Analysis of Unilateral Adrenal Hyperplasia with Primary Aldosteronism from the Aspect of Messenger Ribonucleic Acid Expression for
http://www.100md.com
《内分泌学杂志》
Divisions of Pathology (K.Shig.) and Nephro-Urology (H.S.), Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
Division of Pathology (K.K., J.I.) Nagasaki Municipal Hospital, Nagasaki 850-8555, Japan
Division of Pathology (O.N.) and Urology (A.I.), National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, Japan
Division of Cardiovascular Internal Medicine (J.S.), Fukuoka City Medical Association Hospital, Fukuoka, 814-8522, Japan
Yame General Hospital (K.Shim.), Fukuoka 834-0034, Japan
Divison of Urology (Y.K.) and Pathology (O.T.), Japanese Red-Cross Nagasaki Atomic Bomb Hospital, Nagasaki 852-8511, Japan
Abstract
Unilateral adrenal hyperplasia with primary aldosteronism is very rare and shows similar endocrine features to aldosterone-producing adenoma and bilateral adrenal hyperplasia. In this study, the mRNA expression of steroidogenic enzymes in unilateral adrenal hyperplasia was examined by in situ hybridization. We found subcapsular micronodules composed of spironolactone body-containing cells, which showed intense expression for 3-hydroxysteroid dehydrogenase, 11-hydroxylase, 18-hydroxylase, and 21-hydroxylase but not 17-hydroxylase, indicating aldosterone production. This expression pattern was the same as that in unilateral multiple adrenocortical micronodules, reported recently. Additionally, it was noted that a nodule with active aldosterone production was closely adjacent to one showing intense 17-hydroxylase expression. In the adrenal cortices adhering to aldosterone-producing adenoma, the majority of hyperplastic zona glomerulosa and hyperplastic nodules demonstrated a decreased steroidogenic activity. However, minute nodules indicative of active aldosterone production were found at high frequency. These results suggest that the subcapsular micronodules observed might be the root of aldosterone-producing adenoma. Furthermore, we emphasize the need for long-term follow-up after unilateral adrenalectomy or enucleation of the adenoma because of the possibility that buds with autonomous aldosterone production may still be present in the contralateral or remaining adrenal tissue.
Introduction
PRIMARY ALDOSTERONISM (PA), resulting from an autonomous excessive aldosterone secretion, is clinicopathologically classified as aldosterone-producing adenoma (APA); bilateral adrenal hyperplasia (BAH), also known as idiopathic hyperaldosteronism; unilateral adrenal hyperplasia (UAH); primary adrenal hyperplasia; adrenal cancer; glucocorticoid-suppressible hyperaldosteronism; or familial hyperaldosteronism type II (1, 2, 3). Among these, most patients with PA were previously assumed to have APA (2), but a recent study has shown that most PA is caused by BAH, followed by APA (4). On the other hand, UAH, a surgically curative subset of PA, is quite rare (3, 5, 6).
The synthesis of aldosterone and cortisol in the adrenal cortex requires several enzymes: cholesterol side-chain cleavage, 3-hydroxysteroid dehydrogenase (3-HSD), 21-hydroxylase (21-OH), 11-hydroxylase (11-OH), 17-hydroxylase (17-OH), and 18-hydroxylase (18-OH). 11-OH and 18-OH, encoded by a single gene, CYP11B2 (7), are enzymes for the final step of aldosterone synthesis. In cortisol synthesis, 17-OH and 11-OH, encoded by CYP17 (8, 9, 10) and CYP11B1 (7), respectively, are needed. The localization of genes coding for steroidogenic enzymes in the adrenals obtained from patients with PA has been examined by in situ hybridization (11, 12). However, the detailed localization or expression pattern of steroidogenic enzymes in UAH has not been reported to date.
In this study, we examined the expression of steroidogenic enzymes in three cases of UAH by in situ hybridization and found a characteristic expression of enzymes. Furthermore, we compared the results with those of adrenal cortices adhering to APA, which often show adrenocortical hyperplasia, as seen in UAH.
Materials and Methods
Tissue samples
Of the paraffin-embedded human adrenal tissues listed in the Division of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Japanese Red-Cross Nagasaki Atomic Bomb Hospital, and National Hospital Organization Kyushu Medical Center between 1992 and 2004, 32 cases of PA were collected. The diagnosis of PA was based on elevated plasma-aldosterone concentration and suppressed plasma-renin activity or plasma-renin concentration. After the diagnosis of PA, imaging of the adrenal glands with computerized tomography (CT) and scintigraphy was done for detection of adrenal masses. Furthermore, all cases were examined by adrenal venous sampling to determine the aldosterone to cortisol ratio on the right and left sides. The diagnosis was confirmed by histological examination of the surgical specimens. Three cases exhibited adrenal hyperplasia associated with unilateral adrenal aldosterone production confirmed by adrenal venous sampling and hence were diagnosed as UAH. Table 1 shows their characteristics. Twenty-nine remaining cases were classified as APA. APA and adrenal tissues adhering to APA were also examined in this study. As controls, we used adrenal glands obtained from patients undergoing adrenalectomy together with nephrectomy for advanced renal cancer.
In situ hybridization
Total RNA was extracted from human adrenal tissues using RNeasy Protect kit (QIAGEN, Tokyo, Japan), in accordance with the manufacturer’s recommendations. cDNA fragments of human 3-HSD (nucleotides 560-1386) (13), 17-OH (nucleotides 958-1667) (8), 11-OH (nucleotides 475-1133) (14), 18-OH (nucleotide 1970–2728) (15), and 21-OH (nucleotides 800-1668) (16) were obtained by RT-PCR and subcloned into pT-NOT vector. Antisense strand cRNAs were synthesized using digoxigenin (DIG)-uridine 5-triphosphate (Roche Diagnostics, Tokyo, Japan) with T3 or T7 RNA polymerase (Takara, Otsu, Japan).
In situ hybridization was performed as described previously (17, 18). Briefly, 3-μm-thick sections were hybridized with DIG-labeled cRNA probes at 42 C for 16 h. The tissue sections were washed several times in 4x saline sodium citrate (SSC) and immersed in 50% formamide/2x SSC at 50 C for 30 min. Next, the sections were treated with 20 μg/ml RNase A at 37 C for 30 min and finally washed in 0.2x SSC at 50 C for 20 min. Hybridization signals were immunologically detected with alkaline phosphatase-conjugated anti-DIG Fab fragments (diluted 1:500; Roche Diagnostics) by using nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Dako Cytomation, Kyoto, Japan) as the chromogenic substrate. The relative degree of expression signals was evaluated subjectively by the extent of positive cells, in addition to the intensity of expression signals. In a preliminary study, the specificity of the signals was examined using three negative-control procedures: 1) hybridization with sense cRNA probe, 2) pretreatment of tissue sections with RNase A, and 3) displacement by addition of excess unlabeled antisense probe.
Results
The adrenal cortex, but not medulla, showed mRNA expression for all steroidogenic enzymes examined in this study, but the expression level differed among the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) (Fig. 1, A–I). 3-HSD, 11-OH, and 18-OH mRNAs were intensely expressed in ZG and outer ZF, followed by inner ZF and ZR. 17-OH expression was intense in ZF and ZR and was weak in some ZG cells. The expression of 21-OH was prominent in ZG and weak in ZF and ZR. In APA cells, the expression for 3-HSD, 18-OH, and 21-OH mRNAs was more intense than was 17-OH mRNA (Fig. 1, J–O). There was no evidence of specific hybridization signals in the negative control (Fig. 1, G–I and O).
Case 1 of UAH showed multiple nodules composed of clear-type cells and was diagnosed microscopically as a nodular hyperplasia of the adrenal cortex (Fig. 2), suggesting that the excess aldosterone was produced from these nodules. However, mRNA expression for steroidogenic enzymes was not seen in these nodules, whereas the ZG, which showed mild diffuse hyperplasia and focal microscopic spheroid nodules, demonstrated intense expression for 3-HSD, 11-OH, 18-OH, and 21-OH but not 17-OH.
In Case 2 of UAH, there was no remarkable change histologically, other than focal hyperplasia of ZG and small nodules in ZF and ZR. Hence, the hyperplastic ZG was considered to be the focus of the excess aldosterone production. When the mRNA expression of each enzyme was examined, the majority of ZG exhibited no or only weak expression of all enzymes. However, multiple microscopic subcapsular foci showed intense mRNA expression for 3-HSD, 11-OH, 18-OH, and 21-OH but did not express 17-OH mRNA (Fig. 3, A–F). These nodules, several to hundreds of micrometers in size and spheroid in shape, were composed of compact cells containing spironolactone bodies (Fig. 3A, square box). Arch-like lesions, which were composed of ZG-type cells with clear cytoplasm containing spironolactone bodies (Fig. 3G, square box), showed a similar expression pattern (Fig. 3, G–L). The nodules in ZF and ZR predominantly expressed 17-OH (Fig. 3, M–O).
The adrenal gland in case 3 of UAH demonstrated miscellaneous findings comprising small and large nodules. In addition to the nodules showing the same morphology and expression pattern as seen in case 2, a nodule with a deficiency of only 17-OH expression closely abutted on one showing an intense expression for 17-OH (Fig. 4), suggestive of cortisol production. The former was composed of compact cells containing spironolactone bodies (Fig. 4A, square box).
We classified the adrenal cortices adhering to APA into the following six patterns according to morphology and mRNA expression levels: 1) minute, aldosterone-only-producing nodules, with intense expression of 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs but not 17-OH mRNA; 2) nodules or 3) diffuse hyperplasia indicative of predominantly aldosterone production, with intense expression of 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs, and weak expression of 17-OH mRNA; 4) nodules indicative of predominantly cortisol production, with intense expression of 17-OH mRNA; 5) nodules or 6) diffuse hyperplasia exhibiting a decreased steroidogenic activity, with weak or no expression of mRNAs. As shown in Table 2, 72.4% (21 of 29) of the adrenal nodules and 86.2% (25 of 29) of ZG showing diffuse hyperplasia showed a decreased steroidogenic activity. However, of the adrenal glands adhering to APA, nine cases (31.0%) and 10 cases (34.5%) revealed several minute spheroid nodules suggestive of having only or prominently aldosterone production, respectively (Fig. 5, A–J). Six (66.7%) of nine cases with only aldosterone production contained spironolactone bodies. Four cases (13.8%) of diffuse hyperplasia had functioning aldosterone synthesis. Thirteen of 29 cases (44.8%) showed nodules with prominent 17-OH expression, indicating mainly cortisol production. Furthermore, one case showed the finding that three nodules with similar expression patterns to that in APA existed contiguously (Fig. 5, K–O).
Discussion
We examined the mRNA expression of steroidogenic enzymes in adrenal glands by in situ hybridization. In the control adrenal glands, the mRNAs for all enzymes were expressed in all three zones of the cortex but not in the medulla, although there were zonal differences in mRNA expression. In ZG and outer ZF, 3-HDS, 11-OH, 18-OH, and 21-OH mRNAs were expressed intensely, indicating aldosterone production from these cells. The signals for 18-OH mRNA were also present in ZF and ZR in this study. A previous study using oligonucleotide probes revealed that the expression for 18-OH mRNA was seen only in ZG and not in ZF or ZR (11, 12). The specific coding region for each probe was carefully selected, but the nucleotide sequences of 18-OH and 11-OH are 95% identical in coding regions (7). Unlike with oligonucleotide probes, it may be difficult, with cRNA probes, to completely avoid cross-reactivity between 18-OH and 11-OH. On the other hand, 17-OH expression was observed mainly in ZF and ZR, corresponding with cortisol and/or sex hormone production. The expression of 17-OH was also found in some ZG cells, in disagreement with previous reports (11, 12, 19). The gene sequence of the 17-OH probe used showed no homology with those of other probes, according to a sequence similarity search in genome databases.
The expression signals obtained in this study were specific and not false positive, as demonstrated by three negative-control studies: hybridization with sense-strand probes instead of antisense probes, RNase A pretreatment, and a competition study with an excess of unlabeled antisense probe. In ZG, 17-OH usually appears dormant, but ACTH stimulation has been reported to induce an increase in 17-OH protein and mRNA level in bovine ZG, indicating that steroid biosynthesis in ZG shifts an aldosterone-producing to a cortisol-producing pathway (20, 21). It has been reported that some patients with critical illness, such as malignant tumors and sepsis, showed elevated plasma ACTH and cortisol levels but a reduction in aldosterone production (22, 23, 24). In this study, the adrenal glands obtained from patients with advanced renal cancer were used as the control. Therefore, our finding may reflect a similar situation, to some degree. It is also possible that this discrepancy may be due to the difference in the methods used (immunohistochemistry vs. in situ hybridization) or the sensitivity of probes (oligonucleotide vs. cRNA).
UAH is very rare, with a frequency among PA of less than 1% (3, 5, 6), and shows similar endocrine features to APA and BAH. UAH with only minute change may not be identified as an adrenal abnormality by CT. Indeed, only in case 1 was a unilateral adrenal nodule detected by CT. For diagnosis, adrenal-vein sampling is the most reliable method to determine the exact localization of an overfunctioning adrenal (6, 25). The term UAH includes all forms (diffuse, micronodular, and macronodular) of adrenal hyperplasia associated with unilateral adrenal aldosterone production confirmed by adrenal venous sampling (25, 26). Therefore, from both morphological and functional perspectives, UAH encompasses the entity that has been labeled, by others, unilateral multiple adrenocortical micronodules (27). This is supported by the fact that expression patterns of the microscopic nodules in our cases were similar to those previously reported in unilateral multiple adrenocortical micronodules, with immunoreactivities of 3-HSD, 11-OH, and 21-OH but not 17-OH, consistent with aldosterone production.
Adrenocortical hyperplasia including focal or diffuse hyperplasia of ZG is a common finding in adrenal cortices adhering to APA, in contrast to adrenal cortices adhering to Cushing adenomas, which are atrophic. Hence, we also made a study of the mRNA expression for steroidogenic enzymes in the adrenal cortices adhering to APA. The majority of hyperplastic ZG and hyperplastic nodules indicated a decreased steroidogenic activity. However, minute nodules indicative of active aldosterone production were found in 62.1% (18 of 29) of cases examined, and six of these contained spironolactone bodies. Enberg et al. (12) also showed by autoradiographic analysis of in situ hybridization that six of 11 adrenals (54.5%) obtained from patients with APA contained ZG and/or small (<5 mm) nodules with expression of CYP11B2 gene encoding 11-OH and 18-OH (6). Aiba et al. (28) classified hyperplastic ZG of the adrenal cortices adhering to APA into two groups: Type I pattern with enhanced steroidogenic activity and type II pattern showing restrained steroidogenesis, according to the patterns of activity of cellular dehydrogenases (3-HSD, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate- isocitrate dehydrogenase, and succinate dehydrogenase activity). The type I pattern was observed in spironolactone body-containing cells of adrenal cortices adhering to APA and in hyperplastic adrenocortical cells of BAH, irrespective of the presence or absence of spironolactone bodies. The nodules observed in UAH and the adrenal cortices adhering to APA in this study corresponded to their type I.
Spironolactone, a competitive antagonist of aldosterone receptors (29), is an antihypertensive drug often taken by patients with PA preoperatively to reduce elevated blood pressure and normalize the serum potassium level. There is evidence that spironolactone can directly interfere with the biosynthesis of aldosterone through inhibiting 11-hydroxylation and 18-hydroxylation in bovine and certain human adrenal cortical tissue (30). Therefore, it has been considered that spironolactone body-containing cells of adrenal cortex adhering to APA might reveal abortive steroidogenic activity (28), although the presence of spironolactone bodies was proof of enhanced steroidogenic activity. However, our finding that spironolactone body-containing cells, but not spironolactone bodies themselves, intensely expressed 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs required for aldosterone synthesis would have to be interpreted as indicating that these cells retain their steroidogenic activity. On the other hand, it is also known that chronic administration of spironolactone activates the endogenous renin-angiotensin system (31). Whether the findings in the present study occur before or after the administration of spironolactone remains the subject of ongoing study because the regulation of CYP11B2 but not CYP11B1 is dependent on the renin-angiotensin system (32). However, nodules lacking spironolactone bodies that showed an intense mRNA expression for aldosterone-synthetic enzymes were also recognized in this study. Therefore, the concept that these lesions were present before the use of the drug would have to be accepted, although the possibility that spironolactone treatment might enhance further mRNA expression for aldosterone-synthetic enzymes is not ruled out.
Both UAH and APA show a renin-independent overproduction of aldosterone (3, 27). However, the expression pattern of steroidogenic enzymes in the nodules of our UAH cases was different from that in APA because 17-OH was generally expressed in the latter. Whether the mechanism of pathogenesis in UAH is different from that in APA is unknown. Figure 6 summarizes the three cases of UAH examined in this study. Diffuse hyperplasia of ZG in case 1 and small nodules located under the adrenal capsule were evident in all cases. The same micronodules as seen in UAH were also present in the adrenal cortices adhering to APA at high frequency. Furthermore, the coexistence with nodules showing the expression pattern of enzymes seen in APA was noted in case of adrenal cortex adhering to APA. Taken in conjunction with the observation that a nodule indicating active aldosterone production was found in close apposition to one showing an intense expression of 17-OH, it is necessary to keep in mind the possibility that multiple micronodules burgeoning in the subcapsular region initially might ultimately become an adenoma by this process of repeated growth and fusion.
Unilateral APA and UAH are treated surgically because of the absence of recurrence after unilateral adrenalectomy (6, 25). Some patients with APA may still remain hypertensive after curative surgery, the main contributing factor for which has been suggested to be delayed diagnosis and therapy, as ascertained by analysis of long-term follow-up after unilateral adrenorectomy for PA (6). On the other hand, it has also been reported that the presence of signs such as hypertension and hypokalemia predicted a postoperative relapse in cases with highly abundant expression of CYP11B2 in ZG and small nodules expressing the CYP11B2 gene but not the CYP17 gene (12), as seen in our cases. Recently laparoscopic adrenal-sparing surgery or enucleation of the adenoma with preservation of functional adrenal tissue has been attempted (33, 34, 35, 36). Although enucleation of the adenoma may be curative, with normalization of the adrenal endocrine function (36), long-term follow-up is required because of the possibility that buds with autonomous aldosterone production, with the potential to develop into PA, may still be present in the remaining adrenal tissue.
Acknowledgments
The authors thank Mrs. Amanda Nishida for critical comments and Miss Kanako Egashira for technical assistance.
Footnotes
First Published Online November 10, 2005
Abbreviations: APA, Aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia; CT, computerized tomography; DIG, digoxigenin; 3-HSD, 3-hydroxysteroid dehydrogenase; 11-OH, 11-hydroxylase; 17-OH, 17-hydroxylase; 18-OH, 18-hydroxylase; 21-OH, 21-hydroxylase; PA, primary aldosteronism; SSC, saline sodium citrate; UAH, unilateral adrenal hyperplasia; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis.
Accepted for publication October 26, 2005.
References
Stowasser M, Gordon RD, Tunny TJ, Klemm SA, Finn WL, Krek AL 1992 Familial hyperaldosteronism type II: five families with a new variety of primary aldosteronism. Clin Exp Pharmacol Physiol 19:319–322
Banks WA, Kastin AJ, Biglieri EG, Ruiz AE 1984 Primary adrenal hyperplasia: a new subset of primary hyperaldosteronism. J Clin Endocrinol Metab 58:783–785
Ganguly A 1998 Primary aldosteronism. N Engl J Med 339:1828–1834
Mulatero P, Stowasser M, Loh KC, Fardella CE, Gordon RD, Mosso L, Gomez-Sanchez CE, Veglio F, Young Jr WF 2004 Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab 89:1045–1050
Wheeler MH, Harris DA 2003 Diagnosis and management of primary aldosteronism. World J Surg 27:627–631
Meyer A, Brabant G, Behrend M 2005 Long-term follow-up after adrenalectomy for primary aldosteronism. World J Surg 29:155–159
Mornet E, Dupont J, Vitek A, White PC 1989 Characterization of two genes encoding human steroid 11-hydroxylase (P-450(11)). J Biol Chem 264:20961–20967
Chung BC, Picado-Leonard J, Haniu M, Bienkowski M, Hall PF, Shively JE, Miller WL 1987 Cytochrome P450c17 (steroid 17-hydroxylase/17, 20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc Natl Acad Sci USA 84:407–411
Bradshaw KD, Waterman MR, Couch RT, Simpson ER, Zuber MX 1987 Characterization of complementary deoxyribonucleic acid for human adrenocortical 17-hydroxylase: a probe for analysis of 17-hydroxylase deficiency. Mol Endocrinol 5:348–354
Picado-Leonard J, Miller WL 1987 Cloning and sequence of the human gene for P450c17 (steroid 17-hydroxylase/17, 20 lyase): similarity with the gene for P450c21. DNA 6:439–448
Enberg U, Farnebo LO, Wedell A, Grondal S, Thoren M, Grimelius L, Kjellman M, Backdahl M, Hamberger B 2001 In vitro release of aldosterone and cortisol in human adrenal adenomas correlates to mRNA expression of steroidogenic enzymes for genes CYP11B2 and CYP17. World J Surg 25:957–966
Enberg U, Volpe C, Hoog A, Wedell A, Farnebo LO, Thoren M, Hamberger B 2004 Postoperative differentiation between unilateral adrenal adenoma and bilateral adrenal hyperplasia in primary aldosteronism by mRNA expression of the gene CYP11B2. Eur J Endocrinol 151:73–85
Rheaume E, Lachance Y, Zhao HF, Breton N, Dumont M, deLaunoit Y, Trudel C, Luu-The V, Simard J, LabrieF 1991 Structure and expression of a new complementary DNA encoding the almost exclusive 3-hydroxysteroid dehydrogenase/5-4-isomerase in human adrenals and gonads. Mol Endocrinol 5:1147–1157
Kawamoto T, Shizuta Y 1990 Cloning of cDNA and genomic DNA for human cytochrome P45011b. FEBS Lett 269:345–349
Kawamoto T, Mitsuuchi Y, Ohnishi T, Ichikawa Y, Yokoyama Y, Sumimoto H, Toda K, Miyahara K, Kuribayashi I, Nakao K, Hosoda K, Yamamoto Y, Imura H, Shizuta Y 1990 Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism. Biochem Biophys Res Commun 173:309–316
Matteson KJ, Phillips III JA, Miller WL, Chung BC, Orlando PJ, Frisch H, Ferrandez A, Burr IM 1987 P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia. Proc Natl Acad Sci USA 84:5858–5862
Shigematsu K, Nakatani A, Kawai K, Moriuchi R, Katamine S, Miyamoto T, Niwa M 1996 Two subtypes of endothelin receptors and endothelin peptides are expressed in differential cell types of the rat placenta: in vitro receptor autoradiographic and in situ hybridization studies. Endocrinology 137:738–748
Li A, Sakaguchi S, Shigematsu K, Atarashi R, Roy BC, Nakaoke R, Arima K, Okimura N, Kopacek J, Katamine S 2000 Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration. Am J Pathol 157:1447–1452
Sasano H 1994 Localization of steroidogenic enzymes in adrenal cortex and its disorders. Endocr J 41:471–482
Braly LM, Adler GK, Mortensen RM, Conlin PR, Chen R, Hallahan J, Menachery AI, Williams GH 1992 Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cell: in vitro correlate of hyperreninemic hypoaldosteronism. Endocrinology 131:187–194
Galtier A, Liakos P, Keramidas M, Feige JJ, Chambaz EM, Defaye G 1996 ACTH angiotensin II and TGF participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells. Endocr Res 22:607–612
Stern N, Beck FW, Sowers JR, Tuck M, Hsueh WA, Zipser RD 1983 Plasma corticosteroids in hyperreninemic hypoaldosteronism: evidence for diffuse impairment of the zona glomerulosa. J Clin Endocrinol Metab 57:217–220
Findling JW, Waters VO, Raff H 1987 The dissociation of renin and aldosterone during critical illness. J Clin Endocrinol Metab 64:592–595
Parker LN, Levin ER, Lifrak ET 1985 Evidence for adrenocortical adaptation to severe illness. J Clin Endocrinol Metab 60:947–952
Morioka M, Kobayashi T, Sone A, Furukawa Y, Tanaka H 2000 Primary aldosteronism due to unilateral adrenal hyperplasia: report of two cases and review of the literature. Endocr J 47:443–449
Ganguly A, Zager PG, Luetscher JA 1980 Primary aldosteronism due to unilateral adrenal hyperplasia. J Clin Endocrinol Metab 51:1190–1194
Omura M, Sasano H, Fujiwara T, Yamaguchi K, Nishikawa T 2002 Unique cases unilateral hyperaldosteronemia due to multiple adrenocortical micronodules, which can only be detected by selective adrenal venous sampling. Metabolism 151:350–355
Aiba M, Suzuki H, Kageyama K, Murai M, Tazaki H, Abe O, Saruta T 1981 Spironolactone bodies in aldosteronomas and in the attached adrenals. Am J Pathol 103:404–410
Kagawa CM, Cella JA, Van Arman CG 1957 Action of new steroids in blocking effects of aldosterone and deoxycorticosterone on salt. Science 126:1015–1016
Cheng SC, Suzuki K, Sadee W, Harding BW 1976 Effects of spironolactone, canrenone and canrenoate-K on cytochrome P450, and 11- and 18-hydroxylation in bovine and human adrenal cortical mitochondria. Endocrinology 99:1097–1106
Spark RF, Dale SL, Kahn PC, Melby JC 1969 Activation of aldosterone secretion in primary aldosteronism. J Clin Invest 48:96–104
Kakiki M, Morohashi K, Nomura M, Omura T, Horie T 1997 Regulation of aldosterone synthase cytochrome P450 (CYP11B2) and 11-hydroxylase cytochrome P450 (CYP11B1) expression in rat adrenal zona glomerulosa cells by low sodium diet and angiotensin II receptor antagonists. Biol Pharm Bull 20:962–968
Nakada T, Kubota Y, Sasagawa I, Yagisawa T, Watanabe M, Ishigooka M 1995 Therapeutic outcome of primary aldosteronism: adrenalectomy versus enucleation of aldosterone-producing adenoma. J Urol 153:1775–1780
Kok YKK, Yapp SKS 2002 Laparoscopic adrenal-sparing surgery for primary hyperaldosteronism due to aldosterone-producing adenoma. Surg Endosc 16:108–111
Jeschke K, Janetschek G, Peschel R, Schellander L, Bartsch G, Henning K 2003 Laparoscopic partial adrenalectomy in patients with aldosterone-producing adenomas: indications, technique, and results. Urology 61:69–72
Walz MK, Peitgen K, Diesing D, Petersenn S, Janssen OE, Philipp T, Metz KA, Mann K, Schmid KW, Neumann HP 2004 Partial versus total adrenalectomy by the posterior retroperitoneoscopic approach: early and long-term results of 325 consecutive procedures in primary adrenal neoplasias. World J Surg 28:1323–1329(Kazuto Shigematsu, Kioko Kawai, Junji Ir)
Division of Pathology (K.K., J.I.) Nagasaki Municipal Hospital, Nagasaki 850-8555, Japan
Division of Pathology (O.N.) and Urology (A.I.), National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, Japan
Division of Cardiovascular Internal Medicine (J.S.), Fukuoka City Medical Association Hospital, Fukuoka, 814-8522, Japan
Yame General Hospital (K.Shim.), Fukuoka 834-0034, Japan
Divison of Urology (Y.K.) and Pathology (O.T.), Japanese Red-Cross Nagasaki Atomic Bomb Hospital, Nagasaki 852-8511, Japan
Abstract
Unilateral adrenal hyperplasia with primary aldosteronism is very rare and shows similar endocrine features to aldosterone-producing adenoma and bilateral adrenal hyperplasia. In this study, the mRNA expression of steroidogenic enzymes in unilateral adrenal hyperplasia was examined by in situ hybridization. We found subcapsular micronodules composed of spironolactone body-containing cells, which showed intense expression for 3-hydroxysteroid dehydrogenase, 11-hydroxylase, 18-hydroxylase, and 21-hydroxylase but not 17-hydroxylase, indicating aldosterone production. This expression pattern was the same as that in unilateral multiple adrenocortical micronodules, reported recently. Additionally, it was noted that a nodule with active aldosterone production was closely adjacent to one showing intense 17-hydroxylase expression. In the adrenal cortices adhering to aldosterone-producing adenoma, the majority of hyperplastic zona glomerulosa and hyperplastic nodules demonstrated a decreased steroidogenic activity. However, minute nodules indicative of active aldosterone production were found at high frequency. These results suggest that the subcapsular micronodules observed might be the root of aldosterone-producing adenoma. Furthermore, we emphasize the need for long-term follow-up after unilateral adrenalectomy or enucleation of the adenoma because of the possibility that buds with autonomous aldosterone production may still be present in the contralateral or remaining adrenal tissue.
Introduction
PRIMARY ALDOSTERONISM (PA), resulting from an autonomous excessive aldosterone secretion, is clinicopathologically classified as aldosterone-producing adenoma (APA); bilateral adrenal hyperplasia (BAH), also known as idiopathic hyperaldosteronism; unilateral adrenal hyperplasia (UAH); primary adrenal hyperplasia; adrenal cancer; glucocorticoid-suppressible hyperaldosteronism; or familial hyperaldosteronism type II (1, 2, 3). Among these, most patients with PA were previously assumed to have APA (2), but a recent study has shown that most PA is caused by BAH, followed by APA (4). On the other hand, UAH, a surgically curative subset of PA, is quite rare (3, 5, 6).
The synthesis of aldosterone and cortisol in the adrenal cortex requires several enzymes: cholesterol side-chain cleavage, 3-hydroxysteroid dehydrogenase (3-HSD), 21-hydroxylase (21-OH), 11-hydroxylase (11-OH), 17-hydroxylase (17-OH), and 18-hydroxylase (18-OH). 11-OH and 18-OH, encoded by a single gene, CYP11B2 (7), are enzymes for the final step of aldosterone synthesis. In cortisol synthesis, 17-OH and 11-OH, encoded by CYP17 (8, 9, 10) and CYP11B1 (7), respectively, are needed. The localization of genes coding for steroidogenic enzymes in the adrenals obtained from patients with PA has been examined by in situ hybridization (11, 12). However, the detailed localization or expression pattern of steroidogenic enzymes in UAH has not been reported to date.
In this study, we examined the expression of steroidogenic enzymes in three cases of UAH by in situ hybridization and found a characteristic expression of enzymes. Furthermore, we compared the results with those of adrenal cortices adhering to APA, which often show adrenocortical hyperplasia, as seen in UAH.
Materials and Methods
Tissue samples
Of the paraffin-embedded human adrenal tissues listed in the Division of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Japanese Red-Cross Nagasaki Atomic Bomb Hospital, and National Hospital Organization Kyushu Medical Center between 1992 and 2004, 32 cases of PA were collected. The diagnosis of PA was based on elevated plasma-aldosterone concentration and suppressed plasma-renin activity or plasma-renin concentration. After the diagnosis of PA, imaging of the adrenal glands with computerized tomography (CT) and scintigraphy was done for detection of adrenal masses. Furthermore, all cases were examined by adrenal venous sampling to determine the aldosterone to cortisol ratio on the right and left sides. The diagnosis was confirmed by histological examination of the surgical specimens. Three cases exhibited adrenal hyperplasia associated with unilateral adrenal aldosterone production confirmed by adrenal venous sampling and hence were diagnosed as UAH. Table 1 shows their characteristics. Twenty-nine remaining cases were classified as APA. APA and adrenal tissues adhering to APA were also examined in this study. As controls, we used adrenal glands obtained from patients undergoing adrenalectomy together with nephrectomy for advanced renal cancer.
In situ hybridization
Total RNA was extracted from human adrenal tissues using RNeasy Protect kit (QIAGEN, Tokyo, Japan), in accordance with the manufacturer’s recommendations. cDNA fragments of human 3-HSD (nucleotides 560-1386) (13), 17-OH (nucleotides 958-1667) (8), 11-OH (nucleotides 475-1133) (14), 18-OH (nucleotide 1970–2728) (15), and 21-OH (nucleotides 800-1668) (16) were obtained by RT-PCR and subcloned into pT-NOT vector. Antisense strand cRNAs were synthesized using digoxigenin (DIG)-uridine 5-triphosphate (Roche Diagnostics, Tokyo, Japan) with T3 or T7 RNA polymerase (Takara, Otsu, Japan).
In situ hybridization was performed as described previously (17, 18). Briefly, 3-μm-thick sections were hybridized with DIG-labeled cRNA probes at 42 C for 16 h. The tissue sections were washed several times in 4x saline sodium citrate (SSC) and immersed in 50% formamide/2x SSC at 50 C for 30 min. Next, the sections were treated with 20 μg/ml RNase A at 37 C for 30 min and finally washed in 0.2x SSC at 50 C for 20 min. Hybridization signals were immunologically detected with alkaline phosphatase-conjugated anti-DIG Fab fragments (diluted 1:500; Roche Diagnostics) by using nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Dako Cytomation, Kyoto, Japan) as the chromogenic substrate. The relative degree of expression signals was evaluated subjectively by the extent of positive cells, in addition to the intensity of expression signals. In a preliminary study, the specificity of the signals was examined using three negative-control procedures: 1) hybridization with sense cRNA probe, 2) pretreatment of tissue sections with RNase A, and 3) displacement by addition of excess unlabeled antisense probe.
Results
The adrenal cortex, but not medulla, showed mRNA expression for all steroidogenic enzymes examined in this study, but the expression level differed among the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) (Fig. 1, A–I). 3-HSD, 11-OH, and 18-OH mRNAs were intensely expressed in ZG and outer ZF, followed by inner ZF and ZR. 17-OH expression was intense in ZF and ZR and was weak in some ZG cells. The expression of 21-OH was prominent in ZG and weak in ZF and ZR. In APA cells, the expression for 3-HSD, 18-OH, and 21-OH mRNAs was more intense than was 17-OH mRNA (Fig. 1, J–O). There was no evidence of specific hybridization signals in the negative control (Fig. 1, G–I and O).
Case 1 of UAH showed multiple nodules composed of clear-type cells and was diagnosed microscopically as a nodular hyperplasia of the adrenal cortex (Fig. 2), suggesting that the excess aldosterone was produced from these nodules. However, mRNA expression for steroidogenic enzymes was not seen in these nodules, whereas the ZG, which showed mild diffuse hyperplasia and focal microscopic spheroid nodules, demonstrated intense expression for 3-HSD, 11-OH, 18-OH, and 21-OH but not 17-OH.
In Case 2 of UAH, there was no remarkable change histologically, other than focal hyperplasia of ZG and small nodules in ZF and ZR. Hence, the hyperplastic ZG was considered to be the focus of the excess aldosterone production. When the mRNA expression of each enzyme was examined, the majority of ZG exhibited no or only weak expression of all enzymes. However, multiple microscopic subcapsular foci showed intense mRNA expression for 3-HSD, 11-OH, 18-OH, and 21-OH but did not express 17-OH mRNA (Fig. 3, A–F). These nodules, several to hundreds of micrometers in size and spheroid in shape, were composed of compact cells containing spironolactone bodies (Fig. 3A, square box). Arch-like lesions, which were composed of ZG-type cells with clear cytoplasm containing spironolactone bodies (Fig. 3G, square box), showed a similar expression pattern (Fig. 3, G–L). The nodules in ZF and ZR predominantly expressed 17-OH (Fig. 3, M–O).
The adrenal gland in case 3 of UAH demonstrated miscellaneous findings comprising small and large nodules. In addition to the nodules showing the same morphology and expression pattern as seen in case 2, a nodule with a deficiency of only 17-OH expression closely abutted on one showing an intense expression for 17-OH (Fig. 4), suggestive of cortisol production. The former was composed of compact cells containing spironolactone bodies (Fig. 4A, square box).
We classified the adrenal cortices adhering to APA into the following six patterns according to morphology and mRNA expression levels: 1) minute, aldosterone-only-producing nodules, with intense expression of 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs but not 17-OH mRNA; 2) nodules or 3) diffuse hyperplasia indicative of predominantly aldosterone production, with intense expression of 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs, and weak expression of 17-OH mRNA; 4) nodules indicative of predominantly cortisol production, with intense expression of 17-OH mRNA; 5) nodules or 6) diffuse hyperplasia exhibiting a decreased steroidogenic activity, with weak or no expression of mRNAs. As shown in Table 2, 72.4% (21 of 29) of the adrenal nodules and 86.2% (25 of 29) of ZG showing diffuse hyperplasia showed a decreased steroidogenic activity. However, of the adrenal glands adhering to APA, nine cases (31.0%) and 10 cases (34.5%) revealed several minute spheroid nodules suggestive of having only or prominently aldosterone production, respectively (Fig. 5, A–J). Six (66.7%) of nine cases with only aldosterone production contained spironolactone bodies. Four cases (13.8%) of diffuse hyperplasia had functioning aldosterone synthesis. Thirteen of 29 cases (44.8%) showed nodules with prominent 17-OH expression, indicating mainly cortisol production. Furthermore, one case showed the finding that three nodules with similar expression patterns to that in APA existed contiguously (Fig. 5, K–O).
Discussion
We examined the mRNA expression of steroidogenic enzymes in adrenal glands by in situ hybridization. In the control adrenal glands, the mRNAs for all enzymes were expressed in all three zones of the cortex but not in the medulla, although there were zonal differences in mRNA expression. In ZG and outer ZF, 3-HDS, 11-OH, 18-OH, and 21-OH mRNAs were expressed intensely, indicating aldosterone production from these cells. The signals for 18-OH mRNA were also present in ZF and ZR in this study. A previous study using oligonucleotide probes revealed that the expression for 18-OH mRNA was seen only in ZG and not in ZF or ZR (11, 12). The specific coding region for each probe was carefully selected, but the nucleotide sequences of 18-OH and 11-OH are 95% identical in coding regions (7). Unlike with oligonucleotide probes, it may be difficult, with cRNA probes, to completely avoid cross-reactivity between 18-OH and 11-OH. On the other hand, 17-OH expression was observed mainly in ZF and ZR, corresponding with cortisol and/or sex hormone production. The expression of 17-OH was also found in some ZG cells, in disagreement with previous reports (11, 12, 19). The gene sequence of the 17-OH probe used showed no homology with those of other probes, according to a sequence similarity search in genome databases.
The expression signals obtained in this study were specific and not false positive, as demonstrated by three negative-control studies: hybridization with sense-strand probes instead of antisense probes, RNase A pretreatment, and a competition study with an excess of unlabeled antisense probe. In ZG, 17-OH usually appears dormant, but ACTH stimulation has been reported to induce an increase in 17-OH protein and mRNA level in bovine ZG, indicating that steroid biosynthesis in ZG shifts an aldosterone-producing to a cortisol-producing pathway (20, 21). It has been reported that some patients with critical illness, such as malignant tumors and sepsis, showed elevated plasma ACTH and cortisol levels but a reduction in aldosterone production (22, 23, 24). In this study, the adrenal glands obtained from patients with advanced renal cancer were used as the control. Therefore, our finding may reflect a similar situation, to some degree. It is also possible that this discrepancy may be due to the difference in the methods used (immunohistochemistry vs. in situ hybridization) or the sensitivity of probes (oligonucleotide vs. cRNA).
UAH is very rare, with a frequency among PA of less than 1% (3, 5, 6), and shows similar endocrine features to APA and BAH. UAH with only minute change may not be identified as an adrenal abnormality by CT. Indeed, only in case 1 was a unilateral adrenal nodule detected by CT. For diagnosis, adrenal-vein sampling is the most reliable method to determine the exact localization of an overfunctioning adrenal (6, 25). The term UAH includes all forms (diffuse, micronodular, and macronodular) of adrenal hyperplasia associated with unilateral adrenal aldosterone production confirmed by adrenal venous sampling (25, 26). Therefore, from both morphological and functional perspectives, UAH encompasses the entity that has been labeled, by others, unilateral multiple adrenocortical micronodules (27). This is supported by the fact that expression patterns of the microscopic nodules in our cases were similar to those previously reported in unilateral multiple adrenocortical micronodules, with immunoreactivities of 3-HSD, 11-OH, and 21-OH but not 17-OH, consistent with aldosterone production.
Adrenocortical hyperplasia including focal or diffuse hyperplasia of ZG is a common finding in adrenal cortices adhering to APA, in contrast to adrenal cortices adhering to Cushing adenomas, which are atrophic. Hence, we also made a study of the mRNA expression for steroidogenic enzymes in the adrenal cortices adhering to APA. The majority of hyperplastic ZG and hyperplastic nodules indicated a decreased steroidogenic activity. However, minute nodules indicative of active aldosterone production were found in 62.1% (18 of 29) of cases examined, and six of these contained spironolactone bodies. Enberg et al. (12) also showed by autoradiographic analysis of in situ hybridization that six of 11 adrenals (54.5%) obtained from patients with APA contained ZG and/or small (<5 mm) nodules with expression of CYP11B2 gene encoding 11-OH and 18-OH (6). Aiba et al. (28) classified hyperplastic ZG of the adrenal cortices adhering to APA into two groups: Type I pattern with enhanced steroidogenic activity and type II pattern showing restrained steroidogenesis, according to the patterns of activity of cellular dehydrogenases (3-HSD, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate- isocitrate dehydrogenase, and succinate dehydrogenase activity). The type I pattern was observed in spironolactone body-containing cells of adrenal cortices adhering to APA and in hyperplastic adrenocortical cells of BAH, irrespective of the presence or absence of spironolactone bodies. The nodules observed in UAH and the adrenal cortices adhering to APA in this study corresponded to their type I.
Spironolactone, a competitive antagonist of aldosterone receptors (29), is an antihypertensive drug often taken by patients with PA preoperatively to reduce elevated blood pressure and normalize the serum potassium level. There is evidence that spironolactone can directly interfere with the biosynthesis of aldosterone through inhibiting 11-hydroxylation and 18-hydroxylation in bovine and certain human adrenal cortical tissue (30). Therefore, it has been considered that spironolactone body-containing cells of adrenal cortex adhering to APA might reveal abortive steroidogenic activity (28), although the presence of spironolactone bodies was proof of enhanced steroidogenic activity. However, our finding that spironolactone body-containing cells, but not spironolactone bodies themselves, intensely expressed 3-HSD, 11-OH, 18-OH, and 21-OH mRNAs required for aldosterone synthesis would have to be interpreted as indicating that these cells retain their steroidogenic activity. On the other hand, it is also known that chronic administration of spironolactone activates the endogenous renin-angiotensin system (31). Whether the findings in the present study occur before or after the administration of spironolactone remains the subject of ongoing study because the regulation of CYP11B2 but not CYP11B1 is dependent on the renin-angiotensin system (32). However, nodules lacking spironolactone bodies that showed an intense mRNA expression for aldosterone-synthetic enzymes were also recognized in this study. Therefore, the concept that these lesions were present before the use of the drug would have to be accepted, although the possibility that spironolactone treatment might enhance further mRNA expression for aldosterone-synthetic enzymes is not ruled out.
Both UAH and APA show a renin-independent overproduction of aldosterone (3, 27). However, the expression pattern of steroidogenic enzymes in the nodules of our UAH cases was different from that in APA because 17-OH was generally expressed in the latter. Whether the mechanism of pathogenesis in UAH is different from that in APA is unknown. Figure 6 summarizes the three cases of UAH examined in this study. Diffuse hyperplasia of ZG in case 1 and small nodules located under the adrenal capsule were evident in all cases. The same micronodules as seen in UAH were also present in the adrenal cortices adhering to APA at high frequency. Furthermore, the coexistence with nodules showing the expression pattern of enzymes seen in APA was noted in case of adrenal cortex adhering to APA. Taken in conjunction with the observation that a nodule indicating active aldosterone production was found in close apposition to one showing an intense expression of 17-OH, it is necessary to keep in mind the possibility that multiple micronodules burgeoning in the subcapsular region initially might ultimately become an adenoma by this process of repeated growth and fusion.
Unilateral APA and UAH are treated surgically because of the absence of recurrence after unilateral adrenalectomy (6, 25). Some patients with APA may still remain hypertensive after curative surgery, the main contributing factor for which has been suggested to be delayed diagnosis and therapy, as ascertained by analysis of long-term follow-up after unilateral adrenorectomy for PA (6). On the other hand, it has also been reported that the presence of signs such as hypertension and hypokalemia predicted a postoperative relapse in cases with highly abundant expression of CYP11B2 in ZG and small nodules expressing the CYP11B2 gene but not the CYP17 gene (12), as seen in our cases. Recently laparoscopic adrenal-sparing surgery or enucleation of the adenoma with preservation of functional adrenal tissue has been attempted (33, 34, 35, 36). Although enucleation of the adenoma may be curative, with normalization of the adrenal endocrine function (36), long-term follow-up is required because of the possibility that buds with autonomous aldosterone production, with the potential to develop into PA, may still be present in the remaining adrenal tissue.
Acknowledgments
The authors thank Mrs. Amanda Nishida for critical comments and Miss Kanako Egashira for technical assistance.
Footnotes
First Published Online November 10, 2005
Abbreviations: APA, Aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia; CT, computerized tomography; DIG, digoxigenin; 3-HSD, 3-hydroxysteroid dehydrogenase; 11-OH, 11-hydroxylase; 17-OH, 17-hydroxylase; 18-OH, 18-hydroxylase; 21-OH, 21-hydroxylase; PA, primary aldosteronism; SSC, saline sodium citrate; UAH, unilateral adrenal hyperplasia; ZF, zona fasciculata; ZG, zona glomerulosa; ZR, zona reticularis.
Accepted for publication October 26, 2005.
References
Stowasser M, Gordon RD, Tunny TJ, Klemm SA, Finn WL, Krek AL 1992 Familial hyperaldosteronism type II: five families with a new variety of primary aldosteronism. Clin Exp Pharmacol Physiol 19:319–322
Banks WA, Kastin AJ, Biglieri EG, Ruiz AE 1984 Primary adrenal hyperplasia: a new subset of primary hyperaldosteronism. J Clin Endocrinol Metab 58:783–785
Ganguly A 1998 Primary aldosteronism. N Engl J Med 339:1828–1834
Mulatero P, Stowasser M, Loh KC, Fardella CE, Gordon RD, Mosso L, Gomez-Sanchez CE, Veglio F, Young Jr WF 2004 Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab 89:1045–1050
Wheeler MH, Harris DA 2003 Diagnosis and management of primary aldosteronism. World J Surg 27:627–631
Meyer A, Brabant G, Behrend M 2005 Long-term follow-up after adrenalectomy for primary aldosteronism. World J Surg 29:155–159
Mornet E, Dupont J, Vitek A, White PC 1989 Characterization of two genes encoding human steroid 11-hydroxylase (P-450(11)). J Biol Chem 264:20961–20967
Chung BC, Picado-Leonard J, Haniu M, Bienkowski M, Hall PF, Shively JE, Miller WL 1987 Cytochrome P450c17 (steroid 17-hydroxylase/17, 20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues. Proc Natl Acad Sci USA 84:407–411
Bradshaw KD, Waterman MR, Couch RT, Simpson ER, Zuber MX 1987 Characterization of complementary deoxyribonucleic acid for human adrenocortical 17-hydroxylase: a probe for analysis of 17-hydroxylase deficiency. Mol Endocrinol 5:348–354
Picado-Leonard J, Miller WL 1987 Cloning and sequence of the human gene for P450c17 (steroid 17-hydroxylase/17, 20 lyase): similarity with the gene for P450c21. DNA 6:439–448
Enberg U, Farnebo LO, Wedell A, Grondal S, Thoren M, Grimelius L, Kjellman M, Backdahl M, Hamberger B 2001 In vitro release of aldosterone and cortisol in human adrenal adenomas correlates to mRNA expression of steroidogenic enzymes for genes CYP11B2 and CYP17. World J Surg 25:957–966
Enberg U, Volpe C, Hoog A, Wedell A, Farnebo LO, Thoren M, Hamberger B 2004 Postoperative differentiation between unilateral adrenal adenoma and bilateral adrenal hyperplasia in primary aldosteronism by mRNA expression of the gene CYP11B2. Eur J Endocrinol 151:73–85
Rheaume E, Lachance Y, Zhao HF, Breton N, Dumont M, deLaunoit Y, Trudel C, Luu-The V, Simard J, LabrieF 1991 Structure and expression of a new complementary DNA encoding the almost exclusive 3-hydroxysteroid dehydrogenase/5-4-isomerase in human adrenals and gonads. Mol Endocrinol 5:1147–1157
Kawamoto T, Shizuta Y 1990 Cloning of cDNA and genomic DNA for human cytochrome P45011b. FEBS Lett 269:345–349
Kawamoto T, Mitsuuchi Y, Ohnishi T, Ichikawa Y, Yokoyama Y, Sumimoto H, Toda K, Miyahara K, Kuribayashi I, Nakao K, Hosoda K, Yamamoto Y, Imura H, Shizuta Y 1990 Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism. Biochem Biophys Res Commun 173:309–316
Matteson KJ, Phillips III JA, Miller WL, Chung BC, Orlando PJ, Frisch H, Ferrandez A, Burr IM 1987 P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia. Proc Natl Acad Sci USA 84:5858–5862
Shigematsu K, Nakatani A, Kawai K, Moriuchi R, Katamine S, Miyamoto T, Niwa M 1996 Two subtypes of endothelin receptors and endothelin peptides are expressed in differential cell types of the rat placenta: in vitro receptor autoradiographic and in situ hybridization studies. Endocrinology 137:738–748
Li A, Sakaguchi S, Shigematsu K, Atarashi R, Roy BC, Nakaoke R, Arima K, Okimura N, Kopacek J, Katamine S 2000 Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration. Am J Pathol 157:1447–1452
Sasano H 1994 Localization of steroidogenic enzymes in adrenal cortex and its disorders. Endocr J 41:471–482
Braly LM, Adler GK, Mortensen RM, Conlin PR, Chen R, Hallahan J, Menachery AI, Williams GH 1992 Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cell: in vitro correlate of hyperreninemic hypoaldosteronism. Endocrinology 131:187–194
Galtier A, Liakos P, Keramidas M, Feige JJ, Chambaz EM, Defaye G 1996 ACTH angiotensin II and TGF participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells. Endocr Res 22:607–612
Stern N, Beck FW, Sowers JR, Tuck M, Hsueh WA, Zipser RD 1983 Plasma corticosteroids in hyperreninemic hypoaldosteronism: evidence for diffuse impairment of the zona glomerulosa. J Clin Endocrinol Metab 57:217–220
Findling JW, Waters VO, Raff H 1987 The dissociation of renin and aldosterone during critical illness. J Clin Endocrinol Metab 64:592–595
Parker LN, Levin ER, Lifrak ET 1985 Evidence for adrenocortical adaptation to severe illness. J Clin Endocrinol Metab 60:947–952
Morioka M, Kobayashi T, Sone A, Furukawa Y, Tanaka H 2000 Primary aldosteronism due to unilateral adrenal hyperplasia: report of two cases and review of the literature. Endocr J 47:443–449
Ganguly A, Zager PG, Luetscher JA 1980 Primary aldosteronism due to unilateral adrenal hyperplasia. J Clin Endocrinol Metab 51:1190–1194
Omura M, Sasano H, Fujiwara T, Yamaguchi K, Nishikawa T 2002 Unique cases unilateral hyperaldosteronemia due to multiple adrenocortical micronodules, which can only be detected by selective adrenal venous sampling. Metabolism 151:350–355
Aiba M, Suzuki H, Kageyama K, Murai M, Tazaki H, Abe O, Saruta T 1981 Spironolactone bodies in aldosteronomas and in the attached adrenals. Am J Pathol 103:404–410
Kagawa CM, Cella JA, Van Arman CG 1957 Action of new steroids in blocking effects of aldosterone and deoxycorticosterone on salt. Science 126:1015–1016
Cheng SC, Suzuki K, Sadee W, Harding BW 1976 Effects of spironolactone, canrenone and canrenoate-K on cytochrome P450, and 11- and 18-hydroxylation in bovine and human adrenal cortical mitochondria. Endocrinology 99:1097–1106
Spark RF, Dale SL, Kahn PC, Melby JC 1969 Activation of aldosterone secretion in primary aldosteronism. J Clin Invest 48:96–104
Kakiki M, Morohashi K, Nomura M, Omura T, Horie T 1997 Regulation of aldosterone synthase cytochrome P450 (CYP11B2) and 11-hydroxylase cytochrome P450 (CYP11B1) expression in rat adrenal zona glomerulosa cells by low sodium diet and angiotensin II receptor antagonists. Biol Pharm Bull 20:962–968
Nakada T, Kubota Y, Sasagawa I, Yagisawa T, Watanabe M, Ishigooka M 1995 Therapeutic outcome of primary aldosteronism: adrenalectomy versus enucleation of aldosterone-producing adenoma. J Urol 153:1775–1780
Kok YKK, Yapp SKS 2002 Laparoscopic adrenal-sparing surgery for primary hyperaldosteronism due to aldosterone-producing adenoma. Surg Endosc 16:108–111
Jeschke K, Janetschek G, Peschel R, Schellander L, Bartsch G, Henning K 2003 Laparoscopic partial adrenalectomy in patients with aldosterone-producing adenomas: indications, technique, and results. Urology 61:69–72
Walz MK, Peitgen K, Diesing D, Petersenn S, Janssen OE, Philipp T, Metz KA, Mann K, Schmid KW, Neumann HP 2004 Partial versus total adrenalectomy by the posterior retroperitoneoscopic approach: early and long-term results of 325 consecutive procedures in primary adrenal neoplasias. World J Surg 28:1323–1329(Kazuto Shigematsu, Kioko Kawai, Junji Ir)