Research Article
Protective effect of L-arginine on gentamicin-induced nephrotoxicity in rats . lbrahim Başhan, Perihan Başhan1, Mehmet Ata Seçilmiş1, Ergin Şingirik1
ABSTRACT Introduction: L-arginine has a protective effect on gentamicin-induced renal failure and it may decrease the tubular reabsorption of another cationic substance, gentamicin due to its cationic structure. The aim of this study is to compare the possible protective effects of L-arginine and its inactive isomer D-arginine on gentamicin-induced nephrotoxicity in rats. Materials and Methods: Wistar albino rats were housed in metabolic cages and assigned to six groups as: control group, gentamicin (100 mg/kg), gentamicin + L-arginine (2 g/l), gentamicin + D-arginine (2 g/l), gentamicin + L-arginine + Nv-nitro-L-arginine methyl ester (L-NAME) (100 mg/l) and gentamicin + D-arginine + L-NAME. Gentamicin was administered by subcutaneous injections and the other drugs were added in drinking water for seven consecutive days. The animals were killed by decapitation and intracardiac blood and urine samples were obtained on the seventh day. Blood urea nitrogen, serum creatinine, sodium, potassium, urine gamma glutamyl transferase, creatinine, sodium, potassium and gentamicin levels were measured using High Performance Liquid Chromatography (HPLC) technique. Results: Gentamicin treated group had significant increase in blood urea nitrogen, serum creatinine, fractional Na excretion and urine gamma glutamyl transferase levels, and significant decrease in creatinine clearance compared to the control group. L-arginine and D-arginine reversed these findings. L-NAME abolished the nephroprotective effect of L-arginine. The urinary levels of gentamicin were significantly increased in rats treated with L-arginine or D-arginine compared to those treated with gentamicin. L-arginine and D-arginine reversed the advanced degenerative changes due to gentamicin administration in histopathological examination. Conclusion: Our study revealed the protective effect of L-arginine on gentamicin-induced nephrotoxicity, the contribution of the cationic feature of L-arginine, and the major role of NO in this protective effect.
Mersin University Medical Faculty Department of Medical Education, 33343 Yenisehir, Mersin, 1 Department of Pharmacology, Çukurova University Medical School, 01330, Balcali, Adana, Turkey Received: 06-01-2014 Revised: 31-01-2014 Accepted: 27-07-2014 Correspondence to: Dr. Perihan Başhan, E-mail: [email protected]
KEY WORDS: D-arginine, gentamicin, HPLC, L-arginine, nephrotoxicity
Introduction Aminoglycoside antibiotics are the most commonly used worldwide in the treatment of gram (-)negative bacterial infections. However, aminoglycosides induce nephrotoxicity in observed 10-20% of therapeutic courses.[1] The widespread Access this article online Website: www.ijp-online.com
Quick Response Code:
DOI: 10.4103/0253-7613.144915
608 Indian Journal of Pharmacology | December 2014 | Vol 46 | Issue 6
therapeutic use of the gentamicin is limited because of its nephrotoxic side effect and oxidative damage which can lead to acute renal failure.[1-3] Although the mechanism underlying gentamicin-induced renal cellular damage has not been entirely elucidated, generation of superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radicals has been attributed to its deleterious effect on the kidney.[4,5] An association between nephrotoxicity and oxidative stress has been confirmed in many experimental models.[6,7] The possible involvement of the L-arginine-Nitric oxide (NO) pathway in gentamicin-induced nephrotoxicity has been suggested by Rivas-Cabanero et al. These investigators reported increased glomerular synthesis of NO in rats with gentamicin-induced renal failure.[8]
Başhan, et al.: L-arginine and gentamicin nephrotoxicity
Arginine, an essential amino acid, has a positively charged guanidine group. Arginine is well-designed to bind the phosphate anion, and is often found in the active centers of proteins that bind phosphorylated substrates. As a cation, arginine plays an important role in maintaining the overall charge balance of a protein. L‑Arginine has been intensively investigated, mainly since it is a precursor of NO that is a highly reactive and multifunctional molecule involved in many physiological processes, such as cardiovascular system, nervous system and immunological reactions.[9] On the other hand, D‑arginine is frequently used in studies on L‑arginine/NO pathway is an inactive form of L‑arginine, because the D‑enantiomer is not a substrate for the stereospecific NO synthase.[10‑14] Data from recent studies showed that the cationic proteins and peptides, inhibit the uptake of a nephrotoxic drug, gentamicin, which is highly accumulated in the kidneys.[15,16] Some authors report that L‑arginine might have protected tubular function simply by decreasing gentamicin reabsorption in the proximal tubules.[17] It is known that gentamicin has an organic polycationic structure. L‑arginine consists of a functional cationic group of guanidin and due this cationic structural trait it can reduce gentamicin tubular reabsorption and increase gentamicin urinary excretion via competition. Therefore, in the present study, we aimed to investigate the possible protective effects other than NO synthesis of L‑arginine and its inactive isomere D‑arginine on gentamicin‑induced nephrotoxicity in rats due to their structural similarities using High Performance Liquid Chromatography (HPLC) technique. Materials and Methods Experimental Animals In this study, 42 male Wistar albino rats (appoximately 250–300 g weight) were procured from the Medical Science Experimental Research Center at Cukurova University. Rats were sheltered in metabolic cages at room temperature (24 ± 2ºC) under 12 hours daylight, 12 hours dark conditions. The study was approved by the Medical Science Experimental Research Center Ethics Committee. Experimental Protocol The rats were divided into six groups and each group consisted of seven rats. The groups were as follows: • Group 1(Control group): This group was treated with normal food and tap water • Group 2(Gentamicin Group): This group was treated with (100 mg/kg gentamicin) subcutaneous (s.c.) injection for seven days • Group 3(Gentamicin + L‑arginine Group): This group was treated with gentamicin injection and L‑arginine (2 g/l) was added to their drinking water • Group 4(Gentamicin + D‑ arginine Group): This group was treated with gentamicin injection and D‑arginine (2 g/l) was added to their drinking water • Group 5(Gentamicin + L‑arginine + L‑NAME Group): This group was treated with gentamicin injection and L‑arginine (2 g/l) and L‑NAME (100 mg/l) were added to their drinking water • Group 6 (Gentamicin + D‑arginine + L‑ NAME Group): This group was treated with gentamicin injection and
D‑arginine (2 g/l) and L‑NAME (100 mg/l) was added to in their drinking water. During seven days water intake and urine volume were followed. After the gentamicin injection, urine samples were collected on the first (initial), fourth (middle) and eighth day (final) to compare gentamicin levels. Ketamine (40 mg/kg) was given for anesthesia 24 hours after the final injection and intracardiac blood was obtained and animals were killed by decapitation. Providing the aseptic conditions, left kidney was removed by middle abdominal line incision for histopathological examination. Biochemical Analysis Blood urea nitrogen (BUN), serum creatinine, sodium (Na+), potassium (K+) levels were investigated from the blood of experimental animals. In addition, gama glutamyl transferase (GGT), creatinine, Na + K+ levels in their urine were measured at Central Laboratory of Cukurova University. The clearance of creatinine (CCr) and fractional Na excretion (FENa) were calculated using the standard methods as below. FENa =
Urine Na × PlasmaCr Plasma Na × UrineCr
×100
Histopathology of Kidney The kidneys were treated with paraffine after the formaline (10% solution) fixation and approximately 3-5 µm cross‑sections were dyed with hematoxylin–eosin. Tubular necrosis, degenerative variations and tubular regeneration were found under fluorescence microscope. Measurement of Gentamicin Levels Gentamicin levels of urine samples were evaluated according to the Kafkas et al., (2008) with major modifications.[18] The samples were stored at −20oC until analysis. HPLC analysis was performed in the Subtropical Fruits Research and Experimental Center Laboratory of Cukurova University. Identification and quantification were conducted using gentamicin (Sigma) standards. HPLC technique was used to identification and quantification of urine gentamicin levels. Fort this purpose, Hewlett Packard 1100 series, HP‑1100 UW dedector (210 nm) and Zorbax SB‑C18 (10 cm × 4.6 mm, 1.8 µm, Beckman) column were used and flow speed was applied 1 ml/min. Statistical analysis Data was presented as means ± Standard error of mean (S.E.M) for the groups. A one‑way variance analysis (One‑Way ANOVA) method was used for comparison of results. Comparison of groups were performed using Bonferroni test. The level of significance was set as P
Protective effect of L-arginine on gentamicin-induced nephrotoxicity in rats . lbrahim Başhan, Perihan Başhan1, Mehmet Ata Seçilmiş1, Ergin Şingirik1
ABSTRACT Introduction: L-arginine has a protective effect on gentamicin-induced renal failure and it may decrease the tubular reabsorption of another cationic substance, gentamicin due to its cationic structure. The aim of this study is to compare the possible protective effects of L-arginine and its inactive isomer D-arginine on gentamicin-induced nephrotoxicity in rats. Materials and Methods: Wistar albino rats were housed in metabolic cages and assigned to six groups as: control group, gentamicin (100 mg/kg), gentamicin + L-arginine (2 g/l), gentamicin + D-arginine (2 g/l), gentamicin + L-arginine + Nv-nitro-L-arginine methyl ester (L-NAME) (100 mg/l) and gentamicin + D-arginine + L-NAME. Gentamicin was administered by subcutaneous injections and the other drugs were added in drinking water for seven consecutive days. The animals were killed by decapitation and intracardiac blood and urine samples were obtained on the seventh day. Blood urea nitrogen, serum creatinine, sodium, potassium, urine gamma glutamyl transferase, creatinine, sodium, potassium and gentamicin levels were measured using High Performance Liquid Chromatography (HPLC) technique. Results: Gentamicin treated group had significant increase in blood urea nitrogen, serum creatinine, fractional Na excretion and urine gamma glutamyl transferase levels, and significant decrease in creatinine clearance compared to the control group. L-arginine and D-arginine reversed these findings. L-NAME abolished the nephroprotective effect of L-arginine. The urinary levels of gentamicin were significantly increased in rats treated with L-arginine or D-arginine compared to those treated with gentamicin. L-arginine and D-arginine reversed the advanced degenerative changes due to gentamicin administration in histopathological examination. Conclusion: Our study revealed the protective effect of L-arginine on gentamicin-induced nephrotoxicity, the contribution of the cationic feature of L-arginine, and the major role of NO in this protective effect.
Mersin University Medical Faculty Department of Medical Education, 33343 Yenisehir, Mersin, 1 Department of Pharmacology, Çukurova University Medical School, 01330, Balcali, Adana, Turkey Received: 06-01-2014 Revised: 31-01-2014 Accepted: 27-07-2014 Correspondence to: Dr. Perihan Başhan, E-mail: [email protected]
KEY WORDS: D-arginine, gentamicin, HPLC, L-arginine, nephrotoxicity
Introduction Aminoglycoside antibiotics are the most commonly used worldwide in the treatment of gram (-)negative bacterial infections. However, aminoglycosides induce nephrotoxicity in observed 10-20% of therapeutic courses.[1] The widespread Access this article online Website: www.ijp-online.com
Quick Response Code:
DOI: 10.4103/0253-7613.144915
608 Indian Journal of Pharmacology | December 2014 | Vol 46 | Issue 6
therapeutic use of the gentamicin is limited because of its nephrotoxic side effect and oxidative damage which can lead to acute renal failure.[1-3] Although the mechanism underlying gentamicin-induced renal cellular damage has not been entirely elucidated, generation of superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radicals has been attributed to its deleterious effect on the kidney.[4,5] An association between nephrotoxicity and oxidative stress has been confirmed in many experimental models.[6,7] The possible involvement of the L-arginine-Nitric oxide (NO) pathway in gentamicin-induced nephrotoxicity has been suggested by Rivas-Cabanero et al. These investigators reported increased glomerular synthesis of NO in rats with gentamicin-induced renal failure.[8]
Başhan, et al.: L-arginine and gentamicin nephrotoxicity
Arginine, an essential amino acid, has a positively charged guanidine group. Arginine is well-designed to bind the phosphate anion, and is often found in the active centers of proteins that bind phosphorylated substrates. As a cation, arginine plays an important role in maintaining the overall charge balance of a protein. L‑Arginine has been intensively investigated, mainly since it is a precursor of NO that is a highly reactive and multifunctional molecule involved in many physiological processes, such as cardiovascular system, nervous system and immunological reactions.[9] On the other hand, D‑arginine is frequently used in studies on L‑arginine/NO pathway is an inactive form of L‑arginine, because the D‑enantiomer is not a substrate for the stereospecific NO synthase.[10‑14] Data from recent studies showed that the cationic proteins and peptides, inhibit the uptake of a nephrotoxic drug, gentamicin, which is highly accumulated in the kidneys.[15,16] Some authors report that L‑arginine might have protected tubular function simply by decreasing gentamicin reabsorption in the proximal tubules.[17] It is known that gentamicin has an organic polycationic structure. L‑arginine consists of a functional cationic group of guanidin and due this cationic structural trait it can reduce gentamicin tubular reabsorption and increase gentamicin urinary excretion via competition. Therefore, in the present study, we aimed to investigate the possible protective effects other than NO synthesis of L‑arginine and its inactive isomere D‑arginine on gentamicin‑induced nephrotoxicity in rats due to their structural similarities using High Performance Liquid Chromatography (HPLC) technique. Materials and Methods Experimental Animals In this study, 42 male Wistar albino rats (appoximately 250–300 g weight) were procured from the Medical Science Experimental Research Center at Cukurova University. Rats were sheltered in metabolic cages at room temperature (24 ± 2ºC) under 12 hours daylight, 12 hours dark conditions. The study was approved by the Medical Science Experimental Research Center Ethics Committee. Experimental Protocol The rats were divided into six groups and each group consisted of seven rats. The groups were as follows: • Group 1(Control group): This group was treated with normal food and tap water • Group 2(Gentamicin Group): This group was treated with (100 mg/kg gentamicin) subcutaneous (s.c.) injection for seven days • Group 3(Gentamicin + L‑arginine Group): This group was treated with gentamicin injection and L‑arginine (2 g/l) was added to their drinking water • Group 4(Gentamicin + D‑ arginine Group): This group was treated with gentamicin injection and D‑arginine (2 g/l) was added to their drinking water • Group 5(Gentamicin + L‑arginine + L‑NAME Group): This group was treated with gentamicin injection and L‑arginine (2 g/l) and L‑NAME (100 mg/l) were added to their drinking water • Group 6 (Gentamicin + D‑arginine + L‑ NAME Group): This group was treated with gentamicin injection and
D‑arginine (2 g/l) and L‑NAME (100 mg/l) was added to in their drinking water. During seven days water intake and urine volume were followed. After the gentamicin injection, urine samples were collected on the first (initial), fourth (middle) and eighth day (final) to compare gentamicin levels. Ketamine (40 mg/kg) was given for anesthesia 24 hours after the final injection and intracardiac blood was obtained and animals were killed by decapitation. Providing the aseptic conditions, left kidney was removed by middle abdominal line incision for histopathological examination. Biochemical Analysis Blood urea nitrogen (BUN), serum creatinine, sodium (Na+), potassium (K+) levels were investigated from the blood of experimental animals. In addition, gama glutamyl transferase (GGT), creatinine, Na + K+ levels in their urine were measured at Central Laboratory of Cukurova University. The clearance of creatinine (CCr) and fractional Na excretion (FENa) were calculated using the standard methods as below. FENa =
Urine Na × PlasmaCr Plasma Na × UrineCr
×100
Histopathology of Kidney The kidneys were treated with paraffine after the formaline (10% solution) fixation and approximately 3-5 µm cross‑sections were dyed with hematoxylin–eosin. Tubular necrosis, degenerative variations and tubular regeneration were found under fluorescence microscope. Measurement of Gentamicin Levels Gentamicin levels of urine samples were evaluated according to the Kafkas et al., (2008) with major modifications.[18] The samples were stored at −20oC until analysis. HPLC analysis was performed in the Subtropical Fruits Research and Experimental Center Laboratory of Cukurova University. Identification and quantification were conducted using gentamicin (Sigma) standards. HPLC technique was used to identification and quantification of urine gentamicin levels. Fort this purpose, Hewlett Packard 1100 series, HP‑1100 UW dedector (210 nm) and Zorbax SB‑C18 (10 cm × 4.6 mm, 1.8 µm, Beckman) column were used and flow speed was applied 1 ml/min. Statistical analysis Data was presented as means ± Standard error of mean (S.E.M) for the groups. A one‑way variance analysis (One‑Way ANOVA) method was used for comparison of results. Comparison of groups were performed using Bonferroni test. The level of significance was set as P
