Korean J Physiol Pharmacol 2021; 25(4): 321-331
Published online July 1, 2021 https://doi.org/10.4196/kjpp.2021.25.4.321
Copyright © Korean J Physiol Pharmacol.
Promise M. Emeka1,*, Sahibzada T. Rasool2, Mohamed A. Morsy1,3, Mohamed I. Hairul Islam4, and Muhammad S. Chohan2
Departments of 1Pharmaceutical Sciences and 2Biomedical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia, 3Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt, 4Department of Biological Sciences, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia
Correspondence to:Promise M. Emeka
Vancomycin, an antibiotic used occasionally as a last line of treatment for methicillin-resistant Staphylococcus aureus, is reportedly associated with nephrotoxicity. This study aimed at evaluating the protective effects of lutein against vancomycin-induced acute renal injury. Peroxisome proliferator-activated receptor gamma (PPARγ) and its associated role in renoprotection by lutein was also examined. Male BALB/c mice were divided into six treatment groups: control with normal saline, lutein (200 mg/kg), vancomycin (250 mg/kg), vancomycin (500 mg/kg), vancomycin (250 mg/kg) with lutein, and vancomycin (500 mg/kg) with lutein groups; they were euthanized after 7 days of treatment. Thereafter, samples of blood, urine, and kidney tissue of the mice were analyzed, followed by the determination of levels of N-acetyl-β-D-glucosaminidase (NAG) in the urine, renal creatine kinase; protein carbonyl, malondialdehyde, and caspase-3 in the kidney; and the expression of PPARγ, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor-kappaB (NF-κB) in renal tissue. Results showed that the levels of protein carbonyl and malondialdehyde, and the activity of NAG, creatine kinase and caspase-3, were significantly increased in the vancomycin-treatment groups. Moreover, the levels of Nrf2 significantly decreased, while NF-κB expression increased. Lutein ameliorated these effects, and significantly increased PPARγ expression. Furthermore, it attenuated vancomycin-induced histological alterations such as, tissue necrosis and hypertrophy. Therefore, we conclude that lutein protects against vancomycin-induced renal injury by potentially upregulating PPARγ/Nrf2 expression in the renal tissues, and consequently downregulating the pathways: inflammation by NF-κB and apoptosis by caspase-3.
Keywords: Acute renal injury, Caspase-3, Lutein, PPAR gamma, Vancomycin
Kidneys are the main excretory organ for many drugs, and could easily be exposed to toxins. Nephrotoxicity has been reported to contribute to approximately 8%–40% of all cases of acute renal injury. Since its introduction, vancomycin, a glycopeptide antibiotic often used as the last line of defense against drug resistant gram-positive bacteria, has been associated with acute renal injury [1,2]. Vancomycin is commonly used to treat hospital-acquired methicillin-resistant
Lutein is a carotenoid that has been reported to modulate pro-inflammatory mediators, such as the peroxisome proliferator-activated receptors (PPAR), which is known to generate oxyradicals that alter signaling transduction pathways . PPARγ, a transcription factor and a member of the PPAR family, has been found to be expressed in renal tissues, and is implicated in the regulation of redox hemostasis in the kidney . PPARγ activity is said to be well-regulated during various cellular processes; indeed, nuclear factor-kappaB (NF-κB) stimulation has shown to often suppress PPARγ activity . PPARγ agonists reportedly decrease NF-κB activity in many tissues during inflammation . Activation of PPARγ could be useful against renal damage induced by oxidative stress through the inhibition of NF-κB protein expression, possibly
Vancomycin was supplied by Julphar (Ras Al Khaimah, UAE), and lutein was supplied by Extrasynthese (Genay, France). Assay kits for N-acetyl-β-D-glucosaminidase (NAG), protein carbonyl content, and caspase-3 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against PPARγ, nuclear factor erythroid 2-related factor 2 (Nrf2), and NF-κB p65 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Gels for Western blotting were procured from Bio-Rad (Hercules, CA, USA), and primers for real-time PCR of PPARγ were procured from Gibco, Thermo Fisher Scientific Inc. (Waltham, MA, USA).
Male BALB (Bagg Albino)/c mice with an average weight of 29.5 g, were obtained from the Department of Biological Sciences, College of Science, King Faisal University, Saudi Arabia. The mice were maintained in groups of six per cage in controlled environmental conditions according to the specified standards, with 12 h dark and 12 h light cycle, at 23 ± 1°C. They were allowed access to food and water throughout the study. Animal care and experimental procedures were carried out according to the guidelines of the Research Ethics Committee at King Faisal University (140237/27/2013) and that of the National Committee of Bioethics (NCBE), King Abdulaziz City for Science and Technology (KACST), Saudi Arabia.
The mice were divided into six groups (six mice per group) as follows: control with normal saline, lutein (L: lutein) 200 mg/kg (dissolved in 0.2% dimethyl sulfoxide [DMSO]), vancomycin 250 mg/kg (low-dose vancomycin: VL), 500 mg/kg vancomycin (high-dose vancomycin: VH), L + VL, and L + VH. Both the drugs were administered intraperitoneally and consecutively for 7 days, according to the modified methods described by Qu
NAG activity in the urine samples was evaluated using an enzymatic assay, according to the manufacturer’s instructions. As reported by Horak
Creatine kinase activity was evaluated according to the method described by Hughes . The tissue homogenates of the kidney was incubated in 50 mM Tris buffer (pH 7.5), 7.5 mM MgSO4, 7 mM phosphocreatine, and 3.2 mM ADP medium for 10 min at 37°C. Then, 15 μl of 1 mM p-hydroxymercuribenzoic acid was added after the reaction was stopped for 10 min. To analyze the reaction colorimetrically, 2% α-naphthol and 1% diacetyl were added; the reaction mixture was allowed to develop color for 15 min at 37°C, and then measured at 540 nm.
Protein carbonyl content, which is a marker of oxidative damage, was measured using a previously reported method . The oxidized proteins produced protein carbonyl, which was then reacted with 2,4-dinitrophenylhydrazine to produce 2,4-dinitrophenylhydrazone, which was measured spectrophotometrically at 375 nm.
Lipid peroxidation (assessed based on MDA production) in the tissue homogenates of the kidney was measured by determining the levels of thiobarbituric acid-reactive substances, as described by Ohkawa
Caspase-3 activity was measured using a colorimetric assay, according to the manufacturer’s instructions. The kidney tissues were homogenized in 3 ml of 10 mM phosphate buffer (pH 7.4) . Detection of caspase-3 activity was predicated on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide by caspase-3. The reaction led to the consequent release of the p-nitroaniline component, which was then measured spectrophotometrically at 405 nm.
The protein expression of PPARγ, Nrf2, and NF-κB p65 in the kidney tissues were determined through Western blotting using specific antibodies, according to the method described by Ahn
Quantitative real-time PCR was performed for expression analysis of PPARγ in the kidney tissues. cDNA was synthesized from the samples by using a previously reported method . In brief, total RNA was isolated from kidney tissues using the TRIzol method (Gibco; Thermo Fisher Scientific), according to the manufacturer’s instructions, and stored at –85°C for later use. The oligonucleotide primers for PCR were as follows: PPARγ, forward 5ʹ-CGGTTTCAGAAGTGCCTTG-3ʹ, reverse 5ʹ-GGTTCAGCTGGTCGATATCAC-3ʹ; and β-actin forward 5ʹ-AGCTATGAGCTGCCTGACGG-3ʹ, reverse 5ʹ-CCAGACAGCACTGTGTTGG-3ʹ. Using kinetic analysis of PCR amplification, the amounts of each template of PPARγ/β-actin mRNA were calculated. The PPARγ mRNA expression was standardized with that of β-actin mRNA, which was used for comparison with each sample. The relative expression of PPARγ was represented as fold change and determined by the relative quantification algorithm with the 2−ΔΔ
The kidney tissues were fixed in 10% formaldehyde in phosphate-buffered saline. Later, the tissues were embedded in paraffin for light microscopic evaluation; they were sectioned and stained with hematoxylin and eosin. Slide preparation and a double-blinded evaluation were performed by a histopathologist. The results of pathology were indicated as follows: (–) absence of pathology, (+) mild pathology, (++) moderate pathology, (+++) severe pathology, and (±) recovery.
The results obtained were expressed as mean ± SD; they were analyzed using the GraphPad Prism software version 8.2 (San Diego, CA, USA). Comparisons between all the treatment and control groups were made using the one-way analysis of variance, and the differences between the groups were measured using the Tukey's multiple comparison test. p < 0.05 was considered statistically significant.
Mice treated with VL and VH showed a significant elevation in NAG activity, which was significantly attenuated by co-administration with lutein (Fig. 1A). In Fig. 1B, the activity of creatine kinase was significantly higher in VL- and VH-treatment groups, than that in the control group. In L + VL or L + VH groups, creatine kinase had reduced significantly. As shown in Fig. 1C, the protein carbonyl content in VL and VH groups were significantly higher than those in the control group, and co-administration with lutein significantly reduced the protein carbonyl content in these treatment groups. MDA levels had increased significantly in the vancomycin-treatment groups than those in the control group. However, following co-administration with lutein, these levels had also decreased significantly (Fig. 1D). Caspase-3 activity in the kidney tissues had increased significantly in the VL- and VH-treatment groups than that in the control group. The activity had reduced significantly again, with the co-treatment of vancomycin with lutein in both the treatment groups (Fig. 1E). Our results indicate that lutein effectively increases the antioxidant activity in the kidney tissues. Subsequently, co-administration with lutein attenuated the injurious effects due to the administration of VL and VH, respectively.
In the control group and groups treated with lutein, the glomeruli and renal tubules showed no signs of cellular damage (Fig. 2A, B). In addition, there was no infiltration of inflammatory cells, and renal architecture was normal (Table 1). In the VL-treatment group, we observed abnormal dilation of renal tubules that were filled with leukocytes, in addition to significant infiltration of inflammatory cells into the renal parenchyma (Fig. 2C). However, in the L + VL-treatment group, there were reductions in infiltration of inflammatory cells, and recovery of the renal parenchyma (Fig. 2D and Table 1). Administration of 500 mg/kg vancomycin produced significant renal damage such as epithelial necrosis of the renal tubules, and complete obliteration of the tubules in most cases (Fig. 2E). Lutein administration to this group, significantly attenuated the injurious effects of vancomycin, indicating recovery of the renal tubules (Fig. 2F).
Table 1 . Histopathological scoring of the effect of lutein on low (250 mg/kg) and high (500 mg/kg) dose vancomycin-induced renal injury in mice.
|Group||Abnormal dilatation of the renal tubules||Renal tubular obliteration||Tubular necrosis||Renal tubules filled with leukocytes||Inflammatory cell infiltration in parenchyma|
|Vancomycin 250 mg/kg||+||–||–||+||+|
|Lutein + vancomycin 250 mg/kg||±||–||–||–||±|
|Vancomycin 500 mg/kg||–||+++||+++||–||–|
|Lutein + vancomycin 500 mg/kg||+||–||–||+||+|
–, absence of pathology; +, mild pathology; ++, moderate pathology; +++, severe pathology; ±, recovery.
The expression levels of PPARγ, (protein and mRNA) and Nrf2, determined in the control and treatment groups, are shown in Fig. 3A–C. Western blotting analysis showed a significantly increased PPARγ expression in the VL- and VH-treatment groups than that in the control group. In addition, co-administration with lutein in both VL- and VH-treatment groups further increased PPARγ expression significantly, compared to that in the control group (Fig. 3A). In the mRNA expression analysis across the different treatment groups, the mRNA levels of PPARγ were found to be increased in the VL- and VH-treatment groups than those in the control group. Co-administration of lutein with either VL or VH significantly increased the mRNA expression of PPARγ in these renal tissues (Fig. 3B). Therefore, we conclude that lutein elevates the mRNA levels of PPARγ in the vancomycin-treatment groups. Western blotting analysis also showed a significantly reduced Nrf2 expression in both VL- and VH-treatment groups; however, in both the groups, the expression was restored with lutein treatment (Fig. 3C).
Results showed that NF-κB p65expression was significantly elevated in both VL- and VH-treatment groups compared to that in the control group; however, co-administration with lutein reduced NF-κB p65 expression in both the groups (Fig. 4).
Vancomycin is an antibiotic used clinically to treat drug resistant bacterial infections, particularly by the gram-positive bacteria, MRSA. Reports indicate that, for clinical effectiveness, the daily area under the curve values of vancomycin should achieve plasma concentrations of 400 and 600 mg × h/L, respectively,
In this study, we examined vancomycin-induced acute renal injury and its underlying mechanism. Moreover, we examined various signaling pathways involved in the generation of oxyradicals by vancomycin, and the inhibition of these oxyradicals by lutein—a natural antioxidant. Lutein has been reported to have a variety of biological effects related to its antioxidative properties . The present findings indicate that the administration of low-dose and high-dose vancomycin in mice induces oxyradicals, leading to a significant reduction in the antioxidant activity in the renal tissues. In addition, co-administration of lutein with vancomycin restored the antioxidant levels, and consequently reduced the generation of cellular oxyradicals. Our data also showed that lutein administration led to a significant reduction in NAG excretion that was observed to increase after vancomycin treatment. The significantly increased NAG levels usually indicate cellular damage in the kidney, and this can be a sensitive biological marker for glycopeptide-antibiotic–induced renal toxicity . Our findings are corroborated by an earlier study by Qu
Furthermore, during renal injury, protein carbonyl content is known to usually increase. This can be used as another biomarker for cellular injury. Protein carbonyl content has been reported to be consistent in protein oxidative damage . In this study, we observed that vancomycin treatment leads to elevated protein carbonyl content in a dose-dependent manner. However, our findings also show that lutein administration mitigated these effects, indicating that it has a protective role, in accordance with the findings of Cheng
Through the histopathology examination of photomicrographs of renal tissues, this study confirms that different doses of vancomycin cause nephrotoxicity. We observed various hallmarks of renal injury, including inflammatory cells, leukocytes infiltration, necrosis, renal hypertrophy, and obliteration of renal tubules. However, upon lutein treatment, these effects were considerably reversed. This further goes to confirm that lutein has potent antioxidant and anti-inflammatory properties against oxyradicals. These findings are also consistent with other studies documented by Ouyang
It is well established that oxyradical generators like vancomycin induce oxidative stress . Studies have shown that NF-κB, a transcriptional mediator of the inflammatory process, plays a key role in cellular injury and damage during this process .
In addition, NF-κB has been tagged the “holy grail” of inflammatory processes, activating multiple pathways during cellular oxidative stress . In a study conducted by Xiao
We also show that, vancomycin treatment leads to elevated expression of NF-κB p65 in a dose-dependent manner, and this correlates with tissue damage. These findings are similar to those reported by Qu
Therefore, this study found that lutein supressed NF-κB p65 activation, thereby inhibiting its regulated gene products such as cytokines and caspase 3. This finding is consistent with the reports of Buhrmann
Accumulative evidence shows that PPARγ plays a vital role in redox homeostasis by regulating several signaling pathways . Therefore, the activation of PPARγ in the renal tissues could be critical for kidney functioning . According to documented reports, the activation of PPARγ mitigates inflammation in various tissues; thereby, preventing cellular injury [11,44].
Numerous studies have shown that PPARγ agonists can be renoprotective by ameliorating inflammation . Similarly, it has also been documented that, lutein has antioxidant properties, which suppress inflammatory responses, possibly through PPARγ activation . The increased PPARγ expression upon vancomycin administration, in comparison to that in the control, can be explained as a compensatory measure by renal cells to overcome oxyradical-induced cellular damage. In this study, we also observed that the PPARγ expression increased with lutein administration, mitigating the effects of vancomycin-induced renal injury. These findings appear to be in agreement with those of Diep and Schiffrin ; they suggested that increased PPARγ expression might play a role in cell remodeling under stressful conditions. Kvandova
However, another body of evidence show that PPARγ activation, in fact, regulates the Nrf2 pathway , owing to the evidence of reduced Nrf2 expression in PPARγ-deficient mice . Furthermore, the aforementioned studies strengthen our observation in the present study that lutein, an activator of PPARγ and Nrf2, attenuates oxidative stress by increasing the expression of both. This view has been supported by many studies, indicating that co-activation of these pathways suppresses inflammation and cellular oxidative stress . Moreover the attenuation of cellular expression of NF-κB by PPARγ, and the suppression of pro-inflammatory mediators, is well documented . Our study also showed a reduction in the NF-κB expression on co-administration of lutein. This indicates that lutein may have a role in the mediation of antioxidant and anti-inflammatory activities observed in this study. Based on these results, we suggest that lutein upregulates PPARγ/Nrf2 signaling pathways and downregulates NF-κB signaling pathway. This finding was also corroborated by Makarov . Jin
This study has highlighted the potential of lutein in protecting against vancomycin-induced renal injury, a major drawback of vancomycin treatment. In addition, this study confirms the anti-inflammatory properties of lutein. The antioxidant activity of lutein is crucial in attenuating the effects of oxyradicals generated by vancomycin treatment; thereby, reducing oxidative stress, and consequently, mitigating renal cellular injury in mice. Lutein’s renoprotective effects seem to be associated with increased PPARγ expression, and subsequent co-activation of Nrf2; hence, suppressing renal NF-κB expression thereby inhibiting the activation of apoptotic process. Based on these observations, we suggest that the molecular mechanism of vancomycin-induced renal injury could be associated with the inhibition of PPARγ/Nfr2 signaling pathways. We show that lutein can reactivate PPARγ/Nfr2 signaling to attenuate the harmful effects of oxyradicals generated from the use of escalated doses of vancomycin.
We hereby wish to thank Deanship of Scientific Research, King Faisal University, Saudi Arabia for supporting this research. Also we wish to acknowledge Mr. Tameem Alyahian, our Laboratory Supervisor, for his assistance. We also acknowledge Mr. Sami Al-Qaimi for helping us with the procurement of chemicals.
This research project was funded by the Deanship of Scientific Research, King Faisal University, Saudi Arabia (grant number: 140237).
P.M.E. contributed to conception and design, acquisition and analysis of data, and drafted the manuscript. S.T.R., M.A.M., and M.I.H.I. contributed to the acquisition and analysis of data and critically revised the manuscript for important intellectual content. M.S.C. substantially contributed to the acquisition and analysis of data and drafted the manuscript.
The authors declare no conflicts of interest.
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