Renoprotective effect of Lycopene on Renal Functional and Histopathological changes in Gentamycin Induced Nephrotoxicity in Rats.

 

Anup A. Patil*, Rajendra Doijad, Akshada Koparde

KIMSDU’S Krishna Institute of Pharmacy, Malkapur, Karad.

 

ABSTRACT:

Nephrotoxicity is toxicity in the kidneys. It is a poisonous effect of some substances, both toxic chemicals and medications, on renal function. There are various forms, and some drugs may affect renal function in more than one way. Nephrotoxins are substances displaying nephrotoxicity. The aim of this study is to explore the effect of lycopene inhibition in lesions of gentamicin-induced nephrotoxicity, Mature 24 male mature Sprague-Dawley rats were divided into 4 groups of 6, group1: control, group 2 sham received daily lycopene injection (200 mg/kg) for 12 days, group 3 received daily GS injection (100mg/kg) for 12 days, group 4 received daily GS (100 mg/kg) and lycopene injection (100mg/kg)) for 12 days. After 12 days, rats were anaesthetized, blood sample were obtained and kidneys were removed then stained with hematoxylin and eosin method and then were studied histophatologically. Serum creatinine and urea were measured. lycopene significantly decreased the levels of urea and creatinine in treated group compared with the nephrotoxic untreated group. treatment of nephrotoxic animals with lycopene could significantly inhibit leukocyte infiltration and tubular necrosis in comparison with the nephrotoxic untreated group the findings showed that lycopene alleviates loss of leukocyte infiltration and tubular necrosis and exerts beneficial effects on kidney function test in nephrotoxic group

 

 

 

INTRODUCTION:

Gentamicin is prescribed in life threatening gram–ve infections and its most unavoidable effect is nephrotoxicity despite close attention to the pharmacokinetics and dosing schedule of the drug^(1). Antibiotics have broad antibacterial spectrum ranging from the +ve aerobic cocci to gram–ve. It has been reported that 30% of the patients treated with aminoglycosides show some signs of nephrotoxicity Like other aminoglycosides gentamincin is eliminated by glomerular filtration but as a result of absorptive endocytosis, gentamicin is partially reabsorbed by proximal tubular cells. Gentamicin loaded endocytic vacoules fuse with lysosomes where the drug accumulates^(2,3). This accumulation leads to development of lysosomal phospholipidosis characterized by an impairment of phospholipase and sphingomyelinase activities^(4).

 

This phospholipidosis eventually leads to tubular regeneration^(5). It has been reported that the concomitant injection of daptomycin⁽⁶⁾ poly-L-aspartic acid and⁽⁷⁾ ceftriaxone (8) reduce significantly the renal toxicity of aminoglycosides in experimental animals. Fish oil has also been reported to protect against acetaminophen (paracetamol) induced hepatotoxicity⁽⁹⁾. ethanol induced gastric mucosal injury in rats⁽¹⁰⁾ and in a number of inflammatory diseases^(11). Curcumin⁽¹²⁾ olive leaf extract ⁽¹³⁾ green tea and coenzyme Q10⁽¹⁴⁾

 

A number of natural antioxidant such as vitamin E and phenolic compounds are known to have protective effects on liver injury and nephrotoxicity (15). Chemical drugs as antioxidant have many side effects; therefore, screening for new natural antioxidants is still attractive because they are safe and good alternative for prevention of nephrotoxicity induced by GS. A growing body of research indicates that nutritional deficiencies such as antioxidants contribute to the development of nephrotoxicity lycopene also has such characteristic because most of the antioxidant activity.⁽¹⁶⁾  The possible protective effects of lycopene on structural lesions of kidney tissue in lesions of gentamicin-induced nephrotoxicity male rats have not yet been reported so study was undertaken to check renoprotective effect of lycopene on renal functional and histopathological changes in gentamycin induced nephrotoxicity.

 

Materials and methods:

Materials:

Lycopene purchased form sigma Aldrich was orally administered (100mg-body weight) Gentamicin vials of 2ml, having concentration of gentamicin 80mg in 2ml, obtained from Parkin Remedies, India, were given i.p in one daily dose (80mg/kg b.wt). All the chemicals used were of the highest purity available commercially and were obtained from E-Marck and Qualigens fine chemicals.

 

Animals:

Mature 24 male mature Sprague-Dawley rats (200-220gm) were used in the experiments. All rats were kept at room temperature 22±2ºC in the department animal house. They were maintained on Hindustan Lever food pellets and water ab libitum. The experiment protocol was approved by the Institutional Animal Ethics committee (IAEC). The study was conducted in accordance with the Basic and Clinical Pharmacology and Toxicology policy for experimental and clinical studies

 

Treatment of Animals:

The animals were randomly assigned to 4 groups (6 per group): 1- Control group 2- Sham group treated with 200 mg/kg Lycopene (purchased from Sigma Chemical Company) i.p. for 12 days without treatment with gentamicin. 3- Gentamicin-induced nephrotoxicity received 100mg/kg gentamicin i.p. for 12 days. 4- Nephrotoxicity group received 100mg/kg gentamicin and 200mg/kg Lycopene i.p. for 12 days. After 12 days treatment animals were anesthetized and blood samples of their hearts were obtained and keeps at room temperature for 30 minutes and then centrifuged at 3000 rpm for 15 minutes for serum separation and used for biochemical analysis. Their right kidneys were excised for tissue processing and were fixed in 10% formaldehyde solution after tissue processing of their kidney paraffin sections (5µm thicknesses) were prepared and stained by hematoxylin and eosin (17)

 

Biochemical analysis:

Serum collected was used for various assays

1.     Bonses⁽¹⁹⁾ using a reagent Kitobtained from Span Quantitative determination of urea by Dam method as described by Fingerhunt et al.⁽¹⁸⁾ using a reagent kit from Techno. Pharm. Chem; India

2.     Creatinine estimation was done by method of Taussky and diagnostics Ltd., India.

3.     Estimation of cholesterol content by method of Wybenga and Pillegi⁽²⁰⁾ using reagent Kit from Span diagnostics Ltd., India.

4.     SGOT and SGPT levels were determined by method of Reitman and Frankel⁽²¹⁾ using a kit obtained from Span diagnostics Ltd., India.

 

Renal Histological Analysis:

After tissue processing of rat kidneys paraffin sections (5 µm thicknesses) were prepared and stained by hematoxylin and eosin. Forty regions of proximal convoluted tubules near of each kidney at the magnification of × 400 were observed to consider the histopathological changes such as eosinophilic casts, leukocyte infiltration and tubular necrosis. Caramel method was utilized to assess these changes semiquantitively [22]:

 

0 = no damage; 1 = damage mild, patchy isolated; 2 = damage less than 25%; 3 = damage between 25% - 50%; 4 = more than 50% damage.

 

the results of each change were reported separately.

 

Statistical Analysis:

All values are expressed as mean±SD. Data between groups were first tested Kruskal-Wallis one-way and then between two groups were analyzed by Mann-Whitney U test. Statistical analyses were performed using SPSS-16. A P value < 0.05 was considered statistically significant.

 

Result:

Our study demonstrated that the level of serum urea in untreated nephrotoxic rats was significantly higher than that of the control animals. Treatment of nephrotoxic animals with lycopene could not significantly. inhibit serum urea in comparison with untreated nephrotoxic group. The level of serum urea in sham, similar to the level found in the nephrotoxic treated animals. The level of serum creatinine is shown in Table 1. Our study demonstrated that the level of serum creatinine in untreated nephrotoxic rats was significantly higher than that of the control animals. Treatment of nephrotoxic animals with Lycopene could not significantly inhibit serum creatinine in comparison with untreated nephrotoxic group. Cholesterol mg/dl level of sham group is slightly elevated. No significant difference among SGPT and SGOT in all groups.

 

Table no.1 Circulating levels of various biochemical parameters in serum.

Groups	Urea mg/dl	Creatinine mg/dl	Cholesterol mg/dl	SGOT nmole/min/ml	SGPT nmole/min/ml
Control	44.83 ± 4.71b	0.61 ± 0.07b	107.89±3.69b	25.63±0.220c	30.06±0.158
Sham	51.00 ± 4.02b	0.81 ± 0.03b	125.43±0.48	27.90±0.450c	34.57±0.446
Gentamicin-induced nephrotoxicity	176.43±39.47c	2.71±0.27c	114.25±1.32b	30.36±0.355b	32.76±0.299
Gentamicin-induced nephrotoxicity treated	54.25±20.33b, c	2.93 ± 0.49c	120.25±1.20	29.36±0.345	35.23±0.32
aValues are expressed as Mean ± SEM. bP < 0.05 as compared with gentamicin-induced nephrotoxicity without treatment group. cP < 0.05 as compared with control group.

 

The level of eosinophilic casts is shown in table 2. Our study demonstrated that the level of the eosinophilic casts in untreated nephrotoxic rats was significantly higher than that of the control animals. Treatment of nephrotoxic animals with Lycopene could not significantly inhibit eosinophilic casts in comparison with untreated nephrotoxic group. The level of eosinophilic casts in sham, similar to the level found in the nephrotoxic treated and untreated animals. The level of leukocyte infiltration is shown in Table 2. Our study demonstrated that the level of the leukocyte infiltration in untreated nephrotoxic rats was significantly higher than that of the control animals. Treatment of nephrotoxic animals with Lycopene could not significantly inhibit leukocyte infiltration in comparison with untreated nephrotoxic group

 

Table 2. Effect of Lycopene on Histopathological Parameters During Gentamicin-Induced Nephrotoxicity.

Groups	Eosinophilic Casts	Leukocyte Infiltration	Tubular Necrosis
Control	0.048 ± 0.043b	0.135 ± 0.058b	0.198 ± 0.08b
Sham	3.712 ± 0.0893c	2.472 ± 0.109b, c	2.583 ± 0.123b, c
Gentamicin-induced nephrotoxicity	3.131 ± 0.094	2.984 ± 0.106	3.433 ± 0.094
Gentamicin-induced nephrotoxicity treated	3.225 ± 0.147c	2.827 ± 0.133c	2.512 ± 0.153b, c
a Values are expressed as Mean ± SEM. b P < 0.05 as compared with gentamicin-induced nephrotoxicity without treatment group. c P < 0.05 as compared with control group.

 

 

DISCUSSION:

Strategies for prevention of kidney damage patients for the prevention and slowing kidney damage complications is antioxidant therapy. Protective mechanisms of lycopene were not completely explained by our results. our results showed that lycopene has some beneficial effects in prevention and slowing of kidney damage. According to the findings of this study, it can be concluded that GS injection in rats for 12 days can lead to renal disorder in rats. The con-current administration of GS with lycopene (100mg/kg) for 12 days at least partially resulted in decreased of renal injuries caused by GS therapy. In this study the level of both serum urea and tubular necrosis significantly decreased in gentamicin-induced-nephrotoxicity groups treated with lycopene compared to gentamicin-induced-nephrotoxicity groups none treated with lycopene

 

Although GS has an effective role in treatment of Gram negative bacteria, its administration has been limited because of increasing the chance of acute renal failure in 10%-20% of patients treated with this drug. Gentamicin-induced-nephrotoxicity can cause renal injuries due to ROS generation. Antioxidant compounds such as melatonin vitamin E and probacol, lycopene etc. may be utilized to decrease the rat of renal injuries caused by gentamicin-induced-nephrotoxicity.

 

The beneficial effects of Lycopene are due to their constituent antioxidant properties (16)^. The protective effects of n- 3 fatty acids, eicosapentaenoic and decosahexaenoic acid present have been established in cyclosporine treated patients and nephrotoxic experimental animals^(15). It has been reported that fish oil (fatty acid) reduces circulating lipid levels in animals as well as in human subjects (22).

 

Acute renal failure, induced in animals by gentamicin exposure was manifested by increased serum urea and creatinine levels. The rise of urea and creatinine levels in gentamicin treated animals⁽²³⁾ compared to control treated animals suggest that due to renal injury, glomerular filtration rate (GFR) and reabsorbtion processes have been effected (24)

 

For these studies it can be conclude that the decrease in the level of tubular necrosis after treatment with lycopene may be due to it’s antioxidant characteristic from the current study, we conclude that administration of lycopene and GS in rats can prevent gentamicin-induced renal toxin

 

 

In addition our studies suggest that lycopene is more useful than other treatment groups studied the maximum recovery effect. The data presented in this manuscript could be helpful in formulating human clinical trials to examine the efficacy of lycopene supplementation on nephrotoxicity induced by commonly used antibiotics.

 

CONCLUSION:

The present study object was to investigate the beneficial effects of lycopene on gentamicin induced nephrotoxicity in rats. Therefore, Protective mechanisms of lycopene were not completely explained by our results. our results showed that lycopene has some beneficial effects in prevention and slowing of kidney damage.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Krishna Institute of Medical sciences Deemed to be University Karad for the facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 31.08.2019           Modified on 18.10.2019

Accepted on 17.11.2019         © RJPT All right reserved