Use of monoclonal antibodies to isolate cortical collecting tubule cells: AVP induces PGE release

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Abstract
Splenic lymphocytes from rats immunized with Madin-Darby canine kidney cells were fused with the mouse plasmacytoma SP2/0-Ag14. A hybridoma line (cct-1) secreting a rat Ig IgG2c that reacts with an ecto-antigen of the canine renal collecting tubule was selected and cloned. Plastic culture dishes coated with the purified monoclonal antibody were used to adsorb 107 collecting tubule cells from a mixture of 109 renal cortical cells prepared by treatment of the canine renal cortex with collagenase. Primary, monolayer cultures of canine cortical collecting tubule (CCCT) cells were established from the adsorbed cells. Cultured CCCT cells stained uniformly for NADH diaphorase and glycerol-3 phosphate dehydrogenase (EC 1.1.99.5) but did not stain for succinate dehydrogenase; this pattern of staining is unique for cortical collecting tubules in the dog kidney. When examined by EM, only principal (64%) and intercalated (36%) collecting tubule cells were present. CCCT cells formed hemicysts when grown to confluency in monolayer culture and developed a transcellular potential difference when seeded on Millipore filters. Arginine vasopressin (AVP), prostaglandin E2 (PGE2) and isoproterenol, but not parathyroid hormone or calcitonin, caused the elevation of intracellular cAMP concentrations in CCCT cells. CCCT cells formed immunoreactive PGE2 in response to bradykinin, [Asu1,6,Arg8]vasopressin (DD-AVP) and AVP. Antibodies directed against discrete segments of the renal tubule can be used to isolate apparently homogeneous populations of tubule cells. Isolated CCCT cells, unlike collecting tubule cells derived from the rabbit renal papillae, form PG in response to AVP. Apparently, AVP induces PG synthesis in the cortical but not the papillary collecting tubule.