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Sulfasalazine-induced renal injury in rats and the protective role of thiol-

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 Renal Failure

ISSN: 0886-022X (Print) 1525-6049 (Online) Journal homepage: http://www.tandfonline.com/loi/irnf20

Sulfasalazine-induced renal injury in rats and the

protective role of thiol-reductants

Reza Heidari, Vahid Taheri, Hamid Reza Rahimi, Babak Shirazi Yeganeh,
Hossein Niknahad & Asma Najibi

To cite this article: Reza Heidari, Vahid Taheri, Hamid Reza Rahimi, Babak Shirazi Yeganeh,
Hossein Niknahad & Asma Najibi (2015): Sulfasalazine-induced renal injury in rats and the
protective role of thiol-reductants, Renal Failure, DOI: 10.3109/0886022X.2015.1096731

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! 2015 Taylor & Francis. DOI: 10.3109/0886022X.2015.1096731

LABORATORY STUDY

Sulfasalazine-induced renal injury in rats and the protective role of

thiol-reductants
Reza Heidari1, Vahid Taheri2, Hamid Reza Rahimi3,4, Babak Shirazi Yeganeh5, Hossein Niknahad1,2, and
Asma Najibi1,2
1
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Pharmacology and Toxicology,
School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran, 3Food Control Laboratory, Department of Food and Drug, Shiraz University
of Medical Sciences, Shiraz, Iran, 4Department of Toxicology and Pharmacology, Kerman University of Medical Sciences, Kerman, Iran, and
5
Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Downloaded by [University of Waterloo] at 21:57 19 October 2015

Abstract Keywords
Sulfasalazine is widely used for inflammatory-mediated disorders in human. Renal damage is a Anti-rheumatoid drugs, glutathione, oxidative
serious adverse effect accompanied sulfasalazine administration. No specific therapeutic option stress, renal injury, sulfasalazine
is available against this complication so far. Oxidative stress seems to play a role in
sulfasalazine-induced renal injury. Current investigation was designed to evaluate the effect of History
N-acetyl cysteine (NAC) and dithiothreitol (DTT) as thiol reductants against sulfasalazine-
induced renal injury in rats. Oral administration of sulfasalazine (600 mg/kg for 14 consecutive Received 19 May 2015
days) caused renal injury as judged by increase in serum level of creatinine and blood urea Revised 6 July 2015
nitrogen. Furthermore, the level of reactive oxygen species and lipid peroxidation were raised Accepted 8 September 2015
in kidney tissue after sulfasalazine administration. Additionally, it was also found that renal Published online 15 October 2015
glutathione reservoirs were significantly depleted in sulfasalazine-treated animals.
Histopathological examination of kidney endorsed organ injury in drug-treated rats. Daily
intraperitoneal administration of NAC (250 and 500 mg/kg/day) and/or DTT (15 and 30 mg/kg/
day) effectively alleviated renal damage induced by sulfasalazine. Data suggested that thiol
reductants could serve as potential protective agents with therapeutic capabilities against
sulfasalazine adverse effect toward kidney.

Introduction N-acetyl cysteine (NAC) and dithiothreitol (DTT) are

Renal injury is accompanied by administrations of several
drugs.1 Sulfasalazine is widely administered against inflam-
matory-mediated diseases in human.2 On the other hand,
some investigations indicate decrease in renal function in
sulfasalazine-treated patients.3,4 Sulfasalazine-induced renal
injury has typically been presented as interstitial nephritis,
glomerulonephritis, nephritic syndrome and acute renal
failure.5–7 Sulfasalazine-induced renal injury, might be poten-
tially irreversible.8,9 No specific therapeutic agent has been
developed against sulfasalazine-induced kidney injury so far.
Although there is no precise mechanism known to be
responsible for sulfasalazine-induced renal injury, some
researches indicated the role of reactive metabolites, reactive
oxygen species (ROS) and oxidative stress in the organ injury
induced by this drug.10,11 Moreover, the oxidative stress has
also been reported to be involved in other side effects of
sulfasalazine.10
robust thiol reducing agents which their cytoprotective
properties have been established in different experiments.12
Due to the involvement of oxidative stress in sulfasalazine
renal injury,10,11 it is expected that thiol reducing agents offer
protective properties against this complication.
Material and methods
Animals
Male Sprague-Dawley rats (n ¼ 36) weighing 250–300 g were
obtained from the Laboratory Animal Breeding Center, Shiraz
University of Medical Sciences, Shiraz, Iran. Animals were
housed in an environmental temperature of 22 ± 2  C with a
50–60% of relative humidity. Rats had free access to water
and a normal chow diet. All the experiments were performed
in conformity with the guidance for care and use of
experimental animals approved by an ethic committee in
Shiraz University of Medical Sciences, Shiraz, Iran.

Address correspondence to: Hossein Niknahad, Pharm. D & Professor of

Toxicology, Department of Pharmacology and Toxicology, School Experimental setup
of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
Tel: +9871-32424126; Fax: +9871-32424126; E-mail: niknahadh@ Animals were randomly divided into six groups containing
sums.ac.ir six rats in each. Rats were treated as follows: (1) control
 2 R. Heidari et al.
(vehicle-treated group), (2) sulfasalazine (600 mg/kg/day,
oral); (3) sulfasalazine (600 mg/kg/day, oral) + NAC
(250 mg/kg/day, i.p); (4) sulfasalazine (600 mg/kg/day,
oral) + NAC (500 mg/kg/day, i.p); (5) sulfasalazine (600 mg/
kg/day, oral) + DTT (15 mg/kg/day, i.p) and (6) sulfasalazine
(600 mg/kg/day, oral) + DTT (30 mg/kg/day, i.p).
It has been previously reported that a dose of 600 mg/kg/
day of sulfasalazine for 14 consecutive days caused marked
renal injury in rats.10 None of the investigated thiol reductants
affected markers of organ injury assessed in current investi-
gation, when they were administered alone at the above-
mentioned doses. At the end of experiments, animals were
anesthetized (sodium thiopental, 80 mg/kg, i.p) and their
blood and kidney were collected.
Serum biochemistry and organ histopathology
A Mindray BS-200Õ auto analyzer (MindrayÕ, Shenzhen,
China) and standard kits (Pars AzmunÕ , Tehran, Iran) were
employed to assess serum creatinine (Cr) and blood urea
Downloaded by [University of Waterloo] at 21:57 19 October 2015
nitrogen (BUN).13 For histopathological assessments, samples
of kidney tissue were fixed in buffered formalin solution
(0.4% sodium phosphate monobasic, NaH2PO4, 0.64%
sodium phosphate dibasic, Na2HPO4, and 10% formaldehyde
in distilled water). Paraffin-embedded sections of tissue were
prepared and stained with hematoxylin and eosin (H&E)
before light microscope viewing.14
Reactive oxygen species formation
ROS in kidney tissue was estimated by a method described by
Gupta et al.,15 with some modifications. Kidney samples
(200 mg) were homogenized in ice-cold Tris-HCl buffer
(40 mM, pH ¼ 7.4) (1:10 w/v). Then, samples of tissue
homogenate (100 mL) were mixed with Tris-HCl buffer
(1 mL) and 5 mL of 20 ,70 -dichlorofluorescein diacetate
(10 mM) (Sigma-Aldrich, St. Louis, MO). The mixture was
incubated for 30 min in 37  C (GyromaxÔ incubator shaker).
Finally, the fluorescence intensity of the samples was assessed
using a FLUOstar OmegaÕ multifunctional microplate reader
(lexcitation ¼ 485 nm and lemission ¼ 525 nm) (BMG Labtech,
Germany).15,16
Measurement of lipid peroxidation
Thiobarbituric acid reactive substances were assessed in
kidney as an index of lipid peroxidation. The reaction mixture
was consisted of thiobarbituric acid (0.375%, w/v) (Sigma-
Aldrich, St. Louis, MO), phosphoric acid (1% w/v, pH ¼ 2)
Table 1. Markers of sulfasalazine-induced renal damage and the role of thiol-reductants administration.
Kidney ROS Lipid peroxidation
Treatment (fluorescence intensity, FI) (nmol/mg tissue)
Control (vehicle-treated) 67,941 ± 12,337
+ Sulfasalazine 600 mg/kg/day 139,442 ± 17,832*
+ NAC 250 mg/kg 70,268 ± 9627a
+ NAC 500 mg/kg 53,654 ± 4384a
+ DTT 15 mg/kg 77,418 ± 13,590a
+ DTT 30 mg/kg 44,519 ± 5323a
Data are given as Mean ± SEM for each group (n ¼ 6). NAC, N-acetyl cysteine; DTT, dithiothreitol.
*Significantly different as compared with the control (vehicle-treated) group (p5 0.05).
a
Significantly different as compared with the sulfasalazine-treated group (p5 0.05).
Ren Fail, Early Online: 1–5
(Merck, Darmstadt, Germany), and 500 mM of tissue hom-
ogenate (10% w/v in KCl, 1.15%). The mixture was shaken
well and heated in boiling water (100  C) for 45 min. After the
incubation period, 2 mL of n-butanol (Merck, Darmstadt,
Germany) was added and vigorously shaken. Samples were
centrifuged (3000g for 5 min) and the absorbance of
developed color in n-butanol phase was measured at 532 nm
using an Ultrospec 2000Õ UV spectrophotometer (Pharmacia
Biotech, Sweden).17
Kidney glutathione content
Tissue samples (200 mg) were homogenized in 8 mL of ice-
cooled EDTA (20 mM) (Merck, Darmstadt, Germany). Five
milliliter of the prepared homogenate was mixed with 4 mL of
distilled water and 1 mL of trichloroacetic acid (50% w/v).
The mixture was shaken and centrifuged (10,000 g, 4  C,
25 min). Then, 2 mL of the supernatant was mixed with
4 mL of Tris buffer (pH ¼ 8.9), and 100 ml of Ellman’s
reagent (DTNB, 0.01 M in methanol). The absorbance of
the developed color was measured in 412 nm using an
Ultrospec 2000Õ UV spectrophotometer (Pharmacia Biotech,
Sweden).13
Statistical analysis
Data are given as the Mean ± SEM. Data comparison was
performed by the one-way analysis of variance with Tukey’s
post hoc multiple comparison test. Differences were con-
sidered statistically significant when p50.05.
Results
Sulfasalazine administration (600 mg/kg/day for 14 days)
caused a marked renal impairment as judged by the elevated
level of serum BUN and Cr (Table 1). Moreover, it was found
that sulfasalazine administration caused a significant increase
in tissue level of ROS in rats’ kidney (Table 1). ROS
formation in kidney tissue was accompanied by consequent
lipid peroxidation and decrease in tissue glutathione (GSH)
content (Table 1).
Histopathological examination of renal tissue specimens
revealed sulfasalazine-induced kidney injury which was
detected as interstitial inflammation, tubular atrophy, focal
necrosis and vascular congestion (Figure 1B).
NAC (250 and 500 mg/kg, i.p., for 14 consecutive days)
and DTT (30 mg/kg, i.p, for 14 consecutive days) significantly
diminished elevation in serum BUN and Cr (Table 1). Kidney
ROS level and lipid peroxidation were also significantly lower
Kidney GSH (mmol/mg Serum BUN Serum cratinine
tissue) (mg/dl) (mg/dl)
1.99 ± 0.30 43.36 ± 4.22 25 ± 4 0.25 ± 0.03
5.83 ± 0.49* 11.72 ± 1.21* 91 ± 4* 0.87 ± 0.06*
2.97 ± 0.21a 22.20 ± 2.34a 29 ± 8a 0.4 ± 0.07a
2.13 ± 0.20 25.14 ± 2.3a 32 ± 6a 0.33 ± 0.08a
2.53 ± 0.10a 29.95 ± 1.80a 34 ± 4a 0.43 ± 0.08a
1.9 ± 0.27a 35.97 ± 2.17a 35 ± 6a 0.38 ± 0.03a
 DOI: 10.3109/0886022X.2015.1096731 Sulfasalazine-induced renal injury 3

when NAC and/or DTT was administered to sulfasalazine-
challenged rats (Table 1). Moreover, it was found that NAC
and DTT prevented kidney GSH depletion in sulfasalazine-
treated rats (Table 1). Finally, sulfasalazine-induced kidney
histopathological changes were mitigated when animals
received NAC and/or DTT (Figure 1C–F).
Discussion
Current investigation aimed to evaluate the effect of thiol
reductants against sulfasalazine-induced renal injury in rats.
NAC (250 and 500 mg/kg, i.p) and DTT (15 and 30 mg/kg,
i.p) administration effectively attenuated sulfasalazine-
induced renal injury which was manifested by preventing
the elevation in serum Cr and BUN as markers of renal
impairment. Moreover, NAC and DTT mitigated sulfasala-
zine-induced ROS formation, lipid peroxidation, GSH deple-
tion and finally tissue lesions in rat kidney.
We found that kidney ROS formation as an indicator of
Downloaded by [University of Waterloo] at 21:57 19 October 2015
It is not clear whether the whole molecule of sulfasalazine
and/or its intestinal metabolites are responsible for the renal
injury induced by this drug. Some investigations mentioned
the role of mesalazine in the nephrotoxic reactions after
sulfasalazine administration. Although mesalazine is recog-
nized as a chemical which might induce renal injury,2,5,9 the
administered dose of mesalazine needed to induce renal
injury is much higher than a mesalazine dose which a patient
will receive during sulfasalazine therapy. Hence, other
mechanisms and the whole molecule of sulfasalazine might
be involved in the renal injury induced by this drug.
Sulfapyridine as the other metabolite of sulfasalazine is a
chemical with sulfonamide structure. Sulfonamides are well
known for their adverse effects toward kidney.19 Some
investigations reported the potential role of sulfapyridine in
sulfasalazine-induced renal injury.20 Despite the cause of
renal injury accompanied sulfasalazine administration, it
seems that oxidative stress and its consequent events play a
major role in this complication. In current investigation we

oxidative stress was significantly increased in sulfasalazine-
treated animals. Multiple intracellular targets are subject to
affect by oxidative stress, including proteins, lipids and
nucleotides.18 It has been reported that cellular defense
mechanisms are impaired in sulfasalazine-treated ani-
mals.10,11 The impairment in cellular defense mechanisms
induced by sulfasalazine, might led to generation of excess
ROS production and finally organ dysfunction.
Sulfasalazine is degraded to mesalazine and sulfapyridine
by bacterial azo reductases in large intestine. Sulfapyridine is
almost completely absorbed compared with about 20–30%
absorption for mesalazine. Approximately 10–30% of sulfa-
salazine is also absorbed unchanged from the small intestine.2
found that thiol reducing agents counteracted the deleterious
effects of sulfasalazine on the renal function as ROS
formation and kidney histopathological lesions were miti-
gated by NAC and DTT.
Therapeutic doses of sulfasalazine administered to human
are between 3 and 6 g/day. As a high dose of the drug is
administered in a chronic manner, it might cause cellular
defense mechanisms impairment and continuous oxidative
stress in patients’ kidney. Thiol reducing agents such as NAC
might be capable of to be administered as a supplement agent
in sulfasalazine-treated patients. The proposed mechanism(s)
of sulfasalazine-induced renal injury and the potential thera-
peutic effects of thiol reductants are summarized in Figure 2.

(A) (B) (C) (D) (E)

(F)

Figure 1. Effects of thiol reducing agents administration on kidney histopathological changes in sulfasalazine-treated animals. Rats received
sulfasalazine (600 mg/kg/day, PO) for 14 consecutive days alone and/or in combination with intraperitoneal administration of thiol-reductants. (A)
Photomicrograph of kidney section taken from control (vehicle-treated) rats, representing normal tubules. (B) Sulfasalazine-treated rats, showing
marked tubular atrophy, focal necrosis, interstitial inflammation, and vascular congestion. (C) and (D) Animals received sulfasalazine and NAC (250
and 500 mg/kg, respectively). Tubular atrophy is detected in these groups, but there is no sign of tubular necrosis in animals treated with 500 mg/kg of
NAC. (E) Rats were treated with sulfasalazine and DTT (15 mg/kg). (F) Animals received sulfasalazine and DTT (30 mg/kg). Sulfasalazine-induced
focal necrosis was absent in this group.
 4 R. Heidari et al.
Downloaded by [University of Waterloo] at 21:57 19 October 2015
Figure 2. Sulfasalazine-induced renal damage and the proposed mechanisms of reno-protection provided by thiol-reductants.
Interestingly, the antioxidant and anti-inflammatory prop-
erties of sulfasalazine in kidney are also reported in previous
investigations.21 The reported renoprotective dose of sulfa-
salazine is lower (100 mg/kg for 7 days) than the nephrotoxic
dose which is applied in current investigation. Hence, we
might be able to conclude that sulfasalazine serves as a
renoprotective agent in lower doses which reduce inflamma-
tion and cytotoxic cytokines probably by inhibiting nuclear
factor kB (NF-kB) in kidney. In current investigation, we
found that higher doses of sulfasalazine (600 mg/kg for
14 days) caused oxidative stress in kidney as revealed by
increase in ROS, lipid peroxidation and tissue GSH depletion.
As sulfasalazine (but not its metabolites like sulfapyridine and
5-aminosalisilic acid) is a very strong and specific NF-kB
inhibitor,22 sulfasalazine metabolites might contribute in the
nephrotoxic properties of this drug in higher doses.
In conclusion, we suggest that thiol reducing agents have a
protective role against sulfasalazine-induced renal injury
probably by attenuating oxidative stress and restoring GSH
level in kidney. Further future investigations are needed to
reveal the precise mechanism of sulfasalazine-induced renal
injury and endorse the renoprotective properties of thiol
reductants.
Acknowledgements
The technical facilities providing of Pharmaceutical Sciences
Research Center of Shiraz University of Medical Sciences is
gratefully acknowledged.
Ren Fail, Early Online: 1–5
Declaration of interest
This investigation was financially supported by the office of
the Vice Chancellor of Research Affairs, Shiraz University of
Medical Sciences (#93-01-36–7612). Authors do not have
conflicts of interest.
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