Nephrol Dial Transplant (2009) 24: 73–84doi: 10.1093/ndt/gfn448Advance Access publication 5 August 2008

Original Article

Effect of eplerenone, enalapril and their combination treatmenton diabetic nephropathy in type II diabetic rats

Young Sun Kang1, Gang Jee Ko1, Mi Hwa Lee1, Hye Kyoung Song1, Sang Youb Han2, Kum Hyun Han2,Hyoung Kyu Kim1, Jee Young Han3 and Dae Ryong Cha1

1Department of Internal Medicine, Korea University, Ansan City, 2Department of Internal Medicine, Inje University, Goyang City,Kyungki-Do and 3Department of Pathology, Inha University, Incheon, Korea

AbstractBackground. Recent data suggest that aldosterone antag-onists have beneficial effects on diabetic nephropathy. Inthis study, we investigated the dose-dependent effect ofeplerenone and a combined treatment with eplerenone andenalapril compared with each drug alone on renal functionin type II diabetic rats. To further explore the molecularmechanism of action of combination therapy, we also per-formed in vitro study.Methods. The animals were divided into six groupsas follows: normal control Long-Evans Tokushima Ot-suka (LETO) rats, Otsuka Long-Evans Tokushima Fatty(OLETF) rats, OLETF rats treated with low dose ofeplerenone (50 mg/kg/day), OLETF rats treated with highdose of eplerenone (200 mg/kg/day), OLETF rats treatedwith enalapril (10 mg/kg/day) and OLETF rats treated witha combination of both drugs (eplerenone 200 mg/kg/dayand enalapril 10 mg/kg/day) for 6 months.Results. Treatment of OLETF rats had no significant effecton body weight, kidney weight and blood glucose levels.However, urinary albumin excretion, glomerular filtrationrate and glomerulosclerosis were significantly improvedin the enalapril group and improvement was observed ina dose-dependent manner in the eplerenone groups; themost dramatic decreases were observed in the combinationgroup. In accordance with these findings, renal expressionsof TGF-β1, type IV collagen and PAI-1 were also markedlydecreased in the treatment groups, with the combined treat-ment providing the most significant level of improvement.In cultured mesangial cells, combined treatment resulted inan additive decrease in TGF-β1, PAI-1 and collagen geneexpressions and protein production induced by high glucoseand aldosterone stimulation.Conclusions. Aldosterone receptor antagonism providedadditional benefits beyond blockade of the renin–angiotensin system in type II diabetic nephropathy.

Correspondence and offprint requests to: Dae Ryong Cha, Departmentof Internal Medicine, Korea University Ansan-Hospital, 516 Kojan-Dong, Ansan City, Kyungki-Do, 425-020, Korea. Tel: +82-31-412-6590;Fax: +82-31-412-5574; E-mail: cdragn@unitel.co.kr

Keywords: albuminuria; diabetic nephropathy; enalapril;eplerenone; glomerulosclerosis

Introduction

The renin–angiotensin–aldosterone system (RAAS) hasbeen a focus of research in cardiovascular, renal andatherosclerotic diseases for several decades [1]. Exten-sive research has found that the blockade of RAAS has awidespread protective role by producing anti-inflammatory,anti-proliferative and anti-oxidative effects [1–6]. Cur-rently, angiotensin II is considered the primary media-tor of RAAS, and thus agents that block RAAS, such asangiotensin-converting enzyme inhibitors (ACEI) or an-giotensin II receptor antagonists (ARB), have been devel-oped as powerful strategies to slow the progression of renaldiseases [7]. However, despite the beneficial effects of thesestrategies, their effects are limited with respect to the pre-vention of end-stage renal failure.

Previous reports have demonstrated that the administra-tion of spironolactone, a nonselective aldosterone blocker,has beneficial effects in a variety of animal models of re-nal injury such as the unilateral ureteral obstruction model,cyclosporin nephrotoxicity model and hypertensive renalinjury model [8–10]. In addition, recent data have demon-strated improved renal function in streptozotocin-induceddiabetic rats, with clinical trials suggesting the possibilitythat inhibition of the aldosterone system may have addi-tional beneficial effects independent of renin–angiotensinblockade on diabetic nephropathy [11–14].

Recent evidence also indicates that aldosterone inhibi-tion may have additional renoprotective effects in patientswith early diabetic nephropathy who experience the aldos-terone escape phenomenon during ACE inhibitor treatment[15]. Another recently published study has shown that inpatients with chronic renal disease and persistent protein-uria of >0.5 g/day, despite ideal BP control with an ACE in-hibitor, adding spironolactone to the conventional treatmentproduces beneficial effects on urinary protein excretion,

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74 Y. S. Kang et al.

especially in diabetic patients [16]. Therefore, inhibitionof the aldosterone system as well as the renin–angiotensinsystem (RAS) raises the possibility that a dual blockade ofRAS and aldosterone may result in a more complete block-ade of RAAS than the inhibition of either system alone,providing individuals with increased beneficial effects onthe prevention of progression of diabetic nephropathy. How-ever, it is still unknown whether combined treatment withaldosterone receptor antagonist based on RAS inhibitionprovides increased renal protective effects on the diabeticnephropathy of a type II diabetic animal model.

For these reasons, we investigated whether differentdoses of eplerenone result in a different effect in renal pro-tection, and tested that combined therapy of eplerenone andenalapril provides a more beneficial effect compared witheach treatment alone on the renal function in type II di-abetic nephropathy. In addition, we conducted an in vitrostudy in order to elucidate a renal protective mechanism ofdual blockade, and we evaluated the effect of dual block-ade on high glucose and aldosterone-induced synthesis ofTGF-β1, type IV collagen and PAI-1 in cultured mesangialcells.

Materials and methods

Animals

Male OLETF rats, a model of type II diabetes mellitus,were kindly supplied by the Tokushima Research Institute(Otsuka Pharmaceutical Co, Tokyo, Japan). Male Long-Evans-Tokushima-Fatty (LETO) rats served as a geneticcontrol. All rats were maintained at a controlled tempera-ture (23 ± 2◦C) and humidity (55 ± 5%) under an artificiallight cycle. Animals were given free access to rat chow. At20 weeks of age, rats were divided into six groups (n =6/group). Group 1 consisted of LETO control rats, group2 consisted of OLETF type II diabetes rats, group 3 con-sisted of OLETF rats treated with low dose of eplerenoneat a dose of 50 mg/kg/day (Pfizer Inc., New York, USA)mixed in rat chow, group 4 consisted of OLETF rats treatedwith high dose of eplerenone at a dose of 200 mg/kg/daymixed in chow, the fifth group consisted of OLETF ratstreated with 10 mg/kg/day of enalapril in drinking water(Sigma-Aldrich, St Louis, MO, USA) and the sixth groupconsisted of OLEFT rats treated with both 10 mg/kg/dayof enalapril and 200 mg/kg/day of eplerenone. Adminis-tration of enalapril or eplerenone was begun at 20 weeksof age, which is the time usually required to develop overthyperglycaemia in OLETF rats. The treatment regimen wasmaintained for 6 months. Daily amounts of food and waterintake were checked at regular intervals to affirm the doseof administered drug. Plasma glucose levels were measuredusing a glucose oxidase-based method, plasma potassiumwas measured by flame photometry and serum creatininelevels were determined using a modified Jaffe method. Theamount of urinary albumin was determined with a com-petitive ELISA kit (Shibayagi, Shibukawa, Japan). Urinaryalbumin concentrations were normalized to urinary creati-nine concentrations. Creatinine clearance was used to esti-mate glomerular filtration rate, and was calculated on the

basis of plasma and urinary creatinine concentrations andthe corresponding urine volume; it is expressed as millil-itres per minute per 100 grams body weight. At the endof the study period, systolic blood pressure was measuredusing tail-cuff plethysmography (LE 5001-Pressure Meter,Letica SA, Barcelona, Spain). Rats were killed under anaes-thesia by intraperitoneal injection of sodium pentobarbitalat a dose of 50 mg/kg body weight. Experiments were con-ducted in accordance with the Korea University Guide forLaboratory Animals.

Determination of serum and urinary concentrationsof βig-h3

Serum and urine concentrations of transforming growthfactor-β inducible gene-h3 (βig-h3) were measured withELISA as previously described [17]. Briefly, 96-well flat-bottom plastic microtitre plates (Coster, Cambridge, MA,USA) were coated with 0.5 µg/mL of wild-type recom-binant βig-h3 protein in a 20 mM carbonate–bicarbonatebuffer (pH 9.6) with 0.02% sodium azide overnight at4◦C. The plates were then rinsed three times in PBS-0.05%Tween-20 (PBST) and maintained at 4◦C. Lyophilized cul-ture media was then pre-incubated with anti-βig-h3 antibod-ies (diluted 1:2000 in PBST) in 96-well round-bottom plas-tic microtitre plates for 90 min at 37◦C. The pre-incubatedsamples were then transferred to the pre-coated plates andincubated for 30 min at room temperature. The plates wererinsed three times in PBST and incubated for 90 min at roomtemperature with the peroxidase-conjugated anti-rabbit IgGantibodies (diluted 1:2000 in PBST, Amersham, Arlington,IL, USA). Thereafter, the plates were rinsed again threetimes in PBST and incubated for 60 min at room temper-ature in the dark with a substrate solution (prepared bydissolving O-phenylenediamine in methanol at a concen-tration of 10 mg/mL, diluting 1:100 with deionized waterand adding 0.01 mL of 30% H2O2 per 100 mL of the so-lution). After termination of the reaction with 8 N H2SO4,the absorbance was read at 492 mm. The detection limitof βig-h3 was 10 ng/mL. Concentrations of urinary βig-h3were normalized to urinary creatinine concentrations.

RNA extraction and analysis of gene expressionby real-time quantitative PCR in renal tissuesand mesangial cells

Total RNA was extracted from renal cortical tissues or ex-perimental cells with Trizol reagent and further purifiedusing an RNeasy Mini kit (Qiagen, Valencia, CA, USA)according to the manufacturer’s instruction. The nucleotidesequence of each primer was as follows: TGF-β1, sense5′-ATA CAG GGC TTT CGA TCC AGG-3′ and anti-sense5′-GTC CAG GCT CCA AAT ATA GG-3′; type IV colla-gen, sense 5′- TAG GTG TCA GCA ATT AGG CAG G-3′and anti-sense 5′-CGG ACC ACT ATG CTT GAA GTG A-3′; PAI-1, sense 5′-ATG AGA TCA GTA CTG CGG ACGCCA TCT TTG-3′ and anti-sense 5′- GCA CGG AGATGG TGC TAC CAT CAG ACT TGT-3′; β-actin, sense 5′-TCA TGA GGT AGT CCG TCA GG -3′ and anti-sense5′- TCT AGG CAC CAA GGT GTG -3′. Quantitative gene

Effect of eplerenone, enalapril and their combination treatment 75

expression was performed using a Bio-Rad iCycler system(Bio-Rad, Hercules, CA, USA) using SYBR Green technol-ogy. Total mRNA was reverse-transcribed into cDNA usingan iScript cDNA synthesis kit (Bio-Rad, Hercules, CA,USA). The specificity of each PCR product was evaluatedby melting curve analysis, followed by agarose gel elec-trophoresis to confirm the presence of a single clean band.Real-time RT-PCR was performed by heating reactions for10 min at 50◦C and 5 min at 95◦C, followed by 45 cyclesconsisting of denaturation for 10 s at 95◦C and annealingwith extension for 30 s at 60◦C. At the end of the PCR cy-cle, samples were heated to 95◦C to confirm the presenceof a single, specific PCR product. The mRNA level of eachsample was normalized to that of β-actin mRNA.

Histologic examination

Paraffin-embedded kidney tissues were cut into 4-µm-thickslices and stained with periodic acid-Schiff (PAS). A semi-quantitative score (SI) was used to evaluate the degree ofglomerulosclerosis on PAS-stained sections according tothe method described by Ma et al. [18]. The severity ofsclerosis for each glomerulus was graded from 0 to 4+as follows: 0, no lesion; 1+, sclerosis of <25% of theglomerulus; 2+, 3+ and 4+, sclerosis of 25 to 50%, >50to 75% and >75% of the glomerulus, respectively. Renalpathologist carried out histologic examinations in a blindedmanner, and for each rat no less than 50 glomeruli wereanalysed in each kidney section.

Immunohistochemical staining for TGF-β1, PAI-1 andtype IV collagen

After removal of paraffin and dehydration in xylene fol-lowed by graded alcohols, slides were immerged in dis-tilled water and the free kidney sections were transferredto a 10 mmol/L citrate buffer solution at a pH of 6.0. Var-ious staining conditions were then applied: sections wereheated at 80◦C for 30 min to retrieve antigens for TGF-β1staining, transferred to a 1 M EDTA buffer solution (pH8.0) for type IV collagen staining or transferred to Bio-genex Retrievit (pH 8.0) (InnoGenex, San Ramon, CA,USA) and microwaved for 10–20 min for PAI-1 staining.After washing in water, 3.0% H2O2 in methanol was ap-plied for 20 min in order to block endogenous peroxidaseactivity, and the slides were incubated at room temper-ature for 20 min with normal goat serum (TGF-β1 andtype IV collagen) or 10% powerblock (PAI-1) to preventnonspecific detection. Next, slides were incubated for 1h with a primary antibody against rabbit polyclonal anti-type IV collagen (1:200; Santa Cruz Biotechnology, SantaCruz, CA, USA) or for 2 h with a rabbit polyclonal anti-TGF-β1 antibody (1:200; Santa Cruz Biotechnology). ForPAI-1 staining, slides were incubated at 4◦C overnightwith a rabbit anti-PAI-1 antibody (1:50; American Diag-nostica, Stamford, CT, USA). Slides were incubated ina secondary antibody for 30 min. For colouration, slideswere incubated at room temperature with a mixture of0.05% 3,3′-diaminobenzidine containing 0.01% H2O2 andcounterstained with Mayer’s haematoxylin. Negative con-

trol sections were stained under identical conditions withthe buffer solution substituting for the primary antibody.In order to evaluate the results of immunohistochemicalstaining, glomerular fields were graded semiquantitatively.Briefly, each score reflected both changes in the extent andthe intensity of staining and was graded according to a 5-point scale, with grade 0 representing very weak or absentstaining and no localized increases of staining; grade 1, dif-fuse, weak staining with 1–25% of the glomerulus showingfocally increased staining; grade 2, 25–50% of the glomeru-lus demonstrating focal, strong staining; grade 3, 50–75%of the glomerulus stained strongly in a focal manner andgrade 4, >75% of the glomerulus stained strongly. Between50 and 60 glomeruli were counted under high power fields(×400), and the average score was calculated.

Mesangial cell culture

A part of normal renal cortex of Sprague-Dawley ratswas obtained immediately after surgical nephrectomy.Glomeruli were treated with collagenase (Gibco BRL,Gaitherburg, Md., USA) and plated on culture dishes inDulbecco’s modified Eagle medium (DMEM, Gibco BRL)containing 17% heat inactivated fetal calf serum (SigmaChemical, St Louis, Mo., USA). Mesangial cells were cul-tured in DMEM supplemented with 17% fetal calf serum,2% of each penicillin and streptomycin, 1% HEPES, 2 gsodium bicarbonate and 2 mM L-glutamine at 37◦C in 5%CO2–95% air. Sub-confluent MCs were serum-starved for24 h, and some wells were cultivated under a high glucosecondition (30 mM of D-glucose) and aldosterone (Sigma-Aldrich, St Louis, MO, USA) was added to the culturemedia at a final concentration of 100 nM. When the effectsof enalapril and eplerenone and their combination weretested, enalaprilat (Sigma-Aldrich) that is the active formof enalapril and eplerenone was added to cells at a finalconcentration of 2.5 µM and 10 µM, respectively, 30 minbefore high glucose and aldosterone treatment. All experi-mental groups were cultured in triplicate and harvested at24 h for extraction of the total RNA and protein.

Measurement of secreted collagen and TGF-β1 in culturedMCs

Total soluble collagen was measured in culture super-natants by the SircolTM soluble collagen assay kit (Bio-color, Belfast, Northern Ireland) following the manufac-turer’s instructions. Briefly, 1 mL of Sirius red reagent wasadded to 100 µL of test samples and mixed for 60 min atroom temperature in a mechanical shaker. The collagen–dye complex was precipitated by centrifugation at 14 000 gfor 10 min. To release the bound dye, 1 mL of alkali reagent(0.5 M NaOH) was added to the precipitate; then the ab-sorbance was measured at 540 nm using an ELISA reader.The amount of TGF-β1 secreted into the culture mediumwas quantified using a commercially available ELISA kit(R&D systems, Abingdon, Oxfordshire, UK) following themanufacturer’s instructions. Total (active plus latent frac-tions) TGF-β1 was measured following acid activation with

76 Y. S. Kang et al.

Table 1. Biochemical parameters in experimental animals

LETO OLETF OLT+EPL(50) OLT+EPL(200) OLT+Ena OLT+EPL+Ena

BW (g) 584 ± 17 622 ± 28 612 ± 13 587 ± 28 581 ± 15 570 ± 33KW/BW (mg/g) 0.29 ± 0.02 0.37 ± 0.05a 0.35 ± 0.06a 0.36 ± 0.02a 0.34 ± 0.04 0.33 ± 0.02FPG (mg/dL) 102 ± 4.2 133 ± 8.5b 124 ± 5.5b 135 ± 9.2b 123 ± 9.5b 126 ± 3.5b

S-Cr (mg/dL) 0.54 ± 0.02 0.58 ± 0.05 0.57 ± 0.03 0.64 ± 0.03 0.58 ± 0.03 0.61 ± 0.02UV (mL/day) 11.0 ± 4.1 21.5 ± 2.2a 16.4 ± 4.3 14.2 ± 6.5 13.1 ± 4.4 13.5 ± 4.8SBP (mmHg) 128 ± 8.4 141 ± 8.2a 134 ± 4.8 132 ± 6.8 121 ± 5.5c 119 ± 9.8c

P-Na (mmol/L) 143 ± 5.6 145 ± 7.9 149 ± 8.1 144 ± 5.7 147 ± 6.5 144 ± 5.7P-K (mmol/L) 3.7 ± 0.17 4.12 ± 0.34 4.98 ± 0.38a 5.24 ± 0.43b 4.78 ± 0.45 5.55 ± 0.62b,c

UNa (mmol/day) 1.04 ± 0.03 1.21 ± 0.11 1.16 ± 0.27 1.27 ± 0.44 1.21 ± 0.52 1.31 ± 0.65UK (mmol/day) 2.21 ± 0.33 2.12 ± 0.37 1.85 ± 0.63 1.49 ± 0.54a,c 1.97 ± 0.43 1.43 ± 0.78a,c

UK/Na ratio 1.92 ± 0.23 1.88 ± 0.27 1.63 ± 0.29 1.32 ± 0.34a,c 1.62 ± 0.49 1.33 ± 0.29a,c

Values are expressed as mean ± SEM. OLT, OLETF; EPL, eplerenone; Ena, enalapril; BW, body weight; KW, kidney weight; FPG, fasting plasma glucose;S-Cr, serum creatinine; UV, urine volume; SBP, systolic blood pressure; P-Na, plasma sodium concentration; P-K, plasma potassium concentration;UNa, 24-h urinary sodium excretion; UK, 24-h urinary potassium excretion; UK/Na ratio, 24-h urinary ratio of potassium to sodium.aP < 0.05, versus LETO; bP < 0.01, versus LETO; cP < 0.05, versus OLETF.

1 N HCl for 10 min at room temperature, followed by neu-tralization with 1.2 N NaOH/0.5 M HEPES. Samples wereapplied to microtitre plates that had been pre-coated withTGF-β1. After 2 h of incubation, they were washed threetimes and the HRP-conjugated anti-TGF-β1 antibody wasapplied to each well for 2 h at room temperature. The plateswere then washed five times, and developed with 100 µL ofcolour reagent per well. The intensity of the colour was mea-sured in an enzyme-linked immunosorbent assay (ELISA)reader at 450 nm. To control for the difference in cell num-bers between wells, the concentration was corrected basedon the amount of total cell protein.

Western blotting of PAI-1 in cultured MCs

Cells were lysed in a lysis buffer (150 mM NaCl, 50 mMTris–HCl, pH 8.0, 1% Triton X-100, 1 mM phenyl-methylsulfonylfluoride), and the protein concentration wasmeasured by the BCA protein assay (Pierce, Rockford,IL, USA). Twenty micrograms of each protein samplewere electrophoresed on 10% SDS–polyacrylamide gel un-der denaturing conditions. The proteins were transferredonto a polyvinylidene difluoride (PVDF) membrane for120 min at 250 mA. After the filter was blocked by in-cubating the membranes with blocking solution for 1 hat room temperature, the membrane was hybridized witha rabbit anti-PAI-1 antibody (American Diagnostica) or amouse monoclonal anti-β-actin antibody (Sigma-Aldrich)diluted 1:100 for PAI-1 and 1:5000 for β-actin in a blockingbuffer overnight at 4◦C. The filter was then incubated witha horseradish peroxidase-conjugated secondary antibodydiluted 1:1000 for 60 min at room temperature. Specificsignals were detected using the ECL method (Amersham,Buckinghamshire, UK).

Statistical analysis

We utilized non-parametric analysis due to the relativelysmall sample size of this study. Results were expressedas mean ± SEM. A Kruskall–Wallis test was used forcomparisons between more than two groups, followed by aMann–Whitney U-test for comparison between two groups.

Statistical analysis was performed using a microcomputer-assisted program with SPSS for Windows 10.0 (SPSS Inc.,Chicago, IL, USA). A value of P < 0.05 was consideredstatistically significant.

Results

Effect of eplerenone, enalapril and their combinationon biochemical parameters and urinary albumin excretionin type II diabetic rats

Table 1 shows the various biochemical results for eachtreatment group. Body weights were similar among thesix groups. Kidney weights normalized to body weightswere higher in OLETF rats than in LETO rats, and nei-ther eplerenone nor enalapril had an effect on kidneyweights. All OLETF rats, both treated and non-treated,had higher fasting plasma glucose concentrations comparedwith LETO rats. While serum creatinine levels were similaramong all groups of rats, urine volume was significantly in-creased in OLETF rats. Further, the systolic blood pressuresof OLETF rats were much higher than that of LETO rats.Although eplerenone treatment did not induce significantchanges in systolic blood pressure even in the high-dosegroup, enalapril treatment including combination treatmentinduced significant reduction in systolic blood pressure.However, systolic blood pressure did not show significantdifference between the group treated with enalapril andthe group with combination treatment. Although plasmapotassium levels were not different between the LETO andOLETF groups, their levels were significantly higher inOLETF rats treated with eplerenone and groups with com-bination treatment, whereas plasma sodium concentrationswere the same among all groups. These changes in plasmapotassium levels were associated with decreased urinarypotassium excretion in the eplerenone treatment groups in-cluding the combination group.

Urinary albumin excretion (UAE) in OLETF rats wassignificantly higher than that in LETO rats, and it wasdose-dependently decreased in the groups with eplerenonetreatment. Compared with eplerenone, enalapril treatment

Effect of eplerenone, enalapril and their combination treatment 77

Fig. 1. Effects of eplerenone and enalapril on urinary albumin excretion and creatinine clearance. (A) Twenty-four-hour urinary albumin excretioncorrected by urine creatinine concentration. (B) Creatinine clearance calculated on the basis of plasma and urinary creatinine concentrations and thecorresponding urine volume and is expressed as millilitres per minute per 100 grams body weight. Data are shown as mean ± SEM. EPL, eplerenone;Ena, enalapril; ∗P < 0.05, versus LETO; ∗∗P < 0.01, versus LETO; ∗∗∗P < 0.001, versus LETO; #P < 0.05, versus OLETF; ##P < 0.01, versus OLETF;###P < 0.001, versus OLETF; θP < 0.05, versus OLETF with eplerenone 200 and P < 0.01, versus OLETF with eplerenone 50.

showed more reduced UAE. Interestingly, there was an addi-tive decrease in UAE in the combination group (Figure 1A).In addition, creatinine clearance was averaged 0.56 ±0.14 mL/min/100 g body weight in LETO rats and was sig-nificantly lower in OLETF rats (0.23 ± 0.04 mL/min/100g body weight) at the end of the study period (Figure 1B).Although low dose of eplerenone group did not restore theirlevels to control LETO rats, high dose of eplerenone treat-ment significantly increased creatinine clearance to nearcontrol LETO rats. However, enalapril treatment includingcombination treatment did not show significant changesin creatinine clearance compared with untreated OLETFgroups (Figure 1B).

Effect of eplerenone, enalapril and their combinationon renal histologic changes in type II diabetic rats

Figure 2 shows the histologic findings for the con-trol LETO rats (Figure 2A,G,M,S), diabetic OLETF rats(Figure 2B,H,N,T) and OLETF rats treated with low dose ofeplerenone (Figure 2C,I,O,U) and high dose of eplerenone(Figure 2D,J,P,V), enalapril (Figure 2E,K,Q,W) and thecombination of eplerenone and enalapril (Figure 2F,L,R,X).Diabetic OLETF rats displayed severe glomerulosclerosiscompared with control LETO rats (Figure 2A,B), and whileeplerenone or enalapril treatment alone significantly im-proved the status of glomerulosclerosis, a combination ofboth drugs produced even greater benefits (Figure 2B–F).In eplerenone groups, glomerular pathology was dose-dependently improved. However, tubulointerstitial changeswere not remarkable even in OLETF rats, so significantdifferences were not found among the experimental groups(Figure 2A–F).

We next examined the immunohistochemical staining oftransforming growth factor β1 (TGF-β1), type IV collagenand plasminogen activator inhibitor-1 (PAI-1) in the kidney(Figure 2G–X). In the diabetic kidney, TGF-β1 and typeIV collagen immunoreactivity was markedly increased inglomerular mesangium, although this was not accompaniedby a significant increase in tubulointerstitial area comparedwith that of control LETO rats (Figure 2G–R). For PAI-1staining, the results were more impressive. PAI-1 deposi-tion was hardly observed in the kidneys of control LETOrats; however, diabetic OLETF rat kidneys dramatically ex-hibited strong positive PAI-1 staining detected mainly inglomeruli (Figure 2S–X). Eplerenone or enalapril treatmentinduced significant reductions in renal TGF-β1, PAI-1 andtype IV collagen protein expressions, in accordance with thedegree of glomerulosclerosis. It was of interest that the com-bination of both eplerenone and enalapril further reducedexpressions of TGF-β1, type IV collagen and PAI-1 proteinin diabetic OLETF rats beyond what was observed witheither of the two treatments alone (Figure 2G–X).

Figure 3 shows the quantitative scoring index of glomeru-losclerosis and immunohistochemical staining of TGF-β1,type IV collagen and PAI-1, respectively. The glomeru-losclerosis index was markedly increased in OLETF ratscompared with LETO rats, and it was dose-dependentlydecreased in eplerenone groups. As shown in Figure 3A,glomerulosclerosis was significantly improved in theenalapril group, and most dramatic improvement was ob-served in the combination group (LETO, 0.02 ± 0.003;OLETF, 2.35 ± 0.35; OLETF with low dose eplerenone,1.83 ± 0.31; OLETF with high dose eplerenone, 1.23 ±0.31; OLETF with enalapril, 0.78 ± 0.10; OLETF witheplerenone plus enalapril, 0.34 ± 0.06). In OLETF ratstreated with eplerenone or enalapril alone, the scoring

78 Y. S. Kang et al.

Fig. 2. Representative renal histologic findings and immunohistochemistry in experimental animals. PAS staining (A–F) and immunohistochemicalstaining for TGF-β1 (G–L), type IV collagen (M–R) and PAI-1 (S–X); LETO (A,G,M,S), OLETF (B,H,N,T), OLETF treated with 50 mg/kg/dayof eplerenone (C,I,O,U), OLETF treated with 200 mg/kg/day of eplerenone (D,J,P,V), OLETF with enalapril (E,K,Q,W), OLETF treated with botheplerenone and enalapril (F,L,R,X); original magnification, A–F: ×200; G–X: ×400.

indices of TGF-β1, type IV collagen and PAI-1 were sig-nificantly decreased, whereas the most dramatic decreasewas observed in the combination group (TGF-β1, LETO,0.21 ± 0.06; OLETF, 2.31 ± 0.10; OLETF with low doseof eplerenone, 1.93 ± 0.21; OLETF with high dose ofeplerenone, 1.20 ± 0.31; OLETF with enalapril, 0.91 ±0.10; OLETF with eplerenone plus enalapril, 0.34 ± 0.08;Type IV collagen, LETO, 0.52 ± 0.11; OLETF, 3.12 ±0.10; OLETF with low dose of eplerenone, 2.56 ± 0.59;OLETF with high dose of eplerenone, 1.86 ± 0.29; OLETFwith enalapril, 1.21 ± 0.27; OLETF with eplerenoneplus enalapril, 0.72 ± 0.14; PAI-1, LETO, 0.09 ± 0.02;OLETF, 2.43 ± 0.44; OLETF with low dose of eplerenone,1.80 ± 0.43; OLETF with high dose of eplerenone, 1.21 ±0.23; OLETF with enalapril, 0.88 ± 0.21; OLETF witheplerenone plus enalapril, 0.33 ± 0.11).

To evaluate the gene expressions of TGF-β1, type IVcollagen and PAI-1, we performed real-time PCR usingtissues isolated from rat kidneys. In accordance with thefindings from immunohistochemical staining of the rat kid-neys, TGF-β1 gene expression was significantly higher inOLETF rats compared with control LETO rats. Similarly,gene expressions of type IV collagen and PAI-1 were higherin OLETF rats. On their own, eplerenone or enalapril in-hibited gene expressions of TGF-β1, type IV collagen andPAI-1, and a combination of these two drugs suppressed theexpressions even more dramatically (Figure 4).

Effect of eplerenone, enalapril and their combinationon the serum and urinary excretion of βig-h3 in type IIdiabetic rats

We measured serum and urinary βig-h3, which has beenused to assess the biological activity of TGF-β1 in vari-ous tissues, including the kidney [19]. As demonstrated inFigure 5, the urinary βig-h3 concentration was nine timeshigher in diabetic OLETF rats than in control LETO rats.The effect of eplerenone and enalapril on the excretionof urinary βig-h3 was consistent with the aforementionedresults. Significantly, treatment with either eplerenone orenalapril alone led to reduced urinary βig-h3 excretion indiabetic OLETF rats, more dramatically so in the combina-tion group. There were no differences in serum βig-h3 con-centrations among groups with OLETF rats with or withouttreatment.

Effect of eplerenone, enalaprilat and their combinationon the synthesis of TGFβ1, type IV collagen and PAI-1in cultured mesangial cells

To determine the direct effect of eplerenone, enalapriland their combination on the expressions of high glucoseand aldosterone-induced profibrotic and proinflammatorymolecules in intrinsic renal cells, we performed a real-time quantitative analysis of TGF-β1, type IV collagen

Effect of eplerenone, enalapril and their combination treatment 79

Fig. 3. Glomerulosclerosis index and immunohistochemical staining score. (A) Glomerulosclerosis index, (B) immunohistochemical staining score forTGF-β1, (C) immunohistochemical staining score for type IV collagen, (D) immunohistochemical staining score for PAI-1; data are shown as mean ±SEM. EPL, eplerenone; Ena, enalapril; ∗P < 0.05, versus LETO; ∗∗P < 0.01, versus LETO; ∗∗∗P < 0.001, versus LETO; #P < 0.05, versus OLETF;##P < 0.01, versus OLETF; ###P < 0.001, versus OLETF; θP < 0.01, versus OLETF with eplerenone 200 and < 0.001, versus OLETF with eplerenone50; ωP < 0.05, versus OLETF with eplerenone 200 and <0.01, versus OLETF with eplerenone 50; ψP < 0.05, versus OLETF with enalapril.

and PAI-1 mRNA expressions in mesangial cells. Althoughhigh glucose treatment increased type IV collagen andTGF-β1 gene expressions, PAI-1 expression was not in-creased significantly under high glucose stimuli. However,administration of aldosterone under a high glucose condi-tion induced a marked up-regulation of PAI-1 gene expres-sion (Figure 6). Furthermore, aldosterone administrationinduced a further increase in type IV collagen and TGF-β1 gene expressions under a high glucose condition. Priortreatment of eplerenone or enalaprilat significantly reducedgene expressions of TGF-β1, type IV collagen and PAI-1induced by high glucose and aldosterone stimulation. Inter-estingly, combined treatment with eplerenone and enalapri-lat induced a further decrease in TGF-β1, type IV colla-gen and PAI-1 gene expressions compared with eplerenoneor enalaprilat treatment alone (Figure 6). Next, we mea-sured gene expression in only high glucose-stimulated MCswithout aldosterone stimulation to exclude the effect of al-dosterone on TGF-β1, type IV collagen and PAI-1 mRNAexpressions. As shown in Figure 7, high glucose-inducedTGF-β1 and type IV collagen expressions were slightly de-creased by eplerenone and enalaprilat treatment, althoughnot significant. Combination treatment in MCs also showedthe most profound effect in high glucose-induced TGF-β1, type IV collagen expressions. However, there were

no changes in PAI-1 expression in only high glucose-stimulated MCs (Figure 7).

We further evaluated whether gene expressions of TGF-β1, type IV collagen and PAI-1 were related to their proteinproduction. As shown in Figure 8A–B, secretory proteinlevels for TGF-β1, and total soluble collagen were signif-icantly increased by high glucose and aldosterone stimu-lation, which was significantly inhibited by eplerenone orenalaprilat treatment. In agreement with gene expression,combined treatment with eplerenone and enalaprilat almostcompletely abolished high glucose and aldosterone-inducedTGF-β1 and collagen protein secretion. In addition, PAI-1protein synthesis evaluated by western blot also showed thatthe most dramatic decrease was observed in the combinedtreatment group (Figure 8C).

Discussion

In the present study, we firstly demonstrated the additivebeneficial effects of combined blockade of both eplerenoneand enalapril in a type II diabetic rat model. The renal-protective effect of aldosterone inhibition has been foundin both animal models of renal disease [8–12,20–24] andhuman clinical trials [14–16,25–27]. Here, we extended our

80 Y. S. Kang et al.

Fig. 4. Gene expression in renal cortical tissues in experimental animals. (A) TGF-β1, (B) type IV collagen, (C) PAI-1; data are shown as mean ±SEM. EPL, eplerenone; Ena, enalapril; P < 0.05, versus LETO; ∗∗P < 0.01, versus LETO; ∗∗∗P < 0.001, versus LETO; #P < 0.05, versus OLETF; ##P< 0.01, versus OLETF; ###P < 0.001, versus OLETF; ωP < 0.05, versus OLETF with eplerenone 200 and < 0.01, versus OLETF with eplerenone 50.

Fig. 5. Serum and 24-h urinary levels of βig-h3 in experimental animals.βig-h3 concentration of serum (black bars) and urine (white bars) wasmeasured by the ELISA method in experimental animals. Data are shownas mean ± SEM. EPL, eplerenone; Ena, enalapril; ∗P < 0.05, versusLETO; ∗∗P < 0.01, versus LETO; ∗∗∗P < 0.001, versus LETO; #P < 0.05,versus OLETF; ##P < 0.01, versus OLETF; ###P < 0.001, versus OLETF;ωP < 0.05, versus OLETF with eplerenone 200 and < 0.01, versus OLETFwith eplerenone 50; ψP < 0.05, versus OLETF with enalapril.

previous studies [28,29] by analysing the effect of aldos-terone blockade in addition to ACE inhibition in an animalmodel of type II diabetic nephropathy.

Aldosterone blockade treatment in addition to ACEinhibition additively decreased UAE and improved thestatus of glomerulosclerosis, suggesting that the inhibi-tion of the aldosterone system has additional beneficialeffects independent of renin–angiotensin blockade on di-abetic nephropathy. The effects of combined aldosteroneand RAS blockade have been recently observed in ex-perimental models of hypertensive kidney disease [30,31],radiation-injured renal injury [24] and proteinuric kidneydisease [32]; however, prior to this study, no similar anal-ysis has been performed in an animal model of diabeticnephropathy.

Although there have been many clinical studies on theeffect of aldosterone blockade in diabetic patients [14–16,26,27,33–36], the exact mechanisms responsible forthe beneficial effects of aldosterone inhibition on diabeticnephropathy are still unclear. In this study, we found thateplerenone treatment dose-dependently decreased the ex-pressions of type IV collagen and TGF-β1, both of whichare linked to the development of glomerulosclerosis indiabetic nephropathy. Although we were unable to pro-vide direct evidence that aldosterone induced TGF-β1 up-regulation or that aldosterone synthesis is up-regulated inthe diabetic kidney, recent studies suggest the possibilitythat aldosterone provokes renal TGF-β1 synthesis [37] and

Effect of eplerenone, enalapril and their combination treatment 81

Fig. 6. Effect of eplerenone or enalaprilat and their combination on gene expression in cultured mesangial cells. (A) TGF-β1, (B) type IV collagen,(C) PAI-1; data are shown as mean ± SEM. NG, normal glucose; HG, high glucose; Aldo, aldosterone; EPL, eplerenone; Ena, enalaprilat; ∗P < 0.05,versus NG; ∗∗P < 0.01, versus NG; ∗∗∗P < 0.001, versus NG; #P < 0.05, versus HG+Aldo; ##P < 0.01, versus HG+Aldo; ###P < 0.001, versusHG+Aldo; θP < 0.01, versus HG+Aldo+Ena; ψP < 0.05, versus HG+Aldo+Ena.

that aldosterone synthesis is up-regulated in the diabetickidney [38].

Furthermore, we also found that eplerenone and enalapriltreatment profoundly suppressed the expression of PAI-1 inthe diabetic kidney, and mitigation of the effects of PAI-1by eplerenone suggests that aldosterone may contribute tooverproduction of PAI-1 in the diabetic kidney. Interest-ingly, PAI-1 expression was more drastically amelioratedby eplerenone treatment when compared with its effect onTGF-β1 expression. This result agrees with recent studiesthat show that aldosterone directly increases PAI-1 synthe-sis in mesangial cells [39] and spironolactone therapy de-creases the plasma level of PAI-1 in patients with diabeticnephropathy [40].

In this study, we observed that eplerenone or enalapriltreatment significantly decreased UAE but had no effect onblood glucose levels, body weight or kidney to body weightratios. Systolic blood pressure was not significantly reducedby eplerenone therapy alone even in the high-dose group,whereas enalapril treatment and a combination of these twotherapies significantly lowered systolic blood pressure, sug-gesting that the beneficial effect of eplerenone therapy isindependent of both haemodynamic and metabolic mech-anisms, and that an additional haemodynamic mechanismmay contribute to a beneficial effect in the enalapril groupand in the combination group. However, systolic blood pres-

sure between the enalapril group and the combination groupwas similar, which suggests that non-haemodymic mech-anisms rather than haemodynamic factors may result inthe additive beneficial effect of the combination therapy.In this study, we observed that high dose of eplerenonetreatment significantly increased creatinine clearance andthe enalapril treatment group and the combination groupdid not show significant changes in creatinine clearance,whereas they showed histological improvement. This dis-crepancy might have been caused by several factors. Firstly,the enalapril treatment group and the combination groupshowed significant reduction in systolic blood pressure,which might have reduced renal perfusion pressure thatresulted in decreased creatinine clearance. The second pos-sibility is that the effect of enalapril on glomerular haemo-dynamics such as the preferential effect of enalapril onefferent arteriole might have led to decreased creatinineclearance. Inhibition of the RAS by either an ARB or anACEI have been reported to induce a reversible reductionin intraglomerular pressure in most nephrons [41]. In thecase of defect in autoregulatory capacity for renal bloodflow such as diabetes mellitus, creatinine clearance will bemore profoundly decreased when RAS activity is blockedunder a low systolic blood pressure condition [41]. We alsonoted that plasma potassium concentrations were signifi-cantly higher in eplerenone groups and that the combination

82 Y. S. Kang et al.

Fig. 7. Effect of eplerenone or enalaprilat and their combination on gene expression in high glucose-stimulated mesangial cells. (A) TGF-β1, (B) typeIV collagen, (C) PAI-1; data are shown as mean ± SEM. NG, normal glucose; HG, high glucose; EPL, eplerenone; Ena, enalaprilat; ∗P < 0.05, versusNG; #P < 0.05, versus HG.

treatment further aggravated hyperkalaemia. These resultsindicate that one should be cautious in deciding to treatpatients with combination regimen containing aldosteronereceptor antagonist and RAS inhibitor.

To define the molecular mechanisms of the additive ben-eficial effect of combination therapy and to exclude the ef-fect of haemodynamic mechanism in the combination ther-apy, we performed an in vitro study. The above-mentionedin vivo additive renal protective effects of combination ther-apy are further supported by the in vitro experiments thatdemonstrated an additive inhibitory effect of combinationof eplerenone and enalaprilat on the synthesis of TGF-β1,type IV collagen and PAI-1 induced by high glucose and al-dosterone treatment in cultured mesangial cells. Althoughwe did not test the beneficial effects in other glomerularcells such as podocyte and endothelial cells, our findingsin mesangial cells suggest that the direct cellular effectof combination treatment may contribute to the observedin vivo additive beneficial effects in our study.

In the present study, we also demonstrated the effect ofeplerenone and enalapril on urinary βig-h3 excretion, whichis a biologic marker associated with the activation of TGF-β1 [19]. Although there was no significant difference inserum βig-h3 levels after treatment, urinary excretion ofβig-h3, which reflects the activity of TGF-β1 in the kidney,

was dramatically increased in the diabetic kidney, and whileboth eplerenone and enalapril alone were able to markedlydecrease urinary excretion of βig-h3, treatment with bothdrugs led to a more profound decrease. These results werein accordance with the results of gene expression and im-munohistochemical results for TGF-β1.

During the long-term use of RAS blockade, which in-cludes ACE inhibitors or angiotensin II receptor antago-nists or a combination of both, plasma aldosterone levelshave been shown to increase in some patients (aldosteroneescape phenomenon). Furthermore, the aldosterone escapeduring long-term blockade of the RAS system is associatedwith an enhanced decline in GFR in patients with type Idiabetic patients with nephropathy [42]. The data from thepresent study imply that aldosterone inhibition based onRAS blockade may provide more complete interruption ofRAAS, thereby resulting in enhanced renal protective ef-fects. Indeed, recent clinical trials have demonstrated thatadd-on therapy with spironolactone based on RAS inhibi-tion provides more anti-proteinuric effects in patients withdiabetic nephropathy [30,31,35].

In conclusion, this study suggests that the combinationof aldosterone blockade with RAS inhibition is more effec-tive in preventing diabetic renal damage than either therapyalone. The combined treatment approach was associated

Effect of eplerenone, enalapril and their combination treatment 83

Fig. 8. Effect of eplerenone or enalaprilat and their combination on protein synthesis in cultured mesangial cells. (A) TGF-β1, (B) total soluble collagen,(C) representative western blot for PAI-1; secretory protein levels for TGF-β1 and total soluble collagen were measured using culture supernatantby the ELISA method. Intracelluar PAI-1 levels were determined by western blotting; protein concentrations in supernatant were corrected based onthe amount of total cell protein. Data are shown as mean ± SEM. NG, normal glucose; HG, high glucose; Aldo, aldosterone; EPL, eplerenone; Ena,enalaprilat; ∗P < 0.05, versus NG; ∗∗P < 0.01, versus NG; ∗∗∗P < 0.001, versus NG; #P < 0.05, versus HG+Aldo; ##P < 0.01, versus HG+Aldo;###P < 0.001, versus HG+Aldo; ψP < 0.01, versus HG+Aldo+Ena and HG+Aldo+EPL.

with a reduction of renal production of profibrotic cy-tokines such as TGF-β1, PAI-1 and type IV collagen. Takentogether, these results indicate that blockade of the aldos-terone system based on RAS inhibition may be a new ther-apeutic strategy for retarding the progression of diabeticnephropathy.

Acknowledgements. We thank the Tokushima Research Institute, OtsukaPharmaceutical Co. Ltd for providing Otsuka Long-Evans TokushimaFatty (OLETF) rats. This work was supported by a Korea University grant.

Conflict of interest statement. None declared.

References

1. Schmieder RE, Hilgers KF, Schlaich MP et al. Renin-angiotensinsystem and cardiovascular risk. Lancet 2007; 369: 1208–1219

2. Brunner HR, Gavras H. Angiotensin blockade for hypertension: apromise fulfilled. Lancet 2000; 359: 990–992

3. Griendling KK, Minieri CA, Ollerenshaw JD et al. Angiotensin IIstimulates NADH and NADPH oxidase activity in cultured vascularsmooth muscle cells. Circ Res 1994; 74: 1141–1148

4. Boring L, Gosling J, Cleary M et al. Decreased lesion formationin CCR2−/− mice reveals a role for chemokines in the initiation ofatherosclerosis. Nature 1998; 394: 894–897

5. Rabbani R, Topol EJ. Strategies to achieve coronary arterial plaquestabilization. Cardiovasc Res 1999; 41: 402–417

6. Wolf G. Role of reactive oxygen species in angiotensin II-mediatedrenal growth, differentiation, and apoptosis. Antioxid Redox Signal2005; 7: 1337–1345

7. Gurley SB, Coffman TM. The renin-angiotensin system and diabeticnephropathy. Semin Nephrol 2007; 27: 144–152

8. Trachtman H, Weiser AC, Valderrama E et al. Prevention of renalfibrosis by spironolactone in mice with complete unilateral ureteralobstruction. J Urol 2004; 172(Suppl 4): S1590–S1594

9. Feria I, Pichardo I, Juarez P et al. Therapeutic benefit of spironolac-tone in experimental cyclosporine A nephrotoxicity. Kidney Int 2003;63: 43–52

10. Blasi ER, Rocha R, Rudolph AE et al. Aldosterone/salt induces renalinflammation and fibrosis in hypertensive rats. Kidney Int 2003; 63:1791–1800

11. Fujisawa G, Okada K, Muto S et al. Spironolactone prevents earlyrenal injury in streptozotocin-induced diabetic rats. Kidney Int 2004;66: 1493–1502

12. Miric G, Dallemagne C, Endre Z. Reversal of cardiac and renal fibrosisby pirfenidine and spironolactone in streptozotocin-diabetic rats. Br JPharmacol 2001; 133: 687–694

13. Nitta K, Uchida K, Nihei H. Spironolactone and angiotensin receptorblocker in nondiabetic renal diseases. Am J Med 2004; 117: 444–445

14. Campese VM, Park J. Use of antagonists of aldosterone in patientswith chronic kidney disease: potential advantages and risks. J Hyper-tens 2006; 24: 2157–2159

84 Y. S. Kang et al.

15. Sato A, Hayashi K, Naruse M et al. Effectiveness of aldosteroneblockade in patients with diabetic nephropathy. Hypertension 2003;41: 64–68

16. Sato A, Hayashi K, Saruta T. Antiproteinuric effects of mineralocor-ticoid receptor blockade in patients with chronic renal disease. Am JHypertens 2005; 18: 44–49

17. Cha DR, Kim IS, Kang YS et al. Urinary concentration of transform-ing growth factor-beta-inducible gene-h3(beta ig-h3) in patients withtype 2 diabetes mellitus. Diabet Med 2005; 22: 14–20

18. Ma LJ, Nakamura S, Aldigier JC et al. Regression of glomeruloscle-rosis with high-dose angiotensin inhibition is linked to decreasedplasminogen activator inhibitor-1. J Am Soc Nephrol 2005; 16: 966–976

19. Gilbert RE, Wilkinson BJL, Johnson DW et al. Renal expression oftransforming growth factor-β-inducible gene-h3 (βig-h3) in normaland diabetic rats. Kidney Int 1998; 54: 1052–1062

20. Rocha R, Chander PN, Zuckerman A et al. Role of aldosterone inrenal vascular injury in stroke-prone hypertensive rats. Hypertension1999; 33: 232–237

21. Blasi ER, Rocha R, Rudolph AE et al. Aldosterone/salt induces renalinflammation and fibrosis in hypertensive rats. Kidney Int 2003; 63:1791–1800

22. Feria I, Pichardo I, Juarez P et al. Therapeutic benefit of spironolac-tone in experimental chronic cyclosporine A nephrotoxicity. KidneyInt 2003; 63: 43–52

23. Zhou X, Ono H, Ono Y et al. Aldosterone antagonism amelioratesproteinuria and nephrosclerosis independent of glomerular dynamicsin L-NAME/SHR model. Am J Nephrol 2004; 24: 242–249

24. Brown NJ, Nakamura S, Ma L. Aldosterone modulates plasminogenactivator inhibitor-1 and glomerulosclerosis in vivo. Kidney Int 2000;58: 1219–1227

25. Bianchi S, Bigazzi R, Campese VM. Antagonists of aldosterone andproteinuria in patients with CKD: an uncontrolled pilot study. Am JKidney Dis 2005; 46: 45–51

26. Rossing K, Schjoedt KJ, Smidt UM et al. Beneficial effects of addingspironolactone to recommended antihypertensive treatment in dia-betic nephropathy: a randomized, double-masked, cross-over study.Diabetes Care 2005; 28: 2106–2112

27. Schjoedt KJ, Rossing K, Juhl TR et al. Beneficial impact of spirono-lactone in diabetic nephropathy. Kidney Int 2005; 68: 2829–2836

28. Han KH, Kang YS, Han SY. Spironolactone ameliorates renal injuryand connective tissue growth factor expression in type II diabetic rats.Kidney Int 2006; 70: 111–120

29. Han SY, Kim CH, Kim HS. Spironolactone prevents diabeticnephropathy through an anti-inflammatory mechanism in type 2 dia-betic rats. J Am Soc Nephrol 2006; 17: 1362–1372

30. Sato A, Saruta T, Funder JW. Combination therapy with aldosteroneblockade and renin-angiotensin inhibitors confers organ protection.Hypertens Res 2006; 29: 211–216

31. Onozato ML, Tojo A, Kobayashi N et al. Dual blockade of aldosteroneand angiotensin II additively suppresses TGF-beta and NADPH ox-idase in the hypertensive kidney. Nephrol Dial Transplant 2007; 22:1314–1322

32. Kramer AB, van der Meulen EF, Hamming I et al. Effect of com-bining ACE inhibition with aldosterone blockade on proteinuria andrenal damage in experimental nephrosis. Kidney Int 2007; 71: 417–424

33. van den Meiracker AH, Baggen RG, Pauli S. Spironolactone in type 2diabetic nephropathy: effects on proteinuria, blood pressure and renalfunction. J Hypertens 2006; 24: 2285–2292

34. Schjoedt KJ, Rossing K, Juhl TR. Beneficial impact of spironolactoneon nephrotic range albuminuria in diabetic nephropathy. Kidney Int2006; 70: 536–542

35. Rachmani R, Slavachevsky I, Amit M. The effect of spironolactone,cilazapril and their combination on albuminuria in patients with hy-pertension and diabetic nephropathy is independent of blood pressurereduction: a randomized controlled study. Diabet Med 2004; 21: 471–475

36. Takebayashi K, Matsumoto S, Aso Y et al. Aldosterone blockadeattenuates urinary monocyte chemoattractant protein-1 and oxida-tive stress in patients with type 2 diabetes complicated by diabeticnephropathy. J Clin Endocrinol Metab 2006; 91: 2214–2217

37. Juknevicius I, Segal Y, Kren S et al. Effect of aldosterone on renaltransforming growth factor-β. Am J Physiol Renal Physiol 2004; 286:F1059–F1062

38. Guo C, Martinez-Vaquez D, Mendez GP et al. Mineralocorticoidreceptor antagonist reduces renal injury in rodent models of types 1and 2 diabetes mellitus. Endocrinology 2006; 147: 5363–5373

39. Yuan J, Jia R, Bao Y. Aldosterone up-regulates production of plas-minogen activator inhibitor-1 by renal mesangial cells. J Biochem MolBiol 2007; 40: 180–188

40. Matsumoto S, Takabayashi K, Aso Y. The effect of spironolactone oncirculating adipocytokines in patients with type 2 diabetes mellituscomplicated by diabetic nephropathy. Metabolism 2006; 55: 1645–1652

41. Bakris GL, Weir MR. Angiotensin-converting enzyme inhibitor-associated elevations in serum creatinine: is this a cause for concern?Arch Intern Med 2000; 160: 685–693

42. Schjoedt KJ, Andersen S, Rossing P et al. Aldosterone escape dur-ing blockade of the renin–angiotensin–aldosterone system in diabeticnephropathy is associated with enhanced decline in glomerular filtra-tion rate. Diabetologia 2004; 47: 1936–1939

Received for publication: 8.5.08Accepted in revised form: 11.7.08