Original Article

Endothelin receptor blockade ameliorates renal injury by inhibition of RhoA/Rho-kinase signalling in deoxycorticosterone acetate-salt hypertensive rats Tsung-Ming Lee a,b, Tun-Hui Chung c, Shinn-Zong Lin d,e, and Nen-Chung Chang f,g

Purpose of review: Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed whether selective endothelin receptor blockers can attenuate renal fibrosis by inhibiting RhoA/ROCK axis in DOCA-salt rats. Methods: At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 4 weeks: vehicle, ABT-627 (endothelin-A receptor inhibitor) and A192621 (endothelin-B receptor inhibitor). Results: DOCA-salt was characterized by increased blood pressure, decreased renal function, increased proteinuria, increased glomerulosclerosis and tubulointerstitial fibrosis with myofibroblast accumulation, increased renal endothelin-1 levels and RhoA activity along with increased expression of connective tissue growth factor at both mRNA and protein levels as compared with uninephrectomized control male Wistar rats. Treatment with a selective mineralocorticoid receptor antagonist, eplerenone, ameliorated proteinuria. Impaired renal function and histological changes were overcome by treatment with ABT-627, but not with A192621. The beneficial effects of bosentan, a nonspecific endothelin receptor blocker, on proteinuria, RhoA activity, and connective tissue growth factor levels were similar to ABT-627. Furthermore, in an isolated perfuse kidney, a RhoA inhibitor, C3 exoenzyme, and two ROCK inhibitors, fasudil and Y-27632, significantly attenuated connective tissue growth factor levels. Conclusions: These results indicate that DOCA-salt elevates renal endothelin-1 levels and RhoA activity via activation of mineralocorticoid receptor, resulting in renal fibrosis and proteinuria. Endothelin-A receptor blockade can attenuate DOCA-salt-induced renal fibrosis probably through the inhibition of RhoA/ROCK activity and connective tissue growth factor expression.

factor; DOCA, deoxycorticosterone acetate; ET-1, endothelin-1; GDP, guanosine diphosphate; GTP, guanosine triphosphate; PAS, periodic acid Schiff; ROCK, Rho-kinase; RT-PCR, real-time quantitative reverse transcription-PCR; Unx, uninephrectomized

INTRODUCTION

H

ypertension is a leading risk factor for the development and progression of chronic renal diseases [1]. Hypertension-induced renal injury is characterized by glomerular sclerosis, tubular atrophy, and interstitial fibrosis, a common final pathway of nearly all forms of chronic kidney disease, resulting in irreversible loss of kidney parenchyma [1]. Clinical studies have shown that patients with salt-sensitive hypertension are at a significantly high risk for the development of renal injury [2]. Deoxycorticosterone acetate (DOCA)-salt rats are commonly used as a salt-sensitive hypertensive model. DOCA-salt hypertension is characterized by an overproduction of the endothelin-1 (ET-1) [3]. ET-1 has been shown to increase synthesis of extracellular matrix proteins [4,5]. However, the molecular mechanisms by which ET-1 attenuates renal fibrosis remain unclear. The effects of ET-1 are mediated by two main receptor subtypes: ETA and ETB [6]. ET-1 stimulates the production of cytokines, such as connective tissue growth factor (CTGF), which is a fibrotic mediator that is overexpressed in experimental models of hypertension [7]. Apart from this, DOCA, a precursor of aldosterone, binds to mineralocorticoid receptor and Journal of Hypertension 2014, 32:795–805 a Department of Medicine, Cardiology Section, Tainan Municipal An-Nan HospitalChina Medical University, Tainan, bDepartment of Medicine, China Medical University, Taichung, cDepartment of Medicine, School of Medicine, Fu-Jen Catholic University, d Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, e Department of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical University, Tainan, fDivision of Cardiology, Department of Internal Medicine, Taipei Medical University Hospital and gDepartment of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

Keywords: connective tissue growth factor, deoxycorticosterone acetate-salt hypertensive rats, endothelin, RhoA/ROCK

Correspondence to Dr Nen-Chung Chang, Department of Internal Medicine, School of Medicine, Taipei Medical University, 250, Wu-Hsing Street, Taipei, 11031, Taiwan. E-mail: [email protected]

Abbreviations: a-SMA, a-smooth muscle actin; Ccr, creatinine clearance; CTGF, connective tissue growth

J Hypertens 32:795–805 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Received 17 March 2013 Accepted 27 November 2013

DOI:10.1097/HJH.0000000000000092

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Lee et al.

translocates to the nucleus in order to modulate gene expression. There were conflicting results regarding the role of mineralocorticoid receptor in pathogenesis of renal injury in the DOCA-salt model. It was uncertain whether the profibrogenic effect of DOCA-salt was due to a direct mineralocorticoid receptor-mediated effect or was the result of indirect, for example, hypertension or saltmediated effects. The molecular mechanisms for excessive DOCA-saltinduced collagen synthesis have not been fully clarified. RhoGTPases consist of six subfamilies, including the RhoArelated subfamily, the Rac1-related subfamily, and the Cdc42-related subfamily and others [8]. The RhoA activity and the subsequent Rho-kinase (ROCK) activity were significantly higher in DOCA-salt rats [9]. Very recently, Wang et al. [10] have shown that overexpression of RhoA in podocytes caused albuminuria. High levels of RhoA activity induced by inflammatory processes may cause podocyte injury [11,12]. Thus, ROCK inhibitors attenuate renal damage in a variety of experimental models [11,12]. Myofibroblasts are key effectors of the renal fibrosis process. Fully differentiated myofibroblasts are characterized by the expression of a-smooth muscle actin (a-SMA). The ROCK signaling pathway has been shown to play a key role in the regulation of myofibroblastic transformation [12–15]. In mouse kidneys with unilateral ureteral obstruction, treatment with a ROCK inhibitor attenuated renal interstitial a-SMA expression and fibrosis [12]. In order to address the possibility that RhoA is involved in the pathogenesis of renal injury in DOCA-salt hypertensive rats, we assessed RhoA activity in the kidneys. The purpose of this study was to evaluate whether renal damage is attenuated by administering a selective mineralocorticoid receptor antagonist, eplerenone; to test the differential response to endothelin receptor antagonists in renal injury through attenuated expression of CTGF and myofibroblast infiltration; and to assess the role of RhoA/ROCK in renal fibrosis using a RhoA inhibitor, C3 exoenzyme, and two different types of ROCK inhibitors, fasudil (1-(5-isoquinolinesulfonyl)-homopiperazine) and Y-27632 {trans-4-[(1R)-aminoethyl]-N-4-pyridinylcyclohexan-carboxamide}.

ABT-627 (5 mg/kg per day; Abbott Park, Illinois, USA), a selective ETA receptor antagonist; and A192621 (15 mg/kg per day; Abbott Park), a selective ETB receptor antagonist. The dose of ABT-627, a nonhypotensive dose, has been shown to be effective in reducing proteinuria [16]. The dose of A192621 was given according to previous studies [17]. The drugs were given orally by gastric gavage once a day. All rats were maintained in metabolic cages so that food and water intake could be monitored and urine could be collected. The drug intervention duration was designed to be 4 weeks. The rats in this section underwent blood pressure measurement, proteinuria excretion, biochemical analysis, histological studies, hydroxyproline measurement, western blot, and real-time quantitative reverse transcription-PCR (RT-PCR). Systolic arterial pressure was recorded by tail cuff plethysmography and a Millar catheter (n ¼ 5 or 6/group) as previously described [18].

Animals

Experiment 2 To investigate the multifaceted interactions among mineralocorticoid receptor, endothelin receptor, and ROCK in DOCA-salt hypertensive rats, we compared renoprotection with drugs including eplerenone (a mineralocorticoid receptor antagonist), bosentan (a nonspecific endothelin receptor blocker), and fasudil (a ROCK inhibitor). Four weeks after the start of DOCA-salt treatment same as in experiment 1, these Unx rats were randomly assigned into four groups: vehicle group; eplerenone (30 mg/kg per day); bosentan (10 mg/kg per day; Actelion Pharmaceuticals, Allschwil, Switzerland); and fasudil (136 mg/kg per day; Asahi Kasei, Tokyo, Japan). The therapeutic efficacy of eplerenone, bosentan, and fasudil has been previously demonstrated without hypotensive effects [19–21]. We tested the effect of fasudil because it is the only ROCK inhibitor practically available for long-term in-vivo use. The drugs were given orally by gastric gavage once a day. The drug intervention duration was designed to be 4 weeks. The rats in this section underwent proteinuria excretion measurement and western blot analysis. Animals were sacrificed at the age of 24 weeks, while under ketamine–xylazine (90 mg/kg–9 mg/kg, intraperitoneally). Blood was drawn from the aorta. The right kidney was rapidly excised, rinsed in a 0.9% NaCl solution, blotted dry, and weighed. A mid-coronal section of the kidney was embedded in paraffin for histological studies and hydroxyproline measurement. The remaining tissues were immediately frozen in liquid nitrogen and stored at 808C until further analysis.

Experiment 1 Male Wistar rats aged 16 weeks were anesthetized with ketamine–xylazine (90 mg/kg–9 mg/kg, intraperitoneally), and the left kidney was removed via a left flank incision. From the following day, these rats were treated twice weekly with DOCA suspended in corn oil and administered subcutaneously (25 mg/kg), and 1% NaCl was added to their tap water for drinking. Control rats were uninephrectomized (Unx), but not given DOCA and salt. Four weeks after the start of DOCA-salt treatment, these rats were randomly assigned into three groups: vehicle group;

Experiment 3 Although results of the above study showed that the amount of CTGF was significantly attenuated after administering ABT-627 (see Results section), the involved mechanism remained unclear. To examine whether RhoA/ROCK is involved in the level of CTGF, DOCA-salt rats were treated with C3 exoenzyme, fasudil, and Y-27632 to exclude the nonspecific activation of ROCK by other second messengers such as arachidonic acid and sphingosylphosphorylcholine [22]. Very recently, Rac1 has been shown to play a role in kidney injury in salt-sensitive hypertensive rats [23].

METHODS The study was approved by our institutional animal care committee, and all of the procedures were in accordance with our Institutes of Care and Use of Laboratory Animals.

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Endothelin receptor blockade and renal injury

Thus, to evaluate the importance of other small RhoGTPases in ETA receptor blockade-related CTGF level, toxin B was assessed. C3 is regarded as a RhoA subfamily inhibitor, whereas toxin B inhibits almost all members of the RhoGTPases, including RhoA, Rac1, and Cdc42-related subfamilies [24]. Eight weeks after the start of DOCA-salt treatment and Unx, right kidneys of male Wistar rats were isolated in the Langendorff perfusion and subjected to no treatment (vehicle), ABT-627 (3 mmol/l), C3 exoenzyme (1 mg/ml; Cytoskeleton Inc., Denver, Colorado, USA), toxin B (100 ng/ml; List Biologicals, Campbell, California, USA), Y-27632 (1 mmol/l; Calbiochem, San Diego, California, USA), or fasudil (5 mmol/l; Calbiochem). To establish Langendorff perfusion, the rats were anesthetized with ketamine–xylazine (90 mg/kg–9 mg/kg, intraperitoneally) and placed on a heating pad. Heparin was administered before kidney excision. The abdominal cavity was opened by a midline incision, and right kidney was excised as described previously [25], which can minimize ischemic period of the kidney. Each kidney was perfused through the renal artery with Krebs-Henseleit consisting of the following (in mmol/l), at a constant flow of 6 ml/min by a peristaltic pump: NaCl, 118.5; KCl, 4.7; NaHCO3, 25; KH2PO, 1.2; MgSO4, 1.2; glucose, 11; and CaCl2, 1.5. The oxygenation of Krebs-Henseleit was performed with carbogen (95% O2 and 5% CO2). The time interval between the opening of the thoracic cavity and initiation of perfusate flow to the kidney was less than 2 min. Although the halflife of the CTGF protein is not known exactly, it seems to be short because CTGF is a member of the Ctgf, Cyr61, Nov immediate early gene family [26]. Previous studies have studies have shown that CTGF was expressed as early as 1 h after wounding [27]. Thus, drug perfusion for 60 min was chosen. The doses of ABT-627, C3, toxin B, and fasudil were used as previously described [28–30]. At the end of the study, all kidneys (n ¼ 5, each group) in this section were used for western blot of CTGF.

Morphological injury score After embedding in paraffin, the kidneys were cut into 5-mm sections and stained with periodic acid Schiff (PAS) for scoring glomerulosclerosis as previously described [18]. Sirius red staining was performed for the histological evaluation of the interstitial area occupied by collagen fibrils as described previously [31]. The percentage of red staining, indicative of fibrosis, was measured in 10 fields randomly selected on each section (Image Pro Plus, California, USA). The value was expressed as the ratio of sirius red-stained fibrosis area to total area. All sections were evaluated under blind conditions without prior knowledge as to which section belonged to which rat.

Immunohistochemical analysis of connective tissue growth factor and a-smooth muscle actin An expanded Methods section is available in the Online Supplementary method (http://links.lww.com/HJH/A325). Renal cortical tissues for assessing fibrosis were incubated Journal of Hypertension

with anti-CTGF (Santa Cruz Biotechnology Inc, Santa Cruz, California, USA) and anti-a-SMA (Sigma, St. Louis, Missouri, USA) as described previously [18].

Western blot analysis of RhoA translocation and connective tissue growth factor An expanded Methods section is available in the Online Supplementary method (http://links.lww.com/HJH/A325). Antibodies to RhoA (Santa Cruz Biotechnology Inc) and CTGF (Santa Cruz Biotechnology Inc) were used from samples obtained from the renal cortex as described previously [18].

Real-time RT-PCR of connective tissue growth factor and cyclophilin An expanded Methods section is available in the Online Supplementary method (http://links.lww.com/HJH/A325). RT-PCR was performed as described previously [18]. For CTGF, the primers were 5’-GAGTGGGTGTGTGACGA GCCCAAGG-3’ (sense) and 5’-ATGTCTCCGTACATCTTCC TGTAGT-3’ (antisense). For cyclophilin, the primers were 5’-ATGGTCAACCCCACCGTGTTCTTCG-3’ (sense) and 5’-CGTGTGAAGTCACCACCCTGACACA-3’ (antisense).

Laboratory tests An expanded Methods section is available in the Online Supplementary method (http://links.lww.com/HJH/A325). After completion of tail blood pressure measurement, urine was collected for 24 h in individual metabolic cages. Plasma and urinary creatinine, urinary proteinuria excretion, creatinine clearance (Ccr, ml/min), and tissue hydroxyproline content were measured as described previously [18]. Renal samples for ET-1 measurements were collected and extracted. In brief, the renal cortex was homogenized and extracted in a cold buffer containing 0.1 mol/l Tris (hydroxymethyl)aminomethane HCl, pH 7.4, a protease inhibitor cocktail (Complete; Roche Diagnostics GmbH, Penzberg, Germany), and 3.5 mmol/l EDTA. The tissue extracts and blood samples were then partially purified by ethanol extraction. ET-1 was measured using an immunoassay (R&D Systems, Minneapolis, Minnesota, USA).

Statistics Results were presented as mean  SD. Statistical analysis was performed using the SPSS statistical package (SPSS, version 12.0; Chicago, Illinois, USA). Differences among the groups of rats were tested by a one-way analysis of variance (ANOVA). Subsequently, analysis for significant differences between the two groups was performed with a multiple comparison test (Scheffe’s method). P value less than 0.05 was considered statistically significant.

RESULTS Part 1: in-vivo study Body weight of DOCA-salt rats was lower than that of normotensive controls and was not significantly affected by treatment (Table 1). www.jhypertension.com

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Lee et al. TABLE 1. Comparative data on intra-arterial blood pressure, Ccr, blood and urinary parameters at the end of study DOCA-salt Parameters No. of rats Body weight (g) SBP (mmHg), intra-arterially DBP (mmHg), intra-arterially Ccr (ml/min) Proteinuria (mg/kg per 100 g BW) ET-1 Plasma (pg/ml) Tissue (pg/g)

Unx-control

Vehicle

ABT-627

A192621

10 425  16 103  5 65  6 3.84  0.45 72

10 366  15 162  11 112  9 1.86  0.21 135  18

10 369  14 162  10 105  12 2.96  0.28, 35  17,y

10 384  16 165  12 113  8 1.68  0.29 129  20

1.1  0.4 198  17

1.3  0.3 364  52

1.2  0.3 353  29

2.3  0.5 428  59

BW, body weight; Ccr, creatinine clearance; DOCA, deoxycorticosterone acetate; ET-1, endothelin-1; Unx, uninephrectomized. Values are mean  SD.  P < 0.05 compared with Unx-control group.  P < 0.05 compared with DOCA-salt groups treated with vehicle and A192621.

Deoxycorticosterone acetate-salt treatment causes increased blood pressure Figure 1 shows the evolution of the tail SBP measured by plethysmography. SBP was progressively elevated in the DOCA-salt rats. These findings were confirmed by direct blood pressure measurement from the arterial catheter at the end of the experiment (Table 1). Eight weeks after the start of DOCA-salt treatment, the SBP of vehicle rats was 162  11 mmHg, whereas that of the Unx-control group was 103  5 mmHg. Daily oral administration of ABT-627 or A192621 for 4 weeks did not significantly affect the blood pressure in systole and diastole.

was significantly increased in DOCA-salt-vehicle rats at 20 weeks of age (89  11 mg/day per 100 g body weight), and 24 weeks of age (135  18 mg/day per 100 g body weight) as compared with the Unx-control rats at 20 weeks of age (5  2 mg/day per 100 g body weight), and at 24 weeks of age (7  2 mg/day per 100 g body weight). These functional changes including Ccr and proteinuria were markedly overcome by treatment with ABT-627 in DOCA-salt rats. Rats treated with A192621 did not show improvement of Ccr and proteinuria compared with DOCA-salt-vehicle rats. Similar to ABT-627, eplerenone, bosentan and fasudil showed significantly attenuated proteinuria (Fig. 2b). There were significantly increased levels of ET-1 in the renal cortex in DOCA-salt-vehicle rats compared with Unx-control. A192621 administration had a significant increase of plasma ET-1 level compared with the Unxcontrol rats. Glomerular damage stained by PAS (Fig. 3, upper) and tubular fibrosis stained by sirius red (Fig. 3, lower) in renal tissue were performed. Specifically, the ETA receptor blocker, not the ETB receptor blocker, ameliorated the renal injury in the DOCA-salt model.

Deoxycorticosterone acetate-salt treatment causes kidney damage, which is ameliorated by inhibiting ETA receptor, mineralocorticoid receptor and Rho-kinase Table 1 and Fig. 2 summarize renal functional parameters at the end of the experimental period. Ccr rate was significantly reduced in DOCA-salt-vehicle rats compared with the Unx-control rats. There was no difference in proteinuria among any of the groups at the start of the study. Proteinuria 240

Systolic blood pressure (mmHg)

220 200 180 Unx-control

160

DOCA DOCA-ABT-627

140

DOCA-A19262

120

*

*

*

*

100 Treatment period

80 16

18

20

22

24

Weeks of age FIGURE 1 Time course in SBP measured by plethysmography. Values are mean  SD (n ¼ 5 or 6 per group). DOCA, deoxycorticosterone acetate. P < 0.05 vs. DOCA-salt at the same age.

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Endothelin receptor blockade and renal injury (a)

Proteinuria (mg/day per 100 g BW)

250

16 weeks of age 20 weeks of age

200

24 weeks of age

*

150

*

100

† * 50

0 UNX-control

DOCA-vehicle

DOCA-ABT627

DOCA-A192621

(b) 250

16 weeks of age 20 weeks of age 24 weeks of age

Proteinuria (mg/day per 100 g BW)

200

*

150

100

† *

50

† *

† *

0 UNX-Control

DOCA-Vehicle DOCA-Eplerenone DOCA-Bosentan

DOCA-Fasudil

FIGURE 2 Temporal profile of proteinuria as measured at the beginning of DOCA-salt administration (16 weeks of age or 0 weeks of DOCA-salt administration), 4 weeks (20 weeks of age), and 8 weeks (24 weeks of age) after DOCA-salt administration. (a) Proteinuria was significantly attenuated in rats treated with ABT-627 at the age of 24 weeks. (b) Proteinuria was significantly attenuated in rats treated with eplerenone, bosentan and fasudil at the age of 24 weeks. Values are mean  SD (n ¼ 10 per group). DOCA, deoxycorticosterone acetate. P < 0.05 vs. 20 weeks of age within the same group; yP < 0.05 vs. DOCA-salt-vehicle at the same age.

Quantification of changes in the collagen content by hydroxyproline assay confirmed the pathological observations. Renal collagen level was significantly elevated in DOCA-salt-treated rats compared with Unx-control rats (18.9  1.5 vs. 10.3  1.3 mg/mg dry tissue in Unx-control rats; P < 0.05). ABT-627 significantly reduced the effects of DOCA-salt treatment on renal collagen levels compared with A192621 (11.5  1.7 vs. 21.3  2.6 mg/mg dry tissue in A1921621; P < 0.05). Immunohistochemical analyses Connective tissue growth factor immunoreactivity was detected in the renal tubulointerstitial regions. CTGF immunostaining in DOCA-salt-vehicle rats was more potent than that in the Unx-control rats (Fig. 4, upper). Significant intensity of a-SMA was detected in glomeruli and the tubulointerstitium in DOCA-salt-vehicle rats compared with that in the Unx-control rats (Fig. 4, lower). The immunoreaction intensity for CTGF and a-SMA was significantly reduced in rats treated with ABT-627, but not A1921621. Journal of Hypertension

Effects of ETA receptor and mineralocorticoid receptor blockade on suppression of RhoA/Rhokinase activation The regulation of RhoA activity was studied in membrane and cytosolic protein preparations. DOCA-salt increased membrane RhoA level by 63  11% (Fig. 5), consistent with activation of RhoA. Treatment with ABT-627 decreased RhoA membrane level to 69  12% of vehicle. However, ABT-627 did not affect cytosol RhoA level compared with vehicle (Fig. 5). Apart from this, treatment with eplerenone decreased RhoA membrane level to 82  8% of vehicle (Fig. 6). Connective tissue growth factor protein and mRNA expression Western blot shows that the CTGF levels in the renal cortex were significantly higher in DOCA-salt-vehicle rats than in Unx-control rats (P < 0.0001; Fig. 5). Compared with DOCAsalt-vehicle rats, CTGF levels were significantly lower in DOCA-salt rats treated with ABT-627, eplerenone, bosentan and fasudil (Figs. 5 and 6). www.jhypertension.com

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Lee et al. Veh

UNX

ABT-627

A192621

PAS

Glomerulosclerosis index

2.5





2 1.5 1

**

0.5

*

0 UNX-Control

DOCA-Vehicle

Sirius red area fraction (%)

Sirius red

25



20

† 15 10 5

* 0 UNX-Control

DOCA-ABT-627 DOCA-A192621

DOCA-Vehicle

DOCA-ABT-627 DOCA-A192621

FIGURE 3 Representative light micrographs of PAS-stained renal cortex (upper, magnification 400) and sirius red-stained renal medulla (lower, magnification 400). DOCA-salt-vehicle rats show significant glomerular damage and tubulointerstitial fibrosis with amelioration after treatment with ABT-627. In contrast, treatment with A192621 did not improve structural damage. (a) Unx-control; (b) DOCA-salt-vehicle; (c) DOCA-salt-ABT-627; (d) DOCA-salt-A192621. Bar ¼ 50 mm. Quantitative analysis is presented as mean  SD (n ¼ 9 per group). DOCA, deoxycorticosterone acetate; PAS, periodic acid Schiff. P < 0.05 compared with all DOCA-salt rats; yP < 0.05 compared with DOCA-salt rats treated with ABT-627.

Veh

UNX

ABT-627

A192621

CTGF

CTGF area fraction (%)

5





4 3 2 1

*

0 UNX-Control

DOCA-Vehicle

DOCA-ABT-627

DOCA-A192621

Intensity of a-SMA staining inglomeruli

α-SMA

40

† †

30

20

10

* 0 UNX-Control

DOCA-Vehicle

DOCA-ABT-627

DOCA-A192621

FIGURE 4 Immunohistochemical staining for CTGF (upper, magnification 400) and a-SMA (lower, magnification 400). Normal rat kidney demonstrating few constitutive CTGF peptide levels and a-SMA (brown staining). CTGF was localized mainly in tubular epithelial cells. DOCA was associated with increases in the area of a-SMA-positive staining, a marker of myofibroblasts, in the glomerular tuft. Quantification of a-SMA in the glomeruli by image analysis of immunostained slides after conversion to gray scale. The color intensities are expressed in an arbitrary gray scale of 0–100, with 0 representing zero intensity (white) and 100 representing maximal intensity (black). (a) Unx-control; (b) DOCA-salt-vehicle; (c) DOCA-salt-ABT-627; (d) DOCA-salt-A192621. Bar ¼ 50 mm. Quantitative analysis is presented as mean  SD (n ¼ 9 per group). a-SMA, a-smooth muscle actin; CTGF, connective tissue growth factor; DOCA, deoxycorticosterone acetate. P < 0.05 compared with all DOCA-salt rats; yP < 0.05 compared with DOCA-salt rats treated with ABT-627.

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Endothelin receptor blockade and renal injury Veh

ABT A192621

Mem RhoA Cyt RhoA CTGF β-actin

3

CTGF area fraction (%)

Ctl

*

*

2

1

0

CTGF area fraction (%)

2

1

0

CTGF area fraction (%)

3

* 2

*

1

0 UNX-Control

DOCA-Vehicle

DOCA-ABT-627

DOCA-A192621

FIGURE 5 In-vivo effect of ETA receptor and ETB receptor blockade on RhoA translocation and CTGF levels by western blotting. Relative abundance was obtained by normalizing the protein density against that of b-actin. Each column and bar represents mean  SD. Quantitative analysis is presented as mean  SD (n ¼ 5–6 per group). Each point is an average of three separate experiments. CTGF, connective tissue growth factor; Cyt, cytosolic; DOCA, deoxycorticosterone acetate; Mem, membrane; Unx, uninephrectomized. P < 0.05 compared with Unx-control and DOCA-salt-ABT-627-treated group.

PCR amplification of the cDNA revealed that the CTGF mRNA levels showed a 1.8-fold up-regulation in DOCA-saltvehicle rats compared with Unx-control rats (P < 0.0001; Fig. 7) in the renal cortex. In DOCA-salt rats treated with ABT-627, the CTGF mRNA levels were significantly decreased compared with those in DOCA-salt-vehicle rats. Thus, the mRNA levels of CTGF changed in parallel to the tissue peptide levels, implying that the production of CTGF is a critical regulation step for its local activation.

Part 2: RhoA/Rho-kinase axis up-regulates connective tissue growth factor levels in the perfused rat kidney To study whether inhibition of RhoA membrane level contributes to the down-regulation of CTGF protein by ABT-627, CTGF level was assessed in DOCA-salt rats in the presence and absence of the cell-permeative RhoA inhibitor C3 exoenzyme or the two ROCK inhibitors Y-27632 and fasudil. We found that C3 exoenzyme, Y-27632, and fasudil prevented DOCA-salt-induced increase in CTGF protein content (Fig. 8), a similar effect of ABT-627 on CTGF level. The extent of CTGF inhibited by administering C3 was similar to that in toxin B. These data indicate that activation of RhoA/ROCK pathway, not other small RhoGTPases, is responsible for a relative increase of CTGF level.

DISCUSSION In the present study, we demonstrate that ET-1 binding with the ETA receptor after administering DOCA-salt stimulates Journal of Hypertension

RhoA translocation and activates ROCK, resulting in increased expression of CTGF and collagen formation. Our results were consistent with the beneficial effects of ABT-627 treatment, as documented structurally by reduction in glomerulosclerosis and tubulointerstitial fibrosis, molecularly by renal CTGF protein and mRNA levels, biochemically by tissue ET-1 and hydroxyproline levels, and functionally by improvement of proteinuria and renal clearance. Indeed, our results were consistent with previous findings, showing that ET-1, via ETA receptors, is involved in CTGF synthesis by activation of RhoA/ROCK pathway in vascular smooth cells [32]. The effect of mineralocorticoid receptor, ETA, combined ETA and ETB receptor and ROCK antagonism on attenuated renal injury was supported by the following four lines of evidence (Fig. 9). 1. Many of these adverse effects of DOCA-salt treatment could be prevented by mineralocorticoid receptor antagonism, indicating that this receptor is involved in the pathophysiological processes through blood pressure-independent mechanisms. The dosage of eplerenone (30 mg/kg per day) in the present study was chosen in an attempt to lower the blood pressure only to a minor extent, if at all. The fact that proteinuria, RhoA translocation and CTGF levels were reduced by the mineralocorticoid receptor antagonist indicates that the mineralocorticoid receptor in the kidney – and not the hypertension itself – causes these types of damage. We made exactly this www.jhypertension.com

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Lee et al. Ctl

Veh

Epl

Bos

Fas

Veh CTGF

Cyt RhoA

β-actin

CTGF β-actin

Mem/cyt RhoA ratio

2

*

*

1.5 1 0.5

Normalizedrelative abundance of CTGF

0 2 1.5



1 0.5 0 Vehicle

Eplerenone Bosentan

Fasudil

FIGURE 6 In-vivo effect of eplerenone (a mineralocorticoid receptor antagonist), bosentan (a nonspecific endothelin receptor blocker), and fasudil (a ROCK inhibitor) on RhoA translocation and CTGF levels by western blotting. Relative abundance was obtained by normalizing the protein density against that of b-actin. Quantitative analysis is presented as mean  SD (n ¼ 5–6 per group). Each point is an average of three separate experiments. Bos, bosentan; CTGF, connective tissue growth factor; Cyt, cytosolic; DOCA, deoxycorticosterone acetate; Epl, eplerenone; Fas, fasudil; Mem, membrane; ROCK, Rho-kinase; Unx, uninephrectomized; Veh, vehicle. P < 0.05 compared with Unx-control and DOCA-salt groups treated with eplerenone and bosentan; yP < 0.05 compared with DOCA-salt rats treated with vehicle.

observation in the perfused kidney, where we could show that CTGF levels were independent of hemodynamics. Our results were consistent with previous findings, showing that the effect of mineralocorticoid/salt on renal fibrosis can be blocked by treatment with the selective mineralocorticoid receptor antagonist, eplerenone [33].

C3

Toxin Y-27632 Fasudil

3

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FIGURE 8 In a rat isolated kidney model, the effect of inhibition of RhoGTPases with C3 exoenzyme and toxin B, and ROCK with Y-27632 and fasudil on CTGF protein levels. Each point is an average of three separate experiments. Results are mean  SD (n ¼ 5 per group). CTGF, connective tissue growth factor; ROCK, Rhokinase. P < 0.05 compared with vehicle.

2. DOCA-salt was associated with increased ET-1 and renal injury. The renal endothelin system is indeed activated in our model after DOCA and high-NaCl dietary exposure. Endothelin systems are responsible for renal impairment in terms of functional and DOCA-salt

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FIGURE 7 Renal CTGF mRNA levels. Cyclophilin mRNA levels served as internal standard. All samples were run in triplicate. DOCA-salt-vehicle-treated rats show significant increased CTGF expression with amelioration after treatment with ABT627. Results are mean  SD (n ¼ 9 per group). CTGF, connective tissue growth factor; DOCA, deoxycorticosterone acetate. P < 0.05 compared with all DOCAsalt rats; yP < 0.05 compared with DOCA-salt rats treated with ABT-627.

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Proteinuria FIGURE 9 Reaction sequences leading to attenuated proteinuria. The diagram summarizes the biochemical, immunohistochemical, molecular, and pharmacological evidence presented in this report. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines. CTGF, connective tissue growth factor; ERA, endothelin receptor antagonist; ET-1, endothelin-1; MR, mineralocorticoid receptor.

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Endothelin receptor blockade and renal injury

structural changes in DOCA-salt rats. DOCA-salt hypertensive rats had a drastically impaired renal function compared with Unx-control rats as determined by Ccr and proteinuria. The kidney damage of this model is characterized by glomerulosclerosis and tubulointerstitial fibrosis. Previous studies have shown that selective ETA receptor blockade can promote ETB receptor-mediated effects [34]; thus, the beneficial effect of renal injury with ABT-627 can originate from both mechanisms. However, no further worsening of renal injury in DOCA-salt rats treated with A192621 compared with vehicle was against a role of ETB receptor in the pathogenesis of renal injury. Although we noted that treatment with ABT-627 markedly improved renal functions in DOCA-salt hypertensive rats. On the contrary, Allcock et al. [35] have recently observed that ETA receptor antagonism with A-127722 does not improve the decreased renal function in DOCA-salt hypertensive rats. The reason for this discrepancy is unknown, but differences in experimental protocol (e.g. drug administration in drinking water in prior study vs. by gavage in our study) may influence the efficacy of the drug. 3. RhoA/ROCK pathway participates in regulating CTGF levels. Rats after administering DOCA-salt increase guanosine triphosphate (GTP)-bound Rho activities as shown by increased membrane Rho. RhoA changes localization from the cytosol to the membrane, indicating RhoA/ROCK activation. A significant decrease in RhoA content in the cytosol fraction was not detected in our study, most likely because of the relative abundance of RhoA in the cytosol fraction. These results are in agreement with previous studies indicating that aldosterone induces renal injury associated with ROCK activation [36]. Our ex-vivo studies have demonstrated that DOCA-salt regulated CTGF via RhoA/ROCK activation, as shown by inhibition of RhoA with C3 exoenzyme, and ROCK inhibition with Y-27632 and fasudil. The exact mechanisms of RhoA-mediated CTGF expression after DOCA-salt have not been completely understood. Activation of RhoA and ROCK leads to increased Factin assembly [37]. The resulting depletion of G-actin triggers activation of the transcription factor serum response factor, an important transcriptional activator of CTGF. Disruption of the actin cytoskeleton by cytochalasin D interfered with induction of CTGF [38]. Overexpression of constitutively serum response factor significantly increased CTGF protein synthesis [37]. It suggested that the actin cytoskeleton and serum response factor are involved in RhoA-mediated CTGF synthesis. Further studies are necessary to confirm this speculation. ET-1 activates several intracellular mediators, including small G proteins [39]. The Rho family of GTP-binding proteins contains many geranylgeranylated proteins, such as Rho, Rac, and Cdc42 [8]. In contrast with our findings, Rac1 has been shown to play a role in renal injury in rodent models of salt-sensitive hypertension and Rac1 inhibition prevented hypertension and renal Journal of Hypertension

damage [24]. Our results showed no additional inhibition of CTGF levels in kidneys treated with toxin B compared with C3. RhoGTPase signaling pathways interact with each other and a cross-talk between the small GTPases Rac1 and RhoA was observed [40]. Thus, it is likely that Rac1 may exert its effect though a RhoA-dependent pathway that has to be studied further. 4. Our results suggest that the beneficial effect of ABT627 on renal fibrosis could be attributable to RhoA inhibition. The findings were further confirmed by the analysis of a-SMA immunohistochemical staining, showing that ABT-627 administration significantly attenuated myofibroblast accumulation. The result was consistent with the notion that blockade of RhoGTPase activation results in the inhibition of renal epithelial–myofibroblast transdifferentiation. Experiments were performed with the C3 exoenzyme (a RhoA inhibitor), Y-27632 (a specific ROCK inhibitor), and fasudil (a nonselective ROCK inhibitor) to confirm whether directly blocking RhoA/ROCK pathway leads to antifibrotic cell signaling. The above three treatments inhibited the increases in CTGF levels as those seen after ABT-627 treatment. Previous findings showed that ETA receptor blockade may have a beneficial effect during the initial week of DOCA-salt treatment as suggested by the improved salt and water balance during that period. Our results extended previous findings and showed that ETA receptor antagonism can restore the diminished renal function and structural changes observed at the established hypertensive stage 4 weeks of treatment with DOCA-salt. The role of ETB in the development of renal injury is controversial. We showed that ETB receptor blockade markedly increased circulating ET-1 concentrations and thereby may stimulate the unblocked ETA receptor. To exclude the possibility that many physiological effects ascribed to ETB receptor blockade are actually due to ETA receptor activation, we evaluated the effects of combined (ETA þ ETB) receptor blockade. The combination of ABT-627 and A192621 is best suitable for this study. Unfortunately, because of the limited availability of the ETB receptor antagonist A192621, we can only perform the experiment with bosentan, a nonspecific endothelin blocker. Surprisingly, compared with vehicle, proteinuria was not exacerbated in A192621-treated DOCA-salt rats, indicating lack of a protective role for ETB receptors in proteinuria. Previous studies have shown that selective blockade of ETB exacerbates renal injury in the remnant kidney model and DOCA-salt rats [41]. On the contrary, blocking ETB may be beneficial as well, because ETB is reported to mediate proliferation of tubular epithelial cells [42]. The relation between ETB and renal function was more complex than previously thought and needed further study. Our results showed that mineralocorticoid receptor may contribute to the progression of renal injury through an up-regulation of RhoA which can favor the activation of the CTGF pathway. Mineralocorticoid receptor has been www.jhypertension.com

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Lee et al.

shown to be involved in aldosterone-mediated induction of ET-1 mRNA [43]. The ET-1 gene is a direct aldosteroneregulated target gene in the kidney. Administration of potassium canrenoate, a mineralocorticoid receptor antagonist, blocked induction of ET-1 mRNA in the kidney [43]. Although the role of aldosterone in renal ET-1 production has been firmly established, the role of salt can not be excluded. Previous studies have shown that the expression of endothelin-converting enzyme and ET-1 mRNA was significantly higher in the renal medulla of rats exposed to high salt intake than in rats on normal salt diet [44]. High salt intake is a consistent stimulus for renal ET-1 production. Thus, increased renal ET-1 production as seen in DOCAsalt-treated animal models is likely to be due to the synergistic effect of salt, aldosterone, and other confounding factors rather than to the elevated circulating aldosterone levels alone [45].

Other mechanisms Although the present study suggests that the mechanisms of ETA receptor blockade-induced attenuation of renal injury may be related to attenuated RhoA/ROCK-dependent CTGF expression, other potential mechanisms need to be studied such as direct antioxidant effect and increased nitric oxide. ET-1 rapidly induced the recruitment of c-fos to the activating protein-1 (AP-1) site located next to the 5’ flanking region of the CTGF gene, thus indicating the role elicited by the AP-1 complex in the regulation of CTGF transcription by ET-1 in cardiomyocytes [46]. Thus we cannot exclude the possibility that ABT-627 acting as an antioxidant [47] may attenuate CTGF expression by inactivating the AP-1 complex, independent of the RhoA/ROCK pathway. Apart from this, selective ETA receptor blockade was associated with increased nitric oxide levels [48]. Nitric oxide has been shown to act as a strong repressor of CTGF expression through a cyclic guanosine monophosphate-dependent manner [49]. Thus, ABT-627 may attenuate CTGF expression by increasing nitric oxide.

Clinical implications Although blockade of the angiotensin system led to a dramatic reduction of proteinuria, it was not reduced to normal levels. Mineralocorticoid-dependent hypertension, as found in a variety of human hypertensive disorders, with an overall prevalence of 10–15%, leads to increased CTGF levels and renal damage in the model of the DOCA-salt hypertensive rat. These findings may provide novel insights into the pathogenesis of mineralocorticoid/salt-induced renal injury and may help to explain the beneficial effects of mineralocorticoid receptor inhibition in clinical trials for patients with hyperaldosteronism. Our results point to the need to evaluate new therapeutic approaches that are able to halt renal disease progression in addition to inhibition of the angiotensin II. These results were consistent with previous studies, showing that ETA receptor antagonism can reduce proteinuria on top of that achieved with optimal treatment with inhibitors of the renin–angiotensin system in patients with proteinuric renal disease [50]. Interference with the endothelin system might provide an alternative beneficial approach. Renal fibrosis is a major complication 804

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associated with the development and progression of hypertension. Reversal of the renal fibrotic process after administering ETA receptor blockers is expected to halt the progression of renal failure. Our results were consistent with the data from the diabetic patients with proteinuria, showing that ABT-627 reduced albuminuria [51]. In conclusion, these studies suggest that RhoA functions as a molecular switch in hypertension and thus the signaling pathway leading to and including RhoA/ROCK would be an appropriate target for therapeutic intervention. The results of the present study would provide significant implications on the treatments of fibrotic complications associated with hypertension and further important information for treating hypertensive patients with proteinuria.

ACKNOWLEDGEMENTS We thank Abbott Laboratories for ABT-627 and A192621 compounds. The work was supported by the grants of China Medical University (CMU102-S-01), and National Health Research Institutes (NHRI-EX100-9841SI), Taiwan.

Conflicts of interest There are no conflicts of interest.

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Reviewer’s Summary Evaluation

fibrosis and dysfunction. The main weakness is that the study was conducted in an animal model of severe hypertension so the findings may not be applicable to clinical hypertension.

Referee 2 The major strength of this paper is identification of a novel pathway involved in hypertension-associated renal

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Rho-kinase signalling in deoxycorticosterone acetate-salt hypertensive rats.

Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxyc...
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