Sage Journals: Discover world-class research

Abstract

Background:

Despite being one of the most nephrotoxic drugs, gentamicin (GM) remains a mainstay as a first-choice agent in a vast variety of clinical situations owing to its superlative efficiency as a broad-spectrum antibiotic in treating several life-threatening bacterial infections. This urgently calls for the need for in-depth analysis of the mechanisms governing GM-induced nephrotoxicity and entails the necessity of presenting novel protective agents capable of ameliorating those renal deleterious effects. The reactive oxygen species and redox-sensitive transcription factors in GM-induced nephrotoxicity have recently called attention.

Purpose:

This study has been designed to shed light on the possible mechanisms of GM-induced nephrotoxicity and to provide a consensus set of histopathological, immunohistochemical, genetic and biochemical parameters elucidating the protective role of vitamin D against this nephrotoxicity.

Methods:

Twenty-four adult male albino rats were equally divided into four groups: group I (control group), group II (GM), group III (GM + vitamin D) and group IV (vitamin D only). Kidney function tests, histopathological examination, gene expression of nuclear factor 2, nuclear factor kappa beta (NF-κB) and western blot of NF-κB p65, assessment of glutathione peroxidase and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase by ELISA, as well as immunohistochemical evaluation of inducible nitric oxide, malondialdehyde, 8-hydroxy 2 deoxyguanine and vitamin D receptor, have been carried out.

Results:

The kidney function deterioration, tissue oxidative stress development and the histopathological changes induced by GM were significantly attenuated by vitamin D administration.

Conclusion:

Vitamin D attenuates GM nephrotoxicity through its antioxidant properties and prevention of DNA damage.

Keywords

Gentamicin nephrotoxicity oxidative stress vitamin D

Introduction

Gentamicin (GM) is one of the most important aminoglycoside antibiotics frequently described for the treatment of gram-negative infections.^1,2 The clinical usefulness of GM is limited by the development of nephrotoxicity. Recent studies have tailored a fairly coherent set of ideas about the role of reactive oxygen species (ROS) as crucial mediators of GM-induced nephrotoxicity.^3

–7

GM-induced nephrotoxicity was reported to chiefly affect the renal tubular cells causing direct tubular necrosis¹ accompanied by upregulation of oxidative stress markers⁴ and apoptosis of proximal tubular cells.⁸

Under normal physiological conditions, the excess ROS can be deactivated by the antioxidant enzyme system which detoxifies O^· and H₂O₂ in cells. In oxidative stress conditions, there is a reduction in the antioxidants level due to excessive consumption by the oxygen free radicals. Previously, in vivo and in vitro studies have shown that GM enhances the generation of ROS metabolites, which may cause cellular necrosis via several mechanisms, including lipid peroxidation, protein oxidation and DNA damage. Administration of antioxidants can be used to prevent or ameliorate GM-induced nephrotoxicity.^9,10

One of the agents whose antioxidant properties have recently caught attention is vitamin D. In addition to its role in Ca⁺⁺ homeostasis, vitamin D can participate in many other biological functions and crucial antioxidant pathways.¹¹ The antioxidant activity of vitamin D is achieved via controlling the genes involved in redox balance like glutathione,¹² glutathione peroxidase (GPx)¹³ and NADPH oxidase.¹⁴ Vitamin D can actually act through two different genomic and non-genomic pathways. The non-genomic pathway entails binding of vitamin D to membrane receptors with subsequent enhancement of calcium influx, phospholipase C, adenylate cyclase or mitogen-activated protein.¹⁵ The genomic action occurs via a vitamin D receptor (VDR) which belongs to the nuclear receptors superfamily. VDR becomes activated by 1,25VD3 then dimerizes with the retinoid X receptor.¹⁶ VDR gene expression regulation occurs at different levels and can be affected by environmental, genetic and epigenetic factors.¹⁷

The current study has been designed to provide a cogent structural and molecular assessment of GM-induced nephrotoxicity and to elucidate the potential protective mechanisms of vitamin D on GM-induced renal damage through assessment of different histopathological, molecular and biochemical parameters.

Material and methods

Chemicals

Vitamin D was purchased from Medical Union Pharmaceuticals Company, Egypt. GM was supplied from Memphis Co. for Pharm. and Chem. Ind. (MEMCO), Egypt. Urea and creatinine assay kits were purchased from Diamond Company, Egypt. Polymerase chain reaction (PCR) kits were purchased from Qiagen, Germantown, Maryland, USA. Immunohistochemical kits were purchased from Aboot, USA. NADPH oxidase and GPx ELISA kits were supplied from the Cusabio Company, USA, and NF p65 antibodies for western blot analysis kits were provided from Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA.

Experimental animals

Twenty-four adult Wister male albino rats (180–240 g, 8–10 weeks old) were obtained from the animal house of El-Nahda University, Beni Suef, Egypt. All animals were acclimatized to the laboratory environment for 10 days. Animals were housed in well-ventilated cages with a room temperature of about 25° with 12-h light and 12-h dark cycle and free access to food and water ad libitum was allowed. The experiment was conducted according to the Institutional Animal Care and Use Committee (IACUC).

Experimental design

Animals were randomly divided into four groups, six rats/each group.

Group I: Control group which received normal saline orally with the gastric tube.

Group II: Rats received GM 100 mg/kg body weight by intraperitoneal (IP) injection as a single daily dose for seven successive days.^18,19

Group III: Rats received vitamin D3 1000 IU/kg/day orally by gastric tube for 2 weeks and then combined with gentamycin 100 mg/kg IP for 7 days.^20

–23

Group IV: Rats received vitamin D3 1000 IU/kg/day orally by gastric tube throughout the whole duration of the study.

Samples collection

Samples were obtained from overnight fasted animals through retro-orbital sinus, under ketamine and zylosin anaesthesia into non-heparinized tubes. The collected blood samples were kept for clotting for 30 min at 25°C, followed by centrifugation at 1000 × g, 4°C for 15 min. Sera were collected and stored frozen for determination of urea and creatinine levels. The animals were sacrificed after anaesthesia by cervical dislocation and then the lower abdomen was opened. After the gentle removal of peri-renal fat, the kidneys were weighed using a digital balance to calculate the kidney weight/body weight ratio. The left kidneys were excised immediately, washed in normal saline (0.9% w/v), homogenized in 0.1-M phosphate buffer (pH 7.4) to get 20% w/v tissue homogenates that were then used for biochemical parameters. The right kidneys were used for histopathological and immunohistochemical analyses.

Serum urea and creatinine levels and NADPH oxidase and GPx

Serum urea and creatinine levels were investigated by colorimetric method using commercial Quanti Assay kits according to the manufacturer’s instructions (Bio Diagnostics, Egypt).

The levels of NADPH oxidase and GPx were detected in kidney tissues using ELISA kits (Cusabio, Inc., Houston, Texas, USA) according to the manufacturer’s instructions.

Histological analysis

Haematoxylin and eosin

The kidney tissues of rats were fixed in 10% buffered formalin solution for 24 h then embedded in a paraffin wax. Tissues were sectioned at thickness of 5-μm and stained with haematoxylin and eosin (H&E).

Acute tubular necrosis (ATN) scoring for estimating the percentage of tubules that displayed cell necrosis and cast formation was done as previously described²⁴ (0 = none, 1 = <10%, 2 = 10–25%, 3 = 26–45%, 4 = 46–75% and 5 = >75%). Area percentage of congestion was also carried out. All measurements were estimated under magnification 400 inside the standard measuring frame (85,550 µm²) in 10 non-overlapping random fields in a blinded fashion using the Leica Imaging System (Cambridge, England). Images were captured live on the screen under the light microscope (Olympus Bx-40, Olympus Optical Co. Ltd, Japan), then digitalized using ‘Lecia Qwin 500C’.

Electron microscopic examination

Kidney specimens were immersed in 2.5% glutaraldehyde (pH 7.4). Semithin sections were prepared at the 0.5-µm thickness and stained with 1% toluidine blue in 1% borax. Ultrathin sections were double stained with 4% uranyl acetate and 0.1% lead citrate, examined and photographed by JEOL JEM 1010 transmission electron microscope (Japan).

Immunohistochemical analysis

The deparaffinized sections on slides were used to detect malondialdehyde (MDA), inducible nitric oxide (iNOS), 8-hydroxy 2 deoxyguanine (8-OHDG) and VDR immunohistochemical expression using rabbit polyclonal antibodies (Aboot).

The area and optical density of immunohistochemically positive structures were estimated via the image analyser software program ‘Lecia Qwin 500C’ which is a Leica’s windows-based image analysis tool kit (Leica Imaging System Ltd) fitted to an IBM compatible personal computer (New York, USA) with a colour monitor attached to a Panasonic GP 210 video colour camera (Tokyo, Japan). The Leica Imaging System was first automatically calibrated to convert the measurement units (pixels) produced by the image analyser program into actual micrometre units. Using the measuring field menu, the parameters of interest (the area, area percentage and optical density) were selected. In each chosen field, the tissue was enclosed inside the standard measuring frame measuring (85,550 µm²) and then the brown stained immunohistochemically reactive structures were masked by a blue binary colour to be measured.

Quantitative real time-PCR (qRT-PCR) examination

Total RNA was isolated from kidney tissue homogenate using Qiagen tissue extraction kit (Qiagen) according to the manufacturer’s instructions. The purity and the concentration of RNA were confirmed using spectrophotometry at 260/280 nm ratio (dual wave length Beckman, Spectrophotometer, USA). One microgram of RNA was utilized for cDNA production for reverse transcription-PCR (RT-PCR) using the Super Script III First-Strand Synthesis System as described in the manufacturer’s protocol (Fermentas, Waltham, Massachusetts, USA). Real-time RT-PCR reactions were carried out using TaqMan® gene expression assays for nuclear factor 2 (Nrf2), nuclear factor kappa beta (NF-κB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems). The primer sequences used were as follows: for Nrf2: forward primer 5′-CAC ATT CCC AAA CAA GAT GC-3′, reverse primer 5′-TCT TTT TCC AGC GAG GAG AT-3′; for NF-κB: forward primer 5′-CATTGAGGTGTATTTCACGG-3′, reverse primer: 5′-GGCAAGTGGCCATTGTGTTC-3′; for GADPH: forward primer 5′-AGG TCG GAG TCA ACG GAT TTG GT-3′, reverse primer: 5′-GTT TGC TCT AGA ATG AAC GGT GGA AGG-3′.

In brief, real-time RT-PCR was performed in a 25-μl reaction volume consisting of 2X SYBR Green PCR Master Mix, 900 nM of each primer and 2μl of cDNA. Thermal cycling conditions were used, including a pre-amplification step of 95°C for 10 min, followed by amplification for 40 cycles of 95°C for15 s, 60°C for 1 min, and 72°C for1 min. Relative expression of the studied genes mRNA was calculated using the comparative threshold cycle method. All values were measured as the ratio of the specific genes to GAPDH which was used as an internal control gene and reported as fold change over background levels determined in the different groups where the relative value for the control group was established as one.

Western blot analysis

The methodology has been described previously.²⁵ The antibodies against NF kB p65 (NF p65) (Santa Cruz Biotechnology, Inc.) were used. The levels of NF p65 protein were detected against the house-keeping gene β-actin.

Statistical analysis

Data were summarized using mean and standard error of mean. One-way analysis of variance was used to analyse statistical differences followed by Tukey’s post hoc test for multiple comparisons. Results were expressed as mean ± SEM. p Values< 0.05 and<0.01 were considered statistically and highly statistically significant respectively.

Results

Effect of vitamin D on renal functions

Table 1 shows a significant increase in serum urea and creatinine levels in GM group (79.98 ± 9.78 and 2.60 ± 0.329 mg/dl), respectively, when compared to control group (22.51 ± 1.78 and 0.89 ± 0.041; p < 0.05), and this increase was significantly prevented by vitamin D (23.09 ± 3.81 and 1.17 ± 0.091; p < 0.05).

Table 1.

Serum urea and creatinine among different groups.

	Control	Gentamicin	Vitamin D + gentamicin	Vitamin D
Urea (mg/dl)	22.51 ± 1.78	79.98 ± 9.78^a	23.09 ± 3.81^b	20.82 ± 0.5
Creatinine (mg/dl)	0.89 ± 0.041	2.60 ± 0.329	1.17 ± 0.091	0.81 ± 0.098

^ap < 0.05 Gentamicin group versus control group.

^bp < 0.05 Gentamicin + vitamin D group and vitamin D group versus gentamicin group.

Effect of vitamin D on NADPH oxidase and GPx activities

As shown in the Table 2, NADPH oxidase activity was significantly increased in GM group (687.8 ± 23.3) when compared to control rats (105.2 ± 5.6; p < 0.01), this result was prevented by vitamin D (387.6 ± 19.9; p < 0.01). On the contrary, GPx activity was significantly decreased under the effect of GM administration (83.3 ± 5.2) when compared to control ones (223.7 ± 23.9; p < 0.01), vitamin D avoided this decrease (247.0 ± 22.4; p < 0.01).

Table 2.

NADPH oxidase and GPx level among different groups.

	Control	Gentamicin	Vitamin D + gentamicin	Vitamin D
NADPH oxidase (pg/ml)	105.2 ± 5.60	687.83 ± 23.32^a	387.56 ± 19.8^b	196.33 ± 7.53^b
GPx (mlU/ml)	223.66 ± 23.97	83.33 ± 5.238^a	247.0 ± 22.36^b	397.0 ± 9.84^b

NADPH: Nicotinamide adenine dinucleoide phosphate oxidase; GPx: glutathione peroxidase.

^a p < 0.01 Gentamicin group versus control group.

^bp < 0.01 Gentamicin + vitamin D group and vitamin D group versus gentamicin group.

Histological results

Examination of H&E-stained sections

Kidneys of GM-treated rats displayed pathological glomerular, tubular and vascular abnormalities (Figure 1). The glomeruli appeared lobulated and were surrounded by widened urinary spaces. The renal tubules showed characteristic features of ATN which were displayed in the form of tubular vacuolation, rarefaction and intraluminal exfoliation of the tubular epithelial cells. The vascular changes were displayed in the form of marked thickening of the renal arterioles and obvious dilatation and congestion of renal blood vessels which were frequently surrounded by perivascular exudate. Preservation of glomerular and tubular architectural patterns with prevention of most of these deleterious changes was noticed with vitamin D administration. This was further elucidated by statistically significant improvement in vascular congestion and tubular necrosis scoring (p < 0.05).

Figure 1.

(a) Group I showing normal renal corpuscle composed of a normal glomerulus (G) formed of capillary tufts and surrounded by Bowman’s space (S). The parietal layer of Bowman’s capsule is lined by simple squamous epithelium (arrow heads), PCT (P), DCT (D) and collecting ducts (C). (b) Group II displaying glomerulus (G) with lobulated appearance (L) and widening of the urinary space (S). Renal tubules are seen exhibiting vacuolation (V), rarefaction (R) of their epithelial lining, others displaying obliteration (O) of their lumen and intraluminal exfoliation (Ex) of the tubular cells. (c) Group II exhibiting markedly thickened arteriole with intramuscular vacuolation (arrow head) in its wall. The arteriole is seen surrounded by wide spaces (asterisks) containing loose areolar connective tissue and cellular debris (CD). Dilated tubules lined by flattened epithelium (arrows) are observed with some tubules showing intraluminal casts (thick arrows). Normal sized arterioles are also observed (curved arrows). (d) Group II showing markedly dilated congested blood vessel (arrow) with irregular outline surrounded by areas of exudate (E). (e) Group III showing restoration of the normal architectural pattern of the PCT (P), DCT (D) and CT except for intraluminal epithelial exfoliation (Ex) in some CT. Lobulated glomeruli (G) are also observed. (f) Group IV showing normal glomerulus (G), PCT (P) lined by pyramidal cells with a narrow lumen, DCT (D) lined by cubical cells with a wider lumen and collecting ducts (C). (g) Group IV renal medulla showing normal Loop of Henle (H) and CT lined with simple cuboidal epithelium and exhibiting vesicular rounded nuclei (N) (H&E: ×400). CT: collecting tubules; H&E: haematoxylin and eosin. DCT: Distal convoluted tubules; PCT: Proximal convoluted tubules.

Examination of uranyl acetate and lead citrate-stained ultrathin sections

Ultrastructural examination of the proximal tubular cells of the gentamycin-treated group showed marked degenerative changes which were represented as cytoplasmic vacuolation, mitochondrial dissolution and margination of the nuclear chromatin (Figures 2 and 3). The vitamin D-treated group, on the other hand, displayed very few cytoplasmic vacuoles, preservation of mitochondrial cristae, euchromatic nuclei as well as intact microvilli (Figure 2).

Figure 2.

An electron micrograph of PCT showed disturbed structure in GM group when compared to control one, while pretreatment with vitamin D prevented this disturbance. PCT: Proximal convoluted tubules; GM: gentamicin.

Figure 3.

Electron photomicrograph of the distal convoluted tubule showed disturbed structure in GM group when compared to control one, while pretreatment with vitamin D prevented this disturbance. GM: gentamicin.

Cells lining the distal tubules of GM-treated rats showed swelling, vacuolation and loss of their apical microvilli. Their mitochondria displayed loss of the regular architectural pattern and the nuclei showed marginated heterochromatin and discontinuity of their nuclear envelope. Vitamin D markedly ameliorated these ultrastructural abnormalities where the distal tubular cells appeared healthy with numerous basal infoldings and intact mitochondria.

Immunohistochemical results

Administration of vitamin D caused highly statistically significant reduction of iNOS (Figure 4), MDA (Figure 5) and 8-OHDG (Figure 6) immunohistochemical expression when compared to control group (p < 0.01), while samples from rats received GM showed strong immune reactivity (p < 0.0001). In contrast, VDR showed a statistically significant reduction in its immunoreactivity in the GM-treated groups when compared to control group (p = 0.001). Under the effect of vitamin D, this reactivity showed a strong positive reaction (p = 0.001; Figure 7).

Figure 4.

Photomicrograph of iNOS immunohistochemically stained kidney sections of (a) group I showing mild immunoreactivity, (b) group II displaying strong positive immunoreactivity with high cytoplasmic expression within the renal tubules and the glomeruli, (c) group III showing mild immunoreactivity and (d) group IV showing negative iNOS immunoreactivity. (e) Mean optical density of iNOS (group I vs. II, p < 0.0001; group II vs. III, p < 0.01; group II vs. IV, p < 0.0001). iNOS: inducible nitric oxide.

Figure 5.

Photomicrograph of MDA immunohistochemically stained kidney sections of (a) group I showing very mild immunoreactivity, (b) group II displaying strong positive MDA immunoreactivity within the renal tubules and the glomeruli denoting marked deposition of lipid peroxidation products. (c) group III showing moderate immunoreactivity and (d) group IV showing very mild MDA immunoreactivity. (e) Mean area percent of MDA immunoexpression (p < 0.0001) (MDA immunohistochemical staining: ×400). MDA: malondialdehyde.

Figure 6.

Photomicrograph of 8-OHDG immunohistochemically stained kidney sections of (a) group I showing mild immunoreactivity, (b) group II displaying strong positive 8-OHDG immunoreactivity within the renal tubules and the glomeruli indicating marked oxidative DNA damage, (c) group III showing moderate immunoreactivity and (d) group IV showing very mild 8-OHDG immunoreactivity. (e) Mean area percent of 8-OHDG immunoexpression (p < 0.0001) (8-OHDG immunohistochemical staining: ×400). 8-OHDG: 8-hydroxy 2 deoxyguanine.

Figure 7.

Photomicrograph of VDR immunohistochemically stained kidney sections of (a) group I showing moderate immunoreactivity, (b) group II displaying reduction in immunoreactivity, (c) group III showing restoration of immunoreactivity and (d) group IV showing strong positive immunoreactivity with high expression within the renal tubules and the glomeruli. (e) Mean optical density of VDR (group I vs. II, p < 0.05; group II vs. III p < 0.05; group II vs. IV p = 0.001) (VDR immunohistochemical staining: ×400). VDR: vitamin D receptor.

RT-PCR results

Expression of NF-κB was significantly increased in GM group (11.8 ± 1.1) relative to control group(1.01 ± 0.0068; p < 0,0001) and this result was avoided by vitamin D (5.1 ± 0.8; p < 0,0001), while expression of Nrf2 was significantly decreased in GM group (0.26 ± 0.08) relative to control rats (1.007 ± 0.004; p < 0.001)which was significantly increased by vitamin D (0.8 ± 0.5; p < 0.001; Table 3).

Table 3.

Results of NF-κB and Nrf2 expression among different groups.

	Control	Gentamicin	Gentamicin + Vitamin D	Vitamin D
NF-κB	1.01 ± 0.0068	11.8333 ± 1.1461^a	5.1833 ± 1.6617^b	5.065 ± 0.7445^b
Nrf2	1.0067 ± 0.0049	0.2633 ± 0.0798^c	0.7133 ± 0.0558^d	0.7967 ± 0.494^d

NF-κB: nuclear factor kappa beta; Nrf2: nuclear factor 2.

^ap > 0.0001 in gentamicin group versus control group in NF-κB expression.

^bp < 0.0001 in gentamicin + vitamin D group versus gentamicin group in NF-κB expression.

^cp > 0.001 in gentamicin group versus control group in Nrf2 expression.

^dp < 0.001 in gentamicin + vitamin D group versus gentamicin group in Nrf2 expression.

Western blot result

NF p65 protein expression was found to be upregulated under the effect of GM (3.58 ± 0.07) relative to control rats (1.67 ± 0.046; p < 0.01), vitamin D administration downregulated this protein expression (2.48 ± 0.076; p < 0.01) in comparison to GM group (Figure 8).

Figure 8.

Western blot analysis, nuclear NF p65 expression in GM group showed high significant increase relative to control group (p < 0.01). In GM + vitamin D group, vitamin D significantly downregulated nuclear NFp65 (p < 0.01). GM: gentamicin.

Discussion

GM is a broad-spectrum aminoglycoside antibiotic whose principal drawback is nephrotoxicity. So, GM-induced nephrotoxicity is a good experimental model of drug-induced renal injury. The purpose of this study is to evaluate the protective effect of vitamin D against the experimentally induced renal toxicity with GM in rats.

Concordant with previous literatures,^26,27 the current study demonstrated several histological and ultrastructural changes along with significant deterioration of renal functions upon GM administration. On the other hand, vitamin D prevented most of these functional and histological disturbances. The protective effect of vitamin D against several forms of renal injury and nephrotoxicity has been previously reported.^28
–30

However, Hur et al. reported that vitamin D could not exert a proper protective effect on gentamycin-induced nephrotoxicity. This could be due to the very small dose of vitamin D (0.5 µg) used in that study.³¹ Another possible explanation to those contradictory results could be the fact that the authors used vitamin D concomitant with GM while in our study we used it as prophylaxis providing it as a protective agent for prevention of nephrotoxicity.

On the other hand, some studies have reported a nephrotoxic effect of vitamin D. Wang et al. documented that vitamin D caused nephrotoxicity in refractory rickets patients.³² This finding may be considered as a special state for refractory rickets due to metabolic disturbance results in the development of rickets despite normal vitamin D concentration, while another study recorded the nephrotoxic effect of vitamin D without any explanation for the molecular mechanism of this toxicity.³³

Many studies have proved the important role of vitamin D as a potent antioxidant.³⁴ In our study, as documented by previous studies,²⁶ GM-treated rats showed significant increase in NADPH oxidase level and decrease in GPx level when compared to control rats and these findings were avoided by vitamin D. On the other hand, rats received GM showed strong positive immunoreactivity of iNOS and MDA as compared to control group. Administration of vitamin D resulted in formation of mild reaction. It is well-known that MDA and iNOS are biomarkers for excessive ROS and free radicals formation. Activation of iNOS has been reported to aggravate the renal injury in GM-induced nephrotoxicity.³⁵ Previous studies have also pointed out the role of vitamin D in the regulation of iNOS expression.³⁶

In the present study, the gentamycin-induced upregulation of 8-OHDG expression was ameliorated by vitamin D administration; 8-OHDG is an important biomarker of DNA damage accompanying oxidative stress consequences^11,37 and it was found that even small dose of vitamin D (800 IU/day) considerably helps to reduce of 8-OHDG.³⁸ On the other hand, rats received GM showed a significant decrease in immunoreactivity of VDR while administration of vitamin D showed positive immune reactivity for the receptor. The antioxidant effect of vitamin D depends mainly on VDR activity.^39,40 So, decreased VDR level may aggravate the renal damage induced by GM.

In the current study, we confirmed the modulatory effect of vitamin D on the gene expression of NF-κB and Nrf2. Also, vitamin D was found to downregulate NF p65 as observed by western blot analysis. NF-κB is an important biomarker for oxidative stress and inflammation. Previous studies reported that the reno-protective function of vitamin D is related to its regulation of NF-κB expression.^{41, 42}

An important negative regulator for NF-κB activation is the inhibitor of NF-κB (I κB)⁴³ which binds to p65 subunit in the cytosol causing blockage of the nuclear translocation of the p65/p50 heterodimer. An activated IκB kinase (IκK) causes phosphorylation of IκB leading to degradation of IκB and consequently upregulation of NF-κB.⁴⁴ Vitamin D was found to prevent degradation of IkB with inhibition of nuclear translocation of p65/p50 resulting in inhibition of NF-κB.⁴⁴ In addition, VDR knockout or VDR loss was found to be associated with high expression of 8-OHDG and NF-κB.^45,46

Nrf2 has a powerful cytoprotective effect through binding to the antioxidant response element (ARE), responsible for regulation of the expression of many cytoprotective genes concerned with the cellular antioxidant and anti-inflammatory actions.⁴⁷ The Nrf2/ARE signalling pathway contributes to regulation of about 200 genes involved in the cellular antioxidant and anti-inflammatory defence systems,^47,48 as catalase, superoxide dismutase, haeme oxygenase-1, GPx-2 and glutathione S-transferase.^49,50 Vitamin D was found to be involved in the regulation of Nrf2 expression.⁵¹ It is also noteworthy that Nrf2 can regulate the genes important for vitamin D expression which acts as a positive feed back loop.⁵²

Conclusion

In this study, we detected the protective effect of vitamin D against GM-induced acute renal damage as rats received vitamin D showed significant decrease of urea and creatinine levels which is found to be elevated under the effect of GM. The protective effect of vitamin D is through antioxidant effect as vitamin D increased the expression of Nrf2 and inhibited the expression of NF-κB. Vitamin D also prevents DNA damage related to oxidative stress.

Vitamin D has demonstrated protection against GM-induced renal damage. The mechanism of this protection is through the anti-oxidant effect of vitamin D via activation of Nrf2 and inhibition of NF-κB expressions. Vitamin D can also prevent oxidative stress-induced DNA damage through reduction of 8- OHDG expression.

Footnotes

Acknowledgements

The authors would like to thank Dr Waffaa lecturer of pharmacology and toxicology, Faculty of Pharmacy, Beni Suef University, for her help.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

MA Mohammed

References

Karahan

Ateşşahin

Yılmaz

, et al. Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicology 2005; 215(3): 198–204.

Mwengee

Butler

Mgema

, et al. Treatment of plague with gentamicin or doxycycline in a randomized clinical trial in Tanzania. Clin Infect Dis 2006; 42(5): 614–621.

Christo

Rodrigues

Mouro

, et al. Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats. Nitric Oxide 2011; 24(2): 77–83.

Banday

Farooq

Priyamvada

, et al. Time dependent effects of gentamicin on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in rat kidney tissues. Life Sci 2008; 82(9): 450–459.

Polat

Parlakpinar

Tasdemir

, et al. Protective role of aminoguanidine on gentamicin-induced acute renal failure in rats. Acta Histochem 2006; 108: 365–371.

Ali

. Agents ameliorating or augmenting experimental gentamicin nephrotoxicity: some recent research. Food Chem Toxicol 2003; 41: 1447–1452.

Ozbek

Cekmen

Ilbey

, et al. Atorvastatin prevents gentamicin-induced renal damage in rats through the inhibition of p38-MAPK and NF-kappaB pathways. Ren Fail 2009; 31(5): 382–392.

Quiros

Vicente-Vicente

Morales

, et al. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicol Sci 2010; 119(2): 245–256.

Yanagida

Ito

Komiya

, et al. Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue. Chem Biol Interact 2004; 148: 139–147.

10.

Stojiljkovic

Stoiljkovic

Randjelovic

, et al. Cytoprotective effect of vitamin C against gentamicin-induced acute kidney injury in rats. Exp Toxicol Pathol 2012; 64(1): 69–74.

11.

Bao

Ting

Hsu

, et al. Protective role of 1α, 25-dihydroxyvitamin D₃ against oxidative stress in nonmalignant human prostate epithelial cells. Int J Cancer 2008; 122(12): 2699–2706.

12.

Jain

Micinski

. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem Bioph Res Co 2013; 437(1): 7–11.

13.

Peehl

Shinghal

Nonn

, et al. Molecular activity of 1, 25-dihydroxyvitamin D₃ in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J Steroid Biochem 2004; 92(3): 131–141.

14.

Husain

Ferder

Mizobuchi

, et al. Combination therapy with paricalcitol and enalapril ameliorates cardiac oxidative injury in uremic rats. Am J Nephrol 2009; 29(5): 465–472.

15.

Lehmann

Meurer

. Vitamin D metabolism. Dermatol Ther 2010; 23(1): 2–12.

16.

Berridge

. Vitamin D cell signalling in health and disease. Biochem Bioph Res Co 2015; 460(1): 53–71.

17.

Saccone

Asani

Bornman

. Regulation of the vitamin D receptor gene by environment, genetics and epigenetics. Gene 2015; 561(2): 171–180.

18.

El-Kashef

El-Kenawi

Suddek

, et al. Protective effect of allicin against gentamicin-induced nephrotoxicity in rats. Int Immunopharmacol 2015; 29(2): 679–686.

19.

Abdelsameea

Mohamed

Amer

, et al. Cilostazol attenuates gentamicin-induced nephrotoxicity in rats. Exp Toxicol Pathol 2016; 68(4): 247–253.

20.

Alfawaz

Bhat

Al-Ayadhi

, et al. Protective and restorative potency of vitamin D on persistent biochemical autistic features induced in propionic acid-intoxicated rat pups. BMC Complement Altern Med 2014; 14(1): 416.

21.

Machado Cda

Venancio

Aissa

, et al. Vitamin D3 deficiency increases DNA damage and the oxidative burst of neutrophils in a hypertensive rat model. Mutat Res Genet Toxicol Environ Mutagen 2016; 798: 19–26.

22.

Abdelghany

BaSalamah

Idris

, et al. The fibrolytic potentials of vitamin D and thymoquinone remedial therapies: insights from liver fibrosis established by CCl4 in rats. J Transl Med 2016; 14(1): 281.

23.

Farhangi

Nameni

Hajiluian

, et al. Cardiac tissue oxidative stress and inflammation after vitamin D administrations in high fat-diet induced obese rats. BMC Cardiovasc Disord 2017; 17(1): 161.

24.

Kim

Ravichandran

Ozkok

, et al. The water-soluble triptolide derivative PG490-88 protects against cisplatin-induced acute kidney injury. J Pharmacol Exp Ther 2014; 349(3): 518–525.

25.

Tan

Pan

, et al. Hydrogen sulfide attenuates carbon tetrachloride-induced hepatotoxicity, liver cirrhosis and portal hypertension in rats. PLoS One 2011; 6(10): e25943.

26.

Rashid

Khan

. Fagoniaolivieri prevented hepatorenal injuries induced with gentamicin in rat. Biomed Pharm 2017; 88: 469–479.

27.

Katary

Salahuddin

. Ameliorative effect of gossypin against gentamicin-induced nephrotoxicity in rats. Life Sci 2017; 176: 75–81.

28.

Elmubarak

Özsoy

. Histoprotective effect of vitamin D against carbon tetrachloride nephrotoxicity in rats. Hum Exp Toxicol 2016; 35(7): 713–723.

29.

Deng

Cheng

Shen

. Vitamin D improves diabetic nephropathy in rats by inhibiting renin and relieving oxidative stress. J Endocrinol Invest 2016; 39(6): 657–666.

30.

BaSalamah

Abdelghany

El-Boshy

, et al. Vitamin D alleviates lead induced renal and testicular injuries by immunomodulatory and antioxidant mechanisms in rats. Sci Rep 2018; 8(1): 4853.

31.

Hur

Garip

Camyar

, et al. The effects of vitamin D on gentamicin-induced acute kidney injury in experimental rat model. Int J Endocrinol 2013; 2013: 313528.

32.

Wang

Kwon

, et al. Reduced expression of Na-K-2Cl cotransporter in medullary TAL in vitamin D-induced hypercalcemia in rats. Am J Physiol Renal 2002; 282(1): F34–F44.

33.

Moncrieff

Chance

. Nephrotoxic effect of vitamin D therapy in vitamin D refractory rickets. Arch Dis Child 1969; 44(237): 571.

34.

Tarcin

Yavuz

Ozben

, et al. Effect of vitamin D deficiency and replacement on endothelial function in asymptomatic subjects. J Clin Endocr Metab 2009; 94(10): 4023–4030.

35.

Ghaznavi

Kadkhodaee

. Comparative effects of selective and non-selective nitric oxide synthase inhibition in gentamicin-induced rat nephrotoxicity. Arch Toxicol 2007; 81(6): 453–457.

36.

Dursun

Gezen-Ak

Yilmazer

. The influence of Vitamin D treatment on the inducible nitric oxide synthase (INOS) expression in primary hippocampal neurons. Nöro Psikiyatr Arş 2014; 51(2): 163.

37.

Aydin

Uzun

Sozer

, et al. Effects of atorvastatin therapy on protein oxidation and oxidative DNA damage in hypercholesterolemic rabbits. Pharmacol Res 2009; 59(4): 242–247.

38.

Fedirko

Bostick

Long

, et al. Effects of supplemental vitamin D and calcium on oxidative DNA damage marker in normal colorectal mucosa: a randomized clinical trial. Cancer Epidemiol Biomarkers Prev 2010; 19(1): 280–291.

39.

Uberti

Lattuada

Morsanuto

, et al. Vitamin D protects human endothelial cells from oxidative stress through the autophagic and survival pathways. J Clin Endocr Metab 2013; 99(4): 1367–1374.

40.

Shab-Bidar

Neyestani

Djazayery

. The interactive effect of improvement of vitamin D status and VDR FokI variants on oxidative stress in type 2 diabetic subjects: a randomized controlled trial. Eur J Clin Nutr 2015; 69(2): 216–222.

41.

Morcos

Sayed

Bierhaus

, et al. Activation of tubular epithelial cells in diabetic nephropathy. Diabetes 2002; 51(12): 3532–3544.

42.

Tan

Wen

Liu

. Paricalcitol inhibits renal inflammation by promoting vitamin D receptor-mediated sequestration of NF-κB signaling. J Am Soc Nephrol 2008; 19(9): 1741–1752.

43.

Bonizzi

Karin

. The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004; 25(6): 280–288.

44.

Zhang

Yuan

Sun

, et al. 1,25-Dihydroxyvitamin D3 targeting of NF-κB suppresses high glucose-induced MCP-1 expression in mesangial cells. Kidney Int 2007; 72(2): 193–201.

45.

Kallay

Pietschmann

Toyokuni

, et al. Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage. Carcinogenesis 2001; 22(9): 1429–1435.

46.

Sun

Kong

Duan

, et al. Increased NF-κB activity in fibroblasts lacking the vitamin D receptor. Am J Physiol Endoc M 2006; 291(2): E315–E322.

47.

Lee

Johnson

. An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol 2004; 37(2): 139–143.

48.

Vargas

Johnson

Sirkis

, et al. Nrf2 activation in astrocytes protects against neurodegeneration in mouse models of familial amyotrophic lateral sclerosis. J Neurosci 2008; 28(50): 13574–13581.

49.

Kobayashi

Yamamoto

. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul 2006; 46(1): 113–140.

50.

Kensler

Wakabayashi

Biswal

. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007; 47: 89–116.

51.

Nakai

Fujii

Kono

, et al. Vitamin D activates the Nrf2-Keap1 antioxidant pathway and ameliorates nephropathy in diabetic rats. Am J Hypertens 2013; 27(4): 586–595.

52.

Bobilev

Novik

Levi

, et al. The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells. Cancer Biol Ther 2011; 11(3): 317–329.