Embed Size (px)
Citation preview
O R I G I N A L
A R T I C L E
Effect of tempol (4-hydroxy tempo) ongentamicin-induced nephrotoxicity in rats
Yusuf Karatasa*, M. Ata Secilmisa, _IIbrahim Karayaylalıb, Figen Doranc,Kansu Buyukafsard, Ergin Singirika, Yahya Saglıkerb, Atilla Dikmena
aDepartment of Pharmacology, Medical Faculty, Cukurova University, 01330 Adana, TurkeybDepartment of Nephrology, Medical Faculty, Cukurova University, 01330 Adana, TurkeycDepartment of Pathology, Medical Faculty, Cukurova University, 01330 Adana, TurkeydDepartment of Pharmacology, Medical Faculty, Mersin University Campus Yenisehir, 33169 Mersin, Turkey
I N T R O D U C T I O N
Gentamicin, an aminoglycoside antibiotic has a thera-
peutic value in the treatment of life threatening severe
gram-negative infections. However, the clinical use of
gentamicin is limited by its serious nephrotoxicity [1].
Although the mechanism underlying gentamicin-induced
renal cellular damage has not been entirely elucidated,
generation of superoxide anion, hydrogen peroxide
(H2O2) and hydroxyl radicals has been attributed to its
deleterious effect on the kidney [2,3]. Therefore, scaven-
ging of these radicals may have a protective effect against
gentamicin nephrotoxicity. Consistently, some antioxi-
dants and free radical scavengers such as deferroxamine
[4], polyascorbic acid [5], melatonin [6], probucol and
vitamin E [7], superoxide dismutase (SOD) [8], methimaz-
ole [9] have been reported to attenuate gentamicin-
induced renal failure, although allopurinol, an inhibitor of
xanthine oxidase, which is one of the responsible enzyme
for the production of superoxide anion failed to protect
against gentamicin-induced renal damage [10].
Tempol (4-hydroxy tempo) is an SOD-mimetic mem-
brane-permeable radical scavenger, which is able to
protect against oxidative stress in animals [11]. Tempol
also reduces renal dysfunction and damage by ischemia/
reperfusion [12] and intestinal injury of the rats subjec-
ted to splanchnic artery occlusion [13]. Therefore, the
aim of the present study was to examine the implication
of this membrane-permeable SOD-mimetic agent, tempol
in the prevention of the acute renal failure induced by
gentamicin.
M A T E R I A L A N D M E T H O D S
Animals
Sixty-two male Wistar albino rats, initially 200–250 g,
were acquired from Centre for Medical Research and
Application (TIPDAM) at Cukurova University, Adana,
Keywords
antioxidant,
free oxygen species,
gentamicin,
nephrotoxicity,
oxidative stress,
tempol
Received 23 January 2003;
revised 11 July 2003;
accepted 1 August 2003
*Correspondence and reprints:
A B S T R A C T
We investigated the effects of tempol (4-hydroxy tempo), a membrane-permeable
radical scavenger, on gentamicin-induced renal failure in rats. The rats were given
gentamicin (100 mg/kg/day, i.p., once a day); and gentamicin (100 mg/kg/day, i.p.)
and tempol (3.5, 7 or 14 mg/kg/day, i.p., once a day). At the end of 7 days, the
gentamicin group produced the remarkable nephrotoxicity, characterized by a
significantly decreased creatinine clearance and increased serum creatinine, blood
urea nitrogen (BUN) and daily urine volume when compared with controls. In
control the BUN value was 21.2 ± 0.07 (mg/100 mL); in comparison, it was
96.9 ± 6.03 in gentamicin group (P < 0.05). Renal histopathologic examination
confirmed acute tubular necrosis in this group. In rats treated with gentamicin and
tempol a partial improvement in biochemical and histologic parameters was
observed. BUN values were 96.9 ± 6.03 and 36.3 ± 2.39 in gentamicin, and
gentamicin plus tempol (14 mg/kg) treated groups, respectively (P < 0.05). These
results suggest that the administration of tempol may have a protective effect on
gentamicin-induced nephrotoxicity in rats.
� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 79–83 79
Turkey. They had free access to water and chow
ad libitum. All procedures were in accordance with
the institutional guidelines concerning animal experi-
mentation.
Experimental protocol
The rats were divided into eight different groups and
treated for 7 days as follows:
Group 1 (control) receiving saline (i.p. 0.2 mL/kg,
once a day, n ¼ 10).
Group 2 received gentamicin (i.p. 100 mg/kg, once a
day, n ¼ 10).
Groups 3, 4 and 5 received tempol (i.p. 3.5, 7 or
14 mg/kg, respectively, n ¼ 7 each).
Groups 6, 7 and 8 received gentamicin (i.p. 100 mg/kg)
and tempol (i.p. 3.5, 7 or 14 mg/kg, respectively, n ¼ 7
each).
All groups were treated once a day for seven successive
days. On the last day, a 24-h urine collection was obtained
in metabolic cages for measurement of clearance and
electrolyte studies. Twenty-four hours after last injection
the rats were anesthetized with ether and a blood sample
was immediately taken from the heart. Thereafter, the
kidneys were removed for histopathologic examination.
Histopathologic examination
The right kidney of the killed rats was removed and fixed
in 10% neutral buffered formaldehyde solution, dehy-
drated in graded alcohol, and embedded in paraffin.
Sections at 3–5 lm of thickness were obtained and
stained with hematoxylin–eosin (H & E). Light micro-
scopy was used to evaluate the following:
1. Tubular necrosis.
2. Tubular regenerative changes.
3. Tubulointerstitial mononuclear cell infiltration.
Tubular necrosis was graded (I–III) as follows:
Grade I (mild): Areas of subcapsular tubular necrosis in
small foci.
Grade II (moderate): Tubular necrosis at different foci
throughout the cortex.
Grade III (severe): Extensive and marked tubular necro-
sis throughout the cortex.
Tubular regeneration, presence of tubulointerstitial
mononuclear cell infiltration was graded similarly [14].
Drugs
Tempol (4-hydroxy tempo) was purchased from Sigma
Chemical Co. (St Louis, MO, USA), and gentamicin
sulphate from Deva (_IIstanbul, Turkey). Tempol was
dissolved in saline.
Data analysis
The mean values (±SEM) for each group were calculated
separately. All data were evaluated in a one-way
analysis of variance (ANOVA; Tukey’s b-test), using a
computer program, SPSS. P-values of <0.05 were
considered to be significant.
R E S U L T S
Effects of gentamicin on blood urea nitrogen,
serum creatinine level and clearance
Gentamicin (100 mg/kg, once a day) increased
blood urea nitrogen (BUN) levels from 21.2 ± 0.07
(mg/100 mL, control) to 96.9 ± 6.03 (P < 0.05). Simi-
larly, the level of creatinine went up from 0.5 ± 0.02
(mg/100 mL, in control) to 2.2 ± 0.33 (in gentamicin
group, P < 0.05). Creatinine clearance was decli-
ned in gentamicin group from 0.6 ± 0.02 (control,
mL/min/100 g body weight) to 0.2 ± 0.01 (P < 0.05).
Biochemical parameters were shown in Table I.
Effects of gentamicin on renal histology
Histopathologic examination of the kidney sections from
gentamicin (100 mg/kg) group demonstrated different
degrees of tubular regeneration mononuclear cell infil-
tration in all rats. Grade II acute tubular necrosis was
indicated in this group (Figure 1).
Effects of tempol on biochemical parameters and
renal histology in control and gentamicin group
Tempol alone (3.5, 7 or 14 mg/kg) did not differ BUN and
serum creatinine level and clearance. Nor did it change
renal histology. However, administration of gentamicin
plus tempol (3.5, 7 or 14 mg/kg) normalized the increased
BUN and serum creatinine level, and diminished creati-
nine clearance in a dose-dependent manner (Table I).
Table I Effects of gentamicin (100 mg/kg, once a day, for 7 days),
gentamicin plus tempol (14 mg/kg/day) on the levels of blood urea
nitrogen (BUN, mg/100 mL), creatinine (mg/100 mL), creatinine
clearance (mL/min/100 g body weight) and daily urine volume
(mL/day).
BUN Creatinine
Creatinine
clearance
Urine
volume n
Control 21.2 ± 0.07 0.5 ± 0.02 0.6 ± 0.02 10.1 ± 0.45 10
Gentamicin 96.9 ± 6.03a 2.2 ± 0.33 0.2 ± 0.01 18.7 ± 2.11 10
Gentamicin +
tempol
36.3 ± 2.39b 0.65 ± 0.06 0.47 ± 0.02 12.8 ± 1.28 7
aP < 0.05 different from control.bP < 0.05 different from gentamicin group.
80 Y. Karatas et al.
� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 79–83
Similarly, histopathologic examination of specimens
revealed a tubular regeneration and mononuclear cell
infiltration in gentamicin plus tempol (3.5, 7 and
14 mg/kg) groups (Figure 2a–c). In group 7, tubular nec-
rosis was conspicuously absent, moderate mononuclear
cell infiltration in interstitium and moderate regenerative
changes were observed. In group 8, there were mild
mononuclear cell infiltration and mild regenerative chan-
ges. But tubular necrosis was absent in this group. In
group 6 (data not shown), there were mild necrosis and
slight degenerative changes, tubular cells were flattened
resulting in relatively dilated lumens. Also in necrotic
area, proximal tubular cells often contained myeloid
bodies.
D I S C U S S I O N
In the present study, we have examined whether tempol,
a membrane-permeable SOD-mimetic agent has any
beneficial effect against gentamicin-induced renal failure.
Gentamicin elevated BUN and serum creatinine levels,
and decreased creatinine clearance, indicating a consis-
tent nephrotoxicity. This finding was also confirmed
by the renal histopathologic examination that exhibits
an acute tubular necrosis. It has been reported that
gentamicin induces the generation of hydroxy radicals,
which are strong mediators of tissue injury [15].
Figure 2 Kidney section from gentamicin (100 mg/kg once a day, for 7 days) and (a) tempol (3.5 mg/kg once a day, for 7 days) treated rats;
(b) tempol (7 mg/kg once a day, for 7 days) treated rats and (c) tempol (14 mg/kg once a day, for 7 days) treated rats (H & E ·100).
Figure 1 Histopathologic examination of kidney section from
gentamicin (100 mg/kg once a day, for 7 days) treated rats (H & E,
·100).
Gentamicin nephrotoxicity and tempol 81
� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 79–83
Moreover, it enhances the production of H2O2 by the rat
cortical mitochondria, and hydroxyl scavengers as well
as iron chelators may prevent the nephrotoxicity by
gentamicin [16]. A hydroxyl radical scavenger, metallo-
thionein protects against gentamicin-induced renal
toxicity [17]. Furthermore, free radical scavengers,
vitamin E and selenium administration have been also
found to be protective against its renal toxicity [18].
Accumulation of iron in the cortical segments of the
kidney has been reported after gentamicin treatment
[19]. Fe2+ may play a critical role in initiating the free
radical formation [20]. Thus, antioxidant therapy has
been frequently resorted to ameliorate gentamicin neph-
rotoxicity in experimental studies [4,18,21]. In this
study, too, we have investigated the effect of tempol,
which has been reported to attenuate renal dysfunction
after bacterial lipopolysaccharide treatment in rats, by a
superoxide anion scavenging effect [22]. Indeed, tempol
decreased the renal toxicity of gentamicin in this study.
In support, this agent has been reported to ameliorate
the decreased vasodilatation in the afferent arterioles of
the kidney in experimental diabetes through inactivating
O�2 anion and thereby increasing the bioavailability of
nitric oxide (NO) [23]. O�2 anions are responsible for the
degradation of NO [24], which is an important signaling
molecule in the cross-communication between glomer-
ular cells, contributing to normal glomerular physiology
[25,26].
The superoxide anions can be catalyzed by three
isoforms of SOD. However, native SOD is limited in
membrane permeability due to its large molecular weight
[27]. This is the disadvantage of using native SOD in
experimental studies where intracellular superoxide
onions are needed to be dismutased. In the present study,
however, the membrane-permeable SOD-mimetic agent,
tempol, has been chosen so that it can effectively reduce
the nephrotoxicity by gentamicin. Other mechanisms of
tempol, not to mention its SOD-mimetic activity, may also
contribute to the protection, i.e. enhancing the catalase-
mimicking activity of metmyoglobin (MbFe3+), so facili-
tating hydrogen peroxide dismutation [28]. However,
scavenging of free radicals by tempol may not only be
protective against cellular damage but may also increase
vasodilatation through NO reserving effect.
In conclusion, these results suggest that gentamicin
leads to acute tubular necrosis in the rat kidney, and
administration of tempol may have protective effects on
gentamicin-induced nephrotoxicity in rats. These findings
may also imply that some selected SOD-mimetic agents
could be examined in human nephrotoxicity induced by
the factors that involve superoxide generation in their
nephrotoxic action, such as aminoglycosides.
A C K N O W L E D G E M E N T
This work was supported by Cukurova University,
Research Foundation (TF-2002/BAP/72).
R E F E R E N C E S
1 Chambers H.F., Sande M.A. Antimicrobial agents, the amino-
glycosides, in: Hardman J.G., Goodman Gilman A., Limbird L.E.
(Eds), Goodman & Gilman’s the pharmacological basis of
therapeutics. McGraw-Hill, New York, 1995, pp. 1103–1121.
2 Baliga R., Ueda N., Walker P.D., Shah S.V. Oxidant mechan-
isms in toxic acute renal failure. Am. J. Kidney Dis. (1997) 29
465–477.
3 Walker P.D., Barri Y., Shah S.V. Oxidant mechanisms in
gentamicin nephrotoxicity. Ren. Fail. (1999) 21 433–442.
4 Ben-Ismail T.H., Ali B.H., Bashir A.A. Influence of iron,
deferroxamine and ascorbic acid on gentamicin-induced
nephrotoxicity in rats. Gen. Pharmacol. (1994) 25 1249–1252.
5 Swan S.K., Gilbert D.N., Kohlhepp S.J., Leggett J.E., Kohnen
P.W., Bennett W.M. Duration of the protective effect of
polyascorbic acid on experimental nephrotoxicity. Antimicrob.
Agents Chemother. (1992) 36 2556–2558.
6 Ozbek E., Turkoz Y., Sahna E., Ozugurlu F., Mizrak B., Ozbek M.
Melatonin administration prevents the nephrotoxicity induced
by gentamicin. BJU Int. (2000) 85 742–746.
7 Abdel-Naim A.B., Abdel-Wahab M.H., Attia F.F. Protective
effects of vitamin E and probucol against gentamicin-induced
nephrotoxicity in rat. Pharmacol. Res. (1999) 40 183–187.
8 Ali B.H., Bashir A.K. Effect of superoxide dismutase treatment
on gentamicin nephrotoxicity in rats. Gen. Pharmacol. (1996)
27 349–353.
9 Elfarra A.A., Duescher R.J., Sausen P.J., O’Hara T.M., Cooley
A.J. Methimazole protection of rats against gentamicin-induced
nephrotoxicity. Can. J. Physiol. Pharmacol. (1994) 72 1238–
1244.
10 Smyth B.J., Davis W.G. Allopurinol fails to protect against
gentamicin-induced renal damage in normotensive and protect
against gentamicin-induced renal damage in normotensive and
spontaneously hypertensive rats. Nephron (1994) 68 468–472.
11 Vaziri N.D., Dicus M., Ho N.D., Boroujerdi-Rad L., Sindhu R.K.
Oxidative stress and dysregulation of superoxide dismutase and
NADPH oxidase in renal insufficiency. Kidney Int. (2003) 63
179–85.
12 Chatterjee P.K., Cuzzocrea S., Brown P.A. et al. Tempol, a
membrane-permeable radical scavenger, reduces oxidant
stress-mediated renal dysfunction and injury in the rat. Kidney
Int. (2000) 58 658–673.
13 Cuzzocrea S., McDonald M.C., Mazzon E. et al. Beneficial effects
of tempol, a membrane-permeable radical scavenger, in a
rodent model of splanchnic artery occlusion and reperfusion.
Shock (2000) 14 150–156.
82 Y. Karatas et al.
� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 79–83
14 Can C., Sen S., Boztok N., Tuglular I. Protective effect of
L-arginine administration on gentamicin-induced renal failure
in rats. Eur. J. Pharmacol. (2000) 390 327–334.
15 Ali B.H. Gentamicin nephrotoxicity in humans and animals:
some recent research. Gen. Pharmacol. (1995) 26 1477–1487.
16 Shah S.V., Walker P.D. Reactive oxygen metabolites in toxic
acute renal failure. Renal Failure (1992) 14 363–370.
17 Yang C.L., Du X.H., Zou W.Z., Chen W. Protective effect of
Zn-induced metallothionein synthesis on gentamicin nephro-
toxicity. Ren. Failure (1991) 13 227–232.
18 Ademuyiwa O., Ngaha E.O., Ubah F.O. Vitamin E and selenium
in gentamicin nephrotoxicity. Human Exp. Toxicol. (1990) 9
281–288.
19 Boliga R., Ueda N., Walker P.D., Shah S.V. Oxidant mech-
anisms in toxic acute renal failure. Am. J. Kidney Dis. (1997)
29 465–477.
20 Braughter J.M., Duncan L.A., Chase R.L. The involvement of
iron in lipid peroxidation. J. Biol. Chem. (1986) 261 10282–
10289.
21 Neumann T.V., Paller M.S. Superoxide dismutase protects
proximal tube epithelial cells against anoxic injury in vitro.
Kidney Int. (1990) 37 490.
22 Leach M., Frank S., Olbrich A., Pfeilschifter J., Thiemermann C.
Decline in the expression of copper/zinc superoxide dismutase
in the kidney of rats with endotoxic shock: effects of the
superoxide anion radical scavenger, tempol, on organ injury.
Br. J. Pharmacol. (1998) 125 817–825.
23 Schnackenberg C.G., Wilcox C.S. The SOD mimetic tempol
restores vasodilation in afferent arterioles of experimental
diabetes. Kidney Int. (2001) 59 1859–1864.
24 Gryglewski, R.J., Palmer, R.M.J., Moncada, S. Superoxide anion
is involved in the breakdown of endothelium-derived vascular
relaxing factor. Nature (1986) 15 170–179.
25 Baylis C., Harton P., Engels K. Endothelial derived relaxing
factor controls renal hemodynamics in the normal rat kidney.
J. Am. Soc. Nephrol. (1990) 1 875–881.
26 Radermacher J., Klanke B., Schurek H.J., Stolte H.F., Frolich,
J.C. Importance of NO/EDRF for glomerular and tubular
function: studies in isolated perfused rat kidneys. Kidney Int.
(1992) 41 1549–1559.
27 Schnackenberg C.G. Oxygen radicals in cardiovascular-renal
disease. Curr. Opin. Pharm. (2002) 2 121–125.
28 Krishna M.C., Samuni A., Taira J., Goldstein S., Mitchell J.B.,
Russo A. Stimulation by nitroxides of catalase-like activity of
hemeproteins. Kinetics and mechanism. J Biol. Chem. (1996)
271 26018–26025.
Gentamicin nephrotoxicity and tempol 83
� 2004 Blackwell Publishing Fundamental & Clinical Pharmacology 18 (2004) 79–83
