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.

Immunohistochemical Localization of Mineralocorticoid Receptor in Human Gut.

“…Immunoreactive precipitates were consistently observed in both cytoplasm and nucleus of the epithelial cells of renal tubules and of those of several exocrine glands under various conditions in agreement with the reports of Krozowski et al (1989) and Rundle et al (1989). In the colonic columnar cells as well as renal tubular cells, the cytoplasmic localization of MR may reflect a non-genomic action of the receptor such as association with unknown factors proximally or distally involved in sodium transport.…”

“…Mucosa was separated from the muscular layer of the intestine at the time of sampling. Specimens were homogenized in 3 vol of TM buffer which consisted of 10 mM Tris, 60 mM Na2Mo4, and 1 mM dithiothreitol, pH 7.4, and centrifuged at 100,000 x g for 1 hr at 4°C (Krozowski et al 1989). The cytosol (2.5 ml) was mixed with 100 pl of DEAE-Sepharose previously equibrated in TM buffer for 1 hr at 4°C, and centrifuged at 100 X g for 5 sec.…”

“…Krozowski et al have synthesized an immunogenic segment of MR protein deduced from the cDNA (Krozowski et al 1989). Using polyclonal antibodies obtained by immunization of rabbits with a synthetic peptide, Krozowski et al have elucidated the localization of MR in human and rat kidney by immunohitochemical and western blot analysis (Krozowski et al 1989). In the present study, we have extended immunocytochemical localization of MR to human gut in order to clarify the physiological role of aldosterone in salt and water homeostasis.…”

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule

“…Immunohistochemical, hormone binding, and RT-PCR studies revealed the MR unambiguously along the ASDN (4,7,10,13,32,37,45,53), but MR localization in upstream nephron portions such as the thick ascending limb (TAL) is less clear. Similarly, a convincing demonstration of MR expression in intercalated cells in the kidney in vivo is lacking.…”

Increased Expression of Mineralocorticoid Receptor in Human Ileum after Total Colectomy: Immunohistochemical and Immunoblotting Studies.

“…Specimens were frozen immediately after resection and were subsequently stored at -80°C. Specimens were homogenized in 3 vol of TM buffer which consisted of 10 mM Tris, 60 mM Na2Mo4, and 1 mM dithiothreitol, pH 7.4, and centrifuged at 100,000 x g for 1 hr at 4°C (Krozowski et al 1989). The cytosol (2.5 ml) was mixed with 0.1 ml of DEAE-Sepharose previously equibrated in TM buffer for 1 hr at 4°C, and centrifuged at 1,000 x g for 5 sec.…”