BICARBONATE TRANSPORT BY THE MATURING RENAL TUBULE

说明（改编自申请人的摘要）：膜结合碳 脱水酶（CA）对于近端小管中的HCO转运是必需的， 集合管临床上，新生儿肾脏酸化的问题可能是 由于延迟的膜CA表达。膜CA包括CA同种型IV， 其通过糖基磷脂酰肌醇锚定在腔膜上 (GPI)联动然而，两种抗CA IV肽抗体显示CA IV是 表达于近端小管的顶膜和基底膜， 后者不是GPI锚定蛋白预期的。其他膜CA亚型（CA XII和XIV），可以驻留在基底外侧膜上，并与 这些抗体。基底外侧CA的身份，其机制， 靶向，及其在酸中毒和发展过程中的调节将被检查 在兔子肾脏中。目的1是确定哪种CA亚型存在于 近端小管的基底外侧膜。将使用基于PCR的方法 获得兔CAs XII和XIV的cDNA探针。将制备肽 从这些序列中检测CA IV抗体的特异性， 产生CA XII和XIV特异性抗体。另一种抗CA IV肽 将制备抗体（与CA XII或XIV没有同源性）。顶端 将对基底外侧CA进行比较和表征。目标2审查了 膜CA的靶向。膜CA cDNA将是FLAG标记的， 瞬时转染到永生化小鼠近曲小管细胞中。的 将检查表达的膜CA的极性，并与 C-末端的靶向。这将确定GPI连接是否赋予 顶端极性和C-末端亲水段赋予基底外侧 膜CA的极性。对这些核证机关的规管， 还将检查代谢性酸中毒和成熟。目标3调查 生理相关性HCO基底外侧CA活性的存在 将在灌注的近端小管中检查转运和细胞pH。的 调节基底外侧CA对酸中毒和发育的反应， 研究了最后，酸中毒引起CA耐药的机制 将在外髓集合管中研究抑制，其吸收 HCO尽管管腔不平衡pH。目的是更好地了解 膜碳酸酐酶（CA）在介导肾脏酸碱平衡中的作用 运输

DESCRIPTION (Adapted from the Applicant's Abstract): Membrane-bound carbonic anhydrase (CA) is essential for HCO transport in the proximal tubule and collecting duct. Clinically, problems in neonatal renal acidification may be due to delayed membrane CA expression. Membrane CA includes CA isoform IV, which is anchored to the luminal membrane via a glycosylphosphatidylinositol (GPI) linkage. However, two anti-CA IV peptide antibodies show that CA IV is expressed on both apical and basolateral membranes of the proximal tubule, the latter not expected for a GPI-anchored protein. Other membrane CA isoforms (CA XII, and XIV), could reside on the basolateral membrane and cross-react with these antibodies. The identity of the basolateral CA, the mechanisms for its targeting, and its regulation during acidosis and development will be examined in rabbit kidney. Aim 1 is to determine which CA isoform resides in the basolateral membrane of the proximal tubule. A PCR-based approach will be used to obtain cDNA probes for CAs XII, and XIV in rabbit. Peptides will be prepared from these sequences to test specificity of the CA IV antibodies and to generate CA XII- and XIV-specific antibodies. Another anti-CA IV peptide antibody (with no homology to CAs XII or XIV) will be made. The apical basolateral CAs will be compared and characterized. Aim 2 examines the targeting of membrane CAs. Membrane CA cDNAs will be FLAG-tagged and transiently transfected into immortalized mouse proximal tubule cells. The polarity of expressed membrane CAs will be examined and compared with the targeting of the C-terminus. This will establish whether GPI-linkage confers apical polarity and the C-terminal hydrophilic segment confers basolateral polarity to the membrane CA. The regulation of these CAs in response to metabolic acidosis and maturation will also be examined. Aim 3 investigates physiologic correlations. The presence of basolateral CA activity on HCO transport and cell pH will be examined in perfused proximal tubules. The regulation of basolateral CAs in response to acidosis and development will be studied. Finally, the mechanisms by which acidosis causes resistance to CA inhibition will be studied in outer medullary collecting ducts, which absorb HCO despite a luminal disequilibrium pH. The objective is to better understand the role of membrane carbonic anhydrases (CAs) in mediating renal acid-base transport.