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Article Type: Original Article
Tropisetron Ameliorates Early Diabetic Nephropathy in StreptozotocinInduced Diabetic Rats Anita Barzegar-Fallah1, Houman Alimoradi2, Firouzeh Asadi1, Ahmad Reza Dehpour3, Mojgan Asgari4, Massoumeh Shafiei1*
Short title: Renoprotective effects of tropisetron 1
Department of Pharmacology, School of Medicine, Iran University of Medical Sciences,
Tehran, Iran 2
Department of Pharmacology and Toxicology, University of Otago, P.O. Box 913, Dunedin,
New Zealand
3
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences,
Tehran, Iran
4
Department of Pathology & Oncopathology Research Center, Shahid Hasheminejad Hospital,
Iran University of Medical Sciences, Tehran, Iran
* Corresponding author. Tel: +98 21 88622573; Fax: +98 21 88622696
E-mail address: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12373 This article is protected by copyright. All rights reserved.
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Address: Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. P.O. Box: 14155-6183
Abstract
Oxidative stress and inflammation are well established to be involved in the pathogenesis of diabetic nephropathy. It has been shown that tropisetron exerts anti-inflammatory and immunomodulatory properties. Current study was designed to investigate protective effects of tropisetron on early diabetic nephropathy in streptozotocin (STZ)-induced diabetic rats. Rats were divided into six groups: (1) untreated diabetic, (2) untreated control, (3-6) diabetic and normal rats treated with tropisetron or granisetron (3 mg/kg) beginning at the time of diabetes induction for 2 weeks. At the termination of the experiments, bodyweight, kidney index, urinary albumin excretion (UAE) and glomerular filtration rate were measured. Moreover, the levels of oxidative stress markers and tumor necrosis factor alpha (TNF-α) were determined. STZ-treated animals showed significant loss of body weight, renal enlargement and dysfunction. Diabetic rats also exhibited an increase in malondialdehyde level along with a significant decrease in reduced glutathione level, and superoxide dismutase and catalase activity. Furthermore, the diabetic animals demonstrated a significant rise in renal cortical and urinary TNF-α levels and UAE. Both granisetron and tropisetron decreased blood glucose in diabetic animals however this decrease was not significant for granisetron. Treatment with tropisetron, but not granisetron, prevented enhanced oxidative stress and TNF-α level, decreased urinary cytokine excretion and albuminuria, and improved the renal morphological damage. In conclusion, the present study suggests that tropisetron may be a protective agent in early diabetic nephropathy, whose action can be mediated, at least in part, by the antioxidative and anti-inflammatory mechanisms and these effects appear to be 5-HT3 receptor-independent.
Key words: Diabetic nephropathy; Tropisetron; Oxidative stress; TNF-α; Renal function
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Introduction
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1.
Diabetic nephropathy (DN) is one of the most serious microvascular complications of both insulin-dependent and non-insulin dependent diabetes mellitus (1) and has become the major cause of end-stage renal disease worldwide (2). DN is defined by specific renal morphological and functional alterations. Features of early diabetic renal changes are an increase in kidney size, glomerular and tubular hypertrophy and abnormal renal function as recognized by creatinine clearance (CrCl) or glomerular filtration rate (GFR) (1). It follows by excessive urinary albumin excretion (UAE), thickening of the glomerular basement membrane and mesangial expansion with the accumulation of glomerular extracellular matrix (3). Hyperglycemia, which has a key role in the pathogenesis of DN, leads to the mitochondrial production of reactive oxygen species (ROS), attenuation of antioxidative mechanisms through glycation of the scavenging enzymes and subsequently cell death and kidney dysfunction (4, 5). Besides oxidative stress, inflammation is intimately linked with the development of DN. There is growing evidence that tumor necrosis factor alpha (TNF-α) is one of the major pro-inflammatory cytokines in the pathogenesis of kidney injury and also produced intrinsically in renal cells (6-8).
Moreover, increases in oxidative stress can increase the production of inflammatory cytokines and likewise, an increase in inflammatory cytokines can stimulate the production of free radicals. Hence, targeting oxidative stress-inflammatory cytokine signaling is speculated to provide an opening for development of novel therapies against DN (9). 5-HT3 receptor antagonists including tropisetron and granisetron are safe drugs with large therapeutic index and are widely used as an effective and well tolerated agent to counteract chemotherapy-induced emesis (10). New investigations indicate that tropisetron exerts notable immune modulatory, analgesic and anti-inflammatory properties (11, 12). In this regard, we have recently shown that tropisetron significantly suppressed the elevated pro-inflammatory cytokines interleukin-2 (IL-2) and TNF-α in a rat model of neurotoxicity induced by vincristine (13). In another study, we reported that pretreatment with tropisetron significantly improved neurological deficits, diminished leukocyte transmigration into the brain, suppressed TNF-α level, and also attenuated brain infarction and edema in an embolic model of stroke in rats. The findings of the latter study suggest that the mentioned properties might not be 5-HT3 receptor-dependent since
granisetron, another selective 5-HT3 receptor antagonist, failed to elicit protective effects (14). In another study, we demonstrated that tropisetron exerted cardioprotective effects against
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Figure legends: Fig. 1. Effects of 2 weeks treatment with tropisetron or granisetron on blood glucose concentrations. Blood glucose levels were determined at multiple time points up to 15 days after induction of diabetes. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.001 compared with control group; #P< 0.05 compared
with STZ-treated group. Fig. 2. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular filtration rate (GFR) in normal and diabetic rats. GFRs were determined by calculating the mean of urea nitrogen clearance and creatinine clearance per gram body weight. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. Values are means ± SE, n= 6/ group. *P< 0.05 and ***P< 0.001 compared with control. ##P< 0.01 compared with STZ+ Tropi group. Fig. 3. Effects of 2 weeks treatment with tropisetron or granisetron on oxidative stress markers in renal cortex. (a) malondialdehyde (MDA) level, (b) reduced glutathione (GSH) level, (c) superoxide dismutase (SOD) level, (d) catalase (CAT) level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and ***P< 0.001 compared with control group; #P< 0.05 and ##P< 0.01 compared with STZ-treated group.
Fig. 4. Effects of 2 weeks treatment with tropisetron or granisetron on renal cortex TNF-α. The level of TNF-α in diabetic rats were significantly increased compared with control group, and treatment with granisetron and tropisetron attenuated its level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and **P< 0.01 compared with control group; #P< 0.05 compared with STZ-treated group.
Fig. 5. Effects of 2 weeks treatment with tropisetron or granisetron on urinary TNF-α. Elevated level of TNF-α as a good indicator of early nephrotoxicity significantly increased in all diabetic
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changes, while granisetron with the same dosage did not exert any effects on biochemical markers and histological damage. Regarding the structural similarity of these 5-HT3 receptor
antagonists and the diverse effects of these two compounds in early diabetic nephropathy, we believe that the molecular target for the beneficial activity of tropisetron might be independent of its binding to the 5-HT3 receptor. The same results obtained from our previous studies on the neurotoxicity of vincristine (13) and embolic model of stroke (14) confirm this hypothesis. Tropisetron, a selective 5-HT3 receptor antagonist, is widely used as an effective and well
tolerated agent in counteracting chemotherapy-induced emesis (20). New investigations have shed light on additional applications for this drug (12, 21) and findings of a pioneering study show new mechanistic insights into anti-inflammatory and immune modulatory properties of tropisetron (11). Further, Vega et al. (22) have indicated that tropisetron inhibits antigen-induced proliferation of human peripheral T cells and production of IL-2. Also they proposed that tropisetron exerts its inhibitory effect via blockade of calcineurin/NFAT-dependent signaling pathway. Briefly, hyperglycemia, the main determinant of the pathogenesis of DN, not only generates
more ROS, such as superoxide and hydrogen peroxide, but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes including SOD and CAT (5, 23). This is in agreement with our findings, as diabetic rats exhibited a marked increase in renal MDA content and a decrease in GSH level and the activity of SOD and CAT. Moreover, increased cytoplasmic calcium (Ca2+) concentration which is induced by oxidative
stress activates phosphatase activity of calcineurin, a Ca2+/calmodulin-dependent protein. It subsequently dephosphorylates the nuclear factor of activated T cells (NFAT) which finally regulates numerous cytokines and pro-inflammatory factors (24, 25). TNF-α has been implicated as a potent pro-inflammatory cytokine in the development and progression of renal injury in DN by promoting inflammation, the accumulation of extracellular matrix and damaging the glomerular permeability barrier with the development of albuminuria. Further, some evidence has suggested that urinary TNF-α may be a useful biomarker for detection of kidney injury in the very early stages of DN (8). Our study demonstrated a significant rise in renal cortical and urinary TNF-α levels 2 weeks after induction of diabetes and these findings were correlated with an increase in UAE. Tropisetron treatment was associated with a reduction of albuminuria and
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renal and urinary TNF- α levels. Our findings are in agreement with previous reports by other investigators (7, 26). In the present study, a mild reduction of blood glucose levels was also demonstrated in tropisetron+ STZ- and granisetron + STZ-treated animals. As it was shown that serotonin and in particular the 5-HT3 receptor channel system are involved in modulating insulin release through
pancreatic insulin-producing beta cell line (27), it is possible that blockade of 5-HT3 receptor by
these drugs is responsible for this hypoglycemic effect. Next, GFR was increased in untreated STZ rats, in comparison with control group and treatment of diabetic animals with tropisetron resulted in a further increase in GFR. This finding is consistent with previously published studies by Gooch et al. (17, 28) showing that GFR in the early stages of diabetes was higher than normal, and cyclosporine A treatment resulted in a further increment in urine output and finally GFR. The expression of calcineurin has been shown to increase rapidly in STZ-induced diabetes primarily in the thick ascending limb of Henle. On the other hand, a potent inhibition of calcineurin by cyclosporine A has been demonstrated to alter aquaporin 2 (AQP2) localization and phosphorylation in principal cells of collecting ducts. As a consequence, cyclosporine A treatment resulted in a further increase in urine output compared with diabetes alone, suggesting a functional consequence of inhibiting calcineurinmediated regulation of AQP2 (28). Our data is in agreement with this study, as urine volume in only tropisetron treated animals were increased compared with control group, and also urine excretion in STZ+ tropisetron group was more than STZ group (Table 1). Moreover, it was shown that tropisetron potently inhibited calcineurin activity in cerebellar granule cell culture (18). Therefore, we believe that the main reason for increased GFR in STZ+ tropisetron group is due to inhibition of calcineurin-mediated regulation of AQP2 and very significant increase in the UFR. In conclusion, our results present the beneficial properties of tropisetron in an experimental model of early STZ-induced diabetic nephropathy, possibly, at least in part, through amelioration of inflammation and oxidative stress. Future investigations can unravel the detailed mechanisms of tropisetron-induced renoprotection and whether this effect is fully independent of 5-HT3 receptor.
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4. Methods 4.1.
Ethics
This study was performed according to the guidelines of the US National Institute of Health (NIH publication no.85.23, revised 1985) guides for the care of lab animals. 4.2.
Animal protocol
Experiments were performed on 200-250 g male Wistar rats in Department of Pharmacology, Iran University of Medical Sciences. Animals were housed in a temperature and humidity controlled environment with 12-h light/12-h dark cycle. Food consists of normal rat chow and water ad libitum. Rats were randomly divided into 6 groups of 6 rats/ group. Group 1 or STZ group were injected intravenously (IV) via the jugular vein with a single dose of 55 mg/kg body wt STZ (AppliChem GmbH, Germany) in sodium citrate buffer (pH 4.0) to induce diabetes. Group 2 or control group were similarly injected with sodium citrate buffer alone. Rats in group 3, 4 were injected with STZ and were also given daily intraperitoneally (IP) injections of tropisetron (STZ+ Tropi group) or granisetron (STZ+ Grani group) (3 mg/kg body wt) beginning at the time of diabetes induction till the end of 2th week for 14 days. Group 5, 6 were given daily
injections of tropisetron (Tropi group) or granisetron (Grani group) alone for 14 days. Tropisetron and granisetron were freshly prepared by dissolving in double distilled water. After injection of STZ, rats were subjected to a 48 h fast and then they had unrestricted access to food and were maintained in accordance with Institutional Animal Care and Use Committee procedures. Moreover, blood glucose levels were determined by using a glucometer (Media Smart, Switzerland) with a drop of blood from the tail vein to verify hyperglycemia and periodically thereafter twice a week. Only diabetic rats with a blood glucose levels (non-fast) > 300 mg/dl were included in the present study. The body weights and blood glucose levels of the experimental animals were taken twice a week. In this study, 3 days after STZ injection, which rats showed the first symptoms of diabetes, were considered as the day 0 of the study.
4.3.
Renal function
Two weeks after induction of diabetes (day 14 of study), animals were placed in metabolic cages overnight with unrestricted access to food and water, and the volume of urine were measured.
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The urine was collected for determination of nitrogen, creatinine and albumin levels. On day 15 of the study, rats were weighed, and then anaesthetized using IP injection of ketamine (60 mg/kg) and xylazine (8 mg/kg). After blood collection from heart, both kidneys were removed and total KW was measured with a precise balance. Serum blood urea nitrogen (BUN), serum creatinine levels and UAE were measured (29-31). GFR, an indicator of residual renal function, was determined by mean of urea nitrogen clearance and CrCl per gram BW. The kidney index was expressed as [KW (g)/BW (g)] ×100. A slice of kidney cortex at the pole was fixed in 10% formalin in 0.1 M sodium phosphate buffer, pH 7.4, for preparation of sections for light microscopy and image analyses. A second portion was quick-frozen immediately in liquid nitrogen for measurement of TNF-α and
oxidative stress markers.
4.4.
Assessment of renal cortex oxidative stress
Preparation of renal homogenate Following the decapsulation of kidneys, 100 mg of kidney cortex was minced and a homogenate was prepared with 10% (w/v) phosphate-buffered saline (0.1 mol/L, pH 7.4) using a homogenizer. Then the kidney homogenate was centrifuged at 1000 rpm for 3 min at 4 0C and
the supernatant was collected and divided into two portions. One of which was used for measurement of lipid peroxidation, in terms of MDA content, and GSH and the remaining ◦
supernatant was centrifuged again at 12,000 rpm at 4 C for 20 min and used for the measurement of antioxidant enzyme activities of SOD and CAT and level of TNF-α. The protein content of renal homogenate was measured by the method of Lowry et al (32) using bovine serum albumin as the standard.
Determination of lipid peroxidase Lipid peroxidation was measured through determination of MDA level using a thiobarbituric acid reactive substances method as described before by Ohkawa et al. (33). In brief, 0.1 ml of kidney homogenate was mixed with 20% of 1.5 ml of acetic acid (pH 3.5), 1.5 ml thiobarbituric acid and 0.2 ml sodium dodecyl sulphate (8.1 %). The mixture was then heated at 100 0C for 60
min. The mixture was cooled and 5 ml of n-butanol–pyridine mixture (15:1) was added followed
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by 1 ml of distilled water. After centrifugation of the mixture at 4000 rpm for 10 min, the organic layer was taken and its absorbance was measured at 532 nm. The concentration of MDA formed is expressed as nmol MDA/ mg protein (34).
Estimation of GSH GSH in the kidney was determined using the method described by Ellman et al. (35). The basis of the GSH determination method is the reaction of Ellman’s reagent 5-5'-Dithio-bis-(2nitrobenzoic acid) (DTNB) with thiol group of GSH at pH 8.0 to give yellow color of 5-thiol-2nitrobenzoate anion. The homogenate (0.75 ml) was precipitated with 0.75 ml of 4% sulphosalicylic acid. Samples were centrifuged at 10,000 rpm for 15 min at 4 0C. The assay
mixture contained 0.5 ml supernatant and 4.5 ml of 0.001 mol/ L DTNB (in 0.1mol/L phosphate buffer, pH 8.0). The yellow color developed was read immediately at 412 nm. Results are expressed as nmol GSH/ mg protein (36).
Measurement of SOD activity SOD activity was assayed according to the method described by Misra et al. (37). This method is based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to adrenochrome and other derivatives at alkaline pH. A mixture of 2.80 ml of sodium carbonate (0.05 mM) buffer (pH 10.2), 100 µl of EDTA (1.0 mM) and 20 µl of kidney homogenate or sucrose (blank) were incubated at 30 0C for 45 min. Thereafter, reaction was initiated by adding 100 µl of adrenaline solution (9.0 mM). The change in the absorbance was recorded at 480 nm for 8–12 min. Similarly, SOD calibration curve was prepared by taking 10 units/ ml a standard solution. One unit of SOD produced approximately 50% inhibition of auto-oxidation of adrenaline. Results are expressed as SOD unit/ mg of protein (34). Measurement of CAT activity CAT activity was assayed according to the method of Luck et al. (38), in which the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, 2.25 ml of potassium phosphate buffer (PBS; 65 mM, pH 7.8) and 100 µl of the kidney homogenate were incubated at 25 0C for
30 min. 650 µl H2O2 (7.5 mM) was added to the kidney homogenate to initiate the reaction. The
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change in absorption was measured at 240 nm for 2–3 min and the results are expressed as µmol H2O2 decomposed/ mg of protein (39).
4.5.
Determination of renal and urinary TNF-α
The level of TNF-α in the kidney cortex and urine samples was measured in duplicate by a commercially sandwich enzyme-linked immunosorbent assay (ELISA) kit (Enzo life sciences,
USA) according to the manufacturer’s recommendations.
4.6.
Renal pathology and measurement of glomerular surface area (GSA)
After fixation of the kidney tissues in 10% formalin, they were embedded in paraffin and 5 µm thick sections were made and stained with Masson's trichrome for quantitative histology and with hematoxylin and eosin (H&E) for general histology and morphometric analysis by light microscopy. The GSA was measured to evaluate glomerular hypertrophy. For each animal, 50 glomeruli per mid-transverse section of the kidney cortex were determined using Image-Tool software. The mean GSA was calculated by averaging the maximum width and the maximum length of the glomerulus.
4.7.
Statistical analysis
We expressed all data as mean ± standard error of mean, and used one-way ANOVA followed by the Tukey test (SPSS v.20) to assess the differences between groups. Differences with values of P< 0.05 were considered significant.
Acknowledgment The collaboration of Mrs. Moazzam and Goldar in expert technical assistance is gratefully acknowledged by the authors. This work was a part of a PhD thesis by Anita Barzegar-Fallah and financially supported by Iran University of Medical Sciences, Grant #21851. Authors declare that there are no conflicts of interest.
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Table 1. Effects of 2 weeks treatment with tropisetron or granisetron on control and streptozotocin (STZ)-induced diabetic rats. The kidney index was expressed as [KW (g)/BW (g)] ×100. Values are means ± SE, n= 6/group.
Control
Tropi
Grani
STZ
STZ+ Tropi
STZ+ Grani
BW (g)
KW (g)
KW/BW
273.22±
266.65±
260.80±
235.0 ±
250.40 ±
247.01±
7.43
10.86
9.25
9.96***
13.81
18.15*
1.864±
1.900 ±
1.879±
2.236 ±
1.995±
2.214±
0.064
0.111
0.111
0.243**
0.201#
0.215*
0.683±
0.713 ±
0.722±
0.997±
0.810± 0.120 0.901±
0.034
0.039
0.052
0.099***
*,#
0.117**
73.4±
103.1±
66.8±
2.5± 0.69
8.87***
16.13***
11.61***
1.48±
13.98± 2.09
0.98
***
UFR (ml/day)
2.7± 0.73
UAE
1.66±
(mg/day)
0.40
9.2± 2.55
1.54± 0.36
12.21± 7.71± 1.19 *
1.44***
BW, Body weight; KW, kidney weight; KW/BW, kidney weight/body weight index; UFR, urine flow rate; UAE, urinary albumin excretion; STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron.
*
P< 0.05,
**
P< 0.01 and
***
P< 0.001 compared with control group;
compared with STZ group.
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#
P< 0.05
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Figure legends: Fig. 1. Effects of 2 weeks treatment with tropisetron or granisetron on blood glucose concentrations. Blood glucose levels were determined at multiple time points up to 15 days after induction of diabetes. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.001 compared with control group; #P< 0.05 compared
with STZ-treated group. Fig. 2. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular filtration rate (GFR) in normal and diabetic rats. GFRs were determined by calculating the mean of urea nitrogen clearance and creatinine clearance per gram body weight. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. Values are means ± SE, n= 6/ group. *P< 0.05 and ***P< 0.001 compared with control. ##P< 0.01 compared with STZ+ Tropi group. Fig. 3. Effects of 2 weeks treatment with tropisetron or granisetron on oxidative stress markers in renal cortex. (a) malondialdehyde (MDA) level, (b) reduced glutathione (GSH) level, (c) superoxide dismutase (SOD) level, (d) catalase (CAT) level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and ***P< 0.001 compared with control group; #P< 0.05 and ##P< 0.01 compared with STZ-treated group.
Fig. 4. Effects of 2 weeks treatment with tropisetron or granisetron on renal cortex TNF-α. The level of TNF-α in diabetic rats were significantly increased compared with control group, and treatment with granisetron and tropisetron attenuated its level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and **P< 0.01 compared with control group; #P< 0.05 compared with STZ-treated group.
Fig. 5. Effects of 2 weeks treatment with tropisetron or granisetron on urinary TNF-α. Elevated level of TNF-α as a good indicator of early nephrotoxicity significantly increased in all diabetic
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rats, and tropisetron was able to decrease it however it was not significant compared with STZ group. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.01 compared with control group. Fig. 6. Effects of 2 weeks treatment with tropisetron or granisetron on the kidney histological changes induced by streptozotocin in Masson's trichrome-stained sections of rat kidneys, ×400. (A) Normal glomeruli of control rats. (B) Glomerular hypertrophy and slight glomerular basement membrane thickening of diabetic rats. (C) Normal glomeruli of animals treated with tropisetron alone. (D) Glomeruli of diabetic rats treated with tropisetron, which significantly improved kidney damage resulting from STZ-induced diabetes in the kidney. (E) Normal glomeruli of animals treated with granisetron alone. (F) Glomeruli of diabetic rats treated with granisetron, which did not affect kidney histological damage. Arrows indicate glomerular
changes in the kidney. Fig. 7. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular surface area. It was determined by microscopic examination of hematoxylin- and eosin-stained paraffinembedded cortex sections. The areas of a minimum of 50 glomeruli/ section were determined by using Image-Tool software. Tropisetron suppressed glomerular hypertrophy of the diabetic rats. Values are means ± SE of glomerular surface area, n= 6/ group. GSA, glomerular surface area; STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 compared with control group; #P< 0.05 compared with STZ-treated group.
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Article Type: Original Article
Tropisetron Ameliorates Early Diabetic Nephropathy in StreptozotocinInduced Diabetic Rats Anita Barzegar-Fallah1, Houman Alimoradi2, Firouzeh Asadi1, Ahmad Reza Dehpour3, Mojgan Asgari4, Massoumeh Shafiei1*
Short title: Renoprotective effects of tropisetron 1
Department of Pharmacology, School of Medicine, Iran University of Medical Sciences,
Tehran, Iran 2
Department of Pharmacology and Toxicology, University of Otago, P.O. Box 913, Dunedin,
New Zealand
3
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences,
Tehran, Iran
4
Department of Pathology & Oncopathology Research Center, Shahid Hasheminejad Hospital,
Iran University of Medical Sciences, Tehran, Iran
* Corresponding author. Tel: +98 21 88622573; Fax: +98 21 88622696
E-mail address: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12373 This article is protected by copyright. All rights reserved.
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Address: Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. P.O. Box: 14155-6183
Abstract
Oxidative stress and inflammation are well established to be involved in the pathogenesis of diabetic nephropathy. It has been shown that tropisetron exerts anti-inflammatory and immunomodulatory properties. Current study was designed to investigate protective effects of tropisetron on early diabetic nephropathy in streptozotocin (STZ)-induced diabetic rats. Rats were divided into six groups: (1) untreated diabetic, (2) untreated control, (3-6) diabetic and normal rats treated with tropisetron or granisetron (3 mg/kg) beginning at the time of diabetes induction for 2 weeks. At the termination of the experiments, bodyweight, kidney index, urinary albumin excretion (UAE) and glomerular filtration rate were measured. Moreover, the levels of oxidative stress markers and tumor necrosis factor alpha (TNF-α) were determined. STZ-treated animals showed significant loss of body weight, renal enlargement and dysfunction. Diabetic rats also exhibited an increase in malondialdehyde level along with a significant decrease in reduced glutathione level, and superoxide dismutase and catalase activity. Furthermore, the diabetic animals demonstrated a significant rise in renal cortical and urinary TNF-α levels and UAE. Both granisetron and tropisetron decreased blood glucose in diabetic animals however this decrease was not significant for granisetron. Treatment with tropisetron, but not granisetron, prevented enhanced oxidative stress and TNF-α level, decreased urinary cytokine excretion and albuminuria, and improved the renal morphological damage. In conclusion, the present study suggests that tropisetron may be a protective agent in early diabetic nephropathy, whose action can be mediated, at least in part, by the antioxidative and anti-inflammatory mechanisms and these effects appear to be 5-HT3 receptor-independent.
Key words: Diabetic nephropathy; Tropisetron; Oxidative stress; TNF-α; Renal function
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Introduction
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1.
Diabetic nephropathy (DN) is one of the most serious microvascular complications of both insulin-dependent and non-insulin dependent diabetes mellitus (1) and has become the major cause of end-stage renal disease worldwide (2). DN is defined by specific renal morphological and functional alterations. Features of early diabetic renal changes are an increase in kidney size, glomerular and tubular hypertrophy and abnormal renal function as recognized by creatinine clearance (CrCl) or glomerular filtration rate (GFR) (1). It follows by excessive urinary albumin excretion (UAE), thickening of the glomerular basement membrane and mesangial expansion with the accumulation of glomerular extracellular matrix (3). Hyperglycemia, which has a key role in the pathogenesis of DN, leads to the mitochondrial production of reactive oxygen species (ROS), attenuation of antioxidative mechanisms through glycation of the scavenging enzymes and subsequently cell death and kidney dysfunction (4, 5). Besides oxidative stress, inflammation is intimately linked with the development of DN. There is growing evidence that tumor necrosis factor alpha (TNF-α) is one of the major pro-inflammatory cytokines in the pathogenesis of kidney injury and also produced intrinsically in renal cells (6-8).
Moreover, increases in oxidative stress can increase the production of inflammatory cytokines and likewise, an increase in inflammatory cytokines can stimulate the production of free radicals. Hence, targeting oxidative stress-inflammatory cytokine signaling is speculated to provide an opening for development of novel therapies against DN (9). 5-HT3 receptor antagonists including tropisetron and granisetron are safe drugs with large therapeutic index and are widely used as an effective and well tolerated agent to counteract chemotherapy-induced emesis (10). New investigations indicate that tropisetron exerts notable immune modulatory, analgesic and anti-inflammatory properties (11, 12). In this regard, we have recently shown that tropisetron significantly suppressed the elevated pro-inflammatory cytokines interleukin-2 (IL-2) and TNF-α in a rat model of neurotoxicity induced by vincristine (13). In another study, we reported that pretreatment with tropisetron significantly improved neurological deficits, diminished leukocyte transmigration into the brain, suppressed TNF-α level, and also attenuated brain infarction and edema in an embolic model of stroke in rats. The findings of the latter study suggest that the mentioned properties might not be 5-HT3 receptor-dependent since
granisetron, another selective 5-HT3 receptor antagonist, failed to elicit protective effects (14). In another study, we demonstrated that tropisetron exerted cardioprotective effects against
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Figure legends: Fig. 1. Effects of 2 weeks treatment with tropisetron or granisetron on blood glucose concentrations. Blood glucose levels were determined at multiple time points up to 15 days after induction of diabetes. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.001 compared with control group; #P< 0.05 compared
with STZ-treated group. Fig. 2. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular filtration rate (GFR) in normal and diabetic rats. GFRs were determined by calculating the mean of urea nitrogen clearance and creatinine clearance per gram body weight. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. Values are means ± SE, n= 6/ group. *P< 0.05 and ***P< 0.001 compared with control. ##P< 0.01 compared with STZ+ Tropi group. Fig. 3. Effects of 2 weeks treatment with tropisetron or granisetron on oxidative stress markers in renal cortex. (a) malondialdehyde (MDA) level, (b) reduced glutathione (GSH) level, (c) superoxide dismutase (SOD) level, (d) catalase (CAT) level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and ***P< 0.001 compared with control group; #P< 0.05 and ##P< 0.01 compared with STZ-treated group.
Fig. 4. Effects of 2 weeks treatment with tropisetron or granisetron on renal cortex TNF-α. The level of TNF-α in diabetic rats were significantly increased compared with control group, and treatment with granisetron and tropisetron attenuated its level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and **P< 0.01 compared with control group; #P< 0.05 compared with STZ-treated group.
Fig. 5. Effects of 2 weeks treatment with tropisetron or granisetron on urinary TNF-α. Elevated level of TNF-α as a good indicator of early nephrotoxicity significantly increased in all diabetic
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changes, while granisetron with the same dosage did not exert any effects on biochemical markers and histological damage. Regarding the structural similarity of these 5-HT3 receptor
antagonists and the diverse effects of these two compounds in early diabetic nephropathy, we believe that the molecular target for the beneficial activity of tropisetron might be independent of its binding to the 5-HT3 receptor. The same results obtained from our previous studies on the neurotoxicity of vincristine (13) and embolic model of stroke (14) confirm this hypothesis. Tropisetron, a selective 5-HT3 receptor antagonist, is widely used as an effective and well
tolerated agent in counteracting chemotherapy-induced emesis (20). New investigations have shed light on additional applications for this drug (12, 21) and findings of a pioneering study show new mechanistic insights into anti-inflammatory and immune modulatory properties of tropisetron (11). Further, Vega et al. (22) have indicated that tropisetron inhibits antigen-induced proliferation of human peripheral T cells and production of IL-2. Also they proposed that tropisetron exerts its inhibitory effect via blockade of calcineurin/NFAT-dependent signaling pathway. Briefly, hyperglycemia, the main determinant of the pathogenesis of DN, not only generates
more ROS, such as superoxide and hydrogen peroxide, but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes including SOD and CAT (5, 23). This is in agreement with our findings, as diabetic rats exhibited a marked increase in renal MDA content and a decrease in GSH level and the activity of SOD and CAT. Moreover, increased cytoplasmic calcium (Ca2+) concentration which is induced by oxidative
stress activates phosphatase activity of calcineurin, a Ca2+/calmodulin-dependent protein. It subsequently dephosphorylates the nuclear factor of activated T cells (NFAT) which finally regulates numerous cytokines and pro-inflammatory factors (24, 25). TNF-α has been implicated as a potent pro-inflammatory cytokine in the development and progression of renal injury in DN by promoting inflammation, the accumulation of extracellular matrix and damaging the glomerular permeability barrier with the development of albuminuria. Further, some evidence has suggested that urinary TNF-α may be a useful biomarker for detection of kidney injury in the very early stages of DN (8). Our study demonstrated a significant rise in renal cortical and urinary TNF-α levels 2 weeks after induction of diabetes and these findings were correlated with an increase in UAE. Tropisetron treatment was associated with a reduction of albuminuria and
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renal and urinary TNF- α levels. Our findings are in agreement with previous reports by other investigators (7, 26). In the present study, a mild reduction of blood glucose levels was also demonstrated in tropisetron+ STZ- and granisetron + STZ-treated animals. As it was shown that serotonin and in particular the 5-HT3 receptor channel system are involved in modulating insulin release through
pancreatic insulin-producing beta cell line (27), it is possible that blockade of 5-HT3 receptor by
these drugs is responsible for this hypoglycemic effect. Next, GFR was increased in untreated STZ rats, in comparison with control group and treatment of diabetic animals with tropisetron resulted in a further increase in GFR. This finding is consistent with previously published studies by Gooch et al. (17, 28) showing that GFR in the early stages of diabetes was higher than normal, and cyclosporine A treatment resulted in a further increment in urine output and finally GFR. The expression of calcineurin has been shown to increase rapidly in STZ-induced diabetes primarily in the thick ascending limb of Henle. On the other hand, a potent inhibition of calcineurin by cyclosporine A has been demonstrated to alter aquaporin 2 (AQP2) localization and phosphorylation in principal cells of collecting ducts. As a consequence, cyclosporine A treatment resulted in a further increase in urine output compared with diabetes alone, suggesting a functional consequence of inhibiting calcineurinmediated regulation of AQP2 (28). Our data is in agreement with this study, as urine volume in only tropisetron treated animals were increased compared with control group, and also urine excretion in STZ+ tropisetron group was more than STZ group (Table 1). Moreover, it was shown that tropisetron potently inhibited calcineurin activity in cerebellar granule cell culture (18). Therefore, we believe that the main reason for increased GFR in STZ+ tropisetron group is due to inhibition of calcineurin-mediated regulation of AQP2 and very significant increase in the UFR. In conclusion, our results present the beneficial properties of tropisetron in an experimental model of early STZ-induced diabetic nephropathy, possibly, at least in part, through amelioration of inflammation and oxidative stress. Future investigations can unravel the detailed mechanisms of tropisetron-induced renoprotection and whether this effect is fully independent of 5-HT3 receptor.
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4. Methods 4.1.
Ethics
This study was performed according to the guidelines of the US National Institute of Health (NIH publication no.85.23, revised 1985) guides for the care of lab animals. 4.2.
Animal protocol
Experiments were performed on 200-250 g male Wistar rats in Department of Pharmacology, Iran University of Medical Sciences. Animals were housed in a temperature and humidity controlled environment with 12-h light/12-h dark cycle. Food consists of normal rat chow and water ad libitum. Rats were randomly divided into 6 groups of 6 rats/ group. Group 1 or STZ group were injected intravenously (IV) via the jugular vein with a single dose of 55 mg/kg body wt STZ (AppliChem GmbH, Germany) in sodium citrate buffer (pH 4.0) to induce diabetes. Group 2 or control group were similarly injected with sodium citrate buffer alone. Rats in group 3, 4 were injected with STZ and were also given daily intraperitoneally (IP) injections of tropisetron (STZ+ Tropi group) or granisetron (STZ+ Grani group) (3 mg/kg body wt) beginning at the time of diabetes induction till the end of 2th week for 14 days. Group 5, 6 were given daily
injections of tropisetron (Tropi group) or granisetron (Grani group) alone for 14 days. Tropisetron and granisetron were freshly prepared by dissolving in double distilled water. After injection of STZ, rats were subjected to a 48 h fast and then they had unrestricted access to food and were maintained in accordance with Institutional Animal Care and Use Committee procedures. Moreover, blood glucose levels were determined by using a glucometer (Media Smart, Switzerland) with a drop of blood from the tail vein to verify hyperglycemia and periodically thereafter twice a week. Only diabetic rats with a blood glucose levels (non-fast) > 300 mg/dl were included in the present study. The body weights and blood glucose levels of the experimental animals were taken twice a week. In this study, 3 days after STZ injection, which rats showed the first symptoms of diabetes, were considered as the day 0 of the study.
4.3.
Renal function
Two weeks after induction of diabetes (day 14 of study), animals were placed in metabolic cages overnight with unrestricted access to food and water, and the volume of urine were measured.
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The urine was collected for determination of nitrogen, creatinine and albumin levels. On day 15 of the study, rats were weighed, and then anaesthetized using IP injection of ketamine (60 mg/kg) and xylazine (8 mg/kg). After blood collection from heart, both kidneys were removed and total KW was measured with a precise balance. Serum blood urea nitrogen (BUN), serum creatinine levels and UAE were measured (29-31). GFR, an indicator of residual renal function, was determined by mean of urea nitrogen clearance and CrCl per gram BW. The kidney index was expressed as [KW (g)/BW (g)] ×100. A slice of kidney cortex at the pole was fixed in 10% formalin in 0.1 M sodium phosphate buffer, pH 7.4, for preparation of sections for light microscopy and image analyses. A second portion was quick-frozen immediately in liquid nitrogen for measurement of TNF-α and
oxidative stress markers.
4.4.
Assessment of renal cortex oxidative stress
Preparation of renal homogenate Following the decapsulation of kidneys, 100 mg of kidney cortex was minced and a homogenate was prepared with 10% (w/v) phosphate-buffered saline (0.1 mol/L, pH 7.4) using a homogenizer. Then the kidney homogenate was centrifuged at 1000 rpm for 3 min at 4 0C and
the supernatant was collected and divided into two portions. One of which was used for measurement of lipid peroxidation, in terms of MDA content, and GSH and the remaining ◦
supernatant was centrifuged again at 12,000 rpm at 4 C for 20 min and used for the measurement of antioxidant enzyme activities of SOD and CAT and level of TNF-α. The protein content of renal homogenate was measured by the method of Lowry et al (32) using bovine serum albumin as the standard.
Determination of lipid peroxidase Lipid peroxidation was measured through determination of MDA level using a thiobarbituric acid reactive substances method as described before by Ohkawa et al. (33). In brief, 0.1 ml of kidney homogenate was mixed with 20% of 1.5 ml of acetic acid (pH 3.5), 1.5 ml thiobarbituric acid and 0.2 ml sodium dodecyl sulphate (8.1 %). The mixture was then heated at 100 0C for 60
min. The mixture was cooled and 5 ml of n-butanol–pyridine mixture (15:1) was added followed
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by 1 ml of distilled water. After centrifugation of the mixture at 4000 rpm for 10 min, the organic layer was taken and its absorbance was measured at 532 nm. The concentration of MDA formed is expressed as nmol MDA/ mg protein (34).
Estimation of GSH GSH in the kidney was determined using the method described by Ellman et al. (35). The basis of the GSH determination method is the reaction of Ellman’s reagent 5-5'-Dithio-bis-(2nitrobenzoic acid) (DTNB) with thiol group of GSH at pH 8.0 to give yellow color of 5-thiol-2nitrobenzoate anion. The homogenate (0.75 ml) was precipitated with 0.75 ml of 4% sulphosalicylic acid. Samples were centrifuged at 10,000 rpm for 15 min at 4 0C. The assay
mixture contained 0.5 ml supernatant and 4.5 ml of 0.001 mol/ L DTNB (in 0.1mol/L phosphate buffer, pH 8.0). The yellow color developed was read immediately at 412 nm. Results are expressed as nmol GSH/ mg protein (36).
Measurement of SOD activity SOD activity was assayed according to the method described by Misra et al. (37). This method is based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to adrenochrome and other derivatives at alkaline pH. A mixture of 2.80 ml of sodium carbonate (0.05 mM) buffer (pH 10.2), 100 µl of EDTA (1.0 mM) and 20 µl of kidney homogenate or sucrose (blank) were incubated at 30 0C for 45 min. Thereafter, reaction was initiated by adding 100 µl of adrenaline solution (9.0 mM). The change in the absorbance was recorded at 480 nm for 8–12 min. Similarly, SOD calibration curve was prepared by taking 10 units/ ml a standard solution. One unit of SOD produced approximately 50% inhibition of auto-oxidation of adrenaline. Results are expressed as SOD unit/ mg of protein (34). Measurement of CAT activity CAT activity was assayed according to the method of Luck et al. (38), in which the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, 2.25 ml of potassium phosphate buffer (PBS; 65 mM, pH 7.8) and 100 µl of the kidney homogenate were incubated at 25 0C for
30 min. 650 µl H2O2 (7.5 mM) was added to the kidney homogenate to initiate the reaction. The
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change in absorption was measured at 240 nm for 2–3 min and the results are expressed as µmol H2O2 decomposed/ mg of protein (39).
4.5.
Determination of renal and urinary TNF-α
The level of TNF-α in the kidney cortex and urine samples was measured in duplicate by a commercially sandwich enzyme-linked immunosorbent assay (ELISA) kit (Enzo life sciences,
USA) according to the manufacturer’s recommendations.
4.6.
Renal pathology and measurement of glomerular surface area (GSA)
After fixation of the kidney tissues in 10% formalin, they were embedded in paraffin and 5 µm thick sections were made and stained with Masson's trichrome for quantitative histology and with hematoxylin and eosin (H&E) for general histology and morphometric analysis by light microscopy. The GSA was measured to evaluate glomerular hypertrophy. For each animal, 50 glomeruli per mid-transverse section of the kidney cortex were determined using Image-Tool software. The mean GSA was calculated by averaging the maximum width and the maximum length of the glomerulus.
4.7.
Statistical analysis
We expressed all data as mean ± standard error of mean, and used one-way ANOVA followed by the Tukey test (SPSS v.20) to assess the differences between groups. Differences with values of P< 0.05 were considered significant.
Acknowledgment The collaboration of Mrs. Moazzam and Goldar in expert technical assistance is gratefully acknowledged by the authors. This work was a part of a PhD thesis by Anita Barzegar-Fallah and financially supported by Iran University of Medical Sciences, Grant #21851. Authors declare that there are no conflicts of interest.
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Anjaneyulu M, Chopra K. Quercetin, an anti-oxidant bioflavonoid, attenuates diabetic
nephropathy in rats. Clin. Exp. Pharmacol. Physiol. 2004; 31:244-8.
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Table 1. Effects of 2 weeks treatment with tropisetron or granisetron on control and streptozotocin (STZ)-induced diabetic rats. The kidney index was expressed as [KW (g)/BW (g)] ×100. Values are means ± SE, n= 6/group.
Control
Tropi
Grani
STZ
STZ+ Tropi
STZ+ Grani
BW (g)
KW (g)
KW/BW
273.22±
266.65±
260.80±
235.0 ±
250.40 ±
247.01±
7.43
10.86
9.25
9.96***
13.81
18.15*
1.864±
1.900 ±
1.879±
2.236 ±
1.995±
2.214±
0.064
0.111
0.111
0.243**
0.201#
0.215*
0.683±
0.713 ±
0.722±
0.997±
0.810± 0.120 0.901±
0.034
0.039
0.052
0.099***
*,#
0.117**
73.4±
103.1±
66.8±
2.5± 0.69
8.87***
16.13***
11.61***
1.48±
13.98± 2.09
0.98
***
UFR (ml/day)
2.7± 0.73
UAE
1.66±
(mg/day)
0.40
9.2± 2.55
1.54± 0.36
12.21± 7.71± 1.19 *
1.44***
BW, Body weight; KW, kidney weight; KW/BW, kidney weight/body weight index; UFR, urine flow rate; UAE, urinary albumin excretion; STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron.
*
P< 0.05,
**
P< 0.01 and
***
P< 0.001 compared with control group;
compared with STZ group.
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#
P< 0.05
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Figure legends: Fig. 1. Effects of 2 weeks treatment with tropisetron or granisetron on blood glucose concentrations. Blood glucose levels were determined at multiple time points up to 15 days after induction of diabetes. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.001 compared with control group; #P< 0.05 compared
with STZ-treated group. Fig. 2. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular filtration rate (GFR) in normal and diabetic rats. GFRs were determined by calculating the mean of urea nitrogen clearance and creatinine clearance per gram body weight. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. Values are means ± SE, n= 6/ group. *P< 0.05 and ***P< 0.001 compared with control. ##P< 0.01 compared with STZ+ Tropi group. Fig. 3. Effects of 2 weeks treatment with tropisetron or granisetron on oxidative stress markers in renal cortex. (a) malondialdehyde (MDA) level, (b) reduced glutathione (GSH) level, (c) superoxide dismutase (SOD) level, (d) catalase (CAT) level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and ***P< 0.001 compared with control group; #P< 0.05 and ##P< 0.01 compared with STZ-treated group.
Fig. 4. Effects of 2 weeks treatment with tropisetron or granisetron on renal cortex TNF-α. The level of TNF-α in diabetic rats were significantly increased compared with control group, and treatment with granisetron and tropisetron attenuated its level. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 and **P< 0.01 compared with control group; #P< 0.05 compared with STZ-treated group.
Fig. 5. Effects of 2 weeks treatment with tropisetron or granisetron on urinary TNF-α. Elevated level of TNF-α as a good indicator of early nephrotoxicity significantly increased in all diabetic
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rats, and tropisetron was able to decrease it however it was not significant compared with STZ group. Values are means ± SE, n= 6/ group. STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. ***P< 0.01 compared with control group. Fig. 6. Effects of 2 weeks treatment with tropisetron or granisetron on the kidney histological changes induced by streptozotocin in Masson's trichrome-stained sections of rat kidneys, ×400. (A) Normal glomeruli of control rats. (B) Glomerular hypertrophy and slight glomerular basement membrane thickening of diabetic rats. (C) Normal glomeruli of animals treated with tropisetron alone. (D) Glomeruli of diabetic rats treated with tropisetron, which significantly improved kidney damage resulting from STZ-induced diabetes in the kidney. (E) Normal glomeruli of animals treated with granisetron alone. (F) Glomeruli of diabetic rats treated with granisetron, which did not affect kidney histological damage. Arrows indicate glomerular
changes in the kidney. Fig. 7. Effects of 2 weeks treatment with tropisetron or granisetron on glomerular surface area. It was determined by microscopic examination of hematoxylin- and eosin-stained paraffinembedded cortex sections. The areas of a minimum of 50 glomeruli/ section were determined by using Image-Tool software. Tropisetron suppressed glomerular hypertrophy of the diabetic rats. Values are means ± SE of glomerular surface area, n= 6/ group. GSA, glomerular surface area; STZ, streptozotocin; Tropi, tropisetron; Grani, granisetron. *P< 0.05 compared with control group; #P< 0.05 compared with STZ-treated group.
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