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The Antiproteinuric Effects of Green Tea Extract on Acute Cyclosporine-Induced Nephrotoxicity in Rats
The Antiproteinuric Effects of Green Tea Extract on Acute Cyclosporine-Induced Nephrotoxicity in Rats B.C. Shin, Y.E. Kwon, J.H. Chung, and H.L. Kim ABSTRACT Background. It has been reported that the proteinuria is an early useful marker to detect cyclosporine (CsA) nephrotoxicity. The aim of this study was to investigate the antiproteinuric effects of green tea extract (GTE) on CsA-induced acute renal injury in rats. Methods. The rats (n ⫽ 28) were divided into four groups (n ⫽ 7/group); controls intraperitoneally (IP) injected with 0.9% saline; CsA group IP injected CsA (50 mg/kg); inducible nitric oxide synthase (iNOS) inhibitor group administered in addition NG-nitroL-arginine-methyl ester (12 mmol/L) subcutaneously and CsA-GTE group of CsA IP plus GTE (100 mg/kg) subcutaneously. Results. The 24-hour urine proteins were significantly increased among the CsA (22.6 ⫾ 3.1 mg/d) compared with the control (7.1 ⫾ 1.5 mg/d) and significantly decreased in the CsA-GTE group (8.2 ⫾ 1.8 mg/d, P ⬍ .01). Nitric oxide production induces by CsA treatment was significantly suppressed by GTE and iNOS inhibitor. Renal tissue malondialdehyde level was significantly increased in the CsA compared with controls and significantly decreased in the CsA-GTE group. The antioxidant enzyme activities of superoxide dysmutase and catalase, which were significantly suppressed in the CsA compared with the control group, were restored in the CsA-GTE cohort. Conclusion. GTE treatment of rats showed meaningful antiproteinuric effects through antioxidative activity in kidneys from CsA-induced acute renal injury.
P
ROTEINURIA is an early useful clinical marker to detect cyclosporine (CsA) nephrotoxicity.1 Nitric oxide (NO) produced by CsA plays a role of pathogenesis of the induced nephrotoxicity.2 Green tea extract (GTE), as an antioxidant and free radical scavenger, induces apoptosis in renal interstitial fibroblast cells in vitro.3 We investigated the antiproteinuric effects of GTE on CsA-induced acute renal injury in rats
renal tissue malondialdehyde levels using TCA-TBA-HCl reagent and antioxidant enzyme activities. Rat kidney tissue samples were fixed in 10% buffered formalin, embedded in paraffin, cut at 4 m, and examined histopathologically after staining with periodic acid-Schiff reagent. The statistical differences between the control and the experimental groups were analyzed using Mann-Whitney test and Kruskal-Wallis test (SPSS, Statistical Package for Social Science version 17.0, Chicago, IL, USA). A P value of ⬍ .05 was considered significant.
MATERIALS AND METHODS
RESULTS
GTE was prepared from a hot-water extract of green tea (Boseong, Chonnam, Korea) as reported by Maity et al.4 CsA was purchased from Chong Kun Dang (Cypol, Seoul, Korea). Normal male SpragueDawley rats (n ⫽ 28) weighing 200 to 250 g, were divided into four groups (n ⫽ 7/group): controls intraperitoneally (IP) injected with 0.9% saline; CsA group IP injected with drug (50 mg/kg); inducible NO synthase (iNOS) inhibitor group administered in addition NGnitro-L-arginine-methyl ester (L-NAME; 12 mmol/L) subcutaneously; and CsA-GTE group of CsA IP and GTE (100 mg/kg) subcutaneously. After blood samples were obtained, the two kidneys of each rat were immediately removed. The NO concentration was measured by an NO enzyme-linked immunosorbent assay kit. We also measured
There were no significant differences in serum urea nitrogen, creatinine, or serum CsA levels (P ⬎ .05). The 24-hour urine protein levels were significantly increased in the CsA From the Department of Internal Medicine, Division of Nephrology (B.C.S., J.H.C., H.L.K.), Chosun University Hospital, and Division of Allergy (Y.E.K.), Chosun University Hospital, Gwangju, Republic of Korea. Address reprint requests to Hyun Lee Kim, MD, Department of Internal Medicine, Chosun University, College of Medicine, Gwangju, Republic of Korea. E-mail: [email protected]
0041-1345/12/$–see front matter doi:10.1016/j.transproceed.2012.03.047
© 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1080
Transplantation Proceedings, 44, 1080 –1082 (2012)
ANTIPROTEINURIC EFFECTS OF GREEN TEA EXTRACT
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(22.6 ⫾ 3.1 mg/d) compared with the controls (7.1 ⫾ 1.5 mg/d, P ⬍ .01) and significantly decreased in the CsA-GTE cohort (8.2 ⫾ 1.8 mg/d, P ⬍ .01). The NO production by CsA was significantly suppressed by GTE or the NOS inhibitor cohorts compared with the CsA group (P ⬍ .01). Renal tissue MDA levels, significantly increased in the CsA compared with the control group, were significantly decreased among the CsA-GTE group (P ⬍ .01). The antioxidant superoxide dysmutase and catalase activities, significantly (P ⬍ .05) suppressed in the CsA compared with the control group, were restored in the CsA-GTE group (Fig 1). After CsA injection there was proximal tubular necrosis and mild interstitial inflammation in the kidney of rats but no significant pathological change was evident among the GTE and L-NAME cohorts (Fig 2). DISCUSSION
In this study, we observed that proteinuria produced by CsA-induced nephrotoxicity was associated with lipid peroxidation and NO production. The present study also demonstrated that treatment with GTE showed meaningful antiproteinuric effects through antioxidative actions on kidneys from rats with CsA-induced acute renal injury. Green tea contain various types of catechins, including (⫺)-epigallocatechin 3-O-gallate (EGCG), (⫺)-gallocatechin 3-O-gallate, (⫺)-epicatechin 3-O-gallate, (⫺)-epigallocatechin, (⫹)-gallocatechin, (⫺)-epicatechin, and (⫹)-catechin.5
Fig 2. Light microscopic findings of rat kidney (periodic-acid Schiff staining, ⫻40). Rats were divided into 0.9% saline-injected group (control A), cyclosporine-injected group (B), cyclosporine plus inducible nitric oxide synthase inhibitor group (C), and cyclosporine plus green tea extract group. (D).
EGCG is known to display the most potent antitumor, antimutagenic, hypocholesterolemic, antioxidant, and antidiabetic activities.6 In this experiment, GTE showed antiproteinuric activity in CsA induced acute renal injury in rats. Further
Fig 1. The effects of green tea extracts on 24-hour urine protein amount (A), malondialdehyde level of kidney tissue (B), nitric oxide level (C), and antioxidant enzyme activity of catalase and SOD (D) on cyclosporine-induced nephrotoxicity in rats.
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studies of GTE should be conducted to investigate its effects on CsA-induced nephrotoxicity in humans. REFERENCES 1. Fernandez-Fresnedo G, Escallada R, Rodrigo E, et al: Transplant Proc 33:3373, 2001 2. Wang C, Salahudeen AK: Kidney Int 47:927, 1995
SHIN, KWON, CHUNG ET AL 3. Miyamoto Y, Sano M, Haylor JL, et al: J Toxicol Sci 33:367, 2008 4. Maity S, Vedasiromoni JR, Ganguly DK: Jpn J Pharmacol 78:285, 1998 5. Zhang Q, Kelly AP, Wang L, et al: J Invest Dermatol 126:2607, 2006 6. Yamabe N, Yokozawa T, Oya T, et al: J Pharmacol Exp Ther 319:228, 2006
P
ROTEINURIA is an early useful clinical marker to detect cyclosporine (CsA) nephrotoxicity.1 Nitric oxide (NO) produced by CsA plays a role of pathogenesis of the induced nephrotoxicity.2 Green tea extract (GTE), as an antioxidant and free radical scavenger, induces apoptosis in renal interstitial fibroblast cells in vitro.3 We investigated the antiproteinuric effects of GTE on CsA-induced acute renal injury in rats
renal tissue malondialdehyde levels using TCA-TBA-HCl reagent and antioxidant enzyme activities. Rat kidney tissue samples were fixed in 10% buffered formalin, embedded in paraffin, cut at 4 m, and examined histopathologically after staining with periodic acid-Schiff reagent. The statistical differences between the control and the experimental groups were analyzed using Mann-Whitney test and Kruskal-Wallis test (SPSS, Statistical Package for Social Science version 17.0, Chicago, IL, USA). A P value of ⬍ .05 was considered significant.
MATERIALS AND METHODS
RESULTS
GTE was prepared from a hot-water extract of green tea (Boseong, Chonnam, Korea) as reported by Maity et al.4 CsA was purchased from Chong Kun Dang (Cypol, Seoul, Korea). Normal male SpragueDawley rats (n ⫽ 28) weighing 200 to 250 g, were divided into four groups (n ⫽ 7/group): controls intraperitoneally (IP) injected with 0.9% saline; CsA group IP injected with drug (50 mg/kg); inducible NO synthase (iNOS) inhibitor group administered in addition NGnitro-L-arginine-methyl ester (L-NAME; 12 mmol/L) subcutaneously; and CsA-GTE group of CsA IP and GTE (100 mg/kg) subcutaneously. After blood samples were obtained, the two kidneys of each rat were immediately removed. The NO concentration was measured by an NO enzyme-linked immunosorbent assay kit. We also measured
There were no significant differences in serum urea nitrogen, creatinine, or serum CsA levels (P ⬎ .05). The 24-hour urine protein levels were significantly increased in the CsA From the Department of Internal Medicine, Division of Nephrology (B.C.S., J.H.C., H.L.K.), Chosun University Hospital, and Division of Allergy (Y.E.K.), Chosun University Hospital, Gwangju, Republic of Korea. Address reprint requests to Hyun Lee Kim, MD, Department of Internal Medicine, Chosun University, College of Medicine, Gwangju, Republic of Korea. E-mail: [email protected]
0041-1345/12/$–see front matter doi:10.1016/j.transproceed.2012.03.047
© 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1080
Transplantation Proceedings, 44, 1080 –1082 (2012)
ANTIPROTEINURIC EFFECTS OF GREEN TEA EXTRACT
1081
(22.6 ⫾ 3.1 mg/d) compared with the controls (7.1 ⫾ 1.5 mg/d, P ⬍ .01) and significantly decreased in the CsA-GTE cohort (8.2 ⫾ 1.8 mg/d, P ⬍ .01). The NO production by CsA was significantly suppressed by GTE or the NOS inhibitor cohorts compared with the CsA group (P ⬍ .01). Renal tissue MDA levels, significantly increased in the CsA compared with the control group, were significantly decreased among the CsA-GTE group (P ⬍ .01). The antioxidant superoxide dysmutase and catalase activities, significantly (P ⬍ .05) suppressed in the CsA compared with the control group, were restored in the CsA-GTE group (Fig 1). After CsA injection there was proximal tubular necrosis and mild interstitial inflammation in the kidney of rats but no significant pathological change was evident among the GTE and L-NAME cohorts (Fig 2). DISCUSSION
In this study, we observed that proteinuria produced by CsA-induced nephrotoxicity was associated with lipid peroxidation and NO production. The present study also demonstrated that treatment with GTE showed meaningful antiproteinuric effects through antioxidative actions on kidneys from rats with CsA-induced acute renal injury. Green tea contain various types of catechins, including (⫺)-epigallocatechin 3-O-gallate (EGCG), (⫺)-gallocatechin 3-O-gallate, (⫺)-epicatechin 3-O-gallate, (⫺)-epigallocatechin, (⫹)-gallocatechin, (⫺)-epicatechin, and (⫹)-catechin.5
Fig 2. Light microscopic findings of rat kidney (periodic-acid Schiff staining, ⫻40). Rats were divided into 0.9% saline-injected group (control A), cyclosporine-injected group (B), cyclosporine plus inducible nitric oxide synthase inhibitor group (C), and cyclosporine plus green tea extract group. (D).
EGCG is known to display the most potent antitumor, antimutagenic, hypocholesterolemic, antioxidant, and antidiabetic activities.6 In this experiment, GTE showed antiproteinuric activity in CsA induced acute renal injury in rats. Further
Fig 1. The effects of green tea extracts on 24-hour urine protein amount (A), malondialdehyde level of kidney tissue (B), nitric oxide level (C), and antioxidant enzyme activity of catalase and SOD (D) on cyclosporine-induced nephrotoxicity in rats.
1082
studies of GTE should be conducted to investigate its effects on CsA-induced nephrotoxicity in humans. REFERENCES 1. Fernandez-Fresnedo G, Escallada R, Rodrigo E, et al: Transplant Proc 33:3373, 2001 2. Wang C, Salahudeen AK: Kidney Int 47:927, 1995
SHIN, KWON, CHUNG ET AL 3. Miyamoto Y, Sano M, Haylor JL, et al: J Toxicol Sci 33:367, 2008 4. Maity S, Vedasiromoni JR, Ganguly DK: Jpn J Pharmacol 78:285, 1998 5. Zhang Q, Kelly AP, Wang L, et al: J Invest Dermatol 126:2607, 2006 6. Yamabe N, Yokozawa T, Oya T, et al: J Pharmacol Exp Ther 319:228, 2006