ORIGINAL ARTICLE

Nephroprotective effect of catechin on gentamicin-inducedexperimental nephrotoxicity

Ankush Sardana • Sanjeev Kalra • Deepa Khanna •

Pitchai Balakumar

Received: 25 September 2013 / Accepted: 26 April 2014

� Japanese Society of Nephrology 2014

Abstract

Background Gentamicin is an effective aminoglycoside

antibiotic employed against severe Gram-negative bacterial

infections, but induction of nephrotoxicity limits its fre-

quent clinical use. This study was undertaken to investigate

the effect of catechin hydrate on gentamicin-induced

nephrotoxicity in rats.

Methods Rats were administered nephrotoxic dose of

gentamicin (100 mg/kg/day, i.p.) once daily for 14 days.

Gentamicin-administered rats were treated with catechin

hydrate (50 mg/kg/day, per os), the treatment was started

3 days before the administration of gentamicin while it was

continued for 14 days from the day of gentamicin

administration.

Results Two weeks administration of gentamicin signifi-

cantly increased the serum creatinine and blood urea

nitrogen levels. Renal histopathological examination of

gentamicin-administered rats revealed degenerative chan-

ges in glomeruli and tubules after 2 weeks. These renal

structural and functional abnormalities in gentamicin-

administered rats were accompanied with renal oxidative

stress as assessed in terms of marked decrease in renal-

reduced glutathione (GSH). However, catechin hydrate

treatment showed considerably nephroprotective action

against gentamicin-induced nephrotoxicity in rats by

preventing aforementioned renal structural and functional

abnormalities and oxidative stress.

Conclusion Catechin hydrate has a potential to prevent

gentamicin-induced experimental nephrotoxicity. The ren-

oprotective effect of catechin hydrate against gentamicin-

induced nephrotoxicity might be mediated through its

antioxidant and possible direct nephroprotective actions.

Keywords Gentamicin � Nephrotoxicity � Oxidative

stress � Catechin hydrate � Direct nephroprotection

Introduction

Aminoglycosides are potent broad-spectrum antibiotics that

kill the bacteria by binding to the 30 s subunit of the bacterial

ribosome and reducing the fidelity of protein synthesis [1].

Aminoglycosides are considered as clinically effective

antimicrobial agents used to date since their introduction

long ago. They are commonly used because of their key

properties such as rapid concentration-dependent bacterici-

dal effects, synergism with beta-lactam antibiotics, low rate

of resistance, and particularly low-cost therapy [2, 3]. Gen-

tamicin is an aminoglycoside antibiotic effective against

various Gram-negative bacterial infections. However, its

frequent clinical use is often limited with a criticism of its

adverse action on the renal system and subsequent induction

of nephrotoxicity [4–6]. The selective accumulation of

gentamicin in the renal proximal convoluted tubule results in

the induction of nephrotoxicity [7]. Gentamicin-induced

nephrotoxicity is characterized by tubular necrosis and glo-

merular congestion, resulting in decreased glomerular fil-

tration rate and renal dysfunction [6, 8]. In addition,

induction of oxidative stress and inflammatory cascades

plays a key role in gentamicin nephrotoxicity [6, 9–11].

A. Sardana � S. Kalra � D. Khanna (&)

Cardiovascular Pharmacology Division, Department of

Pharmacology, Institute of Pharmacy, Rajendra Institute of

Technology and Sciences, Sirsa 125 055, Haryana, India

e-mail: 7drdeepa@gmail.com

P. Balakumar

Pharmacology Unit, Faculty of Pharmacy, AIMST University,

Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia

123

Clin Exp Nephrol

DOI 10.1007/s10157-014-0980-3

Numerous pharmacological agents have been identified

to have a potential in preventing gentamicin nephrotoxicity

[6]. However, we do not have a promising intervention

clinically to blunt gentamicin nephrotoxicity. Basic research

in identifying a potent pharmacological intervention to sat-

isfactorily halt gentamicin nephrotoxicity is therefore

underway. Catechins are flavonoids, and green tea is one of

the major sources for catechins [12]. Green tea catechin has

potent antioxidant and anti-inflammatory properties [13–15].

Catechin has been shown to reduce vascular oxidative stress

by suppressing NADPH oxidase activity in rats [16]. Inter-

estingly, catechin was reported to afford renoprotection

against rhabdomyolysis-induced myoglobinuric acute renal

failure through its potent renal antioxidant action [17]. The

renoprotective potential of catechin was further confirmed

by the fact that catechin in 5/6 nephrectomized rats inhibited

the progress of glomerulosclerosis and interstitial fibrosis

[18]. Intriguingly, administration of tea catechin retarded the

progression of functional and morphological changes in the

kidney of diabetic rats [19]. Likewise, a recent study dem-

onstrated the renoprotective effects of catechin in diabetic

rats [20]. These studies certainly suggest a renoprotective

potential of catechin. However, the effect of catechin in

gentamicin-induced nephrotoxicity is not yet known. The

present study has therefore been designed to investigate the

effect of catechin hydrate in gentamicin-induced nephro-

toxicity in rats.

Materials and methods

The experimental protocol employed in the present study

has been approved by the ‘Institutional Animal Ethics

Committee’ in accordance with the guidelines of the

‘Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA)’ Chennai, India. Wi-

star albino rats of either sex weighing about 200–250 g

were employed in the present study. The rats were accli-

matized in the animal house and maintained on rat chow

(Ashirwad Industries, Mohali, Punjab, India) and tap water.

Rats were allowed ad libitum access to food and water.

They were exposed to normal day and night cycles.

Induction of experimental nephrotoxicity

Experimental nephrotoxicity was induced in rats by

administering gentamicin (100 mg/kg/day i.p.) for 14 days

[21, 22].

Assessment of gentamicin-induced nephrotoxicity

The development of nephrotoxicity in rats, 14 days after

the administration of gentamicin, was assessed by

measuring serum creatinine and blood urea nitrogen con-

centration using commercially available kits. In addition,

histopathological studies were performed to assess genta-

micin-induced renal structural abnormalities. Moreover,

renal oxidative stress was assessed by measuring reduced

glutathione (GSH).

Determination of serum creatinine

The serum creatinine concentration was quantified by

modified Jaffe’s method using the commercially available

kit (Transasia Bio-Medicals Ltd., Solan, India). In brief,

100-lL serum sample and 100-lL standard creatinine

solutions (2 mg/dl) were taken separately in glass tubes

and named as test (T) and standard (S), respectively. The

working reagent (1000 lL) containing alkaline picrate

solution was added in both tubes and mixed. The reaction

temperature was kept at 30 �C. The absorbance of T and S

at 20 s (T1, S1) and again at 80 s (T2, S2) was noted against

blank spectrophotometrically. The formation of a colored

complex as a result of a reaction between creatinine present

in serum sample and alkaline picrate present in working

reagent was measured at 505 nm.

The serum creatinine concentration was calculated using

the following formula:

Serum Creatinine mg/dLð Þ

¼ D A of Test

D A of Standard� Concentration of standard mg/dLð Þ

D A ¼ A2 � A1

A1 = Initial absorbance

A2 = Final absorbance

Concentration of the standard solution = 2 mg/dL.

Determination of blood urea nitrogen

The blood urea nitrogen was measured using the com-

mercially available kit (Crest Biosystems, Goa India).

Briefly, 10-lL standard solutions (40 mg/dL) and 10-lL

serum sample were taken separately in standard (S) and test

(T) glass tubes, respectively. The working enzyme reagent

(800 lL) was added in all glass tubes with thorough mix-

ing. All glass tubes were incubated at 37 �C for 5 min.

Then, the working starter reagent (200 lL) (containing

alkaline buffer) was added to all glass tubes, and they were

again incubated at 37 �C for 5 min. The absorbance of test

and standard at 30 s (T1, S1) and again at 60 s (T2, S2)

was noted against blank spectrophotometrically. The

principle involved in this measurement follows. Urease

present in the working enzyme reagent hydrolyzes urea to

ammonia and carbon dioxide. The ammonia formed further

combines with a ketoglutarate and NADH to form

Clin Exp Nephrol

123

Glutamate and NAD. The rate of oxidation of NADH to

NAD is measured as a decrease in absorbance in a fixed

time, which is proportional to the urea concentration in the

sample. The intensity of the color produced was measured

spectrophotometrically at 340 nm.

Blood urea concentration was calculated using the fol-

lowing formula:

Urea þ H2O þ 2 H �!þUREASE 2NHþ4 þ CO2

2NHþ4 þ 2a Ketoglutarate

þ 2NADH �! GLDH2L - glutamate þ 2NADþ

þ 2H2O

Blood urea concentration was calculated using the fol-

lowing formula:

Blood urea concentration mg=dLð Þ

¼ Absorbance of T

Absorbance of S� 40

Blood urea nitrogen BUNð Þ concentration mg=dlð Þ¼ 0:467� Blood urea concentration mg=dLð Þ:

Histopathological study

Gentamicin-induced renal structural changes in glomeruli and

tubules were assessed histologically with the help of Mangalam

Pathological Laboratory, Haryana, India. The kidney was

excised and immersed in 10 % formalin solution. The kidney

was then dehydrated in graded concentration of alcohol,

immersed in xylene and embedded in paraffin. From the par-

affin blocks, sections of 5 lm in thickness were made and

stained with hematoxylin and eosin to assess the pathological

changes that have occurred in glomeruli and tubules using light

microscopy at 40 X (Motic Digital Microscope BA310, USA).

Assessment of renal oxidative stress

A decrease in the level of GSH is an indication of oxidative

stress [23]. The development of oxidative stress in the

kidney was assessed by measuring GSH.

Preparation of renal homogenate

The kidney was excised and washed with ice cold isotonic

saline and weighed. The kidney weight to body weight

ratio (KW/BW, mg/g) was calculated. The kidney was then

minced, and a homogenate (10 % w/v) was prepared in

chilled 1.15 % KCL. The homogenate was used for the

measurement of renal GSH.

Determination of renal GSH

The renal GSH level was measured using the methods descri-

bed by Ellman [24] and Boyne and Ellman [25]. The renal

homogenate was mixed with 10 % w/v trichloroacetic acid in

1:1 ratio and centrifuged at 4 �C for 10 min at 5000 rpm. The

supernatant (0.5 mL) was mixed with 2 mL of 0.3 M disodium

hydrogen phosphate buffer (pH 8.4) and 0.4 mL of distilled

water. Then, 0.25 mL of 0.001 M freshly prepared DTNB [5,

50–dithiobis (2-nitrobenzoic acid) dissolved in 1 % w/v sodium

citrate] was added to the reaction mixture, and incubated for

10 min. The absorbance of the yellow colored complex was

noted spectrophotometrically at 412 nm. A standard curve

using the reduced form of glutathione was plotted to calculate

the concentration of renal GSH. The renal GSH concentration

was expressed as lM/g wet weight of renal tissue.

Statistical analysis

All values were expressed as mean ± S.D. Data obtained

from various groups were statistically analyzed using one

way ANOVA, followed by Tukey’s multiple comparison

test. A p \ 0.05 was considered statistically significant.

Drugs and chemicals

Catechin hydrate and DTNB were obtained from Sigma-

Aldrich Ltd., St. Louis, USA. Gentamicin was purchased

from Parth-Parentral, Kalol, India. Reduced glutathione

was obtained from SD Fine, Mumbai, India. Trichloro-

acetic acid was obtained from Rankem, New Delhi, India.

Thiobarbituric acid was obtained from Otto Chemika-Bi-

ochemica, Mumbai, India. All other chemicals used in the

present study were of analytical grade.

Experimental protocol

Rats were randomly divided into four groups with six rats

each. Catechin hydrate was solubilized in warm distilled

water.

Group 1 (Normal control): rats were maintained on stan-

dard food and water, and no treatment was given. Group 2

(Gentamicin control): rats were administered gentamicin

(100 mg/kg/day, i.p.) for 14 days. Group 3 (Catechin Hydrate

per se): the normal rats were administered catechin hydrate

(50 mg/kg/day, per os) for 14 days. Group 4 (Catechin

Hydrate Treated): rats administered gentamicin (100 mg/kg/

day, i.p., 2 weeks) were treated with catechin hydrate (50 mg/

kg/day, per os), and the treatment was started 3 days before

the administration of gentamicin and it was continued for

2 weeks from the day of administration of gentamicin.

Results

Administration of catechin hydrate (50 mg/kg/day, per os)

to normal rats did not produce statistically significant per

Clin Exp Nephrol

123

se effects on various parameters assessed in normal rats in

the present study.

Effect of catechin hydrate on serum creatinine

The serum creatinine level was noted to be markedly

increased in gentamicin-administered rats as compared to

normal rats. However, treatment with catechin hydrate

significantly reduced gentamicin-induced elevation of

serum creatinine levels (Table 1).

Effect of catechin hydrate on blood urea nitrogen

A significant increase in blood urea nitrogen was noted in

gentamicin-administered rats as compared to normal rats.

However, treatment with catechin hydrate significantly

reduced gentamicin-induced increase in blood urea nitro-

gen (Table 1).

Effect of catechin hydrate on oxidative stress

Gentamicin-administered rats exhibited a marked decrease

in renal concentration of GSH as compared to normal rats.

However, catechin hydrate treatment markedly prevented

gentamicin-induced decrease in renal GSH (Table 1).

Effect of catechin hydrate on renal histopathology

Renal structural pathological abnormalities in the glomer-

ulus and tubules were observed in gentamicin-administered

rats. As compared to normal rats, degeneration in glo-

merular wall and mild hypertrophy in glomerulus, while in

the tubules, mononuclear cell infiltration, degeneration in

epithelial layer, intertubular hemorrhage and hyaline casts

were found in gentamicin-administered rats. Studies

revealed the presence of dilated capillaries indicating

protein desorption material in gentamicin group. The

administration of catechin hydrate markedly reduced these

renal pathological changes and no casts were identified

(Figs. 1 and 2).

Discussion

Gentamicin, an aminoglycoside antibiotic effective against

severe Gram-negative bacterial infections, is known to be

potentially a nephrotoxic agent. In this study, we found

that the major antioxidant component of green tea,

‘catechin’ employed in the form of catechin hydrate could

serve as a preventive agent against gentamicin-induced

nephrotoxicity.

Renal dysfunction is often manifested with elevation in

serum creatinine and blood urea nitrogen [26, 27]. Ele-

vated serum creatinine concentration is indeed an indi-

cation of reduced glomerular filtration rate. The elevated

level of blood urea nitrogen as well is an index of renal

dysfunction because urea formed by the liver is cleared

from the blood by the kidney [28]. We observed in the

present study that gentamicin-administered rats exhibited

a marked elevation in serum creatinine and blood urea

nitrogen concentration. These results suggest an induction

of nephrotoxicity with renal functional abnormalities in

gentamicin-administered rats. These renal functional

abnormalities were noted to be accompanied with renal

oxidative stress as assessed in terms of a marked decrease

in renal GSH. These results suggest that gentamicin-

induced nephrotoxicity is correlated with an induction of

renal oxidative stress.

In the present study, catechin hydrate treatment signifi-

cantly prevented the elevated level of serum creatinine in

gentamicin-administered rats. Moreover, catechin hydrate

treatment significantly reduced high blood urea nitrogen

concentration in gentamicin-administered rats. These

results have certainly pointed out a potent nephroprotective

potential of catechin hydrate against gentamicin-induced

nephrotoxicity. In the present study, catechin hydrate

treatment was noted to improve the diminished level of

renal GSH in gentamicin-administered rats, showing its

potent antioxidant action. A direct renal antioxidant action

of catechin hydrate might have, therefore, chiefly contrib-

uted to its nephroprotective action against gentamicin

nephrotoxicity.

Table 1 Effect of catechin hydrate on serum creatinine, blood urea nitrogen and renal GSH in gentamicin-administered rats

Parameters/assessments Normal control Gentamicin control Catechin hydrate per se Catechin hydrate treated

Serum creatinine (mg/dL) 0.725 ± 0.23 3.58 ± 0.96a 0.71 ± 0.20 1.94 ± 0.72b

Blood urea nitrogen (mg/dL) 14.25 ± 2.65 37.44 ± 7.27a 16.32 ± 1.86 26.72 ± 2.99b

GSH (lM/g wet weight of renal tissue) 0.73 ± 0.09 0.29 ± 0.03a 0.65 ± 0.03 0.53 ± 0.02c

All values were represented as mean ± S.D.a p \ 0.001 versus normal controlb p \ 0.01 versus gentamicin control for serum creatinine and blood urea nitrogenc p \ 0.001 versus gentamicin control for GSH

Clin Exp Nephrol

123

Numerous studies have shown the renoprotective

potential of catechin in different sets of animal experi-

ments. Catechin treatment afforded renoprotection against

rhabdomyolysis-induced myoglobinuric acute renal failure

through its potent renal antioxidant action [17]. In addition,

catechin treatment in 5/6 nephrectomized rats inhibited the

progress of glomerulosclerosis and interstitial fibrosis

through a reduction in Ang II production [18]. Moreover,

tea catechin was reported to retard the progression of

functional and morphological changes in the kidney of

diabetic rats [19]. A recent study confirmed the renopro-

tective effects of catechin in diabetic rats [20]. These

studies strongly support the renoprotective potential of

catechin that was noted in the present study in gentamicin-

induced nephrotoxic rats.

The gentamicin-induced nephrotoxicity occurs due to

selective accumulation of the drug in renal proximal con-

voluted tubules, resulting in loss of brush border integrity.

The gentamicin nephrotoxicity involves renal-free radical

generation, reduction in antioxidant defense mechanisms,

and acute tubular necrosis and glomerular congestion,

leading to renal dysfunction [6, 8, 29–32]. Moreover,

gentamicin-administered rat kidneys are more susceptible

to oxidative damage because of the induction of deficiency

in antioxidant defense enzymes [33, 34]. In the present

study, histopathological analysis revealed renal structural

pathological abnormalities occurred in glomerulus and

tubules of gentamicin-administered rats. As compared to

normal rats, degeneration in glomerular wall and mild

hypertrophy in glomerulus were noted in gentamicin-

administered rats. In addition, in the tubules, mononuclear

cell infiltration, degeneration in epithelial layer, intertu-

bular hemorrhage and hyaline casts were found in genta-

micin-administered rats. However, treatment with catechin

hydrate markedly reduced aforementioned renal patholog-

ical changes in gentamicin-administered rats. In fact, Ha-

rada et al. [35] identified the major antioxidant metabolite

in biological fluids of the rat after the ingestion of catechin.

This study reported that the major antioxidant metabolite

appearing in biological fluids after the oral administration

Fig. 1 Effect of catechin hydrate on pathological changes in the

glomerulus of the Kidney. The kidney of the gentamicin-administered

rat developed pathological changes in the glomerulus such as

degeneration in glomerular wall and mild hypertrophy. Treatment

with catechin hydrate markedly reduced these pathological changes of

the glomerulus

Clin Exp Nephrol

123

of catechin was 5-o-beta-glucuronide [35]. Taken together,

on the fact of gentamicin-induced nephrotoxicity could

largely involve renal oxidative stress [6], the potent anti-

oxidant action of catechin hydrate might explain the pos-

sible mechanism involved in catechin hydrate-mediated

alleviation of gentamicin-induced renal structural and

functional abnormalities. The dose of the catechin hydrate

(50 mg/kg) was selected on the basis of previous studies

showing its different therapeutic actions [36–39].

On the basis of above discussion, it may be concluded

that catechin hydrate might have a therapeutic potential to

prevent gentamicin-induced renal structural and functional

abnormalities. The renoprotective effect of catechin

hydrate against gentamicin-induced experimental nephro-

toxicity might be mediated through its antioxidant action

and direct nephroprotective action.

Acknowledgments We express our gratefulness to Dr. Rajendar

Singh Sra, MD, Chairman, and Shri Om Parkash, Secretary, Rajendra

Institute of Technology and Sciences (RITS), Sirsa, Haryana, India,

for their support.

Conflict of interest The authors have declared no conflict of

interest.

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