Adult male Sprague Dawley rats (190 to 210 g, Harlan Winkelmann, Borchen, Germany), were treated with DOCA-salt or Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) to induce hypertension. To compare the pharmacological potency of ROCK-inhibition with the current anti-hypertension drugs, the individual blood pressure lowering effect of the respective compounds was measured in spontaneous hypertensive rats (SHR). Oral application of SAR407899 at 3 mg/kg lowered blood pressure in conscious telemetred SHR by 26 ± 4 mmHg (n = 10), which was comparable to the action of amlodipine at 3 mg/kg (blood pressure reduction by 33 ± 8 mmHg, n = 10) and ramipril at 1 mg/kg (blood pressure reduction by 21 ± 7 mmHg, n = 10). Therefore, the animals were divided into the following groups: (1) Control; (2) DOCA or LNAME; (3) DOCA or LNAME + SAR407899 at 3 mg/kg; (4) DOCA or LNAME + SAR407899 at 10 mg/kg; (5) DOCA or LNAME + ramipril at 1 mg/kg; and (6) DOCA or LNAME + amlodipine at 3 mg/kg. All DOCA-salt treated animals underwent a unilateral nephrectomy, received a subcutaneous injection of DOCA (30 mg/kg; Sigma Chemical, St. Louis, MO, United States) dissolved in sesame oil once a week and 1% NaCl in the drinking water ad libitum. All LNAME groups received 40 mg/kg per day LNAME in the drinking water ad libitum. After a 4-wk treatment, the animals were placed into metabolic cages and 24 h-urine samples and blood samples were taken to analyze kidney function. Urinary and plasma creatinine levels were determined using standard kits (Roche diagnostics, Basel, Switzerland) on a Hitachi 912 E analyzer (Hitachi, Mountain View, Calif., United States). Urinary albumin was measured using a standard kit from Progen (Mikroflural, Progen, Heidelberg, Germany) and was normalized to urinary creatinine concentrations. After 5 wk of treatment, measurements of: (1) blood pressure (invasively, two hours after the last treatment); (2) organ weights; and (3) endothelial function were taken. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication no. 85-23, revised 1996).

The animals were anaesthetized with an intraperitoneal injection of thiopental (0.1 g/mL per 100 g body weight). The common carotid artery was catheterized with a heparinized microcatheter (diameter 1.6 mm, Vycon, France). Blood pressure was measured with a Hugo Sachs hemodynamic system (March-Hugstetten, Germany) using the software Hemodyn.

Assessment of in vitro vascular function was performed as described earlier[21,25,26]. Heart function was determined using a Langendorff-setup in the working heart perfusion mode. This technique allows the heart to perform its physiological pumping action, i.e., it performs pressure/volume work. The working heart technique therefore provides a complete analysis of heart function. Heart power is an integrative parameter and is calculated by formula (1):

HW (J) = 133.3 [(N/m²)/mmHg] × (ALPmean-PLPmean) (mmHg) × SV (m³) (1)

+ 0.5 × 1.004 (kg/m³) × SV (m³) × {SV (m³)/[π× r² (m²) × ET (s)]}²

where HW-heart power, ALPmean-afterload pressure, PLPmean-preload pressure, SV-stroke volume, and ET-Ejection time. If not otherwise indicated, chemicals were obtained from Sigma (Deisenhofen, Germany).

Real-time quantitative PCR was performed using the QuantiTect Probe RT-PCR Kit (Qiagen, Hilden, Germany). Each sample was assayed in quadruplicate. For relative quantification of gene expression, the ΔCt method was used with GAPDH as a control. Amplification of the target and housekeeping genes was detected simultaneously using differently fluorescent-labeled Taq Man probes (Col1A1: Rn00801665_g1, CD3: Rn01417941_g1 and CD68: Rn01495634_g1), which were obtained from Applera/Applied Biosystems (Foster City, United States). Amplification linearity of the target and housekeeping genes within the multiplex RT-PCR was assessed by performing RT-PCR reactions with dilutions of the templates. RT-PCR reactions and data acquisition was performed using the iCycler-iQ-Thermocycler (Bio-Rad Laboratories GmbH, Munich, Germany). Relative gene expression was calculated as fold induction vs control samples.

Heart and kidneys underwent a standard fixation procedure and standard haematoxylin-eosin and sirius red staining. The hearts and kidneys were analyzed with regard to incidence and extent of fibrosis, inflammatory events, glomerulosclerosis and tubular atrophy. A semi-quantitative score was assigned to each specimen by an experienced pathologist ranging from 1 (minimal changes) to 5 (marked alterations) at a standard magnification of 4 to 20-fold. All histopathological analyses were performed in a blinded fashion. Anti-podocin staining was performed using anti-podocin antibodies (Sigma-Aldrich, United States).

All values are given as the mean and standard error of mean. Normality of the distribution and the homogeneity of variance were checked using the Levene test. For group comparisons, one-way analysis of variance (ANOVA) or two-way ANOVA was performed followed by Dunnett’s post-hoc test using the SAS version 8.2 software. Differences between groups were considered significant if P < 0.05; n represents the number of specimens or animals tested.

The statistical methods of this study were reviewed by and complies to the standard of Sanofi-Aventis GmbH Deutschland.

The long-term effects of SAR407899 in DOCA- and LNAME-induced hypertension were compared to those of the current standard anti-hypertensive drugs, namely ramipril (ACE-inhibitor) and amlodipine (calcium channel blocker, CCB). Figure 1 depicts the effects of SAR407899 on body weight of the DOCA- and LNAME-hypertensive animals. Treatment with SAR407899 was well tolerated and showed a significant protective effect on body weight in both hypertensive animal models (Figure 1A and C). Factors involved in the continuous body weight loss are not known and most likely depend on hypertension related end-organ damage, including proteinuria. Ramipril showed protective effects on body weight only in the LNAME model (Figure 1D), whereas amlodipine significantly protected the DOCA hypertensive animals from body weight loss (Figure 1B).

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Figure 1 Effect of SAR407899 on body weight in deoxycorticosterone acetate and Nω-Nitro-L-arginine methyl ester hydrochloride hypertensive animals. A and B: Body weight of deoxycorticosterone acetate (DOCA) rats. A significant decrease in body weight was observed after 28 d; A: Effect of SAR407899 at 3 mg/kg and 10 mg/kg on body weight. Both doses protected DOCA rats against body weight loss; B: Effect of ramipril at 1 mg/kg and amlodipine at 3 mg/kg on body weight. Only amlodipine showed protective effects on body weight loss; C and D: Body weight of Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) rats. A significant decrease in body weight was observed after 22 d. C: SAR407899 at 10 mg/kg significantly protected LNAME rats from body weight loss; D: Effect of ramipril at 1 mg/kg and amlodipine at 3 mg/kg on body weight. Only ramipril showed significant protective effects on body weight loss.

Figure 2 demonstrates the effects of SAR407899 (3 mg/kg and 10 mg/kg), ramipril (1 mg/kg) and amlodipine (3 mg/kg) on blood pressure in the DOCA- (Figure 2A) and LNAME-treated rats (Figure 2B). SAR407899 effectively reduced blood pressure in both hypertensive models. Because the DOCA-induced hypertensive model is characterized by hypervolemia and resistance to ACE-inhibition, it was not surprising that ramipril had no significant effect on blood pressure. At the dose employed, amlodipine non-significantly lowered blood pressure in the DOCA rats. In the LNAME rats, all treatments significantly lowered blood pressure. In comparison to amlodipine and ramipril, SAR407899 showed superior blood pressure lowering effects at both doses in the LNAME rats. Thus, only SAR407899 was able to control blood pressure efficiently in both models and was therefore superior to the reference substances.

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Figure 2 SAR407899 efficiently lowers blood pressure, proteinuria and mortality in hypertensive rats. Invasive measurement of blood pressure in deoxycorticosterone acetate (DOCA) (A) and in Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) (B) rats. At the given dose, SAR407899 showed superior blood pressure lowering effect in comparison to ramipril and amlodipine in both animal models; C and D: Assessment of kidney function in DOCA and LNAME rats. SAR407899 showed significant protective effects on the kidneys in both models, whereas ramipril reduced albuminuria only in the LNAME model. Amlodipine had no therapeutic effect on kidney in either model.

Kidney function (Figure 2C and D) was assessed by the measurement of albuminuria. In both models, SAR407899 dose-dependently reduced albuminuria. Ramipril significantly reduced albuminuria in only the LNAME model but not in the DOCA model. At the dose administered, amlodipine did not significantly reduce albuminuria in the DOCA or LNAME models. The lack of efficacy of amlodipine has been linked to the inability of CCB’s to dilate renal efferent vessels, which is critical for improvement of the renal microcirculation.

Figure 3 illustrates the effect of SAR407899 and of reference substances on heart function (A and B), measured in isolated Langendorff perfused hearts in the working heart mode and on vessel function in vitro (C and D). Hearts of hypertensive the DOCA rats and the LNAME rats were functionally compromised. At an afterload pressure of 40 mmHg (basal afterload), a similar heart performance was found in all groups; however, at higher afterload pressures (60 and 80 mmHg), a significant reduction was found in heart function from the DOCA- and LNAME-treated rats. Long-term treatment with SAR407899 restored heart function in both groups. In contrast, treatment with either amlodipine or ramipril had no significant effect. Endothelial function was found to be severely compromised in the DOCA rats. Figure 3C and D show the effect of SAR407899 and the reference substances on endothelial function. Long-term treatment with SAR407899 improved endothelial function in a dose-dependent manner (Figure 3C). As expected, ramipril had no effect on endothelial function of arteries from the DOCA-induced hypertensive rats (Figure 3D). Amlodipine had similar protective effects on endothelial function in the DOCA hypertensive rats (Figure 3D). The endothelial function of arteries of the LNAME-treated rats has not been assessed because LNAME exerts a potent and long lasting inhibition of the endothelial nitric oxide synthase.

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Figure 3 Effect of SAR407899 on heart and endothelial function. Measurement of heart function of isolated hearts of deoxycorticosterone acetate (DOCA) (A) and Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME)-treated rats (B). SAR407899 showed a significant improvement of heart function compared to reference substances at both doses and in both models; C and D: Measurement of endothelial function of aortas of DOCA rats treated with SAR407899 (C) or with reference substances (D). Ramipril treatment had no effect on endothelial function. Long-term treatment with SAR407899 improved endothelial function in a dose-dependent fashion. Amlodipine had similar protective effects as SAR407899.

Histological examination of the hearts of the DOCA-(Figure 4A-F) and LNAME-treated (Figure 4G-L) rats revealed a prominent perivascular fibrosis, massive infiltration of leukocytes into the interstitium and sclerotic changes. Figure 4A-C and 4G-I shows sirius red staining of DOCA- (Figure 4B) and LNAME-(Figure 4H) induced heart fibrosis in comparison to control (Figure 4A and G) and SAR407899 treatment (Figure 4C and I). Figure 4D-F and 4J-L shows haematoxylin eosin staining of DOCA- (Figure 4E) and LNAME-(Figure 4K) induced heart fibrosis in comparison to control (Figure 4D and J) and SAR407899 treatment (Figure 4F and L). In the hearts of the DOCA and LNAME rats, SAR407899 treatment at 10 mg/kg abolished the development of fibrosis (protection with SAR407899 at 3 mg/kg was less pronounced and data not shown). Chronic treatment of the LNAME hypertensive rats with SAR407899 attenuated leukocyte infiltration and perivascular fibrosis. Figure 4M and N summarize heart lesions according to the scoring procedure. The heart lesions of the DOCA rats were significantly reduced by SAR407899 at 10 mg/kg. Both reference compounds showed only marginal non-significant effects on heart lesions (Figure 4M). SAR407899 (at 3 and 10 mg/kg) and ramipril significantly diminished heart lesions in the LNAME-treated rats (Figure 4N). Histological analysis of kidneys from the DOCA- (Figure 5A-F) and LNAME- (Figure 5G-L) treated rats revealed severe lesions of glomeruli and tubuli. Figure 5A-C shows haematoxylin eosin staining in kidneys of DOCA- and of LNAME-treated rats (Figure 5G-I). Figure 5D-F shows podocin staining in kidneys of DOCA- and of LNAME-treated rats (Figure 5J-L). In kidneys from the DOCA rats (Figure 5B), severe destruction and sclerotic changes of glomeruli were associated with fibrinous discharges into the capsule, swelling of lobuli and multiple fibrotic events. In comparison to control (Figure 5A), a strong hypertrophy of glomeruli was detectable in the kidneys of the DOCA- treated animals (Figure 5B). Podocin staining (Figure 5D-F)of the DOCA rat kidneys revealed a drastic loss of podocin positive cells (podocytes) upon DOCA treatment (Figure 5E) in comparison to control (Figure 5D). Chronic treatment with SAR407899 attenuated these glomerular (much stronger staining of podocytes), vascular, and interstitial changes (Figure 5F). In contrast to kidneys of control animals (Figure 5G), in the kidneys from the LNAME hypertensive animals (Figure 5H), many changes were detected, including fibrotic changes, infiltration of leukocytes, hypertrophy of glomeruli, marked thickening and inflammation of the vascular wall, hyperplasia and degeneration of tubuli, and tubulointerstitial changes with inflammatory cell infiltration. Again, chronic treatment with SAR407899 attenuated these pathophysiological changes (Figure 5I).

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Figure 4 Histological examination of the effect of SAR407899 on the heart. Sirius red staining of hearts of deoxycorticosterone acetate (DOCA) (A-C) and Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) rats (G-I) showed a strong induction of myocardial fibrosis. Upon treatment with SAR407899 at 10 mg/kg, significant protective effects were observed. Haematoxylin eosin staining of hearts of DOCA (D-F) and LNAME rats (J-L) showed perivascular fibrosis, massive infiltration of leukocytes into the interstitium and sclerotic changes. SAR407899 treatment at 10 mg/kg attenuated multifocal fibrosis, perivascular fibrosis and leukocyte infiltration. M and N: Summary of heart lesions and the effect of SAR407899 and reference substances in DOCA (M) and LNAME rats (N). A, D, G, J: Control; B, E: DOCA; H, K: LNAME; C, F, I, L: SAR407899 10 mg/kg.

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Figure 5 Histological examination of the effect of SAR407899 on the kidney. A-C: Haematoxylin eosin staining of normal glomeruli in control rats (left), sclerotic changes, dilation and hypertrophy of glomeruli of deoxycorticosterone acetate (DOCA) rats (center) and protective effects of SAR407899 at 10 mg/kg (right); D-F: Podocin staining in kidneys. Upon DOCA treatment, massive loss of podocytes can be detected. SAR407899 at 10 mg/kg exerts a protective effect in the kidneys of DOCA rats and rescues podocytes; G-I: Haematoxylin eosin staining of normal glomeruli in control rats (left), severe fibrotic changes, infiltration of leukocytes, and hypertrophy of glomeruli of Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) rats (center), protective effects of SAR407899 at 10 mg/kg (right); J-L: Loss of podocytes upon LNAME treatment. SAR407899 at 10 mg/kg protected against loss of podocytes. Summary of kidney lesions and effect of SAR407899 and reference substances in DOCA (M) and LNAME rats (N). A, D, G, J: Control; B, E: DOCA; H, K: LNAME; C, F, I, L: SAR407899 10 mg/kg.

Figure 5K shows the fading of podocin staining upon LNAME treatment indicating that podocin positive cells are damaged. Figure 5J demonstrates podocin staining in control animals. Long-term treatment with SAR407899 attenuated glomerular, vascular, and interstitial changes (Figure 5L). Figure 5M and N summarize kidney lesions according to the scoring procedure. The kidney lesions of the DOCA rats were significantly reduced by SAR407899 at 3 and 10 mg/ kg. Both reference compounds showed only marginal non-significant effects on kidney lesions (Figure 5M). SAR407899 (at 3 and 10 mg/kg) and ramipril significantly diminished kidney lesions in the LNAME-treated rats (Figure 5N). Expression of genes characteristic of fibrosis and leukocyte infiltration of the kidneys of the DOCA- and LNAME-treated rats was measured using RT PCR (Figure 6A-F). The expression of collagen was significantly increased upon DOCA and LNAME treatment. SAR407899 at 10 mg/kg significantly reduced collagen expression in the kidneys of the DOCA rats and non-significantly in the kidneys of the LNAME rats (Figure 6A and B). Leukocyte infiltration into kidney tissue was quantified by measuring CD3 (T lymphocytes) and CD68 (macrophages) mRNA abundance in the kidneys of the DOCA and LNAME rats. Upon DOCA or LNAME treatment, both parameters were significantly induced, thus indicating significant leukocyte infiltration. SAR407899 at 10 mg/kg efficiently reduced leukocyte infiltration in the kidneys of the DOCA rats and non-significantly in the kidneys of the LNAME rats (Figure 6E and F), presumably by inhibition of leukocyte migration.

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Figure 6 Effect of SAR407899 on the expression of fibrotic and leukocyte genes in the kidney. Determination of collagen, T cell (CD3) and macrophage (CD68) expression in the kidneys of deoxycorticosterone acetate (DOCA) (A, C and E) and Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME) (B, D and F) rats. Expression of collagen was significantly increased upon DOCA and LNAME treatment. Upon DOCA or LNAME treatment, CD3 and CD68 abundance was induced, indicating that leukocytes infiltrated into the kidneys. SAR407899 treatment reduced expression of collagen and leukocyte genes in both models.

Large clinical trials have demonstrated that current treatment with angiotensin converting enzyme-inhibitors, Angiotensinreceptor-blockers or calcium channel blockers only modestly reduces the progression of chronic kidney diseases. Hypertension increases the risk of target organ damage, including heart hypertrophy, heart ischemia, kidney dysfunction or failure, cerebrovascular events and malfunction of the endothelial tissue.

Rho-kinase (ROCK) is considered an important target for a variety of cardiovascular diseases, including hypertension and hypertension-related end-organ damage. Several inhibitors of ROCK have been extensively used to evaluate the role of ROCK in cardiovascular physiology and pathology. However, these inhibitors have a moderate specificity, moderate potency and short duration of action in vivo that limit their clinical use. Therefore, the development of a more potent and specific inhibitor with a better pharmacokinetic profile is needed to explore the potential of ROCK inhibition in the therapy of hypertension and its complications.

The authors have identified a novel, potent and selective inhibitor of ROCK (SAR407899) and characterized its long-term effects in two animal models of hypertension.

ROCK-inhibition by the SAR407899 represents a new therapeutic option for the treatment of hypertension and its complications.

Rho-kinases (ROCK1 and ROCK2) are intimately involved in the transmission of contractile signaling within smooth muscle tissue. Upon activation of the small GTPase RhoA by ligand-bound specific GPCRs, ROCKs, the downstream effectors of RhoA, phosphorylate the myosin light chain phosphatase and the myosin regulatory light-chain itself, resulting in a net increase in activated myosin. This promotes smooth muscle contraction and actin cytoskeleton re-organization. Inhibition of ROCKs leads to relaxation of vascular smooth muscle cells and, consequently, to a decrease in blood pressure.

This article is particular meaningful for hypertension-related end-organ damage.