Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7552-7556, October 1986 Physiological Sciences Analysis by immunocytochemistry and in situ hybridization of and its mRNA in , testis, adrenal, and pituitary of the rat CHRISTIAN F. DESCHEPPER*, SYNTHIA H. MELLONt, FREDERIC CUMINt, JOHN D. BAXTERt, AND WILLIAM F. GANONG* *Department of Physiology and tDepartments of Medicine and Biochemistry and Biophysics and the Metabolic Research Unit, University of California, San Francisco, CA 94143; and fInstitut National de la Sant6 et de la Recherche Mddicale U36, rue du Fer-A-Moulin, 75005 Paris, France Communicated by Rudi Schmid, May 19, 1986

ABSTRACT Renin gene expression in cells and tissues of testes, renin mRNA has been detected by hybridization of the rat was examined by in situ hybridization histochemistry total tissue RNA (21). However, this study failed to detect and immunocytochemistry. By using a mouse cDNA probe, renin mRNA in the pituitary. In addition, it did not provide hybridization histochemistry revealed renin mRNA in the renal information about the type ofcells that actually produce renin juxtaglomerular cells, testicular Leydig cells, adrenal zona in these tissues. To extend this information in the rat and to glomerulosa cells, the intermediate lobe of the pituitary, and clarify these issues, we have in the current studies examined scattered cells of the anterior lobe of the pituitary. With four a number of tissues of the rat by immunocytochemistry and separate antisera to mouse submaxillary renin, there was in situ hybridization to determine the presence and the immunoreactivity in the renal juxtaglomerular cells. However, precise cellular localization of renin and its mRNA. only one of the antisera stained the Leydig cells, a second stained the adrenal zona glomerulosa, a third stained the MATERIALS AND METHODS intermediate lobe of the'pituitary, and a fourth stained scat- tered cells of the anterior lobe of the pituitary that were In Situ Hybridization. cDNA to mouse submandibular identified as gonadotrophs. The variations with the different gland renin mRNA in pSMG213 (22) was used as the antisera in detecting extrarenal renin are unexplained but hybridization probe. It was labeled with deoxycytidine 5'- could imply that posttranslational proteolysis or glycosylation [[35S]thio]triphosphate (specific activity, 1000 Ci/mmol; 1 Ci of preprorenin varies in different tissues with consequent = 37 GBq; Amersham) by nick-translation (23), yielding variations in immunoreactivity. The finding of renin mRNA fragments averaging 50-100 base pairs, as determined by and renin-like immunoreactivity in these tissues supports the electrophoresis on 5% acrylamide gels. The specific activity notion that these tissues are sites for production of renin. was about 108 cpm/,ug of DNA. The method for in situ hybridization was adapted from Gee Renin of renal origin plays an important role in the control of and Roberts (24) and Shivers et al. (25). Rats were killed by blood pressure by cleaving angiotensinogen into decapitation. Tissues were removed immediately, placed in I. It is synthesized as a preprorenin that is converted to OCT compound (Tissue-Tek, Miles), and frozen at -70°C in prorenin and then into active mature renin by proteolytic methanol containing dry ice. Ten-micrometer sections were cleavage (1). In nephrectomized rats, circulating active renin cut on a cryostat at -16°C, collected onto gelatinized glass falls to undetectable levels (2), indicating that most or all of slides, maintained frozen, and fixed for 5 min in a fresh 4% the circulating active renin is of renal origin. However, paraformaldehyde solution in 0.1 M phosphate buffer (pH prorenin is still readily detectable and even increases in the 7.3) containing 200 ,g of diethylpyrocarbonate per liter to plasma of nephrectomized rats (3). These data suggest that prevent mRNA degradation. The slides were then succes- some extrarenal tissues also express the renin gene. sively dehydrated in 70% and 100% ethanol, dried on a warm Studies using renin activity assays of whole tissue extracts plate, and stored in a desiccating environment at -70°C. have detected renin-like activity in many different extrarenal For hybridization, the sections were thawed in a solution tissues. These include the submaxillary glands (4), uterus (5, of proteinase K (2.5 in 0.3 M mM 6), (7), brain (8, 9), (10), testis (11), ,ug/ml) NaCl/30 and adrenals (12, 13) of rats, rabbits, humans, and other citrate (in H20) at 37°C for 12 min. Slides were prehybridized mammalian species. In most ofthese studies, inhibition ofthe at 37°C for 1-2 hr in buffer containing 50% formamide, 0.6 M renin activity by specific renin antibodies and determination NaCl/60 mM sodium citrate, and Denhardt's solution (0.02% of the pH optimum have separated the renin-like activity polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum al- from other enzymes that can generate angiotensin. In some bumin). After aspiration ofbuffer, the sections were covered instances, immunocytochemistry or enzymatic assays in with 20 ,1 of hybridization buffer containing the radiolabeled specific tissue fractions provided information about the probe (100 pg/,ul) previously denatured by boiling for 5 min. localization of renin in the tissue; examples include the rat The sections were incubated overnight at 37°C in a humidified adrenal glomerulosa (14, 15), the inner cortex of the mouse environment. The unhybridized probe was removed by adrenal (16), the Leydig cells of the rat testes (17, 18), the washing the sections for 48 hr in 75 mM NaCl/7.5 mM sodium renal juxtaglomerular cells of rodents (19), and the gonado- citrate at 37°C with frequent changes of buffer. The slides trophs of the rat pituitary (20). were air dried, dipped into photographic emulsion [Ilford L-4 However, the presence of renin in a tissue does not emulsion diluted in H20, 1:1 (vol/vol)], air dried for 2 hr in demonstrate synthesis of the protein in the tissue; a better the dark, and stored desiccated at 4°C. They were developed indication that renin is actually produced is demonstration of after 5-10 days with Kodak D-19 developer and were its mRNA in the tissue. In mouse kidneys, adrenals, and examined and photographed in a Zeiss photomicroscope. The slides were not counterstained, since we observed that The publication costs of this article were defrayed in part by page charge counterstaining generally diminished the intensity of the payment. This article must therefore be hereby marked "advertisement" signal and the structures examined were readily identifiable. in accordance with 18 U.S.C. §1734 solely to indicate this fact. On occasion, phase-contrast or dark-field illumination was 7552 Downloaded by guest on September 25, 2021 Physiological Sciences: Deschepper et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7553 used, either to provide better outlines ofthe tissues examined Renin was purified from the submaxillary glands of male or to intensify the silver grain signal. AKR mice as published (27), using an affinity column of Specificity of the hybridization histochemistry was as- pepstatin-aminohexyl-Sepharose. Renin appeared >90% sessed in two different ways. (i) Sections were incubated with pure on NaDodSO4/polyacrylamide gel electrophoresis. ribonuclease A (final concentration, 25 pg/ml in 0.3 M Polyclonal antibodies to purified renin were raised in four NaCl/30 mM sodium citrate) for 2 hr at 370C prior to rabbits and designated by the following code names: LAR, incubation in proteinase K. (ii) A cold probe (final concen- TAD, CAS, and GUE. These antibodies were used at a tration, 0.1 ug/pl) was incorporated in the hybridization 1:8000 dilution for staining of kidney sections and at a 1:1000 buffer during the prehybridization step. dilution for staining ofextrarenal tissues. Rat LHB antibody, Immunocytochenustry. The immunocytochemical proce- developed by A. F. Parlow, was obtained from the National dures were similar to those described (26). After Hormone and Pituitary Program, National Institute of Ar- pentobarbital anesthesia, the rats were perfused through the thritis, Diabetes, and Digestive and Kidney Diseases, and aorta with heparinized saline followed by Bouin-Holland was used at a 1:8000 dilution. sublimate. The tissues were removed, postfixed overnight in the same fixative, dehydrated in acidified dimethoxypro- pane, cleared in benzene, and embedded in Paraplast RESULTS (Sherwood Medical Industries, St. Louis, MO). Five-micro- The in situ hybridization results are shown in Figs. 1 and 2. meter sections were cut and collected on gelatinized slides. A positive signal was detected in the juxtaglomerular appa- After deparaffination and rehydration, the slides were stained ratus of the kidney, in the cytoplasm of Leydig cells of the according to the avidin-biotin complex method using the kit testis, and in the zona glomerulosa of the . In provided by Vector Laboratories (Burlingame, CA) and the , the high concentrations of silver grains diaminobenzidine as a chromogen. Method specificity was were seen throughout the intermediate lobe and in many cells assessed by observing a gradual inhibition ofthe staining with of the anterior lobe but not in the posterior lobe, where only increasing dilutions of the primary antibody. Antiserum background grains could be seen. We have not yet identified specificity was assessed by observing the extinction of the the exact type of anterior pituitary cells that concentrate the staining after preincubation of the primary antirenin antise- signal. rum with purified mouse submaxillary gland renin (final Figs. 1 and 2 also show the immunocytochemical results concentration, 10 pg/ml). with the various tissues studied. Renin immunoreactivity was

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FIG. 1. Immunocytochemical staining of renin and in situ hybridization of renin mRNA in rat kidney and adrenal. Each procedure was performed in tissue sections from different animals. (A) Immunocytochemical staining ofkidney with the LAR antirenin antiserum. Dark positive staining can be seen at the vascular pole of three glomeruli (see arrows). (x50.) (B) In situ hybridization in rat kidney, photographed with phase-contrast illumination. The positive signal appears as dark grains and is located next to three glomeruli (see arrows). (x50.) (C) Immunocytochemical staining of adrenal with the GUE antirenin antiserum. Dark positive cells are visible in the outer layer of the cortex; the medulla is not visible in this picture. glom, Zona glomerulosa; fasc, . (x30.) (D) In situ hybridization in adrenal. The highest density of dark grains can be seen in the outer layer of the cortex. (x30.) Downloaded by guest on September 25, 2021 7554 Physiological Sciences: Deschepper et al. Proc. Natl. Acad. Sci. USA 83 (1986)

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FIG. 2. Immunocytochemical staining of renin and in situ hybridization of renin mRNA in rat testis and adrenal. Each procedure was performed in tissue sections from different animals. (A) In situ hybridization in testis. The positive signal appears as dark grains concentrated in the cytoplasm of Leydig cells in the interstitial tissue (see arrows). (x60.) (B) Immunocytochemical staining of a rat testis section with the LAR antirenin antiserum. Dark positive staining is seen in the cytoplasm of Leydig cells in the interstitial tissue between unstained seminiferous tubules. (x60.) (C) Immunocytochemical staining of testis with the TAD antirenin antiserum. No positive staining can be detected. (x60.) (D) In situ hybridization of pituitary photographed with dark-field illumination. The positive signal appears as white grains and is detected in the intermediate and anterior lobes. Only background is visible in the neural lobe and the cleft. post, Posterior lobe; int, intermediate lobe; cl, cleft; ant, anterior lobe. (x30.) (E) Immunocytochemical staining of pituitary with the TAD antirenin antiserum. Dark positive staining is detected in a distinct population ofcells ofthe anterior lobe; no staining is detected in the intermediate or posterior lobes. (x30.) (F) Immunocytochemical staining..of pituitary with the CAS antirenin antiserum. Dark positive staining is seen only in the intermediate lobe. (x40.) found in the renal juxtaglomerular cells, the Leydig cells of obtained with the LAR antibody, staining of the adrenal the testis, the zona glomerulosa of the adrenal cortex, the cortex was only obtained with the GUE antibody, staining of intermediate lobe of the pituitary, where -30% of the cells the anterior pituitary lobe was only obtained with the TAD were immunopositive, and scattered cells of the anterior antibody, and staining of the intermediate lobe of the pitu- pituitary. All of the antirenin antisera stained positively the itary was only obtained with the CAS antibody. In all juxtaglomerular cells of the rat kidney. However, there were locations, the staining was abolished by preincubation of the marked differences in their abilities to stain extrarenal tissues diluted antisera with pure renin at a final concentration of 10 (Table 1). Staining of the Leydig cells in the testis was only Ag/ml. The cells of the anterior pituitary were identified as Downloaded by guest on September 25, 2021 Physiological Sciences:, Deschepper et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7555 Table 1. Immunocytochemistry with four different extrarenal tissues (Table 1). The reasons for these differences antirenin antisera were not explored, but the various extrarenal may Pituitary have different posttranslational proteolytic or glycosylytic modifications that affect their recognition by the antibodies. Antiserum Kidney Adrenal Testis Anterior Intermediate Rat renin, like human renin, is a glycoprotein, as assessed by CAS + - -- + its binding to concanavalin A (33). The antisera used in these GUE + + -- - studies have been raised against mouse submaxillary renin, LAR + - + - - which lacks glycosylation sites (1). It is possible that varia- TAD + - - + - tions in glycosylation mask antigenic determinants in the + and - signs refer to presence or absence, respectively, of tissues. Furthermore, mouse renin precursor undergoes sev- detectable immunocytochemical reactivity with the indicated eral proteolytic cleavage modifications (1, 34); variations in antirenin antiserum. such processing might also account for differences in antigenicity. Finally, it is possible that renin in these extra- renal tissues is associated specifically or nonspecifically with gonadotrophs in serial sections consecutively stained with other proteins that affect reactions with the antisera; such a TAD or anti-rat LHI (data not shown). possibility has been suggested for renin in the testes (16). Comparison between in situ hybridization and immunocy- DISCUSSION tochemistry has potential for being a powerful tool, with the possibility of revealing combinations and differences in The current studies demonstrate the presence of renin-like posttranslational processing. In this study, the tissues used immunoreactivity and prorenin mRNA in certain cell types in for immunocytochemistry were prepared separately, be- several tissues of the rat. The finding of mRNA in a tissue cause better morphology can be, achieved in paraffin sections, indicates expression ofthe gene in that tissue. Since there are and the tissue preparation for in situ hybridization is not only rare circumstances in which a mRNA has been present optimal for immunocytochemical detection. However, future in a cell and not expressed, the presence of mRNA most studies might be done with immunocytochemistry and in situ likely reflects its translation as well. The finding of a mRNA hybridization performed on the same section, allowing com- and its peptide translation product in the same cell is strong parisons at the cellular level. evidence that the product is produced in that tissue. The current finding that renin is almost certainly synthe- The immunocytochemical data in kidney and testis confirm sized in several extrarenal tissues raises further the question previous reports (17, 19) and our hybridization histochem- of whether extrarenal renin can contribute to extrarenal istry confirms and extends the data obtained in mice by autocrine or paracrine local renin-angiotensin systems or to blotting of mRNA from whole tissue (21). Our data demon- the production ofplasma prorenin. However, the presence of strate that renin is synthesized in the testicular Leydig cells angiotensin 11 (15, 26, 35) and converting enzyme (28, 35, 36) and the renal juxtaglomerular cells. in these same tissues makes it likely that renin participates in No immunocytochemical data on renin have as yet been the local generation of . published for the rat adrenal; however, the presence ofactive renin has been demonstrated in this tissue (12), and by We thank Dr. Pierre Corvol for help and support, Annette Lowe microdissection, it has been shown to be located mainly in the and Christina Kuroiwa for excellent technical assistance, and Lloyd zona glomerulosa (14, 15). Our data indicate that the zona Dixon and Jen Taylor for typing of the manuscript. C.F.D. was is the main site of renin in the adrenal supported by Public Health Service Grants AM07265, HL29714, and glomerulosa synthesis AM07418. S.H.M. was supported by Public Health Service Grant in rats. Renin mRNA has been demonstrated in whole tissue HL35706 and by a gift from California Biotechnology, Inc. extracts of mouse adrenals (21). However, the site of syn- thesis might be different from rat, since by immunocy- 1. Panthier, J. J., Foote, S., Chambraud, B., Strosberg, A. D., tochemistry, most of the renin-like immunoreactivity in Corvol, P. & Rougeon, F. (1982) Nature (London) 298, 90-92. mouse adrenal has been reported to be in the inner cortical 2. Menard, J. & Catt, K. J. (1972) 90, 422-430. region (16). It is of interest to note that in the rat adrenal, 3. Doi, Y., Franco-Saenz, R. & Mulrow, P. J. (1984) Hyperten- renin, converting enzyme activity (28), angiotensin II (15), sion (Dallas) 6, 627-632. and angiotensin II receptors (29) are all located in the zona 4. Cohen, S., Taylor, J. M., Murakami, K., Michelakis, A. M. & glomerulosa. This suggests that locally synthesized renin and Inagami, T. (1972) Biochemistry 11, 4286-4293. 5. Gross, F., Schaechtelin, G., Ziegler, M. & Berger, M. (1964) converting enzyme may generate intraadrenal angiotensin II. Lancet i, 914-916. The angiotensin II may play a local role in the regulation of 6. Warren, A. Y., Craven, D. J. & Symonds, E. M. (1982) Br. J. secretion (15). Obstet. Gynaecol. 89, 628-632. A previous immunocytochemical study with a different 7. Craven, D. J. & Symonds, E. M. (1979) I. R. C. S. Med. Sci. antirenin antibody reported the presence of renin only in the 7, 196. gonadotrophs of the rat pituitary gland (20), with no staining 8. Ganten, D., Minnich, J. L., Granger, P., Hayduk, K., Brecht, in the intermediate lobe. In our study, one antirenin antise- H. M., Barbeau, A., Boucher, R. & Genest, J. (1971) Science rum stained only the gonadotrophs, whereas another stained 173, 64-65. only the intermediate lobe. Both pituitary locations appeared 9. Hirose, S., Yokosawa, H. & Inagami, T. (1978) Nature (Lon- to be since in both lobes was abolished don) 274, 392-393. specific, staining 10. Hirose, S., Ohsawa, T., Inagami, T. & Murakami, K. (1982) J. when the antisera were preincubated with purified renin. Biol. Chem. 257, 6316-6321. Furthermore, both locations contained renin mRNA by in 11. Naruse, M., Naruse, K., Shizume, K. & Inagami, T. (1984) situ hybridization. Renin in the intermediate lobe might also Proc. Soc. Exp. Biol. Med. 177, 331-342. be involved in the local generation ofangiotensin II, since we 12. Naruse, M. & Inagami, T. (1982) Proc. Natl. Acad. Sci. USA have previously reported that a fraction of the intermediate 79, 3295-3299. lobe cells contains angiotensin II-like immunoreactivity (30). 13. Naruse, M., Sussman, C. R., Naruse, K., Jackson, R. V. & In addition, it is worth noting that the intermediate lobe ofthe Inagami, T. (1983) J. Clin. Endocrinol. Metab. 57, 482-487. rat pituitary contains kallikrein (31), and this enzyme may be 14. Yutaka, D., Atarashi, K., Franco-Saenz, R. & Mulrow, P. J. involved in the processing of prorenin into renin (32). (1984) Hypertension (Dallas) 6, 1124-1129. It is noteworthy that although all of our antibodies stained 15. Nakamaru, M., Misono, K. S., Naruse, M., Workman, R. J. the kidney juxtaglomerular cells, each stained only one ofthe & Inagami, T. (1985) Endocrinology 117, 1772-1778. Downloaded by guest on September 25, 2021 7556 Physiological Sciences: Deschepper et al. Proc. Nati. Acad. Sci. USA 83 (1986)

16. Naruse, M., Naruse, K., Inagaki, T. & Inagami, T. (1984) 27. Evin, G., Devin, J., Castro, B., Menard, J. & Corvol, P. (1984) Hypertension (Dallas) 6, 275-280. Proc. Natl. Acad. Sci. USA 81, 48-52. 17. Pandey, K. N., Melner, M. H., Parmentier, M. & Inagami, T. 28. Plunkett, L. M., Correa, F. M. A. & Saavedra, J. M. (1985) (1984) Endocrinology 115, 1753-1759. Soc. Neurosci. Abstr. 11, p. 543. 18. Parmentier, M., Inagami, T., Pochet, R. & Desclin, J. C. 29. Healy, D. P., Maciejeruski, A. R. & Printz, M. P. (1985) (1983) Endocrinology 112, 1318-1323. Endocrinology 116, 1221-1223. 19. Taugner, R., Hackentral, E., Rix, E., Nobiling, R. & Poulsen, 30. Deschepper, C. F., Bouhnik, J., Cumin, F. & Ganong, W. F. K. (1982) Kidney Int. 22, Suppl. 12, S33-S43. (1985) Fed. Proc. Fed. Am. Soc. Exp. Biol. 44, 1358 (abstr.). 20. Naruse, K., Takii, Y. & Inagami, T. (1981) Proc. Natl. Acad. 31. Powers, C. A. & Nasjletti, A. (1983) Endocrinology 112, Sci. USA 78, 7579-7583. 21. Field, L. J., McGowan, R. A., Dickinson, D. P. & Gross, 1194-1200. K. W. (1984) Hypertension 6, 597-603. 32. Sealy, J. E., Atlas, S. A. & Laragh, J. H. (1978) Am. J. Med. 22. Mullins, J. J., Burt, D. W., Windass, J. D., McTurk, P., 65, 994-1000. George, H. & Brammar, W. J. (1982) EMBO J. 1, 1461-1466. 33. Matoba, T., Murakami, K. & Inagami, T. (1978) Biochim. 23. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) Biophys. Acta 526, 560-571. J. Mol. Biol. 113, 237-251. 34. Pratt, R. E., Ouellette, A. J. & Dzau, V. J. (1983) Proc. Natl. 24. Gee, C. E. & Roberts, J. L. (1983) DNA 2, 157-163. Acad. Sci. USA 80, 6809-6813. 25. Shivers, B. D., Schachter, B. S. & Pfaff, D. W. (1986) Meth- 35. Pandey, K. N., Misono, K. S. & Inagami, T. (1984) Biochem. ods Enzymol. 124, 497-510. Biophys. Res. Commun. 122, 1337-1343. 26. Deschepper, C. F., Seidler, C. D., Steele, M. K. & Ganong, 36. Platia, M., Catt, K. & Aguilera, G. (1985) Fed. Proc. Fed. Am. W. F. (1985) Neuroendocrinology 40, 471-476. Soc. Exp. Biol. 44, 1358 (abstr.). Downloaded by guest on September 25, 2021