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Effect of tacrolimus on the gene expression of renin and endothelin in the rat kidney
Effect of Tacrolimus on the Gene Expression of Renin and Endothelin in the Rat Kidney T. Nakatani, J. Uchida, T. Asai, R. Yoshimura, T. Kim, K. Miura, and T. Kishimoto
T
ACROLIMUS is a superior immunosuppressant, but its adverse characteristic of nephrotoxicity often becomes a clinical problem. Tacrolimus and cyclosporine (CsA) bind with different proteins in the blood but exert their immunosuppressive actions in a similar manner by inhibiting calcineurin, thus lowering IL-2 gene expression in T lymphocytes.1 Clinical studies have shown that tacrolimus differs from CsA in that it causes less systemic vasoconstriction and hypertension, whereas both induce comparable renal vasoconstriction. Renin and endothelin (ET) are already known to be involved in the onset of nephrotoxicity by CsA,2,3 but their involvement in tacrolimus nephrotoxicity is not yet clear. In this investigation, we studied the involvement of gene expression of renin and ET in the kidneys of rats given tacrolimus. MATERIALS AND METHODS Spontaneously hypertensive male rats (SHR), aged 15 to 17 weeks, were used and allowed free access to a standard diet and tap water throughout the examination. In this study, tacrolimus for intravenous injection (Prograf injection), containing 5 mg of tacrolimus hydrate, 200 mg of polyoxyethylene hydrogenated oil, and 0.6 g of ethanol, was used and diluted with physiologic saline before injection. FK 506 (4 mg/kg per day) or vehicle was administrated intramuscularly once per day for 14 days. On day 14, after the last drug administration, rats were placed in individual metabolic cages for 24-hour urine collection, and systolic blood pressure (BP) was determined using a tail-cuff method. Rats were then anesthetized and blood samples were drawn. Plasma and urine samples were stored at ⫺20°C until assays for plasma renin activity (PRA), blood urea nitrogen (BUN), and creatinine concentration were carried out. Using another group of animals administered tacrolimus or vehicle, as in the previously described protocol, 24 hours after the last injection of tacrolimus or vehicle (on day 14) animals were anesthetized and surgically prepared for renal blood flow (RBF) measurement. RBF was measured using a flow probe around the left renal artery that was surgically exposed and the RBF was recorded by an electromagnetic flowmeter. Inulin clearance was measured as previously described.4 Creatinine clearance (CCr) was estimated by inulin clearance. After clearance study, kidneys were excised to examine the effects of tacrolimus on renin and ET mRNA levels in renal cortex and medulla and then stored at ⫺80°C until RNA extraction. PRA was measured by radioimmunoassay. cDNA probes used were as follows: rat renin cDNA (0.76-kb
RsaI–RsaI fragment); rat preproET-1 cDNA (2.0-kb EcoRI–EcoRI fragment); rat ETA cDNA (1.5-kb SacI–XbaI fragment); rat ETB cDNA (1.334-kb EcoRI–EcoRI fragment); and rat glyceraldehyde3-phosphate dehydrogenase (GAPDH; 1.3-kb PstI–PstI fragment). Total RNA was extracted from the renal cortex and medulla by the guanidium thiocyanate-phenol-chloroform method. Twenty micrograms of total RNA was electrophoresed on 1% agarose gel and transferred to a nylon membrane. The membrane was hybridized with aforementioned [32P]-dCTP-labeled cDNA probes, washed, and finally exposed to the imaging plate. To evaluate tissue mRNA levels, autoradiograms were digitized to measure density using a bioimaging analyzer. Data are expressed as mean ⫾ SE. Comparisons between the two groups were made using the Mann–Whitney U test. P ⬍ .05 was considered statistically significant.
RESULTS
BUN and plasma creatinine concentration (PCr) rose significantly 7 days after the start of tacrolimus administration and increased further by day 14. Following daily administration of tacrolimus (4 mg/kg per day) for 2 weeks, PCr was 0.60 ⫾ 0.05 mg/dL in vehicle-treated rats and 0.86 ⫾ 0.04 mg/dL in tacrolimus-treated rats. CCr decreased significantly in tacrolimus-treated rats (vehicle 0.36 ⫾ 0.01 and tacrolimus 0.24 ⫾ 0.02 mL/min/100 g body weight). Fractional excretion of Na (FENa) did not change at 7 days after tacrolimus treatment, but increased significantly by day 14 (vehicle 0.13 ⫾ 0.02%, tacrolimus 0.24 ⫾ 0.02%). RBF was significantly lower in the tacrolimus-treated groups (vehicle 5.7 ⫾ 0.3 and tacrolimus 3.2 ⫾ 0.2 mL/min per gram tissue). Systolic BP before treatment was the same between the two groups. Following completion of the treatments, mean BP (MBP) decreased significantly in the tacrolimus-treated group compared with the corresponding vehicle-treated group (vehicle 171 ⫾ 2, tacrolimus 153 ⫾ 1 mm Hg). Calculated RVR increased with tacrolimus treatment. PRA increased significantly with tacrolimus (vehicle From the Department of Urology (T.N., J.U., T.A., R.Y., T. Kim, T. Kis.) and Department of Pharmacology (K.M.), Osaka City University Medical School, Osaka, Japan. Address reprint request to Dr Tatsuya Nakatani, Department of Urology, Osaka City University Medical School, 1-4-3 Asahimachi Abeno-ku, Osaka 545-8585, Japan. E-mail: nakatani@ msic.med.osaka-cu.ac.jp.
0041-1345/01/$–see front matter PII S0041-1345(01)01996-0
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2296
Transplantation Proceedings, 33, 2296–2297 (2001)
EFFECT OF TACROLIMUS
Fig 1. The levels of mRNA of renin (a), ET-1 (b), and ETB receptor (c) in the renal cortex and medulla following daily administration of vehicle or tacrolimus for 2 weeks. Each bar represents the mean ⫾ SE. *P ⬍ .05 calculated by Mann– Whitney U test. N.S., not significant.
22.6 ⫾ 3.5 and tacrolimus 60.9 ⫾ 11.3 ng AI/mL per hour). PRA correlated positively with FENa and negatively with CCr. The increase in renin mRNA level in the renal cortex was measured using a bioimaging analyzer (Fig 1a). In the renal medulla, the level of renin mRNA was not affected by tacrolimus treatment. As for ET, significant increases in ET-1 mRNA level in the renal cortex and medulla in tacrolimus-treated rats were observed when compared to the vehicle-treated rats (Fig 1b). The signal of ETA receptor mRNA was too weak to analyze. The mRNA level of ETB receptor was not affected by tacrolimus treatment (Fig 1c).
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calcineurin in the T lymphocyte,1 but there were some differences in the mechanism of nephrotoxicity between tacrolimus and CsA. Acute injection of CsA in rats caused ET-dependent renal vasoconstriction and decreased CCr,2,3 whereas acute injection of tacrolimus had no effect on renal hemodynamics and function.5 ET receptor antagonists have also been shown to attenuate hypertension during CsA administration.6 Thus, ET plays an important role in the CsA-induced renal dysfunction and hypertension. As for tacrolimus, Mitamura et al7 suggested that tacrolimus-induced nephrotoxicity in the SHR rats is associated with the release of renin from the juxtaglomerular apparatus. On the other hand, the role of ET in tacrolimusinduced renal dysfunction is still unclear. Goodall et al reported that tacrolimus increased the production of ET in vitro,8 but in other studies it had no effect on ET release.5,9 To date, no in vivo study elucidating the role of ET in tacrolimus-induced renal dysfunction has been reported. In our research, renin mRNA in the renal cortex increased significantly after daily tacrolimus administration for 14 days, and PRA had a negative correlation with CCr. ET-1 mRNA increased following daily tacrolimus administration for 2 weeks, in both the renal cortex and medulla. However, mRNA of ETB receptor was not affected by tacrolimus treatment within the kidney. In conclusion, these results suggest that renin strongly affects the renal dysfunction induced by tacrolimus administration, and ET-1 may also impact the onset and/or progress of tacrolimus-induced nephrotoxicity.
DISCUSSION
REFERENCES
The present results clearly show that this in vivo manifestation of tacrolimus-induced nephrotoxicity is characterized by reduced CCr and RBF, and an increase in FENa. The renal dysfunction in this study was strongly affected by the vasoconstrictors because FENa had a positive correlation with PRA and CCr had a negative one. Concomitantly with the increase in plasma values of vasoconstrictors and renal dysfunction, systemic BP was decreased following the administration of tacrolimus for 2 weeks. Although the reason for BP reduction by tacrolimus remains unclear in this experimental model, we believe that one reason is hypertension in the experimental rats, which existed before initiating tacrolimus treatment. The immunosuppressive mechanism of tacrolimus and CsA is very similar, through inhibition of phosphatase
1. O’Keefe SJ, Tamura J, Kincaid RL, et al: Nature 357:692, 1992 2. Lanese DM, Conger JD: J Clin Invest 91:2144, 1993 3. Lanese DM, Falk SA, Conger JD: Transplantation 58:1371, 1994 4. Matsuura T, Yukimura T, Kim S, et al: Jpn J Pharmacol 71:213, 1996 5. Benigni A, Morigi M, Perico N, et al: Transplantation 54:775, 1992 6. Bartholomeusz B, Hardy KJ, Nelson AS, et al: Hypertension 27:1341, 1996 7. Mitamura T, Yamada A, Ishida H, et al: J Toxicol Sci 19:219, 1994 8. Goodall T, Kind CN, Hammond TG: J Cardiovasc Pharmacol 26(suppl):S482, 1995 9. Nakahama H, Fukunaga M, Kakihara M, et al: J Cardiovasc Pharmacol 17(suppl):S172, 1991
T
ACROLIMUS is a superior immunosuppressant, but its adverse characteristic of nephrotoxicity often becomes a clinical problem. Tacrolimus and cyclosporine (CsA) bind with different proteins in the blood but exert their immunosuppressive actions in a similar manner by inhibiting calcineurin, thus lowering IL-2 gene expression in T lymphocytes.1 Clinical studies have shown that tacrolimus differs from CsA in that it causes less systemic vasoconstriction and hypertension, whereas both induce comparable renal vasoconstriction. Renin and endothelin (ET) are already known to be involved in the onset of nephrotoxicity by CsA,2,3 but their involvement in tacrolimus nephrotoxicity is not yet clear. In this investigation, we studied the involvement of gene expression of renin and ET in the kidneys of rats given tacrolimus. MATERIALS AND METHODS Spontaneously hypertensive male rats (SHR), aged 15 to 17 weeks, were used and allowed free access to a standard diet and tap water throughout the examination. In this study, tacrolimus for intravenous injection (Prograf injection), containing 5 mg of tacrolimus hydrate, 200 mg of polyoxyethylene hydrogenated oil, and 0.6 g of ethanol, was used and diluted with physiologic saline before injection. FK 506 (4 mg/kg per day) or vehicle was administrated intramuscularly once per day for 14 days. On day 14, after the last drug administration, rats were placed in individual metabolic cages for 24-hour urine collection, and systolic blood pressure (BP) was determined using a tail-cuff method. Rats were then anesthetized and blood samples were drawn. Plasma and urine samples were stored at ⫺20°C until assays for plasma renin activity (PRA), blood urea nitrogen (BUN), and creatinine concentration were carried out. Using another group of animals administered tacrolimus or vehicle, as in the previously described protocol, 24 hours after the last injection of tacrolimus or vehicle (on day 14) animals were anesthetized and surgically prepared for renal blood flow (RBF) measurement. RBF was measured using a flow probe around the left renal artery that was surgically exposed and the RBF was recorded by an electromagnetic flowmeter. Inulin clearance was measured as previously described.4 Creatinine clearance (CCr) was estimated by inulin clearance. After clearance study, kidneys were excised to examine the effects of tacrolimus on renin and ET mRNA levels in renal cortex and medulla and then stored at ⫺80°C until RNA extraction. PRA was measured by radioimmunoassay. cDNA probes used were as follows: rat renin cDNA (0.76-kb
RsaI–RsaI fragment); rat preproET-1 cDNA (2.0-kb EcoRI–EcoRI fragment); rat ETA cDNA (1.5-kb SacI–XbaI fragment); rat ETB cDNA (1.334-kb EcoRI–EcoRI fragment); and rat glyceraldehyde3-phosphate dehydrogenase (GAPDH; 1.3-kb PstI–PstI fragment). Total RNA was extracted from the renal cortex and medulla by the guanidium thiocyanate-phenol-chloroform method. Twenty micrograms of total RNA was electrophoresed on 1% agarose gel and transferred to a nylon membrane. The membrane was hybridized with aforementioned [32P]-dCTP-labeled cDNA probes, washed, and finally exposed to the imaging plate. To evaluate tissue mRNA levels, autoradiograms were digitized to measure density using a bioimaging analyzer. Data are expressed as mean ⫾ SE. Comparisons between the two groups were made using the Mann–Whitney U test. P ⬍ .05 was considered statistically significant.
RESULTS
BUN and plasma creatinine concentration (PCr) rose significantly 7 days after the start of tacrolimus administration and increased further by day 14. Following daily administration of tacrolimus (4 mg/kg per day) for 2 weeks, PCr was 0.60 ⫾ 0.05 mg/dL in vehicle-treated rats and 0.86 ⫾ 0.04 mg/dL in tacrolimus-treated rats. CCr decreased significantly in tacrolimus-treated rats (vehicle 0.36 ⫾ 0.01 and tacrolimus 0.24 ⫾ 0.02 mL/min/100 g body weight). Fractional excretion of Na (FENa) did not change at 7 days after tacrolimus treatment, but increased significantly by day 14 (vehicle 0.13 ⫾ 0.02%, tacrolimus 0.24 ⫾ 0.02%). RBF was significantly lower in the tacrolimus-treated groups (vehicle 5.7 ⫾ 0.3 and tacrolimus 3.2 ⫾ 0.2 mL/min per gram tissue). Systolic BP before treatment was the same between the two groups. Following completion of the treatments, mean BP (MBP) decreased significantly in the tacrolimus-treated group compared with the corresponding vehicle-treated group (vehicle 171 ⫾ 2, tacrolimus 153 ⫾ 1 mm Hg). Calculated RVR increased with tacrolimus treatment. PRA increased significantly with tacrolimus (vehicle From the Department of Urology (T.N., J.U., T.A., R.Y., T. Kim, T. Kis.) and Department of Pharmacology (K.M.), Osaka City University Medical School, Osaka, Japan. Address reprint request to Dr Tatsuya Nakatani, Department of Urology, Osaka City University Medical School, 1-4-3 Asahimachi Abeno-ku, Osaka 545-8585, Japan. E-mail: nakatani@ msic.med.osaka-cu.ac.jp.
0041-1345/01/$–see front matter PII S0041-1345(01)01996-0
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2296
Transplantation Proceedings, 33, 2296–2297 (2001)
EFFECT OF TACROLIMUS
Fig 1. The levels of mRNA of renin (a), ET-1 (b), and ETB receptor (c) in the renal cortex and medulla following daily administration of vehicle or tacrolimus for 2 weeks. Each bar represents the mean ⫾ SE. *P ⬍ .05 calculated by Mann– Whitney U test. N.S., not significant.
22.6 ⫾ 3.5 and tacrolimus 60.9 ⫾ 11.3 ng AI/mL per hour). PRA correlated positively with FENa and negatively with CCr. The increase in renin mRNA level in the renal cortex was measured using a bioimaging analyzer (Fig 1a). In the renal medulla, the level of renin mRNA was not affected by tacrolimus treatment. As for ET, significant increases in ET-1 mRNA level in the renal cortex and medulla in tacrolimus-treated rats were observed when compared to the vehicle-treated rats (Fig 1b). The signal of ETA receptor mRNA was too weak to analyze. The mRNA level of ETB receptor was not affected by tacrolimus treatment (Fig 1c).
2297
calcineurin in the T lymphocyte,1 but there were some differences in the mechanism of nephrotoxicity between tacrolimus and CsA. Acute injection of CsA in rats caused ET-dependent renal vasoconstriction and decreased CCr,2,3 whereas acute injection of tacrolimus had no effect on renal hemodynamics and function.5 ET receptor antagonists have also been shown to attenuate hypertension during CsA administration.6 Thus, ET plays an important role in the CsA-induced renal dysfunction and hypertension. As for tacrolimus, Mitamura et al7 suggested that tacrolimus-induced nephrotoxicity in the SHR rats is associated with the release of renin from the juxtaglomerular apparatus. On the other hand, the role of ET in tacrolimusinduced renal dysfunction is still unclear. Goodall et al reported that tacrolimus increased the production of ET in vitro,8 but in other studies it had no effect on ET release.5,9 To date, no in vivo study elucidating the role of ET in tacrolimus-induced renal dysfunction has been reported. In our research, renin mRNA in the renal cortex increased significantly after daily tacrolimus administration for 14 days, and PRA had a negative correlation with CCr. ET-1 mRNA increased following daily tacrolimus administration for 2 weeks, in both the renal cortex and medulla. However, mRNA of ETB receptor was not affected by tacrolimus treatment within the kidney. In conclusion, these results suggest that renin strongly affects the renal dysfunction induced by tacrolimus administration, and ET-1 may also impact the onset and/or progress of tacrolimus-induced nephrotoxicity.
DISCUSSION
REFERENCES
The present results clearly show that this in vivo manifestation of tacrolimus-induced nephrotoxicity is characterized by reduced CCr and RBF, and an increase in FENa. The renal dysfunction in this study was strongly affected by the vasoconstrictors because FENa had a positive correlation with PRA and CCr had a negative one. Concomitantly with the increase in plasma values of vasoconstrictors and renal dysfunction, systemic BP was decreased following the administration of tacrolimus for 2 weeks. Although the reason for BP reduction by tacrolimus remains unclear in this experimental model, we believe that one reason is hypertension in the experimental rats, which existed before initiating tacrolimus treatment. The immunosuppressive mechanism of tacrolimus and CsA is very similar, through inhibition of phosphatase
1. O’Keefe SJ, Tamura J, Kincaid RL, et al: Nature 357:692, 1992 2. Lanese DM, Conger JD: J Clin Invest 91:2144, 1993 3. Lanese DM, Falk SA, Conger JD: Transplantation 58:1371, 1994 4. Matsuura T, Yukimura T, Kim S, et al: Jpn J Pharmacol 71:213, 1996 5. Benigni A, Morigi M, Perico N, et al: Transplantation 54:775, 1992 6. Bartholomeusz B, Hardy KJ, Nelson AS, et al: Hypertension 27:1341, 1996 7. Mitamura T, Yamada A, Ishida H, et al: J Toxicol Sci 19:219, 1994 8. Goodall T, Kind CN, Hammond TG: J Cardiovasc Pharmacol 26(suppl):S482, 1995 9. Nakahama H, Fukunaga M, Kakihara M, et al: J Cardiovasc Pharmacol 17(suppl):S172, 1991