- Email: [email protected]
Effect of cyclosporin A on rat kidney catecholamines
Life Sclences, Vol. 47, pp. 255-261 Printed in the U.S.A.
Pergamon Press
EFFECT OF CYCLOSPORIN A ON RAT KIDNEY CATECHOLAMINES Valentine A Durulbe, Anthony Okonmah, Lloyd Panton, and Gershwln T. Blyden Cllnlcal Pharmacology Research Unit, College of Pharmacy, Florida A&M University and the V.A. Medical Center, 1500 NW 12th Avenue, Miami, Fl (Received in final form May 22, 1990)
Summary The immunosuppressive agent, Cyclosporin A, (CsA) has been associated with nephrotoxiclty and hypertension. The mechanlsm for these effects are not known. We therefore determined the levels of the catecholamines; epinephrine (EPI), norepinephrine (NE) and dopamlne (DA) and some of their metabolites; epinine, dihyroxyphenylacetic acid (DOPAC), homovanilllc acid (HVA), metanephrine (ME) and 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the kidneys of rats treated intraperitoneally with either CsA (120~g/kg/body wt/day) or control vehicle (lml olive o i l / k g body wt/day). Six control or CsA treated rats were sacriflced at 1 hour or 24 hours after a single treatment or after 7 days of daily treatment. Renal catecholamlne levels were determined using HPLCamperometric detector. Treatment with CsA increased renal NE and EPI levels by 59% and 70% respectively within 1 hour. In the rats sacrificed 24 hours after treatment, renal NE, EPI and DA levels were similar to or less than the control levels. Treatment wlth CsA for 7 days resulted in marglnal Increases in renal NE (22%) and EPI (30%). These changes were assoclated with a signiflcant decrease in the levels of catecholamlne metabolites in the CsA treated kldneys as compared to the controls. The above findings suggest that increases in renal catecholamines may be involved in the CsA-induced hypertension and nephrotoxicity, perhaps by increasing renovascular resistance. The immunosuppressive agent CsA is wldely used in transplantation medlcine (1-4) and for the treatment of autoimmune dlseases (5,6). The use of CsA is associated with nephrotoxicity (7) and hypertension (9, 10). The acute and subacute forms of CsA nephrotoxicity have been associated wlth a decrease in renal blood flow (11-13) and are usually reversible (10). The CsA induced hypertension is also usually reverslble (14) and responds to conventional antihypertenslve therapy, Includlng treatment wlth adrenergic receptor blockers (14,15). The mechanlsms of the CsA-induced nephrotoxlcity and hypertension are yet unclear. I t has been suggested that the hypertension may be related to CsA-Induced increase in renovascular resistance and that the resulting decrease In renal blood flow and hypoxia may contrlbute to CsA induced nephrotoxicity (13). 0024-3205/90 $3.00 +.00 Copyrlght (c) 1990 Pergamon Press plc
256
CsA and Renal Catecholamines
Vol. 47, No. 4, 1990
Infuslon wlth NE or EPI was shown to increase renovascular reslstance, decrease renal blood flow (16), and also Induce renal hypertension (16). The role of renal catecholamines in the CsA-lnduced hypertension Is not known. Therefore, we investigated the possible involvement of catecholamines in the CsA-induced nephrotoxicity and hypertension by determlning the levels of NE, EPI and DA as well as the levels of the precursor, L-dopa, and some of the catecholamlne metabolites in the kldney of rats treated acutely or subchronically with CsA. Methods Male Sprague-Dawley rats, welghlng 200-250g each, were obtalned from Charles River Laboratorles, Wilmlngton, MA. Cyclosporin A was supplied by Sandoz Pharmaceutical Company, East Hanover, NJ. The rats were divided into 2 groups of 18 rats each. The control group was treated dally with a single intraperitoneal injection of olive o i i (lml/kg body wt/rat/day). The second group was treated s l m l l a r l y wlth olive o i i containing CsA (60 ~g/ml), to achieve a CsA dose of 120~g/kg body wt, as previously described (17) This dose of CsA was reported to be optimal for inducing prolactin secretion in rats ( 1 8 ) A l l the rats received water and food ad libitum and were housed in a temperature and l l g h t controlled room. Six rats from each group were sacrlficed by decapitation at the end of elther 1 hour, 24 hours or 7 days post i n l t l a t i o n of treatment. Tissue Preparatlon: The kidneys from each rat were excised immediately after the animal was sacrlficed. Each kldney was rinsed in cold d i s t i l l e d water, weighed and then homogenized in 5 vol of cold 0.2 M perchlorlc acid, using a polytron. The homogenate was centrifuged at 2000 x g for 10 min at 4 °C The resulting supernatant was passed through a 0.22 micron f i l t e r to remove large molecular weight peptides. Allquots (10-50 ~ l ) of the f i l t r a t e were used for catecholamlne analysls by means of a HPLCamperometric detector. HPLC Condltions: An ESA model 5700 solvent delivery module equipped wlth an ESA catecholamine HR-80 reverse phase column (PIN 68-0100) was used for the chromatography. The moblle phase used was CATA-PHASE, a phosphate buffer (pH 3.0)/methanol solvent, contalnlng sodium octyl sulfate as an ion palrlng agent (ESA, Bedford, MA). The flow rate was maintained at 1 ml/mln The catecholamines were monitored wlth an ESA Coulochem, Model 5100A amperometric detector, previously optlmlzed for catecholamlne analysis, wlth authentic catecholamine standards. The cells were set as follows: Guard cell (+ 0.25 V), detector 1 (+ 0.4 V) and detector 2 (- 0.35 V). Recording of the peaks was done with a linear 1200 chart recorder set at a chart speed of 1 cm/min. The retention times and recoveries for the d i f f e r e n t catecholamines were determlned by injectlng an aliquot of a supernatant spiked wlth known amounts of authentlc standards and f i l t e r e d as was done for the samples. Supernatants splked with varlous amounts of catecholamlnes and treated s i m l a r l y were used to generate calibration curves. Unspiked supernatant was also injected as blank to establlsh the
Vol. 47, No. 4, 1990
CsA and Renal Catecholamlnes
baseline used to correct the peak heights of the authentic standards. Sample catecholamine concentrations were interpolated from the peak height versus the concentration curves of the known standards, using the sample peak height. The catecholamlne concentratlons were adjusted for recovery and for tissue d i l u t i o n . S t a t i s t i c a l analysis was done by Student's t - t e s t . Results Intraperltoneal administration of CsA (120 #g/kg/day) caused a s i g n i f i c a n t increase in renal catecholamine levels within 1 hour of the i n i t i a t i o n of treatment. This increase in catecholamines was reversible, as there was a decrease in renal catecholamine levels in the CsA treated rats sacrificed 24 hours after the f i r s t treatment, r e l a t i v e to the levels in the time matched control rats. However, the levels of catecholamlnes in the kidney of the CsA treated rats sacrificed at 24 hours were not s i g n l f l c a n t l y d i f f e r e n t from the levels observed in the control rats sacrlflced at the end of 1 hour. As shown in flgure 1, the renal EPI level was increased by 70% over the control level within 1 hour of CsA administration. At the end of 24 hours of i n i t i a l treatment, the renal EPI levels in the CsA treated rats reverted to the same or below the control levels. Subchronic daily administration of CsA for 7 days resulted in an increase in the renal EPI level r e l a t i v e to the levels in the kidneys of time matched control rats. However, the increase in renal EPI level at the end of day 7 (30%) r e l a t i v e to the control, was less than that observed after the single i n l t l a l dose of CsA. Similar patterns were observed in the response of and subchronic treatment with CsA (figure 1). Thus the kidneys of CsA-treated rats sacrificed at 1 hour after or at the end of 7 days of treatment were increased by respectively.
renal NE to acute NE levels in the a single injection 59% and 22%
Like the EPI level, the increase in NE observed one hour after treatment wlth CsA, was reversed within 24 hours. There was no s i g n i f i c a n t difference in the kidney dopamine levels of the control and the CsA treated rats sacrificed after 1 hour (figure 1). The DA levels in the CsA-treated rats remained at or below the control levels 24 hours post treatment. There was an apparent but s t a t i s t i c a l l y i n s i g n l f i c a n t increase in the DA level (45-50%) in the kidneys of rats treated with CsA for 7 days, when compared with the control rats. However, when a l l the control rats were pooled and used for comparlson, no change was apparent In DA levels at any of the times studied (flgure 2). A similar comparison yielded a 30% or 17% increase in NE and a 42% or 31% increase in EPI levels, in the kidneys of rats treated with CsA for 1 hour or 7 days respectlvely (figure 2). The renal catecholamine levels of the CsA treated rats sacrlficed at 24 hours were not s i g n i f i c a n t l y d i f f e r e n t from those of the pooled control rats
257
258
CsA and Renal Catecholamznes
Vol. 47, No. 4, 1990
# P ( 0.15 * P ( 0.05 • * P ( 0.01
50.
40.
# 30, f-
20,
.m J<
10. e-
0
EPI NE DA 1 HOUR
24 HOURS
7 DAYS
DURATION OF TREATMENT
FIG 1 Changes in renal catecholamine, epinephrine (EPI), norepinephrine (NE) and dopamlne (DA) levels following daily single treatment with cyclosporin A (CsA). Rats were sacrificed at the end of the indlcated tlmes. The bars represent the mean ~ S.E. for duplicate assays of 6 kidneys from the control (open) and the CsA (solid) treated rats. The values indicated for EPI are 1/10 the real values. #P(0.1 • P ( 0.05 ** P ( 0.01
200. m
i",oo. m
150.
#
i1
~ 50. 0
HOURS DURATION OF CSA TREATMENT Figure Z
Renal EPI ( I - l ) , NE ( I I ) and t r e a t e d r a t s s a c r i f i c e d at were compared w i t h the mean a l l the c o n t r o l r a t s pooled of sacrifice.
DA ( ~ ] ) l e v e l s in the CsA 1 hour, 24 hours or 7 days catecholamlne l e v e l s f o r regardless of t h e i r time
Vol. 47, No. 4, 1990
CsA and Renal Catecholamines
259
Only a marginal increase was observed in the renal levels of the catecholamine precursor; L-dopa, in the CsA treated rats sacrificed at 1 hour (20%) or 7 days (15%), r e l a t i v e to the time matched controls (figure 3). As also shown in figure 3, CsA had no significant effect on the renal levels of the catecholamine metabolites eplnine, DOPAC, MHPGor ME In the rats sacrificed at 1 hour. However, i t decreased the level of HVA (20%) s i g n i f i c a n t l y withln 1 hour (figure 3). After 7 days of treatment, renal levels of MHPG, DOPAC, epinine, ME, and HVA decreased by 40%, 27%, 22%, 20% and 4% respectively, in the CsA treated rats, as compared with the control rats (figure 3). 150.
# P ( 0.1 * P ( 0.05
I #
,--
100
M
0
' O/'O .
$
1 HOUR 7 DAYS DURATION OF CSA TREATMENT Figure 3
The levels of renal L-dopa (F'I), epinine ( . ) , and the catecholamine metabolites dopac ( [ ] ) , MHPG ( [ ] ) , HVA ( [ ] ) and ME ( [ ] ) were determined as described for the catecholamines in the methods. The bars represent the mean + SE of their levels in the kidneys of rats treated wlth CsA, expressed as a percent of the mean levels in time matched control rat kidneys. The above results suggest that CsA causes an early and transient increase in the levels of renal NE and EPl, and a decrease in the level of some of their metabolites. Discussion The mechanism of CsA-induced hypertension is st111 unclear. The neurogenic and direct vascular involvement in CsA-induced hypertension were previously suggested, but never proven (19). I t is known that this hypertension responds to treatment with beta and alpha adrenergic blockers (14,15). The current observation of an early increase in renal NE and EPI by CsA supports the involvement of adrenergic mechanisms in the hypertensive side effects of this agent. I t has been shown that NE or EPI administration results in an increase in renovascular resistance and decreased renal clearance and blood flow (16). The above study also revealed no consistent difference between the renal cortex and the medulla
260
CsA and Renal Catecholamines
Vol. 47, No. 4, 1990
in t h e l r response to catecholamines. Thus, although we did not study the regional affects of CsA on renal catecholamines, an increase in EPI or NE content of the kidney would be expected to increase the renovascular resistance of both the cortex and the medulla. Other investigators have also demonstrated the existence of adrenergic innervatlon of the kidney, wlth NE being found in a l l the regions (20). The early increase of renal NE and EPI observed In the present study, may provlde a mechanism for the CsA induced increase in renovascular resistance and hypertension. The decline in t h e l r levels 24 hours after a single dose of CsA may explain the r e v e r s l b i l i t y of t h l s hypertension following CsA withdrawal. A better response to antihypertensive therapy occurs with time in CsA treated patlents and t h i s hypertension has been reported to be non progressive (15). This better response to therapy may be p a r t i a l l y explained by the less increase in renal NE and EPI observed durlng chronlc treatment wlth CsA. The observed decrease in renal catecholamlne metabolltes by CsA suggests that t h i s drug may i n h i b i t catecholamine metabollsm. Although the exact mechanism of t h i s i n h l b i t i o n was not investigated, the decreases in renal HVA, ME and MHPG levels by CsA suggest a possible l n h i b l t i o n of methylatlon of catecholamines. The increases in NE and EPI do not appear to be due to increase in t h e i r synthesls since there was no s l g n l f l c a n t change in the level of DA in t h i s study. The generalized decrease in renal levels of the metabolites after 7 days may r e f l e c t a decrease in the catecholamine turnover rate in the CsA treated rats. Although the dose of CsA used to produce the above changes also affected glutathione (17) and prolactln (18) levels markedly, hlgher doses may cause more pronounced increases in the levels of catecholamlnes. The influence of systemic levels of CsA was i n d l r e c t l y investigated by quantltating the levels of renal catecholamines, 24 hours after a slngle injection of CsA. The decllne of the catecholamines to t h e i r control levels after t h l s period, which corresponds approximately to the t 1/2 for CsA (21), suggests that the observed responses were dependent on the systemic a v a i l a b i l i t y of CsA. This was further supported by the fact that the catecholamine levels remained hlgh durlng dally treatments wlth CsA for 7 days. Observatlon of these changes wlth the low dose of CsA used in t h l s study suggest that an increase in renal catecholamines levels may be one of the primary and early effects of t h l s important immunosuppresslve agent. Although the underlying mechanism for the increase in catecholamlnes by CsA is not clear from the present study, the reported observatlons are pertlnent for further elucldatlon of the mechanism of CsA-induced nephrotoxicity and hypertension. In conclusion, the present flndlngs suggest that increase in renal EPI and NE levels may play a s i g n l f l c a n t role in the early CsA induced hypertenslon
References 1. 2. 3.
M. GASCON, Transplant. Proc. 19(40) 3481-3485 (1987). R.BUSUTTIL, L. GOLDSTEIN, G. DANOVITCH, M. AMENT, and L. MEMSIC, Ann I n t . Med. 104:377-389 (1986). A. TATMAN, B. TUCKER, J AMESS, W. CATTELL, and L BAKER, Lancet 1(8597): 1297 (1988).
Vol. 47, No. 4, 1990
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14 15. 16. 17. 18. 19. 20. 21.
CsA and Renal Catecholamines
C. HAMBERGER, S. URIEN, J. BARRE, M. BRANDEBOURGER, M. LEMAIRE and P. LANG, Ther. Drug. Monit. 10(1): 28-33 (1988). B. VON GRAFFENRIED, Dlabetol. Hotel Diul. 135; 4-6 (1987) G. THOENES, T. UMSCHEID, T. SITTER, and K. LANGER, Immunol. Lett. 15(4): 301-6 (1987). P. HEERING, and B. GRABENESEE, DTSCHMED WOCHENSCHR. 113(20): 833-4 (1988). F. DISCHE, J. NEUBERGER, O. KEATING, V PARSONS, R. CALNE, and R. WILLIAMS, Lab-Invest 58(4): 395-402 (1988). M. HAKIM, J WALLWORK, and T ENGLISH, Ann-Thorac-Surg 46(5):495-501 (1988). G. DERAY, P. LE-HOANG, P. CACOUB, B. AUPETIT, A. MERTANI, F. MARTINEZ and J. ROTTEMBOURG, Eur. J. Clln. Pharmaco]. 34(6):601-4 (1988). H.S. CAIRNS, U. RAVAL and G.H. NEILD. Transp. 46(1): 79-82 (1988). B.D. KAHAN, (ed). Transp]. Proc. 17" 185-196 (1985). H MOODY, Lancet 1(8491): 1221-2 (1986). R M. FERGUSON, and B.G. SOMMER, Am. O. Kidney Dis. 6, 296-306 (1985). 8.D KAHAN, Am.J. Kidney Dis. 5, 313-7 (1985). K. AUKLAND, Acta Physiol. Scand 72, 498-509 (1968). V.A. DURUIBE, A. OKONMAHand G.T. BLYDEN, Pharmaco] 39(6):205-212 (1989). S.B. CARDON, D.F. LARSON, and D.H. RUSSELL, Blochem. Biophys. Res. Commun 120,614-618 (1984). THOMPSONet a] Transp]. Proc. 1___5:2573-2577 (1983). O. MCKENNA, and E. ANGELAKOS, E Circ. Res. 22, 345-354 (1968). O. WAGNER, E. SCHREIER, F. HEITZ, and G. MAURER, Drug Metab. Dlsp. 15(3): 377-383 (1987)
261
Pergamon Press
EFFECT OF CYCLOSPORIN A ON RAT KIDNEY CATECHOLAMINES Valentine A Durulbe, Anthony Okonmah, Lloyd Panton, and Gershwln T. Blyden Cllnlcal Pharmacology Research Unit, College of Pharmacy, Florida A&M University and the V.A. Medical Center, 1500 NW 12th Avenue, Miami, Fl (Received in final form May 22, 1990)
Summary The immunosuppressive agent, Cyclosporin A, (CsA) has been associated with nephrotoxiclty and hypertension. The mechanlsm for these effects are not known. We therefore determined the levels of the catecholamines; epinephrine (EPI), norepinephrine (NE) and dopamlne (DA) and some of their metabolites; epinine, dihyroxyphenylacetic acid (DOPAC), homovanilllc acid (HVA), metanephrine (ME) and 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the kidneys of rats treated intraperitoneally with either CsA (120~g/kg/body wt/day) or control vehicle (lml olive o i l / k g body wt/day). Six control or CsA treated rats were sacriflced at 1 hour or 24 hours after a single treatment or after 7 days of daily treatment. Renal catecholamlne levels were determined using HPLCamperometric detector. Treatment with CsA increased renal NE and EPI levels by 59% and 70% respectively within 1 hour. In the rats sacrificed 24 hours after treatment, renal NE, EPI and DA levels were similar to or less than the control levels. Treatment wlth CsA for 7 days resulted in marglnal Increases in renal NE (22%) and EPI (30%). These changes were assoclated with a signiflcant decrease in the levels of catecholamlne metabolites in the CsA treated kldneys as compared to the controls. The above findings suggest that increases in renal catecholamines may be involved in the CsA-induced hypertension and nephrotoxicity, perhaps by increasing renovascular resistance. The immunosuppressive agent CsA is wldely used in transplantation medlcine (1-4) and for the treatment of autoimmune dlseases (5,6). The use of CsA is associated with nephrotoxicity (7) and hypertension (9, 10). The acute and subacute forms of CsA nephrotoxicity have been associated wlth a decrease in renal blood flow (11-13) and are usually reversible (10). The CsA induced hypertension is also usually reverslble (14) and responds to conventional antihypertenslve therapy, Includlng treatment wlth adrenergic receptor blockers (14,15). The mechanlsms of the CsA-induced nephrotoxlcity and hypertension are yet unclear. I t has been suggested that the hypertension may be related to CsA-Induced increase in renovascular resistance and that the resulting decrease In renal blood flow and hypoxia may contrlbute to CsA induced nephrotoxicity (13). 0024-3205/90 $3.00 +.00 Copyrlght (c) 1990 Pergamon Press plc
256
CsA and Renal Catecholamines
Vol. 47, No. 4, 1990
Infuslon wlth NE or EPI was shown to increase renovascular reslstance, decrease renal blood flow (16), and also Induce renal hypertension (16). The role of renal catecholamines in the CsA-lnduced hypertension Is not known. Therefore, we investigated the possible involvement of catecholamines in the CsA-induced nephrotoxicity and hypertension by determlning the levels of NE, EPI and DA as well as the levels of the precursor, L-dopa, and some of the catecholamlne metabolites in the kldney of rats treated acutely or subchronically with CsA. Methods Male Sprague-Dawley rats, welghlng 200-250g each, were obtalned from Charles River Laboratorles, Wilmlngton, MA. Cyclosporin A was supplied by Sandoz Pharmaceutical Company, East Hanover, NJ. The rats were divided into 2 groups of 18 rats each. The control group was treated dally with a single intraperitoneal injection of olive o i i (lml/kg body wt/rat/day). The second group was treated s l m l l a r l y wlth olive o i i containing CsA (60 ~g/ml), to achieve a CsA dose of 120~g/kg body wt, as previously described (17) This dose of CsA was reported to be optimal for inducing prolactin secretion in rats ( 1 8 ) A l l the rats received water and food ad libitum and were housed in a temperature and l l g h t controlled room. Six rats from each group were sacrlficed by decapitation at the end of elther 1 hour, 24 hours or 7 days post i n l t l a t i o n of treatment. Tissue Preparatlon: The kidneys from each rat were excised immediately after the animal was sacrlficed. Each kldney was rinsed in cold d i s t i l l e d water, weighed and then homogenized in 5 vol of cold 0.2 M perchlorlc acid, using a polytron. The homogenate was centrifuged at 2000 x g for 10 min at 4 °C The resulting supernatant was passed through a 0.22 micron f i l t e r to remove large molecular weight peptides. Allquots (10-50 ~ l ) of the f i l t r a t e were used for catecholamlne analysls by means of a HPLCamperometric detector. HPLC Condltions: An ESA model 5700 solvent delivery module equipped wlth an ESA catecholamine HR-80 reverse phase column (PIN 68-0100) was used for the chromatography. The moblle phase used was CATA-PHASE, a phosphate buffer (pH 3.0)/methanol solvent, contalnlng sodium octyl sulfate as an ion palrlng agent (ESA, Bedford, MA). The flow rate was maintained at 1 ml/mln The catecholamines were monitored wlth an ESA Coulochem, Model 5100A amperometric detector, previously optlmlzed for catecholamlne analysis, wlth authentic catecholamine standards. The cells were set as follows: Guard cell (+ 0.25 V), detector 1 (+ 0.4 V) and detector 2 (- 0.35 V). Recording of the peaks was done with a linear 1200 chart recorder set at a chart speed of 1 cm/min. The retention times and recoveries for the d i f f e r e n t catecholamines were determlned by injectlng an aliquot of a supernatant spiked wlth known amounts of authentlc standards and f i l t e r e d as was done for the samples. Supernatants splked with varlous amounts of catecholamlnes and treated s i m l a r l y were used to generate calibration curves. Unspiked supernatant was also injected as blank to establlsh the
Vol. 47, No. 4, 1990
CsA and Renal Catecholamlnes
baseline used to correct the peak heights of the authentic standards. Sample catecholamine concentrations were interpolated from the peak height versus the concentration curves of the known standards, using the sample peak height. The catecholamlne concentratlons were adjusted for recovery and for tissue d i l u t i o n . S t a t i s t i c a l analysis was done by Student's t - t e s t . Results Intraperltoneal administration of CsA (120 #g/kg/day) caused a s i g n i f i c a n t increase in renal catecholamine levels within 1 hour of the i n i t i a t i o n of treatment. This increase in catecholamines was reversible, as there was a decrease in renal catecholamine levels in the CsA treated rats sacrificed 24 hours after the f i r s t treatment, r e l a t i v e to the levels in the time matched control rats. However, the levels of catecholamlnes in the kidney of the CsA treated rats sacrificed at 24 hours were not s i g n l f l c a n t l y d i f f e r e n t from the levels observed in the control rats sacrlflced at the end of 1 hour. As shown in flgure 1, the renal EPI level was increased by 70% over the control level within 1 hour of CsA administration. At the end of 24 hours of i n i t i a l treatment, the renal EPI levels in the CsA treated rats reverted to the same or below the control levels. Subchronic daily administration of CsA for 7 days resulted in an increase in the renal EPI level r e l a t i v e to the levels in the kidneys of time matched control rats. However, the increase in renal EPI level at the end of day 7 (30%) r e l a t i v e to the control, was less than that observed after the single i n l t l a l dose of CsA. Similar patterns were observed in the response of and subchronic treatment with CsA (figure 1). Thus the kidneys of CsA-treated rats sacrificed at 1 hour after or at the end of 7 days of treatment were increased by respectively.
renal NE to acute NE levels in the a single injection 59% and 22%
Like the EPI level, the increase in NE observed one hour after treatment wlth CsA, was reversed within 24 hours. There was no s i g n i f i c a n t difference in the kidney dopamine levels of the control and the CsA treated rats sacrificed after 1 hour (figure 1). The DA levels in the CsA-treated rats remained at or below the control levels 24 hours post treatment. There was an apparent but s t a t i s t i c a l l y i n s i g n l f i c a n t increase in the DA level (45-50%) in the kidneys of rats treated with CsA for 7 days, when compared with the control rats. However, when a l l the control rats were pooled and used for comparlson, no change was apparent In DA levels at any of the times studied (flgure 2). A similar comparison yielded a 30% or 17% increase in NE and a 42% or 31% increase in EPI levels, in the kidneys of rats treated with CsA for 1 hour or 7 days respectlvely (figure 2). The renal catecholamine levels of the CsA treated rats sacrlficed at 24 hours were not s i g n i f i c a n t l y d i f f e r e n t from those of the pooled control rats
257
258
CsA and Renal Catecholamznes
Vol. 47, No. 4, 1990
# P ( 0.15 * P ( 0.05 • * P ( 0.01
50.
40.
# 30, f-
20,
.m J<
10. e-
0
EPI NE DA 1 HOUR
24 HOURS
7 DAYS
DURATION OF TREATMENT
FIG 1 Changes in renal catecholamine, epinephrine (EPI), norepinephrine (NE) and dopamlne (DA) levels following daily single treatment with cyclosporin A (CsA). Rats were sacrificed at the end of the indlcated tlmes. The bars represent the mean ~ S.E. for duplicate assays of 6 kidneys from the control (open) and the CsA (solid) treated rats. The values indicated for EPI are 1/10 the real values. #P(0.1 • P ( 0.05 ** P ( 0.01
200. m
i",oo. m
150.
#
i1
~ 50. 0
HOURS DURATION OF CSA TREATMENT Figure Z
Renal EPI ( I - l ) , NE ( I I ) and t r e a t e d r a t s s a c r i f i c e d at were compared w i t h the mean a l l the c o n t r o l r a t s pooled of sacrifice.
DA ( ~ ] ) l e v e l s in the CsA 1 hour, 24 hours or 7 days catecholamlne l e v e l s f o r regardless of t h e i r time
Vol. 47, No. 4, 1990
CsA and Renal Catecholamines
259
Only a marginal increase was observed in the renal levels of the catecholamine precursor; L-dopa, in the CsA treated rats sacrificed at 1 hour (20%) or 7 days (15%), r e l a t i v e to the time matched controls (figure 3). As also shown in figure 3, CsA had no significant effect on the renal levels of the catecholamine metabolites eplnine, DOPAC, MHPGor ME In the rats sacrificed at 1 hour. However, i t decreased the level of HVA (20%) s i g n i f i c a n t l y withln 1 hour (figure 3). After 7 days of treatment, renal levels of MHPG, DOPAC, epinine, ME, and HVA decreased by 40%, 27%, 22%, 20% and 4% respectively, in the CsA treated rats, as compared with the control rats (figure 3). 150.
# P ( 0.1 * P ( 0.05
I #
,--
100
M
0
' O/'O .
$
1 HOUR 7 DAYS DURATION OF CSA TREATMENT Figure 3
The levels of renal L-dopa (F'I), epinine ( . ) , and the catecholamine metabolites dopac ( [ ] ) , MHPG ( [ ] ) , HVA ( [ ] ) and ME ( [ ] ) were determined as described for the catecholamines in the methods. The bars represent the mean + SE of their levels in the kidneys of rats treated wlth CsA, expressed as a percent of the mean levels in time matched control rat kidneys. The above results suggest that CsA causes an early and transient increase in the levels of renal NE and EPl, and a decrease in the level of some of their metabolites. Discussion The mechanism of CsA-induced hypertension is st111 unclear. The neurogenic and direct vascular involvement in CsA-induced hypertension were previously suggested, but never proven (19). I t is known that this hypertension responds to treatment with beta and alpha adrenergic blockers (14,15). The current observation of an early increase in renal NE and EPI by CsA supports the involvement of adrenergic mechanisms in the hypertensive side effects of this agent. I t has been shown that NE or EPI administration results in an increase in renovascular resistance and decreased renal clearance and blood flow (16). The above study also revealed no consistent difference between the renal cortex and the medulla
260
CsA and Renal Catecholamines
Vol. 47, No. 4, 1990
in t h e l r response to catecholamines. Thus, although we did not study the regional affects of CsA on renal catecholamines, an increase in EPI or NE content of the kidney would be expected to increase the renovascular resistance of both the cortex and the medulla. Other investigators have also demonstrated the existence of adrenergic innervatlon of the kidney, wlth NE being found in a l l the regions (20). The early increase of renal NE and EPI observed In the present study, may provlde a mechanism for the CsA induced increase in renovascular resistance and hypertension. The decline in t h e l r levels 24 hours after a single dose of CsA may explain the r e v e r s l b i l i t y of t h l s hypertension following CsA withdrawal. A better response to antihypertensive therapy occurs with time in CsA treated patlents and t h i s hypertension has been reported to be non progressive (15). This better response to therapy may be p a r t i a l l y explained by the less increase in renal NE and EPI observed durlng chronlc treatment wlth CsA. The observed decrease in renal catecholamlne metabolltes by CsA suggests that t h i s drug may i n h i b i t catecholamine metabollsm. Although the exact mechanism of t h i s i n h l b i t i o n was not investigated, the decreases in renal HVA, ME and MHPG levels by CsA suggest a possible l n h i b l t i o n of methylatlon of catecholamines. The increases in NE and EPI do not appear to be due to increase in t h e i r synthesls since there was no s l g n l f l c a n t change in the level of DA in t h i s study. The generalized decrease in renal levels of the metabolites after 7 days may r e f l e c t a decrease in the catecholamine turnover rate in the CsA treated rats. Although the dose of CsA used to produce the above changes also affected glutathione (17) and prolactln (18) levels markedly, hlgher doses may cause more pronounced increases in the levels of catecholamlnes. The influence of systemic levels of CsA was i n d l r e c t l y investigated by quantltating the levels of renal catecholamines, 24 hours after a slngle injection of CsA. The decllne of the catecholamines to t h e i r control levels after t h l s period, which corresponds approximately to the t 1/2 for CsA (21), suggests that the observed responses were dependent on the systemic a v a i l a b i l i t y of CsA. This was further supported by the fact that the catecholamine levels remained hlgh durlng dally treatments wlth CsA for 7 days. Observatlon of these changes wlth the low dose of CsA used in t h l s study suggest that an increase in renal catecholamines levels may be one of the primary and early effects of t h l s important immunosuppresslve agent. Although the underlying mechanism for the increase in catecholamlnes by CsA is not clear from the present study, the reported observatlons are pertlnent for further elucldatlon of the mechanism of CsA-induced nephrotoxicity and hypertension. In conclusion, the present flndlngs suggest that increase in renal EPI and NE levels may play a s i g n l f l c a n t role in the early CsA induced hypertenslon
References 1. 2. 3.
M. GASCON, Transplant. Proc. 19(40) 3481-3485 (1987). R.BUSUTTIL, L. GOLDSTEIN, G. DANOVITCH, M. AMENT, and L. MEMSIC, Ann I n t . Med. 104:377-389 (1986). A. TATMAN, B. TUCKER, J AMESS, W. CATTELL, and L BAKER, Lancet 1(8597): 1297 (1988).
Vol. 47, No. 4, 1990
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14 15. 16. 17. 18. 19. 20. 21.
CsA and Renal Catecholamines
C. HAMBERGER, S. URIEN, J. BARRE, M. BRANDEBOURGER, M. LEMAIRE and P. LANG, Ther. Drug. Monit. 10(1): 28-33 (1988). B. VON GRAFFENRIED, Dlabetol. Hotel Diul. 135; 4-6 (1987) G. THOENES, T. UMSCHEID, T. SITTER, and K. LANGER, Immunol. Lett. 15(4): 301-6 (1987). P. HEERING, and B. GRABENESEE, DTSCHMED WOCHENSCHR. 113(20): 833-4 (1988). F. DISCHE, J. NEUBERGER, O. KEATING, V PARSONS, R. CALNE, and R. WILLIAMS, Lab-Invest 58(4): 395-402 (1988). M. HAKIM, J WALLWORK, and T ENGLISH, Ann-Thorac-Surg 46(5):495-501 (1988). G. DERAY, P. LE-HOANG, P. CACOUB, B. AUPETIT, A. MERTANI, F. MARTINEZ and J. ROTTEMBOURG, Eur. J. Clln. Pharmaco]. 34(6):601-4 (1988). H.S. CAIRNS, U. RAVAL and G.H. NEILD. Transp. 46(1): 79-82 (1988). B.D. KAHAN, (ed). Transp]. Proc. 17" 185-196 (1985). H MOODY, Lancet 1(8491): 1221-2 (1986). R M. FERGUSON, and B.G. SOMMER, Am. O. Kidney Dis. 6, 296-306 (1985). 8.D KAHAN, Am.J. Kidney Dis. 5, 313-7 (1985). K. AUKLAND, Acta Physiol. Scand 72, 498-509 (1968). V.A. DURUIBE, A. OKONMAHand G.T. BLYDEN, Pharmaco] 39(6):205-212 (1989). S.B. CARDON, D.F. LARSON, and D.H. RUSSELL, Blochem. Biophys. Res. Commun 120,614-618 (1984). THOMPSONet a] Transp]. Proc. 1___5:2573-2577 (1983). O. MCKENNA, and E. ANGELAKOS, E Circ. Res. 22, 345-354 (1968). O. WAGNER, E. SCHREIER, F. HEITZ, and G. MAURER, Drug Metab. Dlsp. 15(3): 377-383 (1987)
261