Ulrike Raff

Differential role of angiotensin II receptor subtypes on endothelial superoxide formation

The physiological role of the angiotensin II AT2 receptor subtype is not fully characterized. We studied whether AT2 receptor could antagonize AT1 mediated superoxide formation in endothelial cells. In quiescent human umbilical vein endothelial cells (HUVEC) superoxide formation was measured after long‐term incubation (6 h) with angiotensin II in the presence or absence of its receptor blocker candesartan (AT1) or PD123319 (AT2) using the cytochrome c assay. In separate experiments, the effects of AT2 mediated effects on activities of cellular phosphates including the src homology 2 domain containing phosphatases (SHP‐1) was studied. The basal superoxide formation (0.19±0.03 nmol superoxide mg protein−1 min−1) in HUVEC was increased by 37.1% after exposure to angiotensin II (100 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 μM) as well as the tyrosine kinase inhibitor genistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the superoxide formation by 73.7% in intact cells. Stimulation of AT2 went along with an increased activity of tyrosine phosphatases in total cell lysates (29.8%) and, in particular, a marked stimulation of src homology 2 domain containing phosphatases (SHP‐1, by 293.4%). The tyrosine phosphatase inhibitor vanadate, in turn, prevented the AT2 mediated effects on superoxide formation. The expression of both angiotensin II receptor subtypes AT1 and AT2 was confirmed by RT–PCR analysis. It is concluded that AT2 functionally antagonizes the AT1 induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.

Renal resistive index in addition to low-grade albuminuria complements screening for target organ damage in therapy-resistant hypertension.

Objective We examined the value of renal resistive index (RRI) for prevalence of cardiovascular target organ damage in therapy-resistant hypertension in comparison to low-grade albuminuria. Methods Eighty-four patients with therapy-resistant hypertension (age 59.7 ± 8.1 years) were screened for cardiovascular target organ damage with coronary computed tomography, cardiac magnetic resonance imaging (MRI), Doppler sonography for the assessment of carotid intima media thickness and, RRI, pulse wave velocity and for low-grade albuminuria of at least 10 mg/day in men and 15 mg/day in women, respectively. Results In patients with RRI greater than 0.7 pulse wave velocity (11.6 ± 3.7 vs. 9.8 ± 2.2 m/s; P = 0.02) intima media thickness (0.85 ± 0.09 vs. 0.76 ± 0.1 mm; P = 0.007) and Agatston score of coronary calcification (640 ± 915 vs. 129 ± 256; P = 0.05) were increased, whereas left ventricular mass (127 ± 24.5 vs. 125 ± 15.0 g; P = 0.70) was similar between the two groups. When patients were categorized according to low-grade albuminuria left ventricular mass was significantly higher in those with low-grade albuminuria (123 ± 25.8 vs. 135 ± 15.7 g; P = 0.01), whereas vascular parameters (intima media thickness, Agatston score, pulse wave velocity) did not differ between the two groups. Conclusion In patients with therapy-resistant hypertension RRI reflects functional and structural vascular parameters, whereas low-grade albuminuria is related to cardiac structural changes. Thus, measurement of RRI in addition to low-grade albuminuria complements screening for target organ damage in therapy-resistant hypertension.

Oxidized LDL and its Compound Lysophosphatidylcholine Potentiate AngII-Induced Vasoconstriction by Stimulation of RhoA

RhoA stimulates vascular tone by increasing smooth muscle Ca(2+) sensitivity, e.g., in atherosclerosis. This study was an investigation of the influence of oxidized LDL (OxLDL), which accumulates in atherosclerotic plaques, on vascular tone induced by angiotensin II (AngII), with particular emphasis on the RhoA pathway. OxLDL had no influence on unstimulated vascular tone of isolated rabbit aorta, but it potentiated contractile responses induced by AngII. The Ca(2+)-antagonist felodipin partially prevented potentiation of contractile responses, whereas the AT(1) receptor antagonist losartan blunted AngII responses in presence and in absence of OxLDL. Rho-kinase inhibition by Y27632 abolished potentiation of contractile responses, and RhoA inhibition by C3-like transferase partially prevented it, suggesting that OxLDL activated RhoA. Activation of RhoA was further analyzed by detection of its translocation to the cell membrane after stimulation with OxLDL. Western blot analysis of aorta homogenates, as well as direct visualization in cultured smooth muscle cells using confocal laser scan microscopy, revealed that OxLDL potently activated RhoA. The effect of OxLDL was mimicked by its compound lysophosphatidylcholine, and C3 inhibited both lysophosphatidylcholine and OxLDL-induced RhoA stimulation. In conclusion, OxLDL stimulates the RhoA pathway, resulting in potentiation of AngII-induced vasoconstriction. Lysophosphatidylcholine mimics the OxLDL effect, consistent with a causal role of this OxLDL compound. Stimulation of RhoA by OxLDL may contribute to vasospasm in atherosclerotic arteries.

Endogenous Erythropoietin and the Association with Inflammation and Mortality in Diabetic Chronic Kidney Disease

BACKGROUND AND OBJECTIVES Anemia and inflammation are prevalent in diabetic patients with chronic kidney disease (CKD). The role of endogenous erythropoietin (EPO) in the pathophysiology of anemia in chronic diseases and its relationship to clinical outcomes remain uncertain. In this cohort study, we aimed to identify factors associated with endogenous EPO levels and investigate their relation to all-cause mortality. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Between 2004 and 2005, 215 patients with type 2 diabetes were enrolled. Exclusion criteria included stage renal disease ESRD and any form of anemia therapy. The association of EPO levels with clinical and laboratory variables was investigated by linear regression modeling. Predictors of all-cause mortality were evaluated by Cox proportional hazards analyses. RESULTS Patients (median age, 67 years; 52% men; median duration of diabetes, 10 years; median estimated GFR, 49 ml/min per 1.73 m²) were followed for up to 7.0 years. Forty-one patients died. Elevated EPO levels were independently associated with elevated C-reactive protein, low ferritin, and hypertension, in a multivariate model that also included age, cardiovascular disease, kidney function, albumin, cholesterol, and hemoglobin. Higher EPO levels were independently predictive for mortality, as were age, low levels of albumin, and cardiovascular disease. CONCLUSIONS In diabetic patients with CKD, elevated endogenous EPO levels were predictive for mortality and were related mainly to markers of inflammation, independent of kidney function, and despite low hemoglobin levels. Understanding the phenomenon of EPO resistance and iron dysregulation caused by inflammation is crucial for effective and safe treatment of anemia in patients with CKD.

Skin Sodium Concentration Correlates with Left Ventricular Hypertrophy in CKD

The pathogenesis of left ventricular hypertrophy in patients with CKD is incompletely understood. Sodium intake, which is usually assessed by measuring urinary sodium excretion, has been inconsistently linked with left ventricular hypertrophy. However, tissues such as skin and muscle may store sodium. Using 23sodium-magnetic resonance imaging, a technique recently developed for the assessment of tissue sodium content in humans, we determined skin sodium content at the level of the calf in 99 patients with mild to moderate CKD (42 women; median [range] age, 65 [23-78] years). We also assessed total body overhydration (bioimpedance spectroscopy), 24-hour BP, and left ventricular mass (cardiac magnetic resonance imaging). Skin sodium content, but not total body overhydration, correlated with systolic BP (r=0.33, P=0.002). Moreover, skin sodium content correlated more strongly than total body overhydration did with left ventricular mass (r=0.56, P<0.001 versus r=0.35, P<0.001; P<0.01 between the two correlations). Linear regression analysis demonstrated that skin sodium content is a strong explanatory variable for left ventricular mass, unaffected by BP and total body overhydration. In conclusion, we found skin sodium content to be closely linked to left ventricular mass in patients with CKD. Interventions that reduce skin sodium content might improve cardiovascular outcomes in these patients.

L-Arginine does not affect renal morphology and cell survival in ischemic acute renal failure in rats.

Background:L-Arginine (L-Arg), a substrate of nitric oxide synthases, improves renal function in ischemic acute renal failure (iARF). We evaluated whether L-Arg improves renal morphology and cell survival in the course of iARF. Methods and Results: iARF was induced in rats by bilateral clamping of renal arteries for 45 min. L-Arg was applied intraperitoneally during clamping, and orally during 14 days of follow-up. Morphology and cell survival of renal cortical and medullar tissue was analyzed on days 1, 3, 7, and 14 of follow-up, using toluidine blue staining and immunohistochemistry of perfusion-fixated tissue, and Western blot analysis of tissue homogenate. Renal tubular injury showed typical features of necrosis and was most severe on days 1 and 3 after clamping, predominantly in S3 segments, with almost complete recovery by day 14. Enhanced medullar monocyte infiltration, determined by ED-1 expression as well as by immunohistochemistry, and enhanced expression of proliferating cell nuclear antigen (PCNA), indicative of proliferation and regeneration, accompanied these morphological changes. Compared to controls, L-Arg had no impact on renal morphology, ED-1, and PCNA expression. Furthermore, expression of markers of apoptosis Bcl-2, Bax, and cleaved caspase-3 was only slightly increased in iARF rats, compared to sham-operated animals, and was also not influenced by L-Arg. Conclusion: Despite its repeatedly reported positive impact on renal function as also shown in our model, L-Arg does not alter cell death and proliferation in the course of iARF in our model. Thus, different mechanisms have to be considered, in particular improved intrarenal hemodynamics.

Reversibility of the Effects of Aliskiren in the Renal Versus Systemic Circulation

BACKGROUND AND OBJECTIVES Renal hemodynamic effects of inhibitors of the renin-angiotensin system can increase the risk of acute kidney injury under certain conditions. The BP-lowering effects of the renin inhibitor aliskiren are sustained 3-4 weeks after withdrawal. In this study, the reversibility of the renal hemodynamic effects of aliskiren was tested. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this open-label study, renal perfusion was measured by 1.5-T magnetic resonance imaging-arterial spin labeling in 34 subjects with arterial hypertension before aliskiren (pre-aliskiren), after 4 weeks of aliskiren treatment (300 mg), and 4-5 days (∼2.5-3.0× plasma half-life) after withdrawal (post-aliskiren). RESULTS Aliskiren reduced systolic BP from 152 ± 14 to 139 ± 16 mmHg (P<0.0001), which was sustained post-aliskiren (136 ± 13 mmHg, P=1.00 versus aliskiren). Aliskiren significantly altered renal perfusion (P=0.005), increasing from 272 ± 25 pre-aliskiren to 287 ± 29 ml/min per 100 g during aliskiren (P=0.03). This increase in renal perfusion was completely reversed post-aliskiren (272 ± 26 ml/min per 100 g, P=0.03 versus aliskiren, P=0.63 versus pre-aliskiren). No changes were noted in urinary angiotensinogen levels. Plasma renin activity was reduced by aliskiren, which was sustained post-aliskiren. Angiotensin II and aldosterone were reduced by aliskiren but recovered post-aliskiren to pre-aliskiren levels. CONCLUSIONS After withdrawal of aliskiren, the effects on BP were sustained, whereas increase in renal perfusion was reversed, which was associated with recovery of angiotensin II and aldosterone to pretreatment levels. Renal hemodynamic effects are more readily reversible than systemic effects of aliskiren.

Prevention of electrocardiographic left ventricular remodeling by the angiotensin receptor blocker olmesartan in patients with type 2 diabetes.

Objective: To assess the ability of olmesartan (OLM) to prevent or delay left ventricular remodeling and hypertrophy in patients with type 2 diabetes. Methods: This prespecified ECG substudy of Randomised OlmesArtan and Diabetes MicroAlbuminuria Prevention (ROADMAP), which compared OLM with placebo, assessed the signs of left ventricular remodeling in patients with a 12-lead ECG at baseline and after at least 2 years. Cornell voltage QRS duration product (primary objective), Cornell voltage index and Sokolow–Lyon index were assessed. Results: In total, 9418 ECG recordings and 1513 patients from ROADMAP were analyzed (placebo, n = 736; OLM, n = 777). Quartiles defined by baseline Cornell voltage QRS duration product were assessed and the proportion of patients in the highest quartile (≥200 mV*ms) increased from 24.0 to 26.5% in the placebo group and decreased from 25.5 to 22.3% in the OLM group [odds ratio (OR) 0.598 (95% confidence interval [CI] 0.440–0.813); P = 0.0011]. The OR did not change after adjustment for baseline parameters. By the end of study, 38.7% of patients in the placebo group and 34.7% in the OLM group shifted from a lower to a higher quartile or remained in the highest quartile of Cornell voltage QRS duration product [OR 0.797 (95% CI 0.637–0.996); P = 0.0465]. This translated into a 20.3% risk reduction with OLM and suggested OLM attenuated the progression of left ventricular remodeling versus placebo. Conclusion: OLM substantially delayed the development of left ventricular remodeling in type 2 diabetes. This effect was not explained by the differences in blood pressure control. Thus, OLM delayed the onset of microalbuminuria, as well as the ECG signs of cardiac structural adaptation in type 2 diabetes.

Postchallenge hyperglycemia is closely related with early vascular damage in overweight and obese patients.

Objective There is ongoing discussion on how best to screen for diabetes mellitus. Previous studies suggest that an abnormal oral glucose tolerance test (OGTT) is better than fasting glucose levels in predicting cardiovascular mortality, which is largely determined by macrovascular complications in type 2 diabetes. We examined the relationship between screening methods for diabetes and indices of vascular damage in young individuals at high risk of type 2 diabetes. Methods Overweight and obese individuals (n = 76, average age 38 ± 6 years) were screened for diabetes by measuring fasting glucose levels, HbA1c and by performing the OGTT. Indices of early vascular damage, including the central augmentation index (cAIx) and pulse pressure amplification (PPA), were assessed by pulse wave analysis (Sphygmocor). Linear regression analyses were performed to identify independent predictors of vascular damage. Results Central SBP and DBP (BPs) were best predicted by age and by peripheral BP levels. cAIx was independently predicted by age (r = +0.324, P = 0.008), DBP (r = +0.294, P = 0.011) and 2-h glucose values of the OGTT (r = +0.390, P = 0.001). PPA was independently predicted by age (r = −0.445, P < 0.001) and 2-h glucose value of the OGTT (r = −0.353, P = 0.003). Conclusions The 2-h value of the OGTT was superior to fasting glucose levels and HbA1c in predicting cAIx and PPA in young individuals at high risk of type 2 diabetes. Cardiovascular mortality is largely determined by macrovascular complications in type 2 diabetes, and these data suggest that diabetes screening by OGTT may help to identify those individuals with the greatest risk of future vascular complications.

Left ventricular structure in patients with mild to moderate chronic kidney disease – an MRI study

Introduction The high burden of left ventricular (LV) abnormalities in patients with advanced chronic kidney disease (CKD) is well established. However, less is known about the prevalence, patterns, and determinants of LV abnormalities in patients with early CKD. Methods We examined LV structure in 290 patients with a median estimated glomerular filtration rate (eGFR) of 51 ml/min per 1.73 m2 by magnetic resonance imaging (MRI). We explored associations with clinical and hemodynamic parameters, hydration (bioimpedance), endothelial function, inflammation (including C-reactive protein and tumor necrosis factor−α and its soluble receptors) and mineral bone disease (MBD) markers (including vitamin D, parathyroid hormone, α-klotho and fibroblast growth factor−23). Results Normal geometry was found in 56% of patients, dilation in 4%, concentric remodeling in 10%, and LV hypertrophy in 29%. Linear regression analysis revealed that greater LV mass was independently associated with male sex, greater body mass index (BMI), and higher 24-hour systolic blood pressure (24-hour SBP). Concentric remodeling was independently associated with age, male sex, higher 24-hour SBP, and greater hemoglobin levels. Surprisingly, neither hydration status, nor endothelial function, nor any of the inflammatory or MBD parameters added significantly to these models. Conclusion Abnormal LV structure was found in almost one-half of the patients. Reducing BMI and 24-hour SBP and avoiding high hemoglobin concentrations appear to be the key factors to prevent abnormal LV remodeling in patients with mild-to-moderate CKD.