CHARACTERIZATION OF ANION-EXCHANGERS IN AN INNER MEDULLARY COLLECTINGDUCT CELL-LINE

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 a re lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3- exit. This cell line functionally res embles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl--dependent, Na+-i ndependent process. It can be driven by Cl- gradients and inhibited by stilbenes such as 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 consisten t with a basolateral localization of AE in cultured cells and in cu-in tercalated cells identified in sections of rat kidney cortex. Immunobl ot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells. rat renal medulla, and fr eshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney ho mogenate and therefore probably represents AE1. The mobility of the 18 0-kD band is similar to that for rat stomach and kidney AE2 and theref ore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that bot h AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE 3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these ce lls was found to be virtually identical to that reported for kidney AE 1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, A E1 and AE2, and both of these exchangers probably mediate the electron eutral Cl--dependent HCO3- transport observed in this cell line.