Lars Rothermund

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.

Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number.

Objective An inherited reduction in nephron number has been implicated in the development of salt-sensitive hypertension and end stage renal disease. The Munich Wistar Frömter (MWF) rat represents a genetic model with a 30–50% reduction of nephrons compared with normal rats. MWF rats develop spontaneous hypertension and increased urinary albumin excretion (UAE). We addressed the question whether the inherited defect in this model leads to salt-sensitive hypertension. Methods At the age of 6 weeks, we started male and female MWF/Fub rats and salt-resistant Lewis (Lew) reference rats on either a normal NaCl (0.2%) or a high NaCl (8%) diet (n = 8, each group). Systolic blood pressure (SBP) and UAE were measured at 14 weeks. Results Under a normal diet, MWF/Fub rats demonstrated significantly elevated SBP compared to Lew rats both in male (165 ± 2 versus 133 ± 3 mmHg, P <0.0001) and female (156 ± 3 versus 134 ± 3 mmHg, P <0.0001) rats. After high NaCl treatment, SBP was significantly higher in both male and female MWF/Fub rats (+55 mmHg and +36 mmHg, P <0.0001, respectively) compared with MWF/Fub under a normal diet. UAE was also significantly higher in male and female MWF/Fub rats after high NaCl excess (P <0.0005, respectively). In contrast, both SBP and UAE remained unchanged in response to high NaCl in Lew rats. Conclusions Our findings demonstrate that both the hypertension and UAE are sensitive to high NaCl loading in female and male MWF/Fub rats. Thus, an inborn nephron deficit may lead to salt-sensitive hypertension and renal dysfunction.

Early onset of chondroitin sulfate and osteopontin expression in angiotensin ii-dependent left ventricular hypertrophy

BACKGROUND Chondroitin sulfate proteoglycan (CSPG) is expressed during embryonic heart development and osteopontin (OPN) is an important mediator of the profibrotic effects of angiotensin II (Ang II). The objective of this study was to analyze extracellular matrix protein (ECMP) expression in Ang II-dependent left ventricular (LV) hypertrophy (LVH), LV dysfunction, and to investigate right ventricular changes. METHODS We used the hypertensive transgenic rat line TGR(mRen2)27 (Ren2), which provides a well-established model of Ang II-driven cardiac remodeling and progressive LV dysfunction and compared young Ren2 rats at the age of 10 weeks with normotensive Sprague-Dawley (SD) rats (n = 15, each group). RESULTS Systolic blood pressure and LV weight were elevated in Ren2 compared to SD rats (P < .001). Left ventricular end-diastolic pressure was not altered in Ren2, but +dP/dt(max) and -dP/dt(max) were decreased in Ren2 compared to SD rats (P < .01). Cardiomyocyte widths, interstitial and perivascular fibrosis were increased in left and right ventricles of Ren2 in comparison to SD rats (P < .05). The LV mRNA expression of atrial natriuretic factor, OPN, and collagen I were increased in Ren2 as compared to SD rats (P < .05, respectively). The LV CSPG, collagen I, collagen III, fibronectin, laminin, and OPN contents were elevated in Ren2 compared to SD rats as measured by image analysis and Western blotting (P < .01). CONCLUSIONS Reactivated expression of CSPG in the adult heart may be an important component of LV ECMP remodeling in LVH. Elevated cardiac OPN expression could mediate the alterations in LV ECMP pattern in Ang II-dependent LVH, thus contributing to the development of contractile dysfunction in young Ren2 rats.

Renal Endothelin ETA/ETB Receptor Imbalance Differentiates Salt-Sensitive From Salt-Resistant Spontaneous Hypertension

It is unclear why a subgroup of patients with essential hypertension develop salt-sensitive hypertension with progression of target organ damage over time. We evaluated the role of the renal endothelin (ET) system in the stroke-prone spontaneously hypertensive rat (SHRSP) model of salt-sensitive spontaneous hypertension (SS-SH) compared with the spontaneously hypertensive rat (SHR) model of salt-resistant spontaneous hypertension (SR-SH). Both strains were studied after either sham-operation on a normal diet (Sham) or after unilateral nephrectomy and high NaCl loading (NX-NaCl) with 4% NaCl in diet for 6 weeks (n=10, respectively). Systolic blood pressure (SBP) increased only in SHRSP-NX-NaCl compared with SHRSP-Sham (250±6 versus 172±5 mm Hg, P <0.0001). SBP remained unchanged in SHR-NX-NaCl compared with SHR-Sham. In SHRSP-NX-NaCl animals, urinary albumin and ET-1 excretion, renal ET-1 mRNA expression, glomerulosclerosis index, and tubulointerstitial damage index were elevated compared with SHRSP-Sham (P <0.05, respectively), whereas no significant changes were found in SHR after NX-NaCl. Urinary sodium excretion (UNa+) was significantly reduced by 38% in SHRSP-NX-NaCl compared with SHR-NX-NaCl (P <0.005, respectively). SHR animals showed a similar increase in both renal ETA and ETB receptor densities after NX-NaCl (2.2-fold, P <0.05). In contrast, SHRSP-NX-NaCl developed a significantly more pronounced increase in ETA compared with ETB binding (4.7-fold versus 2.4-fold, P <0.05, compared with SHRSP-Sham, respectively), resulting in a significant 2.1-fold increase in ETA/ETB receptor ratio only in the SHRSP-NX-NaCl (P <0.05). Thus, activation of the renal ET system together with an increased ETA/ETB receptor ratio may contribute to the development and progression of SS-SH.

Acute blood pressure effects of YC-1-induced activation of soluble guanylyl cyclase in normotensive and hypertensive rats.

We used YC‐1 as a pharmacological tool to investigate the short‐term blood pressure effects of NO‐independent activation of sGC in normotensive and hypertensive rats. Four groups of normotensive Wistar‐Kyoto rats were treated by i.v. injection with vehicle (V), YC‐1 (YC‐1), sodium nitroprusside (SNP), or YC‐1 and SNP (YC‐1+SNP). Hypertension was induced in four additional groups of WKY rats by 3 weeks of oral treatment with L‐NAME. These animals were investigated with the same protocol as the normotensive animals: L‐NAME/V, L‐NAME/YC‐1, L‐NAME/SNP, L‐NAME/YC‐1+SNP. YC‐1 lowered mean arterial blood pressure (MAP) in normotensive and hypertensive animals similarly to SNP alone (P<0.05, respectively). The combination of YC‐1 with SNP caused a strong decrease of MAP in both the hypertensive and normotensive animals (P<0.05, respectively). SNP with YC‐1 also induced a pronounced cyclic GMP increase in the aorta. This study shows for the first time the blood pressure lowering potential of bimodal targeting of the NO‐sGC‐system.

Endothelin-A Receptor Blockade Prevents Left Ventricular Hypertrophy and Dysfunction in Salt-Sensitive Experimental Hypertension

Background—Salt-sensitive hypertension represents a major cause of left ventricular (LV) dysfunction. We therefore explored the potential effects of the selective endothelin-A (ETA) receptor antagonist darusentan on the development of hypertension, LV hypertrophy (LVH), and dysfunction in a genetic rat model of salt-sensitive hypertension. Methods and Results—Animals from the salt-sensitive Sabra rat strain (SBH/y) and the salt-resistant strain (SBN/y) were treated with either normal diet (SBH/y and SBN/y) or with deoxycorticosterone-acetate (DOCA) and salt (SBN/y-DOCA and SBH/y-DOCA). Additional groups were treated with 50 mg · kg−1 · d−1 of darusentan (SBH/y-DOCA-DA and SBN/y-DOCA-DA). Systolic blood pressure and LV weight increased in response to DOCA only in the SBH/y strain (+75 mm Hg and +30%;P <0.05). LV end-diastolic pressure increased and −dP/dtmax decreased in SBH/y-DOCA compared with SBH/y (P <0.05). This was paralleled by a 5-fold upregulation of LV mRNA expression of atrial natriuretic factor (ANF) and a significant reduction of sarcoplasmic reticulum (SR) Ca2+-reuptake and the SR Ca2+-ATPase to phospholamban protein ratio (−30%). Whereas treatment with darusentan in SBH/y-DOCA-DA reduced the SBP increase by 50%, LVH elevation of ANF mRNA and LV dysfunction were completely prevented (P <0.05); this was associated with a normalization of SR Ca2+-reuptake and SR Ca2+-ATPase to phospholamban ratio by darusentan (P <0.05). A moderate elevation of interstitial fibrosis in SBH/y-DOCA (P <0.05) remained unaffected by darusentan treatment. Conclusion—In the Sabra model of salt-sensitive hypertension, ETA-receptor blockade demonstrated striking effects on the prevention of LVH and LV dysfunction beyond its considerable antihypertensive effect.

THE ROLE OF ENDOTHELIN IN HYPERTENSION

The endothelins are 21-ammo-acid peptides which may play a role in the pathogenesis of hypertension. There is increasing evidence that the endothelins have a central function in mediating end-organ damage in hypertension, and that important effects of endothelin in the pathogenesis of hypertension may be based on the interactions of the endothelins and the renin-angiotensin and the nitric oxide systems.

Genetic low nephron number hypertension is associated with dysregulation of the hepatic and renal insulin-like growth factor system during nephrogenesis.

Objective Low nephron number may represent a major determinant of human primary hypertension in adult life. This hypothesis is supported by a genetic rat model, namely the Munich–Wistar–Frömter (MWF) rat, which demonstrates an inherited deficit in nephron number and the development of spontaneous hypertension. Insulin-like growth factor (IGF) I and II exert endocrine and paracrine effects that are required for normal growth and nephron development. We tested the hypothesis that low nephron number is already present during fetal development, and the expression pattern of important molecules of the IGF system is altered in MWF rat during the critical period of kidney development. Methods We compared MWF and normal Wistar rats during nephrogenesis at day 19 of fetal development (E19) and adult rats at postnatal day 100 (D100). Histomorphometric analysis was performed by stereological methods. Quantitative messenger RNA and protein expression was determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results At E19, glomerular density (−32%) and hepatic mRNA (−48%) and protein (−18%) expression of IGF-I were decreased (P < 0.05, respectively), whereas renal mRNA expression of IGF-II receptor (+52%) and IGF binding protein 3 (+113%) were increased in MWF compared with Wistar rats (P < 0.05, respectively). Systolic blood pressure, urinary albumin excretion, and mean glomerular area were significantly elevated in MWF compared with Wistar rats at D100 (P < 0.05, respectively). Conclusions The fetal expression of IGF system molecules in the MWF rat model points towards a link between the decreased availability of active IGF-I and IGF-II and the fetal development of low nephron number, with manifestation of genetic hypertension in adult life.

Effects of angiotensin II subtype 1 receptor blockade on cardiac fibrosis and sarcoplasmic reticulum Ca2+ handling in hypertensive transgenic rats overexpressing the Ren2 gene.

Objective We evaluated the effects of angiotensin II subtype 1 (AT1) receptor antagonism on cardiac fibrosis and sarcoplasmic (SR) Ca2+ handling in a transgenic rat model of renin-dependent left ventricular (LV) hypertrophy (LVH). Methods Hypertensive transgenic rats overexpressing the Ren2 gene (TGR(mRen2)27) were treated between 10 and 30 weeks of age with the angiotensin II subtype 1 (AT1) receptor antagonist, eprosartan, in an antihypertensive (Ren2-E60, 60 mg/kg per day) and a non-antihypertensive (Ren2-E6, 6 mg/kg per day) dose applied intraperitoneally via osmotic-mini-pumps. They were compared to age-matched Ren2 and Sprague–Dawley (SD) control rats receiving 0.9% NaCl as vehicle via osmotic mini-pumps (Ren2-Vehicle, SD-Vehicle, respectively). Results Systolic blood pressure (SBP), LV weight, LV end-diastolic pressure (LVEDP), and cardiac fibrosis were elevated in Ren2-Vehicle, while diastolic function (−d P/d tmax) and sarcoplasmic reticulum (SR) Ca2+ uptake were decreased in Ren2-Vehicle compared to SD-Vehicle (P < 0.05, respectively). SBP was not altered in Ren2-E6, but reduced to normotensive levels in Ren2-E60 compared to Ren2-Vehicle and SD-Vehicle (P < 0.0001). In both Ren2-E6 and Ren2-E60, LV weights were reduced and LVEDP and − d P/d tmaxnormalized compared to Ren2-Vehicle (P < 0.05). SR Ca2+ uptake was normalized in both Ren2-E6 and Ren2-E60. Cardiac fibrosis did not change in Ren2-E6, but perivascular LV fibrosis and hydroxyprolin content were reduced in Ren2-E60 compared to Ren2-Vehicle (P < 0.05, respectively). Conclusions Normalization of LV SR Ca2+ uptake is an important mechanism by which AT1receptor antagonism improves LV diastolic dysfunction independent from a reduction of SBP and cardiac fibrosis in the TGR (mRen2)27 model.

Nephroprotective effects of the endothelin ETA receptor antagonist darusentan in salt-sensitive genetic hypertension

We tested the effect of selective endothelin ET(A) receptor blockade on the development renal damage in the Sabra rat model of genetic salt-sensitivity. Animals from the salt-sensitive (SBH/y) and salt-resistant strains (SBN/y) were either salt-loaded with deoxycorticosterone acetate and salt (DOCA) or fed a normal diet. Additional salt-loaded groups were also treated with the selective ET(A) antagonist darusentan (DA). Salt-loading in SBH/y increased systolic blood pressure by 75 mm Hg and urinary albumin excretion 23-fold (P<0.0001). Darusentan attenuated the rise of systolic blood pressure (50%) and urinary albumin excretion (63%, P<0.01, respectively). Salt-loading in SBH/y was associated with significant increased osteopontin mRNA expression as well as glomerulosclerosis and tubulointerstitial damage in the kidney (P<0.05, respectively). This was either significantly reduced or normalized by darusentan (P<0.05, respectively). Thus, darusentan confers a significant renal protection in the Sabra model of salt-sensitive hypertension.