Carvedilol attenuates ischemia–reperfusion-induced oxidative renal injury in rats

doi: 10.1111/j.1472-8206.2004.00279.x

OR IG INAL

ART ICLE

Carvedilol attenuates ischemia–reperfusion-induced oxidative renal injury in rats

Devinder Singh, Vikas Chander, Kanwaljit Chopra*Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh – 160 014, India

INTRODUCT ION

Despite significant advances in critical care medicine,

acute renal failure (ARF) remains a major clinical

problem, and mortality associated with ARF has not

decreased substantially over the past 50 years [1,2].

Renal ischemia is a major cause of ARF, initiating a

complex and interrelated sequence of events, resulting

in injury to, and the eventual death of renal cells [1,3].

The prognosis is complicated by the fact that reper-

fusion, although essential for the survival of ischemic

renal tissue, causes additional damage (reperfusion

injury) [4], contributing to the renal dysfunction and

injury associated with ischemia/reperfusion (I/R) of the

kidney [1,3,4]. Furthermore, it appears that the prox-

imal tubule (PT) is particularly susceptible to injury

caused by renal I/R [5,6]. Renal I/R injury occurs in

many settings, including shock, vascular surgery, renal

transplantation and as well the early allograft rejection

subsequent to renal transplantation. Increasing evi-

dence has accumulated over the past decade indicating

that production of reactive oxygen species (ROS) such

as hydrogen peroxide, superoxide and hydroxyl radicals

contribute to renal I/R injury (and associated ARF)

[4,5].

Thus, motivated by the fact that previous interven-

tions against ARF have proved to be largely ineffective

and that dialysis still remains the only effective therapy

[2], development of novel therapeutic interventions

targeted at ameliorating renal injury mediated by I/R

Keywords

carvedilol,

ischemia–reperfusion,

oxidative stress

Received 1 March 2004;

revised 8 June 2004;

accepted 11 June 2004

*Correspondence and reprints:

[email protected]

ABSTRACT

There is increasing evidence to suggest that toxic oxygen radicals play a role in the

pathogenesis of ischemia/reperfusion (I/R) injury in the kidney. This study was

designed to investigate the effects of carvedilol (CVD), an antihypertensive drug in

I/R-induced renal failure in rats. The protective effect of CVD against the damage

inflicted by reactive oxygen species (ROS) during renal I/R was investigated in

Sprague–Dawley rats using histopathological and biochemical parameters. In one set

of experiments, animals were unilaterally nephrectomized, and subjected to 45 min

of left renal pedicle occlusion and in another set both the renal pedicles were occluded

for 45 min followed by 24 h of reperfusion. Carvedilol (2 mg/kg, i.p.) was

administered twice, 30 min prior to ischemia and 12 h after the reperfusion period.

At the end of the reperfusion period, rats were killed. Thiobarbituric acid-reactive

substances (TBARS), reduced glutathione (GSH) levels, catalase (CAT) and super-

oxide dismutase (SOD) activities were determined in renal tissue. Serum creatinine

and blood urea nitrogen (BUN) concentrations were measured for the evaluation of

renal function. Ischemic control animals demonstrated severe deterioration of renal

function, renal morphology and a significant renal oxidative stress. Pretreatment of

animals with CVD markedly attenuated renal dysfunction, morphological alterations,

reduced elevated TBARS levels and restored the depleted renal antioxidant enzymes.

The findings imply that ROS play a causal role in I/R-induced renal injury and CVD

exerts renoprotective effects probably by the radical scavenging and antioxidant

activities.

� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 627–634 627

of the kidney, and associated ARF, have been topics of

intense research interest. In the present study, the

antioxidant and renoprotective potential of carvedilol

(CVD) in renal I/R injury is assessed.

Carvedilol is a third-generation, nonselective b-blocker

and is used in the treatment of hypertension, angina and

congestive heart failure. Carvedilol and some of its

metabolites (SB 211475 and SB 209995) are potent

antioxidants and this activity has been attributed to the

carbazole moiety of the drug [7–14]. This antioxidative

function would provide an additional benefit given that

CVD is used in the treatment of patients with reno-

vascular hypertension. However, the mechanism of its

antioxidative action is still unclear. It scavenges the free

radicals as well as forms a complex with iron ion [15].

The protective effects of this drug have been demonstra-

ted in a variety of in vitro and in vivo systems [8,16,17].

METHODS

Animals

Male Sprague–Dawley rats (150–200 g) bred in the

central animal house of Panjab University (Chandigarh,

India) were used. The animals were housed under

standard conditions of light and dark cycle with free

access to food (Hindustan Lever Products, Kolkata, India)

and water. The experimental protocols were approved by

the institutional animal ethics committee of Panjab

University, Chandigarh.

Carvedilol treatment

Carvedilol was dissolved in normal saline and was given

intraperitoneally 30 min before surgery and was repea-

ted 12 h after the institution of reperfusion. In our

preliminary studies dose ranges from 0.5 to 4 mg were

tested, and the range of 1–3 mg was found to be most

effective in preservation of renal function following I/R

with least effect on the hemodynamic parameters. The

higher doses of 3 and 4 mg did not show any advantage

in the renal function preservation; however, they

produced a significant decrease (P < 0.05) in systolic

blood pressure and heart rate.

Experimental protocols

The rats were anesthetized with thiopental sodium

(40 mg/kg, i.p.). The abdominal region was shaved with

a safety razor and sterilized with povidone iodine solution.

A midline incision was made and both the kidneys were

isolated. Renal ischemia was instituted by using two

different sets of protocols. In one, both the renal pedicles

were occluded, whereas, in the other, only the left renal

pedicle was occluded after right nephrectomy. The neph-

rectomy group was employed to delineate the effect of

contralateral nephrectomy on I/R. Ischemia was given for

45 min followed by reperfusion for 24 h. After the surgical

procedures, the midline incision was sutured back with

the local applications of povidone and neosporin. The

animals were allowed to recover from anesthesia.

A total of 40 rats were divided into five groups each

consisting of eight animals. The control group (group I,

C) animals underwent the exposure of both the renal

pedicles, but did not receive any ischemia reperfusion.

Ischemic control group (group II, I/R) animals were

subjected to 45 min of bilateral ischemia plus 24 h

reperfusion. Nephrectomized ischemia control group

(group III, NP + I/R) animals underwent right nephrec-

tomy and after 10 min of stabilization 45 min of left

renal ischemia and 24 h reperfusion. Carvedilol-treated

ischemic group (group IV, CVD + B I/R) animals were

treated the same way as the group II except that 30 min

prior to ischemia they were treated with CVD (2 mg/kg,

i.p.), which was repeated 12 h after the institution of

reperfusion. This dose of CVD was selected on the basis of

extensive literature survey and our preliminary studies.

Carvedilol-treated nephrectomized ischemic group

(group V, CVD + NP + I/R) animals were treated the

same way as the group III animals except that 30 min

prior to ischemia they were treated with CVD (2 mg/kg,

i.p.), which was repeated 12 h after the institution of

reperfusion. At the end of 24 h of reperfusion, the

animals were killed with a high dose of anesthesia and

blood was collected in heparinized centrifuge tubes from

the abdominal aorta. Serum was isolated and was used

for the assessment of renal function tests. A midline

abdominal incision was performed and both the kidneys

were isolated with the left kidney kept in deep frozen

condition for further enzymatic analysis, and the right

kidney stored in 10% formalin for histological sectioning.

In nephrectomized animals, additional groups were

employed for harvesting the left kidney for histological

analysis.

Assessment of renal function

Serum samples were assayed for blood urea nitrogen

(BUN) and serum creatinine by using standard diagnos-

tic kits (Span Diagnostics, Sachin, Gujarat, India).

Postmitochondrial supernatant preparation

After killing the animals, their kidneys were quickly

removed, perfused immediately with ice-cold normal

628 D. Singh et al.

� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 627–634

saline and homogenized in chilled potassium chloride

(1.17%) using a Potter Elvehjem homogenizer (Remi,

Mumbai, India). The homogenate was centrifuged at

800 g for 5 min at 4 �C in a refrigerated centrifuge to

separate the nuclear debris. The supernatant so obtained

was centrifuged at 10 500 g for 20 min at 4 �C to get

the postmitochondrial supernatant (PMS) which was

used to assay reduced glutathione (GSH), glutathione

reductase (GR), catalase (CAT), and superoxide dismu-

tase (SOD) activity.

Estimation of lipid peroxidation

The malondialdehyde (MDA) content, a measure of lipid

peroxidation, was assayed in the form of thiobarbituric

acid-reacting substances (TBARS) [18]. In brief, the

reaction mixture consisted of 0.2 mL of 8.1% sodium

lauryl sulphate, 1.5 mL of 20% acetic acid solution

adjusted to pH 3.5 with sodium hydroxide and 1.5 mL of

0.8% aqueous solution of thiobarbituric acid was added

to 0.2 mL of 10% (w/v) of PMS. The mixture was made

up to 4.0 mL with distilled water and heated at 95 �C for

60 min. After cooling with tap water, 1.0 mL distilled

water and 5.0 mL of the mixture of n-butanol : pyridine

(15 : 1 v/v) was added and centrifuged. The organic

layer was taken out and its absorbance measured at

532 nm. TBARS were quantified using an extinction

coefficient of 1.56 · 105/M/cm and expressed as nano-

grams of TBARS per milligram of protein. Tissue protein

was estimated using the Biuret method [19] of protein

assay and the renal MDA content expressed as nano-

moles of MDA per milligram of protein.

Estimation of antioxidant enzymes (AOE)

The AOE were estimated by the well-established proce-

dures already published elsewhere [20]. The nonprotein

sulfhydryl (NPSH) as a marker for reduced GSH, was

measured by the method of Jollow et al. [21] and the

yellow color developed by the reduction of Ellman’s

reagent by -SH groups of NPSH was read at 412 nm. The

GR activity was measured by the NADPH oxidation

method of Mohandas et al. [22]. The CAT activity was

assayed by the method of Claiborne [23] and the rate of

decomposition of H2O2 was followed at 240 nm. The

SOD activity was assessed by the method of Kono [24].

The nitro blue tetrazolium (NBT) reduction by super-

oxide anion to blue formazon was followed at 560 nm.

Renal histology

The kidneys fixed in a 10% neutral-buffered formalin

solution were embedded in paraffin and were used for

histopathological examination. Five-micrometer-thick

sections were cut, deparaffinized, hydrated and stained

with hematoxylin and eosin. The renal sections were

examined in blindly for tubular cell swelling, cellular

vacuolization, pyknotic nuclei, medullary congestion

and moderate to severe necrosis in all treatments. A

minimum of 10 fields for each kidney slide were

examined and assigned for severity of changes using

0

0.5

1

1.5

2

2.5

3

3.5(a)

(b)

(c)

C I/R NP+I/R CVD+I/R CVD+NP+I/R

Ser

um

cre

atin

ine

(mg

/dL

) *

*

** ***

0

10

20

30

40

50

60

70

80

90


BU

N (

mg

/dL

)

*

*

** ***

0

0.1

0.2

0.3

0.4

0.5

0.6


Cre

atin

ine

clea

ran

ce (

mL

/min

)

*

*

**

***

Figure 1 Effect of carvedilol (2 mg/kg) on serum creatinine (a),

blood urea nitrogen (BUN) (b) and creatinine clearance (c) in rats

exposed to renal I/R. Values expressed as mean ± SEM. *P < 0.05

compared with control group; **P < 0.05 compared with I/R

group; ***P < 0.05 compared with NP + I/R group (one-way

ANOVA followed by Dunnett’s test).

Carvedilol in ischemia–reperfusion renal injury 629


scores on a scale of none ()), mild (+), moderate (++)

and severe (+++) damage.

Statistical analysis

Values are expressed as mean ± SEM. One-way analysis

of variance (ANOVA) followed by Dunnett’s test was

applied to calculate the statistical significance between

various groups. A value of P < 0.05 was considered to

be statistically significant.

RESULTS

Effect of CVD on renal I/R-induced renal

dysfunction

Animals that underwent renal I/R exhibited significant

increase in the serum concentrations of creatinine and

urea nitrogen when compared with control animals,

suggesting a significant degree of glomerular dysfunction

mediated by renal I/R. Renal I/R also produced a

significant reduction in creatinine clearance, which

was used as an indicator of glomerular filtration rate

and thus glomerular function. Treatment of rats with

CVD (2 mg/kg, i.p.) produced a significant reduction in

the serum levels of creatinine and urea nitrogen and a

significant increase in creatinine clearance associated

with I/R (Figure 1a–c).

Effect of CVD on renal I/R-induced lipid

peroxidation

Renal I/R produced a significant increase in TBARS

levels when compared with control animals. Treatment

with CVD (2 mg/kg, i.p.) produced a significant

reduction in TBARS in renal I/R-treated animals

(Figure 2).

Effect of CVD on renal I/R-induced changes

in the antioxidant pool

Renal I/R significantly decreased the enzymatic activity

of GSH, CAT and SOD. This reduction was significantly

improved by 2 mg/kg, i.p. treatment with CVD

(Figure 3a–c).

0

50

100

150

200

250


MD

A (

nm

ol/m

g p

rote

in)

*

*

**

***

Figure 2 Effect of carvedilol (2 mg/kg) on renal I/R-induced lipid

peroxidation (MDA). Values expressed as mean ± SEM. *P < 0.05

compared with control group; **P < 0.05 compared with I/R



0

1

2

3

4

5

6

7

8

9

10(a)

(b)

(c)




GS

H (

mo

l) ×

10–4

* *

**

***

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

CA

T (

k/m

in)

**

**

***

0

2

4

6

8

10

12

SO

D (

un

its/

mg

pro

rein

)

*

*

**

***

Figure 3 Effect of carvedilol (2 mg/kg) on reduced glutathione

(GSH) (a), catalase (b), and superoxide dismutase (SOD) (c), in rats

exposed to renal I/R. Values expressed as mean ± SEM. *P < 0.05

as compared to control group; **P < 0.05 compared with I/R



630 D. Singh et al.


Effect of CVD on renal I/R-induced changes

on renal morphology

The histopathological changes were graded and sum-

marized in Table I. The control group did not show any

morphological changes. By contrast, the kidneys of

untreated ischemic rats showed tubular cell swelling,

interstitial edema, tubular dilatation, hyaline casts

and moderate to severe necrosis. Treatment with CVD

(2 mg/kg, i.p.) preserved the normal morphology of the

kidney (Figure 4), and shows normal glomeruli with

slight edema of the tubular cells.

DISCUSS ION

Renal ischemia causes changes that start with vasocon-

striction and a decrease in glomerular filtration rate, and

that could end up with ARF. Oxygen-free radicals have

been implicated in the pathogenesis of I/R injury in

various organs, such as liver, intestine, and kidney

[25–28]. If free radical-mediated lipid peroxidation

remains uncontrolled, cell death will ultimately result.

ROS have been considered to exert their effects through a

direct toxic action on target cells. For example, ROS

causes DNA damage during I/R and oxidative stress

[29,30] leading to the activation of the nuclear enzyme

poly (ADP-ribose) polymerase (PARP), depletion of NAD

and adenosine 5¢-triphosphate (ATP) and ultimately cell

Table I Effect of carvedilol (2 mg/kg) treatment on morphological

changes as assessed by histopathological examination of kidneys of

the rats exposed to renal I/R.

Group

Tubular

cell swelling

Interstitial

edema

Tubular

dilatation

Necrosis of

epithelium

Hyaline

casts

C ) ) ) ) )

I/R +++ +++ +++ +++ +++

NP + I/R +++ +++ +++ +++ +++

CVD + BI/R ) ) ) ) )

CVD + NP + I/R ) ) ) ) )

C, control; I/R, ischemia/reperfusion; NP + I/R, nephrectomy ischemia/

reperfusion; CVD + BI/R, carvedilol-treated + ischemia/reperfusion;

CVD + NP + I/R, carvedilol-treated + nephrectomy + ischemia/reperfusion.

(a) (b)

(c)

(e)

(d)

Figure 4 Hematoxylin and eosin-stained

sections of rat kidneys: (a) normal kidney

section of control rat; (b) kidney section

of the rat exposed to bilateral renal I/R;

(c) kidney section of the rat exposed to

left renal I/R after right nephrectomy;

(d) kidney section of carvedilol (2 mg/kg) +

bilateral renal I/R-treated rat showing

normal morphology; (e) kidney section of

carvedilol (2 mg/kg) + left renal I/R

after right nephrectomy treated rat

showing normal morphology.



death [19,30]. Furthermore, various antioxidant strat-

egies such as the use of TEMPOL or deferoxamine have

been shown to be beneficial against renal dysfunction

and injury mediated by I/R of the kidney [31]. Experi-

mentally various antioxidant agents such as SOD, CAT,

allopurinol, iloprost, and calcium channel blockers were

used to prevent kidney from I/R injury [25,32,33].

In this study, renal I/R caused an increase in the renal

TBARS levels and depleted the antioxidant enzyme pool,

as evident from the declined levels of reduced GSH, CAT,

GR, and SOD enzymes. Renal I/R-induced oxidative

stress was associated with impaired renal function

leading to a marked increase in serum creatinine, urea

nitrogen and a marked fall in the creatinine clearance.

Moreover, the kidney of rats that underwent I/R

(+ contralateral nephrectomy) showed characteristic

morphological changes such as tubular cell swelling,

cellular vacuolization, pyknotic nuclei, medullary con-

gestion and moderate to severe necrosis. Oxidative stress

can promote the formation of a variety of vasoactive

mediators that can affect renal function directly by

causing renal vasoconstriction or decreasing the glom-

erular capillary ultrafiltration coefficient, and thus

reduce the glomerular filtration rate [34,35]. It was

interesting to notice that pretreatment with CVD pre-

vented the renal I/R-induced lipid peroxidation and

protected the severe depletion of antioxidant enzyme

pool in the renal I/R-treated rats. Furthermore, the renal

functional and morphological damage was significantly

improved and CVD produced no per se hemodynamic

and morphological changes. The half-life of disintegra-

tion of ROS is very short because of their high reactivity.

For the scavenger to be efficient, it is necessary that they

are present on places of origin and action of ROS.

Carvedilol meets this condition because it is bound to

plasmatic proteins and is secreted by kidney. Renal

proximal tubules are the site of the protective effect of

CVD. Based on the results of this study, it is possible to

state that CVD prevents the functional and the morpho-

logical alterations in kidney. The results of our study are

in consistence with those of Necas et al. [36], where the

CVD-fed rats showed a significant morphological protec-

tion and reduced lipid peroxidation after 60 min of

ischemia and 10 min of reperfusion.

Carvedilol inhibited the lipid peroxidation in myocar-

dial cell membranes initiated by oxygen radicals gener-

ated by chemical, enzymatic or cellular systems [8], also

protected endothelial cells from oxygen radical-mediated

injury. Carvedilol inhibited superoxide ion release from

activated neutrophils [10]. Carvedilol preserves the

endogenous antioxidant systems (i.e. vitamin E and

GSH) that are normally consumed when tissue or organs

are exposed to oxidative stress [37]. Carvedilol also

protects against peroxinitrite (ONOO)) toxicity and

reported to increase GSH levels [38].

The antioxidant action of CVD has been speculated to

be because of (1) inhibition of direct cytotoxic actions of

free radicals, (2) prevention of oxygen-free radicals from

activated transcription factors such as NF-jB and

(3) protecting and replenishing the endogenous anti-

oxidant defense mechanisms, GSH and vitamin E.

However, the dynamics of the antioxidant action of

CVD are not known, and Yue et al. [8] have reported

that CVD inhibits lipid peroxidation by scavenging free

radicals, while some others reported that CVD is not a

free-radical scavenger but rather a sequester of ferric ion

[16,39]. Carvedilol is also reported to act as a calcium

channel blocker [40,41].

Whatever could be the mechanism, our results con-

firm that CVD has an antioxidant activity. This activity

appears particularly relevant for the understanding

of the molecular mechanisms that underlie the action

of CVD, but also represents a valid rationale for the use of

CVD in the prevention and therapy of renal I/R injury.

In conclusion, the findings of the present study

strongly suggest the role of oxidative stress in the

pathophysiology of renal I/R injury and that CVD could

be used for the prevention and treatment of renal

I/R-exposing procedures.

ACKNOWLEDGEMENTS

The Senior Research Fellowship of the Council of

Scientific and Industrial Research (CSIR), New Delhi, is

gratefully acknowledged. The authors also thank Zydus-

Medicus for their kind gesture of providing the gift,

carvedilol.

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Carvedilol attenuates ischemia–reperfusion-induced oxidative renal injury in rats