1 Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80317, Taiwan; and 2 Department of Internal Medicine, Ping Tung Christian Hospital, Ping Tung 900, Taiwan
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ABSTRACT |
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To investigate the role of renal endothelin-1 (ET-1) synthesis in water-sodium homeostasis, we measured mRNA expressions, protein levels, enzyme activity, and receptor binding of the renal ET-1 system in a DOCA- and salt-treated rat model. Male Wistar rats were divided into control and DOCA- and salt-treated (DOCA-Salt) groups. The DOCA-Salt group received 25 mg/kg body wt DOCA and was maintained on 1% NaCl drinking water. Rats were killed on days 1, 2, 4, and 10 of the experiment. Urinary ET-1-like immunoreactivity significantly increased from the second day in the DOCA-Salt group and correlated well with the urinary sodium excretion rate (r = 0.81, P < 0.001). Renal endothelin-converting enzyme (ECE) activity, ET-1, and ECE-1 mRNA expressions were significantly increased in the renal medullary area of DOCA-Salt rats. In situ hybridization and immunohistochemical studies showed that the increase in ET-1 synthesis was mainly localized in the inner medullary collecting ducts. The maximum binding of endothelin B receptor also increased from the second day in the renal medulla of the DOCA-Salt group. Our results suggest that renal medullary synthesized ET-1 may be a natriuretic factor and may participate in the intrarenal regulation of water and salt homeostasis in prehypertensive DOCA-and salt-treated rats.
endothelin-converting enzyme-1; endothelin A receptor; endothelin B receptor
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INTRODUCTION |
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ENDOTHELIN-1 (ET-1) was initially discovered as a potent vasoconstrictor produced by cultured endothelial cells (39). Human ET-1 mRNA encodes a 212-amino acid prepropeptide that is cleaved by dibasic pair-specific endopeptidases to yield the 38-amino acid propeptide Big ET-1 (25). Big ET-1 is converted both inside and outside the cell to mature ET-1 by endothelin-converting enzymes (ECEs) (8, 38). The biological activities of ET-1 are mediated by two distinct receptors, endothelin A and B (ETA and ETB) receptors (13, 34). Although ET-1 was originally found to be generated in the endothelium and thought to act primarily on vascular smooth muscle cells, it has now been shown to be produced ubiquitously and to exert actions on different tissues (33). Kidney, especially in inner medullary collecting ducts (IMCDs), is one of the major regions in which ET-1 production takes place (6, 16, 37). Evidence has been reported that the kidney also expresses ECEs, ETA, and ETB receptors, and the receptors were localized in company with ET-1. Thus ET-1 was thought to play some role in renal pathophysiology via autocrine or paracrine manners (17, 18).
ET-1 has potent and complex actions in the kidney, whereby it causes renal vasoconstriction, leading to a reduction of glomerular filtration rate and renal blood flow as well as sodium excretion and urinary volume (15, 17). However, ET-1 also causes both diuretic and natriuretic actions by inhibiting the activities of Na+-K+-ATPase and water channel on renal tubular cells (41). Abnormality of renal ET-1 synthesis and related sodium-regulation disorders has been thought to be one of the etiologies of hypertension (24, 29, 30). Hoffman et al. (12) reported that salt-sensitive hypertensive patients had lower levels of urinary sodium excretion than did salt-resistant hypertensive patients (12). Recently, we found a correlation between the circadian rhythm of urinary sodium excretion and urinary ET-1 excretion in normal subjects and those with essential hypertension (14). Furthermore, our results have showed that the urinary ET-1 excretion rate was lower in patients with essential hypertension compared with normal subjects after normal saline infusion. Therefore, renal synthesized ET-1 might play a role in the handling of sodium excretion and contribute to the pathophysiology of salt-sensitive hypertension.
Almost all cell types in the kidney possess the ability to synthesize ET-1, and the synthesized ET-1 may have different biological functions. Although there are many reports that indicate diverse actions of ET-1 in the kidney (17), the physiological role of ET-1 system in response to volume expansion and salt overloading is still unclear. In this study, to further clarify the role of renal ET-1 synthesis in regulation of electrolyte-fluid homeostasis, we have investigated renal ET-1, ECE-1, ETA, and ETB receptor mRNA expressions, ECE activity, receptor binding activity, and urinary ET-1 excretion in rats with DOCA and salt treatment.
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MATERIALS AND METHODS |
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Animal experiments.
Male Wistar rats weighing ~250 g were housed individually in
metabolic cages during the entire experimental period. All rats were
given a normal rat diet and tap water ad libitum. In the first
experiment, rats were divided into two groups: 1) a DOCA- and salt-treated group (DOCA-Salt group, n = 18), in
which the rats received 25 mg/kg body wt DOCA (Sigma Chemical, St.
Louis, MO) in corn oil per day by subcutaneous injection and were
maintained on 1% NaCl drinking water; and 2) a control
group (n = 12), in which the rats were injected daily
with the same dose of corn oil as were DOCA-Salt rats and fed with tap
water (23). All experimental rats had unlimited access to
food and water. Daily 24-h urine samples were collected, and the amount
of food and water consumed was recorded every 24 h. Rats were
killed by decapitation on the tenth day after experiment, and blood was
collected to measure plasma renin activity and ET-1 levels. This study
was repeated three times, and the data were combined for analysis. In
the second experiment, rats were also divided into control (n = 5) and DOCA-Salt (n = 20) groups
and treated the same as described above, but they were killed on
days 1, 2, 4, and 10 of the
experiment. Aortas and kidneys were obtained for mRNA and ECE activity
analysis. The kidney tissues were divided into cortex and medulla
immediately after removal. Tissues were packaged separately in plastic
tubes and stored at 70°C until RNA and protein extraction.
Blood pressure monitoring. Systolic blood pressure was measured in conscious rats by the rat-tail manometer-tachometer system (model KN-210, Natsume). Rats were prewarmed and held in a warm box mounted on a heating plate maintained at 37°C during the measurement. Six readings were taken for each measurement.
RT-PCR. Total RNA from the aorta, kidney cortex, and kidney medulla/papilla tissues was isolated by a modified guanidinium isothiocyanate method (5) and quantified by its absorbency at 260 nm.
Two micrograms of RNA from each sample were reverse transcripted by incubating with 20 µl RT mixture by using an Advantage RT for PCR kit (Clontech Laboratories). Then, the cDNA was diluted with water to a 1:4 ratio. The resulting first-strand cDNA was used for the PCR procedure. PCR was performed at a final concentration of 1× PCR buffer, 0.2 mM dNTP, 0.4 µmol/l of each pair of primer oligos (Table 1), and 2.0 U of AmpliTaq DNA polymerase (Perkin-Elmer) to a total volume of 50 µl. After an initial denaturation step at 94°C for 1 min, cDNA fragments were amplified with each amplification profile in a Perkin-Elmer Cetus 9600 thermocycler (Perkin-Elmer Cetus, Norwalk, CT).
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Southern blot analysis.
To identify the presence of ET-1, ECE-1, ETA, and
ETB cDNA in the rat aorta and kidney, 15 µl of the PCR
product were electrophoresed on 1.5% agarose gels and transferred to a
nylon membrane (Amersham). The blots were hybridized with each specific
32P-labeled, random-primed cDNA probe for 16 h at
42°C, according to the standard technique. Blots were exposed to
Kodak BMR film (Eastman Kodak, Rochester, NY) at 70°C.
ECE activity assay. Measurements of ECE activity were performed as described by Minamino et al. (28). Excised samples were homogenized in a 10× vol of homogenization buffer [20 mmol/l Tris · HCl, pH 7.5; 5 mmol/l MgCl2; 0.1 mmol/l polymethylsulfonyl fluoride (PMSF); 20 µmol/l pepstatin A; and 20 µmol/l leupeptin] by the use of a polytron homogenizer. The homogenates were centrifuged at 800 g for 10 min, and the supernatant was further centrifuged at 100,000 g for 45 min. The pellets were rinsed in homogenization buffer and recentrifuged. They were then dissolved in homogenization buffer containing 0.5% (wt/vol) Triton X-100 and centrifuged at 100,000 g for 60 min. The supernatant (10 µg protein) was then subjected to enzyme assay. The enzyme reaction was carried out at 37°C in 100 µl of assay buffer (20 mmol/l Tris · HCl, pH 7.0; 0.1% bovine serum albumin; 20 µmol/l pepstatin A; 20 µmol/l leupeptin; and 20 µmol/l E-64) containing 0.1 µmol/l rat Big ET-1 (1---39, Peninsula Laboratories, Belmont, CA). After 4 h of incubation, the reaction was stopped by adding an equal volume of 5 mmol/l EDTA. The concentration of mature ET-1 was determined by use of an ELISA. Tissue ECE activity was expressed as the amount of ET-1 generated per 10 µg protein of tissue extract in the assay. To measure the ET-1 levels, we used a sandwich-type ELISA system (Amersham). The cross-reactivity of this assay system with ET-1 and ET-2 is >100%, <0.001% with ET-3, and 0.07% with Big ET-1.
Radioimmunoassays (RIAs).
Plasma and urinary ET-1-like immunoreactivity (irET-1) was determined
by a specific ET-1 RIA (Peninsula Laboratories) according to our
previous study (22). The plasma and urine samples were applied to Sep-Pak C18 cartridges (Waters Associates,
Milford, MA) and eluted with 60% acetonitrile in 0.1% trifluoroacetic
acid. The eluates were lyophilized and reconstituted for RIA. The
antibody cross-reacted with ET-1 (100%), Big ET-1 (17%), ET-2 (7%),
and ET-3 (7%) but did not react with angiotensin II, vasoactive
intestinal peptide, or -atrial natriuretic pepetide 1---28. Plasma
renin activity was also determined by RIA (DuPont, Billerica, MA).
Concentrations of plasma and urinary sodium were determined in an
automatic analyzer (Nova 5, Nova Biochemical, Newton, MA).
In situ hybridization. In situ hybridization was performed in six kidneys, three from the control group and three from the DOCA-Salt group, on the third day after treatment with DOCA and salt. Briefly, fresh kidney portions containing cortex and medulla were placed on the same block, mounted in optimal cutting temperature (OCT) compound (Tissue-Tek OCT compound; Ames Division, Miles Laboratory, Elkhart, IN) and cut into 10-µm sections. Frozen sections were fixed in 4% paraformaldehyde for 5 min at 4°C. After a series of standard procedures, each section was overlaid with 100 µl of hybridization buffer containing 10 ng of digoxigenin (DIG)-labeled RNA sense or antisense probe and incubated at 42°C overnight in a humid chamber. Sections were then washed and incubated with a suitable dilution of sheep anti-DIG-alkaline phosphatase (1:500), and signal detection was performed by the fast red substrate system (DAKO). Sections were subsequently counterstained with hematoxylin and mounted.
The rat ET-1 sense and antisense RNA probes were prepared by RNA transcription reaction. In brief, a PCR product was obtained by amplification of cDNA from rat kidney by using specific oligonucleotide primers (sense: 5'-ACAAGGAGTGTGTCTACTTC-3'; antisense: 5'-TGTTGCTGATGGCCTCCAAC-3') that were derived from the cloned rat prepro-ET-1 sequence (6). It was then subcloned into pT-Adv vector (Clontech Laboratories). Then, a 348-bp ET-1 cDNA fragment was cut out by EcoR I restriction enzyme and subcloned into the CMV plasmid. The plasmid was subsequently linearized with BamH I or Sal I restriction enzyme. Thereafter, sense and antisense DIG-labeled RNA probes were synthesized by using T7 or T3 RNA polymerase with a DIG RNA labeling kit (Boehringer Mannheim). RNA probes were extracted using phenol-chloroform and ethanol precipitation.Immunohistochemistry. Immunohistochemical detection of ET-1 was performed by the avidin-biotin-peroxidase complex method by using a commercially available kit (DAKO). Kidney samples from the frozen serial sections of in situ hybridization were used. Frozen sections were fixed in ice-cold acetone for 10 min and incubated with 0.3% H2O2 in distilled water, followed by exposure to normal rabbit serum. Sections were then layered with a rabbit anti-ET-1 serum (Peninsula Laboratories) that was diluted at a ratio of 1:500. Biotinylated secondary antibody and an avidin-biotin-peroxidase complex were subsequently applied to the sections; diaminobenzidine (Zymed Laboratories) was used as a peroxidase substrate. All incubations were performed overnight at 4°C, and washing cycles were carried out by using Tris · HCl, pH 7.6. Finally, sections were counterstained with hematoxylin and mounted.
Receptor binding assay.
The binding assay was performed as described previously
(36). Renal tissues (cortex and medulla) were homogenized
with 9 vol of ice-cold 0.25 mol/l sucrose containing 20 mmol/l
Tris · HCl (pH 7.4), 0.2 mmol/l PMSF, 1 µmol/l leupeptin, 1 µmol/l pepstatin, 0.1 mmol/l EDTA, and 0.5 mmol/l EGTA. The
homogenate was centrifuged at 1,000 g for 10 min at 4°C,
and the resulting supernatant was centrifuged again at 48,000 g for 20 min. The sedimentary membranes were washed three
times with the same buffer and stored in aliquots at 70°C until use.
Statistics. Results are expressed as means ± SE. Student's t-test was performed to analyze mRNA levels, ECE activity, and receptor binding activities between control and DOCA-Salt groups. Linear regression analysis was used to test the correlation between the urine sodium excretion and urinary irET-1 excretion.
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RESULTS |
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As shown in Table 2, there were no
significant differences between control and DOCA-Salt-treated groups in
body weight change, kidney weight, and plasma sodium levels. Plasma
renin activity was markedly suppressed in DOCA-Salt rats (0.65 ± 0.30 ng · l1 · h
1,
P < 0.001) compared with control rats (7.20 ± 0.99 ng · l
1 · h
1 ).
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Blood pressure.
Rat-tail blood pressure of control and DOCA-Salt-treated groups was
measured before the first day of DOCA-Salt treatment and on the day of
death. As shown in Table 3, there were no
significant changes pre- and postexperiment in control rats and in rats
after DOCA-salt treatment at 1, 2, 4, and 10 days.
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Urinary flow rates, urinary sodium, and ET-1 excretion.
Urine flow rates were significantly increased from the first day to the
last day of the study period in the DOCA-Salt group (Fig
1A). Urinary sodium excretion
was markedly increased at the second day after DOCA-salt treatment and
remained elevated for the entire 10-day study period (Fig.
1B).
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ET-1 system mRNA expression in kidney and aorta.
To detect changes in the renal ET-1 system during DOCA-salt treatment,
we measured aorta, renal cortex, and medulla ET-1, ECE-1,
ETA, and ETB receptor mRNA expressions by
RT-PCR combined with Southern blotting. The results showed that there
were no significant differences between control and DOCA-Salt groups in aortic ET-1 mRNA expression (Fig. 3,
A and B). In renal medulla, ET-1 mRNA expression
significantly increased from the second day after DOCA-salt treatment
(Fig. 3C). ET-1 mRNA expression in renal cortex decreased on
the first day, and no difference was detected on the other days after
DOCA-salt treatment (Fig. 3D). There were no significant
differences in ECE-1 mRNA levels in aortas and renal cortex of the
study groups (Fig. 3, A, E, and G). In
contrast, renal medulla in the DOCA-Salt group (Fig. 3F)
showed significantly higher expression of ECE-1 mRNA. There were no
differences between control and DOCA-Salt rats in renal cortical and
medullary ETA and ETB receptor mRNA expression
(data not shown).
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ECE-1 enzyme activity in kidney.
To examine whether the alterations of ECE-1 mRNA levels correlated with
enzyme activity, we measured ECE activity in the kidneys of the control
and DOCA-Salt rats. Because ECE-1 is the major isoenzyme responsible
for the cleavage of Big ET-1 at the neutral pH, ECE activity is thought
to reflect the activity of ECE-1 (17). As shown in Fig.
4, ECE activity of the renal cortex in
the DOCA-Salt group was not different compared with the control group,
whereas the medullas from the DOCA-Salt group expressed higher enzyme activity and the result was consistent with the ECE-1 mRNA expression.
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In situ hybridization.
There were no differences in ET-1 mRNA expression in the renal cortex,
including tubular structures, glomeruli (Fig.
5, A and B),
arcuate arteries, interlobular arteries, and veins between the rats
studied. In DOCA-Salt rats, the density of ET-1 mRNA hybridization
signals in collecting tubules and proximal and distal convoluted
tubules of the outer and inner medullary area were more closely packed
than those of the control animals (Fig. 5, C-F). There were
few signals present in capillaries and no variety between the two
groups. Very few scattered positive signals were detectable in the
negative control sections hybridized with the sense probe (data not
shown).
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Immunohistochemistry. The distribution patterns of ET-1 protein paralleled those of mRNA expression. ET-1 immunoreactivity was detected in the renal cortex, medulla, and papilla. In the cortex, we found positive staining present in glomeruli, glomerular arterioles, interlobular arteries, and veins, whereas in the proximal and distal tubules, only a weak staining signal was found. Intense immunostaining was seen in the endothelium of the vasa rectae in the outer medulla, and the most intense staining occurred in the inner medulla collecting ducts. When we examined irET-1 of the control and DOCA-Salt groups, there were no significant differences in the cortex (data not shown). In contrast, ET-1 immunostaining in the DOCA-Salt group was more potent than that in the control group in the renal medullary area (Fig. 5, G and H).
Receptor binding of ETA and ETB.
The results of ETA and ETB receptor binding
assays are shown in Table 4. The maximum
binding (Bmax) of the renal cortical ETA
receptor was 1.64-fold higher than that of the renal medulla. In normal
control rats, the Bmax of the renal medullary
ETB receptor was 3.25-fold higher than that of the renal
cortex. These ratios are similar to the findings of Hocher et al.
(11). The renal cortex and the renal medulla showed no
different ETA receptor binding capacity between the control
and the DOCA-Salt groups. In the DOCA-Salt group, medullary
ETB receptor expressed a higher Bmax at 2, 4, and 10 days than in the control group (P < 0.001). Bmax of cortical ETB receptor was not different
between the control and DOCA-Salt groups.
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DISCUSSION |
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Our present study demonstrates the following points: 1) the urinary ET-1 excretion rate increased from an early stage in the DOCA-Salt group, but there was no difference in plasma ET-1 levels between the control and DOCA-Salt groups; 2) urinary ET-1 excretion correlated well with urinary sodium excretion; 3) the irET-1 increased only in the medullary tubular area after DOCA-salt treatment and corresponded to the results of mRNA expressions; 4) ECE-1 mRNA expression and ECE-1 activity increased in the medulla of the DOCA-Salt group; and 5) Bmax of the renal medullary ETB receptor increased in the DOCA-Salt group. These results indicated that renal medullary ET-1 synthesis was enhanced in volume expansion state and might play some role in sodium homeostasis.
It has been suggested that urinary irET-1 is derived mainly from the kidney. Evidence supporting this conception was based on the finding that only negligible amounts of labeled ET-1 infused into the systemic circulation of normal rats could be recovered in the urine (2). In normal subjects, urinary irET-1 concentrations have been showed to be several times higher than those in plasma, and no significant correlation could be detected among urinary irET-1 excretion, plasma ET-1 levels, and glomerular filtration rate (1, 3). In our study, significant urinary irET-1 excretion accompanied by renal medullary ET-1 gene overexpression was found in DOCA-Salt- treated rats. In situ hybridization analysis and immunohistochemical study showed the increased renal ET-1 synthesis was localized only in the medullary area. These results indicated that renal medullary ET-1 synthesis increased in rats with salt loading and mineralocorticoid excess. Plasma endothelin and renal endothelin are two distinct systems involved in volume homeostasis (35). The fact that there was no elevation of aortic ET-1 mRNA expression and plasma ET-1 levels in this study further suggests that the renal tubular ET-1 system has as its regional functions in the modulation of DOCA- and salt-induced volume expansion in the early stage.
Besides having vasoactive properties, ET-1 also induces proliferation and synthesis of matrix protein in mesangial cells (10). Although ET-1 is thought to have an important role in glomerular diseases, it is interesting to note that the IMCD is the prime location where ET-1 is expressed in the kidney, and its function has not yet been thoroughly clarified. Recently, renal tubular epithelial cells have been found to be able to synthesize and secrete ET-1. Moreover, the renal medullary area has been found to be the most important site of ET-1 binding (19). This discrepancy may be due to two different ET-1 receptors with contrasting functions being present in the kidney with different distributions (17, 18). Kohan (17, 19) indicated that IMCD cells could synthesize and secrete ET-1 and that IMCD cells responded to ET-1 via the ETB receptor to inhibit vasopressin-induced cAMP formation and water permeability and could inhibit Na+-K+-ATPase to regulate urinary water and sodium excretion. When administered in a dosage that does not affect glomerular filtration rate, ET-1 was found to cause diuresis (9). It seems that glomerular ET-1 and tubular ET-1 may be two distinct systems for controlling renal physiology. In the present experiment, we showed that only medullary ET-1 synthesis altered in response to water-salt imbalance. This result confirmed that the renal medullary ET-1 system and the glomerular ET-1 system are differently regulated and may have distinct physiological functions.
The enhanced synthesis of ET-1 in the renal medulla may contribute to water-electrolyte balance. To further clarify this point, we explored the receptor binding of ETA and ETB. ET-1 affects renal sodium homeostasis through multiple sites, and the role of ETA and ETB receptors in mediating the natriuretic and diuretic effects needs to be elucidated. Both receptors are found in the mammalian kidney, although their distribution differs among species (31, 32). Evidence for the physiological roles of the two ET receptor subtypes is often controversial, and the subtype of ET receptors in the nephron has not been completely resolved. mRNA and binding studies indicate that renal tubules predominantly express the ETB receptor (17). Functional studies also implicate that the ETB receptor mediates most of the effects of ET on the renal tubule (17). With minimal effects on renal hemodynamics, specific ETB receptor agonists have been reported to increase urinary sodium and water excretion (4, 40). Recently, in DOCA-salt-treated hypertensive rats, FR-139317 (a selective ETA receptor antagonist) has been found to cause sustained renal vasodilation. Urine flow and urinary sodium excretion increased significantly after drug injection (26). Daily administration of ABT-627 (a selective ETA receptor antagonist) for 2 wk to DOCA-salt-treated hypertensive rats almost abolished any further increases in blood pressure, whereas A-192621 (a selective ETB receptor antagonist) did not affect the development of DOCA-salt-induced hypertension and led to worsening of the renal dysfunction (27). When these data are taken together, it seems that the ETB receptor mediates the diuretic and natriuretic effects, and the ETA receptor mainly modulates vascular functions. Our results show that only the Bmax of the medullary ETB receptor was increased in the DOCA-Salt group. This fact provides evidence that renal medullary synthesis ET-1 may regulate water and sodium metabolism via the ETB receptor.
Although there is little evidence that ECE activity is rate limiting in ET-1 production, ECEs present a notable regulatory role for ET synthesis (17). In our study, we found ECE-1 mRNA overexpression and high ECE-1 enzyme activity in the medullary area in the early stage in DOCA-Salt rats. ECE-1 may contribute to renal medullary ET-1 secretion in prehypertensive DOCA-salt-treated rats.
In the DOCA-salt hypertensive model, investigators have demonstrated the existent high levels of plasma ET-1 and a significant increase in the expression of the prepro-ET-1 gene in blood vessels (20, 21). By using in situ hybridization for localization, significant increases in ET-1 mRNA transcripts were found in the endothelium of renal vessels and in capillary endothelial and mesangial cells of glomeruli (7). However, previous studies have also demonstrated that DOCA and salt do not stimulate ET-1 expression if blood pressure is not significantly elevated (7). This may suggest a role for salt retention and volume expansion in the mechanisms leading to enhanced ET-1 gene expression in hypertension. In our prehypertensive DOCA-salt animal model, we examined the ET-1 system before blood pressure elevation. We found that the plasma ET-1 levels did not significantly increase, which is in agreement with other studies (7). On the contrary, the study presented here demonstrates enhanced urinary ET-1 excretion from the second day after DOCA-salt treatment, indicating that renal ET-1 begins to be synthesized from an early stage in DOCA-salt-treated rats. In our previous studies, urinary ET-1 excretion presented a circadian rhythm phenomenon and correlated with urinary sodium excretion in normal subjects without any excess salt supply (14). The present data also display a good correlation between urinary ET-1 and urinary sodium excretion. These results imply that renal ET-1 modulates diurnal sodium metabolism and suggest that renal medullary ET-1 may play a major physiological role in the natriuretic effect.
In conclusion, renal medullary ET-1 synthesis increased in the early stage of DOCA-salt treatment in rats and correlated well with urinary sodium excretion. Our work suggests that the tubular ET-1 system plays a role in the compensation for sodium overload and volume expansion.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant from the National Science Council of Taiwan. (NSC 89-2314-B-037-018).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J.-H. Tsai, Dept. of Internal Medicine, Kaohsiung Medical Univ., No 100, Shih-Chuan 1st Rd., Kaohsiung 80317, Taiwan (E-mail: hsiehjun{at}ms4.hinet.net).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 27 May 1999; accepted in final form 24 February 2000.
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