8073560

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 func- tionality has seldom been assessed. In this case, immunocytochemistry was used to char- acterize adrenocortical tissue present in the subcapsular region of a nephrectomy speci- men 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 por- tions, the cortex and medulla, within a fibrous cap- sule. Medulla and cortex are morphologically, func- tionally, and embryologically distinct. The cortex arises from coelomic mesodermal epithelium adja- cent to the gonad and kidney. In the human em- bryo it appears by the fourth week of development. Ectodermal cells of the neural crest along the mid- dorsal region penetrate the cortical primordium to give rise to the adrenal medulla.1 Penetration of neural crest chromaffin cells, as well as differentia- tion of the zones of the cortex, continue until the end of the ninth month of gestation.2

Heterotopic or accessory adrenal tissue has been identified in multiple sites. These ectopias may contain both cortex and medulla and are usually present in the retroperitoneum. Schechter2 distin- guished between true heterotopia, in which the primordial adrenal tissue fails to separate from the coelomic epithelium, becoming lodged within an adjacent organ (kidney or liver), and accessory or

quasiheterotopia, the result of fragmentation of an appropriately located gland. True heterotopic 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 tis- sues, often as multiple paraovarian or testicular masses.2,7,8 In these cases, adrenal cortical tissue is thought to have been adherent to primordial go- nadal tissue and to have been carried with the go- nads during their subsequent migration. Accessory adrenal tissue has also been identified in a wide variety of other, nonsteroidogenic tissues, includ- ing localization in lung, in the spinal canal, and at- tached to leptomeninges in the anterior cranial fossa.9-11 Heterotopic adrenal tissue is usually of no clinical significance, but may become impor- tant in certain circumstances, including hyperpla- sia of accessory tissue in adrenalectomized pa- tients; adrenal insufficiency following excision of organs containing true heterotopic adrenal glands; and neoplastic transformation, which may poten- tially cause endocrinologic sequelae.2 In the pres- ent case, heterotopic adrenal cortical tissue was present in the subcapsular region in a right nephrectomy specimen from a 53-year-old man with renal cell carcinoma. Immunohistochemistry was used to characterize the tissue and to assess its potential for steroidogenesis.

*This study was performed by one of the authors (L.C.) while a student at Stuyvesant High School, as part of a Westinghouse Sci- ence Talent Search Project. It was supported by a research grant, DK 39671, National Institutes of Health, granted to V.H.B.

Submitted: August 26, 1993, accepted (with revisions): April 8, 1994

FIGURE 1. This hematoxylin and eosin-stained, paraf- fin-embedded section shows the adrenal cortical tissue (A) lying under the capsule and directly apposed to the kidney tissue (K). Large thin-walled 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 glomeruli (G).

CASE REPORT

A 53-year-old white man 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 diam- eter. At the superior pole of the kidney beneath the capsule was an irregularly shaped bright yellow area, 5.0 x 2.5 x 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. The patient had no history of adrenal insufficiency.

Frozen sections 5 um thick were taken of the yellow tissue and underlying renal cortex. Some

FIGURE 2. This high-power micrograph of a region similar to that shown in Figure 1 clearly demonstrates the lack of connective tissue demarcation between the adrenal and renal tissue. Multiple thin-walled vessels (V) of large diameter occur at the boundary between the two. In the adrenal tissue, the cells closest to the surface are of small diameter and are closely packed They are similar in appearance and location to the cells of the zona glomerulosa (ZG). Beneath these are found lipid-filled cells of larger diameter, the zona fasciculata (ZF). Adjacent to the renal tissue are clusters of cells containing little lipid with quite eosinophilic cytoplasm. the zona reticularis (ZR).

were stained with hematoxylin and eosin and others were stored at -70℃ for future analysis. The remainder of the tissue was fixed in formalde- hyde and embedded in paraffin. Immunocyto- chemical staining of frozen and paraffin-embedded tissues was performed using antibody made against P450scc (CYP11A1) from bovine adrenal mito chondria (Oxygene, Dallas, TX). Reaction product with peroxidase conjugated antirabbit immu noglobulin G was visualized with DAB/H2O2.

Hematoxylin and eosin-stained sections showed unremarkable renal cortical parenchyma (Fig. 1):

FIGURE 3. (A) This frozen section has been incubated with antibody to cytochrome P450scc. All of the cells in the adrenocortical tissue are intensely stained, clearly differentiating this tissue from that of the kidney. (B) This higher magnification micrograph of a region similar to that seen in Figure 3A shows that the paler staining in the zona fas- ciculata-like cells is due to the vacuolated lipid droplets.

A

B

Överlying this, corresponding to the yellow area seen grossly, were closely packed eosinophilic cells whose relative size, lipid content, and architectural arrangement resembled that of the adrenal cortex: zona glomerulosa, zona fasciculata, and zona retic- ularis. The cells in the region closest to the con- nective tissue capsule were small and closely packed, containing some lipid clusters. Beneath these were larger cells that had more abundant lipid. The cells in this region were arranged in ir- regular cords. Deeper cells, immediately bordering the renal tissue were smaller and contained less lipid. Except for a few large capillaries, there was no anatomic separation between the two tissue types (Fig. 2).

Immunocytochemical staining with anti-P450scc highlighted the adrenal tissue (Fig. 3). The most intensely stained regions were the zona glomeru- losa, lying immediately below the connective tissue capsule, and the zona reticularis, adjacent to the renal parenchyma (Fig. 3A). The large, lipid-filled cells of the zona fasciculata were not as darkly stained. The apparent lesser intensity of staining in these cells may be explained by fusion and extrac- tion of lipid droplets during acetone fixation, which left the cells extensively vacuolated (Fig. 3B). There was no reaction with anti-P450scc in the underlying renal tissue. Incubation with non- immune serum showed no reaction.

COMMENT

Because of close juxtaposition during embry- ologic development, adrenal tissue may become incorporated into the liver, kidney, or the gonads. Several cases of ectopic adrenal tissue have been reported.2-11 However, none of these studies used

immunocytochemistry to establish steroidogenic enzyme content.

In this study, sections of kidney containing the ectopic adrenal tissue were incubated with anti- body to cytochrome P450scc, a mitochondrial en- zyme unique to tissues synthesizing steroids. After incubation, the adrenal cortical cells were intensely stained when 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 coworkers8 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 actu- ally 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 tis- sue thus appears to comprise the adrenal cortex on the right side. The left adrenal gland was not available for examination because this was a surgi- cal, 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 immunocy- tochemical 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

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 ves- sels 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 cor- tical cells, of large veins found at the corti- comedullary junction into which adrenocortical capillaries empty. However, in this case, no chro- maffin 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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