European Journal of Pharmacology, 194 (1991) 115-117
115
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100210I
EJP 20786
Short communication
Effect of nifedipine on cyclosporine A-induced nephrotoxicity, urinary endothelin excretion and renal endothelin receptor number David P. Brooks, Eliot H. Ohlstein, Lisa C. C o n t i n o , Barbara Storer, M a r k Pullen, M a d e l y n C a l t a b i a n o 1 and Ponnal Nambi SmithKline Beecham Pharmaceuticals, Departments of Pharmacology and I Cell Sciences, King of Prussia, PA 19406-0939, U.S.A. Received 21 November 1990, accepted 2 January 1991
The aim of the present study was to determine the effect of a calcium channel blocker on renal function, urinary endothelin excretion and endothelin receptor number in rats. Administration of cyclosporine resulted in a significant impairment of renal function when measured by either [14C]inulin or 24 h creatinine clearances. This nephrotoxicity was associated with statistically significant (P < 0.05) increases in urinary endothelin excretion and renal endothelin receptor number. Treatment with nifedipine attenuated cyclosporine A-induced renal dysfunction and reduced urinary endothelin excretion. The data provide further evidence of a role for endothelin in cyclosporine A-induced nephrotoxicity. Cyclosporine; Endothelin; Nephrotoxicity; Ca 2+ channel antagonists; Nifedipine
1. Introduction
2. Materials and methods
Cyclosporine A is an immunosuppressive agent used to prevent allograft rejection and, more recently, to treat some autoimmune diseases. Its use, however, can be complicated by nephrotoxicity which is characterized in its early stages by renal vasoconstriction. Endothelin (ET) is a newly discovered peptide and is one of the most potent endogenous vasoconstrictors yet described (Yanigasawa et al., 1988). There is growing evidence that ET may play a role in cyclosporine A-induced renal insufficiency since endothelin is a potent renal vasoconstrictor (e.g. Gardiner et al., 1990; Goetz et al., 1988; King et al., 1989), circulating endothelin levels are elevated in rats with cyclosporine A-induced nephrotoxicity (Kon et al., 1990), an ET antibody can attenuate cyclosporine A-induced reductions in renal function (Dadan et al., 1990; Kon et al., 1990), and cyclosporine A causes an increase in the number of renal ET receptors (Nambi et al., 1990). In the present report, we have determined the effect of nifedipine, an agent reported to ameliorate cyclosporine A nephrotoxicity in rats (Dieperink et al., 1988), on renal function, urinary ET excretion, and renal ET receptor number in rats treated with cyclosporine A.
2.1. Animal preparation
Correspondence to: D.P. Brooks, SmithKline Beecham Pharmaceuticals, Department of Pharmacology, L521, P.O. Box 1539, King of Prussia, PA 19406-0939, U.S.A.
Male Sprague-Dawley rats weighing approximately 300 g were housed in individual metabolism cages. The animals were placed randomly in four groups and treated daily for 4 days with either nifedipine (0.1 m g / k g per day i.p.) or its vehicle (15% PEG, 15% ethanol, 70% H 2 0 ) and cyclosporine A (Sandimmune, 50 m g / k g i.p.) or its vehicle (olive oil). Daily food intake of the cyclosporine A-treated rats was measured, and the food intake of the olive oil-treated animals adjusted appropriately. Urine was collected for the 24 h period immediately following the last administration of cyclosporine A or vehicle. Animals were then anesthetized with sodium pentobarbitone and serum samples (by cardiac puncture) and renal tissue harvested. Serum and urine samples were assayed for creatinine and urea nitrogen (Synchron AS8 Clinical Analyzer) and 24 h creatinine clearances calculated. In addition, urine was assayed for ET content vide infra. To insure that the 24 h creatinine clearances were providing reasonable estimates of the reductions in glomerular filtration rate induced by cyclosporine A, direct renal function was measured in a group of cyclosporine- and vehicle-treated animals. Animals were anesthetized with inactin and the clearances of [14C]inulin determined using methods described previously (Brooks et al., 1990).
116
2.2. Determination of the E T receptor number Kidney membranes were prepared following the procedure of Platia et al. (1986). [125I]ET-1 binding was performed following the procedure of Nambi et al. (1990). Briefly, 10 /zg of membrane protein was incubated for 1 h at 3 0 ° C in 50 mM Tris/10 m M MgC12, 0.05% BSA, pH. 7.5 buffer in a total volume of 100/~1 and 0.5 nM [125I] ET-1. Non-specific binding was measured in the presence of 100 nM unlabelled ET-1. The reactions were stopped with 3.0 ml of ice-cold 50 mM Tris/10 mM MgC12, free ligand was separated from bound ligand by filtration and washing through Whatman G F / C filters using a Brandel cell harvester, and the filters were counted in a gamma counter with an efficiency of 75%.
2.3. Endothelin assay Approximately 10 ml urine was acidified with 0.1% trifluoroacetic acid and applied to preactivated 'Sep-Pak C18' cartridges (Waters). The absorbed peptides were eluted with 3.0 ml acetonitrile/water/trifluoroacetic acid (60/40/0.1), evaporated to dryness and then reconstituted to 1.0 ml with radioimmunoassay (RIA) buffer. ET levels were determined by a specific RIA. Antiserum specific for ET was prepared in rabbits with an ET-(15-21)-KLH conjugate. Cross-reactivity of this antibody with either porcine big endothelin-(1-39) or human big ET-(1-38) was < 0.001%. The R I A assay buffer was 0.02 M boric acid (pH 7.4) containing 0.1% BSA, 0.1% Tween 20 and 0.02% sodium azide. Standard ET-1 (American Peptides) or sample (100/11) was incubated with 100/~1 of antiserum (final dilution 1 : 100000) for 24 h at 4 ° C , then 100 /~1 [125I]ET-1 (ca. 12000-14000 cpm, New England Nuclear) was added to each tube a n d incubated for 18 h at 4 ° C . Donkey anti-rabbit serum coated onto magnetized polymer particles (Amersham Corp.) was incubated in each tube for 10 min and the antibody bound fraction was separated
magnetically. The sensitivity of the RIA was 1 f m o l / t u b e and the 50% intercept was 5 fmol/tube. Urinary ET excretion was corrected for creatinine clearance and expressed as f m o l / m i n (per creatinine clearance). Data are expressed as means_+ S.E. and were analyzed statistically with a one-way analysis of variance with four levels (one for each treatment group). Orthogonal contrasts were used to test the a priori comparisons between groups.
3. Results Treatment of rats with cyclosporine A for 4 days resulted in a significant impairment of renal function as indicated by a reduction in 24 h creatinine clearance and increases in serum creatinine and blood urea nitrogen levels (table 1) when compared to pair-fed control animals. Coincident with the reduction in renal function was a 3-fold increase in urinary ET excretion and an 84% increase in renal ET receptor number (table 1). Treatment of rats with nifedipine resulted in a significant attenuation of cyclosporine A-induced reductions in renal function and urinary ET excretion but not cyclosporine A-induced renal ET receptor up-regulation (table 1). Nifedipine administration to non-cyclosporine A-treated rats had no effect on any of the parameters measured. Direct measurement of [14C]inulin clearance in inactin-anaesthetized rats indicated that cyclosporine resulted in a 47% decrease in glomerular filtration rate (0.79 +_ 0.03 versus 0.42 + 0.05 m l / m i n per 100 g). This corresponds well with the 43% decrease observed in 24 h creatinine clearance (0.60 + 0.02 versus 0.34 + 0.04 m l / m i n per 100 g).
4. Discussion In the present study, we provide more evidence for a role of ET in cyclosporine A-induced renal dysfunction
TABLE 1 Effect of nifedipine on renal function, urinary endothelin in excretion and renal endothelin binding. Serum creatinine (Scr), blood urea nitrogen (BUN), 24 h creatinine clearance (Ccr), urinary endothelin (ET) excretion (UETV) corrected for creatinine clearance, and endothelin receptor number in rats treated with nifedipine (NIF, 0.1 mg/kg per day i.p.) or its vehicle (VEH) and cyclosporineA (CYA, 50 mg/kg i.p.) or its vehicle (VEH). n = 7-9 rats per group. Group Scr (mg/dl) BUN(mg/dl) Ccr (ml/min per 100 g) UETV (fmol/min per Cer) ET receptors (fmol/mg)
VEH/VEH
NIF/VEH
VEH/CYA
NIF/CYA
0.43 + 0.02 6.0 + 0.4 0.60+ 0.02 0.50-t- 0.10 190 + 79
0.42 + 0.02 6.2 + 0.3 0.59+ 0.03 0.50± 0.10 246 + 72
0.59 d: 0.66 b 12.1 + 3.4b 0.34+ 0.05 b 1.57+ 0.34 b 349 ± 94 b
0.44 + 0.03 c 8.3 + 0.4a 0.51+ 0.05 c 0.84+ 0.24 c 295 + 84
a p < 0.05; b p < 0.01 versus VEH/'VEH or NIF/VEH as appropriate; ~ P < 0.05 versus VEH/CYA.
117 by confirming previous observations of cyclosporine A-induced E T receptor up-regulation ( N a m b i et al., 1990) and demonstrating for the first time increased urinary ET excretion. This cyclosporine-induced up-regulation of E T receptors is mainly due to an increase in m a x i m u m binding with no significant change in affinity ( N a m b i et al., 1990). Since ET is both freely filterable in the glomerulus and can be synthesized in cultured kidney cells (Kosaka et al., 1989), the increased E T excretion likely reflects increased circulating ET levels ( K o n et al., 1990) a n d / o r increased renal ET production. Furthermore, nifedipine, which significantly attenuated the cyclosporine A-induced changes in serum creatinine and creatinine clearance, also significantly reduced urinary ET excretion. Interestingly, the significant increase in renal E T receptor n u m b e r was not altered b y nifedipine treatment. The reason for this is not clear, but may reflect the incomplete protection of renal function afforded by nifedipine or the small (not statistically significant P > 0.05) increase in E T receptor n u m b e r observed in rats receiving only nifedipine. The significant attenuation of cyclosporine A-induced changes in renal function induced by nifedipine supports previous observations in rats that this calcium c h a n n e l b l o c k e r m a y be beneficial in treating cyclosporine A nephrotoxicity (Dieperink et al., 1986). The mechanism for the therapeutic effect of calcium channel blockers m a y be direct renal vasodilation, resulting in a functional antagonism of the renal vasoconstricting effects of cyclosporine A. This would be comparable to the significant reversal of cyclosporine A-induced vasoconstriction observed by administration of fenoldopam, a d o p a m i n e D A 1 agonist with renal vasodilating properties (Brooks et al., 1990). Observations, however, that nifedipine can attenuate b o t h cyclosporine A-induced renal dysfunction and enhanced urinary ET excretion might suggest that the mechanism of action of nifedipine m a y involve reduced ET release in addition to direct renal vasodilation. Furthermore, calcium channel blockers have been shown to attenuate ET-induced renal vasoconstriction (Cao and Banks, 1990). In summary, the present study demonstrates that the reduction in renal function induced by cyclosporine A is accompanied by significant renal ET receptor up-regulation and enhanced urinary excretion of ET. Furthermore, the calcium channel antagonist, nifedipine, was
able to attenuate b o t h the renal dysfunction and enhanced ET excretion.
Acknowledgements We thank Miss Susan Tim and Mrs. Rose Ann Boothroyd for their assistance in preparing the manuscript. Clare Garvie and Sandra Hoffman for superb technical assistance, and Dr. Timothy Halverson for performing the statistical analyses.
References Brooks, D.P., D.J. Drutz and R.R. Ruffolo, 1990, Prevention and complete reversal of cyclosporine A-induced renal vasoconstriction in the rat by fenoldopam, J. Pharmacol. Exp. Ther. 254, 375. Cao, L. and R.O. Banks, 1990, Cardiovascular and renal actions of endothelin: effects of calcium-channel blockers, Am. J. Physiol. 258, F254. Dadan, J., N. Perico and G. Remuzzi, 1990, Role of endothelin in cyclosporine-induced renal vasoconstriction, Kidney Int. 37, 479. Dieperink, H., P.P. Leyssac, H. Starldint, K.A. Jorgensen and E. Kemp, 1986, Antagonist capacities of nifedipine, captopril, phenoxybenzamine, prostacyclin, and indomethacin on cyclosporine A-induced impairment of rat renal function, European J. Clin. Invest. 16, 540. Gardiner, S.M., A.M. Compton and T. Bennett, 1990, Regional haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans and Brattleboro rats, Br. J. Pharmacol. 99, 107. Goetz, K.L., B.C. Wang, J.B. Madwed, J.L. Zhu and R.J. Leadley, Jr., 1988, Cardiovascular, renal and endocrine responses to intravenous endothelin in conscious dogs, Am. J. Physiol. 255, R1064. King, A.J., B.M. Brenner and S. Anderson, 1989, Endothelin: a potent renal and systemic vasoconstrictor peptide, Am. J. Physiol. 256, F1051. Kon, V., M. Sugiura, T. Inagami, B.R. Harvie, I. Ichikawa and R.L. Hoover, 1990, Role of endothelin in cyclosporine-induced glomerular dysfunction, Kidney Int. 37, 1487. Kosaka, T., N. Suzuki, H. Matsumoto, Y. Itoh, T. Yasuhara, H. Onda and M. Fujino, 1989, Synthesis of the vasoconstrictor peptide endothelin in kidney cells, FEBS Lett. 249, 42. Nambi, P., M. Pullen, L.C. Contino and D.P. Brooks, 1990, Upregulation of renal endothelin receptors in rats with cyclosporine A nephrotoxicity, European J. Pharmacol. 187, 113. Platia, M.P., K.J. Catt, G.D. Hadgen and G. Aguileva, 1986, Regulation of primate angiotensin II receptors during altered sodium intake, Hypertension 8, 1121. Yanagisawa, M., H. Kurihar, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, G. Katsutoshi and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 441.
115
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100210I
EJP 20786
Short communication
Effect of nifedipine on cyclosporine A-induced nephrotoxicity, urinary endothelin excretion and renal endothelin receptor number David P. Brooks, Eliot H. Ohlstein, Lisa C. C o n t i n o , Barbara Storer, M a r k Pullen, M a d e l y n C a l t a b i a n o 1 and Ponnal Nambi SmithKline Beecham Pharmaceuticals, Departments of Pharmacology and I Cell Sciences, King of Prussia, PA 19406-0939, U.S.A. Received 21 November 1990, accepted 2 January 1991
The aim of the present study was to determine the effect of a calcium channel blocker on renal function, urinary endothelin excretion and endothelin receptor number in rats. Administration of cyclosporine resulted in a significant impairment of renal function when measured by either [14C]inulin or 24 h creatinine clearances. This nephrotoxicity was associated with statistically significant (P < 0.05) increases in urinary endothelin excretion and renal endothelin receptor number. Treatment with nifedipine attenuated cyclosporine A-induced renal dysfunction and reduced urinary endothelin excretion. The data provide further evidence of a role for endothelin in cyclosporine A-induced nephrotoxicity. Cyclosporine; Endothelin; Nephrotoxicity; Ca 2+ channel antagonists; Nifedipine
1. Introduction
2. Materials and methods
Cyclosporine A is an immunosuppressive agent used to prevent allograft rejection and, more recently, to treat some autoimmune diseases. Its use, however, can be complicated by nephrotoxicity which is characterized in its early stages by renal vasoconstriction. Endothelin (ET) is a newly discovered peptide and is one of the most potent endogenous vasoconstrictors yet described (Yanigasawa et al., 1988). There is growing evidence that ET may play a role in cyclosporine A-induced renal insufficiency since endothelin is a potent renal vasoconstrictor (e.g. Gardiner et al., 1990; Goetz et al., 1988; King et al., 1989), circulating endothelin levels are elevated in rats with cyclosporine A-induced nephrotoxicity (Kon et al., 1990), an ET antibody can attenuate cyclosporine A-induced reductions in renal function (Dadan et al., 1990; Kon et al., 1990), and cyclosporine A causes an increase in the number of renal ET receptors (Nambi et al., 1990). In the present report, we have determined the effect of nifedipine, an agent reported to ameliorate cyclosporine A nephrotoxicity in rats (Dieperink et al., 1988), on renal function, urinary ET excretion, and renal ET receptor number in rats treated with cyclosporine A.
2.1. Animal preparation
Correspondence to: D.P. Brooks, SmithKline Beecham Pharmaceuticals, Department of Pharmacology, L521, P.O. Box 1539, King of Prussia, PA 19406-0939, U.S.A.
Male Sprague-Dawley rats weighing approximately 300 g were housed in individual metabolism cages. The animals were placed randomly in four groups and treated daily for 4 days with either nifedipine (0.1 m g / k g per day i.p.) or its vehicle (15% PEG, 15% ethanol, 70% H 2 0 ) and cyclosporine A (Sandimmune, 50 m g / k g i.p.) or its vehicle (olive oil). Daily food intake of the cyclosporine A-treated rats was measured, and the food intake of the olive oil-treated animals adjusted appropriately. Urine was collected for the 24 h period immediately following the last administration of cyclosporine A or vehicle. Animals were then anesthetized with sodium pentobarbitone and serum samples (by cardiac puncture) and renal tissue harvested. Serum and urine samples were assayed for creatinine and urea nitrogen (Synchron AS8 Clinical Analyzer) and 24 h creatinine clearances calculated. In addition, urine was assayed for ET content vide infra. To insure that the 24 h creatinine clearances were providing reasonable estimates of the reductions in glomerular filtration rate induced by cyclosporine A, direct renal function was measured in a group of cyclosporine- and vehicle-treated animals. Animals were anesthetized with inactin and the clearances of [14C]inulin determined using methods described previously (Brooks et al., 1990).
116
2.2. Determination of the E T receptor number Kidney membranes were prepared following the procedure of Platia et al. (1986). [125I]ET-1 binding was performed following the procedure of Nambi et al. (1990). Briefly, 10 /zg of membrane protein was incubated for 1 h at 3 0 ° C in 50 mM Tris/10 m M MgC12, 0.05% BSA, pH. 7.5 buffer in a total volume of 100/~1 and 0.5 nM [125I] ET-1. Non-specific binding was measured in the presence of 100 nM unlabelled ET-1. The reactions were stopped with 3.0 ml of ice-cold 50 mM Tris/10 mM MgC12, free ligand was separated from bound ligand by filtration and washing through Whatman G F / C filters using a Brandel cell harvester, and the filters were counted in a gamma counter with an efficiency of 75%.
2.3. Endothelin assay Approximately 10 ml urine was acidified with 0.1% trifluoroacetic acid and applied to preactivated 'Sep-Pak C18' cartridges (Waters). The absorbed peptides were eluted with 3.0 ml acetonitrile/water/trifluoroacetic acid (60/40/0.1), evaporated to dryness and then reconstituted to 1.0 ml with radioimmunoassay (RIA) buffer. ET levels were determined by a specific RIA. Antiserum specific for ET was prepared in rabbits with an ET-(15-21)-KLH conjugate. Cross-reactivity of this antibody with either porcine big endothelin-(1-39) or human big ET-(1-38) was < 0.001%. The R I A assay buffer was 0.02 M boric acid (pH 7.4) containing 0.1% BSA, 0.1% Tween 20 and 0.02% sodium azide. Standard ET-1 (American Peptides) or sample (100/11) was incubated with 100/~1 of antiserum (final dilution 1 : 100000) for 24 h at 4 ° C , then 100 /~1 [125I]ET-1 (ca. 12000-14000 cpm, New England Nuclear) was added to each tube a n d incubated for 18 h at 4 ° C . Donkey anti-rabbit serum coated onto magnetized polymer particles (Amersham Corp.) was incubated in each tube for 10 min and the antibody bound fraction was separated
magnetically. The sensitivity of the RIA was 1 f m o l / t u b e and the 50% intercept was 5 fmol/tube. Urinary ET excretion was corrected for creatinine clearance and expressed as f m o l / m i n (per creatinine clearance). Data are expressed as means_+ S.E. and were analyzed statistically with a one-way analysis of variance with four levels (one for each treatment group). Orthogonal contrasts were used to test the a priori comparisons between groups.
3. Results Treatment of rats with cyclosporine A for 4 days resulted in a significant impairment of renal function as indicated by a reduction in 24 h creatinine clearance and increases in serum creatinine and blood urea nitrogen levels (table 1) when compared to pair-fed control animals. Coincident with the reduction in renal function was a 3-fold increase in urinary ET excretion and an 84% increase in renal ET receptor number (table 1). Treatment of rats with nifedipine resulted in a significant attenuation of cyclosporine A-induced reductions in renal function and urinary ET excretion but not cyclosporine A-induced renal ET receptor up-regulation (table 1). Nifedipine administration to non-cyclosporine A-treated rats had no effect on any of the parameters measured. Direct measurement of [14C]inulin clearance in inactin-anaesthetized rats indicated that cyclosporine resulted in a 47% decrease in glomerular filtration rate (0.79 +_ 0.03 versus 0.42 + 0.05 m l / m i n per 100 g). This corresponds well with the 43% decrease observed in 24 h creatinine clearance (0.60 + 0.02 versus 0.34 + 0.04 m l / m i n per 100 g).
4. Discussion In the present study, we provide more evidence for a role of ET in cyclosporine A-induced renal dysfunction
TABLE 1 Effect of nifedipine on renal function, urinary endothelin in excretion and renal endothelin binding. Serum creatinine (Scr), blood urea nitrogen (BUN), 24 h creatinine clearance (Ccr), urinary endothelin (ET) excretion (UETV) corrected for creatinine clearance, and endothelin receptor number in rats treated with nifedipine (NIF, 0.1 mg/kg per day i.p.) or its vehicle (VEH) and cyclosporineA (CYA, 50 mg/kg i.p.) or its vehicle (VEH). n = 7-9 rats per group. Group Scr (mg/dl) BUN(mg/dl) Ccr (ml/min per 100 g) UETV (fmol/min per Cer) ET receptors (fmol/mg)
VEH/VEH
NIF/VEH
VEH/CYA
NIF/CYA
0.43 + 0.02 6.0 + 0.4 0.60+ 0.02 0.50-t- 0.10 190 + 79
0.42 + 0.02 6.2 + 0.3 0.59+ 0.03 0.50± 0.10 246 + 72
0.59 d: 0.66 b 12.1 + 3.4b 0.34+ 0.05 b 1.57+ 0.34 b 349 ± 94 b
0.44 + 0.03 c 8.3 + 0.4a 0.51+ 0.05 c 0.84+ 0.24 c 295 + 84
a p < 0.05; b p < 0.01 versus VEH/'VEH or NIF/VEH as appropriate; ~ P < 0.05 versus VEH/CYA.
117 by confirming previous observations of cyclosporine A-induced E T receptor up-regulation ( N a m b i et al., 1990) and demonstrating for the first time increased urinary ET excretion. This cyclosporine-induced up-regulation of E T receptors is mainly due to an increase in m a x i m u m binding with no significant change in affinity ( N a m b i et al., 1990). Since ET is both freely filterable in the glomerulus and can be synthesized in cultured kidney cells (Kosaka et al., 1989), the increased E T excretion likely reflects increased circulating ET levels ( K o n et al., 1990) a n d / o r increased renal ET production. Furthermore, nifedipine, which significantly attenuated the cyclosporine A-induced changes in serum creatinine and creatinine clearance, also significantly reduced urinary ET excretion. Interestingly, the significant increase in renal E T receptor n u m b e r was not altered b y nifedipine treatment. The reason for this is not clear, but may reflect the incomplete protection of renal function afforded by nifedipine or the small (not statistically significant P > 0.05) increase in E T receptor n u m b e r observed in rats receiving only nifedipine. The significant attenuation of cyclosporine A-induced changes in renal function induced by nifedipine supports previous observations in rats that this calcium c h a n n e l b l o c k e r m a y be beneficial in treating cyclosporine A nephrotoxicity (Dieperink et al., 1986). The mechanism for the therapeutic effect of calcium channel blockers m a y be direct renal vasodilation, resulting in a functional antagonism of the renal vasoconstricting effects of cyclosporine A. This would be comparable to the significant reversal of cyclosporine A-induced vasoconstriction observed by administration of fenoldopam, a d o p a m i n e D A 1 agonist with renal vasodilating properties (Brooks et al., 1990). Observations, however, that nifedipine can attenuate b o t h cyclosporine A-induced renal dysfunction and enhanced urinary ET excretion might suggest that the mechanism of action of nifedipine m a y involve reduced ET release in addition to direct renal vasodilation. Furthermore, calcium channel blockers have been shown to attenuate ET-induced renal vasoconstriction (Cao and Banks, 1990). In summary, the present study demonstrates that the reduction in renal function induced by cyclosporine A is accompanied by significant renal ET receptor up-regulation and enhanced urinary excretion of ET. Furthermore, the calcium channel antagonist, nifedipine, was
able to attenuate b o t h the renal dysfunction and enhanced ET excretion.
Acknowledgements We thank Miss Susan Tim and Mrs. Rose Ann Boothroyd for their assistance in preparing the manuscript. Clare Garvie and Sandra Hoffman for superb technical assistance, and Dr. Timothy Halverson for performing the statistical analyses.
References Brooks, D.P., D.J. Drutz and R.R. Ruffolo, 1990, Prevention and complete reversal of cyclosporine A-induced renal vasoconstriction in the rat by fenoldopam, J. Pharmacol. Exp. Ther. 254, 375. Cao, L. and R.O. Banks, 1990, Cardiovascular and renal actions of endothelin: effects of calcium-channel blockers, Am. J. Physiol. 258, F254. Dadan, J., N. Perico and G. Remuzzi, 1990, Role of endothelin in cyclosporine-induced renal vasoconstriction, Kidney Int. 37, 479. Dieperink, H., P.P. Leyssac, H. Starldint, K.A. Jorgensen and E. Kemp, 1986, Antagonist capacities of nifedipine, captopril, phenoxybenzamine, prostacyclin, and indomethacin on cyclosporine A-induced impairment of rat renal function, European J. Clin. Invest. 16, 540. Gardiner, S.M., A.M. Compton and T. Bennett, 1990, Regional haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans and Brattleboro rats, Br. J. Pharmacol. 99, 107. Goetz, K.L., B.C. Wang, J.B. Madwed, J.L. Zhu and R.J. Leadley, Jr., 1988, Cardiovascular, renal and endocrine responses to intravenous endothelin in conscious dogs, Am. J. Physiol. 255, R1064. King, A.J., B.M. Brenner and S. Anderson, 1989, Endothelin: a potent renal and systemic vasoconstrictor peptide, Am. J. Physiol. 256, F1051. Kon, V., M. Sugiura, T. Inagami, B.R. Harvie, I. Ichikawa and R.L. Hoover, 1990, Role of endothelin in cyclosporine-induced glomerular dysfunction, Kidney Int. 37, 1487. Kosaka, T., N. Suzuki, H. Matsumoto, Y. Itoh, T. Yasuhara, H. Onda and M. Fujino, 1989, Synthesis of the vasoconstrictor peptide endothelin in kidney cells, FEBS Lett. 249, 42. Nambi, P., M. Pullen, L.C. Contino and D.P. Brooks, 1990, Upregulation of renal endothelin receptors in rats with cyclosporine A nephrotoxicity, European J. Pharmacol. 187, 113. Platia, M.P., K.J. Catt, G.D. Hadgen and G. Aguileva, 1986, Regulation of primate angiotensin II receptors during altered sodium intake, Hypertension 8, 1121. Yanagisawa, M., H. Kurihar, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, G. Katsutoshi and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 441.
