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NDT Advance Access originally published online on February 26, 2007
Nephrology Dialysis Transplantation 2007 22(5):1314-1322; doi:10.1093/ndt/gfl780
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dual blockade of aldosterone and angiotensin II additively suppresses TGF-ß and NADPH oxidase in the hypertensive kidney

Maristela Lika Onozato1, Akihiro Tojo1, Naohiko Kobayashi2, Atsuo Goto1, Hiroaki Matsuoka2 and Toshiro Fujita1

1Division of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan and 2Division of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Tochigi, Japan

Correspondence and offprint requests to: Akihiro Tojo, MD, Division of Nephrology and Endocrinology, Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Email: tojyo-2im{at}h.u-tokyo.ac.jp



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Angiotensin II blockade and spironolactone effectively reduces proteinuria in humans. To clarify the mechanisms of the beneficial effect of blockade of both aldosterone and angiotensin II, we associated the aldosterone antagonist eplerenone to an angiotensin-converting enzyme inhibitor (ACEI) and examined the effect on renal transforming growth factor (TGF)-ß expression and oxidative stress by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the Dahl salt-sensitive rat with heart failure (DSHF).

Methods. Dahl salt-resistant control rats and DSHF rats were fed with 8% NaCl diet and at 11 weeks the DSHF rats were treated with vehicle, eplerenone (Epl), trandolapril or a combination of both drugs for 7 weeks.

Results. DSHF rats showed increased NADPH oxidase and decreased superoxide dismutase (SOD) resulting in increased oxidative stress. ACEI and Epl reduced NADPH oxidase showing an additive effect in their combination; ACEI increased manganese SOD (MnSOD) and Epl increased MnSOD, copper–zinc SOD and catalase, resulting in the lowest levels of oxidative stress with the combination therapy. Glomerulosclerosis and proteinuria were increased in the DSHF rats, and Epl suppressed them more effectively than ACEI to levels not different from the combination of both, showing a positive correlation with NADPH oxidase expression and TGF-ß. Renal TGF-ß was specifically suppressed with Epl

Conclusion. The association of Epl to ACEI is beneficial due to further reduction of NADPH oxidase and specific inhibition of TGF-ß resulting in improvement of renal damage.

Keywords: ACE inhibitor; eplerenone; hypertension; NADPH oxidase; oxidative stress, TGF-ß



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
The activation of the renin–angiotensin–aldosterone system (RAAS) is a crucial factor in the development and progression of organ damage in hypertension, diabetes and chronic kidney diseases [1–3]. We have documented that hypertensive renal damage is linked to the increased renal angiotensin II that induced the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and radical production, and that angiotensin-converting enzyme inhibitor (ACEI) suppressed renal angiotensin II and NADPH oxidase-derived radical production [4–6]. In diabetic nephropathy, the blockade of angiotensin II with ACEI or angiotensin II receptor blocker (ARB) ameliorated organ damage by the down-regulation of NADPH oxidase in spite of the suppressed systemic renin-angiotensin-aldosterone system [7–9]. These lines of evidence indicated the importance of the blockade of the local angiotensin II with an ACEI or an ARB for renoprotection. However, a complete protection has not been achieved, suggesting that another mechanism other than local angiotensin II may be acting.

The classical view of aldosterone action is to keep sodium balance by reabsorption of sodium via activation of the apical epithelial sodium channel and basal Na+,K+-ATPase in the distal tubules. The increased sodium reabsorption by aldosterone elevates blood pressure and could increase reactive oxygen species (ROS) production, causing renal damage [10,11]. Recently, non-classical actions of aldosterone other than sodium balance have been considered. The mineralocorticoid receptor has been found in non-epithelial cells in brain, vessels, heart and glomerulus [12–15] and they could induce inflammation, tissue remodelling and fibrosis [16,17]. The importance of aldosterone on end-organ damage has been recognized in the heart and vasculature [18]. In the kidney, aldosterone increases reactive oxygen species [19,20] and also up-regulates transforming growth factor-ß (TGF-ß) that increases fibronectin and renal fibrosis. The administration of antioxidant was shown to decrease TGF-ß expression and renal damage in the Dahl salt-sensitive model [21] as well as in an aldosterone infusion model [22,23]. However, it has not been elucidated if aldosterone blockade is more beneficial in the hypertensive kidney than angiotensin II blockade with an ACEI on NADPH oxidase and TGF-ß expression and glomerular damage. Moreover, the chronic administration of ACEI can lead to aldosterone increase, a phenomenon called ‘aldosterone escape’ [24–27] and the increased aldosterone can stimulate the mineralocorticoid receptor and unleash the events that cause organ damage even in the presence of the ACEI.

Therefore, we investigated whether combination therapy with the mineralocorticoid receptor blocker eplerenone and ACEI could have additive effects on renal NADPH oxidase and oxidative stress and TGF-ß expression and could be responsible for the improvement in renal damage.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Animal preparation
Male, inbred Dahl salt-sensitive rats (DS, n = 40, Eisai Co., Ltd, Tokyo, Japan) and age-matched Dahl salt-resistant rats (Control, n = 10) were weaned and fed a diet containing 0.3% NaCl until the age of 6 weeks. Thereafter, they were fed a diet containing 8% NaCl until the age of 18 weeks. At age 11 weeks, concentric left ventricular hypertrophy develops, with increase in left ventricular weight and end-diastolic diameter and reduction of fractional shortening evolving to left ventricular failure with chamber dilatation at 18 weeks [28]. On the 11th week, DS rats were randomly divided in four groups: (i) rats vehicle-treated [Dahl salt-sensitive rat with heart failure (DSHF), n = 10]; (ii) rats trandolapril-treated (0.3 mg/kg/day in the drinking water, Tanabe Pharmaceutical Co., Tokyo, Japan, DSHF + ACEI, n = 10); (iii) rats eplerenone (Epl) treated (30 mg/kg/day in the 8% NaCl containing diet, DSHF + Epl, n = 10) and (iv) rats given a combination of trandolapril and Epl (DSHF + ACEI + Epl, n = 10) and treated until the age of 18 weeks. Trandolapril dose was chosen according to previous reports in rats [29,30] and the use of equivalent doses in clinical practice. Epl dose and route of administration were chosen following our previous study [31].

All procedures were conducted in accordance with our institutional guidelines for animal research and with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.

Urinary assay of albumin, creatinine, lipid peroxidation products and renal Na+,K+-ATPase
The systolic blood pressure (SBP) was measured by tail-cuff method at the start of 8% NaCl diet and at 1-week interval thereafter. The 24 h urine samples were collected from rats in metabolic cages five weeks after the start of treatment. Creatinine and potassium were analysed by standard methods. Urinary albumin was measured by the enzyme-linked immunosorbent assay kit (Panaform Laboratory, Kumamoto, Japan).

The lipid peroxidation products (LPO) in the urine were measured by the thiobarbituric acid method. After precipitation of proteins, 100 µl of urine was incubated with 100 µl of 4% sodium dodecyl sulphate (SDS), 400 µl of 20% acetic acid at pH 3.5 and 400 µl of 0.8% 2-thiobarbituric acid (TBA, Wako Pure Chemical Industries Ltd., Osaka, Japan) for 60 min at 95°C. The malondialdehyde (MDA) formation was measured by spectrofluorometry (Hitachi F-2000, Tokyo, Japan) with an excitation/emission wavelength at 515/553 nm [7].

Na+,K+-ATPase activity was measured with an ATP-assay kit (Toyo Inki, Tokyo, Japan) in the kidney homogenate with or without 1 mmol/L ouabain.

Reverse-transcription polymerase chain reaction for quantification of NADpolymerase PH oxidase RNA in the kidney
At 18 weeks, five rats in each group were anaesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.) and the kidneys were immediately excised and frozen in liquid nitrogen. Total RNA was prepared and reverse-transcription polymerase chain reaction (RT-PCR) was performed by standard methods using a synthetic gene-specific primer for NADPH oxidase p47phox; upstream primer 5'-GGCAGGACCTGTCGGAGAAGGTGG-3' (132–155) and downstream primer 5'-TGAAGGATGATGGGGCCTGTGATG-3' (513–490); p22phox upstream primer 5'-TGCGGGACGCTTCACGCAGTGG-3' (131–152); and downstream primer 5'-GGTTGGTAGGTGGCTGCTTGATGG-3' (507–484). Parallel amplification of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was done for reference and the intensity of each band was quantified using a densitometry. The intensity of each gene's band was expressed relative to the corresponding densities of the GAPDH bands from the same RNA samples.

Light microscopy morphological study and immunohistochemistry
Five rats from each group were anaesthetized and the kidneys were flushed with phosphate-buffered saline (PBS). The right kidney was removed and frozen for western blot and the left kidney was immersed in paraformaldehyde solution overnight at 4°C. The tissues were embedded in paraffin for Periodic acid Schiff (PAS) staining and light microscopic immunohistochemistry using a monoclonal antibody against NADPH oxidase component p47phox (BD Transduction Laboratories, San Jose, CA, USA), rabbit polyclonal antibody against malondialdehyde (MDA, Alpha Diagnostic International, San Antonio, TX, USA) and polyclonal antibody against fibronectin (Chemicon International, Temecula, CA, USA) as described previously [7,32]. The glomerular damage assessed in the PAS section from five rats in each group was evaluated as glomerulosclerosis score (0–4): grade 0, normal glomeruli; grade 1, sclerosis area up to 25% (mild); grade 2, 25–50% (moderate); grade 3, 50–75% (moderate–severe); grade 4, 75–100% (severe) of all glomeruli in each section [4]. Immunoreactivity for NADPH oxidase p47phox and MDA in glomerulus was scored from five rats in each group as 0 for no staining; 1 for weak, 2 for moderate, 3 for strong staining, and the average of all glomerular scores were calculated in the section of each animal [33].

Western blot
Kidneys were homogenized on ice with a tissue homogenizer in 3 ml of 20 mmol/L Tris and homogenates were centrifuged at 4°C and 12 000 g. for 20 min. The supernatants were diluted in the same volume of SDS buffer and samples containing 25 µg of protein were applied to 4–20% gradient gel (Daiichi Pure Chemicals Co., Tokyo, Japan) and electroblotted to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dried milk for 30 min, and incubated overnight with monoclonal antibody for p47phox, manganese (Mn), copper–zinc (CuZn), and extracellular (ec) superoxide dismutase (SOD) (BD Transduction Laboratory, San Jose, CA, USA), catalase (Sigma, St Louis, MO), 4-hydroxy-2-nonenal (JaICA, Shizuoka, Japan) and polyclonal antibody against TGF-ß (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in a 1:1000 dilution. Primary antibody incubation was followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against mouse or rabbit IgG (Dako, Glostrup, Denmark) in a 1:1000 dilution, and thereafter with 0.8 mmol/L diaminobenzidine (DAB) with 0.01% H2O2 and 3 mmol/L NiCl2 for the detection of blots. The density of the bands was analysed using NIH software.

Statistics
All data were expressed as means ± SE. The mean values were compared among the three groups using ANOVA followed by the Bonferroni post hoc test. P-values <0.05 were required for statistical significance. The correlation coefficients among immunostaining scores of glomerular NADPH oxidase p47phox and MDA, western blot densitometry of renal TFG-ß and Na+,K+-ATPase activity were evaluated using the StatView-J4.5 computer programme.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
Renoprotective effect of ACEI and Epl
At 18 weeks, the DSHF model had severe hypertension and renal damage evaluated by the increase in serum creatinine levels (Table 1), proteinuria and glomerulosclerosis (Figure 1). ACEI reduced blood pressure, serum creatinine, urinary albumin excretion and improved glomerulosclerosis (Figure 1). Epl alone did not change blood pressure (Table 1) but reduced proteinuria and glomerulosclerosis index (Figure 1). The association of ACEI and Epl reduced blood pressure to the same level as ACEI alone (Table 1). Interestingly, combination therapy showed a further decrease in proteinuria, serum creatinine and renal morphological damage compared with the ACEI alone, but it was not significantly different from Epl alone (Figure 1). During the study period, no change in serum potassium levels was observed (Table 1).


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  		Table 1. Blood pressure, serum creatinine and potassium and renal oxidative products

 

Figure 1
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  		Fig. 1. Urinary albumin excretion (A), Glomerulosclerosis score (B), Representative morphological changes (C) by PAS staining. DSHF indicates Dahl salt-sensitive rats fed with 8% NaCl from 6-week until 18-week-old, DSHF + ACEI, DSHF rats treated with trandolapril from 11-week to 18-week-old, DSHF + Epl, DSHF rats treated with eplerenone from 11-week to 18-week-old, DSHF + ACEI + Epl, DSHF treated with trandolapril and eplerenone from 11-week to 18-week-old. *P < 0.05, **P < 0.005 vs Control, {dagger}P < 0.05, {dagger}{dagger}P < 0.005, {dagger}{dagger}{dagger}P < 0.0001 vs DSHF, {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.005 vs DSHF + ACEI + Epl. The bar indicates 100 µm.

 
NADPH oxidase expression in the kidney of heart failure rats
NADPH oxidase p47phox and p22phox mRNA levels were increased in the kidney of DSHF rats (Figure 2). ACEI and Epl reduced mRNA expression of NADPH oxidase p47phox and p22phox in kidney of DSHF rat to the same extent. Combination therapy with ACEI and Epl showed an additive effect at mRNA level (Figure 2).


Figure 2
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  		Fig. 2. The mRNA expression of NADPH oxidase p47phox and p22phox in the kidney evaluated by RT-PCR. *P < 0.05 vs Control, {dagger}P < 0.05 vs DSHF, {ddagger}P < 0.05 vs DSHF + ACEI + Epl.

 
At protein level NADPH oxidase, component p47phox was weakly expressed in the glomerular cells and distal tubules in the kidney of control DR rat, whereas its expression was enhanced in the kidney of DSHF rat by immunohistochemistry and western blot analysis (Figure 3). ACEI or Epl treatment reduced NADPH oxidase protein expression to the same level, however, combination therapy with ACEI and Epl showed an addictive effect on NADPH oxidase p47phox expression in the kidney (Figure 3).


Figure 3
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  		Fig. 3. Western blot analysis of NADPH oxidase p47phox in the kidney. *P < 0.05, **P < 0.005 vs Control, {dagger}P < 0.05, {dagger}{dagger}P < 0.005 vs DSHF, {ddagger}P < 0.05 vs DSHF + ACEI + Epl.

 
Renal SOD and Oxygen radical production
As shown in Figure 4A, the antioxidant defence enzyme MnSOD was decreased in the kidney of DSHF. The renal MnSOD expression was increased by ACEI but it was still significantly different from control. Epl treatment alone or in combination with ACEI reversed MnSOD to the control level (Figure 4A). EcSOD was also decreased in DSHF compared with control, but did not change with treatment (Figure 4B). On the other hand, CuZnSOD did not show a significant change in the kidney of DSHF rats, but it increased with Epl treatment (Figure 4C). Hydrogen peroxide is generated by SOD and degraded by catalase; its expression was decreased in the DSHF. Single or combined administration of Epl increased catalase expression more effectively than ACEI in the kidney of DSHF rats (Figure 4D). The enhanced NADPH oxidase and suppressed MnSOD, ecSOD and catalase expression increased glomerular LPO, renal 4-hydroxy-2-nonenal (4-HNE) and urinary LPO excretion in the DSHF rats (Table 1). Treatment with ACEI or Epl significantly reduced glomerular and urinary LPO and combination therapy of ACEI and Epl showed the lowest level of glomerular and urinary LPO (Table 1). The suppression of oxidative stress with ACEI, eplerenone, or their combination was associated with inhibition of NADPH oxidase and recovery of MnSOD, CuZnSOD and catalase expression.


Figure 4
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  		Fig. 4. Western blot analysis of manganese superoxide dismutase (MnSOD; A), extracellular superoxide dismutase (ecSOD; B), copper-zinc superoxide dismutase (CuZnSOD; C) and catalase (D) in the kidney. *P < 0.05, **P < 0.005 vs Control, {dagger}P < 0.05, {dagger}{dagger}P < 0.005 vs DSHF, {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.005 vs DSHF + ACEI + Epl.

 
TGF-ß expression and relation with renal damage
TGF-ß expression in the kidney was increased in the DSHF (Figure 5A). Single treatment with Epl significantly decreased TGF-ß expression to the same level as combination therapy with Epl and ACEI, whereas a significant decrease was not observed with ACEI (Figure 5A).


Figure 5
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  		Fig. 5. Western blot analysis of TGF-ß in the kidney (A) and glomerular immunoreactivity to fibronectin (B). *P < 0.05, **P < 0.005 vs Control, {dagger}P < 0.05, {dagger}{dagger}P < 0.005 vs DSHF, {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.005 vs DSHF + ACEI + Epl.

 
TGF-ß is a known regulator of fibronectin expression in the kidney and we found an increased fibronectin expression in the DSHF compared with control and observed a decrease with ACEI and Epl treatment (Figure 5B). A discrete improvement was observed with the association of Epl and ACEI compared with each treatment alone (Figure 5B). Glomerulosclerosis was determined mainly by TGF-ß expression in the kidney and also oxidative stress was derived by NADPH oxidase (Table 2). Proteinuria showed the best correlation with glomerulosclerosis (r = 0.82, P < 0.001), implying that it is also determined by both TGF-ß expression and NADPH oxidase (Table 2).


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  		Table 2. Correlation coefficients among renal expression of NADPH oxidase, malondialdehyde (MDA), TGF-ß, Na+,K+-ATPase and renal damage makers

 
Na+,K+-ATPase activity and its protein amount
Na+,K+-ATPase protein and activity were decreased in the kidney of DSHF rat as a result of salt overloading (Figure 6). Combination therapy with ACEI and Epl increased Na+,K+-ATPase activity (Figure 6A), but this effect was not observed with each single treatment (Figure 6A). The protein amount of Na+,K+-ATPase was increased by Epl and there was no additive effect with ACEI (Figure 6B). Na+,K+-ATPase activity had a significant negative correlation with TGF-ß (r = –0.59, P < 0.005) and showed a weak but significant negative correlation with glomerulosclerosis (r = –0.49, P < 0.05, Table 2).


Figure 6
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  		Fig. 6. Na+,K+-ATPase activity (A) and its protein amount (B) in the kidney. *P < 0.05, **P < 0.005 vs Control, {dagger}P < 0.05, {dagger}{dagger}P < 0.005 vs DSHF, {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.005 vs DSHF + ACEI + Epl.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
In this work, we found that combination therapy with ACEI and Epl had an additive effect on NADPH oxidase suppression and that TGF-ß was more susceptive to the blockade of the aldosterone receptor than to ACE inhibition in the kidney. The combination of ACEI and Epl prevented renal damage and reduced proteinuria to the control level in the hypertensive rat with heart failure. We will discuss this mechanism and the benefit of the combination of ACEI and Epl in other kidney diseases.

Renoprotective effect of combination therapy with ACEI and aldosterone blocker
The Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) demonstrated that the selective aldosterone blocker eplerenone used in addition to the base-line antihypertensive treatments which included an ACEI or an angiotensin II AT1 receptor blocker (86%) and a ß-blocker (75%), resulted in significant improvement in survival and morbidity in patients with left ventricular systolic dysfunction and heart failure after acute myocardial infarction [34–36]. In the kidney, the combination of ACE inhibitor and Epl can provide a better renal outcome with reduction of proteinuria and tissue damage rather than single therapy [3,37] [abstract Epstein M et al. J Am Coll Cardiol 2002; 39 (Suppl A)]. To clarify the mechanism of these clinical observations of combination therapy with ACEI and Epl, we investigated an animal model of hypertensive renal disease and heart failure. In this study, proteinuria and morphological glomerular damage additively improved with the combination of ACEI and Epl. Interestingly, Epl alone or with the ACEI reduced proteinuria more effectively than the ACEI alone supporting similar observations made in humans [38,39]. We have recently reported that Epl prevented glomerulosclerosis in a dose-dependent fashion [31]; in the present work we used a dose of Epl that did not change blood pressure, but addition of Epl to ACEI reduced glomerulosclerosis, proteinuria and serum creatinine more effectively compared with ACEI alone.

One limitation of the co-administration of an ACEI and the aldosterone blocker could be hyperkalaemia, however, we did not observe an increase in serum potassium levels in any of the treatment groups. The absence of hyperkalaemia may be the result of preservation of renal function as serum creatinine reversed to the control level with the combination therapy. Moreover, Na+,K+-ATPase activity that was decreased in the DSHF rat was restored to the control level with the combination therapy promoting the urinary excretion of potassium. Therefore, combination therapy of ACEI and Epl was safe and effective to ameliorate renal functional and morphological damage in hypertension.

Mechanism of the additive effect on the suppression of oxidative stress
It is well established that angiotensin II enhances NADPH oxidase causing oxidative stress and renal damage [4,5,7,40] and blockade of angiotensin II with ACEIs or angiotensin II AT1 receptor blocker protects the kidney in hypertension, diabetes and hyperlipidaemia [4,5,7,41] due to inhibition of renal NADPH oxidase. In this model of DSHF, we have reported that renal ACE and renal angiotensin II levels were increased and that ACEI significantly reduced renal angiotensin II and NADPH oxidase [4]. Aldosterone also enhances NADPH oxidase [19] and its activity by translocation of the cytosolic component, p47phox, to the membrane subunits by an ERK1/2-related pathway [19]. Aldosterone has also been shown to stimulate ACE expression [42] and the increase in ACE may stimulate renal angiotensin II production. Therefore, we believe that the association of ACEI and Epl may have down-regulated the NADPH oxidase system by diverse routes which can explain this additive effect on the enzyme expression. Renal antioxidant systems, MnSOD, ecSOD and catalase were suppressed in DSHF rats, ACEI restored MnSOD while Epl and combination therapy restored MnSOD as well as CuZnSOD and catalase. Glomerular and urinary LPO and renal 4-HNE were maximally suppressed with the combination of ACEI and Epl. Glomerular LPO was suppressed more importantly with ACEI compared with Epl indicating that angiotensin II-induced superoxide production is essential in glomeruli as previously observed [4,5,7]. However, for the whole kidney, it is not possible to explain this result merely by the suppression of NADPH oxidase and by the scavenging action of SODs and catalase. It is possible that ACEI and Epl have a diverse action on other systems including mitochondria electron transport chain and xanthine oxidase and that the effect of ACEI and Epl is different in the glomeruli and renal tubules. On the other hand, urinary LPO representing systemic LPO production seems to rely more on aldosterone-stimulated LPO production because Epl alone inhibited urinary LPO and no further decrease was observed with combination therapy. Renal CuZnSOD and catalase may partly have contributed to the reduction of urinary LPO with Epl treatment. The different regulation of SOD isoforms with ACEI and Epl was not clear from this study, and the different activation of systemic vs renal, glomerular vs tubular activation of NADPH oxidase and SODs by angiotensin II and aldosterone need further investigation.

Aldosterone blockade and TGF-ß expression
TGF-ß is linked to the development of glomerulosclerosis in diabetic and hypertensive kidney diseases. In this study, we showed that renal TGF-ß is increased in the kidney of DSHF rat and is related to the increase in fibronectin and with the development of glomerulosclerosis. The direct action of angiotensin II on TGF-ß expression has been demonstrated in cardiac fibroblasts and myocytes [43], however this action seems to be weak in the kidney because ACEI did not significantly reduce TGF-ß in the kidney of DSHF. On the other hand, aldosterone blocker significantly suppressed renal TGF-ß in the DSHF rat indicating that in the kidney the effect of aldosterone on TGF-ß activation is more important. Actually, it has been observed in vivo that aldosterone administration per se induces TGF-ß increase [44] and organ fibrosis independent of blood pressure. In our study, the aldosterone blocker eplerenone did not reduce blood pressure; however, it reduced TGF-ß and fibronectin with a positive correlation with glomerulosclerosis.

In the kidney, the aldosterone-TGF-ß relationship has been verified in the tubules, regulating the reabsorption of sodium. In cultured tubular cells, TGF-ß suppresses the expression and activity of Na+,K+-ATPase [45] and counteracts aldosterone that increases Na+,K+-ATPase activity [46–48]. The decreased Na+,K+-ATPase activity and expression in the kidney of the DSHF rat may be explained by the increased TGF-ß, by the high salt diet-resultant decreased systemic renin-angiotensin-aldosterone and by the endogenous digitalis-like substances which were increased in the Dahl salt-sensitive rats [49–51]. Even though the systemic level of aldosterone had been suppressed by the high salt diet, Epl reversed the decrease in Na+,K+-ATPase protein by the inhibition of renal TGF-ß in the Dahl salt-sensitive rat. Na+,K+-ATPase activity showed a negative correlation with TGF-ß expression suggesting that Na+,K+-ATPase in the renal tubules can have some role in renal fibrosis. Further studies are necessary to elucidate the role of this ion-transporting enzyme in aldosterone blockade.

In conclusion, combination therapy of Epl and ACEI suppressed NADPH oxidase more effectively compared with each single treatment in the DSHF. ACEI increased MnSOD and Epl increased MnSOD, CuZnSOD and catalase, resulting in the lowest levels of oxidative stress with the combination therapy. TGF-ß showed strong correlation with proteinuria and glomerulosclerosis and was inhibited by Epl alone or with the ACEI. The association of Epl to ACEI is beneficial due to a further reduction of NADPH oxidase and specific inhibition of TGF-ß resulting in improvement of renal damage; glomerulosclerosis, proteinuria and serum creatinine were more effectively normalised compared with ACEI alone and were closest to the control levels.



   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by grants PO17-5229 (to O.M.L.) from the Japan Society for the Promotion of Science (JSPS) and C2-16590780 (to T.A.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Conflict of interest statement: None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 30. 7.06
Accepted in revised form: 1.12.06


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