞检测抗体特异性。对石蜡包埋的大鼠肾脏切片进行免疫组织化学分析，结果显示肾脏中AE2a/AE2b标记的分布与先前报道的大鼠肾脏中AE2的分布相似。AE2在髓质厚升支、皮质厚升支（cTAL）和致密斑中高表达。AE2a特异性标记的模式与AE2a/AE2b标记模式不同，因为在终末内髓集合管中观察到的总免疫标记相对更多。氯化铵负荷（0.033 mmol氯化铵/克体重，持续7天）并未改变髓质中AE2亚型的标记，而皮质中的标记增强，且包括cTAL更远端的部分。免疫印迹证实皮质中AE2a/b表达上调。这些结果表明，AE2a和AE2b在大鼠肾脏中的表达和调节存在差异。cTAL中AE2b在氯化铵负荷后的调节表明AE2在该节段的跨上皮重吸收中发挥作用。
