Renin Gene Expression in the Adrenal and Kidney of Patients with Primary Aldosteronism*
HIROSHI SHIONOIRI, NOBUHITO HIRAWA, SHIN-ICHIRO UEDA, HIDEO HIMENO, EIJI GOTOH, KAZUMI NOGUCHI, AKIYOSHI FUKAMIZU, MIN SEOK SEO, AND KAZUO MURAKAMI
Second Department of Internal Medicine and Department of Urology (K.N.), Yokoharta City University School of Medicine, and the Institute of Applied Biochemistry, University of Tsukuba (A.F., M.S.S., K.M.), Yokohama 236, Japan
ABSTRACT. mRNA levels for renin in the adrenal gland and kidney were measured by ribonuclease protection assay (RPA). Renin mRNA was not detected by RPA in aldosteronoma and kidney tissues obtained from two patients with primary aldos- teronism (PA). In these patients, the PRA values, plasma con- centrations of active renin (ARC), and total renin (TRC = ARC + prorenin) were below the assay limit (<0.03 ng/L . s, 2.5 ng/L, and 10 ng/L, respectively). On the other hand, renin mRNA was recognized by RPA in aldosteronoma and kidney tissues ob- tained from two other patients with PA treated with 50 mg/day spironolactone for more than 2 months. Their TRC values were 49.8 and 16.6 ng/L, but their PRA and ARC were undetectable.
Renin mRNA content was greater in normal adrenocortical tissue and in the normal kidneys obtained from three hyperten- sive patients with renal cell carcinoma. In these patients, the mean values of PRA, ARC, and TRC were 0.28 ± 0.03 (mean ± SD) ng/L.s, 18.4 ± 7.8 ng/L, and 110 ± 15 ng/L, respectively.
This is the first report of the lack of renin gene expression in aldosteronoma and kidney tissues obtained from untreated pa- tients with PA. Furthermore, treatment with spironolactone resulted in an increase in the levels of renin mRNA in the aldosteronoma and kidney tissues of patients with PA. (J Clin Endocrinol Metab 74: 103-107, 1992)
R ENIN (EC 3.4.23.15), which was first identified as a pressor substance extracted from renal cortex, catalyzes the rate-limiting step in the formation of phys- iologically active peptide angiotensin-II (AII). AII stim- ulates the biosynthesis of aldosterone, and both AII and aldosterone contribute to cardiovascular homeostasis by causing arteriolar vasoconstriction and increased sodium balance. Recently, a role for the local renin-angiotensin system has been proposed based on the identification of renin mRNA in extrarenal tissues, including adrenal, brain, cultured vascular smooth muscle cells, cardiac atria and ventricles, ovary, pituitary, submandibular gland, testis, and uterus (1-4). Moreover, sensitive spe-
Received March 25, 1991.
Address all correspondence and requests for reprints to: Dr. Hiroshi Shionoiri, Second Department of Internal Medicine, Yokohama City, University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
* This work was supported in part by grants for Joint Research (63044118 and 01044120); the International Scientific Research Pro- gram from the Ministry of Education, Science, and Culture (Japan); the Kihara Memorial Yokohama Foundation for the Advancement of Life Science (Japan); Yokohama City for Special Research Program (Japan); and an award from the Yokohama City University Medical Association (Japan). Part of the material in this paper was presented at the 63rd Annual Scientific Sessions of the American Heart Associ- ation (November 1990, Dallas, TX), and the abstract of this article appeared in Circulation [Suppl 3] 82:III-553, 1990.
cific techniques of ribonuclease protection assay (RPA) have demonstrated renin in the adrenal gland of the rat (5, 6) and mouse (7).
Little is known of the renin mRNA in the human adrenal gland because of the limited availability of hu- man material. Renin activity in plasma has been shown to be suppressed in patients with primary aldosteronism (PA). Excessive secretion of aldosterone from adrenal tumor is a major cause of hypertension by causing sodium retention and volume expansion. These effects of aldo- sterone may be responsible for the suppression of renin secretion. On the other hand, the presence of enzymatic renin activity has been reported in the aldosterone- producing adrenal adenoma (aldosteronoma) (8-10).
In the present study we investigated renin gene expres- sion by sensitive RPA in aldosteronoma and kidney tissues obtained from patients with PA and in normal adrenocortical and kidney tissues obtained from patients with renal cell carcinoma.
Subjects and Methods
Informed consent was obtained from all subjects after full explanation of the study. The subjects were seven hypertensive patients who were hospitalized and received a constant sodium diet (7g NaCl/day) for at least 7 days. This is a mildly restricted
sodium diet at Yokohama City University Hospital. In each patient, systolic pressure was 150 mm Hg or more, and diastolic pressure was 90 mm Hg or more, but no patient showed severe hypertension (Table 1). Four of seven patients were diagnosed as having PA caused by adrenal adenoma by endocrinological and radiological examinations, and the diagnosis was confirmed surgically. The remaining three had renal cell carcinoma. Under full agreement, five of seven subjects did not receive any anti- hypertensive drugs or spironolactone for at least 2 weeks before their surgery in order to exclude the effects of the drugs on renin gene expression. Two of four patients with PA continu- ously received 50 mg/day spironolactone, orally, for more than 2 months.
Blood samples for the determination of PRA and the con- centrations of active renin (ARC), total renin (TRC; ARC plus prorenin), and aldosterone (PAC) were drawn before and 60 min after oral administration of 25 mg captopril in the morning while the patient was in the supine position and fasting, and each plasma sample was kept at -20 C until assay. PRA and PAC were assessed by RIA (11, 12). ARC and TRC were separately measured by direct RIAs using antihuman renin monoclonal antibodies, as reported previously (13, 14).
The clinical characteristics of the patients and plasma levels of PRA, ARC, TRC, PAC, and potassium are summarized in Table 1.
Adrenal and kidney tissue
To measure the steady state mRNA level for renin, aldoster- onoma tissues and open biopsied kidney tissues were obtained from the patients with PA under general anesthesia for adre- nalectomy. The ischemic period of the adrenal gland for adre- nalectomy was less than 10 min. Normal adrenocortical and kidney tissues were obtained from the patients with renal cell carcinoma during radical nephrectomy. The ischemic period of the en bloc radical nephrectomy including the adrenal gland was less than 15 min. Each tissue was immediately frozen with liquid nitrogen and stored separately at -80 C.
RNA preparation and RNase protection analysis
Each frozen aldosteronoma, adrenal gland, or kidney tissue sample was homogenized to a fine powder under liquid nitrogen,
and the total cellular RNA from each tissue was prepared by the guanidinium thiocyanate method (15). Detection of renin mRNA was performed by RPA using a human renin cRNA probe, prepared as illustrated in Fig. 1. A 444-basepair KpnI- BamHI fragment containing the human renin gene transcrip- tion initiation site (16, 17) was subcloned into pGEM4Z vector to generate pGEMHRn 444. This plasmid was linearized with EcoRI and used as a template to make the cRNA probe. The RPA was performed by hybridizing total RNA obtained from the adrenal or the kidney tissues at 60 C for 12 h to the 32P- labeled cRNA probe, which was prepared by transcription in vitro in the presence of T7 RNA polymerase and [32P]CTP, and then the RNAs were digested with single strand-specific RNase-A and -T1, as described by Krieg and Melton (18). The RNAs protected against RNase digestion were extracted with phenol-chloroform, precipitated using tRNA as a carrier, and electrophoresed on a 5% polyacrylamide gel containing 8 M urea. After electrophoresis to remove the urea, the gel was soaked in a solution containing 10% acetic acid for 30 min. Then, the gel was dried and exposed to x-ray film at -70 C for 120 h.
Results
Basal values of PRA and ARC in the two patients with PA without medication were undetectable (<0.03 ng/L· s and 2.5 ng/L, respectively), and the basal values of TRC were 10.0 and 8.9 ng/L (Table 1). In the other two patients with PA treated with spironolactone, PRA and ARC were undetectable, but their basal values of TRC were 49.8 and 16.6 ng/L. In the patients with PA, PRA, ARC, and TRC did not increase after a single dose of 25 mg captopril. On the other hand, in the patients with renal cell carcinoma, PRA, ARC, and TRC increased after captopril administration, from 0.28 ± 0.03 (mean ± SD) to 0.64 ± 0.42 ng/L.s, 18.4 ± 7.8 to 28.2 ± 7.1 ng/ L, and 110 ± 15.0 to 136 ± 15.1 ng/L, respectively. Basal PAC values in the patients with PA were significantly higher than those in the patients with renal cell carci- noma (Table 1).
RPA was performed on RNAs prepared from aldoster-
| Renal cell carcinoma | PA | ||||||
|---|---|---|---|---|---|---|---|
| Without spironolactone | With spironolactone | ||||||
| Case | A | B | C | D | E | F | G |
| Age (yr)/sex | 63/M | 54/M | 48/F | 39/F | 65/F | 52/M | 45/F |
| SBP (mm Hg) | 158 | 154 | 160 | 160 | 168 | 164 | 166 |
| DBP (mm Hg) | 96 | 92 | 95 | 96 | 100 | 98 | 102 |
| Cr (umol/L) | 97 | 106 | 80 | 71 | 97 | 106 | 80 |
| PRA (ng/L·s) | 0.28 | 0.33 | 0.25 | <0.03 | <0.03 | <0.03 | <0.03 |
| ARC (ng/L) | 13.5 | 29.4 | 12.3 | <2.5 | <2.5 | <2.5 | <2.5 |
| TRC (ng/L) | 106 | 130 | 94 | 10.0 | 8.9 | 49.8 | 16.6 |
| PAC (pmol/L) | 277 | 416 | 361 | 1165 | 1082 | 915 | 1054 |
| K (mmol/L) | 4.4 | 4.3 | 4.1 | 2.9 | 3.0 | 3.0 | 2.8 |
SBP, Cystolic blood pressure, DBP, diastolic blood pressure; Cr, creatinine.
Strategy for RNase Protection Assay
EcoRI Kpnl
BamHI
HRn
T7
cRNA probe : 51 Ont
Human renin mRNA
RNase A and T1
Double strand
300 nt
Electrophoresis
510nt-+
Full length probe
300nt -
Protected fragment
onoma, normal adrenocortex, and kidney using human renin cRNA as a probe. The results are shown in Figs. 2 and 3. The presence of renin mRNA is indicated by the detection of a protected fragment with a single band, 300 nucleotides in length. Renin mRNA was most abundant in the kidney tissues obtained from the patients with renal cell carcinoma (in lanes 6 and 9 of Fig. 2, and in lanes 3 and 8 of Fig. 3). No protected fragment of renin mRNA was detected in the kidney tissues biopsied from the two patients with PA without medication (in lanes 7 and 9 of Fig. 3) when same amount (50 µg) of total RNA was applied for the assay. In contrast, renin mRNA was detected in the kidney tissues biopsied from the other two patients with PA treated with spironolactone (in lanes 5 and 6 of Fig. 3). Fairly small amounts of renin mRNA were detected in normal adrenocortical tissues obtained from the patients with renal cell carcinoma (in lanes 4 and 7 of Fig. 2). Although we have consistently detected undegraded total RNA in the aldosteronoma tissues by ethidium bromide staining, no protected frag- ment of renin mRNA was detected in the aldosteronoma tissues obtained from the patients with PA without med- ication (in lanes 5 and 8 of Fig. 2). On the other hand, a small amount of protected fragment of renin mRNA was detected in the aldosteronoma tissue obtained from one
Marker track
tRNA
· cRNA probe
Adrenal(N)
1 Adrenal(PA) ® Kidney(N)
Adrenal(N)
co Adrenal(PA)
« Kidney(N)
Lane
Base-pair
1057
770
612
493
510nt
392
345/341/ 35
297/291
300nt
210
162
patient with PA treated with spironolactone (in lane 4 of Fig. 3). In the aldosteronoma and kidney tissues obtained from the patients with PA without medication, even when 50 µg total RNA from each tissue were applied for the analyses, renin mRNA was not detectable by RPA.
Discussion
This is the first report of the lack of renin gene expression in aldosteronoma and kidney tissues obtained from the patients with PA without any medication. Fur- thermore, treatment with an antialdosterone drug, spi- ronolactone, resulted in an increase in the levels of renin mRNA in the aldosteronoma and kidney tissues of the patients with PA. In addition, the present studies dem- onstrate that human renin mRNA is present in normal adrenocortical tissues obtained from patients with renal cell carcinoma and confirms the presence of renin mRNA in the apparently normal part of the kidney tissue ob- tained from these patients.
The role of the local renin-angiotensin system in the adrenal gland has been demonstrated by enzymatic assay of renin or renin-like activity and by biochemical, im- munological, and histological analyses in various mam- malian species (1-4, 8, 19, 20) and man (8-10, 21). Recent progress in the field of molecular biology has provided new insights into renin gene expression. Using Northern blot analysis, Field and co-workers (1) have identified renin mRNA in the adrenal glands of the mouse in
Marker track
5 CRNA probe
w Kidney ( N)
> Adrenal(PA with SL)
51 Kidney(PA with SL)
/ Kidney(PA with SL)
__ Kidney(PA)
o Kidney(N)
Kidney(PA)
Lane
1
510nt
300nt
addition to the salivary glands and kidneys. Using RPA, rat (5, 6, 22) and mouse (7) renin mRNAs have been demonstrated in several tissues, including the kidney and adrenal, although the level of extrarenal expression is very low. However, little is known of human renin mRNA in these tissues, because of limited availability of human materials.
Renin mRNA in humans has been identified in the kidney (16, 23). The presence of renin in apparently normal adrenocortical tissues has been demonstrated by biochemical and immunological analyses in human adult adrenals (9) and fetal adrenals (21). In the former report (9), the adrenal contained renin-like activity, with par- ticularly high levels in the cortical portion of the gland. However, the adrenal renin-like activity was a hetero- geneous mixture of renin and other renin-like enzymes, because the proportion of specific renin varied from 35- 100% in the adrenals examined, whereas kidney renin was 99% specific. In the latter study (21), 5 of 38 fetal adrenal glands contained renin-like enzymes, and the mean specific activity in the fetal adrenal was signifi- cantly lower (1/750) than that in the fetal kidney.
In the present study we used relatively large amounts of total RNA (50 µg) for the assay of tissue renin mRNA. The presence of renin mRNA in normal human adrenal tissues was recognized by RPA in addition to its detection in normal kidney tissue. However, the content of renin mRNA in the normal adrenal tissues was very low com- pared to that in normal kidney tissues obtained from patients with renal cell carcinoma. No renin mRNA was detected in aldosteronoma tissues or kidney tissues ob- tained from patients with PA not receiving any drugs, while small amounts of renin mRNA were demonstrated in the aldosteronoma and kidney tissues obtained from the patients with PA who were treated with spironolac- tone. Because of the limited amount of specimens, we did not measure the tissue renin activity. However, deg- radation of the renin gene in the tissues of the patients with PA can be excluded, since the ischemic period during surgery was shorter for adrenalectomy than for en bloc radical nephrectomy.
On the other hand, some investigators (8-10) have demonstrated the presence of renin or renin-like en- zymes in human aldosteronoma tissues by biochemical and immunological analyses. However, they did not ex- clude the possibility that the renin-like activity observed in the adrenal tumors resulted from cathepsin-D. In addition, cathepsin-D generates angiotensin-I from renin substrate, and considerable homology has been shown to exist between the genes for renin and cathepsin-D (58%) in the human (24). There is no information about whether the patients with PA (8-10) had discontinued antihypertensive drugs, such as diuretics and/or spiron- olactone, and received a controlled sodium diet before surgery.
It has remained unknown whether tissue renin gene expression is modulated by sodium balance and/or by the antihypertensive drugs. To exclude these influences on renin gene expression, our subjects received a con- trolled sodium diet, and two of four patients with PA discontinued antihypertensive drugs at least 2 weeks before surgery. The remaining two patients with PA continued antihypertensive treatment with spironolac- tone. There were no differences in blood pressure, PRA, ARC, PAC, and serum potassium between the two groups of patients with PA. However, TRC was slightly in- creased, and small amounts of renin mRNA in the tissues were recognized in the patients treated with spironolac- tone.
A single dose of captopril increased plasma renin levels in the patients with renal cell carcinoma, but captopril failed to increase plasma renin in patients with PA, which confirms our previous findings (13, 14). The reg- ulation of the renin secretory response has been studied in detail (for review, see Refs. 25 and 26). Our under- standing of the control of renin gene expression is lim-
ited, but there are reports that sodium depletion (5, 22, 27), converting enzyme inhibition (6, 28), and 3-adrener- gic stimulation (27) all increase the renal renin mRNA level. In contrast, renin mRNA is suppressed in the kidney of hypertensive rats treated with deoxycorticos- terone acetate and high salt (29). On the other hand, administration of deoxycorticosterone acetate alone failed to suppress renin gene expression. In a recent report by Barrett and co-workers (30), chronic treatment with 9a-fludrocortisone and sodium loading resulted in a 60% decrease in renal renin mRNA in mice. Adminis- tration of diuretics or cessation of 9a-fludrocortisone resulted in a slight increase in renin mRNA.
Comparatively little is known about the regulation of extrarenal renin gene expression (5, 6, 22). Brecher and co-workers (22) have reported that salt-deplete rats con- tain twice as much renin mRNA in adrenal zona glomer- ulosa as rats on normal salt diets.
Taken together, all results in experimental models and in patients with PA indicate that excessive mineralocor- ticoid and exaggerated sodium retention may have a significant influence on the suppression of renin gene expression. Further studies on renin mRNA in various tissues under several pathophysiological conditions should improve our understanding of the regulation of renin gene expression in human tissues.
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