Nephrotoxicity is one of the limiting factors for using doxorubicin (DOX). Interleukin 1 has major role in DOX-induced nephrotoxicity, so we investigated the effect of interleukin 1 receptor antagonist diacerein (DIA) on DOX-induced nephrotoxicity. DIA (25 and 50 mg/kg/day) was administered orally to rats for 15 days, in the presence or absence of nephrotoxicity induced by a single intraperitoneal injection of DOX (15 mg/kg) at the 11th day. We measured levels of serum urea, creatinine, renal reduced glutathione (GSH), malondialdehyde (MDA), total nitrites (NO
Drug-induced nephrotoxicity is a major cause of acute kidney injury [
DIA is a new anti-inflammatory, analgesic, and antipyretic drug that was developed specially for the treatment of osteoarthritis [
DIA powder was from Eva Pharma Company and it was dissolved in 1% carboxymethylcellulose. DOX hydrochloride 10 mg vial (Pharmacia Italia, SPA, Italy), polyclonal rabbit/antirat caspase-3, TNF
Adult male Wistar rats weighing about 250–350 g were obtained from the Animal Research Centre, Giza, Egypt. Animals were kept in standard housing conditions in cages and were left to acclimatize for one week. Rats were supplied with laboratory chow and tap water. This work was conducted in the Pharmacology Department, Faculty of Medicine, El-Minia University, Egypt, and the animal experimental protocol was approved by the faculty board.
Rats were randomly assigned into 6 groups ( Group I received vehicle (1% carboxymethylcellulose) for 15 days and ip saline at day 11. Group II was treated with DLD (25 mg/kg/d orally) for 15 days and ip saline at day 11. Group III was treated with DHD (50 mg/kg/d orally) and ip saline at day 11. Group IV was treated with vehicle for 15 days and DOX (15 mg/kg) at day 11. Group V was treated with DLD (25 mg/kg/d orally) for 15 days + ip injection of DOX (15 mg/kg) at day 11. Group VI was treated with DHD (50 mg/kg/d orally) for 15 days + ip injection of DOX (15 mg/kg) at day 11. The doses of DOX and DIA were based on the previous studies [
After 4 days of DOX injection, each rat was weighed then anesthetized with ip injection of urethane (25% in a dose of 1.6 gm/kg) and then sacrificed.
Venous blood samples were collected from the jugular vein.
Serum was collected for biochemical analysis of urea [
After sacrifice, both kidneys were rapidly excised and weighed.
A longitudinal section of the left kidney and one half was fixed in 10% formalin then embedded in paraffin for histopathological and immunohistochemical examinations. The rest of the kidneys were snap frozen in liquid nitrogen and kept at −80°C.
GSH spectrophotometric kit was used. Briefly, the method is based on the fact that sulfhydryl group of GSH reacts with
Assessment of renal catalase antioxidant enzyme activity was determined from the rate of decomposition of H2O2 at 510 nm after the addition of tissue homogenate as described by colorimetric kit. The results were expressed as unit/g tissue [
The assessments of SOD levels were based on the ability of the enzyme to inhibit the phenazine methosulfate-mediated reduction of nitroblue tetrazolium dye and the results were expressed as unit/g tissue [
The renal contents of lipid peroxides were assayed by a spectrophotometric method based on the reaction between MDA and thiobarbituric acid [
The absorbance values of the samples and the blank were determined at 535 nm using a (Beckman DU-64 spectrophotometer, USA) and then blank absorbance value was subtracted from the sample absorbance value. From a standard curve, MDA concentration in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol/gm tissue by multiplying in the tissue dilution factor.
Nitric oxide (NO) in the form of nitrite was determined with spectrophotometric method using Griess reagent systems. The stable oxidation end products of NO, nitrite (
From a standard curve, NO
Renal tissue was fixed in 10% formalin, embedded in paraffin, sectioned by a microtome at 5
The renal tissues were examined in random microscopic areas semiquantitatively under 40 high power fields and the number of changes was assessed by the counting of 3 nonoverlapped fields for the same slide of each animal. The frequency and the severity of lesions in the kidneys were assessed semiquantitatively as follows: Score −: assigned normal, Score +: in between normal and mild, Score ++ (mild level): less than 25% of the examined fields revealed histological alterations, Score +++ (moderate level): less than 50% of the examined fields revealed histological alterations, and Score ++++ (severe level): less than 75% of the total fields examined revealed histological alterations [
The caspase-3, TNF
Data was analyzed by one way ANOVA followed by Dunnett multiple comparison test. The values are represented as means ± SEM. Statistical analysis was done using GraphPad Prism software (version 5). The differences were considered significant when the calculated
Table
Effect of DLD (25 mg/kg/day) and DHD (50 mg/kg/day) on serum creatinine, serum urea, MDA, and
Group | Creatinine (mg/dL) | Urea (mg/dL) | MDA (nmol/g tissue) |
|
---|---|---|---|---|
Control | 0.8828 ± 0.07291 | 50.26 ± 4.450 | 42.27 ± 2.202 | 179.5 ± 11.98 |
DLD | 0.7395 ± 0.06486 | 51.75 ± 1.721 | 44.77 ± 2.098 | 272.8 ± 22.35 |
DHD | 0.9540 ± 0.08678 | 53.73 ± 2.989 | 52.02 ± 5.036 | 295.0 ± 20.25 |
DOX | 1.475 ± 0.0522a | 251.8 ± 12.23a | 250.9 ± 16.37a | 1114 ± 64.29a |
DOX/DLD | 1.211 ± 0.0660ab | 241.7 ± 13.17a | 51.86 ± 4.461b | 322.8 ± 11.33ab |
DOX/DHD | 1.153 ± 0.0209ab | 69.74 ± 4.161b | 46.79 ± 1.39b | 228.5 ± 18.83b |
Values are representation of 4–6 observations as means ± SEM. Results are considered significantly different when
Renal MDA was evaluated as an indicator of kidney lipid peroxidation and nitrites and nitrates as an indicator of renal NO
Treatment with DOX (15 mg/kg) caused significant decrease in renal GSH, SOD, and catalase levels compared with untreated control group (Table
Effect of DLD (25 mg/kg/day) and DHD (50 mg/kg/day) on GSH, catalase, and SOD in DOX (15 mg/kg) induced nephrotoxicity.
Group | GSH (mmol/g tissue) | Catalase (unit/g tissue) | SOD (unit/g tissue) |
---|---|---|---|
Control | 10.32 ± 0.2999 | 92.10 ± 2.835 | 829.7 ± 5.182 |
DLD | 10.22 ± 0.5530 | 91.53 ± 1.860 | 832.0 ± 6.915 |
DHD | 9.085 ± 0.3000 | 91.80 ± 2.127 | 826.6 ± 7.575 |
DOX | 4.814 ± 0.1630a | 72.49 ± 3.662a | 657.6 ± 15.28a |
DOX/DLD | 8.678 ± 0.1985ab | 80.48 ± 4.108 | 722.7 ± 41.13a |
DOX/DHD | 9.215 ± 0.2814b | 8627 ± 4.496b | 807.3 ± 16.96b |
Values are representation of 4–6 observations as means ± SEM. Results are considered significantly different when
The histological study of the rat renal cortical tissue of control group (Figure
Photomicrographs of renal cortex of (a), (b), and (c), control, DLD, and DHD groups, respectively, showing normal lobular organization of the renal cortex; normal renal glomeruli and tubules. (d) DOX treated group showing markedly enlarged and congested vascular renal glomeruli and cytoplasmic vacuolations of corpuscular cells. Inflammatory cell infiltrations are observed. (e) DOX/DLD group showing less cytoplasmic vacuolations of the renal corpuscular cells and tubular cells. (f) DOX/DHD showing apparent normal renal cortex. H&E ×400. Bar = 20
The severity of the morphological changes was assessed semiquantitatively; DOX exposed group showed increase in the glomerular and tubular morphological changes at the light microscopic levels when compared with control group. These changes were suppressed by the administration of both doses of DIA, but the high dose showed marked improvement than the low dose (Table
Scoring of morphological changes observed in control and experimental groups by light microscope (
Findings | Control group | DLD group | DHD group | DOX treated group | DOX/DLD group | DOX/DHD group |
---|---|---|---|---|---|---|
(i) Glomerular vacuolations | − | − | − | ++++ | + | + |
(ii) Enlarged renal corpuscles | − | − | − | ++++ | + | − |
(iii) Tubular cells vacuolations | − | + | − | ++++ | + | + |
(iv) Lumen widening | − | − | − | ++++ | + | − |
(v) Distortion and Degeneration | − | − | − | ++++ | + | − |
(vi) Casts | − | − | − | − | − | − |
Animal groups tested are control untreated group, animals treated with diacerein (25 mg/kg/day, DLD) and diacerein (50 mg/kg/day, DHD), respectively, and animals treated with doxorubicin (DOX, 15 mg/kg), or with DOX together with low or high dose of diacerein (DOX/DLD or DOX/DHD), respectively.
Normal (−), in-between normal and mild (+), mild (++), moderate (+++), and severe (++++) [
Administration of DOX caused significant increase in the immunoreactivity of caspase-3, NF
The effect of DLD and DHD doses on caspase-3, TNF
Group | Caspase-3 | TNF |
NF |
---|---|---|---|
Control | 0.42 ± 0.80 | 0.40 ± 0.78 | 0.40 ± 0.88 |
DLD | 0.60 ± 0.88 | 0.60 ± 0.80 | 0.60 ± 0.80 |
DHD | 0.40 ± 0.40 | 2.40 ± 0.40 | 0.40 ± 0.40 |
DOX | 58.60 ± 8.90a | 80.60 ± 8.90a | 58.60 ± 8.90a |
DOX/DLD | 30.20 ± 7.90a/b | 35.20 ± 7.90a/b | 25.20 ± 7.90a/b |
DOX/DHD | 10.00 ± 6.90b | 5.00 ± 4.90b | 10.00 ± 6.90b |
Animal groups tested are control untreated group, animals treated with low or high doses of DIA alone (DLD or DHD), respectively, and animals treated with DOX or with DOX together with low or high dose of DIA (DOX/DLD or DOX/DHD), respectively.
Photomicrographs of renal cortex immune stained for caspase-3 of (a), (b), and (c), control, DLD, and DHD groups, respectively, showing negative immunoreactivity. (d) DOX treated group showing extensive expression in the renal glomeruli and renal tubules. (e) DOX/DLD group showing moderate expression within the glomeruli and the renal tubules. (f) DOX/DHD group showed marked improvement with no expression in glomeruli and renal tubules. The expression is mainly cytoplasmic, but with some immunopositive nuclei. Immunohistochemistry counter stained with H&E ×400. Bar = 20
Photomicrographs of renal cortex immune stained for NF
Photomicrographs of renal cortex immune stained for TNF
Anticancer therapy usually demolishes the physiological homoeostasis and affects multiple organs during treatment process. Effective anticancer therapy with anthracyclines as DOX is limited because of its toxicity to various organs including kidneys [
Induction of DOX nephrotoxicity was detected in our study by significant elevation of serum urea and creatinine levels which were confirmed by toxic histopathological changes compared to control group. Urea and serum creatinine are the most sensitive markers of nephrotoxicity implicated in the diagnosis of renal injury [
Improvement of DOX-induced nephrotoxicity was previously tried by compounds that partially succeeded in preserving normal renal function and structure probably through their antioxidant and anti-inflammatory effects as caffeic acid phenethyl ester [
DIA could significantly decrease serum urea and creatinine compared to DOX treated group. That is due to the anti-inflammatory and antioxidant effects of DIA which suppress DOX mediated oxidative stress, inflammation, and tissue damage. Our histopathological changes showed that DOX treated group presented with marked damage of renal tubules. This is in agreement with Rashid et al. [
Coadministration of DIA significantly improved the histopathological changes compared to DOX treated group. These results are in agreement with Zhao et al. [
The elevated levels of GSH could effectively provide thiol group for the possible GSH mediated detoxification reactions of GPx (glutathione peroxidase) and GST (glutathione-s-transferase) which is involved in the scavenging of
SOD extensively distributes in all cells and has a significant shielding role against oxidative injury induced by reactive oxygen species [
In our study, the activities of SOD and catalase significantly decreased in DOX treated rats in kidney as compared to control rats. The accumulation of these highly reactive free radicals leads to the reduction of the activity of SOD and catalase which in turn results in damaging effects in the form of loss of cell membrane integrity and function. The decrease in the SOD and catalase activities related to the increase in the intracellular levels of H2O2. Catalase has been reported to be responsible for the detoxification of H2O2, which is an effective inhibitor of SOD. Other researchers reported the same results [
Coadministration of DIA significantly improved SOD, GSH, and catalase levels compared to DOX treated group. These results may be due to antioxidant effect of DIA which was approved previously by Tamura et al. [
Oxidative stress may damage cellular structures via lipid peroxidation of cellular membranes.
Another radical formatting mechanism in such an experimental protocol is NO
Coadministration of DIA significantly decreased MDA and NO
DOX treatment induced p53 phosphorylation. Induction of p53 mediates cell apoptosis through activation of caspase-3 family of proteases and apoptotic cell death [
Coadministration of DIA significantly decreased caspase-3 expression compared to DOX treated group. Our study is in consistence with Torina et al. [
DOX-induced superoxide anion production which was reported to be responsible for TNF
Coadministration of DIA significantly decreased TNF
Moreover, Zhao et al. [
Our results are consistent with Meng et al. [
In conclusion, DIA protected against DOX-induced nephrotoxicity in rats most probably due to its antioxidant and anti-inflammatory activities. However, DHD (50 mg/kg/day) showed more protective effect than DLD (25 mg/kg/day).
The authors reported no conflict of interests regarding the publication of this paper.