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Role of renin and aldosterone in hypertension due to a renin-secreting tumor
Role of Renin and Aldosterone in Hypertension Due to a Renin-Secreting Tumor
M. SCHAMBELAN,
M.D.
E. L. HOWES, Jr.,
M.D.
J. R. STOCKIGT,
M.D.*
C. A. NOAKES,
M.D.
E. G. BIGLIERI, San Francisco.
M.D. California
From the Medical and Anatomic Pathology Services, San Francisco General Hospital, and the Departments of Medicine and Pathology, University of California, San Francisco, California. This study was supported by U.S. Public Health Service Research Grants HE-l 1046 from the National Heart Institute, AM-06415 from the National Institute of Arthritis and Metabolic Diseases, and National Institutes of Health Grant EY-00056 (E.L.H.). The studies were carried out in the General Clinical Research Center, RR-83, at San Francisco General Hospital, supported by Division of Research Resources, National Institutes of Health. They were presented in part at the 4th International Congress of Endocrinology, Washington, D.C., June 1972. Requests for reprints should be addressed to Dr. Morris Schambelan, Clinical Study Center, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, California 94110. Manuscript accepted January 9, 1973. *Present address: Donnie Metabolic Unit, Alfred Hospital, Melbourne, Australia 3181.
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A renin-secreting renal tumor is a rare cause of curable hypertension characterized clinically by hypertension, hypokalemia, hyperaldosteronism and increased plasma renin activity (PRA). Prior subtotal adrenalectomy in such a patient allowed examination of the role of hyperreninemia in the absence of excessive aldosterone secretion. Hypertension was severe, but serum potassium and aldosterone production were normal. PRA was markedly increased under all conditions studied, with a persistent increase noted with changes in posture. Renal vein renin studies were critical in localizing the renal tumor, and unilateral nephrectomy resulted in a prompt fall in PRA to subnormal levels and dramatic cure of the hypertension. Renin content of the renal tumor was more than 500 times higher than in the adjacent kidney, and histologic studies confirmed that the tumor was of juxtaglomerular cell origin. This case demonstrates that severe hypertension can occur in this syndrome despite subtotal adrenalectomy and further illustrates an association of renin overproduction with a specific type of hypertension. In several reports a syndrome of hypertension, hypokalemia, hyperaldosteronism and increased plasma renin activity (PRA) has been described in association with a renin-secreting renal tumor [l-6]. The syndrome is completely reversed by removal of the benign neoplasm, and although the incidence of such a ttimor is unknown, it represents a definite cause of surgically curable hypertension. We describe an additional patient in whom removal of a renin-secreting renal tumor resulted in a rapid and sustained fall in blood pressure to normal levels. Prior subtotal adrenalectomy had reduced aldosterone production to normal levels, but severe hypertension had persisted. Determination of PRA and studies of renal vein renin were critical in making the correct diagnosis. This case demonstrates that severe hypertension can occur in this syndrome without excessive aldosterone secretion and offers further presumptive evidence that gross inappropriate renin overproduction and hypertension are closely associated in such cases. CASE REPORT Hypertension was first noted in this 24 year old white man in December 1966 (160/120 mm Hg); his blood pressure had been within normal limits in 1965. The medical history was unremarkable except for
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Figure 1. A, nephrogram phase from selective right renal arteriogram showing decreased perfusion of an area in the mid upper pole (arrows). 13, right kidney showing a well demarcated nodule in the upper pole. Note hemorrhagic appearance on cut section. enuresis during childhood and lifelong polydypsia and nocturia. Blood urea nitrogen and creatinine were within normal limits, and no abnormalities were detected on the intravenous pyelogram, the radioisotopic renogram and the renal arteriogram. Excretion of vanillylmandelic acid was 8.7 mg/24 hours (normal range 0 to 10 mg), and a phentolamine test was negative. Serum sodium was 141, potassium 3.0, chloride 96 and bicarbonate 22 meq/liter. Urinary protein excretion was 376 mg/24 hours (normal range cl.50 mg). A biopsy specimen of the left kidney showed only mild nephrosclerosis. Blood pressure was controlled with guanethidine and chlorothiazide. In 1968, guanethidine therapy was discontinued because of side effects, and control of blood pressure with alpha-methyldopa was unsatisfactory (170/l 30 mm Hg). Creatinine clearance was 119 ml/min and a rapid sequence intravenous pyelogram was negative. Serum sodium was 140, potassium 2.8, chloride 96 and bicarbonate 35 meq/liter. Aldosterone excretion was 40.8 pg/24 hours (normal range 4 to 17 pg) under control conditions and 36.1 pg/24 hours on the third day of administration of deoxycorticosterone acetate (DOCA), 10 mg intramuscularly every 12 hours. A presumptive diagnosis of primary aldosteronism was made although PRA was not measured. At surgery the adrenal glands appeared normal, and the right (4.8 g) and two thirds of the left adrenal gland (3.4 g) were removed. Microscopic examination revealed no abnormalities. One week postoperatively urinary aldosterone was 6.1 pg/24 hours and 17-hydroxycorticoids 2.9 mg/24 hours. Stimulation with adrenocorticotropin (ACTH) did not increase 17-hydroxycorticoid excretion, and the patient was maintained on oral hydrocortisone therapy, 25 mg every morning and 10 mg every evening. Blood pressure was somewhat lower postoperatively, but was 160/140 mm Hg 1 year later and remained elevated despite treatment with alphamethyldopa, hydrochlorothiazide and spironolactone. On referral to the Clinical Study Center in 1971,
blood pressure was 190/136 mm Hg supine and 160/ 130 mm Hg upright. Physical examination revealed no abnormalities other than mild retinal arteriolar narrowing without hemorrhages or exudates. The hemoglobin level was 17.6 g/100 ml and white blood cell count 11,600/mm3. Blood urea nitrogen was 9, creatinine 1.1, fasting blood sugar 109 and uric acid 4.5 mg/lOO ml. Serum sodium was 141, potassium 3.9, chloride 102 and bicarbonate 26 meq/liter. Creatinine clearance was 121 ml/min, and urinalysis revealed no abnormalities except trace protein. Renal vein renin collections indicated markedly elevated levels of PRA with an apparent source in the right kidney (see “Results”) and, although a midstream renal arteriogram failed to show a definite lesion, a selective right renal arteriogram identified a filling defect in the upper pole of the right kidney (Figure IA). On October 14, 1971, the right kidney and a portion of the left adrenal remnant were removed. Postoperatively, the blood pressure fell immediately to normal levels and remained so except for a transient elevation. On discharge, the blood pressure was 130/90 mm Hg while the patient was on a regimen consisting of only hydrocortisone, 20 mg twice a day, and fludrocortisone acetate, 0.1 mg daily. Eight months postoperatively blood pressure was 125/80 mm Hg without antihypertensive medication, and reduction of fludrocortisone to 0.05 mg/day maintained normal serum electrolyte levels. METHODS Studies were performed while the patient was on a constant diet containing 20 meq sodium and 64 meq potassium daily to which sodium chloride, 6 g, was added daily in the form of weighed capsules. Dexamethasone, 0.5 mg every 12 hours, was given as maintenance glucocorticoid therapy, and antihypertensive therapy with alpha-methyldopa, 500 mg orally 4 times daily, was continued. Aldosterone excretion and PRA were measured in the control period and at the July 1973
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TABLE ___
AND ALDOSTERONE
IN HYPERTENSION-SCHAMBELAN
ET At
Studies in 1971Following Subtotal Adrenalectomy I __~
_-________-
Serum(meq/liter) Maneuver
Potassium
Sodium Inpatient
Control Low sodium diet Control DOCA administration Potassium chloride administration Control ACTH administration Day 1 Day 2 Day 3
3.9 3.8 3.7
... ...
. . ...
134
3.2
3.8 4.2 3.6
Aldosterone Excretion kg/24 hr)
104 81 70 46 59 109
169 183 148 121 128 174
13.6 15.8 14.5 14.6 22.0 13.6
133 94 104
... . . 98
20.1 20.1 19.6
82 176
185 236
18.4 23.2
3-11
5-32
4-17
Studies
134 127 Normal
hr)
Studies
141 134 132 133 136 135
Outpatient
Dexamethasone administration Spironolactone administration
PlasmaReninActivity (m&ml/3 ---Supine Upright
4.7 4.7 Ranges*
* On 120 meq sodium intake. completion of the following maneuvers: (1) sodium restriction for 3 days, effected by removing the sodium chloride capsules from the regimen; (2) administration of DOCA, 10 mg intramuscularly every 12 hours for 3 days; (3) administration of potassium chloride, 1.0 g 4 times daily for 3 days; and (4) administration of ACTH, 25 units intravenously daily as an 8 hour infusion for 3 days. In subsequent studies dexamethasone, 0.5 mg, was administered every 6 hours and spironolactone, 100 mg, 3 times daily for 2 and 3 weeks, respectively, in separate periods on an outpatient basis; at the completion of each period the patient was admitted so that specimens could be collected for aldosterone and renin determinations. Plasma specimens for the determination of renin were collected with the patient in the supine position after overnight recumbency and again after 4 hours of upright activity. Renal vein specimens for the determination of renin were collected as described previously [7]. In the first study, samples were obtained from a peripheral vein, the right and left renal veins, and the caudal inferior vena cava after overnight recumbency. Hydralazine, 20 mg, was administered intravenously as a stimulus to renin secretion, and renal vein and inferior vena cava samples were again obtained after 15 min. In the second study, similar control samples were obtained and an additional segmental sample was collected from the venous drainage of the upper pole of the right kidney. Serum and urinary sodium and potassium were measured by internal standard flame photometry. Aldosterone excretion and secretion were measured by double isotope derivative dilution assay [8]. Plasma renin activity was determined by radioimmunoassay of angiotensin I as described previously [7,9]. Portions of the renal tumor and adjacent normal
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renal tissue were obtained at the time of surgery, rapidly frozen and stored at -20°C. Renin was extracted from 1 g portions of both tumor and normal tissue by the method of Haas et al. [lo]. Serial dilutions of the tissue extracts and standard human renal renin* were incubated for 6 hours with an excess of sheep renin substrate and with inhibitors of angiotensinase and converting enzyme; additional incubations of the same mixtures were carried out after the addition of antirenin antibody* [ll]. Aliquots were removed from the incubation mixtures before and after 2, 4 and 6 hours of incubation, and the renin content was quantitated by comparison of the angiotensin I generation rate of the tissue extracts with that of the standard human renin [9] and expressed as Goldblatt units per gram tissue. Histopathologic studies were carried out on formalin-fixed tissue. In addition, small fragments of fixed tissue were washed in 0.10 M cacodylate buffer, postfixed in 2.0 per cent osmium tetroxide veronal acetate buffer, dehydrated and embedded in araldite. Sections 1 to 2 p in size were stained with methylene blue and azure II and examined by light microscopy; selected blocks were thin-sectioned and examined by electron microscopy. RESULTS
Metabolic Studies (Table I). um diet aldosterone excretion limits (13.6 pg/24 hours) as cretion (114 pg/24 hours, N hours). Aldosterone excretion response to the administration um chloride and spironolactone
On a 120 meq sodiwas within normal was aldosterone se= 60 to 168 pg/24 increased slightly in of ACTH, potassibut, in general, re-
*Generously supplied by Dr. Erwin Haas. Cleveland, Ohio.
RENIN
ET AL.
Tissue
pole
of the
otherwise
normal
right
kidney
(Figure
1B). Grossly, the tumor was slightly mottled grey with foci of hemorrhage and was well demarcated. Microscopically, the surrounding parenchyma was compressed and atrophied, but no invasion was present. Centrally the tumor was composed in part of numerous branching tubules lined by cuboidal epithelium (Figure 3A); the major component of the tumor consisted of clusters of cells of epithelioid appearance (Figures 3A and 3B), usually defined by vascular spaces (Figure 38) and sometimes embedded in a hyalinized stroma. Some intracytoplasmic granules could be identified by periodic acid-Schiff stain after amylase digestion, thioflavin T fluorescence [12] and Bowie stain, but there was considerable variation from cell to cell, the majority having no identifiable granules using these technics. In 1 to 2 p resin-embedded sections stained with methylene blue and azure II, granules could be easily identified in most cells (Figure 4). By electron microscopy, the granules were irregular in shape and staining intensity, and an abundant rough-surfaced endoplasmic reticulum was found in the cytoplasm (Figure 5A). Smaller angular rhomboidshaped granules could also be identified (Figure
Renal Vein Renin Studies Renal Vein Renin (nWW3 W
Date 9/29/71
10/14/71 *Superior
IN HYPERTENSION-SCXAMBELAN
Assay for Renin Content. Suitable dilutions of the tissue extracts were selected to provide an angiotensin generation rate of the order 2 X 10-j Goldblatt units of standard human renal renin. Doubling of the amount of standard renin and the tissue extracts resulted in doubling of the angiotensin generation rate. Angiotensin generation remained linear over the 6 hour incubation period; adequate substrate was therefore present and recovery of added angiotensin I was 94.1 per cent, indicating adequate inhibition of converting enzyme and angiotensinase. The renin content of the tumor tissue was 47.8 Goldblatt units/g and the content for the adjacent kidney was 0.08 Goldblatt units/g. Addition of antirenin antibody to each of the incubation mixtures completely blocked generation of angiotensin. Pathologic Studies. A single cortical nodule, 2.0 cm in greatest diameter, was found in the upper
mained unchanged throughout these studies. PRA was markedly elevated with the patient supine (104 m~g/ml/3 hours, N = 3 to 11 mpg/ml/3 hours) and increased when he assumed the upright position (169 mpg/ml/3 hours, N = 5 to 32 mpg/ml/3 hours). A persistent increase in PRA was seen with postural change except after ACTH administration, but the levels remained essentially the same except for an increase during spironolactone therapy. Blood pressure decreased somewhat during spironolactone treatment, but diastolic pressure remained > 110 mm Hg. Renal Vein Renin Studies (Table II). In the first study the control specimens suggested predominant renin release from the right kidney with a renal vein renin ratio (right:left) of 1.6. Intravenous administration of hydralazine resulted in a further increase in renin levels with persistent lateralization to the right (right:left, 1.5). In the second study the control collections from the main renal veins again lateralized to the right and a segmental sample from the upper pole of the right kidney showed a further increase in renin concentration. In each instance levels in the left renal vein, caudal inferior cava and peripheral vein were comparable, suggesting suppression of renin release from the left kidney [7]. Postoperative Course (Figure 2). Large doses of hydrocortisone were administered in the immediate postoperative period and tapered to maintenance levels by the fifth postoperative day. Blood pressure fell immediately to 120/80 mm Hg, rose briefly to a maximum of 170/130 mm Hg on the second postoperative day, and then gradually fell again to 130/90 mm Hg on discharge. Plasma renin activity was CO.5 mpg/ml/3 hours on the morning after surgery and remained subnormal despite weight loss and negative sodium balance. Normal values of 6.3 (supine) and 13.5 (upright) mpg/ml/3 hours were obtained on the ninth day prior to the institution of fludrocortisone therapy. Aldosterone excretion was 3.2 pg/24 hours on the ninth postoperative day, which may have reflected the reduced adrenal mass as well as the diminished stimulation. TABLE II
AND ALDOSTERONE
Maneuver Control Hydralazine administration Control
Right
Left
Inferior Vena Cava Renin (r%&W3 hr)
79.2 103.4
48.4 67.7
57.2 58.2
55.6 ...
44.0
46.2
44.0
63.8 89.1*
Peripheral Vein Renin (m&ml/3 hr)
branch. July 1973
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I50
SERUM SODIUM
t
13~
E
mEq/L
X0 SERUM POTASSIUM 3.0
.dv
14
’ I5 ’ 16 ’
17
’ 18 ’
19
’
20
’
21
’
22
’
23
’
24
’
25.
’
26
mEq/L
,’
2. Blood pressure, serum and urinary sodium and potassium, plasma renin activity and body weight prior to and immediately after right nephrectomy. Note the subnormal plasma renin /eve/s immediately after surgery and the fall in blood pressure despite discontinuation of antihypertensive drugs. Figure
A, central portion of tumor. Tubular and “epithelioid” components Figure 3. are evident. Hematoxylin and eosin stain; original magnification X 100, reduced by 31 per cent. B, epithelioid component of tumor. Cells have abundant cytoplasm; the syncytial arrangement in clusters is defined by slit-like vascular spaces (arrows). Hematoxylin and eosin stain: original magnification X 400, reduced by 31 per cent.
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One to two micron aralFigure 4. dite section of tumor. Granules are evident in each cell although sparse in some cells; a mast cell is present (arrow). Methylene blue and azure II stain; original magnification X 800. reduced by 3 1 per cent.
RENIN
of Figure 5. Electron micrographs cytoplasm of tumor cells. A, granules of irregular size and electron density are found in cytoplasm of tumor cell. Endoplasmic reticulum is dilated and studded with ribonocleoprotein partic/es (arrow). Original magnification X 9,600, reduced by 3 1 per cent. 6, some granules are small and rhomboid in shape (center). Original magnification X 70,750, reduced by 31 Per cent. These have a crystalline parallel substructure (inset). Original magnification X 106,000, reduced by 3 1 per cent.
AND
ALDOSTERONE
IN HYPERTENSION-SCHAMBELAN
ET AL.
/“_ _.2_ ..-F.p r:,c “1 *t&4
parallel latticework 58), and a basic crystal-like was seen (inset). Often, such granules were within a membrane-bound vesicle. Microscopic examination of the normal kidney tissue disclosed some concentric medial hypertrophy of arteries and arteriolar hyalinization consistent with the changes of benign hypertension. Additionally, an occasional glomerulus had a prominent juxtaglomerular zone, but no specifically Electron microscopy of stainable granules. glomeruli from the normal kidney failed to reveal any specific granules although nonspecific granules were prominent. Examination of portions of the adrenal glands removed in 1969 and 1971 showed a prominence of the zona glomerulosa in otherwise normal glands. COMMENTS Analysis of this and the reported cases [l-6] of a renin-secreting tumor suggest a common clinical syndrome: the patients are young and present with hypertension, hypokalemia and hyperaldosteronism. These abnormalities are present in patients with primary aldosteronism, and failure to obtain renin measurements, which clearly differentiate these lesions, can lead to adrenalectomy which occurred in our case and that of Robertson et al. [I]. In our case the failure to suppress aldosterone levels with DOCA [13] contributed to the initial diagnosis of primary aldosteronism. In other types of secondary aldosteronism associated with normal or elevated PRA, such as renovascular hypertension or hypertension related to estrogen therapy, administration of DOCA results in reduction of urinary aldosterone excretion to normal levels [14]. The failure to suppress elevated aldosterone excretion in a patient with elevated PRA
may be yet another characteristic of renin-secreting tumors. The histogenesis of this type of tumor seems unique to the kidney [l]. The primary cell component appears to be derived from the granule-containing cells in the wall of the afferent arteriole, a modified smooth muscle cell. The ultrastructural characteristics of the granules are similar to those found in cells of the juxtaglomerular apparatus [15,16]. Histopathologically, this tumor partially resembles both a hemangiopericytoma [1,5] and a chemodectoma (paraganglioma or carotid body tumor), but has a tubular component as well. It, therefore, contains multiple elements of the juxtaglomerular apparatus and is thus a form of hamartoma. It is quite similar to the glomangioma, or glomus tumor, another tumor composed of multiple elements, such as nonmyelinated nerves, vessels, and pericytes derived from the Sucquet-Hoyer canal [17]. It would seem most appropriate at present to consider it as a benign tumor of juxtaglomerular apparatus [2,6], rather than solely as a hemangiopericytoma. Except for the Wilm’s tumor described by Mitchell et al. [3], in all previously recorded cases of renin-secreting tumor the pathologic picture is similar. Several lines of evidence support the conclusion that the renal tumor was responsible for the elevated renin levels. The renal vein renin studies indicate a predominant source of renin from the right kidney and furthermore suggest no net release of renin from the left kidney [7]. This conclusion is supported by the markedly subnormal renin levels measured after removal of the right kidney. The increase in renin release from the upper pole of the right kidney seen in the segmental sample and the greater than 500-fold difJuly 1973
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ference in renin content of the tumor and adjacent renal tissue further localize the source of renin within the right kidney to the tumor itself. Recently, Conn et al. [6] showed that such a tumor is capable of secreting renin when incubated in tissue culture, providing further evidence that this tumor may be a source of renin production. It appears that production of renin by the tumor was not completely autonomous. An increase in PRA levels was observed in response to upright posture and intravenous administration of hydralazine, but maneuvers known to influence sodium balance, such as sodium restriction and administration of DOCA, had little effect on renin [6], even though administration of spironolactone appeared to increase renin levels significantly. Although tubular elements were observed in the histologic sections, it seems unlikely that filtration and excretion occurred in the tumor so that macula densa function is unlikely, and the response of the tumor tissue may have been secondary to hemodynamic or sympathomimetic stimuli. The role of the renin-angiotensin system in the pathogenesis of renal hypertension remains controversial, but studies in patients with this syndrome may shed further light on the subject. Prior subtotal adrenalectomy in our patient resulted in only a transient fall in blood pressure despite reduction of aldosterone excretion to normal levels. Aldosterone production from the adrenal remnant was relatively fixed, responding slightly to direct stimulation with ACTH and potassium but showing no response to sodium restriction and DOCA ad-
minis?ration, which presumably are mediated indirectly via the renin-angiotensin system. Although the production of aldosterone may have had a contributory role, the dramatic response to nephrectomy and limited adrenal surgery suggests that the elevated levels of renin and, presumably, angiotensin II were major factors in the maintenance of hypertension. A renin-secreting tumor has only recently been recognized as a cause of hypertension. Although only seven cases have been reported to date, the true incidence of the syndrome is as yet undetermined. Published reports suggest that this diagnosis should be considered particularly in the evaluation of young hypertensive patients with hypokalemia. Plasma renin measurements are necessary to distinguish these patients from those with primary hyperaldosteronism, and the demonstration of unilateral overproduction of renin without gross arteriographic abnormality of the kidneys should alert the clinician to this diagnosis. Removal of the benign neoplasm has invariably been followed by cure of hypertension so that this diagnosis must be considered in the evaluation of any patient with severe hypertension. ACKNOWLEDGMENT We are indebted to Drs. Edward Dennis Murphy and James Anderson for referring this patient for study, to Dr. Morton Glickman who performed the renal vein renin collections and arteriographic studies, and to Drs. F. William Blaisdell and Slate Wilson who performed the nephrectomy.
REFERENCES 1.
2.
3.
4.
5. 6.
7.
8.
92
Robertson PW, Klidjian A, Harding LK, Waiters G, Lee MR, Robb-Smith AHT: Hypertension due to a reninsecreting renal tumour. Amer J Med 43: 963, 1967. Kihara I, Kitamura S, Hoshimo T, Seida H, Watanabe T: A hitherto unreported vascular tumor of the kidney. A proposal of “juxtaglomerular cell tumor.” Acta Path Jap 18: 197,1968. Mitchell JD, Baxter TJ, Blair-West JR, McCardie DA: Renin levels in nephroblastoma (Wilm’s tumour). Report of a renin-secreting tumour. Arch Dis Child 45: 376.1970. Lee MR: Renin-secreting kidney tumours. A rare but remediable cause of serious hypertension. Lancet 2: 254, 1971. Eddy RL, Sanchez SA: Renin-secreting renal neoplasm and hypertension with hypokalemia. Ann Intern Med 75: 725, 1971. Conn JW, Cohen EL, Lucas CP, McDonald WJ, Mayor GH, Blough WM Jr, Eveland WC, Bookstein JJ, Lapides J: Primary reninism. Hypertension, hyperreninemia and secondary aldosteronism due to reninproducing juxtaglomerular cell tumors. Arch Intern Med (Chicago) 130: 682,1972. Stockigt JR, Collins RD, Noakes CA, Schambelan M, Biglieri EG: Renal vein renin in various forms of renal hypertension. Lancet 1: 1194, 1972. New MI, Miller B, Peterson RE: Aldosterone excretion in normal children and in children with adrenal hyJuly 1973
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9.
10.
11.
12. 13.
14.
15.
16.
17.
perplasia. J Clin Invest 45: 412. 1966. Stockigt JR, Collins RD, Biglieri EG: Determination of plasma renin concentration using angiotensin I immunoassay. Circ Res 28 (suppl I I): I l-l 75, 1971. Haas E, Goldblatt H. Gipson EC, Lewis L: Extraction, purification, and assay of human renin free of angiotensinase. Circ Res 19: 739, 1966. Gould AB, Skeggs LT, Kahn JR: Measurement of renin and substrate concentrations in human serum. Lab Invest 15: 1802, 1966. Janigan DT: Fluorochrome staining of juxtaglomerular cell granules. Arch Path 79: 370. 1965. Biglieri EG, Slaton PE Jr, Kronfield SJ, Schambelan M: Diagnosis of an aldosterone-producing adenoma in primary aldosteronism: an evaluative maneuver. JAMA 201: 510,1967. Biglieri EG, Schambelan M, Stockigt JR: Hypertension and hyperaldosteronism: discriminating value of deoxycorticosterone acetate (DOCA) testing. Clin Res 20: 163, 1972. Barajas L: The development and ultrastructure of the juxtaglomerular cell granule. J Ultrastruct Res 15: 400, 1966. Biava CG, West M: Fine structure of normal human juxtaglomerular cells. I I. Specific and nonspecific cytoplasmic granules. Amer J Path 49: 955, 1966. Evans RW: Histological Appearances of Tumours, Baltimore, Williams & Wilkins Co., 1968, p 98.
M. SCHAMBELAN,
M.D.
E. L. HOWES, Jr.,
M.D.
J. R. STOCKIGT,
M.D.*
C. A. NOAKES,
M.D.
E. G. BIGLIERI, San Francisco.
M.D. California
From the Medical and Anatomic Pathology Services, San Francisco General Hospital, and the Departments of Medicine and Pathology, University of California, San Francisco, California. This study was supported by U.S. Public Health Service Research Grants HE-l 1046 from the National Heart Institute, AM-06415 from the National Institute of Arthritis and Metabolic Diseases, and National Institutes of Health Grant EY-00056 (E.L.H.). The studies were carried out in the General Clinical Research Center, RR-83, at San Francisco General Hospital, supported by Division of Research Resources, National Institutes of Health. They were presented in part at the 4th International Congress of Endocrinology, Washington, D.C., June 1972. Requests for reprints should be addressed to Dr. Morris Schambelan, Clinical Study Center, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, California 94110. Manuscript accepted January 9, 1973. *Present address: Donnie Metabolic Unit, Alfred Hospital, Melbourne, Australia 3181.
86
July 1973
A renin-secreting renal tumor is a rare cause of curable hypertension characterized clinically by hypertension, hypokalemia, hyperaldosteronism and increased plasma renin activity (PRA). Prior subtotal adrenalectomy in such a patient allowed examination of the role of hyperreninemia in the absence of excessive aldosterone secretion. Hypertension was severe, but serum potassium and aldosterone production were normal. PRA was markedly increased under all conditions studied, with a persistent increase noted with changes in posture. Renal vein renin studies were critical in localizing the renal tumor, and unilateral nephrectomy resulted in a prompt fall in PRA to subnormal levels and dramatic cure of the hypertension. Renin content of the renal tumor was more than 500 times higher than in the adjacent kidney, and histologic studies confirmed that the tumor was of juxtaglomerular cell origin. This case demonstrates that severe hypertension can occur in this syndrome despite subtotal adrenalectomy and further illustrates an association of renin overproduction with a specific type of hypertension. In several reports a syndrome of hypertension, hypokalemia, hyperaldosteronism and increased plasma renin activity (PRA) has been described in association with a renin-secreting renal tumor [l-6]. The syndrome is completely reversed by removal of the benign neoplasm, and although the incidence of such a ttimor is unknown, it represents a definite cause of surgically curable hypertension. We describe an additional patient in whom removal of a renin-secreting renal tumor resulted in a rapid and sustained fall in blood pressure to normal levels. Prior subtotal adrenalectomy had reduced aldosterone production to normal levels, but severe hypertension had persisted. Determination of PRA and studies of renal vein renin were critical in making the correct diagnosis. This case demonstrates that severe hypertension can occur in this syndrome without excessive aldosterone secretion and offers further presumptive evidence that gross inappropriate renin overproduction and hypertension are closely associated in such cases. CASE REPORT Hypertension was first noted in this 24 year old white man in December 1966 (160/120 mm Hg); his blood pressure had been within normal limits in 1965. The medical history was unremarkable except for
The American Journal of Medicine
Volume 55
RENlN
AND ALD~STERONE
IN HYPERTENSION-SCHAMBELAN
ET AL.
Figure 1. A, nephrogram phase from selective right renal arteriogram showing decreased perfusion of an area in the mid upper pole (arrows). 13, right kidney showing a well demarcated nodule in the upper pole. Note hemorrhagic appearance on cut section. enuresis during childhood and lifelong polydypsia and nocturia. Blood urea nitrogen and creatinine were within normal limits, and no abnormalities were detected on the intravenous pyelogram, the radioisotopic renogram and the renal arteriogram. Excretion of vanillylmandelic acid was 8.7 mg/24 hours (normal range 0 to 10 mg), and a phentolamine test was negative. Serum sodium was 141, potassium 3.0, chloride 96 and bicarbonate 22 meq/liter. Urinary protein excretion was 376 mg/24 hours (normal range cl.50 mg). A biopsy specimen of the left kidney showed only mild nephrosclerosis. Blood pressure was controlled with guanethidine and chlorothiazide. In 1968, guanethidine therapy was discontinued because of side effects, and control of blood pressure with alpha-methyldopa was unsatisfactory (170/l 30 mm Hg). Creatinine clearance was 119 ml/min and a rapid sequence intravenous pyelogram was negative. Serum sodium was 140, potassium 2.8, chloride 96 and bicarbonate 35 meq/liter. Aldosterone excretion was 40.8 pg/24 hours (normal range 4 to 17 pg) under control conditions and 36.1 pg/24 hours on the third day of administration of deoxycorticosterone acetate (DOCA), 10 mg intramuscularly every 12 hours. A presumptive diagnosis of primary aldosteronism was made although PRA was not measured. At surgery the adrenal glands appeared normal, and the right (4.8 g) and two thirds of the left adrenal gland (3.4 g) were removed. Microscopic examination revealed no abnormalities. One week postoperatively urinary aldosterone was 6.1 pg/24 hours and 17-hydroxycorticoids 2.9 mg/24 hours. Stimulation with adrenocorticotropin (ACTH) did not increase 17-hydroxycorticoid excretion, and the patient was maintained on oral hydrocortisone therapy, 25 mg every morning and 10 mg every evening. Blood pressure was somewhat lower postoperatively, but was 160/140 mm Hg 1 year later and remained elevated despite treatment with alphamethyldopa, hydrochlorothiazide and spironolactone. On referral to the Clinical Study Center in 1971,
blood pressure was 190/136 mm Hg supine and 160/ 130 mm Hg upright. Physical examination revealed no abnormalities other than mild retinal arteriolar narrowing without hemorrhages or exudates. The hemoglobin level was 17.6 g/100 ml and white blood cell count 11,600/mm3. Blood urea nitrogen was 9, creatinine 1.1, fasting blood sugar 109 and uric acid 4.5 mg/lOO ml. Serum sodium was 141, potassium 3.9, chloride 102 and bicarbonate 26 meq/liter. Creatinine clearance was 121 ml/min, and urinalysis revealed no abnormalities except trace protein. Renal vein renin collections indicated markedly elevated levels of PRA with an apparent source in the right kidney (see “Results”) and, although a midstream renal arteriogram failed to show a definite lesion, a selective right renal arteriogram identified a filling defect in the upper pole of the right kidney (Figure IA). On October 14, 1971, the right kidney and a portion of the left adrenal remnant were removed. Postoperatively, the blood pressure fell immediately to normal levels and remained so except for a transient elevation. On discharge, the blood pressure was 130/90 mm Hg while the patient was on a regimen consisting of only hydrocortisone, 20 mg twice a day, and fludrocortisone acetate, 0.1 mg daily. Eight months postoperatively blood pressure was 125/80 mm Hg without antihypertensive medication, and reduction of fludrocortisone to 0.05 mg/day maintained normal serum electrolyte levels. METHODS Studies were performed while the patient was on a constant diet containing 20 meq sodium and 64 meq potassium daily to which sodium chloride, 6 g, was added daily in the form of weighed capsules. Dexamethasone, 0.5 mg every 12 hours, was given as maintenance glucocorticoid therapy, and antihypertensive therapy with alpha-methyldopa, 500 mg orally 4 times daily, was continued. Aldosterone excretion and PRA were measured in the control period and at the July 1973
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TABLE ___
AND ALDOSTERONE
IN HYPERTENSION-SCHAMBELAN
ET At
Studies in 1971Following Subtotal Adrenalectomy I __~
_-________-
Serum(meq/liter) Maneuver
Potassium
Sodium Inpatient
Control Low sodium diet Control DOCA administration Potassium chloride administration Control ACTH administration Day 1 Day 2 Day 3
3.9 3.8 3.7
... ...
. . ...
134
3.2
3.8 4.2 3.6
Aldosterone Excretion kg/24 hr)
104 81 70 46 59 109
169 183 148 121 128 174
13.6 15.8 14.5 14.6 22.0 13.6
133 94 104
... . . 98
20.1 20.1 19.6
82 176
185 236
18.4 23.2
3-11
5-32
4-17
Studies
134 127 Normal
hr)
Studies
141 134 132 133 136 135
Outpatient
Dexamethasone administration Spironolactone administration
PlasmaReninActivity (m&ml/3 ---Supine Upright
4.7 4.7 Ranges*
* On 120 meq sodium intake. completion of the following maneuvers: (1) sodium restriction for 3 days, effected by removing the sodium chloride capsules from the regimen; (2) administration of DOCA, 10 mg intramuscularly every 12 hours for 3 days; (3) administration of potassium chloride, 1.0 g 4 times daily for 3 days; and (4) administration of ACTH, 25 units intravenously daily as an 8 hour infusion for 3 days. In subsequent studies dexamethasone, 0.5 mg, was administered every 6 hours and spironolactone, 100 mg, 3 times daily for 2 and 3 weeks, respectively, in separate periods on an outpatient basis; at the completion of each period the patient was admitted so that specimens could be collected for aldosterone and renin determinations. Plasma specimens for the determination of renin were collected with the patient in the supine position after overnight recumbency and again after 4 hours of upright activity. Renal vein specimens for the determination of renin were collected as described previously [7]. In the first study, samples were obtained from a peripheral vein, the right and left renal veins, and the caudal inferior vena cava after overnight recumbency. Hydralazine, 20 mg, was administered intravenously as a stimulus to renin secretion, and renal vein and inferior vena cava samples were again obtained after 15 min. In the second study, similar control samples were obtained and an additional segmental sample was collected from the venous drainage of the upper pole of the right kidney. Serum and urinary sodium and potassium were measured by internal standard flame photometry. Aldosterone excretion and secretion were measured by double isotope derivative dilution assay [8]. Plasma renin activity was determined by radioimmunoassay of angiotensin I as described previously [7,9]. Portions of the renal tumor and adjacent normal
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renal tissue were obtained at the time of surgery, rapidly frozen and stored at -20°C. Renin was extracted from 1 g portions of both tumor and normal tissue by the method of Haas et al. [lo]. Serial dilutions of the tissue extracts and standard human renal renin* were incubated for 6 hours with an excess of sheep renin substrate and with inhibitors of angiotensinase and converting enzyme; additional incubations of the same mixtures were carried out after the addition of antirenin antibody* [ll]. Aliquots were removed from the incubation mixtures before and after 2, 4 and 6 hours of incubation, and the renin content was quantitated by comparison of the angiotensin I generation rate of the tissue extracts with that of the standard human renin [9] and expressed as Goldblatt units per gram tissue. Histopathologic studies were carried out on formalin-fixed tissue. In addition, small fragments of fixed tissue were washed in 0.10 M cacodylate buffer, postfixed in 2.0 per cent osmium tetroxide veronal acetate buffer, dehydrated and embedded in araldite. Sections 1 to 2 p in size were stained with methylene blue and azure II and examined by light microscopy; selected blocks were thin-sectioned and examined by electron microscopy. RESULTS
Metabolic Studies (Table I). um diet aldosterone excretion limits (13.6 pg/24 hours) as cretion (114 pg/24 hours, N hours). Aldosterone excretion response to the administration um chloride and spironolactone
On a 120 meq sodiwas within normal was aldosterone se= 60 to 168 pg/24 increased slightly in of ACTH, potassibut, in general, re-
*Generously supplied by Dr. Erwin Haas. Cleveland, Ohio.
RENIN
ET AL.
Tissue
pole
of the
otherwise
normal
right
kidney
(Figure
1B). Grossly, the tumor was slightly mottled grey with foci of hemorrhage and was well demarcated. Microscopically, the surrounding parenchyma was compressed and atrophied, but no invasion was present. Centrally the tumor was composed in part of numerous branching tubules lined by cuboidal epithelium (Figure 3A); the major component of the tumor consisted of clusters of cells of epithelioid appearance (Figures 3A and 3B), usually defined by vascular spaces (Figure 38) and sometimes embedded in a hyalinized stroma. Some intracytoplasmic granules could be identified by periodic acid-Schiff stain after amylase digestion, thioflavin T fluorescence [12] and Bowie stain, but there was considerable variation from cell to cell, the majority having no identifiable granules using these technics. In 1 to 2 p resin-embedded sections stained with methylene blue and azure II, granules could be easily identified in most cells (Figure 4). By electron microscopy, the granules were irregular in shape and staining intensity, and an abundant rough-surfaced endoplasmic reticulum was found in the cytoplasm (Figure 5A). Smaller angular rhomboidshaped granules could also be identified (Figure
Renal Vein Renin Studies Renal Vein Renin (nWW3 W
Date 9/29/71
10/14/71 *Superior
IN HYPERTENSION-SCXAMBELAN
Assay for Renin Content. Suitable dilutions of the tissue extracts were selected to provide an angiotensin generation rate of the order 2 X 10-j Goldblatt units of standard human renal renin. Doubling of the amount of standard renin and the tissue extracts resulted in doubling of the angiotensin generation rate. Angiotensin generation remained linear over the 6 hour incubation period; adequate substrate was therefore present and recovery of added angiotensin I was 94.1 per cent, indicating adequate inhibition of converting enzyme and angiotensinase. The renin content of the tumor tissue was 47.8 Goldblatt units/g and the content for the adjacent kidney was 0.08 Goldblatt units/g. Addition of antirenin antibody to each of the incubation mixtures completely blocked generation of angiotensin. Pathologic Studies. A single cortical nodule, 2.0 cm in greatest diameter, was found in the upper
mained unchanged throughout these studies. PRA was markedly elevated with the patient supine (104 m~g/ml/3 hours, N = 3 to 11 mpg/ml/3 hours) and increased when he assumed the upright position (169 mpg/ml/3 hours, N = 5 to 32 mpg/ml/3 hours). A persistent increase in PRA was seen with postural change except after ACTH administration, but the levels remained essentially the same except for an increase during spironolactone therapy. Blood pressure decreased somewhat during spironolactone treatment, but diastolic pressure remained > 110 mm Hg. Renal Vein Renin Studies (Table II). In the first study the control specimens suggested predominant renin release from the right kidney with a renal vein renin ratio (right:left) of 1.6. Intravenous administration of hydralazine resulted in a further increase in renin levels with persistent lateralization to the right (right:left, 1.5). In the second study the control collections from the main renal veins again lateralized to the right and a segmental sample from the upper pole of the right kidney showed a further increase in renin concentration. In each instance levels in the left renal vein, caudal inferior cava and peripheral vein were comparable, suggesting suppression of renin release from the left kidney [7]. Postoperative Course (Figure 2). Large doses of hydrocortisone were administered in the immediate postoperative period and tapered to maintenance levels by the fifth postoperative day. Blood pressure fell immediately to 120/80 mm Hg, rose briefly to a maximum of 170/130 mm Hg on the second postoperative day, and then gradually fell again to 130/90 mm Hg on discharge. Plasma renin activity was CO.5 mpg/ml/3 hours on the morning after surgery and remained subnormal despite weight loss and negative sodium balance. Normal values of 6.3 (supine) and 13.5 (upright) mpg/ml/3 hours were obtained on the ninth day prior to the institution of fludrocortisone therapy. Aldosterone excretion was 3.2 pg/24 hours on the ninth postoperative day, which may have reflected the reduced adrenal mass as well as the diminished stimulation. TABLE II
AND ALDOSTERONE
Maneuver Control Hydralazine administration Control
Right
Left
Inferior Vena Cava Renin (r%&W3 hr)
79.2 103.4
48.4 67.7
57.2 58.2
55.6 ...
44.0
46.2
44.0
63.8 89.1*
Peripheral Vein Renin (m&ml/3 hr)
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ET AL.
I50
SERUM SODIUM
t
13~
E
mEq/L
X0 SERUM POTASSIUM 3.0
.dv
14
’ I5 ’ 16 ’
17
’ 18 ’
19
’
20
’
21
’
22
’
23
’
24
’
25.
’
26
mEq/L
,’
2. Blood pressure, serum and urinary sodium and potassium, plasma renin activity and body weight prior to and immediately after right nephrectomy. Note the subnormal plasma renin /eve/s immediately after surgery and the fall in blood pressure despite discontinuation of antihypertensive drugs. Figure
A, central portion of tumor. Tubular and “epithelioid” components Figure 3. are evident. Hematoxylin and eosin stain; original magnification X 100, reduced by 31 per cent. B, epithelioid component of tumor. Cells have abundant cytoplasm; the syncytial arrangement in clusters is defined by slit-like vascular spaces (arrows). Hematoxylin and eosin stain: original magnification X 400, reduced by 31 per cent.
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One to two micron aralFigure 4. dite section of tumor. Granules are evident in each cell although sparse in some cells; a mast cell is present (arrow). Methylene blue and azure II stain; original magnification X 800. reduced by 3 1 per cent.
RENIN
of Figure 5. Electron micrographs cytoplasm of tumor cells. A, granules of irregular size and electron density are found in cytoplasm of tumor cell. Endoplasmic reticulum is dilated and studded with ribonocleoprotein partic/es (arrow). Original magnification X 9,600, reduced by 3 1 per cent. 6, some granules are small and rhomboid in shape (center). Original magnification X 70,750, reduced by 31 Per cent. These have a crystalline parallel substructure (inset). Original magnification X 106,000, reduced by 3 1 per cent.
AND
ALDOSTERONE
IN HYPERTENSION-SCHAMBELAN
ET AL.
/“_ _.2_ ..-F.p r:,c “1 *t&4
parallel latticework 58), and a basic crystal-like was seen (inset). Often, such granules were within a membrane-bound vesicle. Microscopic examination of the normal kidney tissue disclosed some concentric medial hypertrophy of arteries and arteriolar hyalinization consistent with the changes of benign hypertension. Additionally, an occasional glomerulus had a prominent juxtaglomerular zone, but no specifically Electron microscopy of stainable granules. glomeruli from the normal kidney failed to reveal any specific granules although nonspecific granules were prominent. Examination of portions of the adrenal glands removed in 1969 and 1971 showed a prominence of the zona glomerulosa in otherwise normal glands. COMMENTS Analysis of this and the reported cases [l-6] of a renin-secreting tumor suggest a common clinical syndrome: the patients are young and present with hypertension, hypokalemia and hyperaldosteronism. These abnormalities are present in patients with primary aldosteronism, and failure to obtain renin measurements, which clearly differentiate these lesions, can lead to adrenalectomy which occurred in our case and that of Robertson et al. [I]. In our case the failure to suppress aldosterone levels with DOCA [13] contributed to the initial diagnosis of primary aldosteronism. In other types of secondary aldosteronism associated with normal or elevated PRA, such as renovascular hypertension or hypertension related to estrogen therapy, administration of DOCA results in reduction of urinary aldosterone excretion to normal levels [14]. The failure to suppress elevated aldosterone excretion in a patient with elevated PRA
may be yet another characteristic of renin-secreting tumors. The histogenesis of this type of tumor seems unique to the kidney [l]. The primary cell component appears to be derived from the granule-containing cells in the wall of the afferent arteriole, a modified smooth muscle cell. The ultrastructural characteristics of the granules are similar to those found in cells of the juxtaglomerular apparatus [15,16]. Histopathologically, this tumor partially resembles both a hemangiopericytoma [1,5] and a chemodectoma (paraganglioma or carotid body tumor), but has a tubular component as well. It, therefore, contains multiple elements of the juxtaglomerular apparatus and is thus a form of hamartoma. It is quite similar to the glomangioma, or glomus tumor, another tumor composed of multiple elements, such as nonmyelinated nerves, vessels, and pericytes derived from the Sucquet-Hoyer canal [17]. It would seem most appropriate at present to consider it as a benign tumor of juxtaglomerular apparatus [2,6], rather than solely as a hemangiopericytoma. Except for the Wilm’s tumor described by Mitchell et al. [3], in all previously recorded cases of renin-secreting tumor the pathologic picture is similar. Several lines of evidence support the conclusion that the renal tumor was responsible for the elevated renin levels. The renal vein renin studies indicate a predominant source of renin from the right kidney and furthermore suggest no net release of renin from the left kidney [7]. This conclusion is supported by the markedly subnormal renin levels measured after removal of the right kidney. The increase in renin release from the upper pole of the right kidney seen in the segmental sample and the greater than 500-fold difJuly 1973
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ference in renin content of the tumor and adjacent renal tissue further localize the source of renin within the right kidney to the tumor itself. Recently, Conn et al. [6] showed that such a tumor is capable of secreting renin when incubated in tissue culture, providing further evidence that this tumor may be a source of renin production. It appears that production of renin by the tumor was not completely autonomous. An increase in PRA levels was observed in response to upright posture and intravenous administration of hydralazine, but maneuvers known to influence sodium balance, such as sodium restriction and administration of DOCA, had little effect on renin [6], even though administration of spironolactone appeared to increase renin levels significantly. Although tubular elements were observed in the histologic sections, it seems unlikely that filtration and excretion occurred in the tumor so that macula densa function is unlikely, and the response of the tumor tissue may have been secondary to hemodynamic or sympathomimetic stimuli. The role of the renin-angiotensin system in the pathogenesis of renal hypertension remains controversial, but studies in patients with this syndrome may shed further light on the subject. Prior subtotal adrenalectomy in our patient resulted in only a transient fall in blood pressure despite reduction of aldosterone excretion to normal levels. Aldosterone production from the adrenal remnant was relatively fixed, responding slightly to direct stimulation with ACTH and potassium but showing no response to sodium restriction and DOCA ad-
minis?ration, which presumably are mediated indirectly via the renin-angiotensin system. Although the production of aldosterone may have had a contributory role, the dramatic response to nephrectomy and limited adrenal surgery suggests that the elevated levels of renin and, presumably, angiotensin II were major factors in the maintenance of hypertension. A renin-secreting tumor has only recently been recognized as a cause of hypertension. Although only seven cases have been reported to date, the true incidence of the syndrome is as yet undetermined. Published reports suggest that this diagnosis should be considered particularly in the evaluation of young hypertensive patients with hypokalemia. Plasma renin measurements are necessary to distinguish these patients from those with primary hyperaldosteronism, and the demonstration of unilateral overproduction of renin without gross arteriographic abnormality of the kidneys should alert the clinician to this diagnosis. Removal of the benign neoplasm has invariably been followed by cure of hypertension so that this diagnosis must be considered in the evaluation of any patient with severe hypertension. ACKNOWLEDGMENT We are indebted to Drs. Edward Dennis Murphy and James Anderson for referring this patient for study, to Dr. Morton Glickman who performed the renal vein renin collections and arteriographic studies, and to Drs. F. William Blaisdell and Slate Wilson who performed the nephrectomy.
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Robertson PW, Klidjian A, Harding LK, Waiters G, Lee MR, Robb-Smith AHT: Hypertension due to a reninsecreting renal tumour. Amer J Med 43: 963, 1967. Kihara I, Kitamura S, Hoshimo T, Seida H, Watanabe T: A hitherto unreported vascular tumor of the kidney. A proposal of “juxtaglomerular cell tumor.” Acta Path Jap 18: 197,1968. Mitchell JD, Baxter TJ, Blair-West JR, McCardie DA: Renin levels in nephroblastoma (Wilm’s tumour). Report of a renin-secreting tumour. Arch Dis Child 45: 376.1970. Lee MR: Renin-secreting kidney tumours. A rare but remediable cause of serious hypertension. Lancet 2: 254, 1971. Eddy RL, Sanchez SA: Renin-secreting renal neoplasm and hypertension with hypokalemia. Ann Intern Med 75: 725, 1971. Conn JW, Cohen EL, Lucas CP, McDonald WJ, Mayor GH, Blough WM Jr, Eveland WC, Bookstein JJ, Lapides J: Primary reninism. Hypertension, hyperreninemia and secondary aldosteronism due to reninproducing juxtaglomerular cell tumors. Arch Intern Med (Chicago) 130: 682,1972. Stockigt JR, Collins RD, Noakes CA, Schambelan M, Biglieri EG: Renal vein renin in various forms of renal hypertension. Lancet 1: 1194, 1972. New MI, Miller B, Peterson RE: Aldosterone excretion in normal children and in children with adrenal hyJuly 1973
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perplasia. J Clin Invest 45: 412. 1966. Stockigt JR, Collins RD, Biglieri EG: Determination of plasma renin concentration using angiotensin I immunoassay. Circ Res 28 (suppl I I): I l-l 75, 1971. Haas E, Goldblatt H. Gipson EC, Lewis L: Extraction, purification, and assay of human renin free of angiotensinase. Circ Res 19: 739, 1966. Gould AB, Skeggs LT, Kahn JR: Measurement of renin and substrate concentrations in human serum. Lab Invest 15: 1802, 1966. Janigan DT: Fluorochrome staining of juxtaglomerular cell granules. Arch Path 79: 370. 1965. Biglieri EG, Slaton PE Jr, Kronfield SJ, Schambelan M: Diagnosis of an aldosterone-producing adenoma in primary aldosteronism: an evaluative maneuver. JAMA 201: 510,1967. Biglieri EG, Schambelan M, Stockigt JR: Hypertension and hyperaldosteronism: discriminating value of deoxycorticosterone acetate (DOCA) testing. Clin Res 20: 163, 1972. Barajas L: The development and ultrastructure of the juxtaglomerular cell granule. J Ultrastruct Res 15: 400, 1966. Biava CG, West M: Fine structure of normal human juxtaglomerular cells. I I. Specific and nonspecific cytoplasmic granules. Amer J Path 49: 955, 1966. Evans RW: Histological Appearances of Tumours, Baltimore, Williams & Wilkins Co., 1968, p 98.