Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence

Abstract

We have synthesized three peptides corresponding to putative antigenic regions in the immunogenic domain, hinge region, and carboxy-terminus of the protein. A rabbit immunized with a peptide derived from the hinge region of the receptor produced an antiserum which showed 50% displacement with 20 pg peptide at a final serum dilution of 1:35,000. When the antiserum was immunopurified and applied to sections of intact rat and human kidney it stained cells lining segments corresponding to distal tubule, connecting piece, and initial cortical collecting duct, consistent with the known sites of mineralocorticoid action. In both human (formaldehyde-fixed) and rat (Bouin's solution) there was ample evidence for both nuclear and cytoplasmic staining. The thymus, in which previously we have found [3H]aldosterone binding to be below detection limits, showed little or no staining. Western blot analyses demonstrated that the polyclonal antibody recognized an epitope of the expected molecular size. The availability of antibodies to the mineralocorticoid receptor should, thus, facilitate investigation of the steroid specificity-conferring mechanism which allows mineralocorticoids, but not glucocorticoids, access to the nonselective receptor in the kidney.