Roflumilast,a phosphodiesterase 4 inhibitor,attenuates cadmium-induced renal toxicity via modulation of NF-κB activation and induction of NQO1 in rats

Abstract

Objective:

In the present study, the protective effect of Roflumilast (ROF, a selective phosphodiesterase (PDE-4) inhibitor) was investigated against cadmium (Cd)-induced nephrotoxicity in rats.

Methods:

A total of 24 rats were selected and randomly divided into four groups (n = 6). Group 1 served as the control; groups 2–4 administered with CdCl₂ (3 mg/kg, i.p.) for 7 days; groups 3 and 4 were co-administered with ROF in doses of 0.5 and 1.5 mg/kg, orally for 7 consecutive days. Nephrotoxicity was evaluated by measuring urine volume, urea and creatinine levels in urine and serum. Oxidative stress was confirmed by measuring malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) levels in kidney tissue followed by histopathological studies.

Results:

CdCl₂ administration results in a significant (p < 0.01) decrease in urine volume, urea, and creatinine levels in urine, as well as GSH, SOD, and CAT levels in renal tissue. In addition, Cd also produced significantly increased (p < 0.01) urea and creatinine levels in serum and TBARS levels in renal tissues. Rats treated with ROF significantly (p < 0.01) restore the altered levels of kidney injury markers, nonenzymatic antioxidant, as well as depleted enzymes in dose-dependent manner. An increased expression of NF-κB p65 and decreased expression of GST and NQO1 in the Cd only treated group were significantly reversed by high dose of ROF (1.5 mg/kg). Histopathological changes were also ameliorated by ROF administration in Cd-treated groups.

Conclusion:

In conclusion, ROF treatment showed protective effect against renal damage and increased oxidative stress induced by Cd administration.

Keywords

Roflumilast cadmium nephrotoxicity superoxide dismutase NQO1 NF-κB

Introduction

Cadmium (Cd) is one of the most dangerous toxic chemicals in the environment because of its increasing level day by day as a result of agricultural and growing industries all around the world.^1,2 Among the most important sources of human Cd exposure, cigarette smoking is one of them and causes serious toxic effects. Accumulation of Cd in organs has been reported to be one of causative factor to several disorders such as myocardial infarction, renal dysfunction, neuro-degeneration, Parkinson’s disease, Alzheimer’s disease, lung dysfunction, and carcinogenesis.³

The kidney in our body is one of the vital organs to perform a number of important functions including homeostasis maintenance, regulation of the environment outside the cell, such as removal and excretion of toxic metabolites, nitrogenous wastes and drugs from the body in the form of urine.⁴ Because kidney has to deal with the toxicants and toxic drug metabolites, it can be descried as a chief target for the toxicants exposed by exogenous sources. The kidney is the principal organ targeted by Cd on chronic exposure. Other experts believe that the renal tubular dysfunction associated with Cd-toxicity is irreversible.⁵ Nephrotoxic injury suddenly impairs normal kidney function and excretion of the toxic chemicals and/or drugs as a result, which may cause acute renal failure. Furthermore, nephrotoxicity may also induce chronic renal failure, and if unchecked, renal failure can result in death.^6,7

The most possible mechanism of renal injury is release of intracellular Cd which results in production of oxygen free radicals (OFRs), decrease of glutathione content, lipid peroxidation, DNA damage, and finally resulting in oxidative stress–related cell death.⁸ OFR plays a very important role in cellular signaling, disturbing almost all features of cellular physiology including gene expression, propagation, immigration, and apoptosis.⁹ Among the various key factors, tissues GSH depletion and cellular damaging effects of OFR are the key factors that lead to lipid peroxidation.^10,11

Therefore, in the pathogenesis of kidney damage, increased level of apoptosis may underline the main cause. Moreover, oxidative stress and inflammation are closely linked to each other and are distinctive features of kidney problems.¹² Nuclear factor-κB (NF-κB) regulates many genes involved in inflammatory reactions and its activity is inducible in all cell types.¹³ NF-κB is a major factor in pathological conditions of the kidney and can be stimulated by Cd. In previous studies, it has been reported that the inhibition of NF-κB function may be a useful mechanism for anti-inflammatory effects and agents can be used as anti-inflammatory drugs.¹⁴

NADH:quinone oxido-reductase 1 (NQO1), a cytosolic antioxidant flavo-protein, catalyzes the reduction of quinones to hydroquinones by utilizing NADH as an electron donor, which subsequently results in increased intracellular NADP levels.^15,16 In addition, it has also been reported in previous studies that NQO1 has an important role in other biological activities also, including anti-inflammatory processes, the scavenging of superoxide anion radicals, and the stabilization of p53 and other tumor-suppressor proteins.^17

–20

Thus, reducing oxidative stress may be a potential protective mechanism for renal damage and disease induced by Cd. Therefore, in recent research, scientists are focusing on the protective mechanisms against toxins and drug-induced organ toxicities and also looking for biologically active relevant compounds, which can inhibit oxidative stress and inflammatory processes and protect vital organs from unwanted oxidative stress and inflammation induced by Cd exposure.^21,22

From previous literature, it is clear that phosphodiesterase type 4 (PDE4) inhibition results in increased levels of intracellular cAMP, which involves the activation of protein kinase A, and activation of cAMP responsive element binding protein (CREB/ATF-1) family of transcription factors, as well as downregulation of NF-κB transcriptional activity.²³

Roflumilast (ROF), a PDE4 enzyme inhibitor, acts by inhibiting a key enzyme for the degradation of cAMP and thus results in increased cellular cAMP levels, and cAMP inhibits microvascular leakage, trafficking, and the release of cytokines and chemokines from inflammatory cells.²⁴ cAMP and cGMP exhibit many intracellular effects, mediated mainly through stimulation of protein kinases.²⁵

As far as we know, there is no study concerning the effect of ROF against Cd-induced nephrotoxicity. We hypothesized that ROF could suppress Cd-induced nephrotoxicity. Therefore, the present study was designed to investigate the protective effect of ROF against Cd-induced damage in rat’s kidney.

Materials and methods

Chemicals and reagents

The chloride salt of Cd was purchased from Sigma Chemical Co. (St. Luis, Missouri, USA). Primary and secondary antibodies used for Western blotting were obtained from Santa Cruz (Dallas, Texas, USA). Nitrocellulose membrane was purchased from Bio-Rad Laboratories (Hercules, California, USA). Chemiluminescence Western blot detection kits were obtained from GE Healthcare Life Sciences (Piscataway, New Jersey, USA). All other chemicals and reagents used were of analytical grade.

Animals

Twenty-four healthy male Wistar albino rats (200–250 g body weight, 6–8 weeks old) were used in this present study. Animals were procured from Animal care unit, College of Pharmacy, Prince Sattam Bin Abdulaziz University (PSAU), KSA. During acclimatization (7 days), animals were provided standard laboratory conditions, with a standard pellet diet and water ad libitum, under a 12-h light/12-h dark cycle. All experiments were carried out following the guidelines of the animal care at PSAU, KSA. The study was approved by the Ethical Review Committee, College of Pharmacy, Prince Sattam Bin Abdulaziz University, KSA (approval ref no. HAP-01-KJ-050).

Experimental design

Adult male Wistar albino rats were randomly divided into four groups (n = 6): Group 1 (control) receive normal saline only for 7 days, group 2 (CdCl₂ only) serve as toxic control and receive CdCl₂ (3 mg/kg, i.p.) daily for 7 days. Groups 3 and 4 (ROF-treated) were co-administered with CdCl₂ (3 mg/kg, i.p.) and ROF in two doses of 0.5 and 1.5 mg/kg, respectively, once a day orally by gavage, for 7 consecutive days.

After the last dose of treatment, rats were housed in metabolic cages for 24 h to collect urine samples for the assessment of urine volume, urea, and creatinine levels. After 24 h of intoxication with CdCl₂, blood samples were collected from retro orbital plexus of all the animals under light ether anesthesia and serum was separated and stored at −20 °C until analysis for biochemical estimations (urea and creatinine levels). After blood collection, animals were killed under light ether anesthesia and bilateral nephrectomy was done. The right kidney was placed in 10% formalin solution for histopathological examination and the left kidney stored at −80 °C until the analysis of biochemical parameters in kidney tissue (malondialdehyde (MDA), Superoxide dismutase (SOD), catalase (CAT) and Glutathione (GSH)) levels and Western blot analysis.

Urine and serum sample analysis

Functions of kidney were assessed by measuring the urine volume and levels of urea²⁶ and creatinine²⁷ in urine and serum. The levels of urea and creatinine were estimated by using respective diagnostic kits and expressed in milligrams per deciliter.

Biochemical estimations in kidney

One kidney from each animal was immediately removed and washed using saline solution. Tissues were minced and homogenized (10% w/v) using a homogenizer in ice-cold 0.1 M phosphate buffer (pH 7.4) and centrifuged at 12,000 r min⁻¹ for 30 min at 4 °C. The homogenate was used for the estimation of MDA,²⁸ GSH,²⁹ SOD,³⁰ and CAT.³¹

Western blot analysis

Kidney was washed in ice cold PBS, cut into small pieces, and homogenized separately in cold protein lysis buffer and protease inhibitor cocktail.³² Total cellular proteins were obtained by incubating the cell lysates on ice for 1 h, with intermittent vortex mixing every 10 min, followed by centrifugation at 12,000 × g for 10 min at 4 °C. Total protein was measured by the Lowry method.³³ Western blot analysis was performed using a previously described method.³² Briefly, 25–50 µg of protein from each group was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to nitrocellulose membranes (Bio-Rad). Protein blots were blocked overnight at 4 °C, followed by incubation with primary antibodies against GST, NF-κB p65 and NQO1 (Santa Cruz) and peroxidase-conjugated secondary antibodies at room temperature. Bands were visualized using the enhanced chemiluminescence method (GE Health Care, Mississauga, Canada) and quantified relative to β-actin bands using the ImageJ® image processing program (National Institutes of Health, Bethesda, Maryland, USA). Images were taken using a C-Digit chemiluminescent Western blot scanner (LI-COR, Lincoln, Nebraska, USA).

Histopathological studies

Another kidney preserved in 10% buffered formalin was processed for histopathology. The kidney tissues were dehydrated, embedded on paraffin block, and cut in to sections of about 5 µm thickness followed by staining with hematoxylin and eosin. Then, the samples were observed under microscope by an experienced histopathologist.

Statistical analysis

All values were expressed as mean ± standard error mean (n = 6). One-way analysis of variance was applied to test for the significance of biochemical data of the different groups followed by Dunnett’s test. Significance is set at p < 0.01.

Results

Effect of ROF on kidney function markers

Results of kidney function markers in urine and serum are depicted in Tables 1 and 2, respectively. Intraperitoneal administration of Cd resulted in significantly (p < 0.01) decreased levels of urine volume, urea, and creatinine levels in urine (Table 1) as well as elevated levels of urea and creatinine in serum (Table 2). Treatment of Cd-induced nephrotoxic rats with ROF (0.5 and 1.5 mg/kg) significantly and dose dependently prevented the altered levels of urine volume, urea, and creatinine. Therefore, treatment with ROF significantly and dose-dependently ameliorated the Cd-induced renal injuries.

Table 1.

Effect of ROF on kidney function markers in urine.^a

Groups	Urine volume (ml)	Urinary urea (mg/dl)	Urinary creatinine (mg/dl)
Control	9.22 ± 0.68	4.15 ± 0.38	46.22 ± 2.11
CdCl₂ (3 mg/kg)	2.90 ± 0.25^b	1.13 ± 0.09^b	14.15 ± 0.49^b
ROF (0.5 mg/kg) + CdCl₂	6.03 ± 0.29^c	2.83 ± 0.16^c	28.04 ± 1.57^c
ROF (1.5 mg/kg) + CdCl₂	7.23 ± 0.35^c	3.02 ± 0.18^c	33.57 ± 1.09^c

ROF: Roflumilast; SEM: standard error mean; Cd: cadmium.

^aData are presented as mean ± SEM (n = 6).

^bSignificant differences compared with the control group (p < 0.01).

^cSignificant differences compared with the Cd-intoxicated group (p < 0.01).

Table 2.

Effect of ROF on kidney function markers in serum.^a

Groups	Serum urea (mg/dl)	Serum creatinine (mg/dl)
Control	1.06 ± 0.07	2.56 ± 0.12
CdCl₂ (3 mg/kg)	4.59 ± 0.23^b	6.58 ± 0.31^b
ROF (0.5 mg/kg) + CdCl₂	3.28 ± 0.14^c	4.92 ± 0.13^c
ROF (1.5 mg/kg) + CdCl₂	2.85 ± 0.15^c	4.06 ± 0.13^c

ROF: Roflumilast; SEM: standard error mean; Cd: cadmium.

^aData are presented as mean ± SEM (n = 6).

^bSignificant differences compared with the control group (p < 0.01).

^cSignificant differences compared with the Cd-intoxicated group (p < 0.01).

Effect of ROF on antioxidant enzymes and oxidative stress markers

The results of antioxidant enzymes and oxidative stress markers are shown in Table 3. Intraperitoneal administration of Cd enhances the level of lipid peroxidation in terms of MDA as compared to control (0.49 ± 0.02 to 3.73 ± 0.13 nmol/mg). The ROF treatment in doses of 0.5 and 1.5 mg/kg dose dependently decreases the lipid peroxidation in renal tissues of Cd-administered rats (from 3.73 ± 0.13 to 2.55 ± 0.12 and 1.97 ± 0.08 nmol/mg, respectively). In addition, Cd administration resulted in reduced renal tissue antioxidant systems such as SOD (from 2.75 ± 0.21to 0.66 ± 0.04 U/mg protein), CAT (from 52.83 ± 4.87 to 23.50 ± 2.39 U/mg protein), and GSH (from 77.67 ± 3.64 to 31.50 ± 2.51) as compared to the control group. Treatment of Cd-induced nephrotoxic rats with ROF (0.5 and 1.5 mg/kg) significantly replenished the depleted levels, in a dose-dependent manner, of SOD (from 0.66 ± 0.04 to 1.56 ± 0.07 and 1.99 ± 0.09 U/mg protein, respectively), CAT (from 23.50 ± 2.39 to 41.67 ± 3.25 and 45.83 ± 4.09 U/mg protein, respectively), and GSH (from 31.50 ± 2.51to 52.33 ± 3.32 and 69.17 ± 1.92, nmole/mg protein, respectively).

Table 3.

Effect of ROF on antioxidant enzymes and oxidative stress markers.^a

Groups	MDA (nmole/mg protein)	GSH (nmole/mg protein)	SOD (units/mg protein)	CAT (units/mg protein)
Control	0.49 ± 0.02	77.67 ± 3.64	2.75 ± 0.21	52.83 ± 4.87
CdCl₂ (3 mg/kg)	3.73 ± 0.13^b	31.50 ± 2.51^b	0.66 ± 0.04^b	23.50 ± 2.39^b
ROF (0.5 mg/kg) + CdCl₂	2.55 ± 0.12^c	52.33 ± 3.32^c	1.56 ± 0.07^c	41.67 ± 3.25^c
ROF (1.5 mg/kg) + CdCl₂	1.97 ± 0.08^c	69.17 ± 1.92^c	1.99 ± 0.09^c	45.83 ± 4.09^c

ROF: Roflumilast; SEM: standard error mean; Cd: cadmium.

^aData are presented as mean ± SEM (n = 6).

^bSignificant differences compared with the control group (p < 0.01).

^cSignificant differences compared with the CD-intoxicated group (p < 0.01).

Effect of ROF on NF-κB p65, GST, and NQO1 protein expression

On the basis of results of kidney function markers and oxidative markers, we studied the effects of ROF with only high dose (1.5 mg/kg) on protein expression of NF-κB p65, GST, and NQO1. Activation of NF-κB plays a key role in the regulation of inflammatory mediator production. In the present study, Western blotting was performed to investigate the activation of NF-κB p65. Western blotting analyses of lysates generated from kidney tissue revealed that the Cd-treated group had significantly increased NF-κB p65, protein expression as compared to the control group. In the ROF-treated groups (1.5 mg/kg), the expression of NF-κB p65 was significantly suppressed compared to the Cd-injected rats (Figure 1(a)). Furthermore, Cd-injected rats showed reduced GST (Figure 1(b)) and NQO1 (Figure 1(c)) protein expression levels as compared to the control group, whereas it was significantly found to be higher in ROF-treated rats as compared to Cd-treated rats.

Figure 1.

Effect of ROF on NF-κB p65, GST, and NQO1 protein expression in kidney tissues of Cd-intoxicated rats. Immunoblot analysis of (a) NF-κB p65, (b) GST, and (c) NQO1. Results are mean ± SEM (n = 6). ^aSignificant differences compared with the control group (p < 0.05); ^bSignificant differences compared with the Cd-intoxicated group (p < 0.05). ROF: Roflumilast; NF-κB: nuclear factor-κB; Cd: cadmium; SEM: standard error mean.

Effect of ROF on histopathology of Cd-induced kidney damage

Histopathological examination of the control rats showed normal kidney convoluted tubules (Figure 2(a)), glomeruli, and Bowman capsules with normal space (Figure 2(b)), while photomicrographs of Cd-treated kidney tissues indicated abnormal glomeruli (G), degenerated and necrotic convoluted tubules (N), degenerated and almost absence of bowman space (Figure 2(c)), abnormal convoluted tubules showed histological disruption, congestion (C), and necrosis (Figure 2(d)). Treatment with ROF at low dose reinstated the normal glomeruli and normal convoluted tubules except slightly necrosis and hyperemia (Figure 2(e)) and at high dose showed normal tubules of convoluted and calyx area with slightly necrosis and hyperemia (Figure 2(f)). Overall, our results demonstrate that ROF has potential as a protective agent against Cd-induced nephrotoxicity (Figure 2).

Figure 2.

Effect of ROF on histopathological features of Cd-intoxicated kidney tissues (H&E 40×). Photomicrographs of (a and b) normal control kidney tissues (c and d) Cd-intoxicated kidney tissues, (e) ROF-treated kidney tissues at low dose (0.5 mg/kg), and (f) ROF-treated kidney tissues at high dose (1.5 mg/kg). ROF: Roflumilast; Cd: cadmium.

Discussion

Several studies reported and explained the toxic and dangerous effects of the Cd on human health because of its extensive availability in the atmosphere (cigarette smoke, industrial wastes), water (from pipeline and batteries), and many plants.³⁴ Its accumulation in different human body organs results in production of oxidative stress, which weakens natural antioxidant defense system and as an end result contributes to the development of various serious pathological diseases.³⁵ The mechanism of the Cd-induced injury includes the generation of reactive oxygen species (ROS) that change the mitochondrial activity and genetic information, subsequently causes kidney injury attributed to oxidative stress.³⁶ Therefore, on the basis of previous literature, it can be assumed that antioxidants may be one of the important contributory components for the effective treatment of Cd-induced poisoning.³⁷ Hence, the present study was designed to investigate the possible protective effect of ROF on Cd-induced oxidative stress and nephrotoxicity in Wistar albino rats.

Cd-intoxicated renal injury could be evaluated by assessing biochemical markers of kidney damage in serum and urine. Severe renal damage caused by administration of Cd could be associated with marked increase in the activity of urea and creatinine in serum, because of their leakage into the blood circulation, which confirmed the kidney toxicity.³⁸ In this present work also, the creatinine and urea level was found to be significantly increased in serum after administration of Cd, which is clearly shown in Table 1, indicating the impairment in the renal functions. These results are in agreement with previous studies.^39
–41 Among the acute renal function marker, urea is the first one which increases with any type of kidney injuries. Further, creatinine is the most trustable renal marker and increases only when most of the renal function is lost.⁴² In the present study, increased urea and creatinine levels confirmed the severe nephrotoxic effect of Cd. ROF treatment at both doses significantly reversed the Cd-induced renal damage, which was confirmed by the decreased levels of creatinine and urea in serum. Our data also showed a significant decrease in urine volume, urinary urea, and urinary creatinine in Cd-treated rats, and these results were reversed by ROF at both doses in dose-dependent manner.

The diminished antioxidant defense system as a result of Cd-induced renal damage is considered as a critical result. Chronic Cd exposure is characterized by the reduction of nonenzymatic (tissue GSH) as well as circulating enzymatic (SOD, CAT, and GST) antioxidants.⁴³ In the present study, the extent of lipid peroxidation was estimated by the estimation of MDA, and its level was found to be significantly increased in renal tissues of Cd-intoxicated rats, thus, signifying increased oxidative stress. These results are in agreement with Kawamoto et al. who reported that an early and sensitive exposure of Cd results in lipid peroxidation, which is considered as the main cause of its harmful effect on membrane-dependent function.⁴⁴ Our results are also in the agreement with study of Koyuturk et al. who reported a decreased GSH level in the kidney tissues of Cd-treated rats compared to the normal control group.⁴⁵ The causes of decrease GSH contents may be its consumption in the prevention of lipid peroxidation caused by free radical generation⁴⁵ and detoxification of heavy metals.^46,47 Our findings are in conflict with the results of Kamiyama et al. who reported an increased GSH concentration in kidney tissues after the Cd exposure.⁴⁸

In the present study, our findings showed that the ROF at both doses results in a significant increase in the GSH level and decrease in the MDA level. These results coincide with the study of Sayed-Ahmed and Nagi who reported protective effects of thymoquinone (TQ) against gentamycin-induced nephrotoxicity and showed a significant increase in GSH levels and decrease in blood urea nitrogen and creatinine.⁴⁹

SOD and CAT found to be a supportive team of natural defense system against ROS. In the present investigation, Cd-induced oxidative stress in rats also results in decreased activities of SOD and CAT significantly, which corroborates with the findings of Renugadevi and Prabu.⁵⁰ The results of present study showed that ROF treatment with Cd exposure produce potential defensive effect as it successfully reduced the oxidative stresses and reverted CAT and SOD activities to nearly normal level in kidney tissues. The oral administration of ROF in Cd-treated rats caused some significant improvement in Cd-induced antioxidant defense also by normalizing the antioxidant activity of the enzymes (CAT and SOD), total GSH, and lipid peroxidation in renal tissues.

In previous studies, it has been reported that NF-kB activation stimulates apoptosis induced by Cd in the renal tubules of rats.^51,52 In the present study with Western blot analysis, an increased NF-kBp65 protein expression and decreased expression of GST and NQO1 were observed in rats with Cd exposure, which is in agreement with previous studies. ROF treatment in Cd-intoxicated rats significantly reversed the NF-kBp65 protein expression. These results corroborated with previous studies on antioxidants that are able to improve apoptosis-induced renal tubular injuries.^21–22,53 Anti-inflammatory potential of ROF has been confirmed through NF-kB inhibition in Cd-induced renal damage. In addition, ROF treatment also causes increase in GST and NQO1 levels. These results suggest that may be preventive effect of ROF against Cd-induced renal injury through the GST and NQO1 enzymatic activation.

Epidemiological studies have shown that Cd is one of the most dangerous heavy metals to human body; 70% of the ultrafiltered Cd is taken up mostly by the proximal convoluted tubules and is stored mainly in the cortex layer of kidney leading to proximal tubular lesions.⁵⁴ These findings are in agreement with the current study. Because we also observed a significant damage in function and structure of the kidney was assessed and confirmed by the existence of severe damage of glomeruli with severe congestion of the renal blood vessels in Cd-treated rats at a dose of 3 mg/kg (i.p.) daily for 7 days. These results are in agreement with the previous study which also reported some glomerular capillaries atrophy, proximal tubular necrosis, as well as degenerative changes and cytoplasmic vacuolation in the distal convoluted tubules.⁵⁵

Histopathological analysis of kidney tissues confirmed the findings of biochemical estimations in serum and tissue where ROF treatment at both doses prevents Cd-induced degenerative changes in kidney tissues and improved renal function due to its potential antioxidant properties, via attenuating oxidative stress–mediated decline in kidney.

Cortijo et al. previously demonstrated anti-inflammatory and antioxidant effect of ROF against bleomycin-induced lung injury in rats.⁵⁶ In the present study also, treatment with ROF potentially protects the nephrons and normalizes its functions from Cd-induced free radicals and restored the levels of kidney function markers.

The present biochemical and histological results confirmed potential ROF effect in protecting the kidney tissue against oxidative damages and could be used in future as an effective treatment against nephrotoxicity and other kidney problems also.

Conclusion

In summary, it could be concluded that increased MDA levels and decreased GSH, SOD, and CAT levels indicating chronic renal injury produced by Cd treatment were identified. ROF (PDE4 inhibitor) administration in Cd-intoxicated rats succeeded in amelioration and improvement of the altered biochemical and oxidative antioxidant parameters to nearly those of the control group. Histopathological studies of kidney tissues also confirmed that changes caused by Cd-toxicity were significantly ameliorated with ROF treatment. Collectively, we can conclude that ROF can be used as potentially effective therapeutic agents for workers in factories whose products are contaminated with Cd. Our findings of the present study also strongly recommend that pharmacological activation of NQO1 enzyme as a potential strategy and target for the prevention of Cd-induced renal injury.

Footnotes

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

MN Ansari

AM Hamad

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