Immunodissection and culture of rabbit cortical collecting tubule cells

A mouse monoclonal antibody designated IgG₃(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG₃(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10⁶ rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was >96% based on immunocytofluorescent staining with three different anti-collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10⁷ RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of ~32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E₂ (PGE₂), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D₂, or prostaglandin I, and 2) to release PGE₂ in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.