Immunohistochemical demonstration of the mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase-1 and -2, and hexose-6-phosphate dehydrogenase in rat ovary.

An IHC survey using several monoclonal antibodies against different portions of the rat mineralocorticoid receptor (MR) molecule demonstrated significant specific MR immunoreactivity in the ovary, prompting further study of the localization of MR and of determinants of extrinsic MR ligand specificity, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2, and hexose-6-phosphate dehydrogenase (H6PDH). MR expression (real-time RT-PCR and Western blot) did not differ significantly in whole rat ovaries at early diestrus, late diestrus, estrus, and a few hours after ovulation. MR immunostaining was most intense in corporal lutea cells, light to moderate in oocytes and granulosa cells, and least intense in theca cells. Light immunoreactivity for 11beta-HSD2 occurred in most cells, with some mural granulosa cells of mature follicles staining more strongly. The distribution of immunoreactivity for 11beta-HSD1 and H6PDH required to generate NADPH, the cofactor required for reductase activity of 11beta-HSD1, was similar, with the most-intense staining in the cytoplasm of corporal lutea and theca cells and light or no staining in the granulosa and oocytes. MR function in the ovary is as yet unclear, but distinct patterns of distribution of 11beta-HSD1 and -2 and H6PDH suggest that the ligand for MR activation in different cells of the ovary may be differentially regulated.

Mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type II expression in renal cell neoplasms: a tissue microarray and quantitative RT-PCR study.

The kidney is an important target for mineralocorticoids. Aldosterone, the major endogenously secreted mineralocorticoid, acts by binding to mineralocorticoid receptor (MR) in the distal renal tubule. The enzyme 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) prevents the binding of glucocorticoids to the MR by inactivating cortisol to cortisone. Our goal was to determine whether MR and 11beta-HSD2 expression could be used to characterize the major types of renal cell neoplasms. Using immunohistochemistry we analyzed tissue microarray specimens from 132 patients with renal cell neoplasms, stratified into 84 clear cell renal cell carcinomas (CRCC), including 9 cases clear cell carcinoma with predominantly granular cytoplasm; 14 papillary RCC (PRCC); 20 chromophobe RCC (CHRCC); and 14 oncocytomas (OCs). MR and 11beta-HSD2 expression were also quantitated by real-time reverse transcription-polymerase chain reaction. Expression of both MR and 11-betaHSD2 was detected in the distal nephrons of normal kidneys. The CHRCC group stained for 11-betaHSD2 in a membranous and cytoplasmic pattern whereas diffuse cytoplasmic reactivity was seen in OCs. MR and 11beta-HSD2 were coexpressed in most of CHRCC (90% and 95%) and oncocytomas (93% and 100%). No MR staining was detected in CRCC, including clear cell carcinoma with predominantly granular cytoplasm, or in PRCC. Only 2 cases of CRCC (2.6%) showed focal positivity for 11beta-HSD2, whereas all PRCCs were negative. CHRCC and OC demonstrated significantly higher levels of MR and 11beta-HSD2 expression than CRCC and PRCC by real-time polymerase chain reaction. Moreover, CHRCC showed higher expression of MR and 11beta-HSD2, as compared with OC. Our study indicates MR and 11beta-HSD2 are both sensitive and specific markers of the distal nephron and its related neoplasms (CHRCC and OC). Additionally, the staining pattern and the level of MR and 11beta-HSD2 expression seems to be useful in the distinction of CHRCC from OC. MR and 11beta-HSD2 should be considered in the immunohistochemical panel to more accurately subtype renal cell tumors.

Hexose-6-phosphate dehydrogenase and 11beta-hydroxysteroid dehydrogenase-1 tissue distribution in the rat.

Intracellular concentrations of the glucocorticoids cortisol and corticosterone are modulated by the enzymes 11beta-hydroxysteroid dehydrogenase (11beta-HSD) 1 and 2. 11beta-HSD1 is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent microsomal reductase that converts the inactive glucocorticoids cortisone and 11-dehydrocorticosterone to their active forms, cortisol and corticosterone. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme that generates NADPH from oxidized NADP (NADP(+)) within the endoplasmic reticulum. In the absence of NADPH or H6PDH to regenerate NADPH, 11beta-HSD1 acts as a dehydrogenase and inactivates glucocorticoids, as does 11beta-HSD2. A monoclonal antibody against H6PDH was produced to study the possibility that 11beta-HSD1 in the absence of H6PDH may be responsible for hydroxysteroid dehydrogenase activity in tissues that do not express significant amounts of 11beta-HSD2. H6PDH and 11beta-HSD1 expression was surveyed in a variety of rat tissues by real-time RT-PCR, Western blot analysis, and immunohistochemistry. H6PDH was found in a wide variety of tissues, with the greatest concentrations in the liver, kidney, and Leydig cells. Although the brain as a whole did not express significant amounts of H6PDH, some neurons were clearly immunoreactive by immunohistochemistry. H6PDH was amply expressed in most tissues examined in which 11beta-HSD1 was also expressed, with the notable exception of the renal interstitial cells, in which dehydrogenase activity by 11beta-HSD1 probably moderates activation of the glucocorticoid receptor because rat renal interstitial cells do not have significant amounts of mineralocorticoid receptors. This antibody against the H6PDH should prove useful for further studies of enzyme activity requiring NADPH generation within the endoplasmic reticulum.

Disabled-2 is expressed in adrenal zona glomerulosa and is involved in aldosterone secretion.

The differentiation of the adrenal cortex into functionally specific zones is probably due to differential temporal gene expression during fetal growth, development, and adulthood. In our search for adrenal zona glomerulosa-specific genes, we found that Disabled-2 (Dab2) is expressed in the zona glomerulosa of the rat adrenal gland using a combination of laser capture microdissection, mRNA amplification, cDNA microarray hybridization, and real-time RT-PCR. Dab2 is an alternative spliced mitogen-regulated phosphoprotein with features of an adaptor protein and functions in signal transduction, endocytosis, and tissue morphogenesis during embryonic development. We performed further studies to analyze adrenal Dab2 localization, regulation, and role in aldosterone secretion. We found that Dab2 is expressed in the zona glomerulosa and zona intermedia of the rat adrenal cortex. Low-salt diet treatment increased Dab2-long isoform expression at the mRNA and protein level in the rat adrenal gland, whereas high-salt diet treatment did not cause any significant modification. Angiotensin II infusion caused a transient increase in both Dab2 isoform mRNAs in the rat adrenal gland. Dab2 overexpression in H295R human adrenocortical cells caused an increase in aldosterone synthase expression and up-regulated aldosterone secretion under angiotensin II-stimulated conditions. In conclusion, Dab2 is an adrenal gland zona glomerulosa- and intermedia-expressed gene that is regulated by aldosterone secretagogues such as low-salt diet or angiotensin II and is involved in aldosterone synthase expression and aldosterone secretion. Dab2 may therefore be a modulator of aldosterone secretion and be involved in mineralocorticoid secretion abnormalities.

Development of a panel of monoclonal antibodies against the mineralocorticoid receptor.

Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1-18, 64-82, 79-97, and 365-381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.