Obrador G, Yuan H, Shih T M, Wang Y H, Shia M A, Alexander E A, Schwartz J H
Boston Medical Center, and Department of Medicine, Boston University School of Medicine, Massachusetts 02118-2908, USA.
J Am Soc Nephrol. 1998 May;9(5):746-54. doi: 10.1681/ASN.V95746.
Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.
尽管内髓集合管（IMCD）在尿液酸化过程中起主要作用，但该肾单位节段中许多转运系统特定成分的分子鉴定仍很缺乏。利用大鼠IMCD细胞培养系来表征细胞内HCO3排出的介质。该细胞系在功能上类似于α-闰细胞。生理学实验证明，HCO3-转运是一个可逆的、电中性的、依赖Cl-且不依赖Na+的过程。它可由Cl-梯度驱动，并被诸如4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸等芪类物质抑制。使用针对阴离子交换蛋白1（AE1）羧基末端12个氨基酸的兔多克隆抗体进行免疫组织化学分析，结果显示免疫反应性蛋白的分布与培养细胞以及大鼠肾皮质切片中鉴定出的α-闰细胞中AE的基底外侧定位一致。免疫印迹显示，在培养的IMCD细胞、大鼠肾髓质和新鲜分离的IMCD细胞的膜中有两条免疫反应带（大小约为100和180 kD）。较低分子量条带的迁移率与红细胞血影和肾匀浆中AE1的迁移率相似，因此可能代表AE1。180-kD条带的迁移率与大鼠胃和肾中的AE2相似，因此可能代表AE2。对培养的IMCD细胞顶端或基底外侧膜蛋白进行选择性生物素化显示，AE1和AE2都定位于基底外侧膜。Northern印迹分析证明了AE1和AE2的mRNA表达，但未检测到AE3的表达。此外，发现这些细胞表达的AE1和AE2的cDNA序列与报道的肾AE1和大鼠胃AE2的序列几乎相同。得出的结论是，这种大鼠IMCD细胞培养系表达阴离子交换蛋白基因家族的两个成员AE1和AE2，并且这两种交换蛋白可能介导了在该细胞系中观察到的依赖Cl-的电中性HCO3-转运。
