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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 4  |  Page : 227-233

The protective effect of vernonia amygdalina in lead acetate-induced nephrotoxicity in wistar rats


1 Department of Anatomy, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria
2 Department of Biochemistry, Faculty of Sciences, Federal University of Lafia, Nasarawa, Nigeria

Date of Submission15-Jul-2021
Date of Decision25-Jul-2021
Date of Acceptance25-Jul-2021
Date of Web Publication19-May-2022

Correspondence Address:
Dr. Abraham Ehinomhen Ubhenin
Department of Biochemistry, Faculty of Sciences, Federal University of Lafia, Nasarawa
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_29_21

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  Abstract 


Introduction/Background: Inadvertent poisoning from indiscriminate use of lead acetate-containing agents has transformed into an issue of public health concern, most especially in developing countries, coupled with the paucity of potent antidotes. Aims: We investigated the protective effect of Vernonia amygdalina in lead acetate-induced nephrotoxicity in Wistar rats. Materials and Methods: In this study, thirty adult rats of either sex were divided into five groups of six animals each. Groups A and B were administered (daily) distilled water and lead acetate, respectively for 28 days. Groups C, D, and E received (daily) lead acetate at doses of 100 mg/kg body weight and aqueous extract of V. amygdalina at doses of 100, 200, and 250 mg/kg body weight, respectively, for 28 days. Results: The results from the study showed that were significant (P < 0.05) increases in the levels of serum creatinine, urea, sodium Na + and chloride Cl − in lead-intoxicated rats when compared to the control group. There was significant (P < 0.05) decrease in the serum levels of superoxide dismutase, catalase, peroxidase (GPx) Uric acid, URA and reduced glutathione (GSH) as the consequences of lead acetate administration. The histograms of the rats intoxicated with lead acetate were characterized by tubular necrosis and a reduction in myeloid-erythroid cells. Treatment with aqueous extract of V. amygdalina at the doses of 100, 200, and 250 mg/kg body weight significant (P < 0.05) protected against these alterations. The dose of 250 mg/kg exhibited the highest protective activity. Conclusion: Results of the present study may suggest that V. amygdalina possess a potent phytochemical that could be standardized for use in kidney and other related oxidative damage diseases.

Keywords: Erythroid, myeloid, necrosis, nephrotoxicity, pathognomonic, Vernonia amygdalin, Wistar rat


How to cite this article:
Innih SO, Ubhenin AE. The protective effect of vernonia amygdalina in lead acetate-induced nephrotoxicity in wistar rats. Niger J Exp Clin Biosci 2021;9:227-33

How to cite this URL:
Innih SO, Ubhenin AE. The protective effect of vernonia amygdalina in lead acetate-induced nephrotoxicity in wistar rats. Niger J Exp Clin Biosci [serial online] 2021 [cited 2022 Jul 7];9:227-33. Available from: https://www.njecbonline.org/text.asp?2021/9/4/227/345553




  Introduction Top


Lead (Pb) is a heavy metal with a density of about five times that of water and is well-known multi-organ toxicant which enters the body through inhalation, ingestion, and skin absorption.[1],[2],[3] Its toxicity is associated with its ability to induce oxidative stress that is the hallmark in the development of nephrotoxicity.[2] It tends to settle in the proximal tubule of the nephron and its buildup in the proximal tubule can also lead to hyperuricemia and gout, in a mechanism that probably related to the inhibition of uric acid excretion and altered glomerular filtration rate (GFR).[4],[5] Induction of endogenous generation of reactive oxygen species (ROS) which causes depletion of antioxidant defend system has been reported as the mechanism of lead toxicity.[6],[7] Exposure to lead occurs mainly through intake of lead-contaminated food, water, dusts, and paints.[1],[2],[3] lead exposure triggers the generation of ROS, and depletes the cellular antioxidant capacity. This oxidative stress condition damages cellular organelles, antioxidant enzymes, membranes, DNA, proteins, and finally destroys the tissue.[1],[2],[3],[4] Therefore, it is rational that exogenous administration of antioxidant substances would have a beneficial effect on the cells' antioxidant system with the view of combating lead intoxication. To this end, there are growing interests in using natural compounds to treat lead nephrotoxicity and other conditions which may include cardiovascular, reproductive, hematopoietic, gastrointestinal, and nervous systems abnormalities.[5],[6],[7],[8] Lead accumulation has also been reported to be associated with the development of cancer, hypertension, neurodegenerative disease, cognitive impairment, kidney dysfunction.[9],[10],[11],[12],[13],[14] Several studies have shown that lead acetate intoxication can alter biochemical and histological abnormalities in blood, kidney, liver, and brain tissues.[15],[16]

There are conflicting reports on the effects of chronic and sub-lead poisoning in renal function parameters. Some studies reported that lead exposure led to renal hypertrophy and increased GFR.[17],[18] whereas others reported a decreased GFR.[12] Separate studies on low-dose lead intoxication have also showed that there were no significant pathological changes in rats intoxicated with lead.[12],[14] Following these disparities in numerous studies, it is not clear if lead toxicity can be related to the duration and dosage of administration of lead intoxication.

Many plants have been used as a therapy for kidney failure in the traditional systems of medicine throughout the world. In fact, natural products from plants have been are recommended for alleviating renal damage and to avoid kidney-related complications.[19],[20] Vernonia amygdalina is a perennial shrub belonging to the Asteraceae or Compositae family is grows throughout the tropical part of Africa. It is perhaps the most widely cultivated species of the genus Vernonia which has over 1000 species of shrubs. It is popularly called bitter leaves because of its bitter taste which can be attributed to its anti-nutritional components such as tannins, alkaloids, glycosides, flavonoids and saponins.[21],[22],[23],[24],[25] V. amygdalina can be identified in Nigeria by some local names such as “Ewuro” in Yoruba language, “Onugbu” in Igbo language, “Oriwo” in Edo language, “Ityuna” in Tiv language, “Etidot” in Ibiobio and “Chusardoki or fatefate” in Hausa language.[25],[26] Proximal analysis revealed that V. amygdalina, contains crude fiber, crude protein, ash, and carbohydrate as the major nutrients present leaves of the plants.[27],[28],[29],[30],[31] The major ions present in V. amygdalina are nitrogen, phosphorus, calcium, magnesium, sodium, potassium manganese, copper, and cobalt.[27],[28] Studies have also shown that the plant contains appreciable quantities of ascorbic acid and caroteinoids.[29],[30]

The aqueous extract of the leaves of the plant possesses antibacterial, anti-cancer, antioxidant, antidiabetic, hepatoprotective, hypolipidemic, anti-fertility properties[29],[30],[31],[32] The extract of this plant has been widely used as anti-coagulant, anti-cancer, antithrombic, antipyretic, and anti-inflammatory agents.[33],[34],[35],[36],[37] Phytochemical analysis has showed that V. amygdalina contains secondary metabolites such as flavonoids, terpenes, saponins, alkaloids, steroids, coumarins, phenolic acids, lignins, and anthraquinones.[38],[39] The pharmacologic activities of this plant are attributable to some of the bioactive phytochemicals present in the plant.

Therefore, the purpose of the present study is to investigate the pathological and therapeutic effects of lead and V. amygdalina, respectively, on the serum antioxidant defense system, electrolytes, and renal function parameters in Wistar rats.


  Materials and Methods Top


Collection of medicinal plant

The leaves of the plant bitter leave (V. amygdalina) where obtained from the Botanical garden in the University of Benin, Benin City, Edo State. It was identified and authenticated at the herbarium in the Department of Pharmacognosy, Faculty of Pharmacy, and the University of Benin. The leaves of the plant washed under running tap water to remove adhering dirt followed by rinsing with distilled water. The leaves were then shade dried in the laboratory at room temperature, pulverized into powdered from and stored in a cool dry place.

Preparation of extract

The powdered extract was soaked for about 48 h at room temperature. The mixture was filtered into a conical flask with Whatman no 1 filter paper. The filtrate was then concentrated to dryness under reduced temperature and pressure using Rotary evaporator (Sofowora, 1984). The extract was stored in an air-tight container and kept in the refrigerator at 4°C until use.

Experimental animals

Thirty (30) Adult Wistar rats weighing 190–300 g were procured from the Animal House, Department of Anatomy, University of Benin, Benin-City. The rats were allowed to acclimatize for a period of 2 weeks before the commencement of the research. They were housed in standard animal cages and drinking water and standard livestock feed (vital grower's feed, livestock feed company, Benin City were allowed ad libitum. All animals were treated in accordance with the Guide for the care and use of laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health Guide for the Use of Laboratory Animal (NIN, 2002 Production, No. 83-23), Revised 1978.

Acute toxicity studies

Acute oral toxicity of V. amygdalina was determined using Swiss albino mice in a method described by Lorke.[40] The animals were fasted for 12 h (overnight) before the experiment. The animals were divided into six groups of five animals each and were administered with single dose of extracts dissolved in 5% tragacanth orally at doses of 1, 2, 4, 8, 12, and 16 g/kg body weight. The animals were observed for mortality up to 48 h (acute) and for another 14 days for sub-chronic toxicity.

Drugs usage

The plant extract was dissolved in tragacanth and was adequately prepared at doses 100, 200, and 250 mg/kg body weight, whereas lead acetate was also prepared at dose 100 mg/kg body weight.

The grouping of the experimental animals

The rats were divided into five groups, each group consisting of six animals.

Group I: (Control) received 5% tragacanth (1 mL/kg body weight. orally) only daily for 28 days.

Group II: (lead acetate–induced) received lead acetate at dose of 100 mg/kg body daily for 28 days only.

Group III: received V. amygdalina (orally) at the dose of 100 mg/kg body weight and 100 mg/kg body weight of lead acetate daily for 28 days.

Group IV: received V. amygdalina (orally) at dose of 200 mg/kg body weight and 100 mg/kg body weight of lead acetate daily for 28 days.

Group V: received V. amygdalina (orally) at dose of 250 mg/kg body weight and 100 mg/kg body weight of lead acetate daily for 28 days.

After 24 h of the last treatment, all the animals were fasted overnight and were anesthetized with chloroform before blood was collected via cardiac puncture. The blood was put into plain sample tubes and sera was obtain from it by allowing it to stand for 2 h at room temperature before centrifuging at 2000 rpm. The serum was used for the estimation of various biochemical parameters.

Histopathological analysis

The kidney and bone where harvested and fixed in 10% buffered formalin for a period of at least 24 h, dehydrated in several grade (70%–100%) alcohol embedded in paraffin (58°C–60°C) and sectioned at 5 μm thickness. The sections were stained with hematoxylin and eosin in the procedure described by.[41]

Biochemical analysis

Serum creatinine, urea, and uric acid levels

Spectrophotometry technique was used to estimate serum urea, creatinine, and uric acid levels using Commercial kits in accordance with the methods described.[42],[43],[44]

Serum Na+ and Cl levels

Serum concentrations of Na+, and Cl were measured according to the procedure described by[13],[45],[46] respectively.

Malondialdehyde levels

Malondialdehyde which is the final product of lipid peroxidation is estimated calorimetrically by measuring thiobarbituric acid (TBA) reactive substances following the procedure described by Draper and Hadley.[47] Briefly, Aliquots of kidney homogenates were mixed with 1 ml of 5% TCA and centrifuged at 4000 g for 10 min. 1 ml of TBA reagent (TBA, 0.67%) was added to 500 ml of supernatant and the mixture was heated at 95° for 15 min. The mixture was then cooled and was measured at absorbance at 532 nm. The MDA values were calculated using 1, 1, 3, 3-tetraethoxypropane as the standard and expressed as nanomoles of MDA/g of tissue.

Superoxide dismutase (SOD), Catalase (CAT), and Glutathione peroxidase (GPx) activities

The activities of GPX, SOD, and CAT were estimated in the serum of the rats according to the methods described by Kakka et al., Sinha and Rotruct et al.[48],[49],[50]

Reduced glutathione (GSH)

GSH content of the Kidney was determined by the method of Ellman's[51] with a little modification by Jollow et al.[52] 500 ml of homogenate tissues was deproteinized by the addition of 3 ml sulfosalicylic acid (4%). The mixture was centrifuged at 2500 g for about 20 min and the supernatant was decanted immediately. This is then followed by the addition of 500 ml Ellman's reagent to the supernatant. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as μmol/g of tissue.

Statistical analysis

Statistical analysis of the results was performed using one-way analysis of variance using SPSS software (IBM SPSS (Version 25), New York, United States) followed by Dunnet's comparison test for significance. Significance was set at (P < 0.05). Results are presented as Mean ± standard deviation.


  Results Top


The study revealed that there were significant (P < 0.05) increased in the levels of serum creatinine, urea, MDA, Na+ and Cl concentrations following the administration of lead acetate to rats as the dose of 100 mg/kg body weight daily for 28 days as compared to the normal control group. The study reveals that simultaneous treatment with aqueous extract of V. amygdalina at the doses of 100, 200, and 250 mg/kg mg/kg body weight led to decreases in the above parameters.

The study shows that there were significant decreases (P < 0.05) in serum levels of the level of antioxidant body defense system (URA, SOD, CAT, GPX, and GSH). The study also revealed that treatment with aqueous extract of V. amygdalina at the doses of 100, 200, and 250 mg/kg mg/kg body weight led to increases in the above parameters.


  Discussion Top


Lead usually accumulates in many organs or tissues that may include the liver, kidney, and bone. The bone forms the largest residues of lead buildup (up to 90%). The liver and kidneys play a major role in the removal of accumulated lead in the body and this accounts for their toxicities.[15] The kidney is an organ saddled with the responsibilities of removing metabolic wastes and maintaining homeostasis between the body's fluid and electrolytes. Serum levels of urea, creatinine, and electrolytes are frequently used to ascertain the structural and functional state of the kidney.[53],[54] There are compelling evidence that lead toxicities are attributed to their ability to induce excess production of free radicals capable of overwhelming the body's antioxidant defense system. Therefore estimating the level of the antioxidant body defense system and MDA concentration might correlate to the extent of oxidative stress and lipid peroxidation respectively.[51],[55],[56],[57]

Creatinine and urea are metabolic products of creatine and amino acid, respectively. They are regularly used as indicators of renal function. Damage to functioning nephrons leads to rise in serum creatinine and urea levels due to fall in glomerular filtration[51],[58] Serum creatinine and urea are therefore usually used to measure the manifestation of glomerular function and their elevated levels suggest renal failure to excrete them from the blood leading their increase in blood and the concomitant decrease in urine.[58]

The results from the oral toxicity study show that the aqueous extract of V. amygdalina may be regarded as nontoxic and safe for consumption judging from its high LD50 value which was found >5000 mg/kg. The fact that there was also no mortality further confirmed nontoxicity of V. amygdalina.

This study demonstrated that there were significant (P < 0.05) increased in the levels of serum creatinine, urea and MDA concentrations following the administration of lead acetate to rats at the dose of 100 mg/kg body weight daily for 28 days as compared to the normal control group as shown in [Table 1] and [Table 2]. The results indicated that there was kidney dysfunction that led to a fall in GFR. Studies have showed that lead build up in the kidney causes damage in renal tubules leading to reduction in the number of functional nephrons which weakens renal functions. This impairment of the renal functions led to the elevation of blood urea and creatinine levels. The appearance of focal tubular necrosis further from the microgram of the kidney further confirmed the pathognomonic signs of kidney toxicity. The significant increase (P < 0.05) in the MDA concentration suggests the reality of oxidative stress induced by lead-intoxication Concomitantly, there were a significant decrease (P < 0.05) in serum levels of antioxidant body defense system (URA, SOD, CAT, GPX and GSH). The findings are in consonant with reports of several studies.[51],[54],[55],[56],[57] SOD, CAT, and peroxidase (GPx) are among enzymatic body defense systems whereas Uric acid, URA and glutathione GSH represent nonenzymatic defense systems that scavenge free radicals in the body. Antioxidant enzymes are metalloprotein and their decrease in activities in this research might be in connection with the ability of lead to displace trace elements components from their prosthetic groups thereby undermining their scavenging capacities. Thus lead intoxication led to the compromise of structural and functional integrity of the enzymes following the effect it has on their metallic components. Overwhelming the scavenging capacities of the body defense system by the excess free radical production following oxidative stress induced by lead administration is also a major factor responsible for the marked decrease in the activities of these stress markers.
Table 1: Effect of vernonia amygdalina on serum urea, creatinine, uric acid, Na+ and Cl levels in lead acetate-induced nephrotoxicity on rats

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Table 2: Effect of vernonia amygdalina on enzymatic and nonenzymatic antioxidants levels in Lead acetate-induced nephrotoxicity on rats

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Sodium-ion (Na+) is the key cation of extracellular fluids and it plays an important role in muscle contraction. Its level in the urine is correlated to the GFR. Increase in GFR can lead to elevated excretion of Na+. The serum Na+ and Cl levels in the group intoxicated with lead acetate significant (<0.05) increased in this study. Increase in reabsorption of Na+ resulting from fall in GFR may account for this alteration as shown in [Table 1].

The study unambiguously revealed that simultaneous treatment with aqueous extract of V. amygdalina at the doses of 100, 200, and 250 mg/kg mg/kg body weight positively altered these kidney biomarkers and oxidative stress parameters to near-normal levels.

Histopathological changes in the kidney

The histogram from kidney's control group shows normal glomerular tubules and interstitial spaces. There were absent of tubular necrosis as well as congestion in the interstitial space. The overall structural architecture of the kidney's control group was normal. However, the histogram of the rats intoxicated with lead acetate was characterized with tubular necrosis which confirmed the pathognomonic sign of kidney toxicity. Treatment with aqueous extract of V. amygdalina at the doses of 100 and 200 mg/kg body weight does not show any noticeable sign of improvement as tubular necrosis were still observed. Treatment with aqueous extract of V. amygdalina at the dose of 250 mg/kg attenuated the tubular necrosis caused by lead acetate intoxication.

The control group shows normal cellularity with a myeloid-erythroid cells ratio of 30:70 [(a) in [Figure 1]]. There were no atherosclerotic changes in the control group whereas there was a reduction in myeloid-erythroid cells in the histogram of the rats intoxicated with lead acetate. This observation indicated that there was a reduction in primordial white blood cells with a simultaneous increase in primordial red blood cell in the group intoxicated with lead acetate. Treatment with aqueous extract of V. amygdalina at the doses of 100 and 200 and 2500 mg/kg body weight led to increase in myeloid-erythoid ratio. This observation suggested that A. V. amygdalina enhances animal immunity by increasing in primordial white blood cells with a concomitant reduction in primordial red blood.
Figure 1: Histopathological examination of the kidney: The histogram (a) of Rats' kidney in control group shows a normal glomerular tubules and interstitial spaces. (b) The histogram of Rats' kidney given 100mg/kg of Lead acetate only showing; A: focal tubular necrosis (c) The histogram of Rats' kidney given 100 mg/kg extract of Vernonia amygdalina plus 100 mg/kg Lead showing; A: focal tubular necrosis. (d): Rat kidney given 80mg/kg leave extract of Vernonia amygdalina plus 200 mg/kg of Lead acetate showing A, focal tubular necrosis (e) Rat kidney given 250 mg/kg leave extract of Vernonia amygdalina plus 100 mg/kg of Lead acetate showing A: normal renal architecture H an E × 100) (f) Rat bone marrow (control) composed of; bone trabecula, fat vacuoles, megakaryocyte and myeloid-erythroid cells (g) marrow given Lead only showing marked reduction of myeloid-erythroid cells (h) Rat marrow given 100 mg/kg Extract plus Lead showing mild increase in the myeloid-erythroid cells (i) Rat marrow given 200 mg/kg Extract plus Lead showing mild increase in the myeloid-erythroid cells (j) Rat marrow given 250 mg/kg Extract plus Lead showing increase in the myloid-erythroid cells

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  Conclusion Top


This study showed that lead acetate intoxication is associated with oxidative stress and this is identified as the main major culprit in the failure of the antioxidant defense system and as well as the pathogenesis of renal dysfunctions. The present study also reveals that V. amygdalina possess potent phytochemicals that could be standardized for use in either kidney or other related oxidative damage diseases.

Acknowledgement

The authors are appreciative of all the support from Department of Anatomy, School of Basic Medical sciences, College of Medical sciences, University of Benin, Benin City, Nigeria for the use of their research laboratory and Enoghase Raymond Joseph for his technical assistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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