0021-972X/85/6101-0172$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1985 by The Endocrine Society

Vol. 61, No. 1 Printed in U.S.A.

Immunohistological Evidence for Renin in Human Endocrine Tissues* KIYOKO NARUSE, MASANORI MURAKOSHI, R. YOSHIYUKI OSAMURA, MITSUHIDE NARUSE, HIROSHI TOMA, KEIICHI WATANABE, HIROSHI DEMURA, TADASHI INAGAMI, AND KAZUO SHIZUME Institute of Clinical Endocrinology (K.N., M.N., H.D., K.S.) and Department of Urology (H.T.), Tokyo Women's Medical College, Tokyo 162; and the Department of Pathology, Tokai University School of Medicine (M.M., R.Y.O., K.W.), Kanagawa 259-11, Japan; and the Department of Biochemistry and Hypertension Center, Vanderbilt University School of Medicine (T.I.), Nashville, Tennessee 37232

ABSTRACT. The peroxidase-labeled antibody method and the avidin-biotin-complex method with antiserum to purified human kidney renin were used to identify renin in human endocrine tissues. Renin immunoreactivity was found in some large cells of the anterior pituitary, the zona glomerulosa and the zona reticularis of the adrenal, the Leydig cells of the testis,

and the follicular epithelial cells of the thyroid and prostate glands. The specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive renin in each tissue suggests a possible role of renin in the function of these tissues. (J Clin Endocrinol Metab 6 1 : 172, 1985)

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ENIN-like activity has been reported in various extrarenal tissues (1). Although once attributed to the nonspecific proteolytic activity of cathepsin D (2, 3), recent biochemical and immunological studies using specific antirenin antibody distinguished renin from other proteolytic enzymes in various tissues (4-9). Angiotensin II, an active product of the enzymatic action of renin, is well known for its effect on blood pressure, water metabolism, adrenal function, and cell growth (10). In addition, Steele et al. (11) recently reported that angiotensin II modulates the release of anterior pituitary hormones. The presence of renin in extrarenal tissues raises an intriguing hypothesis that there exists a tissue reninangiotensin system which can locally regulate tissue functions. It is not clear, however, whether the renin in extrarenal tissues is synthesized locally or taken up from the circulation. The answer to this question requires electron microscopic and/or incorporation studies. Immunohistochemical localization of renin can supply evidence to exclude the possibility that renin activity determined by biochemical methods in tissue extracts is the result of contamination by plasma renin remaining in the tissue.

In addition, identification of renin in certain tissue cell types may help to support the hypothesis of local synthesis of renin and, furthermore, can provide insight into its physiological role in the tissue. The localization of extrarenal renin to various tissues in mice and rats with antirenin antibody has been demonstrated immunohistochemically (4, 12-15). On the other hand, localization in human tissues has only been achieved for some tissues (16,17). In this study, using immunohistochemical staining techniques, we demonstrate the presence of renin in various human endocrine tissues.

Materials and Methods Antisera Specific antibodies to human kidney renin were produced in Dutch-Belted rabbits using pure human renal renin as antigen. The pure renin was prepared by a previously described method (18). The enzyme preparation satisfied multiple criteria of purity, including single bands upon polyacrylamide gel electrophoresis, sodium dodecyl sulfate-gel electrophoresis, isoelectric focusing, and double immunodiffusion and symmetric chromatographic elution patterns. The specificity of the antiserum was described previously (19).

Received April 24,1984. Address requests for reprints to: Dr. Kiyoko Naruse, Institute of Clinical Endocrinology, Tokyo Women's Medical College, 10 IchigayaKawadacho Shinjuku-Ku, Tokyo 162, Japan. * This work was supported in part by grants for "Specific Disease" from the Japanese Ministry of Health and Welfare and Grant HL14192 from the NIH.

Tissue preparation Various human endocrine tissues were obtained from four men, aged 45, 69, 70, and 79 yr, and one woman, aged 70 yr, within 2 h after death from nonendocrine disease. Tissues from 172

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IMMUNOREACTIVE RENIN IN HUMAN TISSUES pituitary gland, thyroid gland, parathyroid gland, adrenal gland, testis, and prostate were obtained. In addition, testes were obtained from two patients, aged 64 and 82 yr, who had bilateral orchiectomy as part of the treatment for prostate cancer. Tissues were fixed in cold Bouin's fixative, dehydrated, and embedded in paraffin. Four-micron-thick sections were cut, attached to glass, deparaffinized, and rehydrated. Immunohistochemical staining Immunostaining using antihuman kidney renin antibody as first antiserum was carried out, by the method of Nakane and Pierce (20), using peroxidase-labeled goat antirabbit immunoglobulin G (IgG) Fab fragment as second antibody and/or the avidin-biotin complex method (21) using Vectastain ABC kits (Vector Laboratories, Inc., Burlingame, CA). Diaminobenzidine tetrahydrochloride was used as peroxidase substrate. The sections were treated with 0.3% H2O2 in methanol and 3% normal goat serum to reduce nonspecific background staining and block endogenous peroxidase activity. To test the specificity of the immunohistochemical reaction, the following control sera were substituted for the primary antiserum: normal rabbit serum and antihuman renin antiserum absorbed with purified human renin. Preincubation was performed with an excess of renin overnight at 4 C. Additional methodological controls involved omission of one of the stain components: 1) primary antiserum,

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2) secondary antisera {i.e. peroxidase-labeled goat antirabbit IgG Fab fragment or biotinylated antirabbit IgG), 3) avidinbiotinylated horseradish peroxidase complex, and 4) diaminobenzidine tetrahydrochloride.

Results Renin-specific immunohistochemical staining was found in large ovoid cells of the anterior pituitary (Fig. 1) in three of four patients examined. No staining was found in the intermediate or posterior lobes. In the thyroid gland, specific staining was observed in the epithelial cells of the follicle in all patients. The parathyroid gland was negative in all patients. In the adrenal gland, renin was localized in both the zona glomerulosa and the zona reticularis cells (Fig. 2) in three of five patients. Cells in the zona fasciculata and medulla did not stain. In the testis, immunoreactivity was found in the Leydig cells in all six male patients, but not in the cells of seminiferous tubules or in the vascular walls (Fig. 3). Renin staining also was observed in the epithelial cells of prostate glands (Fig. 4) in all four male patients examined. Control studies with antikidney renin antiserum preincubated with purified kidney renin did not show positive

FIG. 1. Human pituitary stained by the avidin-biotin complex method. A, Renin is localized in large cells (Magnification, X720, B, No reaction is seen using preincubated antiserum. Magnification, X720.

using antihuman renin antiserum.

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2B FIG. 2. Human adrenal stained by the peroxidase-labeled antibody method, c, Capsule; g, zona glomerulosa. A, Renin is localized in zona glomerulosa cells using antihuman renin antiserum. Magnification, X300. B, No reaction is seen using control preincubated antiserum. Magnification, X150.

staining in most cells in the tissues examined; in the thyroid epithelial cells and the zona reticularis of the adrenal gland, specific staining was diminished, but not completely abolished, using preincubated antiserum. Replacement of primary antibody by normal rabbit serum or omission of one of the stain components gave a completely negative result in all instances.

Discussion In this study, we demonstrated the presence of intracellular immunoreactive renin in various human endocrine tissues. These observations indicate that renin is not a contaminant of tissue preparations due to entrapped plasma, but is, indeed, endogenous to each tissue. Iwao et al. (22), investigating the distribution of exogenously administered 125I-labeled renin in mice, found no significant uptake of renin in tissues other than the kidney, further indicating endogenous synthesis of renin in these extrarenal tissues. The identification of renin in the human anterior pituitary reported here is in agreement with previous observations in the rat pituitary, where renin was demonstrated immunohistochemically in the LH-containing

cells (13). The existence of renin in the large ovoid cells of the human pituitary is also in agreement with this observation. Slater et al. (16) demonstrated positive staining in many cells of the human anterior pituitary and some cells of the posterior pituitary; however, details about the types of cell were not provided. The reason for the difference between their results and ours in the posterior pituitary is not clear. Of interest, angiotensinlike immunoreactivity has been reported in cells of the rat anterior pituitary gland (23, 24) which were later identified as gonadotrophs and lactotrophs (25). These studies suggest that intracellular synthesis of angiotensin II occurs in the pituitary cells; the intrapituitary reninangiotensin system may play a role in the regulation of gonadotroph and/or lactotroph functions. Goormaghtigh and Handovsky (26) recognized that arterioles of the adrenal capsule contained epitheloid cells morphologically similar to the juxtaglomerular cells of the renal afferent arterioles. In addition, renin-like activity has been reported in the human adrenal gland (1). However, the distinction of these renin-like activities from nonspecific proteolytic activity due to cathepsin D was only recently elucidated (2, 3). Using antirenin an-

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3A FIG. 3. Human testis stained by the peroxidase-labeled antibody method. L, Leydig cells; S, seminiferous tubules. A, Renin-specific staining is seen in Leydig cells, but no staining is observed in the cells of seminiferous tubules. Magnification, X150. B, No immunoreactive material is seen using preincubated antiserum. Magnification, x300.

tibody, we demonstrated the presence of immunoreactive renin in the human adrenal (9). By gross dissection of the cortex from the medulla, renin was found predominantly in the cortex (9). Further, adrenal tissue from patients with adrenocortical diseases, such as primary aldosteronism and Cushing's syndrome, contained more renin than did adrenal tissue from patients with adrenomedullary pheochromocytoma (9). In this study, positive staining was observed in both zona glomerulosa and zona reticularis cells, although the staining in the latter was not completely abolished using antibody preincubated with renin. It is possible that the amount of renin used for preincubation was not sufficient to block staining, since the zona reticularis contained more renin than other positive cells. Recently, we determined the presence of renin in the inner layer of the mouse adrenal cortex (15). The difference between the results in mice and humans may be species dependent. It is well known that angiotensin II stimulates adrenocortical steroidogenesis (10). Furthermore, angiotensin II was reported to be present in the rat adrenal even after bilateral nephrectomy (27, 28), indicating intraadrenal generation of angiotensin. Thus, renin in the adrenal may serve as

a local regulator for adrenal steroidogenesis by generating antiotensin II. Parmentier et al. (14) demonstrated the existence of immunoreactive renin in Leydig cells of the rat testis. Our observation in human testis is consistent with this result. Testicular renin was found to be pituitary dependent in the rat (14). The exclusive localization of renin in Leydig cells of the human testis could indicate that renin is involved in regulation of testosterone production. On the other hand, the observation that mouse submaxillary gland renin appears to be under androgenic control (29, 30) may suggest that testosterone regulates renin production in the testis. Renin also was identified in the epithelial cells of the prostate. Angiotensin-converting enzyme has been found in similar sites (31, 32). Therefore, testicular renin may exert its effect locally by generating angiotensin I, in a manner similar to cultured neuroblastoma cells (33, 34) and cultured juxtaglomerular cells (35). A sustained level of plasma renin in anephric patients (36-38) and aggression-provoked increases in plasma renin in nephrectomized and submaxillary sialoadenectomized male mice (39) implied other sources of plasma renin in addition to

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FIG. 4. Human prostate stained by peroxidase-labeled antibody method. A, Renin is localized in the epithelial cells (—») but not in other cells. Magnification, x 300. B, Control study using preincubated antiserum shows no positive staining in any cells. Magnification, x 300.

kidney and submaxillary gland. Renin has been demonstrated in human seminal blood plasma (40). It can be postulated that, like the kidney enzyme system, renin is secreted into the circulation from male reproductive tissue. The thyroid follicular cells also showed some specific immunoreactive renin in their cytoplasm. The nature and significance of this staining, however, remain to be determined. A broad distribution of renin in human endocrine tissues was demonstrated in this study. This result is of great interest in view of the various pharmacophysiological actions of angiotensin II (10). The biological relationship between extrarenal renin and endocrine functions remains to be determined.

Acknowledgments The authors are indebted to Miss Yuko Kato and Mrs. Reiko Nukumizu for their able technical assistance.

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IMMUNOREACTIVE RENIN IN HUMAN TISSUES 15. Naruse M, Naruse K, Inagaki T, Inagami T 1984 Immunoreactive renin in mouse adrenal: localization in inner cortical region. Hypertension 6:275 16. Slater EE, Defendimi R. Zimmerman EA 1980 Wide distribution of immunoreactive renin in nerve cells of human brain. Proc Natl Acad Sci USA 77:5458 17. Poisner AM, Wood GW, Poisner R, Inagami T 1981 Localization of renin in trophoblasts in human chorion leave at term pregnancy. Endocrinology 109:1150 18. Yokosawa H, Holladay LA, Inagami T, Haas E, Murakami K 1980 Human renal renin: complete purification and characterization. J Biol Chem 255:3498 19. Yokosawa H, Yokosawa N, Inagami T 1980 Specific antibody to human renal renin and its cross reactivity with inactive human plasma prorenin. Proc Soc Exp Biol Med 164:466 20. Nakane PK, Pierce GB 1967 Enzyme-labeled antibody: preparation and application for the localization of antigens. J Histochem Cytochem 14:929 21. Hsu SM, Raine L, Fanger H 1981 A comparative study of the PAP method and avidin-biotin-complex method for studying polypeptide hormones. Am J Clin Pathol 75:734 22. Iwao H, Nakamura N, Ikemoto F, Yamamoto K, Mizuhira V, Ono M, Sugiura Y 1982 Distribution of exogenously administered renin in mouse kidney. Clin Exp Hypertension A4:2449 23. Ganten D, Fuxe K, Phillips MI, Mann JFE, Ganten U 1978 In: Ganong WF, Martin L (eds) Frontiers of Neuroendocrinology. Raven Press, New York, vol 5:61 24. Changaris DG, Severs WB, Keil LC 1978 Localization of angiotensin in rat brain. J Histochem Cytochem 26:593 25. Steele MK, Brownfield MS, Ganong WF 1982 Immunocytochemical localization of angiotensin immunoreactivity in gonadotrops and lactotrops of the rat anterior pituitary gland. Neuroendocrinology 35:155 26. Goormaghtigh N, Handovsky H 1938 Effect of vitamin D2 (calciferol) on dog. Arch Pathol 26:1144 27. Aguilera G, Schirar A, Baukal A, Catt KJ 1981 Circulating angiotensin II and adrenal receptors after nephrectomy. Nature 289:507 28. Mendelsohn FAO 1982 Angiotensin II is concentrated or locally produced in rat adrenal gland. Clin Exp Pharmacol Physiol [Suppl]

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7:3 29. Trautschold I, Werle E, Schmal A, Hendrikoff NG 1966 Die hormonelle Beeinflussung des Isorenin-Spiegels der Submandibularisdriise der wei/Sen Maus und zur Lokalisierung des Enzyms in der Druse. Hoppe Seylers Z Physiol Chem 344:232 30. Michelakis AM, Yoshida H, Menzie J, Murakami K, Inagami T 1974 A radioimmunoassay for the direct measurement of renin in mice and its application to submaxillary gland and kidney studies. Endocrinology 94:1101 31. Cushman DW, Cheung HS 1971 Concentrations of angiotensin converting enzyme in tissues of the rat. Biochim Biophys Acta 250:261 32. Hohlbrugger G, Schweisfurth H, Dahlheim H 1982 Angiotensin I converting enzyme in rat testis, epididymis and vas deferens under different conditions. J Reprod Fertil 65:97 33. Okamura T, Clemens DL, Inagami T 1981 Renin, angiotensins, and angiotensin-converting enzyme in neuroblastoma cells: evidence for intracellular formation of angiotensins. Proc Natl Acad Sci USA 78:6940 34. Fishman MC, Zimmerman EA, Slater EE 1981 Renin and angiotensin: the complete system within the neuroblastoma X glioma cell. Science 20:921 35. Rightsel WA, Okamura T, Inagami T, Pitcock JA, Takii Y, Brooks B, Brown P, Muirhead EE 1982 Juxtaglomerular cells grown as monolayer cell culture contain renin, angiotensin I-converting enzyme, and angiotensin I and II/III. Circ Res 50:822 36. Oparil S, Koerner TJ, Haber E 1974 Effects of pH and enzyme inhibitors on apparent generation of angiotensin I in human plasma. J Clin Endocrinol Metab 39:965 37. Derkx FHM, Gool JMGV, Wenting GJ, Verhoeven RP, Man In't Veld AJ, Schalekamp MADH 1976 Inactive renin in human plasma. Lancet 2:496 38. Sealey JE, White RP, Laragh JH, Rubin AL 1977 Plasma prorenin and renin in anephric patients. Circ Res [Suppl 2] 41:17 39. Bing J, Poulsen K 1979 Aggression-provoked renin release from extrarenal and extra-submaxillary sources in mice. Acta Physiol Scand 107:251 40. Craven DJ, Warren AY, Symonds EM 1981 Occurrence of active and inactive renin in human seminal plasma. Arch Androl 71:63

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