Protective effects of mineralocorticoid receptor

original article

Protective effects of mineralocorticoid receptor blockade against neuropathy in experimental diabetic rats H. Takata, Y. Takeda, A. Zhu, Y. Cheng, T. Yoneda, M. Demura, K. Yagi, S. Karashima & M. Yamagishi Division of Endocrinology and Hypertension, Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

Aims: Mineralocorticoid receptor (MR) blockade is an effective treatment for hypertension and diabetic nephropathy. There are no data on the effects of MR blockade on diabetic peripheral neuropathy (DPN). The aim of this study was to determine whether MRs are present in the peripheral nerves and to investigate the effectiveness of MR blockade on DPN in streptozotocin (STZ)-induced diabetic rats. Methods: Expression of MR protein and messenger RNA (mRNA) was examined in the peripheral nerves using Western blot analysis and RT-PCR. We next studied the effects of the selective MR antagonist eplerenone and the angiotensin II receptor blocker candesartan on motor and sensory nerve conduction velocity (NCV), morphometric changes and cyclooxygenase-2 (COX-2) gene and NF-κB protein expression in the peripheral nerves of STZ-induced diabetic rats. Results: Expression of MR protein and mRNA in peripheral nerves was equal to that in the kidney. Motor NCV was significantly improved by 8 weeks of treatment with either eplerenone (39.1 ± 1.2 m/s) or candesartan (46.4 ± 6.8 m/s) compared with control diabetic rats (33.7 ± 2.0 m/s) (p < 0.05). Sensory NCV was also improved by treatment with candesartan or eplerenone in diabetic rats. Eplerenone and candesartan caused significant improvement in mean myelin fibre area and mean myelin area compared with control diabetic rats (p < 0.05). COX-2 mRNA and NF-κB protein were significantly elevated in the peripheral nerves of diabetic rats compared with control rats, and treatment with eplerenone or candesartan reduced these changes in gene expression (p < 0.05). Conclusion: MR blockade may have neuroprotective effects on DPN. Keywords: aldosterone, angiotensin II, diabetic neuropathy, mineralocorticoid receptor Date submitted 8 July 2011; date of first decision 8 August 2011; date of final acceptance 20 September 2011

Introduction Blockade of the renin–angiotensin–aldosterone system (RAAS) is recognized as an effective treatment for hypertension as well as nephropathy and cardiovascular disease in patients with diabetes. There are several reports on the potential benefits of angiotensin converting enzyme (ACE) inhibitors and type 1 angiotensin II receptor (AT1R) blockers (ARBs) on diabetic neuropathy [1,2]. Coppey et al. [3] have showed that treatment with an ACE inhibitor or ARB attenuated diabetic neuropathy in streptozotocin (STZ)-induced diabetic rats. Two clinical studies in patients with diabetes showed that diabetic neuropathy was improved by treatment with ACE inhibitors [4,5]. However, the effectiveness of aldosterone blockade in diabetic neuropathy has not previously been investigated. Mineralocorticoid receptors(MRs) are present not only in the kidney and colon but also in the brain and cardiovascular tissue [6]. There are several reports that aldosterone acts directly on cardiovascular and renal tissues and can cause deleterious tissue injuries. We previously reported that cardiac fibrosis and hypertrophy induced by aldosterone infusion Correspondence to: Yoshiyu Takeda, Division of Endocrinology and Hypertension, Department of Internal Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan. E-mail: [email protected]

in rats could be partially prevented by treatment with an ARB [7]. A number of experimental models have shown that aldosterone may have a pathogenetic role in mediating renal injury [8,9]. Aldosterone acts directly on the central nervous system and has been shown to prevent glutamateinduced cell death in cultured neurons. This action is inhibited by the MR antagonist spironolactone, suggesting that the protective effect of aldosterone is mediated by activation of MRs [10]. Chronic treatment with spironolactone in strokeprone spontaneously hypertensive rats has beneficial effects on the outcome of acute ischaemic strokes, and these effects appear to be linked to improvements in vessel structure [11]. However, little is known about the effects of aldosterone on the peripheral nervous system. In human beings, aldosterone decreases baroreceptor activity, whereas treatment with MR blockers improves sympathetic nerve activity in patients with congestive heart failure [12]. Diabetic peripheral neuropathy (DPN) commonly complicates diabetes and is the leading cause of non-traumatic lowerlimb amputations and the resulting major impact on quality of life. The pathogenesis of DPN is complex. Several contributing factors, such as increased production of reactive oxygen species (ROS), non-enzymatic glycation, increased glucose flux through the polyol pathway, protein kinase C activation and neurovascular dysfunction have all been proposed. Pop-Busui

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Diabetes, Obesity and Metabolism 14: 155–162, 2012. © 2011 Blackwell Publishing Ltd

original article et al. [13] have reported a link between hyperglycaemia and cyclooxygenase (COX)-2 activation in the pathogenesis of experimental DPN and have showed protective effects of COX2 gene inactivation in DPN [14]. Aldosterone treatment in rats was shown to induce vascular inflammation in the heart, characterized by monocyte and macrophage infiltration and increased expression of the inflammatory marker COX-2 [15]. Resamen et al. [16] showed that aldosterone directly induced COX-2 expression in cardiomyocytes. The aim of this study was to determine whether MRs are present in the peripheral nerves and also to investigate the effects of MR blockade on nerve conduction velocity (NCV), morphometric changes and COX-2 expression in DPN in STZ-induced diabetic rats in comparison with angiotensin II blockade.

Research Design and Methods Animal Experiments All experiments were performed according to the guidelines for the use of experimental animals of the Animal Research Committee of Kanazawa University. Seven-week-old male Wistar rats weighing 200–220 g were divided randomly into four groups. Diabetes was induced in the rats by an intravenous injection of STZ at a dose of 40 mg/kg dissolved in 0.01 mol/l citric acid buffer at pH 4.5 as described previously [17,18]. Diabetes was verified 48 h later by measuring blood glucose levels using glucose-oxidase reagent strips (Bayer HealthCare, Tokyo, Japan) and blood glucose levels were subsequently measured once per week. The rats that were deprived of food for 8 h and with a fasting blood glucose level of 300 mg/dl (16.7 mmol/l) were considered to be diabetic and were used in the study. All rats had free access to water and chow during the study. Three experimental and one control group were studied for 8 weeks: STZ-induced diabetic rats (DM group, n = 8), STZ-induced diabetic rats receiving 0.2 mg/kg/day of candesartan administered intraperitoneally using an ALZET osmotic mini-pump [DURECT Corp., Cupertino, CA, USA (ARB group, n = 8)], and STZ-induced diabetic rats receiving eplerenone synthesized by Pharmacia/Pfizer (100 mg/kg/day), incorporated into the Teklad 22/5 rodent diet at a concentration of 1.0 mg/g of chow as reported previously [19] (Epl group, n = 8), and age-matched non-diabetic rats (Control group, n = 6) with or without eplerenone or candesartan treatment. Blood pressure was determined by a plethysmographic tail-cuff method as reported previously [7]. Urine protein was determined using a commercially available kit (Micro TP-Test Wako, WAKO, Osaka, Japan). Serum sodium and potassium were measured as described elsewhere. Plasma aldosterone concentrations (PAC) were determined by radioimmunoassay following extraction using a Sep-Pak C18 cartridge (Nihon Waters K.K, Tokyo, Japan) as previously reported [19]. Plasma renin activity (PRA) was measured using a commercial RIA kit. Haematocrit was measured using a haematocrit tube as described elsewhere.

Measurement of Nerve Conduction Velocity After 8 weeks, motor nerve conduction velocity (MNCV) was measured in a temperature-controlled environment under

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DIABETES, OBESITY AND METABOLISM

pentobarbital sodium anaesthesia (40 mg/kg) as described previously [20]. The left sciatic nerve was stimulated for 0.3 ms proximally at the sciatic notch and distally at the ankle using bipolar electrodes with supramaximal stimuli (6 mA) applied by a rectangular pulse from a stimulator at 10 Hz on a Neuropack 2 EMG (Nihon Kohden, Tokyo, Japan). The latencies of the compound muscle action potentials were recorded by bipolar electrodes from the first interosseous muscle of the hindpaw and measured from the stimulus artifact to the onset of the negative M-wave deflection. MNCV was calculated by subtracting the distal latency from the proximal latency, and the result was divided into the distance between the stimulating and recording electrodes. Sensory nerve conduction velocity (SNCV) in the tail was determined as described previously [21]. Recording needle electrodes were inserted into the tail 2 and 8 cm distal from the anus, whereas the stimulating electrodes were placed 12 cm distal from the anus. To determine latency, electrical stimulation was applied 50 times at a frequency of 1 Hz. The latency of the average summation of potentials recorded at the two sites was determined (peak-to-peak), and the NCV was calculated by dividing by the distance (6 cm).

Perfusion Experiments Rats from each group were used for experiments involving mesenteric arterial perfusion. After each rat was anaesthetized with pentobarbital, the superior mesenteric artery was immediately cannulated and perfused with Krebs-Ringer solution (pH 7.4) at 37 ◦ C and oxygenated with a 95% O2 –5% CO2 gas mixture at a constant flow rate of 3 ml/min. All connections of the mesenteric vascular bed to the small intestine were carefully dissected as previously reported [19]. Noradrenaline was added to a scaled reservoir that was continuously gassed with 95% O2 –5% CO2 and kept at 37 ◦ C, and from which Krebs solution was continuously perfused through the mesenteric vascular bed. Acetylcholine was injected at a volume of 10 μl into the perfusate in the silicone rubber close to the vascular bed. The perfusion pressure was constantly monitored and recorded by means of a pressure transducer connected to a polygraph (RM 600, Nihon-Koden, Tokyo, Japan).

Quantification of MR and COX-2 mRNA Before animals were killed, they were anaesthetized with pentobarbital (100 mg/kg IP), intubated and mechanically ventilated. The chest was opened by medial sternotomy, and the hearts, aortas, mesenteric arteries, kidneys and sciatic nerves were removed. Total RNA was extracted from each tissue using TRIzol (Invitrogen Japan, Tokyo, Japan) according to the manufacturer’s protocol. Real-time quantitative reverse transcription-PCR was carried out using the TaqMan One-Step RT-PCR Master Mix Reagent Kit with an ABI Prism 7000 HT Detection System (Applied Biosystems Japan, Tokyo, Japan) according to the manufacturers’ protocols. The sequences of the sense and antisense primers for MR were 5 -TGCATGATCTCGTGAGTGAC-3 and 5 -AAGTTCTTCCTGGCCGGTAT-3 , respectively. The sense and antisense primers for MR corresponded to nucleotides

Volume 14 No. 2 February 2012

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3256–3276 and 3425–3445, respectively, of the complementary DNA. The sequences of sense and antisense primers and probe for COX-2 were designed as reported previously [22]. To obtain a calibration curve, serial dilutions of stock standard RNA were used. The relative amount of each mRNA was normalized to the housekeeping gene 18S ribosomal RNA.

Western Blot Analysis Tissue extracts (20 mg protein) from kidney, sciatic nerve, aorta and mesenteric artery were separated by SDS-PAGE on 12% (w/v) polyacrylamide gels and electrotransferred to PVDF membranes using a Trans-blot unit (Bio-Rad Laboratories, Tokyo, Japan) for 1 h at 100 V. The membranes were blocked overnight at 4 ◦ C with 5% (w/v) skimmed milk in TBS (pH 7.4) containing 0.1% (v/v) Tween 20 (PBST) and then incubated for 1 h with primary antibodies at 24 ◦ C. The antibodies used were as follows: anti-MR (sc-11412, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal MR antibody (rMR1-18, a generous gift from Dr Gomez-Sanchez) [23], anti-NF-κB (Cell Signaling Technology Japan, Tokyo, Japan) [24] and anti-βactin (A5441, Sigma Japan, Tokyo, Japan). After incubation with secondary antibodies, the signals on the Western blots were revealed with chemiluminescence, visualized by autoradiography and then quantified by densitometry, followed by correction for β-actin.

Morphometric Analysis Sciatic nerve specimens were obtained from one rat in each group as described elsewhere [20]. Segments of sciatic nerves 5 mm in length were excised and fixed immediately in 2% glutaraldehyde in PBS (pH 7.4). Transverse semithin sections (1 μm thick) were prepared and stained with haematoxylin–eosin for light microscopy. Approximately 200 fibres per animal were randomly selected and analysed using the Image J analysis. Mean myelinated nerve fibre area and myelin area were measured as described previously [25]. Myelinated fibres without axons, redundant

myelin, and fibres with sheaths that were too thick relative to their axonal diameter were considered abnormal fibres and were excluded from this study.

Statistical Analysis Data are expressed as means ± standard error of the mean. Data were compared by two-way anova or Friedman’s test and Fisher’s protected least significance or Scheffe’s F tests were performed when an anova indicated significance. A p value < 0.05 was considered statistically significant.

Results Expression of MR was showed by Western blot analysis of protein extracted from the kidney, peripheral nerves, aorta and mesenteric arteries (figure 1). We confirmed by microscopic examination that no small arteries were present on the peripheral nerves. MR protein was clearly detected in peripheral nerves, suggesting that MR is found not only in the cardiovascular and renal tissue but also in the peripheral nervous system. The same MR band could also be detected using an MR monoclonal antibody (data not shown). Expression of MR mRNA was nearly equal in the kidney and in peripheral nerves (figure 1). Figure 2 shows the morphological changes in each experimental rat peripheral nerve. Fibre size and myelin content were analysed using the Image J analysis (figure S1). Fibre sizes were found to be smaller in the DM group than in controls. The DM group also had a smaller myelin–fibre ratio than the control group. The decreased fibre and myelin content in diabetic rats was improved significantly by treatment with either eplerenone or candesartan (Table 1). Eplerenone and candesartan did not influence body weight (396 ± 10 g, 387 ± 8 g, respectively), plasma glucose concentration (129 ± 7 mg/dl, 118 ± 4 mg/dl, respectively) or systolic blood pressure (112 ± 5 mmHg, 98 ± 6 mmHg, respectively) in control rats.

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Figure 1. The expression levels of protein (right panel) and messenger RNA of mineralocorticoid receptor (MR) (left panel) in rat kidney (K), sciatic nerve (SN), aorta (Ao) and mesenteric arteries (MA). Four samples of each tissue were used. *p < 0.05 versus kidney.


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Control rats

Diabetic rats

Diabetic rats treated with candesartan

Diabetic rats treated with eplerenone

Figure 2. Histology of the sciatic nerve of each experimental rat by staining with haematoxylin–eosin (×400). Table 1. Body weight (BW), plasma glucose concentrations (PG), systolic blood pressure (BP) and light microscopic morphometric data in each experimental rats. Group

BW (g)

PG (mg/dl)

BP (mmHg)

PAC (pg/ml)

PRA (ng/ml/h)

MFA (μm2 )

MMA (μm2 )

Control (n = 6) Diabetic rats (n = 8) Cande (n = 8) Epl (n = 8)

411 ± 9 255 ± 20∗ 295 ± 6∗ 268 ± 17∗

149 ± 7 501 ± 24∗ 447 ± 16∗ 529 ± 8∗

107 ± 3 119 ± 6 121 ± 4 123 ± 2

188 ± 39 175 ± 25 205 ± 27 1237 ± 130#

16 ± 4.6 3.7 ± 0.8∗∗∗ 17 ± 7.9∗∗ 11 ± 1.4∗∗

38.4 ± 2.4 31.2 ± 1.4∗ 36.9 ± 1.5∗∗ 35.8 ± 1.4∗∗

23.8 ± 1.2 17.3 ± 0.6∗ 22.1 ± 0.6∗∗ 21.0 ± 0.6∗∗

Data are mean ± s.e. Cande, STZ-induced diabetic rats treated with candesartan; Epl, STZ-induced diabetic rats treated with eplerenone; PAC, plasma aldosterone concentration; U-prot, urinary protein excretion; MFA, mean fibre area; MMA, mean myelin area. ∗ p < 0.05 versus control rats, ∗∗ p < 0.05 versus diabetic rats, ∗∗∗ p < 0.01 versus control rats, #p < 0.01 versus control, diabetic rats and cande.

Table 1 shows the summary of data on final weight and non-fasting blood glucose levels and light microscopic morphometric data in rats from all four study groups. The body weights of the diabetic rats in the three experimental groups were markedly lower than those of control rats. Treatment with eplerenone or candesartan did not affect blood glucose levels or blood pressure in the STZ-induced diabetic rats. PAC did not differ between control and diabetic rats; however, eplerenone treatment significantly increased PAC in diabetic rats (p < 0.01). PRA was significantly lower in diabetic rats than in controls (p < 0.01). Treatment with candesartan or eplerenone significantly increased PRA. Urinary protein excretion was significantly higher in diabetic rats (28 ± 2.3 mg/day) than in control rats (12 ± 1.8 mg/day) (p < 0.05). Treatment with candesartan or eplerenone mitigated urinary

158 Takata et al.

protein excretion (15 ± 3.0, 12 ± 3.6 mg/day, respectively) in diabetic rats. Serum sodium levels did not differ among experimental groups (data not shown). Serum potassium levels were significantly higher in diabetic rats treated with eplerenone (5.1 ± 0.2 mmol/l) compared with control rats (4.4 ± 0.1 mmol/l), diabetic rats (4.6 ± 0.1 mmol/l) or diabetic rats treated with candesartan (4.7 ± 0.2 mmol/l) (p < 0.05). Haematocrit did not significantly differ between control rats (38 ± 0.3%), diabetic rats (40 ± 0.7%), diabetic rats treated with candesartan (38 ± 0.3%) and diabetic rats treated with eplerenone (39 ± 0.5%). Acetylcholine-induced relaxation was blunted in diabetic rats (ED50, 18 ± 3.4 × 10−8 M) compared with control rats (ED50, 1.9 ± 0.3 × 10−8 M) (figure 3). Treatment with eplerenone (ED50, 5.5 ± 0.7 × 10−8 M) or candesartan (ED50,


original article


p < 0.05

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Figure 3. Endothelium-dependent relaxation to Ach in the mesenteric arteries of eplerenone- and candesartan-treated STZ-induced diabetic rats, diabetic rats and control rats. Vascular relaxation to Ach was significantly attenuated in diabetic rats compared to control rats. Eplerenone or candesartan improved vascular relaxation to Ach. *p < 0.05 versus diabetic rats; **p < 0.01 versus diabetic rats.

7.0 ± 1.6 × 10−8 M) for 8 weeks improved endotheliumdependent relaxation, and no significant difference was seen between these two treatment groups. MNCV and SNCV data from each experimental group are shown in figure 4. MNCV and SNCV were significantly decreased in DM rats (33.7 ± 2.0, 21.1 ± 0.7 m/s, respectively) compared with control rats (55.7 ± 1.7, 28.6 ± 0.9 m/s, respectively) (p < 0.05, p < 0.01, respectively). Treatment with candesartan (46.8 ± 1.8, 25.3 ± 08 m/s, respectively) or eplerenone (39.1 ± 1.2, 24.8 ± 0.6 m/s, respectively) somewhat improved MNCV in diabetic rats (p < 0.05). Figure 5 shows COX-2 mRNA levels and NF-κB protein levels in each experimental group. COX-2 mRNA in peripheral nerves was significantly higher in DM rats than in controls (p < 0.05). Treatment with eplerenone or candesartan significantly decreased expression of COX-2 mRNA in DM rats. Densitometry of NF-κB p65 bands confirmed a significant increase in NF-κB p65 protein levels in nerves from DM rats compared with controls (p < 0.01). Treatment with candesartan or eplerenone reduced the expression of NF-κB p65 (p < 0.05).

Discussion There is increasing evidence that aldosterone acts directly on cardiomyocytes, vascular smooth muscle cells, renal mesangial cells and astrocytes, all of which contain MRs [26]. Miyata et al. [27] reported that aldosterone stimulated the production of ROS through activation of NADPH oxidase in rat mesangial cells. Oyamada et al. [28] showed that MR expression was increased in astrocytes migrating into the ischaemic striatum. Treatment with an MR blocker reduced ROS production and apoptosis in the ischaemic striatum and caused a small but significant reduction in the area of the brain damaged by ischaemia. Girard et al. [29] recently reported that MR and glucocorticoid receptor are expressed in the sciatic nerve. We detected MR mRNA and protein in peripheral nervous tissue at levels almost equal to those found in the


0 C

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Cande Epl

C

(n = 6) (n = 8) (n = 8) (n = 8)

DM

Cande Epl

(n = 6) (n = 8) (n = 8) (n = 8)

Figure 4. The motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) of sciatic–tibial nerves in each experimental group. Cande, candesartan; Epl, eolerenone. MNCV and SNCV were significantly decreased in the diabetic rats compared to control rats (p < 0.05). Treatment with candesartan or eplerenone partially improved MNCV and SNCV compared to measurements in diabetic rats (p < 0.05).

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NF-κB β-Actin

Figure 5. Concentrations of mRNA of COX-2 and protein levels of NF-κB (p65) in the sciatic nerves in each experimental group. Cande, candesartan; Epl, eolerenone. The levels of COX-2 mRNA and NF-κB (p65) protein expression in the peripheral nerve significantly increased in diabetic rats compared to control rats (p < 0.05). Treatment with eplerenone or candesartan significantly decreased the expression of COX-2 mRNA and NF-κB (p65) protein compared to diabetic rats (p < 0.05).

kidney and found that both the MR blocker eplerenone and the ARB candesartan improved electrophysiological and histopathological parameters in this model of diabetic neuropathy, independent of changes in either blood pressure or blood glucose. Cameron and colleagues [2] previously showed that treatment of STZ-induced diabetic rats with an ARB improved nerve function and nerve blood flow. Coppey et al. [3] showed that treatment with an ARB prevented diabetes-induced impairment in vascular relaxation and reduced superoxide levels in the blood vessels. Endothelial dysfunction and oxidative stress play a key role in the pathogenesis of diabetic neuropathy. Schiffrin and coworkers found an increase in the markers of oxidative stress, such as thiobarbituric acid-reactive substances (TBARs) and 8-iso-prostanes in the blood of rats with aldosterone-induced hypertension [30]. Both angiotensin II and aldosterone promote oxidative

doi:10.1111/j.1463-1326.2011.01499.x 159

original article stress through NADPH oxidase activation in cardiovascular tissues [31]. Increased oxidative stress through induction of nuclear factor (NF)-κB and nerve hypoxia may impair nerve perfusion by increasing COX-2 activity and inhibiting prostacyclin synthase activity. Coppey et al. [32] reported that antioxidant treatment of STZ-induced diabetic rats improved MNCV and vascular reactivity of epineurial arteries of the sciatic nerve. Previously, we reported that aldosterone blockade as well as angiotensin II blockade improved both vascular endothelial function and mitigated cardiovascular and renal injury in hypertensive rats [19,33]. In this study, treatment with eplerenone improved diabetic neuropathy as well as vascular endothelial function in diabetic rats. The preventative effects of eplerenone and candesartan treatment against diabetic neuropathy seen in this study may therefore be due in part to a reduction in arterial endothelial dysfunction. In our experiments, diabetic rats showed lower PRA compared to control rats. The beneficial effects of ACE inhibitor, ARBs and aldosteorne blocker in the prevention of diabetic complications suggest that angiotensin II and aldosterone are major mediators of progressive tissue injuries. However, measurement of the activity of components of RAAS has largely indicated suppression in diabetes mellitus [34]. These observations suggest local tissue activation of RAAS in diabetes mellitus and hypertension [6,35]. In this study, STZ-induced diabetic rats did not show hypertension, and treatment with eplerenone or candesartan did not influence blood pressure. The blood pressure changes seen in STZ-induced diabetic rodent models are usually mild [36]. Recently Bidani et al. reported a spontaneously reduced ambient blood pressure load as indicated by chronic blood pressure radiotelemetry [37]. Sugiyama et al. [38] reported that candesartan decreased blood pressure in STZinduced diabetic rats after 25 weeks of treatment but not after 15 weeks. The hypotensive effect of candesartan in STZinduced diabetic rats may depend on blood pressure levels or duration of treatment. Treatment with candesartan did not decrease PAC in the diabetic rats. The reason is unclear, however, aldosterone breakthrough phenomenon caused by chronic blockade of angiotensin II type 1 receptor is reported in diabetic patients and hypertensive rats [39,40]. Treatment with eplerenone improved urinary protein excretion and elevated serum potassium levels in STZ-induced diabetic rats independent of blood pressure. These data suggest that MR controls electrolytes in the kidney and contributes to the pathophysiology of diabetic nephropathy. Various lines of evidence have elucidated the direct effects of aldosterone on vascular and renal cells. Previously, we reported that aldosterone is involved in vascular muscle hypertrophy [41]. Rocha et al. [42] showed that aldosterone/salt treatment induced coronary inflammation characterized by monocyte and macrophage infiltration and by increased expression of COX-2 and other inflammatory markers. Increased COX-2 expression and activity has been described in several models of diabetic renal disease and DPN. However, few animal or human studies have focused on the relationship between aldosterone and the peripheral nervous system. In our experiment, COX-2 mRNA and NF-κB

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protein were increased in the peripheral nerves of diabetic rats, and treatment with an MR blocker or ARB decreased expression. Kellogg et al. [43] showed that diabetic COX-2 knockout mice were protected against the functional and biochemical effects of experimental DPN and against nerve fibre loss. Queisser et al. [44] reported that aldosterone treatment led to a dose-dependent induction of oxidative stress and activation of NF-κB in renal cells and that treatment with eplerenone improved oxidative stress. NF-κB is a pleiotropic transcription factor that is involved in the regulation of genes encoding a diverse group of enzymes, including an inducible nitric oxide synthase, as well as COX-2, and proinflammatory cytokines. An upcoming report provides support for the role of the NF-κB inflammatory cascade in diabetogenesis and in the pathophysiology of DPN [45]. Taken together, our data suggest that MR activity may be partly responsible for COX-2 induction and NF-κB activation in DPN. NCV was partially restored by treatment with candesartan or eplerenone in our study. The decreased fibre and myelin sizes in diabetic rats were also significantly improved by treatment with either eplerenone or candesartan. Tuncer et al. [46] recently reported that demyelination and intraaxonal degeneration with neurofilament depletion were apparent in light photomicrographs of cross sections of sciatic nerves from STZ-diabetic rats at 4 weeks after STZ treatment. Multiple mechanisms are reported to contribute to DPN. Leonelli et al. [47] reported that chronic treatment with progesterone and its derivatives counteracted the impairment of NCV in STZ-induced diabetic rats. Recently, Kakoki et al. [48] reported that a lack of bradykinin B1 and B2 receptors exacerbated diabetic neuropathy in Akita diabetic mice. Currently, the prevention and early management of human diabetic neuropathy is based on achieving better glycaemic control. However, attainment of optimum glycaemic control is often difficult. Blockade of the RAAS has been shown to be the most effective therapeutic intervention for postponing the progression of diabetic angiopathy [49]; however, the effects of blockade of RAAS on diabetic neuropathy are not well understood. ACE inhibitors have been shown to improve diabetic neuropathy in small clinical trials [4,5], and there is also evidence that both ACE inhibitors and ARBs are effective against diabetic neuropathy in STZ-induced diabetic rats. However, no clinical trial has shown the effectiveness of ARB on diabetic neuropathy. The present study in rats showed that treatment with either an MR blocker or an ARB was effective in the attenuation of diabetic neuropathy. We have previously reported that combination therapy with eplerenone and candesartan is more effective for lowering blood pressure and improvement of vascular endothelial function in saltsensitive hypertensive rats than monotherapy [17]. However, another previous clinical study reported a possible deleterious effect of MR blockade in type 2 diabetic patients [50]. Clinical trials are needed to assess the effectiveness of MR blockers and ARBs for the treatment of diabetic neuropathy. In conclusion, the present study showed that MRs are expressed not only in blood vessels but also in peripheral nerves. MR blockade may thus represent an effective treatment for diabetic neuropathy.



Acknowledgement This research was supported by a grant from the Japanese Ministry of Health, Labor and Welfare.

Conflict of Interest Y. T. designed the study. H. T., A. Z., Y. C., T. Y., M. D., K. Y. and S. K. conducted data collection. Y. T. and M. Y. carried out analysis. H. T. and Y. T. wrote the article. The authors do not have any conflict of interest relevant to this manuscript.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Morphologic changes of the sciatic nerve of each experimental rat were analysed using the Image J analysis. Image J converts images to black and white. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References 1. Cameron NE, Copper MA, Robertoson S. Angiotensin converting enzyme inhibition prevents development of muscle and nerve dysfunction and stimulates angiogenesis in streptozotocin-diabetic rats. Diabetologia 1992; 35: 12–18. 2. Maxfield EK, Cameron NE, Cotter MA, Dines KC. Angiotensin II receptor blockade improves nerve function, modulates nerve blood flow and stimulates endoneurial angiogenesis in streptozotocin-diabetic rats. Diabetologia 1993; 36: 1230–1237. 3. Coppey LJ, Davidson EP, Rinehart TW et al. ACE inhibitor or angiotensin II receptor antagonist attenuates diabetic neuropathy in streptozotocininduced diabetic rats. Diabetes 2006; 55: 341–348. 4. Malik RA, Williamson S, Abbott C et al. Effect of angiotensin-convertingenzyme (ACE) inhibitor trandolapril on human diabetic neuropathy: randomized double-blind controlled trial. Lancet 1998; 352: 1978–1981. 5. Reja A, Tesfaye S, Harris ND, Ward JD. Is ACE inhibition with lisinopril helpful in diabetic neuropathy? Diabet Med 1995; 12: 307–309. 6. Takeda Y. Pleiotropic actions of aldosterone and the effects of eplerenone, a selective mineralocorticoid receptor antagonist. Hypertens Res 2004; 27: 781–789. 7. Takeda Y, Yoneda T, Demura M, Usukura M, Mabuchi H. Calcineurin inhibition attenuates mineralocortiocid-induced cardiac hypertrophy. Circulation 2002; 105: 677–679. 8. Rocha R, Chander PN, Zuckerman A, Stier CT, Jr. Role of aldosterone in renal vascular injury in stroke-prone hypertensive rats. Hypertension 1999; 33: 232–237. 9. Epstein M. Aldosterone as a mediator of progressive renal disease: pathogenetic and clinical implications. Am J kidney Dis 2001; 37: 677–688. 10. McCullers DL, Herman JP. Adrenocorticosteroid receptor blockade and excitotoxic challenge regulate adrenocorticosteroid receptor mRNA levels in hippocampus. J Neurosci Res 2001; 64: 277–283. 11. Rigsby CS, Pollock DM, Dorrance AM. Spironolactone improves structure and increases tone in the cerebral vasculature of male spontaneously hypertensive stroke-prone rats. Microvasc Res 2007; 73: 198–205.


original article 12. Kasama S, Toyama T, Kumakura H et al. Effect of spironolactone on cardiac sympathetic nerve activity and left ventricular remodeling in patients with dilated cardiomyopathy. J Am Coll Cardiol 2003; 41: 574–581. 13. Pop-Busui R, Sima A, Stevens M. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 2006; 22: 257–273. 14. Pop-Busui R, Marinescu V, Huysen CV et al. Dissection of metabolic, vascular, and nerve conduction interrelationships in experimental diabetic neuropathy by cyclooxygenase inhibition and acetyl-L-carnitine administration. Diabetes 2002; 51: 2619–2628. 15. Rocha R, Rudolph AE, Frierdich GE et al. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol Heart Circ Physiol 2002; 283: H1802–H1810. 16. Resamen MC, Perrier E, Gerber-Wicht C, Benitah J-P, Lang U. Direct and indirect effects of aldosterone on cyclooxygenase-2 and interluekin-6 expression in rat cardiac cells in culture and after myocardial infarction. Endocrinology 2004; 145: 3135–3142. 17. Takeda Y, Miyamori I, Yoneda Takeda R. Production of endothelin-1 from the mesenteric arteries of streptozotocin-induced diabetic rats. Life Sci 1991; 48: 2553–2556. 18. Cotter MA, Gibson TM, Nagle MR, Cameron NE. Effects of interleukin6 treatment on neurovascular function, nerve perfusion and vascular endothelium in diabetic rats. Diabetes Obes Metab 2010; 12: 689–699. 19. Takeda Y, Zhu A, Yoneda T, Usukura M, Takata H, Yamagishi M. Effects of aldosterone and angiotensin II receptor blockade on cardiac angiotensinogen and angiotensin-converting enzyme 2 expression in experimental salt-sensitive hypertension. Am J Hypertens 2007; 20: 1119–1124. 20. Coppey LJ, Davidson EP, Dunlap JA, Lund DD, Yorek MA. Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve. Int J Exp Diabetes Res 2000; 1: 131–143. 21. Kita T, Kawamata T, Namiki A, Yamakage M. Role of satellite cell-derived L-serine in the dorsal root ganglion in paclitaxel-induced painful peripheral neuropathy. Neuroscience 2011; 174: 190–199. 22. Wiingaarden P, Brereton HM, Gibbins IL, Coster DJ, Williams KA. Kinetics of strain-dependent differential gene expression in oxygen-induced retinopathy in the rat. Exp Eye Res 2007; 85: 508–517. 23. Gomez-Sanchez CE, de Rodriguez AF, Romero DG et al. Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 2006; 147: 1343–1348. 24. Kumar A, Sharma SS. NF-kB inhibitory action of resveratol: a probable mechanism of neuroprotection in experimental neuropathy. Biochem Biophys Res Commun 2010; 394: 360–365. 25. Yagihashi S, Kamijo M, Baba M, Yagihashi N, Nagai K. Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes 1992; 41: 47–52. 26. Takeda Y. Vascular synthesis of aldosterone: role in hypertension. Mol Cel Endocrinol 2004; 217: 75–79. 27. Miyata K, Rahman M, Shokoji T et al. Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells. J Am Soc Nephrol 2005; 16: 2906–2912. 28. Oyamada N, Sone M, Miyashita K et al. The role of mineralocorticoid receptor expression in brain remodeling after cerebral ischemia. Endocrinology 2008; 149: 3764–3777. 29. Girard C, Eychenne B, Schweizer-Groyer G, Cadepond F. Mineralocorticoid and glucocorticoid receptors in sciatic nerve function and regeneration. J Steroid Biochem Mol Biol 2010; 122: 149–158. 30. Touyz RM, Schiffrin EL. Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol 2004; 122: 339–352. 31. Funder JW. Minireview aldosterone and the cardiovascular system: genomic and nongenomic effects. Endocrinology 2006; 147: 5564–5567.

doi:10.1111/j.1463-1326.2011.01499.x 161

original article 32. Coppey LJ, Gellett JS, Davidson EP et al. Effect of antioxidant treatment of streptozotocin-induced diabetic rats on endoneurial blood flow, motor nerve conduction velocity, and vascular reactivity of epineurial arterioles of the sciatic nerve. Diabetes 2001; 50: 1927–1937. 33. Zhu A, Yoneda T, Demura M et al. Effect of mineralocorticoid receptor blockade on the renal renin–angiotensin system in Dahl salt-sensitive hypertensive rats. J Hypertension 2009; 27: 800–805. 34. Carey RM, Siragy HM. The intrarenal renin–angiotensin system and diabetic nephropathy. Trends Endocrinol Metab 2003; 14: 274–281. 35. Hanes DS, Nahar A, Weir MR. The tissue renin–angiotensin–aldosterone system in diabetes mellitus. Curr Hypertens Rep 2004; 6: 98–105. 36. Tesch GH, Allen TJ. Rodent models of streptozotocin-induced diabetic nephropathy. Nephrology 2007; 12: 261–266. 37. Bidani AK, Picken M, Hacioglu R, Williamson G, Griffin KA. Spontaneously reduced blood pressure load in the rat streptozotocin-induced diabetes model: potential pathogenetic relevance. Am J Physiol Renal Physiol 2007; 292: F647–F654. 38. Sugiyama T, Okuno T, Fukuhara M et al. Angiotensin II receptor blocker inhibits abnormal accumulation of advanced glycation end products and retinal damage in a rat model of type 2 diabetes. Exp Eye Res 2007; 85: 406–412. 39. Yoneda T, Takeda Y, Usukura M et al. Aldosterone breakthrough during angiotensin II receptor blockade in hypertensive patients with diabetes mellitus. Am J Hypertens 2007; 20: 1329–1333. 40. Naruse M, Tanabe A, Sato A et al. Aldosterone breakthrough during angiotensin II receptor antagonist therapy in stroke-prone spontaneously hypertensive rats. Hypertension 2002; 40: 28–33. 41. Hatakeyama H, Miyarori I, Fujita T et al. Vascular aldosterone: biosynthesis and a link to angiotensin-induced hypertrophy of vascular smooth muscle cells. J Biol Chem 1994; 269: 24316–24320.

162 Takata et al.


42. Rocha R, Martin-Berger CL, Yang P et al. Selective aldosterone blockade prevents angiotensin II/salt-induced vascular inflammation in the rat heart. Endocrinology 2002; 143: 4828–4836. 43. Kellogg AP, Wiggin TD, Larkin DD, Hayes JM, Stevens MJ, Pop-Busi B. Protective effects of cyclooxygenase-2 gene inactivation against peripheral nerve dysfunction and intraepidermal nerve fiber loss in experimental diabetes. Diabetes 2007; 56: 2997–3005. 44. Queisser N, Oteiza PI, Stopper H et al. Aldosterone induces oxidative stress, osidative DNA damage, and NF-kB-activation in kidney tubule cells. Mol Carcinog 2011; 50: 123–135. 45. Kumer A, Negi G, Sharma SS. JSH-23 targets nuclear factor-kappa B and reverses various deficits in experimental diabetic neuropathy: effect on neuroinflammation and antioxidant defence. Diabetes Obes Metab 2011; 13: 750–758. 46. Tuncer S, Dalkilic N, Esen HH et al. An early diagnostic tool for diabetic neuropathy: conduction velocity distribution. Muscle Nerve 2011; 43: 237–244. 47. Leonelli E, Bianchi R, Cavaletti G et al. Progesterone and its derivatives are neuroprotective agents in experimental neuropathy: a multimodel analysis. Neuroscience 2007; 144: 1293–1304. 48. Kakori M, Sullivan KA, Backus C et al. Lack of both bradykinin B1 and B2 receptors enhances nephropathy, neuropathy, and bone mineral loss in Akita diabetic mice. Proc Natl Acad Sci U S A 2010; 107: 10190–10195. 49. Epstein M. Aldosterone blockade: an emerging strategy for abrogating progressive renal disease. Am J Med 2006; 119: 912–919. 50. Swaminathan K, Davies J, George J, Rajendra NS, Morris AD, Struthers AD. Spironolactone for poorly controlled hypertension in type 2 diabetes: conflicting effects on blood pressure, endothelial function, glycaemic control and hormonal profiles. Diabetologia 2008; 51: 762–768.
