WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Ramadhan et al. World Journal of Pharmacy and Pharmaceutical Sciences

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Volume 13, Issue 11, 1499-1507 Research Article ISSN 2278 – 4357

PROTECTIVE EFFECT OF ANTIOXIDANT SUPPLEMENTS ON

KIDNEY FUNCTION IN VANCOMYCIN-STREPTOZOTOCIN
INDUCED DIABETICKIDNEY DISEASE IN RABBITS

Raghda K. Tuama1, Atheer AL Idan2, Usama H. Ramadhan1* and Falah H. Sheri1

1
Department of Clinical Laboratory Sciences, College of Pharmacy, University of Basrah,
Basrah, Iraq.
2
College of Pharmacy, Almaaqal University, Basrah, Iraq.

ABSTRACT
Article Received on
25 September 2024, Diabetes mellitus is a group of metabolic disorders characterized by
Revised on 15 Oct. 2024, hyperglycaemia and insufficiency in the production or action of
Accepted on 05 Nov. 2024
DOI: 10.20959/wjpps202411-28387 insulin. Long-term elevation in blood glucose levels is associated with
macro-and microvascular complications. Diabetic kidney disease
(DKD) is defined as diabetes with albuminuria or impaired glomerular
filtration rate or both of them. DKD is the single strongest predictor
of mortality in patients with diabetes. Forty male and female rabbits

*Corresponding Author weighing 1000-1300 gm were divided randomly into five groups, n=8
Prof. Dr. Usama H. in each group. Group 1 was normal and group 2 diabetic while group 3
Ramadhan vancomycin, group 4 treated 1 and group 5 treated 2. Diabetes mellitus
Department of Clinical
was induced in groups 2 to 5 after overnight fasting by a single
Laboratory Sciences,
intraperitoneal injection of Streptozotocin 50 mg/kg. After that, the
College of Pharmacy,
University of Basrah, animals began treatment with antioxidants, various antioxidants were
Basrah, Iraq. used orally in different combinations, in the treated 1 group (quercetin
15 mg/kg and L-carnitine 15 mg/kg) and treated 2 group (quercetin 15
mg/kg, L-carnitine 15 mg/kg, Thioctic acid 20 mg/kg and Vitamin C 15 mg/kg). A blood
sample of 3 ml was collected every two weeks, and the following biochemical: blood sugar,
serum urea, serum creatinine and serum LDL-c were measured. There was a non-significant
decrease in serum glucose level in the treated 1 group and treated 2 group compared with the
diabetic group, but a significant decrease in serum urea, creatinine and LDL-c levels in the
treated 1 group and treated 2 group compared with the diabetic group. The role of
antioxidants as adjuvant therapy is to decrease and prevent diabetic kidney disease through

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Ramadhan et al. World Journal of Pharmacy and Pharmaceutical Sciences

the scavenging effect of reactive oxygen species produced by diabetics in kidney tissues.

KEYWORDS: Antioxidant, Diabetic Kidney Disease, L-Carnitine, Quercetin, Thioctic

acid, Vitamin C.

INTRODUCTION
Diabetes mellitus is a metabolic disease characterized by relative or absolute insulin
deficiency, Diabetic kidney disease (DKD) seems to be one of the most common
complications of diabetes mellitus.[1] Diabetic kidney disease is known as Diabetic
Nephropathy (DN) and define as “diabetes with albuminuria (ratio of urine albumin to
creatinine ≥ 30mg/g), impaired glomerular filtration rate (<60 mL/min/1.73m2), or both”.
DKD is the single strongest predictor of mortality in patients with diabetes. Multiple large
trials have demonstrated that improved glycaemic control in patients with type 1 and 2
diabetes reduced microalbuminuria, macro albuminuria, and progression to DKD and
ESRD.[2]

Oxidative stress plays a pivotal role in the diabetes complications development, both
microvascular and cardiovascular. The metabolic abnormalities of diabetes cause
mitochondrial superoxide overproduction in endothelial cells of both large and small
vessels.[3] The damaging effects of oxidative stress are caused by the production of free
radicals of oxygen and reactive oxygen species (ROS). On the other hand, ROS substances
can modified by enzymatic or non-enzymatic antioxidants such as superoxide dismutase,
vitamins, minerals and polyphenols.[4]

There are many antioxidant agents used for delaying diabetic kidney disease progression and
restoring the antioxidant defence system. Thereby preventing ROS-mediated injuries like
quercetin, L-carnitine, Thioctic acid and Vitamin C.[5]

MATERIALS AND METHODS

Animal care
Forty male and female rabbits weighing 1000-1300 gm were obtained from a local market
in Basrah city, the rabbits were separated into five groups (eight rabbits in each group) at
the animal house of the College of Pharmacy / University of Basrah.

Group 1 had normal control, group 2 had diabetic control, group 3 had vancomycin control,
group 4 treated 1 and group 5 treated 2. They were kept for two weeks to acclimate before

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Ramadhan et al. World Journal of Pharmacy and Pharmaceutical Sciences

the experiment began. The rabbits were housed in a light-controlled, air- conditioned
atmosphere at a constant temperature and supplied with food ad libitum. The rabbits were fed
trefoil, bread and lettuce with free access to tap water. Also, stress is reduced by avoiding
noisiness and harsh handling. Animals used in this study were handled by the National
Institute of Health (NIH) guidelines for handling laboratory animals.

Preparation of antioxidants supplement

Antioxidant supplements were purchased from private pharmacies in Basrah as tablets. The
tablets were crushed with a mortar to a powder and then dissolved in distilled water to obtain
a suitable solution or suspension for oral administration to rabbits. The various antioxidants
were used in different combinations to treated 1 group (quercetin 15 mg/kg and L-carnitine
15 mg/kg) and treated 2 group (quercetin 15 mg/kg, L-carnitine 15 mg/kg, Thioctic acid
20mg/kg, and Vitamin C 15mg/kg).

Induction of diabetes mellitus

Diabetes mellitus was induced in the overnight fasted rabbits by a single IP injection of
Streptozotocin 50mg/kg of body weight. Streptozotocin was prepared immediately before
injection by dissolving it in citrate buffer (pH 4.5). After 5 hours of injection, drinking water
is replaced by a glucose solution of 5% for all rabbits injected with Streptozotocin to
overcome the high insulin release that causes hypoglycaemia. Hyperglycaemia in rabbits was
followed up within five days by ear vein tipping using a glucometer Accu-Chek active meter,
advance checks up by blood sampling and analysis for glucose concentration using a glucose
kit. Rabbits with random blood sugar concentrations of more than 200 mg/dl were considered
diabetic.

Preparation of vancomycin
Vancomycin was dissolved in normal saline to obtain a solution concentration of
(200mg/ml). A dose of (200mg /kg) of body weight was injected intraperitoneally (IP) for
each rabbit in specific groups (Diabetic Control group, Vancomycin control group, treated
1 group and treated 2 group).

Treatment
Vancomycin was administered at an initial dose (200mg/kg, IP) after induction of diabetes
mellitus to increase renal injury. Next, the animals began treatment with antioxidants. The
vancomycin dose was repeated after two weeks. Blood samples were taken in a volume of 3

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ml every two weeks 4 times during the study. The blood samples were collected
independently by direct withdrawal from the heart, taking into account the survival of rabbits
alive. The blood sample was placed in a gel-tube and centrifuged at 4,000 rpm for 12 minutes
to obtain serum. The serum was collected and stored in a deep- freezer at -20 °C for later
analysis. Antioxidants treatment was continued for 40 days. Table 1 shows animals’ groups,
drugs doses and duration of treatments.

Table 1: Summary of animal grouping, drug dosing, and duration of treatments.

Duration of
Group Treatment
treatment
Group 1 -citrate buffer 1ml/kg, IP -once only
(Normal -normal saline 1ml/kg, IP -twice only
Control) -Distilled water (4ml/kg/day) orally -40 days
Group 2 -Streptozotocin 1ml/kg (50mg/kg), IP -once only
(Diabetic -Vancomycin 1ml/kg (200mg/kg), IP -twice only
Control) -Distilled water (4ml/kg/day) orally -40 days
Group 3 -citrate buffer solution 1ml/kg, IP -once only
(Vancomycin -vancomycin solution 1ml/kg (200mg/kg), IP -twice only
control) -Distilled water (4ml/kg/day) orally -40 days
-Streptozotocin 1ml/kg(50mg/kg), IP
-once only
-Vancomycin solution1ml/kg(200mg/kg), IP
Treated 1 -twice only
- Solution of two antioxidants quercetin 1ml/kg (15
-40 days
mg/kg) and L-carnitine 1ml/kg (15 mg/kg)
-Streptozotocin 1ml/kg (50mg/kg), IP
-Vancomycin solution 1ml/kg(200mg/kg), IP -once only
Treated 2 -Solution of four antioxidants quercetin 1ml/kg (15 -twice only
mg/kg) + L-carnitine 1ml/kg (15 mg/kg) + Thioctic acid -40 days
1 ml/kg (20mg/kg)+ Vitamin C 1ml/kg (15mg/kg )

Assays
The following serum parameters were measured: glucose, urea, creatinine, and low-
density lipoprotein (LDL-c) cholesterol.

Statistical Analysis
Data represent the Mean ± standard deviation (SD) of samples. Analysis wasmade by using
SPSS (statistical package for social sciences) for Windows (version 25) and Microsoft
Office (2016). Differences among different groups are compared by one- way analysis of
variance (ANOVA). The statistical significance for p level at p ≤ 0.05.[5]

RESULTS
Random blood glucose: Table 2 shows a significant (P<0.001) increase in random blood

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glucose for three diabetic-induced groups (diabetic control, treated one, and treated two)
compared to the normal control group and Vancomycin control group. On the other hand,
non-significant difference (P>0.05) between treated one and treated two groups compared
with the diabetic control group. Also, a non-significant difference between the Vancomycin
control group and the normal control group (P>0.05).

Table 2: Effect of different antioxidants and vancomycin on the serum glucose level.
Serum glucose (mg/dL)
Group
Blood sample 1 Blood sample 2 Blood sample 3 Blood sample 4
Normal control 101.6±8.68 104.9±6.96 106.48±13.81 113.2±11.05
a,c a,c a,c
Diabetic control 236.5±26.14 233.7±25.36 200.1±14.89 180.8±17.10a,c
Vancomycin control 103.7±14.0 102.5±7.45 112.33±17.53 112.3±16.96
a,c a,c a,c
Treated one 232.0±23.74 221.3±22.40 185.6±24.67 173.1±23.21a,c
Treated two 231.6±24.62a,c 201.2±18.16a,c 189.2±26.33a,c 181.9±12.70a,c
Values represent mean + SD. N=8 for each group.
a= significant difference when compared with the normal control group at the same period;
b= significant difference when compared with the diabetic control group;
c= significant difference when compared with the vancomycin control group.

Serum urea levels: Table 3 shows a significant (p<0.001) increase in serum urea for
three diabetic-induced groups (diabetic control, treated one, and treated two) compared to the
normal control group and Vancomycin control group in blood sampling 1 (before starting of
antioxidants and vancomycin). And significant (p<0.001) decrease in serum urea in treated
one and treated two groups compared with diabetic control in blood sampling 2, blood
sampling 3, and blood sampling 4 (after starting of antioxidants). A non-significant difference
(p>0.05) between the Vancomycin control group and the normal control group for each
blood sample.

Table 3: Effect of different antioxidants and vancomycin on serum urea level.

Serum Urea (mg/dL)
Group
Blood sample 1 Blood sample 2 Blood sample 3 Blood sample 4
Normal Control 24.01±3.40 23.39±5.84 33.77±4.97 34.08±4.96
a,c a,c
Diabetic Control 116.8±49.38 132.1±86.6 109.0±52.47a,c 71.37±26.71a,c
Vancomycin control 25.67±4.08 37.79±6.82 40.27±4.34 44.25±5.50
a,c b
Treated one 114.7±46.54 42.62±14.59 40.91±6.46b 44.51±8.09b
a,c b
Treated two 131.8±65.0 34.85±10.81 35.70±3.76b 39.31±10.16b
Values represent mean + SD. N=8 for each group.
a= significant difference when compared with the normal control group at the same period;
b= significant difference when compared with the diabetic control group;

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c= significant difference when compared with the vancomycin control group.

Serum creatinine levels: Table 4 shows a significant increase in serum creatinine for three
Diabetic induced groups; diabetic control (P=0.001), treated one (P=0.005), and treated two
(P<0.001) compared to the normal control group in blood sampling 1 (before starting of
antioxidants and vancomycin). And significant (P<0.001) decrease in serum creatinine in
treated one and treated two groups compared with diabetic control in blood sampling 2,
blood sampling 3, and blood sampling 4 (after starting of antioxidants).

A non-significant difference (P>0.05) between the Vancomycin control group and the
normal control group for each blood sample.

Table 4: Effect of different antioxidants and vancomycin on serum creatinine level.

Creatinine (mg/dL)
Group
Blood sample 1 Blood sample 2 Blood sample 3 Blood sample 4
Normal Control 0.95 ± 0.16 0.88 ± 0.192 0.93 ± 0.12 0.72 ± 0.24
Diabetic Control 2.51 ± 0.93ac 2.73 ± 1.02ac 2.30 ± 0.90ac 1.90 ± 0.83ac
Vancomycin control 0.78 ± 0 .28 0.97 ± 0.28 1.01 ± 0.24 0.83 ± 0.26
Treated one 2.30 ± 0.81ac 1.03 ± 0.15b 0.86 ± 0.32b 0.67 ± 0.33b
Treated two 2.90 ± 1.51ac 0.95 ± 0.23b 0.91 ± 0.16b 0.77 ± 0.25b
Values represent mean + SD. N=8 for each group.
a= significant difference when compared with the normal control group at the sameperiod;
b= significant difference when compared with the diabetic control group;
c= significant difference when compared with the vancomycin control group.

Serum Low-density lipoprotein Cholesterol (LDL-c) shown in Table 5 a significant

(P<0.001) increase in serum LDL-c for three Diabetic induced groups (diabetic control,
treated one, and treated two) compared to the normal control group and Vancomycin control
group in blood sampling 1 (before starting of antioxidants and vancomycin). A significant
difference (P<0.001) between treated one and treated two groups compared with the diabetic
control group in blood sampling 2, blood sampling 3, and blood sampling 4 (after starting of
antioxidants). A significant (P<0.001) increase in serum LDL-c in the vancomycin control
group compared to the normal control group in blood sampling 2, blood sampling 3, and
blood sampling 4 (after starting vancomycin).

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Table 5: Effect of different antioxidants and vancomycin on serum LDL-c level.

Serum (LDL-c) (mg/dL)
Group
Blood sample 1 Blood sample 2 Blood sample 3 Blood sample 4
Normal Control 49.26 ± 27.13 49.58 ± 20.46 59.46 ± 17.02 61.4 ± 28.39
Diabetic Control 108.25 ± 10.66ac 137.86 ± 46.55 158.20 ± 25.87 144.40 ± 39.24
Vancomycin control 45.00 ± 24.48 124.2 ± 47.06a 109.63 ± 25.82ab 113.20 ± 3.99ab
Treated one 96.00 ± 31.18ac 70.26 ± 31.06cb 47.96 ± 36.30cb 82.14 ± 18.12cb
Treated two 105.62 ± 10.14ac 82.92 ± 17.55cb 90.72 ± 7.99ab 93.83 ± 6.19ab
Values represent mean + SD. N=8 for each group.
a= significant difference when compared with the normal control group at the sameperiod;
b= significant difference when compared with the diabetic control group;
c= significant difference when compared with the vancomycin control group.

DISCUSSION
The local and systemic oxidative stress “that underlies the pathological features of diabetic
nephropathy” results from the imbalance in the production of oxidants/ antioxidants. Also,
the powerful antioxidant mechanism is balanced oxidative aggression. Furthermore,
antioxidants have to modulate multiple cell signalling molecules such as pro-inflammatory
cytokines, transcription factors, apoptosis proteins, and various endogenous antioxidants.[6]

The diabetic control group showed increased serum glucose, urea, creatinine, and LDL-c.
There was a non-significant decrease in serum glucose levels in the treated groups
compared with the diabetic control group. The effect of antioxidants in decreasing serum
glucose levels may depend on the dose and duration of the antioxidant treatment. Some
studies showed the role of antioxidants like quercetin that have a positive influence on
glucose metabolism in the skeletal muscle and liver, quercetin is from the flavonoid family
and has the most potent antioxidant activity.[7] There was a significant decrease in serum
(urea, creatinine, and LDL-c) treated groups compared with the diabetic controlgroup.

Many studies showed the effectiveness of antioxidants against diabetic nephropathy and a
decrease in serum urea levels. Antioxidants like vitamin C and vitamin E are effective
clinically for diabetic nephropathy treatment and have a significant decrease in high blood
urea in diabetic patients.[8] On the other hand, the effectiveness of antioxidants in decreasing
serum creatinine like the use of saffron extract (crocin) decreased the high level of serum
creatinine in streptozotocin-induced diabetic rats.[9]

There was a significant increase in serum LDL-c level in the vancomycin control group

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compared with the normal control group. Also, nephrotoxicity is caused by some drugs such
as vancomycin, animal and human studies indicate that nephrotoxicity induced by
vancomycin occurs through glomeruli destruction and accumulation in the proximal renal
tubule leading to cellular necrosis.[10] Also, the role of antioxidants in improving lipid profile
like the use of L-carnitine supplementation to improve lipid profile that include LDL-c
level.[11] Other studies show that quercetin decreases the effect on the levels of lipid
profile.[12]

There are several methods for inducing diabetes, the chemical method of streptozotocin-
induced diabetes represents the most important and preferable experimental model for
diabetic induction, a lower dose of streptozotocin leads to less B cell destruction.[13]

CONCLUSION
The study examined the effect of antioxidant agents and kidney function status in
streptozotocin-induced diabetic rabbits, there was a significant decrease in serum (urea,
creatinine, and LDL-c) treated groups compared with the diabetic control group. The results,
therefore, suggest that the antioxidant agents may be useful in ameliorating the effect of
diabetes and oxidative stress-related kidney dysfunction and the use of antioxidants as
adjuvant therapy in decreasing and preventing diabetic kidney disease.

Conflict of interest: The authors declare there is no conflict of interest.

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