Endothelin Converting Enzyme-1 Gene Expression in the Kidney of Spontaneously Hypertensive Rats
Abstract Abnormal renal handling of water and sodium is implicated in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). Alteration of renal endothelin-1 synthesis is also reported in SHR. Endothelin-1, a potent vasoconstrictor and regulator of sodium reabsorption in the nephron, has a pathophysiological potential in the development of hypertension. Because synthesis of bioactive endothelin-1 requires endothelin converting enzyme-1 (ECE-1), we investigated whether renal ECE-1 gene expression is altered in the kidney of SHR. Kidneys from both 4- and 12-week-old SHR and age-matched Wistar-Kyoto rats (WKY) were studied. ECE-1 mRNA in microdissected nephron segments was assessed by reverse transcription–competitive polymerase chain reaction, and ECE-1 protein level by Western blot. In 4-week-old SHR, ECE-1 mRNA was significantly increased in the proximal straight tubule, medullary thick ascending limb, cortical thick ascending limb, and inner medullary collecting duct. ECE-1 protein level was increased in both the outer and inner medulla. In 12-week-old SHR, ECE-1 gene expression was significantly increased in the proximal straight tubule, medullary thick ascending limb, and also in the glomeruli. Glomerular preproendothelin-1 mRNA expression was not different between the two strains at both 4 and 12 weeks. We conclude that high ECE-1 gene expression in the nephron, via increase of endothelin-1 synthesis, may promote sodium retention that contributes to the development and/or maintenance of hypertension in SHR.
Several lines of evidence from renal transplantation experiments suggest that renal intrinsic defects are involved in hypertension in SHR.1 The defect, not yet identified to date, leads to sodium retention before and during the development of hypertension2 and also to an increased peripheral vascular resistance, the hallmark of established hypertension.
A decade ago, endothelin, a family of 21-amino acid vasoconstrictor peptides was discovered.3 Three isoforms of endothelin, ET-1, ET-2, and ET-3, encoded by three different genes have been identified. Among the three isoforms, ET-1 is the most abundantly expressed in vessels. It is long acting and one of the most potent vasoconstrictors known.4 In rats, infusion of ET-1 induced sustained renal vasoconstriction and decrease of GFR.5 Inappropriate release of such a long-acting substance could conceivably contribute to the increase of regional and systemic vascular resistance. In addition to its vasoactive properties, the observation that ET-1 stimulates sodium and water retention in rats,6 dogs,7 monkeys,8 or humans9 raised interest in its potential pathophysiological significance in the development or maintenance of hypertension.
In contrast to elevated plasma ET-1 levels in patients with hypertension,10 11 12 circulating levels of ET-1 in SHR are either lower or comparable to those in normotensive WKY.13 14 However, the use of anti-ET-1 monoclonal antibodies or ET-1 receptor antagonist was effective in lowering the blood pressure or improving the renal function in SHR, suggesting that ET-1 has an important role in the pathogenesis of hypertension in SHR.15 16 Moreover, ET-1 released from the kidney was increased in SHR.13 The kidney is not only a target of circulating ET-1 but also is an important source of ET-1.17
The gene encoding for ET-1 precursor is preproendothelin-1 gene, but synthesis of bioactive ET-1 requires a recently cloned phosphoramidon-sensitive ECE.18 19 ECE-1 converts an intermediate metabolite, big ET-1, into bioactive ET-1. Its expression has been detected in the kidney.19 With the possibility that some genes involved in the pathogenesis or maintenance of hypertension in SHR may be differentially expressed in certain structures of the kidney, we investigated whether steady state ECE-1 gene expression is altered in the kidney of SHR, especially in the nephron segments.
Pathogen-free male SHR and normotensive WKY were purchased from Hoshino Animals Laboratory (Saitama, Japan). These animals had free access to regular laboratory chow and tap water and were studied at 4 weeks and 12 weeks of age.
Blood Pressure Monitoring
Systolic blood pressure was measured in conscious rats with the tail-cuff method using a programmable sphygmomanome2ter with photoelectric and pulse amplifier (model PS-200, Riken kaihatsu). Rats were prewarmed and held in a warm box mounted on a heating plate maintained between 37°C to 38°C during the measurement. Ten readings were taken for each measurement.
Renal Tubule Microdissection
To determine whether the mRNA expression was different at the nephron level, we used microdissection combined with RT-PCR techniques and Southern blot.20 Animals were anesthetized with pentobarbital 50 mg/kg wt, IP. The abdominal aorta was ligated at two sites, under the right renal artery and 2 cm below the left renal artery. A catheter was then inserted in the abdominal aorta for the perfusion of the left kidney. Perfusion was made first with 10 mL ice-cold solution A and then with 10 mL ice-cold solution B. Solution A contained (mmol/L) NaCl 130, KCl 5.0, NaH2PO4 1.0, MgSO4 1.0, Ca lactate 1.0, Na acetate 2.0, glucose 5.5, HEPES 10, pH 7.4. Solution B was made from solution A by addition of 300 U/mg collagenase type I and 1 g/L BSA.
The left kidney was decapsulated, removed, and cut in thin 1-mm transverse sections. The sections were transferred into tubes containing 3 mL of solution B with 10 mmol/L vanadyl ribonucleotide complex (VRC) and incubated with bubbling oxygen at 37°C under constant agitation. After 30 minutes of incubation, the slices were rinsed with solution A and then transferred to a microdissection dish containing 10 mL of solution A with 10 mmol/L VRC (New England Biolabs).
Microdissection of nephron segments was performed using dissecting needles under a dissection microscope with a cooling plate at 4°C. Superficial and juxtamedullary glomeruli were extracted separately. Identification of tubule segments was based on previously reported criteria.21 The following segments were microdissected: superficial glomeruli (sGlm), juxtamedullary glomeruli (jGlm), proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb of Henle’s loop (MTAL), cortical thick ascending limb of Henle’s loop (CTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). Microdissected segments were measured with a calibrated eyepiece micrometer. A total length of 2 mm was collected for each renal tubule segment, and 20 glomeruli of each type were collected. Microdissected segments were transferred to a clean wash dish containing solution A using pipettes coated with 0.1% BSA. After a washing for VRC and binding debris, cleaned microdissected segments were transferred into individual microcentrifuge tubes containing 10 μL of solution A with 1 U/μL RNase inhibitor and 5 mmol/L DTT.
Microdissected segments were collected at the bottom of the tube by centrifugation at 20 000g for 5 minutes. The supernatant was checked to be free of tubules under a microscope and then discarded. The pellet was dissolved in 3.5 μL of 2% Triton X-100 in the presence of 1U/μL RNase inhibitor and 5 mmol/L DTT; we then proceeded with the reverse transcription using a cDNA synthesis kit (Boehringer Mannheim) according to the instructions of the manufacturer. In brief, a 4.5-μL RT reaction mixture containing RNase inhibitor, deoxynucleotide mixture, avian myeloblastosis virus reverse transcriptase, and random primer was added. The sample was mixed and centrifuged to be collected at the bottom of the tube. The reaction mixture was incubated at 42°C for 60 minutes and heated to 95°C for 5 minutes to denature the RNA-cDNA hybrids and to inactivate the reverse transcriptase activity.
Polymerase Chain Reaction
The resulting RT product was processed by PCR with rat specific primers for ECE-1 and preproET-1. For amplification of preproET-1, the antisense primer was 5′-TCCTGCTCCT CCTTGATGGA-3′ and the sense primer was 5′-AAACACCACGGGGCTCTGTA-3′. The predicted size of cDNA amplification product was 471 bp, extending from base 724 through base 1194. Simultaneously, we performed RT-PCR for the housekeeping gene GAPDH. For amplification of GAPDH, the antisense primer was 5′-TCCCTCAAGATTGTCAGCAA-3′ and the sense primer was 5′-AGATCCACAACGGATACATT-3′. The predicted size of cDNA amplification product was 308 bp, extending from base 506 through base 813.
Including the RT product, the final volume of the PCR reaction was 100 μL, with the following concentrations: 25 U/mLTaq DNA-polymerase, 2 mmol/mL deoxynucleotide, 0.2 pmol/μL sense primer, 0.2 pmol/μL antisense primer, 10 mmol/L Tris HCl, 1.5 mmol/L MgCl2, and 50 mmol/L KCl. The mixture was overlaid with 100 μL of mineral oil and amplified in a DNA thermocycler (ASTEC-Takara). PreproET-1 cDNA fragments were amplified by 32 cycles and GAPDH by 28 cycles with the following amplification profile: 45 seconds at 94°C (denaturation), 45 seconds at 62°C (hybridization of primers), 1 minute 45 seconds at 72°C (elongation), and 7 minutes at 72°C (final elongation). ECE-1 cDNA was coamplified with a constant amount of competitive template by 29 cycles. The antisense primer was 5′-AGCTCAGCAGCCGTGA CTTT-3′ and the sense primer was 5′-AATATCACAGCACCCTGGGC-3′. The predicted size of cDNA amplification product was 389 bp for ECE-1 and 201 bp for the competitive template.
To synthesize this mimic DNA for competitive PCR, we used an in vitro strategy for site-directed deletion.22 The 389-bp portion of ECE-1 cDNA extending from base 676 through base 1064 was analyzed to construct four different primers: left, right, internal left, and internal right primers. The left and right primers were sense and antisense primers used to amplify the 389-bp portion of ECE-1 cDNA extending from base 676 through base 1064. The internal primers of 20-mer were constructed with an additional 20-mer nonsense sequence to the 5′ end. The internal left primer was 5′-TTGGCCAAGGACAACTTCCATACATGGTCCAGCTGGGGAA-3′ extending from base 691 through base 710, and the internal right primer was 5′-TTCCCCAGCTGGACCATGTATGGAAGTTGTCCTTGGCCCA-3′ extending from base 899 through base 918. The 20-mer nonsense sequences are italicized. The full-length internal primers were carefully chosen so that their sequences were complementary. Primary PCRs were performed in separate tubes with the left primer and its internal partner and the right primer with its internal partner. The PCR products were purified separately with Centricon 30 (Amicon Inc) to eliminate the primers. After purification, the two primary PCR products were recombined, and a zipping PCR reaction was performed with the outermost left and right primers to produce a recombinant competitor DNA of 201 bp, with a deletion of the 188-bp portion between 710 and 899 bp. A second purification with Centricon 30 was performed, and the amplified product was quantified by photometry.
The initial amount of ECE-1 cDNA before amplification was quantified from the densitometric ratio of ECE-1 cDNA to mimic DNA in ethidium bromide–stained gel.23 After coamplification of ECE-1 cDNA with a dilution series of mimic DNA, a linear plot of the ratio of ECE-1 cDNA to mimic DNA versus mimic DNA concentration was drawn on an arithmetic scale. To correct for differences in molecular weight between ECE-1 cDNA and mimic DNA, the densitometric ratio obtained from the ethidium bromide–stained gel was multiplied by 389/201 as reported elsewhere with modification.24
The identity of PCR products was confirmed by Southern hybridization after size fractionation by 2% agarose gel electrophoresis. The cDNA bands were stained with ethidium bromide, visualized, and photographed under UV illumination. After destaining and denaturation, the PCR product was transferred to a positively charged nylon membrane by capillary blotting. After 30 minutes of prehybridization, hybridization was conducted at 37°C for 6 hours with digoxigenin-labeled oligonucleotide probes using a Dig Easy Hyb kit (Boehringer Mannheim). Posthybridization washes were performed twice in 2× SSC, 0.1% SDS for 15 minutes at room temperature and then twice in 0.1× SSC, 0.1% SDS at 68°C. Hybridized probes were immunodetected with antidigoxigenin, Fab fragments conjugated to alkaline phosphatase, and then visualized by chemiluminescence with disodium phenyl phosphate (DIG luminescence detection kit, Boehringer Mannheim). Chemilumigraphs were produced with Fuji film and analyzed by densitometer.
Renal Membrane Fraction Extraction
Rat kidneys were quickly removed, decapsulated, and cooled in ice-cold PBS. The cortex, the outer medulla, and the inner medulla were dissected out, finely minced on a chilled petri dish, and homogenized separately in a lysis buffer (20 mmol/L HEPES buffer, pH 7.4, 0.05 mmol/L sodium vanadate, 1 mmol/L EDTA, 5 mmol/L EGTA, 2 mmol/L DTT, 1 mmol/L PMSF, 10 pg/L aprotinine, 10 pg/L leupeptine, 5 mmol/L β-glycerophosphate) by five strokes in a 5-mL Teflon glass homogenizer with the piston rotating at 1000 rpm. The homogenate was centrifuged at 5000g for 15 minutes at 4°C, and the resulting supernatant was further centrifuged at 20 000g for 30 minutes as reported elsewhere with little modification.25 The resulting membrane fraction pellet was resuspended into 50 to 100 μL of lysis buffer, and a portion was taken for protein assay by spectrophotometer using BSA for standards.
Samples of 6 μg protein in an SDS buffer (0.5% bromophenol blue, 5% 2-mercaptoethanol, 12 mg/L DTT, 10% glycerol, 3% SDS, 0.5 mol/L Tris, pH 6.8) were resolved on a gradient SDS-polyacrylamide gel (Daiichi Pure Chemicals Co) using a discontinuous buffer electrophoresis system. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore Corp) with a semidry blotting apparatus. The membrane was cut into several strips and blocked in Tris-buffered saline solution, pH 7.6, containing 0.1% Tween-20 (TBS-T) and 5% nonfat dry milk at room temperature for 1 hour. The membrane strips were incubated at 4°C overnight with an anti-rat ECE-1 monoclonal antibody (Sankyo Co) diluted 1:1000 with 5% milk TBS-T and subsequently incubated with a sheep anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham Life Science) for 1 hour at room temperature. Blots were washed in TBS-T for 15 minutes once, then for 5 minutes twice after blocking and after incubation with each antibody. Immunological bands were visualized by an enhanced chemiluminescence (ECL) detection system (Amersham Life Science). Chemilumigraphs produced with Fuji medical x-ray film were analyzed by densitometer.
Immunoblotting for Glomerular ECE-1
Microdissected glomeruli were dissolved in 110 μL lysis buffer, and the protein was processed for immunoblot after ethanol precipitation.
Data are presented as mean±SEM unless otherwise stated. Significance of difference in mean values was evaluated by the Wilcoxon rank-sum test. Values of P<.05 were considered significant.
SHR were studied before and after development of hypertension. At 4 weeks, SHR and WKY had similar blood pressure (109±3 versus 109±4 mm Hg, n=8, NS), but as shown in Fig 1⇓, SHR had higher systolic blood pressure than WKY (137±9 versus 231±12 mm Hg, n=8, P<.05) at 12 weeks of age.
ECE-1 mRNA Expression in the Glomeruli
The amplification product of reverse-transcribed ECE-1 mRNA was detected as a single band of 389 bp, concordant with the predicted size. Southern blot confirmed the nature of the PCR product. When the sample was not reverse transcribed, PCR did not yield any detectable band. RT combined with competitive PCR analysis of ECE-1 mRNA in glomeruli showed that the expression of ECE-1 mRNA in both superficial and juxtamedullary glomeruli of 4-week-old SHR and that in age-matched WKY were comparable (Fig 2A⇓). At 12 weeks, however, SHR showed significantly higher expression of ECE-1 mRNA in both superficial and juxtamedullary glomeruli (Fig 2B⇓).
ECE-1 Protein Level in the Glomeruli
Fig 3⇓ shows the ECE-1 protein level in glomeruli of 4- and 12-week-old SHR and WKY by Western blot. At 4 weeks, ECE-1 protein level was not significantly different between SHR and WKY; at 12 weeks, SHR had significantly higher glomerular ECE-1 protein level.
ECE-1 mRNA Expression in the Renal Tubule
In 4-week-old SHR, the expression of ECE-1 mRNA in renal tubules was significantly increased in PST, MTAL, CTAL, and IMCD (Fig 4A⇓). In 12-week-old rats, the pattern of ECE-1 expression was similar, but ECE-1 mRNA was no more significantly increased in CTAL and in IMCD (Fig 4B⇓). Both SHR and WKY had low expression of ECE-1 in PCT. Although ECE-1 mRNA expression in PCT of SHR was not statistically different from that of WKY, a trend toward lower ECE-1 mRNA was observed in the PCT of SHR.
Renal ECE-1 Protein Level Distribution
Western blot analysis of renal membrane fractions from 4- and 12-week-old SHR and WKY with anti-rat ECE-1 monoclonal antibody demonstrated a single band of 125 kD. Fig 5⇓ shows the ECE-1 protein level distribution in kidney at 4 and 12 weeks in both SHR and WKY by Western blot. We found that compared with age-matched WKY, ECE-1 protein level was significantly higher in both the inner and outer medulla of SHR at 4 weeks but not at 12 weeks of age.
PreproET-1 mRNA Expression in the Glomeruli
Because preproET-1 is a substantial determinant of ET-1 synthesis, we also studied preproET-1 mRNA expression. After RT-PCR, preproET-1 cDNA was detected at the expected 471-bp size. As shown in Fig 6⇓, analysis of glomerular preproET-1 mRNA by RT-PCR in both 4- and 12-week-old WKY and SHR showed no difference between the two strains.
To our knowledge, this is the first report on renal expression of ECE-1 gene in an animal model of hypertension. We observed that glomerular ECE-1 expression of 12-week-old SHR was higher than that of WKY, but that of 4-week-old SHR was comparable to that of age-matched WKY. We also found that steady state ECE-1 mRNA was more abundantly expressed in the tubule segments of both 4- and 12-week-old SHR than in those of age-matched WKY.
To assess whether this increase in ECE-1 mRNA also resulted in an increase of protein synthesis, ECE-1 protein was also determined. We analyzed ECE-1 protein level in both the superficial and juxtamedullary glomeruli by Western blot. We found that at 4 weeks, glomerular ECE-1 protein level of SHR was not yet significantly different from that of WKY rats. But at 12 weeks, ECE-1 protein level in superficial as well as in juxtamedullary glomeruli was higher in SHR. High ECE-1 transcripts resulted into a high protein level. We observed that ECE-1 mRNA expression was increased in renal tubule segments in SHR and also found that the ECE-1 protein level in the outer and inner medulla was high at 4 weeks in SHR. Determination of ECE-1 protein levels in renal tubule segments was not performed because it would have required too great a length of tubular segments, but it is likely that the high expression of ECE-1 mRNA in the renal tubule segments of SHR was also translated into ECE-1 protein to contribute to the high ECE-1 protein levels observed in both the outer and inner medulla of young SHR.
It was surprising to find that ECE-1 protein level in the overall cortex of SHR was comparable to WKY at 12 weeks, while glomerular ECE-1 level was rather high in 12-week-old SHR. This observation suggests that within the renal cortex of SHR, some structures have rather low ECE-1 expression. This is supported by the trend toward lower ECE-1 mRNA expression in the PCT of SHR. We consider that the increase in the expression of ECE-1 gene in SHR may be tissue-specific, involving certain segments of the nephron among various structures of the kidney.
ECE-1 mRNA in PST and MTAL was consistently high in both 4- and 12-week-old SHR, and ECE-1 gene expression increased in glomeruli of 12-week-old SHR. This change of ECE-1 gene expression might be induced by high blood pressure or related to other changes induced by aging. However, high ECE-1 expression in glomeruli or renal tubule has some pathophysiological potential.
First, the pathophysiological implication of a high tubular ECE-1 level has to be considered. Localization of ET-1 binding sites in rat kidney,26 detection of ETB receptor mRNA in renal tubules,20 and a physiological effect of ET-1 on fluid transport27 28 29 support a direct action of ET-1 on renal tubules. Young and adult SHR had high ECE-1 mRNA expression in renal tubules. Increased ECE-1 expression in the renal tubules will yield abundant local ET-1 synthesis, a condition promoting sodium retention. Indeed, pathological levels of ET-1 as found in either hypertension or renal failure are antinatriuretic.7 9 Either ET-1 specific antibodies or ET-1 receptor antagonists improved renal function and sodium excretion in SHR.15 16 Moreover, the administration of ET-1–specific antibodies to rats on a salt-restricted diet induced natriuresis.6 However, some reports indicated a natriuretic effect of ET-1. A pharmacological dose of ET-1 induced natriuresis in rats.5 ET-1, 10−8 to 10−10 mol/L, was natriuretic in CCD28 and diuretic in IMCD27 with use of the isolated tubule perfusion method. Following these reports about the natriuretic effect of ET-1, the biphasic effect of ET-1 on fluid absorption in the proximal straight tubule has been demonstrated, with 10−12 to 10−13 mol/L ET-1 increasing fluid reabsorption in the PST and 10−8 to 10−9 mol/L decreasing it.29 The local concentration of ET-1 in the tissue is not known, but in physiological or pathological conditions, ET-1 plasma concentration is in a 10−12 to 10−13 mol/L range in rats30 or humans10 31 and in a 10−11 to 10−12 mol/L range in ET-1–overexpressing rats.32 In addition, ET-1 concentration in renal perfusate is estimated to be in a 10−13 mol/L range in SHR.13 Altogether, these data support the view that ET-1 is antinatriuretic in a physiological or pathological concentration range and natriuretic in a high pharmacological concentration range. Proximal tubule and thick ascending limb play a major role in sodium reabsorption. PST and MTAL, with increased ECE-1 gene expression both in 4- and 12-week-old SHR, appear to be fitting targets of ET-1–induced sodium reabsorption.
Second, the pathophysiological implication of high glomerular ECE-1 level has to be considered. Twelve-week-old SHR had higher glomerular ECE-1 expression. Because no difference was found between SHR and WKY for glomerular preproET-1 mRNA, the rate of glomerular ET-1 synthesis is likely increased in SHR. ET-1 exerts a predominant vasoconstriction on the afferent arteriole with less effect on the efferent arteriole.33 It also induces the contraction of glomerular mesangial cells.34 By decreasing GFR in an autocrine or paracrine fashion, increased ET-1 will cause sodium retention and may contribute to the increase of blood pressure in SHR. Vasoconstriction at the juxtamedullary glomerulus level would not particularly influence the whole kidney GFR because juxtamedullary glomeruli make up only a limited amount of the population of glomeruli. However, blood flow to the renal medulla is supplied primarily from efferent arterioles of juxtamedullary nephrons. A decrease of medullary blood flow will favor sodium retention,35 36 but we do not know yet how important ET-1 is in regulating medullary blood flow, which is subject to a complex interplay of substances such as nitric oxide, angiotensin II, arginine-vasopressin, kinins, and prostaglandins.37 Although it is not clear whether the increase of glomerular ECE-1 expression observed in 12 week-old SHR is either a primary or a secondary phenomenon, this increase may potentially contribute to the maintenance of hypertension.
From both of the above considerations, increased ECE-1 expression has a potential to promote sodium retention via ET-1 by increasing tubular reabsorption, decreasing GFR, and medullary blood flow. These mechanisms may operate either separately or in various combinations.
The importance of ET-1 in modulating the blood pressure in SHR is demonstrated by the effectiveness of endothelin-specific antibodies to decrease the blood pressure in this strain.15 Similarly, the blockade of ECE-1 with specific inhibitors should decrease the blood pressure in SHR. Phosphoramidon, an ECE-1 inhibitor, was effective in reducing the blood pressure in SHR in one study38 but not in another.39 In both studies, pressor response to exogenous big ET-1 was abolished by phosphoramidon. ECE-1 is on the cell surface as well as in the cell secretory pathway into which access to phosphoramidon is limited.19 The incomplete blockade of intracellular ECE-1 can explain these conflicting results. In the present study, the change of glomerular ECE-1 gene expression in the presence of unchanged glomerular preproET-1 expression indicates that the ECE-1 gene could be important in the regulation of ET-1 synthesis in SHR. An absence of upregulation of preproET-1 gene expression in the glomeruli of 12-week-old SHR could also mean that major glomerular injury had not yet occurred in these rats, since preproET-1 gene expression is increased in the presence of glomerular injury.40
Our results demonstrate that the ECE-1 gene is differentially expressed in the kidney of SHR, especially at the nephron level. SHR have increased ECE-1 expression in some tubule segments at 4 and 12 weeks of age, and also in the glomeruli at 12 weeks. This high ECE-1 expression implies an increase of ET-1 synthesis in nephron segments that will promote sodium retention to contribute to the development and/or maintenance of hypertension. Our study did not address the mechanism by which ECE-1 gene expression is increased in SHR. At which level the transcription is regulated remains to be studied. Furthermore, because differential gene expression could be either a cause of hypertension or secondary to high blood pressure, or could be just one of the strain differences, genomic linkage analysis is important in drawing a final conclusion regarding a potential role for ECE-1 gene in the pathogenesis of hypertension in SHR.
Selected Abbreviations and Acronyms
|CCD||=||cortical collecting duct|
|CTAL||=||cortical thick ascending limb of Henle’s loop|
|ECE||=||endothelin converting enzyme|
|GFR||=||glomerular filtration rate|
|IMCD||=||inner medullary collecting duct|
|MTAL||=||medullary thick ascending limb of Henle’s loop|
|OMCD||=||outer medullary collecting duct|
|PCT||=||proximal convoluted tubule|
|PST||=||proximal straight tubule|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by a grant-in aid for scientific research (08671291, 07457242, and 06671133), a grant-in aid for the development of scientific research (B1, 05557053 and 05557054), and a grant-in aid from Houn-sha Foundation from the Japanese Ministry of Education, Science, and Culture. We also are indebted to Professor Dr Tatsuo SATO for his helpful support.
Correspondance to Tumba Disashi, MD, Third Department of Internal Medicine, Kumamoto University School of Medicine, 1–1-1 Honjo, Kumamoto 860, Japan.
- Received May 27, 1997.
- Revision received June 16, 1997.
- Accepted July 9, 1997.
Harrap S, Doyle A. Renal haemodynamics and total body sodium in immature spontaneously hypertensive and Wistar-Kyoto rats. J Hypertens. 1986;4(suppl 3):S249–S252.
King A, Brenner B, Anderson S. Endothelin: a potent renal and systemic vasoconstrictor peptide. Am J Physiol. 1989;256:F1051–F1058.
Yamada K, Yoshida S. Role of endogenous endothelin on renal functions in rats. Am J Physiol. 1991;260:F34–F38.
Lerman A, Hildebrand F, Aarhus L, Burnett J. Endothelin has biological actions at pathophysiological concentrations. Circulation. 1991;83:1808–1814.
Shichiri M, Hirata Y, Ando K, Emori T, Ohta K, Kimoto S, Ogura M, Inoue A, Marumo F. Plasma endothelin levels in hypertension and chronic renal failure. Hypertension. 1990;15:493–496.
Hughes A, Cline R, Kohan D. Alterations in renal endothelin-1 production in the spontaneously hypertensive rat. Hypertension. 1992;20:666–673.
Kato T, Kassab S, Wilkins FJ, Kirchner K, Keiser J, Granger J. Endothelin antagonists improve renal function in spontaneously hypertensive rats. Hypertension. 1995;25:883–887.
Abassi Z, Klein H, Golomb E, Keiser H. Urinary endothelin: a possible biological marker of renal damage. Am J Hypertens. 1993;6:1046–1054.
Shimada K, Takahashi M, Tanzawa K. Cloning and functional expression of endothelin-converting enzyme from rat endothelial cells. J Biol Chem. 1994;269:18275–18278.
Terada Y, Tomita K, Nonoguchi H, Marumo F. Different localization of two types of endothelin receptor mRNA in microdissected rat nephron segments using reverse transcription and polymerase chain reaction assay. J Clin Invest. 1992;90:107–112.
Morel F, Chabardes D, Imbert-Teboul M. Methodology for enzymatic studies of isolated tubular segment: adenylate cyclase. Methods Pharmacol. 1978;4B:297–323.
Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl Acids Res. 1988;16:7351–7367.
Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A.. 1990;87:2725–2729.
Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T, Tomita K. Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. J Clin Invest. 1995;96:1768–1778.
Oishi R, Nonoguchi H, Tomita K, Marumo F. Endothelin-1 inhibits vasopressin-stimulated osmotic water permeability in inner medullary collecting duct of rat. Am J Physiol. 1991;261:F951–F956.
Tomita K, Nonoguchi H, Terada Y, Marumo F. Effects of ET-1 on water and chloride transport in cortical collecting ducts of the rat. Am J Physiol. 1993;264:F690–F696.
Garcia NH, Garvin JL. Endothelin’s biphasic effect on fluid absorption in the proximal straight tubule and its inhibitory cascade. J Clin Invest. 1994;93:2572–2577.
Kohno M, Murakawa K, Horio T, Yokokawa K, Yasunari K, Fukui T, Takeda T. Plasma immunoreactive endothelin-1 in experimental malignant hypertension. Hypertension. 1991;18:93–100.
Loutzenhiser R, Epstein M, Hayashi K, Horton C. Direct visualization of effects of endothelin on the renal microvasculature. Am J Physiol. 1990;258:F61–F68.
Badr KF, Murray JJ, Breyer MD, Takahashi K, Inagami T, Harris RC. Mesangial cell, glomerular and renal vascular responses to endothelin in rat kidney: elucidation of signal transduction pathways. J Clin Invest. 1989;83:336–342.
Lu S, Mattson DL, Cowlery AW Jr. Renal medullary captopril delivery lowers blood pressure in spontaneously hypertensive rats. Hypertension. 1994;23:337–345.
Larson TS, Lockhart JC. Restoration of vasa recta hemodynamics and pressure natriuresis in SHR by l-arginine. Am J Physiol. 1995;268:F907–F912.
Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard LM, Mitchell KD. Paracrine regulation of the renal microcirculation. Physiol Rev. 1996;76:425–536. Review.
McMahon E, Palomo M, Moore W. Phosphoramidon blocks the pressor activity of big endothelin[1–39] and lowers blood pressure in spontaneously hypertensive rats. J Cardiovasc Pharmacol. 1991;17(suppl 7):S29–S33.