Immunocytochemical characterization of the high-affinity thiazide diuretic receptor in rabbit renal cortex

Thiazide diuretics increase urinary NaCl excretion primarily by inhibiting Na and Cl transport across the apical membrane of cells in the renal distal tubule. Although these diuretics bind to a membrane protein that couples transport of Na and Cl directly, the molecular nature of this transporter and its localization in the mammalian kidney remain controversial. The present experiments were designed to develop monoclonal antibodies to the high-affinity thiazide diuretic receptor to investigate its molecular characteristics and its cellular and subcellular localization in rabbit kidney. Mice were immunized with high-affinity thiazide diuretic receptors that had been partially purified from rabbit kidney cortex. Resulting hybridomas were screened for the ability to immunoprecipitate thiazide diuretic receptors that were labeled with the thiazide-like diuretic [3H]metolazone. A single hybridoma (MAb JM5) produced antibodies capable of immunoprecipitating up to 80% of the labeled thiazide receptors from solubilized renal cortical membranes. MAb JM5 reacted with a 125-kDa protein on Western blots of solubilized renal cortical apical membranes. It stained the apical membrane of cells in the distal convoluted and connecting tubule but did not stain proximal tubules, glomeruli, or interstitial structures. Less intense staining of apical membranes of principal cells in the collecting tubule and a subpopulation of cells in the thick ascending limb were also present. These results indicate that the high-affinity thiazide diuretic receptor comprises a 125-kDa protein that localizes to the apical membrane of cells in the renal distal tubule.