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Determining the Protective Effect of L-Arginine Against Amikacin-Induced Nephrotoxicity in Normal African Green Monkey Kidney Epithelial Cells by Evaluating Oxidative Stress Parameters
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Elahe Gharehkhani1    , Marzieh Megharad2 , Mahboube Rahmati Kukandeh1 , Mohammad Shokrzadeh *3 |
1- Ph.D in Toxicology, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
2- Pharmacy Student, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Ramsar, Iran.
3- Professor, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. Pharmaceutical Sciences Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran. , mshokrzadeh@mazums.ac.ir |
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Keywords: Oxidative Stress [MeSH], Vero Cells [MeSH], Amikacin [MeSH], Arginine [MeSH]
Article ID: Vol27-37 |
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Type of Study: Original Articles |
Subject:
Pharmacology
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Abstract: (60 Views) |
Extended Abstract
Introduction
The pathophysiology of nephrotoxicity is complex and multifactorial, involving processes such as oxidative stress, inflammation, apoptosis, and impaired renal hemodynamics. These mechanisms are exacerbated by underlying conditions, such as hypertension, diabetes, and cardiovascular diseases.
Oxidative stress represents a pivotal pathophysiological mechanism in nephrotoxicity, playing a central role in the initiation and progression of renal injury. Due to high metabolic activity and rich blood supply, the kidneys are highly vulnerable to oxidative stress. Nephrotoxic agents, such as cisplatin, aminoglycosides, and radiocontrast agents induce the production of reactive oxygen species (ROS) in renal tubular cells, leading to lipid peroxidation, protein oxidation, and mitochondrial dysfunction, ultimately stimulating apoptosis and contributing to the decline in renal function observed in nephrotoxicity.
Among the most well-known nephrotoxic agents are aminoglycosides (e.g., amikacin), non-steroidal anti-inflammatory drugs (NSAIDs), cisplatin, and radiocontrast agents.
Once established, renal damage-particularly in cases of chronic kidney disease-can be irreversible. Therefore, there is a pressing need to develop therapeutic interventions capable of preventing the onset of nephrotoxicity or, at the very least, mitigating its severity and long-term impact on renal function. Consequently, despite the discovery of various antioxidant agents, the search for novel nephroprotective antioxidants remains a critical necessity.
L-arginine is a semi-essential amino acid that serves as a vital precursor for various metabolic pathways, most notably the synthesis of nitric oxide-a key signaling molecule in numerous physiological processes. Following ingestion, L-arginine is absorbed in the small intestine and transported via the bloodstream to various tissues, where it plays a fundamental role in protein synthesis and the urea cycle. The levels of L-arginine-nitric oxide pathway derivatives, such as nitrite, nitrate, and potentially L-citrulline, in biological fluids may serve as clinical biomarkers for monitoring specific pathological conditions and their therapeutic progression.
Nitric oxide is a highly reactive molecule with a short biological half-life. Within the vasculature, nitric oxide diffuses from endothelial cells into adjacent vascular smooth muscle cells (VSMCs). In these cells, it activates soluble guanylate cyclase (sGC), leading to the synthesis of cyclic guanosine monophosphate (cGMP). The elevation of cGMP levels induces vasodilation and subsequently increases blood flow. Furthermore, nitric oxide exerts potent anti-inflammatory and anti-atherogenic effects by inhibiting leukocyte and platelet adhesion and activation, reducing oxidative stress, and preventing the proliferation of vascular smooth muscle cells.
Nitric oxide serves as a critical modulator of the tubuloglomerular feedback (TGF) response. Nitric oxide synthesized in the macula densa, mediated by neuronal nitric oxide synthase, attenuates TGF-induced afferent arteriolar constriction. Furthermore, elevated nitric oxide production may account for the impaired autoregulatory efficiency of medullary blood flow during volume expansion. Consequently, L-arginine may be considered a nephroprotective agent.
This study was conducted to determine the protective effect of L-arginine against amikacin-induced nephrotoxicity in normal African green monkey kidney epithelial cells (Vero) by evaluating oxidative stress parameters.
Methods
This descriptive-analytical in vitro study was conducted on Vero cell lines purchased from the National Genetic Resources Cell Bank at the Cell Culture Laboratory of the Faculty of Pharmacy, Mazandaran University of Medical Sciences.
For each cell line, the classification consisted of 7 groups as follows:
1. Control group: No treatment.
2. Amikacin (653.2 µg/mL) group.
3. L-arginine (108 µM) + Amikacin (653.2 µg/mL) group.
4. L-arginine (216 µM) + Amikacin (653.2 µg/mL) group.
5. L-arginine (430 µM) + Amikacin (653.2 µg/mL) group.
6. L-arginine (860 µM) + Amikacin (653.2 µg/mL) group.
7. L-arginine (860 µM) group.
The cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cultures were maintained in an incubator at 37°C with adequate humidity and 5% carbon dioxide (CO2). For implementing various tests, once the cells reached at least 70-80% cell growth, they were detached using trypsin-ethylenediaminetetraacetic acid (EDTA) without any mechanical agitation or shaking of the flask. The cell suspension was then centrifuged at 2000 rpm for 5 minutes. The resulting cell pellet was resuspended in 1 mL of culture medium. Cell viability was determined by mixing equal volumes of the cell suspension and Trypan blue dye, followed by counting using a hemacytometer under an optical microscope. After ensuring the absence of contamination, cells with a viability of over 80–90% were utilized for the test. Cell viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. To measure intracellular ROS levels, cells were cultured at a density of 10 cells in 6-well plates. Following a 24-hour incubation period, the cells were treated with various concentrations of L-arginine (108, 216, 430, and 860 µM) and subsequently exposed to Amikacin. After 4 hours, the cells were washed with phosphate-buffered saline (PBS) and treated with a lysis buffer. Following this, dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added, and the samples were incubated for 30 minutes at 37°C in the dark. Finally, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
To evaluate lipid peroxidation levels, cells were cultured at a density of 10 cells in 6-well plates and incubated for 24 hours. Subsequently, the cells were treated with various concentrations of L-arginine (108, 216, 430, and 860 µM) and then exposed to Amikacin. Following a 4-hour incubation period with the aforementioned compounds, lipid peroxidation levels were quantified using the thiobarbituric acid reactive substances (TBARS) assay.
Results
The Effect of L-arginine on the Viability of the Vero Cell Line in Amikacin-Induced Cytotoxicity: The viability of cells treated solely with amikacin (at the IC50 concentration) was significantly reduced compared to the control group (P<0.001). Subsequently, treatment of the toxicity-induced cells with L-arginine (at concentrations of 108, 216, 430, and 860 µM) resulted in a significant increase in cell viability (P<0.001). Furthermore, cell viability increased with higher L-arginine concentrations.
The Effect of L-arginine on Amikacin-Induced Peroxidation in the Vero Cell Line: Malondialdehyde (MDA) levels in cells treated with Amikacin (at IC50 concentration) significantly increased compared to the control group (P<0.001). The reduction in MDA levels in the the group receiving 108 µM of L-arginine was not significant compared to the group receiving Amikacin alone. However, MDA levels in cells treated with
216 µM (P<0.050), 430 µM (P<0.001), and 860 µM (P<0.001) of L-arginine showed a significant decrease compared to the group receiving Amikacin alone. Furthermore, MDA levels decreased with higher concentrations of L-arginine.
The Effect of L-Arginine on Amikacin-Induced Cellular Reactive Oxygen Species Production in the Vero Cell Line: ROS levels in cells treated solely with Amikacin (at the IC50 concentration) exhibited a significant increase compared to the control group (P<0.001). Moreover, ROS production in toxicity-induced cells treated with L-arginine at concentrations of 108, 216, 430, and 860 µM showed a significant reduction compared to cells receiving Amikacin alone (P<0.001). The concentration of produced ROS decreased with higher concentrations of
L-arginine.
Conclusion
According to the results of this study, in assays measuring ROS levels and cell viability, all tested concentrations of
L-arginine (108, 216, 430, and 860 µM) culminated in a significant reduction in ROS levels and a significant increase in cell viability. Regarding the lipid peroxidation assay, while the cells receiving 108 µM of L-arginine produced no significant decrease in MDA levels, all other concentrations (216, 430, and 860 µM) led to a significant reduction in MDA.
The significant protective effects of L-arginine in acute kidney injury (AKI) are primarily mediated through mechanisms involving nitric oxide synthesis, as well as the regulation of oxidative stress and inflammation. In the context of ischemia-reperfusion injury-a leading cause of AKI-the pre-injury administration of L-arginine improves renal outcomes by increasing the bioavailability of nitric oxide. Nitric oxide, synthesized from L-arginine by nitric oxide synthase, is a vital mediator of vasodilation that enhances renal blood flow, thereby mitigating ischemic injury. Furthermore, L-arginine upregulates the expression of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2–related factor 2 (Nrf2), both of which serve protective roles against oxidative stress by bolstering antioxidant defenses, such as superoxide dismutase (SOD) and glutathione (GSH).
Chronic nitric oxide deficiency, a hallmark of chronic kidney disease (CKD), leads to the exacerbation of hypertension, glomerulosclerosis, and tubular atrophy-all of which are detrimental to long-term renal function. L-arginine helps ameliorate these conditions by serving as a substrate for nitric oxide synthesis. Studies have demonstrated that L-arginine supplementation in CKD models reduces the expression of profibrotic factors, such as transforming growth factor-beta (TGF-β) and fibronectin, which are critical drivers of renal fibrosis. Moreover, L-arginine upregulates the expression of endothelial nitric oxide synthase, which is essential for maintaining the equilibrium between vasodilatory and vasoconstrictive forces within the renal vasculature. This restoration of nitric oxide levels not only improves hemodynamic flow but also inhibits the activation of fibrotic pathways, thereby slowing the progression of CKD.
Ethical Statement
This study was approved by the Ethics Committee at Mazandaran University of Medical Sciences (IR.MAZUMS.RIB.REC.1402.061).
Funding
This article has been derived from the doctoral dissertation (approval code: 17917) by Marzieh Megharad in Doctor of Pharmacy (Pharm.D) at Ramsar Self-Governing Campus, Mazandaran University of Medical Sciences.
Authors' Contributions
Elahe Gharehkhani (Ph.D): Project execution, Data analysis, Interpretation of the results, Drafting of the initial manuscript.
Marzieh Megharad: Project execution, Data collection.
Mahboube Rahmati Kukandeh (Ph.D): Data analysis, Interpretation of the results.
Mohammad Shokrzadeh (Ph.D): Project administration and design, Approval of the final manuscript.
Conflicts of Interest
No conflicts of interest.
Acknowledgement
The authors would like to thank the research authorities of the Faculty of Pharmacy at Mazandaran University of Medical Sciences and the thesis examination committee for their invaluable support and guidance in conducting and improving the quality of this study.
Key Message: L-arginine was effective in improving renal cell viability parameters and increasing GSH levels across all tested concentrations (108, 216, 430, and 860 μM). Furthermore, at concentrations of 216, 430, and 860 μM, L-arginine significantly reduced lipid peroxidation.
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Gharehkhani E, Megharad M, Rahmati Kukandeh M, Shokrzadeh M. Determining the Protective Effect of L-Arginine Against Amikacin-Induced Nephrotoxicity in Normal African Green Monkey Kidney Epithelial Cells by Evaluating Oxidative Stress Parameters. J Gorgan Univ Med Sci 2025; 27 (4) :63-72 URL: http://goums.ac.ir/journal/article-1-4540-en.html
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