Evaluation of Nephroprotective effect of Ubiquinol on Ifosfamide Induced Nephrotoxicity in Albino Wistar Rats

 

Vinayaka Anuhya1, Amberkar Mohanbabu Vittalrao2, Meena Kumari Kamalkishore3*,

Brij Mohan Kumar Singh4, Gangaparameswari Soundarrajan1

1Postgraduate, Department of Pharmacology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Karnataka, Manipal, 576104, India.

2Professor, Department of Pharmacology, The Oxford Medical College,

Hospital and Research Centre, Bangalore, Karnataka, India.

3Associate Professor, Department of Pharmacology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Karnataka, Manipal, 576104, India.

4Associate Professor, Department of Pathology, Kasturba Medical College, Manipal,

Manipal Academy of Higher Education, Karnataka, Manipal, 576104, India.

*Corresponding Author E-mail: meena.kumari@manipal.edu

 

ABSTRACT:

To determine nephroprotective activity of ubiquinol on ifosfamide induced renal damage by measuring the oxidative stress, biochemical parameters and histopathological examination. Thirty-six rats were divided into 6 groups, Group 1 rats were treated with 2ml of corn oil (vehicle) p.o., Group 2 and 3 received only ubiquinol of 10mg/kg/day and 50mg/kg/day respectively. Ubiquinol was prepared as oral suspensions with 2ml of corn oil. Group 4 received ifosfamide 80mg/kg/day, i.p. for 3 days.  Groups 5 and 6 also received ubiquinol 10mg/kg/day and 50mg/kg/day respectively p.o. for 14 days. Then, we added ifosfamide to both the groups and dosed concomitantly along with ubiquinol from 8th -10th day. Ubiquinol proved to be an effective renoprotectant by decreasing the kidney to body weight ratio in ubiquinol treated IFO groups as compared to IFO group (p<0.05). Biochemical tests done on day 9 revealed that there was no significant difference in levels of Sr.Creatinine and total protein, but the levels of BUN were found to be significantly higher in IFO treated groups as compared to corn oil group (p<0.05). On day 15, it was seen that significant improvement in Sr.Creatinine, BUN and total protein levels of ubiquinol treated IFO groups as compared to IFO group (p<0.01). The levels of MDA and GSH were reversed in ubiquinol treated IFO groups as compared to IFO group (p<0.01). Histopathological examination supported that ubiquinol preserved the normal architecture of kidney in ubiquinol treated IFO groups. Ubiquinol proved to be an effective nephroprotective agent against ifosfamide induced nephrotoxicity.

 

KEYWORDS: Renal Injury, Oxidative phosphorylation, Co-enzyme Q10, Anti-cancer drug.

 

 


INTRODUCTION: 

Ifosfamide (IFO), is an alkylating oxazaphosphorine antitumor prodrug that is synthetically developed.1 It is a close congener of cyclophosphamide with structural isomerism that has been approved for the concomitant use of other chemotherapy drugs like cisplatin, etoposide,  or vinca alkaloids like Vinblastine.

 

for management of metastatic germ-cell testicular cancer, sarcomas, and certain forms of lymphomas as well1,2,3. The direct tubular injury is a common impediment seen associated with if osfamide and is increased most in organs toxicity as well4. Cyclophosphamide and if osfamide are closely related based on structural similarities, and it is observed that cyclophosphamide has virtually no nephrotoxicity1.

 

The mechanism by which IFO causes renal injury and the protective agents that may reverse or prevent the renal damage remains unclear. IFO-induced Fanconi syndrome is seen in patients and demonstrated in animal models as well5. It presents as renal injury at proximal tubules with decreased glomerular filtration rate, glucosuria, phosphaturia, and aminoaciduria5. Concomitant use of MESNA as an uroprotective drug fails to decrease the renal injury in most of the cases6. Itis observed that the prodrug of ifosfamide is not toxic, but its metabolites are7.

 

Oxidation of Ifosfamide occurs initially by the production of 4-OH-IFO and further formation of metabolites like isophosphoramide mustard and acrolein takes place7. There is another pathway of dechlorethylation, leading to the formation of chloroacetaldehyde(CAA)8. Fifty percent of IFO metabolism occurs from the second part of oxidation. Cytochrome enzymes like CYP3A5 and CYP2B6 mediate this process of oxidation9,10. The activity of these  cytochrome enzymes is increased mostly in organs like kidney and liver. CAA is often seen to be formed in proximal renal tubules, the primary site for production of ifosfamide-induced renal injury7.

 

The assumption of CAA-induced renal injury is primarily by disrupting the oxidative phosphorylation of mitochondria that damages energy production, thus subsequently causing  numerous metabolic irregularities and injury at the cellular level7. A marked improvement in mitochondrial oxygen consumption and fatty acid oxidation is believed to cause attenuation or prevention of nephrotoxicity by preserving the integrity of the electron transport chain in the mitochondria9. NADH: ubiquinone oxidoreductase is the major complex of the mitochondrial respiratory chain, also called as Complex I. Defects of Complex I frequently cause mitochondrial respiratory diseases and myopathies11,9. It is hypothesised that inhibiting the function of Complex I is the chief target of ifosfamide-induced nephrotoxicity, then supplementation of a drug that up-regulates Complex I function might reduce and even inhibit the failure of energy metabolism.

 

Co-enzyme Q10 is a vitamin-like essential nutrient required for the electron transport chain in the mitochondria to control the production of energy in the eukaryotic cell.12 With the assistance of CoQ10 every human cell transforms nutrition into energy in the mitochondria. In the last decade, the use of  CoQ10 as a dietary and nutraceutical supplement has increased for the prevention of cardiovascular diseases13,14. In this study we have used the reduced form of Co-enzyme Q10 called ubiquinol (rQ10), to evaluate its nephroprotective role against the toxicity produced by ifosfamide.

 

MATERIALS AND METHODS:

Chemicals:

rQ10 was purchased from Jarrow Formulas, USA. Ifosfamide was purchased from hospital pharmacy stores. For the analysis of biochemical parameters semi-analyzer kits, other chemicals, and for dissection of kidneys surgical instruments were usedfrom the pharmacology department lab.

 

Animals and Experimental Design:

Thirty-six male and female Albino Wistar rats were selected for the study, which are locally bred in the Central animal house research facility. The study was undertaken after taking approval from the institutional animal ethics committee (IAEC/KMC/90/2016 dated 13.09.2016). The animal handling protocol was designed to minimize pain or discomfort to the animals. These animals are 200-250g weight and six months old, housed under controlled conditions of temperature 23±2°C, humidity of 50±5% and 10-14 hour of light and dark cycle respectively. The animals were housed individually in polypropylene cages containing sterile paddy husk (procured locally) as bedding throughout the study and were given free access to sterile food (VRK nutritional solutions, Pune, India) and water ad libitum. The animals were acclimatized two weeks prior to experimentation. For intragastric and intrapleural administration of drugs, measures were taken appropriate to animal size. Animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection.

 

Thirty-six male and female Albino Wistar rats were selected, six groups of six rats each. Group 1 was treated with 2ml of the vehicle, i.e., corn oil p.o (per os). for 14 consecutive days. Group 2 and 3 received ubiquinol of 10mg/kg/day and 50mg/kg/day respectively p.o. for 14 days. For the administration of ubiquinol, corn oil is used as a vehicle.  Groups 4-6 received ifosfamide 80mg/kg i.p. from day 8 to day 10, of these groups 5 and 6 also received ubiquinol 10mg/kg/day and 50mg/kg/day respectively p.o. for 14 days.

 

Figure 1: Schematic representation of the experimental study design. The above arrows indicate the six treatment arms starting from Day 1 to Day 14. Corn oil group, rQ10 (10 and 50 mg/kg) groups indicate that the dosing of rats was done for 14 consecutive days. In IFO groups, group 4 indicate that IFO was administered alone at a dose of 80mg/kg from Day 8-10; Groups 5 and 6 indicate that rQ10 (10 and 50 mg/kg) was administered for 14 days with IFO at a dose of 80mg/kg from Day 8-10. On Day 9 and 15 indicate that the blood samples were collected for serum analysis and on Day 15 the rats were sacrificed for weighing the kidney size and histopathological examination.

 

Evaluation of Renal Function:

On day 9 and on day 15, 2mL blood was collected by retro-orbital puncture for biochemical assays from the serum. Serum was separated by centrifuging the blood collected at 2500rpm for 15min. The rats were sacrificed by cervical dislocation on day 15. The kidneys were isolated and its weight (on an analytical balance) and volume (using water displacement method in a glass measuring cylinder), which are expressed per 100g of body weight were measured. Sections of the kidney were preserved in 10% formalin and analyzed using hematoxylin and eosin stain for histopathological changes.

 

Estimation of Serum Creatinine, BUN and Total Protein:

Quantitative determination of serum creatinine was done by using modified Jaffe’s method. The levels were measured on day 9 and at the end of the study on day 15. Quantitative determination of BUN was done by using Urease/GLDH (glutamate dehydrogenase) method15. The levels were measured on day 9 and at the end of the study on day 15. Quantitative determination of total protein was done by using biuret method16. The levels were measured on day 9 and at the end of the study on day 15.

 

Estimation of Renal MDA and Glutathione Reductase:

Malondialdehyde (MDA) measurement was done in kidney homogenate17. End products of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a coloured complex, a complex that has maximum absorbance at 532nm. The levels were measured at the end of the study on day 15. Glutathione reductase measurement was done in kidney homogenate. Glutathione was measured by its reaction with DTNB (5, 5’dithio 2-nitrobenzoic acid) to give a compound that absorbs light at 412nm. The levels were measured at the end of the study on day 1518.

 

Histopathological sections of kidney:

After sacrificing the rats, kidneys were dissected and kept in 10% formalin which were further sent for histopathological sectioning. The slides were stained by haematoxylin and eosin method and observed under 400X magnification.

 

Statistical Analysis:

The data was analyzed using one-way ANOVA (analysis of variance) followed by post hoc analysis using Tukey’s test in SPSS Ver. 15 Software. The level of statistical significance for any measure was set at p<0.05 at a confidence interval of 95%. The data is expressed as mean ± standard error ofmean.

 

 

RESULTS:

Effect of rQ10 on Kidney % of total body weight: At the end of the study, no significant differences in the kidney weight as a percentage of the total body weight were observed between the corn oil and rCoQ10 groups. There was a statistically significant decrease (p<0.05) in kidney weight as a percentage of the total body weight of ubiquinol treated groups as compared to the ifosfamide group (Figure 2).

 

Figure 2

 

Effect of rQ10 on Sr. Creatinine, BUN and Total Protein levels in ifosfamide induced nephrotoxicity on Day 9:

 

Table 1: Effect of rQ10 on renal parameters on Day 9

Groups

Sr. Creatinine

(mg/dL)

BUN

(mg/dL)

Total Protein

(g/dL)

Corn oil

0.51 ±0.02

9.04 ±0.63

6.27 ±0.13

rQ10 (10mg/kg)

0.46 ±0.02

8.63 ±0.35

6.31 ±0.40

rQ10 (50mg/kg)

0.57 ±0.04

8.53 ±0.36

6.77 ±0.31

IFO

0.77 ±0.09

29.06 ±1.32*

5.44 ±0.27

IFO+ rQ10 (10mg/kg)

0.54 ±0.55

24.44 ±2.65*

6.35 ±0.28

IFO+ rQ10 (50mg/kg)

0.63 ±0.07

25.62 ±2.58*

5.93 ±0.31

All values are expressed as mean±SEM; n=6 in each group

*p<0.05 Vs. Corn oil

 

Results of Sr. Creatinine, BUN and Total protein are summarized in Table 1. Rats which received only ifosfamide (80mg/kg, i.p.) showed a significant increase in BUN levels on day 9 compared to control group. Concomitant administration of rQ10 in both doses (10 and 50mg/kg) of ifosfamide groups also showed significant increase in BUN levels as compared to control group on day 9. However, comparing these ifosfamide groups treated with rQ10 to the toxic group did not show any significant decrease in BUN levels. The levels of Sr. Creatinine and total protein did not show any significant difference between the groups.

 

 

 

 

 

Effect of rQ10 on Sr. Creatinine, BUN and Total Protein levels in ifosfamide induced nephrotoxicity on Day 15:

Table 2: Effect of rQ10 on renal parameters on Day 15

Groups

Sr. Creatinine

(mg/dL)

BUN

(mg/dL)

Total Protein

(g/dL)

Corn oil

0.52 ±0.02

8.93 ±0.51

6.17 ±0.09

rQ10 (10mg/kg)

0.46 ±0.02

8.56 ±0.39

6.29 ±0.39

rQ10 (50mg/kg)

0.56 ±0.04

8.38 ±0.39

6.81 ±0.27

IFO

2.33 ±0.14*

71.92 ±2.57*

2.82 ±0.37*

IFO+ rQ10 (10mg/kg)

1.42 ±0.19a

57.72 ±2.91

5.49 ±0.23b

IFO+ rQ10 (50mg/kg)

1.19 ±0.08b

51.43 ±4.05a

5.32 ±0.36b

All values are expressed as mean±SEM; n=6 in each group

* p<0.01 Vs. Corn oil,a p<0.05 Vs. IFO,b p<0.01 Vs. IFO

 

Results of Sr. Creatinine, BUN and Total protein are summarized in Table 2. Rats receiving only ifosfamide (80mg/kg, i.p.) showed a significant increase in Sr. Creatinine and BUN levels as compared to control group. Concomitant administration of rQ10 in both doses (10 and 50mg/kg) has shown a significant decrease in Sr. Creatinine and BUN levels, and a significant increase in total protein levels as compared to the toxic group.

 

Effect of rQ10 on Renal MDA and GSH levels:

Table 3: Effect of rQ10 on Renal MDA and GSH levels on Day 15

Groups

MDA

(nmol/g tissue)

GSH

(mmol/g tissue)

Corn oil

6.09 ± 0.22

0.31 ± 0.009

rQ10 (10mg/kg)

5.90 ± 0.14

0.30 ± 0.005

rQ10 (50mg/kg)

6.00 ± 0.26

0.30 ± 0.004

IFO

14.76 ± 0.31*

0.13 ± 0.009*

IFO+ rQ10 (10mg/kg)

10.16 ± 0.38b

0.27 ± 0.005b

IFO+ rQ10 (50mg/kg)

9.87 ± 0.32b

0.29 ± 0.004b

All values are expressed as mean±SEM; n=6 in each group

* p<0.01 Vs. Corn oil,a p<0.05 Vs. IFO,b p<0.01 Vs. IFO

 

Results of MDA and GSH levels are summarized in Table 3. Rats receiving only ifosfamide (80mg/kg, i.p.) showed a significant increase in MDA levels and decrease in GSH levels as compared to control group. Concomitant administration of rQ10 in both doses (10 & 50mg/kg) has shown a significant improvement in MDA and GSH levels.

 

Effect of rQ10 on Histopathological sections of Kidney:

Microphotographs of rat kidneys stained by Haematoxylin and Eosin. 400X

 

Corn oil

 

rQ10 (10mg/kg)  rQ10 (10mg/kg)

 

rQ10 (50mg/kg)

Figures A, B, C: Normal kidney architecture with presence of uniform brush border cells, and uniform distribution of epithelial cells

 

IFO

Figure D shows uniform loss of Brush border cells with tubular atrophy (Black arrow)

IFO + rQ10 (10mg/kg)

 

IFO + rQ10 (50mg/kg)

Figure E and F shows glomeruli maintained with preserved brush border cells in few areas (Black arrow)

 

DISCUSSION:

Wide clinical application of chemotherapy in the management of cancer is facing numerous hurdles; one of it being drug-induced nephrotoxicity that affects the patients’ quality of life and adds to the morbidities of patients. Ifosfamide is the chosen chemotherapeutic for this study to produce nephrotoxicity in rats. Ubiquinol, the reduced form of Co-enzyme Q10 is used for testing the nephroprotective effects at both preventive and therapeutic levels.

 

Ifosfamide induced renal injury is temporary, and renal function can resume to normalize after cessation of the drug. Nevertheless, analyzing adult survivors of pediatric malignancies for long term, who were managed with ifosfamide has shown to cause a permanent renal damage8.Sometimes this injury can be progressive causing end-stage renal disease8.Risk factors like elderly, concomitant use of other chemotherapeutics like cisplatin has been found to increase the burden19,20. Ifosfamide is oxidized by enzymes like CYP3A5 and CYP2B6 oxidase6. One of the metabolites, choloracetaldehyde is thought to be the culprit to cause renal damage by disrupting the oxidative phosphorylation in mitochondria of renal proximal tubules7,10. We hypothesized that supplementation of ubiquinol might increase the mitochondrial oxygen consumption and fatty acid oxidation because Coenzyme Q10 is a part of electron transport chain, that may reduce or inhibit the nephrotoxicity by preserving the integrity of the respiratory chain.

 

There was no study done prior to the best of our knowledge where ubiquinol was used to reduce the nephrotoxicity produced by ifosfamide. Nissim et al. have demonstrated that chloroacetaldehyde, a metabolite of ifosfamide is the root cause for the development of nephrotoxicity and they have proven that agmatine can be used to increase oxidative phosphorylation and β-oxidation that would inhibit renal injury9. Nissim et al. also stated that there was a significant rise in the levels of NADH, depletion of NAD by inhibition of complex-I9. Agmatine has prevented the ifosfamide induced inhibition of complex I9.

In our study, the results of ubiquinol treated groups did show Reno protective activity compared to ifosfamide group which were similar to that of Nissim et al. It was observed that pretreatment of ubiquinol 7 days prior to the anticancer drug, did not show any significant difference in the biochemical parameters. But later after continuing for another 7 days on day 15, there was a significant improvement in the renoprotection as seen by decreased sr. creatinine and BUN levels in the ubiquinol treated groups. Total protein levels were found to be elevated significantly higher (p<0.001) in the ubiquinol treated groups as compared to the ifosfamide group. The Reno protective activity seen by ubiquinol has prevented renal injury because, probably both ubiquinol and agmatine share similar Reno protective mechanism as shown by Nissim et al. On day 15 there was no statistical difference between the two doses of ubiquinol in the reversal of toxicity produced by ifosfamide.

 

Histopathological sections of kidney were taken, which showed that the control groups had a normal looking architecture with uniform distribution of brush border cells and epithelial cells. No signs of tubular injury were seen in the sections of control groups. The sections of ifosfamide group showed loss of brush border cells, signs of tubular atrophy like increased vacuolization in the epithelial cells with desquamation in few areas and presence of reactive changes in the epithelial cells with prominent nucleoli. Those groups treated with ubiquinol have preserved the brush border cells, minimal reactive changes in epithelial cells and no vacuolization or desquamation of cells.

 

MDA levels estimated in the kidney homogenate is considered as an indicator for lipid peroxidation and presence of renal GSH, the active antioxidant plays an effective role in the detoxification, conjugation and excretion of toxic substances21,22. Ifosfamide group has shown a significant increase in MDA and decrease in GSH levels demonstrating oxidative stress producing renal damage. Ubiquinol treated groups of ifosfamide have shown decreased lipid peroxidation by decreased MDA levels and improved GSH levels, compared to anticancer drugs proving ubiquinol as protective factor against the oxidative stress produced by the anti-cancer drugs.  Kidney weight as percentage of total bodyweight was calculated. There were no significant differences in kidney weight percentage between the corn oil and ubiquinol groups. Ifosfamide group showed significant increase in the kidney weight percentage of total body weight. Ubiquinol treated groups of ifosfamide showed significant decrease in kidney weight percentage as compared to their respective toxic groups, proving that ubiquinol prevented the anti-cancer induced renal damage.

 

CONCLUSION:

Ubiquinol has reversed the levels of Sr. Creatinine, BUN, total protein and histopathological changes seen due to administration of ifosfamide. There was no significant difference in changes seen with respect to the two doses of ubiquinol (10mg/kg and 50mg/kg) that were used. This effect is achieved by the antioxidant property of ubiquinol as demonstrated by increase in tissue levels of non-enzymatic antioxidants like GSH and reduction in lipid peroxidation marker like MDA. Additionally, the reversal of toxicity in ifosfamide groups suggeststhat supplementation of ubiquinol could cause an up regulation of energy metabolism, which is usually the chief target of ifosfamide induced nephrotoxicity. Pretreatment with ubiquinol did not prevent the nephrotoxicity produced by ifosfamide. Nevertheless, based on the observation that ubiquinol offered potent decrease in nephrotoxicity induced by ifosfamide, ascertaining that it may offer new possibilities in managing the renal adverse effects due to cancer chemotherapy.

 

CONFLICT OF INTEREST:

The authors state that there is no conflict of interest related to the manuscript.

 

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Received on 30.08.2023            Modified on 14.01.2024

Accepted on 05.03.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(5):2309-2314.

DOI: 10.52711/0974-360X.2024.00362