Axial distribution of band 3-positive intercalated cells in the collecting duct of control and ammonium chloride-loaded rabbits.

Recent studies have suggested that less than 10% of intercalated cells in the rabbit outer cortical collecting duct (CCD) [1, 2] and less than 3% in the connecting segment (CNT) [3] are identifiable by functional criteria as acid-secreting (type A or alpha) intercalated cells. Other studies, using peanut lectin-binding and the absence of apical endocytic activity to identify bicarbonate-secreting (type B or beta) intercalated cells, have suggested that acid-loading increases the percentage of alpha intercalated cells in the CCD. Because our preliminary observations of band 3 immunoreactivity suggest that the percentages of alpha intercalated cells in the rabbit outer CCD and the CNT are underestimated by physiologic studies and are not altered by chronic acid-loading, we quantified the percentage of alpha intercalated cells in various segments of the collecting duct using light microscopic immunohistochemistry in kidneys of rabbits receiving tap water (control) or 75 mM NH4Cl for 12 days plus 8 daily gavages of 2 to 6 mEq NH4Cl/kg body wt. Mean urine pH values were 5.96 in acid-loaded animals versus 8.47 in controls. Kidneys were preserved by in vivo perfusion with periodatelysine-paraformaldehyde fixation and processed for immunohistochemical colocalization using sequential labeling with monoclonal antibodies and peanut lectin, followed by immunoperoxidase detection. Colocalization of band 3 and carbonic anhydrase II immunoreactivity revealed the following percentages of band 3-positive intercalated cells in control versus NH4Cl rabbits: CNT, 49.0 versus 52.8; initial collecting tubule (ICT), 27.2 versus 34.5; outer CCD, 33.5 versus 30.3; inner CCD, 38.2 versus 41.8; corticomedullary CD, 67.9 versus 58.8. There were no differences between the groups for all comparisons. Similar results were obtained using band 3 protein immunoreactivity and peanut lectin-binding to identify intercalated cell subtypes. However, in NH4Cl-loaded rabbits, peanut lectin-binding was observed in band 3 positive intercalated cells in the outer medullary CD. We conclude that: (1) the percentage of alpha intercalated cells in rabbit CCD subsegments are approximately 50% in the CNT, 30% in the ICT and the outer CCD, 40% in the inner CCD, and 60% in the corticomedullary CD; (2) the percentage of alpha intercalated cells is not altered by chronic NH4Cl-loading; (3) peanut lectin is not a specific marker of beta intercalated cells.