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.
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