Cardioprotective role of peroxisome proliferator-activated receptor-γ agonist, rosiglitazone in a unique murine model of diabetic cardiopathy

Life Sciences 162 (2016) 1–13

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Cardioprotective role of peroxisome proliferator-activated receptor-γ agonist, rosiglitazone in a unique murine model of diabetic cardiopathy Suresh R. Giri a,b,⁎, Bibhuti Bhoi a, Mukul R. Jain a, Madhumanjiri M. Gatne b a b

Department of Pharmacology & Toxicology, Zydus Research Centre, Cadila Healthcare Limited, Sarkhej-Bavla N.H. No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India Department of Pharmacology & Toxicology, Bombay Veterinary College, Parel, Mumbai 400012, India

a r t i c l e

i n f o

Article history: Received 7 April 2016 Received in revised form 9 August 2016 Accepted 12 August 2016 Available online 13 August 2016 Keywords: Amiloride Diabetic cardiopathy PPAR gamma agonist Rosiglitazone Type 2 diabetes mellitus

a b s t r a c t Aims: Rosiglitazone (RSZ), a PPARγ agonist was potent efficacious insulin sensitizing blockbuster drug for treatment of Type 2 diabetes mellitus (T2DM) but the benefit of PPARγ activation in congestive heart failure (CHF) was controversial. The present work was planned to study the role of RSZ in diabetic cardiopathy. Main methods: Zucker fa/fa rats, the genetic model of T2DM were subjected to constriction of suprarenal abdominal aorta so that they represent a combined model of diabetes and cardiopathy. The development cardiopathy was assessed biochemically (plasma BNP and aldosterone levels), using echocardiography and expression angiotensin II receptor type 1a gene in heart and Endothelin-1 gene in aorta. Rats were treated with RSZ and in combination with amiloride for four weeks and were assessed to evaluate the effect of RSZ or amiloride or its combination on antidiabetic activity, adverse or toxic effects and congestive heart failure status. Key findings: RSZ shows its anti-diabetic effect from 0.3 mg/kg dose onwards and at 3 mg/kg dose levels it caused beneficial effects (reduction of blood pressure) on cardiovascular system and at highest (30 mg/kg) dose it starts showing adverse effects like body weight gain, edema, left ventricular hypertrophy. However, when highest dose of RSZ animals were treated with amiloride (ENaC inhibitor) at 2 mg/kg the reversal of the adverse effects was evident, indicating the combination of RSZ and amiloride is beneficial in diabetic cardiopathy model. Significance: RSZ and amiloride combination appeared promising treatment in diabetic patients with cardiopathy without any side effect. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The number of people with diabetes mellitus is increasing day by day due to population growth, aging, urbanization, and increasing prevalence of obesity, stress and physical inactivity. The most common Abbreviations: AAALAC International, Association for Assessment and Accreditation of Laboratory Animal Care International; AC, aortic constriction; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT-1R, angiotensin II receptor type 1a; AUC, area under curve; BNP, brain natriuretic peptide; CHF, congestive heart failure; CK-MB, creatine kinase-MB; CPCSEA, Committee for the Purpose of Control and Supervision of Experiments on Animals; CVD, cardiovascular diseases; DBP, diastolic blood pressure; ENaC, epithelial Na channel; eNOS, endothelial nitric oxide synthase; ET-1, Endothelin-1; HOMA-IR, homeostasis model assessment-insulin resistance; HsCRP, high sensitive C-reactive protein; MABP, mean arterial blood pressure; MI, myocardial infarction; NEFA, esterified fatty acids; NF-κB, nuclear factor-kappa B; NIN, National Institute of Nutrition; OGTT, oral glucose tolerance test; PPARs, peroxisome proliferator activator receptors; RAS, renin-angiotensin system; RSZ, rosiglitazone; SBP, systolic blood pressure; T2DM, Type 2 diabetes mellitus; TGF-beta, transforming growth factor; TZD, thiazolidinedione; VEGF, vascular endothelial growth factor; ZRC, Zydus Research Centre. ⁎ Corresponding author at: Departmenmt of Pharmacology & Toxicology, Zydus Research Centre, Sarkhej-Bavla Highway No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India. E-mail address: [email protected] (S.R. Giri).

http://dx.doi.org/10.1016/j.lfs.2016.08.013 0024-3205/© 2016 Elsevier Inc. All rights reserved.

complication of Type 2 diabetes mellitus (T2DM) is the cardiovascular diseases (CVD), the leading cause of morbidity and mortality, accounting for 80% of deaths. The incidence of CVD is three to five times greater in diabetic patients than in those without diabetes [1]. One of the most promising developments in the treatment of T2DM has been the introduction of thiazolidinedione (TZD) class of drugs (e.g., rosiglitazone and pioglitazone) which exert their effects via activation of peroxisome proliferator activator receptors (PPARs). Since its introduction in 1999, rosiglitazone (RSZ) was a major blockbuster drug, till 2007 when Nissen and Wolski published a meta-analysis report in the New England Journal of Medicine and stated that RSZ was associated with increase in myocardial infarction (MI) and a non-statistically significant increase in death from all cardiovascular diseases, which led to its withdrawal in Europe and tight restrictions on its use in the US [2]. The PPAR subfamily of nuclear receptors includes three isotypes, namely PPARα, PPARγ and PPARβ/δ which are encoded by three distinct genes [3]. PPARγ agonists have been reported to have number of cardioprotective properties but the benefit of PPARγ activation in congestive heart failure (CHF) is controversial. The clear-cut evidence of PPAR agonists inducing the cardiac adverse effects was not available. Some reports mentioned that they are beneficial in CHF, possibly due to the attenuation of the ET-1 induced vasoconstriction [4], inhibition

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of the nuclear factor-kappa B (NF-κB) [5] and increased nitric oxide [6]. On the other hand, there is a stream of opinion that PPARγ agonists may worsen heart failure in patients with T2DM. PPARγ agonists or TZD treatments are consistently accompanied by a modest but significant increase in body mass due to both an increase in adipose tissue and body fluid expansion. This fluid expansion is associated with hemodilution, peripheral edema and may thus potentially increase the risk of cardiac insufficiency. Thus, TZDs are contraindicated in patients at risk of cardiac failure and fluid retention can be considered as the main limiting adverse event [7,8]. It is proved through various studies that the fluid retention caused by PPARγ agonists may be attributed to sodium retention affected through the collecting duct epithelial Na channel (ENaC) [9,10]. Thus, it was hypothesized that if amiloride (ENaC Inhibitor) is administered in combination with PPARγ agonist, it may be helpful to reduce the mortality caused by CHF in diabetic patients. Hence, the present work was planned to study the role of PPAR agonist, RSZ in diabetic cardiopathy, induced by aortic constriction in Zucker fa/fa rats, to develop a suitable model that would mimic combined features of T2DM and congestive heart failure for preclinical studies and to evaluate the role of the combination of PPARγ agonist (RSZ) with ENaC inhibitor (amiloride) in a congestive heart failure animal model.

2.3. Study design and treatment groups Following two weeks of surgery, operated animals were subjected to pretreatment (day-0) blood collection and then to oral glucose tolerance test. Animals were confirmed for hypertrophy mediated dilated cardiomyopathy by measuring the plasma brain natriuretic peptide (BNP). The animals which showed significant increase (BNP N 50 pg/ml) over the SHAM control were confirmed for cardiomyopathy progression [13]. They were then divided in various treatment groups, based on their pretreatment glucose AUC (area under curve), hematocrit and body weights, in such a way that these parameters did not differ significantly between the groups as described in Table 1. The oral treatment continued for minimum four weeks (28 days), when animals were sacrificed terminally for the evaluation of various parameters. 2.3.1. Drugs and chemicals RSZ was obtained from Cadila Healthcare Ltd., Thane, India. Amiloride was procured from Sigma-Aldrich Co. LLC. Required quantity of RSZ (purity 100.4% as calculated on anhydrous basis) and amiloride (purity N 98%) was weighed and oral formulation was prepared in polyethylene glycol (PEG) 400 and 0.5% sodium carboxymethyl cellulose (Na-CMC) at 10:90 ratio so that final volume of administration was 2 ml/kg body weight of rats.

2. Materials and methods 2.4. Collection of blood for assessing various parameters 2.1. Experimental animals and environmental conditions Zucker fa/fa rats which are known to exhibit mild hyperglycaemia, insulin resistance, glucose intolerance, hyperlipidaemia, hyperinsulinaemia and moderate hypertension [11] were recruited in the experiments to produce diabetic cardiopathy. Seventy-six of male Zucker fa/fa rats of 8–12 weeks age was used from animal breeding facility of Zydus Research Centre (ZRC), registered under Rule 5(a) for the breeding and Experiments on Animals control and supervision rules 1998 (Registration no. 77/1999). Animals were kept in an environmentally controlled room designed to maintain a temperature of 22 ± 3 °C and a relative humidity of 30% to 70% with at least 14 fresh air changes per hour, in a facility accredited by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International). During the acclimatization period as well as during the biological phase of the study the experimental animals were maintained on normal chow pelleted diet (Standard Rodent Diet, NIN, Hyderabad, India). All the study protocols were approved by Institutional Animal Ethical Committee as per the guidance of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

2.2. Development of congestive heart failure (CHF) by aortic constriction Zucker fatty (fa/fa) rats (70 out of 76) were subjected to aortic constriction (AC) surgery as described previously [12]. Under general anesthesia with ketamine (60 mg/kg i.p.) and xylazine (10 mg/kg i.p.), animal was fixed on surgical table at dorsal recumbent position, the kidney was palpated and incision was made just above it on midline and abdomen was opened, the abdominal aorta was surgically dissected and isolated from the inferior vena cava above the renal arteries and below the mesenteric artery. A 0.7-mm gauge blunt needle covered with PE10 catheter tube was placed along the side of the isolated suprarenal abdominal aorta segment. Then, a 3-0 suture was tightly tied around the aorta and the overlying needle. The needle was removed, thus producing aortic constriction above the renal arteries. Six Zucker fatty (fa/fa) rats were assigned to the sham-operated group underwent the same procedures without constriction of the aorta. After surgery all animals were kept under observation for two weeks.

Blood was collected under light ether anesthesia from orbital sinus at pretreatment (day-0) and on day-28 of treatment, in nonfasted animals. Blood was collected in three separate microcentrifuge tubes, first for serum where approx. 0.35–0.40 ml blood and second tube containing heparin (25 IU/ml) was for plasma where 0.25 to 0.30 ml blood was collected and third tube containing 2% sodium EDTA (10 μl) where 0.10 ml blood was collected for hematocrit measurement. 2.5. Determination of serum/plasma biochemical parameters for assessing various parameters Serum parameters like triglycerides (TG), glucose, non-esterified fatty acids (NEFA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatine kinase-MB (CK-MB), and high sensitive C-reactive protein (Hs-CRP) were measured using Cobas c 311 clinical chemistry autoanalyser (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland) and diagnostic kits from (Roche Diagnostics kits, GmbH, D-68298 Mannheim, Germany). Serum electrolytes sodium, potassium and chloride were estimated using Cobas c 311 clinical chemistry autoanalyser and ISE (ion-selective electrode) module. Serum insulin levels were measured using the ELISA kit from Linco Research, (Linco Research, Inc., St Charles, MO, USA). Plasma BNP and aldosterone levels were as measured by enzyme linked immunoassay from Rat BNP-32 ELISA kit (Assay Pro Inc., Blvd St. Charles, MO, USA) and aldosterone EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA) respectively. 2.6. Oral glucose tolerance test (OGTT) For glucose tolerance testing, overnight fasted rats were orally administered 3 g/kg glucose and the blood samples were collected from retro orbital sinus under light anesthesia at 0, 30, 60 and 120 min after glucose load administration. OGTT was performed at pretreatment and, on day 29-post treatment to evaluate glucose intolerance if any and antidiabetic activity. The post treatment OGTT was done an hour after the last dose of vehicle or compound administration.

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Table 1 Details of treatment groups. Group no.

Group details

Treatment (oral)

No. of animals

1 2 3 4 5 6 7

Diabetic control − Zucker fa/fa rats (SHAM fa/fa rat) Diabetic Zucker fa/fa rats + aortic constriction (CHF) control (AC-fa/fa rat vehicle control) Diabetic Zucker fa/fa rats + aortic constriction (CHF) with rosiglitazone (RSZ) low dose Diabetic Zucker fa/fa rats + aortic constriction (CHF) with rosiglitazone (RSZ) mid dose Diabetic Zucker fa/fa rats + aortic constriction (CHF) with rosiglitazone (RSZ) high dose Diabetic Zucker fa/fa rats + aortic constriction (CHF) with rosiglitazone (RSZ) + amiloride (AMI) Diabetic Zucker fa/fa rats + aortic constriction (CHF) with amiloride (AMI)

Vehicle Vehicle RSZ 0.3 mg/kg RSZ 3 mg/kg RSZ 30 mg/kg RSZ 3 mg/kg + AMI 2 mg/kg AMI 2 mg/kg

6 6 6 6 6 6 6

2.7. Plasma volume measurement On day 32, an hour after completion of the treatment, the plasma volume was measured in all animals by Evans blue dye dilution method as described by Chen et al. [14].

Green kit (Cat. No. 204054, Qiagen, Germantown, MD, USA). Rat Ribosomal Acidic Protein gene was used as an internal control for normalization of the results. The details of genes and primer sequences used as given in Table 2.

2.8. Echocardiography

2.12. Statistical analysis

The two-dimensional (2D) echocardiography was performed on three animals of each group, after three days of plasma volume measurement (on day 35) to evaluate non-invasive parameters like left ventricular mass, ejection fraction, left ventricular interventricular septum thickness at systole, left ventricular internal diameter at systole and cardiac output as described by Lindsay et al. [15]. Rats were anaesthetized with Ketamine (60 mg/kg) and xylazine (10 mg/kg) by intraperitoneal route and two-dimensional echocardiography was performed using pediatric 12 MHz frequency probe in GE Healthcare, VOLUSON® 730 – Pro machine.

Data are expressed as mean ± S.E.M. The difference among the experimental groups were analyzed by one-way analysis of variance (ANOVA) or two way ANOVA, followed by Dunnett's test or Bonferroni posttests respectively, to evaluate the statistical difference between various treatment groups. The difference between SHAM fa/fa rats and Aortic constriction fa/fa rats vehicle control group was compared using ‘t-test’. p b 0.05 was considered significant. All analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla CA).

2.9. Blood pressure Systolic, diastolic and mean arterial blood pressure was measured just before termination in all treated animals, by cannulating the left carotid artery with a PE-10 polyethylene catheter, and pulse waveforms were monitored by BIOPAC system under urethane anesthesia (1 g/kg i.p. of 10% solution). 2.10. Left ventricle weight and heart weight After completion of the evaluation of the above parameters all treated animals were euthanized by carbon dioxide asphyxiation, heart was removed and weighed. Both the atria and the right ventricle were trimmed away, and the remaining left ventricle and septum were weighed as left ventricular weight. Immediately heart samples were stored in liquid nitrogen for further estimation. In order to get relative organ weight, absolute organ weights were divided by body weight taken on the day of sacrifice of animals. Heart weight to body weight ratio as well as left ventricle weight to body weight ratio (relative organ weights) were also considered to assess the extent of cardiac hypertrophy. 2.11. mRNA isolation and gene expression in heart, aorta and kidney Total RNA was extracted from the heart, aorta and kidney samples by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the supplier's instructions. From each sample 1 μg of total RNA was taken for first-strand cDNA synthesis using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA; Part No. 4322171). An equal amount of cDNA from each sample was taken for quantitative real-time PCR using ABIprism-7300 (Applied Biosystems, Foster City, CA, USA). Gene expression of various genes in heart (PPARα, PPARγ, AT II receptor and TGF-beta), aorta (ET-1, eNOS and VEGF), and kidney (ENaC-α and Na, K-ATPase) was determined using SYBR Green quantitative real-time PCR and QIAGEN QuantiFast SYBR

3. Results Zucker fa/fa rats, the genetic model of T2DM were subjected to constriction of suprarenal abdominal aorta. Out of seventy-six Zucker fa/fa rats, seventy animals which were subjected for aortic constriction 50 animals survived after two weeks, that showed 71% of operated animals survived the surgical procedure and most of mortality was observed in first week after surgery. Following two weeks of surgery, the blood samples were collected for BNP estimation. Rats showing significantly (N6 fold) higher BNP compared to SHAM were further subjected to OGTT, so that they would represent a combined model of diabetes and cardiopathy. Various treatments were offered to grouped rats by oral route once daily as mentioned in Table 1. and treatment was continued for four weeks. After four weeks of treatment various parameters were assessed to evaluate the effect of RSZ or amiloride or its combination on 1) antidiabetic activity, 2) adverse or toxic effects and 3) congestive heart failure status. Table 2 Sense and antisense primers for the gene of interest. Name

Primer sequence

AT 1R AT 1R TGFβ-1 TGFβ-1 PPAR-α PPAR-α PPAR-γ-1 PPAR-γ-1 ET-1 ET-1 eNos-3 eNos-3 VEFG-α-1 VEFG-α-1 Na-K ATPase-α-1 Na-K ATPase-α-1 ENaC-α ENaC-α

Forward primer: 5′-GGCTCTCTCAGCTCTGCCACAT-3′ Reverse primer: 5′-CCTGGTGATCACTTTCTGGGAGGG-3′ Forward primer: 5′-CTGGAAAGGGCTCAACACC-3′ Reverse primer: 5′-GTAGACGATGGGCAGTGGCT-3′ Forward primer: 5′-CGGGTCATACTCGCAGGAAAG-3′ Reverse primer: 5′-TGGCAGCAGTGGAAGAATCG-3′ Forward primer: 5′-GGAAGCCCTTTGGTGACTTTATGG-3′ Reverse primer: 5′-GCAGCAGGTTGTCTTGGATGTC-3′ Forward primer: 5′-ACCCCGCAGGTCCAAGCGTT-3′ Reverse primer: 5′-TCCATACGGGACGACGCGCTC-3′ Forward primer: 5′-TTCTGGCAAGACCGATTACACGACAT-3′ Reverse primer: 5′-AAAGGCGGAGAGGACTTGTCCAAA-3′ Forward primer: 5′-CAGCGCAGCTATTGCCGTCCA-3′ Reverse primer: 5′-CGCACACCGCATTAGGGGCA-3′ Forward primer: 5′-TAGTCTCCAGCCACAGGACCCG-3′ Reverse primer: 5′-CATGTTCTGATACAGCTGCGGGCT-3′ Forward primer: 5′-AGAGCTGGACCGCATCACGGA-3′ Reverse primer: 5′-CTGCAGGGGGTGCGGGAAAG-3′

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Table 3 Effect on non-fasting parameters in aortic constricted Zucker fa/fa rats after 4 weeks of treatment with rosiglitazone and its combination with amiloride.

Body weight (g) Serum glucose (mg/dl) Serum insulin (ng/ml) HOMA index Serum TG (mg/dl) Serum NEFA (mmol/l) Serum ALT (U/l) Serum AST (U/l) Serum urea (mg/dl) Serum CK-MB (U/l) Serum Hs-CRP (mg/l) Serum sodium (mmol/l) Serum potassium (mmol/l) Serum Chloride (mmol/l) Plasma volume (ml/kg) Blood hematocrit (%) Plasma BNP (pg/ml) Plasma aldosterone (pg/ml)

SHAM fa/fa rats

Aortic constriction fa/fa rats

Vehicle

Vehicle control

616.13 ± 13.15 169.45 ± 32.13 12.87 ± 2.79 97.90 ± 24.24 512.14 ± 180.27 1.09 ± 0.05 73.21 ± 9.21 190.64 ± 24.10 33.25 ± 1.95 2650.50 ± 262.71 0.05 ± 0.01 144.28 ± 1.38 5.44 ± 0.09 96.70 ± 1.19 39.60 ± 0.20 41.90 ± 0.85 16.27 ± 4.71 189.65 ± 52.56

599.78 ± 13.54 199.85 ± 53.39 8.15 ± 3.07 62.50 ± 26.12 395.52 ± 32.10 1.10 ± 0.14 90.68 ± 18.73 213.88 ± 35.84 36.67 ± 2.62 2467.77 ± 208.88 0.05 ± 0.01 144.45 ± 1.77 5.67 ± 0.12 97.07 ± 0.81 41.28 ± 0.69# 41.92 ± 0.70 123.55 ± 21.14# 628.19 ± 222.17#

Rosiglitazone (mg/kg) 0.3

3

30

649.95 ± 14.54 130.72 ± 9.24⁎ 3.18 ± 0.59 22.83 ± 4.52 333.70 ± 28.75 0.77 ± 0.07 100.70 ± 23.17 186.67 ± 34.67 41.67 ± 1.23 2194.52 ± 342.91 0.04 ± 0.01 148.25 ± 2.79 5.47 ± 0.14 101.27 ± 1.35⁎ 43.12 ± 0.93 41.07 ± 0.89 74.49 ± 20.55 491.04 ± 94.57

711.95 ± 30.75⁎ 153.68 ± 11.74 3.34 ± 0.92 21.94 ± 5.86 216.95 ± 19.07 0.47 ± 0.02⁎ 141.58 ± 17.19⁎ 229.68 ± 27.41 50.00 ± 2.12⁎

726.88 ± 36.88⁎ 127.34 ± 7.88⁎ 1.49 ± 0.09 10.40 ± 0.72 112.68 ± 20.12⁎ 0.38 ± 0.06⁎ 157.10 ± 24.67⁎ 277.44 ± 41.40 53.20 ± 3.68⁎

1476.95 ± 193.73⁎ 0.04 ± 0.01 150.80 ± 3.95 5.16 ± 0.16⁎ 101.67 ± 1.22⁎ 45.46 ± 1.14⁎

1348.22 ± 177.65⁎ 0.05 ± 0.01 156.44 ± 7.01⁎ 4.75 ± 0.16⁎ 102.64 ± 0.98⁎ 51.51 ± 0.80⁎

40.35 ± 0.91 63.48 ± 17.47 144.14 ± 19.65

39.62 ± 0.61 55.08 ± 5.39⁎ 156.02 ± 35.31

Amiloride (2 mg/kg) RSZ 30 + AMI (2)

AMI (2)

673.72 ± 19.32 168.88 ± 13.56 2.47 ± 0.48 16.69 ± 3.94 139.55 ± 9.71⁎ 0.52 ± 0.09⁎ 134.85 ± 12.65 210.67 ± 23.52 51.83 ± 1.68⁎

608.07 ± 19.68 193.97 ± 30.05 15.61 ± 5.20 114.94 ± 32.47 461.38 ± 85.98 1.10 ± 0.16 109.82 ± 43.02 118.58 ± 29.97 36.83 ± 2.24 493.70 ± 60.86⁎ 0.07 ± 0.01 143.75 ± 1.30 5.84 ± 0.14 97.90 ± 1.41 38.58 ± 0.79 42.87 ± 0.88 78.04 ± 26.00 3292.72 ± 1000.21⁎

1346.02 ± 192.21⁎ 0.03 ± 0.01 147.38 ± 3.63 5.80 ± 0.21 98.72 ± 1.60 40.17 ± 0.50 42.28 ± 0.53 24.73 ± 4.83⁎ 1079.51 ± 185.83

Data are means ± SEM of six animals per group. The significant differences between SHAM fa/fa rats and aortic constriction fa/fa rats vehicle control group given as #, p b 0.05 (‘t-test’) and significant differences between aortic constriction fa/fa rats was given as *, p b 0.05 (One way ANOVA followed by Dunnett's test).

3.1. Effect on parameters evaluated for anti-diabetic activity Antidiabetic activity was evaluated based on OGTT, serum glucose, insulin levels, calculating homeostasis model assessment insulin resistance (HOMA-IR) index, serum triglycerides (TG), NEFA levels (Table 3). There was significant improvement in glucose tolerance

which was indicated by 38 and 43% reduction in AUC glucose during OGTT at 3 and 30 mg/kg RSZ (Fig. 1A and B). In group treated with RSZ at 0.3 mg/kg and another group treated with combination of RSZ at 30 mg/kg + amiloride 2 mg/kg, though there was improvement in glucose tolerance (17 and 28%), it was (statistically) non-significant, whereas animals treated with amiloride 2 mg/kg did not show any

Fig. 1. Effect of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment on parameters evaluated for anti-diabetic activity. (A) serum glucose levels and (B) AUC-glucose in oral glucose tolerance test on day 29. Non-fasting serum triglycerides and NEFA level was measured on pretreatment and on day-28 one hour post dose and % change vs vehicle control are given in (C) and (D) respectively. All values are expressed as mean ± SEM (n = 6). *p b 0.05 vs AC-fa/fa rat vehicle control by one-way ANOVA followed by Dunnett's test except (A) where two-way ANOVA was applied followed by Bonferroni's post test.

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effect on glucose tolerance. There was 32, 34 and 39% mean decrease in serum glucose levels at RSZ 0.3, 3 and 30 mg/kg respectively, whereas RSZ in combination with amiloride showed only 20% decrease in glucose. Mean serum insulin levels were decreased in all the RSZ treatment groups viz., 2, 55, 57 and 59% at 0.3, 3, 30 and combination group respectively. HOMA-IR index reduction estimated in 0.3, 3, 30 mg/kg RSZ and RSZ + amiloride combination group and it was 63, 65, 83 and 73% respectively, amiloride 2 mg/kg alone caused 82% increase in HOMA-IR index. Serum triglycerides levels reduced dose dependently in RSZ treated groups and reduction was 28 and 73% at 3 and 30 mg/kg respectively (Fig. 1C). The combination of RSZ and amiloride also showed 67% decrease in triglycerides. In amiloride alone group there was 45% increase in mean triglycerides levels. Mean serum NEFA levels showed significant and dose dependent (17, 44 and 59%) reduction at 0.3, 3 and 30 mg/kg dose of RSZ respectively (Fig. 1D). The combination of RSZ and amiloride treatment also showed 48% decrease in NEFA levels which was slightly less than that caused by RSZ alone. In amiloride alone group there was 12% increase in NEFA levels, indicating possible role of amiloride in causing less decrease in NEFA levels in RSZ + amiloride combination group. The treatment with RSZ did not have any significant effect on serum total cholesterol levels. 3.2. Effect on parameters evaluated for adverse or toxic effects The adverse and toxic effects of RSZ were evaluated considering various parameters like measurement of body weight, serum AST, ALT, creatinine, urea, CK-MB, Hs-CRP, serum electrolytes like Na+, Cl− and K+ (Table 3). Effect of RSZ on edema was estimated by measurement of plasma volume and hematocrit levels. Serum osmolality was calculated from serum sodium, potassium, glucose, and urea concentrations. Oral RSZ dosing for four weeks caused significant gain in body weights at dose starting from 3 mg/kg, increase in mean body weights

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were 9, 15 and 16% at 0.3, 3 and 30 mg/kg dose of RSZ respectively (Fig. 2A). When amiloride was given in combination with RSZ, the increase in body weight was only 5% which indicated that there was 67% reversal of body weight gain caused by RSZ alone at 30 mg/kg. Treatment with RSZ at 30 mg/kg exhibited non-significant (48%) increase in AST levels which were reversed by RSZ and amiloride combination treatment (Fig. 2B). Dose dependent increase of 5, 19, and 64% in serum ALT was observed at RSZ 0.3, 3 and 30 mg/kg respectively (Fig. 2C). Treatment with RSZ did not have any significant effect on serum creatinine. Serum urea levels were dose dependently increased by 11, 31 and 46% at 0.3, 3 and 30 mg/kg dose of RSZ respectively. The combination of RSZ with amiloride did not affect the serum urea levels. Serum CK-MB levels showed dose dependent reduction which was significant at 3 and 30 mg/kg dose. The combination of RSZ and amiloride also showed significant (41%) reduction in serum CK-MB levels. Amiloride alone treatment caused 74% reduction in serum CK-MB levels. The treatment with RSZ did not have any significant effect on Hs-CRP, amiloride treatment caused 38% statistically non-significant increase but combination of RSZ and amiloride treatment showed 46% statistically non-significant decrease in serum Hs-CRP (Fig. 2D). The treatment with RSZ at 0.3, 3 and 30 mg/kg caused dose dependent (3, 5 and 8%) increase in serum sodium and chloride levels (3, 3 and 5%). Both sodium and chloride levels (Fig. 3A and B) were significantly increased at 30 mg/kg dose indicative of sodium resorption and consequent plasma volume expansion. Serum potassium levels were decreased by 9 and 14% at 3 and 30 mg/kg dose (Fig. 3C). When amiloride was administered with RSZ it caused only 2 and 2% increase in sodium and chloride levels and no decrease of serum potassium levels, indicating that amiloride caused reversal of sodium and chloride retention caused by RSZ. The retention of sodium results into increase in plasma volume and decrease in hematocrit. The plasma volume measurement by dye dilution technique showed that RSZ caused dose

Fig. 2. Effect of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment on parameters evaluated for assessing adverse or toxic effects. The % change vs vehicle control are (A) body weight gain (B) serum AST levels (C) serum ALT levels (D) serum Hs-CRP levels. The % change in body weight gain was calculated on comparing body weight on pretreatment and on day-28 vs vehicle control group. Non-fasting serum AST, ALT and Hs-CRP level were measured on pretreatment and on day-28 one hour post dose and values are calculated as percent change vs vehicle control group. The bars represent the fold change in the treatment groups compared with the vehicle control group, mean ± SEM (n = 6).

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Fig. 3. Effect of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment serum electrolytes and plasma volume. Serum electrolytes level were measured on pretreatment and on day-28 one hour post dose and values are calculated as percent change vs vehicle control group given as (A) sodium (B) chloride (C) potassium. On day 32, all animals were subjected for plasma volume measurement by Evans blue dye dilution and percentage change over control is depicted in figure (D) and % change in hematocrit measured on day-28 was shown in figure (E). The bars represent the percent change in the treatment groups compared with the vehicle control group, mean ± SEM (n = 6).

dependent increase in plasma volume at 0.3, 3 and 30 mg/kg dose which was 4, 10 and 25% respectively (Fig. 3D), but the increase was significant only at 3 and 30 mg/kg dose. RSZ with amiloride showed 3% decrease in plasma volume, indicating that amiloride caused complete reversal of plasma volume expansion caused by RSZ. Hematocrit is the indirect measure of plasma volume; RSZ caused dose dependent decrease in hematocrit which was 3, 5 and 8% at 0.3, 3 and 30 mg/kg dose respectively (Fig. 3E). Combination of RSZ and amiloride showed only 1% decrease in hematocrit. The decrease in hematocrit was the indirect effect resulting from increase in plasma volume caused due to sodium retention by RSZ but when RSZ was combined with amiloride, it caused complete blockage of ENaC and prevented sodium retention and increased in plasma volume and decrease in hematocrit caused by high (30 mg/kg) dose of RSZ.

3.3. Effect on parameters evaluated for congestive heart failure The effects of RSZ on cardiovascular parameters were evaluated by measurement of BNP and aldosterone in plasma, two-dimensional echocardiography, systolic and diastolic blood pressure, weight of heart and left ventricle weight. The two-dimensional (2D) echocardiography was performed to measure non-invasive parameters like left ventricular mass, ejection fraction, left ventricular interventricular septum thickness at systole, left ventricular internal diameter at systole and cardiac output (Table 4). The determination of terminal systolic and diastolic blood pressure was done and rats were sacrificed for weighing left ventricle and heart weight for evaluation of effect on cardiac weight (Table 5). Pre-treatment plasma BNP levels of SHAM operated animals were N6 fold higher than aortic constricted animals which confirmed the

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Table 4 Effect on two-dimensional echocardiographic parameters in aortic constricted Zucker fa/fa rats after 4 weeks of treatment with rosiglitazone and its combination with amiloride.

Left ventricular mass (g) IVSs (cm) LVIDs (cm) Ejection fraction (%) Cardiac output (ml/min)

SHAM fa/fa rats

Aortic constriction fa/fa rats

Vehicle

Vehicle control

0.603 ± 0.05 0.200 ± 0.01 0.305 ± 0.10 88.06 ± 2.1 84.96 ± 1.83

0.683 ± 0.49# 0.183 ± 0.01 0.423 ± 0.02 70.01 ± 1.09# 59.83 ± 9.69#

Rosiglitazone (mg/kg) 0.3

3

30

Amiloride (2 mg/kg) RSZ 30 + AMI (2)

AMI (2)

0.808 ± 0.01 0.200 ± 0.01 0.480 ± 0.07 68.01 ± 9.07 78.45 ± 1.91

1.001 ± 0.10⁎ 0.240 ± 0.01 0.453 ± 0.04 74.29 ± 4.84 85.77 ± 4.95

0.960 ± 0.01⁎ 0.235 ± 0.03 0.505 ± 0.09 62.88 ± 13.56 72.19 ± 4.81

0.654 ± 0.10 0.210 ± 0.02 0.245 ± 0.04 92.31 ± 4.16 77.32 ± 15.62

0.843 ± 0.02 0.210 ± 0.01 0.380 ± 0.01 79.72 ± 3.44 66.85 ± 2.55

Data are means ± SEM of six animals per group. The significant differences between SHAM fa/fa rats and aortic constriction fa/fa rats vehicle control group given as #, p b 0.05 (‘t-test’) and significant differences between aortic constriction fa/fa rats was given as *, p b 0.05 (One way ANOVA followed by Dunnett's test).

progression of cardiomyopathy in aortic constricted animals. Treatment with RSZ at 3 and 30 mg/kg dose showed 33% decrease in BNP levels and amiloride alone treatment caused 32% decrease whereas combination of RSZ and amiloride caused additive effect, 67% reduction in BNP levels (Fig. 4A). Plasma aldosterone levels exhibited 77 and 75% decrease at 3 and 30 mg/kg RSZ (Fig. 4B) which may be due to sodium resorption caused by RSZ. Amiloride alone treatment caused very significant (5-fold) increase in aldosterone due to potassium sparing effect and combination of RSZ and amiloride caused only 72% (1.7-fold) increase in aldosterone levels indicating that amiloride is reversing sodium resorption caused by RSZ. The two-dimensional echocardiography showed that RSZ at 0.3, 3 and 30 mg/kg caused dose dependent (18, 47 and 41%) increase in left ventricular mass as compared to aortic constriction (AC-fa/fa Rat Vehicle Control) control which was completely reversed (−4%) by the RSZ and amiloride combination treatment (Table 5 and Fig. 5). Aortic constriction vehicle treated group showed 21% decrease in ejection fraction as compared to SHAM fa/fa rats and treatment with RSZ and amiloride combination showed 32% increase in ejection fraction as compared to the control group. Left ventricular interventricular septum thickness was increased by 9, 31 and 28% by RSZ treatment at 0.3, 3 and 30 mg/kg dose respectively and combination of RSZ with amiloride showed 15% increase. This indicates that combination reduced interventricular septum thickness by 47%. RSZ at 30 mg/kg caused 19% increase in left ventricular internal diameter but combination of amiloride and RSZ showed 42% decrease in ventricular internal diameter as compared to control animals treated with vehicle. Aortic constriction had shown 29% decrease in cardiac output as compared to SHAM fa/fa rats and treatment with RSZ and amiloride combination showed 29% increase in cardiac output as compared to the control. The systolic and diastolic blood pressure measured by cannulating left carotid artery and monitored by BIOPAC system showed that RSZ at 3 and 30 mg/kg caused 14 and 31% reduction in systolic blood pressure (SBP) and 19 and 52% decrease in diastolic blood pressure (DBP) respectively. Combination of RSZ with amiloride showed 22% reduction in systolic blood pressure and 49% decrease in diastolic blood pressure.

Amiloride alone treatment also showed 24% decrease in SBP and 29% decrease in DBP. The mean arterial blood pressure (MABP) showed dose dependent (17 and 45%) reduction by RSZ treatment at 3 and 30 mg/kg, whereas combination of RSZ and amiloride showed 33% reduction in mean arterial blood pressure and amiloride alone showed 28% reduction in mean arterial blood pressure. Aortic constriction in Zucker fa/fa rat caused 26% increase in left ventricle weights. The left ventricle weights were increased by RSZ in dose dependent manner (12, 10, and 33% at 0.3, 3 and 30 mg/kg dose respectively) but combination of RSZ with amiloride caused only 15% increase in left ventricle weight which was approx. 50% less than that caused by RSZ at 30 mg/kg dose (Fig. 6A). Aortic constriction in Zucker fa/fa rat caused only 10% increase in heart weights as compared to 26% increase in left ventricle weights (Fig. 6B), clearly showing that aortic constriction mainly causes left ventricle hypertrophy. When RSZ treatment was given to aortic constricted Zucker fa/fa rats, heart weights were dose dependently increased by 15, 36, and 71% at 0.3, 3 and 30 mg/kg dose, however in amiloride combination the increase in heart weights was only 15% which is approx. 80% less than that caused by RSZ alone at 30 mg/kg dose (Fig. 6B). Amiloride alone treatment did not show any significant change in heart weights. 3.4. Effect on gene expression in heart, aorta and kidney The effect of aortic constriction and then various treatments was mechanistically evaluated by measuring the mRNA expression for various genes in heart, aorta and kidney (Table 6). Aortic constriction caused 6-fold upregulation of angiotensin II receptor type 1a (AT-1R) mRNA levels in heart of Zucker fa/fa rats (Fig. 7A). It also caused 2.8fold statistically non-significant upregulation of PPARγ-1 gene after aortic constriction. In this study RSZ treatment showed downregulation of PPARα which was statistically non-significant but clear trend of dose dependent (Fig. 7B) was observed which might be due to pressure overload induced cardiac hypertrophy and reversal of fetal metabolic genotype in myocardium as reported by Barger et al. [16] and Chen et al. [17]. But when RSZ was combined with amiloride it reduced the plasma

Table 5 Effect on blood pressure and heart and left ventricle weight in aortic constricted Zucker fa/fa rats after 4 weeks of treatment with rosiglitazone and its combination with amiloride.

SBP (mm Hg) DBP (mm Hg) MABP (mm Hg) Heart weight (g) Left Ventricle weight (g)

SHAM fa/fa rats

Aortic constriction fa/fa rats

Vehicle

Vehicle control

112.33 ± 6.71 82.17 ± 6.70 98.17 ± 6.90 1.23 ± 0.06 0.585 ± 0.027

122.40 ± 6.31 93.40 ± 8.66 110.40 ± 6.87 1.35 ± 0.06 0.738 ± 0.054#

Rosiglitazone (mg/kg) 0.3

3

30

Amiloride (2 mg/kg) RSZ 30 + AMI (2)

AMI (2)

120.67 ± 14.47 92.67 ± 17.13 100.83 ± 12.18 1.56 ± 0.09 0.827 ± 0.109

105.33 ± 4.91 76.00 ± 2.08 91.33 ± 3.53 1.84 ± 0.19⁎ 0.815 ± 0.047

84.33 ± 6.33⁎ 44.33 ± 5.36⁎ 60.75 ± 4.13⁎ 2.31 ± 0.22⁎ 0.988 ± 0.089⁎

95.50 ± 1.50 47.50 ± 7.50 74.50 ± 1.50 1.55 ± 0.14 0.848 ± 0.045

93.25 ± 5.02⁎ 66.00 ± 4.71 79.75 ± 4.92⁎ 1.43 ± 0.07 0.762 ± 0.043

Data are means ± SEM of six animals per group. The significant differences between SHAM fa/fa rats and aortic constriction fa/fa rats vehicle control group given as #, p b 0.05 (‘t-test’) and significant differences between aortic constriction fa/fa rats was given as *, p b 0.05 (One way ANOVA followed by Dunnett's test).

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Fig. 4. Effect of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment on (A) plasma BNP and (B) plasma aldosterone. Plasma BNP was measured on pretreatment and on day-28 one hour post dose and plasma aldosterone was measured on day-28. The bars represent the values on day-28, mean ± SEM (n = 6). *p b 0.05 vs AC-fa/fa rat vehicle control by one-way ANOVA followed by Dunnett's test.

volume expansion and ultimately pressure overload, there was 50% reversal of PPARα expression. Amiloride treatment also caused statistically non-significant decrease in PPARγ expression and as reported by Chen et al. [17] increase in expression of in PPARγ can lead to cardiac dysfunction. The combination of amiloride with RSZ proved to be beneficial as its dose dependently increases the PPARα expression. In aorta of Zucker fa/fa rats there was 2.9-fold upregulation of Endothelin-1 (ET-1) gene expression. In this study aortic constriction in Zucker rats caused 2.9 fold upregulation of ET-1 and treatment with RSZ caused dose dependent reduction in ET-1 expression (Fig. 7C) along with down regulation of Ang II type 1 receptor which might be causing vasodilation resulting into reduced mean, systolic and diastolic blood pressure as reported by Chen et al. [17]. Treatment of RSZ caused statistically non-significant but clear cut dose dependent decrease in AT1R mRNA levels in heart and statistically significant increase in ET-1 mRNA levels in aorta. It also caused statistically non-significant but clear cut dose dependent increase in ENaC-α and Na\\K ATPase-α-1 mRNA levels in kidney (Fig. 7D and E). In combination of RSZ and amiloride group there was no adverse or beneficial effect caused by RSZ in heart and aorta but combination caused further beneficial effect by causing downregulation of Na\\K ATPase-α-1 and ENaC-α mRNA levels in kidney. These changes in gene expression support the hypothesis that RSZ is causing suppression of overtly activated renin-angiotensin system (RAS) in aortic constricted condition and it

also further causes vasodilation by inhibition of ET-1 in aortic tissue and when it was combined with amiloride it also reversed the adverse effects caused due to increased plasma volume (edema and body weight gain) which makes the combination of RSZ and amiloride very beneficial and it may be helpful to reduce the mortality caused by CHF in diabetic patients. 4. Discussion Cardiovascular diseases (CVD) are the most common complication of T2DM and are a leading cause of morbidity and mortality [1]. The contradiction of effect of blockbuster drug, RSZ (PPARγ agonist) in CVD was an inspiration to work on this topic, wherein an attempt was made to develop a rat model that will exhibit the characteristics of T2DM and congestive heart failure or cardiopathy simultaneously. In genetically diabetic Zucker fa/fa rats [11,18] congestive heart failure was developed by performing aortic constriction at suprarenal abdominal aorta [12,19] and the effects of RSZ were assessed in the animal model exhibiting diabetes and congestive heart failure simultaneously. In this study, as compared to SHAM treated, aortic constriction in Zucker fa/fa rats caused significant increase in plasma BNP and aldosterone levels, as reported by Tabbi-Anneni et al. [13] in Wistar rats and Iwanaga et al. [20] in patients with chronic heart failure. Echocardiographic measurement of aortic constriction induced rats showed that

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A: AC-fa/fa Vehicle Control

B: RSZ 30 mg/kg

C: RSZ 30/kg + AMI 2 mg/kg

D: AMI 2 mg/kg

E: Typical 2D Echocardiographic representation (Source- Juric et al., 2007 [21])

Fig. 5. Two-dimensional echocardiographs of representative rat done on day 35 of treatment. Various echocardiograms are (A) aortic constricted Zucker fa/fa rat from vehicle treated control group (B) aortic constricted Zucker fa/fa rat treated with rosiglitazone 30 mg/kg dose (C) aortic constricted Zucker fa/fa rat treated combination of rosiglitazone 30 mg/kg and amiloride 2 mg/kg dose (D) aortic constricted Zucker fa/fa rat treated with amiloride 2 mg/kg alone (E) is typical 2-dimensional M-mode echocardiographic representation of left ventricular (LV) interventricular septal dimension (IVS), LV internal dimension (LVID), and LV posterior wall dimensions (LVPW) at systole (s) and diastole (d).

there was significant increase in left ventricular mass, decrease in ejection fraction and cardiac output as compared to SHAM treated Zucker fa/fa rats. These findings are consistent with those reported by Juric et al. [21]. Aortic constricted Zucker fa/fa rats showed significant increase in plasma volume and increase in systolic, diastolic and mean arterial blood pressure similar to reports of Jacob et al. [22], there was significant 26.2% increase in left ventricle weight and 10.4% increase in heart weight similar to that reported by Kurosawa et al. [23] and Jacob et al. [22]. Aortic constriction caused significant six-fold upregulation of AT-1R mRNA in heart of Zucker fa/fa rats. There was statistically non-significant but 2.8-fold upregulation of PPARγ-1 gene and 2.9 fold upregulation of ET-1 gene expression observed in heart and aorta respectively. All these observations suggest that aortic constriction

exhibited marked left ventricular hypertrophy with increased expression AT-1R in heart and ET-1 in aorta along with increased plasma BNP and aldosterone levels demonstrating the hyper-activation of RAS. This condition with a pressure overload (increased plasma volume) caused due to RSZ then exhibited the clinical syndrome of congestive heart failure in preclinical studies. In aortic constricted Zucker fa/fa rats, RSZ started showing anti-diabetic activity at 0.3 mg/kg and these effects reached peak levels at dose of 3 mg/kg. This is consistent with findings from previous reports from Pickavance et al. [24], Gaikwad [25] and Chang et al. [26]. Amiloride is known to inhibit insulin sensitivity by impaired activation of insulin receptor tyrosine kinase and decreased binding but overall glucose homeostasis was not affected due to its compensatory insulin release

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Fig. 6. Effect of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment on (A) heart weight and (B) left ventricle weight. The bars represent the percent change in the treatment groups compared with the vehicle control group, mean ± SEM (n = 6).

[27,28]. In the present study, when RSZ was administered in combination with amiloride it did not cause any significant effect on reduction in NEFA levels but caused less reduction in AUC-glucose as compared

to RSZ alone. Treatment with amiloride alone showed increase in HOMA-IR, which may be due to inhibition of insulin sensitivity and compensatory insulin release as reported in literature [27,28].

Table 6 Effect on mRNA expression of genes in heart, aorta and kidney tissue in aortic constricted Zucker fa/fa rats after 4 weeks of treatment with rosiglitazone and its combination with amiloride (The results are normalized to Ribosomal Acidic Protein expression and presented as -fold change vs AC-fa/fa rat vehicle control). SHAM fa/fa rats

Aortic constriction fa/fa rats

Vehicle

Vehicle control

Rosiglitazone (mg/kg) 0.3

3

30

Amiloride (2 mg/kg) RSZ 30 + AMI (2)

AMI (2)

Heart AT 1R TGF beta-1 PPAR-α PPAR-γ-1

0.16 ± 0.04 0.87 ± 0.08 1.40 ± 0.06 0.44 ± 0.01

1.00 ± 0.07# 1.12 ± 0.36 1.13 ± 0.37 1.25 ± 0.46

0.66 ± 0.49 0.83 ± 0.41 0.73 ± 0.22 0.78 ± 0.01

0.46 ± 0.29 1.11 ± 0.11 0.51 ± 0.17 1.16 ± 0.30

0.37 ± 0.22 0.49 ± 0.27 0.39 ± 0.01 0.71 ± 0.22

0.49 ± 0.36 0.50 ± 0.07 0.69 ± 0.22 0.43 ± 05

10.01 ± 3.21⁎ 0.73 ± 0.01 1.10 ± 0.05 0.42 ± 0.18

Aorta ET-1 eNos-3 VEFG-α-1

0.47 ± 0.04 0.88 ± 0.24 1.33 ± 0.45

1.35 ± 0.57 1.10 ± 0.31 1.03 ± 0.45

0.56 ± 0.12 0.75 ± 0.36 0.84 ± 0.36

0.48 ± 0.15 0.83 ± 0.16 0.80 ± 0.31

0.33 ± 0.01⁎ 0.76 ± 0.22 0.93 ± 0.44

0.73 ± 0.09 1.06 ± 0.21 1.21 ± 0.38

0.58 ± 0.06 0.84 ± 0.25 1.69 ± 0.96

Kidney Na-K ATPase-α-1 ENaC-α

0.99 ± 0.06 1.73 ± 0.92

1.02 ± 0.16 1.00 ± 0.03

1.32 ± 0.16 1.44 ± 0.34

1.43 ± 0.47 1.56 ± 0.36

1.76 ± 0.37 1.97 ± 0.38

0.55 ± 0.41 1.19 ± 0.26

1.29 ± 0.28 1.04 ± 0.27

Data are means ± SEM of six animals per group. The significant differences between SHAM fa/fa rats and aortic constriction fa/fa rats vehicle control group given as #, p b 0.05 (‘t-test’) and significant differences between aortic constriction fa/fa rats was given as *, p b 0.05 (One way ANOVA followed by Dunnett's test).

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Fig. 7. Effect on gene expression of rosiglitazone and its combination with amiloride in aortic constricted Zucker fa/fa rats after 4 weeks of treatment. Relative mRNA expression of genes (A) angiotensin II receptor Type 1a and (B) PPAR-α in heart (C) Endothelin-1 in aorta and (D) ENaC-α and (E) Na+-K+-ATPase-α-1 and in kidney. The results are normalized to Ribosomal Acidic Protein expression and presented as -fold change vs vehicle control (Mean ± SEM, n = 6). *p b 0.05 vs AC-fa/fa rat vehicle control by one-way ANOVA followed by Dunnett's test.

Four weeks of oral RSZ treatment caused statistically significant gain in body weights from 3 mg/kg dose onwards. The increase in body weight found in the present study was similar to the one reported by Pickavance et al. [24] and Sotiropoulos et al. [29]. RSZ has shown statistically significant effect on serum electrolytes like retention of sodium, chloride and loss of potassium, which resulted into significant increase in plasma volume at 3 and 30 mg/kg dose. Increase in plasma volume resulted in corresponding decrease in hematocrit levels. These finding are consistent with previous reports that PPARγ agonist causes body weight gain, induces sodium retention and potassium loss and increase in plasma volume and which infers in decrease of hematocrit [10,14,30, 31]. The adverse effects like body weight gain and plasma volume increase, which are closely (mechanistically) related to its anti-diabetic activity, were observed at 3 mg/kg dose. The general toxic effects like elevation of serum AST, ALT and urea levels were prominent at 30 mg/kg dose. These effects might be secondary to increased pressure overload caused due to aortic constriction and RSZ treatment at high dose, as reported by de-Hoyos et al. [32] and Alvarez and Mukherjee [33] who stated that increased AST and ALT levels may be secondary to CHF, hypertrophic cardiomyopathy or increased filling pressure and hepatic congestion. Therefore in the present study, RSZ might be aggravating the above conditions at high (30 mg/kg) dose. However, when the adverse and toxic effects of high (30 mg/kg) dose of RSZ

were compared with the group which received combination of RSZ 30 mg/kg and amiloride at 2 mg/kg dose, the retention of sodium and chloride and loss of potassium was completely reversed and there was 67% reversal of body weight gain caused by high dose of RSZ. As amiloride reduced the increased plasma volume, the preload caused by fluid retention and thereby the stress on heart might have reduced and this might have been reflected in reversal of increased AST and ALT levels in combination group. Corresponding changes were also observed in hematocrit, and hemoglobin levels (increased) in combination group. Results of the present study are in line with all literature reports which conclude that RSZ causes dose dependent decrease in mean systolic and diastolic blood pressures [17,29,30,34,35]. An echocardiography observation had shown that there was thickening of interventricular septum and there was non-significant increase in cardiac output found at all doses of RSZ treatment. At 3 mg/kg dose, where cardiac hypertrophy was not significant, ejection fraction was not affected and a higher increase in cardiac output was evident. Combination of RSZ and amiloride has shown to cause increase in ejection fraction and cardiac output which may be due to the reversal of left ventricular hypertrophy caused by amiloride that prevented the increase in plasma volume caused by RSZ treatment. This beneficial effect of combination therapy was also confirmed by a 67% reduction in plasma BNP levels.

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The evaluation of the mRNA expression for various genes in heart, aorta and kidney showed that aortic constricted Zucker fa/fa rats showed marked left ventricular hypertrophy with increased expression of AT-1R in heart and ET-1 in aorta along with increased plasma BNP and aldosterone levels demonstrating the hyper-activation of RAS [21]. RSZ treatment caused statistically non-significant but clear cut dose dependent decrease in AT-1R and PPARα mRNA levels in heart and significant decrease in ET-1 mRNA levels in aorta. It also caused statistically non-significant but clear cut dose dependent increase in ENaCα and Na\\K ATPase-α-1 mRNA levels in kidney. Down-regulation of AT1 receptor gene expression indicated that RSZ caused suppression of overtly activated RAS and down-regulation of ET-1 gene in aorta might be one of the possible mechanisms responsible for decrease in blood pressure caused by RSZ treatment [4,36]. In this study RSZ treatment showed downregulation of PPARα which might be due to pressure overload induced cardiac hypertrophy and reversal of fetal metabolic genotype in myocardium as report by Chen et al. [17] and Barger et al. [16]. But when RSZ was combined with amiloride which reduces the plasma volume expansion and ultimately pressure overload, there was 50% reversal of PPARα expression. Amiloride treatment also caused decrease in PPARγ expression. As reported by Chen et al. [17] increase in expression of PPARγ can lead to cardiac dysfunction, so combination of amiloride with RSZ proved to be beneficial as it increased the PPARα expression. In contrast to these beneficial effects, in kidney RSZ caused statistically non-significant but clear cut dose dependent upregulation of Na+-K+-ATPase-α-1 and ENaC-α gene which might be responsible for increased sodium, chloride retention and resultant increased plasma volume and hemodilution [37] these findings are consistent to as reported by Song et al. [30], Will et al. [38] and Vallon et al. [39]. The RSZ induced increased expression of ENaC-α was similar to as reported by Will et al. [38] but Vallon et al. [39] did not find any change in renal expression of the ENaC subunits. This study indicated that RSZ was causing the Na+ retention and fluid accumulation which was prevented when amiloride was administered with RSZ. 5. Conclusions In conclusion, the findings of present study suggest that the vasodilatory hemodynamic effects and improvement in glycemic control with RSZ are unlikely to have significantly influenced the effect of amiloride on attenuating RSZ-induced plasma volume expansion. However in RSZ and amiloride combination group, amiloride caused further beneficial effects by reversing the sodium retention. This resulted in reversal of the adverse effects caused due to increased plasma volume (edema and body weight gain) and RSZ and amiloride combination appeared promising in reducing the mortality caused by CHF in diabetic patients. Conflicts of interest statement Following authors, Suresh R Giri, Bibhuti Bhoi and Mukul R. Jain are employees of Cadila Healthcare Ltd., Ahmedabad, India, who has financially supported this work and has no conflict of interest. Dr. M.M. Gatne is a Professor of Pharmacology & Toxicology, Bombay Veterinary College, Parel, Mumbai, has no conflict of interest. Acknowledgements This work was financially supported by Cadila Healthcare Limited, Ahmedabad, India. The authors are thankful to Mr. Pankaj Patel, CMD, Cadila Healthcare Limited and Associate Dean, Bombay Veterinary College, Mumbai. The authors are also thankful to Dr. Vikas Karande and all staff at Dept. of Pharmacology and Toxicology, Bombay Veterinary College and Zydus Research Centre, Ahmedabad for supporting the work.

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