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Effect of nifedipine in cyclosporine-induced nephrotoxicity in rats: roles of the thromboxane and endothelin systems
Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 63(5), 263^269 & 2000 Harcourt Publishers Ltd doi:10.1054/plef.2000.0213, available online at http://www.idealibrary.com on
Effect of nifedipine in cyclosporineinduced nephrotoxicity in rats: roles of the thromboxane and endothelin systems I. E. Darlametsos, E. N. Papanikolaou, D. D.Varonos Centre Franco-Helle¤nique de Recherches Biome¤dicales, General Hospital of Agrinion, 30100 Agrinion, Greece
Summary Cyclosporine (CsA) (45 mg/kg/day for 7 days) administration in female Wistar rats induced significant decrease in creatinine clearance (Ccr) and body weight loss (BWL).Urine volume (V) was not altered and proteinuria (PU) not provoked.These changes were associated with increased urinary endothelin1 (ET-1) and thromboxane B2 (TXB2) concentrations, and decreased urinary ratios of prostaglandin (6ketoPGF1a and PGE2) toTXB2 excretions. Nifedipine (NFD) (0.1 mg/kg/day for 7 days), a calcium channel blocker, administrated in addition to CsA, to another group of animals, significantlyaugmented CcrandurineV but did not prevent BWLin comparison to CsA-only treated rats.The urinary ET-1 and TXB2 concentrations displayed significant and non-significant decrease respectively, while the urinary excretion ratios of 6ketoPGF1a /TXB2 and PGE2/TXB2 were significantly enhanced. These observations indicate that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary prostanoid ratios of renal vasodilators (6ketoPGF1a and PGE2) to vasoconstrictor (TXB2) excretions, and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date.In a previous study, we found that NFD only slightly prevented structural renal damage, induced by CsA. So, the NFD protection refers only to functional toxicity and not to structural damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. However, other nephrotoxic factors and additional mechanisms could also be implicated in this CsA-induced syndrome. & 2000 Harcourt Publishers Ltd
INTRODUCTION Cyclosporine (CsA) is an important immunosuppressive agent used not only in the prevention of allograft rejection but also in the treatment of various autoimmune diseases. However, the use of this drug is complicated by diverse side-effects like hepatotoxicity,1 hypertension and other organ toxicity among which nephrotoxicity (NT)2 is the most common and grave. Numerous adverse effects of CsA on renal function have been described. The functional toxicity consists of a dose-
Received 21January 2000 Accepted 20 June 2000 Correspondence to: I. E. Darlametsos, Centre Franco-Helle¤nique de Recherches Biome¤dicales, Panagopoulou 11, 30100 Agrinion, Greece. Tel.:/Fax: +30(0)64145 760
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dependent reduction of renal plasma flow (RPF) and glomerular filtration rate (GFR), which are reversible upon dose diminution.2,3 Morphological changes to the kidney, consisting of tubular diffuse vacuolization, single cell necrosis4 and microcalcification, are fully reversible.5 Experimental studies in spontaneously hypertensive rats revealed a nephrotoxicity similar to that observed in man.6 Furthermore, haemolytic uraemic syndrome7 and irreversible functional and morphological changes have been induced by this agent when it was administered for long periods of time.5 It has been observed that renal dysfunction induced by CsA is associated with increased production of vasoconstrictor agents like renine-angiotensin (R-A),8 noradrenaline (NA),9 thromboxane A2 (TXA2),10 endothelin-1 (ET1),11 which belongs to the newly-recognized 21 amino acid peptide family containing four isoforms ET-1, ET-2,
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ET-3 and the vasoactive intestinal contractor (VIC),12 and diminished synthesis of vasodilator prostaglandins E2 and I2 (PGE2 and PGI2).13 It is well established that the R-A system does not play an important role in the development of acute renal failure (ARF)14–16 and that of NA is a controversial subject.15,17,18 Thus, in this study concerning the prevention of CsA nephrotoxicity (NT) with the use of nifedipine (NFD), a calcium channel blocker, we examined all those mentioned above, with the exception of R-A and NA, vasoactive agents participation, i.e. TXB2 and 6ketoPGF1a (6kPGF1a) the chemically stable metabolites of TXA2 and PGI2, respectively, PGE2 and ET-1. Our results suggest that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary ratios of renal vasodilators (6kPGF1a and PGE2) to vasoconstrictor (TXB2) excretions with reference to arachidonic acid metabolites and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date.19 This protection refers only to functional toxicity and not to structural damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. However, other nephrotoxic factors and additional mechanisms could also be implicated in this syndrome. MATERIALS AND METHODS The study was performed on 27 female Wistar rats weighing 189+6 g. Animals were housed between 228C and 258C, and humidity was 35–40%. For convenience, lighting was controlled by an automatic timer which allowed 12 h of light per day. Tap water and standard rat chow were available ad libitum. The animals were randomly allocated to three groups. The nine rats, of each group, were injected for 7 days as follows: . Normal rats: 1 ml/kg per day (d) saline intramusculary (i.m.) and 1.8 ml/kg/d olive oil intraperitoneally (i.p.). Saline and olive oil were the vehicles of NFD and CsA respectively . CsA group: 45 mg/kg/d CsA, dissolved in 1.8 ml olive oil, i.p. and 1 ml/kg/d saline, i.m. . CsAþNFD group: 45 mg/kg/d CsA, dissolved in 1.8 ml olive oil, i.p. and 0.1 mg/kg/d NFD, dissolved in 1 ml saline, i.m. On the 7th day of the experiment all the animals were placed into individual metabolic cages for 24 h urine collection. After that, the rats were anaesthetized and about 3 ml of blood was collected via a femorial artery. CsA, Sandimmun, (50 mg/ml) injectable and NFD (Adalat, 5 mg/50 ml) were provided by Sandoz, Basel, Switzerland and Bayer, Leverkousen, Germany, respectively.
The following parameters were measured: . Urinary and serum creatinine concentrations by a method using Fuller’s earth in order to eliminate chromogenes 20 . Creatinine clearance (Ccr) was utilized to express glomerular filtration rate (GFR)21,22 . Urine protein concentration by the method of Goodwin23 . Urinary TXB2, 6kPGF1a and PGE2 by the direct radioimmunoassay (RIA) used in our laboratory24 . A two-site enzyme immunometric method was used for the determination of endothelin concentrations in chromatographically purified urine samples.
Sandwich enzyme immunoassay for ET-1 A sandwich-type enzyme immunometric assay was established for the determination of ET-1 in chromatographically purified rat urines. This indirect EIA uses two differing solid-phase and tracer anti-ET-1 monoclonal antibodies (mAbs) and is based on C. Cre´minon et al’s.25 method, which permits the direct determination of ET-1 in human plasma. We decided on the purification of rat urine because, when using unpurified samples, all the obtained ET-1 values were very low (5–6 pg/ml or less) near the detection limit of the method. We obtained from Cayman Chemical, USA: a) 96-well microtiter plates coated with a capture mAb directed against the C-terminal moiety of ET-1; b) the tracer (mAbAChE), which was a mAb, against the N-terminal of the peptide, coupled to acetylcholinesterase (AChE); c) the standard ET-1; and d) the Ellman’s reagent for the spectrophotometric measurement of AChE activity. The EIA-buffer, used in this immunoassay, was a 0.1 M phosphate buffer (pH¼7,4) containing 0.15 M NaCl, 1 mM EDTA, 0.1% BSA, 0.01% NaN3 and 0.1% Tween 20.
Urine samples One ml urine was diluted 1:4 with 4% acetic acid and applied to cartridges preactivated with 5 ml methanol and 5 ml ultrapure water (Waters, Millipore, USA). Thereinafter the cartridges were washed with 10 ml of 0.1% trifluoroacetic acid (TFA). The absorbed peptides were eluted with 3 ml of methanol:water:TFA (90:10:0.1), evaporated to dryness under a stream of pure N2 and then reconstituted to 1 ml with EIA-buffer. The recovery of ET-1 was 64.5+6.4% (n¼6) when 25 pg ET-1 was added to 1 ml of rat urine.
Incubations Using one-step protocol (standard or sample were added together with mAb-AChE conjugate) even for purified urine samples, the obtained ET-1 values were near the
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detection limit of the method. For the annulment of this problem, possibly due to crossreaction of the tracer antibody with endogenous sample factors like potentially big-ET-1,26 we elected to apply the following two-step procedure, in which the two different mAbs were not performed simultaneously. In a first step, 100 ml of standard ET-1 or purified sample were left to react with the capture mAb for 18 h. After that the plates were washed in a microplate washer (SLT 812 SW 2) and so the existing crossreacting factors were eliminated. As a second step, 100 ml of mAb-AChE conjugate were added for a new 18 h incubation.
Absorbance measurement Subsequently, the microplates were again washed and, after the addition of 200 ml of Ellman’s reagent, which reacted for 1 h, the enzyme activity was measured in a spectrophotometer (SLT 340 ATTC) at 414 nm. The measured absorbance units (AU) in the wells B0 (0 pg standard ET-1) and BB (abscence of tracer) were 0.125 AU and 0.115 AU, respectively. The standard curve of the method is that of Figure 1. The minimum detectable concentration was 2.2 pg/ml while the within and between assay coefficient of variation was CVW%¼14.3 (n¼62) and CVB%¼15.4 (n¼46), respectively. The ‘parallelism’ test of the method was made as follows. We added 100 pg standard ET-1 in 1 ml rat urine and after 6-fold dilution, chromatography and application of the foretold protocole, we measured the ET-1 concentrations in the six dilutions. The calculated curve, log pg found vs log pg expected, was the straight line y¼0.191þ0.976x (Fig. 2). The fact that the slope is close to the unit (0.976) is indicative of parallelism.27 This assay crossreacts 100% with ET-1 related peptides such as ET-2, ET-3, VIC and sarafotoxin 6-b.25 Thus, our measurements represent not only the urinary ET-1 levels but also the potential crossreacting peptide concentrations existing in our samples.
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Fig. 2 Parallelism test of the ET-1sandwich EIA.The fact that the slope of the calculated curve, straight line y¼0.191þ 0.976x, is close to the unit is indicative of parallelism.
Statistical study Statistical analysis was made using Student’s t-test and P50.05 was considered to be significant. All values are expressed as the mean value + SEM.
RESULTS
Effects of cyclosporine on rat renal function The administration of CsA in dose, inducing ARF,4,24 45 mg (37.4 mmol)/kg/d for 7 days in the CsA group, diminished Ccr and provoked body weight loss (BWL) while it did not alter urine volume (V) and did not exhibit proteinouria (PU) (Table 1) in comparison to normal rats. These changes were associated with significant alterations in urinary prostanoids and ET-1 excretion. So, there was observed augmentation of TXB2 (P50.001) and ET-1 (P50.01) and diminution of both PGE2 (P50.001) and 6kPGF1a (P50.05) (Table 2). Moreover, very significant decreases were noticed in the ratios of PGE2/TXB2 and 6kPGF1a/TXB2 vs the normal rats (Table 3).
Effects of nifedipine on cyclosporine treated rat renal function
Fig. 1 Standard curve of ET-1in the sandwich EIA.
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In order to find the optimal dose of NFD and to observe its effects on normal rats, we administered it alone and in combination with CsA in three progressively increasing doses (0.050, 0.075 and 0.1 mg/kg) in two groups of animals. In the first group, the treatment with NFD had no effect on GFR (Ccr) but in the second one, the administration of NFD in combination with CsA (45 mg/ kg) induced a dose-dependent increase on GFR. This increase was significant only at the dose 0.1 mg (289 nmol)/kg and so, we concluded in the injection of this daily dose of NFD. Thus administration of 0.1 mg/kg NFD for 7 days, in the CsAþNFD group, prevented diminution of Ccr but not BWL vs the CsA group. Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 63(5), 263^269
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Table 1 Urine volume (V), creatinine clearance (Ccr), body weight loss (BWL) and proteinouria (PU) alterations in NR, CsA and CsAþNFD groups of treated animals.Values are means+SEM; n=9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.01and **P50.001 Group
V ml/Kg/24 h
Ccr ml/Kg/min
BWL %
PU mg/24 h
NR CsA CsAþNFD
21.0+2.1 23.9+3.2 46.1+7.1*
2.53+0.14** 1.19+0.10 2.63+0.31**
(þ) 0.63+0.44** 712.50+1.96 711.16+2.64
6.73+2.51 11.75+4.23 15.91+2.53
Table 2 Urinary 6kPGF1a,PGE2,TXB2 and ET-1excretionsin NR,CsA and CsAþNFD groups of treaded animals.Values are mean + SEM; n¼9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.05, **P50.01and ***P50.001 Group
6kPGF1a pM/Kg/24 h
PGE2 pM/Kg/24 h
TXB2 pM/Kg/24 h
ET-1 fM/Kg/24 h
NR CsA CsAþNFD
406+30 * 314+30 413+47 *
1087+70 *** 512+70 793+81 **
297+27 *** 806+86 650+162
121+13 ** 468+120 206+16 *
Table 3 Ratios of urinary 6kPGF1a / TXB2 and PGE2/TXB2 excretions in NR, CsA and CsAþNFD groups of treaded animals. Values are means + SEM; n¼9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.05 and **P50.001 Group
6kPGF1a / TXB2
PGE2/TXB2
NR CsA CsAþNFD
1.52+0.26** 0.42+0.05 0.90+0.21*
4.13+0.71** 0.67+0.09 1.72+0.41*
Regarding to urine V and PU, they enhanced significantly and non-significantly, respectively (Table 1). On the other hand, both urinary PGE2 (P50.01) and 6kPGF1a (P50.05) were significantly augmented while the TXB2 decrease was not significant (Table 2). However, the urinary ratios of PGE2/TXB2 and 6kPGF1a/TXB2 significantly increased (Table 3) vs the CsA group. Analogous comparison between the two groups gave significant reduction of the excreted ET-1 (P50.05) (Table 2). DISCUSSION It is well established that afferent arteriole contractions lead to Ccr decrease, which is strongly implicated in the pathogenesis of renal dysfunction.2,28 These contractions could be provoked by increased release of vasoconstrictor substances and/or diminished release of vasodilator agents. CsA nephrotoxicity (NT) was found to be associated with the enhanced production of many vasoconstrictor factors such as renine-angiotensin (R-A),8 noradrenaline (NA),9 TXA2,10,24 ET11 and the reduced release of vasodilator prostaglandins (PGE2, 6kPGF1a).13,24 How-
ever, contrasting experimental data display CsA nephrotoxicity irrespective of changes in prostanoid (PGE2 and 6kPGF1a and TXB2) syntheses, potentially due to variation of rat strains.29,30 By now, the R-A system has been found not to play an important role or secondary one in the development of acute renal failure (ARF).14–16 Moreover, the NA involvement in ARF is a controversial subject.15,17,18 Thus, in this study concerning the prevention of CsA NT with the use of NFD, a calcium channel blocker, we examined the role of all the above mentioned, with the exception of R-A and NA, related vasoactive agents. Our results indicate that CsA administration in rats provoked Ccr reduction (Table 1). The noted renal dysfunction was accompanied by the rise of urinary concentrations of both potent vasoconstrictors ET-1 and TXB2, but also by the diminished release of both important vasodilators PGE2 and 6kPGF1a (Table 2). The documentation of urinary ET-1 origin requires the reminder of certain experimental data. The plasma rat levels of ET-1 remained unchangeable after renal mass ablation, in a model of chronic renal desease,31 or after the long-time administration of a nitric oxide (NO) synthesis inhibitor32 while in both cases, as observed in rat, the urinary ET-1 excretion significantly augmented. These findings suggest the increased urinary levels of the peptide must be due to its renal synthesis. On the other hand, increase of the circulating ET levels was observed in the CsA induced NT.33 Since this peptide has both libre infiltration in the glomerulus and possibility of renal synthesis, findings as to urinary endothelin excretion could reflect events occurring in the systemic circulation and/or renal ET synthesis. However, Benigni et al.31 found that less than 0.3% of the i.v. infused radiolabeled ET into
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rats was recovered in their urine. All these data suggest the possibility that our observation as to enhanced urinary ET-1 could reflect its renal synthesis rather than its plasma levels. The origin of urinary prostaglandins is a more unambiguous matter, for as much as they reflect their renal production.34 The vascular bed of the kidney is a target for the endothelin constrictor actions.35 So, our finding of the urinary ET-1 rise induced by CsA, which is compatible with the observation made by others,36 suggests that this peptide may play a pivotal role in the CsA-induced nephrotoxicity (NT). On the other hand, in our experiments we found that CsA enhanced the urinary vasoconstrictor TXB2 (TXA2). Besides, it is well known that the TXA2 rise is the major cause of renal vasoconstriction induced by different chemical compounds like glycerol (GL), HgCl2, gentamicin (GM)37–39 and the development of ARF. However, we expected the CsA-induced NT, by decreasing the vasodilator PG (PGE2 and 6kPGF1a), to be more serious than that induced by other nephrotoxic agents like GL, HgCl2 and GM, which were found to enhance both TXA2 and PG production. This discrepancy demands further investigation. Since the discovery of endothelin by Yanagisawa, it has been suggested that ET may induce its biological actions by facilitating the intracellular Ca2þ influx through dihydropyridine-sensitive Ca2þ channels of the target cells.40 In the current study, we observed that NFD (a dihydropyridine channel blocker) administration in animals restored Ccr fall provoked by CsA (Table 2). This partial protection against the CsA-induced renal dysfunction was associated with the significant decrease of urinary vasoconstrictor ET-1. This is very important because endothelin can contract afferent arterioles,41 resulting in NT. Besides, it has been shown that glomerular vasoconstriction, following CsA administration, was prevented by the infusion anti-ET serum.33 Furthermore, in this study NFD was found to increase the ratios of vasodilator prostanoids (PGE2 and 6kPGF1a) to vasoconstrictor (TXB2) one (Table 3). This is consistent with the observations that infusion of PGI2 and PGE242–45 or inhibition of TXA2 synthesis protected against the development of ARF.16,28 In the present study, we referred only to the functional toxicity provoked by CsA. Our previous studies have shown24 that CsA nephrotoxicity was accompanied by renal morphological changes of all CsA-treated (nine) rats, i.e. tubular, diffuse or focal, vacuolization (nine rats), brush border loss (four rats), single cell necrosis (four rats), tubular casts (one rat) and interstitial oedema (two rats). But the NFD administration in rats receiving CsA only slightly prevented both TXB2 increase (Table 2) and renal damage since light microscopic sections showed that the kidneys of seven out of nine rats were affected by & 2000 Harcourt Publishers Ltd
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diffuse or focal tubular vacuolization (seven rats), brush border loss (two rats), single cell necrosis (one rat) and interstitial oedema (two rats).24 On the other hand, we obtained partial protection against the CsA-induced NT as much by the addition of evening primrose oil (EPO), an essential fatty acid precursor of PGE1 synthesis, in rat diet as by the administration either of ketanserine (KTS), an antagonist of S2 serotonergic–a1 adrenergic–H1 histaminergic receptors, or OKY-046, a TXA2 synthetase inhibitor in rats.4,24,46 These protective means prevented not only TXB2 rise and functional aggravation of rat kidneys, but also structural renal damage since, in any case, only five out of nine rats were affected by lesions of minor importance than those observed in the NFD protection. Notably, EPO and KTs were the only means which prevented the BWL of the animals.24 Now the fact that NFD only slightly prevented the CsA-induced increase of TXB2 levels could explain why this protection refers mainly to functional toxicity and hardly to structural renal damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. Besides, it is well known that TXB2, in addition to being a potent vasoconstrictor,47,48 is also platelet aggregator agonist.49 Platelet aggregation is also implicated in glomerular injury.50–53 TXA2 is a leukocyte chemoattractant too; therefore, the well known tissue damaging effect of stimulated leukocytes could come into play.54,55 The eventual contribution of ET-1 in the structural renal damage associated with CsA must be excluded, because the ET-1 cut, induced by the NFD administration in animals receiving CsA, did not protect them from renal morphological changes. This fact shows that ET-1 is linked to vasoconstriction and not to structural damage, which is consistent with the observation made by Kon et al.56 Our results suggest that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary ratios of renal vasodilators (6ketoPGF1a and PGE2) to vasoconstrictor (TXB2) excretions with reference to arachidonic acid metabolites, and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date. This protection refers only to functional toxicity and not to structural renal damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. Thus the mechanism for the protective effect of NFD may be due to redistribution of vasoactive agents for the benefit of the vasodilation in kidney perturbed by the renal vasoconstricting effects of CsA. It remains unclear whether the irreversible renal morphological changes like interstitial fibrosis, induced mainly in long-term CsA treatment, are dependent on haemodynamic modifications, tubular damage or both. However, other vasoactive factors known and/or unknown and additional mechanisms implicated in this syndrome could explain why this
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drug is detrimental in the treatment of some autoimmune diseases.
ACKNOWLEDGEMENTS This work was supported by a grant from the Municipality of Agrinion. We thank especially Mr Th. Sokos, mayor of the town, for his generous support. We thank also Mrs D. Markopoulou for her secretarial assistance, Mr A. Tsourapes for his administrative assistance and Mr P. Papadogeorgopoulos for his help with the English.
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39. Papanikolaou N., Peros G., Morphake P. et al. Does gentamicin induce acute renal failure by increasing renal TXA2 synthesis? Prost Leuk EFAs 1992; 45: 131–136. 40. Yanagisawa M., Masaki T. Endothelin, a novel endotheliumderived peptide. Pharmacological activities, regulation and possible roles in cardiovascular control. Biochem Pharmacol 1989; 38(12): 1877–1883. 41. Loutzenhiser R., Epstein M., Hayashi K., Horton C. Direct visualization of effects of endothelin on the renal microvasculature. Am J Physiol 258 (Renal Fluid Electrolyte Physiol) 1990; 270: F61–F68. 42. Lifschitz M., Barnes J. Prostaglandin I2 attenuates ischemic acute renal failure in the rat. Am J Physiol 1984; 247: F714–F717. 43. Mendal A., Miller J. Protection against ischemic acute renal failure by prostaglandin I2 infusion. Leuk Med 1982; 8: 361–373. 44. Papanikolaou N., Callard P., Bariety J., Milliez P. The effect of indomethacin and PGE2 on renal failure due to glycerol in saline loaded rats. Clin Sci 1975; 49: 507–510. 45. Werb R., Clark W., Lindsay R., Jones E., Turnbull D., Linton A. Protective effect of PGE2 in glycerol-induced acute renal failure in rats. Clin Sci 1978; 55: 505–507. 46. Morphake P., Bariety J., Darlametsos I. et al. Alteration of Cyclosporine (CsA)-Induced Nephrotoxicity by Gamma Linolenic Acid (GLA) and Eicosapentaenoic Acid (EPA) in Wistar Rats. Prost Leuk EFAs 1994; 50: 29–35. 47. Ellis E. F., Oelz O., Roberts L. T. Coronary arterial smooth muscle contraction by a substance released from platelets. Evidence that it is TXA2. Science 1976; 193: 1135–1137.
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48. Svensson J., Hamberg M. Thromboxane A2 and prostaglandin H2. Potent stimulators of swine coronary artery. Prostaglandins 1976; 12: 943–950. 49. Hamberg M., Svensson J., Samuelsson B. Thromboxane a new group of biologically active compounds derived from prostaglandin endoperoxidasa. Proc Natl Acad Sci USA 1975; 72: 2994–2998. 50. Clark W. F., Friesen M., Linton A. L., Lindsay R. M. The platelet as a mediator of tissue damage in immune complex glomerulonephritis. Clin Nephrol 1976; 6: 287–289. 51. Kincaid-Smith P. Coagulation and renal disease. Kidney Int 1972; 2: 183–190. 52. Kniker W. T., Cochrane C. G. The localization of circulating immune complexes in experimental serum sickness. J Exp Med 1968; 127: 119–135. 53. Parbtani A., Frampton G., Cameron J. S. Platelet and plasma serotonin concentration in glomerulonephritis. Part II. Clin Nephrol 1980; 14: 112–123. 54. Hawkins D. Neutrophilic leukocytes in immunologic reactions: evidence for the selective release of lysosomal constituents. J Immunol 1972; 108: 310–317. 55. Boot J. R., Dawson W., Kichen E. A. The chemotactic activity of TXB2. A possible role in inflammation. J Physiol 1976; 257: 47–49. 56. Kon V., Hunley E., Fogo A. Combined antagonism of endothelin A/B receptors links endothelin to vasoconstriction whereas angiotensin II effects fibrosis. Transplantation 1995; 60: 89–95.
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Effect of nifedipine in cyclosporineinduced nephrotoxicity in rats: roles of the thromboxane and endothelin systems I. E. Darlametsos, E. N. Papanikolaou, D. D.Varonos Centre Franco-Helle¤nique de Recherches Biome¤dicales, General Hospital of Agrinion, 30100 Agrinion, Greece
Summary Cyclosporine (CsA) (45 mg/kg/day for 7 days) administration in female Wistar rats induced significant decrease in creatinine clearance (Ccr) and body weight loss (BWL).Urine volume (V) was not altered and proteinuria (PU) not provoked.These changes were associated with increased urinary endothelin1 (ET-1) and thromboxane B2 (TXB2) concentrations, and decreased urinary ratios of prostaglandin (6ketoPGF1a and PGE2) toTXB2 excretions. Nifedipine (NFD) (0.1 mg/kg/day for 7 days), a calcium channel blocker, administrated in addition to CsA, to another group of animals, significantlyaugmented CcrandurineV but did not prevent BWLin comparison to CsA-only treated rats.The urinary ET-1 and TXB2 concentrations displayed significant and non-significant decrease respectively, while the urinary excretion ratios of 6ketoPGF1a /TXB2 and PGE2/TXB2 were significantly enhanced. These observations indicate that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary prostanoid ratios of renal vasodilators (6ketoPGF1a and PGE2) to vasoconstrictor (TXB2) excretions, and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date.In a previous study, we found that NFD only slightly prevented structural renal damage, induced by CsA. So, the NFD protection refers only to functional toxicity and not to structural damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. However, other nephrotoxic factors and additional mechanisms could also be implicated in this CsA-induced syndrome. & 2000 Harcourt Publishers Ltd
INTRODUCTION Cyclosporine (CsA) is an important immunosuppressive agent used not only in the prevention of allograft rejection but also in the treatment of various autoimmune diseases. However, the use of this drug is complicated by diverse side-effects like hepatotoxicity,1 hypertension and other organ toxicity among which nephrotoxicity (NT)2 is the most common and grave. Numerous adverse effects of CsA on renal function have been described. The functional toxicity consists of a dose-
Received 21January 2000 Accepted 20 June 2000 Correspondence to: I. E. Darlametsos, Centre Franco-Helle¤nique de Recherches Biome¤dicales, Panagopoulou 11, 30100 Agrinion, Greece. Tel.:/Fax: +30(0)64145 760
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dependent reduction of renal plasma flow (RPF) and glomerular filtration rate (GFR), which are reversible upon dose diminution.2,3 Morphological changes to the kidney, consisting of tubular diffuse vacuolization, single cell necrosis4 and microcalcification, are fully reversible.5 Experimental studies in spontaneously hypertensive rats revealed a nephrotoxicity similar to that observed in man.6 Furthermore, haemolytic uraemic syndrome7 and irreversible functional and morphological changes have been induced by this agent when it was administered for long periods of time.5 It has been observed that renal dysfunction induced by CsA is associated with increased production of vasoconstrictor agents like renine-angiotensin (R-A),8 noradrenaline (NA),9 thromboxane A2 (TXA2),10 endothelin-1 (ET1),11 which belongs to the newly-recognized 21 amino acid peptide family containing four isoforms ET-1, ET-2,
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ET-3 and the vasoactive intestinal contractor (VIC),12 and diminished synthesis of vasodilator prostaglandins E2 and I2 (PGE2 and PGI2).13 It is well established that the R-A system does not play an important role in the development of acute renal failure (ARF)14–16 and that of NA is a controversial subject.15,17,18 Thus, in this study concerning the prevention of CsA nephrotoxicity (NT) with the use of nifedipine (NFD), a calcium channel blocker, we examined all those mentioned above, with the exception of R-A and NA, vasoactive agents participation, i.e. TXB2 and 6ketoPGF1a (6kPGF1a) the chemically stable metabolites of TXA2 and PGI2, respectively, PGE2 and ET-1. Our results suggest that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary ratios of renal vasodilators (6kPGF1a and PGE2) to vasoconstrictor (TXB2) excretions with reference to arachidonic acid metabolites and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date.19 This protection refers only to functional toxicity and not to structural damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. However, other nephrotoxic factors and additional mechanisms could also be implicated in this syndrome. MATERIALS AND METHODS The study was performed on 27 female Wistar rats weighing 189+6 g. Animals were housed between 228C and 258C, and humidity was 35–40%. For convenience, lighting was controlled by an automatic timer which allowed 12 h of light per day. Tap water and standard rat chow were available ad libitum. The animals were randomly allocated to three groups. The nine rats, of each group, were injected for 7 days as follows: . Normal rats: 1 ml/kg per day (d) saline intramusculary (i.m.) and 1.8 ml/kg/d olive oil intraperitoneally (i.p.). Saline and olive oil were the vehicles of NFD and CsA respectively . CsA group: 45 mg/kg/d CsA, dissolved in 1.8 ml olive oil, i.p. and 1 ml/kg/d saline, i.m. . CsAþNFD group: 45 mg/kg/d CsA, dissolved in 1.8 ml olive oil, i.p. and 0.1 mg/kg/d NFD, dissolved in 1 ml saline, i.m. On the 7th day of the experiment all the animals were placed into individual metabolic cages for 24 h urine collection. After that, the rats were anaesthetized and about 3 ml of blood was collected via a femorial artery. CsA, Sandimmun, (50 mg/ml) injectable and NFD (Adalat, 5 mg/50 ml) were provided by Sandoz, Basel, Switzerland and Bayer, Leverkousen, Germany, respectively.
The following parameters were measured: . Urinary and serum creatinine concentrations by a method using Fuller’s earth in order to eliminate chromogenes 20 . Creatinine clearance (Ccr) was utilized to express glomerular filtration rate (GFR)21,22 . Urine protein concentration by the method of Goodwin23 . Urinary TXB2, 6kPGF1a and PGE2 by the direct radioimmunoassay (RIA) used in our laboratory24 . A two-site enzyme immunometric method was used for the determination of endothelin concentrations in chromatographically purified urine samples.
Sandwich enzyme immunoassay for ET-1 A sandwich-type enzyme immunometric assay was established for the determination of ET-1 in chromatographically purified rat urines. This indirect EIA uses two differing solid-phase and tracer anti-ET-1 monoclonal antibodies (mAbs) and is based on C. Cre´minon et al’s.25 method, which permits the direct determination of ET-1 in human plasma. We decided on the purification of rat urine because, when using unpurified samples, all the obtained ET-1 values were very low (5–6 pg/ml or less) near the detection limit of the method. We obtained from Cayman Chemical, USA: a) 96-well microtiter plates coated with a capture mAb directed against the C-terminal moiety of ET-1; b) the tracer (mAbAChE), which was a mAb, against the N-terminal of the peptide, coupled to acetylcholinesterase (AChE); c) the standard ET-1; and d) the Ellman’s reagent for the spectrophotometric measurement of AChE activity. The EIA-buffer, used in this immunoassay, was a 0.1 M phosphate buffer (pH¼7,4) containing 0.15 M NaCl, 1 mM EDTA, 0.1% BSA, 0.01% NaN3 and 0.1% Tween 20.
Urine samples One ml urine was diluted 1:4 with 4% acetic acid and applied to cartridges preactivated with 5 ml methanol and 5 ml ultrapure water (Waters, Millipore, USA). Thereinafter the cartridges were washed with 10 ml of 0.1% trifluoroacetic acid (TFA). The absorbed peptides were eluted with 3 ml of methanol:water:TFA (90:10:0.1), evaporated to dryness under a stream of pure N2 and then reconstituted to 1 ml with EIA-buffer. The recovery of ET-1 was 64.5+6.4% (n¼6) when 25 pg ET-1 was added to 1 ml of rat urine.
Incubations Using one-step protocol (standard or sample were added together with mAb-AChE conjugate) even for purified urine samples, the obtained ET-1 values were near the
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detection limit of the method. For the annulment of this problem, possibly due to crossreaction of the tracer antibody with endogenous sample factors like potentially big-ET-1,26 we elected to apply the following two-step procedure, in which the two different mAbs were not performed simultaneously. In a first step, 100 ml of standard ET-1 or purified sample were left to react with the capture mAb for 18 h. After that the plates were washed in a microplate washer (SLT 812 SW 2) and so the existing crossreacting factors were eliminated. As a second step, 100 ml of mAb-AChE conjugate were added for a new 18 h incubation.
Absorbance measurement Subsequently, the microplates were again washed and, after the addition of 200 ml of Ellman’s reagent, which reacted for 1 h, the enzyme activity was measured in a spectrophotometer (SLT 340 ATTC) at 414 nm. The measured absorbance units (AU) in the wells B0 (0 pg standard ET-1) and BB (abscence of tracer) were 0.125 AU and 0.115 AU, respectively. The standard curve of the method is that of Figure 1. The minimum detectable concentration was 2.2 pg/ml while the within and between assay coefficient of variation was CVW%¼14.3 (n¼62) and CVB%¼15.4 (n¼46), respectively. The ‘parallelism’ test of the method was made as follows. We added 100 pg standard ET-1 in 1 ml rat urine and after 6-fold dilution, chromatography and application of the foretold protocole, we measured the ET-1 concentrations in the six dilutions. The calculated curve, log pg found vs log pg expected, was the straight line y¼0.191þ0.976x (Fig. 2). The fact that the slope is close to the unit (0.976) is indicative of parallelism.27 This assay crossreacts 100% with ET-1 related peptides such as ET-2, ET-3, VIC and sarafotoxin 6-b.25 Thus, our measurements represent not only the urinary ET-1 levels but also the potential crossreacting peptide concentrations existing in our samples.
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Fig. 2 Parallelism test of the ET-1sandwich EIA.The fact that the slope of the calculated curve, straight line y¼0.191þ 0.976x, is close to the unit is indicative of parallelism.
Statistical study Statistical analysis was made using Student’s t-test and P50.05 was considered to be significant. All values are expressed as the mean value + SEM.
RESULTS
Effects of cyclosporine on rat renal function The administration of CsA in dose, inducing ARF,4,24 45 mg (37.4 mmol)/kg/d for 7 days in the CsA group, diminished Ccr and provoked body weight loss (BWL) while it did not alter urine volume (V) and did not exhibit proteinouria (PU) (Table 1) in comparison to normal rats. These changes were associated with significant alterations in urinary prostanoids and ET-1 excretion. So, there was observed augmentation of TXB2 (P50.001) and ET-1 (P50.01) and diminution of both PGE2 (P50.001) and 6kPGF1a (P50.05) (Table 2). Moreover, very significant decreases were noticed in the ratios of PGE2/TXB2 and 6kPGF1a/TXB2 vs the normal rats (Table 3).
Effects of nifedipine on cyclosporine treated rat renal function
Fig. 1 Standard curve of ET-1in the sandwich EIA.
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In order to find the optimal dose of NFD and to observe its effects on normal rats, we administered it alone and in combination with CsA in three progressively increasing doses (0.050, 0.075 and 0.1 mg/kg) in two groups of animals. In the first group, the treatment with NFD had no effect on GFR (Ccr) but in the second one, the administration of NFD in combination with CsA (45 mg/ kg) induced a dose-dependent increase on GFR. This increase was significant only at the dose 0.1 mg (289 nmol)/kg and so, we concluded in the injection of this daily dose of NFD. Thus administration of 0.1 mg/kg NFD for 7 days, in the CsAþNFD group, prevented diminution of Ccr but not BWL vs the CsA group. Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 63(5), 263^269
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Table 1 Urine volume (V), creatinine clearance (Ccr), body weight loss (BWL) and proteinouria (PU) alterations in NR, CsA and CsAþNFD groups of treated animals.Values are means+SEM; n=9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.01and **P50.001 Group
V ml/Kg/24 h
Ccr ml/Kg/min
BWL %
PU mg/24 h
NR CsA CsAþNFD
21.0+2.1 23.9+3.2 46.1+7.1*
2.53+0.14** 1.19+0.10 2.63+0.31**
(þ) 0.63+0.44** 712.50+1.96 711.16+2.64
6.73+2.51 11.75+4.23 15.91+2.53
Table 2 Urinary 6kPGF1a,PGE2,TXB2 and ET-1excretionsin NR,CsA and CsAþNFD groups of treaded animals.Values are mean + SEM; n¼9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.05, **P50.01and ***P50.001 Group
6kPGF1a pM/Kg/24 h
PGE2 pM/Kg/24 h
TXB2 pM/Kg/24 h
ET-1 fM/Kg/24 h
NR CsA CsAþNFD
406+30 * 314+30 413+47 *
1087+70 *** 512+70 793+81 **
297+27 *** 806+86 650+162
121+13 ** 468+120 206+16 *
Table 3 Ratios of urinary 6kPGF1a / TXB2 and PGE2/TXB2 excretions in NR, CsA and CsAþNFD groups of treaded animals. Values are means + SEM; n¼9. All values were compared against those obtained in the second group. Significantly different from CsA group values at: *P50.05 and **P50.001 Group
6kPGF1a / TXB2
PGE2/TXB2
NR CsA CsAþNFD
1.52+0.26** 0.42+0.05 0.90+0.21*
4.13+0.71** 0.67+0.09 1.72+0.41*
Regarding to urine V and PU, they enhanced significantly and non-significantly, respectively (Table 1). On the other hand, both urinary PGE2 (P50.01) and 6kPGF1a (P50.05) were significantly augmented while the TXB2 decrease was not significant (Table 2). However, the urinary ratios of PGE2/TXB2 and 6kPGF1a/TXB2 significantly increased (Table 3) vs the CsA group. Analogous comparison between the two groups gave significant reduction of the excreted ET-1 (P50.05) (Table 2). DISCUSSION It is well established that afferent arteriole contractions lead to Ccr decrease, which is strongly implicated in the pathogenesis of renal dysfunction.2,28 These contractions could be provoked by increased release of vasoconstrictor substances and/or diminished release of vasodilator agents. CsA nephrotoxicity (NT) was found to be associated with the enhanced production of many vasoconstrictor factors such as renine-angiotensin (R-A),8 noradrenaline (NA),9 TXA2,10,24 ET11 and the reduced release of vasodilator prostaglandins (PGE2, 6kPGF1a).13,24 How-
ever, contrasting experimental data display CsA nephrotoxicity irrespective of changes in prostanoid (PGE2 and 6kPGF1a and TXB2) syntheses, potentially due to variation of rat strains.29,30 By now, the R-A system has been found not to play an important role or secondary one in the development of acute renal failure (ARF).14–16 Moreover, the NA involvement in ARF is a controversial subject.15,17,18 Thus, in this study concerning the prevention of CsA NT with the use of NFD, a calcium channel blocker, we examined the role of all the above mentioned, with the exception of R-A and NA, related vasoactive agents. Our results indicate that CsA administration in rats provoked Ccr reduction (Table 1). The noted renal dysfunction was accompanied by the rise of urinary concentrations of both potent vasoconstrictors ET-1 and TXB2, but also by the diminished release of both important vasodilators PGE2 and 6kPGF1a (Table 2). The documentation of urinary ET-1 origin requires the reminder of certain experimental data. The plasma rat levels of ET-1 remained unchangeable after renal mass ablation, in a model of chronic renal desease,31 or after the long-time administration of a nitric oxide (NO) synthesis inhibitor32 while in both cases, as observed in rat, the urinary ET-1 excretion significantly augmented. These findings suggest the increased urinary levels of the peptide must be due to its renal synthesis. On the other hand, increase of the circulating ET levels was observed in the CsA induced NT.33 Since this peptide has both libre infiltration in the glomerulus and possibility of renal synthesis, findings as to urinary endothelin excretion could reflect events occurring in the systemic circulation and/or renal ET synthesis. However, Benigni et al.31 found that less than 0.3% of the i.v. infused radiolabeled ET into
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rats was recovered in their urine. All these data suggest the possibility that our observation as to enhanced urinary ET-1 could reflect its renal synthesis rather than its plasma levels. The origin of urinary prostaglandins is a more unambiguous matter, for as much as they reflect their renal production.34 The vascular bed of the kidney is a target for the endothelin constrictor actions.35 So, our finding of the urinary ET-1 rise induced by CsA, which is compatible with the observation made by others,36 suggests that this peptide may play a pivotal role in the CsA-induced nephrotoxicity (NT). On the other hand, in our experiments we found that CsA enhanced the urinary vasoconstrictor TXB2 (TXA2). Besides, it is well known that the TXA2 rise is the major cause of renal vasoconstriction induced by different chemical compounds like glycerol (GL), HgCl2, gentamicin (GM)37–39 and the development of ARF. However, we expected the CsA-induced NT, by decreasing the vasodilator PG (PGE2 and 6kPGF1a), to be more serious than that induced by other nephrotoxic agents like GL, HgCl2 and GM, which were found to enhance both TXA2 and PG production. This discrepancy demands further investigation. Since the discovery of endothelin by Yanagisawa, it has been suggested that ET may induce its biological actions by facilitating the intracellular Ca2þ influx through dihydropyridine-sensitive Ca2þ channels of the target cells.40 In the current study, we observed that NFD (a dihydropyridine channel blocker) administration in animals restored Ccr fall provoked by CsA (Table 2). This partial protection against the CsA-induced renal dysfunction was associated with the significant decrease of urinary vasoconstrictor ET-1. This is very important because endothelin can contract afferent arterioles,41 resulting in NT. Besides, it has been shown that glomerular vasoconstriction, following CsA administration, was prevented by the infusion anti-ET serum.33 Furthermore, in this study NFD was found to increase the ratios of vasodilator prostanoids (PGE2 and 6kPGF1a) to vasoconstrictor (TXB2) one (Table 3). This is consistent with the observations that infusion of PGI2 and PGE242–45 or inhibition of TXA2 synthesis protected against the development of ARF.16,28 In the present study, we referred only to the functional toxicity provoked by CsA. Our previous studies have shown24 that CsA nephrotoxicity was accompanied by renal morphological changes of all CsA-treated (nine) rats, i.e. tubular, diffuse or focal, vacuolization (nine rats), brush border loss (four rats), single cell necrosis (four rats), tubular casts (one rat) and interstitial oedema (two rats). But the NFD administration in rats receiving CsA only slightly prevented both TXB2 increase (Table 2) and renal damage since light microscopic sections showed that the kidneys of seven out of nine rats were affected by & 2000 Harcourt Publishers Ltd
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diffuse or focal tubular vacuolization (seven rats), brush border loss (two rats), single cell necrosis (one rat) and interstitial oedema (two rats).24 On the other hand, we obtained partial protection against the CsA-induced NT as much by the addition of evening primrose oil (EPO), an essential fatty acid precursor of PGE1 synthesis, in rat diet as by the administration either of ketanserine (KTS), an antagonist of S2 serotonergic–a1 adrenergic–H1 histaminergic receptors, or OKY-046, a TXA2 synthetase inhibitor in rats.4,24,46 These protective means prevented not only TXB2 rise and functional aggravation of rat kidneys, but also structural renal damage since, in any case, only five out of nine rats were affected by lesions of minor importance than those observed in the NFD protection. Notably, EPO and KTs were the only means which prevented the BWL of the animals.24 Now the fact that NFD only slightly prevented the CsA-induced increase of TXB2 levels could explain why this protection refers mainly to functional toxicity and hardly to structural renal damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. Besides, it is well known that TXB2, in addition to being a potent vasoconstrictor,47,48 is also platelet aggregator agonist.49 Platelet aggregation is also implicated in glomerular injury.50–53 TXA2 is a leukocyte chemoattractant too; therefore, the well known tissue damaging effect of stimulated leukocytes could come into play.54,55 The eventual contribution of ET-1 in the structural renal damage associated with CsA must be excluded, because the ET-1 cut, induced by the NFD administration in animals receiving CsA, did not protect them from renal morphological changes. This fact shows that ET-1 is linked to vasoconstriction and not to structural damage, which is consistent with the observation made by Kon et al.56 Our results suggest that the partial protection of NFD in CsA-induced nephrotoxicity could be attributed to augmented urinary ratios of renal vasodilators (6ketoPGF1a and PGE2) to vasoconstrictor (TXB2) excretions with reference to arachidonic acid metabolites, and also to reduced release of rather renal origin ET-1, the most potent mamalian vasoconstrictor peptide known to date. This protection refers only to functional toxicity and not to structural renal damage, mediated at least in part by the preservation of relatively high renal TXB2 levels. Thus the mechanism for the protective effect of NFD may be due to redistribution of vasoactive agents for the benefit of the vasodilation in kidney perturbed by the renal vasoconstricting effects of CsA. It remains unclear whether the irreversible renal morphological changes like interstitial fibrosis, induced mainly in long-term CsA treatment, are dependent on haemodynamic modifications, tubular damage or both. However, other vasoactive factors known and/or unknown and additional mechanisms implicated in this syndrome could explain why this
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drug is detrimental in the treatment of some autoimmune diseases.
ACKNOWLEDGEMENTS This work was supported by a grant from the Municipality of Agrinion. We thank especially Mr Th. Sokos, mayor of the town, for his generous support. We thank also Mrs D. Markopoulou for her secretarial assistance, Mr A. Tsourapes for his administrative assistance and Mr P. Papadogeorgopoulos for his help with the English.
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