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C A S E REPORT

1992;5:566-569

Concomitant Release of Renin, Angiotensin I, and Angiotensin II During Supervision of Human Juxtaglomerular Cell Tumor Francesco Fallo, Domenico D'Agostino, Decio Armanini, Lorenza Caregaro, ana Franco

Increasing evidence indicates that angiotensin II can be formed by juxtaglomerular cells (JGC) and cosecreted with renin. We investigated the existence of this local renin-angiotensin system in a human JGC tumor, using an in vitro superfusion. The JGC tumor was found concomitantly to release renin and angiotensin I and II. Sequential addition of atrial natriuretic peptide, dopamine, and a somatostatin analog in the superfusion did not affect

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renin-secreting juxtaglomerular tumor of the kidney (reninoma) is a rare cause of surgically remediable hypertension in humans. The availability of the tumoral tissue represents a useful tool to investigate the mechanisms regulating the synthesis, storage, and release of renin by the juxtaglomerular cells (JGC). Studies in animals have indicated that angiotensin II is formed by JGC and cosecreted with renin. There is also evidence for the existence of a local renin-angiotensin system in the human kidney, which may regulate several renal functions by autocrine or paracrine mechanisms. The aim of our study was to investigate the presence of such a system using superfusion of a renin-producing JGC tumor surgically removed from a hypertensive patient. 1

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MATERIALS AND M E T H O D S Case Report A 41-year-old woman was studied for arterial hypertension resistant to standard triple antihyReceived December 9, 1991. Accepted April 28, 1992. From the Institute of Semeiotica Medica (FF, DD, DA, FM) and Clinica Medica II (LC), University of Padova, Italy. Address correspondence and reprint requests to Francesco Fallo, MD, Institute of Semeiotica Medica, University of Padova, Via Ospedale 105, 35126 Padova, Italy.

© 1992 by the American Journal of Hypertension, Inc.

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renin or angiotensin I and II release. The vide evidence that the human JGC tumor generates angiotensin II, and supports its role as a local in vivo regulator of kidney Am J Hypertens 1992;5:566-569

data proin vitro possible function.

KEY WORDS: Renin, angiotensin, juxtaglomerular cell tumor.

pertensive therapy. Laboratory examinations were normal except for slight hypokalemia. Upright plasma renin activity, angiotensin II, and aldosterone were elevated. Renal angiography showed an avascular formation within the lower pole of the right kidney 1 cm in diameter; selective sampling of blood from renal veins showed monolateral secretion of renin. After surgical removal of the formation, light and electron microscopy revealed the presence of endocrine and vascular smooth muscle cells, with transitional cells containing both secretory granules and myofilaments. The ultrastructure of these cells showed the presence of characteristic romboidal protogranules and mature granules. Immunohistochemistry (performed by P. Corvol, Paris) using fluorescent mouse monoclonal antihuman renin antibodies confirmed that these granules contained renin. After the operation, the patient's clinical and biochemical indices returned to normal. Superfusion Experiment After removal, we quickly placed the tumor tissue in an ice-cold superfusion medium. Two 1-mL volume superfusion chambers were run simultaneously as a control (blank channel without tissue) or experimental preparation, with Bio-Gel P-2 (Bio Rad, Richmond, CA) as a support matrix. Tissue 0895-7061/'92/'$5.00

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(200 mg wet weight) was finely minced and placed in one of the chambers. The medium for superfusion was constantly gassed with 9 5 % C 0 - 5 % O and consisted of medium MEM-Earle containing 0 . 1 % bovine serum albumin (RIA Grade; Sigma Chemicals Co, St. Louis, MO) with pH adjusted between 7.3 to 7.4. Bovine serum albumin was heat-inactivated, so that it contained no detectable renin. The medium, at 37°C, was delivered to each chamber at a flow rate of 0.5 mL/min. After a initial 30-min period, the superfusate was automatically collected for 300 min during 2-min successive intervals in prechilled tubes with 5 mmol/L EDTA, 0.1 mmol/L Captopril (E. R. Squibb, Princeton, NJ), and 0.01 mmol/ L renin-inhibitor SR 43845 (Sanofi Recherche, Montpellier, France). The 2-min fractions were immediately frozen in dry ice and pooled by five consecutive addi­ tions in a 15-mL polyethylene test tube, lyophilized under vacuum, and the residue stored at —80C until assay for renin, angiotensin I, and angiotensin II. After 60 min of baseline collection period, one of the two chambers was superfused consecutively for 60 min with 10~ mol/L a-human atrial natruiretic peptide (hANP 1-28; Bissendorf, GmbH, Wedemark, Germany), 1 0 " mol/L dopamine, (3-hydroxytyramine; Sigma) and 1 0 " mol/L somatostatin analog SMS 201-995 (Sandostatin; Sandoz, Milan, Italy). A 30-min interval period between the first two substances was left for basal col­ lection of medium alone. 2

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We also assayed fractions for lactic dehydrogenase activity (Sigma test kits) as an index for cell damage. Lactic dehydrogenase activity in the superfusate frac­ tions did not show significant variations over the time of superfusions, in either control or samples treated with the three agents. Assays For the hormone assay, we redissolved dried samples in 2 mL of buffer. Active renin was measured by an immunoradiometric assay kit (Diagnostics Pas­ teur, Marnes La Coquette, France), using two monoclo­ nal human antirenin antibodies, 3E8 and 4 G 1 . Because of the expected very high levels of renin, a 10-//L sample in duplicate, diluted to 250 //L with buffer, was used for the assay. The limit of detection was 2 pg/mL, the intraassay coefficient of variation (CV) 6%, and the inter­ assay CV 10%. Angiotensin I and II were assayed using a double-antibody radioimmunoassay (RIA) system, as previously described in detail. The first antibody (Arnel Products Inc., New York) raised in rabbits crossreacted 100% each with the Des-Asp-angiotensin I and DesAsp-angiotensin II. The second antibody, a donkey antirabbit immunoglobulin, was coupled to magnetic beads (Amersham International P i c , Amersham, Buck­ inghamshire, England). The lowest concentrations of angiotensin I and II detected were 1 pg/mL and 2 pg/ mL, respectively. Intraassay CV was 7% and 9 % (N = 5

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12), and interassay CV was 1 0 % and 1 3 % (N = 18) for angiotensin I and II, respectively. Because we did not perform a previous chromatographic separation of an­ giotensins in the perfusate samples, the terms angioten­ sin I and II refer to the immunoreactive peptides. Statistics During superfusion, we compared mean ± SEM basal variations of renin and angiotensin I and II (ie, fractions collected at 1 to 6 0 , 1 3 0 to 150, and 220 to 240 min) with variations during hANP, dopamine, and SMS 201-995 administration (ie, fractions at 70 to 120, 160 to 210, and 250 to 300 min, respectively) using the Mann-Whitney U test. The relationship between the variations of renin or angiotensin I with angiotensin II was investigated by calculating the nonparametric Spearman's correlation coefficient between pairs over superfusion time. Significance was defined as Ρ < .05. RESULTS During superfusion, renin release ranged from 275 to 120 ng/10 min, showing a progressive decline over time. Release of angiotensin I ranged from 9 to 72 pg/10 min (mean 33 ± 6), and angiotensin II from 10 to 152 pg/10 min fraction (mean 60 ± 12). The addition in the superfusion fluid of hANP, dopamine, or SMS 201-995 did not significantly affect the rates of renin, angioten­ sin I and II release (Figure 1). The rate of release of angiotensin I was directly related to the rate of angio­ tensin II (r = 0.82, Ρ < .01), but renin was not. DISCUSSION Immunohistochemical and biochemical studies have demonstrated that renin and angiotensin I and II coexist in the JGC of rat kidney, suggesting that angiotensin II is formed locally through intracellular pathways. In con­ trast, other studies in rats showed a lack of immunostaining for angiotensinogen and angiotensin convert­ ing enzyme (ACE) in juxtaglomerular epitheloid cells, whereas angiotensins have been found in the interstitial renal fluid, indicating that angiotensin II is generated in the extracellular space and then endocytozed in the in­ tracellular secretory granules. Paracrine secretion of angiotensin II may exert a local regulatory influence on renal functions independent from circulating angioten­ sin II. Our results show that immunoreactive angioten­ sin I and II are released concomitantly with active renin from human JGC tumor during superfusion. We did not measure prorenin (ie, inactive renin), but the proportion of active renin on the total renin after short-term culture of renin-secreting tumor is known to be higher than that found in long-term culture studies, because of the prev­ alence of the regulated on the secretory pathway of renin secretion. High levels of active renin in our brief superfusion experiment confirm these data. Dispropor2

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FIGURE 1. Renin (top panel) and angiotensin I

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and II (bottom panel) concentrations during in vitro superfusion of human juxtaglomerular cells tumor, and response to human atrial natriuretic peptide (ANP), dopamine (DOPA), and somatostatin analog SMS 201-995.

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tionally low levels of angiotensin I and II compared with renin are not surprising. In fact, the concentration of angiotensinogen in renal interstitium has been found to be similar to that of plasma, ie, lower than the Michaelis constant for renin ; in this case, angiotensinogen is rate-limiting in angiotensin I generation. Because we did not use an isolated human JGC prepa­ ration, our data did not allow us to distinguish between an intracellular and an extracellular generation of an­ giotensin I and II. However, it has been reported that cells from human juxtaglomerular tumor were not la­ beled by indirect immunofluorescence with ACE and angiotensinogen antiserum. Furthermore, tumoral renin-secreting cells do not produce angiotensinogen or ACE in primary culture. These data make the presence of a functional intracellular renin-angiotensin system within the JGC cells unlikely, but they suggest that an­ giotensinogen and ACE could reside at different tissue sites and reach renin and angiotensin I extracellularly. In fact, an in vivo intrarenal production of angiotensin I 10

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and I I , probably derived from renin secreted by JGC into the renal interstitium, has been shown in humans. The three pulses of agents sequentially administered during superfusion of tumoral JGC tissue did not affect the renin or angiotensin I and II release. Although most studies show that the in vitro effect of dopamine on renin release is stimulatory, atrial natriuretic peptide in­ hibits renin production from rat JGC in primary cul­ ture. No previous data are available on the effect of somatostatin on renin secretion, but somatostatin ana­ logs have the potential to inhibit the tumoral secretion of different peptides. Lack of secretion changes in our superfusion experiment during administration of renin modulators confirm other studies using renin-secreting tumoral cells in culture. No response of plasma renin and angiotensins to physiologic or pharmacologic agents are observed in vivo, and this behavior has been attributed to the autonomous tumoral production of renin by the pathologic tissue. In conclusion, our data demonstrate concomitant re12

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lease of renin and angiotensin I and II during superfusion of human JGC tumor. The findings provide in vitro evidence for a direct generation of angiotensin II by human JGC tumor, and further support its possible role as a local in vivo regulator of kidney function.

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Cantin M, Gutkowska J, Lacasse, et al: Ultrastructural immunocytochemical localization of renin and angiotensin II in the juxtaglomerular cells of the ischemic kidney in experimental renal hypertension. Am J Pathol 1984;115:212-224.

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Seikaly MG, Arant BS, Jr., Seney FD, Jr.: Endogenous angiotensin concentrations in specific intrarenal fluid compartment of the rats. J Clin Invest 1990;86:13521357.

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Galen FX, Devaux C, Houot AM, et al: Renin biosynthesis by human juxtaglomerular cells. Evidence for a renin precursor. J Clin Invest 1984;73:1144-1155.

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Horky K, Rojo-Ortega JM, Rodriguez J, et al: Renin, renin substrate, and angiotensin I-converting enzyme in the lymphs of rats. Am J Physiol 1971;220:307-311.

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Camilleri JP, Hinglais N, Bruneval P, et al: Renin storage and cell differentiation in a juxtaglomerular cell tumor: study of three cases. Hum Pathol 1984;15:1069-1079.

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Admiraal PJJ, Derkx FHM, Danser AHJ, et al: Intrarenal de novo production of angiotensin I in subjects with renal artery stenosis. Hypertension 1990;16:555-563.

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6. Kifor I, Moore TJ, Fallo F, et al: Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically dispersed cells of the zona glomerulosa. Endocrinology 1991;129:823-831.