European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Q1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats Mohamed A. Morsy a,n, Gehan H. Heeba b, Magda E. Mahmoud c a
Department of Pharmacology, Faculty of Medicine, Minia University, 61511 El-Minia, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Minia University, 61511 El-Minia, Egypt c Department of Agricultural Chemistry, Faculty of Agriculture, Minia University, 61511 El-Minia, Egypt b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 15 January 2015 Accepted 19 January 2015
Diabetic nephropathy becomes the single most frequent cause of end-stage renal disease. The present study aimed therefore to investigate possible protective effect of eprosartan, an angiotensin II type 1 receptor blocker, on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats and various mechanisms underlie this effect. Male Wistar rats with type 2 diabetes induced by high-fat diet/streptozotocin were treated with eprosartan at the dose levels of 30 and 60 mg/kg daily for 5 weeks. Eprosartan induced a nephroprotective effect as evident by the significant decrease in serum creatinine, urea, total cholesterol, triglycerides and glucose levels, urinary albumin excretion and kidney index as well as renal levels of malondialdehyde and nitric oxide products (nitrite/nitrate), in addition to angiotensin II, inducible nitric oxide synthase, transforming growth factor-β1 and collagen IV expressions with a concurrent increase in renal levels of reduced glutathione and catalase activity compared to diabetic untreated rats. Histopathological examination confirmed the renoprotective effect of eprosartan. In conclusion, eprosartan protects rats against high-fat diet/streptozotocininduced early diabetic nephropathy possibly, in part, through its antioxidant effect as well as by abrogating the overexpression of angiotensin II, inducible nitric oxide synthase, transforming growth factor-β1 and collagen IV. & 2015 Published by Elsevier B.V.
Keywords: Eprosartan Diabetic nephropathy Angiotensin II Inducible nitric oxide synthase Transforming growth factor-β1 Collagen IV
1. Introduction Diabetic nephropathy is the leading cause of end-stage renal disease and its prevalence is increasing because of the global epidemic of diabetes (Hajhosseiny et al., 2014). It is characterized clinically by progressive albuminuria and histologically by definite changes including progressive accumulation of extracellular matrix components (e.g. collagen IV) that lead to renal fibrosis and failure (Kolset et al., 2012). Dyslipidemia is considered as a risk factor for the progression of diabetic nephropathy (Ravid et al., 1998). Hyperglycemia increases the production of angiotensin (Ang) II that generates reactive oxygen species by activation of angiotensin II type 1 receptors resulting in oxidative stress (Welch, 2008). Oxidative stress is considered as a key component in the development of diabetic complications including diabetic nephropathy (Niedowicz and Daleke, 2005). Ang II is also a potent growth modulator and proinflammatory peptide (Vieitez et al., 2008). Similar to Ang II, hyperglycemia increases the production of transforming growth factor (TGF)-β in mesangial cells (Poczatek et al., 2000). TGF-β induces the accumulation of extracellular matrix and increases reactive oxygen species production in tubular epithelial cells (Chiarelli et al., 2009). n
Corresponding author. Tel.: þ 20 1112197640; fax: þ 20 862342813. E-mail address: [email protected] (M.A. Morsy).
Nitric oxide (NO) is an important gasotransmitter molecule that mediates a variety of physiological and pathological processes in kidney. Limiting NO production decreases glomerular injury and subsequent glomerulosclerosis (Narita et al., 1995). Inducible NO synthase (iNOS) produces large amounts of NO in many cell types in response to several stimuli (Förstermann and Sessa, 2012). Alternatively, high concentrations of glucose have been shown to increase iNOS-induced NO production and promote extracellular matrix accumulation in rat mesangial cells (Noh et al., 2002). Studies have shown that several angiotensin receptor blockers via their pleiotropic effects are beneficial in controlling the development and progression of diabetic nephropathy (Si et al., 2014; Zhou et al., 2014). Therefore, eprosartan, an angiotensin II type 1 receptor blocker, seems conceivable to be implicated in amelioration of diabetic nephropathy. The aim of the present study was to investigate the effects of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats and the underlying mechanisms.
2. Materials and methods 2.1. Chemicals Eprosartan mesylate (purity 498%) was purchased from Matrix Laboratories Ltd. (Hyderabad, India). Streptozotocin was purchased
http://dx.doi.org/10.1016/j.ejphar.2015.01.027 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
from Sigma-Aldrich Corp. (St. Louis, MO, USA). Antibodies against iNOS and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals were of analytical grade and were obtained from commercial sources. 2.2. Animals Male Wistar rats weighing 170–190 g were used after 1 week for proper acclimatization to the animal house conditions (12 h lighting cycle and 257 2 1C temperature) and had free access to standard rodent chow (25% protein, 5% fat, 40% starch, 10% sugar, 6% fibers, 8% ash, 5% minerals and 1% vitamins) and water. Procedures involving animals and their care were conducted according to ethical guidelines of Minia University, Egypt and EU Directive 2010/63/EU. 2.3. Experimental induction of diabetes Rats had free access to high-fat diet which was prepared as previously described (Ouwens et al., 2005) by mixing casein (30%), raw beef fat (suet, 40%), wheat flour (7%), glucose (10%), salt mixture (6%), vitamin mixture (3%), and bran (4%) and sufficient water to form a consistent paste. After 4 weeks, type 2 diabetes was induced in overnight fasted rats by intraperitoneal injection of freshly prepared streptozotocin (40 mg/kg, dissolved in 0.1 M cold citrate buffer, pH 4.5) (Srinivasan et al., 2005). Three days after streptozotocin injection, rats with fasting blood glucose level Z200 mg/dl measured by a glucometer (Accu-Chek, Roche, Basel, Switzerland) were considered diabetic and selected for the study. 2.4. Experimental procedures Diabetic rats were randomized into 3 groups of 6 animals each, namely non-treated diabetic group, low-dose eprosartan (30 mg/kg/ day, orally) group (Mukaddam-Daher et al., 2009), and high-dose eprosartan (60 mg/kg/day, orally) group (Abrahamsen et al., 2002). Normal-diet non-diabetic rats served as control group (n¼ 6). Our preliminary experiments showed that eprosartan alone at both low and high doses did not alter renal function markers so only normaldiet non-diabetic rats treated with the high dose of eprosartan were presented and served as eprosartan control group (n¼6). Eprosartan was suspended in 1% aqueous solution of carboxymethyl cellulose. All groups received equivalent volumes of the above-mentioned vehicles. High-fat diet/streptozotocin-induced diabetic rats were left for 5 weeks untreated to induce early diabetic nephropathy (Honoré et al., 2012). The treatment was initiated one week after the induction of diabetes and continued for 4 weeks. The animals were placed in individual metabolic cages for 24 h to collect urine before they were killed. At the end of the experiment, rats were fasted overnight and each rat was weighed then killed. Rats were dissected to obtain the kidneys, which were weighted. Kidney index ([kidney weight/body weight] 1000) that reveals the profile of kidney hypertrophy (Liu et al., 2003) was calculated. Blood samples were collected and centrifuged at 3000g for 10 min to obtain clear sera. Both kidneys were rapidly dissected out and weighed. The longitudinal section of the right kidney from each animal was used for histological examination. The renal cortices of the rest of the kidneys were snap frozen in liquid nitrogen and stored at 80 1C. 2.5. Biochemical analysis Using commercially available kits, serum creatinine (Diamond Diagnostics, Egypt), urea and glucose (Biodiagnostic, Egypt), cholesterol and triglycerides (Spectrum Diagnostics, Egypt), urinary albumin (BioSystems, Spain), as well as renal reduced glutathione
and catalase (Biodiagnostic, Egypt) levels were quantified according to the manufacturers' guidelines. In addition, urinary albumin excretion (mg/24 h) was calculated. The renal cortex content of lipid peroxides was determined by biochemical assessment of thiobarbituric acid reacting substance through spectrophotometric measurement of color at 535 nm, using 1,1,3,3-tetramethoxypropane as standard. The results were expressed as equivalents of malondialdehyde in nmol/g tissue (Buege and Aust, 1978). Renal cortex nitric oxide level was measured as total nitrite/nitrate, the stable degradation products of nitric oxide, by reduction of nitrate into nitrite using copperized cadmium, followed by color development with Griess reagent in acidic medium (Sastry et al., 2002). 2.6. Expression of Ang II, TGF-β1, and collagen IV by real-time PCR Total RNA was isolated from kidney tissue homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. Total RNA (5 μg) was denatured at 70 1C for 2 min. Denatured RNA was reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA): transcription mixture containing 50 mM KCl, 50 mM Tris HCl (pH 8.3), 0.5 mM of deoxyribonucleotide triphosphate (dNTP), 3 mM MgCl2, 1 U/ml RNase inhibitor and 200 units of moloney murine leukemia virus reverse transcriptase. The reaction tube was placed at 42 1C for 1 h, followed by heating to 92 1C to stop the reaction. For real-time quantitative PCR, 5 μl of first-strand cDNA was used in a total volume of 25 μl, containing 12.5 μl 2 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 200 ng of each primer. Sequences of the primers were as follows: Ang II forward primer, 50 -CATCATGAGGAGACGGGG-30 and reverse primer, 50 -TCCAAGTGGACAGGTAAGCC-30 ; TGF-β1 forward primer, 50 -AGGGCTACCATGCCACTTC-30 and reverse primer, 50 -GCGGC ACGCAGCACGGTGAT-30 ; collagen IV forward primer, 50 -TGGTCCCCAAG GTGTCAAAG-30 and reverse primer, 50 -GGGGGTCCTGGGTTACCATTA30 ; and β-actin forward primer, 50 -CCTTCCTGGGCATGGAG;TCCT-30 and reverse primer, 50 -GGAGCAATGATCTTGATCTTC-30 . PCR reactions consisting of 95 1C for 10 min (1 cycle), 94 1C for 15 s, and 60 1C for 1 min (40 cycles) were performed on a StepOnePlus System (Applied Biosystems, Foster City, CA, USA). Data were analyzed with the ABI Prism 7500 Sequence Detection System software and quantified using the version 1.7 Sequence Detection Software from PE Biosystems (Foster City, CA, USA). Relative expression of studied genes was calculated using the comparative threshold cycle method. All values were normalized to the β-actin genes (Livak and Schmittgen, 2001). 2.7. Western blot analysis Kidney tissue samples were homogenized in lysis buffer (20 mM Tris–HCl pH 7.5, 50 mM 2-mercaptoethanol, 5 mM EGTA, 2 mM EDTA, 1% NP40, 0.1% SDS, 0.5% deoxycholic acid, 10 mM NaF, 1 mM PMSF, 25 mg/ml leupeptin, 2 mg/ml aprotinin) and protein concentrations were determined using a total protein kit (Biodiagnostic, Egypt). For direct immunoblotting, aliquots of lysate were mixed with loading buffer containing 2-mercaptoethanol and maintained at 100 1C for 10 min before loading on 10% SDSPAGE. Following SDS-PAGE separation, proteins were transferred to polyvinyl difluoride membrane. Membranes were blocked in Tris-buffered saline with Tween-20 containing 5% (w/v) non-fat milk and dried for 1 h at room temperature. Membrane strips were incubated with primary antibodies (1:1000 for iNOS and β-actin) overnight at 4 1C. Following extensive washing, membrane strips were incubated with anti-rabbit IgG (1:5000; Cell Signaling Technology Inc., MA, USA) conjugated to horseradish peroxidase for 1 h. Protein bands were detected by a standard enhanced chemiluminescence method and densitometry measurements were made using ImageJ software (freeware; rsbweb.nih.gov/ij).
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
(Fig. 2A and B). On the other hand, the untreated diabetic group 67 showed focal interstitial nephritis and cystic dilatation of renal tubules 68 (Fig. 2C). Treatment with the low dose eprosartan (30 mg/kg) improved 69 Renal tissue samples were fixed in 10% neutral buffered formalin, renal histology (Fig. 2D). The high dose eprosartan (60 mg/kg) reversed 70 embedded in paraffin, sectioned by a microtome at 5 μm thickness, renal histopathological damage induced by high-fat diet/streptozoto71 and stained with hematoxylin and eosin for histological examination cin-induced early diabetic nephropathy, that was accompanied only Q2 72 using light microscopy. 73 with slight congestion of glomerular tufts (Fig. 2E; Table 1). 74 2.9. Statistical analysis 75 3.3. Effects of eprosartan on renal malondialdehyde, nitrite/nitrate, 76 reduced glutathione and catalase activity levels The data are normally distributed and are expressed as mean7S. 77 E.M. Statistical analysis was performed by one-way ANOVA followed 78 Oxidative stress was assessed through measuring renal malondialby Tukey–Kramer postanalysis test for multiple comparisons with 79 dehyde, nitrite/nitrate, reduced glutathione and catalase activity levels. Po0.05 being considered as statistically significant. 80 Renal malondialdehyde was evaluated as an index of renal lipid 81 peroxidation and nitrite/nitrate as an indicator of renal NO level. 82 3. Results Eprosartan (30 and 60 mg/kg) treatment significantly suppressed both 83 lipid peroxidation and increased NO levels in comparison with diabetic 84 3.1. Effects of eprosartan on renal functions untreated group (Fig. 3A and B). Eprosartan at both dose levels caused 85 significant increase in renal reduced glutathione levels compared to 86 Serum creatinine as well as urea levels, 24-h urinary albumin non-treated diabetic rats (Fig. 3C). The high dose of eprosartan caused 87 excretion and kidney index was assessed as markers of renal significant increase in renal catalase activity (Fig. 3D). 88 functions. Diabetic rats showed significant increase in serum creati89 nine and urea levels as well as 24-h urinary albumin excretion and 3.4. Effects of eprosartan on serum glucose, total cholesterol 90 kidney index compared to normal group (Fig. 1A–D). Eprosartan and triglycerides levels 91 treatments at both doses significantly decreased these markers of 92 renal functions compared to non-treated diabetic rats (Fig. 1A–D). At the end of experiment, serum glucose and triglycerides 93 levels were lower in the eprosartan (30 and 60 mg/kg)-treated 94 3.2. Effects of eprosartan on renal histological changes diabetic group compared to the untreated diabetic group (Fig. 4A 95 and C). Eprosartan only in the high dose caused a significant 96 Histopathological examination revealed that both the control decrease in serum total cholesterol level compared to diabetic rats 97 and eprosartan (60 mg/kg)-treated groups had normal appearance without treatment (Fig. 4B). 98 99 100 1.5 80 101 *† 102 *† 60 103 1.0 †‡ ‡ 104 †‡ 105 40 ‡ 106 0.5 107 20 108 109 0.0 0 110 111 112 113 114 115 40 10 *† 116 *† 117 8 30 118 *†‡ 119 ‡ 6 *†‡ *†‡ 20 120 121 4 122 10 123 2 124 0 125 0 126 127 128 129 130 Fig. 1. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on serum creatinine (A) and urea (B) levels as well as urinary albumin excretion (C), and 131 kidney index (D) of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean7 S.E.M. of 6 rats. *, †, ‡Significantly different from control, 132 Epro-60, and EDN groups, respectively, at Po 0.05. +E pr o60
pr o30
+E pr o30 ED N +E pr o60
ED N
ED N
Ep ro -6 0
C
on tr
ol
60
o-
pr
+E
ED N
ED N
+E
pr o30
ED N
-6 0
Ep ro
C
on t
ro l
Kidney index
ED N
ED N
+E
C
ED N
ED N
ED N
Ep ro -6 0
on t
ro l
pr o60
+E
+E
pr o30
ED N
-6 0
Ep ro
C
on tr ol
Serum urea mg/dl
Serum creatinine mg/dl
2.8. Histological examination
Urinary albumin excretion mg/24 h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
3
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Fig. 2. Effect of eprosartan on kidney histopathological picture of high-fat diet/streptozotocin-induced early diabetic nephropathy in rats (H&E 400). (A) and (B) Sections from control and eprosartan (60 mg/kg)-treated groups show normal histological structure of renal parenchyma. (C) Early diabetic nephropathy group shows focal interstitial nephritis and cystic dilatation of renal tubules (arrows) and interstitial fibroblasts proliferation (arrows in inset). (D) Kidney tissue from early diabetic nephropathyþ eprosartan (30 mg/kg) group showing vacuolization of epithelial lining renal tubules (arrow). (E) Kidney tissue from early diabetic nephropathyþ eprosartan (60 mg/kg) group showing slight congestion of glomerular tufts. Table 1 Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on severity of histopathological lesions of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Histopathological alterations
Control
Epro-60
EDN
EDNþ Epro-30
EDN þ Epro-60
Tubular degeneration Tubular necrosis Tubular dilation Glomerular damage Dilated Bowman's spaces Congestion of renal blood vessels Leukocyte infiltrates Vacuolation of endothelium and congestion of glomerular tuft Interstitial fibroblasts proliferation
– – – – – – – – –
– – – – – – – – –
þþ þþ þþ – þ þþþ þþþ þþ þ
þþ – þ – – þþ þ þ –
þ – – – – þ – þ –
Score level – is considered normal. Scores þ , þ þ and þ þ þ are mild, moderate and severe levels, revealing less than 25%, 50% and 75% histopathological alterations of total fields examined, respectively.
3.5. Effects of eprosartan on renal mRNA Ang II, TGF-β1 and collagen IV expressions Eprosartan (30 and 60 mg/kg)-treated diabetic groups showed significant reductions in the mRNA expressions of renal Ang II and collagen IV compared to the untreated diabetic group
(Fig. 5A and C). On the other hand, only the high dose of eprosartan caused a significant down-regulation in renal TGF-β1 mRNA expression compared to the untreated diabetic group (Fig. 5B). Eprosartan alone, without high-fat diet/streptozotocin treatment, had no effect on these parameters compared to control (Fig. 5A–C).
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*†
400
*†‡
60
Nitrite/nitrate nmol/g tissue
*†‡§ 40 20
*†‡
200
ठ100
0
ED N
ED N
+E pr o60
pr o30
ED N
+E
C
on t
Ep ro -6 0
ro l
+E pr o30 +E pr o60 ED N
ED N
ED N
ol on tr C
Ep ro -6 0
0
15
8
‡
10
Catalase U/g tissue
*†‡ *†
5
‡§
6
*†
4
*† 2
0
pr o60
pr o30
ED N
ED N
+E
+E
C on t
ro
l
+E pr o30 ED N +E pr o60
ED N
ED N
Ep ro -6 0
C
on t
ro l
0 ED N
Reduced glutathione mol/g tissue
*†
300
Ep ro -6 0
Malondialdehyde nmol/g tissue
80
Fig. 3. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on renal malondialdehyde (A), nitric oxide (nitrite/nitrate) (B), reduced glutathione (C), and catalase (D) levels of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7 S.E.M. of 6 rats. *, †, ‡, §Significantly different from control, Epro-60, EDN, and EDN þEpro-30 groups, respectively, at Po 0.05.
*†
300
*†‡
Serum total cholesterol mg/dl
400
*†‡
200 100
200
*†
*†
150
*†‡
100 50
Serum triglycerides mg/dl
200
pr o60
ED N +E
pr o30
*†‡
ED N +E
on tr ol C
+E pr o30 ED N +E pr o60
ED N
*†‡
ED N
Ep ro -6 0
ro l on t C
ED N
0
0
Ep ro -6 0
Serum glucose mg/dl
*†
150 100 50
+E p
ro -6 0
ro -3 0 ED N
ED N
+E p
ED N
ro -6 0 Ep
ol
0
C on tr
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
5
Fig. 4. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on serum glucose (A), total cholesterol (B), and triglycerides (C) levels of high-fat diet/ streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7S.E.M. of 6 rats. n, †, ‡Significantly different from control, Epro-60, and EDN groups, respectively, at P o 0.05.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*†
1.0
4
0.8 0.6
*†‡ *†‡§
0.4 0.2
*†
3
‡
2 1
ED N
ED N
ED N
*†
2.0
Relative expression of collagen IV mRNA
+E pr o-
ED N
60
C on tr ol
0 +E pr o
-3
-6
0
N
+E pr o
ED N
ED
60 Ep ro -
on tr ol C
30 +E pr o60
0
0.0
Ep ro -
Relative expression of Ang II mRNA
1.5 1.0
*†‡
*†‡
0.5
pr o60
30
N ED
ED
N
N
+E
+E
pr
o-
ED
0 -6 ro Ep
on tr ol
0.0
C
Fig. 5. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on angiotensin (Ang) II (A), transforming growth factor (TGF)-β1 (B), and collagen IV mRNA expressions of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7S.E.M. of 6 rats. *, †, ‡, §Significantly different from control, Epro-60, EDN, and EDNþ Epro-30 groups, respectively, at P o0.05.
3.6. Effect of eprosartan on renal iNOS protein expression
‡
0.1
‡
ED
60 pr +E
N ED
+E
pr
o-
o-
30
N ED N
Ep
ro
-6
l
0
0.0 tro
High-fat diet/streptozotocin-induced type 2 diabetic rats develop hyperglycemia and hyperlipidemia that are comparable to those seen in human type 2 diabetes. In the present study, eprosartan significantly decreased the markers of renal functions and improved renal histopathological damage induced by high-fat diet/streptozotocininduced early diabetic nephropathy. Consistent with these results, treatment with eprosartan maintained glomerular filtration rate level in subjects with cardiovascular stress (Frank et al., 2003). Eprosartan was found to be renoprotective in 5/6 nephrectomized rats (Gandhi et al., 1999). Moreover, Abrahamsen et al. (2002) found that eprosartan can preserve renal structure and function in spontaneously hypertensive stroke-prone rats. Since hyperglycemia-induced oxidative stress plays an essential role in the development and progression of early diabetic nephropathy, several oxidative stress parameters were consequently assessed. In the current study, the ability of eprosartan to increase renal reduced glutathione level and catalase activity in addition to decrease in malondialdehyde level is in agreement with the finding of Labiós et al. (2008) who found that eprosartan corrects
0.2
on
4. Discussion
*†
0.3
C
The results showed that iNOS protein was highly up-regulated in the kidney of diabetic rats compared with normal group. Eprosartan both in the low and high doses significantly downregulated renal iNOS protein level in diabetic groups compared to diabetic rats without treatment. Eprosartan alone had no significant effect on renal iNOS levels compared to control (Fig. 6).
Relative densities iNOS/ β -actin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Relative expression of TGF- β 1 mRNA
6
Fig. 6. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on inducible nitric oxide synthase (iNOS) protein expression (A) and its relative density (B) of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean7 S.E.M. of 6 rats. *, †, ‡Significantly different from control, Epro-60, and EDN groups, respectively, at P o 0.05.
oxidative disturbances in hypertensive patients. Reduced glutathione plays a central role in the antioxidant defense directly through scavenging reactive oxygen species as well as indirectly through functioning as a cofactor of antioxidant enzymes
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Q3 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
(Franco et al., 2007). On the other hand, the inhibitory effect of eprosartan on lipid peroxidation could be secondary to its antioxidant activity. In the present study eprosartan significantly inhibited diabeticinduced elevation of both iNOS and NO levels. Noh et al. (2002) reported that exposure of rat mesangial cells to high glucose concentration led to increase in iNOS expression and NO production. Moreover, Lee et al. (2002) reported that Ang II mediates the enhancement of iNOS expression through angiotensin II type 1 receptor in rat diabetic nephropathy. In addition, candesartan (another angiotensin receptor blocker) attenuated iNOS expression in type 2 diabetic KK/Ta mouse kidneys (Fan et al., 2004). Prabhakar (2004) postulated that early diabetic nephropathy is associated with increased intrarenal NO production which may contribute to hyperfiltration and microalbuminuria that characterizes early diabetic nephropathy. Christo et al. (2011) reported that NO has a role in the acute renal failure because of its free radical nature that might contribute to tubular damage. Furthermore, NO increases renal injury through its reaction with superoxide radical and generation of the cytotoxic peroxynitrite (Walker et al., 2000). Additionally, Ang II increases NO conversion to peroxynitrite (Sowers, 2002). Moreover, in the current study, sustained iNOS-mediated NO generation may mediate lipid peroxidation (Goligorsky et al., 2002). Ang II, TGF-β1 and collagen IV have been reported as major mediators that cause many of pathophysiological changes associated with diabetic nephropathy. In the present study, eprosartan significantly reduced diabetic-induced elevation of these mediators. In line with our findings, Leehey et al. (2008) found that glomerular Ang II level was significantly increased in streptozotocin-diabetic rats. Moreover, Ang II in the supernatant of cultured glomeruli was inhibited significantly in candesartan-treated nephritic rats (Kinoshita et al., 2011). Considerable evidence indicates increased sympathetic nervous activity in renal disease (Hagiwara et al., 2012). Recently, we found that carvedilol, a nonselective β-blocker as well as a selective α1blocker, protects rats against streptozotocin-induced early diabetic nephropathy (Morsy et al., 2014). Ang II enhances catecholamine release from the sympathetic nerve endings (Yasuda et al., 1987) and stimulates the sympathetic nervous activity. The sympathoinhibitory potency is superior for eprosartan compared with the other angiotensin receptor blockers (Balt et al., 2001). Therefore, this interaction raises a possibility that the eprosartan exerts its renoprotective effect through inhibiting the sympathetic nervous activity by blocking Ang II action. Ang II has been shown to stimulate TGF-β1 expression directly in renal glomeruli (Vieitez, 2008) or indirectly by enhancing the development of proteinuria (Remuzzi et al., 1997). On the other hand, it was found that eprosartan abrogated the increased TGF-β mRNA expression in 5/6 nephrectomized (Wong et al., 2000) and spontaneously hypertensive stroke prone (Abrahamsen et al., 2002) rats. Zhai et al. (2013) demonstrated that in rat mesangial cells, high glucose causes oxidative stress and NO overproduction partly via increasing iNOS, which is partially mediated by the TGF-β1. In harmony with the present study, several previous studies denoted similar results regarding the ability of angiotensin receptor blockers to decrease collagen IV level (Yanagi et al., 2013; Zhou et al., 2014) consequently; it seems that it is a class effect. Xia et al. (2006) reported that reactive oxygen species are required for mesangial cell accumulation of collagen IV in high glucose and the mechanism may involve TGF-β1 or a direct effect on the collagen IV promoter-specific transcription factors. Treatment with angiotensin receptor blockers has been associated with a beneficial effect on both glucose homeostasis and lipid profile. However, important differences among available angiotensin receptor blockers emerged from clinical studies pointing to different pharmacodynamics and pharmacokinetic properties. Hence, generalization of results obtained with a specific angiotensin receptor blocker to all available angiotensin receptor blockers may be misleading (Kyvelou et al., 2006; Rizos and Elisaf, 2014). In the present study, eprosartan
7
caused modest, but significant decrease in serum glucose level. Thus, it appears that part of eprosartan nephroprotection may be secondary to its glycemic control. Oh et al. (2011) reported that in bovine aortic endothelial cells, eprosartan dose dependently improved the insulin binding capacity which was reduced by Ang II. On the other hand, telmisartan, but not eprosartan, significantly improved plasma levels of total cholesterol and triglycerides in hypertensive, type 2 diabetic patients (Derosa et al., 2004) although in the current study, eprosartan significantly decreased these parameters. Hence, we cannot exclude that the ameliorative effect of eprosartan on dyslipidemia is a consequence of reduction of the sequelae of diabetes rather than of direct reduction of dyslipidemia. Lastly, Salman et al. (2011) reported an early role for the renal sympathetic innervation in the pathogenesis of diabetic kidney disease. Therefore, inhibition of sympathetic outflow by eprosartan (Balt et al., 2001) may offer potential advantages in patients with hypertension and renal disease. In addition, diabetes mellitus and hypertension commonly coexist. Patients with both diabetes and hypertension are at high risk for the development of nephropathy. Therefore, eprosartan, an antihypertensive drug, could be a valuable preventive therapeutic option for these patients. In conclusion, the angiotensin II type 1 receptor blocker, eprosartan has renoprotective effects on early diabetic nephropathy in rats by decreasing oxidative stress as well as by controlling the increase of Ang II, iNOS, TGF-β1 and collagen IV expressions.
Acknowledgments The authors wish to thank Dr. Kawkab A. Ahmed at Department of Pathology, College of Veterinary Medicine, Cairo University, Egypt, for her cooperation in the histopathological study. References Abrahamsen, C.T., Barone, F.C., Campbell Jr., W.G., Nelson, A.H., Contino, L.C., Pullen, M.A., Grygielko, E.T., Edwards, R.M., Laping, N.J., Brooks, D.P., 2002. The angiotensin type 1 receptor antagonist, eprosartan, attenuates the progression of renal disease in spontaneously hypertensive stroke-prone rats with accelerated hypertension. J. Pharmacol. Exp. Ther. 301, 21–28. Balt, J.C., Mathy, M.J., Pfaffendorf, M., van Zwieten, P.A., 2001. Inhibition of facilitation of sympathetic neurotransmission and angiotensin II-induced pressor effects in the pithed rat: comparison between valsartan, candesartan, eprosartan and embusartan. J. Hypertens. 19, 2241–2250. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Chiarelli, F., Gaspari, S., Marcovecchio, M.L., 2009. Role of growth factors in diabetic kidney disease. Horm. Metab. Res. 41, 585–593. Christo, J.S., Rodrigues, A.M., Mouro, M.G., Cenedeze, M.A., Simões Mde, J., Schor, N., Higa, E.M., 2011. Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats. Nitric Oxide 24, 77–83. Derosa, G., Ragonesi, P.D., Mugellini, A., Ciccarelli, L., Fogari, R., 2004. Effects of telmisartan compared with eprosartan on blood pressure control, glucose metabolism and lipid profile in hypertensive, type 2 diabetic patients: a randomized, double-blind, placebo-controlled 12-month study. Hypertens. Res. 27, 457–464. Fan, Q., Liao, J., Kobayashi, M., Yamashita, M., Gu, L., Gohda, T., Suzuki, Y., Wang, L.N., Horikoshi, S., Tomino, Y., 2004. Candesartan reduced advanced glycation endproducts accumulation and diminished nitro-oxidative stress in type 2 diabetic KK/Ta mice. Nephrol. Dial. Transplant. 19, 3012–3020. Förstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829–837 (837a–837d). Franco, R., Schoneveld, O.J., Pappa, A., Panayiotidis, M.I., 2007. The central role of glutathione in the pathophysiology of human diseases. Arch. Physiol. Biochem. 113, 234–258. Frank, H., Schobel, H.P., Vitkowsky, J., Schmieder, R.E., Heusser, K., 2003. Effects of angiotensin II receptor antagonism on the renal hemodynamic response to cardiovascular stress. Kidney Int. 63, 617–623. Gandhi, M., Meyer, T.W., Brooks, D.P., 1999. Effects of eprosartan on glomerular injury in rats with reduced renal mass. Pharmacology 59, 89–94. Goligorsky, M.S., Brodsky, S.V., Noiri, E., 2002. Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int. 61, 855–861. Hagiwara, S., Kantharidis, P., Cooper, M.E., 2012. What are new avenues for renal protection, in addition to RAAS inhibition? Curr. Hypertens. Rep. 14, 100–110.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Hajhosseiny, R., Khavandi, K., Jivraj, N., Mashayekhi, S., Goldsmith, D.J., Malik, R.A., 2014. Have we reached the limits for the treatment of diabetic nephropathy? Expert Opin. Investig. Drugs 23, 511–522. Honoré, S.M., Cabrera, W.M., Genta, S.B., Sánchez, S.S., 2012. Protective effect of yacon leaves decoction against early nephropathy in experimental diabetic rats. Food Chem. Toxicol. 50, 1704–1715. Kinoshita, Y., Kondo, S., Urushihara, M., Suga, K., Matsuura, S., Takamatsu, M., Shimizu, M., Nishiyama, A., Kawachi, H., Kagami, S., 2011. Angiotensin II type I receptor blockade suppresses glomerular renin-angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis. Transl. Res. 158, 235–248. Kolset, S.O., Reinholt, F.P., Jenssen, T., 2012. Diabetic nephropathy and extracellular matrix. J. Histochem. Cytochem. 60, 976–986. Kyvelou, S.M., Vyssoulis, G.P., Karpanou, E.A., Adamopoulos, D.N., Zervoudaki, A.I., Pietri, P.G., Stefanadis, C.I., 2006. Effects of antihypertensive treatment with angiotensin II receptor blockers on lipid profile: an open multi-drug comparison trial. Hell. J. Cardiol. 47, 21–28. Labiós, M., Martínez, M., Gabriel, F., Guiral, V., Ruiz-Aja, S., Beltrán, B., Muñoz, A., 2008. Effects of eprosartan on mitochondrial membrane potential and H2O2 levels in leucocytes in hypertension. J. Hum. Hypertens. 22, 493–500. Lee, H.Y., Noh, H.J., Gang, J.G., Xu, Z.G., Jeong, H.J., Kang, S.W., Choi, K.H., Han, D.S., 2002. Inducible nitric oxide synthase (iNOS) expression is increased in lipopolysaccharide (LPS)-stimulated diabetic rat glomeruli: effect of ACE inhibitor and angiotensin II receptor blocker. Yonsei Med. J. 43, 183–192. Leehey, D.J., Singh, A.K., Bast, J.P., Sethupathi, P., Singh, R., 2008. Glomerular renin angiotensin system in streptozotocin diabetic and Zucker diabetic fatty rats. Transl. Res. 151, 208–216. Liu, B.C., Chen, Q., Luo, D.D., Sun, J., Phillips, A.O., Ruan, X.Z., Liu, N.F., 2003. Mechanisms of irbesartan in prevention of renal lesion in streptozotocininduced diabetic rats. Acta Pharmacol. Sin. 24, 67–73. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2( Delta Delta C(T)) method. Methods 25, 402–408. Morsy, M.A., Ibrahim, S.A., Amin, E.F., Kamel, M.Y., Abdelwahab, S.A., Hassan, M.K., 2014. Carvedilol ameliorates early diabetic nephropathy in streptozotocininduced diabetic rats. Biomed. Res. Int. 2014, 105214. Mukaddam-Daher, S., Menaouar, A., Paquette, P.A., Jankowski, M., Gutkowska, J., Gillis, M.A., Shi, Y.F., Calderone, A., Tardif, J.C., 2009. Hemodynamic and cardiac effects of chronic eprosartan and moxonidine therapy in stroke-prone spontaneously hypertensive rats. Hypertension 53, 775–781. Narita, I., Border, W.A., Ketteler, M., Noble, N.A., 1995. Nitric oxide mediates immunologic injury to kidney mesangium in experimental glomerulonephritis. Lab. Investig. 72, 17–24. Niedowicz, D.M., Daleke, D.L., 2005. The role of oxidative stress in diabetic complications. Cell Biochem. Biophys. 43, 289–330. Noh, H., Ha, H., Yu, M.R., Kang, S.W., Choi, K.H., Han, D.S., Lee, H.Y., 2002. High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production. Nephron 90, 78–85. Oh, S.J., Ha, W.C., Lee, J.I., Sohn, T.S., Kim, J.H., Lee, J.M., Chang, S.A., Hong, O.K., Son, H.S., 2011. Angiotensin II inhibits insulin binding to endothelial cells. Diabetes Metab. J. 35, 243–247. Ouwens, D.M., Boer, C., Fodor, M., de Galan, P., Heine, R.J., Maassen, J.A., Diamant, M., 2005. Cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats. Diabetologia 48, 1229–1237. Poczatek, M.H., Hugo, C., Darley-Usmar, V., Murphy-Ullrich, J.E., 2000. Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1. Am. J. Pathol. 157, 1353–1363.
Prabhakar, S.S., 2004. Role of nitric oxide in diabetic nephropathy. Semin. Nephrol. 24, 333–344. Ravid, M., Brosh, D., Ravid-Safran, D., Levy, Z., Rachmani, R., 1998. Main risk factors for nephropathy in type 2 diabetes mellitus are plasma cholesterol levels, mean blood pressure, and hyperglycemia. Arch. Intern. Med. 158, 998–1004. Remuzzi, G., Ruggenenti, P., Benigni, A., 1997. Understanding the nature of renal disease progression. Kidney Int. 51, 2–15. Rizos, C.V., Elisaf, M.S., 2014. Antihypertensive drugs and glucose metabolism. World J. Cardiol. 6, 517–530. Salman, I.M., Ameer, O.Z., Sattar, M.A., Abdullah, N.A., Yam, M.F., Abdullah, G.Z., Abdulkarim, M.F., Khan, M.A., Johns, E.J., 2011. Renal sympathetic nervous system hyperactivity in early streptozotocin-induced diabetic kidney disease. Neurourol. Urodyn. 30, 438–446. Sastry, K.V., Moudgal, R.P., Mohan, J., Tyagi, J.S., Rao, G.S., 2002. Spectrophotometric determination of serum nitrite and nitrate by copper–cadmium alloy. Anal. Biochem. 306, 79–82. Si, X., Li, P., Zhang, Y., Zhang, Y., Lv, W., Qi, D., 2014. Renoprotective effects of olmesartan medoxomil on diabetic nephropathy in streptozotocin-induced diabetes in rats. Biomed. Rep. 2, 24–28. Sowers, J.R., 2002. Hypertension, angiotensin II, and oxidative stress. N. Engl. J. Med. 346, 1999–2001. Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C.L., Ramarao, P., 2005. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313–320. Vieitez, P., Gómez, O., Uceda, E.R., Vera, M.E., Molina-Holgado, E., 2008. Systemic and local effects of angiotensin II blockade in experimental diabetic nephropathy. J. Renin Angiotensin Aldosterone Syst. 9, 96–102. Walker, L.M., Walker, P.D., Imam, S.Z., Ali, S.F., Mayeux, P.R., 2000. Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine. J. Pharmacol. Exp. Ther. 295, 417–422. Welch, W.J., 2008. Angiotensin II-dependent superoxide: effects on hypertension and vascular dysfunction. Hypertension 52, 51–56. Wong, V.Y., Laping, N.J., Contino, L.C., Olson, B.A., Grygielko, E., Brooks, D.P., 2000. Gene expression in rats with renal disease treated with the angiotensin II receptor antagonist, eprosartan. Physiol. Genomics 4, 35–42. Xia, L., Wang, H., Goldberg, H.J., Munk, S., Fantus, I.G., Whiteside, C.I., 2006. Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression. Am. J. Physiol. Ren. Physiol. 290, F345–F356. Yanagi, M., Tamura, K., Fujikawa, T., Wakui, H., Kanaoka, T., Ohsawa, M., Azushima, K., Maeda, A., Kobori, H., Umemura, S., 2013. The angiotensin II type 1 receptor blocker olmesartan preferentially improves nocturnal hypertension and proteinuria in chronic kidney disease. Hypertens. Res. 36, 262–269. Yasuda, G., Shionoiri, H., Kubo, T., Misu, Y., 1987. Presynaptic angiotensin II receptors and captopril-induced adrenergic transmission failure probably not via converting enzyme inhibition in guinea-pig pulmonary arteries. J. Hypertens. Suppl. 5, S39–S45. Zhai, Y.P., Lu, Q., Liu, Y.W., Cheng, Q., Wei, Y.Q., Zhang, F., Li, C.L., Yin, X.X., 2013. Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose. Acta Pharmacol. Sin. 34, 507–514. Zhou, G., Cheung, A.K., Liu, X., Huang, Y., 2014. Valsartan slows the progression of diabetic nephropathy in db/db mice via a reduction in podocyte injury, and renal oxidative stress and inflammation. Clin. Sci. 126, 707–720.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Q1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats Mohamed A. Morsy a,n, Gehan H. Heeba b, Magda E. Mahmoud c a
Department of Pharmacology, Faculty of Medicine, Minia University, 61511 El-Minia, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Minia University, 61511 El-Minia, Egypt c Department of Agricultural Chemistry, Faculty of Agriculture, Minia University, 61511 El-Minia, Egypt b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 15 January 2015 Accepted 19 January 2015
Diabetic nephropathy becomes the single most frequent cause of end-stage renal disease. The present study aimed therefore to investigate possible protective effect of eprosartan, an angiotensin II type 1 receptor blocker, on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats and various mechanisms underlie this effect. Male Wistar rats with type 2 diabetes induced by high-fat diet/streptozotocin were treated with eprosartan at the dose levels of 30 and 60 mg/kg daily for 5 weeks. Eprosartan induced a nephroprotective effect as evident by the significant decrease in serum creatinine, urea, total cholesterol, triglycerides and glucose levels, urinary albumin excretion and kidney index as well as renal levels of malondialdehyde and nitric oxide products (nitrite/nitrate), in addition to angiotensin II, inducible nitric oxide synthase, transforming growth factor-β1 and collagen IV expressions with a concurrent increase in renal levels of reduced glutathione and catalase activity compared to diabetic untreated rats. Histopathological examination confirmed the renoprotective effect of eprosartan. In conclusion, eprosartan protects rats against high-fat diet/streptozotocininduced early diabetic nephropathy possibly, in part, through its antioxidant effect as well as by abrogating the overexpression of angiotensin II, inducible nitric oxide synthase, transforming growth factor-β1 and collagen IV. & 2015 Published by Elsevier B.V.
Keywords: Eprosartan Diabetic nephropathy Angiotensin II Inducible nitric oxide synthase Transforming growth factor-β1 Collagen IV
1. Introduction Diabetic nephropathy is the leading cause of end-stage renal disease and its prevalence is increasing because of the global epidemic of diabetes (Hajhosseiny et al., 2014). It is characterized clinically by progressive albuminuria and histologically by definite changes including progressive accumulation of extracellular matrix components (e.g. collagen IV) that lead to renal fibrosis and failure (Kolset et al., 2012). Dyslipidemia is considered as a risk factor for the progression of diabetic nephropathy (Ravid et al., 1998). Hyperglycemia increases the production of angiotensin (Ang) II that generates reactive oxygen species by activation of angiotensin II type 1 receptors resulting in oxidative stress (Welch, 2008). Oxidative stress is considered as a key component in the development of diabetic complications including diabetic nephropathy (Niedowicz and Daleke, 2005). Ang II is also a potent growth modulator and proinflammatory peptide (Vieitez et al., 2008). Similar to Ang II, hyperglycemia increases the production of transforming growth factor (TGF)-β in mesangial cells (Poczatek et al., 2000). TGF-β induces the accumulation of extracellular matrix and increases reactive oxygen species production in tubular epithelial cells (Chiarelli et al., 2009). n
Corresponding author. Tel.: þ 20 1112197640; fax: þ 20 862342813. E-mail address: [email protected] (M.A. Morsy).
Nitric oxide (NO) is an important gasotransmitter molecule that mediates a variety of physiological and pathological processes in kidney. Limiting NO production decreases glomerular injury and subsequent glomerulosclerosis (Narita et al., 1995). Inducible NO synthase (iNOS) produces large amounts of NO in many cell types in response to several stimuli (Förstermann and Sessa, 2012). Alternatively, high concentrations of glucose have been shown to increase iNOS-induced NO production and promote extracellular matrix accumulation in rat mesangial cells (Noh et al., 2002). Studies have shown that several angiotensin receptor blockers via their pleiotropic effects are beneficial in controlling the development and progression of diabetic nephropathy (Si et al., 2014; Zhou et al., 2014). Therefore, eprosartan, an angiotensin II type 1 receptor blocker, seems conceivable to be implicated in amelioration of diabetic nephropathy. The aim of the present study was to investigate the effects of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats and the underlying mechanisms.
2. Materials and methods 2.1. Chemicals Eprosartan mesylate (purity 498%) was purchased from Matrix Laboratories Ltd. (Hyderabad, India). Streptozotocin was purchased
http://dx.doi.org/10.1016/j.ejphar.2015.01.027 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
from Sigma-Aldrich Corp. (St. Louis, MO, USA). Antibodies against iNOS and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals were of analytical grade and were obtained from commercial sources. 2.2. Animals Male Wistar rats weighing 170–190 g were used after 1 week for proper acclimatization to the animal house conditions (12 h lighting cycle and 257 2 1C temperature) and had free access to standard rodent chow (25% protein, 5% fat, 40% starch, 10% sugar, 6% fibers, 8% ash, 5% minerals and 1% vitamins) and water. Procedures involving animals and their care were conducted according to ethical guidelines of Minia University, Egypt and EU Directive 2010/63/EU. 2.3. Experimental induction of diabetes Rats had free access to high-fat diet which was prepared as previously described (Ouwens et al., 2005) by mixing casein (30%), raw beef fat (suet, 40%), wheat flour (7%), glucose (10%), salt mixture (6%), vitamin mixture (3%), and bran (4%) and sufficient water to form a consistent paste. After 4 weeks, type 2 diabetes was induced in overnight fasted rats by intraperitoneal injection of freshly prepared streptozotocin (40 mg/kg, dissolved in 0.1 M cold citrate buffer, pH 4.5) (Srinivasan et al., 2005). Three days after streptozotocin injection, rats with fasting blood glucose level Z200 mg/dl measured by a glucometer (Accu-Chek, Roche, Basel, Switzerland) were considered diabetic and selected for the study. 2.4. Experimental procedures Diabetic rats were randomized into 3 groups of 6 animals each, namely non-treated diabetic group, low-dose eprosartan (30 mg/kg/ day, orally) group (Mukaddam-Daher et al., 2009), and high-dose eprosartan (60 mg/kg/day, orally) group (Abrahamsen et al., 2002). Normal-diet non-diabetic rats served as control group (n¼ 6). Our preliminary experiments showed that eprosartan alone at both low and high doses did not alter renal function markers so only normaldiet non-diabetic rats treated with the high dose of eprosartan were presented and served as eprosartan control group (n¼6). Eprosartan was suspended in 1% aqueous solution of carboxymethyl cellulose. All groups received equivalent volumes of the above-mentioned vehicles. High-fat diet/streptozotocin-induced diabetic rats were left for 5 weeks untreated to induce early diabetic nephropathy (Honoré et al., 2012). The treatment was initiated one week after the induction of diabetes and continued for 4 weeks. The animals were placed in individual metabolic cages for 24 h to collect urine before they were killed. At the end of the experiment, rats were fasted overnight and each rat was weighed then killed. Rats were dissected to obtain the kidneys, which were weighted. Kidney index ([kidney weight/body weight] 1000) that reveals the profile of kidney hypertrophy (Liu et al., 2003) was calculated. Blood samples were collected and centrifuged at 3000g for 10 min to obtain clear sera. Both kidneys were rapidly dissected out and weighed. The longitudinal section of the right kidney from each animal was used for histological examination. The renal cortices of the rest of the kidneys were snap frozen in liquid nitrogen and stored at 80 1C. 2.5. Biochemical analysis Using commercially available kits, serum creatinine (Diamond Diagnostics, Egypt), urea and glucose (Biodiagnostic, Egypt), cholesterol and triglycerides (Spectrum Diagnostics, Egypt), urinary albumin (BioSystems, Spain), as well as renal reduced glutathione
and catalase (Biodiagnostic, Egypt) levels were quantified according to the manufacturers' guidelines. In addition, urinary albumin excretion (mg/24 h) was calculated. The renal cortex content of lipid peroxides was determined by biochemical assessment of thiobarbituric acid reacting substance through spectrophotometric measurement of color at 535 nm, using 1,1,3,3-tetramethoxypropane as standard. The results were expressed as equivalents of malondialdehyde in nmol/g tissue (Buege and Aust, 1978). Renal cortex nitric oxide level was measured as total nitrite/nitrate, the stable degradation products of nitric oxide, by reduction of nitrate into nitrite using copperized cadmium, followed by color development with Griess reagent in acidic medium (Sastry et al., 2002). 2.6. Expression of Ang II, TGF-β1, and collagen IV by real-time PCR Total RNA was isolated from kidney tissue homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. Total RNA (5 μg) was denatured at 70 1C for 2 min. Denatured RNA was reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA): transcription mixture containing 50 mM KCl, 50 mM Tris HCl (pH 8.3), 0.5 mM of deoxyribonucleotide triphosphate (dNTP), 3 mM MgCl2, 1 U/ml RNase inhibitor and 200 units of moloney murine leukemia virus reverse transcriptase. The reaction tube was placed at 42 1C for 1 h, followed by heating to 92 1C to stop the reaction. For real-time quantitative PCR, 5 μl of first-strand cDNA was used in a total volume of 25 μl, containing 12.5 μl 2 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 200 ng of each primer. Sequences of the primers were as follows: Ang II forward primer, 50 -CATCATGAGGAGACGGGG-30 and reverse primer, 50 -TCCAAGTGGACAGGTAAGCC-30 ; TGF-β1 forward primer, 50 -AGGGCTACCATGCCACTTC-30 and reverse primer, 50 -GCGGC ACGCAGCACGGTGAT-30 ; collagen IV forward primer, 50 -TGGTCCCCAAG GTGTCAAAG-30 and reverse primer, 50 -GGGGGTCCTGGGTTACCATTA30 ; and β-actin forward primer, 50 -CCTTCCTGGGCATGGAG;TCCT-30 and reverse primer, 50 -GGAGCAATGATCTTGATCTTC-30 . PCR reactions consisting of 95 1C for 10 min (1 cycle), 94 1C for 15 s, and 60 1C for 1 min (40 cycles) were performed on a StepOnePlus System (Applied Biosystems, Foster City, CA, USA). Data were analyzed with the ABI Prism 7500 Sequence Detection System software and quantified using the version 1.7 Sequence Detection Software from PE Biosystems (Foster City, CA, USA). Relative expression of studied genes was calculated using the comparative threshold cycle method. All values were normalized to the β-actin genes (Livak and Schmittgen, 2001). 2.7. Western blot analysis Kidney tissue samples were homogenized in lysis buffer (20 mM Tris–HCl pH 7.5, 50 mM 2-mercaptoethanol, 5 mM EGTA, 2 mM EDTA, 1% NP40, 0.1% SDS, 0.5% deoxycholic acid, 10 mM NaF, 1 mM PMSF, 25 mg/ml leupeptin, 2 mg/ml aprotinin) and protein concentrations were determined using a total protein kit (Biodiagnostic, Egypt). For direct immunoblotting, aliquots of lysate were mixed with loading buffer containing 2-mercaptoethanol and maintained at 100 1C for 10 min before loading on 10% SDSPAGE. Following SDS-PAGE separation, proteins were transferred to polyvinyl difluoride membrane. Membranes were blocked in Tris-buffered saline with Tween-20 containing 5% (w/v) non-fat milk and dried for 1 h at room temperature. Membrane strips were incubated with primary antibodies (1:1000 for iNOS and β-actin) overnight at 4 1C. Following extensive washing, membrane strips were incubated with anti-rabbit IgG (1:5000; Cell Signaling Technology Inc., MA, USA) conjugated to horseradish peroxidase for 1 h. Protein bands were detected by a standard enhanced chemiluminescence method and densitometry measurements were made using ImageJ software (freeware; rsbweb.nih.gov/ij).
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
(Fig. 2A and B). On the other hand, the untreated diabetic group 67 showed focal interstitial nephritis and cystic dilatation of renal tubules 68 (Fig. 2C). Treatment with the low dose eprosartan (30 mg/kg) improved 69 Renal tissue samples were fixed in 10% neutral buffered formalin, renal histology (Fig. 2D). The high dose eprosartan (60 mg/kg) reversed 70 embedded in paraffin, sectioned by a microtome at 5 μm thickness, renal histopathological damage induced by high-fat diet/streptozoto71 and stained with hematoxylin and eosin for histological examination cin-induced early diabetic nephropathy, that was accompanied only Q2 72 using light microscopy. 73 with slight congestion of glomerular tufts (Fig. 2E; Table 1). 74 2.9. Statistical analysis 75 3.3. Effects of eprosartan on renal malondialdehyde, nitrite/nitrate, 76 reduced glutathione and catalase activity levels The data are normally distributed and are expressed as mean7S. 77 E.M. Statistical analysis was performed by one-way ANOVA followed 78 Oxidative stress was assessed through measuring renal malondialby Tukey–Kramer postanalysis test for multiple comparisons with 79 dehyde, nitrite/nitrate, reduced glutathione and catalase activity levels. Po0.05 being considered as statistically significant. 80 Renal malondialdehyde was evaluated as an index of renal lipid 81 peroxidation and nitrite/nitrate as an indicator of renal NO level. 82 3. Results Eprosartan (30 and 60 mg/kg) treatment significantly suppressed both 83 lipid peroxidation and increased NO levels in comparison with diabetic 84 3.1. Effects of eprosartan on renal functions untreated group (Fig. 3A and B). Eprosartan at both dose levels caused 85 significant increase in renal reduced glutathione levels compared to 86 Serum creatinine as well as urea levels, 24-h urinary albumin non-treated diabetic rats (Fig. 3C). The high dose of eprosartan caused 87 excretion and kidney index was assessed as markers of renal significant increase in renal catalase activity (Fig. 3D). 88 functions. Diabetic rats showed significant increase in serum creati89 nine and urea levels as well as 24-h urinary albumin excretion and 3.4. Effects of eprosartan on serum glucose, total cholesterol 90 kidney index compared to normal group (Fig. 1A–D). Eprosartan and triglycerides levels 91 treatments at both doses significantly decreased these markers of 92 renal functions compared to non-treated diabetic rats (Fig. 1A–D). At the end of experiment, serum glucose and triglycerides 93 levels were lower in the eprosartan (30 and 60 mg/kg)-treated 94 3.2. Effects of eprosartan on renal histological changes diabetic group compared to the untreated diabetic group (Fig. 4A 95 and C). Eprosartan only in the high dose caused a significant 96 Histopathological examination revealed that both the control decrease in serum total cholesterol level compared to diabetic rats 97 and eprosartan (60 mg/kg)-treated groups had normal appearance without treatment (Fig. 4B). 98 99 100 1.5 80 101 *† 102 *† 60 103 1.0 †‡ ‡ 104 †‡ 105 40 ‡ 106 0.5 107 20 108 109 0.0 0 110 111 112 113 114 115 40 10 *† 116 *† 117 8 30 118 *†‡ 119 ‡ 6 *†‡ *†‡ 20 120 121 4 122 10 123 2 124 0 125 0 126 127 128 129 130 Fig. 1. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on serum creatinine (A) and urea (B) levels as well as urinary albumin excretion (C), and 131 kidney index (D) of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean7 S.E.M. of 6 rats. *, †, ‡Significantly different from control, 132 Epro-60, and EDN groups, respectively, at Po 0.05. +E pr o60
pr o30
+E pr o30 ED N +E pr o60
ED N
ED N
Ep ro -6 0
C
on tr
ol
60
o-
pr
+E
ED N
ED N
+E
pr o30
ED N
-6 0
Ep ro
C
on t
ro l
Kidney index
ED N
ED N
+E
C
ED N
ED N
ED N
Ep ro -6 0
on t
ro l
pr o60
+E
+E
pr o30
ED N
-6 0
Ep ro
C
on tr ol
Serum urea mg/dl
Serum creatinine mg/dl
2.8. Histological examination
Urinary albumin excretion mg/24 h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
3
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Fig. 2. Effect of eprosartan on kidney histopathological picture of high-fat diet/streptozotocin-induced early diabetic nephropathy in rats (H&E 400). (A) and (B) Sections from control and eprosartan (60 mg/kg)-treated groups show normal histological structure of renal parenchyma. (C) Early diabetic nephropathy group shows focal interstitial nephritis and cystic dilatation of renal tubules (arrows) and interstitial fibroblasts proliferation (arrows in inset). (D) Kidney tissue from early diabetic nephropathyþ eprosartan (30 mg/kg) group showing vacuolization of epithelial lining renal tubules (arrow). (E) Kidney tissue from early diabetic nephropathyþ eprosartan (60 mg/kg) group showing slight congestion of glomerular tufts. Table 1 Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on severity of histopathological lesions of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Histopathological alterations
Control
Epro-60
EDN
EDNþ Epro-30
EDN þ Epro-60
Tubular degeneration Tubular necrosis Tubular dilation Glomerular damage Dilated Bowman's spaces Congestion of renal blood vessels Leukocyte infiltrates Vacuolation of endothelium and congestion of glomerular tuft Interstitial fibroblasts proliferation
– – – – – – – – –
– – – – – – – – –
þþ þþ þþ – þ þþþ þþþ þþ þ
þþ – þ – – þþ þ þ –
þ – – – – þ – þ –
Score level – is considered normal. Scores þ , þ þ and þ þ þ are mild, moderate and severe levels, revealing less than 25%, 50% and 75% histopathological alterations of total fields examined, respectively.
3.5. Effects of eprosartan on renal mRNA Ang II, TGF-β1 and collagen IV expressions Eprosartan (30 and 60 mg/kg)-treated diabetic groups showed significant reductions in the mRNA expressions of renal Ang II and collagen IV compared to the untreated diabetic group
(Fig. 5A and C). On the other hand, only the high dose of eprosartan caused a significant down-regulation in renal TGF-β1 mRNA expression compared to the untreated diabetic group (Fig. 5B). Eprosartan alone, without high-fat diet/streptozotocin treatment, had no effect on these parameters compared to control (Fig. 5A–C).
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*†
400
*†‡
60
Nitrite/nitrate nmol/g tissue
*†‡§ 40 20
*†‡
200
ठ100
0
ED N
ED N
+E pr o60
pr o30
ED N
+E
C
on t
Ep ro -6 0
ro l
+E pr o30 +E pr o60 ED N
ED N
ED N
ol on tr C
Ep ro -6 0
0
15
8
‡
10
Catalase U/g tissue
*†‡ *†
5
‡§
6
*†
4
*† 2
0
pr o60
pr o30
ED N
ED N
+E
+E
C on t
ro
l
+E pr o30 ED N +E pr o60
ED N
ED N
Ep ro -6 0
C
on t
ro l
0 ED N
Reduced glutathione mol/g tissue
*†
300
Ep ro -6 0
Malondialdehyde nmol/g tissue
80
Fig. 3. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on renal malondialdehyde (A), nitric oxide (nitrite/nitrate) (B), reduced glutathione (C), and catalase (D) levels of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7 S.E.M. of 6 rats. *, †, ‡, §Significantly different from control, Epro-60, EDN, and EDN þEpro-30 groups, respectively, at Po 0.05.
*†
300
*†‡
Serum total cholesterol mg/dl
400
*†‡
200 100
200
*†
*†
150
*†‡
100 50
Serum triglycerides mg/dl
200
pr o60
ED N +E
pr o30
*†‡
ED N +E
on tr ol C
+E pr o30 ED N +E pr o60
ED N
*†‡
ED N
Ep ro -6 0
ro l on t C
ED N
0
0
Ep ro -6 0
Serum glucose mg/dl
*†
150 100 50
+E p
ro -6 0
ro -3 0 ED N
ED N
+E p
ED N
ro -6 0 Ep
ol
0
C on tr
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
5
Fig. 4. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on serum glucose (A), total cholesterol (B), and triglycerides (C) levels of high-fat diet/ streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7S.E.M. of 6 rats. n, †, ‡Significantly different from control, Epro-60, and EDN groups, respectively, at P o 0.05.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*†
1.0
4
0.8 0.6
*†‡ *†‡§
0.4 0.2
*†
3
‡
2 1
ED N
ED N
ED N
*†
2.0
Relative expression of collagen IV mRNA
+E pr o-
ED N
60
C on tr ol
0 +E pr o
-3
-6
0
N
+E pr o
ED N
ED
60 Ep ro -
on tr ol C
30 +E pr o60
0
0.0
Ep ro -
Relative expression of Ang II mRNA
1.5 1.0
*†‡
*†‡
0.5
pr o60
30
N ED
ED
N
N
+E
+E
pr
o-
ED
0 -6 ro Ep
on tr ol
0.0
C
Fig. 5. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on angiotensin (Ang) II (A), transforming growth factor (TGF)-β1 (B), and collagen IV mRNA expressions of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean 7S.E.M. of 6 rats. *, †, ‡, §Significantly different from control, Epro-60, EDN, and EDNþ Epro-30 groups, respectively, at P o0.05.
3.6. Effect of eprosartan on renal iNOS protein expression
‡
0.1
‡
ED
60 pr +E
N ED
+E
pr
o-
o-
30
N ED N
Ep
ro
-6
l
0
0.0 tro
High-fat diet/streptozotocin-induced type 2 diabetic rats develop hyperglycemia and hyperlipidemia that are comparable to those seen in human type 2 diabetes. In the present study, eprosartan significantly decreased the markers of renal functions and improved renal histopathological damage induced by high-fat diet/streptozotocininduced early diabetic nephropathy. Consistent with these results, treatment with eprosartan maintained glomerular filtration rate level in subjects with cardiovascular stress (Frank et al., 2003). Eprosartan was found to be renoprotective in 5/6 nephrectomized rats (Gandhi et al., 1999). Moreover, Abrahamsen et al. (2002) found that eprosartan can preserve renal structure and function in spontaneously hypertensive stroke-prone rats. Since hyperglycemia-induced oxidative stress plays an essential role in the development and progression of early diabetic nephropathy, several oxidative stress parameters were consequently assessed. In the current study, the ability of eprosartan to increase renal reduced glutathione level and catalase activity in addition to decrease in malondialdehyde level is in agreement with the finding of Labiós et al. (2008) who found that eprosartan corrects
0.2
on
4. Discussion
*†
0.3
C
The results showed that iNOS protein was highly up-regulated in the kidney of diabetic rats compared with normal group. Eprosartan both in the low and high doses significantly downregulated renal iNOS protein level in diabetic groups compared to diabetic rats without treatment. Eprosartan alone had no significant effect on renal iNOS levels compared to control (Fig. 6).
Relative densities iNOS/ β -actin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Relative expression of TGF- β 1 mRNA
6
Fig. 6. Effect of low (30 mg/kg) and high (60 mg/kg) doses of eprosartan (Epro) on inducible nitric oxide synthase (iNOS) protein expression (A) and its relative density (B) of high-fat diet/streptozotocin-induced early diabetic nephropathy (EDN) in rats. Data are mean7 S.E.M. of 6 rats. *, †, ‡Significantly different from control, Epro-60, and EDN groups, respectively, at P o 0.05.
oxidative disturbances in hypertensive patients. Reduced glutathione plays a central role in the antioxidant defense directly through scavenging reactive oxygen species as well as indirectly through functioning as a cofactor of antioxidant enzymes
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Q3 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
(Franco et al., 2007). On the other hand, the inhibitory effect of eprosartan on lipid peroxidation could be secondary to its antioxidant activity. In the present study eprosartan significantly inhibited diabeticinduced elevation of both iNOS and NO levels. Noh et al. (2002) reported that exposure of rat mesangial cells to high glucose concentration led to increase in iNOS expression and NO production. Moreover, Lee et al. (2002) reported that Ang II mediates the enhancement of iNOS expression through angiotensin II type 1 receptor in rat diabetic nephropathy. In addition, candesartan (another angiotensin receptor blocker) attenuated iNOS expression in type 2 diabetic KK/Ta mouse kidneys (Fan et al., 2004). Prabhakar (2004) postulated that early diabetic nephropathy is associated with increased intrarenal NO production which may contribute to hyperfiltration and microalbuminuria that characterizes early diabetic nephropathy. Christo et al. (2011) reported that NO has a role in the acute renal failure because of its free radical nature that might contribute to tubular damage. Furthermore, NO increases renal injury through its reaction with superoxide radical and generation of the cytotoxic peroxynitrite (Walker et al., 2000). Additionally, Ang II increases NO conversion to peroxynitrite (Sowers, 2002). Moreover, in the current study, sustained iNOS-mediated NO generation may mediate lipid peroxidation (Goligorsky et al., 2002). Ang II, TGF-β1 and collagen IV have been reported as major mediators that cause many of pathophysiological changes associated with diabetic nephropathy. In the present study, eprosartan significantly reduced diabetic-induced elevation of these mediators. In line with our findings, Leehey et al. (2008) found that glomerular Ang II level was significantly increased in streptozotocin-diabetic rats. Moreover, Ang II in the supernatant of cultured glomeruli was inhibited significantly in candesartan-treated nephritic rats (Kinoshita et al., 2011). Considerable evidence indicates increased sympathetic nervous activity in renal disease (Hagiwara et al., 2012). Recently, we found that carvedilol, a nonselective β-blocker as well as a selective α1blocker, protects rats against streptozotocin-induced early diabetic nephropathy (Morsy et al., 2014). Ang II enhances catecholamine release from the sympathetic nerve endings (Yasuda et al., 1987) and stimulates the sympathetic nervous activity. The sympathoinhibitory potency is superior for eprosartan compared with the other angiotensin receptor blockers (Balt et al., 2001). Therefore, this interaction raises a possibility that the eprosartan exerts its renoprotective effect through inhibiting the sympathetic nervous activity by blocking Ang II action. Ang II has been shown to stimulate TGF-β1 expression directly in renal glomeruli (Vieitez, 2008) or indirectly by enhancing the development of proteinuria (Remuzzi et al., 1997). On the other hand, it was found that eprosartan abrogated the increased TGF-β mRNA expression in 5/6 nephrectomized (Wong et al., 2000) and spontaneously hypertensive stroke prone (Abrahamsen et al., 2002) rats. Zhai et al. (2013) demonstrated that in rat mesangial cells, high glucose causes oxidative stress and NO overproduction partly via increasing iNOS, which is partially mediated by the TGF-β1. In harmony with the present study, several previous studies denoted similar results regarding the ability of angiotensin receptor blockers to decrease collagen IV level (Yanagi et al., 2013; Zhou et al., 2014) consequently; it seems that it is a class effect. Xia et al. (2006) reported that reactive oxygen species are required for mesangial cell accumulation of collagen IV in high glucose and the mechanism may involve TGF-β1 or a direct effect on the collagen IV promoter-specific transcription factors. Treatment with angiotensin receptor blockers has been associated with a beneficial effect on both glucose homeostasis and lipid profile. However, important differences among available angiotensin receptor blockers emerged from clinical studies pointing to different pharmacodynamics and pharmacokinetic properties. Hence, generalization of results obtained with a specific angiotensin receptor blocker to all available angiotensin receptor blockers may be misleading (Kyvelou et al., 2006; Rizos and Elisaf, 2014). In the present study, eprosartan
7
caused modest, but significant decrease in serum glucose level. Thus, it appears that part of eprosartan nephroprotection may be secondary to its glycemic control. Oh et al. (2011) reported that in bovine aortic endothelial cells, eprosartan dose dependently improved the insulin binding capacity which was reduced by Ang II. On the other hand, telmisartan, but not eprosartan, significantly improved plasma levels of total cholesterol and triglycerides in hypertensive, type 2 diabetic patients (Derosa et al., 2004) although in the current study, eprosartan significantly decreased these parameters. Hence, we cannot exclude that the ameliorative effect of eprosartan on dyslipidemia is a consequence of reduction of the sequelae of diabetes rather than of direct reduction of dyslipidemia. Lastly, Salman et al. (2011) reported an early role for the renal sympathetic innervation in the pathogenesis of diabetic kidney disease. Therefore, inhibition of sympathetic outflow by eprosartan (Balt et al., 2001) may offer potential advantages in patients with hypertension and renal disease. In addition, diabetes mellitus and hypertension commonly coexist. Patients with both diabetes and hypertension are at high risk for the development of nephropathy. Therefore, eprosartan, an antihypertensive drug, could be a valuable preventive therapeutic option for these patients. In conclusion, the angiotensin II type 1 receptor blocker, eprosartan has renoprotective effects on early diabetic nephropathy in rats by decreasing oxidative stress as well as by controlling the increase of Ang II, iNOS, TGF-β1 and collagen IV expressions.
Acknowledgments The authors wish to thank Dr. Kawkab A. Ahmed at Department of Pathology, College of Veterinary Medicine, Cairo University, Egypt, for her cooperation in the histopathological study. References Abrahamsen, C.T., Barone, F.C., Campbell Jr., W.G., Nelson, A.H., Contino, L.C., Pullen, M.A., Grygielko, E.T., Edwards, R.M., Laping, N.J., Brooks, D.P., 2002. The angiotensin type 1 receptor antagonist, eprosartan, attenuates the progression of renal disease in spontaneously hypertensive stroke-prone rats with accelerated hypertension. J. Pharmacol. Exp. Ther. 301, 21–28. Balt, J.C., Mathy, M.J., Pfaffendorf, M., van Zwieten, P.A., 2001. Inhibition of facilitation of sympathetic neurotransmission and angiotensin II-induced pressor effects in the pithed rat: comparison between valsartan, candesartan, eprosartan and embusartan. J. Hypertens. 19, 2241–2250. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Chiarelli, F., Gaspari, S., Marcovecchio, M.L., 2009. Role of growth factors in diabetic kidney disease. Horm. Metab. Res. 41, 585–593. Christo, J.S., Rodrigues, A.M., Mouro, M.G., Cenedeze, M.A., Simões Mde, J., Schor, N., Higa, E.M., 2011. Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats. Nitric Oxide 24, 77–83. Derosa, G., Ragonesi, P.D., Mugellini, A., Ciccarelli, L., Fogari, R., 2004. Effects of telmisartan compared with eprosartan on blood pressure control, glucose metabolism and lipid profile in hypertensive, type 2 diabetic patients: a randomized, double-blind, placebo-controlled 12-month study. Hypertens. Res. 27, 457–464. Fan, Q., Liao, J., Kobayashi, M., Yamashita, M., Gu, L., Gohda, T., Suzuki, Y., Wang, L.N., Horikoshi, S., Tomino, Y., 2004. Candesartan reduced advanced glycation endproducts accumulation and diminished nitro-oxidative stress in type 2 diabetic KK/Ta mice. Nephrol. Dial. Transplant. 19, 3012–3020. Förstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829–837 (837a–837d). Franco, R., Schoneveld, O.J., Pappa, A., Panayiotidis, M.I., 2007. The central role of glutathione in the pathophysiology of human diseases. Arch. Physiol. Biochem. 113, 234–258. Frank, H., Schobel, H.P., Vitkowsky, J., Schmieder, R.E., Heusser, K., 2003. Effects of angiotensin II receptor antagonism on the renal hemodynamic response to cardiovascular stress. Kidney Int. 63, 617–623. Gandhi, M., Meyer, T.W., Brooks, D.P., 1999. Effects of eprosartan on glomerular injury in rats with reduced renal mass. Pharmacology 59, 89–94. Goligorsky, M.S., Brodsky, S.V., Noiri, E., 2002. Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int. 61, 855–861. Hagiwara, S., Kantharidis, P., Cooper, M.E., 2012. What are new avenues for renal protection, in addition to RAAS inhibition? Curr. Hypertens. Rep. 14, 100–110.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
M.A. Morsy et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Hajhosseiny, R., Khavandi, K., Jivraj, N., Mashayekhi, S., Goldsmith, D.J., Malik, R.A., 2014. Have we reached the limits for the treatment of diabetic nephropathy? Expert Opin. Investig. Drugs 23, 511–522. Honoré, S.M., Cabrera, W.M., Genta, S.B., Sánchez, S.S., 2012. Protective effect of yacon leaves decoction against early nephropathy in experimental diabetic rats. Food Chem. Toxicol. 50, 1704–1715. Kinoshita, Y., Kondo, S., Urushihara, M., Suga, K., Matsuura, S., Takamatsu, M., Shimizu, M., Nishiyama, A., Kawachi, H., Kagami, S., 2011. Angiotensin II type I receptor blockade suppresses glomerular renin-angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis. Transl. Res. 158, 235–248. Kolset, S.O., Reinholt, F.P., Jenssen, T., 2012. Diabetic nephropathy and extracellular matrix. J. Histochem. Cytochem. 60, 976–986. Kyvelou, S.M., Vyssoulis, G.P., Karpanou, E.A., Adamopoulos, D.N., Zervoudaki, A.I., Pietri, P.G., Stefanadis, C.I., 2006. Effects of antihypertensive treatment with angiotensin II receptor blockers on lipid profile: an open multi-drug comparison trial. Hell. J. Cardiol. 47, 21–28. Labiós, M., Martínez, M., Gabriel, F., Guiral, V., Ruiz-Aja, S., Beltrán, B., Muñoz, A., 2008. Effects of eprosartan on mitochondrial membrane potential and H2O2 levels in leucocytes in hypertension. J. Hum. Hypertens. 22, 493–500. Lee, H.Y., Noh, H.J., Gang, J.G., Xu, Z.G., Jeong, H.J., Kang, S.W., Choi, K.H., Han, D.S., 2002. Inducible nitric oxide synthase (iNOS) expression is increased in lipopolysaccharide (LPS)-stimulated diabetic rat glomeruli: effect of ACE inhibitor and angiotensin II receptor blocker. Yonsei Med. J. 43, 183–192. Leehey, D.J., Singh, A.K., Bast, J.P., Sethupathi, P., Singh, R., 2008. Glomerular renin angiotensin system in streptozotocin diabetic and Zucker diabetic fatty rats. Transl. Res. 151, 208–216. Liu, B.C., Chen, Q., Luo, D.D., Sun, J., Phillips, A.O., Ruan, X.Z., Liu, N.F., 2003. Mechanisms of irbesartan in prevention of renal lesion in streptozotocininduced diabetic rats. Acta Pharmacol. Sin. 24, 67–73. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2( Delta Delta C(T)) method. Methods 25, 402–408. Morsy, M.A., Ibrahim, S.A., Amin, E.F., Kamel, M.Y., Abdelwahab, S.A., Hassan, M.K., 2014. Carvedilol ameliorates early diabetic nephropathy in streptozotocininduced diabetic rats. Biomed. Res. Int. 2014, 105214. Mukaddam-Daher, S., Menaouar, A., Paquette, P.A., Jankowski, M., Gutkowska, J., Gillis, M.A., Shi, Y.F., Calderone, A., Tardif, J.C., 2009. Hemodynamic and cardiac effects of chronic eprosartan and moxonidine therapy in stroke-prone spontaneously hypertensive rats. Hypertension 53, 775–781. Narita, I., Border, W.A., Ketteler, M., Noble, N.A., 1995. Nitric oxide mediates immunologic injury to kidney mesangium in experimental glomerulonephritis. Lab. Investig. 72, 17–24. Niedowicz, D.M., Daleke, D.L., 2005. The role of oxidative stress in diabetic complications. Cell Biochem. Biophys. 43, 289–330. Noh, H., Ha, H., Yu, M.R., Kang, S.W., Choi, K.H., Han, D.S., Lee, H.Y., 2002. High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production. Nephron 90, 78–85. Oh, S.J., Ha, W.C., Lee, J.I., Sohn, T.S., Kim, J.H., Lee, J.M., Chang, S.A., Hong, O.K., Son, H.S., 2011. Angiotensin II inhibits insulin binding to endothelial cells. Diabetes Metab. J. 35, 243–247. Ouwens, D.M., Boer, C., Fodor, M., de Galan, P., Heine, R.J., Maassen, J.A., Diamant, M., 2005. Cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats. Diabetologia 48, 1229–1237. Poczatek, M.H., Hugo, C., Darley-Usmar, V., Murphy-Ullrich, J.E., 2000. Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1. Am. J. Pathol. 157, 1353–1363.
Prabhakar, S.S., 2004. Role of nitric oxide in diabetic nephropathy. Semin. Nephrol. 24, 333–344. Ravid, M., Brosh, D., Ravid-Safran, D., Levy, Z., Rachmani, R., 1998. Main risk factors for nephropathy in type 2 diabetes mellitus are plasma cholesterol levels, mean blood pressure, and hyperglycemia. Arch. Intern. Med. 158, 998–1004. Remuzzi, G., Ruggenenti, P., Benigni, A., 1997. Understanding the nature of renal disease progression. Kidney Int. 51, 2–15. Rizos, C.V., Elisaf, M.S., 2014. Antihypertensive drugs and glucose metabolism. World J. Cardiol. 6, 517–530. Salman, I.M., Ameer, O.Z., Sattar, M.A., Abdullah, N.A., Yam, M.F., Abdullah, G.Z., Abdulkarim, M.F., Khan, M.A., Johns, E.J., 2011. Renal sympathetic nervous system hyperactivity in early streptozotocin-induced diabetic kidney disease. Neurourol. Urodyn. 30, 438–446. Sastry, K.V., Moudgal, R.P., Mohan, J., Tyagi, J.S., Rao, G.S., 2002. Spectrophotometric determination of serum nitrite and nitrate by copper–cadmium alloy. Anal. Biochem. 306, 79–82. Si, X., Li, P., Zhang, Y., Zhang, Y., Lv, W., Qi, D., 2014. Renoprotective effects of olmesartan medoxomil on diabetic nephropathy in streptozotocin-induced diabetes in rats. Biomed. Rep. 2, 24–28. Sowers, J.R., 2002. Hypertension, angiotensin II, and oxidative stress. N. Engl. J. Med. 346, 1999–2001. Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C.L., Ramarao, P., 2005. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313–320. Vieitez, P., Gómez, O., Uceda, E.R., Vera, M.E., Molina-Holgado, E., 2008. Systemic and local effects of angiotensin II blockade in experimental diabetic nephropathy. J. Renin Angiotensin Aldosterone Syst. 9, 96–102. Walker, L.M., Walker, P.D., Imam, S.Z., Ali, S.F., Mayeux, P.R., 2000. Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine. J. Pharmacol. Exp. Ther. 295, 417–422. Welch, W.J., 2008. Angiotensin II-dependent superoxide: effects on hypertension and vascular dysfunction. Hypertension 52, 51–56. Wong, V.Y., Laping, N.J., Contino, L.C., Olson, B.A., Grygielko, E., Brooks, D.P., 2000. Gene expression in rats with renal disease treated with the angiotensin II receptor antagonist, eprosartan. Physiol. Genomics 4, 35–42. Xia, L., Wang, H., Goldberg, H.J., Munk, S., Fantus, I.G., Whiteside, C.I., 2006. Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression. Am. J. Physiol. Ren. Physiol. 290, F345–F356. Yanagi, M., Tamura, K., Fujikawa, T., Wakui, H., Kanaoka, T., Ohsawa, M., Azushima, K., Maeda, A., Kobori, H., Umemura, S., 2013. The angiotensin II type 1 receptor blocker olmesartan preferentially improves nocturnal hypertension and proteinuria in chronic kidney disease. Hypertens. Res. 36, 262–269. Yasuda, G., Shionoiri, H., Kubo, T., Misu, Y., 1987. Presynaptic angiotensin II receptors and captopril-induced adrenergic transmission failure probably not via converting enzyme inhibition in guinea-pig pulmonary arteries. J. Hypertens. Suppl. 5, S39–S45. Zhai, Y.P., Lu, Q., Liu, Y.W., Cheng, Q., Wei, Y.Q., Zhang, F., Li, C.L., Yin, X.X., 2013. Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose. Acta Pharmacol. Sin. 34, 507–514. Zhou, G., Cheung, A.K., Liu, X., Huang, Y., 2014. Valsartan slows the progression of diabetic nephropathy in db/db mice via a reduction in podocyte injury, and renal oxidative stress and inflammation. Clin. Sci. 126, 707–720.
Please cite this article as: Morsy, M.A., et al., Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.01.027i
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
