Immunocytochemical characterizationof intrarenal adrenal tissue

CASE REPORT

IMMUNOCYTOCHEMICAL CHARACTERIZATION OF INTRARENAL ADRENAL TISSUE* LISA CHIN RACHEL I. BRODY, M.D., PH.D. PABLO MORALES, M.D. VIRGINIA H. BLACK, PH.D. From the Departments of Cell Biology, Pathology, Urology, and the Kaplan Cancer Center, New York University School of Medicine, New York, New York

ABSTRACT--Heterotopic adrenal tissue has been reported in multiple sites, but its functionality has seldom been assessed. In this case, immunocytochemistry was used to characterize adrenocortical tissue present in the subcapsular region of a nephrectomy specimen and to determine its potential for steroidogenesis. Immunodetectable cytochrome P450scc was detectable in the adrenal gland, but not in the renal tissue, clarifying the demarcation between the two tissue types. The high level of this key enzyme in steroid synthesis in the adrenocortical cells suggested that they were capable of producing steroids.

The adrenal gland consists of two distinct porions, the cortex and medulla, within a fibrous capiule. Medulla and cortex are morphologically, funcionally, and embryologically distinct. The cortex rises from coelomic mesodermal epithelium adjalent to the gonad and kidney. In the h u m a n emiry0 it appears by the fourth week of development. ~ctodermal cells of the neural crest along the mid[orsal region penetrate the cortical p r i m o r d i u m to ive rise to the adrenal medulla. 1 Penetration of eural crest Chromaffin cells, as well as differentiaof the zones of the cortex, continue until the inC~of the ninth m o n t h of gestation. 2 Heterotopic or accessory adrenal tissue has been dentified in multiple sites. These ectopias may ~ontain both cortex and medulla and are usually i~resent in the retroperitoneum. Schechter 2 distinguished between true heterotopia, in which the ~rimordial adrenal tissue fails to separate from the ~coelomic e p ithelium, becomin g lod g ed within an iladjacent organ (kidney or liver), and accessory or

;!!~.?a~jaent~:Tstudy Fhis was perfi,ormed b'y one oJf the authors (L. C.) while a at Stuyvesant High School as part of a Westinghouse Sci:~ce Talent Search Project It was'supported by a research grant, ~K 39671 National Insti~tes of Health granted to V.H.B. ~:~ubrnitted'. Aueust 26 1993 acce~ted "(with revisions) A ril 8 ~ .

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quasiheterotopia, the result of fragmentation of an a p p r o p r i a t e l y located gland. True h e t e r o t o p i c adrenal tissue has been seen in the liver, pancreas, gallbladder, and kidney. 3-6 Rests of adrenal cortical tissue have been noted in other steroidogenic tissues, often as multiple paraovarian or testicular masses. 2,7,8 In these cases, adrenal cortical tissue is thought to have been adherent to primordial gonadal tissue and to have been carried with the gonads during their subsequent migration. Accessory adrenal tissue has also been identified in a wide variety of other, nonsteroidogenic tissues, including localization in lung, in the spinal canal, and attached to leptomeninges in the anterior cranial fossa. 9-11 Heterotopic adrenal tissue is usually of no clinical significance, but may become important in certain circumstances, including hyperplasia of accessory tissue in adrenalectomized patients; adrenal insufficiency following excision of organs containing true heterotopic adrenal glands; and neoplastic transformation, which may potentially cause endocrinologic sequelae. 2 In the present case, heterotopic adrenal cortical tissue was p r e s e n t in the s u b c a p s u l a r r e g i o n in a r i g h t n e p h r e c t o m y specimen from a 53-year-old m a n with renal cell carcinoma. Immunohistochemistry was used to characterize the tissue and to assess its potential for steroidogenesis. 429

FIGURE 1. This hematoxylin and eosin-stained, paraffin-embedded section shows the adrenal cortical tissue (A) lying under the capsule and directly apposed to the kidney tissue (K). Large thin-wafted vessels (V) occur at the junction between the two parenchymal types but there is no connective tissue demarcation. The adrenal tissue is characterized by closely packed epithelioid cells, the renal tissue by tubules (T) and glomeruft (G).

CASE REPORT A 53-year-old white m a n underwent partial right nephrectomy of the inferior pole of the kidney for renal cell carcinoma. His surgery and postoperative course were unremarkable for 2 years, when a right renal mass was detected on a follow-up computed tomography (CT) scan. A right nephrectomy was performed, and the specimen showed renal cell carcinoma, grade II/IV, 1.8 cm in its greatest diameter. At the superior pole of the kidney beneath the capsule was an irregularly shaped bright yellow area, 5.0 x 2.5 × 0.1 cm. Cut sections showed the yellow tissue focally infiltrating the renal cortex. The adrenal glands were not involved in the surgery. However, no adrenal tissue per se was identified on the right side at the time of surgery. The CT scan showed no enlargement of the left adrenal gland. T h e p a t i e n t h a d no h i s t o r y of adrenal insufficiency. Frozen sections 5 [~m thick were taken of the yellow tissue and underlying renal cortex. Some 430

FIGURE 2.

This high-power micrograph of a regi~ similar to that shown in Figure 1 clearly demonstra the lack of connective tissue demarcation between i adrenal and renal tissue. Multiple thin-walled ves~ (V) of large diameter occur at the boundary betw~ the two. In the adrenal tissue, the cells closest to i surface are of small diameter and are closely packi They are similar in appearance and location to the ce of the zona glomerulosa (ZG). Beneath these are f o ~ ftpid-rifted cells of larger diameter, the zona fascicula! (ZF). Adjacent to the renal tissue are clusters of ce containing little lipid with quite eosinophilic cytoplail the zona reticularis (ZR).

were stained with h e m a t o x y l i n and eosin a! others were stored at -70°C for future analy' The remainder of the tissue was fixed in formal{ hyde and e m b e d d e d in paraffin. Immunocyt chemical staining of frozen and paraffin-embedd' tissues was performed using antibody made again P450scc (CYPllA1) from bovine adrenal mit! chondria (Oxygene, Dallas, TX). Reaction prodU! w i t h p e r o x i d a s e c o n j u g a t e d arltirabbit irnrn! noglobulin G was visualized with DAB/H202. Hematoxylin and eosin-stained sections shoV~i unremarkable renal cortical parenchyrna (Fig. ! UROLOGY~ /

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(,4) This frozen section has been incubated with antibody to cytochrorne P450scc. All of the cells in the ~enocortical tissue are intensely stained, clearly differentiating this tissue from that of the kidney. (B) This higher agnification rnicrograph of a region sirnflar to that seen in Figure 3A shows that the paler staining in the zona fasculata-like cells is due to the vacuolated lipid droplets. GURE 3.

tverlying this, corresponding to the yellow area hen grossly, were closely packed eosinophilic ceils }hose relative size, lipid content, and architectural }rangement resembled that of the adrenal cortex: bna glomerulosa, zona fasciculata, and zona reticlaris. The cells in the region closest to the conective tissue capsule were small and closely acked, containing some lipid clusters. Beneath ~hese were larger ceils that had more a b u n d a n t id. The ceils in this region were arranged in irregular cords. Deeper cells, immediately bordering ghe renal tissue were smaller and contained less Lpid.Except for a few large capillaries, there was no ~atomic separation between the two tissue types :ig. 2). Immunocytochemical staining with anti-P450scc ighlighted the adrenal tissue (Fig. 3). The most ~tensely stained regions were the zona glomeru.~sa, lyingimmediately below the connective tissue ~a " ' " psule, and the zona reucularls, adjacent to the !enal parenchyma (Fig. 3A). The large, lipid-filled !ells of the zona fasciculata were not as darkly l::stained. The apparent lesser intensity of staining in !ihese cells may be explained by fusion and extrac!tion of lipid droplets d u r i n g acetone fixation {Which left the Cehs extensively vacuolated (Fig'. ~3B). There was no reaction with anti-P450scc in l~e underlying renal tissue Incubation with noniimmune serum showed no reaction.

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COMMENT }i Because of close juxtaposition during embry~;0logic development, adrenal tissue may become Incorporated into the liver, kidney, or the gonads. iS.everal cases of ectopic adrenal tissue have been Ceported. >n However, none of these studies used .[JR©LoGY~ /

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immunocytochemistry to establish steroidogenic enzyme content. In this study, sections of kidney containing the ectopic adrenal tissue were incubated with antibody to cytochrome P450scc, a mitochondrial enzyrne unique to tissues synthesizing steroids. After incubation, the adrenal cortical ceils were intensely stained w h e n compared with the nonreactive cells of the kidney, making it easy to distinguish the two tissue types. The intense staining of the adrenal cortical cells indicated that there was a high level of cytochrome P450scc present. Possession of this key enzyme in steroid synthesis suggested that these ectopic adrenal cortical cells were capable of producing steroids. In a recent article Clark and coworkers 8 showed that adrenal rests in the testes were biochemically competent for steroidogenesis. However, neither the immunocytochemical nor the biochemical results prove that the cells were actually functioning in situ. In this case, no adrenal gland beyond the ectopic tissue in the kidney was identified on the right side at the time of surgery; the heterotopic adrenal tissue thus appears to comprise the adrenal cortex on the right side. The left adrenal gland was not available for examination because this was a surgical, not an autopsy, specimen, but no enlargement of the left adrenal gland was noted in the CT scan. In addition, the patient had no history of adrenal insufficiency. Taken together with the immunocytochemical data presented here, these observations strongly suggest that the heterotopic adrenal tissue on the right side was functional. The absence of an adrenal gland on the right side indicates that the adrenal tissue within the kidney was a true heterotopia. Despite its location 431

within the kidney, zonation of the adrenal cortical cells developed appropriately. Thin-walled blood vessels of wide diameter were the only demarcation found between the adrenal and kidney tissue. Under normal conditions, there are no blood vessels of this diameter so near to the surface of the kidney. These vessels are, however, reminiscent in both their size and their location beneath the cortical cells, of large veins f o u n d at the corticomedullary junction into which adrenocortical capillaries empty. However, in this case, no chromaffin tissue was found in association with the vessels. Complete zonation of heterotopic adrenal cortical tissue has not been previously reported. The vascular arrangement noted may provide a key to its development. Virginia H. Black, Ph.D.

Department of Cell Biology New York University School of Medicine 550 First Avenue New York, New York 10016 ACKNOWLEDGMENTS.To Jody Culkin and Frank Forcino for photographic assistance.

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REFERENCES :~e 1. Corliss CE: Patten'sHuman Embryology. New Yorl~ Graw-Hill, 1976, pp 336-338. 2. Schechter DC: Aberrant adrenal tissue. Ann Surg li 421-426, 1968. 3. Sommers SC: Adrenal glands, in Anderson WAD, Kissane JM (Eds): Pathology, 7th ed. St. Louis, CV M'os~ 1977, p 1660. 4. Nelson AA: Accessory adrenal cortical tissue. Af Pathol 27: 955-965, 1939. 5. Busuttil A: Ectopic adrenal within the gallbladder w ~ J Pathol 113: 231-233, 1974. 6. Weller CV: Heterotopia of adrenal in liver and kidi~ ~(mJ Med Sci 169: 696-712, 1925. ~ 7. Wild RA, Albert RD, Zaino RJ, and Abrams CS: Vir ing paraovarian tumors: a consequence of Nelson's syndro~ Obstet Gynecol 71: 1053-1056, 1988. 8. Clark RV, Albertson BD, Munabi A, Cassorla F, Agui] G, Warren DW, Sherins RJ, and Loriaux DL Steroidoge enzyme activities, morphology, and receptor studies of a ticular adrenal rest in a patient with congenital adrenal perplasia. J Clin Endocrinol Metab 70: 1408-1413, 1990i 9. Kepes JJ, O'Boynick P, Jones S, Baum D, McMfllal and Adams ME: Adrenal cortical adenoma in the spinal c of an 8-year-old girl. AmJ Surg Pathol 14: 481-484, 19901 10. Bozic C: Ectopic fetal adrenal cortex in the lung~ newborn. Virchows Arch A Pathol Anat Histopathol 3i 371-374, 1974. i 11. Weiner MF, and Dallgaard SA: Intracranial adre~ gland. Arch Pathol 67: 228-233, 1959.

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