(Hypertension. 1996;27:1140-1148.)
© 1996 American Heart Association, Inc.
Articles |
Effects of Cyclosporin A on the Synthesis, Excretion, and Metabolism of Endothelin in the Rat
From the Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md, and Istituto di Clinica Medica e Terapia Medica, Universita' di Milano, and Centro di Fisiologia Clinica e Ipertensione, Ospedale Maggiore, Milan, Italy (F.P.).
Correspondence to Harry R. Keiser, MD, Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, Building 10, Room 8C103, 10 Center Dr, MSC 1754, Bethesda, MD 20892-1754.
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Abstract |
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Abstract Increasing evidence suggests that endothelin, a potent vasoconstrictor, is implicated in cyclosporin A (CsA)–induced nephrotoxicity. Increased levels of urinary and circulating endothelin have been described in CsA-treated humans and animals. The exact mechanisms by which CsA induces these increases are still unknown, and no data indicate whether these elevated levels reflect increased synthesis or decreased clearance of endothelin. In the present study, we investigated the effects of CsA administration (50 mg/kg per day IP for 6 days) to rats on plasma and urinary levels of endothelin; expression of endothelin-1 (ET-1), ET-3, and endothelin-converting enzyme in renal tissue; clearance of infused 125I–ET-1; and degradation of 125I–ET-1 by recombinant neutral endopeptidase. Rats given CsA for 6 days developed severe renal insufficiency, as shown by a 74% decrease in creatinine clearance rate (Ccr) (P<.006). Ccr was remarkably improved in CsA-treated rats that received bosentan, the combined antagonist of both endothelin A and endothelin B receptors. Urinary excretion of endothelin increased from an undetectable level to 31.7±6.0 pg/24 h (P<.001), and plasma levels of endothelin were unchanged (2.8±0.2 to 3.1±0.2 pg/mL). Reverse transcription followed by quantitative polymerase chain reaction revealed that ET-1 mRNA in the renal medulla increased by 59% (P<.006), whereas the expression of both ET-3 and endothelin-converting enzyme was unchanged. In other rats, neither acute nor chronic treatment with CsA affected either the clearance of 125I–ET-1 from the blood or the renal and pulmonary uptake of the peptide. Moreover, CsA did not affect the degradation of 125I–ET-1 by highly purified recombinant neutral endopeptidase, a well-known endothelinase. Taken together, these data suggest that the elevated urinary endothelin levels obtained after CsA treatment originate from the kidney and reflect increased renal synthesis of ET-1. Moreover, the production of endothelin appears to be regulated at the mRNA transcription level, and expressions of ET-1 and ET-3 are regulated independently.
Key Words: endothelins • metabolic clearance rate • cyclosporine • endopeptidase
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Introduction |
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The immunosuppressive agent CsA is widely used to prevent allograft rejection. However, it has adverse side effects, such as acute and chronic nephrotoxicity and hypertension, that hamper its clinical use.1 2 The precise mechanisms by which CsA contributes to these phenomena are not well characterized.3 4 In the kidney, CsA induces contraction and hyperplasia of afferent arterioles, leading to decreases in renal blood flow and GFR and increases in renal vascular resistance.5 6 7 CsA-induced vasoconstriction has been attributed to several factors, including activation of the sympathetic nervous system,8 activation of the renin-angiotensin system,9 impairment of nitric oxide–mediated vasodilation,10 11 increased calcium uptake by smooth muscle cells,12 and even endothelial injury.13
The discovery of endothelin, a 21–amino acid vasoconstrictor peptide,14 has provided further insight into the mechanisms of CsA-induced nephrotoxicity and vasoconstriction. Endothelin is generated from an intermediate precursor composed of 38 to 39 amino acids, big endothelin, through a unique proteolytic cleavage of Trp21-Val22 by a putative ECE. The kidney, mainly the medulla,15 is both a major site of endothelin production and very susceptible to the actions of endothelin. Since endothelin and CsA exert similar changes in the renal circulation, it has been suggested that endothelin mediates CsA-induced nephrotoxicity. CsA administration to renal transplant patients16 or normal rats17 results in increased UETV and is accompanied by elevated blood pressure and impaired renal function. Furthermore, elevated PET has been reported in CsA-treated patients18 19 and experimental animals.20 However, there is great uncertainty regarding the mechanisms by which CsA stimulates endothelin release. Although such stimulation may reflect increased endothelin synthesis, it could also be due to impairment in endothelin clearance and degradation. The latter became more likely in light of the finding that CsA inhibits endothelin degradation by rat kidney membranes.21 Therefore, the aims of the present study were to examine the effects of CsA on (1) the expression of ECE, ET-1, and ET-3 in the kidneys of CsA-treated rats; (2) the clearance of labeled endothelin in vivo; and (3) the degradation of labeled endothelin by highly purified NEP, a key enzyme in endothelin metabolism.22
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Methods |
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Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Harlan Farms, Indianapolis, Ind) weighing 300 to 350 g were used and given standard commercial rat chow and tap water ad libitum.
In Vivo Studies
Effects of CsA Treatment on UETV
A group of six rats was housed in individual
metabolic cages and treated with CsA (50 mg/kg per day IP,
Sandoz Pharmaceutical Corp) for 6 days. Urine samples were collected
daily into tubes containing 100 µL of 6N HCl and stored at
-20°C until analyzed. After CsA treatment, rats were
decapitated and their blood was collected into precooled tubes
containing potassium EDTA (3 mg/mL) and aprotinin (500 000 IU/mL).
Blood samples were immediately centrifuged (4°C, 3000 rpm),
and plasma was removed and stored at -20°C. Urine and plasma
samples were assayed for ET-1, creatinine, and sodium
concentration. Plasma renin activity was determined in some plasma
samples (see below). A group of control rats (n=6) received an
intraperitoneal dose (0.4 mL) of olive oil daily
for 6 days.
Effects of CsA Treatment on Expression of ECE, ET-1, and ET-3 in
the Renal Medulla
The effects of chronic CsA administration on mRNA levels
of ECE, ET-1, and ET-3 in the renal cortex and medulla were examined.
Control and CsA-treated rats were decapitated and their kidneys removed
and immediately placed in liquid nitrogen. Kidneys were bisected, and
the lower two thirds of the medulla and the outer cortex were
dissected. RNA was extracted separately from the renal cortex and
medulla as described by Chomczynski and Sacchi23 with a
commercial solution (RNAzol B, Tel-Test Inc) and quantified by
spectrophotometry. Quantitative RT followed by quantitative PCR were
performed as follows.
ECE cDNA was synthesized from 2 µg total RNA with the specific primer 5'-GGTCTCAAAATCCAGGATCCTGC-3' (bases 867-888, see Reference 2424 ) synthesized at the National Institutes of Health, Bethesda, Md. Avian myeloblastosis virus reverse transcriptase (AMV-RT, 8 U per reaction; Promega) was used for RT, along with the reaction mixture recommended by the enzyme manufacturer, in a volume of 20 µL. PCR was then performed with the use of 2 µL of the resulting cDNA and a GeneAmp kit (Cetus Perkin-Elmer), with the upstream primer 5'-TGACCAGCTCCATCCTAAACTCC-3' (bases 272-294, see Reference 2424 ) and the downstream primer used for RT. Each PCR reaction mixture contained 200 µmol/L dATP, dGTP, and dTTP; 100 µmol/L "cold" dCTP; and 0.8 µCi 32P-labeled dCTP (NEN). Primers were chosen to span introns that would distinguish by size PCR products derived from cDNA from those derived from genomic DNA contaminants. In a preliminary study, we found that a minimum of 23 PCR cycles was necessary to obtain a visible product on an acrylamide gel and that the quantity of product available after 23 PCR cycles was directly proportional to the amount of cDNA used. In contrast, mRNA levels of ECE in the renal cortex were undetectable after 30 cycles of amplification. After an initial denaturation step at 94°C for 3 minutes, cycles of annealing at 56°C for 1 minute, elongation at 72°C for 1 minute, and denaturation at 94°C for 1 minute were performed with 10% of the cDNA described above. The expected size of the ECE PCR product was 618 bp.
ET-1 cDNA was synthesized from 2 µg total RNA with the specific primer 5'-AAGATCCCAGCCAGCATGGAGAGCG-3' (bases 675-699, see Reference 2525 ). PCR was then performed with the use of 2 µL of the resulting cDNA and a GeneAmp kit, with the upstream primer 5'-CGTTGCTCCTGCTCCTCCTTGATGG-3' (bases 157-182, see Reference 2525 ) and the downstream primer used for RT. In a preliminary study, we found that a minimum of 25 PCR cycles was necessary to obtain a visible product on an acrylamide gel for the renal medulla and that the quantity of the product available after 25 PCR cycles was directly proportional to the amount of cDNA used. In contrast to the renal medulla, ET-1 mRNA levels in the renal cortex were undetectable after 30 cycles of amplification. After an initial denaturation step at 94°C for 3 minutes, cDNA was amplified as described above. The cDNA amplification product was predicted to be 543 bp in length.
ET-3 cDNA was synthesized from 2 µg total RNA with the specific primer 5'-GCTGGTGGACTTTATCTGTCC-3' (bases 479-499, see Reference 2525 ). PCR was then performed with the use of 2 µL of the resulting cDNA and a GeneAmp kit, with the upstream primer 5'-TTCTCGGGCTCACAGTGACC-3' (bases 23-42, see Reference 2525 ) and the downstream primer used for RT. In a preliminary study, we found that a minimum of 25 PCR cycles was necessary to obtain a visible product on an acrylamide gel for the renal medulla and that the quantity of the product available after 25 PCR cycles was directly proportional to the amount of cDNA used. Similar to ET-1 mRNA, ET-3 mRNA levels were not detectable in the renal cortex after 30 cycles of cDNA amplification. After an initial denaturation step at 94°C for 3 minutes, cDNA was amplified as described above. The predominant cDNA amplification product was predicted to be 477 bp in length.
The RT-PCR product of the gene encoding ß-actin served as a quantity control. The ß-actin primers were upstream, 5'-GGTATGGGTCAGAAGGACTCC-3' (bases 138-156) and downstream, 5'-TGATCTTCATGGTGCTAGGAGCC-3' (bases 960-981) and were designed to span an 844-bp region that contains an intron in the genomic structure. Negative controls for the PCR reaction included tubes lacking either template or AMV-RT.
Eight microliters of the PCR product was electrophoresed on a 4% to 20% Tris-glycine gel (Novex). The resulting gel was exposed to x-ray film for several hours until clear bands were visible.
ECE, ET-1, ET-3, and ß-actin mRNAs were quantified by densitometric analysis (Image 1.55, National Institutes of Health), and the ratios of ECE, ET-1, or ET-3 to ß-actin were calculated for each rat.
Effects of CsA Treatment on Clearance of Labeled
Endothelin
To evaluate the effects of acute or chronic CsA treatment on the
clearance of labeled ET-1, we performed the following experimental
protocols.
For acute studies, rats (n=5) were anesthetized with 100 mg/kg thiobutabarbital IP (Byk-Gulden) and prepared for clearance studies. After tracheotomy, polyethylene catheters (PE-50) were inserted into the jugular vein for infusion of various solutions and into the carotid artery for periodic blood sampling. The urinary bladder was catheterized via a suprapubic incision for urine collection. A solution of 0.9% NaCl was infused at the rate of 1.5 mL/h throughout the experiment. The rats were given a loading dose of CsA (50 mg/kg) over 10 minutes followed by a constant infusion of the drug (50 mg/kg per hour) throughout the rest of the experiment. Then the rats were given a single intravenous injection of 1 µCi of 125I-labeled ET-1 (labeled at Tyr13, 2000 Ci/mmol, Amersham), and the catheter was flushed rapidly with 0.2 mL saline. Arterial blood samples, each 200 µL, were obtained at 0.5, 1, 2, 3, 5, 10, 20, 45, and 60 minutes after administration of the labeled peptide. Urine was collected for two consecutive 30-minute periods. At the end of the experiment, the kidneys and lungs were removed for determination of the radioactivity in these organs as well as in the collected urine. Blood samples were centrifuged, and 100-µL plasma samples were treated with 500 µL of 10% TCA at 4°C and centrifuged. The supernatants were removed and the pellets resuspended in 500 µL TCA and recentrifuged. Radioactivity in either the combined supernatants (TCA-soluble) or the pellet (TCA-precipitable) was measured with a gamma counter (Beckman Instruments). TCA-soluble radioactivity was shown by high-performance liquid chromatography (HPLC) to be mainly free 125I or hydrolytic products, whereas TCA-precipitable radioactivity was mainly intact 125I-labeled ET-1.22 Normal rats (n=5) that were not treated with CsA served as controls.
For chronic studies, five rats were placed in individual metabolic cages and each rat received CsA (50 mg/kg per day IP) for 6 days. On the day of the experiment, rats were anesthetized and prepared for pharmacokinetic studies. The clearance of labeled ET-1 by these rats was performed as described above. Rats that received a daily dose of olive oil served as controls (n=5) for this experimental group.
For pharmacokinetic analysis, we used the program MKMODEL (version 4.40; Holford, N Biosoft) to estimate pharmacokinetic parameters for each rat. For all rats, the last portion (10 to 60 minutes) of the plasma disappearance curve of TCA-precipitable radioactivity, which represents mostly entrapped free iodine, was not used for calculation of the pharmacokinetic parameters. These were calculated from the plasma disappearance curve of TCA-precipitable 125I radioactivity by use of a two-compartment model,26 with parameterization of the model as follows:
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where V is the volume of the central compartment,
L1 is the distribution or rapid disposition
constant, L2 is the elimination or slow
disposition constant, and Ex2 is
L1xL2xV/clearance. The
half-life (t
) of the rapid distribution phase
can be calculated as
t=log(2)/L1. The
L2 parameter was shown to be
significantly different from zero (P=.0001), which implies
that a two-compartment model is more appropriate than a
one-compartment model.
Effects of Endothelin Receptor Blockade on GFR in CsA-Treated
Rats
To evaluate the role of endothelin in CsA-induced renal
hypofiltration, we performed the following experiment. Six rats were
identically treated with CsA as described above; however, they also
received a combined ETA/ETB receptor
antagonist, bosentan (kindly provided by Dr Martin Clozel,
Hoffmann–La Roche, Basel, Switzerland), at a daily oral doze of 100
mg/kg with both powdered chow and drinking water beginning 2 days
before CsA administration and continuing throughout the experiment.
This dose was shown to prevent ET-1– and big ET-1–induced
vasoconstriction.27 Urine samples were collected daily as
described above. After bosentan treatment, rats were decapitated, their
blood was collected immediately and centrifuged, and plasma was
removed and stored at -20°C. Urine samples were assayed for
creatinine.
In Vitro Studies
Effect of CsA on Hydrolysis of Labeled ET-1 by rNEP
rNEP was kindly provided by Khepri Pharmaceuticals, Inc. The
full-length cDNA coding for human NEP was cloned originally from a
human placental cDNA library,28 and transfection of
mammalian cells with an expression plasmid containing this cDNA
resulted in the production of the enzymatically active
protein,29 which was purified by modification of a
standard procedure and quantified in terms of protein catalytic
activity. To evaluate the effects of CsA on the degradation of ET-1 by
rNEP, we performed the following protocol. 125I-labeled
ET-1 (0.5 µCi, labeled at Tyr13, 2000 Ci/mmol) was
incubated with 1 µg of the rNEP at 37°C in 1 mL of a solution of 50
mmol/L HEPES, pH 7.4, and 50 mmol/L NaCl for 120 minutes. Either CsA,
at concentrations of 20 or 100 µmol/L, or SQ-28,603
(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]ß-alanine;
Bristol-Myers Squibb Pharmaceuticals), at a concentration of 100
µmol/L, was added to the solution before the enzyme. Each reaction
was terminated by the addition of acetic acid to a final pH of 4.0 and
heating at 60°C for 3 minutes. Fifty microliters of each reaction
mixture was analyzed by HPLC (C18 µBondapak, 3.9x300-mm
column, Waters Associates) with a linear gradient of 10% to 60%
acetonitrile in 0.1% TCA over 30 minutes at a flow rate of 1 mL/min.
The column was calibrated with 125I-labeled ET-1.
Radioactivity eluting from the column was monitored with a gamma
counter.
Analytic Methods
The sodium concentration in urine was measured by an
ion-selective electrode (Synchron EL-ISE, Electrolyte System,
Beckman Instruments). Creatinine in either plasma or urine
was measured by a modification of the Jaffe reaction with the
Roche-Cobas-Mira analyzer. PET and UETV
were determined by radioimmunoassay with a specific kit for ET-1 and
ET-2 (Amersham). Plasma renin activity was determined by Endocrine
Sciences.
Data were analyzed statistically by either Student's t test for unpaired values or ANOVA for repeated measurements followed by Fisher's post hoc test. A value of P<.05 was considered significant.
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In Vivo
Renal and Hormonal Status
Rats that received CsA for 6 days lost 15% of their body weight. Urinary sodium excretion decreased significantly (Table 1
) and urine volume was unchanged.
Creatinine clearance declined 74% in CsA-treated rats (Fig 1). This severe renal insufficiency was accompanied by a
remarkable increase in UETV from nondetectable pretreatment
levels to 31.7±6.0 pg/d. In contrast, PET remained
unchanged after CsA treatment. Plasma renin activity was significantly
higher in CsA-treated than in control rats. Creatinine
clearance in rats that received CsA or CsA plus bosentan is shown in
Fig 1. In control rats, creatinine clearance was 1.05±0.2
mL/min and significantly decreased to 0.27±0.05 mL/min in CsA-treated
rats. In bosentan-treated rats, creatinine clearance
significantly increased to 0.50±0.03 mL/min.
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Expression of ECE, ET-1, and ET-3
The effects of chronic CsA treatment on the expression of ECE,
ET-1, and ET-3 are depicted in Fig 2. Single bands of
the expected lengths for ECE (618), ET-1 (543), ET-3 (477), and the
internal control ß-actin (840) were detected by PCR amplification
(data not shown). ET-1 mRNA levels in the renal medulla of CsA-treated
rats (ratio of ET-1 mRNA to ß-actin mRNA: 2.0±0.14) were
significantly (P=.005) higher than those in control rats
(ratio of ET-1 mRNA to ß-actin mRNA: 1.3±0.10). CsA did not
affect the mRNA levels of ECE or ET-3 in the renal medulla (Fig 2). In
contrast, in the renal cortex, mRNA levels of ET-1 and ET-3 were
undetectable after 30 cycles of amplification. Moreover, ECE mRNA
levels in the renal cortex were not significantly different between
CsA-treated and control rats (data not shown).
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Clearance of Labeled ET-1
Fig 3 depicts the effects of both acute and chronic
CsA treatment on the clearance of 125I–ET-1. In control
rats, the initial plasma levels of intact 125I–ET-1, ie,
TCA-precipitable radioactivity, decreased in a time-dependent
manner, reaching a plateau about 5 minutes after injection of the
peptide (Fig 3a and 3b). Neither acute (Fig 3a) nor chronic (Fig 3b)
CsA treatment affected the disappearance rate of the intact peptide
from plasma. Furthermore, CsA given acutely or chronically had no
effect on either the volume of distribution, metabolic
clearance rate, or half-life of 125I–ET-1 (Table 2).
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About 3% and 12% of the radioactivity infused into control rats were
recovered in the kidneys and lungs, respectively. These values were not
altered by either acute or chronic CsA treatment (Fig 4a and 4b), suggesting that CsA does not influence either the
renal or pulmonary uptake of endothelin.
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Only a small amount of the total radioactivity injected as
125I–ET-1 was recovered in the urine of control rats, that
is, 0.06±0.03% in the first period and 0.14±0.05% in the second
period (Fig 5a). In rats acutely treated with CsA,
urinary radioactivity, 0.06±0.01% in the first period and
0.07±0.03% in the second period, was not significantly different from
that of controls. In contrast, urinary radioactivity in chronically
CsA-treated rats decreased significantly (P<.003), from
0.07±0.003% and 0.22±0.04% in the first and second periods,
respectively, in rats given the vehicle alone to 0.003±0.001% and
0.015±0.005%, respectively (Fig 5b). The reduction in urinary
radioactivity after chronic CsA treatment might be attributed to the
impaired Ccr (Table 1
). Nevertheless, HPLC analysis revealed
that free iodine accounted for all the radioactivity in the urine of
both control and CsA-treated rats, suggesting that CsA did not affect
the degradation of 125I–ET-1.
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In Vitro
Degradation of Labeled ET-1 by rNEP
125I–ET-1 eluted with a retention time of 23 minutes
(Fig 6a). Incubation of 125I–ET-1 with 1
µg/mL rNEP for 120 minutes resulted in total degradation of the
peptide (Fig 6b). CsA at both low (20 µmol/L) and high (100 µmol/L)
concentrations did not affect the cleavage of ET-1 by rNEP (Fig 6c and 6d), and SQ-28,603, a specific NEP inhibitor, fully
protected ET-1 from degradation (Fig 6e). These findings clearly
demonstrate that CsA does not inhibit the degradation of ET-1 by NEP, a
well-known endothelinase.
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Discussion |
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The present study demonstrates that CsA (1) induces a significant increase in UETV but not PET, (2) increases the concentration of ET-1 mRNA but not that of ECE or ET-3 in renal medulla, (3) does not affect the clearance of endothelin or the renal and pulmonary uptake of the peptide, and (4) has no effect on the degradation of ET-1 by rNEP.
The possibility that endothelin is involved in CsA-induced nephrotoxicity has gained special interest in the last few years because of the following observations: (1) CsA-induced glomerular hypoperfusion is associated with a remarkable increase in UETV16 17 and in some cases in PET19 ; (2) CsA stimulates endothelin production by several types of renal and nonrenal cells, such as endothelial, epithelial, mesangial, and smooth muscle cells30 31 32 33 ; and (3) CsA-related glomerular hypoperfusion/hypofiltration and exaggerated cellular proliferation can be ameliorated by agents that block endothelin actions, such as anti-endothelin antibodies34 or selective endothelin antagonists.35
Our finding that CsA-induced renal insufficiency was accompanied by increased UETV but not PET is in agreement with those of Benigni et al,17 who showed that CsA administration to normal rats (50 mg/kg per day for 30 days) resulted in a threefold increase of UETV and no change in PET. These effects were associated with parallel reductions in renal blood flow and GFR. Similar findings were also reported in CsA-treated patients with renal transplantation.16 The lack of a marked effect of CsA on PET indicates that CsA treatment has minimal effects on the endothelium of the cardiovascular system. However, it should be noted that PET is not a reliable marker of the endothelial production of the hormone because endothelin secretion is predominantly polarized toward the basolateral side of the blood vessels,36 and plasma PET is widely believed to be uncontrolled spillover of the peptide. In addition, the rapid clearance of endothelin from the circulation by either enzymatic degradation (ie, NEP) or uptake by the lungs, liver, and kidneys could mask the release of excessive amounts of endothelin after chronic CsA treatment.22 37 Interestingly, plasma renin activity in CsA-treated rats was 3.5 times higher than that of control rats. The mechanism of this CsA-induced increase in plasma renin activity is unclear; however, sodium depletion, which is caused by anorexia or a decrease in food intake as indicated by major decreases in both sodium excretion and body weight of CsA-treated rats, may be one mechanism. The low urinary excretion of sodium by these rats may also stem from avid sodium retention as a result of the severe decrease in GFR. This possibility is gleaned from our finding and several other reports suggesting that CsA causes preglomerular vasoconstriction presumably through effects of endothelin. Our findings that the elevated levels of ET-1 mRNA and UETV in CsA-treated rats were accompanied by decreased urinary sodium excretion deviate from the well-known natriuretic action of renal endothelin. However, it should be emphasized that CsA also activated the main sodium-retaining system, ie, the renin-angiotensin-aldosterone system, which most likely masks the natriuretic effect of the endothelin system. The activation of the renin-angiotensin-aldosterone and endothelin systems may contribute to the hypertension and hyperkalemia that hamper the clinical use of CsA in humans. These systems have a direct bearing on these complications through either their vasoconstrictive properties or their metabolic and renal effects.
The source of the elevated urinary endothelin levels measured after CsA treatment has not been determined. Previously, we22 and others37 have demonstrated that under normal conditions, urinary endothelin is of renal origin. However, it is unclear from the literature whether this is also true for pathophysiological conditions characterized by renal dysfunction, such as CsA nephrotoxicity. The latter became even more relevant in light of the report by Janas et al21 that CsA inhibits endothelin degradation by highly purified renal endothelinase, suggesting that CsA may be nephrotoxic because of either an increase in endothelin synthesis in the kidney or a decrease in endothelin degradation. Our data from radiolabeled endothelin suggest that in normal rats, only a very small portion of total infused endothelin is excreted in the urine. This portion was even smaller in CsA-treated rats, most likely because of the well-established inhibitory effect of CsA on GFR. Moreover, CsA given acutely or chronically did not affect the composition of the urinary radioactivity, which was mainly free iodine. Taken together, these findings provide strong evidence that the enhanced UETV after CsA treatment is also of renal origin. The findings that neither acute nor chronic CsA treatment affects the volume of distribution, metabolic clearance rate, and half-life of endothelin in vivo and that CsA did not alter the degradation of endothelin by rNEP in vitro cast doubts on the earlier theory of Janas et al21 that an attenuated clearance/degradation rate of endothelin after CsA treatment might contribute to the elevated urinary and plasma levels of this peptide.
Our findings confirm and extend previous reports dealing with the effects of CsA treatment on the renal expression of several components of the endothelin system. Iwasaki et al38 showed that CsA administration to normal rats induced the expression of preproET-1 in the renal medulla but not in the cortex. Unfortunately, these authors did not examine the effect of CsA on either UETV or mRNA expression of ET-3, an abundant endothelin isoform in rat kidney.25 The current study clearly demonstrates that the elevated UETV in rats given CsA was associated with a significant increase in ET-1 expression, whereas ET-3 mRNA levels remained unchanged in the renal medulla. This suggests that ET-1 and not ET-3 is the mediator of CsA-induced renal dysfunction and that the increased UETV reflects increased renal synthesis of ET-1. In addition, the observation that the mRNAs for ET-1 and ET-3 were undetectable in the renal cortex of CsA-treated rats indicates that the medulla rather than the cortex is the principle source of urinary endothelin. This observation is consistent with previous reports that the highest concentrations of immunoreactive endothelin were found in the medulla of the kidney.15 Although vascular endothelium was originally considered the main site of endothelin production, recent studies demonstrate that renal tubules synthesize remarkable amounts of endothelin.39 Endothelin is generated by mesangial cells40 as well as different epithelial cell lines of renal origin, such as proximal tubule–like LLC-PK1 cells41 and distal tubule–like MDCK cells.42 In addition, the inner medullary collecting duct cells produce the greatest amounts of endothelin,39 which is in good agreement with our findings that the expression of ET-1 and ET-3 is most abundant in the renal medulla.
Although the stimulatory effects of CsA on renal ET-1 mRNA levels could be due to a direct effect of the drug, it could also be secondary to CsA-induced renal hypoperfusion/hypofiltration and resultant renal ischemia. In this regard, Firth and Ratcliffe43 reported that ET-1 mRNA but not that of ET-3 increased in rat kidney after acute ischemia. Furthermore, Nir et al44 demonstrated that acute moderate hypoxia increased renal tubular endothelin immunoreactivity, suggesting that the endothelin system is activated after exposure to low oxygen pressure. Nevertheless, this hypothesis (see above) is at odds with the well-documented finding that CsA also directly stimulates the expression and secretion of endothelin by different types of cultured cells, such as endothelial cells,30 renal cortical epithelial cells,31 and rat vascular smooth muscle cells.32 Moreover, the elevation in endothelin mRNA after CsA treatment is not confined to the kidney. Takeda et al45 demonstrated increased concentrations of endothelin mRNA in the mesenteric arteries of CsA-treated rats. Taken together, these data show it is likely that CsA directly provoked endothelin production in renal tissue, although the contribution of CsA-induced renal hemodynamic changes cannot be excluded.
The present finding that CsA increased the expression of ET-1 but not ET-3 or ECE indicates that (1) the expression of endothelin isoforms is subject to different regulation and (2) the production of endothelin is regulated at the mRNA transcription level rather than the ECE level. Although the first conclusion is supported by several previously discussed studies, the latter is based on the fact that endothelin is released (at least by vascular endothelium) constitutively without additional regulation at the level of secretion. However, the conversion of big endothelin to endothelin, which may be involved in the regulation of endothelin production, was ignored mainly because of our poor understanding of the nature of the enzyme that catalyzes this process (ie, ECE). Most recently, the gene encoding ECE was sequenced24 in different species, a crucial step that enabled us to evaluate the expression of this enzyme during physiological and pathophysiological conditions. To the best of our knowledge, the present study is the first to examine the expression of renal ECE in general and after CsA treatment in particular. Our finding that ECE expression was not affected by CsA provides scientific support for the previously baseless assumption that the production of endothelin is regulated through modulation of mRNA levels46 and not by regulation of ECE levels. This study also demonstrates that blockade of the endothelin system at the level of both ETA and ETB receptors remarkably improves the renal function of CsA-treated rats. The beneficial effects of bosentan in ameliorating the renal dysfunction after CsA administration appears to be due to the antagonism of endothelin-induced vasoconstriction. Several groups have shown that the renal hypoperfusion and hypofiltration that occur after CsA treatment are preventable by blocking endothelin actions with endothelin antagonists or endothelin antibodies.34 35 Most recently, Kon et al47 have shown that bosentan but not enalapril prevented the profound and progressive deterioration in GFR caused by CsA. The remarkable preservation of GFR in their CsA-treated rats required 3 to 5 weeks of bosentan treatment. Our findings are in line with these observations; however, the preservation of GFR in the present study was less impressive than in theirs. The differences between these two studies appear to be due to the short duration (1 week) of bosentan treatment in our study compared with 3 to 5 weeks in that of Kon et al.
In summary, we have demonstrated that the increased UETV observed after CsA treatment was accompanied by enhanced renal synthesis of ET-1. Taking into account the fact that CsA did not affect either the clearance or degradation of endothelin, one can conclude that this increase in UETV most likely originated from the kidney and reflects augmented renal production of this hormone. Nevertheless, it is possible that CsA induces renal damage that could lead to uncontrolled local release of endothelin. Our observation that CsA selectively upregulated ET-1 but not ET-3 indicates that the former is an important mediator of CsA-induced renal dysfunction.
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Selected Abbreviations and Acronyms |
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Received August 1, 1995; first decision September 5, 1995; accepted December 26, 1995.
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