Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney.

Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney N. FARMAN, M. E. OBLIN, M. LOMBES, F. DELAHAYE, H. M. WESTPHAL, J. P. BONVALET, AND J. M. GASC Institut National de la Sant6 et de la Recherche Mkdicale (INSERM) U. 246, Departement de Biologie, Service de Biologie Cellulaire, CEN Saclay, 91191 Gif-sur- Yvette; INSERM U. 33, 94270 KremlinBic&re, France; and Institut fiir Molekularbiologie, D-3550 Marburg, Federal Republic of Germany

FARMAN, N., M. E. OBLIN, M. LOMBES, F. DELAHAYE, H. M. WESTPHAL, J. P. BONVALET, AND J. M. GASC. Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney. Am. J. Physiol. 260 (Cell Physiol. 29): C226-C233, 1991.--The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium. aldosterone; renal tubule

anti-idiotypic

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THE KIDNEY is composed of several transporting epithelia that are submitted to various hormonal regulations. Both aldosterone and glucocorticoids play a major regulatory role on renal functions. Several studies (reviewed in Ref. 29) have been devoted to the distribution of the specific binding sites of these two classes of hormones along the nephron. However, several questions remain open regarding the mineralo- or glucocorticoid specificity of the observed binding of corticosteroid hormones in the varC226

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ious parts of the nephron (29). One major cause of uncertainties resides in the coexistence of mineralo- and glucocorticoid actions at the same tubular sites. The cross-occupancy of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) by these two classes of corticosteroids (5) makes it difficult to distinguish unambiguously between the respective contributions of MR and GR for the observed effects. On the other hand, interactions between the physiological effects of the two hormones (for example, an indirect effect of one hormone in a segment located downstream from its direct site of action) hinder the interpretation of transport studies. The localization of MR and GR by using specific antibodies allows us to circumvent these difficulties. Recently, Lombes et al. (26) produced an anti-idiotypic antibody, which is an internal image of aldosterone. The epitope recognized by this monoclonal antibody is localized in the steroid-binding domain of MR. It has been demonstrated that this antibody specifically detects MR in the rabbit kidney (27). Using this antibody, we have previously determined the localization of MR along the rabbit nephron (27). On the other hand, specific antibodies directed against the immunological domain of the glucocorticoid receptor are available (16,41). The aim of the present study was to compare the respective distribution of MR and GR in the rabbit kidney. For this purpose, further experimental series were performed where immunohistochemistry was applied to serial histological sections with the anti-idiotypic anti-MR antibody HlOE (26) and the anti-GR antibody IGR 49/4 (41). The influence of adrenalectomy on the immunodetection of MR and GR was examined. Results show that MR and GR are coexpressed in the same cells along the distal nephron, whereas immunostaining was absent in proximal tubular cells with both anti-MR and anti-GR antibodies. MATERIALS

AND

METHODS

Experiments were performed on seven New Zealand female rabbits, l-2 kg body wt. Three animals were bilaterally adrenalectomized under anesthesia. Two received 1.9 mg/kg deoxycorticosterone acetate (Syncortyl, Roussel Uclaf, Romainville, France) ip at the time of

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surgery and for the next 2 days and were given 0.9% saline to drink. Experiments were done 6 days after adrenalectomy. One rabbit did not receive any hormonal substitution. Rabbits were anesthetized with 25-50 mg pentobarbital sodium (Nembutal) iv and perfused through the abdominal aorta with 500 ml of Zamboni’s solution [2% (wt/vol) paraformaldehyde, 15% (vol/vol) saturated picric acid solution, and 85% (vol/vol) 300 mM sodium phosphate buffer, pH 7.41 at 37OC. The perfusion procedure was done according to Kaissling and Le Hir (19). At the end of the perfusion, the kidneys were removed, cut into slices and pyramids (about 2 mm thick), and postfixed for 24 h in the same fixative. The kidney slices and pyramids were washed in 70% ethanol, dehydrated in graded ethanol, cleared in l-butanol and embedded in Paraplast. Sections (7 mm) were cut, mounted on histological slides, and processed for immunohistochemistry. Immunohistochemical technique. A routine procedure of indirect immunostaining was used (12, 13). Briefly, after deparaffinization and rehydration in 10 mM Na phosphate buffer, 150 mM NaCl, pH 7.4 (PBS), sections were incubated with 3% normal horse serum. After 10 min the excess of fluid was blotted and replaced by the monoclonal anti-idiotypic antibody HlOE, a mouse IgGl immunoglobulin, used as diluted ascite fluid (26). The antibody was used at a concentration of -1-5 mg/ml. MOPC 3X, an IgGl (Sigma, St. Louis, MO), was used as a control. After the primary antibody, a horse biotinylated anti-mouse IgG antibody (Vector Laboratories, Burlingame, CA) and the avidin-biotin-peroxidase complex (ABC-Elite, Vector) was used as a detection system. After each incubation step, the slides were rinsed in PBS. The peroxidase activity was revealed by diaminobenzidine tetrahydrochloride (0.5 mg/ml) in the presence of 0.01% H,Oz. Sections were then dehydrated and mounted in Canada balsam without counterstaining. The competition experiments were performed by preincubating sections in PBS containing 1 mM aldosterone (Sigma), or anti-hormone [ RU486 (RousselUclaf), SC 9420 (Searle Laboratories, Chicago, IL)] for 30 min before incubation with HlOE antibody (l-5 mg/ ml or -30 nM on the basis of a mol wt 150,000 for an IgG) together with the steroid or the antihormone. The same protocol was used for detection of GR. IGR 49/4 is a monoclonal mouse antibody prepared against the purified rat liver cytosolic receptor (41). It was used as an ammonium sulfate precipitate of ascites fluid at the concentration of 0.1-l mg/ml. The specificity of the GR immunostaining was established by substitution of MOPC 3lC for the IGR 49/4 antibody, by dilution of the antibody up to extinction (10m5 dilution), and by presaturation of the antibody (0.1 mg/ml) with a purified preparation of rat liver GR (1 mg/ml, a gift from J. A. Gustafsson). The extinction of immunostaining after presaturation shows the specificity of the recognition of an epitope common to the rat and rabbit GRs. Although the IGR 49/4 antibody was originally described as reacting poorly with the native form of the rabbit liver GR (41), we have obtained a very good immunostaining under our fixation conditions. This may be due to the

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optimization of these conditions (other fixation protocols tested did not allow a good preservation of immunoreactivity) and also to a discrepancy between the affinity of the antibody for the native receptor (used to establish the specificity of the antibody in the original article) and for the denatured-fixed forms of GR detected in histological sections. Such an inconsistency of recognition of these two forms of the same antigen by the same antibody is a frequent observation. It can be explained by masking, unmasking, and denaturation of the corresponding epitope. It is likely that the antibody has a better affinity for the fixed receptor in solid phase than for the native receptor in liquid phase. RESULTS

Glomerulus and proximal tubule. No staining was apparent in the cells of these structures with either HlOE (Fig. 1A) or IGR 49/4 (Fig. 10). In the glomerulus, none of the different cell types was stained. In the proximal tubule, both convoluted and straight parts were devoid of immunostaining. Loop of Henle. Nuclei of the thin parts of the loop were stained by both antibodies. In the medullary portion of the thick ascending limb (MTAL), a nuclear and cytoplasmic staining was present in all cells. A comparison of the staining by the anti-MR and the anti-GR antibodies in the outer medulla is shown in Fig. 1 on two serial sections. It appears that the staining by anti-GR antibody was more intense in the MTAL than in the outer medullary collecting duct (OMCD) (Fig. lE), whereas the reverse occurred with the anti-MR antibody (Fig. 1B). In the cortical part of the thick ascending limb (CTAL), both MR and GR were found in cell cytoplasm and nuclei. Early distal tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). In each of these structures, a strong immunostaining was observed with both antibodies, as shown in Figs. l-3. The staining affected both cytoplasm and nuclei with some differences from one cell to another: it was either predominant in the cytoplasm or equally distributed between the nucleus and the cytoplasm. In the DCT, close to the glomerulus, all cells were stained by either of the antibodies (Fig. 1, A and D, Fig. 3A). By contrast, -15-20% of cells were unstained in CNT and CCD (Fig. 1, A and D, Fig. 2). From both the relative distribution of stained and unstained cells and the morphological aspect of the latter ones, it seems likely that unstained cells correspond to intercalated cells, although no definite conclusion can be drawn from light microscopy observation. The comparison of serial sections incubated with IGR 49/4 or HlOE suggests that the same cell population is concerned, whatever the antibody applied. It is to be noticed that some differences are visible in the relative intensity of immunostaining by either HlOE or IGR 49/4 along these distal cortical structures; the staining by HlOE was of similar intensity in the three structures, but IGR 49/4 gave a weaker staining in CCD than in DCT or CNT. In situ competition studies assessed the specificity of the staining by HlOE; when slides were incubated in the


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FIG. 1. Immunolocalization of mineralocorticoid receptors (MR) (A-C) and glucocorticoid receptors (GR) (D-F) in cortex (A and D), medulla (B and E), and papilla (C and 5’) of rabbit kidney, with antibodies to receptors: HlOE for MR and IGR 49/4 for GR. G, glomerulus; P, proximal tubule: TL. thin limbs of Henle’s loop; MTAL, medullary portion of thick ascending limb of Henle’s loop; D, distal tubule: C. connecting tubule: OMCD, outer medullary collect& duct; IMCD, inner medullary collecting duct; EP. epithelium lining papilla: IC. interstitial cells. Glomeruli-and proximal tubules are devoid of immunostaining, whereas all other tubular segments from Henle’s loop to medullary collecting tubule stain for both MR and GR. Magnification, X330.

presence of an aldosterone excess, the staining was abolished (Fig. 2). A similar observation was noted with an excess of SC 9420 (not shown). RU486, a glucocorticoid ligand that does not bind to MR (33), did not modify the pattern of immunostaining (Fig. 2). With regard to IGR 49/4, the antibody presaturation with purified GR preparation almost completely extinguished the staining (Fig. 3).

No clear difference was apparent between experiments in intact and adrenalectomized rabbits for both HlOE (Fig. 2, A and D) and IGR 49/4 (Fig. 3, A and C). Medullary collecting tubule (MCD). With both antibodies, cells of the medullary collecting tubule were immunostained along this segment, from OMCD to the end of the papillary collecting duct (IMCD) (Fig. 1). Both cytoplasm and nuclei were stained. No unstained cells were present. As mentioned above, the staining by HlOE predominates in OMCD compared with the neighboring MTAL (Fig. lB), whereas the reverse was observed with IGR 49/4 (Fig. 1E). Of interest, the staining of GR gradually decreased along the MCD; in contrast, staining of MR remained high all along the collecting tubule. Papillary surface epithelium. As for MCD, an intense

staining by HlOE, comparable or superior to that of the CCD, was present in cytoplasm and nuclei of this epithelium (Fig. 1C). Staining by IGR 49/4 was also observed (Fig. lF), but it was much lower than in the CCD. Papillary interstitial cells. These cells were stained by both antibodies (Fig. 1, C and F). Nuclei were clearly positive. Because of the difficulty in delineating without doubt the cytoplasmic areas, the staining remained often uncertain in this cell compartment. DISCUSSION

Up to now, the precise localization of the sites of action of mineraloand glucocorticoid hormones along the nephron was hindered by methodological problems. Binding experiments on whole kidney homogenates preclude the attribution of the observed binding to a welldefined cell type. The early biochemical (2, 6, 24) and autoradiographic studies (7, 8) on isolated tubular segments constituted a significant progress, However, the cross-binding of mineralo- and glucocorticoids to MR and GR limited the possibility to attribute unambiguously the binding to one of the two classes of receptors.


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FIG. 2. Immunostaining of MR by HlOE in kidney cortex of a normal rabbit (A). Staining is almost completely abolished when antibody is competed for by 1 mM aldosterone (B, a natural ligand of MR), but not by RU 486 (C, a synthetic ligand with no affinity for MR). HlOE antibody reveals MR in both cell cytoplasm and nuclei, whether animal is normal (A) or adrenalectomized (D). Abbreviations as in Fig. 1. Magnification, x400.

In addition, it has been recently proposed (11) that tissue mineralocorticoid specificity depends on the presence of llP-hydroxysteroid dehydrogenase, which converts cortisol or corticosterone to metabolites with low affinity for MR. This notion may bring further insights for establishing the mineralo- or glucocorticoid sensitivity of tissues with regard to MR and GR. Lombes et al. (26) recently obtained an anti-MR antiidiotypic antibody, HlOE. This antibody is an internal image of aldosterone, and it has been demonstrated to bind specifically to MR without interfering with glucocorticoid binding sites in the rabbit kidney cytosol (26). The present study confirms the specificity of HlOE immunostaining (27): complete extinction of the immunostaining due to HlOE by an aldosterone excess or by SC

FIG. 3. Immunostaining of GR by IGR 49/4 in kidney cortex of a normal (A) or adrenalectomized (C) rabbit. When antibody is presaturated with a purified preparation of GR, staining is abolished (B), IGR 49/4 reveals GR in cell nuclei and cytoplasm independently of endocrine state of animal (normal, A, or adrenalectomized, C). Abbreviations as in Fig. 1. Magnification, x400.


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9420, a spirolactone *antagonist of aldosterone, and absence of competition by the glucocorticoid antagonist RU486 (33). The specificity of IGR 49/4 for GR was evidenced in the present study by extinction of the immunostaining after presaturation of the antibody with purified GR. The intrarenal distribution of MR and GR is schematically summarized in Fig. 4. The presence of MR and GR immunostaining in the cortical collecting tubule is in agreement with the current knowledge of corticosteroid sensitivity of this epithelium (29). In addition, this paper shows that MR and GR are both expressed in tubular segments, such as the loop of Henle and the medullary collecting tubule, where an exclusive specificity with regard to mineralo- or glucocorticoids was under discussion. Furthermore, new, unexpected target cells for corticosteroids have been found. Concerning the presence of mineralocorticoid receptors in the CCD, our results are in agreement with a whole body of evidence along this line based on studies on aldosterone binding (7), aldosterone-modulated electrolyte transports (9, 37), and trophic and metabolic effects of aldosterone (19, 20, 30). The data concerning the other parts of the distal nephron were less established or even controversial, as discussed by Marver (29). The present study demon-

FIG. 4. Schematic representation of distribution of MR and GR ;dong nephron, as determined by immunostaining with HlOE and IGR 49/4. In boxes is given a semiquantitative estimation of immunoreacrepresented by signs + to +++ according to t ivity alon, 0 nephron, staining intensity obtained with each antibody; -, no immunostaining. GLOM, glomerulus; PCT, proximal convoluted tubule; TL, thin limbs of‘ Henle’s loop; medullary (MTAL) and cortical (CTAL) portions of thick ascending limb of Henle’s loop; DCT, distal tubule; CNT, connecting tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct; PAP.EP, epit helium lining the papilla.

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strates the presence of both MR and GR in the DCT, the loop of Henle, and the medullary collecting tubule. Using an antibody directed against the hinge region of the human mineralocorticoid receptor, Krozowski et al. (23) and Rundle et al. (35) recently reported the presence of MR in the DCT and initial CCD of rat and humans, in agreement with our data in the rabbit, but did not discuss the other parts of the nephron, in particular the medulla. With regard to glucocorticoid receptors, autoradiographic studies with tritiated dexamethasone (8) and biochemical studies with tritiated corticosterone (24) demonstrated their presence all along the distal nephron. In addition, physiological effects of glucocorticoids in various parts of the distal nephron have been reported (29), although it is somewhat difficult to dissociate direct specific effects of these hormones from indirect ones. Considering all this evidence, our observations are compatible with specific effects of glucocorticoids all along the distal nephron. MR and GR coexist within the same cells. Although it is hazardous to draw quantitative estimations from immunostaining data, it is of interest to note that the relative distribution of GRs and MRs along the distal nephron differs somewhat (Fig. 4). Whereas MR immunostaining by HlOE seems approximately of similar intensity all along the distal nephron, GR immu .noreactivity is inte nse in the medu .llary portion of the loop of Henle and in the initial part of the distal tubule; then it progressively decreases along the collecting tu .bule to become very weak in the papillary portion of the collecting duct. The present experiments point out two elements as yet undescribed. First, the cells of the epithelium lining the papilla possess both GR and MR (Fig. 1). The physiological relevance of this observation is yet unknown. Second, papillary interstitial cells are also stained by both antibodies (Fig. 1). Whereas the modulation of prostaglandin synthesis by glucocorticoids (1) suggests a role of glucocorticoids in these cells, the eventual role of aldosterone is unknown . Cell heterogeneity in the cortical collecting tubule. In this segment, 1520% of cells were unstained by both antibodies. This observation confirms our previous report with HlOE (27) and the recent data of Krosowski et al. (23) and Rundle et al. (35), obtained with another antibody. Examination of serial sections shows that the same cells lack both MR and GR. Morphological and functional cell heterogeneity in the CCD is well documented (19); principal cells constitute the major part of the cell population, and intercalated cells [subdivided in at least two groups (19, 36)] are a minority. Although it is difficult to clearly distinguish between cell types at the light microscopy level, both the morphological aspect of the cell and the frequency distribution of immunostaining suggest that unstained cells should correspond to intercalated cells (or to one class of intercalated cells). Along this line, the decrease in the number of unstained cells from the connecting tubule to the medullary portions of the collecting tubule correlates with the gradual


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disappearance of intercalated cells along this structure (19) SzLbcellular distribution of immunostaining. Concerning the subcellular distribution of corticosteroid receptors, both GR and MR immunostaining were found in the cytoplasmic and nuclear compartments. However, this distribution seems to be variable from one cell to another, with a predominance of cytoplasmic staining in some cells and a nucleocytoplasmic staining occurring in other cells. The subcellular immunolocalization of corticosteroid receptors is clearly different from the one described for estrogen and progesterone receptors that are found exclusively in the nucleus, even in the absence of hormone (13, 15, 22). Actually, in our system, no obvious difference in the nucleocytoplasmic distribution of corticosteroid receptors could be evidenced between normal and adrenalectomized animals, a pattern previously observed for MR in the rabbit (27) and the rat kidney (23). As a matter of fact, there is no general consensus for a hormone-dependent nuclear translocation of GR. As pointed out by Gustafsson (16), the subcellular localization of steroid receptors seems to be highly dependent on the experimental conditions used, i.e., tissue sections or cell preparations, perfusion and/or immersion of the tissue, and use of detergent such as Triton X-100. Additional experiments are necessary to further assessthe intracellular distribution of corticosteroid receptors and its physiological significance in view of the receptors’ molecular mechanism of action. Difficulties in establishing the presence of GR in the proximal tubule of rat and rabbit appear in data from the literature. Two studies (24, 38) with [“Hlcorticosterone detected no or very little binding to the proximal tubule. In rat isolated tubules, Lee et al. (24) found that the specific binding of corticosterone (without separation of nuclear from cytoplasmic binding) was IO-fold lower in proximal than in cortical collecting tubule. Strum et al. (38) reported that specific binding of [“Hlcorticosterone was restricted to collecting tubules in autoradiographs of rat kidney sections. Using autoradiography with [“Hldexamethasone on rabbit microdissected tubules, Farman et al. (8) reported specific nuclear labeling in distal structures; no nuclear labeling was found in proximal tubules, which exhibited a high extranuclear labeling that was only partially displaceable. Mishina et al. (31) described specific [“HI triamcinolone binding in both proximal and distal suspensions of rat cortical tubules. However, contamination of proximal suspensions by distal tubules was important (25%). Thus nuclear glucocorticoid binding to type II and/or to type III (corticosterone-specific) sites has been constantly found in the distal nephron, whereas it remains questionable in the proximal tubule. A similar pattern is apparent from immunostaining with IGR 49/4. The low or undetectable level of GR in the proximal tubule, as observed by several methodological approaches, could indicate that the GR in these cells may differ from the classical GR of hepatic and renal distal cells. Such a possibility is compatible with our previous observation (8) of an important and partially displaceable binding of [3H]dexamethasone in extra nuclear struc-

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tures of the terminal parts of the proximal tubule. The existence of [“Hldexamethasone receptor complexes, unable to translocate into the nucleus, could suggest the presence of a nonclassical GR that differs from either type II and/or type III receptors and not recognized by IGR 49/4: it may be, for example, an isoreceptor such as IB (28). An alternative possibility would be that the epitope of the proximal GR recognized by IGR 49/4 could be masked, thus preventing immunodetection by this monoclonal antibody. Because immunostaining is observed in distal structures, this would imply that the proximal receptor differs in some manner from that of the distal nephron. Finally, the possibility remains that the classical GR is expressed at a low level, or even nonexpressed, in proximal tubular cells. Such a hypothesis leads to the question of a nonclassical mechanism of action of glucocorticoids in this tissue, as previously evoked in other cell types (4). For example, binding of glucocorticoids to plasma membranes has been reported in rat brain (40) and liver (17). In L-cells, at least two major cellular responses to glucocorticoids that do not seem to involve an interaction between the activated hormone-receptor complex and chromatin have been described (21). Membrane receptor-mediated electrophysiological effects of glucocorticoids have been reported in mammalian neurons (18). The presence of extra nuclear binding of [“HI dexamethasone in the proximal tubule, as previously reported (8), may correspond to such membrane receptor-mediated effects of glucocorticoids. Interestingly, it has been reported that steroids modify the composition and fluidity of plasma membranes, resulting in modifications of transmembrane transports (3, 34). Recently, it has been demonstrated that such effects could be obtained in vitro on brush-border membrane vesicles from proximal tubule; phosphate transport is directly affected by l-25dihydroxy vitamin D3 (39) and even by cholesterol (29, the precursor of steroid hormones. On the whole, the absence of immunolocalization of GR in the proximal tubule, associated with several evidences of nonclassical binding and/or action of glucocorticoids, requires further studies to elucidate the mechanism of glucocorticoid action in these cells. Conclusion. The present study compares the intrarenal distribution of MRs and GRs along the nephron, as determined by immunohistochemistry on serial histological sections. The glomerulus and the proximal tubule were not stained by either antibody. Considering the evidence of glucocorticoid action in the proximal tubule, the absence of staining by the anti-GR antibody may correspond to either an actual absence of GR in these cells (implicating the existence of nonreceptor mediated effects of glucocorticoids) or to the presence of a receptor that differs in some way from those present in the liver and distal structures. Both immunodetected GR and MR are restricted to the distal nephron, from the beginning of the loop of Henle to the end of the medullary collecting duct. They coexist in the same cells. MR predominates in the distal tubule and the collecting duct, either cortical or medullary. GR predominates in the ascending limb of the loop of Henle and in the distal tubule. Cell hetero-


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geneity, with regard to MR as well as GR, was present in the CCD. In addition to tubular cells, papillary interstitial cells and cells of the epithelium lining the papilla also possess MRs and GRs. The coexpression of both receptors in the same cell may be an important factor in view of the integrated regulation of ion transport by corticosteroid hormones, as previously suggested (14). We are indebted to F. Wanstok, who performed rabbit adrenalectomies, and N. Koechlin, for help in kidney fixation. We acknowledge M. H. Badoureaux for secretarial assistance. Address for reprint requests: N. Farman, U246 INSERM, Universite Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. Received

5 February

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25 September

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20. 21.

1990. 22.

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Mineralocorticoid and glucocorticoid receptors in human kidney.

Chromatographic demonstration of mineralocorticoid-specific receptors in rat kidney.

Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary.

16alpha, 18-Dihydroxydeoxycorticosterone and the binding of aldosterone to mineralocorticoid receptors in kidney of adrenalectomized rats.

Glucocorticoid and mineralocorticoid receptors in gut mucosa.

Aldosterone and vascular mineralocorticoid receptors: regulators of ion channels beyond the kidney.

Aldosterone and Mineralocorticoid Receptors-Physiology and Pathophysiology.

Immunolocalization of androgen receptor and estrogen receptors in skin tags.

Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney.

Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney.

Mineralocorticoid receptors guide spatial and stimulus-response learning in mice.

Gluco- and antimineralocorticoid effects on human sleep: a role of central corticosteroid receptors.

Mineralocorticoid receptor signaling: crosstalk with membrane receptors and other modulators.

Glial cells express both mineralocorticoid and glucocorticoid receptors.

Atrial receptors in the dog and rabbit.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries.

Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action.

Myeloid mineralocorticoid receptor activation contributes to progressive kidney disease.

Binding characteristics of mineralocorticoid and glucocorticoid receptors in dog brain and pituitary.

Efficacy and safety of mineralocorticoid receptors in mild to moderate arterial hypertension.

Aldosterone receptors and the evaluation of plasma mineralocorticoid activity in normal and hypertensive states.

Expression of mineralocorticoid and glucocorticoid receptors in preautonomic neurons of the rat paraventricular nucleus.

The Role of the Mineralocorticoid Receptor in Inflammation: Focus on Kidney and Vasculature.

Mineralocorticoids and mineralocorticoid receptors in mononuclear leukocytes in patients with pregnancy-induced hypertension.